Patent Application
20180305451
Naïve T cells must receive two independent signals from antigen-presenting cells (APC) in order to become productively activated. The first, Signal 1, is antigen-specific and occurs when T cell antigen receptors encounter the appropriate antigen-MHC complex on the APC. The fate of the immune response is determined by a second, antigen-independent signal (Signal 2) which is delivered through a T cell costimulatory molecule that engages its APC-expressed ligand. This second signal could be either stimulatory (positive costimulation) or inhibitory (negative costimulation or coinhibition). In the absence of a costimulatory signal, or in the presence of a coinhibitory signal, T-cell activation is impaired or aborted, which may lead to a state of antigen-specific unresponsiveness (known as T-cell anergy), or may result in T-cell apoptotic death.
Costimulatory molecule pairs usually consist of ligands expressed on APCs and their cognate receptors expressed on T cells. The prototype ligand/receptor pairs of costimulatory molecules are B7/CD28 and CD40/CD40L. The B7 family consists of structurally related, cell-surface protein ligands, which may provide stimulatory or inhibitory input to an immune response. Members of the B7 family are structurally related, with the extracellular domain containing at least one variable or constant immunoglobulin domain.
Both positive and negative costimulatory signals play critical roles in the regulation of cell-mediated immune responses, and molecules that mediate these signals have proven to be effective targets for immunomodulation. Based on this knowledge, several therapeutic approaches that involve targeting of costimulatory molecules have been developed, and were shown to be useful for prevention and treatment of cancer by turning on, or preventing the turning off, of immune responses in cancer patients and for prevention and treatment of autoimmune diseases and inflammatory diseases, as well as rejection of allogenic transplantation, each by turning off uncontrolled immune responses, or by induction of “off signal” by negative costimulation (or coinhibition) in subjects with these pathological conditions.
Manipulation of the signals delivered by B7 ligands has shown potential in the treatment of autoimmunity, inflammatory diseases, and transplant rejection. Therapeutic strategies include blocking of costimulation using monoclonal antibodies to the ligand or to the receptor of a costimulatory pair, or using soluble fusion proteins composed of the costimulatory receptor that may bind and block its appropriate ligand. Another approach is induction of co-inhibition using soluble fusion protein of an inhibitory ligand. These approaches rely, at least partially, on the eventual deletion of auto- or allo-reactive T cells (which are responsible for the pathogenic processes in autoimmune diseases or transplantation, respectively), presumably because in the absence of costimulation (which induces cell survival genes) T cells become highly susceptible to induction of apoptosis. Thus, novel agents that are capable of modulating costimulatory signals, without compromising the immune system's ability to defend against pathogens, are highly advantageous for treatment and prevention of such pathological conditions.
Costimulatory pathways play an important role in tumor development. Interestingly, tumors have been shown to evade immune destruction by impeding T cell activation through inhibition of co-stimulatory factors in the B7-CD28 and TNF families, as well as by affecting regulatory T cells, which inhibit anti-tumor T cell responses (see Wang (2006), “Immune Suppression by Tumor Specific CD4+ Regulatory T cells” in Cancer. Semin. Cancer. Biol. 16:73-79; Greenwald, et al. (2005), “The B7 Family Revisited”, Ann. Rev. Immunol. 23:515-48; Watts (2005) “TNF/TNFR Family Members in Co-stimulation of T Cell Responses”, Ann. Rev. Immunol. 23:23-68; Sadum, et al. (2007), “Immune Signatures of Murine and Human Cancers Reveal Unique Mechanisms of Tumor Escape and New Targets for Cancer Immunotherapy”, Clin. Canc. Res. 13(13): 4016-4025). Such tumor expressed co-stimulatory molecules have become attractive cancer biomarkers and may serve as tumor-associated antigens (TAAs). Furthermore, costimulatory pathways have been identified as immunologic checkpoints that attenuate T cell dependent immune responses, both at the level of initiation and effector function within tumor metastases. As engineered cancer vaccines continue to improve, it is becoming clear that such immunologic checkpoints are a major barrier to the vaccines' ability to induce therapeutic anti-tumor responses. In that regard, costimulatory molecules can serve as adjuvants for active (vaccination) and passive (antibody-mediated) cancer immunotherapy, providing strategies to thwart immune tolerance and stimulate the immune system.
In addition, such agents could be of use in other types of cancer immunotherapy, such as adoptive immunotherapy, in which tumor-specific T cell populations are expanded and directed to attack and kill tumor cells. Agents capable of augmenting such anti-tumor response have great therapeutic potential and may be of value in the attempt to overcome the obstacles to tumor immunotherapy. Recently, novel agents that modulate several costimulatory pathways were indeed introduced to the clinic as cancer immunotherapy.
Regulating costimulation using agonists and/or antagonists to various costimulatory proteins has been extensively studied as a strategy for treating autoimmune diseases, graft rejection, allergy and cancer. This field has been clinically pioneered by CTLA4-Ig (Abatacept, Orencia®) which is approved for treatment of RA, mutated CTLA4-Ig (Belatacept, Nulojix®) for prevention of acute kidney transplant rejection and by the anti-CTLA4 antibody (Ipilimumab, Yervoy®), recently approved for the treatment of melanoma. Other costimulation regulators are currently in advanced stages of clinical development including anti-PD-1 antibody (BMS-936558) which is in development for treatment of Non-Small Cell Lung cancer and other cancers. Furthermore, such agents are also in clinical development for viral infections, for example the anti PD-1 Ab, MDX-1106, which is being tested for treatment of hepatitis C, and the anti-CTLA-4 Ab CP-675,206 (tremelimumab) which is in a clinical trial in hepatitis C virus-infected patients with hepatocellular carcinoma.
In addition recently researchers have developed compounds which target regulatory T cells (iTregs) for use in immunotherapy. With respect thereto it is known that inducible regulatory T cells (iTregs) are commonly seen in many tumors, and form the major subset of immune suppressor cells in the tumor tissue and moreover represent a major tumor resistance mechanism from immune surveillance. Accordingly, iTregs are therefore viewed as important cellular targets for cancer therapy.
Multiple immune-checkpoint receptors, such as CTLA4, PD-1, TIM3 and LAG3, are expressed at high levels on the surface of iTregs and directly promote Treg cell-mediated suppression of effector immune responses. Therefore, some immune-checkpoint antibodies may, in addition to increasing CTL immunity, further block the immunosuppressive activity of iTregs and thereby enhance anti-tumor immunity. For example, CTLA4 blockade by ipilimumab both enhances effector T cell activity, and inhibits Treg immunosuppressive activity.
B cells play a critical role in recognition of foreign antigens and they produce the antibodies necessary to provide protection against various types of infectious agents. T cell help to B cells is a pivotal process of adaptive immune responses. Follicular helper T (Tfh) cells are a subset of CD4+ T cells specialized in B cell help (reviewed by Crotty, Annu. Rev. Immunol. 29: 621-663, 2011). Tfh cells express the B cell homing chemokine receptor, CXCR5, which drives Tfh cell migration into B cell follicles within lymph nodes in a CXCL13-dependent manner. The requirement of Tfh cells for B cell help and T cell-dependent antibody responses indicates that this cell type is of great importance for protective immunity against various types of infectious agents, as well as for rational vaccine design. In addition, regulatory B cells (Bregs) have a role in impairing effective clearance of tumors. The mechanisms for Bregs effects in cancer are not well understood, however one proposed mechanism is through inhibition of cytotoxic CD8+ T cells.
NK cells are effector lymphocytes of the innate immune system that are known to be involved in killing of pathological or diseased cells such as cancer and infected cells and pathogens. Natural killer cells have the capacity to kill cellular targets and produce cytokines without prior specific sensitization. NK cells are unique, as they have the ability to recognize stressed cells in the absence of antibodies and MHC, allowing for a much faster immune reaction. This role is especially important because harmful cells that are missing MHC 1 markers cannot be detected and destroyed by other immune cells, such as T cells.
Induction of immune tolerance has long been considered the “holy grail” for autoimmune disease therapy. The immune system has the reciprocal tasks to protect the host against invading pathogens, but simultaneously to prevent damage resulting from unwanted reactions to self-antigens. The latter part is known as immune tolerance and performed by a complex set of interactive and complementary pathways, which regulate immune responses. T cells have the ability to react to a variety of antigens, both self and nonself. Therefore, there are many mechanisms that exist naturally to eliminate potentially self-reactive responses—this is known as natural tolerance. The main mechanism for eliminating potential autoreactive T cells occurs in the thymus and is known as central tolerance. Some potentially autoreactive T cells escape central tolerance and, therefore, peripheral tolerance mechanisms also exist. Despite these mechanisms, some self-reactive T cells may ‘escape’ and be present in the repertoire; it is believed that their activation may lead to autoimmune disease.
Studies on therapeutic tolerance have attempted to induce and amplify potent physiological mechanisms of tolerance in order to eliminate or neutralize self-reactive T cells and prevent or treat autoimmune diseases. One way to induce tolerance is by manipulation of the interaction between costimulatory ligands and receptors on antigen presenting cells (APCs) and lymphocytes.
CTLA-4 is the most extensively studied costimulatory molecule which down-regulates immune responses. The attributes of immunosuppressive qualities and capacity to induce tolerance have made its recognition as a potential immuno-therapeutic agent for autoimmune mediated inflammatory disorders. Abatacept (commercial name: Orencia) is a fusion protein composed of the ECD (extracellular domain) of CTLA-4 fused to the Fc fragment of hIgG1. Abatacept is believed to induce costimulation blockade, which has been approved for treating patients with rheumatoid arthritis, by effectively interfering with the inflammatory cascade.
Induction of disease control with the current therapies, followed by progressive withdrawal in parallel with re-establishing immune tolerance, may be an attractive approach in the future of autoimmune therapies. Furthermore, due to their immune specificity, in the absence of global immunosuppression, such therapies should be safe for chronic use.
Certain mechanisms are known to be widespread in various autoimmune diseases. T helper type 1 (Th1) cells are induced by IL-12 and produce IFN-γ; while T helper type 2 (Th2) cells secrete IL-4, IL-5 and IL-13. Th1 cells can mediate proinflammatory or cell-mediated immune responses, whereas Th2 cells mainly promote certain types of humoral immunity. Some immune related diseases, such as autoimmune reactions, inflammation, chronic infection and sepsis, are characterized by a dysregulation of the pro-versus anti-inflammatory tendencies of the immune system, as well as an imbalance in the Th1 versus Th2 cytokine balance. During inflammation, induction of a shift in the balance from Th1 to Th2 protects the organism from systemic ‘overshooting’ with Th1/pro-inflammatory cytokines, by reducing the inflammatory tendencies of the immune system. Immunomodulatory therapies that are associated with a Th1 to Th2 immune shift have protective effects in Th1-mediated autoimmune diseases, such as multiple sclerosis and rheumatoid arthritis. For example, Laquinimod, which has demonstrated efficacy in animal models of several autoimmune diseases including MS, shows immunomodulatory effects through Th1/Th2 shift, and does not lead to immunosuppression. Glatiramer acetate (Copaxone®) also induces Th1/Th2 shift with decreased secretion of proinflammatory cytokines, and increased secretion of antiinflammatory cytokines. Furthermore, glatiramer acetate-specific Th2 cells are able to migrate across the blood-brain barrier and cause in situ bystander suppression of autoaggressive Th1 T cells.
Certain immune cells and immune cell signal transduction pathways are promising targets for new agents for treating immune disorders. For example Th1, Th17, Th2 and regulatory T cells (Tregs) play important roles in modulating autoimmunity and inflammation. Mounting evidence from numerous studies shows the importance of these immune cells in disorders such as rheumatoid arthritis, inflammatory bowel disease, multiple sclerosis, psoriasis, lupus erythematosus, type 1 diabetes and uveitis. Most existing therapies target only one pathway at a time.
Accordingly, there is a need for additional agonists and antagonists of immune checkpoint pathways.
According to at least some embodiments, the present invention provides for a method of activating T cells of a patient comprising administering an anti-HIDE1 antibody to said patient, wherein a subset of the T cells of said patient are activated.
According to at least some embodiments, the present invention provides for a method of activating cytotoxic T cells (CTLs) of a patient comprising administering an anti-HIDE1 antibody to said patient, wherein a subset of the CTLs of said patient are activated.
According to at least some embodiments, the present invention also provides for a method of activating NK cells of a patient comprising administering an anti-HIDE1 antibody to said patient, wherein a subset of the NK cells of said patient are activated.
According to at least some embodiments, the present invention also provides for a method of activating γδ T cells of a patient comprising administering an anti-HIDE1 antibody to said patient, wherein a subset of the γδ T cells of said patient are activated.
According to at least some embodiments, the present invention also provides for a method of activating Th1 cells of a patient comprising administering an anti-HIDE1 antibody to said patient, wherein a subset of the Th1 cells of said patient are activated.
According to at least some embodiments, the present invention also provides for a method of decreasing or eliminating cell number and/or activity of at least one of regulatory T cells (Tregs) in a patient comprising administering an anti-HIDE1 antibody to said patient.
According to at least some embodiments, the present invention also provides for a method of increasing interferon-γ production and/or pro-inflammatory cytokine secretion in a patient comprising administering an anti-HIDE1 antibody to said patient.
According to at least some embodiments, the present invention also provides for a method of modulating myeloid cell polarization in a patient comprising administering an anti-HIDE1 antibody to said patient.
According to at least some embodiments, the present invention also provides for a method of modulating myeloid cell shifting toward a pro-inflammatory response in a patient comprising administering an anti-HIDE1 antibody to said patient.
According to at least some embodiments, the present invention also provides for a method of shifting myeloid from M2 toward M1 phenotype in a patient comprising administering an anti-HIDE1 antibody to said patient.
According to at least some embodiments, the present invention also provides for a method of modulating myeloid cell in the TME to support anti-cancer immune response in a patient comprising administering an anti-HIDE1 antibody to said patient.
According to at least some embodiments, the present invention also provides for a method of restricting the pro-tumorigenic effects of the myeloid cells in the tumor microenvironment in a patient comprising administering an anti-HIDE1 antibody to said patient.
According to at least some embodiments, the present invention also provides for a method to elicit one or more of the following effects on immunity in a patient by administering an anti-HIDE1 antibody to said patient, wherein said effect is selected from the group consisting of: i) increases immune response, (ii) increases T cell activity, (iii) increases activation of αβ and/or γδ T cells, (iv) increases cytotoxic T cell activity, (v) increases NK and/or NKT cell activity, (vi) alleviates and/or γδ T-cell suppression, (vii) increases pro-inflammatory cytokine secretion, (viii) increases IL-2 secretion; (ix) increases interferon-γ production, (x) increases Th1 response, (xi) decrease Th2 response, (xii) decreases or eliminates cell number and/or activity of at least one of regulatory T cells (Tregs), myeloid derived suppressor cells (MDSCs), iMCs, mesenchymal stromal cells, TIE2-expressing monocytes, (xiii) reduces regulatory cell activity, and/or the activity of one or more of myeloid derived suppressor cells (MDSCs), iMCs, mesenchymal stromal cells, TIE2-expressing monocytes, (xiv) decreases or eliminates M2 macrophages, (xv) reduces M2 macrophage pro-tumorigenic activity, (xvi) decreases or eliminates N2 neutrophils, (xvii) reduces N2 neutrophils pro-tumorigenic activity, (xviii) reduces inhibition of T cell activation, (xix) reduces inhibition of CTL activation, (xx) reduces inhibition of NK and/or NKT cell activation, (xxi) reverses αβ and/or γδ T cell exhaustion, (xxii) increases αβ and/or γδ T cell response, (xxiii) increases activity of cytotoxic cells, (xxiv) stimulates antigen-specific memory responses, (xxv) elicits apoptosis or lysis of cancer cells, (xxvi) stimulates cytotoxic or cytostatic effect on cancer cells, (xxvii) induces direct killing of cancer cells, (xxviii) increases Th17 activity and (xxix) modulating myeloid cell polarization, (xxx) modulating myeloid cell shifting toward a pro-inflammatory response, (xxxi) shifting myeloid from M2 toward M1 phenotype, (xxxii) modulating myeloid cell in the TME to support anti-cancer immune response, (xxxiii) restricting the pro-tumorigenic effects of the myeloid cells in the TME, (xxxiv) enhancing myeloid and lymphoid infiltration into the tumor cite thereby shifting the tumor into more immunogenic, (xxxv) induces complement dependent cytotoxicity and/or antibody dependent cell-mediated cytotoxicity.
According to at least some embodiments, the present invention also provides for a method of depleting myeloid cells or other circulating tumor cells expressing HIDE1 from a patient or patient sample, said method comprising: i) contacting said patient or said patient sample with an anti-HIDE1 antibody, wherein said anti-HIDE1 antibody binds to HIDE1 expressing cells, ii) identifying cells to which said anti-HIDE1 antibody has bound, and iii) removing said cells in step ii) from said patient or said patient sample.
According to at least some embodiments, the present invention also provides for a method of treating cancer in a patient, comprising administering an anti-HIDE1 antibody to said patient, wherein said cancer is treated. In some embodiments said patient has cancer. In some embodiments, the cancer is selected from the group consisting of Acute Myeloid Leukemia, Acute Myeloid Leukemia Induction Failure, Acute Lymphoblastic Leukemia, Diffuse Large B-cell Lymphoma, Malignant Lymphoma, Non-Hodgkin Lymphoma, Diffuse Large B-Cell Lymphoma, Glioblastoma multiforme, Mesothelioma, Thymoma, Testicular Germ Cell Tumors, Kidney renal clear cell carcinoma, Sarcoma, Brain Lower Grade Glioma, Chronic Lymphocytic Leukemia, Non-Hodgkin Lymphoma-Follicular Lymphoma, Uterine Carcinosarcoma, Pediatric Brain Tumors, Lung adenocarcinoma, Cervical squamous cell carcinoma, endocervical adenocarcinoma, Pancreatic adenocarcinoma, Skin Cutaneous Melanoma, Kidney renal papillary cell carcinoma, Liver hepatocellular carcinoma; Bladder Urothelial Carcinoma, Colon adenocarcinoma, Head and Neck squamous cell carcinoma, Lung squamous cell carcinoma, Rectum adenocarcinoma, and Stomach adenocarcinoma. In some embodiments, the cancer is a cancer having high immune infiltrate of myeloid cells expressing HIDE1.
In some embodiments, the anti-HIDE1 antibody is selected from the group consisting of CPA.12.001 human IgG4, CPA.12.002 human IgG4, CPA.12.003 human IgG4, CPA.12.004 human IgG4, CPA.12.005 human IgG4, CPA.12.006 human IgG4, CPA.12.007 human IgG4, CPA.12.008 human IgG4, CPA.12.009 human IgG4, CPA.12.011 human IgG4, CPA.12.012 human IgG4, CPA.12.013 human IgG4, CPA.12.014 human IgG4, and CPA.12.015 human IgG4. In some embodiments, the anti-HIDE1 antibody is selected from the group consisting of CPA.12.006-H4, CPA.12.007-H4, and CPA.12.0012-H4.
In some embodiments, the anti-HIDE1 antibody is selected from the group consisting of CPA.12.001, CPA.12.002, CPA.12.003, CPA.12.004, CPA.12.005, CPA.12.006, CPA.12.007, CPA.12.008, CPA.12.009, CPA.12.011, CPA.12.012, CPA.12.013, CPA.12.014, and CPA.12.015. In some embodiments, the anti-HIDE1 antibody is selected from the group consisting of CPA.12.006, CPA.12.007, and CPA.12.0012.
According to at least some embodiments, the present invention also provides for a method of diagnosing cancer comprising: a) contacting a tissue from a patient with an anti-HIDE1 antibody; and b) determining the presence of over-expression of HIDE1 in said tissue as an indication of the presence of cancer. In some embodiments, the tissue is a blood sample. In some embodiments, the tissue is a biopsy of a solid tumor.
In some embodiments of the diagnostic method, the anti-HIDE1 antibody is labeled. In some embodiments, a second labeled antibody that binds to said anti-HIDE1 antibody is contacted with said sample.
According to at least some embodiments, the present invention also provides for an anti-HIDE1 antigen-binding domain comprising: a) a heavy chain variable domain comprising a vhCDR1, vhCDR2, and vhCDR3 from an anti-HIDE1 antibody; and b) a light chain variable domain comprising a vlCDR1, vlCDR2, and vlCDR3 from said anti-HIDE1 antibody; wherein said anti-HIDE1 antibody is selected from the group consisting of CPA.12.001 human IgG4, CPA.12.002 human IgG4, CPA.12.003 human IgG4, CPA.12.004 human IgG4, CPA.12.005 human IgG4, CPA.12.006 human IgG4, CPA.12.007 human IgG4, CPA.12.008 human IgG4, CPA.12.009 human IgG4, CPA.12.011 human IgG4, CPA.12.012 human IgG4, CPA.12.013 human IgG4, CPA.12.014 human IgG4, and CPA.12.015 human IgG4.
According to at least some embodiments, the present invention also provides for an anti-HIDE1 antigen-binding domain comprising: a) a heavy chain variable domain comprising a vhCDR1, vhCDR2, and vhCDR3 from an anti-HIDE1 antibody; and b) a light chain variable domain comprising a vlCDR1, vlCDR2, and vlCDR3 from said anti-HIDE1 antibody; wherein said anti-HIDE1 antibody is selected from the group consisting of CPA.12.001, CPA.12.002, CPA.12.003, CPA.12.004, CPA.12.005, CPA.12.006, CPA.12.007, CPA.12.008, CPA.12.009, CPA.12.011, CPA.12.012, CPA.12.013, CPA.12.014, and CPA.12.015.
In some embodiments, the anti-HIDE1 antigen binding domain of the antibody is a single chain Fv (scFv), wherein said heavy chain variable domain and said light chain variable domain are covalently attached via a scFv linker.
According to at least some embodiments, the present invention also provides for an anti-HIDE1 antibody comprising: a) a heavy chain variable domain comprising a vhCDR1, vhCDR2, and vhCDR3 from an anti-HIDE1 antibody; and b) a light chain variable domain comprising a vlCDR1, vlCDR2, and vlCDR3 from said anti-HIDE1 antibody; wherein said anti-HIDE1 antibody is selected from the group consisting of CPA.12.001 human IgG4, CPA.12.002 human IgG4, CPA.12.003 human IgG4, CPA.12.004 human IgG4, CPA.12.005 human IgG4, CPA.12.006 human IgG4, CPA.12.007 human IgG4, CPA.12.008 human IgG4, CPA.12.009 human IgG4, CPA.12.011 human IgG4, CPA.12.012 human IgG4, CPA.12.013 human IgG4, CPA.12.014 human IgG4, and CPA.12.015 human IgG4.
According to at least some embodiments, the present invention also provides for an anti-HIDE1 antibody comprising: a) a heavy chain variable domain comprising a vhCDR1, vhCDR2, and vhCDR3 from an anti-HIDE1 antibody; and b) a light chain variable domain comprising a vlCDR1, vlCDR2, and vlCDR3 from said anti-HIDE1 antibody; wherein said anti-HIDE1 antibody is selected from the group consisting of CPA.12.001, CPA.12.002, CPA.12.003, CPA.12.004, CPA.12.005, CPA.12.006, CPA.12.007, CPA.12.008, CPA.12.009, CPA.12.011, CPA.12.012, CPA.12.013, CPA.12.014, and CPA.12.015.
According to at least some embodiments, the present invention also provides for an anti-HIDE1 antibody that competes for binding with an antibody comprising: a) a heavy chain variable domain comprising a vhCDR1, vhCDR2, and vhCDR3 from an anti-HIDE1 antibody; and b) a light chain variable domain comprising a vlCDR1, a vlCDR2 and vlCDR3, from said anti-HIDE1 antibody; wherein said anti-HIDE1 antibody is selected from the group consisting of CPA.12.001 human IgG4, CPA.12.002 human IgG4, CPA.12.003 human IgG4, CPA.12.004 human IgG4, CPA.12.005 human IgG4, CPA.12.006 human IgG4, CPA.12.007 human IgG4, CPA.12.008 human IgG4, CPA.12.009 human IgG4, CPA.12.011 human IgG4, CPA.12.012 human IgG4, CPA.12.013 human IgG4, CPA.12.014 human IgG4, and CPA.12.015 human IgG4.
According to at least some embodiments, the present invention also provides for an anti-HIDE1 antibody that competes for binding with an antibody comprising: a) a heavy chain variable domain comprising a vhCDR1, vhCDR2, and vhCDR3 from an anti-HIDE1 antibody; and b) a light chain variable domain comprising a vlCDR1, a vlCDR2 and vlCDR3, from said anti-HIDE1 antibody; wherein said anti-HIDE1 antibody is selected from the group consisting of CPA.12.001, CPA.12.002, CPA.12.003, CPA.12.004, CPA.12.005, CPA.12.006, CPA.12.007, CPA.12.008, CPA.12.009, CPA.12.011, CPA.12.012, CPA.12.013, CPA.12.014, and CPA.12.015.
According to at least some embodiments, the present invention also provides for an anti-HIDE1 antibody that competes for binding with an antibody comprising: a) a heavy chain variable domain comprising a vhCDR1, vhCDR2, and vhCDR3 from an anti-HIDE1 antibody; and b) a light chain variable domain comprising a vlCDR1, a vlCDR2 and vlCDR3, from said anti-HIDE1 antibody; wherein said anti-HIDE1 antibody is selected from the group consisting of AB-506, AB-507, AB-508, AB-509, and AB-510.
According to at least some embodiments, the present invention also provides for an anti-HIDE1 antibody that competes for binding with an antibody comprising: a) a heavy chain variable domain comprising a vhCDR1, vhCDR2, and vhCDR3 from an anti-HIDE1 antibody; and b) a light chain variable domain comprising a vlCDR1, a vlCDR2 and vlCDR3, from said anti-HIDE1 antibody; wherein said anti-HIDE1 antibody is selected from the group consisting of 33B4, 36C1, and 39A7.
According to at least some embodiments, the present invention also provides for an anti-HIDE1 antibody that competes for functional activity with an antibody comprising: a) a heavy chain variable domain comprising a vhCDR1, vhCDR2, and vhCDR3 from an anti-HIDE1 antibody; and b) a light chain variable domain comprising a vlCDR1, a vlCDR2 and vlCDR3, from said anti-HIDE1 antibody; wherein said anti-HIDE1 antibody is selected from the group consisting of CPA.12.001 human IgG4, CPA.12.002 human IgG4, CPA.12.003 human IgG4, CPA.12.004 human IgG4, CPA.12.005 human IgG4, CPA.12.006 human IgG4, CPA.12.007 human IgG4, CPA.12.008 human IgG4, CPA.12.009 human IgG4, CPA.12.011 human IgG4, CPA.12.012 human IgG4, CPA.12.013 human IgG4, CPA.12.014 human IgG4, and CPA.12.015 human IgG4.
According to at least some embodiments, the present invention also provides for an anti-HIDE1 antibody that competes for functional activity with an antibody comprising: a) a heavy chain variable domain comprising a vhCDR1, vhCDR2, and vhCDR3 from an anti-HIDE1 antibody; and b) a light chain variable domain comprising a vlCDR1, a vlCDR2 and vlCDR3, from said anti-HIDE1 antibody; wherein said anti-HIDE1 antibody is selected from the group consisting of CPA.12.001, CPA.12.002, CPA.12.003, CPA.12.004, CPA.12.005, CPA.12.006, CPA.12.007, CPA.12.008, CPA.12.009, CPA.12.011, CPA.12.012, CPA.12.013, CPA.12.014, and CPA.12.015.
According to at least some embodiments, the present invention also provides for an anti-HIDE1 antibody that competes for functional activity with an antibody comprising: a) a heavy chain variable domain comprising a vhCDR1, vhCDR2, and vhCDR3 from an anti-HIDE1 antibody; and b) a light chain variable domain comprising a vlCDR1, a vlCDR2 and vlCDR3, from said anti-HIDE1 antibody; wherein said anti-HIDE1 antibody is selected from the group consisting of AB-506, AB-507, AB-508, AB-509, and AB-510.
According to at least some embodiments, the present invention also provides for an anti-HIDE1 antibody that competes for functional activity with an antibody comprising: a) a heavy chain variable domain comprising a vhCDR1, vhCDR2, and vhCDR3 from an anti-HIDE1 antibody; and b) a light chain variable domain comprising a vlCDR1, a vlCDR2 and vlCDR3, from said anti-HIDE1 antibody; wherein said anti-HIDE1 antibody is selected from the group consisting of 33B4, 36C1, and 39A7.
In some embodiments, in the composition comprising an anti-HIDE1 antibody comprise an antibody selected from the group consisting of CPA.12.001 human IgG4, CPA.12.002 human IgG4, CPA.12.003 human IgG4, CPA.12.004 human IgG4, CPA.12.005 human IgG4, CPA.12.006 human IgG4, CPA.12.007 human IgG4, CPA.12.008 human IgG4, CPA.12.009 human IgG4, CPA.12.011 human IgG4, CPA.12.012 human IgG4, CPA.12.013 human IgG4, CPA.12.014 human IgG4, and CPA.12.015 human IgG4. In some embodiments, in the composition comprising an anti-HIDE1 antibody, the antibody is selected from the group consisting CPA.12.006-H4, CPA.12.007-H4, and CPA.12.0012-H4.
In some embodiments, in the composition comprising an anti-HIDE1 antibody comprise an antibody selected from the group consisting of CPA.12.001, CPA.12.002, CPA.12.003, CPA.12.004, CPA.12.005, CPA.12.006, CPA.12.007, CPA.12.008, CPA.12.009, CPA.12.011, CPA.12.012, CPA.12.013, CPA.12.014, and CPA.12.015. In some embodiments, in the composition comprising an anti-HIDE1 antibody, the antibody is selected from the group consisting CPA.12.006-H4, CPA.12.007-H4, and CPA.12.0012-H4.
According to at least some embodiments, the present invention also provides for a nucleic acid composition comprising: a) a first nucleic acid encoding a heavy chain variable domain comprising a vhCDR1, vhCDR2, and vhCDR3 from an anti-HIDE1 antibody; and b) a second nucleic acid encoding a light chain variable domain comprising a vlCDR1, vlCDR2, and vlCDR3 from said anti-HIDE1 antibody; wherein said anti-HIDE1 antibody is selected from the group consisting of CPA.12.001 human IgG4, CPA.12.002 human IgG4, CPA.12.003 human IgG4, CPA.12.004 human IgG4, CPA.12.005 human IgG4, CPA.12.006 human IgG4, CPA.12.007 human IgG4, CPA.12.008 human IgG4, CPA.12.009 human IgG4, CPA.12.011 human IgG4, CPA.12.012 human IgG4, CPA.12.013 human IgG4, CPA.12.014 human IgG4, and CPA.12.015 human IgG4.
According to at least some embodiments, the present invention also provides for a nucleic acid composition comprising: a) a first nucleic acid encoding a heavy chain variable domain comprising a vhCDR1, vhCDR2, and vhCDR3 from an anti-HIDE1 antibody; and b) a second nucleic acid encoding a light chain variable domain comprising a vlCDR1, vlCDR2, and vlCDR3 from said anti-HIDE1 antibody; wherein said anti-HIDE1 antibody is selected from the group consisting of CPA.12.001, CPA.12.002, CPA.12.003, CPA.12.004, CPA.12.005, CPA.12.006, CPA.12.007, CPA.12.008, CPA.12.009, CPA.12.011, CPA.12.012, CPA.12.013, CPA.12.014, and CPA.12.015.
According to at least some embodiments, the present invention also provides for an expression vector composition comprising: a) a first expression vector comprising a first nucleic acid as described above and herein; and b) a second expression vector comprising said second nucleic as described above and herein. In some embodiments, the expression vector composition comprises an expression vector comprising the first nucleic acid of as described above and herein and the second nucleic acid of as described above and herein.
In some embodiments, the invention provides a host cell comprising the expression vector composition as described above and herein.
According to at least some embodiments, the present invention also provides for a method of making an anti-HIDE1 antibody comprising: a) culturing the host cell as described above and herein under conditions wherein said antibody is expressed; and b) recovering said antibody.
According to at least some embodiments, the present invention also provides for a method of activating cytotoxic T cells (CTLs) of a patient comprising administering an anti-HIDE1 antibody to said patient, wherein a subset of the CTLs of said patient are activated. In some embodiments, the antibody is optionally an antibody as described above and herein.
According to at least some embodiments, the present invention also provides for a method of activating NK cells of a patient comprising administering an anti-HIDE1 antibody to said patient, wherein a subset of the NK cells of said patient are activated. In some embodiments, the antibody is optionally an antibody as described above and herein.
According to at least some embodiments, the present invention also provides for a method of activating γδ T cells of a patient comprising administering an anti-HIDE1 antibody to said patient, wherein a subset of the γδ T cells of said patient are activated. In some embodiments, the antibody is optionally an antibody as described above and herein.
According to at least some embodiments, the present invention also provides for a method of activating Th1 cells of a patient comprising administering an anti-HIDE1 antibody to said patient, wherein a subset of the Th1 cells of said patient are activated. In some embodiments, the antibody is optionally an antibody as described above and herein.
According to at least some embodiments, the present invention also provides for a method of decreasing or eliminating cell number and/or activity of at least one of regulatory T cells (Tregs) in a patient comprising administering an anti-HIDE1 antibody to said patient. In some embodiments, the antibody is optionally an antibody as described above and herein.
According to at least some embodiments, the present invention also provides for a method of increasing interferon-γ production and/or pro-inflammatory cytokine secretion in a patient comprising administering an anti-HIDE1 antibody to said patient. In some embodiments, the antibody is optionally an antibody as described above and herein.
According to at least some embodiments, the present invention also provides for a method of activating monocytes of a patient comprising administering an anti-HIDE1 antibody to said patient, wherein a subset of the monocyte cells of said patient are activated. In some embodiments, the antibody is optionally an antibody as described above and herein.
According to at least some embodiments, the present invention also provides for a method of treating cancer in a patient comprising administering an anti-HIDE1 antibody to said patient. In some embodiments, the antibody is optionally an antibody as described above and herein.
In some embodiments, the treatment is an increase in immune response. In some embodiments, the treatment is an increase in activation of and/or γδ T cells. In some embodiments, the treatment is an increase in cytotoxic T cell activity. In some embodiments, the treatment is an increase in natural killer (NK) and/or NKT cell activity. In some embodiments, the treatment is an increase in and/or γδ T-cell activity. In some embodiments, the treatment is an increase in pro-inflammatory cytokine secretion. In some embodiments, the treatment is increase in IL-2 secretion. In some embodiments, the treatment is an increase in interferon-γ production. In some embodiments, the treatment is an increase in Th1 response. In some embodiments, the treatment is a decrease in the cell number and/or activity of regulatory T cells. In some embodiments, the treatment decreases cell number and/or activity of at least one or more cells selected from the group consisting of regulatory T cells (Tregs), myeloid derived suppressor cells (MDSCs), iMCs, mesenchymal stromal cells, and TIE2-expressing monocytes. In some embodiments, the treatment is decreases the cell activity, and/or the activity of one or more cells selected from the group consisting of myeloid derived suppressor cells (MDSCs), iMCs, mesenchymal stromal cells, and TIE2-expressing monocytes. In some embodiments, the said treatment is a decrease in M2 macrophages. In some embodiments, the treatment is a decrease in M2 macrophage activity. In some embodiments, the treatment is a decrease in N2 neutrophils. In some embodiments, the treatment is a decrease in N2 neutrophils activity. In some embodiments, the treatment is a decrease in inhibition of T cell activation. In some embodiments, the treatment is a decrease in inhibition of CTL activation. In some embodiments, the treatment is a decrease in inhibition of NK cell activation. In some embodiments, the treatment is a decrease in and/or γδ T cell exhaustion. In some embodiments, the treatment is an increase in and/or γδ T cell response. In some embodiments, the treatment is an increase in activity of cytotoxic cells. In some embodiments, the treatment is an induction of antigen-specific memory responses. In some embodiments, the treatment induces apoptosis or lysis of cells. In some embodiments, the treatment is an increase in cytotoxic or cytostatic effect on cells. In some embodiments, the treatment induces direct killing of cells. In some embodiments, the treatment is an increase in Th17 activity. In some embodiments, the treatment induces complement dependent cytotoxicity and/or antibody dependent cell-mediated cytotoxicity.
According to at least some embodiments, the present invention also provides for a method of treating an immune disorder, comprising administering to a patient a composition comprising an enhancer of HIDE1 associated immune suppression, to effect treatment. In some embodiments, the treatment is a decrease in immune response. In some embodiments, the treatment is a decrease in activation of and/or γδ T cells. In some embodiments, the treatment is a decrease in cytotoxic T cell activity. In some embodiments, the treatment is a decrease in NK and/or NKT cell activity. In some embodiments, the treatment is a decrease of and/or γδ T-cell activity. In some embodiments, the treatment is a decrease in pro-inflammatory cytokine secretion. In some embodiments, the treatment is a decrease in IL-2 secretion. In some embodiments, the treatment is a decrease in interferon-γ production. In some embodiments, the treatment is a decrease in Th1 response. In some embodiments, the treatment is a decrease in Th2 response. In some embodiments, the treatment is an increase in inhibition of T cell activity. In some embodiments, the treatment is an increase in inhibition of CTL activity. In some embodiments, the treatment is an increase in inhibition of NK cell activity. In some embodiments, the treatment is an increase in αβ and/or γδ T cell exhaustion. In some embodiments, the treatment is a decrease in αβ and/or γδ T cell response. In some embodiments, the treatment is a decrease in activity of cytotoxic cells. In some embodiments, the treatment is a reduction in antigen-specific memory responses. In some embodiments, the treatment is an inhibition of apoptosis or lysis of cells. In some embodiments, the treatment is a decrease in cytotoxic or cytostatic effect on cells. In some embodiments, the treatment is a reduction in direct killing of cells. In some embodiments, the treatment is a decrease in Th17 activity. In some embodiments, the treatment is a reduction of complement dependent cytotoxicity and/or antibody dependent cell-mediated cytotoxicity.
According to at least some embodiments, the present invention also provides a method to elicit one or more of the following effects on immunity in a patient by administering a HIDE1 peptide to said patient, wherein said effect is selected from the group consisting of: i) decreases immune response, (ii) decreases αβ and/or γδ T cell activation, (iii) decreases T cell activity, (iv) decreases cytotoxic T cell activity, (v) decreases natural killer (NK) and/or NKT cell activity, (vi) decreases αβ and/or γδ T-cell activity, (vii) decreases pro-inflammatory cytokine secretion, (viii) decreases IL-2 secretion; (ix) decreases interferon-γ production, (x) decreases Th1 response, (xi) decreases Th2 response, (xii) increases cell number and/or activity of regulatory T cells, (xiii) increases regulatory cell activity and/or one or more of myeloid derived suppressor cells (MDSCs), iMCs, mesenchymal stromal cells, TIE2-expressing monocytes, (xiv) increases regulatory cell activity and/or the activity of one or more of myeloid derived suppressor cells (MDSCs), iMCs, mesenchymal stromal cells, TIE2-expressing monocytes, (xv) increases M2 macrophages, (xvi) increases M2 macrophage activity, (xvii) increases N2 neutrophils, (xviii) increases N2 neutrophils activity, (xix) increases inhibition of T cell activation, (xx) increases inhibition of CTL activation, (xxi) increases inhibition of NK cell activation, (xxii) increases αβ and/or γδ T cell exhaustion, (xxiii) decreases αβ and/or γδ T cell response, (xxiv) decreases activity of cytotoxic cells, (xxv) reduces antigen-specific memory responses, (xxvi) inhibits apoptosis or lysis of cells, (xxvii) decreases cytotoxic or cytostatic effect on cells, (xxviii) reduces direct killing of cells, (xxix) decreases Th17 activity, (xxx) modulates myeloid cell polarization, and/or modulates myeloid cell shifting toward an anti-inflammatory response, (xxxi) reduces complement dependent cytotoxicity and/or antibody dependent cell-mediated cytotoxicity.
According to at least some embodiments, the present invention also provides for a method of treating an immune disorder, comprising administering to a patient a composition comprising an enhancer of HIDE1 associated immune suppression to effect treatment. In some embodiments, the enhancer is a HIDE1 peptide. In some embodiments, the HIDE1 peptide is a HIDE1 ECD. In some embodiments, the HIDE1 peptide is a HIDE1 polypeptide consisting of a HIDE1 polypeptide ECD domain having at least 95% identity to the ECD domain of an amino acid sequence selected from the group consisting of the sequences depicted in
According to at least some embodiments, the present invention also provides for a method of activating cytotoxic T cells (CTLs) of a patient comprising administering the HIDE1 peptide as described above and herein to said patient, wherein a subset of the CTLs of said patient are inhibited.
According to at least some embodiments, the present invention also provides for a method of activating NK cells of a patient comprising administering the HIDE1 peptide as described above and herein to said patient, wherein a subset of the NK cells of said patient are inhibited.
According to at least some embodiments, the present invention also provides for a method of activating γδ T cells of a patient comprising administering the HIDE1 peptide as described above and herein to said patient, wherein a subset of the γδ T cells of said patient are inhibited.
According to at least some embodiments, the present invention also provides for a method of activating Th1 cells of a patient comprising administering the HIDE1 peptide as described above and herein to said patient, wherein a subset of the Th1 cells of said patient are inhibited.
According to at least some embodiments of the present invention also provides for a method of increasing cell number and/or activity of at least one of regulatory T cells (Tregs) in a patient comprising administering the HIDE1 peptide as described above and herein to said patient.
According to at least some embodiments, the present invention also provides for a method of decreasing interferon-γ production and/or pro-inflammatory cytokine secretion in a patient comprising administering the HIDE1 peptide as described above and herein to said patient.
According to at least some embodiments, the present invention also provides for a method of treating an autoimmune disease in a patient comprising administering the HIDE1 peptide as described above and herein to said patient. In some embodiments, the patient has an immune disorder. In some embodiments, the immune disorder is selected from the group consisting of an autoimmune disease, organ transplant rejection and inflammation. In some embodiments, the autoimmune disease is selected from the group consisting of rheumatoid arthritis, lupus, Inflammatory bowel disease, psoriasis, multiple sclerosis and diabetes type I.
In some embodiments, the enhancer of HIDE1 is selected from the group consisting of a protein and a nucleic acid. In some embodiments, the protein comprises an extracellular domain (ECD) of HIDE1. In some embodiments, the protein is a fusion protein comprising said ECD and a fusion partner. In some embodiments, the fusion partner is selected from the group consisting of a human IgG Fc domain and a human serum albumin (HSA).
According to at least some embodiments, the present invention also provides for a composition comprising an isolated HIDE1 polypeptide consisting of a HIDE1 polypeptide ECD domain having at least 95% identity to the ECD domain of an amino acid sequence selected from the group consisting of the sequences depicted in
According to at least some embodiments, the present invention also provides for a composition comprising a HIDE1 fusion polypeptide comprising: a) an ECD from a HIDE1 polypeptide; and b) a covalently attached fusion partner moiety. In some embodiments of the composition, the fusion partner moiety is selected from the group consisting of a human IgG Fc domain, a human serum albumin (HSA) and a polyethylene glycol (PEG). In some embodiments of the composition, the ECD has an amino acid sequence selected from the group consisting of the sequences depicted in
In some embodiments of the composition, the composition comprises a pharmaceutically acceptable carrier.
According to at least some embodiments, the present invention also provides for a method of suppressing T cell activation of a patient comprising administering a composition as described above and herein to said patient such that said patient's immune response is suppressed as a result of treatment. In some embodiments, the patient has an immune disorder. In some embodiments, the immune disorder is selected from the group consisting of an autoimmune disease, and organ transplant rejection. In some embodiments, the autoimmune disease is selected from the group consisting of rheumatoid arthritis, lupus, Inflammatory bowel disease, psoriasis, multiple sclerosis, and Diabetes type I.
Plates were coated with anti-CD3 mAb (2 μg/mL) in the presence of 10 μg/ml HIDE1-Fc or control mIgG2a as described in materials and methods. Wells were plated with 1×105 CD4+CD25− mouse T cells per well in the presence of 2 ug/ml of soluble anti-CD28. (A) The expression of CD69 was analyzed by flow cytometry at 48 h post-stimulation. (B-C) Culture supernatants were collected at 48 h post-stimulation and mouse IFNγ or IL-2 levels were analyzed by ELISA. Results are shown as Mean±Standard errors of duplicate samples.
Cancer can be considered as an inability of the patient to recognize and eliminate cancerous cells. In many instances, these transformed (e.g. cancerous) cells counteract immunosurveillance. There are natural control mechanisms that limit T-cell activation in the body to prevent unrestrained T-cell activity, which can be exploited by cancerous cells to evade or suppress the immune response. Restoring the capacity of immune effector cells—especially T cells—to recognize and eliminate cancer is the goal of immunotherapy. The field of immuno-oncology, sometimes referred to as “immunotherapy” is rapidly evolving, with several recent approvals of T cell checkpoint inhibitory antibodies such as Yervoy, Keytruda and Opdivo. These antibodies are generally referred to as “checkpoint inhibitors” because they block normally negative regulators of T cell immunity. It is generally understood that a variety of immunomodulatory signals, both costimulatory and coinhibitory, can be used to orchestrate an optimal antigen-specific immune response. Generally, these antibodies bind to checkpoint inhibitor proteins such as CTLA-4 and PD-1, which under normal circumstances prevent or suppress activation of cytotoxic T cells (CTLs). By inhibiting the checkpoint protein, for example through the use of antibodies that bind these proteins, an increased T cell response against tumors can be achieved. That is, these cancer checkpoint proteins suppress the immune response; when the proteins are blocked, for example using antibodies to the checkpoint protein, the immune system is activated, leading to immune stimulation, resulting in treatment of conditions such as cancer and infectious disease.
According to at least some embodiments of the present invention is directed to the use of antibodies to HIDE1. HIDE1 is expressed on the cell surface of myeloid cells including but not limited to monocytes, dendritic cells, macrophages, M1/M2 tumor associated macrophages, neutrophils, Myeloid-derive suppressor cells (MDSC), and shares several similarities to other known immune checkpoints.
Functional effects of HIDE1 blocking antibodies on myeloid cells including, for example, but not limited to monocytes, dendritic cells, macrophages, M1/M2 tumor associated macrophages, neutrophils, Myeloid-derive suppressor cells (MDSC), and/or on NK and T-cells can be assessed in vitro (and in some cases in vivo, as described more fully below) by measuring changes in the following parameters: proliferation, cytokine release and cell-surface makers. For NK cells, increases in cell proliferation, cytotoxicity (ability to kill target cells as measured by increases in CD107a, granzyme, and perforin expression, or by directly measuring target cells killing), cytokine production (e.g. IFN-γ and TNF), and cell surface receptor expression (e.g., CD25) is indicative of immune modulation, e.g. enhanced killing of cancer cells. For T-cells, increases in proliferation, increases in expression of cell surface markers of activation (e.g., CD25, CD69, CD137, and PD1), cytotoxicity (ability to kill target cells), and cytokine production (e.g., IL-2, IL-4, IL-6, IFNγ, TNF-a, IL-10, IL-17A) are indicative of immune modulation, e.g. enhanced killing of cancer cells. For myeloid cells: effect on myeloid cells polarization, such as M2 to M1 shift, improvement of antigen presentation by myeloid cells including more efficient cross-presentation by professional as well as non-professional antigen-presenting cells, enhanced antigen uptake and processing by antigen-presenting cells. Additional effects can include relief of T cell suppression (i.e. indirect effect on T cell activation), effect on cell recruitment (i.e. influx of immune cells and shift to more “inflamed tumors”).
In some embodiments, the anti-HIDE1 antibody is a depleting HIDE1 antibody. In some embodiments, a depleting anti-HIDE1 antibody binds to cell surface HIDE1. In some embodiments, the anti HIDE1 depleting antibody preferably is able to deplete HIDE1 expressing cells including but not limited to monocytes, dendritic cells, macrophages, M1/M2 tumor associated macrophages, neutrophils, Myeloid-derive suppressor cells (MDSC) and as a result reduce the number of HIDE1 expressing cells in a patient treated with the anti HIDE1 depleting antibody. Such depletion may be achieved via various mechanisms such as antibody-dependent cell mediated cytotoxicity (ADCC) and/or complement dependent cytotoxicity (CDC), inhibition of HIDE1 expressing cells proliferation and/or induction of HIDE1+ cell death (e.g. via apoptosis).
HIDE1 expressing cell depleting anti HIDE1 antibody might optionally be conjugated with or fused to a cytotoxic agent.
“Complement dependent cytotoxicity” or “CDC” refers to the lysis of a target cell in the presence of complement. Activation of the classical complement pathway is initiated by the binding of the first component of the complement system to antibodies which are bound to their cognate antigen. To assess complement activation, a CDC assay, e.g. as described in Gazzano-Santoro et al. (1997), or any other CDC assay known in the art, can be performed or employed with the anti-HIDE1 antibodies of the invention.
“Antibody-dependent cell-mediated cytotoxicity” or “ADCC” refers to a form of cytotoxicity in which secreted antibodies bound onto Fc receptors (FcRs) present on certain cytotoxic cells (e.g., Natural Killer (NK) cells, neutrophils, monocytes and macrophages) allow the cytotoxic effector cells to bind specifically to an antigen-bearing target cell and subsequently kill the target cell.
Accordingly, in at least some embodiments of the present invention provides antibodies, including antigen binding domains, that bind to human HIDE1 and methods of activating T cells and/or NK cells and/or methods of activating myeloid cells which induce activatory, migratory, secretory effect enhancing T/NK cell function and migration to treat diseases such as cancer and infectious diseases, and other conditions where increased immune activity results in treatment. According to at least some embodiments of the present invention also provides antibodies, including antigen binding domains, that bind to human HIDE1 for use in methods of depleting myeloid cells, or other circulating tumor cells, in order to treat diseases such as cancer.
Furthermore, without wishing to be limited by a single hypothesis, HIDE1 shows potentiating effects on the following immune functions: induction or differentiation and proliferation of inducible T regulatory or suppressor cells (iTregs). These cells are known to be involved in eliciting tolerance to self-antigens and to suppress anti-tumor immunity. Again without wishing to be limited by a single hypothesis, HIDE1 contributes to a non-functional phenotype of CD8 T cell from the tumor environment, also known as T cell exhaustion.
The flip side of immuno-oncology is the suppression of T cell activation in conditions where the immune system is too active, or is launching an immune response to an auto-antigen, etc. Thus, by providing HIDE1 proteins (for example as fusion proteins, as discussed below), treatment of immune conditions such as auto-immune disease, inflammation and allergic diseases can be treated. That is, as HIDE1 has an inhibitory effect on specific immune cells such as CD4+ T cells, CD8+ T cells or CTLs, and NK cells, which cells are known to be involved in the pathology of certain immune conditions such as autoimmune and inflammatory disorders, as well as eliciting a potentiating effect on Tregs or immuno-suppressive myeloid cells, HIDE1 polypeptides which potentiate or agonize the effects of HIDE1 on immunity may optionally be used for treating conditions wherein the suppression of T cell or NK mediated immunity and/or the induction of immune tolerance or prolonged suppression of antigen-specific immunity is therapeutically desirable, e.g., the treatment of autoimmune, inflammatory or allergic conditions, and/or the suppression of undesired immune responses such as to cell or gene therapy, adverse immune responses during pregnancy, and adverse immune responses to transplanted heterologous, allogeneic or xenogeneic cells, organs and tissues and for inhibiting or preventing the onset of graft versus host disease (GVHD) after transplant.
Therefore, in one embodiment the present invention broadly relates to the development of novel “immunomodulatory proteins” wherein this includes HIDE1 polypeptides that antagonize or block the effects of HIDE1 on immunity and particularly the effects of HIDE1, on specific types of immune cells and cytokine production (i.e., immunostimulatory HIDE1 polypeptides or fusion proteins and/or immunostimulatory HIDE1 antibodies).
Additionally, the invention relates to HIDE1 polypeptides that agonize or mimic the effects of HIDE1 on immunity and particularly the effects of HIDE1 on specific types of immune cells and cytokine production (i.e., “immunoinhibitory HIDE1 polypeptides or fusion proteins” or HIDE1 polypeptides that increase and/or enhance in immune suppression), as well as immune suppressing polypeptides which mimic HIDE1 immune suppressive activity through binding to the HIDE1 binding/signalling partner and can be referred to as enhancers of HIDE1 associated immune suppression.
Accordingly, as discussed herein, HIDE1 is an immune checkpoint protein, sometimes referred to as “an immuno-oncology protein”. As has been shown for PD-1 and CTLA-4, among others, immune checkpoint proteins can be exploited in several ways, to either immunopotentiate the immune system to increase immune activity, such as through the activation of T cells for treatments of diseases such as cancer and infectious disease, or through immunoinhibition, where immunosuppression is desired, for example in allergic reactions, autoimmune diseases and inflammation. HIDE1 as shown herein exhibits negative signaling on the immune system, by suppressing T cell activation and other pathways as outlined herein. Thus, by reducing the activity of HIDE1, for example by inhibiting its binding ability to its ligand (i.e., inhibiting binding to its binding partner or signaling partner), the suppression is decreased and the immune system can be activated or stimulated to treat cancer, for example. Conversely, by increasing the activity of HIDE1 (“stimulating” the activity with a “stimulator” and/or with an enhancer of HIDE1 associated immune suppression), for example by adding recombinant HIDE1 ECD that mimics HIDE1 immune suppressive activity through binding to HIDE1 counterpart (a “stimulator of HIDE1” or an enhancer of HIDE1 associated immune suppression; i.e., HIDE1 binding and/or signaling partner) to a host in the form of a soluble ECD (and optionally a fusion protein), the suppression is increased and the immune system is suppressed, allowing for treatment of diseases associated with increased immune function such as autoimmune diseases and others outlined herein. As shown in the Example section, HIDE1 tetramers have been shown to bind to activated T cells and TILs, activation status of T cells has been manifested by increased PD-1 and CD137 expression. Additionally, introduction of HIDE1 (for example, as an ECD Fc-fusion protein) was shown to inhibit the activation of T cells, as shown in the Examples. Accordingly, anti-HIDE1 antibodies can be used to treat conditions for which T cell or NK cell activation is desired such as cancer.
Accordingly, in some embodiments of the present invention is directed to compounds that either suppress the signaling pathway triggered by the binding interaction of HIDE1 and its binding and/or signaling partner (leading to increased T cell and NK cell activation, among other things, leading to treatment of diseases such as cancer and pathogen infection), or activate the signaling pathway triggered by the binding interaction of HIDE1 and its binding and/or signaling partner (leading to decreased T cell and NK cell activation, among other things), leading to treatment of diseases such as autoimmune diseases and inflammation.
Thus, specific mechanisms of action are provided for the immunostimulatory actions of, for example, anti-HIDE1 antibodies, that are useful for increasing immune function, for example for the treatment of cancer. These include, but are not limited to, (i) increases immune response, (ii) increases T cell activity, (iii) increases activation of αβ and/or γδ T cells, (iv) increases cytotoxic T cell activity, (v) increases NK and/or NKT cell activity, (vi) alleviates αβ and/or γδ T-cell suppression, (vii) increases pro-inflammatory cytokine secretion, (viii) increases IL-2 secretion; (ix) increases interferon-γ production, (x) increases Th1 response, (xi) decrease Th2 response, (xii) decreases or eliminates cell number and/or activity of at least one of regulatory T cells (Tregs), myeloid derived suppressor cells (MDSCs), iMCs, mesenchymal stromal cells, TIE2-expressing monocytes, (xiii) reduces regulatory cell activity, and/or the activity of one or more of myeloid derived suppressor cells (MDSCs), iMCs, mesenchymal stromal cells, TIE2-expressing monocytes, (xiv) decreases or eliminates M2 macrophages, (xv) reduces M2 macrophage pro-tumorigenic activity, (xvi) decreases or eliminates N2 neutrophils, (xvii) reduces N2 neutrophils pro-tumorigenic activity, (xviii) reduces inhibition of T cell activation, (xix) reduces inhibition of CTL activation, (xx) reduces inhibition of NK and/or NKT cell activation, (xxi) reverses αβ and/or γδ T cell exhaustion, (xxii) increases αβ and/or γδ T cell response, (xxiii) increases activity of cytotoxic cells, (xxiv) stimulates antigen-specific memory responses, (xxv) elicits apoptosis or lysis of cancer cells, (xxvi) stimulates cytotoxic or cytostatic effect on cancer cells, (xxvii) induces direct killing of cancer cells, (xxviii) increases Th17 activity and/or (xxix) modulating myeloid cell polarization, (xxx) modulating myeloid cell shifting toward a pro-inflammatory response, (xxxi) shifting myeloid from M2 toward M1 phenotype, (xxxii) modulating myeloid cell in the TME to support anti-cancer immune response, (xxxiii) restricting the pro-tumorigenic effects of the myeloid cells in the TME, (xxxiv) enhancing myeloid and lymphoid infiltration into the tumor cite thereby shifting the tumor into more immunogenic, (xxxv) induces complement dependent cytotoxicity and/or antibody dependent cell-mediated cytotoxicity.
Functional effects of HIDE1 blocking antibodies on myeloid cells including but not limited to monocytes, dendritic cells, macrophages, M1/M2 tumor associated macrophages, neutrophils, Myeloid-derive suppressor cells (MDSC), and/or on NK and T-cells can be assessed in vitro (and in some cases in vivo, as described more fully below) by measuring changes in the following parameters: proliferation, cytokine release and cell-surface makers. For NK cells, increases in cell proliferation, cytotoxicity (ability to kill target cells as measured by increases in CD107a, granzyme, and perforin expression, or by directly measuring target cells killing), cytokine production (e.g. IFN-γ and TNF), and cell surface receptor expression (e.g. CD25) is indicative of immune modulation, e.g. enhanced killing of cancer cells. For T-cells, increases in proliferation, increases in expression of cell surface markers of activation (e.g. CD25, CD69, CD137, and PD1), cytotoxicity (ability to kill target cells), and cytokine production (e.g. IL-2, IL-4, IL-6, IFNγ, TNF-α, IL-10, IL-17A) are indicative of immune modulation, e.g. enhanced killing of cancer cells. For monocytes, increases in monocyte differentiation, M1-M2 skuwing and vise versa detected by specific differentiating markers (M1-iNOS, IL-12p35, TNFa, IL-1b; M2-IL-10, OH-1, CCL17, CCL22, Msr2, MRC1); Monocyte activation detected by cell surface markers of activation (e.g. MHC, CD80/86, PDL1) and cytokine and chemokine secretion (CCL19/21). The aforementioned effects could be examined upon differentiating primary myeloid cells as well as cellslines towards macrophage-like cells using M1/M2 polarizing conditions. Indirect suppressive effect of HYDE1-expressing myeloid cells on effector immune cells, including but not limited to CD4+ T cells, CD8+ T cells, NK cells, NKT cells, could be reversed with HIDE1-blocking Ab upon poly-clonal or antigen-specific activation of immune effector cells in the presence of HIDE1-expressing myeloid cells.
Accordingly, in some embodiments the present invention provides antibodies, including antigen binding domains, that bind to human HIDE1 and methods of activating myeloid cells including but not limited to monocytes, dendritic cells, macrophages, M1/M2 tumor associated macrophages, neutrophils, Myeloid-derive suppressor cells (MDSC), and/or on, T cells and/or NK cells to treat diseases such as cancer and infectious diseases, and other conditions where increased immune activity results in treatment. According to at least some embodiments of the present invention also provides antibodies, including antigen binding domains, that bind to human HIDE1 for use in methods of depleting myeloid cells, or other circulating tumor cells, in order to treat diseases such as cancer.
According to at least some embodiments, the present invention provides immunoinhibitory HIDE1 therapeutic agents (e.g., a compound, including but not limited to a HIDE1 peptide, that mimics HIDE1 immune suppressive activity through binding to HIDE1 counterpart, i.e., the HIDE1 binding and/or signalling partner; such compounds can also be referred to as enhancers of HIDE1 associated immune suppression), wherein said agents are used for treatment of immune related diseases and/or for reducing the undesirable immune activation that follows gene therapy, and wherein said agents mediate at least one of the following immune effects: (i) decreases immune response, (ii) decreases and/or γδ T cell activation, (iii) decreases T cell activity, (iv) decreases cytotoxic T cell activity, (v) decreases natural killer (NK) and/or NKT cell activity, (vi) decreases and/or γδ T-cell activity, (vii) decreases pro-inflammatory cytokine secretion, (viii) decreases IL-2 secretion; (ix) decreases interferon-γ production, (x) decreases Th1 response, (xi) decreases Th2 response, (xii) increases cell number and/or activity of regulatory T cells, (xiii) increases regulatory cell activity and/or one or more of myeloid derived suppressor cells (MDSCs), iMCs, mesenchymal stromal cells, TIE2-expressing monocytes, (xiv) increases regulatory cell activity and/or the activity of one or more of myeloid derived suppressor cells (MDSCs), iMCs, mesenchymal stromal cells, TIE2-expressing monocytes, (xv) increases M2 macrophages, (xvi) increases M2 macrophage activity, (xvii) increases N2 neutrophils, (xviii) increases N2 neutrophils activity, (xix) increases inhibition of T cell activation, (xx) increases inhibition of CTL activation, (xxi) increases inhibition of NK cell activation, (xxii) increases αβ and/or γδ T cell exhaustion, (xxiii) decreases αβ and/or γδ T cell response, (xxiv) decreases activity of cytotoxic cells, (xxv) reduces antigen-specific memory responses, (xxvi) inhibits apoptosis or lysis of cells, (xxvii) decreases cytotoxic or cytostatic effect on cells, (xxviii) reduces direct killing of cells, (xxix) decreases Th17 activity, and/or (xxx) modulates myeloid cell polarization, and/or modulates myeloid cell shifting toward an anti-inflammatory response, (xxxi) reduces complement dependent cytotoxicity and/or antibody dependent cell-mediated cytotoxicity. Again without wishing to be limited by a single hypothesis, HIDE1 shows potentiating effects on the following immune functions: induction or differentiation and proliferation of inducible T regulatory or suppressor cells (iTregs). These cells are known to be involved in eliciting tolerance to self-antigens and to suppress anti-tumor immunity.
Again without wishing to be limited by a single hypothesis, HIDE1 contributes to a non-functional phenotype of CD8 T cell from the tumor environment, also known as T cell exhaustion.
The flip side of immuno-oncology is the suppression of T cell activation in conditions where the immune system is too active, or is launching an immune response to an auto-antigen, etc. Thus, by providing HIDE1 proteins (for example as fusion proteins, as discussed below), treatment of immune conditions such as auto-immune disease, inflammation and allergic diseases can be treated. That is, as HIDE1 has an inhibitory effect on specific immune cells such as CD4+ T cells, CD8+ T cells or CTLs, and NK cells, which cells are known to be involved in the pathology of certain immune conditions such as autoimmune and inflammatory disorders, as well as eliciting a potentiating effect on Tregs or immuno-suppressive myeloid cells, HIDE1 polypeptides which potentiate or agonize the effects of HIDE1 on immunity may optionally be used for treating conditions wherein the suppression of T cell or NK mediated immunity and/or the induction of immune tolerance or prolonged suppression of antigen-specific immunity is therapeutically desirable, e.g., the treatment of autoimmune, inflammatory or allergic conditions, and/or the suppression of undesired immune responses such as to cell or gene therapy, adverse immune responses during pregnancy, and adverse immune responses to transplanted heterologous, allogeneic or xenogeneic cells, organs and tissues and for inhibiting or preventing the onset of graft versus host disease (GVHD) after transplant.
Furthermore, in some embodiments a HIDE1 ECD (for example, in the form of an Fc fusion, for example) binds to the HIDE1 binding and/or signaling and interrupts one or more inhibitory signals (via HIDE1) and thus acts in an immunoinhibitory manner (including increasing and/or enhancing immune suppression). These include, but are not limited to, i) decreases immune response, (ii) decreases and/or γδ T cell activation, (iii) decreases T cell activity, (iv) decreases cytotoxic T cell activity, (v) decreases natural killer (NK) and/or NKT cell activity, (vi) decreases and/or γδ T-cell activity, (vii) decreases pro-inflammatory cytokine secretion, (viii) decreases IL-2 secretion; (ix) decreases interferon-γ production, (x) decreases Th1 response, (xi) decreases Th2 response, (xii) increases cell number and/or activity of regulatory T cells, (xiii) increases regulatory cell activity and/or one or more of myeloid derived suppressor cells (MDSCs), iMCs, mesenchymal stromal cells, TIE2-expressing monocytes, (xiv) increases regulatory cell activity and/or the activity of one or more of myeloid derived suppressor cells (MDSCs), iMCs, mesenchymal stromal cells, TIE2-expressing monocytes, (xv) increases M2 macrophages, (xvi) increases M2 macrophage activity, (xvii) increases N2 neutrophils, (xviii) increases N2 neutrophils activity, (xix) increases inhibition of T cell activation, (xx) increases inhibition of CTL activation, (xxi) increases inhibition of NK cell activation, (xxii) increases and/or γδ T cell exhaustion, (xxiii) decreases and/or γδ T cell response, (xxiv) decreases activity of cytotoxic cells, (xxv) reduces antigen-specific memory responses, (xxvi) inhibits apoptosis or lysis of cells, (xxvii) decreases cytotoxic or cytostatic effect on cells, (xxviii) reduces direct killing of cells, (xxix) decreases Th17 activity, (xxx) modulates myeloid cell polarization, and/or modulates myeloid cell shifting toward an anti-inflammatory response, and/or (xxxi) reduces complement dependent cytotoxicity and/or antibody dependent cell-mediated cytotoxicity.
Therefore, in one embodiment the present invention broadly relates to the development of novel immunomodulatory proteins wherein this includes HIDE1 polypeptides that antagonize or block the effects of HIDE1 on immunity and particularly the effects of HIDE1, on specific types of immune cells and cytokine production (i.e., immunostimulatory HIDE1 polypeptides or fusion proteins and/or immunostimulatory HIDE1 antibodies).
Additionally, the invention relates to HIDE 1 polypeptides that agonize or mimic the effects of HIDE1 on immunity and particularly the effects of HIDE1 on specific types of immune cells and cytokine production (i.e., immunoinhibitory HIDE1 polypeptides or fusion proteins or HIDE1 polypeptides that increase and/or enhance in immune suppression), as well as immune enhancing polypeptides which mimi HIDE1 immune suppressive activity through binding to the HIDE1 binding/signalling partner and can be referred to as enhancers of HIDE1 associated immune suppression.
Accordingly, in some embodiments the present invention provides methods of screening for modulators of the interaction of HIDE1 and the binding and/or signaling partner for HIDE1, which either leads to immunostimulation or immunoinhibition, as outlined herein. For example, compounds that inhibit the interaction of HIDE1 and the binding and/or signaling partner for HIDE1, which normally leads to the suppression of myeloid cells including but not limited to monocytes, dendritic cells, macrophages, tumor associated macrophages, neutrophils, Myeloid-derive suppressor cells (MDSC), and/or T cell and/or NK cell activation and migration and thus increase the immune response to allow for the ultimate administration to patients for the treatment of cancer and pathogen infections, for example. Conversely, compounds that increase the signaling due to the interaction of HIDE1 and the binding and/or signaling partner for HIDE1, lead to increased suppression of myeloid cells including but not limited to monocytes, dendritic cells, macrophages, tumor associated macrophages, neutrophils, Myeloid-derive suppressor cells (MDSC), and/or T cell and/or NK cell activation and migration, thus resulting in decreased immune responses, to allow for the ultimate administration to patients for the treatment of diseases associated with increased immune function such as autoimmune diseases and inflammation. In this latter case, the increase of signaling is termed “stimulation of binding”, which can be effected, for example, by adding a compound (a “stimulator”) such as the ECD of HIDE1, resulting in stimulated binding of the ECD to the endogenous binding and/or signaling partner for HIDE1 and triggering the signaling pathway.
A. Cell Based Assays
Accordingly, in one embodiment, the invention provides assays to screen for inhibitors of the binding association of HIDE1 and the binding and/or signaling partner for HIDE1.
In one embodiment, the methods of screening provide cells that comprise an exogenous recombinant nucleic acid encoding a human HIDE1 protein, generally the full length protein including the transmembrane domain, such that the HIDE1 protein is expressed in the correct orientation, resulting in the extracellular domain (ECD) being on the surface of the cell. By “exogenous” in this context herein is meant that the gene (and any required expression vector sequences) is not endogenous (naturally occurring in the genome) to the cell. In the case of non-human cell lines to be used in the assays herein, this means that the non-human cell line has a human gene transfected into the cell. In the case where human cell lines are used (preferable in most instances), and thus contain an endogenous HIDE1 gene, the cells contain at least an additional, recombinant human gene, if not additional copies as well.
In this embodiment, cells expressing exogenous HIDE1 are contacted with candidate agent(s) as is more fully outlined below, and a labeled binding and/or signaling partner for HIDE1 (generally the ECD domain). By comparing the results to a reference standard not including the candidate agent, where binding is known to occur, the lack of bound label means the candidate agent binds to the HIDE1 in such a way as to prevent binding to the binding and/or signaling partner for HIDE1.
In one embodiment, the methods are reversed, and thus use cells that comprise an exogenous recombinant nucleic acid encoding a binding and/or signaling partner for HIDE1, generally the full length protein including the transmembrane domain, such that the binding and/or signaling partner for HIDE1 protein is expressed in the correct orientation, resulting in the extracellular domain (ECD) being on the surface of the cell.
In this embodiment, cells expressing exogenous binding and/or signaling partner for HIDE1 are contacted with candidate agent(s) as is more fully outlined below, and a labeled binding and/or signaling partner for HIDE1 (generally the ECD domain). By comparing the results to a reference standard not including the candidate agent, where binding is known to occur, the lack of bound label means the candidate agent binds to the binding and/or signaling partner for HIDE1 in such a way as to prevent binding to binding and/or signaling partner for HIDE1.
As will be appreciated by those in the art, these assays can be done on surfaces such as in microtiter plates.
B. Support Based Assays
In one embodiment, the screening assay is a solid support assay, where one or the other of HIDE1 and/or the binding and/or signaling partner for HIDE1 is attached, for example to a microtiter plate. Candidate agents and labeled proteins, e.g. the “other” of the HIDE1 and/or the binding and/or signaling partner for HIDE1 is added. If the candidate agent blocks binding, this can be determined using the read out. For example, in one embodiment, HIDE1 is attached to the solid support, generally at discrete locations. A candidate agent and the binding and/or signaling partner for HIDE1 is added, for example that is either directly labeled (for example with a fluorophore as outlined below), or indirectly labeled (for example using a labeled anti-binding and/or signaling partner for HIDE1 antibody). After allowing a sufficient period of time and after washing, if the candidate agent blocks the interaction of HIDE1 and the binding and/or signaling partner for HIDE1, no signal will be seen. If the agent does not, the signal will be generated and bound to the support. Similarly, this can be done using attachment of the binding and/or signaling partner for HIDE1 to the solid support and adding labeled HIDE1 and candidate agents.
C. FRET Based Assays
In some embodiments, this binding assay can be done using fluorescent resonance energy transfer (FRET) assays, as is well known in the art, where one of the receptor-ligand pair of HIDE1 and the binding and/or signaling partner for HIDE1 has a FRET donor and the other has a FRET acceptor. Upon binding of the two, FRET occurs. If the candidate agent prevents binding, the FRET signal will be lost. This is also useful in competition assays, to determine whether the binding of the agent to HIDE1 is stronger than the binding of the binding and/or signaling partner for HIDE1.
D. Functional Assays
These identified candidate agents that bind and block the interaction of HIDE1 and/or the binding and/or signaling partner for HIDE1 can then be further tested to see their effect on the signaling pathway. That is, the binding/blocking agents can be run in assays that measure immuno-suppressive function of myeloid cells including but not limited to monocytes, dendritic cells, macrophages, M1/M2 tumor associated macrophages, neutrophils, Myeloid-derive suppressor cells (MDSC), and/or T cell or NK cell activation and or migration, for example, to determine whether the blocking agents are immunostimulatory (increasing immune function such that diseases such as cancer can be treated) or immunoinhibitory (decreasing immune function to treat autoimmune diseases and inflammation or increasing or enhancing immune suppression).
According to at least some embodiments of the present invention also provides antibodies, including antigen binding domains, that bind to human HIDE1 for use in methods of depleting myeloid cells, or other circulating tumor cells, in order to treat diseases such as cancer.
In addition, the assays below can also be used to assess treatment efficacy, as is more further outlined below.
In some embodiments, the functional assay uses CTLs. The CTLs express a T cell receptor (TCR) recognizing a specific antigen (Ag) presented on an MHC molecule. Upon TCR Ag engagement, CTLs undergo activation as manifested by cell proliferation, up-regulation of activation markers (e.g. CD25, CD137 etc.), and cytokine secretion (e.g. interferon gamma, IL2, TNFα etc.) and cytotoxic activity. Upon contact with the binding and/or signaling partner for HIDE1 expressed on cancer cells or antigen presenting cells, HIDE1 mediates a negative signal to CTLs thereby causing down-regulation of CTL activation as manifested by the above readouts. Thus, contacting candidate agents that have shown binding and/or inhibition of receptor-ligand binding with CTLs will to interrupt the HIDE1 and/or the binding and/or signaling partner for HIDE1 interaction and thereby release the negative signal mediated by HIDE1 and enhances antigen specific CTL activation as manifested by cell proliferation, up-regulation of activation markers (e.g. CD25, CD137 etc.), and cytokine secretion (e.g. interferon gamma, IL2, TNF alfa etc.).
Similarly, in some embodiments, the functional assay uses NK cells. The NK cells express various activating and inhibitory receptors. The execution of NK cytotoxic activity is determined by the balance between the activatory and inhibitory signals derived from these receptors. Upon engagement of NK cells with certain target cells, NK cells undergo activation as manifested by cell proliferation, cytokine secretion (e.g. interferon gamma, IL2, TNF alfa etc.) and cytotoxic activity. Upon contact with the HIDE1 and/or the binding and/or signaling partner expressed on cancer target cells, HIDE1 mediates a negative signal to NK cells thereby causing down-regulation of NK cell activation as manifested by the above readouts. Contacting of candidate agents with NK cells will interrupt the HIDE1 and/or the binding and/or signaling partner for HIDE1 interaction and thereby release the negative signal mediated by HIDE1 and enhances NK cell activation as manifested by cell proliferation, cytokine secretion (e.g. interferon gamma, IL2, TNF alfa etc.) and cytotoxic activity.
In one embodiment, the signaling pathway assay measures increases or decreases in immune response as measured for an example by phosphorylation or de-phosphorylation of different factors, or by measuring other post translational modifications. An increase in activity indicates immunostimulatory activity and a decrease indicates immunoinhibitory activity (or an increase and/or enhancement in immune suppression). Appropriate increases or decreases in activity are outlined below.
In one embodiment, the signaling pathway assay measures increases or decreases in activation of αβ and/or γδ T cells as measured for an example by cytokine secretion or by proliferation or by changes in expression of activation markers like for an example CD137, CD107a, PD1, etc. An increase in activity indicates immunostimulatory activity and a decrease indicates immunoinhibitory activity (or an increase and/or enhancement in immune suppression). Appropriate increases or decreases in activity are outlined below.
In one embodiment, the signaling pathway assay measures increases or decreases in cytotoxic T cell activity as measured for an example by direct killing of target cells like for an example cancer cells or by cytokine secretion or by proliferation or by changes in expression of activation markers like for an example CD137, CD107a, PD1, etc. An increase in activity indicates immunostimulatory activity and a decrease indicates immunoinhibitory activity (or an increase and/or enhancement in immune suppression). Appropriate increases or decreases in activity are outlined below.
In one embodiment, the signaling pathway assay measures increases or decreases in NK and/or NKT cell activity as measured for an example by direct killing of target cells like for an example cancer cells or by cytokine secretion or by changes in expression of activation markers like for an example CD107a, etc. An increase in activity indicates immunostimulatory activity and a decrease indicates immunoinhibitory activity (or an increase and/or enhancement in immune suppression). Appropriate increases or decreases in activity are outlined below. In one embodiment, the signaling pathway assay measures increases or decreases in αβ and/or γδ T-cell suppression. as measured for an example by cytokine secretion or by proliferation or by changes in expression of activation markers like for an example CD137, CD107a, PD1, etc. An increase in activity indicates immunostimulatory activity and a decrease indicates immunoinhibitory activity (or an increase and/or enhancement in immune suppression). Appropriate increases or decreases in activity are outlined below.
In one embodiment, the signaling pathway assay measures increases or decreases in pro-inflammatory cytokine secretion as measured for example by ELISA or by Luminex or by Multiplex bead based methods or by intracellular staining and FACS analysis or by Alispot etc. An increase in activity indicates immunostimulatory activity and a decrease indicates immunoinhibitory activity (or an increase and/or enhancement in immune suppression). Appropriate increases or decreases in activity are outlined below.
In one embodiment, the signaling pathway assay measures increases or decreases in IL-2 secretion as measured for example by ELISA or by Luminex or by Multiplex bead based methods or by intracellular staining and FACS analysis or by Alispot etc. An increase in activity indicates immunostimulatory activity and a decrease indicates immunoinhibitory activity (or an increase and/or enhancement in immune suppression).
In one embodiment, the signaling pathway assay measures increases or decreases in interferon-γ production as measured for example by ELISA or by Luminex or by Multiplex bead based methods or by intracellular staining and FACS analysis or by Alispot etc. An increase in activity indicates immunostimulatory activity and a decrease indicates immunoinhibitory activity (or an increase and/or enhancement in immune suppression). Appropriate increases or decreases in activity are outlined below.
In one embodiment, the signaling pathway assay measures increases or decreases in Th1 response as measured for an example by cytokine secretion or by changes in expression of activation markers. An increase in response indicates immunostimulatory activity and a decrease indicates immunoinhibitory activity (or an increase and/or enhancement in immune suppression). Appropriate increases or decreases in activity are outlined below.
In one embodiment, the signaling pathway assay measures increases or decreases in Th2 response as measured for an example by cytokine secretion or by changes in expression of activation markers. An increase in response indicates immunostimulatory activity and a decrease indicates immunoinhibitory activity (or an increase and/or enhancement in immune suppression). Appropriate increases or decreases in activity are outlined below.
In one embodiment, the signaling pathway assay measures increases or decreases cell number and/or activity of at least one of regulatory T cells (Tregs), as measured for example by flow cytometry or by IHC. A decrease in response indicates immunostimulatory activity and an increase indicates immunoinhibitory activity (or an increase and/or enhancement in immune suppression). Appropriate increases or decreases in activity are outlined below.
In one embodiment, the signaling pathway assay measures increases or decreases in M2 macrophages cell numbers, as measured for example by flow cytometry or by IHC. A decrease in response indicates immunostimulatory activity and an increase indicates immunoinhibitory activity (or an increase and/or enhancement in immune suppression). Appropriate increases or decreases in activity are outlined below.
In one embodiment, the signaling pathway assay measures increases or decreases in M2 macrophage pro-tumorigenic activity, as measured for an example by cytokine secretion or by changes in expression of activation markers. A decrease in response indicates immunostimulatory activity and an increase indicates immunoinhibitory activity (or an increase and/or enhancement in immune suppression).
In one embodiment, the signaling pathway assay measures increases or decreases in N2 neutrophils increase, as measured for example by flow cytometry or by IHC. A decrease in response indicates immunostimulatory activity and an increase indicates immunoinhibitory activity (or an increase and/or enhancement in immune suppression). Appropriate increases or decreases in activity are outlined below.
In one embodiment, the signaling pathway assay measures increases or decreases in N2 neutrophils pro-tumorigenic activity, as measured for an example by cytokine secretion or by changes in expression of activation markers. A decrease in response indicates immunostimulatory activity and an increase indicates immunoinhibitory activity (or an increase and/or enhancement in immune suppression). Appropriate increases or decreases in activity are outlined below.
In one embodiment, the signaling pathway assay measures increases or decreases in inhibition of T cell activation, as measured for an example by cytokine secretion or by proliferation or by changes in expression of activation markers like for an example CD137, CD107a, PD1, etc. An increase in response indicates immunostimulatory activity and a decrease indicates immunoinhibitory activity (or an increase and/or enhancement in immune suppression). Appropriate increases or decreases in activity are outlined below.
In one embodiment, the signaling pathway assay measures increases or decreases in inhibition of CTL activation as measured for an example by direct killing of target cells like for an example cancer cells or by cytokine secretion or by proliferation or by changes in expression of activation markers like for an example CD137, CD107a, PD1, etc. An increase in response indicates immunostimulatory activity and a decrease indicates immunoinhibitory activity (or an increase and/or enhancement in immune suppression). Appropriate increases or decreases in activity are outlined below.
In one embodiment, the signaling pathway assay measures increases or decreases in αβ and/or γδ T cell exhaustion as measured for an example by changes in expression of activation markers. A decrease in response indicates immunostimulatory activity and an increase indicates immunoinhibitory activity (or an increase and/or enhancement in immune suppression). Appropriate increases or decreases in activity are outlined below.
In one embodiment, the signaling pathway assay measures increases or decreases αβ and/or γδ T cell response as measured for an example by cytokine secretion or by proliferation or by changes in expression of activation markers like for an example CD137, CD107a, PD1, etc. An increase in activity indicates immunostimulatory activity and a decrease indicates immunoinhibitory activity (or an increase and/or enhancement in immune suppression). Appropriate increases or decreases in activity are outlined below.
In one embodiment, the signaling pathway assay measures increases or decreases in stimulation of antigen-specific memory responses as measured for an example by cytokine secretion or by proliferation or by changes in expression of activation markers like for an example CD45RA, CCR7 etc. An increase in activity indicates immunostimulatory activity and a decrease indicates immunoinhibitory activity (or an increase and/or enhancement in immune suppression). Appropriate increases or decreases in activity are outlined below.
In one embodiment, the signaling pathway assay measures increases or decreases in apoptosis or lysis of cancer cells as measured for an example by cytotoxicity assays such as for an example MTT, Cr release, Calcine AM, or by flow cytometry based assays like for an example CFSE dilution or propidium iodide staining etc. An increase in activity indicates immunostimulatory activity and a decrease indicates immunoinhibitory activity (or an increase and/or enhancement in immune suppression). Appropriate increases or decreases in activity are outlined below.
In one embodiment, the signaling pathway assay measures increases or decreases in stimulation of cytotoxic or cytostatic effect on cancer cells. as measured for an example by cytotoxicity assays such as for an example MTT, Cr release, Calcine AM, or by flow cytometry based assays like for an example CFSE dilution or propidium iodide staining etc. An increase in activity indicates immunostimulatory activity and a decrease indicates immunoinhibitory activity (or an increase and/or enhancement in immune suppression). Appropriate increases or decreases in activity are outlined below.
In one embodiment, the signaling pathway assay measures increases or decreases direct killing of cancer cells as measured for an example by cytotoxicity assays such as for an example MTT, Cr release, Calcine AM, or by flow cytometry based assays like for an example CFSE dilution or propidium iodide staining etc. An increase in activity indicates immunostimulatory activity and a decrease indicates immunoinhibitory activity (or an increase and/or enhancement in immune suppression). Appropriate increases or decreases in activity are outlined below.
In one embodiment, the signaling pathway assay measures increases or decreases Th17 activity as measured for an example by cytokine secretion or by proliferation or by changes in expression of activation markers. An increase in activity indicates immunoinhibitory activity and a decrease indicates immunostimulatory activity (or an increase and/or enhancement in immune suppression). Appropriate increases or decreases in activity are outlined below.
In one embodiment, the signaling pathway assay measures increases or decreases in induction of complement dependent cytotoxicity and/or antibody dependent cell-mediated cytotoxicity, as measured for an example by cytotoxicity assays such as for an example MTT, Cr release, Calcine AM, or by flow cytometry based assays like for an example CFSE dilution or propidium iodide staining etc. An increase in activity indicates immunostimulatory activity and a decrease indicates immunoinhibitory activity (or an increase and/or enhancement in immune suppression). Appropriate increases or decreases in activity are outlined below.
In one embodiment, the assay measures increases or decreases in cell proliferation as a function of activation or inhibition, using well known methodologies such as thymidine incorporation and CFSE dilution.
Appropriate increases in activity or response (or decreases, as appropriate as outlined above), are increases of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 98 to 99% percent over the signal in either a reference sample or in control samples, for example test samples that do not contain an anti-HIDE1 antibody of the invention. Similarly, increases of at least one-, two-, three-, four- or five-fold as compared to reference or control samples show efficacy.
E. Candidate Agents
The assays are run by contacting candidate agents with the HIDE1 and the binding and/or signaling partner for HIDE1. By “candidate agent”, “candidate bioactive agent” or “candidate drugs” or grammatical equivalents herein is meant any molecule, e.g. proteins (which herein includes proteins, polypeptides, and peptides), small organic or inorganic molecules, polysaccharides, polynucleotides, etc. which are to be tested for binding to HIDE1, inhibition of the HIDE1 and the binding and/or signaling partner for HIDE1 interaction, or activation of the binding and/or signaling partner for HIDE1. Candidate agents encompass numerous chemical classes. In a preferred embodiment, the candidate agents are organic molecules, particularly small organic molecules, comprising functional groups necessary for structural interaction with proteins, particularly hydrogen bonding, and typically include at least an amine, carbonyl, hydroxyl or carboxyl group, preferably at least two of the functional chemical groups. The candidate agents often comprise cyclical carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more chemical functional groups.
Candidate agents are obtained from a wide variety of sources, as will be appreciated by those in the art, including libraries of synthetic or natural compounds. As will be appreciated by those in the art, in some embodiments the present invention provides a rapid and easy method for screening any library of candidate agents, including the wide variety of known combinatorial chemistry-type libraries.
In a preferred embodiment, candidate agents are synthetic compounds. Any number of techniques are available for the random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides. Alternatively, a preferred embodiment utilizes libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts that are available or readily produced.
Additionally, natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means. Known pharmacological agents may be subjected to directed or random chemical modifications, including enzymatic modifications, to produce structural analogs.
In a preferred embodiment, candidate bioactive agents include proteins, nucleic acids, and chemical moieties.
In a preferred embodiment, the candidate bioactive agents are proteins. By “protein” herein is meant at least two covalently attached amino acids, which includes proteins, polypeptides, oligopeptides and peptides. The protein may be made up of naturally occurring amino acids and peptide bonds, or synthetic peptidomimetic structures. Thus “amino acid”, or “peptide residue”, as used herein means both naturally occurring and synthetic amino acids. For example, homo-phenylalanine, citrulline and norleucine are considered amino acids for the purposes of the invention. “Amino acid” also includes imino acid residues such as proline and hydroxyproline. The side chains may be in either the (R) or the (S) configuration. In the preferred embodiment, the amino acids are in the (S) or L-configuration. If non-naturally occurring side chains are used, non-amino acid substituents may be used, for example to prevent or retard in vivo degradations.
In a preferred embodiment, the candidate bioactive agents are naturally occurring proteins or fragments of naturally occurring proteins. Thus, for example, cellular extracts containing proteins, or random or directed digests of proteinaceous cellular extracts, may be attached to beads as is more fully described below. In this way libraries of procaryotic and eucaryotic proteins may be made for screening against any number of targets. Particularly preferred in this embodiment are libraries of bacterial, fungal, viral, and mammalian proteins, with the latter being preferred, and human proteins being especially preferred.
In many embodiments, the candidate agents are antibodies to HIDE1, generated as is known in the art and outlined herein.
In many embodiments, the candidate agents are ECDs of HIDE1, including fusion proteins and variants, as is known in the art and more fully outlined herein.
In a preferred embodiment, the candidate bioactive agents are peptides of from about 2 to about 50 amino acids, with from about 5 to about 30 amino acids being preferred, and from about 8 to about 20 being particularly preferred. The peptides may be digests of naturally occurring proteins as is outlined above, random peptides, or “biased” random peptides. By “randomized” or grammatical equivalents herein is meant that each nucleic acid and peptide consists of essentially random nucleotides and amino acids, respectively. Since generally these random peptides (or nucleic acids, discussed below) are chemically synthesized, they may incorporate any nucleotide or amino acid at any position. The synthetic process can be designed to generate randomized proteins or nucleic acids, to allow the formation of all or most of the possible combinations over the length of the sequence, thus forming a library of randomized candidate bioactive proteinaceous agents.
The library should provide a sufficiently structurally diverse population of randomized agents to effect a probabilistically sufficient range of diversity to allow binding to a particular target. Accordingly, an interaction library must be large enough so that at least one of its members will have a structure that gives it affinity for the target. Although it is difficult to gauge the required absolute size of an interaction library, nature provides a hint with the immune response: a diversity of 107-108 different antibodies provides at least one combination with sufficient affinity to interact with most potential antigens faced by an organism. Published in vitro selection techniques have also shown that a library size of 107 to 108 is sufficient to find structures with affinity for the target. A library of all combinations of a peptide 7 to 20 amino acids in length, such as generally proposed herein, has the potential to code for 207 (109) to 2020. Thus, with libraries of 107 to 108 different molecules the present methods allow a “working” subset of a theoretically complete interaction library for 7 amino acids, and a subset of shapes for the 2020 library. Thus, in a preferred embodiment, at least 10.sup.6, preferably at least 107, more preferably at least 108 and most preferably at least 109 different sequences are simultaneously analyzed in the subject methods. Preferred methods maximize library size and diversity.
In one embodiment, the library is fully randomized, with no sequence preferences or constants at any position. In a preferred embodiment, the library is biased. That is, some positions within the sequence are either held constant, or are selected from a limited number of possibilities. For example, in a preferred embodiment, the nucleotides or amino acid residues are randomized within a defined class, for example, of hydrophobic amino acids, hydrophilic residues, sterically biased (either small or large) residues, towards the creation of cysteines, for cross-linking, prolines for SH-3 domains, serines, threonines, tyrosines or histidines for phosphorylation sites, etc., or to purines, etc.
In some embodiments, the present invention provides a method of screening for inhibitors of the binding association of HIDE1 polypeptide with a HIDE1 binding and/or signaling partner polypeptide, said method comprising: a) providing a surface comprising a first ligand protein comprising HIDE1 polypeptide or HIDE1 binding and/or signaling partner polypeptide; b) contacting said surface with a candidate agent under physiological conditions, wherein if said candidate agent binds to said first ligand protein it forms a first binding complex, c) contacting said surface with a second ligand protein comprising the other of HIDE1 polypeptide or HIDE1 binding and/or signaling partner polypeptide; and d) determining whether said HIDE1 polypeptide and said HIDE1 binding and/or signaling partner polypeptide are bound as an indication of whether said candidate agent inhibits said binding association.
In some embodiments, the present invention provides a method of screening for inhibitors of the binding association of HIDE1 polypeptide with HIDE1 binding and/or signaling partner polypeptide, said method comprising: a) providing a cell comprising an exogeneous recombinant nucleic acid encoding a human HIDE1 polypeptide, wherein said cell expresses said human HIDE1 polypeptide; b) contacting said cell with a candidate agent and a labeled HIDE1 binding and/or signaling partner polypeptide; c) determining whether said HIDE1 polypeptide binds to HIDE1 binding and/or signaling partner polypeptide as an indication of whether said candidate agent inhibits the binding of HIDE1 polypeptide with HIDE1 binding and/or signaling partner polypeptide.
In some embodiments, the present invention provides a method of screening for inhibitors of the binding association of HIDE1 polypeptide with HIDE1 binding and/or signaling partner polypeptide, said method comprising: a) providing a cell comprising an exogeneous recombinant nucleic acid encoding a human HIDE1 binding and/or signaling partner polypeptide, wherein said cell expresses said human HIDE1 binding and/or signaling partner polypeptide; b) contacting said cell with a candidate agent and a labeled HIDE1 polypeptide; c) determining whether said HIDE1 binding and/or signaling partner polypeptide binds to HIDE1 as an indication of whether said candidate agent inhibits the binding of HIDE1 polypeptide with HIDE1 binding and/or signaling partner polypeptide.
In some embodiments, the present invention provides a method of screening for inhibitors of the binding association of HIDE1 polypeptide with HIDE1 binding and/or signaling partner polypeptide, said method comprising: a) providing a test solution comprising: i) a HIDE1 polypeptide comprising a first FRET label; ii) a HIDE1 binding and/or signaling partner polypeptide comprising a second FRET label; c) providing a candidate agent; d) detecting a FRET signal between said first and second label, wherein a difference in said FRET signal in the presence or absence of said candidate agent indicates that the candidate agent inhibits said binding association. In some embodiments of the method, a plurality of candidate agents are tested. In some embodiments of the method, the candidate agent is a protein. In some embodiments of the method, the protein is an anti-HIDE1 antibody. In some embodiments of the method, the protein comprises an extracellular domain (ECD) of HIDE1. In some embodiments of the method, the protein is a fusion protein comprising said ECD and a fusion partner.
In some embodiments, the fusion partner is selected from the group consisting of a human IgG Fc domain and a human serum albumin (HSA).
In some embodiments of the invention, said the method further comprises: a) contacting said candidate agent with a population of cytotoxic T cells (CTLs) under conditions wherein said CTLs would normally be activated; and b) determining the effect of said agent on said activation.
In some embodiments of the invention, the method further comprises: a) contacting said candidate agent with a population of cytotoxic T cells (CTLs); an b) determining the effect of said agent on IFNγ production.
In some embodiments of the invention, the method further comprises: a) contacting said candidate agent with a population of γδ T cells under conditions wherein said γδ T cells would normally be activated; and b) determining the effect of said agent on said activation.
In some embodiments of the invention, the method further comprises: a) contacting said candidate agent with a population of Th1 cells under conditions wherein said Th1 cells would normally be activated; and b) determining the effect of said agent on said activation.
In some embodiments of the invention, the method further comprises: a) contacting said candidate agent with a population of regulatory T cells (Tregs) under conditions and determining the effect of said agent on Treg cell number or activity.
In some embodiments of the invention, the determination is done by measuring the presence or absence of increased expression of a protein selected from the group consisting of IFNg, TNFα, GM-CSF, CD25, CD137, CD69, PD1, CD107A, HLA-DR, IL-2, IL-6, IL-4, IL-5, IL-10 and IL-13, wherein increased expression is an indication of activation.
According to at least some embodiments of the present invention is directed to the use of human HIDE1, as outlined below. As used herein, the term “HIDE1” or “HIDE1 protein” or “HIDE1 polypeptide” may optionally include any such protein, or variants, conjugates, or fragments thereof, including but not limited to known or wild type HIDE1, as described herein, including but not limited to SEQ ID NO:1, and also other HIDE1 variants, including but not limited to SEQ ID NOs: 7-11, as well as any soluble HIDE1 protein, including but not limited to any of SEQ ID NOs: 2,3,6,19-47, and/or variants thereof possessing at least 80% sequence identity, more preferably at least 90% sequence identity therewith and even more preferably at least 95, 96, 97, 98 or 99% sequence identity therewith, and/or fusions and or conjugates thereof, and/or polynucleotides encoding same. As used herein, the term “HIDE1” or “HIDE1 protein” or “HIDE1 polypeptide” may optionally include any such protein, or variants, conjugates, or fragments thereof, including but not limited to non-human HIDE1 orthologs, such as for example, mouse HIDE1 protein as set forth in SEQ ID NO: 12, and/or its corresponding extracellular domain, as set forth in any of SEQ ID NOs: 13-16.
The HIDE1 sequence, as described herein under SEQ ID NO:1, is copied below.
The HIDE1 ECD sequence, as described herein under SEQ ID NO:2, copied below.
The HIDE1 ECD sequence, as described herein under SEQ ID NO:3, copied below.
The term “soluble” form of HIDE1 is also used interchangeably with the terms “soluble ectodomain (ECD)” or “ectodomain” or “fragments of HIDE1 polypeptides” or “extracellular domain”, and which may refer broadly to one or more of the following optional polypeptides:
Optionally, the HIDE1 ECD proteins and fragments thereof refer to any one of the polypeptide sequences listed in any of SEQ ID NOs: 2-6, 21-47, and/or variants thereof possessing at least 80% sequence identity, more preferably at least 90% sequence identity therewith and even more preferably at least 95, 96, 97, 98 or 99% sequence identity therewith, and/or mouse ortholog thereof listed in any one of SEQ ID NOs: 13-16, and/or fusions and or conjugates thereof, and/or polynucleotides encoding same.
Optionally, the fragment is of at least about 95 and so forth amino acids of the extracellular domain of HIDE1 protein, set forth in SEQ ID NO: 2, up to 115 amino acids of the HIDE1 protein extracellular domain, optionally including any integral value between 95 and 115 amino acids in length. Preferably, the fragment is of at least about 101 and up to 109 amino acids of the HIDE1 protein extracellular domain, optionally including any integral value between 101 and 109 amino acids in length. Also preferably the fragment is of at least about 103 up to 105 amino acids of the HIDE1 protein extracellular domain, optionally including any integral value between 103 and 105 amino acids in length. More preferably, the fragment is about 105 amino acids. The HIDE1 fragment protein according to at least some embodiments of the present invention may or may not include a signal peptide sequence, and may or may not include 1, 2, 3, 4, or 5 contiguous amino acids from the HIDE1 transmembrane domain.
In particular, the fragments of the extracellular domain of HIDE1 can include any sequence corresponding to any portion of or comprising the Ig domain of the extracellular domain of HIDE1, having any sequence corresponding to residues of HIDE1 (SEQ ID NO:1) starting from any position between 17 and 21 and ending at any position between 109 and 113.
Without wishing to be limited by a single hypothesis, the HIDE1 proteins contain an immunoglobulin domain within the extracellular domain. The Ig domain may optionally be responsible for receptor binding, by analogy to the other B7 family members. The Ig domain of the extracellular domain includes one disulfide bond formed between intra domain cysteine residues, as is typical for this fold and may optionally be important for structure-function. In SEQ ID NO: 2 these cysteines are located at residues 19 and 69.
In one embodiment, there is provided a soluble fragment of HIDE1; as described in greater detail below with regard to the section on fusion proteins, such a soluble fragment may optionally be described as a first fusion partner. Useful fragments are those that alone or when comprised in fusion proteins or multimerized retain the ability to bind to their natural molecular partner or partners, e.g., expressed on antigen presenting, T and NK cells, and/or which modulate T cell and/or NK cell activation. A HIDE1 polypeptide that is a fragment of full-length HIDE1 typically has at least 20 percent, 30 percent, 40 percent, 50 percent, 60 percent, 70 percent, 80 percent, 90 percent, 95 percent, 98 percent, 99 percent, 100 percent, or even more than 100 percent of the ability to bind its natural molecular partner(s) and/or of the modulation (preferably enhancing and/or agonizing of one or more of the functional effects of HIDE1 on immunity and on specific immune cells as compared to full-length HIDE1; optionally such a percentage may be any integral value between 20 and 100 percent.
Soluble HIDE1 polypeptide fragments are fragments of HIDE1 polypeptides that may optionally be shed, secreted or otherwise extracted from the producing cells. In other embodiments, the soluble fragments of HIDE1 polypeptides include fragments of the HIDE1 extracellular domain that retain HIDE1 biological activity, such as fragments that retain the ability to bind to their natural receptor or receptors and/or which modulate T or NK cell activation. The extracellular domain can include 1, 2, 3, 4, or 5 contiguous amino acids from the transmembrane domain, and/or 1, 2, 3, 4, or 5 contiguous amino acids from the signal sequence. Alternatively, the extracellular domain can have 1, 2, 3, 4, 5 or more amino acids removed from the C-terminus, N-terminus, or both.
In some embodiments the HIDE1 extracellular domain polypeptide comprises the amino acid sequence of the Ig domain as set forth in any one of SEQ ID NO: 6, 24-47, or fragments or variants thereof. In other embodiments the HIDE1 extracellular domain polypeptide consists essentially of the amino acid sequence of the Ig domain as set forth in any one of SEQ ID NOs: 6, 24-47.
Generally, the SEQ ID NOs:6, 24-47 polypeptide fragments are expressed from nucleic acids that include sequences that encode a signal sequence. The signal sequence is generally cleaved from the immature polypeptide to produce the mature polypeptide lacking the signal sequence. The signal sequence of HIDE1 can be replaced by the signal sequence of another polypeptide using standard molecule biology techniques to affect the expression levels, secretion, solubility, or other property of the polypeptide. The signal peptide sequence that is used to replace the HIDE1 signal peptide sequence can be any known in the art.
In one embodiment such “soluble ectodomain (ECD)” or “ectodomain” or “soluble” form of HIDE1 will modulate (preferably enhance and/or agonize) one or more of HIDE1's effects on immunity and specific types of immune cells such as T helper, cytotoxic or effector T cells, Tregs NK cells and antigen presenting cells.
Optionally, the HIDE1 ECD fragments refer also to any one of the polypeptide sequences listed in any of SEQ ID NOs: 19-20, which are reasonably expected to comprise functional regions of the HIDE1 protein. This expectation is based on a systematic analysis of a set of protein complexes with solved 3D structures, which contained complexes of Ig proteins (for example PDB ID 1i85 which describe the complex of CTLA4 and CD86). The intermolecular contact residues from each co-structure were collected and projected on the sequence of HIDE1. Several regions with clusters of interacting residues supported by several contact maps were identified and are reasonably expected to mimic the structure of the intact full length protein and thereby modulate one or more of the effects of HIDE1 on immunity and on specific immune cell types. The HIDE1 extracellular domain polypeptides according to at least some embodiments of the present invention are expected to be useful for treatment of autoimmune diseases which have proved resistant to other drug treatments, because, without wishing to be limited by a single hypothesis, the mode of action of these HIDE1 extracellular domain polypeptides is expected to enable them to overcome such resistance. Methods of treatment are further described herein, below under the methods of using section.
According to at least some embodiments there is provided an isolated or recombinant HIDE1 new variant polypeptide or a fragment thereof, consisting essentially of an amino acid sequence as set forth in any of SEQ ID NOs: 7-11, or variant thereof that possesses at least 90% or 95%, 96%, 97%, 98%, or 99% sequence identity therewith.
According to at least some embodiments there is provided an isolated or recombinant HIDE1 polypeptide comprising a HIDE1 ECD, consisting essentially of an amino acid sequence as set forth in any of SEQ ID NOs: 2-5, 21-23, or a fragment thereof or variant thereof that possesses at least 90% or 95%, 96%, 97%, 98%, or 99% sequence identity therewith, or mouse ortholog corresponding to amino acid sequence as set forth in any one of SEQ ID NOs: 13-16.
According to at least some embodiments there is provided an isolated or recombinant HIDE1 ECD fragment, consisting essentially of an amino acid sequence as set forth in any of SEQ ID NOs: 6, 19-20, 24-47 or variant thereof that possesses at least 90% or 95%, 96%, 97%, 98%, or 99% sequence identity therewith.
According to at least some embodiments there is provided an isolated or recombinant HIDE1 polypeptide comprising a multimer or a fusion polypeptide comprising a HIDE1 ECD, consisting essentially of an amino acid sequence as set forth in any of SEQ ID NOs: 2-5, 21-23 or variant thereof that possesses at least 90% or 95%, 96%, 97%, 98%, or 99% sequence identity therewith, or mouse ortholog corresponding to amino acid sequence as set forth in any one of SEQ ID NOs: 13-16, or HIDE1 ECD fragment thereof, which fragment consists essentially of an amino acid sequence as set forth in any of SEQ ID NOs: 6, 19-20, 24-47 or variant thereof that possesses at least 90% or 95%, 96%, 97%, 98%, or 99% sequence identity therewith, wherein such multimer or fusion polypeptide may optionally comprise one or more of such HIDE1 ECD polypeptides or fragments thereof, e.g., 1-10 fragments, directly linked or attached to one another or fused via a linker or multimerization domain.
According to at least some embodiments there is provided a fusion protein comprising the polypeptide comprising a HIDE1 ECD, consisting essentially of an amino acid sequence as set forth in any of SEQ ID NOs:2-5, 21-23, or variant thereof that possesses at least 90% or 95%, 96%, 97%, 98%, or 99% sequence identity therewith, or mouse ortholog corresponding to amino acid sequence as set forth in any one of SEQ ID NOs: 13-16, or HIDE1 ECD fragment thereof, which fragment consists essentially of an amino acid sequence as set forth in any of SEQ ID NOs: 6, 19-20, 24-47 or variant thereof that possesses at least 90% or 95%, 96%, 97%, 98%, or 99% sequence identity therewith, joined to a second fusion partner composed of a heterologous sequence (i.e., a non-HIDE1 polypeptide), fused together directly or indirectly via a peptide linker sequence or a chemical linker. Optionally, the heterologous sequence comprises at least a portion of an immunoglobulin molecule. Optionally and preferably, the immunoglobulin molecule portion is an immunoglobulin heavy chain constant region Fc fragment. Optionally and more preferably, the immunoglobulin heavy chain constant region is derived from an immunoglobulin isotype selected from the group consisting of an IgG1, IgG2, IgG3, IgG4, IgM, IgE, IgA and IgD. Optionally, the fusion protein has the amino acid sequence set forth in any one of SEQ ID NOs: 17, 18, and also optionally modulates immune cell response in vitro or in vivo.
According to at least some embodiments, the subject invention provides isolated nucleic acid sequences encoding any one of the foregoing HIDE1 polypeptides comprising a HIDE1 ECD, consisting essentially of an amino acid sequence as set forth in any of SEQ ID NOs: 2-5, 21-23, or variant thereof that possesses at least 90% or 95%, 96%, 97%, 98%, or 99% sequence identity therewith, or mouse ortholog corresponding to amino acid sequence as set forth in any one of SEQ ID NOs: 13-16, or HIDE1 ECD fragment thereof, which fragment consists essentially of an amino acid sequence as set forth in any of SEQ ID NOs: 6, 19-20, 24-47 or variant thereof that possesses at least 90% or 95%, 96%, 97%, 98%, or 99% sequence identity therewith, or multimers or fusion proteins thereof
According to at least some embodiments, there is provided an expression vector or a virus, containing at least one isolated nucleic acid sequence as described herein. According to at least some embodiments, there is provided a recombinant cell comprising an expression vector or a virus containing an isolated nucleic acid sequence as described herein, wherein the cell constitutively or inducibly expresses the polypeptide encoded by the DNA segment. According to at least some embodiments, there is provided a method of producing any one of the foregoing HIDE1 polypeptides comprising a HIDE1 ECD, consisting essentially of an amino acid sequence as set forth in any of SEQ ID NOs: 2-5, 21-23, or variant thereof that possesses at least 90% or 95%, 96%, 97%, 98%, or 99% sequence identity therewith, or mouse ortholog corresponding to amino acid sequence as set forth in any one of SEQ ID NOs: 13-16, or HIDE1 ECD fragment thereof, which fragment consists essentially of an amino acid sequence as set forth in any of SEQ ID NOs: 6, 19-20, 24-47, or variant thereof that possesses at least 90% or 95%, 96%, 97%, 98%, or 99% sequence identity therewith, or fusion proteins thereof, comprising culturing the recombinant cell as described herein, under conditions whereby the cell expresses the polypeptide encoded by the DNA segment or nucleic acid and recovering said polypeptide.
In some embodiments, the invention uses HIDE1 polypeptides in the form of fusion proteins, wherein the HIDE1 polypeptide (generally an ECD) is fused, recombinantly in frame to a fusion partner.
A. Fusion Partners
In many embodiments, the HIDE1 polypeptide is fused to a “fusion partner” (also referred to herein as a “fusion partner moiety”), either directly or indirectly through the use of a linker as is more fully described below. As will be appreciated by those in the art, the fusion partner can be any moiety that is fused to the HIDE1 polypeptide for any number of biochemical and/or biological reasons. In some embodiments, the fusion partner moiety increases the half life of the HIDE1 fusion protein as is described below. In some embodiments, the fusion partner moiety adds an additional biologic or biochemical function to the HIDE1 polypeptide.
In some embodiments, the fusion partner is generally linked at either the N-terminus or the C-terminus of the HIDE1 polypeptide, optionally using a linker as described herein, such that the fusion protein has a formula selected from the group consisting of NH2-HIDE1 polypeptide-fusion partner-COOH, HIDE1 polypeptide, NH2-fusion partner-L-HIDE1 polypeptide-COOH, and NH2-fusion partner-HIDE1-polypeptide-COOH.
1. HSA
In some embodiments, the HIDE1 fusion partner is a human serum albumin (HSA), as is known in the art. In particular, fusions to HSA are known to increase serum half life of the fusion protein, as compared to the protein itself. These can include standard flexible linkers such as described herein and shown in
2. Fc Domains
In some embodiments, the HIDE1 polypeptide is fused to a fusion partner that is an Fc domain. By “Fc domain” herein is meant the CH2-CH3 domains of an antibody, as is known in the art, optionally including some or all of the hinge region residues. The Fc domain is generally derived from a human IgG protein, generally IgG1, IgG2, IgG3 or IgG4, the sequences of which are shown in (
In addition, there are a number of Fc domain variants that can be optionally and independently included as amino acid substitutions. By “amino acid substitution” or “substitution” herein is meant the replacement of an amino acid at a particular position in a parent polypeptide sequence with a different amino acid. In particular, in some embodiments, the substitution is to an amino acid that is not naturally occurring at the particular position, either not naturally occurring within the organism or in any organism. For example, the substitution E272Y refers to a variant polypeptide, in this case an Fc variant, in which the glutamic acid at position 272 is replaced with tyrosine. For clarity, a protein which has been engineered to change the nucleic acid coding sequence but not change the starting amino acid (for example exchanging CGG (encoding arginine) to CGA (still encoding arginine) to increase host organism expression levels) is not an “amino acid substitution”; that is, despite the creation of a new gene encoding the same protein, if the protein has the same amino acid at the particular position that it started with, it is not an amino acid substitution.
In some embodiments, amino acid substitutions can be made in the Fc region, in general for altering binding to FcγR receptors. By “Fc gamma receptor”, “FcγR” or “FcgammaR” as used herein is meant any member of the family of proteins that bind the IgG antibody Fc region and is encoded by an FcγR gene. In humans this family includes but is not limited to FcγRI (CD64), including isoforms FcγRIa, FcγRIb, and FcγRIc; FcγRII (CD32), including isoforms FcγRIIa (including allotypes H131 and R131), FcγRIIb (including FcγRIIb-1 and FcγRIIb-2), and FcγRIIc; and FcγRIII (CD16), including isoforms FcγRIIIa (including allotypes V158 and F158) and FcγRIIIb (including allotypes FcγRIIIb-NA1 and FcγRIIIb-NA2) (Jefferis et al., 2002, Immunol Lett 82:57-65, entirely incorporated by reference), as well as any undiscovered human FcγRs or FcγR isoforms or allotypes. An FcγR may be from any organism, including but not limited to humans, mice, rats, rabbits, and monkeys. Mouse FcγRs include but are not limited to FcγRI (CD64), FcγRII (CD32), FcγRIII-1 (CD16), and FcγRIII-2 (CD16-2), as well as any undiscovered mouse FcγRs or FcγR isoforms or allotypes.
There are a number of useful Fc substitutions that can be made to alter binding to one or more of the FcγR receptors. Substitutions that result in increased binding as well as decreased binding can be useful. For example, it is known that increased binding to FcγRIIIa generally results in increased ADCC (antibody dependent cell-mediated cytotoxicity; the cell-mediated reaction wherein nonspecific cytotoxic cells that express FcγRs recognize bound antibody on a target cell and subsequently cause lysis of the target cell. Similarly, decreased binding to FcγRIIb (an inhibitory receptor) can be beneficial as well in some circumstances. Amino acid substitutions that find use in the present invention include those listed in U.S. Ser. No. 11/124,620 (particularly
In addition, the antibodies of the invention are modified to increase its biological half-life. Various approaches are possible. For example, one or more of the following mutations can be introduced: T252L, T254S, T256F, as described in U.S. Pat. No. 6,277,375 to Ward. Alternatively, to increase the biological half-life, the antibody can be altered within the CH1 or CL region to contain a salvage receptor binding epitope taken from two loops of a CH2 domain of an Fc region of an IgG, as described in U.S. Pat. Nos. 5,869,046 and 6,121,022 by Presta et al. Additional mutations to increase serum half life are disclosed in U.S. Pat. Nos. 8,883,973, 6,737,056 and 7,371,826, and include 428L, 434A, 434S, and 428L/4345.
In yet other embodiments, the Fc region is altered by replacing at least one amino acid residue with a different amino acid residue to alter the effector functions of the antibody. For example, one or more amino acids selected from amino acid residues 234, 235, 236, 237, 297, 318, 320 and 322 can be replaced with a different amino acid residue such that the antibody has an altered affinity for an effector ligand but retains the antigen-binding ability of the parent antibody. The effector ligand to which affinity is altered can be, for example, an Fc receptor or the C1 component of complement. This approach is described in further detail in U.S. Pat. Nos. 5,624,821 and 5,648,260, both by Winter et al.
In another example, one or more amino acids selected from amino acid residues 329, 331 and 322 can be replaced with a different amino acid residue such that the antibody has altered C1q binding and/or reduced or abolished complement dependent cytotoxicity (CDC). This approach is described in further detail in U.S. Pat. No. 6,194,551 by Idusogie et al.
In another example, one or more amino acid residues within amino acid positions 231 and 239 are altered to thereby alter the ability of the antibody to fix complement. This approach is described further in PCT Publication WO 94/29351 by Bodmer et al.
In yet another example, the Fc region is modified to increase the ability of the antibody to mediate antibody dependent cellular cytotoxicity (ADCC) and/or to increase the affinity of the antibody for an Fcγ receptor by modifying one or more amino acids at the following positions: 238, 239, 248, 249, 252, 254, 255, 256, 258, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294, 295, 296, 298, 301, 303, 305, 307, 309, 312, 315, 320, 322, 324, 326, 327, 329, 330, 331, 333, 334, 335, 337, 338, 340, 360, 373, 376, 378, 382, 388, 389, 398, 414, 416, 419, 430, 434, 435, 437, 438 or 439. This approach is described further in PCT Publication WO 00/42072 by Presta. Moreover, the binding sites on human IgG1 for FcγRI, FcγRII, FcγRIII and FcRn have been mapped and variants with improved binding have been described (see Shields, R. L. et al. (2001) J. Biol. Chem. 276:6591-6604). Specific mutations at positions 256, 290, 298, 333, 334 and 339 are shown to improve binding to FcγRIII Additionally, the following combination mutants are shown to improve FcγRIII binding: T256A/S298A, S298A/E333A, S298A/K224A and S298A/E333A/K334A. Furthermore, mutations such as M252Y/S254T/T256E or M428L/N434S improve binding to FcRn and increase antibody circulation half-life (see Chan C A and Carter P J (2010) Nature Rev Immunol 10:301-316).
In still another embodiment, the glycosylation of an Fc domain is modified. For example, an aglycosylated Fc domain can be made (i.e., the antibody lacks glycosylation). Glycosylation can be altered to, for example, increase the affinity of the antibody for antigen or reduce effector function such as ADCC. Such carbohydrate modifications can be accomplished by, for example, altering one or more sites of glycosylation within the antibody sequence, for example N297. For example, one or more amino acid substitutions can be made that result in elimination of one or more variable region framework glycosylation sites to thereby eliminate glycosylation at that site.
Additionally or alternatively, an Fc domain can be made that has an altered type of glycosylation, such as a hypofucosylated antibody having reduced amounts of fucosyl residues or an Fc domain having increased bisecting GlcNac structures. Such altered glycosylation patterns have been demonstrated to increase the ADCC ability of antibodies. Such carbohydrate modifications can be accomplished by, for example, expressing the antibody in a host cell with altered glycosylation machinery. Cells with altered glycosylation machinery have been described in the art and can be used as host cells in which to express recombinant antibodies according to at least some embodiments of the invention to thereby produce an antibody with altered glycosylation. For example, the cell lines Ms704, Ms705, and Ms709 lack the fucosyltransferase gene, FUT8 (α (1,6) fucosyltransferase), such that antibodies expressed in the Ms704, Ms705, and Ms709 cell lines lack fucose on their carbohydrates. The Ms704, Ms705, and Ms709 FUT8 cell lines are created by the targeted disruption of the FUT8 gene in CHO/DG44 cells using two replacement vectors (see U.S. Patent Publication No. 20040110704 by Yamane et al. and Yamane-Ohnuki et al. (2004) Biotechnol Bioeng 87:614-22). As another example, EP 1,176,195 by Hanai et al. describes a cell line with a functionally disrupted FUT8 gene, which encodes a fucosyl transferase, such that antibodies expressed in such a cell line exhibit hypofucosylation by reducing or eliminating the α 1,6 bond-related enzyme. Hanai et al. also describe cell lines which have a low enzyme activity for adding fucose to the N-acetylglucosamine that binds to the Fc region of the antibody or does not have the enzyme activity, for example the rat myeloma cell line YB2/0 (ATCC CRL 1662). PCT Publication WO 03/035835 by Presta describes a variant CHO cell line, Lec13 cells, with reduced ability to attach fucose to Asn(297)-linked carbohydrates, also resulting in hypofucosylation of antibodies expressed in that host cell (see also Shields, R. L. et al. (2002) J. Biol. Chem. 277:26733-26740). PCT Publication WO 99/54342 by Umana et al. describes cell lines engineered to express glycoprotein-modifying glycosyl transferases (e.g., β(1,4)-N-acetylglucosaminyltransferase III (GnTIII)) such that antibodies expressed in the engineered cell lines exhibit increased bisecting GlcNac structures which results in increased ADCC activity of the antibodies (see also Umana et al. (1999) Nat. Biotech. 17:176-180). Alternatively, the fucose residues of the antibody may be cleaved off using a fucosidase enzyme. For example, the fucosidase α-L-fucosidase removes fucosyl residues from antibodies (Tarentino, A. L. et al. (1975) Biochem. 14:5516-23).
3. Polyethylene Glycol Fusion Partners
In some embodiments, the fusion partner moiety is one or more polyethylene glycol (PEG) moieties. As is well known in the art, the modification of therapeutic protein drugs, such as erythropoietin, GM-CSF, interferon alpha and beta and human growth hormone, is frequently done to increase to alter a number of pharmacological properties, including, but not limited to, increased solubility, extended serum half-life, decreased dosage frequency, increased stability, decreased immunogenicity and enhanced protection from proteases.
As is known in the art, generally a number of PEG molecules are “loaded” onto each protein, depending on a number of factors, and the PEG molecules may be of varying length.
In general, the PEG moieties are covalently attached directly to the amino acid side chains of the HIDE1 polypeptide, using activated PEG derivatives as is well known in the art. That is, generally no additional linkers are used, e.g. there are no extra atoms between the PEG and the amino acid side chain. In other embodiments, linkers such as those outlined below are used.
4. Additional Heterologous Fusion Partners
In addition to half life extension fusion partner moieties, HIDE1 polypeptides can be fused (generally but optionally using linkers as outlined herein), with heterologous polypeptide that give additional biochemical functionalities to the HIDE1 polypeptides. These heterologous fusion partner moieties including, but are not limited to, receptors, hormones, cytokines, antigens, B-cell targets, NK cell targets, T cell targets, TNF receptor superfamily members, Hedgehog family members, a receptor tyrosine kinases, a proteoglycan-related molecules, a TGF-β superfamily members, Wnt-related molecules, receptor ligands, dendritic cell targets, myeloid cell targets, monocyte/macrophage cell targets or angiogenesis targets.
In some embodiments HIDE1 polypeptides or fusion proteins according to the present invention will comprise a T cell target selected from the group consisting of 2B4/SLAMF4, IL-2 Rα, 4-1BB/TNFRSF9, IL-2Rβ, ALCAM, B7-1/CD80, IL-4R, B7-H3, BLAME/SLAMF8, BTLA, IL-6R, CCR3, IL-7 Rα, CCR4, CXCR1/IL-8 RA, CCR5, CCR6, IL-10 R α, CCR7, IL-10 Rβ, CCR8, IL-12 Rβ1, CCR9, IL-12 Rβ2, CD2, IL-13Rα1, IL-13, CD3, CD4, ILT2/CD85j, ILT3/CD85k, ILT4/CD85d, ILT5/CD85a, Integrin α 4/CD49d, CD5, IntegrinαE/CD103, CD6, Integrin a M/CD11b, CD8, Integrin α X/CD11c, Integrin β2/CD18, KIR/CD158, CD27/TNFRSF7, KIR2DL1, CD28, KIR2DL3, CD30/TNFRSF8, KIR2DL4/CD158d, CD31/PECAM-1, KIR2DS4, CD40 Ligand/TNFSF5, LAG-3, CD43, LAIR1, CD45, LAIR2, CD83, Leukotriene B4 R1, CD84/SLAMF5, NCAM-L1, CD94, NKG2A, CD97, NKG2C, CD229/SLAMF3, NKG2D, CD2F-10/SLAMF9, NT-4, CD69, NTB-A/SLAMF6, Common γ Chain/IL-2 Rγ, Osteopontin, CRACC/SLAMF7, PD-1, CRTAM, PSGL-1, CTLA-4, RANK/TNFRSF11A, CX3CR1, CX3CL1, L-Selectin, CXCR3, SIRP β1, CXCR4, SLAM, CXCR6, TCCR/WSX-1, DNAM-1, Thymopoietin, EMMPRIN/CD147, TIM-1, EphB6, TIM-2, Fas/TNFRSF6, TIM-3, Fas Ligand/TNFSF6, TIM-4, Fcγ RIII/CD16, TIM-6, GITR/TNFRSF18, TNF R1/TNFRSF1A, Granulysin, TNF R11/TNFRSF1B, HVEM/TNFRSF14, TRAIL R1/TNFRSF10A, ICAM-1/CD54, TRAIL R2/TNFRSF10B, ICAM-2/CD102, TRAIL R3/TNFRSF10C, IFN-γR1, TRAIL R4/TNFRSF10D, IFN-γR2, TSLP, IL-1 RI and TSLP R.
In some embodiments HIDE1 polypeptides or fusion proteins according to the present invention will comprise at least one monocyte/macrophage cell target selected from the group consisting of B7-1/CD80, ILT4/CD85d, B7-H1, ILT5/CD85a, Common β Chain, Integrin α 4/CD49d, BLAME/SLAMF8, Integrin α X/CD11c, CCL6/C10, Integrin β 2/CD18, CD155/PVR, Integrin β 3/CD61, CD31/PECAM-1, Latexin, CD36/SR-B3, Leukotriene B4 R1, CD40/TNFRSF5, LIMPII/SR-B2, CD43, LMIR1/CD300A, CD45, LMIR2/CD300c, CD68, LMIR3/CD300LF, CD84/SLAMF5, LMIR5/CD300LB, CD97, LMIR6/CD300LE, CD163, LRP-1, CD2F-10/SLAMF9, MARCO, CRACC/SLAMF7, MD-1, ECF-L, MD-2, EMMPRIN/CD147, MGL2, Endoglin/CD105, Osteoactivin/GPNMB, FcγR1/CD64, Osteopontin, Fcγ RIIB/CD32b, PD-L2, FcγRIIC/CD32c, Siglec-3/CD33, Fcγ RIIA/CD32a, SIGNR1/CD209, Fcγ RIII/CD16, SLAM, GM-CSF R α, TCCR/WSX-1, ICAM-2/CD102, TLR3, IFN-γ R1, TLR4, IFN-γ R2, TREM-1, IL-1 RII, TREM-2, ILT2/CD85j, TREM-3, ILT3/CD85k, TREML1/TLT-1, 2B4/SLAMF4, IL-10 R α, ALCAM, IL-10 R β, Aminopeptidase N/ANPEP, ILT2/CD85j, Common β Chain, ILT3/CD85k, C1q R1/CD93, ILT4/CD85d, CCR1, ILT5/CD85a, CCR2, Integrin α 4/CD49d, CCR5, Integrin α M/CD11b, CCR8, Integrin α X/CD11c, CD155/PVR, Integrin β 2/CD18, CD14, Integrin β 3/CD61, CD36/SR-B3, LAIR1, CD43, LAIR2, CD45, Leukotriene B4 R1, CD68, LIMPII/SR-B2, CD84/SLAMF5, LMIR1/CD300A, CD97, LMIR2/CD300c, CD163, LMIR3/CD300LF, Coagulation Factor III/Tissue Factor, LMIR5/CD300LB, CX3CR1, CX3CL1, LMIR6/CD300LE, CXCR4, LRP-1, CXCR6, M-CSF R, DEP-1/CD148, MD-1, DNAM-1, MD-2, EMMPRIN/CD147, MMR, Endoglin/CD105, NCAM-L1, FcγR1/CD64, PSGL-1, FcγRIII/CD16, RP105, G-CSF R, L-Selectin, GM-CSF R α, Siglec-3/CD33, HVEM/TNFRSF14, SLAM, ICAM-1/CD54, TCCR/WSX-1, ICAM-2/CD102, TREM-1, IL-6 R, TREM-2, CXCR1/IL-8 RA, TREM-3 and TREML1/TLT-1.
In some embodiments HIDE1 polypeptides or fusion proteins according to the present invention will comprise at least one Dendritic cell target is selected from the group consisting of CD36/SR-B3, LOX-1/SR-E1, CD68, MARCO, CD163, SR-AI/MSR, CD5L, SREC-I, CL-P1/COLEC12, SREC-II, LIMPII/SR-B2, RP105, TLR4, TLR1, TLR5, TLR2, TLR6, TLR3, TLR9, 4-1BB Ligand/TNFSF9, IL-12/IL-23 p40, 4-Amino-1,8-naphthalimide, ILT2/CD85j, CCL21/6Ckine, ILT3/CD85k, 8-oxo-dG, ILT4/CD85d, 8D6A, ILT5/CD85a, A2B5, Integrin α 4/CD49d, Aag, Integrin β 2/CD18, AMICA, Langerin, B7-2/CD86, Leukotriene B4 R1, B7-H3, LMIR1/CD300A, BLAME/SLAMF8, LMIR2/CD300c, C1q R1/CD93, LMIR3/CD300LF, CCR6, LMIR5/CD300LB, CCR7, LMIR6/CD300LE, CD40/TNFRSF5, MAG/Siglec-4a, CD43, MCAM, CD45, MD-1, CD68, MD-2, CD83, MDL-1/CLEC5A, CD84/SLAMF5, MMR, CD97, NCAM-L1, CD2F-10/SLAMF9, Osteoactivin/GPNMB, Chem 23, PD-L2, CLEC-1, RP105, CLEC-2, Siglec-2/CD22, CRACC/SLAMF7, Siglec-3/CD33, DC-SIGN, Siglec-5, DC-SIGNR/CD299, Siglec-6, DCAR, Siglec-7, DCIR/CLEC4A, Siglec-9, DEC-205, Siglec-10, Dectin-1/CLEC7A, Siglec-F, Dectin-2/CLEC6A, SIGNR1/CD209, DEP-1/CD148, SIGNR4, DLEC, SLAM, EMMPRIN/CD147, TCCR/WSX-1, FcγR1/CD64, TLR3, FcγRIIB/CD32b, TREM-1, FcγRIIC/CD32c, TREM-2, FcγRIIA/CD32a, TREM-3, FcγRIII/CD16, TREML1/TLT-1, ICAM-2/CD102 and Vanilloid R1.
In some embodiments HIDE1 polypeptides or fusion proteins according to the present invention will comprise at least one TNF receptor superfamily member is selected from the group consisting of 4-1BB/TNFRSF9, NGF R/TNFRSF16, BAFF R/TNFRSF13C, Osteoprotegerin/TNFRSF11B, BCMA/TNFRSF17, OX40/TNFRSF4, CD27/TNFRSF7, RANK/TNFRSF11A, CD30/TNFRSF8, RELT/TNFRSF19L, CD40/TNFRSF5, TACI/TNFRSF13B, DcR3/TNFRSF6B, TNF RI/TNFRSF1A, DcTRAIL R1/TNFRSF23, TNF RII/TNFRSF1B, DcTRAIL R2/TNFRSF22, TRAIL R1/TNFRSF10A, DR3/TNFRSF25, TRAIL R2/TNFRSF10B, DR6/TNFRSF21, TRAIL R3/TNFRSF10C, EDAR, TRAIL R4/TNFRSF10D, Fas/TNFRSF6, TROY/TNFRSF19, GITR/TNFRSF18, TWEAK R/TNFRSF12, HVEM/TNFRSF14, XEDAR, Lymphotoxin β R/TNFRSF3, 4-1BB Ligand/TNFSF9, Lymphotoxin, APRIL/TNFSF13, Lymphotoxin β/TNFSF3, BAFF/TNFSF13C, OX40 Ligand/TNFSF4, CD27 Ligand/TNFSF7, TL1A/TNFSF15, CD30 Ligand/TNFSF8, TNF-α/TNFSF1A, CD40 Ligand/TNFSF5, TNF-β/TNFSF1B, EDA-A2, TRAIL/TNFSF10, Fas Ligand/TNFSF6, TRANCE/TNFSF11, GITR Ligand/TNFSF18, TWEAK/TNFSF12 and LIGHT/TNFSF14.
In some embodiments HIDE1 polypeptides or fusion proteins according to the present invention will comprise at least one Hedgehog family member selected from the group consisting of Patched and Smoothened.
In some embodiments HIDE1 polypeptides or fusion proteins according to the present invention will comprise at least one receptor tyrosine kinase selected from the group consisting of Ax1, FGF R4, C1q R1/CD93, FGF R5, DDR1, Flt-3, DDR2, HGF R, Dtk, IGF-I R, EGF R, IGF-II R, Eph, INSRR, EphA1, Insulin R/CD220, EphA2, M-CSF R, EphA3, Mer, EphA4, MSP R/Ron, EphA5, MuSK, EphA6, PDGF R α, EphA7, PDGF R β, EphA8, Ret, EphB1, ROR1, EphB2, ROR2, EphB3, SCF R/c-kit, EphB4, Tie-1, EphB6, Tie-2, ErbB2, TrkA, ErbB3, TrkB, ErbB4, TrkC, FGF R1, VEGF R1/Flt-1, FGF R2, VEGF R2/Flk-1, FGF R3 and VEGF R3/Flt-4.
In some embodiments HIDE1 polypeptides or fusion proteins according to the present invention will comprise at least one Transforming Growth Factor (TGF)-β superfamily member selected from the group consisting of Activin RIA/ALK-2, GFR α-1, Activin RIB/ALK-4, GFR α2, Activin RHA, GFR α-3, Activin RIIB, GFR α-4, ALK-1, MIS RII, ALK-7, Ret, BMPR-IA/ALK-3, TGF-betβa R1/ALK-5, BMPR-IB/ALK-6, TGF-β RII, BMPR-II, TGF-β RIIb, Endoglin/CD 105 and TGF-β RIII.
In some embodiments HIDE1 polypeptides or fusion proteins according to the present invention will comprise at least one Wnt-related molecule selected from the group consisting of Frizzled-1, Frizzled-8, Frizzled-2, Frizzled-9, Frizzled-3, sFRP-1, Frizzled-4, sFRP-2, Frizzled-5, sFRP-3, Frizzled-6, sFRP-4, Frizzled-7, MFRP, LRP 5, LRP 6, Wnt-1, Wnt-8a, Wnt-3a, Wnt-10b, Wnt-4, Wnt-11, Wnt-5a, Wnt-9a and Wnt-7a.
In some embodiments HIDE1 polypeptides or fusion proteins according to the present invention will comprise at least one receptor ligand selected from the group consisting of 4-1BB Ligand/TNFSF9, Lymphotoxin, APRIL/TNFSF13, Lymphotoxin β/TNFSF3, BAFF/TNFSF13C, OX40 Ligand/TNFSF4, CD27 Ligand/TNFSF7, TL1A/TNFSF15, CD30 Ligand/TNFSF8, TNF-α/TNFSF1A, CD40 Ligand/TNFSF5, TNF-β/TNFSF1B, EDA-A2, TRAIL/TNFSF10, Fas Ligand/TNFSF6, TRANCE/TNFSF11, GITR Ligand/TNFSF18, TWEAK/TNFSF12, LIGHT/TNFSF14, Amphiregulin, NRG1 isoform GGF2, Betacellulin, NRG1 Isoform SMDF, EGF, NRG1-α/HRG1-α, Epigen, NRG1-β 1/HRG1-β 1, Epiregulin, TGF-α, HB-EGF, TMEFF1/Tomoregulin-1, Neuregulin-3, TMEFF2, IGF-I, IGF-II, Insulin, Activin A, Activin B, Activin AB, Activin C, BMP-2, BMP-7, BMP-3, BMP-8, BMP-3b/GDF-10, BMP-9, BMP-4, BMP-15, BMP-5, Decapentaplegic, BMP-6, GDF-1, GDF-8, GDF-3, GDF-9, GDF-5, GDF-11, GDF-6, GDF-15, GDF-7, Artemin, Neurturin, GDNF, Persephin, TGF-β, TGF-β 2, TGF-β 1, TGF-β 3, LAP (TGF-β 1), TGF-β 5, Latent TGF-β 1, Latent TGF-β bp1, TGF-β 1.2, Lefty, Nodal, MIS/AMH, FGF acidic, FGF-12, FGF basic, FGF-13, FGF-3, FGF-16, FGF-4, FGF-17, FGF-5, FGF-19, FGF-6, FGF-20, FGF-8, FGF-21, FGF-9, FGF-23, FGF-10, KGF/FGF-7, FGF-11, Neuropilin-1, P1GF, Neuropilin-2, P1GF-2, PDGF, PDGF-A, VEGF, PDGF-B, VEGF-B, PDGF-C, VEGF-C, PDGF-D, VEGF-D and PDGF-AB.
In some embodiments HIDE1 polypeptides or fusion proteins according to the present invention will comprise at least one tumor antigen selected from the group consisting of Squamous Cell Carcinoma Antigen 1 (SCCA-1), (PROTEIN T4-A), Squamous Cell Carcinoma Antigen 2 (SCCA-2), Ovarian carcinoma antigen CA125 (1A1-3B; KIAA0049), MUCIN 1 (TUMOR-ASSOCIATED MUCIN; Carcinoma-Associated Mucin; Polymorphic Epithelial Mucin; PEM; PEMT; EPISIALIN; Tumor-Associated Epithelial Membrane Antigen; EMA; H23AG; Peanut-Reactive Urinary Mucin; PUM; and Breast Carcinoma-Associated Antigen DF3), CTCL tumor antigen se1-1, CTCL tumor antigen se14-3, CTCL tumor antigen se20-4, CTCL tumor antigen se20-9, CTCL tumor antigen se33-1, CTCL tumor antigen se37-2, CTCL tumor antigen se57-1, CTCL tumor antigen se89-1, Prostate-specific membrane antigen, 5T4 oncofetal trophoblast glycoprotein, Orf73 Kaposi's sarcoma-associated herpesvirus, MAGE-C1 (cancer/testis antigen CT7), MAGE-B1 ANTIGEN (MAGE-XP Antigen; DAM10), MAGE-B2 Antigen (DAME), MAGE-2 ANTIGEN, MAGE-4a antigen, MAGE-4b antigen, Colon cancer antigen NY-CO-45, Lung cancer antigen NY-LU-12 variant A, Cancer associated surface antigen, Adenocarcinoma antigen ART1, Paraneoplastic associated brain-testis-cancer antigen (onconeuronal antigen MA2; paraneoplastic neuronal antigen), Neuro-oncological ventral antigen 2 (NOVA2), Hepatocellular carcinoma antigen gene 520, Tumor-Associated Antigen CO-029, Tumor-associated antigen MAGE-X2, Synovial sarcoma, X breakpoint 2, Squamous cell carcinoma antigen recognized by T cell, Serologically defined colon cancer antigen 1, Serologically defined breast cancer antigen NY-BR-15, Serologically defined breast cancer antigen NY-BR-16, Chromogranin A, parathyroid secretory protein 1, DUPAN-2, CA 19-9, CA 72-4, CA 195 and L6.
In some embodiments HIDE1 polypeptides or fusion proteins according to the present invention will comprise at least one B cell target selected from the group consisting of CD10, CD19, CD20, CD21, CD22, CD23, CD24, CD37, CD38, CD39, CD40, CD72, CD73, CD74, CDw75, CDw76, CD77, CD78, CD79a/b, CD80, CD81, CD82, CD83, CD84, CD85, CD86, CD89, CD98, CD126, CD127, CDw130, CD138 and CDw150.
In some embodiment HIDE1 polypeptides or fusion proteins according to the present invention will comprise at least one angiogenesis target is selected from the group consisting of Angiopoietin-1, Angiopoietin-like 2, Angiopoietin-2, Angiopoietin-like 3, Angiopoietin-3, Angiopoietin-like 7/CDT6, Angiopoietin-4, Tie-1, Angiopoietin-like 1, Tie-2, Angiogenin, iNOS, Coagulation Factor III/Tissue Factor, nNOS, CTGF/CCN2, NOV/CCN3, DANCE, OSM, EDG-1, Plfr, EG-VEGF/PK1, Proliferin, Endostatin, ROBO4, Erythropoietin, Thrombospondin-1, Kininostatin, Thrombospondin-2, MFG-E8, Thrombospondin-4, Nitric Oxide, VG5Q, eNOS, EphA1, EphA5, EphA2, EphA6, EphA3, EphA7, EphA4, EphA8, EphB1, EphB4, EphB2, EphB6, EphB3, Ephrin-A1, Ephrin-A4, Ephrin-A2, Ephrin-A5, Ephrin-A3, Ephrin-B1, Ephrin-B3, Ephrin-B2, FGF acidic, FGF-12, FGF basic, FGF-13, FGF-3, FGF-16, FGF-4, FGF-17, FGF-5, FGF-19, FGF-6, FGF-20, FGF-8, FGF-21, FGF-9, FGF-23, FGF-10, KGF/FGF-7, FGF-11, FGF R1, FGF R4, FGF R2, FGF R5, FGF R3, Neuropilin-1, Neuropilin-2, Semaphorin 3A, Semaphorin 6B, Semaphorin 3C, Semaphorin 6C, Semaphorin 3E, Semaphorin 6D, Semaphorin 6A, Semaphorin 7A, MMP, MMP-11, MMP-1, MMP-12, MMP-2, MMP-13, MMP-3, MMP-14, MMP-7, MMP-15, MMP-8, MMP-16/MT3-MMP, MMP-9, MMP-24/MT5-MMP, MMP-10, MMP-25/MT6-MMP, TIMP-1, TIMP-3, TIMP-2, TIMP-4, ACE, IL-13 R a 1, IL-13, C1q R1/CD93, Integrin α 4/CD49d, VE-Cadherin, Integrin β 2/CD18, CD31/PECAM-1, KLF4, CD36/SR-B3, LYVE-1, CD151, MCAM, CL-P1/COLEC12, Nectin-2/CD112, Coagulation Factor III/Tissue Factor, E-Selectin, D6, P-Selectin, DC-SIGNR/CD299, SLAM, EMMPRIN/CD147, Tie-2, Endoglin/CD105, TNF RI/TNFRSF1A, EPCR, TNF RII/TNFRSF1B, Erythropoietin R, TRAIL R1/TNFRSF10A, ESAM, TRAIL R2/TNFRSF10B, FABP5, VCAM-1, ICAM-1/CD54, VEGF R2/Flk-1, ICAM-2/CD102, VEGF R3/Flt-4, IL-1 RI and VG5Q.
B. Linkers
In many embodiments of fusion proteins comprising a HIDE1 polypeptide and a fusion partner moiety, optional flexible linkers are used to join the sequences in frame. A “flexible linker” herein refers to a peptide or polypeptide containing two or more amino acid residues joined by peptide bond(s) that provides increased rotational freedom for two polypeptides linked thereby than the two linked polypeptides would have in the absence of the flexible linker. Such rotational freedom allows each component of the fusion protein to interact with its intended target without hindrance. Generally these linkers are mixtures of glycine and serine, such as -(GGGS)n-, where n is from 1, 2, 3, 4, or 5 (SEQ ID NO:1).
Other suitable peptide/polypeptide linker domains optionally include naturally occurring or non-naturally occurring peptides or polypeptides. Peptide linker sequences are at least 2 amino acids in length. Optionally the peptide or polypeptide domains are flexible peptides or polypeptides. Exemplary flexible peptides/polypeptides include, but are not limited to, the amino acid sequences Gly-Ser (SEQ ID NO:55), Gly-Ser-Gly-Ser (SEQ ID NO:56), Ala-Ser (SEQ ID NO:57), Gly-Gly-Gly-Ser (SEQ ID NO:58), Gly4-Ser (SEQ ID NO:59), (Gly4-Ser)2 (SEQ ID NO:60), (Gly4-Ser)3 (SEQ ID NO:61), (Gly4-Ser)4 (SEQ ID NO: 62), [Gly4-Ser]2 Gly-Ala-Gly-Ser-Gly4-Ser (SEQ ID NO: 72), Gly-(Gly4-Ser)2 (SEQ ID NO:73), Gly4-Ser-Gly (SEQ ID NO:74), Gly-Ser-Gly2 (SEQ ID NO:75) and Gly-Ser-Gly2-Ser (SEQ ID NO:76). Additional flexible peptide/polypeptide sequences are well known in the art. Other suitable peptide linker domains optionally include the TEV linker ENLYFQG, a linear epitope recognized by the Tobacco Etch Virus protease. Exemplary peptides/polypeptides include, but are not limited to, GSENLYFQGSG (SEQ ID NO:68). Other suitable peptide linker domains include helix forming linkers such as Ala-(Glu-Ala-Ala-Ala-Lys)n-Ala (n=1-5). Additional helix forming peptide/polypeptide sequences are well known in the art. Non-limiting examples of such linkers are depicted in SEQ ID NOs:63-67, 69-76.
In some embodiments HIDE1 polypeptides or fusion proteins according to the present invention will comprise a HIDE1 polypeptide, and at least one heterologous polypeptide and/or binding moiety or HIDE1 polypeptides are linked to one another by an amino acid spacer.
In some embodiments HIDE1 polypeptides or fusion proteins according to the present invention and at least one heterologous polypeptide and/or or binding moiety or HIDE1 polypeptides are linked to one another by an amino acid spacer of sufficient length of amino acid residues so that the different moieties can successfully bind to their individual targets.
In some embodiments HIDE1 polypeptides or fusion proteins according to the present invention will comprise 2-10 of any of the HIDE1 ECD polypeptide fragments disclosed herein.
In some embodiments HIDE1 polypeptides or fusion proteins according to the present invention will comprise one or more of HIDE1 polypeptide(s) and at least one heterologous polypeptide optionally intervened by a heterologous linker which optionally comprises a polypeptide that is not a fragment of a HIDE1 polypeptide.
In some embodiments HIDE1 polypeptides or fusion proteins according to the present invention will comprise a linker which is a peptide comprising 5-50 amino acid residues, more preferably 5-25 amino acid residues.
In some embodiments HIDE1 polypeptides or fusion proteins according to the present invention will comprise a linker which comprises, consists essentially of, glycine, serine, and/or alanine residues.
In some embodiments HIDE1 polypeptides or fusion proteins according to the present invention will comprise a linker which comprises 5-50, 5-25, 5-15, 4-14, 4-12, or more amino acid residues, e.g., which may optionally include or consist of glycine, serine, and/or alanine residues.
In some optionally embodiments HIDE1 fragments, e.g., ECD fragments, are linked to each other (multimers) and/or one or more HIDE1 fragments, e.g., ECD fragments, are linked to a heterologous polypeptide such as an immunoglobulin or fragment thereof, especially an immunoglobulin heavy chain or fragment thereof by a peptide linker, preferably a “flexible linker” sequence. The linker sequence should allow effective positioning of the HIDE1 fragments and the heterologous polypeptide such as an immunoglobulin polypeptide or domains thereof to allow functional activity of both moieties and the domains thereof. Successful presentation of the polypeptide fusion can modulate the activity of a cell either to induce or to inhibit T-cell proliferation, or to initiate or inhibit an immune response to a particular site. This can be determined in appropriate assays such as disclosed herein below, including the in vitro assays that includes sequential steps of culturing T cells to proliferate same, and contacting the T cells with a fusion polypeptide according to the present invention or a cell expressing same and then evaluating whether the fusion polypeptide promotes or inhibits T cell proliferation.
As used herein, the phrase “effective positioning of the heterologous polypeptide and the HIDE1 polypeptide”, or other similar phrase, is intended to mean that the domains of these moieties are positioned so that HIDE1 domains and heterologous polypeptide domains are capable of interacting with immune or other target cells, e.g., HIDE1 expressing cells to initiate or inhibit an immune reaction, or to inhibit or stimulate cell development.
With respect to HIDE1-Ig fusion proteins the linker sequence also preferably permits effective positioning of the Fc domain and HIDE1 domains to allow functional activity of each domain. In certain embodiments, the Fc domains are effectively positioned to allow proper fusion protein complex formation and/or interactions with Fc receptors on immune cells or proteins of the complement system to stimulate Fc-mediated effects including opsonization, cell lysis, degranulation of mast cells, basophils, and eosinophils, and other Fc receptor-dependent processes; activation of the complement pathway; and enhanced in vivo half-life of the fusion protein complex.
Linker sequences are discussed supra in connection with fusion proteins according to some embodiments of the present invention. Linker sequences can optionally be used to link two or more HIDE1 polypeptides of the biologically active polypeptide to generate a single-chain molecule with the desired functional activity. In some preferred embodiments the linker sequence comprises from about 5 to 20 amino acids, more preferably from about 7 or 8 to about 16 amino acids. The linker sequence is preferably flexible so as not hold the HIDE1 polypeptide and moiety linked thereto, e.g., an effector molecule in a single undesired conformation. The linker sequence can be used, e.g., to space the recognition site from the fused molecule. Specifically, the peptide linker sequence can be positioned between the biologically active HIDE1 polypeptide and the effector molecule, e.g., to chemically cross-link same and to provide molecular flexibility. The linker in some embodiments will predominantly comprise amino acids with small side chains, such as glycine, alanine and serine, to provide for flexibility. Preferably about 80 or 90 percent or greater of the linker sequence comprise glycine, alanine or serine residues, particularly glycine and serine residues. Other suitable linker sequences include flexible linker designs that have been used successfully to join antibody variable regions together, see Whitlow, M. et al., (1991) Methods: A Companion to Methods in Enzymology 2:97-105. In some examples, for covalently linking an effector molecule to a HIDE1 molecule, the amino sequence of the linker should be capable of spanning a suitable distance from the C-terminal residue of the HIDE1 polypeptide to the N-terminal residue of the effector molecule. Suitable linker sequences can be readily identified empirically. Additionally, suitable size and sequences of linker sequences also can be determined by known computer modeling techniques based on the predicted size and shape of the fusion polypeptide. Other linker sequences are discussed supra in connection with fusion proteins according to some embodiments of the present invention.
Optionally a polypeptide as described herein comprises 2-20 of any of the HIDE1 ECD polypeptide fragments linked together, such that the polypeptide has less than 95% homology to any of the HIDE1 sequence as described herein.
Optionally the fragments are intervened by a heterologous linker which optionally comprises a polypeptide that is not a fragment of a HIDE1 polypeptide.
Optionally the linker is a peptide comprising 5-50 amino acid residues, more preferably 5-25 amino acid residues.
Optionally the linker comprises, consists essentially of, or consists of 4-12 glycine, serine, and/or alanine residues.
1. Dimerization, Multimerization and Targeting Domains
The fusion proteins disclosed herein optionally contain a dimerization or multimerization or oligomerization domain that functions to dimerize, oligomerize or multimerize two or more fusion proteins, which may optionally be the same or different (heteromultimers or homomultimers). For example a HIDE1 fusion protein may optionally be attached to another HIDE1 fusion protein or another moiety, e.g. another costimulatory fusion protein. The domain that functions to dimerize or multimerize the fusion proteins can either be a separate domain, or alternatively can be contained within one of the other domains (HIDE1 polypeptide, second polypeptide, or peptide/polypeptide linker domain) of the fusion protein.
Dimerization or multimerization can occur between or among two or more fusion proteins through dimerization or multimerization domains. Alternatively, dimerization or multimerization of fusion proteins can occur by chemical crosslinking. The dimers or multimers that are formed can be homodimeric/homomultimeric or heterodimeric/heteromultimeric. The second polypeptide “partner” in the HIDE1 fusion polypeptides may optionally be comprised of one or more other proteins, protein fragments or peptides as described herein, including but not limited to any immunoglobulin (Ig) protein or portion thereof, preferably the Fc region, or a portion of a biologically or chemically active protein such as the papillomavirus E7 gene product, melanoma-associated antigen p97), and HIV env protein (gp120). The “partner” is optionally selected to provide a soluble dimer/multimer and/or for one or more other biological activities as described herein.
A “dimerization domain” is formed by the association of at least two amino acid residues or of at least two peptides or polypeptides (which may have the same, or different, amino acid sequences). The peptides or polypeptides may optionally interact with each other through covalent and/or non-covalent associations). Optional dimerization domains contain at least one cysteine that is capable of forming an intermolecular disulfide bond with a cysteine on the partner fusion protein. The dimerization domain can contain one or more cysteine residues such that disulfide bond(s) can form between the partner fusion proteins. In one embodiment, dimerization domains contain one, two or three to about ten cysteine residues. In a further embodiment, the dimerization domain is the hinge region of an immunoglobulin.
Additional exemplary dimerization domains can be any known in the art and include, but not limited to, coiled coils, acid patches, zinc fingers, calcium hands, a CH1-CL pair, an “interface” with an engineered “knob” and/or “protuberance” as described in U.S. Pat. No. 5,821,333, leucine zippers (e.g., from jun and/or fos) (U.S. Pat. No. 5,932,448), and/or the yeast transcriptional activator GCN4, SH2 (src homology 2), SH3 (src Homology 3) (Vidal, et al, Biochemistry, 43, 7336-44 ((2004)), phosphotyrosine binding (PTB) (Zhou, et al., Nature, 378:584-592 (1995)), WW (Sudol, Prog. Biochys. Mol Biol., 65:113-132 (1996)), PDZ (Kim, et al., Nature, 378: 85-88 (1995); Komau, et al, Science, 269. 1737-1740 (1995)) 14-3-3, WD40 (Hu5 et al., J Biol Chem., 273: 33489-33494 (1998)) EH, Lim, “An isoleucine zipper, a receptor dimer pair (e.g., interleukin-8 receptor (IL-8R); and integrin heterodimers such as LFA-I and GPIIIb/IIIa), or the dimerization region(s) thereof, dimeric ligand polypeptides (e.g., nerve growth factor (NGF), neurotrophin-3 (NT-3), interleukin-8 (IL-8), vascular endothelial growth factor (VEGF), VEGF-C, VEGF-D, PDGF members, and brain-derived neurotrophic factor (BDNF) (Arakawa, et al., J Biol. Chem., 269(45): 27833-27839 (1994) and Radziejewski, et al., Biochem., 32(48): 1350 (1993)) and can also be variants of these domains in which the affinity is altered. The polypeptide pairs can be identified by methods known in the art, including yeast two hybrid screens. Yeast two hybrid screens are described in U.S. Pat. Nos. 5,283,173 and 6,562,576. Affinities between a pair of interacting domains can be determined using methods known in the art, including as described in Katahira, et al., J. Biol Chem, 277, 9242-9246 (2002)). Alternatively, a library of peptide sequences can be screened for heterodimerization, for example, using the methods described in WO 01/00814. Useful methods for protein-protein interactions are also described in U.S. Pat. No. 6,790,624.
A “multimerization domain” or “oligomerization domain” referred to herein is a domain that causes three or more peptides or polypeptides to interact with each other through covalent and/or non-covalent association(s). Suitable multimerization or oligomerization domains include, but are not limited to, coiled-coil domains. A coiled-coil is a peptide sequence with a contiguous pattern of mainly hydrophobic residues spaced 3 and 4 residues apart, usually in a sequence of seven amino acids (heptad repeat) or eleven amino acids (undecad repeat), which assembles (folds) to form a multimeric bundle of helices. Coiled-coils with sequences including some irregular distribution of the 3 and 4 residues spacing are also contemplated. Hydrophobic residues are in particular the hydrophobic amino acids Val, Ile, Leu, Met, Tyr, Phe and Trp. “Mainly hydrophobic” means that at least 50% of the residues must be selected from the mentioned hydrophobic amino acids.
The coiled coil domain may optionally be derived from laminin. In the extracellular space, the heterotrimeric coiled coil protein laminin plays an important role in the formation of basement membranes. Apparently, the multifunctional oligomeric structure is required for laminin function. Coiled coil domains may optionally also be derived from the thrombospondins in which three (TSP-I and TSP-2) or five (TSP-3, TSP-4 and TSP-5) chains are connected, or from COMP (COMPcc) (Guo, et at., EMBO J, 1998, 17: 5265-5272) which folds into a parallel five-stranded coiled coil (Malashkevich, et al., Science, 274: 761-765 (1996)). Additional non-limiting examples of coiled-coil domains derived from other proteins, and other domains that mediate polypeptide multimerization are known in the art such as the vasodilator-stimulated phosphoprotein (VASP) domain, matrilin-1 (CMP), viral fusion peptides, soluble NSF (N-ethylmaleimide-sensitive factor) Attachment Protein receptor (SNARE) complexes, leucine-rich repeats, certain tRNA synthetases, are suitable for use in the disclosed fusion proteins.
In another embodiment, HIDE1 polypeptides, fusion proteins, or fragments thereof can be induced to form multimers by binding to a second multivalent polypeptide, such as an antibody. Antibodies suitable for use to multimerize HIDE1 polypeptides, fusion proteins, or fragments thereof include, but are not limited to, IgM antibodies and cross-linked, multivalent IgG, IgA, IgD, or IgE complexes.
Dimerization or multimerization can occur between or among two or more fusion proteins through dimerization or multimerization domains, including those described above. Alternatively, dimerization or multimerization of fusion proteins can occur by chemical crosslinking. Fusion protein dimers can be homodimers or heterodimers. Fusion protein multimers can be homomultimers or heteromultimers. Fusion protein dimers as disclosed herein are of formula II:
N-R1-R2-R3-C
N-R4-R5-R6-C or, alternatively, are of formula III:
N-R1-R2-R3-C
C-R4-R5-R6-N wherein the fusion proteins of the dimer provided by formula II are defined as being in a parallel orientation and the fusion proteins of the dimer provided by formula III are defined as being in an antiparallel orientation. Parallel and antiparallel dimers are also referred to as cis and trans dimers, respectively. “N” and “C” represent the N- and C-termini of the fusion protein, respectively. The fusion protein constituents “R1”, “R2” and “R3” are as defined above with respect to formula I. With respect to both formula II and formula III, “R4” is a HIDE1 polypeptide or a second polypeptide, “R5” is an optional peptide/polypeptide linker domain, and “R6” is a HIDE1 polypeptide or a second polypeptide, wherein “R6” is a HIDE1 polypeptide when “R4” is a second polypeptide, and “R6′” is a second polypeptide when “R4” is a HIDE1 polypeptide. In one embodiment, “R1” is a HIDE1 polypeptide, “R4” is also a HIDE1 polypeptide, and “R3” and “R6” are both second polypeptides.
Fusion protein dimers of formula II are defined as homodimers when “R1”=“R4”, “R2”=“R5” and “R3”=“R6”. Similarly, fusion protein dimers of formula III are defined as homodimers when “R1”=“R6”, “R2”=“R5” and “R3”=“R4”. Fusion protein dimers are defined as heterodimers when these conditions are not met for any reason. For example, heterodimers may optionally contain domain orientations that meet these conditions (i.e., for a dimer according to formula II, “R1” and “R4” are both HIDE1 polypeptides, “R2” and “R5” are both peptide/polypeptide linker domains and “R3” and “R6” are both second polypeptides), however the species of one or more of these domains is not identical. For example, although “R3” and “R6” may optionally both be HIDE1 polypeptides, one polypeptide may optionally contain a wild-type HIDE1 amino acid sequence while the other polypeptide may optionally be a variant HIDE1 polypeptide. An exemplary variant HIDE1 polypeptide is HIDE1, polypeptide that has been modified to have increased or decreased binding to a target cell, increased activity on immune cells, increased or decreased half-life or stability. Dimers of fusion proteins that contain either a CH1 or CL region of an immunoglobulin as part of the polypeptide linker domain preferably form heterodimers wherein one fusion protein of the dimer contains a CH1 region and the other fusion protein of the dimer contains a CL region.
Fusion proteins can also be used to form multimers. As with dimers, multimers may optionally be parallel multimers, in which all fusion proteins of the multimer are aligned in the same orientation with respect to their N- and C-termini. Multimers may optionally be antiparallel multimers, in which the fusion proteins of the multimer are alternatively aligned in opposite orientations with respect to their N- and C-termini. Multimers (parallel or antiparallel) can be either homomultimers or heteromultimers.
The fusion protein is optionally produced in dimeric form; more preferably, the fusion is performed at the genetic level as described below, by joining polynucleotide sequences corresponding to the two (or more) proteins, portions of proteins and/or peptides, such that a joined or fused protein is produced by a cell according to the joined polynucleotide sequence. A description of preparation for such fusion proteins is described with regard to U.S. Pat. No. 5,851,795 to Linsley et al, which is hereby incorporated by reference as if fully set forth herein as a non-limiting example only.
2. Targeting Domains
The HIDE1 polypeptides and fusion proteins can contain a targeting domain to target the molecule to specific sites in the body. Optional targeting domains target the molecule to areas of inflammation. Exemplary targeting domains are antibodies, or antigen binding fragments thereof that are specific for inflamed tissue or to a proinflammatory cytokine including but not limited to IL17, IL-4, IL-6, IL-12, IL-21, IL-22, IL-23, MIF, TNF-α, and TNF-β and combinations thereof. In the case of neurological disorders such as Multiple Sclerosis, the targeting domain may optionally target the molecule to the CNS or may optionally bind to VCAM-I on the vascular epithelium. Additional targeting domains can be peptide aptamers specific for a proinflammatory molecule. In other embodiments, the HIDE1 fusion protein can include a binding partner specific for a polypeptide displayed on the surface of an immune cell, for example a T cell. In still other embodiments, the targeting domain specifically targets activated immune cells. Optional immune cells that are targeted include Th0, Th1, Th 17, Th2 and Th22 T cells, other cells that secrete, or cause other cells to secrete inflammatory molecules including, but not limited to, IL-10, TNF-α, TGF-β, IFN-γ, IL-17, IL-6, IL-23, IL-22, IL-21, and MMPs, and Tregs. For example, a targeting domain for Tregs may optionally bind specifically to CD25.
Other targeting moieties or heterologous polypeptides that optionally may optionally be attached or contained within HIDE1 polypeptides or fusion proteins according to some embodiments of the present invention are discussed supra in connection with the synthesis of exemplary fusion proteins according to some embodiments of the present invention.
The above changes are intended as illustrations only of optional changes and are not meant to be limiting in any way. Furthermore, the above explanation is provided for descriptive purposes only, without wishing to be bound by a single hypothesis.
C. Addition of Groups
If a protein according to some embodiments of the present invention is a linear molecule, it is possible to place various functional groups at various points on the linear molecule which are susceptible to or suitable for chemical modification. Functional groups can be added to the termini of linear forms of the protein according to at least some embodiments of the present invention. In some embodiments, the functional groups improve the activity of the protein with regard to one or more characteristics, including but not limited to, improvement in stability, penetration (through cellular membranes and/or tissue barriers), tissue localization, efficacy, decreased clearance, decreased toxicity, improved selectivity, improved resistance to expulsion by cellular pumps, and the like. For convenience sake and without wishing to be limiting, the free N-terminus of one of the sequences contained in the compositions according to at least some embodiments of the present invention will be termed as the N-terminus of the composition, and the free C-terminal of the sequence will be considered as the C-terminus of the composition. Either the C-terminus or the N-terminus of the sequences, or both, can be linked to a carboxylic acid functional groups or an amine functional group, respectively.
Non-limiting examples of suitable functional groups are described in Green and Wuts, “Protecting Groups in Organic Synthesis”, John Wiley and Sons, Chapters 5 and 7, (1991), the teachings of which are incorporated herein by reference. Preferred protecting groups are those that facilitate transport of the active ingredient attached thereto into a cell, for example, by reducing the hydrophilicity and increasing the lipophilicity of the active ingredient, these being an example for “a moiety for transport across cellular membranes”.
These moieties can optionally and preferably be cleaved in vivo, either by hydrolysis or enzymatically, inside the cell. (Ditter et al., J. Pharm Sci. 57:783 (1968); Ditter et al., J. Pharm. Sci. 57:828 (1968); Ditter et al., J. Pharm. Sci. 58:557 (1969); King et al., Biochemistry 26:2294 (1987); Lindberg et al., Drug Metabolism and Disposition 17:311 (1989); and Tunek et al., Biochem. Pharm. 37:3867 (1988), Anderson et al., Arch. Biochem. Biophys. 239:538 (1985) and Singhal et al., FASEB J. 1:220 (1987)). Hydroxyl protecting groups include esters, carbonates and carbamate protecting groups. Amine protecting groups include alkoxy and aryloxy carbonyl groups, as described above for N-terminal protecting groups. Carboxylic acid protecting groups include aliphatic, benzylic and aryl esters, as described above for C-terminal protecting groups. In one embodiment, the carboxylic acid group in the side chain of one or more glutamic acid or aspartic acid residue in a composition according to some embodiments of the present invention is protected, preferably with a methyl, ethyl, benzyl or substituted benzyl ester, more preferably as a benzyl ester.
Non-limiting, illustrative examples of N-terminal protecting groups include acyl groups (—CO-R1) and alkoxy carbonyl or aryloxy carbonyl groups (—CO—O-R1), wherein R1 is an aliphatic, substituted aliphatic, benzyl, substituted benzyl, aromatic or a substituted aromatic group. Specific examples of acyl groups include but are not limited to acetyl, (ethyl)-CO—, n-propyl-CO—, iso-propyl-CO—, n-butyl-CO—, sec-butyl-CO—, t-butyl-CO—, hexyl, lauroyl, palmitoyl, myristoyl, stearyl, oleoyl phenyl-CO—, substituted phenyl-CO—, benzyl-CO— and (substituted benzyl)-CO—. Examples of alkoxy carbonyl and aryloxy carbonyl groups include CH3-O—CO—, (ethyl)-O—CO—, n-propyl-O—CO—, iso-propyl-O—CO—, n-butyl-O—CO—, sec-butyl-O—CO—, t-butyl-O—CO—, phenyl-O—CO—, substituted phenyl-O—CO— and benzyl-O—CO—, (substituted benzyl)-O—CO—, Adamantan, naphtalen, myristoleyl, toluen, biphenyl, cinnamoyl, nitrobenzoy, toluoyl, furoyl, benzoyl, cyclohexane, norbornane, or Z-caproic. In order to facilitate the N-acylation, one to four glycine residues can be present in the N-terminus of the molecule.
The carboxyl group at the C-terminus of the compound can be protected, for example, by a group including but not limited to an amide (i.e., the hydroxyl group at the C-terminus is replaced with —NH 2, —NHR2 and —NR2R3) or ester (i.e., the hydroxyl group at the C-terminus is replaced with —OR2). R2 and R3 are optionally independently an aliphatic, substituted aliphatic, benzyl, substituted benzyl, aryl or a substituted aryl group. In addition, taken together with the nitrogen atom, R2 and R3 can optionally form a C4 to C8 heterocyclic ring with from about 0-2 additional heteroatoms such as nitrogen, oxygen or sulfur. Non-limiting suitable examples of suitable heterocyclic rings include piperidinyl, pyrrolidinyl, morpholino, thiomorpholino or piperazinyl. Examples of C-terminal protecting groups include but are not limited to —NH2, —NHCH3, —N(CH3)2, —NH(ethyl), —N(ethyl)2, —N(methyl) (ethyl), —NH(benzyl), —N(C1-C4 alkyl)(benzyl), —NH(phenyl), —N(C1-C4 alkyl) (phenyl), —OCH3, —O-(ethyl), —O-(n-propyl), —O-(n-butyl), —O-(iso-propyl), —O-(sec-butyl), —O-(t-butyl), —O-benzyl and —O-phenyl.
D. Substitution by Peptidomimetic Moieties
A “peptidomimetic organic moiety” can optionally be substituted for amino acid residues in the composition of this invention both as conservative and as non-conservative substitutions. These moieties are also termed “non-natural amino acids” and may optionally replace amino acid residues, amino acids or act as spacer groups within the peptides in lieu of deleted amino acids. The peptidomimetic organic moieties optionally and preferably have steric, electronic or configurational properties similar to the replaced amino acid and such peptidomimetics are used to replace amino acids in the essential positions, and are considered conservative substitutions. However such similarities are not necessarily required. According to some embodiments of the present invention, one or more peptidomimetics are selected such that the composition at least substantially retains its physiological activity as compared to the native protein according to some embodiments of the present invention.
Peptidomimetics may optionally be used to inhibit degradation of the peptides by enzymatic or other degradative processes. The peptidomimetics can optionally and preferably be produced by organic synthetic techniques. Non-limiting examples of suitable peptidomimetics include D amino acids of the corresponding L amino acids, tetrazol (Zabrocki et al., J. Am. Chem. Soc. 110:5875-5880 (1988)); isosteres of amide bonds (Jones et al., Tetrahedron Lett. 29: 3853-3856 (1988)); LL-3-amino-2-propenidone-6-carboxylic acid (LL-Acp) (Kemp et al., J. Org. Chem. 50:5834-5838 (1985)). Similar analogs are shown in Kemp et al., Tetrahedron Lett. 29:5081-5082 (1988) as well as Kemp et al., Tetrahedron Lett. 29:5057-5060 (1988), Kemp et al., Tetrahedron Lett. 29:4935-4938 (1988) and Kemp et al., J. Org. Chem. 54:109-115 (1987). Other suitable but exemplary peptidomimetics are shown in Nagai and Sato, Tetrahedron Lett. 26:647-650 (1985); Di Maio et al., J. Chem. Soc. Perkin Trans., 1687 (1985); Kahn et al., Tetrahedron Lett. 30:2317 (1989); Olson et al., J. Am. Chem. Soc. 112:323-333 (1990); and Garvey et al., J. Org. Chem. 56:436 (1990). Further suitable exemplary peptidomimetics include hydroxy-1,2,3,4-tetrahydroisoquinoline-3-carboxylate (Miyake et al., J. Takeda Res. Labs 43:53-76 (1989)); 1,2,3,4-tetrahydro-isoquinoline-3-carboxylate (Kazmierski et al., J. Am. Chem. Soc. 133:2275-2283 (1991)); histidine isoquinolone carboxylic acid (HIC) (Zechel et al., Int. J. Pep. Protein Res. 43 (1991)); (2S,S)-methyl-phenylalanine, (2S,3R)-methyl-phenylalanine, (2R,3S)-methyl-phenylalanine and (2R,3R)-methyl-phenylalanine (Kazmierski and Hruby, Tetrahedron Lett. (1991)).
Exemplary, illustrative but non-limiting non-natural amino acids include β-amino acids (β3 and β2), homo-amino acids, cyclic amino acids, aromatic amino acids, Pro and Pyr derivatives, 3-substituted Alanine derivatives, Glycine derivatives, ring-substituted Phe and Tyr Derivatives, linear core amino acids or diamino acids. They are available from a variety of suppliers, such as Sigma-Aldrich (USA).
E. Protein Chemical Modifications
In the present invention, according to at least some embodiments, any part of a protein according to at least some embodiments of the present invention may optionally be chemically modified, i.e. changed by addition of functional groups. For example the side amino acid residues appearing in the native sequence may optionally be modified, although as described below alternatively other parts of the protein may optionally be modified, in addition to or in place of the side amino acid residues. The modification may optionally be performed during synthesis of the molecule if a chemical synthetic process is followed, for example by adding a chemically modified amino acid. However, chemical modification of an amino acid when it is already present in the molecule (“in situ” modification) is also possible.
The amino acid of any of the sequence regions of the molecule can optionally be modified according to any one of the following exemplary types of modification (in the peptide conceptually viewed as “chemically modified”). Non-limiting exemplary types of modification include carboxymethylation, acylation, phosphorylation, glycosylation or fatty acylation. Ether bonds can optionally be used to join the serine or threonine hydroxyl to the hydroxyl of a sugar. Amide bonds can optionally be used to join the glutamate or aspartate carboxyl groups to an amino group on a sugar (Garg and Jeanloz, Advances in Carbohydrate Chemistry and Biochemistry, Vol. 43, Academic Press (1985); Kunz, Ang. Chem. Int. Ed. English 26:294-308 (1987)). Acetal and ketal bonds can also optionally be formed between amino acids and carbohydrates. Fatty acid acyl derivatives can optionally be made, for example, by acylation of a free amino group (e.g., lysine) (Toth et al., Peptides: Chemistry, Structure and Biology, Rivier and Marshal, eds., ESCOM Publ., Leiden, 1078-1079 (1990)).
As used herein the term “chemical modification”, when referring to a protein or peptide according to some embodiments of the present invention, refers to a protein or peptide where at least one of its amino acid residues is modified either by natural processes, such as processing or other post-translational modifications, or by chemical modification techniques which are well known in the art. Examples of the numerous known modifications typically include, but are not limited to: acetylation, acylation, amidation, ADP-ribosylation, glycosylation, GPI anchor formation, covalent attachment of a lipid or lipid derivative, methylation, myristoylation, pegylation, prenylation, phosphorylation, ubiquitination, or any similar process.
Other types of modifications optionally include the addition of a cycloalkane moiety to a biological molecule, such as a protein, as described in PCT Application No. WO 2006/050262, hereby incorporated by reference as if fully set forth herein. These moieties are designed for use with biomolecules and may optionally be used to impart various properties to proteins.
Furthermore, optionally any point on a protein may be modified. For example, pegylation of a glycosylation moiety on a protein may optionally be performed, as described in PCT Application No. WO 2006/050247, hereby incorporated by reference as if fully set forth herein. One or more polyethylene glycol (PEG) groups may optionally be added to 0-linked and/or N-linked glycosylation. The PEG group may optionally be branched or linear. Optionally any type of water-soluble polymer may be attached to a glycosylation site on a protein through a glycosyl linker.
F. Altered Glycosylation
Proteins according to at least some embodiments of the present invention may optionally be modified to have an altered glycosylation pattern (i.e., altered from the original or native glycosylation pattern). As used herein, “altered” means having one or more carbohydrate moieties deleted, and/or having at least one glycosylation site added to the original protein.
Glycosylation of proteins is typically either N-linked or O-linked. N-linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences, asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline, are the recognition sequences for enzymatic attachment of the carbohydrate moiety to the asparagine side chain. Thus, the presence of either of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the attachment of one of the sugars N-acetylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine may optionally also be used.
Addition of glycosylation sites to proteins according to at least some embodiments of the present invention is conveniently accomplished by altering the amino acid sequence of the protein such that it contains one or more of the above-described tripeptide sequences (for N-linked glycosylation sites). The alteration may optionally also be made by the addition of, or substitution by, one or more serine or threonine residues in the sequence of the original protein (for O-linked glycosylation sites). The protein's amino acid sequence may optionally also be altered by introducing changes at the DNA level.
Another means of increasing the number of carbohydrate moieties on proteins is by chemical or enzymatic coupling of glycosides to the amino acid residues of the protein. Depending on the coupling mode used, the sugars may optionally be attached to (a) arginine and histidine, (b) free carboxyl groups, (c) free sulfhydryl groups such as those of cysteine, (d) free hydroxyl groups such as those of serine, threonine, or hydroxyproline, (e) aromatic residues such as those of phenylalanine, tyrosine, or tryptophan, or (f) the amide group of glutamine. These methods are described in WO 87/05330, and in Aplin and Wriston, CRC Crit. Rev. Biochem., 22: 259-306 (1981).
Removal of any carbohydrate moieties present on proteins according to at least some embodiments of the present invention may optionally be accomplished chemically or enzymatically. Chemical deglycosylation requires exposure of the protein to trifluoromethanesulfonic acid, or an equivalent compound. This treatment results in the cleavage of most or all sugars except the linking sugar (N-acetylglucosamine or N-acetylgalactosamine), leaving the amino acid sequence intact.
Chemical deglycosylation is described by Hakimuddin et al., Arch. Biochem. Biophys., 259: 52 (1987); and Edge et al., Anal. Biochem., 118: 131 (1981). Enzymatic cleavage of carbohydrate moieties on proteins can be achieved by the use of a variety of endo- and exo-glycosidases as described by Thotakura et al., Meth. Enzymol., 138: 350 (1987).
The disclosed HIDE1 fusion proteins optionally contain a peptide or polypeptide linker domain that separates the HIDE1 polypeptide from the second polypeptide (see,
The hinge can be further shortened to remove amino acids 1, 2, 3, 4, 5, or combinations thereof of any one of SEQ ID NOs: 48-50. In one embodiment, amino acids 1-5 of any one of SEQ ID NOs:48-50 are deleted. Exemplary HIDE1 fusion polypeptides comprised of the hinge, CH2 and CH3 regions of a human immunoglobulin Cγ1 chain with the Cys at position 220 replaced with a Ser are set forth in SEQ ID NO:17.
In another embodiment, the HIDE1 fusion polypeptides contain the CH2 and CH3 regions of human immunoglobulin Cγ1 chain having N297A mutation (SEQ ID NO:50) or the human Fc carrying the C220S, C226 and C229S mutations (SEQ ID NO:86).
In another embodiment, HIDE1 fusion polypeptides contain the CH2 and CH3 regions of a human immunoglobulin Cγ1 chain having at least 85%, 90%, 95%, 99% or 100% sequence homology to amino acid sequence set forth in SEQ ID NO:51:
In another embodiment, the HIDE1 fusion polypeptides contain the hinge, CH2 and CH3 regions of a murine immunoglobulin Cγ2a chain at least 85%, 90%, 95%, 99% or 100% sequence homology to amino acid sequence set forth in SEQ ID NO: 52:
In another embodiment, the HIDE1 fusion polypeptides contain the CH2 and CH3 regions of a murine immunoglobulin Cγ2a chain having N297A mutation (SEQ ID NO:53) or the murine Fc without the Hinge (SEQ ID NO:54).
In another embodiment, the linker domain optionally contains a hinge region of an immunoglobulin as described above, and further includes one or more additional immunoglobulin domains.
A. Peptide or Polypeptide Linker Domain
The disclosed HIDE1 fusion proteins optionally contain a peptide or polypeptide linker domain that separates the HIDE1 polypeptide from the second polypeptide. Various non-limiting examples of such linker domains are described herein. In one embodiment, the linker domain contains the hinge region of an immunoglobulin. In a further embodiment, the hinge region is derived from a human immunoglobulin. Suitable human immunoglobulins that the hinge can be derived from include IgG, IgD and IgA. In a further embodiment, the hinge region is derived from human IgG. Amino acid sequences of immunoglobulin hinge regions and other domains are well known in the art. In one embodiment, HIDE1 fusion polypeptides contain the hinge, CH2 and CH3 regions of a human immunoglobulin Cγ1 chain, optionally with the Cys at position 220 (according to full length human IgG1, position 5 in SEQ ID NO:48) replaced with a Ser having at least 85%, 90%, 95%, 99% or 100% sequence homology to amino acid sequence set forth in SEQ ID NO:49:
The hinge can be further shortened to remove amino acids 1, 2, 3, 4, 5, or combinations thereof of any one of SEQ ID NOs: 48-50. In one embodiment, amino acids 1-5 of any one of SEQ ID NOs: 48-50 are deleted. Exemplary HIDE1 fusion polypeptides comprised of the hinge, CH2 and CH3 regions of a human immunoglobulin Cγ1 chain with the Cys at position 220 replaced with a Ser are set forth in SEQ ID NO: 17.
In another embodiment, the HIDE1 fusion polypeptides contain the CH2 and CH3 regions of human immunoglobulin Cγ1 chain having N297A mutation (SEQ ID NO:50) or the human Fc carrying the C220S, C226 and C229S mutations (SEQ ID NO:86).
In another embodiment, HIDE1 fusion polypeptides contain the CH2 and CH3 regions of a human immunoglobulin Cγ1 chain having at least 85%, 90%, 95%, 99% or 100% sequence homology to amino acid sequence set forth in SEQ ID NO:51:
In another embodiment, the HIDE1 fusion polypeptides contain the hinge, CH2 and CH3 regions of a murine immunoglobulin Cγ2a chain at least 85%, 90%, 95%, 99% or 100% sequence homology to amino acid sequence set forth in SEQ ID NO: 52:
In another embodiment, the HIDE1 fusion polypeptides contain the CH2 and CH3 regions of a murine immunoglobulin Cγ2a chain having N297A mutation (SEQ ID NO:53) or the murine Fc without the Hinge (SEQ ID NO:54).
In another embodiment, the linker domain optionally contains a hinge region of an immunoglobulin as described above, and further includes one or more additional immunoglobulin domains.
Nucleic acid compositions encoding the HIDE1 polypeptides of the invention are also provided, as well as expression vectors containing the nucleic acids and host cells transformed with the nucleic acid and/or expression vector compositions.
The nucleic acid compositions that encode the HIDE1 polypeptides are generally put into a single expression vectors is known in the art, transformed into host cells, where they are expressed to form the HIDE1 proteins (or fusion proteins) of the invention. The nucleic acids can be put into expression vectors that contain the appropriate transcriptional and translational control sequences, including, but not limited to, signal and secretion sequences, regulatory sequences, promoters, origins of replication, selection genes, etc.
For example, to express the protein DNA, DNAs can be obtained by standard molecular biology techniques (e.g., PCR amplification or gene synthesis) and the DNAs can be inserted into expression vectors such that the genes are operatively linked to transcriptional and translational control sequences. In this context, the term “operatively linked” is intended to mean that an antibody gene is ligated into a vector such that transcriptional and translational control sequences within the vector serve their intended function of regulating the transcription and translation of the antibody gene. The expression vector and expression control sequences are chosen to be compatible with the expression host cell used. The protein genes are inserted into the expression vector by standard methods (e.g., ligation of complementary restriction sites on the gene fragment and vector, or blunt end ligation if no restriction sites are present). Additionally or alternatively, the recombinant expression vector can encode a signal peptide that facilitates secretion of the protein (including fusion proteins) from a host cell. The gene can be cloned into the vector such that the signal peptide is linked in-frame to the amino terminus of the gene. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide from a non-immunoglobulin protein).
In addition to the protein genes, the recombinant expression vectors according to at least some embodiments of the invention carry regulatory sequences that control the expression of the genes in a host cell. The term “regulatory sequence” is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signals) that control the transcription or translation of the genes. Such regulatory sequences are described, for example, in Goeddel (“Gene Expression Technology”, Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990)). It will be appreciated by those skilled in the art that the design of the expression vector, including the selection of regulatory sequences, may depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, etc. Preferred regulatory sequences for mammalian host cell expression include viral elements that direct high levels of protein expression in mammalian cells, such as promoters and/or enhancers derived from cytomegalovirus (CMV), Simian Virus 40 (SV40), adenovirus, (e.g., the adenovirus major late promoter (AdMLP) and polyoma. Alternatively, nonviral regulatory sequences may be used, such as the ubiquitin promoter or β-globin promoter. Still further, regulatory elements composed of sequences from different sources, such as the SR α. promoter system, which contains sequences from the SV40 early promoter and the long terminal repeat of human T cell leukemia virus type 1 (Takebe, Y. et al. (1988) Mol. Cell. Biol. 8:466-472).
In addition to the protein genes and regulatory sequences, the recombinant expression vectors according to at least some embodiments of the invention may carry additional sequences, such as sequences that regulate replication of the vector in host cells (e.g., origins of replication) and selectable marker genes. The selectable marker gene facilitates selection of host cells into which the vector has been introduced (see, e.g., U.S. Pat. Nos. 4,399,216, 4,634,665 and 5,179,017, all by Axel et al.). For example, typically the selectable marker gene confers resistance to drugs, such as G418, hygromycin or methotrexate, on a host cell into which the vector has been introduced. Preferred selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use in dhfr− host cells with methotrexate selection/amplification) and the neo gene (for G418 selection).
For expression of the proteins of the invention, an expression vector encoding the protein is transfected into a host cell by standard techniques. The various forms of the term “transfection” are intended to encompass a wide variety of techniques commonly used for the introduction of exogenous DNA into a prokaryotic or eukaryotic host cell, e.g., electroporation, calcium-phosphate precipitation, DEAE-dextran transfection and the like. Although it is theoretically possible to express the proteins according to at least some embodiments of the invention in either prokaryotic or eukaryotic host cells, expression of antibodies in eukaryotic cells, and most preferably mammalian host cells, is the most preferred.
Preferred mammalian host cells for expressing the recombinant proteins according to at least some embodiments of the invention include Chinese Hamster Ovary (CHO cells) (including dhfr− CHO cells, described in Urlaub and Chasin, (1980) Proc. Natl. Acad. Sci. USA 77:4216-4220, used with a DHFR selectable marker, e.g., as described in R. J. Kaufman and P. A. Sharp (1982) Mol. Biol. 159:601-621), NSO myeloma cells, COS cells and SP2 cells. In particular, for use with NSO myeloma cells, another preferred expression system is the GS gene expression system disclosed in WO 87/04462, WO 89/01036 and EP 338,841. When recombinant expression vectors encoding protein genes are introduced into mammalian host cells, the proteins are produced by culturing the host cells for a period of time sufficient to allow for expression of the protein in the host cells or, more preferably, secretion of the protein into the culture medium in which the host cells are grown. Antibodies can be recovered from the culture medium using standard protein purification methods.
HIDE1 protein coding sequences of interest include those encoded by native sequences, as well as nucleic acids that, by virtue of the degeneracy of the genetic code, are not identical in sequence to the disclosed nucleic acids, and variants thereof. Variant polypeptides can include amino acid substitutions as discussed herein. Techniques for in vitro mutagenesis of cloned genes are known. Also included in the subject invention are polypeptides that have been modified using ordinary molecular biological techniques so as to improve their resistance to proteolytic degradation or to optimize solubility properties or to render them more suitable as a therapeutic agent.
The invention further provides nucleic acids which encode a HIDE1 protein according to the invention, or a fragment or conjugate thereof. The nucleic acids may be present in whole cells, in a cell lysate, or in a partially purified or substantially pure form. A nucleic acid is “isolated” or “rendered substantially pure” when purified away from other cellular components or other contaminants, e.g., other cellular nucleic acids or proteins, by standard techniques, including alkaline/SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis and others well known in the art. See, F. Ausubel, et al., ed. (1987) Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York. A nucleic acid according to at least some embodiments of the invention can be, for example, DNA or RNA and may or may not contain intronic sequences.
Nucleic acid compositions encoding the anti-HIDE antibodies of the invention are also provided, as well as expression vectors containing the nucleic acids and host cells transformed with the nucleic acid and/or expression vector compositions. As will be appreciated by those in the art, the protein sequences depicted herein can be encoded by any number of possible nucleic acid sequences, due to the degeneracy of the genetic code.
The nucleic acid compositions that encode the HIDE1 antibodies will depend on the format of the antibody. For traditional, tetrameric antibodies containing two heavy chains and two light chains are encoded by two different nucleic acids, one encoding the heavy chain and one encoding the light chain. These can be put into a single expression vector or two expression vectors, as is known in the art, transformed into host cells, where they are expressed to form the antibodies of the invention. In some embodiments, for example when scFv constructs are used, a single nucleic acid encoding the variable heavy chain-linker-variable light chain is generally used, which can be inserted into an expression vector for transformation into host cells. The nucleic acids can be put into expression vectors that contain the appropriate transcriptional and translational control sequences, including, but not limited to, signal and secretion sequences, regulatory sequences, promoters, origins of replication, selection genes, etc.
Preferred mammalian host cells for expressing the recombinant antibodies according to at least some embodiments of the invention include Chinese Hamster Ovary (CHO cells), PER.C6, HEK293 and others as is known in the art. The nucleic acids may be present in whole cells, in a cell lysate, or in a partially purified or substantially pure form. A nucleic acid is “isolated” or “rendered substantially pure” when purified away from other cellular components or other contaminants, e.g., other cellular nucleic acids or proteins, by standard techniques, including alkaline/SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis and others well known in the art.
To create a scFv gene, the VH- and VL-encoding DNA fragments are operatively linked to another fragment encoding a flexible linker, e.g., encoding the amino acid sequence (Gly4-Ser)3 (SEQ ID NO: 61), such that the VH and VL sequences can be expressed as a contiguous single-chain protein, with the VL and VH regions joined by the flexible linker (see e.g., Bird et al. (1988) Science 242:423-426; Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883; McCafferty et al., (1990) Nature 348:552-554).
The therapeutic compositions used in the practice of the foregoing methods can be formulated into pharmaceutical compositions comprising a carrier suitable for the desired delivery method. Suitable carriers include any material that when combined with the therapeutic composition retains the anti-tumor function of the therapeutic composition and is generally non-reactive with the patient's immune system. Examples include, but are not limited to, any of a number of standard pharmaceutical carriers such as sterile phosphate buffered saline solutions, bacteriostatic water, and the like (see, generally, Remington's Pharmaceutical Sciences 16th Edition, A. Osal., Ed., 1980). Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, acetate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl orbenzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; sweeteners and other flavoring agents; fillers such as microcrystalline cellulose, lactose, corn and other starches; binding agents; additives; coloring agents; salt-forming counter-ions such as sodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™, or polyethylene glycol (PEG).
In a preferred embodiment, the pharmaceutical composition that comprises the antibodies of the invention may be in a water-soluble form, such as being present as pharmaceutically acceptable salts, which is meant to include both acid and base addition salts. “Pharmaceutically acceptable acid addition salt” refers to those salts that retain the biological effectiveness of the free bases and that are not biologically or otherwise undesirable, formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like, and organic acids such as acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid and the like. “Pharmaceutically acceptable base addition salts” include those derived from inorganic bases such as sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts and the like. Particularly preferred are the ammonium, potassium, sodium, calcium, and magnesium salts. Salts derived from pharmaceutically acceptable organic non-toxic bases include salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, and ethanolamine. The formulations to be used for in vivo administration are preferrably sterile. This is readily accomplished by filtration through sterile filtration membranes or other methods.
In some embodiments of, administration of the pharmaceutical composition comprising antibodies of the present invention, preferably in the form of a sterile aqueous solution, may be done in a variety of ways, including, but not limited to subcutaneously and intravenously. Subcutaneous administration may be preferable in some circumstances because the patient may self-administer the pharmaceutical composition. Many protein therapeutics are not sufficiently potent to allow for formulation of a therapeutically effective dose in the maximum acceptable volume for subcutaneous administration. This problem may be addressed in part by the use of protein formulations comprising arginine-HCl, histidine, and polysorbate (see WO 04091658). Fc polypeptides of some embodiments of the present invention may be more amenable to subcutaneous administration due to, for example, increased potency, improved serum half-life, or enhanced solubility.
As is known in the art, protein therapeutics are often delivered by IV infusion or bolus. In some embodiments, the antibodies of the present invention may also be delivered using such methods. For example, administration may venious be by intravenous infusion with 0.9% sodium chloride as an infusion vehicle.
In addition, any of a number of delivery systems are known in the art and may be used to administer the Fc variants of the present invention in various embodiments. Examples include, but are not limited to, encapsulation in liposomes, microparticles, microspheres (e.g. PLA/PGA microspheres), and the like. Alternatively, an implant of a porous, non-porous, or gelatinous material, including membranes or fibers, may be used. Sustained release systems may comprise a polymeric material or matrix such as polyesters, hydrogels, poly(vinylalcohol), polylactides, copolymers of L-glutamic acid and ethyl-L-gutamate, ethylene-vinyl acetate, lactic acid-glycolic acid copolymers such as the LUPRON DEPOT®, and poly-D-(−)-3-hydroxyburyric acid. The antibodies disclosed herein may also be formulated as immunoliposomes. A liposome is a small vesicle comprising various types of lipids, phospholipids and/or surfactant that is useful for delivery of a therapeutic agent to a mammal. Liposomes containing the antibody are prepared by methods known in the art, such as described in Epstein et al., 1985, Proc Natl Acad Sci USA, 82:3688; Hwang et al., 1980, Proc Natl Acad Sci USA, 77:4030; U.S. Pat. No. 4,485,045; U.S. Pat. No. 4,544,545; and PCT WO 97/38731. Liposomes with enhanced circulation time are disclosed in U.S. Pat. No. 5,013,556. The components of the liposome are commonly arranged in a bilayer formation, similar to the lipid arrangement of biological membranes. Particularly useful liposomes can be generated by the reverse phase evaporation method with a lipid composition comprising phosphatidylcholine, cholesterol and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through filters of defined pore size to yield liposomes with the desired diameter. A chemotherapeutic agent or other therapeutically active agent is optionally contained within the liposome (Gabizon et al., 1989, J National Cancer Inst 81:1484).
The antibodies may also be entrapped in microcapsules prepared by methods including but not limited to coacervation techniques, interfacial polymerization (for example using hydroxymethylcellulose or gelatin-microcapsules, or poly-(methylmethacylate) microcapsules), colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules), and macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed., 1980. Sustained-release preparations may be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymer, which matrices are in the form of shaped articles, e.g. films, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (for example poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and gamma ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOT® (which are injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), poly-D-(−)-3-hydroxybutyric acid, and ProLease® (commercially available from Alkermes), which is a microsphere-based delivery system composed of the desired bioactive molecule incorporated into a matrix of poly-DL-lactide-co-glycolide (PLG).
The dosing amounts and frequencies of administration are, in a preferred embodiment, selected to be therapeutically or prophylactically effective. As is known in the art, adjustments for protein degradation, systemic versus localized delivery, and rate of new protease synthesis, as well as the age, body weight, general health, sex, diet, time of administration, drug interaction and the severity of the condition may be necessary, and will be ascertainable with routine experimentation by those skilled in the art.
The concentration of the antibody in the formulation may vary from about 0.1 to 100 weight %. In a some embodiments, the concentration of the Fc variant is in the range of 0.003 to 1.0 molar. In order to treat a patient, a therapeutically effective dose of the Fc variant of the present invention may be administered. By “therapeutically effective dose” herein is meant a dose that produces the effects for which it is administered. The exact dose will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques. Dosages may range from 0.0001 to 100 mg/kg of body weight or greater, for example 0.1, 1, 10, or 50 mg/kg of body weight, with 1 to 10 mg/kg being preferred.
In some embodiments, the present invention provides a composition, e.g., a pharmaceutical composition, containing one or a combination of the HIDE1 therapeutic agent, according to at least some embodiments of the present invention. Thus, the present invention features a pharmaceutical composition comprising a therapeutically effective amount of a therapeutic agent according to at least some embodiments of the present invention.
The pharmaceutical composition according to at least some embodiments of the present invention is further preferably used for the treatment of immune related disorder.
A composition is said to be a “pharmaceutically acceptable carrier” if its administration can be tolerated by a recipient patient. As used herein, “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion).
Such compositions include sterile water, buffered saline (e.g., Tris-HCl, acetate, phosphate), pH and ionic strength and optionally additives such as detergents and solubilizing agents (e.g., Polysorbate 20®, Polysorbate 80®), antioxidants (e.g., ascorbic acid, sodium metabisulfite), preservatives (e.g., Thimersol®, benzyl alcohol) and bulking substances (e.g., lactose, mannitol). Non-aqueous solvents or vehicles may optionally also be used as detailed below.
Examples of suitable aqueous and nonaqueous carriers that may optionally be employed in the pharmaceutical compositions according to at least some embodiments of the present invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Depending on the route of administration, the active compound, i.e., soluble polypeptide, fusion protein or conjugate containing the ectodomain of the HIDE1 antigen, may optionally be coated in a material to protect the compound from the action of acids and other natural conditions that may optionally inactivate the compound. The pharmaceutical compounds according to at least some embodiments of the present invention may optionally include one or more pharmaceutically acceptable salts. A “pharmaceutically acceptable salt” refers to a salt that retains the desired biological activity of the parent compound and does not impart any undesired toxicological effects (see e.g., Berge, S. M., et al. J. Pharm. Sci. 66: 1-19. (1977)). Examples of such salts include acid addition salts and base addition salts. Acid addition salts include those derived from nontoxic inorganic acids, such as hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydriodic, phosphorous and the like, as well as from nontoxic organic acids such as aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxy alkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids and the like. Base addition salts include those derived from alkaline earth metals, such as sodium, potassium, magnesium, calcium and the like, as well as from nontoxic organic amines, such as N,N′-dibenzylethylenediamine, N-methylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, procaine and the like.
A pharmaceutical composition according to at least some embodiments of the present invention also may optionally include a pharmaceutically acceptable anti-oxidant. Examples of pharmaceutically acceptable antioxidants include: (1) water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, α-tocopherol, and the like; and (3) metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.
These compositions may optionally also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of presence of microorganisms may optionally be ensured both by sterilization procedures, supra, and by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may optionally also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may optionally be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.
Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. The use of such media and agents for pharmaceutically active substances is known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the pharmaceutical compositions according to at least some embodiments of the present invention is contemplated. Supplementary active compounds can also be incorporated into the compositions.
Therapeutic compositions typically must be sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, liposome, or other ordered structure suitable to high drug concentration. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin. Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by sterilization microfiltration. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying (lyophilization) that yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by sterilization microfiltration. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying (lyophilization) that yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the subject being treated, and the particular mode of administration. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will generally be that amount of the composition which produces a therapeutic effect. Generally, out of one hundred percent, this amount will range from about 0.01 percent to about ninety-nine percent of active ingredient, preferably from about 0.1 percent to about 70 percent, most preferably from about I percent to about 30 percent of active ingredient in combination with a pharmaceutically acceptable carrier.
Dosage regimens are adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus may optionally be administered, several divided doses may optionally be administered over time or the dose may optionally be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms according to at least some embodiments of the present invention are dictated by and directly dependent on (a) the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active compound for the treatment of sensitivity in individuals.
For fusion proteins as described herein, optionally a similar dosage regimen is followed; alternatively, the fusion proteins may optionally be administered in an amount between 0. 0001 to 100 mg/kg weight of the patient/day, preferably between 0. 001 to 10. 0 mg/kg/day, according to any suitable timing regimen. A therapeutic composition according to at least some embodiments of the present invention can be administered, for example, three times a day, twice a day, once a day, three times weekly, twice weekly or once weekly, once every two weeks or 3, 4, 5, 6, 7 or 8 weeks. Moreover, the composition can be administered over a short or long period of time (e.g., 1 week, 1 month, 1 year, 5 years).
Alternatively, therapeutic agent can be administered as a sustained release formulation, in which case less frequent administration is required. Dosage and frequency vary depending on the half-life of the therapeutic agent in the patient. The half-life for fusion proteins may vary widely. The dosage and frequency of administration can vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic applications, a relatively low dosage is administered at relatively infrequent intervals over a long period of time. Some patients continue to receive treatment for the rest of their lives. In therapeutic applications, a relatively high dosage at relatively short intervals is sometimes required until progression of the disease is reduced or terminated, and preferably until the patient shows partial or complete amelioration of symptoms of disease. Thereafter, the patient can be administered a prophylactic regime.
In some embodiments, the actual dosage levels of the active ingredients in the pharmaceutical compositions of the present invention may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. In some embodiments, he selected dosage level will depend upon a variety of pharmacokinetic factors including the activity of the particular compositions of the present invention employed, or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.
In some embodiments, a composition of the present invention can be administered via one or more routes of administration using one or more of a variety of methods known in the art. As will be appreciated by the skilled artisan, the route and/or mode of administration will vary depending upon the desired results. Routes of administration for therapeutic agents according to at least some embodiments of the present invention include intravascular delivery (e.g., injection or infusion), intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal, oral, enteral, rectal, pulmonary (e.g., inhalation), nasal, topical (including transdermal, buccal and sublingual), intravesical, intravitreal, intraperitoneal, vaginal, brain delivery (e.g. intra-cerebroventricular, intra-cerebral, and convection enhanced diffusion), CNS delivery (e.g., intrathecal, perispinal, and intra-spinal) or parenteral (including subcutaneous, intramuscular, intravenous and intradermal), transmucosal (e.g., sublingual administration), administration or administration via an implant, or other parenteral routes of administration, for example by injection or infusion, or other delivery routes and/or forms of administration known in the art. The phrase “parenteral administration” as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion. In a specific embodiment, a protein, a therapeutic agent or a pharmaceutical composition according to at least some embodiments of the present invention can be administered intraperitoneally or intravenously. Alternatively, a HIDE1 therapeutic agent can be administered via a non-parenteral route, such as a topical, epidermal or mucosal route of administration, for example, intranasally, orally, vaginally, rectally, sublingually or topically.
The active compounds can be prepared with carriers that will protect the compound against rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods for the preparation of such formulations are patented or generally known to those skilled in the art. See, e.g., Sustained and Controlled Release Drug Delivery Systems, J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.
Therapeutic compositions can be administered with medical devices known in the art. For example, in a preferred embodiment, a therapeutic composition according to at least some embodiments of the present invention can be administered with a needles hypodermic injection device, such as the devices disclosed in U.S. Pat. Nos. 5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824; or 4,596,556. Examples of well-known implants and modules useful in some embodiments of the present invention include: U.S. Pat. No. 4,487,603, which discloses an implantable micro-infusion pump for dispensing medication at a controlled rate; U.S. Pat. No. 4,486,194, which discloses a therapeutic device for administering medicaments through the skin; U.S. Pat. No. 4,447,233, which discloses a medication infusion pump for delivering medication at a precise infusion rate; U.S. Pat. No. 4,447,224, which discloses a variable flow implantable infusion apparatus for continuous drug delivery; U.S. Pat. No. 4,439,196, which discloses an osmotic drug delivery system having multi-chamber compartments; and U.S. Pat. No. 4,475,196, which discloses an osmotic drug delivery system. These patents are incorporated herein by reference. Many other such implants, delivery systems, and modules are known to those skilled in the art.
In certain embodiments, HIDE1 soluble proteins, ectodomains, and/or fusion proteins, can be formulated to ensure proper distribution in vivo. For example, the blood-brain barrier (BBB) excludes many highly hydrophilic compounds. To ensure that the therapeutic compounds according to at least some embodiments of the present invention cross the BBB (if desired), they can be formulated, for example, in liposomes. For methods of manufacturing liposomes, see, e.g., U.S. Pat. Nos. 4,522,811; 5,374,548; and 5,399,331. The liposomes may optionally comprise one or more moieties which are selectively transported into specific cells or organs, thus enhance targeted drug delivery (see, e.g., V. V. Ranade J. Clin. Pharmacol. 29:685 (1989)). Exemplary targeting moieties include folate or biotin (see, e.g., U.S. Pat. No. 5,416,016 to Low et al.); mannosides (Umezawa et al., Biochem. Biophys. Res. Commun. 153:1038 (1988)); antibodies (P. G. Bloeman et al. FEBS Lett. 357:140 (1995); M. Owais et al. Antimicrob. Agents Chemother. 39:180 (1995)); surfactant protein A receptor (Briscoe et al.) Am. J Physiol. 1233:134 (1995)); p 120 (Schreier et al. J. Biol. Chem. 269:9090) (1994); see also K. Keinanen; M. L. Laukkanen FEBS Lett. 346:123 (1994); and Killion and Fidler Immunomethods 4:273 (1994).
A. Formulations for Parental Administration
In a further embodiment, compositions disclosed herein, including those containing peptides and polypeptides, are administered in an aqueous solution, by parenteral injection. The formulation may optionally also be in the form of a suspension or emulsion. In general, pharmaceutical compositions are provided including effective amounts of a peptide or polypeptide, and optionally include pharmaceutically acceptable diluents, preservatives, solubilizers, emulsifiers, adjuvants and/or carriers. Such compositions optionally include one or more for the following: diluents, sterile water, buffered saline of various buffer content (e.g., Tris-HCl, acetate, phosphate), pH and ionic strength; and additives such as detergents and solubilizing agents (e.g., TWEEN 20® (polysorbate-20), TWEEN 80® (polysorbate-80)), anti-oxidants (e.g., water soluble antioxidants such as ascorbic acid, sodium metabisulfite, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite; oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, α-tocopherol; and metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid), and preservatives (e.g., Thimersol, benzyl alcohol) and bulking substances (e.g., lactose, mannitol). Examples of non-aqueous solvents or vehicles are ethanol, propylene glycol, polyethylene glycol, vegetable oils, such as olive oil and corn oil, gelatin, and injectable organic esters such as ethyl oleate. The formulations may optionally be freeze dried (lyophilized) or vacuum dried and redissolved/resuspended immediately before use. The formulation may optionally be sterilized by, for example, filtration through a bacteria retaining filter, by incorporating sterilizing agents into the compositions, by irradiating the compositions, or by heating the compositions.
B. Formulations for Topical Administration
HIDE1 polypeptides, fragments, fusion polypeptides, nucleic acids, and vectors disclosed herein can be applied topically. Topical administration does not work well for most peptide formulations, although it can be effective especially if applied to the lungs, nasal, oral (sublingual, buccal), vaginal, or rectal mucosa.
Compositions can be delivered to the lungs while inhaling and traverse across the lung epithelial lining to the blood stream when delivered either as an aerosol or spray dried particles having an aerodynamic diameter of less than about 5 microns. A wide range of mechanical devices designed for pulmonary delivery of therapeutic products can be used, including but not limited to nebulizers, metered dose inhalers, and powder inhalers, all of which are familiar to those skilled in the art. Some specific examples of commercially available devices are the Ultravent nebulizer (Mallinckrodt Inc., St. Louis, Mo.); the Acorn II nebulizer (Marquest Medical Products, Englewood, Colo.); the Ventolin metered dose inhaler (Glaxo Inc., Research Triangle Park, N.C.); and the Spinhaler powder inhaler (Fisons Corp., Bedford, Mass.). Nektar, Alkermes and Mannkind all have inhalable insulin powder preparations approved or in clinical trials where the technology could be applied to the formulations described herein.
Formulations for administration to the mucosa will typically be spray dried drug particles, which may optionally be incorporated into a tablet, gel, capsule, suspension or emulsion. Standard pharmaceutical excipients are available from any formulator. Oral formulations may optionally be in the form of chewing gum, gel strips, tablets or lozenges.
Transdermal formulations may optionally also be prepared. These will typically be ointments, lotions, sprays, or patches, all of which can be prepared using standard technology. Transdermal formulations will require the inclusion of penetration enhancers.
C. Controlled Delivery Polymeric Matrices
HIDE1 polypeptides, fragments, fusion polypeptides, nucleic acids, and vectors disclosed herein may optionally also be administered in controlled release formulations. Controlled release polymeric devices can be made for long term release systemically following implantation of a polymeric device (rod, cylinder, film, disk) or injection (microparticles). The matrix can be in the form of microparticles such as microspheres, where peptides are dispersed within a solid polymeric matrix or microcapsules, where the core is of a different material than the polymeric shell, and the peptide is dispersed or suspended in the core, which may optionally be liquid or solid in nature. Unless specifically defined herein, microparticles, microspheres, and microcapsules are used interchangeably. Alternatively, the polymer may be cast as a thin slab or film, ranging from nanometers to four centimeters, a powder produced by grinding or other standard techniques, or even a gel such as a hydrogel.
Either non-biodegradable or biodegradable matrices can be used for delivery of polypeptides or nucleic acids encoding the polypeptides, although biodegradable matrices are preferred. These may be natural or synthetic polymers, although synthetic polymers are preferred due to the better characterization of degradation and release profiles. The polymer is selected based on the period over which release is desired. In some cases linear release may be most useful, although in others a pulse release or “bulk release” may provide more effective results. The polymer may be in the form of a hydrogel (typically in absorbing up to about 90% by weight of water), and can optionally be crosslinked with multivalent ions or polymers.
The matrices can be formed by solvent evaporation, spray drying, solvent extraction and other methods known to those skilled in the art. Bioerodible microspheres can be prepared using any of the methods developed for making microspheres for drug delivery, for example, as described by Mathiowitz and Langer, J. Controlled Release, 5:13-22 (1987); Mathiowitz, et al., Reactive Polymers, 6:275-283 (1987); and Mathiowitz, et al., J. Appl Polymer Sci, 35:755-774 (1988).
The devices can be formulated for local release to treat the area of implantation or injection—which will typically deliver a dosage that is much less than the dosage for treatment of an entire body—or systemic delivery. These can be implanted or injected subcutaneously, into the muscle, fat, or swallowed.
Accordingly, the invention provides anti-HIDE1 antibodies. The antibodies of the invention are specific for the HIDE1 extracellular domain as more fully outlined herein.
As is discussed below, the term “antibody” is used generally. Antibodies that find use in some embodiments of the present invention can take on a number of formats as described herein, including traditional antibodies as well as antibody derivatives, fragments and mimetics, described below. In general, the term “antibody” includes any polypeptide that includes at least one antigen binding domain, as more fully described below. Antibodies may be polyclonal, monoclonal, xenogeneic, allogeneic, syngeneic, or modified forms thereof, as described herein, with monoclonal antibodies finding particular use in many embodiments. In some embodiments, antibodies of the invention bind specifically or substantially specifically to HIDE1 molecules. The terms “monoclonal antibodies” and “monoclonal antibody composition”, as used herein, refer to a population of antibody molecules that contain only one species of an antigen-binding site capable of immunoreacting with a particular epitope of an antigen, whereas the term “polyclonal antibodies” and “polyclonal antibody composition” refer to a population of antibody molecules that contain multiple species of antigen-binding sites capable of interacting with a particular antigen. A monoclonal antibody composition, typically displays a single binding affinity for a particular antigen with which it immunoreacts.
Traditional full length antibody structural units typically comprise a tetramer. Each tetramer is typically composed of two identical pairs of polypeptide chains, each pair having one “light” (typically having a molecular weight of about 25 kDa) and one “heavy” chain (typically having a molecular weight of about 50-70 kDa). Human light chains are classified as kappa and lambda light chains. According to at least some embodiments of the present invention is directed to the IgG class, which has several subclasses, including, but not limited to IgG1, IgG2, IgG3, and IgG4. Thus, “isotype” as used herein is meant any of the subclasses of immunoglobulins defined by the chemical and antigenic characteristics of their constant regions. While the exemplary antibodies herein designated below and in
SAWVFGGGTQLTVLG
SAWVFGGGTQLTVLGQPKAAPSVTLFPPSSEELQANKATLVC
QQYNYYPITFGQGTRLEIKR
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The amino-terminal portion of each chain includes a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition, generally referred to in the art and herein as the “Fv domain” or “Fv region”. In the variable region, three loops are gathered for each of the V domains of the heavy chain and light chain to form an antigen-binding site. Each of the loops is referred to as a complementarity-determining region (hereinafter referred to as a “CDR”), in which the variation in the amino acid sequence is most significant. “Variable” refers to the fact that certain segments of the variable region differ extensively in sequence among antibodies. Variability within the variable region is not evenly distributed. Instead, the V regions consist of relatively invariant stretches called framework regions (FRs) of 15-30 amino acids separated by shorter regions of extreme variability called “hypervariable regions”.
Each VH and VL is composed of three hypervariable regions (“complementary determining regions,” “CDRs”) and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.
The hypervariable region generally encompasses amino acid residues from about amino acid residues 24-34 (LCDR1; “L” denotes light chain), 50-56 (LCDR2) and 89-97 (LCDR3) in the light chain variable region and around about 31-35B (HCDR1; “H” denotes heavy chain), 50-65 (HCDR2), and 95-102 (HCDR3) in the heavy chain variable region, although sometimes the numbering is shifted slightly as will be appreciated by those in the art; Kabat et al., SEQUENCES OF PROTEINS OF IMMUNOLOGICAL INTEREST, 5 th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991) and/or those residues forming a hypervariable loop (e.g. residues 26-32 (LCDR1), 50-52 (LCDR2) and 91-96 (LCDR3) in the light chain variable region and 26-32 (HCDR1), 53-55 (HCDR2) and 96-101 (HCDR3) in the heavy chain variable region; Chothia and Lesk (1987) J Mol. Biol. 196:901-917. Specific CDRs of the invention and/or for use in the methods of the invention are described above and shown in
The carboxy-terminal portion of each chain defines a constant region primarily responsible for effector function. Kabat et al. collected numerous primary sequences of the variable regions of heavy chains and light chains. Based on the degree of conservation of the sequences, they classified individual primary sequences into the CDR and the framework and made a list thereof (see SEQUENCES OF IMMUNOLOGICAL INTEREST, 5 th edition, NIH publication, No. 91-3242, E. A. Kabat et al., entirely incorporated by reference).
In the IgG subclass of immunoglobulins, there are several immunoglobulin domains in the heavy chain. By “immunoglobulin (Ig) domain” herein is meant a region of an immunoglobulin having a distinct tertiary structure. Of interest in some embodiments of the present invention are the heavy chain domains, including, the constant heavy (CH) domains and the hinge domains. In the context of IgG antibodies, the IgG isotypes each have three CH regions. Accordingly, “CH” domains in the context of IgG are as follows: “CH1” refers to positions 118-220 according to the EU index as in Kabat. “CH2” refers to positions 237-340 according to the EU index as in Kabat, and “CH3” refers to positions 341-447 according to the EU index as in Kabat.
Accordingly, the invention provides variable heavy domains, variable light domains, heavy constant domains, light constant domains and Fc domains to be used as outlined herein. By “variable region” as used herein is meant the region of an immunoglobulin that comprises one or more Ig domains substantially encoded by any of the Vκ or Vλ, and/or VH genes that make up the kappa, lambda, and heavy chain immunoglobulin genetic loci respectively. Accordingly, the variable heavy domain comprises vhFR1-vhCDR1-vhFR2-vhCDR2-vhFR3-vhCDR3-vhFR4, and the variable light domain comprises vlFR1-vlCDR1-vlFR2-vlCDR2-vlFR3-vlCDR3-vlFR4. By “heavy constant region” herein is meant the CH1-hinge-CH2-CH3 portion of an antibody. By “Fc” or “Fc region” or “Fc domain” as used herein is meant the polypeptide comprising the constant region of an antibody excluding the first constant region immunoglobulin domain and in some cases, part of the hinge. Thus Fc refers to the last two constant region immunoglobulin domains of IgA, IgD, and IgG, the last three constant region immunoglobulin domains of IgE and IgM, and the flexible hinge N-terminal to these domains. For IgA and IgM, Fc may include the J chain. For IgG, the Fc domain comprises immunoglobulin domains Cγ2 and Cγ3 (Cγ2 and Cγ3) and the lower hinge region between Cγ1 (Cγ1) and Cγ2 (Cγ2). Although the boundaries of the Fc region may vary, the human IgG heavy chain Fc region is usually defined to include residues C226 or P230 to its carboxyl-terminus, wherein the numbering is according to the EU index as in Kabat. In some embodiments, as is more fully described below, amino acid modifications are made to the Fc region, for example to alter binding to one or more FcγR receptors or to the FcRn receptor.
Thus, “Fc variant” or “variant Fc” as used herein is meant a protein comprising an amino acid modification in an Fc domain. In some embodiments, the Fc variants of the present invention are defined according to the amino acid modifications that compose them. Thus, for example, N434S or 434S is an Fc variant with the substitution serine at position 434 relative to the parent Fc polypeptide, wherein the numbering is according to the EU index. Likewise, M428L/N434S defines an Fc variant with the substitutions M428L and N434S relative to the parent Fc polypeptide. The identity of the WT amino acid may be unspecified, in which case the aforementioned variant is referred to as 428L/434S. It is noted that the order in which substitutions are provided is arbitrary, that is to say that, for example, 428L/434S is the same Fc variant as M428L/N434S, and so on. For all positions discussed in the present invention that relate to antibodies, unless otherwise noted, amino acid position numbering is according to the EU index.
By “Fab” or “Fab region” as used herein is meant the polypeptide that comprises the VH, CH1, VL, and CL immunoglobulin domains. Fab may refer to this region in isolation, or this region in the context of a full length antibody, antibody fragment or Fab fusion protein. By “Fv” or “Fv fragment” or “Fv region” as used herein is meant a polypeptide that comprises the VL and VH domains of a single antibody. As will be appreciated by those in the art, these generally are made up of two chains.
Throughout the present specification, either the IMTG numbering system or the Kabat numbering system is generally used when referring to a residue in the variable domain (approximately, residues 1-107 of the light chain variable region and residues 1-113 of the heavy chain variable region) (e.g., Kabat et al., supra (1991)). EU numbering as in Kabat is generally used for constant domains and/or the Fc domains.
The CDRs contribute to the formation of the antigen-binding, or more specifically, epitope binding site of antibodies. “Epitope” refers to a determinant that interacts with a specific antigen binding site in the variable region of an antibody molecule known as a paratope. Epitopes are groupings of molecules such as amino acids or sugar side chains and usually have specific structural characteristics, as well as specific charge characteristics. A single antigen may have more than one epitope.
The epitope may comprise amino acid residues directly involved in the binding (also called immunodominant component of the epitope) and other amino acid residues, which are not directly involved in the binding, such as amino acid residues which are effectively blocked by the specifically antigen binding peptide; in other words, the amino acid residue is within the footprint of the specifically antigen binding peptide.
Epitopes may be either conformational or linear. A conformational epitope is produced by spatially juxtaposed amino acids from different segments of the linear polypeptide chain. A linear epitope is one produced by adjacent amino acid residues in a polypeptide chain. Conformational and nonconformational epitopes may be distinguished in that the binding to the former but not the latter is lost in the presence of denaturing solvents.
An epitope typically includes at least 3, and more usually, at least 5 or 8-10 amino acids in a unique spatial conformation. Antibodies that recognize the same epitope can be verified in a simple immunoassay showing the ability of one antibody to block the binding of another antibody to a target antigen, for example “binning”. Specific bins are described below.
Included within the definition of “antibody” is an “antigen-binding portion” of an antibody (also used interchangeably with “antigen-binding fragment”, “antibody fragment” and “antibody derivative”). That is, for the purposes of the invention, an antibody of the invention has a minimum functional requirement that it bind to a HIDE1 antigen. As will be appreciated by those in the art, there are a large number of antigen fragments and derivatives that retain the ability to bind an antigen and yet have alternative structures, including, but not limited to, (i) the Fab fragment consisting of VL, VH, CL and CH1 domains, (ii) the Fd fragment consisting of the VH and CH1 domains, (iii) F(ab′)2 fragments, a bivalent fragment comprising two linked Fab fragments (vii) single chain Fv molecules (scFv), wherein a VH domain and a VL domain are linked by a peptide linker which allows the two domains to associate to form an antigen binding site (Bird et al., 1988, Science 242:423-426, Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883, entirely incorporated by reference), (iv) “diabodies” or “triabodies”, multivalent or multispecific fragments constructed by gene fusion (Tomlinson et. al., 2000, Methods Enzymol. 326:461-479; WO94/13804; Holliger et al., 1993, Proc. Natl. Acad. Sci. USA 90:6444-6448, all entirely incorporated by reference), (v) “domain antibodies” or “dAb” (sometimes referred to as an “immunoglobulin single variable domain”, including single antibody variable domains from other species such as rodent (for example, as disclosed in WO 00/29004), nurse shark and Camelid V-HH dAbs, (vi) SMIPs (small molecule immunopharmaceuticals), camelbodies, nanobodies and IgNAR.
Still further, an antibody or antigen-binding portion thereof (antigen-binding fragment, antibody fragment, antibody portion) may be part of a larger immunoadhesion molecules (sometimes also referred to as “fusion proteins”), formed by covalent or noncovalent association of the antibody or antibody portion with one or more other proteins or peptides. Examples of immunoadhesion molecules include use of the streptavidin core region to make a tetrameric scFv molecule and use of a cysteine residue, a marker peptide and a C-terminal polyhistidine tag to make bivalent and biotinylated scFv molecules. Antibody portions, such as Fab and F(ab′)2 fragments, can be prepared from whole antibodies using conventional techniques, such as papain or pepsin digestion, respectively, of whole antibodies. Moreover, antibodies, antibody portions and immunoadhesion molecules can be obtained using standard recombinant DNA techniques, as described herein.
In general, the anti-HIDE1 antibodies of the invention are recombinant. “Recombinant” as used herein, refers broadly with reference to a product, e.g., to a cell, or nucleic acid, protein, or vector, indicates that the cell, nucleic acid, protein or vector, has been modified by the introduction of a heterologous nucleic acid or protein or the alteration of a native nucleic acid or protein, or that the cell is derived from a cell so modified. Thus, for example, recombinant cells express genes that are not found within the native (non-recombinant) form of the cell or express native genes that are otherwise abnormally expressed, under expressed or not expressed at all.
The term “recombinant antibody”, as used herein, includes all antibodies that are prepared, expressed, created or isolated by recombinant means, such as (a) antibodies isolated from an animal (e.g., a mouse) that is transgenic or transchromosomal for human immunoglobulin genes or a hybridoma prepared therefrom (described further below), (b) antibodies isolated from a host cell transformed to express the human antibody, e.g., from a transfectoma, (c) antibodies isolated from a recombinant, combinatorial human antibody library, and (d) antibodies prepared, expressed, created or isolated by any other means that involve splicing of human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies have variable regions in which the framework and CDR regions are derived from human germline immunoglobulin sequences. In certain embodiments, however, such recombinant human antibodies can be subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, while derived from and related to human germline VH and VL sequences, may not naturally exist within the human antibody germline repertoire in vivo.
A. Optional Antibody Engineering
The antibodies of the invention can be modified, or engineered, to alter the amino acid sequences by amino acid substitutions.
By “amino acid substitution” or “substitution” herein is meant the replacement of an amino acid at a particular position in a parent polypeptide sequence with a different amino acid. In particular, in some embodiments, the substitution is to an amino acid that is not naturally occurring at the particular position, either not naturally occurring within the organism or in any organism. For example, the substitution E272Y refers to a variant polypeptide, in this case an Fc variant, in which the glutamic acid at position 272 is replaced with tyrosine. For clarity, a protein which has been engineered to change the nucleic acid coding sequence but not change the starting amino acid (for example exchanging CGG (encoding arginine) to CGA (still encoding arginine) to increase host organism expression levels) is not an “amino acid substitution”; that is, despite the creation of a new gene encoding the same protein, if the protein has the same amino acid at the particular position that it started with, it is not an amino acid substitution.
As discussed herein, amino acid substitutions can be made to alter the affinity of the CDRs for the HIDE1 protein (including both increasing and decreasing binding, as is more fully outlined below), as well as to alter additional functional properties of the antibodies. For example, the antibodies may be engineered to include modifications within the Fc region, typically to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding, and/or antigen-dependent cellular cytotoxicity. Furthermore, an antibody according to at least some embodiments of the invention may be chemically modified (e.g., one or more chemical moieties can be attached to the antibody) or be modified to alter its glycosylation, again to alter one or more functional properties of the antibody. Such embodiments are described further below. The numbering of residues in the Fc region is that of the EU index of Kabat.
In one embodiment, the hinge region of CH1 is modified such that the number of cysteine residues in the hinge region is altered, e.g., increased or decreased. This approach is described further in U.S. Pat. No. 5,677,425 by Bodmer et al. The number of cysteine residues in the hinge region of CH1 is altered to, for example, facilitate assembly of the light and heavy chains or to increase or decrease the stability of the antibody.
In another embodiment, the Fc hinge region of an antibody is mutated to decrease the biological half-life of the antibody. More specifically, one or more amino acid mutations are introduced into the CH2-CH3 domain interface region of the Fc-hinge fragment such that the antibody has impaired Staphylococcyl protein A (SpA) binding relative to native Fc-hinge domain SpA binding. This approach is described in further detail in U.S. Pat. No. 6,165,745 by Ward et al.
In some embodiments, amino acid substitutions can be made in the Fc region, in general for altering binding to FcγR receptors. By “Fc gamma receptor”, “FcγR” or “FcgammaR” as used herein is meant any member of the family of proteins that bind the IgG antibody Fc region and is encoded by an FcγR gene. In humans this family includes but is not limited to FcγRI (CD64), including isoforms FcγRIa, FcγRIb, and FcγRIc; FcγRII (CD32), including isoforms FcγRIIa (including allotypes H131 and R131), FcγRIIb (including FcγRIIb-1 and FcγRIIb-2), and FcγRIIc; and FcγRIII (CD16), including isoforms FcγRIIIa (including allotypes V158 and F158) and FcγRIIIb (including allotypes FcγRIIIb-NA1 and FcγRIIIb-NA2) (Jefferis et al., 2002, Immunol Lett 82:57-65, entirely incorporated by reference), as well as any undiscovered human FcγRs or FcγR isoforms or allotypes. An FcγR may be from any organism, including but not limited to humans, mice, rats, rabbits, and monkeys. Mouse FcγRs include but are not limited to FcγRI (CD64), FcγRII (CD32), FcγRIII-1 (CD16), and FcγRIII-2 (CD16-2), as well as any undiscovered mouse FcγRs or FcγR isoforms or allotypes.
There are a number of useful Fc substitutions that can be made to alter binding to one or more of the FcγR receptors. Substitutions that result in increased binding as well as decreased binding can be useful. For example, it is known that increased binding to FcγRIIIa generally results in increased ADCC (antibody dependent cell-mediated cytotoxicity; the cell-mediated reaction wherein nonspecific cytotoxic cells that express FcγRs recognize bound antibody on a target cell and subsequently cause lysis of the target cell. Similarly, decreased binding to FcγRIIb (an inhibitory receptor) can be beneficial as well in some circumstances. Amino acid substitutions that find use in some embodiments of the present invention include those listed in U.S. Ser. No. 11/124,620 (particularly
In addition, the antibodies of the invention are modified to increase its biological half-life. Various approaches are possible. For example, one or more of the following mutations can be introduced: T252L, T254S, T256F, as described in U.S. Pat. No. 6,277,375 to Ward. Alternatively, to increase the biological half-life, the antibody can be altered within the CH1 or CL region to contain a salvage receptor binding epitope taken from two loops of a CH2 domain of an Fc region of an IgG, as described in U.S. Pat. Nos. 5,869,046 and 6,121,022 by Presta et al. Additional mutations to increase serum half life are disclosed in U.S. Pat. Nos. 8,883,973, 6,737,056 and 7,371,826, and include 428L, 434A, 434S, and 428L/4345.
In yet other embodiments, the Fc region is altered by replacing at least one amino acid residue with a different amino acid residue to alter the effector functions of the antibody. For example, one or more amino acids selected from amino acid residues 234, 235, 236, 237, 297, 318, 320 and 322 can be replaced with a different amino acid residue such that the antibody has an altered affinity for an effector ligand but retains the antigen-binding ability of the parent antibody. The effector ligand to which affinity is altered can be, for example, an Fc receptor or the C1 component of complement. This approach is described in further detail in U.S. Pat. Nos. 5,624,821 and 5,648,260, both by Winter et al.
In another example, one or more amino acids selected from amino acid residues 329, 331 and 322 can be replaced with a different amino acid residue such that the antibody has altered C1q binding and/or reduced or abolished complement dependent cytotoxicity (CDC). This approach is described in further detail in U.S. Pat. No. 6,194,551 by Idusogie et al.
In another example, one or more amino acid residues within amino acid positions 231 and 239 are altered to thereby alter the ability of the antibody to fix complement. This approach is described further in PCT Publication WO 94/29351 by Bodmer et al.
In yet another example, the Fc region is modified to increase the ability of the antibody to mediate antibody dependent cellular cytotoxicity (ADCC) and/or to increase the affinity of the antibody for an Fcγ receptor by modifying one or more amino acids at the following positions: 238, 239, 248, 249, 252, 254, 255, 256, 258, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294, 295, 296, 298, 301, 303, 305, 307, 309, 312, 315, 320, 322, 324, 326, 327, 329, 330, 331, 333, 334, 335, 337, 338, 340, 360, 373, 376, 378, 382, 388, 389, 398, 414, 416, 419, 430, 434, 435, 437, 438 or 439. This approach is described further in PCT Publication WO 00/42072 by Presta. Moreover, the binding sites on human IgG1 for FcγRI, FcγRII, FcγRIII and FcRn have been mapped and variants with improved binding have been described (see Shields, R. L. et al. (2001) J. Biol. Chem. 276:6591-6604). Specific mutations at positions 256, 290, 298, 333, 334 and 339 are shown to improve binding to FcγRIII Additionally, the following combination mutants are shown to improve FcγRIII binding: T256A/S298A, S298A/E333A, S298A/K224A and S298A/E333A/K334A. Furthermore, mutations such as M252Y/S254T/T256E or M428L/N434S improve binding to FcRn and increase antibody circulation half-life (see Chan C A and Carter P J (2010) Nature Rev Immunol 10:301-316).
In still another embodiment, the antibody can be modified to abrogate in vivo Fab arm exchange. Specifically, this process involves the exchange of IgG4 half-molecules (one heavy chain plus one light chain) between other IgG4 antibodies that effectively results in bispecific antibodies which are functionally monovalent. Mutations to the hinge region and constant domains of the heavy chain can abrogate this exchange (see Aalberse, R C, Schuurman J., 2002, Immunology 105:9-19).
In still another embodiment, the glycosylation of an antibody is modified. For example, an aglycosylated antibody can be made (i.e., the antibody lacks glycosylation). Glycosylation can be altered to, for example, increase the affinity of the antibody for antigen or reduce effector function such as ADCC. Such carbohydrate modifications can be accomplished by, for example, altering one or more sites of glycosylation within the antibody sequence, for example N297. For example, one or more amino acid substitutions can be made that result in elimination of one or more variable region framework glycosylation sites to thereby eliminate glycosylation at that site.
Additionally or alternatively, an antibody can be made that has an altered type of glycosylation, such as a hypofucosylated antibody having reduced amounts of fucosyl residues or an antibody having increased bisecting GlcNac structures. Such altered glycosylation patterns have been demonstrated to increase the ADCC ability of antibodies. Such carbohydrate modifications can be accomplished by, for example, expressing the antibody in a host cell with altered glycosylation machinery. Cells with altered glycosylation machinery have been described in the art and can be used as host cells in which to express recombinant antibodies according to at least some embodiments of the invention to thereby produce an antibody with altered glycosylation. For example, the cell lines Ms704, Ms705, and Ms709 lack the fucosyltransferase gene, FUT8 (α (1,6) fucosyltransferase), such that antibodies expressed in the Ms704, Ms705, and Ms709 cell lines lack fucose on their carbohydrates. The Ms704, Ms705, and Ms709 FUT8 cell lines are created by the targeted disruption of the FUT8 gene in CHO/DG44 cells using two replacement vectors (see U.S. Patent Publication No. 20040110704 by Yamane et al. and Yamane-Ohnuki et al. (2004) Biotechnol Bioeng 87:614-22). As another example, EP 1,176,195 by Hanai et al. describes a cell line with a functionally disrupted FUT8 gene, which encodes a fucosyl transferase, such that antibodies expressed in such a cell line exhibit hypofucosylation by reducing or eliminating the a 1,6 bond-related enzyme. Hanai et al. also describe cell lines which have a low enzyme activity for adding fucose to the N-acetylglucosamine that binds to the Fc region of the antibody or does not have the enzyme activity, for example the rat myeloma cell line YB2/0 (ATCC CRL 1662). PCT Publication WO 03/035835 by Presta describes a variant CHO cell line, Lec13 cells, with reduced ability to attach fucose to Asn(297)-linked carbohydrates, also resulting in hypofucosylation of antibodies expressed in that host cell (see also Shields, R. L. et al. (2002)1 Biol. Chem. 277:26733-26740). PCT Publication WO 99/54342 by Umana et al. describes cell lines engineered to express glycoprotein-modifying glycosyl transferases (e.g., β(1,4)-N-acetylglucosaminyltransferase III (GnTIII)) such that antibodies expressed in the engineered cell lines exhibit increased bisecting GlcNac structures which results in increased ADCC activity of the antibodies (see also Umana et al. (1999) Nat. Biotech. 17:176-180). Alternatively, the fucose residues of the antibody may be cleaved off using a fucosidase enzyme. For example, the fucosidase α-L-fucosidase removes fucosyl residues from antibodies (Tarentino, A. L. et al. (1975) Biochem. 14:5516-23).
Another modification of the antibodies herein that is contemplated by the invention is pegylation or the addition of other water soluble moieties, typically polymers, e.g., in order to enhance half-life. An antibody can be pegylated to, for example, increase the biological (e.g., serum) half-life of the antibody. To pegylate an antibody, the antibody, or fragment thereof, typically is reacted with polyethylene glycol (PEG), such as a reactive ester or aldehyde derivative of PEG, under conditions in which one or more PEG groups become attached to the antibody or antibody fragment. Preferably, the pegylation is carried out via an acylation reaction or an alkylation reaction with a reactive PEG molecule (or an analogous reactive water-soluble polymer). As used herein, the term “polyethylene glycol” is intended to encompass any of the forms of PEG that have been used to derivative other proteins, such as mono (C1-C10) alkoxy- or aryloxy-polyethylene glycol or polyethylene glycol-maleimide. In certain embodiments, the antibody to be pegylated is an aglycosylated antibody. Methods for pegylating proteins are known in the art and can be applied to the antibodies according to at least some embodiments of the invention. See for example, EP 0 154 316 by Nishimura et al. and EP 0 401 384 by Ishikawa et al.
In addition to substitutions made to alter binding affinity to FcγRs and/or FcRn and/or increase in vivo serum half life, additional antibody modifications can be made, as described in further detail below.
In some cases, affinity maturation is done. Amino acid modifications in the CDRs are sometimes referred to as “affinity maturation”. An “affinity matured” antibody is one having one or more alteration(s) in one or more CDRs which results in an improvement in the affinity of the antibody for antigen, compared to a parent antibody which does not possess those alteration(s). In some cases, although rare, it may be desirable to decrease the affinity of an antibody to its antigen, but this is generally not preferred.
In some embodiments, one or more amino acid modifications are made in one or more of the CDRs of the VISG1 antibodies of the invention. In general, only 1 or 2 or 3-amino acids are substituted in any single CDR, and generally no more than from 1, 2, 3. 4, 5, 6, 7, 8 9 or 10 changes are made within a set of CDRs. However, it should be appreciated that any combination of no substitutions, 1, 2 or 3 substitutions in any CDR can be independently and optionally combined with any other substitution.
Affinity maturation can be done to increase the binding affinity of the antibody for the HIDE1 antigen by at least about 10% to 50-100-150% or more, or from 1 to 5 fold as compared to the “parent” antibody. Preferred affinity matured antibodies will have nanomolar or even picomolar affinities for the HIDE1 antigen. Affinity matured antibodies are produced by known procedures. See, for example, Marks et al., 1992, Biotechnology 10:779-783 that describes affinity maturation by variable heavy chain (VH) and variable light chain (VL) domain shuffling. Random mutagenesis of CDR and/or framework residues is described in: Barbas, et al. 1994, Proc. Nat. Acad. Sci, USA 91:3809-3813; Shier et al., 1995, Gene 169:147-155; Yelton et al., 1995, J. Immunol. 155:1994-2004; Jackson et al., 1995, J. Immunol. 154(7):3310-9; and Hawkins et al, 1992, J. Mol. Biol. 226:889-896, for example.
Alternatively, amino acid modifications can be made in one or more of the CDRs of the antibodies of the invention that are “silent”, e.g. that do not significantly alter the affinity of the antibody for the antigen. These can be made for a number of reasons, including optimizing expression (as can be done for the nucleic acids encoding the antibodies of the invention).
Thus, included within the definition of the CDRs and antibodies of the invention are variant CDRs and antibodies; that is, the antibodies of the invention can include amino acid modifications in one or more of the CDRs of the enumerated antibodies of the invention. In addition, as outlined below, amino acid modifications can also independently and optionally be made in any region outside the CDRs, including framework and constant regions.
B. HIDE1 Antibodies
According to at least some embodiments of the present invention provides anti-HIDE1 antibodies. (For convenience, “anti-HIDE1 antibodies” and “HIDE1 antibodies” are used interchangeably). The anti-HIDE1 antibodies of the invention specifically bind to human HIDE1, and preferably the ECD of human HIDE1, as depicted in
Specific binding for HIDE1 or a HIDE1 epitope can be exhibited, for example, by an antibody having a KD of at least about 10−4 M, at least about 10−5 M, at least about 10−6 M, at least about 10−7 M, at least about 10−8 M, at least about 10−9 M, alternatively at least about 10−10 M, at least about 10−11 M, at least about 10−12 M, or greater, where KD refers to a dissociation rate of a particular antibody-antigen interaction. Typically, an antibody that specifically binds an antigen will have a KD that is 20-, 50-, 100-, 500-, 1000-, 5,000-, 10,000- or more times greater for a control molecule relative to the HIDE1 antigen or epitope
However, as shown in the Examples, for optimal binding to HIDE1 expressed on the surface of myeloid cells, the antibodies preferably have a KD less 50 nM and most preferably less than 1 nM, with less than 0.1 nM and less than 1 pM and 0.1 pM finding use in the methods of the invention.
Also, specific binding for a particular antigen or an epitope can be exhibited, for example, by an antibody having a KA or Ka for a HIDE1 antigen or epitope of at least 20-, 50-, 100-, 500-, 1000-, 5,000-, 10,000- or more times greater for the epitope relative to a control, where KA or Ka refers to an association rate of a particular antibody-antigen interaction.
In some embodiments, the anti-HIDE1 antibodies of the invention bind to human HIDE1 with a KD of 100 nM or less, 50 nM or less, 10 nM or less, or 1 nM or less (that is, higher binding affinity), or 1 pM or less, wherein KD is determined by known methods, e.g. surface plasmon resonance (SPR, e.g. Biacore assays), ELISA, KinExA, and most typically SPR at 25° or 37° C.
C. Specific Anti-HIDE1 Antibodies
The invention provides antigen binding domains, including full length antibodies, which contain a number of specific, enumerated sets of 6 CDRs, including for use in the methods of the invention. See,
As above, these sets of CDRs may also be amino acid variants as described above.
In addition, the framework regions of the variable heavy and variable light chains can be humanized as is known in the art (with occasional variants generated in the CDRs as needed), and thus humanized variants of the VH and VL chains of
In particular, as is known in the art, murine VH and VL chains can be humanized as is known in the art, for example, using the IgBLAST program of the NCBI website, as outlined in Ye et al. Nucleic Acids Res. 41:W34-W40 (2013), herein incorporated by reference in its entirety for the humanization methods. IgBLAST takes a murine VH and/or VL sequence and compares it to a library of known human germline sequences. As shown herein, for the humanized sequences, the databases that can be used are IMGT human VH genes (F+ORF, 273 germline sequences) and IMGT human VL kappa genes (F+ORF, 74 germline sequences). CDRs were and will be defined according to the AbM definition (see, the World Wide Web at bioinfo.org.uk/abs).
Specific humanized antibodies of CPA antibodies include those shown in
In some embodiments, the anti-HIDE1 antibodies of the present invention include anti-HIDE1 antibodies wherein the VH and VL sequences of different anti-HIDE1 antibodies can be “mixed and matched” to create other anti-HIDE1 antibodies. HIDE1 binding of such “mixed and matched” antibodies can be tested using the binding assays described above. e.g., ELISAs). In some embodiments, when VH and VL chains are mixed and matched, a VH sequence from a particular VH/VL pairing is replaced with a structurally similar VH sequence. Likewise, in some embodiments, a VL sequence from a particular VH/VL pairing is replaced with a structurally similar VL sequence. For example, the VH and VL sequences of homologous antibodies are particularly amenable for mixing and matching.
Accordingly, the antibodies of the invention comprise CDR amino acid sequences selected from the group consisting of (a) sequences as listed herein; (b) sequences that differ from those CDR amino acid sequences specified in (a) by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid substitutions; (c) amino acid sequences having 90% or greater, 95% or greater, 98% or greater, or 99% or greater sequence identity to the sequences specified in (a) or (b); (d) a polypeptide having an amino acid sequence encoded by a polynucleotide having a nucleic acid sequence encoding the amino acids as listed herein.
Additionally included in the definition of HIDE1 antibodies are antibodies that share identity to the HIDE1 antibodies enumerated herein. That is, in certain embodiments, an anti-HIDE1 antibody according to the invention comprises heavy and light chain variable regions comprising amino acid sequences that are homologous to isolated anti-HIDE1 amino acid sequences of preferred anti-HIDE1 immune molecules, respectively, wherein the antibodies retain the desired functional properties of the parent anti-HIDE1 antibodies. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % homology=# of identical positions/total # of positions X 100), taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm, as described in the non-limiting examples below.
The percent identity between two amino acid sequences can be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci., 4:11-17 (1988)) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. In addition, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J. Mol. Biol. 48:444-453 (1970)) algorithm which has been incorporated into the GAP program in the GCG software package (available commercially), using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.
Additionally or alternatively, in some embodiments the protein sequences of the present invention can further be used as a “query sequence” to perform a search against public databases to, for example, identify related sequences. Such searches can be performed using the XBLAST program (version 2.0) of Altschul, et al. (1990) J Mol. Biol. 215:403-10. BLAST protein searches can be performed with the XBLAST program, score=50, wordlength=3 to obtain amino acid sequences homologous to the antibody molecules according to at least some embodiments of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Res. 25(17):3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used.
In general, the percentage identity for comparison between HIDE1 antibodies is at least 75%, at least 80%, at least 90%, with at least about 95, 96, 97, 98 or 99% percent identity being preferred. The percentage identity may be along the whole amino acid sequence, for example the entire heavy or light chain or along a portion of the chains. For example, included within the definition of the anti-HIDE1 antibodies of the invention are those that share identity along the entire variable region (for example, where the identity is 95 or 98% identical along the variable regions), or along the entire constant region, or along just the Fc domain.
In addition, also included are sequences that may have the identical CDRs but changes in the variable domain (or entire heavy or light chain). For example, HIDE1 antibodies of the invention and/or for use in the invention, include those with CDRs identical to those shown in
According to at least some embodiments of the present invention provides not only the enumerated antibodies but additional antibodies that compete with the enumerated antibodies (the
1. Generation of Additional Antibodies
Additional antibodies to human HIDE1 can be done as is well known in the art, using well known methods such as those outlined in the examples. Thus, additional anti-HIDE1 antibodies can be generated by traditional methods such as immunizing mice (sometimes using DNA immunization, for example, such as is used by Aldevron), followed by screening against human HIDE1 protein and hybridoma generation, with antibody purification and recovery.
D. Nucleic Acid Compositions
Nucleic acid compositions encoding the anti-HIDE1 antibodies of the invention are also provided, as well as expression vectors containing the nucleic acids and host cells transformed with the nucleic acid and/or expression vector compositions. As will be appreciated by those in the art, the protein sequences depicted herein can be encoded by any number of possible nucleic acid sequences, due to the degeneracy of the genetic code.
The nucleic acid compositions that encode the HIDE1 antibodies will depend on the format of the antibody. For traditional, tetrameric antibodies containing two heavy chains and two light chains are encoded by two different nucleic acids, one encoding the heavy chain and one encoding the light chain. These can be put into a single expression vector or two expression vectors, as is known in the art, transformed into host cells, where they are expressed to form the antibodies of the invention. In some embodiments, for example when scFv constructs are used, a single nucleic acid encoding the variable heavy chain-linker-variable light chain is generally used, which can be inserted into an expression vector for transformation into host cells. The nucleic acids can be put into expression vectors that contain the appropriate transcriptional and translational control sequences, including, but not limited to, signal and secretion sequences, regulatory sequences, promoters, origins of replication, selection genes, etc.
Preferred mammalian host cells for expressing the recombinant antibodies according to at least some embodiments of the invention include Chinese Hamster Ovary (CHO cells), PER.C6, HEK293 and others as is known in the art.
The nucleic acids may be present in whole cells, in a cell lysate, or in a partially purified or substantially pure form. A nucleic acid is “isolated” or “rendered substantially pure” when purified away from other cellular components or other contaminants, e.g., other cellular nucleic acids or proteins, by standard techniques, including alkaline/SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis and others well known in the art.
To create a scFv gene, the VH- and VL-encoding DNA fragments are operatively linked to another fragment encoding a flexible linker, e.g., encoding the amino acid sequence (Gly4-Ser)3 (SEQ ID NO:61), such that the VH and VL sequences can be expressed as a contiguous single-chain protein, with the VL and VH regions joined by the flexible linker (see e.g., Bird et al. (1988) Science 242:423-426; Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883; McCafferty et al., (1990) Nature 348:552-554).
E. Formulations of Anti-HIDE1 Antibodies
The therapeutic compositions used in the practice of the foregoing methods can be formulated into pharmaceutical compositions comprising a carrier suitable for the desired delivery method. Suitable carriers include any material that when combined with the therapeutic composition retains the anti-tumor function of the therapeutic composition and is generally non-reactive with the patient's immune system. Examples include, but are not limited to, any of a number of standard pharmaceutical carriers such as sterile phosphate buffered saline solutions, bacteriostatic water, and the like (see, generally, Remington's Pharmaceutical Sciences 16th Edition, A. Osal., Ed., 1980). Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, acetate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl orbenzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; sweeteners and other flavoring agents; fillers such as microcrystalline cellulose, lactose, corn and other starches; binding agents; additives; coloring agents; salt-forming counter-ions such as sodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).
In some embodiments, the pharmaceutical composition that comprises the antibodies of the invention may be in a water-soluble form, such as being present as pharmaceutically acceptable salts, which is meant to include both acid and base addition salts. “Pharmaceutically acceptable acid addition salt” refers to those salts that retain the biological effectiveness of the free bases and that are not biologically or otherwise undesirable, formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like, and organic acids such as acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid and the like. “Pharmaceutically acceptable base addition salts” include those derived from inorganic bases such as sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts and the like. Particularly preferred are the ammonium, potassium, sodium, calcium, and magnesium salts. Salts derived from pharmaceutically acceptable organic non-toxic bases include salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, and ethanolamine. The formulations to be used for in vivo administration are preferrably sterile. This is readily accomplished by filtration through sterile filtration membranes or other methods.
In some embodiments, administration of the pharmaceutical composition comprising antibodies of the present invention, preferably in the form of a sterile aqueous solution, may be done in a variety of ways, including, but not limited to subcutaneously and intravenously. Subcutaneous administration may be preferable in some circumstances because the patient may self-administer the pharmaceutical composition. Many protein therapeutics are not sufficiently potent to allow for formulation of a therapeutically effective dose in the maximum acceptable volume for subcutaneous administration. This problem may be addressed in part by the use of protein formulations comprising arginine-HCl, histidine, and polysorbate (see WO 04091658). In some embodiments, Fc polypeptides of the present invention may be more amenable to subcutaneous administration due to, for example, increased potency, improved serum half-life, or enhanced solubility.
As is known in the art, protein therapeutics are often delivered by IV infusion or bolus. In some embodiments, the antibodies of the present invention may also be delivered using such methods. For example, administration may venious be by intravenous infusion with 0.9% sodium chloride as an infusion vehicle.
In addition, any of a number of delivery systems are known in the art and may be used to administer the Fc variants of the various embodiments of the present invention. Examples include, but are not limited to, encapsulation in liposomes, microparticles, microspheres (e.g. PLA/PGA microspheres), and the like. Alternatively, an implant of a porous, non-porous, or gelatinous material, including membranes or fibers, may be used. Sustained release systems may comprise a polymeric material or matrix such as polyesters, hydrogels, poly(vinylalcohol), polylactides, copolymers of L-glutamic acid and ethyl-L-gutamate, ethylene-vinyl acetate, lactic acid-glycolic acid copolymers such as the LUPRON DEPOT®, and poly-D-(−)-3-hydroxyburyric acid. The antibodies disclosed herein may also be formulated as immunoliposomes. A liposome is a small vesicle comprising various types of lipids, phospholipids and/or surfactant that is useful for delivery of a therapeutic agent to a mammal. Liposomes containing the antibody are prepared by methods known in the art, such as described in Epstein et al., 1985, Proc Natl Acad Sci USA, 82:3688; Hwang et al., 1980, Proc Natl Acad Sci USA, 77:4030; U.S. Pat. No. 4,485,045; U.S. Pat. No. 4,544,545; and PCT WO 97/38731. Liposomes with enhanced circulation time are disclosed in U.S. Pat. No. 5,013,556. The components of the liposome are commonly arranged in a bilayer formation, similar to the lipid arrangement of biological membranes. Particularly useful liposomes can be generated by the reverse phase evaporation method with a lipid composition comprising phosphatidylcholine, cholesterol and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through filters of defined pore size to yield liposomes with the desired diameter. A chemotherapeutic agent or other therapeutically active agent is optionally contained within the liposome (Gabizon et al., 1989, J National Cancer Inst 81:1484).
The antibodies may also be entrapped in microcapsules prepared by methods including but not limited to coacervation techniques, interfacial polymerization (for example using hydroxymethylcellulose or gelatin-microcapsules, or poly-(methylmethacylate) microcapsules), colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules), and macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed., 1980. Sustained-release preparations may be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymer, which matrices are in the form of shaped articles, e.g. films, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (for example poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and gamma ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOT® (which are injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), poly-D-(−)-3-hydroxybutyric acid, and ProLease® (commercially available from Alkermes), which is a microsphere-based delivery system composed of the desired bioactive molecule incorporated into a matrix of poly-DL-lactide-co-glycolide (PLG).
The dosing amounts and frequencies of administration are, in a preferred embodiment, selected to be therapeutically or prophylactically effective. As is known in the art, adjustments for protein degradation, systemic versus localized delivery, and rate of new protease synthesis, as well as the age, body weight, general health, sex, diet, time of administration, drug interaction and the severity of the condition may be necessary, and will be ascertainable with routine experimentation by those skilled in the art.
The concentration of the antibody in the formulation may vary from about 0.1 to 100 weight %. In a preferred embodiment, the concentration of the Fc variant is in the range of 0.003 to 1.0 molar. In order to treat a patient, in some embodiments a therapeutically effective dose of the Fc variant of the present invention may be administered. By “therapeutically effective dose” herein is meant a dose that produces the effects for which it is administered. The exact dose will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques. Dosages may range from 0.0001 to 100 mg/kg of body weight or greater, for example 0.1, 1, 10, or 50 mg/kg of body weight, with 1 to 10 mg/kg being preferred.
F. Methods of Using Anti-HIDE1 Antibodies Compositions
1. Therapeutic Uses
A “therapeutically effective dosage” of HIDE1 soluble protein or HIDE1 ectodomain or fusion protein containing same, according to at least some embodiments of the present invention preferably results in a decrease in severity of disease symptoms, an increase in frequency and duration of disease symptom-free periods, an increase in lifespan, disease remission, or a prevention or reduction of impairment or disability due to the disease affliction. Alternatively, this property of a composition can be evaluated by examining the ability of the compound to inhibit, such inhibition in vitro by assays known to the skilled practitioner. A therapeutically effective amount of a therapeutic compound can decrease tumor size, or otherwise ameliorate symptoms in a subject.
One of ordinary skill in the art would be able to determine a therapeutically effective amount based on such factors as the subject's size, the severity of the subject's symptoms, and the particular composition or route of administration selected.
The anti-HIDE1 antibodies of the invention find use in treating patients, such as human subjects, generally with a condition associated with HIDE1. The term “treatment” as used herein, refers to both therapeutic treatment and prophylactic or preventative measures, which in this example relates to treatment of cancer; however, also as described below, uses of antibodies and pharmaceutical compositions are also provided for treatment of infectious disease, sepsis, and/or autoimmune conditions, and/or for inhibiting an undesirable immune activation that follows gene therapy. Those in need of treatment include those already with cancer as well as those in which the cancer is to be prevented. Hence, the mammal to be treated herein may have been diagnosed as having the cancer or may be predisposed or susceptible to the cancer. As used herein the term “treating” refers to preventing, delaying the onset of, curing, reversing, attenuating, alleviating, minimizing, suppressing, halting the deleterious effects or stabilizing of discernible symptoms of the above-described cancerous diseases, disorders or conditions. It also includes managing the cancer as described above. By “manage” it is meant reducing the severity of the disease, reducing the frequency of episodes of the disease, reducing the duration of such episodes, reducing the severity of such episodes, slowing/reducing cancer cell growth or proliferation, slowing progression of at least one symptom, amelioration of at least one measurable physical parameter and the like. For example, immunostimulatory anti-HIDE1 immune molecules should promote myeloid cell, T cell or NK or cytokine immunity against target cells, e.g., cancer, infected or pathogen cells and thereby treat cancer or infectious diseases by depleting the cells involved in the disease condition. Conversely, immunoinhibitory anti-HIDE1 immune molecules should reduce myeloid cell, T cell or NK activity and/or or the secretion of proinflammatory cytokines which are involved in the disease pathology of some immune disease such as autoimmune, inflammatory or allergic conditions and thereby treat or ameliorate the disease pathology and tissue destruction that may be associated with such conditions (e.g., joint destruction associated with rheumatoid arthritis conditions).
The HIDE1 antibodies of the invention are provided in therapeutically effective dosages. A “therapeutically effective dosage” of an anti-HIDE1 immune molecule according to at least some embodiments of the present invention preferably results in a decrease in severity of disease symptoms, an increase in frequency and duration of disease symptom-free periods, an increase in lifespan, disease remission, or a prevention or reduction of impairment or disability due to the disease affliction. For example, for the treatment of HIDE1 positive tumors (including, for example tumors that express HIDE1 on the cellular membrane as well as tumors that express HIDE 1 in the tumor microenvironment, also referred to as the TME), a “therapeutically effective dosage” preferably inhibits cell growth or tumor growth by at least about 20%, more preferably by at least about 40%, even more preferably by at least about 60%, and still more preferably by at least about 80% relative to untreated subjects. The ability of a compound to inhibit tumor growth can be evaluated in an animal model system predictive of efficacy in human tumors. Alternatively, this property of a composition can be evaluated by examining the ability of the compound to inhibit, such inhibition in vitro by assays known to the skilled practitioner. A therapeutically effective amount of a therapeutic compound can decrease tumor size, or otherwise ameliorate symptoms in a subject.
One of ordinary skill in the art would be able to determine a therapeutically effective amount based on such factors as the subject's size, the severity of the subject's symptoms, and the particular composition or route of administration selected.
a. Cancer Treatment
The HIDE1 antibodies of the invention find particular use in the treatment of cancer. In general, the antibodies of the invention are immunomodulatory, in that rather than directly attack cancerous cells, the anti-HIDE1 antibodies of the invention stimulate the immune system, generally by inhibiting the action of HIDE1. Thus, unlike tumor-targeted therapies, which are aimed at inhibiting molecular pathways that are crucial for tumor growth and development, and/or depleting tumor cells, cancer immunotherapy is aimed to stimulate the patient's own immune system to eliminate cancer cells, providing long-lived tumor destruction. Various approaches can be used in cancer immunotherapy, among them are therapeutic cancer vaccines to induce tumor-specific T cell responses, and immunostimulatory antibodies (i.e., antagonists of inhibitory receptors=immune checkpoints) to remove immunosuppressive pathways.
Clinical responses with targeted therapy or conventional anti-cancer therapies tend to be transient as cancer cells develop resistance, and tumor recurrence takes place. However, the clinical use of cancer immunotherapy in the past few years has shown that this type of therapy can have durable clinical responses, showing dramatic impact on long term survival. However, although responses are long term, only a small number of patients respond (as opposed to conventional or targeted therapy, where a large number of patients respond, but responses are transient).
By the time a tumor is detected clinically, it has already evaded the immune-defense system by acquiring immunoresistant and immunosuppressive properties and creating an immunosuppressive tumor microenvironment through various mechanisms and a variety of immune cells.
Accordingly, the anti-HIDE1 antibodies of the invention are useful in treating cancer. Due to the nature of an immuno-oncology mechanism of action, HIDE1 does not necessarily need to be overexpressed on or correlated with a particular cancer type; that is, the goal is to have the anti-HIDE1 antibodies de-suppress myeloid, T cell and NK cell activation, such that the immune system will go after the cancers.
“Cancer,” as used herein, refers broadly to any neoplastic disease (whether invasive or metastatic) characterized by abnormal and uncontrolled cell division causing malignant growth or tumor (e.g., unregulated cell growth.) The term “cancer” or “cancerous” as used herein should be understood to encompass any neoplastic disease (whether invasive, non-invasive or metastatic) which is characterized by abnormal and uncontrolled cell division causing malignant growth or tumor, non-limiting examples of which are described herein. This includes any physiological condition in mammals that is typically characterized by unregulated cell growth. Examples of cancer are exemplified in the working examples and also are described within the specification.
Non-limiting examples of cancer that can be treated using anti-HIDE1 antibodies include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More particular examples of such cancers include squamous cell cancer, lung cancer (including small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung), cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer (including gastrointestinal cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, liver cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma and various types of head and neck cancer, as well as B-cell lymphoma (including low grade/follicular non-Hodgkin's lymphoma (NHL); small lymphocytic (SL) NHL; intermediate grade/follicular NHL; intermediate grade diffuse NHL; high grade immunoblastic NHL; high grade lymphoblastic NHL; high grade small non-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenström's Macroglobulinemia); chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); Hairy cell leukemia; chronic myeloblastic leukemia; multiple myeloma and post-transplant lymphoproliferative disorder (PTLD).
In some embodiments, other cancers amenable for treatment by the present invention include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. More particular examples of such cancers include colorectal, bladder, ovarian, melanoma, squamous cell cancer, lung cancer (including small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung), cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer (including gastrointestinal cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, liver cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma and various types of head and neck cancer, as well as B-cell lymphoma (including low grade/follicular non-Hodgkin's lymphoma (NHL); small lymphocytic (SL) NHL; intermediate grade/follicular NHL; intermediate grade diffuse NHL; high grade immunoblastic NHL; high grade lymphoblastic NHL; high grade small non-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenström's Macroglobulinemia); chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); Hairy cell leukemia; chronic myeloblastic leukemia; and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal vascular proliferation associated with phakomatoses, edema (such as that associated with brain tumors), and Meigs' syndrome. Preferably, the cancer is selected from the group consisting of colorectal cancer, breast cancer, rectal cancer, non-small cell lung cancer, non-Hodgkin's lymphoma (NHL), renal cell cancer, prostate cancer, liver cancer, pancreatic cancer, soft-tissue sarcoma, Kaposi's sarcoma, carcinoid carcinoma, head and neck cancer, melanoma, ovarian cancer, mesothelioma, and multiple myeloma. In an exemplary embodiment the cancer is an early or advanced (including metastatic) bladder, ovarian or melanoma. In another embodiment the cancer is colorectal cancer. In some embodiments, the cancerous conditions amenable for treatment of the present invention include cancers that express or do not express HIDE1 and further include non-metastatic or non-invasive as well as invasive or metastatic cancers wherein HIDE1 expression by immune, stromal or diseased cells suppress antitumor responses and anti-invasive immune responses. The method of the present invention is particularly suitable for the treatment of vascularized tumors.
As shown in the Examples, HIDE1 is over expressed and/or correlates with tumor leukocyte infiltration (as demonstrated by correlation to CSFR1, CD68, CD86, CD11b, IL-4, IL-10, IL-13 and IFN-γ expression) in a number of different tumors of various origins, and thus is useful in treating any cancer, including but not limited to, prostate cancer, liver cancer (HCC), colorectal cancer, ovarian cancer, endometrial cancer, breast cancer, pancreatic cancer, stomach cancer, cervical cancer, head and neck cancer, thyroid cancer, testis cancer, urothelial cancer, lung cancer, melanoma, non melanoma skin cancer (squamous and basal cell carcinoma), glioma, renal cancer (RCC), lymphoma (non-Hodgkins' lymphoma (NHL) and Hodgkin's lymphoma (HD)), Acute myeloid leukemia (AML), T cell Acute Lymphoblastic Leukemia (T-ALL), Diffuse Large B cell lymphoma, testicular germ cell tumors, mesothelioma and esophageal cancer. In some embodiments, the tumors are myeloid or circulating tumor types. In some embodiments, the cancer includes but is not limited to Acute Myeloid Leukemia, Acute Myeloid Leukemia Induction Failure, Acute Lymphoblastic Leukemia, Diffuse Large B-cell Lymphoma, Malignant Lymphoma, Non-Hodgkin Lymphoma, Diffuse Large B-Cell Lymphoma, Glioblastoma multiforme, Mesothelioma, Thymoma, Testicular Germ Cell Tumors, Kidney renal clear cell carcinoma, Sarcoma, Brain Lower Grade Glioma, Chronic Lymphocytic Leukemia, Non-Hodgkin Lymphoma—Follicular Lymphoma, Uterine Carcinosarcoma, Pediatric Brain Tumors, Lung adenocarcinoma, Cervical squamous cell carcinoma, endocervical adenocarcinoma, Pancreatic adenocarcinoma, Skin Cutaneous Melanoma, Kidney renal papillary cell carcinoma, Liver hepatocellular carcinoma; Bladder Urothelial Carcinoma, Colon adenocarcinoma, Head and Neck squamous cell carcinoma, Lung squamous cell carcinoma, Rectum adenocarcinoma, and Stomach adenocarcinoma.
“Cancer therapy” herein refers to any method which prevents or treats cancer or ameliorates one or more of the symptoms of cancer. Typically such therapies will comprises administration of immunostimulatory anti-HIDE1 antibodies (including antigen-binding fragments) either alone or in combination with chemotherapy or radiotherapy or other biologics and for enhancing the activity thereof, i.e., in individuals wherein expression of HIDE1 suppresses antitumor responses and the efficacy of chemotherapy or radiotherapy or biologic efficacy.
In some embodiments, the cancer for treatment is a cancer having high immune infiltrate of myeloid cells expressing HIDE1 wherein said cancer could be treated by administering HIDE1 antibodies. In some embodiments, said cancer is a myeloid cancer. In some embodiments, the myeloid cells include but are not limited to monocytes, dendritic cells, macrophages, M1/M2 tumor associated macrophages, neutrophils, and myeloid-derive suppressor cells (MDSC). In some embodiments, the anti-HIDE1 antibody for use in cancer treatment is a depleting HIDE1 antibody. In some embodiments, a depleting anti-HIDE1 antibody binds to cell surface HIDE1. In some embodiments, the anti HIDE1 depleting antibody preferably is able to deplete HIDE1 expressing cells including but not limited to monocytes, dendritic cells, macrophages, M1/M2 tumor associated macrophages, neutrophils, Myeloid-derive suppressor cells (MDSC) and as a result reduce the number of HIDE1 expressing cells in a patient treated with the anti HIDE1 depleting antibody. Such depletion may be achieved via various mechanisms such as antibody-dependent cell mediated cytotoxicity (ADCC) and/or complement dependent cytotoxicity (CDC), inhibition of HIDE1 expressing cells proliferation and/or induction of HIDE1+ cell death (e.g., via apoptosis).
b. Assessment of Treatment
Generally the anti-HIDE1 antibodies of the invention are administered to patients with cancer, and efficacy is assessed, in a number of ways as described herein. Thus, while standard assays of efficacy can be run, such as cancer load, size of tumor, evaluation of presence or extent of metastasis, etc., immuno-oncology treatments can be assessed on the basis of immune status evaluations as well. This can be done in a number of ways, including both in vitro and in vivo assays. For example, evaluation of changes in immune status (e.g. presence of ICOS+ CD4+ T cells following ipi treatment) along with “old fashioned” measurements such as tumor burden, size, invasiveness, LN involvement, metastasis, etc. can be done. Thus, any or all of the following can be evaluated: the inhibitory effects of HIDE1 on CD4+ T cell activation or proliferation, CD8+ T (CTL) cell activation or proliferation, CD8+ T cell-mediated cytotoxic activity and/or CTL mediated cell depletion, NK cell activity and NK mediated cell depletion, the potentiating effects of HIDE1 on Treg cell differentiation and proliferation and Treg- or myeloid derived suppressor cell (MDSC)-mediated immunosuppression or immune tolerance, and/or the effects of HIDE1 on proinflammatory cytokine production by immune cells, e.g., IL-2, IFN-γ or TNF-α production by T or other immune cells.
In some embodiments, assessment of treatment is done by evaluating immune cell proliferation, using for example, CFSE dilution method, Ki67 intracellular staining of immune effector cells, and 3H-Thymidine incorporation method,
In some embodiments, assessment of treatment is done by evaluating the increase in gene expression or increased protein levels of activation-associated markers, including one or more of: CD25, CD69, CD137, ICOS, PD1, GITR, OX40, and cell degranulation measured by surface expression of CD107A.
In general, gene expression assays are done as is known in the art. See for example Goodkind et al., Computers and Chem. Eng. 29(3):589 (2005), Han et al., Bioinform. Biol. Insights Nov. 15, 2015 9(Suppl. 1):29-46, Campo et al., Nod. Pathol. 2013 January; 26 suppl. 1:S97-S110, the gene expression measurement techniques of which are expressly incorporated by reference herein.
In general, protein expression measurements are also similarly done as is known in the art, see for example, Wang et al., Recent Advances in Capillary Electrophoresis-Based Proteomic Techniques for Biomarker Discovery, Methods. Mol. Biol. 2013:984:1-12; Taylor et al., BioMed Res. Volume 2014, Article ID 361590, 8 pages, Becerk et al., Mutat. Res 2011 June 17:722(2): 171-182, the measurement techniques of which are expressly incorporated herein by reference.
In some embodiments, assessment of treatment is done by assessing cytotoxic activity measured by target cell viability detection via estimating numerous cell parameters such as enzyme activity (including protease activity), cell membrane permeability, cell adherence, ATP production, co-enzyme production, and nucleotide uptake activity. Specific examples of these assays include, but are not limited to, Trypan Blue or PI staining, 51Cr or 35S release method, LDH activity, MTT and/or WST assays, Calcein-AM assay, Luminescent based assay, and others.
In some embodiments, assessment of treatment is done by assessing T cell activity measured by cytokine production, measure either intracellularly in culture supernatant using cytokines including, but not limited to, IFNγ, TNFα, GM-CSF, IL2, IL6, IL4, IL5, IL10, IL13 using well known techniques.
Accordingly, assessment of treatment can be done using assays that evaluate one or more of the following: (i) increases immune response, (ii) increases T cell activity, (iii) increases activation of αβ and/or γδ T cells, (iv) increases cytotoxic T cell activity, (v) increases NK and/or NKT cell activity, (vi) alleviates αβ and/or γδ T-cell suppression, (vii) increases pro-inflammatory cytokine secretion, (viii) increases IL-2 secretion; (vix) increases interferon-γ production, (x) increases Th1 response, (xi) decrease Th2 response, (xii) decreases or eliminates cell number and/or activity of at least one of regulatory T cells (Tregs), myeloid derived suppressor cells (MDSCs), iMCs, mesenchymal stromal cells, TIE2-expressing monocytes, (xiii) reduces regulatory cell activity, and/or the activity of one or more of myeloid derived suppressor cells (MDSCs), iMCs, mesenchymal stromal cells, TIE2-expressing monocytes, (xiv) decreases or eliminates M2 macrophages, (xv) reduces M2 macrophage pro-tumorigenic activity, (xvi) decreases or eliminates N2 neutrophils, (xvii) reduces N2 neutrophils pro-tumorigenic activity, (xviii) reduces inhibition of T cell activation, (xix) reduces inhibition of CTL activation, (xx) reduces inhibition of NK and/or NKT cell activation, (xxi) reverses αβ and/or γδ T cell exhaustion, (xxii) increases αβ and/or γδ T cell response, (xxiii) increases activity of cytotoxic cells, (xxiv) stimulates antigen-specific memory responses, (xxv) elicits apoptosis or lysis of cancer cells, (xxvi) stimulates cytotoxic or cytostatic effect on cancer cells, (xxvii) induces direct killing of cancer cells, (xxviii) increases Th17 activity and/or (xxix) modulating myeloid cell polarization, (xxx) modulating myeloid cell shifting toward a pro-inflammatory response, (xxxi) shifting myeloid from M2 toward M1 phenotype, (xxxii) modulating myeloid cell in the TME to support anti-cancer immune response, (xxxiii) restricting the pro-tumorigenic effects of the myeloid cells in the TME, (xxxiv) enhancing myeloid and lymphoid infiltration into the tumor cite thereby shifting the tumor into more immunogenic, (xxxv) induces complement dependent cytotoxicity and/or antibody dependent cell-mediated cytotoxicity.
Accordingly, assessment of treatment can be done using assays that evaluate one or more of the following: (i) decreases immune response, (ii) decreases αβ and/or γδ T cell activation, (iii) decreases T cell activity, (iv) decreases cytotoxic T cell activity, (v) decreases natural killer (NK) and/or NKT cell activity, (vi) decreases αβ and/or γδ T-cell activity, (vii) decreases pro-inflammatory cytokine secretion, (viii) decreases IL-2 secretion; (ix) decreases interferon-γ production, (x) decreases Th1 response, (xi) decreases Th2 response, (xii) increases cell number and/or activity of regulatory T cells, (xiii) increases regulatory cell activity and/or one or more of myeloid derived suppressor cells (MDSCs), iMCs, mesenchymal stromal cells, TIE2-expressing monocytes, (xiv) increases regulatory cell activity and/or the activity of one or more of myeloid derived suppressor cells (MDSCs), iMCs, mesenchymal stromal cells, TIE2-expressing monocytes, (xv) increases M2 macrophages, (xvi) increases M2 macrophage activity, (xvii) increases N2 neutrophils, (xviii) increases N2 neutrophils activity, (xix) increases inhibition of T cell activation, (xx) increases inhibition of CTL activation, (xxi) increases inhibition of NK cell activation, (xxii) increases αβ and/or γδ T cell exhaustion, (xxiii) decreases αβ and/or γδ T cell response, (xxiv) decreases activity of cytotoxic cells, (xxv) reduces antigen-specific memory responses, (xxvi) inhibits apoptosis or lysis of cells, (xxvii) decreases cytotoxic or cytostatic effect on cells, (xxviii) reduces direct killing of cells, (xxix) decreases Th17 activity, and/or (xxx) modulates myeloid cell polarization, and/or modulates myeloid cell shifting toward an anti-inflammatory response, (xxxi) reduces complement dependent cytotoxicity and/or antibody dependent cell-mediated cytotoxicity. Again without wishing to be limited by a single hypothesis, HIDE1 shows potentiating effects on the following immune functions: induction or differentiation and proliferation of inducible T regulatory or suppressor cells (iTregs). These cells are known to be involved in eliciting tolerance to self-antigens and to suppress anti-tumor immunity.
c. Assays to Measure Efficacy
In some embodiments, T cell activation is assessed using a Mixed Lymphocyte Reaction (MLR) assay as is described in Example 2. An increase in activity indicates immunostimulatory activity. Appropriate increases in activity are outlined below.
In one embodiment, the signaling pathway assay measures increases or decreases in immune response as measured for an example by phosphorylation or de-phosphorylation of different factors, or by measuring other post translational modifications. An increase in activity indicates immunostimulatory activity. Appropriate increases in activity are outlined below.
In one embodiment, the signaling pathway assay measures increases or decreases in activation of αβ and/or γδ T cells as measured for an example by cytokine secretion or by proliferation or by changes in expression of activation markers like for an example CD137, CD107a, PD1, etc. An increase in activity indicates immunostimulatory activity. Appropriate increases in activity are outlined below.
In one embodiment, the signaling pathway assay measures increases or decreases in cytotoxic T cell activity as measured for an example by direct killing of target cells like for an example cancer cells or by cytokine secretion or by proliferation or by changes in expression of activation markers like for an example CD137, CD107a, PD1, etc. An increase in activity indicates immunostimulatory activity. Appropriate increases in activity are outlined below.
In one embodiment, the signaling pathway assay measures increases or decreases in NK and/or NKT cell activity as measured for an example by direct killing of target cells like for an example cancer cells or by cytokine secretion or by changes in expression of activation markers like for an example CD107a, etc. An increase in activity indicates immunostimulatory activity. Appropriate increases in activity are outlined below.
In one embodiment, the signaling pathway assay measures increases or decreases in myeloid cell-mediated suppression of T or NK cell activity as measured for an example by direct killing of target cells like for an example cancer cells or by cytokine secretion or by changes in expression of activation markers like for an example CD107a, etc. An increase in activity indicates immunostimulatory activity. Appropriate increases in activity are outlined below
In one embodiment, the signaling pathway assay measures increases or decreases in myeloid cell-mediated suppression of T or NK cell migration as measured for an example by T or NK cell migration in two-chamber assay system. An increase in migration indicates immunostimulatory activity.
In one embodiment, the signaling pathway assay measures increases or decreases in pro-inflammatory cytokine secretion as measured for example by ELISA or by Luminex or by Multiplex bead based methods or by intracellular staining and FACS analysis or by Alispot etc. An increase in activity indicates immunostimulatory activity. Appropriate increases in activity are outlined below.
In one embodiment, the signaling pathway assay measures increases or decreases in IL-2 secretion as measured for example by ELISA or by Luminex or by Multiplex bead based methods or by intracellular staining and FACS analysis or by Alispot etc. An increase in activity indicates immunostimulatory activity. Appropriate increases in activity are outlined below.
In one embodiment, the signaling pathway assay measures increases or decreases in interferon-γ production as measured for example by ELISA or by Luminex or by Multiplex bead based methods or by intracellular staining and FACS analysis or by Alispot etc. An increase in activity indicates immunostimulatory activity. Appropriate increases in activity are outlined below.
In one embodiment, the signaling pathway assay measures increases or decreases in Th1 response as measured for an example by cytokine secretion or by changes in expression of activation markers. An increase in activity indicates immunostimulatory activity. Appropriate increases in activity are outlined below.
In one embodiment, the signaling pathway assay measures increases or decreases in Th2 response as measured for an example by cytokine secretion or by changes in expression of activation markers. An increase in activity indicates immunostimulatory activity. Appropriate increases in activity are outlined below.
In one embodiment, the signaling pathway assay measures increases or decreases cell number and/or activity of at least one of regulatory T cells (Tregs), as measured for example by flow cytometry or by IHC. A decrease in response indicates immunostimulatory activity. Appropriate decreases are the same as for increases, outlined below.
In one embodiment, the signaling pathway assay measures increases or decreases in M2 macrophages cell numbers, as measured for example by flow cytometry or by IHC. A decrease in response indicates immunostimulatory activity. Appropriate decreases are the same as for increases, outlined below.
In one embodiment, the signaling pathway assay measures increases or decreases in M2 macrophage pro-tumorigenic activity, as measured for an example by cytokine secretion or by changes in expression of activation markers. A decrease in response indicates immunostimulatory activity. Appropriate decreases are the same as for increases, outlined below.
In one embodiment, the signaling pathway assay measures increases or decreases in N2 neutrophils increase, as measured for example by flow cytometry or by IHC. A decrease in response indicates immunostimulatory activity. Appropriate decreases are the same as for increases, outlined below.
In one embodiment, the signaling pathway assay measures increases or decreases in N2 neutrophils pro-tumorigenic activity, as measured for an example by cytokine secretion or by changes in expression of activation markers. A decrease in response indicates immunostimulatory activity. Appropriate decreases are the same as for increases, outlined below.
In one embodiment, the signaling pathway assay measures increases or decreases in inhibition of T cell activation, as measured for an example by cytokine secretion or by proliferation or by changes in expression of activation markers like for an example CD137, CD107a, PD1, etc. An increase in activity indicates immunostimulatory activity. Appropriate increases in activity are outlined below.
In one embodiment, the signaling pathway assay measures increases or decreases in inhibition of CTL activation as measured for an example by direct killing of target cells like for an example cancer cells or by cytokine secretion or by proliferation or by changes in expression of activation markers like for an example CD137, CD107a, PD1, etc. An increase in activity indicates immunostimulatory activity. Appropriate increases in activity are outlined below.
In one embodiment, the signaling pathway assay measures increases or decreases in αβ and/or γδ T cell exhaustion as measured for an example by changes in expression of activation markers. A decrease in response indicates immunostimulatory activity. Appropriate decreases are the same as for increases, outlined below.
In one embodiment, the signaling pathway assay measures increases or decreases αβ and/or γδ T cell response as measured for an example by cytokine secretion or by proliferation or by changes in expression of activation markers like for an example CD137, CD107a, PD1, etc. An increase in activity indicates immunostimulatory activity. Appropriate increases in activity are outlined below.
In one embodiment, the signaling pathway assay measures increases or decreases in stimulation of antigen-specific memory responses as measured for an example by cytokine secretion or by proliferation or by changes in expression of activation markers like for an example CD45RA, CCR7 etc. An increase in activity indicates immunostimulatory activity. Appropriate increases in activity are outlined below.
In one embodiment, the signaling pathway assay measures increases or decreases in apoptosis or lysis of cancer cells as measured for an example by cytotoxicity assays such as for an example MTT, Cr release, Calcine AM, or by flow cytometry based assays like for an example CFSE dilution or propidium iodide staining etc. An increase in activity indicates immunostimulatory activity. Appropriate increases in activity are outlined below.
In one embodiment, the signaling pathway assay measures increases or decreases in stimulation of cytotoxic or cytostatic effect on cancer cells. as measured for an example by cytotoxicity assays such as for an example MTT, Cr release, Calcine AM, or by flow cytometry based assays like for an example CFSE dilution or propidium iodide staining etc. An increase in activity indicates immunostimulatory activity. Appropriate increases in activity are outlined below.
In one embodiment, the signaling pathway assay measures increases or decreases direct killing of cancer cells as measured for an example by cytotoxicity assays such as for an example MTT, Cr release, Calcine AM, or by flow cytometry based assays like for an example CFSE dilution or propidium iodide staining etc. An increase in activity indicates immunostimulatory activity. Appropriate increases in activity are outlined below.
In one embodiment, the signaling pathway assay measures increases or decreases Th17 activity as measured for an example by cytokine secretion or by proliferation or by changes in expression of activation markers. An increase in activity indicates immunostimulatory activity. Appropriate increases in activity are outlined below.
In one embodiment, the signaling pathway assay measures increases or decreases in induction of complement dependent cytotoxicity and/or antibody dependent cell-mediated cytotoxicity, as measured for an example by cytotoxicity assays such as for an example MTT, Cr release, Calcine AM, or by flow cytometry based assays like for an example CFSE dilution or propidium iodide staining etc. An increase in activity indicates immunostimulatory activity. Appropriate increases in activity are outlined below.
In one embodiment, T cell activation is measured for an example by direct killing of target cells like for an example cancer cells or by cytokine secretion or by proliferation or by changes in expression of activation markers like for an example CD137, CD107a, PD1, etc. For T-cells, increases in proliferation, cell surface markers of activation (e.g. CD25, CD69, CD137, PD1), cytotoxicity (ability to kill target cells), and cytokine production (e.g. IL-2, IL-4, IL-6, IFNγ, TNF-α, IL-10, IL-17A) would be indicative of immune modulation that would be consistent with enhanced killing of cancer cells.
In one embodiment, NK cell activation is measured for example by direct killing of target cells like for an example cancer cells or by cytokine secretion or by changes in expression of activation markers like for an example CD107a, etc. For NK cells, increases in proliferation, cytotoxicity (ability to kill target cells and increases CD107a, granzyme, and perforin expression), cytokine production (e.g. IFNγ and TNF), and cell surface receptor expression (e.g. CD25) would be indicative of immune modulation that would be consistent with enhanced killing of cancer cells.
In one embodiment, γδ T cell activation is measured for example by cytokine secretion or by proliferation or by changes in expression of activation markers.
In one embodiment, Th1 cell activation is measured for example by cytokine secretion or by changes in expression of activation markers.
Appropriate increases in activity or response (or decreases, as appropriate as outlined above), are increases of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 98 to 99% percent over the signal in either a reference sample or in control samples, for example test samples that do not contain an anti-HIDE1 antibody of the invention. Similarly, increases of at least one-, two-, three-, four- or five-fold as compared to reference or control samples show efficacy.
2. Combinations
As is known in the art, combination therapies comprising a therapeutic antibody targeting an immunotherapy target and an additional therapeutic agent, specific for the disease condition, are showing great promise. For example, in the area of immunotherapy, there are a number of promising combination therapies using a chemotherapeutic agent (either a small molecule drug or an anti-tumor antibody) with immuno-oncology antibodies like anti-PD-1, and as such, the anti-HIDE1 antibodies outlined herein can be substituted in the same way. Any chemotherapeutic agent exhibiting anticancer activity can be used according to various embodiments of the present invention; various non-limiting examples are described in the specification.
The underlying scientific rationale for the dramatic increased efficacy of combination therapy claims that immune checkpoint blockade as a monotherapy will induce tumor regressions only when there is pre-existing strong anti-tumor immune response to be “unleashed” when the pathway is blocked. However, in most patients and tumor types the endogenous anti-tumor immune responses are weak, and thus the induction of anti-tumor immunity is required for the immune checkpoint blockade to be effective, as shown in the
The terms “in combination with” and “co-administration” are not limited to the administration of said prophylactic or therapeutic agents at exactly the same time. Instead, it is meant that the anti-HIDE1 antibody and the other agent or agents are administered in a sequence and within a time interval such that they may act together to provide a benefit that is increased versus treatment with only either anti-HIDE1 antibody of the various embodiments of the present invention or the other agent or agents. It is preferred that the anti-HIDE1 antibody and the other agent or agents act additively, and especially preferred that they act synergistically. Such molecules are suitably present in combination in amounts that are effective for the purpose intended. The skilled medical practitioner can determine empirically, or by considering the pharmacokinetics and modes of action of the agents, the appropriate dose or doses of each therapeutic agent, as well as the appropriate timings and methods of administration.
Accordingly, in some embodiments, the antibodies of the present invention may be administered concomitantly with one or more other therapeutic regimens or agents. The additional therapeutic regimes or agents may be used to improve the efficacy or safety of the anti-HIDE1 antibody. Also, the additional therapeutic regimes or agents may be used to treat the same disease or a comorbidity rather than to alter the action of the HIDE1 antibody. For example, a HIDE1 antibody of the various embodiments of the present invention may be administered to the patient along with chemotherapy, radiation therapy, or both chemotherapy and radiation therapy.
The HIDE1 antibodies of the various embodiments of the present invention may be administered in combination with one or more other prophylactic or therapeutic agents, including but not limited to cytotoxic agents, chemotherapeutic agents, cytokines, growth inhibitory agents, anti-hormonal agents, kinase inhibitors, anti-angiogenic agents, cardioprotectants, immunostimulatory agents, immunosuppressive agents, agents that promote proliferation of hematological cells, angiogenesis inhibitors, protein tyrosine kinase (PTK) inhibitors, or other therapeutic agents.
According to at least some embodiments, the anti-HIDE1 immune molecules could be used in combination with any of the known in the art standard of care cancer treatment (as can be found, for example, on the World Wide Web at cancer.gov/cancertopics).
In some embodiments, conventional/classical anti-cancer agents suitable for use with the present invention include but are not limited to platinum based compounds, antibiotics with anti-cancer activity, Anthracyclines, Anthracenediones, alkylating agents, antimetabolites, Antimitotic agents, Taxanes, Taxoids, microtubule inhibitors, Folate antagonists and/or folic acid analogs, Topoisomerase inhibitors, Aromatase inhibitors, GnRh analogs, inhibitors of 5α-reductase, bisphosphonates; pyrimidine analogs, purine analogs and related inhibitors, vinca alkaloids, epipodophyllotoxins, antibiotics, L-Asparaginase, topoisomerase inhibitor, interferons, platinum coordination complexes, anthracenedione substituted urea, methyl hydrazine derivatives, adrenocortical suppressant, adrenocorticosteroids, progestins, estrogens, antiestrogen, androgens, antiandrogen, and gonadotropin-releasing hormone analog.
Specific but non-limiting examples of these categories of drugs are as follows: platinum based compounds such as oxaliplatin, cisplatin, carboplatin; Antibiotics with anti-cancer activity, such as dactinomycin, bleomycin, mitomycin-C, mithramycin and Anthracyclines, such as doxorubicin, daunorubicin, epirubicin, idarubicin; Anthracenediones, such as mitoxantrone; Alkylating agents, such as dacarbazine, melphalan, cyclophosphamide, temozolomide, chlorambucil, busulphan, nitrogen mustard, nitrosoureas; Antimetabolites, such as fluorouracil, raltitrexed, gemcitabine, cytosine arabinoside, hydroxyurea and Folate antagonists, such as methotrexate, trimethoprim, pyrimethamine, pemetrexed; Antimitotic agents such as polokinase inhibitors and Microtubule inhibitors, such as Taxanes and Taxoids, such as paclitaxel, docetaxel; Vinca alkaloids such as vincristine, vinblastine, vindesine, vinorelbine; Topoisomerase inhibitors, such as etoposide, teniposide, amsacrine, topotecan, irinotecan, camptothecin; Cytostatic agents including Antiestrogens such as tamoxifen, fulvestrant, toremifene, raloxifene, droloxifene, iodoxyfene, Antiandrogens such as bicalutamide, flutamide, nilutamide and cyproterone acetate, Progestogens such as megestrol acetate, Aromatase inhibitors such as anastrozole, letrozole, vorozole, exemestane; GnRH analogs, such as leuprorelin, goserelin, buserelin, degarelix; inhibitors of 5α-reductase such as finasteride.
More preferably, the chemotherapeutic agent is selected from the group consisting of 5-fluorouracil (5-FU), leucovorin (LV), irenotecan, oxaliplatin, capecitabine, paclitaxel and doxetaxel. Two or more chemotherapeutic agents can be used in a cocktail to be administered in combination with administration of the anti-VEGF antibody. One preferred combination chemotherapy is fluorouracil-based, comprising 5-FU and one or more other chemotherapeutic agent(s). Suitable dosing regimens of combination chemotherapies are known in the art and described in, for example, Saltz et al. (1999) Proc ASCO 18:233a and Douillard et al. (2000) Lancet 355:1041-7. The biologic may optionally be another immune potentiators such as antibodies to PD-L1, PD-L2, CTLA-4, or VISTA as well as PD-L1, PD-L2, CTLA-4 or VISTA fusion proteins as well as cytokines, growth factor antagonists and agonists, hormones and anti-cytokine antibodies.
According to at least some embodiments of the present invention, targeted therapies used as agents for combination with anti HIDE1 antibodies for treatment of cancer are selected from the group consisting of but not limited to: histone deacetylase (HDAC) inhibitors, such as vorinostat, romidepsin, panobinostat, belinostat, mocetinostat, abexinostat, entinostat, resminostat, givinostat, quisinostat, sodium butyrate; Proteasome inhibitors, such as bortezomib, carfilzomib, disulfiram; mTOR pathway inhibitors, such as temsirolimus, rapamycin, everolimus; PI3K inhibitors, such as perifosine, CAL101, PX-866, IPI-145, BAY 80-6946; B-raf inhibitors such as vemurafenib, sorafenib; JAK2 inhibitors, such as lestaurtinib, pacritinib; tyrosine kinase inhibitors (TKIs), such as erlotinib, imatinib, sunitinib, lapatinib, gefitinib, sorafenib, nilotinib, toceranib, bosutinib, neratinib, vatalanib, regorafenib, cabozantinib; other protein kinase inhibitors, such as crizotinib; inhibitors of serine/threonine kinases for example Ras/Raf signalling inhibitors such as farnesyl transferase inhibitors; inhibitors of serine proteases for example matriptase, hepsin, urokinase; inhibitors of intracellular signaling such as tipifarnib, perifosine; Inhibitors of cell signalling through MEK and/or AKT kinases; aurora kinase inhibitors such as AZD1152, PH739358, VX-680, MLN8054, R763, MP235, MP529, VX-528, AX39459; cyclin dependent kinase inhibitors such as CDK2 and/or CDK4 inhibitors; inhibitors of survival signaling proteins including Bcl-2, Bcl-XL, such as ABT-737; HSP90 inhibitors; therapeutic monoclonal antibodies, such as anti-EGFR mAbs cetuximab, panitumumab, nimotuzumab, anti-ERBB2 mAbs trastuzumab, pertuzumab, anti-CD20 mAbs such as rituximab, ofatumumab, veltuzumab and mAbs targeting other tumor antigens such as alemtuzumab, labetuzumab, adecatumumab, oregovomab, onartuzumab; TRAIL pathway agonists, such as dulanermin (soluble rhTRAIL), apomab, mapatumumab, lexatumumab, conatumumab, tigatuzumab; antibody fragments, bi-specific antibodies and bi-specific T-cell engagers (BiTEs), such as catumaxomab, blinatumomab; antibody drug conjugates (ADC) and other immunoconjugates, such as ibritumomab triuxetan, tositumomab, brentuximab vedotin, gemtuzumab ozogamicin, clivatuzumab tetraxetan, pemtumomab, trastuzumab emtansine; anti-angiogenic therapy such as bevacizumab, etaracizumab, volociximab, ramucirumab, aflibercept, sorafenib, sunitinib, regorafenib, axitinib, nintedanib, motesanib, pazopanib, cediranib; metalloproteinase inhibitors such as marimastat; inhibitors of urokinase plasminogen activator receptor function; inhibitors of cathepsin activity.
Other therapeutic antibodies which may optionally be used in combination with an immunostimulatory antibody according to at least some embodiments of the present invention include but are not limited to cetuximab, panitumumab, nimotuzumab, trastuzumab, pertuzumab, rituximab, ofatumumab, veltuzumab, alemtuzumab, labetuzumab, adecatumumab, oregovomab, onartuzumab; apomab, mapatumumab, lexatumumab, conatumumab, tigatuzumab, catumaxomab, blinatumomab, ibritumomab triuxetan, tositumomab, brentuximab vedotin, gemtuzumab ozogamicin, clivatuzumab tetraxetan, pemtumomab, trastuzumab emtansine, bevacizumab, etaracizumab, volociximab, ramucirumab, aflibercept.
Therapeutic agents targeting immunosuppressive cells such as Tregs and/or MDSCs which may optionally be used in combination with an immunostimulatory antibody according to at least some embodiments of the present invention include but are not limited to antimitotic drugs, cyclophosphamide, gemcitabine, mitoxantrone, fludarabine, thalidomide, thalidomide derivatives, COX-2 inhibitors, depleting or killing antibodies that directly target Tregs through recognition of Treg cell surface receptors, anti-CD25 daclizumab, basiliximab, ligand-directed toxins, denileukin diftitox (Ontak), a fusion protein of human IL-2 and diphtheria toxin, or LMB-2, a fusion between an scFv against CD25 and the pseudomonas exotoxin, antibodies targeting Treg cell surface receptors, TLR modulators, agents that interfere with the adenosinergic pathway, ectonucleotidase inhibitors, or inhibitors of the A2A adenosine receptor, TGF-β inhibitors, chemokine receptor inhibitors, retinoic acid, all-trans retinoic acid (ATRA), Vitamin D3, phosphodiesterase 5 inhibitors, sildenafil, ROS inhibitors and nitroaspirin.
Other immunostimulatory antibodies which may according to at least some embodiments optionally be used in combination with an immunostimulatory antibody according to at least some embodiments of the present invention include but are not limited to agonistic or antagonistic antibodies targeting one or more of CTLA4, PD-1, PDL-1, LAG-3, TIM-3, BTLA, B7-H4, B7-H3, VISTA, and/or agonistic or antagonistic antibodies targeting one or more of CD40, CD137, OX40, GITR, CD27, CD28 or ICOS, or fusion proteins containing any of the foregoing or fragments thereof which function as immune agonists or antagonists.
As described infra, without wishing to be limited by a single hypothesis, the HIDE1 protein apparently interacts with a receptor expressed by NK cells. Accordingly, the subject immunostimulatory antibody or immunostimulatory antigen-binding fragments may optionally be used on combination or coupled to an antibody or antigen-binding fragment thereof, or other moiety which specifically binds to an NK cell receptor. Such moieties which specifically bind to an NK cell receptor may optionally agonize or antagonize the effect of said NK cell receptor. Various non-limiting examples are given herein. Such NK receptors include those of unknown function, as well as those known to inhibit NK cell activity such as KIR2DL1, KIR2DL2/3, KIR2DL4, KIR2DL5A, KIR2DL5B, KIR3DL1, KIR3DL2, KIR3DL3, LILRB1, NKG2A, NKG2C, NKG2E and LILRB5 and those known to promote or activate NK cell activity such as NKp30, NKp44, NKp46, NKp46, NKG2D, KIR2DS4 CD2, CD16, CD69, DNAX accessory molecule-1 (DNAM-1), 2B4, NK1.1; a killer immunoglobulin (Ig)-like activating receptors (KAR); ILTs/LIRs; NKRP-1, CD69; CD94/NKG2C and CD94/NKG2E heterodimers, NKG2D homodimer KIR2DS and KIR3DS.
Therapeutic cancer vaccines may also optionally be used in combination with an immunostimulatory antibody or fragment thereof according to at least some embodiments of the present invention, including but not limited to exogenous cancer and infectious agent vaccines including proteins or peptides used to mount an immunogenic response to a tumor antigen or an infectious agent, recombinant virus and bacteria vectors encoding tumor antigens, DNA-based vaccines encoding tumor antigens, proteins targeted to dendritic cells, dendritic cell-based vaccines, whole tumor cell vaccines, gene modified tumor cells expressing GM-CSF, ICOS and/or Flt3-ligand, oncolytic virus vaccines.
Cytokines which according to at least some embodiments may be used in combination with an immunostimulatory antibody according to at least some embodiments of the present invention include but are not limited to one or more cytokines such as interferons, interleukins, colony stimulating factors, and tumor necrosis factors such as IL-2, IL-7, IL-12, IL-15, IL-17, IL-18, IL-21, IL-23, IL-27, GM-CSF, IFNα (interferon α), IFNα-2b, IFNβ, IFNγ, TNF-α, TNF-β and combinations thereof.
Adoptive cell transfer therapy according to at least some embodiments that may optionally be used in combination with an immunostimulatory antibody according to at least some embodiments of the present invention include but are not limited to an ex vivo treatment selected from expansion of the patient autologous naturally occurring tumor specific T cells or genetic modification of T cells to confer specificity for tumor antigens.
a. Treatment of Pathogen Infections
According to at least some embodiments, anti-HIDE1 antibodies may optionally be used for treating infectious disease, for the same reasons that cancer can be treated: chronic infections are often characterized by varying degrees of functional impairment of virus-specific T-cell responses, and this defect is a principal reason for the inability of the host to eliminate the persisting pathogen. Although functional effector T cells are initially generated during the early stages of infection, they gradually lose function during the course of the chronic infection as a result of persistent exposure to foreign antigen, giving rise to T cell exhaustion. Exhausted T cells express high levels of multiple co-inhibitory receptors such as CTLA-4, PD-1, and LAG3 (Crawford et al., Curr Opin Immunol. 2009; 21:179-186; Kaufmann et al., J Immunol 2009; 182:5891-5897, Sharpe et al., Nat Immunol 2007; 8:239-245). PD-1 overexpression by exhausted T cells was observed clinically in patients suffering from chronic viral infections including HIV, HCV and HBV (Crawford et al., Curr Opin Immunol 2009; 21:179-186; Kaufmann et al., J Immunol 2009; 182:5891-5897, Sharpe et al., Nat Immunol 2007; 8:239-245). There has been some investigation into this pathway in additional pathogens, including other viruses, bacteria, and parasites (Hofmeyer et al., J Biomed Biotechnol. Vol 2011, Art. ID 451694, Bhadra et al., Proc Natl. Acad Sci. 2011; 108(22):9196-201). For example, the PD-1 pathway was shown to be involved in controlling bacterial infection using a sepsis model induced by the standard cecal ligation and puncture method. The absence of PD-1 in knockout mice protected from sepsis-induced death in this model (Huang et al., PNAS 2009: 106; 6303-6308).
T cell exhaustion can be reversed by blocking co-inhibitory pathways such as PD-1 or CTLA-4 (Rivas et al., J Immunol. 2009; 183:4284-91; Golden-Mason et al., J Virol. 2009; 83:9122-30; Hofmeyer et al., J Biomed Biotechnol. Vol 2011, Art. ID 451694), thus allowing restoration of anti-viral immune function. The therapeutic potential of co-inhibition blockade for treating viral infection was extensively studied by blocking the PD-1/PD-L1 pathway, which was shown to be efficacious in several animal models of infection including acute and chronic simian immunodeficiency virus (SIV) infection in rhesus macaques (Valu et al., Nature 2009; 458:206-210) and in mouse models of chronic viral infection, such as lymphocytic choriomeningitis virus (LCMV) (Barber et al., Nature. 2006; 439:682-7), and Theiler's murine encephalomyelitis virus (TMEV) model in SJL/J mice (Duncan and Miller PLoS One. 2011; 6:e18548). In these models PD-1/PD-L1 blockade improved anti-viral responses and promoted clearance of the persisting viruses. In addition, PD-1/PD-L1 blockade increased the humoral immunity manifested as elevated production of specific anti-virus antibodies in the plasma, which in combination with the improved cellular responses leads to decrease in plasma viral loads and increased survival.
As used herein the term “infectious disorder and/or disease” and/or “infection” used interchangeably, includes any disorder, disease and/or condition caused by presence and/or growth of pathogenic biological agent in an individual host organism. As used herein the term “infection” comprises the disorder, disease and/or condition as above, exhibiting clinically evident illness (i.e., characteristic medical signs and/or symptoms of disease) and/or which is asymtomatic for much or all of it course. As used herein the term “infection” also comprises disorder, disease and/or condition caused by persistence of foreign antigen that lead to exhaustion T cell phenotype characterized by impaired functionality which is manifested as reduced proliferation and cytokine production. As used herein the term “infectious disorder and/or disease” and/or “infection”, further includes any of the below listed infectious disorders, diseases and/or conditions, caused by a bacterial infection, viral infection, fungal infection and/or parasite infection.
Anti-HIDE1 antibodies can be administered alone or in combination with one or more additional therapeutic agents used for treatment of bacterial infections, viral infection, fungal infections, optionally as described herein.
That is, an infected subject is administered an anti-HIDE1 antibodies that antagonizes at least one HIDE1 mediated effect on immunity, e.g., its inhibitory effect on myeloid cell function, cytotoxic T cells or NK activity and/or its inhibitory effect on the production of proinflammatory cytokines, or inhibits the stimulatory effect of HIDE1 on TRegs thereby prompting the depletion or killing of the infected cells or the pathogen, and potentially resulting in disease remission based on enhanced killing of the pathogen or infected cells by the subject's immune cells.
In at least some embodiments, the present invention provides at least on of the HIDE1-specific immunostimulatory antibody, antigen-binding fragment or conjugate or composition containing according to at least some embodiments of the present invention, pharmaceutical compositions, and/or uses thereof for treatment and/or diagnosis of infectious disease, wherein said infectious disease is e.g., a disease caused by bacterium, virus, fungus or yeast, mycoplasm or a parasite or sepsis associated therewith.
As used herein the term “viral infection” comprises any infection caused by a virus, optionally including but not limited to Retroviridae (e.g., human immunodeficiency viruses, such as HIV-1 or HIV-2, acquired immune deficiency (AIDS) also referred to as HTLV-III, LAV or HTLV-III/LAV, or HIV-III; and other isolates, such as HIV-LP; Picornaviridae (e.g., polio viruses, hepatitis A virus; enteroviruses, human coxsackie viruses, rhinoviruses, echoviruses); Calciviridae (e.g., strains that cause gastroenteritis); Togaviridae (e.g., equine encephalitis viruses, rubella viruses); Flaviridae (e.g., dengue viruses, encephalitis viruses, yellow fever viruses); Coronaviridae (e.g., coronaviruses); Rhabdoviridae (e.g., vesicular stomatitis viruses, rabies viruses); Filoviridae (e.g., ebola viruses); Paramyxoviridae (e.g., parainfluenza viruses, mumps virus, measles virus, respiratory syncytial virus); Orthomyxoviridae (e.g., influenza viruses); Bungaviridae (e.g., Hantaan viruses, bunga viruses, phleboviruses and Nairo viruses); Arena viridae (hemorrhagic fever virus); Reoviridae (e.g., reoviruses, orbiviruses and rotaviruses); Birnaviridae; Hepadnaviridae (Hepatitis B virus); Parvoviridae (parvoviruses); Papovaviridae (papilloma viruses, polyoma viruses); Adenoviridae (most adenoviruses); Herpesviridae (herpes simplex virus (HSV) 1 and 2, varicella zoster virus, cytomegalovirus (CMV), herpes viruses); Poxviridae (variola viruses, vaccinia viruses, pox viruses); and Iridoviridae (e.g., African swine fever virus); and unclassified viruses (e.g., the etiological agents of Spongiform encephalopathies, the agent of delta hepatitides (thought to be a defective satellite of hepatitis B virus), the agents of non-A, non-B hepatitis (class 1—internally transmitted; class 2—parenterally transmitted (i.e., Hepatitis C); Norwalk and related viruses, and astroviruses) as well as Severe acute respiratory syndrome virus and respiratory syncytial virus (RSV).
As used herein the term “fungal infection” comprises any infection caused by a fungus, optionally including but not limited to Cryptococcus neoformans, Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis, Chlamydia trachomatis, and Candida albicans.
As used herein the term “parasite infection” comprises any infection caused by a parasite, optionally including but not limited to protozoa, such as Amebae, Flagellates, Plasmodium falciparum, Toxoplasma gondii, Ciliates, Coccidia, Microsporidia, Sporozoa; helminthes, Nematodes (Roundworms), Cestodes (Tapeworms), Trematodes (Flukes), Arthropods, and aberrant proteins known as prions.
An infectious disorder and/or disease caused by bacteria may optionally comprise one or more of Sepsis, septic shock, sinusitis, skin infections, pneumonia, bronchitis, meningitis, Bacterial vaginosis, Urinary tract infection (UCI), Bacterial gastroenteritis, Impetigo and erysipelas, Erysipelas, Cellulitis, anthrax, whooping cough, lyme disease, Brucellosis, enteritis, acute enteritis, Tetanus, diphtheria, Pseudomembranous colitis, Gas gangrene, Acute food poisoning, Anaerobic cellulitis, Nosocomial infections, Diarrhea, Meningitis in infants, Traveller's diarrhea, Hemorrhagic colitis, Hemolytic-uremic syndrome, Tularemia, Peptic ulcer, Gastric and Duodenal ulcers, Legionnaire's Disease, Pontiac fever, Leptospirosis, Listeriosis, Leprosy (Hansen's disease), Tuberculosis, Gonorrhea, Ophthalmia neonatorum, Septic arthritis, Meningococcal disease including meningitis, Waterhouse-Friderichsen syndrome, pseudomonas infection, Rocky Mountain spotted fever, Typhoid fever type salmonellosis, Salmonellosis with gastroenteritis and enterocolitis, Bacillary dysentery/Shigellosis, Coagulase-positive staphylococcal infections: Localized skin infections including Diffuse skin infection (Impetigo), Deep localized infections, Acute infective endocarditis, Septicemia, Necrotizing pneumonia, Toxinoses such as Toxic shock syndrome and Staphylococcal food poisoning, Cystitis, Endometritis, Otitis media, Streptococcal pharyngitis, Scarlet fever, Rheumatic fever, Puerperal fever, Necrotizing fasciitis, Cholera, Plague (including Bubonic plague and Pneumonic plague), as well as any infection caused by a bacteria selected from but not limited to Helicobacter pyloris, Boreliai burgdorferi, Legionella pneumophila, Mycobacteria sps (e.g., M. tuberculosis, M. avium, M. Intracellulare, M. kansaii, M. gordonae), Staphylococcus aureus, Neisseria gonorrhoeae, Neisseria meningitidis, Listeria monocytogenes, Streptococcus pyogenes (Group A Streptococcus), Streptococcus agalactiae (Group B Streptococcus), Streptococcus (viridans group), Streptococcus faecalis, Streptococcus bovis, Streptococcus (anaerobic sps.), Streptococcus pneumoniae, pathogenic Campylobacter sp., Enterococcus sp., Haemophilus influenzae, Bacillus anthracis, corynebacterium diphtherias, corynebacterium sp., Erysipelothrix rhusiopathiae, Clostridium perfringens, Clostridium tetani, Enterobacter aerogenes, Klebsiella pneumoniae, Pasteurella multocida, Bacteroides sp., Fusobacterium nucleatum, Streptobacillus moniliformis, Treponema pallidum, Treponema pertenue, Leptospira, and Actinomyces israelii.
Non limiting examples of infectious disorder and/or disease caused by virus is selected from the group consisting of but not limited to acquired immune deficiency (AIDS), West Nile encephalitis, coronavirus infection, rhinovirus infection, influenza, dengue, hemorrhagic fever; an otological infection; severe acute respiratory syndrome (SARS), acute febrile pharyngitis, pharyngoconjunctival fever, epidemic keratoconjunctivitis, infantile gastroenteritis, infectious mononucleosis, Burkitt lymphoma, acute hepatitis, chronic hepatitis, hepatic cirrhosis, hepatocellular carcinoma, primary HSV-1 infection, (gingivostomatitis in children, tonsillitis & pharyngitis in adults, keratoconjunctivitis), latent HSV-1 infection (herpes labialis, cold sores), aseptic meningitis, Cytomegalovirus infection, Cytomegalic inclusion disease, Kaposi sarcoma, Castleman disease, primary effusion lymphoma, influenza, measles, encephalitis, postinfectious encephalomyelitis, mumps, hyperplastic epithelial lesions (common, flat, plantar and anogenital warts, laryngeal papillomas, epidermodysplasia verruciformis), croup, pneumonia, bronchiolitis, Poliomyelitis, Rabies, bronchiolitis, pneumonia, German measles, congenital rubella, Hemorrhagic Fever, Chickenpox, Dengue, Ebola infection, Echovirus infection, EBV infection, Fifth Disease, Filovirus, Flavivirus, Hand, foot and mouth disease, Herpes Zoster Virus (Shingles), Human Papilloma Virus Associated Epidermal Lesions, Lassa fever, Lymphocytic choriomeningitis, Parainfluenza Virus Infection, Paramyxovirus, Parvovirus B19 Infection, Picornavirus, Poxviruses infection, Rotavirus diarrhea, Rubella, Rubeola, Varicella, Variola infection.
An infectious disorder and/or disease caused by fungi optionally includes but is not limited to allergic bronchopulmonary aspergillosis, Aspergilloma, Aspergillosis, Basidiobolomycosis, Blastomycosis, Candidiasis, Chronic pulmonary aspergillosis, Chytridiomycosis, Coccidioidomycosis, Conidiobolomycosis, covered smut (barley), Cryptococcosis, Dermatophyte, Dermatophytid, Dermatophytosis, Endothrix, Entomopathogenic fungus, Epizootic lymphangitis, Epizootic ulcerative syndrome, Esophageal candidiasis, Exothrix, Fungemia, Histoplasmosis, Lobomycosis, Massospora cicadina, Mycosis, Mycosphaerella fragariae, Myringomycosis, Paracoccidioidomycosis, Pathogenic fungi, Penicilliosis, Thousand cankers disease, Tinea, Zeaspora, Zygomycosis. Non limiting examples of infectious disorder and/or disease caused by parasites is selected from the group consisting of but not limited to Acanthamoeba, Amoebiasis, Ascariasis, Ancylostomiasis, Anisakiasis, Babesiosis, Balantidiasis, Baylisascariasis, Blastocystosis, Candiru, Chagas disease, Clonorchiasis, Cochliomyia, Coccidia, Chinese Liver Fluke Cryptosporidiosis, Dientamoebiasis, Diphyllobothriasis, Dioctophyme renalis infection, Dracunculiasis, Echinococcosis, Elephantiasis, Enterobiasis, Fascioliasis, Fasciolopsiasis, Filariasis, Giardiasis, Gnathostomiasis, Hymenolepiasis, Halzoun Syndrome, Isosporiasis, Katayama fever, Leishmaniasis, lymphatic filariasis, Malaria, Metagonimiasis, Myiasis, Onchocerciasis, Pediculosis, Primary amoebic meningoencephalitis, Parasitic pneumonia, Paragonimiasis, Scabies, Schistosomiasis, Sleeping sickness, Strongyloidiasis, Sparganosis, Rhinosporidiosis, River blindness, Taeniasis (cause of Cysticercosis), Toxocariasis, Toxoplasmosis, Trichinosis, Trichomoniasis, Trichuriasis, Trypanosomiasis, and Tapeworm infection.
Some optional but particular examples of infectious disease include a disease caused by any of hepatitis B, hepatitis C, infectious mononucleosis, EBV, cytomegalovirus, AIDS, HIV-1, HIV-2, tuberculosis, malaria and schistosomiasis.
The therapeutic agents and/or a pharmaceutical composition comprising same, as recited herein, can be administered in combination with one or more additional therapeutic agents used for treatment of bacterial infections, including, but not limited to, antibiotics including Aminoglycosides, Carbapenems, Cephalosporins, Macrolides, Lincosamides, Nitrofurans, penicillins, Polypeptides, Quinolones, Sulfonamides, Tetracyclines, drugs against mycobacteria including but not limited to Clofazimine, Cycloserine, Cycloserine, Rifabutin, Rifapentine, Streptomycin and other antibacterial drugs such as Chloramphenicol, Fosfomycin, Metronidazole, Mupirocin, and Tinidazole.
The therapeutic agents and/or a pharmaceutical composition comprising same, as recited herein, can be administered in combination with one or more additional therapeutic agents used for treatment of viral infections, including, but not limited to, antiviral drugs such as oseltamivir (brand name Tamiflu®) and zanamivir (brand name Relenza®) Arbidol®-adamantane derivatives (Amantadine®, Rimantadine®)—neuraminidase inhibitors (Oseltamivir®, Laninamivir®, Peramivir®, Zanamivir®) nucleotide analog reverse transcriptase inhibitor including Purine analogue guanine (Aciclovir®/Valacyclovir®, Ganciclovir®/Valganciclovir®, Penciclovir®/Famciclovir®) and adenine (Vidarabine®), Pyrimidine analogue, uridine (Idoxuridine®, Trifluridine®, Edoxudine®), thymine (Brivudine®), cytosine (Cytarabine®); Foscarnet; nucleoside analogues/NARTIs: Entecavir, Lamivudine®, Telbivudine®, Clevudine®; nucleotide analogues/NtRTIs: Adefovir®, Tenofovir; nucleic acid inhibitors such as Cidofovir®; InterferonInterferon alfa-2b, Peginterferon α-2a; Ribavirin®/Taribavirin®; antiretroviral drugs including zidovudine, lamivudine, abacavir, lopinavir, ritonavir, tenofovir/emtricitabine, efavirenz each of them alone or a various combinations, gp41 (Enfuvirtide), Raltegravir®, protease inhibitors such as Fosamprenavir®, Lopinavir® and Atazanavir®, Methisazone®, Docosanol®, Fomivirsen®, and Tromantadine®.
The therapeutic agents and/or a pharmaceutical composition comprising same, as recited herein, can be administered in combination with one or more additional therapeutic agents used for treatment of fungal infections, including, but not limited to, antifungal drugs of the Polyene antifungals, Imidazole, triazole, and thiazole antifungals, Allylamines, Echinocandins or other anti-fungal drugs.
b. Treatment of Sepsis
According to at least some embodiments, anti-HIDE1 antibodies be used for treating sepsis. As used herein, the term “sepsis” or “sepsis related condition” encompasses Sepsis, Severe sepsis, Septic shock, Systemic inflammatory response syndrome (SIRS), Bacteremia, Septicemia, Toxemia, Septic syndrome.
Upregulation of inhibitory proteins has lately emerged as one of the critical mechanisms underlying the immunosuppression in sepsis. The PD-1/PDL-1 pathway, for example, appears to be a determining factor of the outcome of sepsis, regulating the delicate balance between effectiveness and damage by the antimicrobial immune response. During sepsis in an experimental model, peritoneal macrophages and blood monocytes markedly increased PD-1 levels, which was associated with the development of cellular dysfunction (Huang et al 2009 PNAS 106:6303-6308). Similarly, in patients with septic shock the expression of PD-1 on peripheral T cells and of PDL-1 on monocytes was dramatically upregulated (Zhang et al 2011 Crit. Care 15:R70). Recent animal studies have shown that blockade of the PD-1/PDL-1 pathway by anti-PD1 or anti-PDL1 antibodies improved survival in sepsis (Brahmamdam et al 2010 J. Leukoc. Biol. 88:233-240; Zhang et al 2010 Critical Care 14:R220; Chang et al 2013 Critical Care 17:R85). Similarly, blockade of CTLA-4 with anti-CTLA4 antibodies improved survival in sepsis (Inoue et al 2011 Shock 36:38-44; Chang et al 2013 Critical Care 17:R85). Taken together, these findings suggest that blockade of inhibitory proteins, including negative costimulatory molecules, is a potential therapeutic approach to prevent the detrimental effects of sepsis (Goyert and Silver, J Leuk. Biol., 88(2): 225-226, 2010).
According to some embodiments, the invention provides treatment of sepsis using anti-HIDE1 antibodies, either alone or in combination with known therapeutic agent effective for treating sepsis, such as those therapies that block the cytokine storm in the initial hyperinflammatory phase of sepsis, and/or with therapies that have immunostimulatory effect in order to overcome the sepsis-induced immunosuppression phase.
Combination with standard of care treatments for sepsis, as recommended by the “International Guidelines for Management of Severe Sepsis and Septic Shock” (Dellinger et al 2013 Intensive Care Med 39:165-228), some of which are described below.
Optionally the sepsis is selected from sepsis, severe sepsis, septic shock, systemic inflammatory response syndrome (SIRS), bacteremia, septicemia, toxemia, and septic syndrome.
Optionally the treatment is combined with another moiety useful for treating sepsis.
According to at least some embodiments there is provided a diagnostic method for determining whether to perform the use or to administer an antibody composition as described herein, comprising performing the diagnostic method as described herein.
According to at least some embodiments of the present invention, there is provided use of a combination of the therapeutic agents and/or a pharmaceutical composition comprising same, as recited herein, can be combined with standard of care or novel treatments for sepsis, with therapies that block the cytokine storm in the initial hyperinflammatory phase of sepsis, and/or with therapies that have immunostimulatory effect in order to overcome the sepsis-induced immunosuppression phase.
Combination with standard of care treatments for sepsis, as recommended by the “International Guidelines for Management of Severe Sepsis and Septic Shock” (Dellinger et al 2013 Intensive Care Med 39:165-228), some of which are described below.
Broad spectrum antibiotics having activity against all likely pathogens (bacterial and/or fungal—treatment starts when sepsis is diagnosed, but specific pathogen is not identified)—example Cefotaxime (Claforan®), Ticarcillin and clavulanate (Timentin®), Piperacillin and tazobactam (Zosyn®), Imipenem and cilastatin (Primaxin®), Meropenem (Merrem®), Clindamycin (Cleocin), Metronidazole (Flagyl®), Ceftriaxone (Rocephin®), Ciprofloxacin (Cipro®), Cefepime (Maxipime®), Levofloxacin (Levaquin®), Vancomycin or any combination of the listed drugs.
Vasopressors: example Norepinephrine, Dopamine, Epinephrine, vasopressin
Steroids: example: Hydrocortisone, Dexamethasone, or Fludrocortisone, intravenous or otherwise
Inotropic therapy: example Dobutamine for sepsis patients with myocardial dysfunction
Recombinant human activated protein C (rhAPC), such as drotrecogin alfa (activated) (DrotAA).
β-blockers additionally reduce local and systemic inflammation.
Metabolic interventions such as pyruvate, succinate or high dose insulin substitutions.
Combination with novel potential therapies for sepsis:
Selective inhibitors of sPLA2-IIA (such as LY315920NA/S-5920). Rationale: The Group IIA secretory phospholipase A2 (sPLA2-IIA), released during inflammation, is increased in severe sepsis, and plasma levels are inversely related to survival.
Phospholipid emulsion (such as GR270773). Rationale: Preclinical and ex vivo studies show that lipoproteins bind and neutralize endotoxin, and experimental animal studies demonstrate protection from septic death when lipoproteins are administered. Endotoxin neutralization correlates with the amount of phospholipid in the lipoprotein particles.
anti-TNF-α antibody: Rationale: Tumor necrosis factor-α (TNF-α) induces many of the pathophysiological signs and symptoms observed in sepsis
anti-CD14 antibody (such as IC14). Rationale: Upstream recognition molecules, like CD14, play key roles in the pathogenesis. Bacterial cell wall components bind to CD14 and co-receptors on myeloid cells, resulting in cellular activation and production of proinflammatory mediators. An anti-CD14 monoclonal antibody (IC14) has been shown to decrease lipopolysaccharide-induced responses in animal and human models of endotoxemia.
Inhibitors of Toll-like receptors (TLRs) and their downstream signaling pathways. Rationale: Infecting microbes display highly conserved macromolecules (e.g., lipopolysaccharides, peptidoglycans) on their surface. When these macromolecules are recognized by pattern-recognition receptors (called Toll-like receptors [TLRs]) on the surface of immune cells, the host's immune response is initiated. This may optionally contribute to the excess systemic inflammatory response that characterizes sepsis. Inhibition of several TLRs is being evaluated as a potential therapy for sepsis, in particular TLR4, the receptor for Gram-negative bacteria outer membrane lipopolysaccharide or endotoxin. Various drugs targeting TLR4 expression and pathway have a therapeutic potential in sepsis (Wittebole et al 2010 Mediators of Inflammation Vol 10 Article ID 568396). Among these are antibodies targeting TLR4, soluble TLR4, Statins (such as Rosuvastatin®, Simvastatin®), Ketamine, nicotinic analogues, eritoran (E5564), resatorvid (TAK242). In addition, antagonists of other TLRs such as chloroquine, inhibition of TLR-2 with a neutralizing antibody (anti-TLR-2).
Lansoprazole through its action on SOCS1 (suppressor of cytokine secretion)
Talactoferrin or Recombinant Human Lactoferrin. Rationale: Lactoferrin is a glycoprotein with anti-infective and anti-inflammatory properties found in secretions and immune cells. Talactoferrin alfa, a recombinant form of human lactoferrin, has similar properties and plays an important role in maintaining the gastrointestinal mucosal barrier integrity. Talactoferrin showed efficacy in animal models of sepsis, and in clinical trials in patients with severe sepsis (Guntupalli et al Crit Care Med. 2013; 41(3):706-716).
Milk fat globule EGF factor VIII (MFG-E8)—a bridging molecule between apoptotic cells and phagocytes, which promotes phagocytosis of apoptotic cells.
Agonists of the ‘cholinergic anti-inflammatory pathway’, such as nicotine and analogues. Rationale: Stimulating the vagus nerve reduces the production of cytokines, or immune system mediators, and blocks inflammation. This nerve “circuitry”, called the “inflammatory reflex”, is carried out through the specific action of acetylcholine, released from the nerve endings, on the α7 subunit of the nicotinic acetylcholine receptor (α7nAChR) expressed on macrophages, a mechanism termed ‘the cholinergic anti-inflammatory pathway’. Activation of this pathway via vagus nerve stimulation or pharmacologic α7 agonists prevents tissue injury in multiple models of systemic inflammation, shock, and sepsis (Matsuda et al 2012 J Nippon Med Sch. 79:4-18; Huston 2012 Surg. Infect. 13:187-193).
Therapeutic agents targeting the inflammasome pathways. Rationale: The inflammasome pathways greatly contribute to the inflammatory response in sepsis, and critical elements are responsible for driving the transition from localized inflammation to deleterious hyperinflammatory host response (Cinel and Opal 2009 Crit. Care Med. 37:291-304; Matsuda et al 2012 J Nippon Med Sch. 79:4-18).
Stem cell therapy. Rationale: Mesenchymal stem cells (MSCs) exhibit multiple beneficial properties through their capacity to home to injured tissue, activate resident stem cells, secrete paracrine signals to limit systemic and local inflammatory response, beneficially modulate immune cells, promote tissue healing by decreasing apoptosis in threatened tissues and stimulating neoangiogenesis, and exhibit direct antimicrobial activity. These effects are associated with reduced organ dysfunction and improved survival in sepsis animal models, which have provided evidence that MSCs may optionally be useful therapeutic adjuncts (Wannemuehler et al 2012 J. Surg. Res. 173:113-26).
Combination of anti-HIDE1 antibody with other immunomodulatory agents, such as immunostimulatory antibodies, cytokine therapy, immunomodulatory drugs. Such agents bring about increased immune responsiveness, especially in situations in which immune defenses (whether innate and/or adaptive) have been degraded, such as in sepsis-induced hypoinflammatory and immunosuppressive condition. Reversal of sepsis-induced immunoparalysis by therapeutic agents that augments host immunity may optionally reduce the incidence of secondary infections and improve outcome in patients who have documented immune suppression (Hotchkiss et al 2013 Lancet Infect. Dis. 13:260-268; Payen et al 2013 Crit Care. 17:118).
Immunostimulatory antibodies promote immune responses by directly modulating immune functions, i.e. blocking other inhibitory proteins or by enhancing costimulatory proteins. Experimental models of sepsis have shown that immunostimulation by antibody blockade of inhibitory proteins, such as PD-1, PDL-1 or CTLA-4 improved survival in sepsis (Brahmamdam et al 2010 J. Leukoc. Biol. 88:233-240; Zhang et al 2010 Critical Care 14:R220; Chang et al 2013 Critical Care 17:R85; Inoue et al 2011 Shock 36:38-44), pointing to such immunostimulatory agents as potential therapies for preventing the detrimental effects of sepsis-induced immunosuppression (Goyert and Silver J Leuk. Biol. 88(2):225-226, 2010). Immunostimulatory antibodies include: 1) Antagonistic antibodies targeting inhibitory immune checkpoints include anti-CTLA4 mAbs (such as ipilimumab, tremelimumab), Anti-PD-1 (such as nivolumab BMS-936558/MDX-1106/ONO-4538, AMP224, CT-011, lambrozilumab MK-3475), Anti-PDL-1 antagonists (such as BMS-936559/MDX-1105, MEDI4736, RG-7446/MPDL3280A); Anti-LAG-3 such as IMP-321), Anti-TIM-3, Anti-BTLA, Anti-B7-H4, Anti-B7-H3, anti-VISTA. 2) Agonistic antibodies enhancing immunostimulatory proteins include Anti-CD40 mAbs (such as CP-870,893, lucatumumab, dacetuzumab), Anti-CD137 mAbs (such as BMS-663513 urelumab, PF-05082566), Anti-OX40 mAbs (such as Anti-OX40), Anti-GITR mAbs (such as TRX518), Anti-CD27 mAbs (such as CDX-1127), and Anti-ICOS mAbs.
Cytokines which directly stimulate immune effector cells and enhance immune responses can be used in combination with anti-GEN antibody for sepsis therapy: IL-2, IL-7, IL-12, IL-15, IL-17, IL-18 and IL-21, IL-23, IL-27, GM-CSF, IFNα (interferon α), IFNβ, IFNγ. Rationale: Cytokine-based therapies embody a direct attempt to stimulate the patient's own immune system. Experimental models of sepsis have shown administration of cytokines, such as IL-7 and IL-15, promote T cell viability and result in improved survival in sepsis (Unsinger et al 2010 J Immunol. 184:3768-3779; Inoue et al 2010 J Immunol. 184:1401-1409). Interferon-γ (IFNγ) reverses sepsis-induced immunoparalysis of monocytes in vitro. An in vivo study showed that IFNγ partially reverses immunoparalysis in vivo in humans. IFNγ and granulocyte-macrophage colony-stimulating factor (GM-CSF) restore immune competence of ex vivo stimulated leukocytes of patients with sepsis (Mouktaroudi et al Crit Care. 2010; 14: P17; Leentjens et al Am J Respir Crit Care Med Vol 186, pp 838-845, 2012).
Immunomodulatory drugs such as thymosin al. Rationale: Thymosin a 1 (Tal) is a naturally occurring thymic peptide which acts as an endogenous regulator of both the innate and adaptive immune systems. It is used worldwide for treating diseases associated with immune dysfunction including viral infections such as hepatitis B and C, certain cancers, and for vaccine enhancement. Notably, recent development in immunomodulatory research has indicated the beneficial effect of Tal treatment in septic patients (Wu et al. Critical Care 2013, 17:R8).
In the above-described sepsis therapies preferably a subject with sepsis or at risk of developing sepsis because of a virulent infection, e.g., one resistant to antibiotics or other drugs, will be administered an immunostimulatory anti-HIDE1 antibody or antigen-binding fragment according to at least some embodiments of the present invention, which antibody antagonizes at least one HIDE1 mediated effect on immunity, e.g., its inhibitory effect on myeloid cell function, cytotoxic T cells or NK activity and/or its inhibitory effect on the production of proinflammatory cytokines, or inhibits the stimulatory effect of HIDE1 on TRegs thereby promoting the depletion or killing of the infected cells or the pathogen, and potentially resulting in disease remission based on enhanced killing of the pathogen or infected cells by the subject's endogenous immune cells. Because sepsis may optionally rapidly result in organ failure, in this embodiment it may optionally be beneficial to administer anti-HIDE1 antibody fragments such as Fabs rather than intact antibodies as they may optionally reach the site of sepsis and infection quicker than intact antibodies. In such treatment regimens antibody half-life may optionally be of lesser concern due to the sometimes rapid morbidity of this disease.
c. Treatment of Autoimmune Diseases
According to at least some embodiments, HIDE1 therapeutic agents and/or a pharmaceutical composition comprising same, as described herein, which function as HIDE1 agonizing therapeutic agents, may optionally be used for treating an immune system related disease. In some instances the immune system related condition comprises an immune related condition, including but not limited to autoimmune, inflammatory or allergic diseases such as recited herein, transplant rejection and graft versus host disease.
In some instances the immune condition is selected from autoimmune disease, inflammatory disease, allergic disease, transplant rejection, undesired gene or cell therapy immune responses, or graft versus host disease.
In some embodiments the treatment is combined with another moiety useful for treating immune related condition. Non limiting examples thereof include immunosuppressants such as corticosteroids, cyclosporin, cyclophosphamide, prednisone, azathioprine, methotrexate, rapamycin, tacrolimus, leflunomide or an analog thereof; mizoribine; mycophenolic acid; mycophenolate mofetil; 15-deoxyspergualine or an analog thereof; biological agents such as TNF-α blockers or antagonists, or any other biological agent targeting any inflammatory cytokine, nonsteroidal antiinflammatory drugs/Cox-2 inhibitors, hydroxychloroquine, sulphasalazopryine, gold salts, etanercept, infliximab, mycophenolate mofetil, basiliximab, atacicept, rituximab, cytoxan, interferon β-1a, interferon β-1b, glatiramer acetate, mitoxantrone hydrochloride, anakinra and/or other biologics and/or intravenous immunoglobulin (IVIG), interferons such as IFN-β1a (REBIF®. AVONEX® and CINNOVEX®) and IFN-β1b (BETASERON®); EXTAVIA®, BETAFERON®, ZIFERON®); glatiramer acetate (COPAXONE®), a polypeptide; natalizumab (TYSABRI®), mitoxantrone (NOVANTRONE®), a cytotoxic agent, a calcineurin inhibitor, e.g. Cyclosporin A or FK506; an immunosuppressive macrolide, e.g. Rapamycin or a derivative thereof; e.g. 40—O-(2-hydroxy)ethyl-rapamycin, a lymphocyte homing agent, e.g. FTY720 or an analog thereof, corticosteroids; cyclophosphamide; azathioprene; methotrexate; leflunomide or an analog thereof; mizoribine; mycophenolic acid; mycophenolate mofetil; 15-deoxyspergualine or an analog thereof; immunosuppressive monoclonal antibodies, e.g., monoclonal antibodies to leukocyte receptors, e.g., MHC, CD2, CD3, CD4, CD11a/CD18, CD7, CD25, CD27, B7, CD40, CD45, CD58, CD137, ICOS, CD150 (SLAM), OX40, 4-1BB or their ligands; or other immunomodulatory compounds, e.g. CTLA4-Ig (abatacept, ORENCIA®, belatacept), CD28-Ig, B7-H4-Ig, or other costimulatory agents, or adhesion molecule inhibitors, e.g. mAbs or low molecular weight inhibitors including LFA-1 antagonists, Selectin antagonists and VLA-4 antagonists, or another immunomodulatory agent.
In particular, treatment of multiple sclerosis using HIDE1 immunoinhibitory proteins according to some embodiments of the present invention may optionally e.g., be combined with, any therapeutic agent or method suitable for treating multiple sclerosis. Non-limiting examples of such known therapeutic agent or method for treating multiple sclerosis include interferon class, IFN-β-1a (REBIF®. AVONEX® and CINNOVEX®) and IFN-β-1b (BETASERON®, EXTAVIA®, BETAFERON®, ZIFERON®); glatiramer acetate (COPAXONE®), a polypeptide; natalizumab (TYSABRI®); and mitoxantrone (NOVANTRONE®), a cytotoxic agent, Fampridine (AMPYRA®). Other drugs include corticosteroids, methotrexate, cyclophosphamide, azathioprine, and intravenous immunoglobulin (IVIG), inosine, Ocrelizumab (R1594), Mylinax (Caldribine®), alemtuzumab (Campath®), daclizumab (Zenapax®), Panaclar/dimethyl fumarate (BG-12), Teriflunomide (HMR1726), fingolimod (FTY720), laquinimod (ABR216062), as well as Hematopoietic stem cell transplantation, NeuroVax®, Rituximab (Rituxan®) BCG vaccine, low dose naltrexone, helminthic therapy, angioplasty, venous stents, and alternative therapy, such as vitamin D, polyunsaturated fats, medical marijuana.
Similarly, treatment of rheumatoid arthritis, using HIDE1 immunoinhibitory proteins according to some embodiments of the present invention may optionally be combined with, for example, any therapeutic agent or method suitable for treating rheumatoid arthritis. Non-limiting examples of such known therapeutic agents or methods for treating rheumatoid arthritis include glucocorticoids, nonsteroidal anti-inflammatory drug (NSAID) such as salicylates, or cyclooxygenase-2 inhibitors, ibuprofen and naproxen, diclofenac, indomethacin, etodolac Disease-modifying antirheumatic drugs (DMARDs)-Oral DMARDs: Auranofin (Ridaura®), Azathioprine (Imuran®), Cyclosporine (Sandimmune®, Gengraf, Neoral, generic), D-Penicillamine (Cuprimine), Hydroxychloroquine (Plaquenil®), IM gold Gold sodium thiomalate (Myochrysine®) Aurothioglucose (Solganal®), Leflunomide (Arava®), Methotrexate (Rheumatrex®), Minocycline (Minocin®), Staphylococcal protein A immunoadsorption (Prosorba column), Sulfasalazine (Azulfidine®). Biologic DMARDs: TNF-α blockers including Adalimumab (Humira®) Etanercept (Enbrel®), Infliximab (Remicade®), golimumab (Simponi®), certolizumab pegol (Cimzia®), and other biological DMARDs, such as Anakinra (Kineret®), Rituximab (Rituxan®), Tocilizumab (Actemra®), CD28 inhibitor including Abatacept (Orencia®) and Belatacept.
Thus, treatment of IBD, using the agents according to at least some embodiments of the present invention may optionally be combined with, for example, any known therapeutic agent or method for treating IBD. Non-limiting examples of such known therapeutic agents or methods for treating IBD include immunosuppression to control the symptom, such as prednisone, Mesalazine (including Asacol®, Pentasa®, Lialda®, Aspiro®, azathioprine (Imuran®), methotrexate, or 6-mercaptopurine, steroids, Ondansetron®, TNF-α blockers (including infliximab, adalimumab golimumab, certolizumab pegol), Orencia® (abatacept), ustekinumab (Stelara®), Briakinumab (ABT-874), Certolizumab pegol (Cimzia®), ITF2357 (Givinostat®), Natalizumab (Tysabri®), Firategrast® (SB-683699), Remicade® (infliximab), vedolizumab (MLN0002), other drugs including GSK1605786 CCX282-B (Traficet-EN®), AJM300, Stelara® (ustekinumab), Semapimod® (CNI-1493) tasocitinib (CP-690550), LMW Heparin MMX, Budesonide MMX, Simponi® (golimumab), MultiStem®, Gardasil® HPV vaccine, Epaxal® (virosomal hepatitis A vaccine), surgery, such as bowel resection, strictureplasty or a temporary or permanent colostomy or ileostomy; antifungal drugs such as nystatin (a broad spectrum gut antifungal) and either itraconazole (Sporanox) or fluconazole (Diflucan); alternative medicine, prebiotics and probiotics, cannabis, Helminthic therapy or ova of the Trichuris suis helminth.
Thus, treatment of psoriasis, using the agents according to at least some embodiments of the present invention may optionally be combined with, for example, any known therapeutic agent or method for treating psoriasis. Non-limiting examples of such known therapeutics for treating psoriasis include topical agents, typically used for mild disease, phototherapy for moderate disease, and systemic agents for severe disease. Non-limiting examples of topical agents: bath solutions and moisturizers, mineral oil, and petroleum jelly; ointment and creams containing coal tar, dithranol (anthralin), corticosteroids like desoximetasone (Topicort), Betamethasone, fluocinonide, vitamin D3 analogues (for example, calcipotriol), and retinoids. Non-limiting examples of phototherapy: sunlight; wavelengths of 311-313 nm, psoralen and ultraviolet A phototherapy (PUVA). Non-limiting examples of systemic agents: biologics, such as interleukin antagonists, TNF-α blockers including antibodies such as infliximab (Remicade®), adalimumab (Humira®), golimumab, certolizumab pegol, and recombinant TNF-α decoy receptor, etanercept (Enbrel®); drugs that target T cells, such as efalizumab (Xannelim®/Raptiva®), alefacept (Ameviv®), dendritic cells such Efalizumab; monoclonal antibodies (MAbs) targeting cytokines, including anti-IL-12/IL-23 (ustekinumab (Stelara®)) and anti-Interleukin-17; Briakinumab® (ABT-874); small molecules, including but not limited to ISA247; immunosuppressants, such as methotrexate, cyclosporine; vitamin A and retinoids (synthetic forms of vitamin A); and alternative therapy, such as changes in diet and lifestyle, fasting periods, low energy diets and vegetarian diets, diets supplemented with fish oil rich in vitamin A and vitamin D (such as cod liver oil), fish oils rich in the two omega-3 fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) and contain vitamin E, ichthyotherapy, hypnotherapy, and cannabis.
Thus, treatment of type 1 diabetes, using the agents according to at least some embodiments of the present invention may optionally be combined with, for example, any known therapeutic agent or method for treating type 1 diabetes. Non-limiting examples of such known therapeutics for treating type 1 diabetes include insulin, insulin analogs, islet transplantation, stem cell therapy including PROCHYMAL®, non-insulin therapies such as i1-1β inhibitors including Anakinra (Kineret®), Abatacept (Orencia®), Diamyd, alefacept (Ameviv®), Otelixizumab, DiaPep277 (Hsp60 derived peptide), a 1-Antitrypsin, Prednisone, azathioprine, and Cyclosporin, E1-INT (an injectable islet neogenesis therapy comprising an epidermal growth factor analog and a gastrin analog), statins including Zocor®, Simlup®, Simcard®, Simvacor®, and Sitagliptin® (dipeptidyl peptidase (DPP-4) inhibitor), anti-CD3 mAb (e.g., Teplizumab®); CTLA4-Ig (abatacept), anti-IL-1β (Canakinumab), Anti-CD20 mAb (e.g., rituximab) and combinations thereof.
Thus, treatment of uveitis, using the agents according to at least some embodiments of the present invention may optionally be combined with, for example, any known therapeutic agent or method for treating uveitis. Non-limiting examples of such known therapeutics for treating uveitis include corticosteroids, topical cycloplegics, such as atropine or homatropine, or injection of PSTTA (posterior subtenon triamcinolone acetate), antimetabolite medications, such as methotrexate, TNF-α blockers (including infliximab, adalimumab, etanercept, golimumab, and certolizumab pegol).
Thus, treatment for Sjögren's syndrome, using the agents according to at least some embodiments of the present invention may optionally be combined with, for example, any known therapeutic agent or method for treating for Sjögren's syndrome. Non-limiting examples of such known therapeutics for treating for Sjögren's syndrome include Cyclosporine, pilocarpine (Salagen®) and cevimeline (Evoxac®), Hydroxychloroquine (Plaquenil), cortisone (prednisone and others) and/or azathioprine (Imuran®) or cyclophosphamide (Cytoxan®), Dexamethasone, Thalidomide, Dehydroepiandrosterone, NGX267, Rebamipide®, FID 114657, Etanercept®, Raptiva®, Belimumab, MabThera® (rituximab); Anakinra®, intravenous immune globulin (IVIG), Allogeneic Mesenchymal Stem Cells (AlloMSC®), and Automatic neuro-electrostimulation by “Saliwell Crown”.
Thus, treatment for systemic lupus erythematosus, using the agents according to at least some embodiments of the present invention may optionally be combined with, for example, any known therapeutic agent or method for treating for systemic lupus erythematosus. Non-limiting examples of such known therapeutics for treating for systemic lupus erythematosus include corticosteroids and Disease-modifying antirheumatic drugs (DMARDs), commonly anti-malarial drugs such as plaquenil and immunosuppressants (e.g. methotrexate and azathioprine) Hydroxychloroquine, cytotoxic drugs (e.g., cyclophosphamide and mycophenolate), Hydroxychloroquine (HCQ), Benlysta® (belimumab), nonsteroidal anti-inflammatory drugs, Prednisone, Cellcept®, Prograf®, Atacicept®, Lupuzor®, Intravenous Immunoglobulins (IVIGs), CellCept® (mycophenolate mofetil), Orencia®, CTLA4-IgG4m (RG2077), rituximab, Ocrelizumab, Epratuzumab, CNTO 136, Sifalimumab (MEDI-545), A-623 (formerly AMG 623), AMG 557, Rontalizumab, paquinimod (ABR-215757), LY2127399, CEP-33457, Dehydroepiandrosterone, Levothyroxine, abetimus sodium (LW 394), Memantine®, Opiates, Rapamycin®, renal transplantation, stem cell transplantation and combinations of any of the foregoing.
The immunoinhibitory HIDE1 therapeutic agents and/or a pharmaceutical composition comprising same, as recited herein, according to at least some embodiments of the present invention, may optionally be administered as the sole active ingredient or together with other drugs in immunomodulating regimens or other anti-inflammatory agents e.g. for the treatment or prevention of allo- or xenograft acute or chronic rejection or inflammatory or autoimmune disorders, or to induce tolerance.
D. Use of the Hide1 Agents and/or Pharmaceutical Compositions for Adoptive Immunity:
One of the cardinal features of some models of tolerance is that once the tolerance state has been established, it can be perpetuated to naive recipients by the adoptive transfer of donor-specific regulatory cells. Such adoptive transfer studies have also addressed the capacity of T-cell subpopulations and non-T cells to transfer tolerance. Such tolerance can be induced by blocking costimulation or upon engagement of a co-inhibitory B7 with its counter receptor. This approach, that has been successfully applied in animals and is evaluated in clinical trials in humans, (Scalapino K J and Daikh D I. PLoS One, 4(6):e6031 (2009); Riley et al., Immunity 30(5): 656-665 (2009)) provides a promising treatment option for autoimmune disorders and transplantation. According to at least some embodiments of the present invention, HIDE1 secreted or soluble form or ECD and/or variants, and/or orthologs, and/or fusions and/or conjugates thereof, are used for adoptive immunotherapy. Thus, according to at least some embodiments, the present invention provides methods for in vivo or ex vivo tolerance induction, comprising administering effective amount of HIDE1 secreted or soluble form or ECD and/or variants, and/or orthologs, and/or fusions and/or conjugates thereof, to a patient or to leukocytes isolated from the patient, in order to induce differentiation of tolerogenic regulatory cells; followed by ex-vivo enrichment and expansion of said cells and reinfusion of the tolerogenic regulatory cells to said patient.
In another embodiment, a method of inhibiting immune responses involves isolating immune cells from a patient, transfecting them with a nucleic acid molecule encoding a form of HIDE1, such that the cells express all or a portion of the HIDE1 polypeptide according to various embodiments of the present invention on their surface, and reintroducing the transfected cells into the patient. The transfected cells have the capacity to inhibit immune responses in the patient.
3. Diagnostic Uses
The anti-HIDE1 antibodies provided also find use in the in vitro or in vivo diagnosis, including imaging, of tumors that over-express HIDE1. It should be noted, however, that as discussed herein, HIDE1, as an immuno-oncology target protein, is not necessarily overexpressed on cancer cells rather within the immune infiltrates in the cancer. In some instances it is; rather, the mechanism of action, activation of immune cells such as T cells and NK cells, that results in cancer diagnosis. Accordingly, anti-HIDE1 antibodies can be used to diagnose cancer.
In particular, immune cells infiltrating the tumors that over express HIDE1, and thus can be diagnosed by anti-HIDE1 antibodies, include, but are not limited to, squamous cell cancer, lung cancer (including small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung), cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer (including gastrointestinal cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, liver cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma and various types of head and neck cancer, as well as B-cell lymphoma (including low grade/follicular non-Hodgkin's lymphoma (NHL); small lymphocytic (SL) NHL; intermediate grade/follicular NHL; intermediate grade diffuse NHL; high grade immunoblastic NHL; high grade lymphoblastic NHL; high grade small non-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenström's Macroglobulinemia); chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); Hairy cell leukemia; chronic myeloblastic leukemia; multiple myeloma and post-transplant lymphoproliferative disorder (PTLD).
Generally, diagnosis can be done in several ways. In one embodiment, a tissue from a patient, such as a biopsy sample, is contacted with a HIDE1 antibody, generally labeled, such that the antibody binds to the endogenous HIDE1. The level of signal is compared to that of normal non-cancerous tissue either from the same patient or a reference sample, to determine the presence or absence of cancer. The biopsy sample can be from a solid tumor, a blood sample (for lymphomas and leukemias such as ALL, T cell lymphoma, etc).
In general, in this embodiment, the anti-HIDE1 is labeled, for example with a fluorophore or other optical label, that is detected using a fluorometer or other optical detection system as is well known in the art. In an alternate embodiment, a secondary labeled antibody is contacted with the sample, for example using an anti-human IgG antibody from a different mammal (mouse, rat, rabbit, goat, etc.) to form a sandwich assay as is known in the art. Alternatively, the anti-HIDE1 mAb could be directly labeled (i.e. biotin) and detection can be done by a secondary Ab directed to the labeling agent in the art.
Once over-expression of HIDE1 is seen, treatment can proceed with the administration of an anti-HIDE1 antibody according to the invention as outlined herein.
In other embodiments, in vivo diagnosis is done. Generally, in this embodiment, the anti-HIDE1 antibody (including antibody fragments) is injected into the patient and imaging is done. In this embodiment, for example, the antibody is generally labeled with an optical label or an MRI label, such as a gadolinium chelate, radioactive labeling of mAb (including fragments).
In some embodiments, the antibodies described herein are used for both diagnosis and treatment, or for diagnosis alone. When anti-HIDE1 antibodies are used for both diagnosis and treatment, some embodiments rely on two different anti-HIDE1 antibodies to two different epitopes, such that the diagnostic antibody does not compete for binding with the therapeutic antibody, although in some cases the same antibody can be used for both. For example, this can be done using antibodies that are in different bins, e.g., that bind to different epitopes on HIDE1, such as outlined herein. Thus included in the invention are compositions comprising a diagnostic antibody and a therapeutic antibody, and in some embodiments, the diagnostic antibody is labeled as described herein. In addition, the composition of therapeutic and diagnostic antibodies can also be co-administered with other drugs as outlined herein.
Particularly useful antibodies for use in diagnosis include, but are not limited to these enumerated antibodies, or antibodies that utilize the CDRs with variant sequences
In many embodiments, a diagnostic antibody is labeled. By “labeled” herein is meant that the antibodies disclosed herein have one or more elements, isotopes, or chemical compounds attached to enable the detection in a screen or diagnostic procedure. In general, labels fall into several classes: a) immune labels, which may be an epitope incorporated as a fusion partner that is recognized by an antibody, b) isotopic labels, which may be radioactive or heavy isotopes, c) small molecule labels, which may include fluorescent and colorimetric dyes, or molecules such as biotin that enable other labeling methods, and d) labels such as particles (including bubbles for ultrasound labeling) or paramagnetic labels that allow body imagining. Labels may be incorporated into the antibodies at any position and may be incorporated in vitro or in vivo during protein expression, as is known in the art.
Diagnosis can be done either in vivo, by administration of a diagnostic antibody that allows whole body imaging as described below, or in vitro, on samples removed from a patient. “Sample” in this context includes any number of things, including, but not limited to, bodily fluids (including, but not limited to, blood, urine, serum, lymph, saliva, anal and vaginal secretions, perspiration and semen), as well as tissue samples such as result from biopsies of relevant tissues.
In some embodiments, in vivo imaging is done, including but not limited to ultrasound, CT scans, X-rays, Mill and PET scans, as well as optical techniques, such as those using optical labels for tumors near the surface of the body.
In vivo imaging of diseases associated with HIDE1 may be performed by any suitable technique. For example, 99Tc-labeling or labeling with another .beta.-ray emitting isotope may be used to label anti-HIDE1 antibodies. Variations on this technique may include the use of magnetic resonance imaging (MRI) to improve imaging over gamma camera techniques.
In one embodiment, the present invention provides an in vivo imaging method wherein an anti-HIDE1 antibody is conjugated to a detection-promoting agent, the conjugated antibody is administered to a host, such as by injection into the bloodstream, and the presence and location of the labeled antibody in the host is assayed. Through this technique and any other diagnostic method provided herein, in some embodiments the present invention provides a method for screening for the presence of disease-related cells in a human patient or a biological sample taken from a human patient.
For diagnostic imaging, radioisotopes may be bound to an anti-HIDE1 antibody either directly, or indirectly by using an intermediary functional group. Useful intermediary functional groups include chelators, such as ethylenediaminetetraacetic acid and diethylenetriaminepentaacetic acid (see for instance U.S. Pat. No. 5,057,313), in such diagnostic assays involving radioisotope-conjugated anti-HIDE1 antibodies, the dosage of conjugated anti-HIDE1 antibody delivered to the patient typically is maintained at as low a level as possible through the choice of isotope for the best combination of minimum half-life, minimum retention in the body, and minimum quantity of isotope, which will permit detection and accurate measurement.
In addition to radioisotopes and radio-opaque agents, diagnostic methods may be performed using anti-HIDE1 antibodies that are conjugated to dyes (such as with the biotin-streptavidin complex), contrast agents, fluorescent compounds or molecules and enhancing agents (e.g. paramagnetic ions) for magnetic resonance imaging (MRI) (see, e.g., U.S. Pat. No. 6,331,175, which describes Mill techniques and the preparation of antibodies conjugated to a Mill enhancing agent). Such diagnostic/detection agents may be selected from agents for use in magnetic resonance imaging, and fluorescent compounds.
In order to load an anti-HIDE1 antibody with radioactive metals or paramagnetic ions, it may be necessary to react it with a reagent having a long tail to which are attached a multiplicity of chelating groups for binding the ions. Such a tail may be a polymer such as a polylysine, polysaccharide, or other derivatized or derivatizable chain having pendant groups to which can be bound chelating groups such as, e.g., porphyrins, polyamines, crown ethers, bisthiosemicarbazones, polyoximes, and like groups known to be useful for this purpose.
Chelates may be coupled to anti-HIDE1 antibodies using standard chemistries. A chelate is normally linked to an anti-HIDE1 antibody by a group that enables formation of a bond to the molecule with minimal loss of immunoreactivity and minimal aggregation and/or internal cross-linking.
Examples of potentially useful metal-chelate combinations include 2-benzyl-DTPA and its monomethyl and cyclohexyl analogs, used with diagnostic isotopes in the general energy range of 60 to 4,000 keV, such as 125I, 123I, 124I, 62Cu, 64Cu, 18F, 111In, 67Ga, 99Tc, 94Tc, 11C, 13N, 5O, and 76Br, for radio-imaging.
Labels include a radionuclide, a radiological contrast agent, a paramagnetic ion, a metal, a fluorescent label, a chemiluminescent label, an ultrasound contrast agent and a photoactive agent. Such diagnostic agents are well known and any such known diagnostic agent may be used. Non-limiting examples of diagnostic agents may include a radionuclide such as 110In, 111In, 177Lu, 18F, 52Fe, 62Cu, 64Cu, 67Cu, 67Ga, 68Ga, 86Y, 90Y, 89Zr, 94mTc, 99Tc, 99MTc, 120I, 123I, 124I, 125I, 131I, 154-158Gd, 32P, 11C, 13N, 15O, 186Re, 188Re, 51Mn, 52mMn, 55Co, 72As, 75Br, 76Br, 82mRb, 83Sr, or other .gamma.-, .beta.-, or positron-emitters.
Paramagnetic ions of use may include chromium (III), manganese (II), iron (III), iron (II), cobalt (II), nickel (III), copper (III), neodymium (III), samarium (III), ytterbium (III), gadolinium (III), vanadium (II), terbium (III), dysprosium (III), holmium (III) or erbium (III), Metal contrast agents may include lanthanum (III), gold (III), lead (II) or bismuth (III).
Ultrasound contrast agents may comprise liposomes, such as gas filled liposomes. Radiopaque diagnostic agents may be selected from compounds, barium compounds, gallium compounds, and thallium compounds.
These and similar chelates, when complexed with non-radioactive metals, such as manganese, iron, and gadolinium may be useful for MRI diagnostic methods in connection with anti-HIDE1 antibodies. Macrocyclic chelates such as NOTA, DOTA, and TETA are of use with a variety of metals and radiometals, most particularly with radionuclides of gallium, yttrium, and copper, respectively. Such metal-chelate complexes may be made very stable by tailoring the ring size to the metal of interest. Other ring-type chelates such as macrocyclic polyethers, which are of interest for stably binding nuclides, such as 223Ra may also be suitable in diagnostic methods.
Thus, in some embodiments the present invention provides diagnostic anti-HIDE1 antibody conjugates, wherein the anti-HIDE1 antibody conjugate is conjugated to a contrast agent (such as for magnetic resonance imaging, computed tomography, or ultrasound contrast-enhancing agent) or a radionuclide that may be, for example, a .gamma.-, .beta.-, .alpha.-, Auger electron-, or positron-emitting isotope.
Anti-HIDE1 antibodies may also be useful in, for example, detecting expression of an antigen of interest in specific cells, tissues, or serum. For diagnostic applications, the antibody typically will be labeled with a detectable moiety for in vitro assays. As will be appreciated by those in the art, there are a wide variety of suitable labels for use in in vitro testing. Suitable dyes for use in this aspect of the invention include, but are not limited to, fluorescent lanthanide complexes, including those of Europium and Terbium, fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrosin, coumarin, methyl-coumarins, quantum dots (also referred to as “nanocrystals”; see U.S. Ser. No. 09/315,584, hereby incorporated by reference), pyrene, Malacite green, stilbene, Lucifer Yellow, Cascade Blue™, Texas Red, Cy dyes (Cy3, Cy5, etc.), alexa dyes (including Alexa, phycoerythin, bodipy, and others described in the 6th Edition of the Molecular Probes Handbook by Richard P. Haugland, hereby expressly incorporated by reference.
Stained tissues may then be assessed for radioactivity counting as an indicator of the amount of HIDE1-associated peptides in the tumor. The images obtained by the use of such techniques may be used to assess biodistribution of HIDE1 in a patient, mammal, or tissue, for example in the context of using HIDE1 as a biomarker for the presence of invasive cancer cells.
4. Diagnostic Uses of HIDE1 Polypeptides
Soluble HIDE1 polypeptides according to at least some embodiments of the present invention may optionally also be modified with a label capable of providing a detectable signal, either directly or indirectly, including, but not limited to, radioisotopes and fluorescent compounds. Such labeled polypeptides can be used for various uses, including but not limited to, diagnosis, prognosis, prediction, screening, early diagnosis, determination of progression, therapy selection and treatment monitoring of disease, disorder and/or an indicative condition, as detailed herein.
As used herein the term “diagnosis” refers to the process of identifying or aiding in the identification of a medical condition or disease by its signs, symptoms, and in particular from the results of various diagnostic procedures, including e.g., using labeled HIDE1 polypeptides according to at least some embodiments of the present invention, as described herein. Furthermore, as used herein the term “diagnosis” encompasses screening for a disease, detecting a presence or a severity of a disease, providing prognosis of a disease, monitoring disease progression or relapse, as well as assessment of treatment efficacy and/or relapse of a disease, disorder or condition, as well as selecting a therapy and/or a treatment for a disease, optimization of a given therapy for a disease, monitoring the treatment of a disease, and/or predicting the suitability of a therapy for specific patients or subpopulations or determining the appropriate dosing of a therapeutic product in patients or subpopulations. The diagnostic procedure can be performed in vivo or in vitro.
According to at least some embodiments, the present invention provides a method for imaging an organ or tissue, the method comprising: (a) administering to a subject in need of such imaging, a labeled polypeptide; and (b) detecting the labeled polypeptide to determine where the labeled polypeptide is concentrated in the subject. When used in imaging applications, the labeled polypeptides according to at least some embodiments of the present invention typically have an imaging agent covalently or monovalently attached thereto. Suitable imaging agents include, but are not limited to, radionuclides, detectable tags, fluorophores, fluorescent proteins, enzymatic proteins, and the like. One of skill in the art will be familiar with other methods for attaching imaging agents to polypeptides. For example, the imaging agent can be attached via site-specific conjugation, e.g., covalent attachment of the imaging agent to a peptide linker such as a polyarginine moiety having five to seven arginines present at the carboxyl-terminus of and Fc fusion molecule. The imaging agent can also be directly attached via non-site specific conjugation, e.g., covalent attachment of the imaging agent to primary amine groups present in the polypeptide. One of skill in the art will appreciate that an imaging agent can also be bound to a protein via noncovalent interactions (e.g., ionic bonds, hydrophobic interactions, hydrogen bonds, Van der Waals forces, dipole-dipole bonds, etc.).
In certain instances, the polypeptide is radiolabeled with a radionuclide by directly attaching the radionuclide to the polypeptide. In certain other instances, the radionuclide is bound to a chelating agent or chelating agent-linker attached to the polypeptide. Suitable radionuclides for direct conjugation include, without limitation, 18F, 124I, 125I, 131I, and mixtures thereof. Suitable radionuclides for use with a chelating agent include, without limitation 47Sc, 64Cu, 67Cu, 89Sr, 86Y, 87Y, 90Y, 105Rh, 111Ag, 111In, 149P, 117mSn, 149Pm, 153Sm 166Ho, 177Lu, 186Re, 188Re, 211At, 212Bi and mixtures thereof.
Preferably, the radionuclide bound to a chelating agent is 64Cu, 90Y, 111In, or mixtures thereof. Suitable chelating agents include, but are not limited to, DOTA, BAD, TETA, DTPA, EDTA, NTA, HDTA, their phosphonate analogs, and mixtures thereof. One of skill in the art will be familiar with methods for attaching radionuclides, chelating agents, and chelating agent-linkers to polypeptides of various embodiments of the present invention. In particular, attachment can be conveniently accomplished using, for example, commercially available bifunctional linking groups (generally heterobifunctional linking groups) that can be attached to a functional group present in a non-interfering position on the polypeptide and then further linked to a radionuclide, chelating agent, or chelating agent-linker.
Non-limiting examples of fluorophores or fluorescent dyes suitable for use as imaging agents include Alexa Fluor® dyes (Invitrogen Corp.; Carlsbad, Calif.), fluorescein, fluorescein isothiocyanate (FITC), Oregon Green™; rhodamine, Texas red, tetrarhodamine isothiocynate (TRITC), CyDye™ fluors (e.g., Cy2, Cy3, Cy5), and the like.
Examples of fluorescent proteins suitable for use as imaging agents include, but are not limited to, green fluorescent protein, red fluorescent protein (e.g., DsRed), yellow fluorescent protein, cyan fluorescent protein, blue fluorescent protein, and variants thereof (see, e.g., U.S. Pat. Nos. 6,403,374, 6,800,733, and 7,157,566). Specific examples of GFP variants include, but are not limited to, enhanced GFP (EGFP), destabilized EGFP, the GFP variants described in Doan et al., Mol. Microbiol., 55:1767-1781 (2005), the GFP variant described in Crameri et al., Nat. Biotechnol., 14:315-319 (1996), cerulean fluorescent proteins described in Rizzo et al., Nat. Biotechnol, 22:445 (2004) and Tsien, Annu. Rev. Biochem., 67:509 (1998), and the yellow fluorescent protein described in Nagal et al., Nat. Biotechnol., 20:87-90 (2002). DsRed variants are described in, e.g., Shaner et al., Nat. Biotechnol., 22:1567-1572 (2004), and include mStrawberry, mCherry, mOrange, mBanana, mHoneydew, and mTangerine. Additional DsRed variants are described in, e.g., Wang et al., Proc. Natl. Acad. Sci. U.S.A, 101:16745-16749 (2004) and include mRaspberry and mPlum. Further examples of DsRed variants include mRFPmars described in Fischer et al., FEBS Lett., 577:227-232 (2004) and mRFPruby described in Fischer et al., FEBS Lett., 580:2495-2502 (2006).
In other embodiments, the imaging agent that is bound to a polypeptide according to at least some embodiments of the present invention comprises a detectable tag such as, for example, biotin, avidin, streptavidin, or neutravidin. In further embodiments, the imaging agent comprises an enzymatic protein including, but not limited to, luciferase, chloramphenicol acetyltransferase, β-galactosidase, β-glucuronidase, horseradish peroxidase, xylanase, alkaline phosphatase, and the like.
Any device or method known in the art for detecting the radioactive emissions of radionuclides in a subject is suitable for use in the various embodiments of the present invention. For example, methods such as Single Photon Emission Computerized Tomography (SPECT), which detects the radiation from a single photon γ-emitting radionuclide using a rotating γ camera, and radionuclide scintigraphy, which obtains an image or series of sequential images of the distribution of a radionuclide in tissues, organs, or body systems using a scintillation gamma camera, may optionally be used for detecting the radiation emitted from a radiolabeled polypeptide of the various embodiments of the present invention. Positron emission tomography (PET) is another suitable technique for detecting radiation in a subject. Miniature and flexible radiation detectors intended for medical use are produced by Intra-Medical LLC (Santa Monica, Calif.). Magnetic Resonance Imaging (MRI) or any other imaging technique known to one of skill in the art is also suitable for detecting the radioactive emissions of radionuclides. Regardless of the method or device used, such detection is aimed at determining where the labeled polypeptide is concentrated in a subject, with such concentration being an indicator of disease activity.
Non-invasive fluorescence imaging of animals and humans can also provide in vivo diagnostic information and be used in a wide variety of clinical specialties. For instance, techniques have been developed over the years for simple ocular observations following UV excitation to sophisticated spectroscopic imaging using advanced equipment (see, e.g., Andersson-Engels et al., Phys. Med. Biol., 42:815-824 (1997)). Specific devices or methods known in the art for the in vivo detection of fluorescence, e.g., from fluorophores or fluorescent proteins, include, but are not limited to, in vivo near-infrared fluorescence (see, e.g., Frangioni, Curr. Opin. Chem. Biol., 7:626-634 (2003)), the Maestro™ in vivo fluorescence imaging system (Cambridge Research & Instrumentation, Inc.; Woburn, Mass.), in vivo fluorescence imaging using a flying-spot scanner (see, e.g., Ramanujam et al., IEEE Transactions on Biomedical Engineering, 48:1034-1041 (2001), and the like.
Other methods or devices for detecting an optical response include, without limitation, visual inspection, CCD cameras, video cameras, photographic film, laser-scanning devices, fluorometers, photodiodes, quantum counters, epifluorescence microscopes, scanning microscopes, flow cytometers, fluorescence microplate readers, or signal amplification using photomultiplier tubes.
5. Theranostics
The term theranostics describes the use of diagnostic testing to diagnose the disease, choose the correct treatment regime according to the results of diagnostic testing and/or monitor the patient response to therapy according to the results of diagnostic testing. Theranostic tests can be used to select patients for treatments that are particularly likely to benefit them and unlikely to produce side-effects. They can also provide an early and objective indication of treatment efficacy in individual patients, so that (if necessary) the treatment can be altered with a minimum of delay. For example: DAKO and Genentech together created HercepTest® and Herceptin® (trastuzumab) for the treatment of breast cancer, the first theranostic test approved simultaneously with a new therapeutic drug. In addition to HercepTest (which is an immunohistochemical test), other theranostic tests are in development which use traditional clinical chemistry, immunoassay, cell-based technologies and nucleic acid tests. PPGx's recently launched TPMT (thiopurine S-methyltransferase) test, which is enabling doctors to identify patients at risk for potentially fatal adverse reactions to 6-mercaptopurine, an agent used in the treatment of leukemia. Also, Nova Molecular pioneered SNP genotyping of the apolipoprotein E gene to predict Alzheimer's disease patients' responses to cholinomimetic therapies and it is now widely used in clinical trials of new drugs for this indication. Thus, the field of theranostics represents the intersection of diagnostic testing information that predicts the response of a patient to a treatment with the selection of the appropriate treatment for that particular patient.
The term theranostics describes the use of diagnostic testing to diagnose the disease, choose the correct treatment regime according to the results of diagnostic testing and/or monitor the patient response to therapy according to the results of diagnostic testing. Theranostic tests can be used to select patients for treatments that are particularly likely to benefit them and unlikely to produce side-effects. They can also provide an early and objective indication of treatment efficacy in individual patients, so that (if necessary) the treatment can be altered with a minimum of delay. For example: DAKO and Genentech together created HercepTest® and Herceptin® (trastuzumab) for the treatment of breast cancer, the first theranostic test approved simultaneously with a new therapeutic drug. In addition to HercepTest® (which is an immunohistochemical test), other theranostic tests are in development which use traditional clinical chemistry, immunoassay, cell-based technologies and nucleic acid tests. PPGx's recently launched TPMT (thiopurine S-methyltransferase) test, which is enabling doctors to identify patients at risk for potentially fatal adverse reactions to 6-mercaptopurine, an agent used in the treatment of leukemia. Also, Nova Molecular pioneered SNP genotyping of the apolipoprotein E gene to predict Alzheimer's disease patients' responses to cholinomimetic therapies and it is now widely used in clinical trials of new drugs for this indication. Thus, the field of theranostics represents the intersection of diagnostic testing information that predicts the response of a patient to a treatment with the selection of the appropriate treatment for that particular patient.
As described herein, the term “theranostic” may optionally refer to first testing the subject, such as the patient, for a certain minimum level of HIDE1, for example optionally in the cancerous tissue and/or in the immune infiltrate, as described herein as a sufficient level of HIDE1 expression. Testing may optionally be performed ex vivo, in which the sample is removed from the subject, or in vivo.
If the cancerous tissue and/or the immune infiltrate have been shown to have the minimum level of HIDE1, then an anti-HIDE1 antibody, alone or optionally with other treatment modalities as described herein, may optionally be administered to the subject.
6. Surrogate Markers—HIDE1 Antibodies
A surrogate marker is a marker, that is detectable in a laboratory and/or according to a physical sign or symptom on the patient, and that is used in therapeutic trials as a substitute for a clinically meaningful endpoint. The surrogate marker is a direct measure of how a patient feels, functions, or survives which is expected to predict the effect of the therapy. The need for surrogate markers mainly arises when such markers can be measured earlier, more conveniently, or more frequently than the endpoints of interest in terms of the effect of a treatment on a patient, which are referred to as the clinical endpoints. Ideally, a surrogate marker should be biologically plausible, predictive of disease progression and measurable by standardized assays (including but not limited to traditional clinical chemistry, immunoassay, cell-based technologies, receptor occupancy assay nucleic acid tests and imaging modalities).
The therapeutic compositions (e.g., human antibodies, multispecific and bispecific molecules and immunoconjugates) according to at least some embodiments of the present invention which have complement binding sites, such as portions from IgG1, IgG2, or IgG3 or IgM which bind complement, can also be used in the presence of complement. In one embodiment, ex vivo treatment of a population of cells comprising target cells with a binding agent according to at least some embodiments of the present invention and appropriate effector cells can be supplemented by the addition of complement or serum containing complement. Phagocytosis of target cells coated with a binding agent according to at least some embodiments of the present invention can be improved by binding of complement proteins. In another embodiment target cells coated with the compositions (e.g., human antibodies, multispecific and bispecific molecules) according to at least some embodiments of the present invention can also be lysed by complement. In yet another embodiment, the compositions according to at least some embodiments of the present invention do not activate complement.
The therapeutic compositions (e.g., human antibodies, multispecific and bispecific molecules and immunoconjugates) according to at least some embodiments of the present invention can also be administered together with complement. Thus, according to at least some embodiments of the present invention there are compositions, comprising human antibodies, multispecific or bispecific molecules and serum or complement. These compositions are advantageous in that the complement is located in close proximity to the human antibodies, multispecific or bispecific molecules. Alternatively, the human antibodies, multispecific or bispecific molecules according to at least some embodiments of the present invention and the complement or serum can be administered separately.
In some embodiments, the present invention provides a method for determining whether an anti-HIDE1 antibody has produced a desired immunomodulatory effect in a human (e.g., a cancer patient). The method includes detecting an increase or decrease of at least one immunomodulatory biomarker (sometimes referred to herein as an “anti-HIDE1 antibody-associated immunomodulatory biomarker”) described herein in a blood sample obtained from a patient who has been administered an anti-HIDE1 antibody to thereby determine whether the anti-HIDE1 antibody has produced an immunostimulatory effect. The immunostimulatory effect can be characterized by a change (e.g., an increase or a decrease) in at least one biomarker, e.g., an anti-HIDE1 antibody-associated immunomodulatory biomarker described herein, the change selected from the group consisting of: (i) a reduced concentration of regulatory T cells, relative to the concentration of regulatory T cells of the same histological type in the human prior to the first administration of the antibody; (ii) an increased concentration of CTL cells, relative to the concentration of CTL cells of the same histological type in the human prior to the first administration of the antibody; (iii) an increased concentration of activated T cells, relative to the concentration of activated T cells of the same histological type in the human prior to the first administration of the antibody; (iv) an increased concentration of NK cells, relative to the concentration of NK cells of the same histological type in the human prior to the first administration of the antibody; (v) a ratio of percent activated T cells to percent regulatory T cells (T regs) of at least 2:1 (e.g., at least 3:1, at least 4:1, at least 5:1, at least 6:1, or at least 7:1), relative to the ratio of activated T cells to T regs in the human prior to the first administration of the antibody; (vii) a changed level of HIDE1 expression by a plurality of leukocytes in a biological sample obtained from a patient prior to administration to the patient of an anti-HIDE1 antibody, relative to the level of HIDE1 expression by a plurality of leukocytes of the same histological type in a biological sample from the patient prior to administration of the antibody; viii) increasing T and/or NK cell infiltration of tumors, (vix) alleviating myeloid-cell suppression, (vx) eliminating (depleting) immuno-suppressive myeloid cells, such as MDSCs (Myeloid-derived suppressor cells), TAMs (Tumor-associated macrophages).
It is understood that in some embodiments, a change in expression can be a change in protein expression or a change in mRNA expression. That is, for example, the methods can interrogate a population of leukocytes from a patient to determine if a reduction in the level of HIDE1 mRNA and/or HIDE1 protein expression has occurred, relative to a control level of mRNA and/or protein expression. Methods for measuring protein and mRNA expression are well known in the art and described herein.
In some embodiments, any of the methods described herein (e.g., the methods for determining whether an anti-HIDE1 has produced a desired immunostimulatory effect in a human) can include measuring the concentration of the specified cell type (e.g. CD4+ T cells, CTLs, NK cells etc.), or quantifying the level of expression of a specified expression marker on a specified cell type (e.g. Foxp3, CD25, CD69, etc.), in a biological sample obtained from the human prior to administration of the antibody.
7. Surrogate Markers—HIDE1 Peptides
The HIDE1 protein compositions according to at least some embodiments of the present invention can be used as a surrogate marker. A surrogate marker is a marker, that is detectable in a laboratory and/or according to a physical sign or symptom on the patient, and that is used in therapeutic trials as a substitute for a clinically meaningful endpoint. The surrogate marker is a direct measure of how a patient feels, functions, or survives which is expected to predict the effect of the therapy. The need for surrogate markers mainly arises when such markers can be measured earlier, more conveniently, or more frequently than the endpoints of interest in terms of the effect of a treatment on a patient, which are referred to as the clinical endpoints. Ideally, a surrogate marker should be biologically plausible, predictive of disease progression and measurable by standardized assays (including but not limited to traditional clinical chemistry, immunoassay, cell-based technologies, nucleic acid tests and imaging modalities).
HIDE1 protein compositions according to at least some embodiments of the present invention which have complement binding sites, such as portions from IgG1, IGg2, or IgG3 or IgM which bind complement, can also be used in the presence of complement. In one embodiment, ex vivo treatment of a population of cells comprising target cells with a binding agent according to at least some embodiments of the present invention and appropriate effector cells can be supplemented by the addition of complement or serum containing complement. Phagocytosis of target cells coated with a binding agent according to at least some embodiments of the present invention can be improved by binding of complement proteins. In another embodiment target cells coated with the compositions according to at least some embodiments of the present invention can also be lysed by complement. In yet another embodiment, the compositions according to at least some embodiments of the present invention do not activate complement.
The HIDE1 protein compositions according to at least some embodiments of the present invention can also be administered together with complement. Thus, according to at least some embodiments of the present invention there is any of the HIDE1 protein composition, comprising human HIDE1 soluble protein and serum or complement. These compositions are advantageous in that the complement is located in close proximity to the human HIDE1 soluble molecules. Alternatively, the human HIDE1 soluble molecules according to at least some embodiments of the present invention and the complement or serum can be administered separately.
In some embodiments the present invention provides the use of an immunostimulatory antibody, antigen-binding fragment or conjugate thereof according to at least some embodiments of the present invention or a pharmaceutical composition containing the same, to perform one or more of the following in a subject in need thereof: (a) upregulating pro-inflammatory cytokines; (b) increasing T-cell proliferation and/or expansion; (c) increasing interferon-γ or TNF-α production by T-cells; (d) increasing IL-2 secretion; (e) stimulating antibody responses; (f) inhibiting cancer cell growth; (g) promoting antigenic specific T cell immunity; (h) promoting CD4+ and/or CD8+ T cell activation; (i) alleviating T-cell suppression; (j) promoting NK cell activity; (k) promoting apoptosis or lysis of cancer cells; (1) cytotoxic or cytostatic effect on cancer cells, (m) increasing T and/or NK cell infiltration of tumors, (n) alleviating myeloid-cell suppression, (o) eliminating (depleting) immuno-suppressive myeloid cells, such as MDSCs (Myeloid-derived suppressor cells), TAMs (Tumor-associated macrophages).
In some embodiments the present invention provides the use of an immunostimulatory antibody, antigen-binding fragment or conjugate according to at least some embodiments of the present invention for diagnosing a disease in a subject, or for aiding in the diagnosis of a disease, wherein the disease is selected from the group consisting of cancer, wherein the diagnostic method is performed ex vivo, by contacting a tissue or other sample from the subject with the immune molecule or antibody as described herein ex vivo and detecting specific binding thereto.
In other embodiments the present invention provides the use of an immunostimulatory antibody, antigen-binding fragment or conjugate according to at least some embodiments of the present invention in diagnostic methods for diagnosing or aiding in the diagnosis of a disease in a subject, wherein the disease is selected from the group consisting of cancer, and/or an infectious disease wherein the diagnostic method is performed in vivo, comprising administering the immune molecule or antibody as described herein to the subject, preferably labeled with a detectable agent such as a radionuclide, or fluorophore and detecting specific binding of the immunostimulatory antibody, antigen-binding fragment or conjugate as described herein to a tissue of the subject. Alternatively the method may optionally be performed in vitro in a sample taken from the subject.
Optionally such diagnostic method may be performed before concurrent or after administering an immunostimulatory antibody, antigen-binding fragment or conjugate or composition containing according to at least some embodiments of the present invention.
Optionally the diagnostic use or method further comprises determining the level of HIDE1 in a tissue of the subject before administering the immunostimulatory antibody, antigen-binding fragment or conjugate or composition containing according to at least some embodiments of the present invention to the subject. In some embodiments the immunostimulatory antibody, antigen-binding fragment or conjugate or composition containing according to at least some embodiments of the present invention is only administered to the subject if said HIDE1 level is at a threshold level deemed to be “sufficient” for the HIDE1 antibody to elicit a significant therapeutic benefit, e.g., it is expressed at higher than normal levels or it is expressed at detectable levels by the treated disease cells, e.g., specific types of cancer or immune or stromal cells at the site of the disease, or is expressed at a level that based on in vitro or in vivo studies indicates that the antibody is likely to elicit a significant therapeutic benefit.
In some embodiments the expression level of HIDE1 is detected upon initial diagnosis prior to the initiation of cancer therapy, or alternatively after the start of cancer therapy, such as a combination therapy including use of an immunostimulatory antibody, antigen-binding fragment or conjugate according to at least some embodiments of the present invention and another active such as a chemotherapeutic, therapeutic enzyme, radionuclide or radiation or another biologic.
In some embodiments the use or method further comprises determining said level of HIDE1 according to the expression level of said HIDE1.
In some embodiments the expression level of the HIDE1 is determined by use of an IHC (immunohistochemistry) assay or a gene expression assay in a subject's tissue sample.
In some embodiments said IHC assay may optionally comprise determining if the level of HIDE1 expression is at least 1 on a scale of 0 to 3, e.g., in a tissue sample comprising cancer cells and/or immune infiltrate and/or on immune and/or on stromal cells.
In some embodiments the level of HIDE1 may optionally be determined in a tissue by contacting the tissue with an immunostimulatory antibody, antigen-binding fragment or conjugate or composition containing according to at least some embodiments of the present invention and detecting specific binding thereto.
In some embodiments the present invention provides assays for diagnosing or aiding in the diagnosis of a disease in a tissue sample taken from a subject, comprising use of an immunostimulatory antibody, antigen-binding fragment or conjugate as described herein and at least one reagent for diagnosing a disease selected from the group consisting of cancer, infectious disease, and/or sepsis.
In some embodiments the present invention provides the use of anti-HIDE1 antibody, antigen-binding fragment or conjugate or composition containing same according to at least some embodiments of the present invention for screening for a disease or aiding in the diagnosis of a disease (particularly one involving immunosuppression), detecting a presence or a severity of a disease, providing prognosis of a disease, monitoring disease progression or relapse, as well as assessment of treatment efficacy and/or relapse of a disease, disorder or condition, as well as selecting a therapy and/or a treatment for a disease, optimization of a given therapy for a disease, monitoring the treatment of a disease, and/or predicting the suitability of a therapy for specific patients or subpopulations or determining the appropriate dosing of a therapeutic product in patients or subpopulations.
In some embodiments, the present invention provides anti-HIDE1 immunostimulatory immune molecule or composition containing same according to at least some embodiments of the present invention, and/or uses thereof for treatment and/or diagnosis of cancer, wherein the cancer, and/or immune cells infiltrating the cancer, and/or stromal cells of the subject express HIDE1, e.g. prior to, or following cancer therapy, and wherein said cancer is e.g., selected from the group consisting of but not limited to breast cancer, cervical cancer, ovary cancer, endometrial cancer, melanoma, uveal melanoma, bladder cancer, lung cancer, pancreatic cancer, colorectal cancer, prostate cancer, leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, B-cell lymphoma, Burkitt's lymphoma, multiple myeloma, Non-Hodgkin's lymphoma, myeloid leukemia, acute myelogenous leukemia (AML), chronic myelogenous leukemia, thyroid cancer, thyroid follicular cancer, myelodysplastic syndrome (MDS), fibrosarcomas and rhabdomyosarcomas, teratocarcinoma, neuroblastoma, glioma, glioblastoma, benign tumor of the skin, keratoacanthomas, renal cancer, anaplastic large-cell lymphoma, esophageal cancer, follicular dendritic cell carcinoma, seminal vesicle tumor, epidermal carcinoma, spleen cancer, bladder cancer, head and neck cancer, stomach cancer, liver cancer, bone cancer, brain cancer, cancer of the retina, biliary cancer, small bowel cancer, salivary gland cancer, cancer of uterus, cancer of testicles, cancer of connective tissue, myelodysplasia, Waldenström's macroglobinaemia, nasopharyngeal, neuroendocrine cancer, mesothelioma, angiosarcoma, Kaposi's sarcoma, carcinoid, fallopian tube cancer, peritoneal cancer, papillary serous mullerian cancer, malignant ascites, gastrointestinal stromal tumor (GIST), Li-Fraumeni syndrome, Von Hippel-Lindau syndrome (VHL), and other cancers as described herein, and cancer of unknown origin either primary or metastatic, wherein such cancers may be non-metastatic, invasive, or metastatic.
In some embodiments the present invention provides the use of immunostimulatory anti-HIDE1 immune molecule or composition containing according to at least some embodiments of the present invention in treating and/or detecting or aiding in the diagnosis of cancers that express HIDE1 at levels higher than other cancers.
8. Methods of Using HIDE1 Peptide Compositions
As discussed herein, HIDE1 is involved in the immuno-oncology pathway, which means that manipulating certain signaling pathways can have two different effects. On one hand, the HIDE1 protein suppresses T cell activation and one or more of a number of other pathways, through binding to binding and/or signaling partner. Thus, by inhibiting the interaction of HIDE1 and its binding and/or signaling partner, for example using antibodies to HIDE1, the suppression is alleviated, thereby increasing an immune response to allow treatment of conditions for which a stronger immune response is desired, such as cancer and pathogen infection. This is referred to as a “immuno-stimulatory” response. On the other hand, by increasing the amount of HIDE1 in a patient, such as by adding HIDE1 ECD polypeptides, the suppression is increased, thereby decreasing the immune response to allow treatment of conditions for which a decreased immune response is desired, such as autoimmune diseases and inflammation.
Accordingly, once made, the HIDE1 proteins of the invention find use in a variety of applications, including using them in screening assays for additional immunomodulatory agents, as well as treatment of patients as is more fully outlined below.
With regard to the immuno-stimulatory treatments using anti-HIDE1 antibodies and HIDE1 proteins, reference is made to U.S. Ser. No. 62/191,775, filed Jul. 13, 2015, entitled “ANTI-HIDE1 IMMUNE MOLECULES AND THE USE THEREOF IN THERAPY AND DIAGNOSIS” and U.S. Ser. No. 62/191,804, filed Jul. 13, 2015, entitled “HIDE1 POLYPEPTIDES AND USES THEREOF IN THERAPY”, all of which is expressly incorporated by reference in its entirety herein.
G. Treatment
According to at least some embodiments of the present invention provides methods of treating a number of diseases and/or conditions associated with an immune condition. An “immune condition” includes patients who would benefit from immunostimulatory action, such as cancer or pathogen infection, as well as patients who would benefit from immunoinhibitory action, such as autoimmune diseases and inflammation.
The term “autoimmune disease” as used herein should be understood to encompass any autoimmune disease and further includes chronic inflammatory conditions. In some embodiments, the HIDE1 polypeptides of the invention are used to treat autoimmune diseases. Suitable autoimmune diseases include but are not limited to multiple sclerosis, including relapsing-remitting multiple sclerosis, primary progressive multiple sclerosis, and secondary progressive multiple sclerosis, progressive relapsing multiple sclerosis, chronic progressive multiple sclerosis, transitional/progressive multiple sclerosis, rapidly worsening multiple sclerosis, clinically-definite multiple sclerosis, malignant multiple sclerosis, also known as Marburg's Variant, acute multiple sclerosis, conditions relating to multiple sclerosis, psoriasis, rheumatoid arthritis, psoriatic arthritis, gout and pseudo-gout, juvenile idiopathic arthritis, Still's disease, rheumatoid vasculitis, conditions relating to rheumatoid arthritis, discoid lupus erythematosus, lupus arthritis, lupus pneumonitis, lupus nephritis, conditions relating to systemic lupus erythematosus include osteoarticular tuberculosis, antiphospholipid antibody syndrome, systemic lupus erythematosus (SLE); discoid lupus erythematosus, inflammatory bowel disease, ulcerative colitis, Crohn's disease, benign lymphocytic angiitis, thrombocytopenic purpura, idiopathic thrombocytopenia, idiopathic autoimmune hemolytic anemia, pure red cell aplasia, Sjögren's syndrome, rheumatic disease, connective tissue disease, inflammatory rheumatism, degenerative rheumatism, extra-articular rheumatism, juvenile rheumatoid arthritis, arthritis uratica, muscular rheumatism, chronic polyarthritis, polymyalgia rheumatica, mixed connective tissue disease, systemic juvenile idiopathic arthritis, reactive arthritis, cryoglobulinemic vasculitis, ANCA-associated vasculitis, antiphospholipid syndrome, myasthenia gravis, autoimmune hemolytic anaemia, Guillain-Barré syndrome, autoimmune thyroiditis, Hashimoto's thyroiditis, primary myxedema, sympathetic ophthalmia, autoimmune uveitis, anterior uveitis (or iridocyclitis), intermediate uveitis (pars planitis), posterior uveitis (or chorioretinitis), panuveitic form, hepatitis, chronic action hepatitis, collagen diseases, ankylosing spondylitis, periarthritis humeroscapularis, panarteritis nodosa, chondrocalcinosis, Wegener's granulomatosis, microscopic polyangiitis, chronic urticaria, bullous skin disorders, pemphigoid, bullous pemphigoid, cicatricial pemphigoid, vitiligo, atopic eczema, eczema, chronic urticaria, autoimmune urticaria, normocomplementemic urticarial vasculitis, hypocomplementemic urticarial vasculitis, autoimmune lymphoproliferative syndrome, Devic's disease, sarcoidosis, pernicious anemia, childhood autoimmune hemolytic anemia, idiopathic autoimmune hemolytic anemia, Refractory or chronic Autoimmune Cytopenias, Prevention of development of Autoimmune Anti-Factor VIII Antibodies in Acquired Hemophilia A, Cold Agglutinin Disease, Neuromyelitis Optica, Stiff Person Syndrome, gingivitis, periodontitis, pancreatitis, myocarditis, vasculitis, gastritis, gout, gouty arthritis, and inflammatory skin disorders, selected from the group consisting of psoriasis, atopic dermatitisrosacea, urticaria, acne, inflammation of various parts of the heart, such as pericarditis, myocarditis, and endocarditis, lung and pleura inflammation, pleuritis, pleural effusion, chronic diffuse interstitial lung disease, pulmonary hypertension, pulmonary emboli, pulmonary hemorrhage, and shrinking lung syndrome, lupus headache, idiopathic pericarditis, myositis, demyelinating syndrome, mononeuropathy, mononeuritis multiplex, myelopathy, cranial neuropathy, polyneuropathy, collagenous colitis, lymphocytic colitis, ischaemia colitis, diversion colitis, indeterminate colitis, idiopathic autoimmune hemolytic anemia, anti-synthetase syndrome, scleritis, macrophage activation syndrome, Behçet's Syndrome, PAPA Syndrome, Blau's Syndrome, gout, gouty arthritis, inflammatory skin disorders selected from the group consisting of psoriasis, Non pustular Psoriasis including Psoriasis vulgaris and Psoriatic erythroderma (erythrodermic psoriasis), Pustular psoriasis including Generalized pustular psoriasis (pustular psoriasis of von Zumbusch), Pustulosis palmaris et plantaris (persistent palmoplantar pustulosis, pustular psoriasis of the Barber type, pustular psoriasis of the extremities), Annular pustular psoriasis, Acrodermatitis continua, Impetigo herpetiformis, drug-induced psoriasis, Inverse psoriasis, Napkin psoriasis, Seborrheic-like psoriasis, Guttate psoriasis, Nail psoriasis, adult and juvenile Still's disease, cryropyrinopathy, chronic immune polyneuropathy, idiopathic diabetes, juvenile type 1 diabetes, maturity onset diabetes of the young, latent autoimmune diabetes in adults, gestational diabetes, conditions relating to type 1 diabetes, membranous glomerulonephropathy, autoimmune gastritis, Muckle-Wells syndrome, familial cold-induced auto-inflammatory syndrome, neonatal onset multisystemic inflammatory disease, familial Mediterranean fever, chronic infantile neurologic, cutaneous and articular syndrome, Reiter's syndrome, rheumatic fever, relapsing polychondritis, Raynaud's phenomenon, vasculitis, cryoglobulinemic vasculitis, temporal arteritis, giant cell arteritis, Takayasu arteritis, Behçet's disease, chronic inflammatory demyelinating polyneuropathy, insulin dependent diabetes mellitus, type I diabetes, Addison's disease, membranous glomerulonephropathy, polyglandular autoimmune syndromes, Goodpasture's disease, autoimmune gastritis, autoimmune atrophic gastritis, pernicious anaemia, pemphigus, pemphigus vulgaris, cirrhosis, primary biliary cirrhosis, idiopathic pulmonary fibrosis, myositis, dermatomyositis, juvenile dermatomyositis, polymyositis, fibromyositis, myogelosis, celiac disease, celiac sprue dermatitis, immunoglobulin A nephropathy, Henoch-Schönlein purpura, Evans syndrome, dermatitis, atopic dermatitis, psoriasis, psoriasis vulgaris, psoriasis arthropathica, Graves' disease, Graves' ophthalmopathy, scleroderma, systemic scleroderma, progressive systemic scleroderma, diffuse scleroderma, localized scleroderma, Crest syndrome, asthma, allergic asthma, allergy, primary biliary cirrhosis, fibromyalgia, chronic fatigue and immune dysfunction syndrome (CFIDS), autoimmune inner ear disease, Hyper IgD syndrome, Schnitzler's syndrome, autoimmune retinopathy, age-related macular degeneration, atherosclerosis, chronic prostatitis, alopecia, alopecia areata, alopecia universalis, alopecia totalis, autoimmune thrombocytopenic purpura, idiopathic thrombocytopenic purpura, pure red cell aplasia, and TNF receptor-associated periodic syndrome (TRAPS).
In some preferred embodiments, the autoimmune disease includes but is not limited to any of the types and subtypes of any of multiple sclerosis, rheumatoid arthritis, type I diabetes, psoriasis, systemic lupus erythematosus, inflammatory bowel disease, uveitis, or Sjögren's syndrome or related conditions thereof.
Of particular interest in some embodiments is the treatment of rheumatoid arthritis, lupus, inflammatory bowel disease, psoriasis, multiple sclerosis and diabetes type I.
As described herein, HIDE1 polypeptides which modulate (preferably inhibit) immunity may optionally be used to treat or detect “immune related diseases (or disorders or conditions)”. These phrases or terms are used interchangeably and encompass any disease, disorder or condition selected from the group including but not limited to autoimmune diseases, inflammatory disorders, allergic disorders, e.g., chronic allergic disorders such as asthma, and immune disorders associated with graft transplantation rejection, such as acute and chronic rejection of organ or tissue transplantation, allogenic stem cell transplantation, autologous stem cell transplantation, bone marrow transplantation, and graft versus host disease.
As further noted, the present HIDE1 polypeptides which modulate (preferably inhibit) immunity may optionally be used to treat “inflammatory disorders” and/or “inflammation”. These phrases or terms are used interchangeably herein and include e.g., inflammatory abnormalities characterized by dysregulated immune response to harmful stimuli, such as pathogens, damaged cells, or irritants. Inflammatory disorders underlie a vast variety of human diseases. Diseases with etiological origins in inflammatory processes include but are not limited to atherosclerosis, inflammatory destruction of neurons and axons and ischemic heart disease. Examples of disorders associated with inflammation include but are not limited to: Glomerulonephritis, Hypersensitivities, Pelvic inflammatory disease, Reperfusion injury, Sarcoidosis, Interstitial cystitis, normocomplementemic urticarial vasculitis, pericarditis, myositis, anti-synthetase syndrome, scleritis, macrophage activation syndrome, Behçet's Syndrome, PAPA Syndrome, Blau's Syndrome, gout, adult and juvenile Still's disease, cryropyrinopathy, Muckle-Wells syndrome, familial cold-induced auto-inflammatory syndrome, neonatal onset multisystemic inflammatory disease, familial Mediterranean fever, chronic infantile neurologic, cutaneous and articular syndrome, systemic juvenile idiopathic arthritis, Hyper IgD syndrome, Schnitzler's syndrome, TNF receptor-associated periodic syndrome (TRAPSP), gingivitis, periodontitis, hepatitis, cirrhosis, pancreatitis, myocarditis, inflammatory skin disorders, selected from the group consisting of psoriasis, atopic dermatitis, eczema, rosacea, urticaria, and acne.
In some embodiments the treatment may be optionally combined with another moiety useful for treating immune related condition, e.g., a moiety useful for treating immune related condition is selected from immunosuppressants such as corticosteroids, cyclosporin, cyclophosphamide, prednisone, azathioprine, methotrexate, rapamycin, tacrolimus, leflunomide or an analog thereof; mizoribine; mycophenolic acid; mycophenolate mofetil; 15-deoxyspergualine or an analog thereof; biological agents such as TNF-α blockers or antagonists, or any other biological agent targeting any inflammatory cytokine, nonsteroidal antiinflammatory drugs/Cox-2 inhibitors, hydroxychloroquine, sulphasalazopryine, gold salts, etanercept, infliximab, mycophenolate mofetil, basiliximab, atacicept, rituximab, cytoxan, interferon β-1a, interferon β-1b, glatiramer acetate, mitoxantrone hydrochloride, anakinra and/or other biologics and/or intravenous immunoglobulin (IVIG), interferons such as IFN-β-1a (REBIF®, AVONEX® and CINNOVEX®) and IFN-β-1b (BETASERON®); EXTAVIA®, BETAFERON®, ZIFERON®); glatiramer acetate (COPAXONE®), a polypeptide; natalizumab (TYSABRI®), mitoxantrone (NOVANTRONE®), a cytotoxic agent, a calcineurin inhibitor, e.g., cyclosporin A or FK506; an immunosuppressive macrolide, e.g. rapamycin or a derivative thereof; e.g. 40—O-(2-hydroxy) ethyl-rapamycin, a lymphocyte homing agent, e.g. FTY720 or an analog thereof, corticosteroids; cyclophosphamide; azathioprene; methotrexate; leflunomide or an analog thereof; mizoribine; mycophenolic acid; mycophenolate mofetil; 15-deoxyspergualine or an analog thereof; immunosuppressive monoclonal antibodies, e.g., monoclonal antibodies to leukocyte receptors, e.g., MEW, CD2, CD3, CD4, CD11a/CD18, CD7, CD25, CD27, B7, CD40, CD45, CD58, CD137, ICOS, CD150 (SLAM), OX40, 4-1BB or their ligands; or other immunomodulatory compounds, e.g. CTLA4-Ig (abatacept, ORENCIA®, belatacept), CD28-Ig, B7-H4-Ig, or other costimulatory agents, or adhesion molecule inhibitors, e.g. mAbs or low molecular weight inhibitors including LFA-1 antagonists, Selectin antagonists and VLA-4 antagonists, or another immunomodulatory agent.
According to at least some embodiments of the present invention, the method of immunotherapy in a patient optionally, comprises one of the following:
According to at least some embodiments, any one of the foregoing therapeutic agents according to the present invention can be used for adoptive immunotherapy. Immune tolerance or immunological tolerance or prolonged immunosuppression is the process by which the immune system does not attack an antigen. It can be either ‘natural’ or ‘self-tolerance’, where the body does not mount an immune response to self-antigens, or ‘induced tolerance’, where tolerance to external antigens can be created by manipulating the immune system. It occurs in three forms: central tolerance, peripheral tolerance and acquired tolerance. Without wishing to be bound by a single theory, tolerance employs regulatory immune cells—including Tregs—or potentially other immunosuppressive cells such as MDSCs, iMSCs, monocytes, neutrophils, macrophages, that directly suppress autoreactive cells, as well as several other immune cell subsets with immunoregulatory properties—including CD8+ T cells and other types of CD4+ T cells (Tr1, Th3), Th17 cells, in addition to natural killer (NK), NKT cells, dendritic cells (DC) and B cells.
Tolerance or prolonged immunosuppression can be induced by blocking costimulation or upon engagement of a co-inhibitory B7 with its counter receptor. Transfer of tolerance involves isolation of the cells that have been induced for tolerance either in vivo (i.e., prior to cell isolation) or ex-vivo, enrichment and expansion of these cells ex vivo, followed by reinfusion of the expanded cells to the patient. This method can be used for treatment of autoimmune diseases as recited herein, immune related diseases as recited herein, transplantation and graft rejection. Thus, according to at least some embodiments, the present invention optionally provides methods for tolerance induction, comprising in vivo or ex vivo treatment administration of effective amount of any one of isolated soluble HIDE1 polypeptide, or a polypeptide comprising the extracellular domain of HIDE1, or fragment thereof, or a fusion thereof to a heterologous sequence, to a patient or to leukocytes isolated from the patient, in order to induce differentiation of tolerogenic regulatory cells, followed by ex-vivo enrichment and expansion of said cells and reinfusion of the tolerogenic regulatory cells to said patient.
1. Treating by Inhibiting the Interaction of HIDE1 and the HIDE1 Binding and/or Signaling Partner
In some embodiments, the invention provides methods of treating subjects by inhibiting the interaction of HIDE1 and the HIDE1 binding and/or signaling partner. As both HIDE1 and the HIDE1 binding and/or signaling partner contain transmembrane domain, the inhibition can be done by preventing the binding of the two, such as by using anti-HIDE1 antibodies. Alternately, by administering a soluble HIDE1 polypeptide, that will interact with the HIDE1 binding and/or signaling partner, preventing it from binding to the membrane bound endogenous HIDE1, thus preventing signaling (either by the loss of signaling due to the lack of HIDE1 signaling, or by the prevention of free HIDE1 binding and/or signaling partner binding to another of its signaling partners).
In some embodiments, the invention provides methods of treating patients by modulating the interaction of HIDE1 and the HIDE1 binding and/or signaling partner by administering a HIDE1 polypeptide as outlined herein.
In some embodiments, the invention provides methods of treating patients by inhibiting the Interaction of HIDE1 and the HIDE1 binding and/or signaling partner by inhibiting the binding of HIDE1 and the HIDE1 binding and/or signaling partner by administering an anti-HIDE1 antibody.
According to at least some embodiments, HIDE1 therapeutic agents and/or a pharmaceutical composition comprising same, as described herein, which function as HIDE1 agonizing therapeutic agents, may optionally be used for treating an immune system related disease. In some instances the immune system related condition comprises an immune related condition, including but not limited to autoimmune, inflammatory or allergic diseases such as recited herein, transplant rejection and graft versus host disease.
In some instances the immune condition is selected from autoimmune disease, inflammatory disease, allergic disease, transplant rejection, undesired gene or cell therapy immune responses, or graft versus host disease.
According to at least some embodiments, HIDE1 therapeutic agents and/or a pharmaceutical composition comprising same, as described herein, which function as HIDE1 agonizing therapeutic agents, may optionally be used for treating an immune system related disease. In some instances the immune system related condition comprises an immune related condition, including but not limited to autoimmune, inflammatory or allergic diseases such as recited herein, transplant rejection and graft versus host disease.
In some instances the immune condition is selected from autoimmune disease, inflammatory disease, allergic disease, transplant rejection, undesired gene or cell therapy immune responses, or graft versus host disease.
In some embodiments the treatment is combined with another moiety useful for treating immune related condition. Non limiting examples thereof include immunosuppressants such as corticosteroids, cyclosporin, cyclophosphamide, prednisone, azathioprine, methotrexate, rapamycin, tacrolimus, leflunomide or an analog thereof; mizoribine; mycophenolic acid; mycophenolate mofetil; 15-deoxyspergualine or an analog thereof; biological agents such as TNF-α blockers or antagonists, or any other biological agent targeting any inflammatory cytokine, nonsteroidal antiinflammatory drugs/Cox-2 inhibitors, hydroxychloroquine, sulphasalazopryine, gold salts, etanercept, infliximab, mycophenolate mofetil, basiliximab, atacicept, rituximab, cytoxan, interferon β-1a, interferon β-1b, glatiramer acetate, mitoxantrone hydrochloride, anakinra and/or other biologics and/or intravenous immunoglobulin (IVIG), interferons such as IFN-β1a (REBIF®. AVONEX® and CINNOVEX®) and IFN-β1b (BETASERON®); EXTAVIA®, BETAFERON®, ZIFERON®); glatiramer acetate (COPAXONE®), a polypeptide; natalizumab (TYSABRI®), mitoxantrone (NOVANTRONE®), a cytotoxic agent, a calcineurin inhibitor, e.g. Cyclosporin A or FK506; an immunosuppressive macrolide, e.g. Rapamycin or a derivative thereof; e.g. 40—O-(2-hydroxy)ethyl-rapamycin, a lymphocyte homing agent, e.g. FTY720 or an analog thereof, corticosteroids; cyclophosphamide; azathioprene; methotrexate; leflunomide or an analog thereof; mizoribine; mycophenolic acid; mycophenolate mofetil; 15-deoxyspergualine or an analog thereof; immunosuppressive monoclonal antibodies, e.g., monoclonal antibodies to leukocyte receptors, e.g., MEW, CD2, CD3, CD4, CD11a/CD18, CD7, CD25, CD27, B7, CD40, CD45, CD58, CD137, ICOS, CD150 (SLAM), OX40, 4-1BB or their ligands; or other immunomodulatory compounds, e.g. CTLA4-Ig (abatacept, ORENCIA®, belatacept), CD28-Ig, B7-H4-Ig, or other costimulatory agents, or adhesion molecule inhibitors, e.g. mAbs or low molecular weight inhibitors including LFA-1 antagonists, Selectin antagonists and VLA-4 antagonists, or another immunomodulatory agent.
In particular, treatment of multiple sclerosis using HIDE1 immunoinhibitory proteins according to the various embodiments of the present invention may optionally e.g., be combined with, any therapeutic agent or method suitable for treating multiple sclerosis. Non-limiting examples of such known therapeutic agent or method for treating multiple sclerosis include interferon class, IFN-β-1a (REBIF®. AVONEX® and CINNOVEX®) and IFN-β-1b (BETASERON®, EXTAVIA®, BETAFERON®, ZIFERON®); glatiramer acetate (COPAXONE®), a polypeptide; natalizumab (TYSABRI®); and mitoxantrone (NOVANTRONE®), a cytotoxic agent, Fampridine (AMPYRA®). Other drugs include corticosteroids, methotrexate, cyclophosphamide, azathioprine, and intravenous immunoglobulin (IVIG), inosine, Ocrelizumab (R1594), Mylinax (Caldribine®), alemtuzumab (Campath®), daclizumab (Zenapax®), Panaclar/dimethyl fumarate (BG-12), Teriflunomide (HMR1726), fingolimod (FTY720), laquinimod (ABR216062), as well as Hematopoietic stem cell transplantation, NeuroVax®, Rituximab (Rituxan®) BCG vaccine, low dose naltrexone, helminthic therapy, angioplasty, venous stents, and alternative therapy, such as vitamin D, polyunsaturated fats, medical marijuana.
Similarly, treatment of rheumatoid arthritis, using HIDE1 immunoinhibitory proteins according to the various embodiments of the present invention may optionally be combined with, for example, any therapeutic agent or method suitable for treating rheumatoid arthritis. Non-limiting examples of such known therapeutic agents or methods for treating rheumatoid arthritis include glucocorticoids, nonsteroidal anti-inflammatory drug (NSAID) such as salicylates, or cyclooxygenase-2 inhibitors, ibuprofen and naproxen, diclofenac, indomethacin, etodolac Disease-modifying antirheumatic drugs (DMARDs)-Oral DMARDs: Auranofin (Ridaura®), Azathioprine (Imuran®), Cyclosporine (Sandimmune®, Gengraf, Neoral, generic), D-Penicillamine (Cuprimine), Hydroxychloroquine (Plaquenil®), IM gold Gold sodium thiomalate (Myochrysine®) Aurothioglucose (Solganal®), Leflunomide (Arava®), Methotrexate (Rheumatrex®), Minocycline (Minocin®), Staphylococcal protein A immunoadsorption (Prosorba column), Sulfasalazine (Azulfidine®). Biologic DMARDs: TNF-α blockers including Adalimumab (Humira®) Etanercept (Enbrel®), Infliximab (Remicade®), golimumab (Simponi®), certolizumab pegol (Cimzia®), and other biological DMARDs, such as Anakinra (Kineret®), Rituximab (Rituxan®), Tocilizumab (Actemra®), CD28 inhibitor including Abatacept (Orencia®) and Belatacept.
Thus, treatment of IBD, using the agents according to at least some embodiments of the present invention may optionally be combined with, for example, any known therapeutic agent or method for treating IBD. Non-limiting examples of such known therapeutic agents or methods for treating IBD include immunosuppression to control the symptom, such as prednisone, Mesalazine (including Asacol®, Pentasa®, Lialda®, Aspiro®, azathioprine (Imuran®), methotrexate, or 6-mercaptopurine, steroids, Ondansetron®, TNF-α blockers (including infliximab, adalimumab golimumab, certolizumab pegol), Orencia® (abatacept), ustekinumab (Stelara®), Briakinumab (ABT-874), Certolizumab pegol (Cimzia®), ITF2357 (Givinostat®), Natalizumab (Tysabri®), Firategrast® (SB-683699), Remicade® (infliximab), vedolizumab (MLN0002), other drugs including GSK1605786 CCX282-B (Traficet-EN®), AJM300, Stelara® (ustekinumab), Semapimod® (CNI-1493) tasocitinib (CP-690550), LMW Heparin MMX, Budesonide MMX, Simponi® (golimumab), MultiStem®, Gardasil® HPV vaccine, Epaxal® (virosomal hepatitis A vaccine), surgery, such as bowel resection, strictureplasty or a temporary or permanent colostomy or ileostomy; antifungal drugs such as nystatin (a broad spectrum gut antifungal) and either itraconazole (Sporanox) or fluconazole (Diflucan); alternative medicine, prebiotics and probiotics, cannabis, Helminthic therapy or ova of the Trichuris suis helminth.
Thus, treatment of psoriasis, using the agents according to at least some embodiments of the present invention may optionally be combined with, for example, any known therapeutic agent or method for treating psoriasis. Non-limiting examples of such known therapeutics for treating psoriasis include topical agents, typically used for mild disease, phototherapy for moderate disease, and systemic agents for severe disease. Non-limiting examples of topical agents: bath solutions and moisturizers, mineral oil, and petroleum jelly; ointment and creams containing coal tar, dithranol (anthralin), corticosteroids like desoximetasone (Topicort), Betamethasone, fluocinonide, vitamin D3 analogues (for example, calcipotriol), and retinoids. Non-limiting examples of phototherapy: sunlight; wavelengths of 311-313 nm, psoralen and ultraviolet A phototherapy (PUVA). Non-limiting examples of systemic agents: biologics, such as interleukin antagonists, TNF-α blockers including antibodies such as infliximab (Remicade®), adalimumab (Humira®), golimumab, certolizumab pegol, and recombinant TNF-α decoy receptor, etanercept (Enbrel®); drugs that target T cells, such as efalizumab (Xannelim®/Raptiva®), alefacept (Ameviv®), dendritic cells such Efalizumab; monoclonal antibodies (MAbs) targeting cytokines, including anti-IL-12/IL-23 (ustekinumab (Stelara®)) and anti-Interleukin-17; Briakinumab® (ABT-874); small molecules, including but not limited to ISA247; immunosuppressants, such as methotrexate, cyclosporine; vitamin A and retinoids (synthetic forms of vitamin A); and alternative therapy, such as changes in diet and lifestyle, fasting periods, low energy diets and vegetarian diets, diets supplemented with fish oil rich in vitamin A and vitamin D (such as cod liver oil), fish oils rich in the two omega-3 fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) and contain vitamin E, ichthyotherapy, hypnotherapy, and cannabis.
Thus, treatment of type 1 diabetes, using the agents according to at least some embodiments of the present invention may optionally be combined with, for example, any known therapeutic agent or method for treating type 1 diabetes. Non-limiting examples of such known therapeutics for treating type 1 diabetes include insulin, insulin analogs, islet transplantation, stem cell therapy including PROCHYMAL®, non-insulin therapies such as i1-1β inhibitors including Anakinra (Kineret®), Abatacept (Orencia®), Diamyd, alefacept (Ameviv®), Otelixizumab, DiaPep277 (Hsp60 derived peptide), a 1-Antitrypsin, Prednisone, azathioprine, and Cyclosporin, E1-INT (an injectable islet neogenesis therapy comprising an epidermal growth factor analog and a gastrin analog), statins including Zocor®, Simlup®, Simcard®, Simvacor®, and Sitagliptin® (dipeptidyl peptidase (DPP-4) inhibitor), anti-CD3 mAb (e.g., Teplizumab®); CTLA4-Ig (abatacept), anti-IL-1β (Canakinumab), Anti-CD20 mAb (e.g., rituximab) and combinations thereof.
Thus, treatment of uveitis, using the agents according to at least some embodiments of the present invention may optionally be combined with, for example, any known therapeutic agent or method for treating uveitis. Non-limiting examples of such known therapeutics for treating uveitis include corticosteroids, topical cycloplegics, such as atropine or homatropine, or injection of PSTTA (posterior subtenon triamcinolone acetate), antimetabolite medications, such as methotrexate, TNF-α blockers (including infliximab, adalimumab, etanercept, golimumab, and certolizumab pegol).
Thus, treatment for Sjögren's syndrome, using the agents according to at least some embodiments of the present invention may optionally be combined with, for example, any known therapeutic agent or method for treating for Sjögren's syndrome. Non-limiting examples of such known therapeutics for treating for Sjögren's syndrome include Cyclosporine, pilocarpine (Salagen®) and cevimeline (Evoxac®), Hydroxychloroquine (Plaquenil), cortisone (prednisone and others) and/or azathioprine (Imuran®) or cyclophosphamide (Cytoxan®), Dexamethasone, Thalidomide, Dehydroepiandrosterone, NGX267, Rebamipide®, FID 114657, Etanercept®, Raptiva®, Belimumab, MabThera® (rituximab); Anakinra®, intravenous immune globulin (IVIG), Allogeneic Mesenchymal Stem Cells (AlloMSC®), and Automatic neuro-electrostimulation by “Saliwell Crown”.
Thus, treatment for systemic lupus erythematosus, using the agents according to at least some embodiments of the present invention may optionally be combined with, for example, any known therapeutic agent or method for treating for systemic lupus erythematosus. Non-limiting examples of such known therapeutics for treating for systemic lupus erythematosus include corticosteroids and Disease-modifying antirheumatic drugs (DMARD5), commonly anti-malarial drugs such as plaquenil and immunosuppressants (e.g. methotrexate and azathioprine) Hydroxychloroquine, cytotoxic drugs (e.g., cyclophosphamide and mycophenolate), Hydroxychloroquine (HCQ), Benlysta® (belimumab), nonsteroidal anti-inflammatory drugs, Prednisone, Cellcept®, Prograf®, Atacicept®, Lupuzor®, Intravenous Immunoglobulins (IVIGs), CellCept® (mycophenolate mofetil), Orencia®, CTLA4-IgG4m (RG2077), rituximab, Ocrelizumab, Epratuzumab, CNTO 136, Sifalimumab (MEDI-545), A-623 (formerly AMG 623), AMG 557, Rontalizumab, paquinimod (ABR-215757), LY2127399, CEP-33457, Dehydroepiandrosterone, Levothyroxine, abetimus sodium (LJP 394), Memantine®, Opiates, Rapamycin®, renal transplantation, stem cell transplantation and combinations of any of the foregoing.
The immunoinhibitory HIDE1 therapeutic agents and/or a pharmaceutical composition comprising same, as recited herein, according to at least some embodiments of the present invention, may optionally be administered as the sole active ingredient or together with other drugs in immunomodulating regimens or other anti-inflammatory agents e.g. for the treatment or prevention of allo- or xenograft acute or chronic rejection or inflammatory or autoimmune disorders, or to induce tolerance.
Specifically incorporated by reference herein is U.S. Ser. No. 62/191,775, filed Jul. 13, 2015, and U.S. Ser. No. 62/191,804, filed Jul. 13, 2015, in their entirety, and in particular for the Examples therein, and for the associated Figures and Legends.
The presented dry examples 2-6 below suggest a set of functional assays to evaluate the effect of anti-HIDE1 on T cell function. In particular, the suggested assays will be used to evaluate the immuno-modulatory effect of anti-HIDE1 antibodies (agnostic or antagonistic) that can be used to target monocytes, Tumor associated macrophages (TAMs) or other myeloid cells and screen for various functional activities, including modulating the interaction between HIDE-1 and its putative receptor(s), modulation of HIDE1 levels or direct signaling and attenuation of negative signaling and/or depletion of HIDE1 positive cells. Such recombinant antibodies may be used as modulatory molecules to decrease or prevent HIDE1 from interacting with inhibitory receptor(s) on T cells or other cells in the tumor microenvironment, thereby releasing T cells or other functional cells from HIDE1 check point (“break”)/suppressive signaling
A mixed lymphocyte reaction will be employed to demonstrate the effect of blocking the HIDE1 pathway to lymphocyte effector cells. T cells in the assay will be tested for proliferation and cytokine secretion in the presence or absence of an anti-HIDE1 human monoclonal antibodies. Human CD4+ or CD8+ T-cells will be purified from PBMC using a CD4+ or CD8+. Alloreactivite dendritic cells will be derived from purified monocytes cultured with 1000 U/ml of IL-4 and 500 U/ml of GM-CSF (R&D Biosystems) for seven days. Monocytes will be prepared using a monocyte negative selection kit (Mitenyi Biotech). Each culture will contain 105 purified T-cells and 104 allogeneic dendritic cells in a total volume of 200 μl. Anti-HIDE1 blocking or agonistic mAb at varying concentrations will be added to each culture at different antibody concentrations. Either no antibody or an isotype control antibody will be used as a negative control. After day 5, the effect of anti-HIDE1 antibodies on T cell proliferation (CFSE dilution) and cytokine secretion (ELISA or TH1/2/17 CBA kits) in culture supernatants will be assessed.
96-well flat-bottom plates will be coated with mouse anti-human CD3 antibody (1 μg/ml in PBS; Clone HIT3a; BD Pharmingen Cat 555336) overnight at 4° C. The next day, Jurkat cells (50,000) or CFSE-labeled primary human CD4+ or CD8+ T cells will be plated in the pre-coated plates. Mytomicin C treated (50 μg/ml, 1 hr) THP-1 cells, which express HIDE1, (50,000) will be added to the culture in the presence of HIDE1 blocking or agonistic mAb at varying concentrations. After 1-5d at 37° C. and 5.0% CO2, the effect of anti-HIDE1 antibodies on T cell proliferation (CFSE dilution) and cytokine secretion (ELISA or TH1/2/17 CBA kits) in culture supernatants will be assessed.
Purified human T cells will be labeled with CFSE and cultured with autologous monocyte-derived DCs in the presence of tetanus toxoid (TT) or CMV antigens. The effect of the inclusion of HIDE1 blocking or agonistic mAb on TT or CMV specific T cell response will be evaluated. After 5-10d at 37° C. and 5.0% CO2, the effect of anti-HIDE1 antibodies on T cell proliferation (CFSE dilution) and cytokine secretion (ELISA or TH1/2/17 CBA kits) in culture supernatants will be assessed.
Purified human T cells will be CFSE labeled and stimulated with anti-CD3 Ab (OKT3, 1 μg/ml) together with plate-coated HIDE1-Fc or control protein (FLAG-Fc). Control or HIDE1 mAb was added during cell culture. Cells were gated on CD8+ T cells, and their division was analyzed based on the dilution of CFSE. Inclusion of a HIDE1 neutralizing mAb is expected to enhance the T cell function which will indicate that HIDE1 interacts with putative receptor(s) expressed on T cells to inhibit T cell proliferation.
CFSE-labeled T cells will be stimulated with stimulator cells (CHO cells expressing expressing membrane-bound anti-CD3 mAb fragments). CHOS-stimulator cells expressing human HIDE1 and control stimulator cells (empty vector) treated with mitomycin C before co-cultured with CFSE-labeled human T cells at the ratio of 1:5. After 5d at 37° C. and 5.0% CO2, the effect of anti-HIDE1 antibodies on T cell proliferation (CFSE dilution) and cytokine secretion (ELISA or TH1/2/17 CBA kits) in culture supernatants will be assessed.
The purpose of this example was to demonstrate that HIDE1 is correlated to CSF1R expression in cancer samples of both human and mouse. Solid tumors contain a significant population of tumor-infiltrating myeloid cells (TIM). TIMs are now recognized as important mediators of not only tumor progression and metastasis, but also therapeutic resistance, through promoting angiogenesis and suppressing antitumor immune responses. The pro-tumorigenic role of “alternatively” activated macrophages, which are part of the TIM population, has been well established. Recently, another specific subtype of TIMs, namely myeloid-derived suppressor cells (MDSC; (Myeloid-Derived Suppressor Cells—an additional population of myeloid cells that is tumor promoting Journal for ImmunoTherapy of Cancer 2013, 1:10)), is receiving great attention in cancer research. MDSCs comprise a heterogeneous population of immature myeloid cells that originate in the bone marrow and are recruited to the tumor by a diverse array of cytokine and chemokine signals. Similar to tumor-associated macrophages (TAM), MDSCs have been shown to generate an environment favorable for tumors by heightening immunosuppression, angiogenesis, and invasion. Various cell surface markers are used to identify TIM subsets: TAMs can be identified by CD11b and F4/80, and MDSCs by CD11b and Gr-1 coexpression in murine models. Macrophage colony-stimulating factor (M-CSF or CSF1) is a potent growth factor that promotes the differentiation, proliferation, and migration of monocytes/macrophages via signaling through its receptor tyrosine kinase CSF1R (cFMS). It was recently showed that TAMs and MDSCs form a spectrum of bone marrow-derived myeloid cells dependent on CSF1/CSF1R signaling for recruitment into the tumor and that they play critical roles in tumor growth. In addition, DeNardo and colleagues highlighted the importance of CSF1/CSF1R signaling in the recruitment of TAMs in various cancers and further showed that CSF1R blockade can inhibit TAMs and improve treatment outcome (Cancer Res. 2013 May 1; 73(9):2782-94).
As can be seen in Table 18, HIDE1 was found to be highly correlated to CSF1R in various mouse tumor models. This finding was also validated in human tumors by correlating HIDE1 and CSF1R using The Cancer Genome Atlas (TCGA) (http://cancergenome.nih.gov/) data.
The analysis was performed using both human and mouse tumor expression data sets from Gene Expression Omnibus (GEO) (www.ncbi.nlm.nih.gov/GEO, the platform used were GPL570, GPL6244 for human, and GPL1261 and GPL6246 for mouse). The methodology of analysis is described in the “methodology section”. HIDE1 was found to be highly correlated to CSF1R and is a type I membrane protein (http://www.uniprot.org/uniprot/A8MVS5) in murine tumors. Table 18 shows the correlation of AB124611 (the murine orthologue of human HIDE1) in several cancer models in mice.
Raw data is downloaded from the GEO site in SOFT format. In case that the raw data is in MASS format data is taken without manipulation. If the data is Log MASS then the Log is powered to linear data. If the data is in RMA format CEL files are downloaded and re analyzed using MASS. If such data is not available the RMA format is used.
Data is then normalized by multiplicative according to the 95th percentile for Affy data.
Datasets analyzed: GSE49910, GSE47855, GSE39397, GSE36765, GSE27928, GDS4343, GDS4617, GDS4371, GDS3953, GSE35398, GSE21927.
The purpose of this example is to present the involvement of HIDE1 in autoimmune disorders.
The role of myeloid cells in autoimmune disease has been elucidated in recent years. Of the myeloid cell population Myeloid-Derived Suppressor Cells (MDSCs) seem to have a major role in autoimmunity. Understanding of the origination and functions of MDSCs has come mainly from studies in tumor models and from cancer patients. MDSCs are involved in a number of different autoimmune disorders, including multiple sclerosis (MS), type 1 diabetes, rheumatoid arthritis (RA), inflammatory bowel disease (IBD) and autoimmune hepatitis. In steady state conditions, MDSCs reside primarily in the bone marrow. Under pathological conditions, MDSC populations expand and can be detected in the spleen, lymph nodes, cancerous tumors, and bloodstream (World J Immunol 2014 Mar. 27; 4(1): 26-33). In the chronic inflammatory condition of IBD, there are complex interactions between several immune cells infiltrating into the intestinal mucosa, with epithelial cells even ignoring the effects of microbiota. Among them, myeloid cells, including neutrophils, macrophages, and MDSCs, have been a focus of study due to their divergent role in inflammation. In particular, the immunosuppressive function of MDSCs was suggested in several mouse models of IBD. It was reported that CD11b+Gr-1+ MDSCs were accumulated in a murine colitis model, and they expressed nitric oxide synthase 2 and arginase, which are known to be critical functional mediators of MDSCs. As well as in a model of IBD, CD14+HLA-DRlow MDSCs with suppressive functions were reported to be increased in the peripheral blood of IBD patients (Intest Res. 2015 April; 13(2): 105-111.). The expression of murine HIDE1 is induced in colon tissues derived from 2 different mouse DSS models of IBD (
Expression analysis of the Genotype Tissue Expression (GTEx) data (http://www.nature.com/ng/journal/v45/n6/full/ng.2653.html; http://www.gtexportal.org/home/) showed high expression of HIDE1 in blood cells and tissues with enriched blood cells such as the spleen (
Recombinant stable pools of cell lines overexpressing HIDE1 human proteins were generated, for use in determining the effects of HIDE1 on immunity, for HIDE1 characterization, anti-HIDE1 antibody discovery, and to obtain cross-species anti-HIDE1 immune molecules.
Human HIDE1 Reagents
DNA expression constructs:
Human HIDE1 flag pUC57 (Genscript)
Human HIDE1 flag pMSCV (in house)
Recombinant Cells:
HEK293 Human HIDE1 flag pMSCV (in house)
SK-MEL-5 Human HIDE1 flag pMSCV (in house)
HIDE1 Specific Antibody:
Anti c19orf38 (HIDE1), sigma cat. HPA012150
Rabbit anti human HIDE1 pAb (GenScript, #488536_4)
Mouse anti human HIDE1 mAb (Biotem #33B4-2F7)
Mouse anti human HIDE1 mAb (Biotem #36C1-2F6)
Mouse anti human HIDE1 mAb (Biotem #39A7-3A10-3G8)
Full length cloning of human HIDE1 was performed at Genscript by gene synthesis using codon optimized sequence in pUC57 vector and subcloned into a retroviral expression vector.
Full length cloning of human HIDE1 flag was performed by gene synthesis in pUC57 vector and subcloned into a retroviral expression vector, pMSCV, to create the expression plasmid.
The resulting expression constructs were transduced to cells and stable pool cell lines were generated as detailed below. The protein sequences encoded by the expression constructs are detailed herein.
GP2-293 packaging cell line (Clontech cat#631458) was transfected with pMSCV-human HIDE1 Flag or with pMSCV-empty vector using Lipofectamin 2000 reagent (Invitrogen, cat No: 11668-019). 48 hours post transfection supernatants containing virions were collected, and directly used for infection of PT67 stable virus producing cells (Clontech #631510). 48 hour post infection Puromycin antibiotic was added at concentration of 2 μg/ml, and resistant colonies were selected for stable pool generation. After selection of PT67, stably human HIDE1 virions producing cells, supernatant containing virions were collected and used for infection of HEK293 cell line. 48 hour post infection, Puromycin antibiotic was added at concentration of 1 μg/ml, and resistant colonies were selected for stable pool generation.
GP2-293 packaging cell line (Clontech cat#631458) was transfected with pMSCV− human HIDE1 Flag or with pMSCV− empty vector using Lipofectamin 2000 reagent (Invitrogen, cat No: 11668-019). 48 hours post transfection supernatants containing virions were collected, and directly used for infection of PT67 stable virus producing cells (Clontech #631510). 48 hour post infection Puromycin antibiotic was added at concentration of 2 μg/ml, and resistant colonies were selected for stable pool generation. After selection of PT67, stably human HIDE1 virions producing cells, supernatant containing virions were collected and used for infection of SK-Mel-5 cell line. 48 hour post infection, Puromycin antibiotic was added at concentration of 0.5 μg/ml, and resistant colonies were selected for stable pool generation.
Expression Validation by Flow Cytometry (FACS)
In order to validate the cell surface expression of the human HIDE1 protein in the recombinant stable pools, 2×105 cells were stained with Fixable viability stain 450 (BD, cat #562247) diluted 1:1000 in PBS, for 15 min at R.T. followed by cells washing with PBS. Antibodies were used as following:
In order to verify cell surface expression of human HIDE1 Flag protein, the cells, described above were analyzed by FACS using Rabbit anti human-HIDE1 pAb (GenScript) as described in Material and Methods. As shown in
The surface expression of human HIDE1 Flag protein on HEK293 cells transduced with human HIDE1 Flag pMSCV vector or HEK293 cells transduced with the empty vector were further analyzed using mouse anti-human HIDE1 mAb (Biotem) as described in Material and Methods above. As shown in
A. SK-MEL-5 Cells Overexpressing Human HIDE1-Flag pMSCV Vector
SK-MEL-5 cells transduced with human HIDE1 Flag pMSCV vector or with the empty vector were analyzed by FACS for their cell surface expression using Rabbit anti human-HIDE1 pAb (GenScript) as described in Material and Methods. As shown in
B. Generation and Characterization of Stable Cell Pools Over-Expressing HIDE1 Protein
DNA expression constructs:
Human HIDE1 flag pUC57 (Genscript)
Human HIDE1 flag pCDNA3.1 (Genscript)
Mouse HIDE1 flag pUC57
Mouse HIDE1 flag pCDNA3.1
Mouse HIDE1 flag pMSCV
HEK293 Human HIDE1 flag pCDNA3.1 (GenScript)
HEK293 Mouse HIDE1 flag pMSCV
EL4 Mouse HIDE1 flag pMSCV
Commercial pAb, rabbit anti c19orf38 (HIDE1), sigma cat. HPA012150
Custom pAb, rabbit anti mouse HIDE1 (GenScript, #488536_13)
Full length cloning of human and mouse HIDE1 was performed at Genscript by gene synthesis using codon optimized sequence in pUC57 vector and subcloned into a mammalian or retroviral expression vector.
Full length cloning of human HIDE1 flag was performed by gene synthesis in pUC57 vector and subcloned into a mammalian expression vector, pcDNA3.1, to create the expression plasmid (done by GenScript)
The cloning of mouse HIDE1-Flag retroviral expressing construct is described in example 8 section A.
Full length cloning of cyno HIDE1 untagged was performed by gene synthesis in pUC57 vector and subcloned into a mammalian expression vector, pcDNA3.1, to create the expression plasmid (done by GenScript)
The resulting expression constructs were transfected or transduced to cells and stable pool cell lines were generated as detailed below. The protein sequences encoded by the expression constructs are detailed below.
HEK293 cells over expressing human HIDE1 flag were generated by GenScript
Supernatants from 48 hours culture of PT67 mouse HIDE1 virus producing cells or PT67 pMSCV empty vector were collected and used for infection of HEK293 and EL4 cell lines. Four days post infection, puromycin antibiotic was added at a concentration of 1 μg/ml for HEK293 cells or 4 μg/ml for EL4 cells, and resistant colonies were selected for stable pool generation.
Stable pool of CHO-S transduced with OKT3, were electroporated by AMAXA device with Bug of pcDNA3.1_human HIDE1 Flag construct generated at GS. 24 hr post electroporation, G-418 antibiotic was added at a concentration of 300 μg/ml in order to generate stable pool cells. 2 weeks after selection process was finished, sorting for cells with highly expressed human HIDE1 protein was performed.
The resulting expression constructs were transfected to cells and stable pool cell lines were generated as detailed below. The protein sequences encoded by the expression constructs are detailed below.
MPWTILLLAAGSLAIPRPSIRLVPPHPSNQEDPIHIACMAPGNFLGANFT
LYRGGQVVQILQAHGDQRGVTFNLNGSSSEASGEPFHCQYGVLGELSQPQ
LSDLSEPVNVSFPVPTWILVLSLSLAGAVFLLAGLVAVVLVVRRVKLKNL
QKKRDRESCWAQINFNSPDMSFDNSLFTVSGKTMPEEDPATLDDHSGTTA
TPSNSRTRKRPTSTSSLPEIPEFSTFRACQ
HEK-293 (ATCC, CRL-1573) cells were transfected with each of the constructs described above or with the empty vector (pcDNA3.1) as negative control, using polyplus JetPrime, transfection reagent (polyplus transfection, catalog number 114-15). Geneticin, G418 (Gibco, catalog number: 11811-031) resistant colonies were selected for stable pool generation.
Whole cell extracts of cells transfected or transduced with human or mouse HIDE1 protein (35 μg of total protein) were analyzed for HIDE1 protein expression by WB. As a negative control, whole cell extracts of cells transfected or transduced with an empty vector were used.
Antibodies were used as following:
The polyclonal antibodies described above were followed by a secondary antibody goat anti-Rabbit—Peroxidase (Jackson, cat no. 111-035-003) diluted 1:20,000 in 5% milk/TTBS solution.
In order to validate the cell surface expression of the human and mouse HIDE1 protein in the recombinant stable pools, 2×105 cells were stained with Fixable viability stain 450 (BD, cat #562247) diluted 1:1000 in PBS, for 10 min at R.T. followed by cells washing with PBS. Antibodies were used as following:
HEK293 Cells Over Expressing Human HIDE1-Flag pCDNA3.1 Vector (GenScript):
To verify expression of the HIDE1 protein in the stably transfected HEK293 pools (generated by GenScript), whole cell extracts were analyzed by WB using anti-flag antibody and commercial anti HIDE1 antibody (Sigma, cat #HPA012150), as described in Material and Methods. As shown in
In order to verify cell surface expression of the HIDE1 Flag protein, the same cells described above were analyzed by FACS using Rabbit anti-HIDE1 pAb (GenScript, #488536_13) as described in Material and Methods. No cell surface expression was observed in these cells (data is not shown).
HEK293 Cells Overexpressing Mouse HIDE1-Flag pMSCV Vector
To verify the expression of the mouse HIDE1 protein in the stably transduced HEK293 cell pools, whole cell extracts of HEK293 stable pools transduced with pMSCV mouse HIDE1 flag vector or of HEK293 stable pools transduced with pMSCV empty vector, were analyzed by WB using anti-mouse HIDE1 pAb (GenScript #488536_13) as described in Material and Methods. The results, shown in
These cells were further analyzed by FACS for cell surface expression using Rabbit anti mouse-HIDE1 pAb (Genscript t#488536_13) as described in Material and Methods. As shown in
EL4 Cells Overexpressing Mouse HIDE1-Flag pMSCV Vector
To verify membrane expression, the cells were analyzed by FACS using Rabbit anti mouse-HIDE1 pAb (GenScript#488536_13) as described in Material and Methods. As shown in
CHO-S OKT3 Cells Overexpressing Human HIDE1-Flag pcDNA3.1 Vector
In order to verify cell surface expression of human HIDE1 Flag protein, CHO-S OKT3 cells transfected with human HIDE1 construct (described in Material & Methods) were analyzed by FACS using Mouse anti-human HIDE1 monoclonal Ab (Biotem, 33B4-2F7-Alexa 647) as described in Material and Methods. As shown in
HEK293 Cells Over Expressing Cyno HIDE1-Flag pCDNA3.1 Vector
To verify the expression of the cyno HIDE1 protein in the stably transfected HEK293 cell pools, the cells were analyzed by FACS for cell surface expression using mouse anti human HIDE1 monoclonal Ab (Biotem 33B4-2F7-Alexa 647) as described in Material and Methods. As shown in
HIDE1 mECD-mIg fusion protein (SEQ ID NO:18) batch #195, was generated in CHO-DG44 cells by culturing stable cell pools for 10 days, followed by Protein A mouse HIDE1 fused to the Fc of mouse IgG2a, was produced at ProBioGen (Germany purification of cell harvest and preparative SEC purification for aggregate removal. The final product was formulated in 10 mM Na/K phosphate+140 mM NaCl pH 6.0+0.01% Tween.
HIDE1 mECD-mIgfusion protein (SEQ ID NO:18, batch #233), composed of the ECD of mouse HIDE1 fused to the Fc of mouse IgG2a, was produced at ProBioGen (Germany) in CHO-DG44 cells by culturing stable cell pools for 12 days, followed by Protein A purification of cell harvest and preparative SEC purification for aggregate removal. The final product was formulated in 5 mM Na citrate, 5 mM Na/K phosphate, 140 mM NaCl, 0.01% Tween pH5.5.
Expression vector used was ProBioGen's PBG-GPEX6. HIDE1 gene was driven by CMV/EF1 hybrid promoter followed by polyadenylation signal pA-1. The vector contained puromycin N-acetyl-transferase gene that allows selection of transfected cells using puromycin, as well as dehydrofolate reductase gene that allows selection of transfected cells using methotrexate (MTX).
HIDE1 mECD-mIg fusion protein (SEQ ID NO:18 batch #72), composed of the ECD of mouse HIDE1 fused to the Fc of mouse IgG2a, was produced at ExcellGene (Switzeland) by transient transfection in CHO-DG44 cells using Excellgene's proprietary vector system. Cells were cultured for 10 days, followed by Protein A purification of cell harvest. The final product was formulated in 10 mM Na/K phosphate+140 mM NaCl pH 6.0+0.02% Tween.
HIDE1 hECD-hIg fusion protein (SEQ ID NO:17, batch #48), composed of the ECD of human HIDE1 fused to the Fc of human IgG1 bearing C220 to S mutation at the hinge, was produced at ExcellGene (Switzeland) by transient transfection in CHO-DG44 cells using Excellgene's proprietary vector system. Cells were cultured for 10 days, followed by Protein A purification of cell harvest. The final product was formulated in 0.1M Glycine pH 6.
HIDE1 hECD-hIg fusion protein (SEQ ID NO:17 batch #259), composed of the ECD of human HIDE1 fused to the Fc of human IgG1 bearing C220 to S mutation at the hinge, was produced at GenScript (China) by transient transfection in CHO-3E7 cells using GenScript's vector system. Cells were cultured for 6 days, followed by Protein A purification of cell harvest. The final product was formulated in PBS pH 7.2.
HIDE1 hECD-hIg fusion protein (SEQ ID NO:17 batch #280), composed of the ECD of human HIDE1 fused to the Fc of human IgG1 bearing C220 to S mutation at the hinge, was produced at GenScript (China) by transient transfection in CHO-3E7 cells using GenScript's vector system. Cells were cultured for 6 days, followed by Protein A purification of cell harvest. The final product was formulated in 10 mM NaPhosphate, 150 mM NaCl pH 7.0+0.01% tween 20.
The aim of this example was to raise polyclonal and monoclonal antibodies specific to HIDE1 protein.
Rabbit polyclonal antibodies were raised at GeneScript using the GenScript PolyExpress™ custom polyclonal antibody production service.
Generation of rabbit polyclonal antibodies was performed at GenScript (USA). Antibodies against the human HIDE1 protein were raised by using recombinant FC fused protein of human HIDE1 ECD fused to FC domain of human IgG1 (batch #280). Antibodies against the mouse HIDE1 protein were raised by using the recombinant Fc fusion protein of mouse HIDE1 ECD fused to FC domain of mouse IgG2a (batch #195).
Reagents Used in this Study:
Anti Human or Mouse HIDE1 pAbs Production
Production of rabbit polyclonal antibody against Fc fusion protein of mouse or human HIDE1 included immunizations of two rabbits, production bleeds and antibody purification, using GenScript's proprietary fast immunization protocol. The immunoreactivity of the test bleeds was analyzed following the third immunization by ELISA.
Analysis of the pAbs Performance in WB Application
Whole cell extracts of cells (35 ug of total protein) expressing human or mouse HIDE1 protein were used to analyzed the pAbs anti-human or anti-mouse HIDE1 performance in WB. As negative control, whole cell extracts of cells transfected with the empty vector were used.
Antibodies were used as following:
Analysis of the pAbs Performance in FACS Application
The performance of the rabbit pAbs, anti-human or anti-mouse HIDE1 in FACS application was tested using stable cells expressing the mouse or the human HIDE1. Cells (2×105) were stained with Fixable viability stain diluted 1:1000 in PBS, for 10 min at R.T followed by cells washing with PBS. The pAbs were then added to cells (5 μg/ml were stained with Fixable viability stain diluted 1:1000 in PBS, for 10 min at R.T followed by cells washi
The sera of the immunized rabbits were analyzed by ELISA against the respective Fc fusion proteins (human or mouse HIDE1). The negative control was pre immune serum. ELISA results indicated binding of serum to the fusion proteins (data not shown).
Analysis of the Purified Rabbit Polyclonal Anti Human or Anti Mouse HIDE1 by WB
The performance of the rabbit pAbs, anti-human or anti-mouse HIDE1 for WB application was tested using whole cell extracts of HEK293 cells expressing human or mouse HIDE1 protein. As negative control, whole cell extracts of cells transfected with the empty vector were used.
As shown in
The mouse HIDE1 protein expressed on HEK293 cells could be detected by the tested antibody, rabbit anti mouse HIDE1 pAb (C lane 3) but not by the other anti-human HIDE1 antibodies (commercial, D lane 3 or the custom B lane 3) or by the anti-Flag antibody (A lane 3).
Analysis of the Purified Rabbit Polyclonal Anti Human HIDE1 by FACS
The performance of the purified polyclonal antibody against human HIDE1 Fc fusion protein, anti-human HIDE1 pAb (GenScript, ID 488536_4) in FACS application was tested using HEK293 cell expressing human HIDE1 Flag or SKMEL-5 cells expressing human HIDE1 Flag. Rabbit IgG (Jackson) was used an IgG control.
As shown in
Analysis of the Purified Rabbit Polyclonal Anti Mouse HIDE1 by FACS
The performance of the purified polyclonal antibody against mouse HIDE1 Fc fusion protein, anti-mouse HIDE1 pAb (GenScript, ID 488536_13) for FACS application was tested using HEK293 cell expressing mouse HIDE1 Flag. HEK293 cells transfected with an empty vector were used as a negative control.
As shown in
Generation of Mouse Monoclonal Antibodies Against Human HIDE1 Protein
Mouse monoclonal antibodies were raised at BIOTEM (France) using BIOTEM R.A.D® (Rapid Antibody Development) protocol which allows the breaking of tolerance towards self-antigens and the generation of more hybridomas than classical methods in a short time.
Study Design
Generation of murine monoclonal antibodies against the extra-cellular domain of human HIDE1 were performed at BIOTEM. The Antibodies were raised using the human HIDE1 Fc fusion protein, ECD of human HIDE1 fused to human IgG1 (batch #295) for immunizations.
Reagents Used in this Study:
mAbs Anti Human HIDE1 Production
Production of mouse monoclonal antibodies includes the following steps.
The first phase of the project to raise anti-HIDE1 mAbs included immunization of 2 BALB/c mice with human HIDE1 ECD fused to human IgG1.
The second phase of the protocol included fusion of the lymphocytes from the immunized mice with Sp2/O-Ag14 myeloma cells and plating out on 10 microtiter 96-well plates.
Mature clones were screened by ELISA using the HIDE1 H:H fusion proteins. FACS analysis was subsequently carried out with the culture supernatant of ELISA-positive hybridoma clones, using anti mouse-PE (Jackson, cat. 115-116-146) as secondary Ab for detection.
The third phase included hybridoma cloning by limiting dilution and stabilization.
The fourth phase included further processing for production and purification. Antibodies were produced by in vitro production and purification using protein A affinity chromatography purification.
Analysis of the mAbs Performance in WB Application
Whole cell extracts of cells (35 ug of total protein) expressing human or mouse HIDE1 protein were used to analyzed the mAbs anti-human HIDE1 performance for WB. As negative control, whole cell extracts of cells transfected with the empty vector were used.
Antibodies were used as following:
The performance of the mouse mAbs, anti-human HIDE1 for FACS application was tested using stable cells expressing the human HIDE1. Cells (2×105) were stained with Fixable viability stain diluted 1:1000 in PBS, for 10 min at R.T followed by cells washing with PBS. The mAbs were then added to cells (10 μg/ml diluted in FACS buffer) followed by staining with Goat anti mouse-PE (diluted 1:75 in FACS buffer).
The sera of the immunized mouse were analyzed by ELISA against the respective Fc fusion proteins (human HIDE1). The negative control was pre immune serum. ELISA results indicated binding of serum to the fusion proteins (data is not shown).
Analysis of the Purified Mouse mAbs Anti Human HIDE1 by WB
The performance the mouse mAbs, anti-human HIDE1 (Biotem) in WB application was tested using whole cell extracts of HEK293 cells expressing human HIDE1 protein. As negative control, whole cell extracts of cells transfected with the empty vector were used.
As shown in
Analysis of the Purified Mouse mAbs Anti Human HIDE1 by FACS
The performance of the purified monoclonal antibodies against human HIDE1 Fc fusion protein, anti-human HIDE1 mAbs (Biotem) for FACS application was tested using HEK293 cell expressing human HIDE1 Flag. HEK293 cells transfected with an empty vector were used as a negative control.
As shown in
Schematic representation of Biotem's antibodies (33B4-2F7, 36C1-2F6, 39A7-39) are shown in
Generation of Fab's Against Human and Mouse HIDE1 Protein
Fab's were raised at AbD Serotec (Bio Rad, Germany) using Human Combinatorial Antibody Library (HuCAL®) production service. The HuCAL® library is based on the human IgG1 Fab format, which consists of the first two domains of the antibody heavy chain and the complete light chain.
Generation of Fab's against HIDE1 was performed at AbD Serotec (Bio Rad, Germany). Antibodies against the human and mouse HIDE1 protein were raised using the HuCAL® phage library, using 3 rounds of enrichment and counter selection against non-related human IgG1 fusion protein for the depletion of unspecific antibodies. After the panning, the enriched antibody pool from the phage display vector was subcloned into expression vector to determine the final Fab format. The selected Fab format is Fab-A-FH (Bivalent Fab bacterial alkaline phosphatase fusion antibody (FLAG®- and His-6-tags)).
The Antibodies were raised using the human HIDE1 Fc fusion protein, ECD of human HIDE1 fused to human IgG1 (batch #295) and mouse HIDE1 Fc fusion protein, ECD of mouse HIDE1 fused to mouse IgG2a (batch #233).
Reagents Used in this Study:
Fab's generation at AbD Serotec included the following steps:
Whole cell extracts of HEK293 transfected cells over expressing the human or mouse HIDE1 flag protein or whole cell extracts of HEK293 cells transduced with an empty vector were analyzed by WB using the custom Fab's (AbD Serotec) described above at a final concentration of 1-7 μg/ml in 1% TTBS/BSA. The commercial pAb anti-human HIDE1 (Sigma, cat #HPA012150) was diluted 1:100 in TTBS/1% BSA.
Staining with the custom and commercial antibodies described above was followed by a secondary antibody Anti Fab-HRP (Bio Rad) diluted 1:5000 in 5% Milk/TTBS and goat anti Rabbit IgG—Peroxidase (Jackson, cat no. 111-035-003) diluted 1:10,000 in TTBS/5% milk solution.
The performance of the Fab's, anti-human HIDE1 and anti-mouse HIDE1 for FACS application were tested using stable cells over expressing the human HIDE1 or mouse HIDE1 or on cyno HIDE1 an empty vector transduced cells. Cells (2×105) were stained with Fixable viability stain diluted 1:1000 in PBS, for 10 min at R.T followed by cells washing with PBS.
The Fab's were then added to cells (10 μg/ml diluted in FACS buffer) followed by staining with Goat Anti Human IgG F(ab′)2-PE (diluted 1:100 in FACS buffer).
Affinity measurements were done by AbD Serotec using the ForteBio Octer RED384 instrument.
Antibody concentrations: bivalent Fab antibodies in Fab-A-FH format (Mw: 198 kDa): 9.9 μg/ml (50 nM), 1:2 dilution series. The model used to fit the data: (ForteBio Data Analysis software 8.2.0.7)
Epitope Binning measurements were done by AbD Serotec using the ForteBio Octer RED384 instrument. Antibodies were grouped into diffrents bins by using the “in tandem” measurements.
Reformatting of the Fab's into Full Length Immunoglobulin
The conversion to human IgG1 was done to 5 Fab's by AbD Serotec (AbD25751, AbD25753, AbD25754, AbD25755, AbD25760).
Analysis of the Reformatted Fab's (Full hIgG1 Abs) Performance in Flow Cytometry (FACS) Application
The performance of the antibodies, anti-human HIDE1, for FACS application were tested using stable cells over expressing the human HIDE1, mouse HIDE1 or cyno HIDE1 or an empty vector transduced cells. Cells (5×105) were stained with Fixable viability stain diluted 1:1000 in PBS, for 10 min at R.T followed by cells washing with PBS.
The Abs were then added to cells (10 μg/ml diluted in FACS buffer) followed by staining with Goat Anti Human-PE (diluted 1:100 in FACS buffer).
The performance of the antibodies, anti-human HIDE1, for the affinity measurements (KD) using FACS application were tested using stable cells over expressing the
The panning was performed at AbD Serotec using the following proteins:
Panning 1: Human HIDE1 ECDhIgG1 protein batch #280—for generation of anti-human Fab's.
Panning 2: Mouse HIDE1 ECDmIgG2A protein batch #233—for generation of anti-mouse Fab's.
Panning 3: Mouse HIDE1 ECDmIgG2a protein batch #233 (1st and 3rd panning round)/Human HIDE1 ECDhIgG1 protein batch #280 (2nd panning round)—for generation of cross reactive Fab's.
Serotec screened ˜360 clones by ELISA on human HIDE1 ECDhIgG1 protein batch #280 and mouse HIDE1 ECDmIgG2A protein batch #233 and H:H_Internal ECDhIgG1 protein batch #279 as control for the human panning or mouse M:M_Internal ECDmIgG2A protein batch #214 as control for the mouse panning.
In the human HIDE1 panning 122 clones were positive, in the mouse HIDE1 panning 61 clones were positive and in the mouse/human panning 55 clones were positive.
In secondary screening 88 positive clones were screened from panning 1, 33 positive clones were screened from panning 2 and 55 positive clones were screened from panning 3 by FACS on cells overexpressing the human HIDE1 and mouse HIDE1 and on empty vector transduced cells.
The secondary screen resulted in 43 positive clones from panning 1, 15 positive clones from panning 2 and 29 positive clones from panning 3.
Sequencing was done on 20 positive clones from panning 1 which results in 16 unique Fab's, on 15 positive clones from panning 2 which results in 9 unique Fab's and on 25 positive clones from panning 3 which results in 4 unique Fab's.
The purified Fab's were analyzed by ELISA against the respective Fc fusion protein (human HIDE1 or mouse HIDE1). ELISA results indicated binding of the purified Fab's to the fusion proteins (data not shown).
The purified Fab's were also analyzed by WB. The antibodies are not applicable for WB (data is not shown).
The performance of the purified Serotec's Fab's against human HIDE1 and mouse HIDE1, in FACS application were tested using HEK293 cell over-expressing human HIDE1 Flag or mouse HIDE1. HEK293 cells transduced with an empty vector were used as a negative control.
As shown in
As shown in
As shown in
The purified Fab's were also measured for affinity by AbD Serotec. Table 19 and Table 20 summarize the obtained avidity for anti human HIDE1 and anti mouse HIDE1 Fab's.
The purified Fab's were also subjected to epitope binning by AbD Serotec.
Analysis of the Reformatted Fab's (Full hIgG1 Abs) Performance in Flow Cytometry (FACS)
The performance of the reformatted Serotec's antibodies against human HIDE1, in FACS application were tested using HEK293 cell over-expressing human HIDE1 Flag, mouse HIDE1 or cyno HIDE1. HEK293 cells transduced/transfected with an empty vector were used as a negative control
As shown in
The affinity The affinity measurements of the reformatted Serotec's Ab's (full hIgG1 Abs) against human HIDE1, using FACS application were tested using CHO-S cell over-expressing human HIDE1. CHO-S cells transduced with an empty vector were used as a negative control.
As shown in
The following custom antibodies generated were validated and used for further target characterization:
Fab's anti-human HIDE1; Serotec (ID: AB-326, AB-327, AB-328, AB-329, AB-331, AB-332, AB-333, AB-335, AB-336, AB-337, AB-338, AB-340, see Table 21) and reformatted Fab's anti-human HIDE1; Serotec (ID: AB-506, AB-507, AB-508, AB-509, AB-510, see Table 21) validated for FACS application on ectopic expression, with no cross reactivity to mouse HIDE1 protein, and with cross reactivity of four Fab's (ID: AB-327, AB-332, AB-333, AB-338, see Table 21) and three reformatted antibodies (ID: AB-508, AB-509, AB-510, see Table 21) to cyno HIDE1 protein.
Fab's anti-mouse anti-HIDE1 antibody; Serotec (ID: AB-341, AB-342, AB-343, AB-344, AB-345, AB-354, AB-357, AB-359, see Table 22) validated for FACS application on ectopic expression, with cross reactivity of two antibodies (ID: AB-344, AB-345) to human HIDE1 protein.
Five reformatted Fab's anti-human HIDE1; Serotec were characterized for affinity. Two of the five antibodies (AB-507 and AB-508) reached saturation at the concentrations that were measured.
The aim of this study was to identify human and mouse cancer cell lines that endogenously express HIDE1.
RNA (1-5 ug) extraction of human and mouse cell lines (detailed above in Tables 23 and 24) was preformed according to manufactures protocols.
cDNA was prepared according to manufactures protocols (1 ug RNA diluted in 20 ul cDNA mix reaction).
Two Different Approaches were Used:
qRT-PCR Using SYBR Green
cDNA, prepared as described above, diluted 1:10 (representing 25 ng RNA per reaction) in DDW was used as a template for qRT-PCR reactions using the SYBR Green I assay (PE Applied Biosystem) with specific primers (listed in Table 27). Detection was performed using the PE Applied Biosystem SDS 7000 or QuantStudio 12k flex device.
The cycle in which the reactions reached a threshold level of fluorescence (Ct=Threshold Cycle,) was registered and was used to calculate the relative transcript quantity in the RT reactions. The relative quantity was calculated using the equation Q=Efficiency ̂−Ct. The efficiency of the PCR reaction was calculated from a standard curve, created by using serial dilutions of mouse commercial cDNA mixed panels (Cat no. C8334501 and C8334502, Biochain), and for human efficiency calculations, several cDNA from tissues and cell lines that were prepared in house, created for each primer set.
The resulting quantities were normalized to quantities of a housekeeping gene (hHPRT1 or mHPRT1).
qRT-PCR Using TaqMan:
cDNA, prepared as described above, diluted 1:10 (representing 25 ng RNA per reaction) in DDW, was used as a template for qRT-PCR reactions, using a gene specific TaqMan probes (detailed in Materials above). Detection was performed using QuantStudio 12k flex device.
The cycle in which the reactions achieved a threshold level of fluorescence (Ct=Threshold Cycle) was registered and was used to calculate the relative transcript quantity in the RT reactions.
The absolute gene quantity was calculated by using the equation Q=2 ̂−ΔCt.
The resulting quantities were normalized to quantities of human or mouse housekeeping gene, hRPL19 or mRPL19 relatively.
The expression of HIDE1 in human and mouse cell lines was analyzed by WB using whole cell extracts (50-75 μg for the cancer cell lines, and 30 μg for the over expressing cell line and negative control cell line)
For human HIDE1 protein detection:
For Mouse HIDE1 Protein Detection:
The cell surface expression of HIDE1 protein was analyzed by FACS. Human or mouse cell lines were stained with VioBlue reagent diluted 1:1000 in PBS. Cells were incubated 10 min at R.T. and then washed twice with FACS buffer (0.5% BSA in PBS). Cell lines for endogenous protein analysis were pre-incubated with the Fc receptor blocking solutions listed above in material section (2.5 μl/reaction of human blocker and 0.5 mg/ml of mouse blocker were used according to the manufactures procedures). To detect the human HIDE1 protein, cells were stained with a custom mouse monoclonal anti human HIDE1 mAbs (BIOTEM, detailed in material section above) diluted to a concentration of 10 μg/ml or IgG1 Isotype control at the same concentration followed by Goat anti mouse PE conjugated Ab. To detect the mouse HIDE1 protein, cells were stained with a custom rabbit polyclonal anti-mouse HIDE1 pAb (GenScript, ID#488536_13) diluted to a concentration of 10 μg/ml or rabbit IgG whole molecule as isotypes control at the same concentration followed by Donkey anti Rabbit-PE conjugated Ab diluted 1:100.
To detect the mouse HIDE1 protein, cells were stained with a custom rabbit polyclonal anti-mouse HIDE1 pAb (GenScript, ID#488536_13) diluted to a concentration of 10 μg/ml or rabbit IgG whole molecule as isotypes control at the same concentration followed by Donkey anti Rabbit-PE conjugated Ab diluted 1:100.
Knock down of endogenous mouse or human HIDE1 was carried out by transient transfection of siRNA. Transfection of 50 pmol HIDE1 siRNA pool or scrambled siRNA performed by electroporation using Amaxa nucleofector device and Amaxa nucleofector kits as listed above and according to the manufacture procedure. 48 hours post transfection, cells were collected for further analysis by qRT-PCR and FACS. EL4 cell line was further tested by FACS 72 hours post transfection.
Endogenous Expression of HIDE1 Transcripts in Human Cell Lines Detected by qRT-PCR
In order to verify the presence of the HIDE1 transcript in human cell lines (listed in Table 23), qRT-PCR was performed using a specific primers (Table 27 and
As shown in
WB analysis for endogenous expression of HIDE1 protein was carried out on various human cancer cell lines lysates as detailed in Table 23 using commercial anti human HIDE1 pAb (Sigma, HPA012150) or custom anti human HIDE1 mAb (BIOTEM, 36C1-2F6) as described in Materials & Methods above. As a positive control, whole cell extract of stable HEK293T cell pool over-expressing HIDE1 was used while cells transfected with an empty vector served as the negative control.
WB results related to positive and negative controls were as expected. WB results for endogenous expression were inconclusive as multiple bands were detected using the pAb, and no signal was observed using the mAb (data is not shown).
To verify the cell-surface endogenous expression of human HIDE1, various cell lines (detailed in Table 23) were tested as described in Material & Methods above. The cell lines were stained with the custom monoclonal anti human-HIDE1 mAbs: BIOTEM, 33B4-2F7, 36C1-2F6 (
Further analysis for endogenous confirmation of human HIDE1 in THP1 and U937 cell lines, was done by testing various anti-human HIDE1 F(ab′)2, produced by Serotec.
Both cell lines were stained with anti-human HIDE1 custom F(ab′)2s (Serotec, detailed in Material & Methods)(
No expression of human HIDE1 was observed in THP-1 human cell line by either of the various F(ab′)2 (data not shown).
Knock Down of Human HIDE1 by siRNA in Human Cancer Cell Lines
In order to further confirm endogenous expression of HIDE1 protein in HL-60 and U937 cell lines, human HIDE1 siRNA pool was used for knock down as described in Material & Methods.
48 hours post siRNA transfection, cells were harvested for further analysis by qRT-PCR or by FACS. As shown in
Further analysis of human HIDE1 membrane expression in the same siRNA transfected cells was performed by FACS. As shown in
Confirmation of HIDE1 endogenous expression was also tested in THP1 cells transfected with HIDE1 siRNA as described in M&M section. Analysis of human HIDE1 membrane expression in the siRNA transfected cells was performed by FACS. As shown in
Additional knock down experiment of human HIDE1 was performed in U937 cell line. Confirmation of human HIDE1 membrane expression in U937 cell line was tested by using the positive Serotec F(ab′)2 (Ab329, Ab332 and Ab333 have already showed expression in U937 cell line in
As shown in
Endogenous Expression of HIDE1 Transcripts in Mouse Cell Lines Detected by qRT-PCR
In order to verify the presence of the HIDE1 transcript in mouse cell lines (listed in Table 24), qRT-PCR experiment was performed using specific primers (
As shown in
WB analysis for endogenous expression of HIDE1 protein was carried out on various mouse cancer cell lines as detailed in Table 24, Using GenScript anti mouse HIDE1 pAb, 488536_13 described in Materials & Methods above. As a positive control, whole cell extract of stable HEK293 cell pool. ectopically expressing mouse HIDE1 was used while cells transfected with an empty vector served as the negative control. WB results related to positive and negative controls were as expected. WB results for endogenous expression were inconclusive as multiple bands were detected (data is not shown).
To verify the cell-surface endogenous expression of mouse HIDE1 protein, various cell lines (detailed in Table 24, were tested as described in Material & Methods above. The mouse cell lines were stained with a rabbit anti mouse HIDE1 pAb (Genescript, ID 488536_13) (Orange) or with the Isotype control (light blue) followed by Goat anti rabbit PE Ab (Jackson cat #711-116-152). As additional negative control, cells were stained with the secondary antibody only and represented by a red line. Analysis was performed by FACS. As shown in
Further analysis of mouse HIDE1 endogenous expression in EL4 and J774A.2 cell lines, was done by FACS using various F(ab′)2 produced by Serotec.
Both cell lines were stained with anti-mouse HIDE1 custom F(ab′)2s produced by Serotec (
Expression of mouse HIDE1 was observed also in J774A.2 mouse cell line by the following F(ab′)2 as shown in
In order to confirm endogenous expression of mouse HIDE1 protein in EL4 cell line, mouse HIDE1 siRNA pool was used for knock down experiment as described in Material & Methods.
48 hours post siRNA transfection, cells were harvested for further analysis by qRT-PCR and FACS. As shown in
48 or 72 hours post siRNA transfection, cells were harvested and were analysed by FACS for membrane expression of mouse HIDE1. As shown in
72 hours post siRNA transfection membrane expression of mouse HIDE1 was very similar to the fold in cells transfected with the HIDE1 siRNA (data not shown).
This example includes preliminary data on HIDE1 endogenous expression in cell lines both at the RNA level and the protein level in human and mouse cell lines.
Various human cancer cell lines were tested by qRT-PCR, WB and FACS for endogenous expression of HIDE1.
Cell surface expression was observed in HL-60, U937, and THP1 cell lines using the mouse monoclonal Ab (BIOTEM, 33B4-2F7) as shown in
Various mouse cell lines were tested by qRT-PCR, WB and FACS for endogenous expression of HIDE1. In the transcript level, presence of HIDE1 was observed in J774A.1, EL4, YAC-1 and A20 cell lines as well as in RAW264.7 and P3881)1 cell lines as shown in
Tables 25 and 26 below indicate the summary of the findings described in this report, highlighting the cell lines showing correlation between qPCR and FACS, confirmed by knock down. Further analyses are currently on going.
To evaluate the expression of human HIDE1 on human peripheral mononuclear cells (PBMCs) using 2 monoclonal antibodies for HIDE1 by flow cytometry.
Isolation of PBMCs: Buffy coats were obtained from Stanford Blood Bank from healthy human donors. PBMCs were isolated by Ficoll gradient separation, washed with medium, and cryo-preserved for future experiments.
Antibodies:
The following antibodies were used. Alexa flour 647 labeled mouse IgG1 isotype control (lot. Fl150528d-2695, BIOTEM), Alexa flour 647 labeled anti-human HIDE1 antibodies (BIOTEM, 33B4-2f7 and 36-C1-2F6). Brilliant violet 510 labeled anti-CD3 (Biolegend, cat. 344828), PE-labeled anti-CD14 (Biolegend, cat. 301806) and PercP-labeled anti-CD19 (Biolegend, cat. 302228).
PBMCs cells were thawed and stained with viability dye (BD Horizon; Cat#562247, BD biosciences), washed and pre-blocked with Fc blocking solution (cat#422302, Biolegend; 1:20) and/or human Ig Fc (Jackson; 200 μg/ml; cat#009-000-008) to avoid nonspecific binding via Fc receptors. Cells were then stained with anti-hHIDE1 mAbs or mIgG1 isotype control in the presence or absence of lymphocytes or monocytes surface marker. All samples were run on a MACSQuant analyzer (Miltenyi) and data was analyzed using Tree Star FlowJo software. Cells were first gated for lymphocytes or monocyte (FSC-A vs. SSC-A), and further gated for live cells before analyzed for HIDE1 expression by flow cytometry.
Freshly thawed PBMCs from 2 donors were thawed and stained with viability dye, washed and pre-blocked with Fc blocking solution as described in material and methods. HIDE1 expression was observed on CD14+ cells but not on CD3+ cells (
HIDE1 expression on human monocytes was observed using 2 anti-human HIDE1 mAb in 4 different donors. The expression was excluded to monocytes. No expression on T cells or B cells was observed. Further experiment are ongoing in order to analyze the expression of HIDE1 on other leukocytes population such as neutrophils and dendritic cells.
Reagents Used in this Study
7 wk old female BALB/c mice were purchased from ENVIGO laboratories and kept in specific pathogen-free conditions at the Tel Aviv University animal facility. Experiments respected institutional guidelines and were approved by the Tel Aviv University ethics committee
The murine CT26, an N-nitroso-N methylurethane—induced undifferentiated colon carcinoma cell line, was obtained from American Type Culture Collection (CRL-2638). CT26 cells (5×105) were injected subcutaneously at day 0 in the right flank of BALB/c mice (female, 7 wk). Tumor growth was monitored twice a week using a caliper.
Treatments were given twice and started at day 8, when tumor area reached 50 mm2 and on day 12 post tumor inoculation. Treatments were injected intraperitoneal at 10 mg/Kg (9 mice/treatment) of either anti PDL-1 mIgG1 (YWW243.58.870, LOT #40315,) or mIgG1 isotype control (MOPC-21).
Whole tumor and spleens total RNA was extracted using gentleMACS™ Octo Dissociator (Miltenyi Biotec), and tissues were homogenized with M tubes (#130-096-335).
For tumor single cell suspension, tumors were enzymatically digested with 1 μg/ml Collagenase D (Worthington Biomedical Corporations) 0.5 μg/ml Deoxyribonuclease I (Worthington Biomedical Corporations) in RPMI-1670 (Biological Industries) for 1 hr in 370c. Remains of undigested tumor tissues were gently agitated for full a dissociation.
Thy1.2+ T lymphocytes were purified from tumors using a CD90.2+ positive isolation kit (EasySep, #18751) and purified Thy1.2+T lymphocytes and the negative fraction cells were lysed in RLT to extract total RNA.
CD11b+ cells were purified from tumors using a CD11b+ positive isolation kit (EasySep, #18770) and purified CD11b+ cells and the negative fraction cells were lysed in RLT to extract total RNA
Quantitative RT-PCR (qRT-PCR)
RNA (1-5 ug) extraction of mouse cells, derived from Ex-vivo experiments (346-MA-455 & 459-MA) was preformed according to manufactures protocols.
cDNA was prepared according to manufactures protocols (1 ug RNA diluted in 20 ul cDNA mix reaction).
cDNA, prepared as described above, diluted 1:10 (representing 25 ng RNA per reaction), was used as a template for qRT-PCR reactions, using a gene specific TaqMan probes (detailed in Reagents 3&4) Detection was performed using QuantStudio 12k device.
The cycle in which the reactions achieved a threshold level of fluorescence (Ct=Threshold Cycle) was registered and was used to calculate the relative transcript quantity in the RT reactions.
The absolute quantity was calculated by using the equation Q=2 ̂−Ct.
The resulting relative quantities were normalized to a relative quantities of housekeeping gene, mRPL19, mSDHA and mPBGD.
In order to verify the presence of the HIDE1 transcript in tumor derived cell, qRT-PCR was performed using a specific TaqMan probe as describe above in Material & Methods.
As shown in
In order to verify the presence of the HIDE1 transcript in tumor derived cell, qRT-PCR was performed using a specific TaqMan probe as describe above in Material & Methods.
As shown in
Reagents Used in this Study
7 wk old female BALB/c mice were purchased from ENVIGO laboratories and kept in specific pathogen-free conditions at the Tel Aviv University animal facility. Experiments respected institutional guidelines and were approved by the Tel Aviv University ethics committee
The murine CT26, an N-nitroso-N methylurethane—induced undifferentiated colon carcinoma cell line, was obtained from American Type Culture Collection (CRL-2638). CT26 cells (5×105) were injected subcutaneously at day 0 in the right flank of BALB/c mice (female, 7 wk). Tumor growth was monitored twice a week using a caliper.
Treatments were given twice and started at day 8, when tumor area reached 50 mm2 and on day 12 post tumor inoculation. Treatments were injected intraperitoneal at 10 mg/Kg (9 mice/treatment) of either anti PDL-1 mIgG1 (YWW243.58.870, LOT #40315,) or mIgG1 isotype control (MOPC-21).
Whole tumor and spleens total RNA was extracted using gentleMACS™ Octo Dissociator (Miltenyi Biotec), and tissues were homogenized with M tubes (#130-096-335).
For tumor single cell suspension, tumors were enzymatically digested with 1 μg/ml Collagenase D (Worthington Biomedical Corporations) 0.5 μg/ml Deoxyribonuclease I (Worthington Biomedical Corporations) in RPMI-1670 (Biological Industries) for 1 hr in 370c. Remains of undigested tumor tissues were gently agitated for full a dissociation.
Thy1.2+T lymphocytes were purified from tumors using a CD90.2+ positive isolation kit (EasySep, #18751) and purified Thy1.2+T lymphocytes and the negative fraction cells were lysed in RLT to extract total RNA.
CD11b+ cells were purified from tumors using a CD11b+ positive isolation kit (EasySep, #18770) and purified CD11b+ cells and the negative fraction cells were lysed in RLT to extract total RNA
Quantitative RT-PCR (qRT-PCR)
RNA (1-5 μg) extraction of mouse cells, derived from Ex-vivo experiments (346-MA-455 & 459-MA) was preformed according to manufactures protocols.
cDNA was prepared according to manufactures protocols (1 ug RNA diluted in 20 ul cDNA mix reaction).
cDNA, prepared as described above, diluted 1:10 (representing 25 ng RNA per reaction), was used as a template for qRT-PCR reactions, using a gene specific TaqMan probes (detailed in Reagents 3&4) Detection was performed using QuantStudio 12k device.
The cycle in which the reactions achieved a threshold level of fluorescence (Ct=Threshold Cycle) was registered and was used to calculate the relative transcript quantity in the RT reactions.
The absolute quantity was calculated by using the equation Q=2 ̂−Ct.
The resulting relative quantities were normalized to a relative quantities of housekeeping gene, mRPL19.
Endogenous Expression of HIDE1 in Intestine Populations Derived from C57 Black Mouse Cells
Endogenous Expression of HIDE1 in Intestine Populations Derived from C57 Black Mouse Cells (Ex Vivo#466)
In order to verify the presence of the HIDE1 transcript in intestine population cells derived from C57 black mouse, qRT-PCR was performed using a specific TaqMan probe as describe above in Material & Methods.
As shown in
The aim of these experiments was to evaluate binding of HIDE1 tetramer to resting and activated human isolated primary CD4+ and CD8+ T cells, as well as TILs (Tumor Infiltrating Lymphocytes) isolated from human melanoma samples and propagated in the presence of melanoma specific antigens and IL2.
Human HIDE1 was produced as a monomer, including the extracellular domain (ECD), a biotinylation signal and a His-tag, (batch #502). Following enzymatic biotinylation of this protein performed by Genscript, this protein (batch #503) was used for tetramer production and binding studies. As negative and positive controls for binding studies, similar biotinylated monomeric proteins of human EGFR (EGR-H82E7, Acrobiosystems) and human B7H4 (B74-H8222, Acrobiosystems), were used for tetramer production, respectively.
Streptavidin, R-Phycoerythrin Conjugate (SA-PE)—premium grade (Molecular probes, S21388, 1 mg/ml) was used for the production of tetramers. This streptavidin-PE is a high-purity product, ensuring a uniform material with a constant molecular weight (300 kDa).
Similar weights of HIDE′ (batch #503), EGFR and B7H4 were used for tetramer production, Tetramers were created by mixing the above proteins with SA-PE at a 4:1 molar ratio. SA-PE was added by gradual titration into the proteins in 15 portions. After each portion was added, the mixture was incubated for 5 minutes. In the first five portions, a lower amount of SA-PE was added (1.5-fold less than the following ten portions), in order to saturate the streptavidin sites. The entire procedure was carried out at room temperature (SA-PE was kept on ice) in the dark, and tetramers were stored afterwards at 4 degrees. Similar concentration (by weight per volume) of the different tetramers was used for the binding experiments,
in this series of experiments cells from two different donors, obtained from Astarte Biologics were used:
Human primary cells (>95% purity), were thawed 24 h prior to beginning of experiment. Cells were thawed in RPMI complete medium (RPMI+10% FBS+1% Glutamax+1% Na-Pyruvate+1% Pen-Strep) supplemented with 300 U/ml of rhIL2 (Biolegend 509129). Cells were left to recover for 24 hours.
In this series of experiments, two different TILs from resected metastases of three melanoma patients, were used:
Human TILs (>95% CD8+), were thawed 24 h prior to beginning of experiment. Cells were thawed in 12 ml of TIL medium (IMDM+10% human serum+1% Glutamax+1% Na-Pyruvate+1% non-essential amino acids+1% Pen-Strep) supplemented with 300 U/ml of rhIL2 (Biolegend 509129). Cells were left to recover for 24 hours.
The following human T cell lines were used: H9 (ATCC, HTB-176, clonal derivative of HUT-78), Jurkat (ATCC, TIB-152, human acute T cell leukemia) and U937 (ATCC, CRL-1593.2 monocyte).
Cells were cultured in complete media: RPMI (Biological Industries Cat#01-100-1A) supplemented with 10% FBS (Biological Industries Cat#04-001-1A), 1% Glutamax (Life technologies Cat#35050-038).
After recovery, cells were activated using a polyclonal activation of T cells, with 1 μg/ml of plate bound anti CD3 antibody (BD-pharmingen clone Ucht-1, cat-555329), 2 μg/ml of anti CD28 ab (eBioscience clone CD28.2 cat-16-0289-85) and 300 U/ml of IL2.
Activation was carried out for 24 h, 48 h, 72 h and 144 h.
Cells were harvested and washed with PBS. Cells were stained at room temperature for 10 minutes with PBS supplemented with 1/1000 of fixable viability stain efluor 450 (BD horizon cat-562247). After staining, cells were washed twice with PBS and stained with the Abs at the concentrations listed in Table 9 for 30 minutes at room temperature (RT) in FACS buffer (PBS+0.5% BSA+2 mM EDTA+0.05% Azide) and concentrations that are listed in Table 28.
For tetramer binding, cells were incubated for 30-40 min at RT with the tetramer proteins and controls at 3 μg/well (50 μl/well=60 μg/ml) in FACS buffer (PBS+0.5% BSA+2 mM EDTA).
After binding, cells were washed three times and re-suspended in FACS buffer for analysis.
The binding of HIDE1 tetramer to various human cell lines was tested, as described in Materials and Methods (M&M). HIDE1 exhibited binding to H9 and Jurkat, at comparable levels (
The binding of HIDE1 to activated primary human T cells and TILs was evaluated next. T cells from two different donors and TILs were left untreated (resting) or polyclonal stimulated for various timepoints as described in M&M. Cell activation state was evaluated by detection of surface expression of CD137 and PD-1 at each time point compared to isotype control (FMO), as shown for activated CD8+, CD4+ T cells and TILs (
Results show binding of HIDE1 to activated CD4+ and CD8+ T cells between 24 and 72 hours (
In addition, HIDE1 tetramer binding to activated Mart1 and 209 TILs was detected at 24 h after activation, and sustained to 72 h of activation (
The study presented here demonstrates the binding of HIDE1 tetramers to human T cell lines, as well as, human CD4+ and CD8+ T cells and TILs, which were polyclonal activated. Results points to expression of the putative molecular counterpart of HIDE1 on these target cells. Binding of HIDE1 tetramer was detected only upon activation of CD4+ and CD8+ cells, reaching a peak at 72 h post activation. This data suggests the expression of a HIDE1 counterpart on T cells and its up-regulation upon TCR-dependent T cell activation.
In these experiments the immunomodulatory activities of the recombinant fused protein HIDE1-ECD-Ig was investigated on mouse T cell activation.
In order to evaluate the activity of HIDE1 protein on T cell activation, recombinant protein was produced comprising the mouse extracellular domain (ECD) of the mouse HIDE1 fused to the Fc of mouse IgG2a (designated HIDE1-ECD Ig M:M, SEQ ID NO:18). The effect of the Fc fused protein co-immobilized with anti-CD3 on mouse CD4 T cell functions, as manifested by activation markers and cytokines secretion was investigated. Mouse IgG2a (clone MOPC-173; Biolegend or C1.18.4; BioXcell) was used as isotype control.
The Fc fusion protein, HIDE1-ECD-Ig (batches #72 and #195, SEQ ID NO:18) were tested. Mouse IgG2a (clone MOPC-173; Biolegend or C1.18.4, BioXcell) was used as isotype control.
Untouched CD4+CD25− T cells were isolated from pools of spleens of BALB/C mice using a T cell isolation Kit (Miltenyi Cat#130-093-227) according to the manufacturer's instructions. The purity obtained was >90%.
Anti-mouse CD3-ε mAb (clone 145-2C11; BD Biosciences) 2 μg/ml together with HIDE1-ECD-Ig protein (SEQ ID NO:18) or control Ig at various concentration (1, 3 or 10 μg/ml), were co-immobilized for 3 hr at 37° C., on 96-well flat bottom tissue culture plates (Sigma, Cat. #Z707910). Control Ig was added to each well in order to complete a total protein concentration of 1.2 μg/ml per well. Wells were washed 3 times with PBS and plated with 1×105 purified CD4+CD25− T cells per well and kept in a humidified, 5% CO2, 37° C. incubator. In some experiments, soluble anti-CD28 (clone: 37.51; eBioscience; 1 μg/ml) was added. Culture supernatants were collected at the indicated times post stimulation and analyzed for mouse IFNγ or IL-2 secretion by ELISA kits (R&D Systems). The effect of HIDE1-ECD-Ig protein (SEQ ID NO:18) on the expression of the activation marker CD69 on mouse CD4+ T cells was analyzed by flow cytometry. Cells were stained 48 h post stimulation with a cocktail of antibodies including PerCP-anti-CD4 (clone G41.5; Biolegend), FITC or PE-anti-CD69 (clone H1.2F3; Biolegend), in the presence of anti-CD16/32 (clone 2.4g2; BD Biosciences) for blocking of Fcγ-receptors. Cells were evaluated using MACSQuant analyzer 9 (Miltenyi) and data analyzed using BD CellQuest or by MACSQuantify Software. Data was analyzed using Excel or Prism4 software.
HIDE1-ECD-Ig (SEQ ID NO:18) inhibits T cell activation in a concentration-dependent manner when the reagent is co-immobilized with anti-CD3 on plates. Maximal inhibitory effect was observed at 10 g/ml of HIDE1-ECD-Ig (SEQ ID NO:18).
The results demonstrate the inhibitory effect of HIDE1-ECD-Ig on mouse T cells activation, manifested by reduced cytokine secretion, and suppression of activation marker CD69 upregulation. This inhibition of T cell activation, supports the therapeutic potential of immunoinhibitory HIDE1-ECD polypeptides and/or fusion proteins thereof according to at least some embodiments of the present invention in treating T cell-driven autoimmune diseases, such as rheumatoid arthritis, multiple sclerosis, psoriasis and inflammatory bowel disease, as well as for other immune related diseases and/or for reducing the undesirable immune activation that follows gene therapy.
In order to evaluate the activity of HIDE1 protein on T cell activation, recombinant protein comprising the extracellular domain (ECD) of the mouse HIDE1 protein fused to the Fc of mouse IgG2a (designated HIDE1-Fc) was used. The effect of the HIDE1-Fc fused proteins co-immobilized with anti-CD3 on mouse CD4 T cell functions, as manifested by activation markers and cytokines secretion, was investigated.
Mouse IgG2a (clone MOPC-173; Biolegend) was used as isotype control. During assay set-up, B7H4-Ig (Cat. 4206-B7, R&D), a Fc fusion protein comprising the extracellular domain (ECD) of mouse B7H4 fused to the Fc of mouse IgG2a, was used as a positive control. Table 29, above.
Untouched CD4+CD25− T cells were isolated from pools of spleens of BALB/C mice using a T cell isolation Kit (Miltenyi Cat#130-093-227) according to the manufacturer's instructions. The purity obtained was >90%. Following isolation, cells were used either fresh or after freezing (in 90% FCS, 10% DMSO) and thawing.
Anti-mouse CD3-ε mAb (clone 145-2C11; BD Biosciences) 2 μg/ml of PBS, alone or together with HIDE1-Fc protein or control Ig at various ratios, was co-immobilized for 3 hr at 37° C., on 96-well flat bottom tissue culture plates (Sigma, Cat. #Z707910). Wells were washed 3 times with PBS and plated with 1×105 purified CD4+CD25− T cells per well in the presence of soluble anti-CD28 (clone: 37.51; eBioscience; 2 μg/ml) and kept in a humidified, 5% CO2, 37° C. incubator. Culture supernatants were collected 48 h post stimulation and analyzed for mouse IFNγ or IL-2 secretion by ELISA kits (R&D Systems), The effect of CGEN-Fc proteins on the expression of the activation marker CD69 on mouse CD4+ T cells was analyzed by flow cytometry. Cells were stained 48 h post stimulation with a cocktail of antibodies including PerCP-anti-CD4 (clone G41.5; Biolegend), FITC or PE-anti-CD69 (clone H1.2F3; Biolegend), in the presence of anti-CD16/32 (clone 2.4g2; BD Biosciences) for blocking of Fcγ-receptors. Cells were evaluated using MACSQuant analyzer 9 (Miltenyi) and data analyzed using BD CellQuest or by MACSQuantify™ Software. Data was analyzed using Excel or Prism4 software.
We established a functional assay to evaluate the activity of tested protein on mouse CD4 T cells function. The assay evaluates T cell function following activation with plate-bound anti-CD3 mAb, co-immobilized with tested Fc fusion proteins, as reported before for other immune checkpoint regulators, such as B7E14 and VISTA (Ref 1, 2). B7H4-Ig was used as a positive control in the assay (inhibitory ligand) while mouse IgG2a was used as a negative control. Schematic illustration of the experimental system and the effect of B7H4-Ig on T cell activation are described in
In order to evaluate the immunoinodulatory effect of HIDE1 on mouse CD4+ cells,
HIDE1-Fc (batch #72 and #195) was co-immobilized with anti-CD3, as described in material and methods. The effect of two batches of HIDE1.-Fc on mouse CD4 T cell activation, in the presence of soluble anti-CD28, upon co-immobilization on microplates with anti-CD3 was evaluated in two separate experiments using fresh or thawed isolated CD4 T cells. (
No effect of HIDE1-Fc on T cells activity was observed when 1 or 3 ug/ml of HIDE1-Fc were used. Nevertheless, at 10 ug/ml, HIDE1-Fc (batch #72 but not #195) inhibits CD69 expression and IFNγ secretion (Table 30).
Two experiments were performed with HIDE1-Fc, co-immobilized with anti-CD3, aiming at assessment of its effect on CD4 T cells activity. In the first experiment, performed with freshly isolated CD4 T cells, both batches of HIDE1-Fc suppressed mouse CD4 T cell activation. In the second experiments, performed with thawed CD4 T cells, the two batched inhibited T cell activity for different extent. Overall, the maximal inhibitory effect of HIDE1-Fc on cytokine secretion was observed at 10 ug/ml and the magnitude of the inhibition was in the range of 50-60%.
Materials and Methods
In these experiments the effects of HIDE1 on human T cells which were activated using anti-CD3 and anti-CD28 in the presence of autologous PBMCS was evaluated.
HIDE1 hECD-hIg fusion protein (SEQ ID NO:17, batch #48), composed of the ECD of human HIDE1 fused to the Fc of human IgG1 bearing C220 to S mutation at the hinge, was produced at ExcellGene (Switzeland) by transient transfection in CHO-DG44 cells using Excellgene's proprietary vector system. Cells were cultured for 10 days, followed by Protein A purification of cell harvest. The final product was formulated in 0.1M Glycine pH 6.
Expression vector used was Mammalian Expression Vector pTT5, in which HIDE1 gene is driven by CMV promoter.
CD4+ Human T cell Isolation Kit II is purchased from Miltenyi (Cat. #130-094-131). hIgG1 control (Synagis®) is obtained from Medimmune Inc. Anti-human CD3 Ab (OKT3, Cat#16-0037) and anti-human CD28 Ab (clone CD28. 2; Cat#16-0289) were purchased from eBioscience. Dynabeads M-450 Epoxy (Cat. #140. 11) were purchased from Invitrogen. Buffy coats of human blood were obtained from LifeSource. Ficoll-Paque Plus (Cat. #17-1440-02), was purchased from GE HealthCare.
Isolation of PBMCs from Buffy Coats Using Ficoll Separation
Total PBMCs were suspended in Ex-Vivo 20 medium, and irradiated at 3000 rad. Naïve CD4+ T cells were isolated from buffy coats of three healthy human donors' blood using CD4+ Human T cell Isolation Kit II (Miltenyi) according to manufacturer's instructions and co-cultured with irradiated autologous PBMCs at a ratio of 1:1 (1. 5×105 T cells with 1. 5×105 irradiated PBMCs per well). The cultures were activated with anti-CD3 (0. 5 μg/ml) and anti-CD28 (0. 5 μg/ml) antibodies. HIDE1 hECD-hIg (SEQ ID NO: 17) or hIgG1 control Ig (Synagis®) were added to the culture at the indicated concentrations. After 24 hr in culture, cells were pulsed with H3-thymidine. Cells were harvested after 72 hours in culture.
As shown in
The experiments described in this example evaluated the effect of ectopic expression of human HIDE1 on different melanoma cell lines on their ability to activate CTLs (cytotoxic T lymphocytes) and serve as targets for killing by these cells.
Materials & Methods:
Three human melanoma cell lines which present the MART-1 antigen in HLA-A2 context (SK-MEL-23, Mel-624 and Mel-624.38) were used as targets for CTLs. Mel-888 which does not express HLA-A2, served as a negative control.
Ectopic Expression of Human HIDE1 on Cytotoxic T Lymphocytes (CTLs).
In order to express human HIDE1 in peripheral blood leukocyte (PBL) cultures, the cDNA encoding for HIDE1 was amplified using specific primers and cloned into an MSCV-based retroviral vector (pMSGV1). Verification of the cloning was done first using restriction enzyme digestion and subsequently by sequencing. Upon sequence confirmation, large amounts of the retroviral vector (Maxi-prep) were produced for subsequent use.
Peripheral blood leukocytes of healthy human donors were transduced with the retroviral constructs encoding HIDE1 or with the retroviral vectors encoding for NGFR1 or an empty vector, as a negative control. Transduction was carried out using a retronectin-based protocol; briefly, retroviral supernatant was produced in 293GP cells (a retroviral packaging cell line) following transfection with the retroviral vector and an amphotropic envelop gene (VSV-G). The retroviral supernatant was plated on retronectin-coated plates prior to the transduction to enable the binding of virions to the plate, and the PBLs were added to the plate for 6 hours. After that, the cells were replenished in a new culture vessel. An antibody against an intracellular FLAG sequence (cat. No 637309; Biolegend) was used in order to evaluate transduction efficiency. Expression of NGFR was detected with commercial anti-NGFR (Cat.No 345108; BioLegend). Rabbit IgG (Sigma Cat. No. 15006) was used as isotype control, and as secondary antibody we used APC-conjugated anti-rabbit IgG (Jackson, Cat. No. 711-136-152).
In order to obtain effector lymphocytes that express the MART-1-specific F4 TCR, specifically recognizing MART-126-35-/HLA-A2 peptide-MHC complex, freshly isolated human PBLs previously transduced to express either with HIDE1, NGFR or an empty vector were stimulated with PHA and cultured for 5-10 days, and subsequently transduced with in vitro-transcribed mRNA encoding both α and β chains from the MART-1-specific F4 TCR. The transduced lymphocytes were cultured in lymphocyte medium (Bio target medium, fetal bovine serum (10%), L Glutamine Penicillin/Streptomycin (100 units/m1), IL-2 300 IU), replenished every 2-3 days. F4 TCR expression levels were verified by FACS staining using a specific monoclonal antibody that recognizes the extra-cellular domain of the beta-chain from the transduced specific TCR (TCR-Vb12-PE, (Cat.No IM2291; Beckman Coulter).
Cytokine Secretion from HIDE1, NGFR or an Empty Vector and F4-TCR Transduced Lymphocytes Upon Co-Culture with Melanoma Cells
PBLs expressing HIDE1 or NGFR along with F4-TCR were co-cultured with un-manipulated melanoma cells. 105 transduced PBLs were co-cultured with 105 melanoma target cells for 16 hours. In order to assess the response of the effector CD8 T cells to the different tumor cell lines, cytokine secretion (IFN-γ IL-2 and TNF-α) was measured by ELISA in culture supernatants (IFN-γ (Cat.No DY285E), IL-2 (Cat.No DY202E), TNF-α (Cat.No DY210E) R&D SYSTEMS), diluted to be in the linear range of the ELISA assay.
In order to analyze HIDE1 effect on T cell activation and CD137 surface expression in particular, cells were collected after 18 hr co-culture (E:T 1:1) and stained them with APC-anti-CD8a, FITC-anti-137 (Biolegend) in FACS buffer made of PBS, 0.5% BSA, and 0.02% sodium azide. All samples were analyzed by FACS.
In the experimental system described herein (depicted in
Human PBLs were transduced with a retroviral vector encoding the HIDE1 or an empty vector as negative control, as described in Materials & Methods. The levels of HIDE1 were assessed by flow cytometry at 48 hrs after transduction, and compared to cells transduced with an empty vector. Representative data showing HIDE1 expression detected by intra-cellular anti-FLAG staining is shown in
To perform functional assays with human CTLs, we used PBLs engineered to express the F4 TCR, which recognizes HLA-A2+/MART1+ melanoma cells, as described in Materials & Methods.
HIDE1 or Empty-vector and F4-transduced PBLs were co-cultured with melanoma cell lines. The levels of IFNγ secretion were measured at 16-hours of co-culture. The magnitude of inhibition of cytokine secretion due to over-expression is in the range of 90%. Representative data showing the effect of HIDE1 expressed on F4 expressing PBLs co-cultured with Mel-624, Mel-624.38 and SK-Mel23 on IFNγ secretion is presented in
HIDE1 or an empty vector and F4-transduced PBLs were co-cultured with different melanoma cell lines. The levels of TNFα secretion were assessed at 16-hours of co-culture. The magnitude of inhibition of cytokine secretion due to HIDE1 expression ranged between 60 to 80%. Representative data showing the effect of HIDE1 expressed on F4 expressing PBLs co-cultured with Mel-624, Mel-624.38, SK-Mel23 and MEL-526 on TNFα secretion is presented in
HIDE1 or an empty vector and F4-transduced PBLs were co-cultured with different melanoma cell lines. As shown in
Without wishing to be limited by a single hypothesis, the results presented herein indicate that overexpression on primary lymphocytes results in reduced cytokine secretion by CTLs, suggesting that HIDE1 has an inhibitory effect on CTLs, further supporting the therapeutic potential of the immunoinhibitory HIDE1-ECD polypeptides and/or fusion proteins thereof for agonizing HIDE1 inhibitory effect on immunity and treatment of autoimmune diseases.
In order to evaluate the immunomodulatory effect of HIDE1 on CD8+ T cell activation, the full length protein was ectopically expressed on EL4 cells which were pulsed with gp10025-33 peptide and then served as specific target cells for pre-activated pmel-1 transgenic CD8+ T cells, expressing H-2Db/gp10025-33. The effect of the HIDE1 on antigen-specific activity of pmel-1 CD8+ T cells was studied using several T cell activation readouts.
EL4 (TIB-39, ATCC) cells were transduced to express mouse HIDE1 protein using pMSCV retroviral vector encoding for HIDE1 codon-optimized cDNA. Following selection with Puromycin (4 μg/ml, SIGMA), HIDE1-expressing cells were generated. Mock-transduced EL4 cells were produced in parallel and further used as control in all experiments described below. Expression of HIDE1 in EL4 cells was confirmed applying staining with pAbs anti-mouse HIDE1 (GenScript, #488536_13), Detection was done using Donkey Anti Rabbit PE conjugated (Jackson, cat #711-116-152 (1:100)), followed by FACS analysis. To ensure comparable expression levels of H2-Db in HIDE1- and empty vector-transduced cells, staining with anti-H2Db (Cat#111508, Biolegends) antibody and subsequent FACS analysis were used.
Spleens were harvested from pmel-1 mice (6-12 weeks male or female).
Splenocytes were cultured in the presence of 1 μg/ml of gp100-peptide (human gp10025-33 peptide, Sigma) and 25 ng/ml of IL-2 (Cat#589106, Biolegend) for 4 days in 6 well plate at 1.5×106 cells/well (Cat#6570160, Greiner). Culture media was RPMI+10% fetal bovine serum and the following supplements from Gibco: 2-mercaptoethanol (dilution of 1:1000), Gluta-MAX, sodium pyruvate, penicillin/streptomycin, and non-essential amino acids (all at a dilution of 1:100) (splenocytes medium). Cells were diluted 1:2 on the next day with full culture media supplemented with gp100-peptide (1 μg/ml) and IL2 (25 ng/ml).
Pmel-1 splenocytes were collected and CD8+ cells were isolated using CD8a (ly-2) microbead (Miltenyi, 130-049-401). CD8+ pmel-1 cells were cultured in the presence of 25 ng/ml of IL-2 for O.N in 6 well plate at 1×106 cells/well in splenocytes medium. The purity of CD8+ pmel-1 cells was analyzed by FACS (>88% purity).
Secondary Stimulation: Co-Culture of Pre-Activated CD8+ Pmel-1 with Target Cells (Day 6)
Pmel-1 cultures were spun down and washed twice with fresh media. EL4 cells were pulsed with gp-100 peptide (0.3, 1 ng/ml) for 1 hour at 37° C. in serum free media. Following washing, peptide-pulsed targets cells were over-night co-cultured with 50,000 cell/well pmel-1 CD8+ T cells at an effector to target (E:T) ratio of 2:1. T cell activity was assessed based on detection of cytokines secretion (IFN-gamma, TNF-alfa, IL-2 and IL-10) in co-culture supernatants and by FACS analysis of surface expression of 41-BB (CD137) and CD25 (IL-2Ra).
16-24 hours post co-culture, cells were stained with viability dye (BD Horizon; Cat#562247, BD biosciences) followed by surface staining with FITC-anti-CD8a (Cat #100706, Biolegend), PE-anti-CD137 (Cat #106106, Biolegend) and APC-anti-CD25 (Cat #102012, Biolegend) in the presence of anti-CD16/32 (clone 2.4g2; Cat #101302, Biolegend) for blocking of Fcγ-receptors. All samples were run on a MACSQuant analyzer (Miltenyi) and data was analyzed using Tree Star FlowJo software. Culture supernatants were collected and analyzed for mouse Th1/Th2/Th17 cytokine CBA kit (Cat#560485, BD biosciences).
In order to evaluate the immunomodulatory effect of HIDE1 on CD8+ pmel-1 T cell function, the mouse protein was ectopically expressed on EL4 cells which served as specific target cells to pmel-1 CD8+ cells following pulsing with gp-10025-33 peptide (H2-Db restricted peptide).
PDL1 and MHC class I (H2-Db) on mock or PDL1-overexpressing clones. Similar H2-Db expression levels were observed for mock and PDL1-transduced EL4 clones cells (variation of H2-Db expression of PDL1-overexpressing cells compare to mock cells in three different experiments: (−3)-(+20) %, experiment 349-023 excluded: use of polyclonal PDL1-overexpressing cells express 43% more H2-Db compare to polyclonal mock cells. The folds of PDL1 expression compare to mock clones cells varies between 9.5 to 25 (in four different experiments).
In order to validate this assay for both target testing and functional Abs screen for the target, the inhibitory effect of PDL1 on pmel-1 CD8+ cells activity and the reverse effect of blocking anti-PDL1 (20 μg/ml, YWW243.55.S70, Inc.) was assessed first upon co-culture of pre-activated pmel-1 CD8+ cells with gp100-pulsed EL4 cells with forced expression of PDL1 (2:1, E:T)—PDL1 did reduced pmel-1 CD8+ cells activity, this inhibition was also restored by anti-PDL1 (CD137 expression is shown) (
Four experiments were performed to evaluate the proposed immuno-modulatory effect of ectopic expression of HIDE1 in target cells, specific to TCR-transgenic pmel-1 CD8+ T cells. The inhibitory effect of HIDE1, as well as of PDL1 as positive control, on activation of Ag-specific CD8+ T cells (pmel-1) following co-culture with target cells (EL4) pulsed with gp10025-33 peptide, was observed in 4 out of 4 experiments, as manifested by reduced cytokines secretion and by suppression of the activation-induced expression of activation markers CD137 and CD25. Both, for HIDE1 and PDL1, the inhibitory effect was greater at a concentration of 0.3 ng/ml gp100. In the second experiment (349-020) magnitude of inhibition was lesser than the two other experiments, but since that was also the case for PDL 1 and since trends of inhibition remain, we can assume that some technical fault occurred in this experiment. In the fourth experiment (349-023) PDL1 over-expressing cells express 43% more H2-Db vs. mock, that could be the reason for the relatively low percent of inhibition by PDL1 in this experiment. This assay design is used to test the ability of HIDE1-ECD polypeptides and/or fusion proteins thereof to agonise HIDE1 mediated inhibition.
Efficacy of HIDE1-ECD fusion protein in autoimmunity model supports immunoinhibitory function of HIDE1, which without wishing to be limited by a single theory, might allow tumor cells escape from immune surveillance. Activating immunoinhibitory HIDE1 protein with immunoinhibitory antibodies will be efficient for treatment of MS and other autoimmune disorders
To investigate further the immunoinhibitory HIDE1 properties and he therapeutic potential of immunoinhibitory HIDE1 targeting antibodies which agonize HIDE1 activity for treatment of autoimmune diseases, HIDE1-ECD-Ig is was tested in a mouse model of Multiple Sclerosis; Relapsing Remitting Experimental Autoimmune Encephalomyelitis (R-EAE).
Female SJL mice 6 weeks old were purchased from Harlan and maintained in the CCM facility for 1 week prior to beginning the experiment. Mice were randomly assigned into groups of 10 animals and primed with 50 μg PLP139-151/CFA on day 0. Mice received 6 i.p. injections of 100 μg/dose of immunoinhibitory HIDE1 targeting antibody, mIgG2a isotype control, or CTLA4-Ig (mouse ECD fused to mouse IgG2a Fc) as positive control. Treatments began at the time of disease induction (preventive mode) or at onset of disease remission (therapeutic mode) and were given 3 times per week for at least 2 weeks. Mice were scored for disease symptoms on a 0-5 disease score scale: 0, no abnormality; 1, limp tail; 2, limp tail and hind limb weakness; 3, hind limb paralysis; 4, hind limb paralysis and forelimb weakness; and 5, moribund.
On day 35, (during the peak of disease relapse) 5 mice of each group are assayed for DTH (delayed type hypersensitivity) response to disease inducing epitope (PLP139-151) and to relapse-associated myelin epitope (PLP178-191) via injection of 10 μg of PLP139-151 in one ear and PLP178-191 into the opposite ear. The level of ear swelling are assayed at 24 hours post challenge.
The results presented in
In a similar model, the dose dependency of the efficacy of HIDE1-ECD-Ig (SEQ ID NO:18) as well as its mode of action in the PLP-induced R-EAE model is evaluated. Disease is induced as described above and mice are treated from onset of disease remission with 100, 30 or 10 μg/dose HI DE1-ECD-Ig (SEQ ID NO: 18) 3 times per week over two weeks. The level of disease severity is evaluated and is anticipated to be reduced in a dose dependent manner.
HIDE1-ECD-Ig (SEQ ID NO:18) fusion proteins are also anticipated to inhibit DTH responses to spread epitopes PLP178-191 and MBP84-104 on days 45 and 76. Furthermore, recall responses analysis is carried out on day 45 and day 76 splenocytes and cervical lymph node cells, to PLP139-151, PLP178-191 and MBP84-104 in which proliferation as well as cytokine secretion (IFNγ, IL-4, IL-17, TNFα, GM-CSF, and others) are analyzed. Without being bound by a single hypothesis, modulatory effects on Th1 Th17 and/or Th2 cytokines are anticipated following treatment with HIDE1-ECD-Ig. In addition, CNS, draining (cervical) lymph nodes and spleens of mice treated with HIDE1-ECD-Ig are evaluated for infiltration of immune cells.
Surface expression of HIDE1 by healthy PBLs was evaluated using whole blood taken from 3 different healthy donors using anti-Hide1 mAbs 33B4-2F7 and Ab 36C1-2F6 as compared to staining with isotype control.
Prominent staining with anti HIDE1 mAb 33B4-2F7 was observed on monocytes (
Similarly, prominent HIDE1 expression was detected on myeloid blasts in AML while lymphocytes were essentially negative (
The aim of the presented functional assay was to evaluate the effect HIDE1 Knock down by siRNA or overexpression on T cell function as well as the effect of anti-HIDE1 antibodies on T cell function. In particular, to evaluate the immuno-modulatory effect of anti-HIDE1 antibodies that can be used to target monocytes, Tumor associated macrophages (TAMs), or other myeloid cells and screen for various functional activities, including modulating the interaction between HIDE-1 and its putative receptor(s), modulation of HIDE1 levels or direct signaling and attenuation of negative signaling. Such recombinant antibodies may be used as modulatory molecules to decrease or prevent HIDE1 from interacting with inhibitory receptor(s) on T cells or other cells in the tumor microenvironment, thereby releasing T cells or other functional cells from HIDE1 check point (“break”)/suppressive signaling.
THP1 cell line expressing HIDE1 were knocked down (KD) by specific siRNA to HIDE1 or scrambled (SCR) control or PDL1. 24 h post KD, cells were treated with 500 units of IFNγ for another 24 h to induce PDL1 expression and cell maturation. Prior to entering into co-culture assay, levels of HIDE1 or PDL1 KD compared to control siRNA (SCR) were evaluated amongst other markers as pan HLA-I/II or CD86 which were compared to their isotype control. THP1 cells treated with Mitomycin C (SIGMA; 50 ug/ml for 1 hr), were co-cultured at 1:1 ratio with CD3 T cells labeled with CFSE in 96 well flat bottom immobilized with 1000 ng/ml CD3 (#555329 UCHT1) for 5 days. In order to assess the response of the effector CD3 T cells to the different THP1 KD effects, proliferation was measured by the dilution of CFSE and cytokine secretion (IFN-γ & TNF-α) was measured by TH-1/2/17 CBA kit (BD biosciences) in culture supernatants.
THP1 cell line expressing HIDE1 were pre-treated with 500 units of INFγ for 24 h. 24 h post INFγ treatment cells were treated with mitomycin C (SIGMA; 50 ug/ml for 1 hr) and co-cultured with CFSE labeled CD3+ cells at 1:1 ratio in the presence of immobilized anti-CD3 (1 ug/ml; UCHT-1). Anti-HIDE1 blocking or agonistic mAb at 20 ug/ml were added to each culture. Either no antibody or an isotype control antibody was used as a negative control. After day 5, the effect of anti-HIDE1 antibodies on cytokine secretion was assessed from the supernatant using TH-1/2/17 CBA kit (BD biosciences).
A mixed lymphocyte reaction was employed to demonstrate the effect of blocking the HIDE1 pathway to lymphocyte effector cells. T cells in the assay were tested for proliferation and cytokine secretion in the presence or absence of an anti-HIDE1 human monoclonal antibodies (14 Abs with hIgG4 and 5 Abs with hIgG1 backbone). Human CD3 T-cells were purified from PBMC derived from Buffy coats using negative selection isolation kits (EasySep™, STEMCELLS technologies). Alloreactivite dendritic cells in a total volume of 200 μl. Anti-HIDE1 blocking or agonistic mAb at 20 ug/ml were added to each culture. Either no antibody or an isotype control antibody was used as a negative control. After day 5, the effect of anti-HIDE1 antibodies on T cell proliferation (CFSE dilution) and cytokine secretion (ELISA or TH1/2/17 CBA kits) in culture supernatants were assessed.
CFSE-labeled T cells were stimulated with stimulator cells (CHO cells expressing membrane-bound anti-CD3 mAb fragments). CHOS-stimulator cells expressing human HIDE1 and control stimulator cells (empty vector) treated with mitomycin C (50 μg/ml for 1 h) before co-cultured with CFSE-labeled human T cells at the ratio of 1:5. After 5 days at 37° C. and 5.0% CO2, the effect of anti-HIDE1 antibodies (20 ug/ml) on T cell proliferation (CFSE dilution) and cytokine secretion (ELISA or TH1/2/17 CBA kits) in culture supernatants was assessed. All samples were acquired in MACSQuant analyzer (Miltenyi) and data was analyzed using FlowJo software (v10.0.8). Culture supernatants were collected and analyzed for cytokine secretion by CBA kit (Cat #560484, BD).
THP1 cells were knocked down (KD) by specific siRNA to HIDE1 or scrambled (SCR) control or PDL1. 24 post KD, cells were treated with 500 units of IFNγ for another 24 h which induced PDL1 expression and cell maturation. Knock down levels of HIDE1 and PDL1 were 73% and 61% respectively, sufficient for entering into assay. Moreover, no variations in levels of HLA-I, HLA-II and CD86 compared to their isotype control suggesting KD doesn't effect THP1-1 cell state (
IFNγ-treated THP1 cells were co-cultured with CD3+ cells at 1:1 ratio in the presence of 1 ug/ml immobilized anti-CD3 and the effect of anti-HIDE1 antibodies at 20 ug/ml on cytokine secretion 5d post co-culture was tested (
CHOS-OKT3 overexpressing HIDE1 were co-cultured with CD3+ cells at 1:1 ratio in the presence of immobilized anti-CD3 and the effect of anti-HIDE1 antibodies at 20 ug/ml on cytokine secretion 5d post co-culture was tested (
The effect of anti-HIDE1 blocking or agonistic mAb in MLR assay was assessed 5 days post co-culture with allogeneic imDC. Either no antibody or HIDE1 isotype control antibody was used as a negative control. After day 5, the effect of anti-HIDE1 antibodies on T cell proliferation (CFSE dilution) gating on CD4 or CD8+ cells was evaluated (
Several anti-HIDE1 antibodies augmented T cell function in several functional assays.
In THP-1 co-culture assay, CPA.12.006-H4, CPA.12.007-H4, CPA.12.0012-H4, Ab-507 & AB-508 enhance IFNγ & TNFα secretion levels in at least 1 donor (
Aim: To evaluate the effect of HIDE1 on function of human Tumor-infiltrating lymphocytes (TIL) upon co-culture with HIDE1-transduced target cells.
Mel-526 and Mel-624 cell lines are MART-1+, HLA-A2+ melanoma cell line. All cell lines were cultured in complete medium consisting of DMEM supplemented with 10% FCS, 25 mmol/L HEPES, 2 mmol/L glutamine, and combined antibiotics (all from Invitrogen Life Technologies).
Tumor-infiltrating lymphocyte (TIL) micro-cultures were initiated and expanded from tumor specimens taken from resected metastases of two melanoma patients, as described (Uzana et al, JI 2012). Briefly, TILs were cultured in complete medium consisting of RPMI 1640 supplemented with 10% heat-inactivated human AB serum, 6000 IU/ml human rIL-2 (rhIL-2; Chiron, Amsterdam, The Netherlands), 2 mM L-glutamine, 1 mM sodium pyruvate, 25 mM HEPES, 50 mM 2-ME, and combined antibiotics (Invitrogen Life Technologies). On day 14 of TIL initiation, the lymphocytes were washed with PBS, resuspended in PBS supplemented with 0.5% BSA, and stained with FITC-conjugated HLA-A*0201/MART-126-35dextramer (Immudex, Copenhagen, Denmark) for 30 min at 4° C. The lymphocytes were then incubated with allophycocyanin-conjugated mouse anti-human CD8 (eBioscience) for an additional 30 min at 4° C. CD8+ lymphocytes, positively stained by the dextramer (CD8+/dextramer+ cells), were sorted by a BD FACSAria (BD Biosciences) and directly cloned at one or two cells/well in 96-well plates in the presence of ortho-anti-CD3 (30 ng/ml; eBioscience), rhIL-2 (6000 IU/ml), and 4 Gy-irradiated allogeneic PBMCs as feeder cells. Five days later, rhIL-2 (6000 IU/ml) was added and renewed every 2d thereafter. On day 14, the clones were assayed for IFNγ secretion in a peptide-specific manner following their co-incubation with MART-126-35-pulsed T2 cells (peptides were commercially synthesized and purified [>95%] by reverse-phase HPLC) using commercially available ELISA reagents (R&D Systems, Minneapolis, Minn.). The MART-126-35-reactive clones were further expanded in a second-round exposure to ortho-anti-CD3 (30 ng/ml) and (6000 IU/ml) rhIL-2 in the presence of 50-fold excess of irradiated feeder cells. A similar protocol was performed to obtain a gp100154-162 and gp100209-217 reactive clones. MART-126-35 and gp100154-162-reactive clones were derived from patient 412 while gp100209-217-reactive clones were derived from patient 209. Following TILs clones expansion the cells were stain with FITC or PE-conjugated HLA-A*0201/MART-126-35, HLA-A*0201/gp100154-162 or HLA-A*0201/gp100209-217 dextramer and analyzed by FACS. The study was approved by the Institutional Review Board at Sharett Institute of Oncology, Hadassah Medical Organization, Israel, and all patients gave their informed consent prior to initiation of melanoma and lymphocyte cell cultures. The TILs, which were sent to Compugen LTD under service agreement, kept in liquid nitrogen (10-20×106/vial) and thawed one day before co-culture with target cells.
Ectopic Expression of hPDL1 and hHIDE1 on Target Cells
Mel-526 and Mel-624 cells were transduced to express the human HIDE1 with a flag-tag in the C-terminus, using pMSGV1. retroviral vector or human PD-L1, using a pMSCV retroviral vector. Expression of hPDL1 and hHIDE1 on Mel cells was confirmed by flow cytometry following surface staining with a specific antibody to hPDL1 (Cat#329708, Biolegend) and hHIDE1 (in-house). The expression levels of HLA-A2 in hPDL1 and hHIDE1 was compared to the levels in the mock transduced cells by using anti-HLA-A2 antibody (Cat#343306, Biolegend).
Mel-526 and Mel-624 cells were transduced to express the human HIDE1 with a flag-tag in the C-terminus, using pMSGV1 retroviral vector. Expression of hPDL1 and hHIDE1 was validated as indicated in section A following sorting of the cells for higher HIDE1 expression.
To enlarge folds of expression of the transduced human HIDE1 Mel-526 and Mel-624 cell lines, cells were purified using anti-PE beads (Cat#130-048-801, Milteniy,) following staining by PE anti-hHIDE1 (Cat#33B4-2F7, Biotem) followed by Goat anti-mouse PE (cat#115-116-146, Jackson). The purified cells were used in herein assay.
Co-Culture of TILs with Target Cells
One day prior to co-culture, TILs were thawed and cultured with full IMDM media (cat#01-052-1A, Biological industries ltd (BI)) supplemented with 10% human serum (Sigma, H3667), 1% glutamax (Life technologies, 35050-038), 1% MEM eagle (cat#01-340-1B, BI), 1% Sodium pyruvate (cat#03-042-1B, BI), 1% PenStrep (cat#03-031-1B, BI) in the presence of 300 IU/ml of rhIL-2 (Cat#589106, Biolegend). Target cells and TILs were harvest and co-cultured at effector to target (E:T) ratios of 1:1 (1×10̂5 cells from each/well) or 3:1 (1×10̂5 TIL: 3×10̂4 target cells/well) in full IMDM media.
16 hours post co-culture, cells were stained with viability dye (BD Horizon; Cat#562247, BD biosciences) followed by surface staining with FITC-anti-CD8a (Cat #300906, Biolegend), PE-anti-CD137 (Cat #30984, Biolegend) and APC-anti-CD107a (Cat #328620, Biolegend) in the presence of Fc blocking solution (cat#422302, Biolegend). All samples were run on a MACSQuant analyzer (Miltenyi) and data was analyzed using Tree Star FlowJo software. Cells were first gated for lymphocytes (FSC-A vs. SSC-A), followed by singlets gate (FSC-H vs. FSC-A), and further gated for live cells. CD137, CD107a surface expression on gated CD8+ was analyzed. Culture supernatants were collected and analyzed for mouse Th1/Th2/Th17 cytokine CBA kit (Cat#560485, BD biosciences).
In order to investigate immune-modulatory effect of HIDE1 gp100 or MART-1 specific TILs were co-cultured with over expressing HIDE1 and HLA-A2+ target cells. The effect on T cells was assessed by analyzing activation markers surface expression and cytokine secretion. Target cells HLA-A2 and HIDE1/PD-L1 expression was validated prior to experiments (
hPDL1 transduced Mel-526 and Mel-624 cells were used as positive control in this assay. At the first experiment two ratios of E:T were tested, 1:1 and 1:3, as well as two target cells 526 and 624. The inhibitory effect of PDL1 was observed in co-culture of TILs with both target cells and was manifested in reduced IFNg secretion and surface expression of CD137. The inhibitory effect was most pronounced in co-culture of TILs with 624 cells and so we did not proceed to test 526 cells in the second experiment. hPDL1 had an inhibitory effect on all TILs tested, gp100154-162(designated TIL-154), gp100209-217 (designated TIL-209) and MART-126-35 (designated MART-1), though the inhibitory effect by hPDL1 was less pronounced with TIL 154 (
−54% *
−71% *
In the experimental system described herein, gp100 or MART-1-reactive TILs were co-cultured with HLA-A2+ target cells (624 and 526 Mels), which ectopically express HIDE1 and were sorted for higher expression of HIDE1 (149 and 54 folds vs. empty vector), and the effect on TILs effector function compare to mock-transduced cells, as manifested by activation markers and cytokines secretion; was investigated. Cells were validated before each experiment for their levels of target and HLA-A2 expression and were found out to be within the tolerable limit of target expression (more than 5 folds of expression) and of HLA-A2 level (differential expression between mock and hHIDE1/hPDL1 transduced cell lines of ±30%) (
This study was designed to isolate human antibodies with high affinity and specificity for the HIDE1 immuno-oncology target. This was achieved by panning a human antibody phage display library against a recombinant protein comprising the human HIDE1 extracellular domain (ECD) fused to human IgG1Fc (HIDE1-HH-1).
Preparation of Biotinylated HIDE1:
HIDE1-HH-1 (batch #280) was biotinylated using a Sulfo-NHS-LC-Biotin kit (Pierce). Free biotin was removed from the reactions by dialysis against PBS pH 7.2, 0.01% Tween 20. Synagis hIgG1 was also biotinylated to use for depletion steps in panning experiments.
General Method for ELISA:
Biotinylated proteins were captured on the wells of streptavidin-coated 96-well plates (Pierce). Antigen captured plate wells were blocked with PBS-milk (PBS pH 7.4, 5% skim milk powder). Blocking buffer was removed and a primary antibody solution was added and incubated for 1 hr at room temperature (RT). Plates were washed with PBS-T (PBS pH 7.4, 0.05% Tween20) followed by PBS. HRP-conjugated secondary antibody (dependent on the assay system) was added and incubated at RT for 1 hr, and plates were washed again. In some cases, a HRP-conjugated primary antibody (or other detection protein) was used directly. ELISA signals were developed using Sureblue TMB substrate (KPL Inc). Assay signals were read on 96-well plate reader at absorbance 450 nm.
Phage display and Fab screening: A single campaign was divided into four sub-campaigns with different washes (Table 37). Panning reactions were carried out in solution using streptavidin-coated magnetic beads to capture the biotinylated antigens. Beads were recovered using a magnetic rack (Promega).
Preparation of phage library for panning: All phage panning experiments used the XOMA031 human Fab antibody phage display library (XOMA Corporation, Berkeley, Calif.) blocked with 5% skim milk powder.
Antigen coupling to streptavidin beads: For each sub-campaign, biotinylated HIDE1-HH-1 was captured on Dynal streptavidin-coated magnetic beads (Life Technologies). Beads used for subtractive panning were coupled with the ‘depletion’ antigen (biotinylated Synagis hIgG1).
Depletion of human IgG Fc and streptavidin bead binders from the phage library: It was necessary to remove unwanted binders to streptavidin beads and the Fc region of HIDE1-HH-1 during the panning process. To achieve this, a phage aliquot was mixed with beads coupled to the Synagis hIgG1 depletion antigen and incubated at RT for 30 mins. Two depletion reactions were applied to all sub-campaigns. The depletion beads were then discarded.
Phage Panning:
Four rounds of phage panning were performed for each sub-campaign. For the first round, the blocked and depleted phage library was mixed with magnetic beads coupled to biotinylated HIDE1-HH-1 and incubated at RT for 1 hr. Non-specific phage were removed by washing with PBS-T and PBS at various stringency levels, depending on the specific panning sub-campaign (Table 37). After washing, bound phage were eluted by incubation with triethylamine (TEA) (EMD) and the eluate was neutralized by adding Tris-HCl pH 8.0 (Teknova). Second and later rounds were conducted the same way, except that the rescued phage supernatant from the previous round was used in place of the phage library.
Phage Rescue:
The phage eluate was infected into TG1 E. coli, which transformed the cells with the XOMA031 phagemid. Transformed cells were then spread on selective agar plates (carbenicillin) and incubated overnight at 30° C.
The resulting E. coli lawns were scraped and re-suspended in liquid growth media. A small aliquot of re-suspended cells was inoculated into a 50 mL culture (2YT with 2% glucose and carbenicillin) and grown at 37° C. until the OD at 600 nm reached 0.5. This culture was infected with M13K07 helper phage (New England Biolabs) and spun down. The cell pellet was resuspended in 50 ml media (2YT with carbenicillin and kanamycin, which is selection antibiotic for M13K07). The culture was then maintained at 25° C. to allow phage packaging. An aliquot of the culture supernatant was carried over for a subsequent round of panning.
Fab PPE Production:
The XOMA031 library is based on phagemid constructs that also function as IPTG inducible Fab expression vectors. Eluted phage pools from panning rounds 3 and 4 were diluted and infected into TG1 E. coli cells (Lucigen) so that single colonies were generated when spread on an agar plate. Individual clones were grown in 1 mL cultures (2YT with glucose and ampicillin) and protein expression was induced by adding IPTG (Teknova). Expression cultures were incubated overnight at 25° C. Fab proteins secreted into the E. coli periplasm were then extracted for analysis. Each plate of samples also included duplicate ‘blank PPE’ wells to serve as negative controls. These were created from non-inoculated cultures processed the same way as the Fab PPEs.
ELISA Binding Assays:
PPEs from the sub-campaigns were tested for binding to the panning antigen (biotinylated HIDE1-HH-1) and negative control antigens, PVR-hIgG1 Fc fusion and Synagis hIgG1. The ELISA followed the general protocol in paragraph [001056] (“General method for ELISA”). The primary antibody was replaced with a 50 μL aliquot of Fab or blank PPE and the secondary antibody was a HRP-conjugated anti-human F(ab′)2 antibody (Jackson Immunoresearch). ELISA binding was expressed as the ratio of Fab PPE binding signal: blank PPE signal. Positive hits were identified as those giving a ratio of at least two.
FACS Screening of PPE:
FACS analyses were conducted using adherent HEK-293T cells over-expressing the human HIDE1 (293T-hHIDE1). All analyses included negative control parental HEK-293T cells.
Reagent preparation and wash steps were carried out in FACS buffer (PBS with 1% BSA). Fab and blank PPEs were mixed with an aliquot of cells, incubated for 1 hr at 4° C. and then washed with FACS buffer. Cells were then mixed with an anti-C-myc primary antibody (Roche). After the same incubation and wash step cells were stained with an anti-mouse IgG Fc AlexaFlour-647 antibody (Jackson Immunoresearch). After a final incubation and wash cells were fixed in 2% paraformaldehyde made up in FACS buffer. Samples were read on a FACS Calibur (BD Bioscience) or HTFC screening system (Intellicyt). Data were analyzed using FCS Express (De Novo Software, CA, USA) or FloJo (De Novo Software, CA, USA). Results were expressed as the ratio of mean fluorescence intensity (MFI) of 293T-hHIDE1 cells: MFI signal of 293T control cells (MFI ratio). Positive hits were identified as those giving an MFI ratio of at least five.
Preliminary Fab binding kinetics were determined using Fab PPEs produced as described in paragraph [001064] (“Fab PPE production”)[001064]. Studies were conducted using Biacore 3000 and ProteOn XPR 36 instruments at 22° C.
A high density anti-human Fab antibody surface was prepared over one flow cell of a CM5 chip using a Biacore Human Fab Capture Kit (GE Healthcare) according to manufacturer's instructions. One surface of the CM5 chip was activated and blocked without coupling any anti-Fab antibody to act as a reference surface. Antibody immobilization was performed with HEPES-buffered saline, 0.005% polysorbate 20, pH 7.4 (HBS-P).
The molar concentrations of Fabs in PPE samples were quantitated using a Biacore 3000 instrument. Each Fab was diluted 10-fold and injected for 1 minute at 5 μL/min over the Fab capture and reference surfaces described above. A standard human Fab at a known concentration (Bethyl Laboratories, Inc.) was then injected over the anti-Fab surface under the same conditions as the Fab supernatants. The association slopes of each sensorgram from each Fab supernatant were fit against the association slope of the standard human Fab of known concentration using CLAMP 3.40 software to estimate the molar concentrations of each Fab in supernatant (Table 39).
A high density goat anti-human Fc antibody (Invitrogen) was immobilized by standard amine coupling over all lanes of a GLC chip using a ProteOn XPR 36 biosensor (Biorad). This surface was used to capture the recombinant HIDE1-H:H fusion protein (GenScript Lot 393383-21, batch #280) or a control Fc fusion protein (CD155, Sino Biologicals) in the vertical flow-cell direction at a concentration of ˜0.5 μg/mL for 90 seconds.
Each anti-HIDE1 Fab PPE sample was then injected at three concentrations (undiluted fab supernatant and two three-fold serial dilutions) in the horizontal direction over the captured fusion proteins. The dilutions were made using degassed PBS with 0.05% Tween 20 and 0.01% BSA. PPEs were injected for two minutes followed by 10 minutes of dissociation at a flow rate of 50 μL/min. The starting concentration range for each Fab is recorded in Table 39. The anti-human fc capture surfaces were regenerated with two 30-second pulses of 146 mM phosphoric acid after each cycle.
The resulting sensorgrams were processed and double-referenced using a ProteOn version of Scrubber 2.0 (BioLogic Software). Where appropriate, the sensorgrams were fit with a simple 1:1 kinetic binding model (with a term for mass transport) using ProteOn Scrubber 2.0. Sensorgrams and their respective kinetic fits are shown in
HIDE1 binding Fabs were converted to full length human IgGs by sub-cloning the relevant VH and VL domains into separate heavy and light chain expression vectors that already contained the appropriate heavy or light chain constant regions: pUNO3-H4 (human IgG4 heavy chain vector), pUNO3-HK (human kappa light chain vector) or pUNO3-HL (human lambda light chain vector). The base pUNO3 vector was sourced from Invivogen. Matched heavy and light chain constructs were co-transfected into Expi293 cells using Expifectamine and Opti-MEM (Life Technologies). Expression cultures were incubated at 37° C. for a further six days and supernatants were harvested by centrifugation. IgGs were purified from the supernatants using an AKTA Pure FPLC (GE Healthcare Bio-Sciences) and HiTrap MabSelect Sure affinity columns (GE Healthcare Bio-Sciences).
FACS screening of reformatted IgG4 antibodies: The cell lines described in paragraph [001066] (“FACS screening of PPE”) were mixed with FACS buffer containing the purified anti-HIDE1 IgGs or isotype controls. After incubation for 1 hr at 4° C., cells were washed with FACS buffer and stained with an anti-human IgG Fab AlexaFluor-647 antibody (Jackson Immunoresearch) for 45 mins at 4° C. Cells were washed again and fluorescence was detected using an Intellicyt HTFC screening system (Intellicyt). Data were analyzed using FCS Express (DeNovo) and plotted for affinity calculations (KD) in GraphPad Prism (GraphPad Software, Inc.). Note that each anti-HIDE1 antibody was tested over a titration range from 0-80 μg/mL.
Fab clones drawn from the four sub-campaigns were tested for binding by FACS and ELISA. Hits (defined as described in paragraphs [001065] and [001066]; “ELISA binding assays” and “FACS screening of PPE”) were sequenced to eliminate redundant Fabs. General screening outcomes are presented in Table 38. The ELISA and FACS screening hit rates from the 3rd Rd outputs ranged from 30% to 50% and 15% to 19% respectively. A sub-set of Fabs had binding activity that correlated between FACS and ELISA (35%-57%, depending on the sub-campaign). Sequence diversity ranged from 27% to 38%, which represents unique HCDR-3 families. A HCDR-3 family contains Fabs with the same heavy chain CDR-3 sequence, but minor differences in the heavy or light chain framework regions.
The ELISA and FACS screening hit rates from the 4th Rd outputs ranged from 15% to 36% and 7% to 16% respectively. A sub-set of Fabs had binding activity that correlated between FACS and ELISA (23%-75%, depending on the sub-campaign). Sequence diversity ranged from 18% to 41%.
The HCDR-3 families selected from different sub campaigns were highly redundant for both rounds 3 and 4. However, there were some additional low-frequency clones associated with specific sub-campaigns or panning rounds. Overall, the ELISA and FACS hit rates decreased from the third round to the fourth round. This suggests that optimum phage enrichment occurred in round three. Nonetheless, positive hits from the both rounds were included for the subsequent step.
A sub-set of the ELISA and FACS positive hits were subject to SPR kinetic screening. At least one member of each HCDR-3 family was included. Most Fabs displayed 1:1 kinetic binding (Table 39) to HIDE1-HH-1 (
The overall panning and screening exercise (ELISA, FACS and SPR) yielded 14 Fabs that showed sufficient binding activity to move forward into IgG reformatting. The identified Fabs include 12 HCDR-3 families, two of which have 2 members.
IgG Reformatting and Characterization:
The Fab binders described in paragraphs [001076]-[001079] (“Fab PPE screening”) were cloned into expression vectors for production as human IgG4 molecules. All of these antibodies were successfully expressed and purified. Thirteen were shown to bind the 293T-huHIDE1 cell line (Table 40). None of these antibodies were cross-reactive against the mouse HIDE1 ortholog.
This example provides characterization data for a panel of human antibodies raised against the HIDE1 antigen by phage display. A phage panning campaign was conducted using an ECD-Fc fusion protein. Overall, 13 IgGs binding to human HIDE1 were identified. These are recommended for further characterization of binding on cells lines that express HIDE1 endogenously, as well as in vitro functional assays
Table 38: Summary of ELISA and FACS Screening of Fab PPEs.
FACS results measure binding against the 293T-huHIDE1 cell line. ELISA results measure binding against recombinant HIDE1HH-1. FACS/ELISA correlation refers to the proportion of Fabs that bound in both assays (FACS and ELISA) compared to the total number of Fabs showing binding in either assay. Sequence diversity refers to the number of different Fabs present among the analyzed binders (i.e. 30% would indicate 30 different Fab sequences found within a set of 100 HIDE1 binders).
Table 39:
Preliminary Fab binding kinetics determine from PPE samples. The Fabs are listed in order of decreasing KD. Asterisks indicate the values should be taken with some caution, with an explanation in the comments column.
Table 40:
Overview of FACS binding results for human IgG4 antibodies reformatted from Fab screening hits. FACS binding was tested against the 293T-huHIDE1 (human) and 293T-moHIDE1 (mouse) cell. The results are expressed as a ‘Preliminary FACS KD’ calculated from titration curves described herein (“SPR Kinetic Screen of 24 Anti-HIDE1 Fab PPE”). IgGs from the same H-CDR3 family are indicated by the same color, either blue or green. CPA.12.003 & CPA.12.008 have identical heavy chain CDR-3 regions and minor differences in framework regions or other CDRs. The same holds for CPA.12.004 & CPA.12.013. N.B. not binding.
RNA extraction was performed with High Pure Paraffin Kit by JHU (Roche, cat#03270289001).
cDNA was produced using High Capacity cDNA Reverse Transcription Kit by JHU (Applied Biosystems cat#4368814).
Custom Taqman PreAmp pool (Life Technologies, cat #29233051).
TaqMan® PreAmp Master Mix Kit ×2 (Life Technologies, cat#4384266).
HIDE1 TaqMan probes: Hs01128131_m1 and Hs01128129_m1, Life technologies.
TaqMan probes for Housekeeping genes (HSKG) (Life technologies) human GUSB: Hs99999908_m1, human GAPDH: Hs99999905_m1, human RPL19: Hs01577060_gH and human HPRT1: Hs02800695_m1.
TaqMan Custom Arrays, 96A TLDA, Life Technologies, cat #4342259.
Tumor tissues were collected at the Johns Hopkins Hospital (Baltimore, Md.) from patients with primary sporadic colorectal cancer and free of prior chemotherapy. Assessment of MSI was done using the length of a panel of microsatellite markers in the tumor and a normal reference (either normal mucosa or germline) by using fragment analysis of PCR products labeled with fluorescent dyes. Fragment analysis determined the expression level of the proteins in charge of maintaining the integrity of microsatellite tracts. Differences in the length of two or more markers (the standard Bethesda panel uses five markers) were indicative of MSI status. 3 patients tested as MSI (microsatellite instable) positive and 3 patients as MSS (microsatellite stable); all patients' details are indicated in Table 42.
FFPE and hematoxylin and eosin-stained tissue sections (5 μm) were used for the LCM procedure using the Leica LMD 7000 system. For each patient, tissue sections were microdissected from three defined areas (TIL, Invasive Front, and Stroma) and directly collected in tissue lysis buffer for RNA extraction. RNA was isolated following the manufacturer's instructions. RNA was converted to cDNA using the High-Capacity RNA-to-cDNA Kit. The cDNA was used as undiluted or diluted 1:4, and a step of preamplification was performed using a pool of TaqMan probes and a preamplification master mix kit, followed by TaqMan qPCR.
Quantitative PCR (qPCR)
Preamplified cDNA, prepared as described above, was used as a template for qPCR reactions, using a gene specific TaqMan probes (detailed in Reagents 5&6) Detection was performed using QuantStudio 12k device.
The cycle in which the reactions achieved a threshold level of fluorescence (Ct=Threshold Cycle) was registered and was used to calculate the relative transcript quantity in the PCR reactions.
The absolute quantity was calculated by using the equation Q=2 ̂−Ct. Samples were normalized by the average Ct of the housekeeping genes: hGUSB, hRPL19, hGAPDH and hHPRT1, set across all samples to 25.
Undetectable value, was assigned as 40 cycles.
Endogenous Expression of HIDE1 in MSS and MSI Derived from Colorectal Cancer Patients
Endogenous Expression of HIDE1 in MSI and MSS Derived from Colorectal Cancer Patients Tested by qPCR
in order to verify the presence of the HIDE1 transcript in colorectal cancer derived cells, qRT-PCR was performed using a specific TaqMan probes as describe above in Material & Methods. qRT-PCR was performed by using undiluted and diluted 1:4 cDNA samples.
As shown in
Table 41. Represent normalized Ct values of undiluted and diluted (1:4) samples. Human HIDE1 transcript was observed using two specific TaqMan probes describes in M&M, in cell derived from three areas (TIL, IF, Stroma) from MSI and MSS colorectal cancer patients.
0.95325
indicates data missing or illegible when filed
The examples set forth above are provided to give those of ordinary skill in the art a complete disclosure and description of how to make and use the embodiments of the compositions, systems and methods of the invention, and are not intended to limit the scope of what the inventors regard as their invention. Modifications of the above-described modes for carrying out the invention that are obvious to persons of skill in the art are intended to be within the scope of the following claims. All patents and publications mentioned in the specification are indicative of the levels of skill of those skilled in the art to which the invention pertains. All references cited in this disclosure are incorporated by reference to the same extent as if each reference had been incorporated by reference in its entirety individually.
All headings and section designations are used for clarity and reference purposes only and are not to be considered limiting in any way. For example, those of skill in the art will appreciate the usefulness of combining various aspects from different headings and sections as appropriate according to the spirit and scope of the invention described herein.
All references cited herein are hereby incorporated by reference herein in their entireties and for all purposes to the same extent as if each individual publication or patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety for all purposes.
Many modifications and variations of this application can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. The specific embodiments and examples described herein are offered by way of example only.
This application claims priority under 35 U.S.C. § 119 to U.S. Ser. No. 62/191,775, filed Jul. 13, 2015, and U.S. Ser. No. 62/191,804, filed Jul. 13, 2015, both of which are expressly incorporated herein by reference in their entirety.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/IB2016/001079 | 7/13/2016 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62191775 | Jul 2015 | US | |
| 62191804 | Jul 2015 | US |
