Patent Application
20210269547
This application relates to targeted immune cell engaging agents for treating cancer.
The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Dec. 4, 2020, is named 030258-092180WOPT_Sequence_Listing.txt and is 71,065 bytes in size.
Cancer creates significant loss of life, suffering, and economic impact. Immunotherapeutic strategies for targeting cancer have been an active area of translational clinical research.
A variety of other approaches have been explored for immunotherapy, but many of these prior approaches lack sufficient specificity to particular cancer cells. For example, demibodies have been designed each having an scFv portion binding to different antigens on a target cell, an Fc domain allowing pairing to a complementary demibody, and a binding partner capable of forming an association to another binding partner on a complementary demibody. WO 2007/062466. These demibodies, however, are not necessarily specific to cancer cells and could bind and have activity on other cells expressing the same antigens. See also WO 2013/104804, which provides a first polypeptide with a targeting moiety binding to a first antigen and a first fragment of a functional domain, along with a second polypeptide with a targeting moiety binding to a second antigen and a second fragment of a functional domain that is complementary to the first fragment of the functional domain. Likewise, this approach is not necessarily specific to cancer cells and could bind and have activity on other cells expressing the same antigens.
Bispecific T-cell Engaging Antibodies (BiTEs) have been proposed by others; however, these constructs are often not sufficiently specific to the tumor environment. Additionally, BiTEs also can activate regulatory T cells (Tregs), promoting undesired Treg activity at the tumor site. For example, stimulating Tregs has been associated, in certain patients, with high levels of proliferation of suppressive Tregs and rapid cancer progression, termed hyperprogressive disease (see Kamada et al., PNAS 116(20):9999-10008 (2019)). Specific instances of hyperprogressive disease have been seen in patients treated with anti-PD-1 antibodies, which activates and expands certain tumor-infiltrating PD-1+ Treg cells, but concerns exist that other means of stimulating Tregs could have similar unwanted effects in a minority of patients.
Other approaches employing more specificity so that T cells are targeted to cancer cells do not have any means for selecting which T cells arrive at or are activated at the site of the cancer. WO 2017/087789. Activating all T cells, including T cells that do not benefit an immunooncology approach for treating the patient's cancer.
There are two problems with the current bi-specific antibody approach of activating T cells via CD3. The first of these is the over-activation of the immune response. Although not widely discussed, these agents are incredibly potent and are given at extremely low doses compared with whole antibody therapies. This will be partly due to the fact that these reagents can theoretically activate every T cell by binding to CD3. When someone has a viral infection, around 1-10% of their T cells are activated and they feel lethargic and ill because of the immune response. When more T cells are activated, this can lead to larger problems including cytokine release syndrome (CRS) and death in rare cases. CRS can be triggered by release of cytokines from cells targeted by biologics, as well as by cytokine release from recruited immune effector cells. Therefore, there is a need to limit the total number of T cells that are activated using these systems.
The second problem with current BiTE therapies is the CD3-specific activation of any T cell that is in the vicinity of the BiTE-bound target cell. Many immune cells respond to CD3 activation, including CD4 T cells (helper, regulatory, TH17, etc) and CD8 T cells, depending on which cells bind to the BiTE. This may mean that the efficacy of the BiTE is lost because activation of unwanted T cells such as regulatory T cells and TH17 T cells, inhibiting the cytolytic function of T cells such as CD8 T cells and cytotoxic CD4 T cells. Therapies could also be improved if they only activated particular types of T cells, such as only activating CD8+ T cells. The art has not previously proposed a solution to this problem. Only with this invention have we discovered the benefit of a system whereby the tumor-targeting was present to provide specificity for the unwanted and a second moiety was present to selectively bind to desirable immune cells which could combine at the site of the unwanted cancer cells and kill them.
In accordance with the description, this application describes agents and methods of treatment of cancer using antibody tumor-targeting assembly complexes (ATTACs).
In some embodiments, an agent for treating cancer in a patient comprises: (a) a first component comprising a targeted immune cell binding agent comprising: (i) a targeting moiety capable of targeting the cancer; and (ii) a first immune cell engaging domain capable of immune engaging activity when binding a second immune cell engaging domain, wherein the second immune cell engaging domain is not part of the first component; (b) a second component comprising a selective immune cell binding agent comprising: (i) an immune cell capable of selectively targeting an immune cell; and (ii) a second immune cell engaging domain capable of immune cell engaging activity when binding the first immune cell engaging domain, wherein the first and second immune cell engaging domains are capable of binding when neither is bound to an inert binding partner, wherein at least one of the first immune cell engaging domain or the second immune cell engaging domain is bound to an inert binding partner such at the first and second immune cell engaging domains are not bound to each other unless the inert binding partner is removed; and further comprising a cleavage site separating the first inert binding partner and the immune cell engaging domain to which it binds, wherein the cleavage site is: (i) cleaved by an enzyme expressed by the cancer cells; (ii) cleaved through a pH-sensitive cleavage reaction inside the cancer cell; (iii) cleaved by a complement-dependent cleavage reaction; or (iv) cleaved by a protease that is colocalized to the cancer cell by a targeting moiety that is the same or different from the targeting moiety in the agent.
In some embodiments, the first component is not covalently bound to the second component. In some embodiments, the first component is covalently bound to the second component.
In some embodiments, the immune cell engaging domains, when bound to each other, are capable of binding an antigen expressed on the surface of the immune cell. In some embodiments, the immune cell selection moiety capable of selectively targeting an immune cell selectively targets a T cell, a macrophage, a natural killer cell, a neutrophil, an eosinophil, a basophil, a γδ T cell, a natural killer T cell (NKT cells), or an engineered immune cell.
In some embodiments, the immune cell selection moiety capable of selectively targeting an immune cell selectively targets a T cell. In some embodiments, the T cell is a cytotoxic T cell. In some embodiments, the cytotoxic T cell is a CD8+ T cell. In some embodiments, the T cell is a helper T cell. In some embodiments, the helper T cell is a CD4+ T cell. In some embodiments, the immune cell selection moiety targets CD8, CD4, or CXCR3. In some embodiments, the immune cell selection moiety does not specifically bind regulatory T cells. In some embodiments, the immune cell selection moiety does not specifically bind TH17 cells. In some embodiments, the immune cell engaging domains, when bound to each other, are capable of binding CD3. In some embodiments, the immune cell engaging domains, when bound to each other, are capable of binding TCR.
In some embodiments, the immune cell selection moiety capable of selectively targeting an immune cell selectively targets a natural killer cell. In some embodiments, the immune cell selection moiety targets CD2 or CD56. In some embodiments, the immune cell engaging domains, when bound to each other, are capable of binding NKG2D, CD16, NKp30, NKp44, NKp46 or DNAM.
In some embodiments, the immune cell selection moiety capable of selectively targeting an immune cell selectively targets a macrophage. In some embodiments, the immune cell selection moiety targets CD14, CD11b, or CD40. In some embodiments, the immune cell engaging domains, when bound to each other, are capable of binding CD89 (Fc alpha receptor 1), CD64 (Fc gamma receptor 1), CD32 (Fc gamma receptor 2A) or CD16a (Fc gamma receptor 3A).
In some embodiments, the immune cell selection moiety capable of selectively targeting an immune cell selectively targets a neutrophil. In some embodiments, the immune cell selection moiety targets CD15. In some embodiments, the immune cell engaging domains, when bound to each other, are capable of binding CD89 (FcαR1), FcγRI (CD64), FcγRIIA (CD32), FcγRIIIA (CD16a), CD11b (CR3, αMβ2), TLR2, TLR4, CLEC7A (Dectin1), formyl peptide receptor 1 (FPR1), formyl peptide receptor 2 (FPR2), or formyl peptide receptor 3 (FPR3).
In some embodiments, the immune cell selection moiety capable of selectively targeting an immune cell selectively targets an eosinophil. In some embodiments, the immune cell selection moiety targets CD193, Siglec-8, or EMR1. In some embodiments, the immune cell engaging domains, when bound to each other, are capable of binding CD89 (Fc alpha receptor 1), FcεRI, FcγRI (CD64), FcγRIIA (CD32), FcγRIIIB (CD16b), or TLR4.
In some embodiments, the immune cell selection moiety capable of selectively targeting an immune cell selectively targets a basophil. In some embodiments, the immune cell selection moiety targets 2D7, CD203c, or FcεRIα. In some embodiments, the immune cell engaging domains, when bound to each other, are capable of binding CD89 (Fc alpha receptor 1) or FcεRI.
In some embodiments, the immune cell selection moiety capable of selectively targeting an immune cell selectively targets a γδ T cell. In some embodiments, the immune cell selection moiety targets γδ TCR. In some embodiments, the immune cell engaging domains, when bound to each other, are capable of binding γδ TCR, NKG2D, CD3 Complex (CD3ε, CD3γ, CD3δ, CD3ζ, CD3η), 4-1BB, DNAM-1, or TLRs (TLR2, TLR6).
In some embodiments, the immune cell selection moiety capable of selectively targeting an immune cell selectively targets a natural killer T cell. In some embodiments, the immune cell selection moiety targets Vα24 or CD56. In some embodiments, the immune cell engaging domains, when bound to each other, are capable of binding αβTCR, NKG2D, CD3 Complex (CD3ε, CD3γ, CD3δ, CD3ζ, CD3η), 4-1BB, or IL-12R.
In some embodiments, the immune cell selection moiety capable of selectively targeting an immune cell selectively targets an engineered immune cell. In some embodiments, the engineered immune cell is a CAR T cell, natural killer cell, natural killer T cell, or γδ T cell. In some embodiments, the immune cell selection moiety targets the CAR or a marker expressed on the immune cell. In some embodiments, the immune selection moieties targets LNGFR or CD20. In some embodiments, the immune cell engaging domains, when bound to each other, are capable of binding an antigen expressed by the engineered immune cell. In some embodiments, the antigen expressed by the engineered immune cell is CD3.
In some embodiments, the immune cell selection moiety comprises an antibody or antigen-specific binding fragment thereof. In some embodiments, the antibody or antigen-specific binding fragment thereof specifically binds an antigen on a T cell. In some embodiments, the antibody or antigen-specific binding fragment thereof specifically binds an antigen on a cytotoxic or helper T cell. In some embodiments, the antibody or antigen-specific binding fragment thereof specifically binds an antigen on a macrophage. In some embodiments, the antibody or antigen-specific binding fragment thereof specifically binds an antigen on a natural killer cell. In some embodiments, the antibody or antigen-specific binding fragment thereof specifically binds an antigen on a neutrophil. In some embodiments, the antibody or antigen-specific binding fragment thereof specifically binds an antigen on an eosinophil. In some embodiments, the antibody or antigen-specific binding fragment thereof specifically binds an antigen on a γδ T cell. In some embodiments, the antibody or antigen-specific binding fragment thereof specifically binds an antigen on a natural killer T cell. In some embodiments, the antibody or antigen-specific binding fragment thereof specifically binds an antigen on an engineered immune cell. In some embodiments, the engineered immune cell is a CAR T cell, natural killer cell, natural killer T cell, or γδ T cell.
In some embodiments, the immune selection moiety comprises an aptamer. In some embodiments, the aptamer specifically binds an antigen on a T cell. In some embodiments, the aptamer specifically binds an antigen on a cytotoxic or helper T cell. In some embodiments, the aptamer specifically binds an antigen on a macrophage. In some embodiments, the aptamer specifically binds an antigen on a natural killer cell. In some embodiments, the aptamer specifically binds an antigen on a neutrophil. In some embodiments, the aptamer specifically binds an antigen on an eosinophil. In some embodiments, the aptamer specifically binds an antigen on a γδ T cell. In some embodiments, the aptamer specifically binds an antigen on a natural killer T cell. In some embodiments, the aptamer specifically binds an antigen on an engineered immune cell. In some embodiments, the engineered immune cell is a CAR T cell, natural killer cell, natural killer T cell, or γδ T cell.
In some embodiments, the aptamer comprises DNA. In some embodiments, the aptamer comprises RNA. In some embodiments, the aptamer is single-stranded. In some embodiments, the aptamer is a selective immune cell binding-specific aptamer chosen from a random candidate library.
In some embodiments, the targeting moiety is an antibody or antigen-specific binding fragment. In some embodiments, the antibody or antigen-specific binding fragment thereof specifically binds a cancer antigen. In some embodiments, the targeting moiety is an aptamer. In some embodiments, the aptamer specifically binds a cancer antigen. In some embodiments, the aptamer comprises DNA. In some embodiments, the aptamer comprises RNA. In some embodiments, the aptamer is single-stranded. In some embodiments, the aptamer is a target cell-specific aptamer chosen from a random candidate library. In some embodiments, the aptamer is an anti-EGFR aptamer. In some embodiments, the anti-EGFR aptamer comprises any one of SEQ ID NOs: 95-164. In some embodiments, the aptamer binds to the cancer on the cancer cell with a Kd from 1 picomolar to 500 nanomolar. In some embodiments, the aptamer binds to the cancer with a Kd from 1 picomolar to 100 nanomolar.
In some embodiments, the targeting moiety comprises IL-2, IL-4, IL-6, α-MSH, transferrin, folic acid, EGF, TGF, PD1, IL-13, stem cell factor, insulin-like growth factor (IGF), or CD40. In some embodiments, the targeting moiety comprises a full-length sequence of IL-2, IL-4, IL-6, α-MSH, transferrin, folic acid, EGF, TGF, PD1, IL-13, stem cell factor, insulin-like growth factor (IGF), or CD40. In some embodiments, the targeting moiety comprises a truncated form, analog, variant, or derivative of IL-2, IL-4, IL-6, α-MSH, transferrin, folic acid, EGF, TGF, PD1, IL-13, stem cell factor, insulin-like growth factor (IGF), or CD40. In some embodiments, the targeting moiety binds a target on the cancer comprising IL-2 receptor, IL-4, IL-6, melanocyte stimulating hormone receptor (MSH receptor), transferrin receptor (TR), folate receptor 1 (FOLR), folate hydroxylase (FOLH1), EGF receptor, PD-L1, PD-L2, IL-13R, CXCR4, IGFR, or CD40L.
In some embodiments, one immune cell engaging domain comprises a VH domain and the other immune cell engaging domain comprises a VL domain. In some embodiments, the first immune cell binding partner is bound to the inert binding partner and separated from it by a cleavage site.
In some embodiments, the second immune cell binding partner is bound to the inert binding partner and separated from it by a cleavage site.
This application also describes an agent, wherein the first immune cell binding partner is bound to the inert binding partner and separated from it by a first cleavage site and the second immune cell binding partner is bound to the inert binding partner and separated from it by a second cleavage site.
In some embodiments, the first cleavage site and the second cleavage site are the same cleavage site. In some embodiments, the first cleavage site and the second cleavage site are different cleavage sites.
In some embodiments, at least one cleavage site is a protease cleavage site.
In some embodiments, at least one enzyme expressed by the cancer cells is a protease.
In some embodiments, at least one inert binding partner specifically binds the immune cell engaging domain. In some embodiments, at least one inert binding partner is a VH or VL domain.
In some embodiments, when the immune cell engaging domain is a VH domain, the inert binding partner is a VL domain, and when the immune cell engaging domain is VL domain, the inert binding partner is a VH domain.
This application also describes an agent for use in a two-component system for treating cancer comprising a a selective immune cell binding agent comprising: (a) a first component comprising a targeted immune cell binding agent comprising: (i) a targeting moiety capable of targeting the cancer; (ii) a first immune cell engaging domain capable of immune engaging activity when binding a second immune cell engaging domain, wherein the second immune cell engaging domain is not part of the first component; (b) a cleavage site separating the first immune cell engaging domain and the inert binding partner, wherein the cleavage site is: (i) cleaved by an enzyme expressed by the cancer cells; (ii) cleaved through a pH-sensitive cleavage reaction inside the cancer cell; (iii) cleaved by a complement-dependent cleavage reaction; or (iv) cleaved by a protease that is colocalized to the cancer cell by a targeting moiety that is the same or different from the targeting moiety in the agent, wherein cleavage of the cleavage site causes loss of the inert binding partner and allows for binding to the second immune cell engaging domain that is not part of the agent.
In some embodiments, the first component is covalently bound to the second component by a linker comprising a cleavage site.
In some embodiments, the cleavage site is a protease cleavage site.
In some embodiments, the protease cleavage site is cleavable in blood. In some embodiments, the protease cleavage site is a cleavage site for thrombin, neutrophil elastase, or furin.
In some embodiments, the protease cleavage site is cleavable by a tumor-associated protease. In some embodiments, the tumor-associated protease cleavage site comprises any one of SEQ ID NOs: 1-84.
This application also describes a set of nucleic acid molecules encoding the first and second component of the agent.
This application also describes a nucleic acid molecule encoding the selective immune cell binding agent.
This application also describes methods of treating cancer in a patient comprising administering the agent described herein.
In some embodiments, if the patient has regulatory T cells in the tumor, the selective immune cell binding agent does not target markers present on regulatory immune cells (including, but not limited to CD4 and CD25).
In some embodiments, the selective immune cell binding agent does not target markers present on TH17 cells. In some embodiments, the selective immune cell binding agent activates T cells that will target the tumor cells for lysis.
In some embodiments, if the patient has regulatory T cells in the tumor, the immune cell selection moiety targets CD8+ T cells by specifically binding CD8.
In some embodiments, if the patient has regulatory T cells in the tumor, the immune cell selection moiety targets CD8+ T cells and CD4+ T cells by specifically binding CXCR3.
In some embodiments, the cancer is any one of breast cancer, ovarian cancer, endometrial cancer, cervical cancer, bladder cancer, renal cancer, melanoma, lung cancer, prostate cancer, testicular cancer, thyroid cancer, brain cancer, esophageal cancer, gastric cancer, pancreatic cancer, colorectal cancer, liver cancer, leukemia, myeloma, nonHodgkin lymphoma, Hodgkin lymphoma, acute myeloid leukemia, acute lymphoblastic leukemia, chronic lymphoblastic leukemia, lymphoproliferative disorder, myelodysplastic disorder, myeloproliferative disease or premalignant disease.
This application also describes a method of targeting an immune response of a patient to cancer comprising administering an agent described herein to a patient.
Additional objects and advantages will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice. The objects and advantages will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims.
It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the claims.
The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate one (several) embodiment(s) and together with the description, serve to explain the principles described herein.
Table 1A provides a listing of certain sequences referenced herein. Table 1B provides a listing of certain construct sequences used herein.
KKDGGVKLDETGGGLVQPGGAMKLSCVTSGFT
DQVQLVESGGGVVQPGRSLRLSCAASGFTFSSY
*
GSGGADDIVMTQTPLSLSVTPGQPASISCKSSQSI
*
GGGGSELVMTQSPSSLTVTAGEKVTMSCKSSQS
EGTSTGSG
AIPVSLR
GSGGSGGADQVQLVESGG
*
EGTSTGSGGSGGSGGADQVQLVESGGGVVQPG
*
GSGGSGGADQVQLVESGGGVVQPGRSLRLSCA
*
GSDVQINQSPSFLAASPGETITINCRTSRSISQYLA
SLR
GSGGSGGADQVQLVESGGGVVQPGRSLRLS
*
GGSGGGGSDIVMTQSPDSLAVSLGERVTMNCK
*
GGSGGGGSGGGGSDIVMTQTPLSLSVTPGQPAS
The term ATTAC refers to a antibody tumor-targeting assembly complex. By using the word complex, the application refers to the need to have both a first component and a second component to make a complete functional molecule (i.e., the “complex”). The term complex also refers to the Boolean operator logic based upon (i) antigen expression on cancer cells, (ii) protease locations, and (iii) immune cell markers on desired immune cells. By applying logic gating, we obviate many of the current challenges with T-cell engaging antibodies.
ATTACs refer to using one ATTAC component that binds to a cancer antigen and one ATTAC component that does not bind to a cancer antigen, but instead selectively targets an immune cell. Thus, the ATTAC components do not have a parallel configuration (as in prior agents where both members of the ATTAC pair bound to cancer antigens), but instead have a trans configuration.
In an ATTAC component or pair, a first component comprising (a) a targeted immune cell binding agent comprises:
At least one of the first immune cell engaging domain or the second immune cell engaging domain is bound to an inert binding partner such at the first and second immune cell engaging domains are not bound to each other unless the inert binding partner is removed. The inert binding partner, when present, is bound to the immune cell engaging domain by a cleavage site separating the inert binding partner and the immune cell engaging domain to which it binds, wherein the cleavage site is:
A. Single Polypeptide Chain or Two Components
In some embodiments, the first component is covalently bound to the second component. In some embodiments, the first component is not covalently bound to the second component.
In some embodiments, the ATTAC is comprised of two separate components.
In other words, the ATTAC can be comprised of a first and second component that are separate polypeptides.
In some components, the ATTAC is comprised of a single polypeptide chain. In some embodiments, the first and second components are contained within a single amino acid sequence.
When the ATTAC is comprised of a single polypeptide chain, the first and second components may be separated by a linker. In some embodiments, this linker covalently binds the first and second components. In some embodiments, this linker comprises a cleavable linker. In some embodiments, the cleavable linker between the first and second components comprises a protease cleavage site.
In some embodiments, a cleavage site comprised within a linker covalently binding a first component and the second component is a protease cleavage site. SEQ ID NOs: 1-84 list some exemplary protease cleavage sites that may be used, but the invention is not limited to this set of proteases cleavage sites and other protease cleavage sites may be employed.
In some embodiments, a cleavage site comprised within a linker covalently binding a first component and the second component is a tumor-associated protease cleavage site. A tumor associated protease is one that is associated with a tumor. In some embodiments, a tumor-associated protease has higher expression in the tumor versus other regions of the body. Table 3A provides examples of tumor-associated proteases, although any protease with expression in a tumor may be used to select a tumor-associated protease cleavage site for the invention.
In some embodiments, a cleavage site comprised within a linker covalently binding a first component and the second component is a cleavage site for a protease found in the blood. Exemplary proteases found in the blood include thrombin, neutrophil elastase, and furin.
B. Immune Cell Selection Moiety
In some embodiments, an ATTAC comprises an immune cell selection moiety specific for a particular immune cell. In some embodiments, the immune cell selection moiety is specific for CD8+ T cells, CD4+ T cells, natural killer (NK) cells, macrophages, neutrophils, eosinophils, basophils, γδ T cells, natural killer T cells (NKT cells), or engineered immune cells. Engineered immune cells refers to immune cells with engineered receptors with new specificity. Examples of engineered immune cells include chimeric antigen receptor (CAR) T cells, NK, NKT, or γδ T cells.
In some embodiments, the immune cell selection moiety targets an immune cell marker that is not a tumor antigen. In some embodiments, the immune cell selection moiety allows targeting of an ATTAC to an immune cell, wherein the immune cell is not a cancer cell. In some embodiments, the immune cell selection moiety does not target the ATTAC to a lymphoma, myeloma, or leukemia. In some embodiments, the ATTAC targets a solid tumor (in other words any tumor not of an immune cell).
In some embodiments, the immune cell selection moiety does not specifically bind regulatory T cells. In some embodiments, the immune cell selection moiety does not specifically bind TH17 cells. In some embodiments, the selective immune cell binding agent does not target markers present on regulatory immune cells (including, but not limited to CD4 and CD25).
Table 2 lists some representative immune cell selection moieties for different desired immune cells.
Comparative
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C. Targeting Moiety Capable of Targeting the Cancer
The targeting moiety functions in the first component comprising a targeted immune cell engaging agent by delivering the agent to the local environment of the cancer cells, enabling a localized treatment strategy. In certain embodiments, the targeting moiety targets the cancer cells by specifically binding to the cancer cells. In some instances, the targeting moiety specifically binds the cancer cells even while the inert binding partner is binding the first immune cell engaging domain.
In certain embodiments, the targeting moiety is an antibody or antigen-binding fragment thereof. By antigen-binding fragment, we mean any antibody fragment that retains its binding activity to the target on the cancer cell, such as an scFv or other functional fragment including an immunoglobulin devoid of light chains, VHH, VNAR, Fab, Fab′, F(ab′)2, Fv, antibody fragment, diabody, scAB, single-domain heavy chain antibody, single-domain light chain antibody, Fd, CDR regions, or any portion or peptide sequence of the antibody that is capable of binding antigen or epitope. VHH and VNAR are alternatives to classical antibodies and even though they are produced in different species (camelids and sharks, respectively), we will also include them in antigen-binding fragments of antibodies. Unless specifically noted as “full length antibody,” when the application refers to antibody it inherently includes a reference to an antigen-binding fragment thereof.
Certain antibody targets (with examples of cancer cell types in parentheses) may include: Her2/Neu (Epithelial malignancies); CD22 (B cells, autoimmune or malignant); EpCAM (CD326) (Epithelial malignancies); EGFR (epithelial malignancies); PSMA (Prostate Carcinoma); CD30 (B cell malignancies); CD20 (B cells, autoimmune, allergic or malignant); CD33 (Myeloid malignancies); membrane lgE (Allergic B cells); lgE Receptor (CD23) (Mast cells or B cells in allergic disease), CD80 (B cells, autoimmune, allergic or malignant); CD86 (B cells, autoimmune, allergic or malignant); CD2 (T cell or NK cell lymphomas); CA125 (multiple cancers including Ovarian carcinoma); Carbonic Anhydrase IX (multiple cancers including Renal Cell Carcinoma); CD70 (B cells, autoimmune, allergic or malignant); CD74 (B cells, autoimmune, allergic or malignant); CD56 (T cell or NK cell lymphomas); CD40 (B cells, autoimmune, allergic or malignant); CD19 (B cells, autoimmune, allergic or malignant); c-met/HGFR (Gastrointestinal tract and hepatic malignancies; TRAIL-R1 (multiple malignancies including ovarian and colorectal carcinoma); DRS (multiple malignancies including ovarian and colorectal carcinoma); PD-1 (B cells, autoimmune, allergic or malignant); PD1L (Multiple malignancies including epithelial adenocarcinoma); IGF-1R (Most malignancies including epithelial adenocarcinoma); VEGF-R2 (The vasculature associated with the majority of malignancies including epithelial adenocarcinomas; Prostate stem cell antigen (PSCA) (Prostate Adenocarcinoma); MUC1 (Epithelial malignancies); CanAg (tumors such as carcinomas of the colon and pancreas); Mesothelin (many tumors including mesothelioma and ovarian and pancreatic adenocarcinoma); P-cadherin (Epithelial malignancies, including breast adenocarcinoma); Myostatin (GDF8) (many tumors including sarcoma and ovarian and pancreatic adenocarcinoma); Cripto (TDGF1) (Epithelial malignancies including colon, breast, lung, ovarian, and pancreatic cancers); ACVRL 1/ALK1 (multiple malignancies including leukemias and lymphomas); MUC5AC (Epithelial malignancies, including breast adenocarcinoma); CEACAM (Epithelial malignancies, including breast adenocarcinoma); CD137 (B cells or T cells, autoimmune, allergic or malignant); CXCR4 (B cells or T cells, autoimmune, allergic or malignant); Neuropilin 1 (Epithelial malignancies, including lung cancer); Glypicans (multiple cancers including liver, brain and breast cancers); HER3/EGFR (Epithelial malignancies); PDGFRa (Epithelial malignancies); EphA2 (multiple cancers including neuroblastoma, melanoma, breast cancer, and small cell lung carcinoma); CD38 (Myeloma); CD138 (Myeloma); α4-integrin (AML, myeloma, CLL, and most lymphomas).
In certain modes, antibodies include an anti-epidermal growth factor receptor antibody such as Cetuximab, an anti-Her2 antibody, an anti-CD20 antibody such as Rituximab, an anti-CD22 antibody such as Inotuzumab, G544 or BU59, an anti-CD70 antibody, an anti-CD33 antibody such as hp67.6 or Gemtuzumab, an anti-MUC1 antibody such as GP1.4 and SM3, an anti-CD40 antibody, an anti-CD74 antibody, an anti-P-cadherin antibody, an anti-EpCAM antibody, an anti-CD138 antibody, an anti-E-cadherin antibody, an (anti-CEA antibody, an anti-FGFR3 antibody, and an anti a4-integrin antibody such as natalizumab.
Table 3A provides nonlimiting examples of cancer types, possible targeting moieties, and proteases that are expressed by those cancer types. A protease associated with a cancer may be termed a tumor-associated protease. In order to prepare an ATTAC, the cancer may be identified, and a target chosen for the targeting moiety (as desired), and one or two proteases chosen for the cancer type, as well (as desired).
Table 3B provide additional information about cancers that may be targeting with different targeting moieties, including the fact that some targeting moieties may be able to target a number of different types of cancer. In an ATTAC, the first component would comprise a targeting moiety capable of targeting a cancer.
Antibodies that have bind tumor antigens and that have specificity for tumor cells are well-known in the art. Table 3C summarizes selected publications on exemplary antibodies that bind tumor antigens and that could be used as targeting moieties in the invention.
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The FDA maintains listings of approved antibody drugs for treating cancer, many of which bind to cancer antigens and can be employed in this context. See The Orange Book Online or Drugs@FDA on the FDA website. The FDA also maintains listings of clinical trials in progress in the clinicaltrials.gov database, which may be searched by disease names. Table 3D provides a representative list of approved antibodies with specificity for tumor cells. Table 3E provides a representative list of antibodies in development with specificity for tumor cells.
Other antibodies well-known in the art may be used as targeting moieties to target to a given cancer. The antibodies and their respective antigens include nivolumab (anti-PD-1 Ab), TA99 (anti-gp75), 3F8 (anti-GD2), 8H9 (anti-B7-H3), abagovomab (anti-CA-125 (imitation)), adecatumumab (anti-EpCAM), afutuzumab (anti-CD20), alacizumab pegol (anti-VEGFR2), altumomab pentetate (anti-CEA), amatuximab (anti-mesothelin), AME-133 (anti-CD20), anatumomab mafenatox (anti-TAG-72), apolizumab (anti-HLA-DR), arcitumomab (anti-CEA), bavituximab (anti-phosphatidylserine), bectumomab (anti-CD22), belimumab (anti-BAFF), besilesomab (anti-CEA-related antigen), bevacizumab (anti-VEGF-A), bivatuzumab mertansine (anti-CD44 v6), blinatumomab (anti-CD19), BMS-663513 (anti-CD137), brentuximab vedotin (anti-CD30 (TNFRSF8)), cantuzumab mertansine (anti-mucin CanAg), cantuzumab ravtansine (anti-MUC1), capromab pendetide (anti-prostatic carcinoma cells), carlumab (anti-MCP-1), catumaxomab (anti-EpCAM, CD3), cBR96-doxorubicin immunoconjugate (anti-Lewis-Y antigen), CC49 (anti-TAG-72), cedelizumab (anti-CD4), Ch.14.18 (anti-GD2), ch-TNT (anti-DNA associated antigens), citatuzumab bogatox (anti-EpCAM), cixutumumab (anti-IGF-1 receptor), clivatuzumab tetraxetan (anti-MUC1), conatumumab (anti-TRAIL-R2), CP-870893 (anti-CD40), dacetuzumab (anti-CD40), daclizumab (anti-CD25), dalotuzumab (anti-insulin-like growth factor I receptor), daratumumab (anti-CD38 (cyclic ADP ribose hydrolase)), demcizumab (anti-DLL4), detumomab (anti-B-lymphoma cell), drozitumab (anti-DR5), duligotumab (anti-HER3), dusigitumab (anti-ILGF2), ecromeximab (anti-GD3 ganglioside), edrecolomab (anti-EpCAM), elotuzumab (anti-SLAMF7), elsilimomab (anti-IL-6), enavatuzumab (anti-TWEAK receptor), enoticumab (anti-DLL4), ensituximab (anti-5AC), epitumomab cituxetan (anti-episialin), epratuzumab (anti-CD22), ertumaxomab (anti-HER2/neu, CD3), etaracizumab (anti-integrin αvβ3), faralimomab (anti-Interferon receptor), farletuzumab (anti-folate receptor 1), FBTA05 (anti-CD20), ficlatuzumab (anti-HGF), figitumumab (anti-IGF-1 receptor), flanvotumab (anti-TYRP1(glycoprotein 75)), fresolimumab (anti-TGF β), futuximab (anti-EGFR), galiximab (anti-CD80), ganitumab (anti-IGF-I), gemtuzumab ozogamicin (anti-CD33), girentuximab (anti-carbonic anhydrase 9 (CA-IX)), glembatumumab vedotin (anti-GPNMB), guselkumab (anti-IL13), ibalizumab (anti-CD4), ibritumomab tiuxetan (anti-CD20), icrucumab (anti-VEGFR-1), igovomab (anti-CA-125), IMAB362 (anti-CLDN18.2), IMC-CS4 (anti-CSF1R), IMC-TR1 (TGFβRII), imgatuzumab (anti-EGFR), inclacumab (anti-selectin P), indatuximab ravtansine (anti-SDC1), inotuzumab ozogamicin (anti-CD22), intetumumab (anti-CD51), ipilimumab (anti-CD152), iratumumab (anti-CD30 (TNFRSF8)), KM3065 (anti-CD20), KW-0761 (anti-CD194), LY2875358 (anti-MET) labetuzumab (anti-CEA), lambrolizumab (anti-PDCD1), lexatumumab (anti-TRAIL-R2), lintuzumab (anti-CD33), lirilumab (anti-KIR2D), lorvotuzumab mertansine (anti-CD56), lucatumumab (anti-CD40), lumiliximab (anti-CD23 (IgE receptor)), mapatumumab (anti-TRAIL-R1), margetuximab (anti-ch4D5), matuzumab (anti-EGFR), mavrilimumab (anti-GMCSF receptor a-chain), milatuzumab (anti-CD74), minretumomab (anti-TAG-72), mitumomab (anti-GD3 ganglioside), mogamulizumab (anti-CCR4), moxetumomab pasudotox (anti-CD22), nacolomab tafenatox (anti-C242 antigen), naptumomab estafenatox (anti-5T4), narnatumab (anti-RON), necitumumab (anti-EGFR), nesvacumab (anti-angiopoietin 2), nimotuzumab (anti-EGFR), nivolumab (anti-IgG4), nofetumomab merpentan, ocrelizumab (anti-CD20), ocaratuzumab (anti-CD20), olaratumab (anti-PDGF-R α), onartuzumab (anti-c-MET), ontuxizumab (anti-TEM1), oportuzumab monatox (anti-EpCAM), oregovomab (anti-CA-125), otlertuzumab (anti-CD37), pankomab (anti-tumor specific glycosylation of MUC1), parsatuzumab (anti-EGFL7), pascolizumab (anti-IL-4), patritumab (anti-HER3), pemtumomab (anti-MUC1), pertuzumab (anti-HER2/neu), pidilizumab (anti-PD-1), pinatuzumab vedotin (anti-CD22), pintumomab (anti-adenocarcinoma antigen), polatuzumab vedotin (anti-CD79B), pritumumab (anti-vimentin), PRO131921 (anti-CD20), quilizumab (anti-IGHE), racotumomab (anti-N-glycolylneuraminic acid), radretumab (anti-fibronectin extra domain-B), ramucirumab (anti-VEGFR2), rilotumumab (anti-HGF), robatumumab (anti-IGF-1 receptor), roledumab (anti-RHD), rovelizumab (anti-CD11 & CD18), samalizumab (anti-CD200), satumomab pendetide (anti-TAG-72), seribantumab (anti-ERBB3), SGN-CD19A (anti-CD19), SGN-CD33A (anti-CD33), sibrotuzumab (anti-FAP), siltuximab (anti-IL-6), solitomab (anti-EpCAM), sontuzumab (anti-episialin), tabalumab (anti-BAFF), tacatuzumab tetraxetan (anti-alpha-fetoprotein), taplitumomab paptox (anti-CD19), telimomab aritox, tenatumomab (anti-tenascin C), teneliximab (anti-CD40), teprotumumab (anti-CD221), TGN1412 (anti-CD28), ticilimumab (anti-CTLA-4), tigatuzumab (anti-TRAIL-R2), TNX-650 (anti-IL-13), tositumomab (anti-CS20), tovetumab (anti-CD140a), TRBS07 (anti-GD2), tregalizumab (anti-CD4), tremelimumab (anti-CTLA-4), TRU-016 (anti-CD37), tucotuzumab celmoleukin (anti-EpCAM), ublituximab (anti-CD20), urelumab (anti-4-1BB), vantictumab (anti-Frizzled receptor), vapaliximab (anti-AOC3 (VAP-1)), vatelizumab (anti-ITGA2), veltuzumab (anti-CD20), vesencumab (anti-NRP1), visilizumab (anti-CD3), volociximab (anti-integrin α5β1), vorsetuzumab mafodotin (anti-CD70), votumumab (anti-tumor antigen CTAA16.88), zalutumumab (anti-EGFR), zanolimumab (anti-CD4), zatuximab (anti-HER1), ziralimumab (anti-CD147 (basigin)), RG7636 (anti-ETBR), RG7458 (anti-MUC16), RG7599 (anti-NaPi2b), MPDL3280A (anti-PD-L1), RG7450 (anti-STEAP1), and GDC-0199 (anti-Bcl-2).
Antibodies that bind these antigens may also be used as targeting moieties, especially for the types of cancers noted: aminopeptidase N (CD13), annexin A1, B7-H3 (CD276, various cancers), CA125 (ovarian cancers), CA15-3 (carcinomas), CA19-9 (carcinomas), L6 (carcinomas), Lewis Y (carcinomas), Lewis X (carcinomas), alpha fetoprotein (carcinomas), CA242 (colorectal cancers), placental alkaline phosphatase (carcinomas), prostate s7pecific antigen (prostate), prostatic acid phosphatase (prostate), epidermal growth factor (carcinomas), CD2 (Hodgkin's disease, NHL lymphoma, multiple myeloma), CD3 epsilon (T-cell lymphoma, lung, breast, gastric, ovarian cancers, autoimmune diseases, malignant ascites), CD19 (B cell malignancies), CD20 (non-Hodgkin's lymphoma, B-cell neoplasmas, autoimmune diseases), CD21 (B-cell lymphoma), CD22 (leukemia, lymphoma, multiple myeloma, SLE), CD30 (Hodgkin's lymphoma), CD33 (leukemia, autoimmune diseases), CD38 (multiple myeloma), CD40 (lymphoma, multiple myeloma, leukemia (CLL)), CD51 (metastatic melanoma, sarcoma), CD52 (leukemia), CD56 (small cell lung cancers, ovarian cancer, Merkel cell carcinoma, and the liquid tumor, multiple myeloma), CD66e (carcinomas), CD70 (metastatic renal cell carcinoma and non-Hodgkin lymphoma), CD74 (multiple myeloma), CD80 (lymphoma), CD98 (carcinomas), CD123 (leukemia), mucin (carcinomas), CD221 (solid tumors), CD22 (breast, ovarian cancers), CD262 (NSCLC and other cancers), CD309 (ovarian cancers), CD326 (solid tumors), CEACAM3 (colorectal, gastric cancers), CEACAM5 (CEA, CD66e) (breast, colorectal and lung cancers), DLL4 (A-like-4), EGFR (various cancers), CTLA4 (melanoma), CXCR4 (CD 184, heme-oncology, solid tumors), Endoglin (CD 105, solid tumors), EPCAM (epithelial cell adhesion molecule, bladder, head, neck, colon, NHL prostate, and ovarian cancers), ERBB2 (lung, breast, prostate cancers), FCGR1 (autoimmune diseases), FOLR (folate receptor, ovarian cancers), FGFR (carcinomas), GD2 ganglioside (carcinomas), G-28 (a cell surface antigen glycolipid, melanoma), GD3 idiotype (carcinomas), heat shock proteins (carcinomas), HER1 (lung, stomach cancers), HER2 (breast, lung and ovarian cancers), HLA-DR10 (NHL), HLA-DRB (NHL, B cell leukemia), human chorionic gonadotropin (carcinomas), IGF1R (solid tumors, blood cancers), IL-2 receptor (T-cell leukemia and lymphomas), IL-6R (multiple myeloma, RA, Castleman's disease, IL6 dependent tumors), integrins (αvβ3, α5β1, α6β4, α11β3, α5β5, αvβ5, for various cancers), MAGE-1 (carcinomas), MAGE-2 (carcinomas), MAGE-3 (carcinomas), MAGE 4 (carcinomas), anti-transferrin receptor (carcinomas), p97 (melanoma), MS4A1 (membrane-spanning 4-domains subfamily A member 1, Non-Hodgkin's B cell lymphoma, leukemia), MUC1 (breast, ovarian, cervix, bronchus and gastrointestinal cancer), MUC16 (CA125) (ovarian cancers), CEA (colorectal cancer), gp100 (melanoma), MARTI (melanoma), MPG (melanoma), MS4A1 (membrane-spanning 4-domains subfamily A, small cell lung cancers, NHL), nucleolin, Neu oncogene product (carcinomas), P21 (carcinomas), nectin-4 (carcinomas), paratope of anti-(N-glycolylneuraminic acid, breast, melanoma cancers), PLAP-like testicular alkaline phosphatase (ovarian, testicular cancers), PSMA (prostate tumors), PSA (prostate), ROB04, TAG 72 (tumour associated glycoprotein 72, AML, gastric, colorectal, ovarian cancers), T-cell transmembrane protein (cancers), Tie (CD202b), tissue factor, TNFRSF10B (tumor necrosis factor receptor superfamily member 10B, carcinomas), TNFRSF13B (tumor necrosis factor receptor superfamily member 13B, multiple myeloma, NHL, other cancers, RA and SLE), TPBG (trophoblast glycoprotein, renal cell carcinoma), TRAIL-R1 (tumor necrosis apoptosis inducing ligand receptor 1, lymphoma, NHL, colorectal, lung cancers), VCAM-1 (CD106, Melanoma), VEGF, VEGF-A, VEGF-2 (CD309) (various cancers). Some other tumor associated antigen targets have been reviewed (Gerber, et al, mAbs 2009 1:247-253; Novellino et al, Cancer Immunol Immunother. 2005 54:187-207, Franke, et al, Cancer Biother Radiopharm. 2000, 15:459-76, Guo, et al., Adv Cancer Res. 2013; 119: 421-475, Parmiani et al. J Immunol. 2007 178:1975-9). Examples of these antigens include Cluster of Differentiations (CD4, CDS5, CD6, CD7, CD8, CD9, CD10, CD11a, CD11b, CD11c, CD12w, CD14, CD15, CD16, CDw17, CD18, CD21, CD23, CD24, CD25, CD26, CD27, CD28, CD29, CD31, CD32, CD34, CD35, CD36, CD37, CD41, CD42, CD43, CD44, CD45, CD46, CD47, CD48, CD49b, CD49c, CD53, CD54, CD55, CD58, CD59, CD61, CD62E, CD62L, CD62P, CD63, CD68, CD69, CD71, CD72, CD79, CD81, CD82, CD83, CD86, CD87, CD88, CD89, CD90, CD91, CD95, CD96, CD100, CD103, CD105, CD106, CD109, CD117, CD120, CD127, CD133, CD134, CD135, CD138, CD141, CD142, CD143, CD144, CD147, CD151, CD152, CD154, CD156, CD158, CD163, CD166, CD168, CD184, CDw186, CD195, CD202 (a, b), CD209, CD235a, CD271, CD303, CD304), annexin A1, nucleolin, endoglin (CD105), ROB04, amino-peptidase N, -like-4 (DLL4), VEGFR-2 (CD309), CXCR4 (CD184), Tie2, B7-H3, WT1, MUC1, LMP2, HPV E6 E7, EGFRvIII, HER-2/neu, idiotype, MAGE A3, p53 nonmutant, NY-ESO-1, GD2, CEA, MelanA/MART1, Ras mutant, gp100, p53 mutant, proteinase3 (PR1), bcr-abl, tyrosinase, survivin, hTERT, sarcoma translocation breakpoints, EphA2, PAP, ML-IAP, AFP, EpCAM, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, ALK, androgen receptor, cyclin B 1, polysialic acid, MYCN, RhoC, TRP-2, GD3, fucosyl GM1, mesothelin, PSCA, MAGE A1, sLe(a), CYPIB I, PLAC1, GM3, BORIS, Tn, GloboH, ETV6-AML, NY-BR-1, RGS5, SART3, STn, carbonic anhydrase IX, PAXS, OY-TES1, sperm protein 17, LCK, HMWMAA, AKAP-4, SSX2, XAGE 1, B7H3, legumain, Tie 2, Page4, VEGFR2, MAD-CT-1, FAP, PDGFR-β, MAD-CT-2, and Fos-related antigen 1.
In some embodiments, the targeting moiety capable of targeting a cancer is not an antibody, but is another type of targeting moiety. A wide range of targeting moieties capable of targeting cancer are known, including DNA aptamers, RNA aptamers, albumins, lipocalins, fibronectins, ankyrins, CH1/2/3 scaffolds (including abdurins (IgG CH2 scaffolds)), fynomers, Obodies, DARPins, knotins, avimers, atrimers, anticallins, affilins, affibodies, bicyclic peptides, cys-knots, FN3 (adnectins, centryrins, pronectins, TN3), and Kunitz domains. These and other non-antibody scaffold structures may be used for targeting to a cancer cell. Smaller non-antibody scaffolds are rapidly removed from the bloodstream and have a shorter half-life than monocolonal antibodies. They also show faster tissue penetration owing to fast extravasation from the capillary lumen through the vascular endothelium and basement membrane. See Vazquez-Lombardi et al., Drug Discovery Today 20(1):1271-1283 (2015). A number of non-antibody scaffolds targeting cancer are already under clinical development, with other candicates in the preclinical stage. See Vazquez-Lombardi, Table 1.
In another embodiment, a targeting moiety may be a binding partner for a protein known to be expressed on the cancer cell. Such expression levels may include overexpression. For example, the binding partners described in Table 4 may bind to the following targets on a cancer cell:
The binding partner need not comprise the full length or wildtype sequence for the binding partners listed in Table 4B. All that is required is that the binding partner bind to the target on the cancer cell and can thus include truncated forms, analogs, variants, and derivatives that are well known in the art.
Additionally, in some embodiments, the binding partner may be an aptamer that is capable of binding to a protein known to be expressed on the cancer cell. Aptamers that bind cancer cells, such as cancer cells, are well known and methods for designing them are known.
Cell-based SELEX systems may be used to select a panel of target cell-specific aptamers from a random candidate library. A ssDNA or ssRNA pool may be dissolved in binding buffer and denatured and then incubated with target cells. After washing the bound DNAs or RNAs may be eluted by heating and then incubated with negative cells (if desired), centrifuged, and the supernatant removed. The supernatant may be amplified by PCR with biotin labeled primers. The selected sense ssDNA or ssRNA may be separated from the antisense biotinylated strand using streptavidin coated beads. To increase affinity, washing strength may be increased through increasing washing time, volume of buffer, and number of washes. After the desired rounds of selection, the selected ssDNA or ssRNA pool may be PCR amplified and cloned into E. coli and sequenced. See Shangguan et al., Aptamers evolved from live cells as effective molecular probes for cancer study, PNAS 103(32:11838-11843 (2006); Lyu et al, Generating Cell Targeting Aptamers for Nanotherapeutics Using Cell-SELEX, Theranostics 6(9):1440-1452 (2016); see also Li et al., Inhibition of Cell Proliferation by an Anti-EGFR Aptamer, PLoS One 6(6):e20229 (2011). The specific approaches for designing aptamers and specific aptamers binding to cancer cells in these references are hereby incorporated by reference.
For example, an aptamer may comprise SEQ ID NO: 94 to 164. In some embodiments, an aptamer may comprise SEQ ID NO: 95. These aptamers are directed to EGFR and are provided only as representative of the aptamers that can bind to targets presented on cancer cells. Other aptamers against other targets on cancer cells are equally part of the description herein and incorporated by reference as described in Zhu et al., Progress in Aptamer Mediated Drug Delivery Vehicles for Cancer Targeting, Theranostics 4(9):931-944 (2014).
In some embodiments, aptamers for use herein bind to the target on the cancer cell with a Kd in the nanomolar to picomolar range (such as 1 picomolar to 500 nanomolar or 1 picomolar to 100 nanomolar).
Additional specific targeting moieties include those provided in Table 4C.
D. Immune Cell Engaging Domain
The immune cell engaging domain functions are capable of immune cell engaging activity when a first immune cell engaging domain binds to a second immune cell engaging domain. When the first and second immune cell engaging domains are paired together, when the inert binding partner is removed, they can bind to an immune cell. This binding can lead to activation of the immune cell.
In the absence of pairing of the first and second immune cell engaging domain, neither the first nor the second immune cell engaging domain alone can bind to an immune cell.
In some embodiments, the immune cell is a T cell, natural killer cell, macrophage, neutrophil, eosinophil, basophil, γδ T cell, NKT cell, or engineered immune cell. In some embodiments, the first and second immune cell engaging domains when paired together can activate an immune cell.
1. T-cell Engaging Domains
In some embodiments, the immune cell engaging domain is a T-cell engaging domain. The targeted T-cell engaging agent comprises a first T-cell engaging domain that is unable of engaging a T-cell alone. Instead, the first T-cell engaging domain is capable of activity when binding a second T-cell engaging domain, which is not part of the targeted T-cell engaging agent. Thus, the first and second T-cell engaging domains may be any two moieties that do not possess T-cell engaging activity alone, but do possess it when paired with each other. In other words, the first and second T-cell engaging domains are complementary halves of a functional active protein.
When the two T-cell engaging domains are associated together in the two-component system, they may bind to the CD3 antigen and/or T-cell receptor on the surface of the T-cell as these activate T cells. CD3 is present on all T cells and consists of subunits designated γ, δ, ε, ζ, and η. The cytoplasmic tail of CD3 is sufficient to transduce the signals necessary for T cell activation in the absence of the other components of the TCR receptor complex. Normally, activation of T cell cytotoxicity depends first on binding of the TCR with a major histocompatibility complex (MHC) protein, itself bound to a foreign antigen, located on a separate cell. In a normal situation, only when this initial TCR-MHC binding has taken place can the CD3 dependent signally cascade responsible for T cell clonal expansion and, ultimately, T cell cytotoxicity ensue. In some of the present embodiments, however, when the two-component system binds to CD3 and/or the TCR, activation of cytotoxic T cells in the absence of independent TCR-MHC can take place by virtue of the crosslinking of the CD3 and/or TCR molecules mimicking an immune synapse formation. This means that T cells may be cytotoxically activated in a clonally independent fashion, i.e. in a manner that is independent of the specific TCR clone carried by the T cell. This allows for activation of the entire T cell compartment rather than only specific T cells of a certain clonal identity.
In some embodiments, the first T-cell engaging domain is a VH domain and the second T-cell engaging domain is a VL domain. In other embodiments, the first T-cell engaging domain is a VL domain and the second T-cell engaging domain is a VH domain. In such embodiments, when paired together the first and second T-cell engaging domains may comprise an scFv (by this we mean equivalent to an scFv but for the fact that the VH and VL are not in a single-chain configuration).
If the first and second T-cell engaging domains are a pair of VH and VL domains, the VH and VL domains may be specific for an antigen expressed on the surface of a T cell, such as CD3 or TCR. If the antigen is CD3, one potential T-cell engaging domain may be derived from muromonab (muromonab-CD3 or OKT3), otelixizumab, teplizumab, visilizumab, foralumab, or SP34. One skilled in the art would be aware of a wide range of anti-CD3 antibodies, some of which are approved therapies or have been clinically tested in human patients (see Kuhn and Weiner Immunotherapy 8(8):889-906 (2016)). Table 5 presents selected publications on exemplary anti-CD3 antibodies.
Diabetes. 54(6): 1763-9 (2005).
Clin Immunol. 149(3): 268-78 (2013) (abstract).
Expert Opin Biol Ther. 10(3): 459-65 (2010).
Clin Immunol. 149(3): 268-78 (2013) (abstract).
Antibodies with specificity to the TCR, including the αβ and γδ TCRs, are also well-known. Table 6 presents selected publications on exemplary anti-TCR antibodies.
J Immunol. 184: 2156-2165 (2010).
J Immunol. 171(7): 3394-400 (2003).
2. Natural Killer Cell Engaging Domains
In some embodiments, the immune cell engaging domain is a natural killer cell engaging domain. When the two natural killer cell engaging domains are associated together in the two-component system, they may bind to an antigen on the surface of the NK cell to engage these cells. In some embodiments, the antigen on the surface of the NK cell may be NKG2D, CD16, NKp30, NKp44, NKp46 or DNAM.
In some embodiments, having one half of the two-component system bind to a surface protein on the natural killer cell and having the other half of the system bind to cancer cells allows specific engagement of natural killer cells. Engagement of natural killer cells can lead to their activation and induce natural killer cell-mediated cytotoxicity and cytokine release.
When the two natural killer cell engaging domains are associated together in the ATTAC, the natural killer cell may specifically lyse the cancer cells bound by the cancer-specific ATTAC component. Killing of a cancer cell may be mediated by either the perforin/granzyme system or by FasL-Fas engagement. As well as this potential cytotoxic function, natural killer cells are also able to secrete pro-inflammatory cytokines including interferon gamma and tumor necrosis factor alpha which can activate macrophages and dendritic cells in the immediate vicinity to enhance the anti-cancer immune response.
In some embodiments, the first natural killer cell engaging domain is a VH domain and the second natural killer cell engaging domain is a VL domain. In other embodiments, the first natural killer cell engaging domain is a VL domain and the second natural killer cell engaging domain is a VH domain. In such embodiments, when paired together the first and second natural killer cell engaging domains may comprise an scFv (by this we mean equivalent to an scFv but for the fact that the VH and VL are not in a single-chain configuration).
If the first and second natural killer cell engaging domains are a pair of VH and VL domains, the VH and VL domains may be specific for an antigen expressed on the surface of a natural killer cell, such as NKG2D, CD16, NKp30, NKp44, NKp46 and DNAM.
Table 7 presents selected publications on some exemplary antibodies specific for an antigen expressed on the surface of a natural killer cell.
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World J Hepatol. 9(25): 1073-1080 (2017).
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3. Macrophage Engaging Domains
In some embodiments, the immune cell engaging domain is a macrophage engaging domain. As used herein, a “macrophage” may refer to any cell of the mononuclear phagocytic system, such as grouped lineage-committed bone marrow precursors, circulating monocytes, resident macrophages, and dendritic cells (DC). Examples of resident macrophages can include Kupffer cells and microglia.
When the two macrophage engaging domains are associated together in the two-component system, they may bind to an antigen on the surface of the macrophage to engage these cells. In some embodiments, the antigen on the surface of the macrophage may be CD89 (Fc alpha receptor 1), CD64 (Fc gamma receptor 1), CD32 (Fc gamma receptor 2A) or CD16a (Fc gamma receptor 3A).
In some embodiments, having one half of the two-component system bind to a surface protein on the macrophage and having the other half of the system bind to cancer cells allows specific engagement of macrophages. Engagement of macrophages can lead the macrophage to phagocytose the cancer cell.
In some embodiments, inducing macrophage phagocytosis via binding to an antigen on the surface of the macrophages is independent of Fc receptor binding, which has been shown previously to be a method of tumor cell killing by macrophages. Normally, cancer cells are bound by whole antibodies and the Fc portion of the antibody binds to the Fc receptor and induces phagocytosis.
In some embodiments, engagement of toll-like receptors on the macrophage surface (see patent application US20150125397A1) leads to engagement of macrophages.
When the two macrophage engaging domains are associated together in the ATTAC, they may induce the macrophage to phagocytose the cancer cell bound by the cancer-specific ATTAC component.
In some embodiments, the first macrophage engaging domain is a VH domain and the second macrophage engaging domain is a VL domain. In other embodiments, the first macrophage engaging domain is a VL domain and the second macrophage engaging domain is a VH domain. In such embodiments, when paired together the first and second macrophage engaging domains may comprise an scFv (by this we mean equivalent to an scFv but for the fact that the VH and VL are not in a single-chain configuration).
If the first and second macrophage engaging domains are a pair of VH and VL domains, the VH and VL domains may be specific for an antigen expressed on the surface of a macrophage, such as CD89 (Fc alpha receptor 1), CD64 (Fc gamma receptor 1), CD32 (Fc gamma receptor 2A) and CD16a (Fc gamma receptor 3A), or toll-like receptors.
Table 8 presents selected publications on some exemplary antibodies specific for an antigen expressed on the surface of a macrophage.
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4. Neutrophil Engaging Domains
In some embodiments, the immune cell engaging domain is a neutrophil engaging domain. When the two neutrophil engaging domains are associated together in the two-component system, they may bind to an antigen on the surface of the neutrophil to engage these cells. In some embodiments, the antigen on the surface of the neutrophil may be CD89 (FcαR1), FcγRI (CD64), FcγRIIA (CD32), FcγRIIIA (CD16a), CD11b (CR3, αMβ2), TLR2, TLR4, CLEC7A (Dectin1), formyl peptide receptor 1 (FPR1), formyl peptide receptor 2 (FPR2), or formyl peptide receptor 3 (FPR3).
In some embodiments, having one half of the two-component system bind to a surface protein on the neutrophil and having the other half of the system bind to cancer cells allows specific engagement of neutrophils. Engagement of neutrophils can lead to phagocytosis and cell uptake.
When the two neutrophil engaging domains are associated together in the ATTAC, the neutrophil may engulf the target cells.
In some embodiments, the first neutrophil engaging domain is a VH domain and the second neutrophil engaging domain is a VL domain. In other embodiments, the first neutrophil engaging domain is a VL domain and the second neutrophil engaging domain is a VH domain. In such embodiments, when paired together the first and second neutrophil engaging domains may comprise an scFv (by this we mean equivalent to an scFv but for the fact that the VH and VL are not in a single-chain configuration).
If the first and second neutrophil engaging domains are a pair of VH and VL domains, the VH and VL domains may be specific for an antigen expressed on the surface of a neutrophil, such as CD89 (FcαR1), FcγRI (CD64), FcγRIIA (CD32), FcγRIIIA (CD16a), CD11b (CR3, αMβ2), TLR2, TLR4, CLEC7A (Dectin1), FPR1, FPR2, or FPR3.
Table 9 presents selected publications on some exemplary antibodies specific for an antigen expressed on the surface of a neutrophil.
Journal of Biological Chemistry 292(10):
Cancer Immunol Immunother. 50(2):
Front Immunol. 5: 461 (2014)
Front Immunol. 5: 461 (2014)
5. Eosinophil Engaging Domains
In some embodiments, the immune cell engaging domain is an eosinophil engaging domain. When the two eosinophil engaging domains are associated together in the two-component system, they may bind to an antigen on the surface of the eosinophil to engage these cells. In some embodiments, the antigen on the surface of the eosinophil may be CD89 (Fc alpha receptor 1), FcεRI, FcγRI (CD64), FcγRIIA (CD32), FcγRIIIB (CD16b), or TLR4.
In some embodiments, having one half of the two-component system bind to a surface protein on the eosinophil and having the other half of the system bind to cancer cells allows specific engagement of eosinophils. Engagement of eosinophils can lead to degranulation and release of preformed cationic proteins, such as EPO, major basic protein 1 (MBP1), and eosinophil-associated ribonucleases (EARs), known as ECP and eosinophil-derived neurotoxin.
When the two neutrophil engaging domains are associated together in the ATTAC, the neutrophil may phagocytose the target cell or secrete neutrophil extracellular traps (NETs); finally, they may activate their respiratory burst cascade to kill phagocytosed cells.
In some embodiments, the first eosinophil engaging domain is a VH domain and the second eosinophil engaging domain is a VL domain. In other embodiments, the first eosinophil engaging domain is a VL domain and the second eosinophil engaging domain is a VH domain. In such embodiments, when paired together the first and second eosinophil engaging domains may comprise an scFv (by this we mean equivalent to an scFv but for the fact that the VH and VL are not in a single-chain configuration).
If the first and second eosinophil engaging domains are a pair of VH and VL domains, the VH and VL domains may be specific for an antigen expressed on the surface of an eosinophil, such as CD89 (Fc alpha receptor 1), FcεRI, FcγRI (CD64), FcγRIIA (CD32), FcγRIIIB (CD16b), or TLR4.
Table 10 presents selected publications on some exemplary antibodies specific for an antigen expressed on the surface of an eosinophil.
Arch Immunol Ther Exp (Warsz). 49(3): 217-29 (2001)
J Allergy Clin Immunol. 125(2 Suppl 2): S73-80 (2010)
Curr Opin Immunol. 19(2): 239-45 (2007)
Curr Opin Immunol. 19(2): 239-45 (2007)
Curr Opin Immunol. 19(2): 239-45 (2007)
6. Basophil Engaging Domains
In some embodiments, the immune cell engaging domain is a basophil engaging domain. When the two basophil engaging domains are associated together in the two-component system, they may bind to an antigen on the surface of the basophil to engage these cells. In some embodiments, the antigen on the surface of the basophil may be CD89 (Fc alpha receptor 1) or FcεRI.
In some embodiments, having one half of the two-component system bind to a surface protein on the basophil and having the other half of the system bind to cancer cells allows specific engagement of basophils. Engagement of basophils can lead to the release of basophil granule components such as histamine, proteoglycans, and proteolytic enzymes. They also secrete leukotrienes (LTD-4) and cytokines.
When the two basophil engaging domains are associated together in the ATTAC, the basophil may degranulate.
In some embodiments, the first basophil engaging domain is a VH domain and the second basophil engaging domain is a VL domain. In other embodiments, the first basophil engaging domain is a VL domain and the second basophil engaging domain is a VH domain. In such embodiments, when paired together the first and second basophil engaging domains may comprise an scFv (by this we mean equivalent to an scFv but for the fact that the VH and VL are not in a single-chain configuration).
If the first and second basophil engaging domains are a pair of VH and VL domains, the VH and VL domains may be specific for an antigen expressed on the surface of a basophil, such as CD89 (Fc alpha receptor 1) or FcεRI.
Table 11 presents selected publications on some exemplary antibodies specific for an antigen expressed on the surface of a basophil.
Arch Immunol Ther Exp (Warsz). 49(3): 217-29 (2001)
J Allergy Clin Immunol. 125(2 Suppl 2): S73-80 (2010)
7. γδ T cells
In some embodiments, the immune cell engaging domain is a γδ T-cell engaging domain. As used herein, a γδ T cell refers to a T cell having a TCR made up of one gamma chain (γ) and one delta chain (δ).
When the two γδ T-cell engaging domains are associated together in the two-component system, they may bind to an antigen on the surface of the γδ T cell to engage these cells. In some embodiments, the antigen on the surface of the γδ T cell may be γδ TCR, NKG2D, CD3 Complex (CD3ε, CD3γ, CD3δ, CD3ζ, CD3ζ), 4-1BB, DNAM-1, or TLRs (e.g., TLR2, TLR6).
In some embodiments, having one half of the two-component system bind to a surface protein on the γδ T cell and having the other half of the system bind to cancer cells allows specific engagement of γδ T cells. Engagement of γδ T cell can lead to cytolysis of the target cell and release of proinflammatory cytokines such as TNFα and IFNγ.
When the two γδ T-cell engaging domains are associated together in the ATTAC, the γδ T cell may kill the target cell.
In some embodiments, the first γδ T-cell engaging domain is a VH domain and the second γδ T-cell engaging domain is a VL domain. In other embodiments, the first γδ T-cell engaging domain is a VL domain and the second γδ T-cell engaging domain is a VH domain. In such embodiments, when paired together the first and second γδ T-cell engaging domains may comprise an scFv (by this we mean equivalent to an scFv but for the fact that the VH and VL are not in a single-chain configuration).
If the first and second γδ T-cell engaging domains are a pair of VH and VL domains, the VH and VL domains may be specific for an antigen expressed on the surface of a γδ T cell, such as γδ TCR, NKG2D, CD3 Complex (CD3ε, CD3γ, CD3δ, CD3ζ, CD3η), 4-1BB, DNAM-1, or TLRs (TLR2, TLR6).
Table 12 presents selected publications on some exemplary antibodies specific for an antigen expressed on the surface of a γδ T cell.
Eur J Immunol. 39(5): 1361-8 (2009)
8. Natural Killer T Cells (NKT Cells)
In some embodiments, the immune cell engaging domain is a NKT engaging domain. NKT cells refers to T cells that express the Vα24 and Vβ11 TCR receptors.
When the two NKT engaging domains are associated together in the two-component system, they may bind to an antigen on the surface of the NKT to engage these cells. In some embodiments, the antigen on the surface of the NKT may be αβTCR, NKG2D, CD3 Complex (CD3ε, CD3γ, CD3δ, CD3ζ, CD3η), 4-1BB, or IL-12R.
In some embodiments, having one half of the two-component system bind to a surface protein on the NKT and having the other half of the system bind to cancer cells allows specific engagement of NKT. Engagement of NKTs can lead to cytolysis of the target cell.
When the two NKT engaging domains are associated together in the ATTAC, the NKT may cytolysis of the target cell and the release of proinflammatory cytokines.
In some embodiments, the first NKT engaging domain is a VH domain and the second NKT engaging domain is a VL domain. In other embodiments, the first NKT engaging domain is a VL domain and the second NKT engaging domain is a VH domain. In such embodiments, when paired together the first and second NKT engaging domains may comprise an scFv (by this we mean equivalent to an scFv but for the fact that the VH and VL are not in a single-chain configuration).
If the first and second NKT engaging domains are a pair of VH and VL domains, the VH and VL domains may be specific for an antigen expressed on the surface of a NKT, such as αβTCR, NKG2D, CD3 Complex (CD3ε, CD3γ, CD3δ, CD3ζ, CD3η), 4-1BB, or IL-12R.
Table 13 presents selected publications on some exemplary antibodies specific for an antigen expressed on the surface of a NKT.
Annu. Rev. Immunol. 16: 523-544 (1998)
Eur J Cancer. 54: 112-119 (2016)
9. Engineered Immune Cells
In some embodiments, the immune cell engaging domain is an engineered immune cell engaging domain.
In some embodiments, the engineered immune cell is a chimeric antigen receptor (CAR) cell. In some embodiments, the CAR comprises an extracellular domain capable of tightly binding to a tumor antigen (for example, an scFv), fused to a signaling domain partly derived from a receptor naturally expressed by an immune cell. Exemplary CARs are described in Facts about Chimeric Antigen Receptor (CAR) T-Cell Therapy, Leukemia and Lymphoma Society, December 2017. CARs may comprise an scFV region specific for a tumor antigen, an intracellular co-stimulatory domain, and linker and transmembrane region. For example, a CAR in a CAR T cell may comprise an extracellular domain of a tumor antigen fused to a signaling domain partly derived from the T cell receptor. A CAR may also comprise a co-stimulatory domain, such as CD28, 4-1 BB, or OX40. In some embodiments, binding of the CAR expressed by an immune cell to a tumor target antigen results in immune cell activation, proliferation, and target cell elimination. Thus, a range of CARs may be used that differ in their scFV region, intracellular co-stimulatory domains, and linker and transmembrane regions to generate engineered immune cells.
Exemplary engineered immune cells include CAR T cells, NK cells, NKT cells, and γδ cells. In some embodiments, engineered immune cells are derived from the patient's own immune cells. In some embodiments, the patient's tumor expresses a tumor antigen that binds to the scFV of the CAR.
Potential CAR targets studied so far include CD19, CD20, CD22, CD30, CD33, CD123, ROR1, Igk light chain, BCMA, LNGFR, and NKG2D. However, the CAR technology would be available for developing engineered immune cells to a range of tumor antigens.
In some embodiments, the engineered immune cell is a genetically engineered immune cell.
When the two engineered immune cell engaging domains are associated together in the two-component system, they may bind to an antigen on the surface of the engineered immune cell to engage these cells. In some embodiments, the antigen on the surface of the engineered immune cell may be be an engagement domain recited in this application with specificity for T cells, NK cells, NKT cells, or γδ cells.
In some embodiments, having one half of the two-component system bind to a surface protein on the engineered immune cell and having the other half of the system bind to cancer cells allows specific engagement of engineered immune cells. Engagement of engineered immune cells can lead to activation of the effector response of these cells such as cytolysis of their target and release of cytokines.
When the two engineered immune cell engaging domains are associated together in the ATTAC, the engineered immune cell may kill the target cell.
In some embodiments, the first engineered immune cell engaging domain is a VH domain and the second engineered immune cell engaging domain is a VL domain. In other embodiments, the first engineered immune cell engaging domain is a VL domain and the second engineered immune cell engaging domain is a VH domain. In such embodiments, when paired together the first and second engineered immune cell engaging domains may comprise an scFv (by this we mean equivalent to an scFv but for the fact that the VH and VL are not in a single-chain configuration).
If the first and second engineered immune cell engaging domains are a pair of VH and VL domains, the VH and VL domains may be specific for an antigen expressed on the surface of an engineered immune cell, based on the type of cell used for the engineering.
E. Inert Binding Partner
The ATTAC also comprises at least one inert binding partner capable of binding the immune cell engaging domain to which it binds and preventing it from binding to another immune engaging domain unless certain conditions occur. When an immune cell engaging domain is bound to the at least one inert binding partner, it does not possess immune cell engaging activity.
In other words, the at least one inert binding partner cripples the function of an immune engaging domain by blocking it from binding its complementary pair (the other immune cell engaging domain) and preventing the two domains from joining together to have immune cell engaging activity. As such, the inert binding partner binds to an immune cell engaging domain such that the immune cell engaging domain does not bind to the other immune cell engaging domain unless the inert binding partner is removed. By does not bind, the application does not exclude nonspecific binding or low levels of binding (for example, ≤1%, ≤5%, ≤10%).
In some embodiments, the first immune cell engaging domain is bound to an inert binding partner. The inert binding partner bound to the first immune cell engaging domain prevents the first immune cell engaging domain from binding to the second immune cell binding domain.
In some embodiments, the second immune cell engaging domain is bound to an inert binding partner. The inert binding partner bound to the second immune cell engaging domain prevents the second immune cell engaging domain from binding to the first immune cell binding domain.
In some embodiments, the first and the second immune cell engaging domain are both bound to an inert binding partner. The inert binding partners bound to the first and the second immune cell engaging domain prevents the two immune cell engaging domain from binding to each other.
In some embodiments, the inert binding partner binds specifically to the immune cell engaging domain.
In some embodiments, the at least one inert binding partner is a VH or VL domain. In some embodiments, when the immune cell engaging domain in the ATTAC is a VH domain, the inert binding partner may be a VL domain and when the first immune cell engaging domain is a VL domain, the inert binding partner may be a VH domain.
If a first component comprises a targeting moiety and a VL immune cell engaging domain and a VH inert binding partner, in some embodiments, the VH inert binding partner has an equilibrium dissociation constant for binding to the VL immune cell engaging domain, which is greater than the equilibrium dissociation constant of the VL immune cell engaging domain for its partner VH immune cell engaging domain in the second component. In some embodiments, the prior sentence is equally true when VH is switched for VL and vice versa.
It is believed that using the inert binding partner as a mispairing partner with the immune cell engaging domain in the construct results in constructs that are more stable and easier to manufacture. In some embodiments, both the first and second immune binding domains may be bound to an inert binding partner as described herein. In some embodiments, only one of the immune binding domains is bound to an inert binding partner.
1. Inactivated VH or VL Domains as Inert Binding Partners
In some embodiments when an immune cell engaging domain is a VH or VL domain, the inert binding partner has homology to a corresponding VL or VH domain that can pair with the immune cell binding domain to form a functional antibody and bind to an immune cell antigen. This immune cell antigen may be an antigen present on any immune cell, including a T cell, a macrophage, a natural killer cell, a neutrophil, eosinophil, basophil, γδ T cell, natural killer T cell (NKT cells), or engineered immune cell. In some embodiments, this immune cell antigen is CD3.
In some embodiments, the inert binding partner is a VH or VL that cannot specifically bind an antigen when paired with its corresponding VL or VH of the immune cell engaging domain because of one or more mutations made in the inert binding partner to inhibit binding to the target antigen. In some embodiments, the VH or VL of the inert binding partner may differ by one or more amino acids from a VH or VL specific for an immune cell antigen. In other words, one or more mutations may be made to a VH or VL specific for a target immune cell antigen to generate an inert binding partner.
These mutations may be, for example, a substitution, insertion, or deletion in the polypeptide sequence of a VH or VL specific for an immune cell antigen to generate an inert binding partner. In some embodiments, the mutation in a VH or VL specific for an immune cell antigen may be made within CDR1, CDR2, or CDR3 to generate an inert binding partner. In some embodiments, an VH or VL used as an inert binding partner may retain the ability to pair with an immune cell engaging domain, but the resulting paired VH/VL domains have reduced binding to the immune cell antigen. In some embodiments, an inert binding partner has normal affinity to bind its corresponding immune cell engaging domain, but the paired VH/VL has lower binding affinity for the immune cell antigen compared to a paired VH/VL that does not comprise the mutation of the inert binding partner. For example, this lower affinity may be a 20-fold, 100-fold, or 1000-fold lower binding to an immune cell antigen.
In some embodiments, the first immune cell binding domain is a VH specific for an immune cell antigen and the inert binding partner is a VL domain for the same antigen that has one or more mutations such that the paired VH/VL has decreased or no binding to the antigen. In some embodiments, the first immune cell binding domain is a VL specific for an immune cell antigen and the inert binding partner is a VH domain for the same antigen that has one or more mutations such that the paired VH/VL has decreased or no binding to the antigen.
In some embodiments, the second immune cell binding domain is a VH specific for an immune cell antigen and the inert binding partner is a VL domain for the same antigen that has one or more mutations such that the paired VH/VL has decreased or no binding to the antigen. In some embodiments, the second immune cell binding domain is a VL specific for an immune cell antigen and the inert binding partner is a VH domain for the same antigen that has one or more mutations such that the paired VH/VL has decreased or no binding to the antigen.
2. Inert Binding Partners Obtained from Unrelated Antibodies
In some embodiments, a VH or VL used as an inert binding partner is unrelated to the VL or VH of the immune cell engaging domain. In other words, the inert binding partner may have little or no sequence homology to the corresponding VH or VL that normally associates with the VL or VH of the immune cell engaging domain. In some embodiments, the VH or VL used as an inert binding partner may be from a different antibody or scFv than the VL or VH used as the immune cell engaging domain.
If both components have inert binding partner, in some embodiments, the VH inert binding partner of one component and the VL inert binding partner of the other component may be from different antibodies.
F. Cleavage Site
By way of overview, the cleavage site may be (i) cleaved by an enzyme expressed by the cancer cells; (ii) cleaved through a pH-sensitive cleavage reaction inside the cancer cell; (iii) cleaved by a complement-dependent cleavage reaction; or (iv) cleaved by a protease that is colocalized to the cancer cell by a targeting moiety that is the same or different from the targeting moiety in the agent. In some embodiments, the cleavage site is a protease cleavage site.
The cleavage sites function to release the inert binding partner from the first immune cell engaging domain. The cleavage sites can function in different ways to release the inert binding partner from one or both immune cell engaging domains in the microenvironment of the cancer cells. The cleavage may occur inside the cancer cell or outside the cancer cell, depending on the strategy employed. If cleavage occurs outside the cancer cell, the immune cell engaging domain can be presented without first being internalized into a cell and being engaged in the classical antigen-processing pathways.
In certain embodiments, at least one cleavage site may be cleaved by an enzyme expressed by the cancer cells. Cancer cells, for instance, are known to express certain enzymes, such as proteases, and these may be employed in this strategy to cleave the ATTAC's one or more cleavage site. By way of nonlimiting example, cathepsin B cleaves FR, FK, VA and VR amongst others; cathepsin D cleaves PRSFFRLGK (SEQ ID NO: 45), ADAM28 cleaves KPAKFFRL (SEQ ID NO: 1), DPAKFFRL (SEQ ID NO: 2), KPMKFFRL (SEQ ID NO: 3) and LPAKFFRL (SEQ ID NO: 4); and MMP2 cleaves AIPVSLR (SEQ ID NO: 46), SLPLGLWAPNFN (SEQ ID NO: 47), HPVGLLAR (SEQ ID NO: 48), GPLGVRGK (SEQ ID NO: 49), and GPLGLWAQ (SEQ ID NO: 50), for example. Other cleavage sites listed in Table 1A or 3A may also be employed. Protease cleavage sites and proteases associated with cancer are well known in the art. Oncomine (www.oncomine.org) is an online cancer gene expression database, so when the agent of the invention is for treating cancer, the skilled person may search the Oncomine database to identify a particular protease cleavage site (or two protease cleavage sites) that will be appropriate for treating a given cancer type. Alternative databases include the European Bioinformatic Institute (www.ebi.ac.uk), in particular (www.ebi.ac.uk/gxa). Protease databases include ExPASy Peptide Cutter (ca.expasy.org/tools/peptidecutter) and PMAP.Cut DB (cutdb.burnham.org).
In some embodiments, at least one cleavage site may be cleaved through a pH-sensitive cleavage reaction inside the cancer cell. If the ATTAC is internalized into the cell, the cleavage reaction may occur inside the cell and may be triggered by a change in pH between the microenvironment outside the cancer cell and the interior of the cell. Specifically, some cancer types are known to have acidic environments in the interior of the cancer cells. Such an approach may be employed when the interior cancer cell type has a characteristically different pH from the extracellular microenvironment, such as particularly the glycocalyx. Because pH cleavage can occur in all cells in the lysozymes, selection of a targeting agent when using a pH-sensitive cleavage site may require, when desired, more specificity. For example, when a pH-sensitive cleavage site is used, a targeting agent that binds only or highly preferably to cancer cells may be desired (such as, for example, an antibody binding to mesothelin for treatment of lung cancer).
In certain embodiments, at least one cleavage site may be cleaved by a complement-dependent cleavage reaction. Once the ATTAC binds to the cancer cell, the patient's complement cascade may be triggered. In such a case, the complement cascade may also be used to cleave the inert binding partner from the first immune cell engaging domain by using a cleavage site sensitive to a complement protease. For example, C1r and C1s and the C3 convertases (C4B,2a and C3b,Bb) are serine proteases. C3/C5 and C5 are also complement proteases. Mannose-associated binding proteins (MASP), serine proteases also involved in the complement cascade and responsible for cleaving C4 and C2 into C4b2b (a C3 convertase) may also be used. For example, and without limitation, C1s cleaves YLGRSYKV and MQLGRX. MASP2 is believed to cleave SLGRKIQI. Complement component C2a and complement factor Bb are believed to cleave GLARSNLDE.
In some embodiments, at least one cleavage site may be cleaved by a protease that is colocalized to the cancer cell by a targeting moiety that is the same or different from the targeting moiety in the ATTAC. For example, any protease may be simultaneously directed to the microenvironment of the cancer cells by conjugating the protease to a targeting agent that delivers the protease to that location. The targeting agent may be any targeting agent described herein. The protease may be affixed to the targeting agent through a peptide or chemical linker and may maintain sufficient enzymatic activity when bound to the targeting agent.
In some embodiments, both the first component and second component are mispaired with an inert binding partner. In some embodiments, the protease cleavage site in the first component and the second component are the same. In other embodiments, the protease cleavage sites in the first component and the second component are different cleavage sites for the same protease. In other embodiments, the protease cleavage sites in the first component and the second component are cleavage sites for different proteases. In some embodiments employing two different proteases, the cancer cell expresses both proteases.
In some embodiments, in a first component, the inert binding partner in an uncleaved state interferes with the specific binding of a VL or VH immune engaging domain to its partner VH or VL, respectively, immune cell engaging domain in a second component. In some embodiments, the inert binding partner in an uncleaved state inhibits the binding of the VL or VH immune cell engaging domain to its partner VH or VL, respectively, immune cell engaging domain in a second component such that the dissociation constant (Kd) of the VL or VH immune cell engaging domain to its partner VH or VL, respectively, immune cell engaging domain in a second component in an uncleaved state is at least 100 times greater than the Kd of the VL or VH immune cell engaging domain to its partner VH or VL, respectively, immune cell engaging domain in a second component in a cleaved state.
G. Linkers
In addition to the cleavage site, linkers may optionally be used to attach the separate parts of the ATTAC together. By linker, we include any chemical moiety that attaches these parts together. In some embodiments, the linkers may be flexible linkers. Linkers include peptides, polymers, nucleotides, nucleic acids, polysaccharides, and lipid organic species (such as polyethylene glycol). In some embodiments, the linker is a peptide linker. Peptide linkers may be from about 2-100, 10-50, or 15-30 amino acids long. In some embodiments, peptide linkers may be at least 10, at least 15, or at least 20 amino acids long and no more than 80, no more than 90, or no more than 100 amino acids long. In some embodiments, the linker is a peptide linker that has a single or repeating GGGGS (SEQ ID NO: 85), GGGS (SEQ ID NO: 86), GS (SEQ ID NO: 87), GSGGS (SEQ ID NO: 88), GGSG (SEQ ID NO: 89), GGSGG (SEQ ID NO: 90), GSGSG (SEQ ID NO: 91), GSGGG (SEQ ID NO: 92), GGGSG (SEQ ID NO: 93), and/or GSSSG (SEQ ID NO: 94) sequence(s).
In some embodiments, the linker is a maleimide (MPA) or SMCC linker.
H. Methods of Making
The ATTACs as described herein can be made using genetic engineering techniques. Specifically, a nucleic acid may be expressed in a suitable host to produce an ATTAC. For example, a vector may be prepared comprising a nucleic acid sequence that encodes the ATTAC including all of its component parts and linkers and that vector may be used to transform an appropriate host cell.
Various regulatory elements may be used in the vector as well, depending on the nature of the host and the manner of introduction of the nucleic acid into the host, and whether episomal maintenance or integration is desired.
Chemical linkage techniques, such as using maleimide or SMCC linkers, may also be employed.
In instances where the binding partner is an aptamer, a person of ordinary skill in the art would appreciate how to conjugate an aptamer to a protein, namely the immune cell engaging domain. Aptamers may be conjugated using a thiol linkage or other standard conjugation chemistries. A maleimide, succinimide, or SH group may be affixed to the aptamer to attach it to the immune cell engaging domain.
The ATTACs may be employed as pharmaceutical compositions. As such, they may be prepared along with a pharmaceutically acceptable carrier. If parenteral administration is desired, for instance, the ATTACs may be provided in sterile, pyrogen-free water for injection or sterile, pyrogen-free saline. Alternatively, the ATTACs may be provided in lyophilized form for resuspension with the addition of a sterile liquid carrier.
The ATTACs described herein may be used in a method of treating a disease in a patient characterized by the presence of cancer cells comprising administering an ATTAC comprising at least a first and a second component to the patient, as each of the components have been described in detail in various embodiments above. Additionally, the agents described herein may also be used in a method of targeting a patient's own immune response to cancer cells comprising administering an ATTAC to the patient.
In some embodiments, the patient has cancer or a recognized pre-malignant state. In some embodiments, the patient has undetectable cancer, but is at high risk of developing cancer, including having a mutation associated with an increased risk of cancer. In some embodiments, the patient at high risk of developing cancer has a premalignant tumor with a high risk of transformation. In some embodiments, the patient at high risk of developing cancer has a genetic profile associated with high risk. In some embodiments, the presence of cancer or a pre-malignant state in a patient is determined based on the presence of circulating tumor DNA (ctDNA) or circulating tumor cells. In some embodiments, treatment is pre-emptive or prophylactic. In some embodiments, treatment slow or blocks the occurrence or reoccurrence of cancer.
The amount of the agent administered to the patient may be chosen by the patient's physician so as to provide an effective amount to treat the condition in question. The first component and the second component of the ATTAC may be administered in the same formulation or two different formulations within a sufficiently close period of time to be active in the patient.
The patient receiving treatment may be a human. The patient may be a primate or any mammal. Alternatively, the patient may be an animal, such as a domesticated animal (for example, a dog or cat), a laboratory animal (for example, a laboratory rodent, such as a mouse, rat, or rabbit), or an animal important in agriculture (such as horses, cattle, sheep, or goats).
The cancer may be a solid or non-solid malignancy, The cancer may be any cancer such as breast cancer, ovarian cancer, endometrial cancer, cervical cancer, bladder cancer, renal cancer, melanoma, lung cancer, prostate cancer, testicular cancer, thyroid cancer, brain cancer, esophageal cancer, gastric cancer, pancreatic cancer, colorectal cancer, liver cancer, leukemia, myeloma, nonHodgkin lymphoma, Hodgkin lymphoma, acute myeloid leukemia, acute lymphoblastic leukemia, chronic lymphoblastic leukemia, lymphoproliferative disorder, myelodysplastic disorder, myeloproliferative disease and premalignant disease.
In some embodiments, a patient treated with an ATTAC has a tumor characterized by the presence of high levels of regulatory T cells (see Fridman W H et al., Nature Reviews Cancer 12:298-306 (2012) at Table 1). In patients with tumors characterized by a high presence of regulatory T cells, ATTAC therapy may be advantageous over other therapies that non-selectively target T cells, such as unselective BiTEs. In some embodiments, ATTAC therapy avoids engagement of regulatory T cells. In some embodiments, at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% of activated T cells are not regulatory T cells. In some embodiments, no regulatory T cells are activated by ATTAC therapy.
In some embodiments, the presence of a biomarker is used to select patients for receiving the ATTAC. A wide variety of tumor markers are known in the art, such as those described at www.cancer.gov/about-cancer/diagnosis-staging/diagnosis/tumor-markers-fact-sheet. In some embodiments, the tumor marker is ALK gene rearrangement or overexpression; alpha-fetoprotein; beta-2-microglobulin; beta-human chorionic gonadotropin; BRCA1 or BRCA2 gene mutations; BCR-ABL fusion genes (Philadelphia chromosome); BRAF V600 mutations; C-kit/CD117; CA15-3/CA27.29; CA19-9; CA-125; calcitonin; carcinoembryonic antigen (CEA); CD20; chromogranin A (CgA); chromosomes 3, 7, 17, or 9p21; circulating tumor cells of epithelial origin (CELLSEARCH®); cytokeratin fragment 21-1; EGFR gene mutation analysis; estrogen receptor (ER)/progesterone receptor (PR); fibrin/fibrinogen; HE4; HER2/neu gene amplification or protein overexpression; immunoglobulins; KRAS gene mutation analysis; lactate dehydrogenase; neuron-specific enolase (NSE); nuclear matrix protein 22; programmed death ligand 1 (PD-L1); prostate-specific antigen (PSA); thyroglobulin; urokinase plasminogen activator (uPA); plasminogen activator inhibitor (PAI-1); 5-protein signature (OVA1®); 21-gene signature (Oncotype DX®); or 70-gene signature (Mammaprint®).
The ATTAC may be administered alone or in conjunction with other forms of therapy, including surgery, radiation, traditional chemotherapy, or immunotherapy.
In some embodiments, the immunotherapy is checkpoint blockade. Checkpoint blockade refers to agents that inhibit or block inhibitory checkpoint molecules that suppress immune functions. In some embodiments, the checkpoint blockade targets CTLA4, PD1, PD-L1, LAG3, CD40, TIGIT, TIM3, VISTA or HLA-G.
In some embodiments, the immunotherapy is immune cytokines or cytokine fusions. Cytokines refer to cell-signaling proteins naturally made by the body to activate and regulate the immune system. Cytokine fusions refer to engineered molecules comprising all or part of a cytokine. For example, a cytokine fusion may comprise all or part of a cytokine attached to an antibody that allows targeting to a tumor such as Darleukin (see Zegers et al. (2015) Clin. Cancer Res., 21, 1151-60), Teleukin (see WO2018087172).
In some embodiments, the immunotherapy is cancer treatment vaccination. In some embodiments, cancer treatment vaccination boosts the body's natural defenses to fight cancer. These can either be against shared tumor antigens (such as E6, E7, NY-ESO, MUC1, or HER2) or against personalized mutational neoantigens.
To facilitate initial testing of the ATTAC platform and to show proof of concept, a model system employing FITC was used. Immune cells were stained with FITC-labelled antibodies against immune cell markers and anti-FITC ATTAC components were used for initial testing.
Thus, in this model, the anti-FITC ATTAC component (SEQ ID NO: 165) acts as an adapter ATTAC component whereby firstly, FITC-labelled antibodies can be used to label different target antigens on the immune cells of interest. Using an adapter ATTAC component means a large number of antigens on the immune cell surface can be assayed using one ATTAC component that constitutes half of the required two components. Immune cells would then be labelled with the anti-FITC ATTAC component, only if the FITC-labelled antibody bound to the cells of interest. The anti-FITC ATTAC component would contain one half of the immune cell activating domain with the second half of the immune cell activating domain coming from a second ATTAC component bound to an antigen on the unwanted tumor cells.
In this experiment, we counted T cells (4×106) and washed twice in RPMI+10% NBS. Re-suspended T cells to 2.6×106 per ml and added 95 μl to 15 ml Falcon tubes and added 5 μl FITC antibodies (do not add anything to untreated T cells), then incubated at room temperature for 30 minutes.
Washed off excess antibody by adding 5 mls media and spinning down. Removed supernatant and re-suspended cells in residual media (around 80 ul). Added 100 ul media to each tube.
Added 20 l anti-FITC ATTAC component (SEQ ID NO: 165-300 μg/ml) to each tube so there was a final concentration of 30 μg/ml—incubated at room temperature for 30 minutes.
Washed off excess ATTAC component by adding 5 mls media and spinning down. Removed supernatant and re-suspended cells to 0.3×106 per ml and add 100 μl per well of 96 well U-bottom plate.
The T cells then were labelled with CD3-VL (from the 20G6 anti-CD3 clone) through the anti-FITC ATTAC component.
The unwanted tumor cells are labelled with either a combination of ATTAC or T-cell engaging antibodies (TEAC) components that bind to EpCAM and once processed at the cell surface, will re-combine to produce a functional anti-CD3 activating domain. TEACs refer to a kit or composition wherein both components target to a cancer cell (see WO2017/087789). TEACs lack an immune cell selection moiety, which is comprised in an ATTAC. This pairing was used as a positive control as this pairing generates a T cell response by cytokine secretion.
To pair with the anti-FITC ATTAC component, the unwanted tumor cells were labelled with an ATTAC component that bound to EpCAM on the tumor cell and once processed at the cell surface expressed the corresponding CD3 domain to the anti-FITC ATTAC component so that once the T cells with the anti-FITC ATTAC component and the tumor cells with the anti-EpCAM ATTAC component are mixed together, there is a functional anti-CD3 VH-VL domain to activate the wanted subset of T cells. Counted MCF-7 cells (12×106) and washed twice in RPMI+10% NBS.
Re-suspended in media so there are 300,000 cells per 160 μl and added 2.56 ml to two 15 ml Falcon tubes labelled (i) EpCAM VH TEAC component (SEQ ID NO: 166) and EpCAM VL TEAC component (SEQ ID NO: 167) (the components form a TEAC [used as a control] when both components target to the cancer cell and neither component contains an immune cell selection moiety) and (ii) EpCAM VH ATTAC component (SEQ ID NO: 166) only. Also added 160 ul to another two Falcon tubes (iii) BiTE labelled (SEQ ID NO: 168) and (iv) untreated.
Mixed 320 μl EpCAM-20G6 VL TEAC component (300 μg/ml) and 320 μl EpCAM-20G6 VH TEAC component (300 μg/ml) together and added 640 ul to tube (i). Added 320 ul EpCAM-20G6 VH ATTAC component (300 μg/ml) to tube (ii). Final concentration of each ATTAC/TEACcomponent was 30 μg/ml. Incubated at room temperature for 30 minutes.
Washed off excess ATTAC/TEAC component by adding 5 mls media and spinning down. Removed supernatant and re-suspended cells to 1×106 per ml and added 100 ul per well already containing the T cells (see above).
In tube (i), tumor cells were labelled with TEAC components containing both VH and VL. In tube (ii), the tumor cells were only labelled with the EpCAM ATTAC component containing the VH domain of the anti-CD3 and this can complement the VL domain of the anti-CD3 which can be found on the T cells.
As a positive control, tumor cells were labelled with BiTE (SEQ ID NO: 168) to demonstrate that if a complete anti-CD3 molecule is on the surface of the tumor cell, T cells can become activated. As a negative control, T cells were incubated with untreated tumor cells to demonstrate that there is no T cell activation if there is no anti-CD3 molecules on the tumor cell surface.
For BiTE treated cells, added 20 μl BiTE (SEQ ID NO: 168-20 μg/ml). Final concentration of BiTE was 2 μg/ml. Incubated at room temperature for 30 minutes.
Washed off excess BiTE by adding 5 mls media and spinning down. Removed supernatant and re-suspended cells to 1×106 per ml and add 100 ul per well.
For untreated target cells, nothing was added. Incubated at room temperature for 30 minutes.
Added 5 mls media and spun down. Removed supernatant and re-suspended cells to 1×106 per ml and added 100 ul per well.
Incubated plate at 37° C. overnight and used 100 μl supernatant for IFN-gamma ELISA and then pool cells from triplicate wells and use for FACS staining.
For the IFN-gamma ELISA assay, a kit from ThermoFisher (Cat #88-7316-77) was used.
Background of IFNγ Assays Generally: Expression of cytokine markers in vitro, such as IFNγ expression, is known to have a predictive value for T cell responses and, thus, predicts in vivo results. As described in Ghanekar et al., Clin Diag Lab Immunol j8(3):628-31 (2001), IFNγ expression in CD8+ T cells measured by cytokine flow cytometry (CFC) is a surrogate marker for the response of cytotoxic T lymphocytes. Ghanekar at 628. Prior work showed that there is a strong correlation between the expression of IFNγ by CD8+ T cells and the activity of CTL effector cells. Ghanekar at 630. Prior work shows that the use of data on IFNγ expression allows greater accuracy in assessing CD8+ T-cell responses in a clinical setting. Id. at 631. This demonstrates that the cytokine expression assays herein were known to have predictive value for in vivo and clinical responses. While the methods herein do not follow the exact method steps of Ghanekar because there are multiple ways to assess IFNγ expression, Ghanekar demonstrates that IFNγ expression is a proxy for T-cell activity.
Cells were washed in 3 ml FACS buffer (PBS+2% serum) and the supernatant discarded. Cells were stained with antibodies against CD3, CD4, CD8 and CD69 (T cell activation marker) for 30 minutes. Excess antibody was washed off using FACS buffer. The cells were filtered prior to running on the flow cytometer.
The controls worked well with strong T cell activation by BiTE and there is no T cell activation when they are incubated with unlabeled target cells (no anti-CD3 on the cell surface). Thus, more specifically, this control experiment shows that TEACs are not selective between CD4 and CD8 and that using an FITC model did not alter the expected results. The use of the FITC model does not prevent T cell activation. The results seen in
Therefore, even though all T cells express the listed proteins on their cell surface (see
T cells stained with the FITC-conjugated antibodies prior to running the experiment to demonstrate that FITC will be on the T cell surface for the anti-FITC ATTAC component to bind to.
The results in
Results in
Target cells have both EpCAM-CD3VH and EpCAM-CD3VL (therefore have both halves of the anti-CD3 molecule).
The controls in
As expected, there is very similar CD4 T cell activation across all antibody labelled T cells as both TEAC components have been bound to the tumor cells.
Activation of CD4 T cells was only seen when bound with the CD52 and CXCR3 antibodies, and no activation of CD4 T cells was seen when bound with other antibodies including the CD8 antibodies.
These data support specific activation of CD8 T cells and not CD4 T cells using the CD8 FITC antibody and the anti-FITC ATTAC component as a means of getting the anti-CD3 VL on the T cell surface where it can pair with the anti-CD3 VH which is present on the tumor cell surface from binding of the EpCAM ATTAC component.
Experiments were performed with direct targeting to immune cells, instead of using a model system employing FITC.
An ATTAC comprises two components. In these examples, for convenience, a first component comprising a targeted immune cell binding agent is referred to as an ATTAC1, and a second component comprising a selected immune cell binding agent is referred to as an ATTAC2.
In some experiments, a component that comprises a targeting moiety capable of targeting the cancer was used together with a second component that also comprises a targeting moiety capable of targeting the cancer to generate a TEAC. The TEACs are used herein as a control. The TEAC control shows activity induced when both components target the cancer cell.
MDA-MB-231 cells over-expressing EpCAM were labelled with anti-EpCAM ATTAC1 (containing the anti-CD3 VH domain (SEQ ID NO: 166)) and excess ATTAC component removed by washing.
Peripheral blood mononuclear cells (PBMCs) from healthy donor were labelled with the anti-CD8 ATTAC2 (containing the anti-CD3 VL domain (SEQ ID NO: 170)) and excess ATTAC component was removed by washing.
Control cells were labelled with anti-EpCAM TEACs. For experiments where anti-EpCAM TEACs were used (SEQ ID NOs: 166 and 167), both components will bind EpCAM on the tumor cells, without a targeting moiety that binds to an immune cell. In this control experiment, the TEAC pair thus will not confer specificity with an immune cell selection moiety.
The PBMCs were then co-cultured with the tumor cells at a PBMC to tumor cell ratio of 1:2. The ATTACs are proteolytically activatable by addition of an exogenous protease (enterokinase) with the protease added or not to the mixed cells. The co-cultured cells were then incubated overnight at 37° C.
After incubation, co-cultured cells were washed in FACS buffer (PBS+2% serum) and labelled for flow cytometry using CD3 APC-Cy7, CD4 PE, CD8 APC and CD69 FITC to ascertain the level of T cell activation (measured by an increase in CD69 staining) of the CD4 and CD8 T cell subsets.
An increase in activation of CD8 T cells was seen after treatment with anti-EpCAM ATTAC1 and anti-CD8 ATTAC2 when enterokinase (protease) is added (
When both components of a TEAC are bound to the tumor cell (control wherein a TEAC component pair both bind to EpCAM) to form a functional anti-CD3 moiety at the tumor cell surface, both CD4 T cells (
These results show that treatment with the EpCAM ATTAC VH (ATTAC1) plus CD8 ATTAC VL (ATTAC2) activates CD8 T cells in the presence of a protease, without activating CD4 T cells. In contrast, treatment with an EpCAM TEAC component pair activates both CD4 and CD8 T cells.
Thus, ATTACs can be used to specifically activate CD8 T cells, which are critical for successful anti-tumor immune responses.
Interferon gamma release was also used to evaluate activity of an ATTAC1 targeting a tumor cell antigen and an ATTAC2 targeting an immune cell antigen. In this example, ATTAC1 comprises a targeting moiety capable of targeting the cancer by targeting EpCAM expressed on the tumor cells and an anti-CD3 VH domain. ATTAC2 comprises an immune cell selection moiety capable of selectively targeting an immune cell by targeting CD8 and an anti-CD3 VH domain.
Tumor cells were labelled with increasing concentrations of anti-EpCAM ATTAC1 (containing both the an anti-EpCAM function and an anti-CD3 VH domain (SEQ ID NO: 166); termed “EpCAM VH”) and excess ATTAC component removed by washing. PBMCs from a healthy donor (
Following co-culture, the supernatant was assayed for the presence of interferon gamma (IFN-gamma), which denotes cytokine release by activated T cells. There was a dose-dependent increase in interferon gamma release by both PBMCs (
The results in
In the experimental controls, there was a lack of T cell activation, as measured by interferon gamma release, when T cells (
The concentration dependence of an ATTAC pair was tested, wherein the ATTAC1 targeted a tumor cell antigen and an ATTAC2 targeted an immune cell antigen.
Tumor cells were labelled with increasing concentrations of anti-EpCAM ATTAC1 (containing the anti-CD3 VH domain; SEQ ID NO: 166) and excess ATTAC component removed by washing. PBMCs from a healthy donor (
The data demonstrates strong T cell activation when the concentrations of ATTAC1 and 2 increase in equimolar concentrations (
Selective activation of T cell subsets was also tested using a model system employing FITC.
Tumor cells were labelled with an anti-EpCAM ATTAC1 (containing the anti-CD3 VH domain (SEQ ID NO: 166)), and excess ATTAC component was removed by washing. PBMCs from healthy donor were labelled with FITC-conjugated antibodies against CD4, CD8, or CD19 with excess antibody removed by washing. The PBMCs were further labelled with an anti-FITC ATTAC2 (containing the anti-CD3 VL domain (SEQ ID NO: 165)), and excess ATTAC component was removed by washing. The PBMCs were then co-cultured with the tumor cells at a PBMC to tumor cell ratio of 1:2. The ATTACs were proteolytically activatable by addition of an exogenous protease (enterokinase) with the protease added to the mixed cells. The co-cultured cells were then incubated overnight at 37° C.
In these experiments, FITC-labeled CD19 cells are a negative control, because CD19-expressing cells do not normally express CD3. Thus, binding of an anti-FITC ATTAC component to a CD19-positive cell would not lead to activation via a paired anti-CD3 VH/VL from an ATTAC component pair.
After incubation, co-cultured cells were washed in FACS buffer (PBS+2% serum) and labelled for flow cytometry using CD3 APC-Cy7, CD4 PE, CD8 APC and CD69 BV421 to ascertain the level of T cell activation (measured by the increase in CD69 staining) of CD4 and CD8 T cell subsets. Excess antibodies were removed by washing and the cells were analyzed by flow cytometry. CD4 T cells were only significantly activated (compared with the background activation of untreated T cells) when the PBMCs were labelled with the anti-CD4 FITC antibody (
In contrast, CD8 T cells were only significantly activated (compared with the background activation of untreated T cells) when the PBMCs were labelled with the anti-CD8 FITC antibody (
These data show the ability of ATTACs to activate a specific subset of T cell within a more complex mix of T cells. As shown
Selective activation of a specific subset of immune cells could be therapeutically useful. For example, ATTACs that activate only cytotoxic T cells could avoid activation of unwanted T cells, such as regulatory T cells. Further, use of ATTACs that require cleavage by a tumor-associated protease can allow activation of immune cells within the tumor microenvironment. In this way, ATTACs could provide specificity for activating specific subsets of immune cells within the tumor microenvironment.
Peripheral blood mononuclear cells are labelled with the anti-CD8 ATTAC component and the excess ATTAC component removed by washing. The anti-CD8 ATTAC component contains one half of the anti-CD3 activating domain (VL). Unwanted tumor cell line would be labelled with an anti-EpCAM ATTAC component that contains the corresponding half of the anti-CD3 activating domain (VH) (SEQ ID NO: 166). The ATTAC would then be able to activate CD3 specifically on the CD8 T cells within the peripheral blood mononuclear cells. The activation of the CD8 T cells can be assayed by ELISA for IFN gamma secretion or by flow cytometry assaying for activation markers such as CD69 and CD38.
Peripheral blood mononuclear cells are labelled with the anti-CD4 ATTAC component and the excess ATTAC component removed by washing. The anti-CD4 ATTAC component contains one half of the anti-CD3 activating domain (VL) (SEQ ID NO: 166). Unwanted tumor cell line would be labelled with an anti-EpCAM ATTAC component that contains the corresponding half of the anti-CD3 activating domain (VH). The ATTAC would then be able to activate CD3 specifically on the CD4 T cells within the peripheral blood mononuclear cells. The activation of the CD4 T cells can be assayed by ELISA for IFN gamma secretion or by flow cytometry assaying for activation markers such as CD69 and CD38.
The following numbered items provide embodiments as described herein, though the embodiments recited here are not limiting.
Item 1. An agent for treating cancer in a patient comprising:
a. a first component comprising a targeted immune cell binding agent comprising:
b. a second component comprising a selective immune cell binding agent comprising:
Item 2. The agent of item 1, wherein the first component is not covalently bound to the second component.
Item 3. The agent of item 1, wherein the first component is covalently bound to the second component.
Item 4 The agent of any one of items 1-3, wherein the immune cell engaging domains, when bound to each other, are capable of binding an antigen expressed on the surface of the immune cell.
Item 5. The agent of any one of items 1-4, wherein the immune cell selection moiety capable of selectively targeting an immune cell selectively targets a T cell, a macrophage, a natural killer cell, a neutrophil, an eosinophil, a basophil, a γδ T cell, a natural killer T cell (NKT cells), or an engineered immune cell.
Item 6. The agent of item 5, wherein the immune cell selection moiety capable of selectively targeting an immune cell selectively targets a T cell.
Item 7. The agent of item 6, wherein the T cell is a cytotoxic T cell.
Item 8. The agent of item 7, wherein the cytotoxic T cell is a CD8+ T cell.
Item 9. The agent of item 6, wherein the T cell is a helper T cell.
Item 10. The agent of item 9, wherein the helper T cell is a CD4+ T cell.
Item 11. The agent of any one of items 6-10, wherein the immune cell selection moiety targets CD8, CD4, or CXCR3.
Item 12. The agent of any one of items 6-11, wherein the immune cell selection moiety does not specifically bind regulatory T cells.
Item 13. The agent of any one of items 6-12, wherein the immune cell selection moiety does not specifically bind TH17 cells.
Item 14. The agent of any one of items 6-13, wherein the immune cell engaging domains, when bound to each other, are capable of binding CD3.
Item 15. The agent of any one of items 6-13, wherein the immune cell engaging domains, when bound to each other, are capable of binding TCR.
Item 16. The agent of item 5, wherein the immune cell selection moiety capable of selectively targeting an immune cell selectively targets a natural killer cell.
Item 17. The agent of item 16, wherein the immune cell selection moiety targets CD2 or CD56.
Item 18. The agent of any one of items 16-17, wherein the immune cell engaging domains, when bound to each other, are capable of binding NKG2D, CD16, NKp30, NKp44, NKp46 or DNAM.
Item 19. The agent of item 5, wherein the immune cell selection moiety capable of selectively targeting an immune cell selectively targets a macrophage.
Item 20. The agent of item 19, wherein the immune cell selection moiety targets CD14, CD11b, or CD40.
Item 21. The agent of any one of items 19-20, wherein the immune cell engaging domains, when bound to each other, are capable of binding CD89 (Fc alpha receptor 1), CD64 (Fc gamma receptor 1), CD32 (Fc gamma receptor 2A) or CD16a (Fc gamma receptor 3A).
Item 22. The agent of item 5, wherein the immune cell selection moiety capable of selectively targeting an immune cell selectively targets a neutrophil.
Item 23. The agent of item 22, wherein the immune cell selection moiety targets CD15.
Item 24. The agent of any one of items 22-23, wherein the immune cell engaging domains, when bound to each other, are capable of binding CD89 (FcαR1), FcγRI (CD64), FcγRIIA (CD32), FcγRIIIA (CD16a), CD11b (CR3, αMβ2), TLR2, TLR4, CLEC7A (Dectin1), formyl peptide receptor 1 (FPR1), formyl peptide receptor 2 (FPR2), or formyl peptide receptor 3 (FPR3).
Item 25. The agent of item 5, wherein the immune cell selection moiety capable of selectively targeting an immune cell selectively targets an eosinophil.
Item 26. The agent of item 25, wherein the immune cell selection moiety targets CD193, Siglec-8, or EMR1.
Item 27. The agent of any one of items 25-26, wherein the immune cell engaging domains, when bound to each other, are capable of binding CD89 (Fc alpha receptor 1), FcεRI, FcγRI (CD64), FcγRIIA (CD32), FcγRIIIB (CD16b), or TLR4.
Item 28. The agent of item 5, wherein the immune cell selection moiety capable of selectively targeting an immune cell selectively targets a basophil.
Item 29. The agent of item 28, wherein the immune cell selection moiety targets 2D7, CD203c, or FcεRIα.
Item 30. The agent of any one of items 28-29, wherein the immune cell engaging domains, when bound to each other, are capable of binding CD89 (Fc alpha receptor 1) or FcεRI.
Item 31. The agent of item 5, wherein the immune cell selection moiety capable of selectively targeting an immune cell selectively targets a γδ T cell.
Item 32. The agent of item 31, wherein the immune cell selection moiety targets γδ TCR.
Item 33. The agent of any one of items 31-32, wherein the immune cell engaging domains, when bound to each other, are capable of binding γδ TCR, NKG2D, CD3 Complex (CD3ε, CD3γ, CD3δ, CD3ζ, CD3η), 4-1BB, DNAM-1, or TLRs (TLR2, TLR6).
Item 34. The agent of item 5, wherein the immune cell selection moiety capable of selectively targeting an immune cell selectively targets a natural killer T cell.
Item 35. The agent of item 34, wherein the immune cell selection moiety targets Va24 or CD56.
Item 36. The agent of any one of items 34-35, wherein the immune cell engaging domains, when bound to each other, are capable of binding αβTCR, NKG2D, CD3 Complex (CD3ε, CD3γ, CD3δ, CD3ζ, CD3η), 4-1BB, or IL-12R.
Item 37. The agent of item 5, wherein the immune cell selection moiety capable of selectively targeting an immune cell selectively targets an engineered immune cell.
Item 38. The agent of item 37, wherein the engineered immune cell is a chimeric antigen receptor (CAR) T cell, natural killer cell, natural killer T cell, or γδ T cell.
Item 39. The agent of item 37-38, wherein the immune cell selection moiety targets the CAR or a marker expressed on the immune cell.
Item 40. The agent of item 37-39, wherein the immune selection moieties targets LNGFR or CD20.
Item 41. The agent of item 37-40, wherein the immune cell engaging domains, when bound to each other, are capable of binding an antigen expressed by the engineered immune cell.
Item 42. The agent of item 37-41, wherein the antigen expressed by the engineered immune cell is CD3.
Item 43. The agent of any one of items 1-42, wherein the immune cell selection moiety comprises an antibody or antigen-specific binding fragment thereof.
Item 44. The agent of item 43, wherein the antibody or antigen-specific binding fragment thereof specifically binds an antigen on a T cell.
Item 45. The agent of any one of items 43, wherein the antibody or antigen-specific binding fragment thereof specifically binds an antigen on a cytotoxic or helper T cell.
Item 46. The agent of item 43, wherein the antibody or antigen-specific binding fragment thereof specifically binds an antigen on a macrophage.
Item 47. The agent of item 43, wherein the antibody or antigen-specific binding fragment thereof specifically binds an antigen on a natural killer cell.
Item 48. The agent of item 43, wherein the antibody or antigen-specific binding fragment thereof specifically binds an antigen on a neutrophil.
Item 49. The agent of item 43, wherein the antibody or antigen-specific binding fragment thereof specifically binds an antigen on an eosinophil.
Item 50. The agent of item 43, wherein the antibody or antigen-specific binding fragment thereof specifically binds an antigen on a γδ T cell.
Item 51. The agent of item 43, wherein the antibody or antigen-specific binding fragment thereof specifically binds an antigen on a natural killer T cell.
Item 52. The agent of item 43, wherein the antibody or antigen-specific binding fragment thereof specifically binds an antigen on an engineered immune cell.
Item 53. The agent of item 43, wherein the engineered immune cell is a CAR T cell, natural killer cell, natural killer T cell, or γδ T cell.
Item 54. The agent of any one of items 1-42, wherein the immune selection moiety comprises an aptamer.
Item 55. The agent of item 54, wherein the aptamer specifically binds an antigen on a T cell.
Item 56. The agent of item 55, wherein T cell is a cytotoxic or helper T cell.
Item 57. The agent of item 54, wherein the aptamer specifically binds an antigen on a macrophage.
Item 58. The agent of item 54, wherein the aptamer specifically binds an antigen on a natural killer cell.
Item 59. The agent of item 54, wherein the aptamer specifically binds an antigen on a neutrophil.
Item 60. The agent of item 54, wherein the aptamer specifically binds an antigen on an eosinophil.
Item 61. The agent of item 54, wherein the aptamer specifically binds an antigen on a γδ T cell.
Item 62. The agent of item 54, wherein the aptamer specifically binds an antigen on a natural killer T cell.
Item 63. The agent of item 54, wherein the aptamer specifically binds an antigen on an engineered immune cell.
Item 64. The agent of item 54, wherein the engineered immune cell is a CAR T cell, natural killer cell, natural killer T cell, or γδ T cell.
Item 65. The agent of any one of items 54-64, wherein the aptamer comprises DNA.
Item 66. The agent of any one of items 54-64, wherein the aptamer comprises RNA.
Item 67. The agent of any one of items 65-66, wherein the aptamer is single-stranded.
Item 68. The agent of any one of items 54-67, wherein the aptamer is a selective immune cell binding-specific aptamer chosen from a random candidate library.
Item 69. The agent of any one of items 1-68, wherein the targeting moiety is an antibody or antigen-specific binding fragment.
Item 70. The agent of item 69, wherein the antibody or antigen-specific binding fragment thereof specifically binds a cancer antigen.
Item 71. The agent of any one of items 1-68, wherein the targeting moiety is an aptamer.
Item 72. The agent of item 71, wherein the aptamer specifically binds a cancer antigen.
Item 73. The agent of any one of items 71-72, wherein the aptamer comprises DNA.
Item 74. The agent of any one of items 71-72, wherein the aptamer comprises RNA.
Item 75. The agent of any one of items 73-74, wherein the aptamer is single-stranded.
Item 76. The agent of any one of items 71-75, wherein the aptamer is a target cell-specific aptamer chosen from a random candidate library.
Item 77. The agent of any one of items 71-76, wherein the aptamer is an anti-EGFR aptamer.
Item 78. The agent of any one of items 77, wherein the anti-EGFR aptamer comprises any one of SEQ ID NOs: 95-164.
Item 79. The agent of any one of items 71-78, wherein the aptamer binds to the cancer on the cancer cell with a Kd from 1 picomolar to 500 nanomolar.
Item 80. The agent of any one of items 71-79, wherein the aptamer binds to the cancer with a Kd from 1 picomolar to 100 nanomolar.
Item 81. The agent of any one of items 1-68, wherein the targeting moiety comprises IL-2, IL-4, IL-6, α-MSH, transferrin, folic acid, EGF, TGF, PD1, IL-13, stem cell factor, insulin-like growth factor (IGF), or CD40.
Item 82. The agent of any one of items 1-68, wherein the targeting moiety comprises a full-length sequence of IL-2, IL-4, IL-6, α-MSH, transferrin, folic acid, EGF, TGF, PD1, IL-13, stem cell factor, insulin-like growth factor (IGF), or CD40.
Item 83. The agent of any one of items 1-68, wherein the targeting moiety comprises a truncated form, analog, variant, or derivative of IL-2, IL-4, IL-6, α-MSH, transferrin, folic acid, EGF, TGF, PD1, IL-13, stem cell factor, insulin-like growth factor (IGF), or CD40.
Item 84. The agent of any one of items 1-68, wherein the targeting moiety binds a target on the cancer comprising IL-2 receptor, IL-4, IL-6, melanocyte stimulating hormone receptor (MSH receptor), transferrin receptor (TR), folate receptor 1 (FOLR), folate hydroxylase (FOLH1), EGF receptor, PD-L1, PD-L2, IL-13R, CXCR4, IGFR, or CD40L.
Item 85. The agent of any one of items 1-84, wherein one immune cell engaging domain comprises a VH domain and the other immune cell engaging domain comprises a VL domain.
Item 86. The agent of any one of items 1-85, wherein the first immune cell binding partner is bound to an inert binding partner and separated from it by a cleavage site.
Item 87. The agent of any one of items 1-86, wherein the second immune cell binding partner is bound to an inert binding partner and separated from it by a cleavage site.
Item 88. The agent of any one of items 1-87, wherein
Item 89. The agent of item 88, wherein the first cleavage site and the second cleavage site are the same cleavage site.
Item 90. The agent of item 88, wherein the first cleavage site and the second cleavage site are different cleavage sites.
Item 91. The agent of any one of items 1-90, wherein at least one cleavage site is a protease cleavage site.
Item 92. The agent of any one of items 1-91, wherein at least one enzyme expressed by the cancer cells is a protease.
Item 93. The agent of any one of items 1-92, wherein at least one inert binding partner specifically binds the immune cell engaging domain.
Item 94. The agent of item 93, wherein at least one inert binding partner is a VH or VL domain.
Item 95. The agent of item 94, wherein
Item 96. The agent of item 3, wherein the first component is covalently bound to the second component by a linker comprising a cleavage site.
Item 97. The agent of item 96, wherein the cleavage site is a protease cleavage site.
Item 98. The agent of items 97, wherein the protease cleavage site is cleavable in blood.
Item 99. The agent of item 98, wherein the protease cleavage site is a cleavage site for thrombin, neutrophil elastase, or furin.
Item 100. The agent of item 97, wherein the protease cleavage site is cleavable by a tumor-associated protease.
Item 101. The agent of item 100, wherein the tumor-associated protease cleavage site comprises any one of SEQ ID NOs: 1-84.
Item 102. An agent for treating cancer in a patient comprising a selective immune cell binding agent comprising:
Item 103. A set of nucleic acid molecules encoding the first and second component of the agent of any one of items 1-101.
Item 104. A nucleic acid molecule encoding the selective immune cell binding agent of item 102.
Item 105. A method of treating cancer in a patient comprising administering the agent of any one of items 1-101.
Item 106. The method of item 105, wherein if the patient has regulatory T cells in the tumor, the selective immune cell binding agent does not target markers present on regulatory immune cells (including, but not limited to CD4 and CD25).
Item 107. The method of any one of items 105-106, wherein the selective immune cell binding agent does not target markers present on TH17 cells.
Item 108. The method of any one of items 105-107, wherein the selective immune cell binding agent activates T cells that will target the tumor cells for lysis.
Item 109. The method of any one of items 105-108, wherein if the patient has regulatory T cells in the tumor, the immune cell selection moiety targets CD8+ T cells by specifically binding CD8.
Item 110. The method of any one of items 105-108, wherein if the patient has regulatory T cells in the tumor, the immune cell selection moiety targets CD8+ T cells and CD4+ T cells by specifically binding CXCR3.
Item 111. The method of any one of items 105-110, wherein the cancer is any one of breast cancer, ovarian cancer, endometrial cancer, cervical cancer, bladder cancer, renal cancer, melanoma, lung cancer, prostate cancer, testicular cancer, thyroid cancer, brain cancer, esophageal cancer, gastric cancer, pancreatic cancer, colorectal cancer, liver cancer, leukemia, myeloma, nonHodgkin lymphoma, Hodgkin lymphoma, acute myeloid leukemia, acute lymphoblastic leukemia, chronic lymphoblastic leukemia, lymphoproliferative disorder, myelodysplastic disorder, myeloproliferative disease or premalignant disease.
Item 112. A method of targeting an immune response of a patient to cancer comprising administering the agent of any one of items 1-101 to the patient.
The foregoing written specification is considered to be sufficient to enable one skilled in the art to practice the embodiments. The foregoing description and Examples detail certain embodiments and describes the best mode contemplated by the inventors. It will be appreciated, however, that no matter how detailed the foregoing may appear in text, the embodiment may be practiced in many ways and should be construed in accordance with the appended claims and any equivalents thereof.
As used herein, the term about refers to a numeric value, including, for example, whole numbers, fractions, and percentages, whether or not explicitly indicated. The term about generally refers to a range of numerical values (e.g., +/−5-10% of the recited range) that one of ordinary skill in the art would consider equivalent to the recited value (e.g., having the same function or result). When terms such as at least and about precede a list of numerical values or ranges, the terms modify all of the values or ranges provided in the list. In some instances, the term about may include numerical values that are rounded to the nearest significant figure.
This application is a 371 National Phase Entry of International Patent Application No. PCT/US2019/040336 filed Jul. 2, 2019, which claims benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application No. 62/693,125 filed Jul. 2, 2018, the contents of which are incorporated herein by reference in their entirety.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2019/040336 | 7/2/2019 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62693125 | Jul 2018 | US |
