Patent Application
20210008113
The present application generally relates to the technical field of Guidance and Navigation Control (GNC) proteins with multi-specific binding activities against surface molecules on both immune cells and tumor cells, and more particularly relates to making and using GNC proteins.
Cancer cells develop various strategies to evade the immune system. One of the underlying mechanisms for the immune escape is the reduced recognition of cancer cells by the immune system. Defective presentation of cancer specific antigens or lack of thereof results in immune tolerance and cancer progression. In the presence of effective immune recognition tumors use other mechanisms to avoid elimination by the immune system. Immunocompetent tumors create suppressive microenvironments to downregulate the immune response. Multiple players are involved in shaping the suppressive tumor microenvironment, including tumor cells, regulatory T cells, Myeloid-Derived Suppressor cells, stromal cells, and other cell types. The suppression of immune response can be executed in a cell contact-dependent format as well as in a contact-independent manner, via secretion of immunosuppressive cytokines or elimination of essential survival factors from the local environment. Cell contact-dependent suppression relies on molecules expressed on the cell surface, e.g. Programmed Death Ligand 1 (PD-L1), T-lymphocyte-associated protein 4 (CTLA-4) and others (Dunn, Old et al. 2004, Adachi and Tamada 2015).
As the mechanisms by which tumors evade recognition by the immune system continue to be better understood, new treatment modalities that target these mechanisms have recently emerged. On Mar. 25, 2011, the U. S. Food and Drug Administration (FDA) approved ipilimumab injection (Yervoy, Bristol-Myers Squibb) for the treatment of unresectable or metastatic melanoma. Yervoy binds to cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) expressed on activated T cells and blocks the interaction of CTLA-4 with CD80/86 on antigen-presenting cells thereby blocking the negative or inhibitory signal delivered into the T cell through CTLA-4 resulting in re-activation of the antigen-specific T cell leading to, in many patients, eradication of the tumor. A few years later in 2014 the FDA approved Keytruda (Pembrolizumab, Merck) and Opdivo (Nivolumab, Bristol-Myers Squibb) for treatment of advanced melanoma. These monoclonal antibodies bind to PD-1 which is expressed on activated and/or exhausted T cells and block the interaction of PD-1 with PD-L1 expressed on tumors thereby eliminating the inhibitory signal through PD-1 into the T cell resulting in re-activation of the antigen-specific T cell leading to again, in many patients, eradication of the tumor. Since then additional clinical trials have been performed comparing the single monoclonal antibody Yervoy to the combination of the monoclonal antibodies Yervoy and Opdivo in the treatment of advanced melanoma which showed improvement in overall survival and progression-free survival in the patients treated with the combination of antibodies. (Hodi, Chesney et al. 2016, Hellmann, Callahan et al. 2018). However, as many clinical trials have shown a great benefit of treating cancer patients with monoclonal antibodies that are specific for one or more immune checkpoint molecules data has emerged that only those patients with a high mutational burden that generates a novel T cell epitope(s) which is recognized by antigen-specific T cells show a clinical response (Snyder, Makarov et al. 2014). Those patients that have a low tumor mutational load mostly do not show an objective clinical response (Snyder, Makarov et al. 2014, Hellmann, Callahan et al. 2018).
In recent years other groups have developed an alternate approach that does not require the presence of neoepitope presentation by antigen-presenting cells to activate T cells. One example is the development of a bi-specific antibody where the binding domain of an antibody which is specific for a tumor associated antigen, e.g., CD19, is linked to an antibody binding domain specific for CD3 on T cells thus creating a bi-specific T cell engager or BiTe molecule. In 2014, the FDA approved a bi-specific antibody called Blinatumumab for the treatment of Precursor B-Cell Acute Lymphoblastic Leukemia. Blinatumumab links the single-chain variable fragment (scFv) specific for CD19 expressed on leukemic cells with the scFv specific for CD3 expressed on T cells (Benjamin and Stein 2016). However, despite an initial response rate of >50% in patients with relapsed or refractory ALL many patients are resistant to Blinatumumab therapy or relapse after successful treatment with Blinatumumab. Evidence is emerging that the resistance to Blinatumumab or relapse after Blinatumumab treatment is attributable to the expression of immune checkpoint inhibitory molecules expressed on tumor cells, such as PD-L1 that drives an inhibitory signal through PD-1 expressed on activated T cells (Feucht, Kayser et al. 2016). In a case study of a patient who was resistant to therapy with Blinatumumab, a second round of Blinatumumab therapy was performed but with the addition of a monoclonal antibody, pembrolizumab (Keytruda, Merck). Pembrolizumab specifically binds to PD-1 and blocks the interaction of T cell-expressed PD-1 with tumor cell expressed PD-L1, which resulted in a dramatic response and reduction of tumor cells in the bone marrow from 45% to less than 5% in this one patient (Feucht, Kayser et al. 2016). These results show that combining a bi-specific BiTe molecule with one or more monoclonal antibodies can significantly increase clinical activity compared to either agent alone. Despite the promising outcome, the cost leading to the combined therapy must be high due to multiple clinical trials and the difficulty in recruiting representative populations.
Adoptive cell therapy with chimeric antigen receptor T cells (CAR-T) is another promising immunotherapy for treating cancer. The clinical success of CAR-T therapy has revealed durable complete remissions and prolonged survival of patients with CD19-positive treatment-refractory B cell malignancies (Gill and June 2015). However, the cost and complexity associated with the manufacture of a personalized and genetically modified CAR-T immunotherapy has restricted their production and use to specialized centers for treating relatively small numbers of patients. Cytokine release syndrome (CRS), also known as cytokine storm, is considered as the major adverse effect after the infusion of engineered CAR-T cells (Bonifant, Jackson et al. 2016). In many cases, the onset and severity of CRS seems to be personally specific to the patient. Current options of mitigating CRS are mainly focused on rapid response and management care because the option of controlling CRS prior to T cell infusion is limited.
While the efficacy of CAR-T therapy specific for a CD19-positive B cell malignancy is now clearly established, the efficacy of CAR-T therapy against solid tumors has not been unequivocally demonstrated to date. Currently, many clinical trials are in progress to explore a variety of solid tumor-associated antigens (TAA) for CAR-T therapy. Inefficient T cell trafficking into the tumors, an immunosuppressive tumor micro-environment, suboptimal antigen recognition specificity, and lack of control over treatment-related adverse events are currently considered as the main obstacles in solid tumor CAR-T therapy (Li, Li et al. 2018). The option of managing the therapeutic effect, as well as any adverse effect before and after the CAR-T cell infusion, is limited.
The application provides, among others, methods for generating therapeutic compositions containing a guidance and navigation (GNC) proteins, methods for treating cancer conditions using a guidance and navigation control (GNC) proteins, and therapeutic compositions containing GNC proteins or therapeutic cells having cytotoxic cells coated (or bound) with GNC proteins.
In one aspect, the application provides therapeutic compositions. In one embodiment, the therapeutic composition comprises a cytotoxic cell, a GNC protein, and a therapeutic cell. The therapeutic cell comprises the GNC protein bound to the cytotoxic cell through the binding interaction with the cytotoxic cell receptor, and the therapeutic cell composition is substantially free exogenous of viral and non-viral DNA and RNA.
In one embodiment, the therapeutic composition may further comprise a second GNC protein, a second therapeutic cell, or a combination thereof, wherein the second therapeutic cell comprises the cytotoxic cells with the second GNC protein bound thereupon or with both the first and the second GNC proteins bound thereupon.
GNC protein includes a cytotoxic binding moiety and a cancer targeting moiety. The cytotoxic binding moiety has a binding specificity to a cytotoxic cell receptor and is configured to activate the cytotoxic cell through the binding with the cytotoxic cell receptor. The cancer targeting moiety has a binding specificity to a cancer cell receptor.
In one embodiment, the GNC protein includes a binding domain for T-cell receptors. Examples T-cell receptor include without limitation CD3, CD28, PDL1, PD1, OX40, 4-1BB, GITR, TIGIT, TIM-3, LAG-3, CTLA4, CD40L, VISTA, ICOS, BTLA, Light, CD30, NKp30, CD28H, CD27, CD226, CD96, CD112R, A2AR, CD160, CD244, CECAM1, CD200R, TNFRSF25 (DR3), or a combination thereof. In one embodiment, the GNC protein is capable of activating a T-cell by binding the T-cell binding moiety to a T-cell receptor on the T-cell. In one embodiment, the GNC protein is capable of activating a T-cell by binding multiple T-cell binding moieties on the T-cell.
In one embodiment, the GNC protein includes a binding domain for a NK cell receptor. Examples NK cell receptor include, without limitation, receptors for activation of NK cell such as CD16, NKG2D, KIR2DS1, KIR2DS2, KIR2DS4, KIR3DS1, NKG2C, NKG2E, NKG2H; agonist receptors such as NKp30a, NKp30b, NKp46, NKp80, DNAM-1, CD96, CD160, 4-1BB, GITR, CD27, OX-40, CRTAM; and antagonist receptors such as KIR2DL1, KIR2DL2, KIR2DL3, KIR3DL1, KIR3DL2, KIR3DL3, NKG2A, NKp30c, TIGIT, SIGLEC7, SIGLEC9, LILR, LAIR-1, KLRG1, PD-1, CTLA-4, CD161.
In one embodiment, the GNC protein includes a binding domain for a macrophage receptor. Examples macrophage receptor include, without limitation, agonist receptor on macrophage such as TLR2, TLR4, CD16, CD64, CD40, CD80, CD86, TREM-1, TREM-2, ILT-1, ILT-6a, ILT-7, ILT-8, EMR2, Dectin-1, CD69; antagonist receptors such as CD32b, SIRPa, LAIR-1, VISTA, TIM-3, CD200R, CD300a, CD300f, SIGLEC1, SIGLEC3, SIGLEC5, SIGLEC7, SIGLEC9, ILT-2, ILT-3, ILT-4, ILT-5, LILRB3, LILRB4, DCIR; and other surface receptors such as CSF-1R, LOX-1, CCR2, FRP, CD163, CR3, DC-SIGN, CD206, SR-A, CD36, MARCO.
In one embodiment, the GNC protein includes a binding domain for a dendritic cell receptor. Examples dendritic cell receptor include, without limitation, agonist receptors on dendritic cell such as TLR, CD16, CD64, CD40, CD80, CD86, HVEM, CD70; antagonist receptors such as VISTA, TIM-3, LAG-3, BTLA; and other surface receptors such as CSF-1R, LOX-1, CCR7, DC-SIGN, GM-CSF-R, IL-4R, IL-10R, CD36, CD206, DCIR, RIG-1, CLEC9A, CXCR4.
In one embodiment, the GNC protein may include a T-cell binding moiety and a cancer-targeting moiety. In one embodiment, the T-cell binding moiety has a binding specificity to a T-cell receptor comprising CD3, CD28, PDL1, PDL2, PD1, OX40, 4-1BB, GITR, TIGIT, TIM-3, LAG-3, CTLA4, CD40L, VISTA, ICOS, BTLA, Light, CD30, CD27, or a combination thereof. In one embodiment, the cancer targeting moiety has a binding specificity to a cancer cell receptor. In one embodiment, the cancer cell receptor may include BCMA, CD19, CD20, CD33, CD123, CD22, CD30, ROR1, CEA, HER2, EGFR, EGFRvIII, LMP1, LMP2A, Mesothelin, PSMA, EpCAM, glypican-3, gpA33, GD2, TROP2, as yet to be discovered tumor associated antigens or a combination thereof.
In one embodiment, the GNC protein may have multi-specific antigen binding activities to the surface molecules of a T cell and a tumour cell. In one embodiment, the guidance and navigation control (GNC) protein comprises a binding domain for a T cell activating receptor, a binding domain for a tumor associated antigen, a bind domain for an immune checkpoint receptor, and a binding domain for a T cell co-stimulating receptor.
In one embodiment, the binding domain for the tumor associated antigen is not adjacent to the binding domain for the T cell co-stimulating receptor. In one embodiment, the binding domain for the T cell activating receptor is adjacent to the binding domain for the tumor associated antigen (TAA). The T cell activating receptor may include without limitation CD3. The T cell co-stimulating receptor may include without limitation 4-1BB, CD28, OX40, GITR, CD40L, ICOS, Light, CD27, CD30, or a combination thereof. The immune checkpoint receptor may include without limitation PD-L1, PD-1, TIGIT, TIM-3, LAG-3, CTLA4, BTLA, VISTA, PDL2, or a combination thereof.
The tumor associated antigen (TAA) may include without limitation ROR1, CD19, EGFRVIII, BCMA, CD20, CD33, CD123, CD22, CD30, CEA, HER2, EGFR, LMP1, LMP2A, Mesothelin, PSMA, EpCAM, glypican-3, gpA33, GD2, TROP2, or a combination thereof. In one embodiment, the tumor associated antigen may be ROR1. In one embodiment, the tumor associated antigen may be CD19. In one embodiment, the tumor associated antigen may be EGFRVIII.
In one embodiment, the guidance and navigation control (GNC) protein may be an antibody or an antibody monomer or a fragment thereof. In one embodiment, the GNC protein may be a tri-specific antibody. In one embodiment, the GNC protein may be a tetra-specific antibody. In one embodiment, the GNC protein includes Fc domain or a fragment thereof. Any Fc domain from an antibody may be used. Example Fc domains may include Fc domains from IgG, IgA, IgD, IgM, IgE, or a fragment or a combination thereof. Fc domain may be natural or engineered. In one embodiment, the Fc domain may contain an antigen binding site.
In one embodiment, the GNC protein comprises a bi-specific antibody, a tri-specific antibody, a tetra-specific antibody, or a combination thereof yielding up to eight binding motifs on the GNC protein. Examples of antibodies, antibody monomers, antigen-binding fragment thereof are disclosed herein. In one embodiment, GNC proteins may include an immunoglobulin G (IgG) moiety with two heavy chains and two light chains, and at least two scFv moieties being covalently connected to either C or N terminals of the heavy or light chains. The IgG moiety may provide stability to the scFv moiety, and a tri-specific GNC protein may have two moieties for binding the surface molecules on T cells.
In one embodiment, the guidance and navigation control (GNC) protein may be an antibody. In one embodiment, the tumor associated antigen comprises ROR1, CD19, or EGRFVIII. In on embodiment, the T cell activating receptor comprises CD3 and the binding domain for CD3 may be linked to the binding domain for the tumor associated (TAA) antigen through a linker to form a CD3-TAA pair. In one embodiment, the IgG Fc domain may intermediate the CD3-TAA pair and the binding domain for the immune checkpoint receptor. In one embodiment, the immune checkpoint receptor may be PD-L1.
The linker may be a covalent bond or a peptide linker. In one embodiment, the peptide linker may have from about 2 to about 100 amino acid residues.
In on embodiment, the guidance and navigation control (GNC) protein has a N-terminal and a C-terminal, comprising in tandem from the N-terminal to the C-terminal, the binding domain for CD3, the binding domain for EGFRVI, IgG Fc domain, the bind domain for PD-L1, and the binding domain for 41-BB. In one embodiment, the guidance and navigation control (GNC) protein has a N-terminal and a C-terminal, comprising in tandem from the N-terminal to the C-terminal, the binding domain for 4-1BB, the binding domain for PD-L1, IgG Fc domain, the bind domain for ROR1, and the binding domain for CD3. In one embodiment, the guidance and navigation control (GNC) protein has a N-terminal and a C-terminal, comprising in tandem from the N-terminal to the C-terminal, the binding domain for CD3, the binding domain for CD19, IgG Fc domain, the bind domain for PD-L1, and the binding domain for 4-1BB.
In one embodiment, the GNC protein comprises an amino acid having a percentage homology to SEQ ID NO. 50, 52, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, and 110. The percentage homology is not less than 70%. 80%, 90%, 95%, 98% or 99%.
In another aspect, the application provides nucleic acid sequences encoding the GNC protein or its fragments disclosed thereof. In one embodiment, the nucleic acid has a percentage homology to SEQ ID NO. 49, 51, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, and 109. The percentage homology is not less than 70%. 80%, 90%, 95%, 98% or 99%.
In another aspect, the application provides methods for generating a therapeutic composition. In one embodiment, the method may include the steps of providing a cell material comprising a cytotoxic cell, incubating the cell material with a first GNC protein to provide an activated cell composition, and formulating the activated cell composition to provide a therapeutic composition. The activated cell composition contains a first therapeutic cell. The first therapeutic cell comprises the first GNC protein bound to the cytotoxic cell through the binding interaction with the first cytotoxic cell receptor. The therapeutic composition is substantially free of exogenous viral and non-viral DNA or RNA.
In one embodiment, the cell material may include or be derived from PBMC.
The first GNC protein may include a first cytotoxic binding moiety and a first cancer targeting moiety. The first cytotoxic binding moiety has a specificity to a first cytotoxic cell receptor and is configured to activate the first cytotoxic cell through the binding with the first cytotoxic cell receptor. The first cancer targeting moiety has a specificity to a first cancer cell receptor.
In one embodiment, the method may repeat the incubating step by incubating a second GNC protein with the activated cell composition. The second GNC protein comprising a second cytotoxic binding moiety and a second cancer targeting moiety, the second cytotoxic binding moiety has a specificity to a second cytotoxic cell receptor, and the second cancer targeting moiety has a specificity to a second cancer cell receptor. The activated cell composition comprises a second therapeutic cell, and the second therapeutic cell comprises the second GNC protein bound to the cytotoxic cell or the first therapeutic cell through the binding interaction with the second cytotoxic cell receptor.
In one embodiment, the first and the second cancer-targeting moiety independently has a specificity for CD19, PDL1, or a combination thereof. In one embodiment, the first and the second cytotoxic binding moiety independently has a specificity for CD3, PDL1, 41BB, or a combination thereof.
The method may further include the repeated incubating steps by incubating additional GNC proteins with the activated composition. The additional GNC proteins may be a third GNC protein, a fourth GNC protein, etc. to provide addition therapeutic cells, each having the additional protein bound to the cytotoxic cell.
The first, second, and the additional GNC protein may be the same or may be different. The therapeutic cells may have one GNC protein, multiple same GNC proteins, or multiple different GNC proteins bound thereupon. In one embodiment, the therapeutic cell may have the first GNC protein bound thereupon. In one embodiment, the therapeutic cell may have both the first and the second GNC proteins bound thereupon. In one embodiment, the therapeutic cell may have the first, the second and the additional GNC proteins bound thereupon.
In one embodiment, the therapeutic cell comprises the cytotoxic cell having at least one bound GNC protein. In one embodiment, the therapeutic cell comprises the cytotoxic cell having at least 10, 20, 50, 100, 200, 300, 400 bound GNC proteins.
The therapeutic composition may include the first therapeutic cell, the first GNC protein, the cytotoxic cell, or a combination thereof. In one embodiment, the therapeutic composition may include the second therapeutic cell, the second GNC protein, comprises the first therapeutic cell, the first GNC protein, the cytotoxic cell, or a combination thereof. In one embodiment, the therapeutic composition may include additional GNC proteins and additional therapeutic cells.
In one embodiment, the incubating step may serve to expand the therapeutic cells. In one embodiment, expanding the therapeutic cell may include incubating the therapeutic cells with an additional amount of the GNC protein to provide an expanded cell population. In one embodiment, the expanded cell population comprises at least 102, at least 103, at least 104, at least 105, at least 106, at least 107, at least 108, at least 109, at least 1010 cells per ml. In one embodiment, the expanded cell population comprises the GNC bound cell, the GNC protein, the cytotoxic cell, or a combination thereof. In one embodiment, in order to deplete PD-1+ T cells, a GNC protein may be added to the expansion culture that redirects killing to PD-1+ T cells therefore resulting in reduction in PD-1+ exhausted T cells. In one embodiment, in order to preferentially support PD-1+ T cells, a GNC protein may be added to the expansion culture that relieves checkpoint signaling through PD-1 on T cells therefore resulting in functional improvement of PD-1+ T cells. In one embodiment, in order to isolate 4-1BB mediated co-stimulation through 3rd gen CAR-T, a GNC protein may be added to the expansion culture that redirects killing to 4-1BB+ T cells or resulting in therapeutic composition with controlling level of 4-1BB stimulation in the therapeutic cells, such as CAR-T cells.
In one embodiment, the cancer targeting moiety has the specificity against B cell, and the therapeutic composition is substantially free of B cell. Therefore, the methods disclosed herein couple the activation and purification functions for the therapeutic cells, which allows the methods to produce B cell free therapeutic composition without the need to introduce any foreign materials (such as beads) nor any foreign genetic materials (such as viral and non-viral DNA or RNA vectors).
In one embodiment, the ratio of the GNC protein and the cytotoxic cell is at least 30 to 1 when incubating the cell material with the GNC protein.
In one embodiment, the therapeutic composition may include at least 107 cells per ml.
In a further aspect, the application provides methods for using guidance and navigation control (GNC) proteins for cancer treatment. In one embodiment, the method of treating a subject having a cancer, comprises providing a cytotoxic cell, combining a GNC protein with the cytotoxic cell to provide a therapeutic cell, optionally expanding the therapeutic cell to provide an expanded cell population, and administering the therapeutic cell or the expanded cell population to the subject.
In one embodiment, the method include the step of providing a cell material comprising a cytotoxic cell, incubating the cell material with a first GNC protein to provide an activated cell composition, wherein the activated cell composition comprises a first therapeutic cell, formulating the activated cell composition to provide a therapeutic composition, wherein the therapeutic composition is substantially free exogenous of viral and non-viral DNA or RNA, and administering the therapeutic composition to the subject.
In one embodiment, the method may further include the steps of incubating a second GNC protein with the activated cell composition to provide the activated cell composition further comprising a second therapeutic cell. In one embodiment, the method may further include the step of incubating additional GNC proteins with the activated cell composition to provide the activated cell composition further comprising additional therapeutic cells.
In one embodiment, the method may further comprise isolating the cytotoxic cell from peripheral blood mononuclear cells (PBMC) before providing the cytotoxic cell. In one embodiment, the method may further comprise isolating the peripheral blood mononuclear cells (PBMC) from a blood. In one embodiment, the blood is from the subject. In one embodiment, the blood is not from the subject. In one embodiment, the cytotoxic cells may be from the patient that is under treatment or a different individual, such as a universal donor.
In one embodiment, the cytotoxic cell may be an autologous T cell, an alloreactive T cell, or a universal donor T cell. In one embodiment, when autologous donor T cells are used, in order to prevent infusion of contaminating cancer cells, a GNC protein may be added to the expansion culture that redirects killing to tumor antigens, example tumor antigen may include CD19 for B cell malignancies, Epcam for Breast carcinoma, MCP1 for melanoma.
In one embodiment, the method includes steps of providing a blood from the subject, isolating peripheral blood mononuclear cells (PBMC) from the blood, isolating a cytotoxic cell from the PBMC, combining a GNC protein with the cytotoxic cell to provide a therapeutic cell, optionally expanding the therapeutic cell to provide an expanded cell population, and administering the therapeutic cell or the expanded cell population to the subject.
In one embodiment, the method further comprises administering additional GNC protein to the subject after administering the therapeutic composition to the subject. In one embodiment, the cytotoxic cell may include CD3+ T cell, NK cell, or a combination thereof.
In one embodiment, the isolating of the cytotoxic cell comprises isolating at least one subpopulation of cytotoxic cells to provide the therapeutic T cells. In one embodiment, the subpopulation of cytotoxic cells comprises CD4+ cells, CD8+ cells, CD56+ cells, CD69+ cells, CD107a+ cells, CD45RA+ cells, CD45RO+ cells, CD2+ cells, CD178+ cells, Granzyme+ cells, or a combination thereof.
In one embodiment, the combining of a GNC protein with the cytotoxic cell comprises incubating the GNC protein with the cytotoxic cell for a period of time from about 2 hours to about 14 days, from about 1 day to about 7 days, from about 8 hours to about 24 hours, from about 4 days to about 7 days, or from about 10 days to about 14 days. In one embodiment, the incubating period may be more than 14 days. In one embodiment, the incubating period may be less than 2 hours.
In one embodiment, the ratio between the GNC protein and the cytotoxic cell is at least 600 to 1, 500 to 1, 400 to 1, 300 to 1, 200 to 1, 100 to 1, or 1 to 1. In one embodiment, the ratio between the GNC protein and the cytotoxic cell is from about 1 to 1, 10 to 1, 100 to 1, or to about 1000 to 1 ratio.
In one embodiment, the method may further comprise evaluating therapeutic efficacy after the administering step. In one embodiment, the evaluating therapeutic efficacy includes checking one or more biomarkers of the cancer, monitoring the life span of the therapeutic cells, or a combination thereof. In one embodiment, evaluating therapeutic efficacy comprises checking one or more biomarkers of the cancer, monitoring the life span of the therapeutic cells, or a combination thereof. In one embodiment, the biomarker comprises a tumor antigen, release of cytokines e.g., gamma interferon, IL-2, IL-8, and/or chemokines, and/or CD markers on the surface of various cell types e.g., CD69, PD-1, TIGIT, and/or mutated nucleic acid released into the bloodstream by tumors upon death, circulating tumor cells and their associated nucleic acid, or exosome associated nucleic acid, host inflammatory mediators, or tumor derived analytes, or a combination thereof. In one embodiment, the biomarker comprises a tumor antigen, tumor-associated apoptotic bodies, small molecule metabolites, release of cytokines, lymphocyte surface marker expression, phosphorylated/dephosphorylated signaling molecules, transcription factors, or a combination thereof.
The method disclosed herein is free of the step of transfecting the cytotoxic cell with a DNA vector or a viral vector. In one embodiment, the therapeutic cell or the expanded cell population is substantially free of a DNA vector or a viral vector.
The method may be used to treat a human subject suffering from cancer. In one embodiment, the cancer comprises cells expressing ROR1, CEA, HER2, EGFR, EGFRvIII, LMP1, LMP2A, Mesothelin, PSMA, EpCAM, glypican-3, gpA33, GD2, TROP2, BCMA, CD20, CD33, CD123, CD22, CD30, CD19, as yet to be identified tumor associated antigens, or a combination thereof. In one embodiment, the method may be used to treat mammals.
Varieties of cancer may be treated using the methods disclosed herein. Example cancers includes without limitation breast cancer, colorectal cancer, anal cancer, pancreatic cancer, gallbladder cancer, bile duct cancer, head and neck cancer, nasopharyngeal cancer, skin cancer, melanoma, ovarian cancer, prostate cancer, urethral cancer, lung cancer, non-small lung cell cancer, small cell lung cancer, brain tumor, glioma, neuroblastoma, esophageal cancer, gastric cancer, liver cancer, kidney cancer, bladder cancer, cervical cancer, endometrial cancer, thyroid cancer, eye cancer, sarcoma, bone cancer, leukemia, myeloma or lymphoma.
In one embodiment, the method may further include administering an effective amount of a therapeutic agent after the administering the therapeutic cell or the expanded cell population to the subject. In one embodiment, the therapeutic agent comprises a monoclonal antibody, a chemotherapy agent, an enzyme, a protein, a co-stimulator, or a combination thereof. In one embodiment, the co-stimulator is configured to increase the amount of cytotoxic T cells in the subject.
The application further provides a solution comprising an effective concentration of the GNC protein. In one embodiment, the solution is blood plasma in the subject under treatment. In one embodiment, the solution includes the GNC protein bound cells. In one embodiment, the solution includes a GNC cluster including a GNC protein, a T-cell bound to the T-cell binding moiety of the GNC protein, and a cancer cell is bound to the caner-targeting moiety of the GNC protein.
The objectives and advantages of the present application will become apparent from the following detailed description of preferred embodiments thereof in connection with the accompanying drawings.
The foregoing and other features of this disclosure will become more fully apparent from the following description and appended claims, taken in conjunction with the accompanying drawings.
Understanding that these drawings depict only several embodiments arranged in accordance with the disclosure and are, therefore, not to be considered limiting of its scope, the disclosure will be described with additional specificity and detail through use of the accompanying drawings, in which:
In the following detailed description, reference is made to the accompanying drawings, which form a part hereof. In the drawings, similar symbols typically identify similar components, unless context dictates otherwise. The illustrative embodiments described in the detailed description, drawings, and claims are not meant to be limiting. Other embodiments may be utilized, and other changes may be made, without departing from the spirit or scope of the subject matter presented herein. It will be readily understood that the aspects of the present disclosure, as generally described herein, and illustrated in the Figures, can be arranged, substituted, combined, separated, and designed in a wide variety of different configurations, all of which are explicitly contemplated herein.
In one embodiment, the guidance navigation control (GNC) proteins are characterized by their composition of multiple antigen-specific binding domains (AgBDs) and by their ability of directing T cells (or other effector cells) to cancer cells (or other target cells such as bystander suppressor cells) through the binding of multiple surface molecules on a T cell and a tumor cell. In one embodiment, GNC proteins are composed of Moiety 1 for binding at least one surface molecule on a T cell and Moiety 2 for binding at least one surface antigen on a cancer cell as shown in TABLE 1.
In a T cell therapy, the cytotoxic T cells are regulated by T cell receptor complex proteins, as well as co-stimulation signaling proteins via either agonist receptors or antagonist receptors on their surface. To regulate this signaling, as well as the interaction between a T cell and a cancer cell, multiple AgBDs may compose Moiety 1 and Moiety 2, respectively. Examples of molecules that can be targeted by agonistic or antagonistic binding domains in Moiety 1 and 2 are shown in TABLE 1. In one embodiment, the GNC proteins may have at least one linker to link Moiety 1 and Moiety 2. In one example GNC protein, any linker molecule can be used to link two or more AgBDs together either in vitro or in vivo by using complementary linkers of DNA/RNA or protein-protein interactions, including but not limited to, that of biotin-avidin, leucine-zipper, and any two-hybrid positive protein. In some embodiments, the linkers may be an antibody backbone structure or antibody fragments, so that GNC protein and GNC antibody may have the same meaning, e.g. the structure of the example tetra-specific GNC antibody in
GNC proteins or antibodies are capable of directing a T cell to a cancer cell, in vivo or ex vivo, through the binding function of multiple AgBDs (
The present application relates to methods of making GNC-activated therapeutic cell composition. Multiple AgBDs can be divided into Moiety 1 and Moiety 2 due to their interface with a T cell and a cancer cell, respectively (TABLE 1). A GNC protein with two AgBDs may simultaneously bind to a surface molecule, such as CD3 on a T cell, and a tumor antigen, such as ROR1 on a tumor cell, for re-directing the T cell to the tumor cell.
The addition of a third AgBD, for example, one that specifically binds to 41BB, may help enhance anti-CD3-induced T cell activation because 41BB is a co-stimulation factor and the binding stimulates its agonist activity to activated T cells. The addition of a fourth AgBD to a GNC protein, for example, one that specifically binds to PD-L1 on a tumor cell, may block the inhibitory pathway of PD-L1 on tumor cells or that is mediated through its binding to PD-1 on the T cells.
In some embodiments, with these basic principles, GNC proteins are constructed to acquire multiple AgBDs specifically for binding unequal numbers of T cell antagonists and agonists, not only to re-direct activated T cells to tumor cells but also to control their activity in vivo (TABLE 2). Therefore, in some embodiments, GNC proteins may be bi-specific, tri-specific, tetra-specific, penta-specific, hexa-specific, hepta-specific, or octa-specific proteins.
In one embodiment, the application relates to a GNC-T cell therapy where GNC proteins are used to expand the T cells ex vivo prior to adoptive transfer (
In one aspect, the application relates to the production of therapeutic GNC-T cells. In comparison with and to distinguish from the production of therapeutic CAR-T cells, their general processes are shown in
The CAR-T therapy cellular material must undergo the gene transfer that involves the preparation and transfection of CAR-T vector DNA, which results in genetically modifying the genome of the T cells. Furthermore, these genetically modified T cells may undergo another round of T cell expansion before being transferred back into the patient. The random integration of CAR-T vector DNA carries a risk of transformation of the T cells leading to primary leukemogenesis or introduction of the CAR-T vector to leukemia cells increasing the risk of relapse by mechanism of internal sequestration of the CAR target antigen (Zhang, Liu et al. 2017).
In contrast, GNC-T cell therapy has the advantages of not involving the transfection of any vector DNA, therefore there is no risk of genetic modification prior to adoptive transfer, which provides one of the significant advantages and technical improvements over the existing CAR-T therapy. Besides the advantage of GNC-T cell therapy being free of exogenous generic material contamination and cancer risk, the efficacy of GNC-T cell therapy may be improved when PBMC or different T cell subsets are being primed and activated ex vivo as shown in
In some embodiments, it may be beneficial to remove leukemia or other cancer cells from the cellular material prior to cell expansion (
In one embodiment, to enable the production of therapeutic T cells primed (or coated or bound) with GNC proteins, a tetra-specific antibody is produced and used as the GNC protein. In one embodiment, the tetra-specific antibody/GNC protein comprises 4 different binding domains linked by antibody fragments as its backbone. One binding domain is specific for CD3 on T cells, a second binding domain is specific for a tumor associated antigen, including but not limited to ROR1, CEA, HER2, EGFR, EGFRvIII, LMP1, LMP2A, Mesothelin, PSMA, EpCAM, glypican-3, gpA33, GD2, TROP2, BCMA, CD19, CD20, CD33, CD123, CD22, CD30, and a third and fourth binding domains are specific for two distinct immune checkpoint modulators such as PD-L1, PD-L2, PD-1, OX40, 4-1BB, GITR, TIGIT, TIM-3, LAG-3, CTLA4, CD40L, VISTA, ICOS, BTLA, Light, etc.
Without being bound by theory, the advantages of GNC protein-mediated GNC-T cell therapy over conventional CAR-T therapies include, but are not limited to, first, that inclusion of an IgG Fc domain may confer the characteristic of a longer half-life in serum compared to a bi-specific BiTe molecule; second, that inclusion of two binding domains specific for immune checkpoint modulators may inhibit the suppressive pathways and engage the co-stimulatory pathways at the same time; third, that cross-linking CD3 on T cells with tumor associated antigens re-directs and guides T cells to kill the tumor cells without the need of removing T cells from the patient and genetically modifying them to be specific for the tumor cells before re-introducing them back into the patient, also known as chimeric antigen receptor T cells (CAR-T) therapy; and fourth, that GNC protein-mediated antibody therapy or T cell therapy does not involve genetic modification of T cells, the latter of which may carry the risk of transforming modified T cells to clonal expansion, i.e. T cell leukemia.
The present disclosure may be understood more readily by reference to the following detailed description of specific embodiments and examples included herein. Although the present disclosure has been described with reference to specific details of certain embodiments thereof, it is not intended that such details should be regarded as limitations upon the scope of the disclosure.
While the following examples are provided by way of illustration only and not by way of limitation. Those of skill in the art will readily recognize a variety of non-critical parameters that could be changed or modified to yield essentially the same or similar results.
In the present application, the examples of GNC proteins are classes of tetra-specific GNC antibodies, of which 4 AgBDs are covalently linked using an IgG antibody as its backbone (
Of tetra-specific GNC antibodies, the SI-35E class comprises targets an anti-human CD3 binding domain (SEQ IDs 1-4), an anti-human PD-L1 (SEQ IDs 5-12), an anti-human 4-1BB (SEQ IDs 13-24), and targets a human ROR1 (SEQ IDs 25-32), i.e. a TAA. In this context, the classes of SI-38E and SI-39E target CD19 (SEQ IDs 47-50) and EGFR (SEQ ID 51-54), respectively.
To construct tetra-specific GNC antibodies, AgBDs were converted to scFv and VLVH for placement at the N-terminal Domain 1 (D1) or scFv and VHVL for placement at the C-terminal Domains 3 (D3) and 4 (D4) of the GNC protein. All scFv molecules described herein contain a 20 amino acid flexible gly-gly-gly-gly-ser (G4S) X4 linker that operably links the VH and VL, regardless of the V-region orientation (LH or HL). The remaining position in the tetra-specific GNC antibody, Domain 2 (D2), consists of an IgG1 heavy chain, VH-CH1-Hinge-CH2-CH3, and its corresponding light chain, VL-CL, which can be either a kappa or lambda chain. D1 and D2 are genetically linked through a 10 amino acid (G4S)×2 linkers, as are D2, D3 and D4 resulting in a contiguous ˜150 kDa heavy chain monomer peptide. When co-transfected with the appropriate light chain, the final symmetric tetra-specific GNC peptide can be purified through the IgG1 Fc (Protein A/Protein G) and assayed to assess functional activity. Heavy and light chain gene “cassettes” were previously constructed such that V-regions could be easily cloned using either restriction enzyme sites (HindIII/NhelI for the heavy chain and HindIII/BsiWI for the light chain) or “restriction-free cloning” such as Gibson Assembly (SGI-DNA, La Jolla, Calif.), Infusion (Takara Bio USA) or NEBuilder (NEB, Ipswich, Mass.), the latter of which was used here.
The tetra-specific GNC antibodies can be produced through a process that involves design of the intact molecule, synthesis and cloning of the nucleotide sequences for each domain, expression in mammalian cells and purification of the final product. Herein, nucleotide sequences were assembled using the Geneious 10.2.3 software package (Biomatters, Auckland, NZ) and broken up into their component domains for gene synthesis (Genewiz, South Plainsfield, N.J.). In this example, SI-35E18 (SEQ ID 65 and 67) was split into its component domains where the anti-41BB scFv, VL-VH, occupies D1, anti-human PD-L1 clone PL230C6 occupies D2 (Fab position), anti-human ROR1 Ig domain-specific clone 323H7 VHVL scFv occupies D3, and anti-human CD3 scFv, VHVL, occupies the C-terminal D4. Using NEBuilder web-based tools, 5′ and 3′ nucleotides were appended to each of the domains depending on their position in the larger protein so that each domain overlaps its flanking domains by 20-30 nucleotides which direct site-specific recombination, thus genetically fusing each domain in a single gene assembly step. Due to the high number of homologous regions in the tetra-specific nucleotide sequence, the N-terminal domains 1 and 2 are assembled separately from the C-terminal D3 and D4. The N- and C-terminal fragments were then assembled together in a second NEBuilder reaction. A small aliquot was transformed into E. coli DH10b (Invitrogen, Carlsbad, Calif.) and plated on TB+carbenicillin 100 ug/ml plates (Teknova, Hollister, Calif.) and incubated at 37° C. overnight. Resultant colonies were selected and 2 mL overnight cultures inoculated in TB+carbenicillin. DNA was prepared (Thermo-Fisher, Carlsbad, Calif.) from overnight cultures and subsequently sequenced (Genewiz, South Plainsfield, N.J.) using sequencing primers (Sigma, St. Louis, Mo.) flanking each domain. All DNA sequences were assembled and analyzed in Geneious.
In another tetra-specific GNC protein, SI-38E17 targeting human CD19 (SEQ IDs 47-50), multiple AgBDs carry an anti-human 4-1BB (scFv 466F6, SEQ IDs 17-20) as well as an anti-human PD-L1 (scFv PL221G5 SEQ IDs 9-13), and an anti-human CD3 binding domain (SEQ IDs 1-4). The methods and procedures for producing this tetra-specific antibody were the same.
GNC proteins are composed of Moiety 1 for binding at least one surface molecule on a T cell and Moiety 2 for binding at least one surface antigen on a cancer cell (TABLE 1A). The tetra-specific GNC antibodies can be used to directly engage the body's endogenous T cells to kill tumor cells independent of tumor antigen presentation by MHC to the antigen specific T cell receptors. This is in contrast to therapies based solely on immune checkpoint blockade, which have been limited by antigen recognition. In context, the immune checkpoint modulating component may be constructed as a part of tetra-specific GNC antibodies, which may provide benefits similar to that in a standard checkpoint blockade therapy.
In addition to T cells, other cytotoxic cells may also be targeted by GNC proteins for cancer killing or preventing purposes. TABLE 1B shows the example compositions of functional moieties (Moiety 1 and Moiety 2) and antigen binding domain in GNC proteins with NK cell binding domains. TABLE 1C shows the example compositions of functional moieties (Moiety 1 and Moiety 2) and antigen binding domain in GNC proteins with macrophage binding domains. TABLE 1D shows the example compositions of functional moieties (Moiety 1 and Moiety 2) and antigen binding domain in GNC proteins with dendritic cell binding domains.
GNC proteins are constructed to acquire multiple AgBDs specifically for binding unequal numbers of T cell antagonists and agonists. In this way, GNC proteins may re-direct activated T cells to tumor cells with certain levels of control of their activity in vivo (TABLE 2). Therefore, GNC proteins may be bi-specific, tri-specific, tetra-specific, penta-specific, hexa-specific, hepta-specific, or even octa-specific proteins. In the present invention, three classes of tetra-specific GNC antibodies, i.e. SI-39E, SI-35E, and SI-38E, were created to enable GNC-T cell therapy, of which antibody domains and its specificity is listed in TABLE 3. The structures of tetra-specific GNC antibodies targeting EGFRvIII (SI-39E), ROR1 (SI-35E), and CD19 (SI-38E) are listed in TABLE 4.
The SI-35 class listed in Table 4 were tested for their ability to activate and induce proliferation of different cell types, such as CD4+ and/or CD8+ T cells and/or CD56+ natural killer cells (NK) within PBMC. The tetra-specific GNC antibodies were prepared at 2× final concentration and titrated in 1:10 serial dilutions across 6 wells of a 96 well plate in 200 ul of RPMI+10% FBS. Human PBMC were purified by standard Ficoll density gradient from a “leukopak” which is an enriched leukapheresis product collected from normal human peripheral blood. In the final destination 96 well plate, the PBMC and serially titrated GNC proteins were combined by adding 100 μL of PBMC (100,000), and 100 μL of each antibody dilution to each well of the assay. The assay plate was incubated at 37° C. for approximately 72 hours and then the contents of each assay well were harvested and analyzed by FACS for the number of CD4+ T cells, CD8+ T cells, and CD56+NK cells. Cells were harvested from each well and transferred to a new 96 well V-bottom plate then centrifuged at 400×g for 3 minutes. Supernatant was transferred to a 96 well plate for analysis of IL-2 and Granzyme B. Cells were re-suspended in 200 μL of 2% FBS/PBS of FACS antibodies and incubated on ice for 30 minutes. The plate was centrifuged at 400×g for 3 minutes and the supernatant was aspirated. This wash step was repeated once more and then the cells were re-suspended in 100 μL 2% FBS/PBS and analyzed on a BD LSR FORTESSA.
As shown in
Proliferation and production of gamma interferon was measured from cultures of CD3+ or naïve CD8+ T cells (70,000 cells/well) stimulated for 5 days with a panel of SI-35 class antibodies. Human CD3+ or CD8+CD45RA+naïve T cells were enriched from peripheral blood mononuclear cells from a normal donor using the EasySep™ Human CD3+ or Naïve CD8+ T Cell Isolation Kits (StemCell Technologies) as per the manufacturer protocols. The final cell populations were determined to be >98% CD3+ or CD8+CD45RA+ T cells by flow cytometry. Proliferation in the culture was measured after stain with Alamar blue (ThermoFisher Cat. No. DAL1100) for 1 hour at 37° C., and then read on a Spectramax plus 384 well reader (Molecular Devices). Proliferation of GNC-expanded CD3+ T cells was expressed as a fold increase in cell number over background of CD3+ T cells in cell culture without GNC (
The manufacture of GNC-activated and -coated T cells at clinically significant dosage of 10E9 was achieved after 7 days culture. Human PBMC were isolated from LRS cone leukocytes by standard Ficoll density gradient from leukopaks which are enriched leukapheresis product collected from normal human peripheral blood. After collection the cells were frozen at −80° C. and then later thawed before putting in culture. Using the G-Rex plate and bioreactor culture systems, the growth of SI-38E17 GNC-stimulated PBMC cultures was monitored for up to 14 days. The culture medium consisted of RPMI 1640, 10% fetal calf serum, 1% non-essential amino acids, 1% GlutaMax, 0.6% glutamine-alanine supplement, 15 ng/mL human IL-2, and 1 nM GNC protein. The 6-well G-Rex cultures tolerated seeding densities of 25-100 million PBMC/well for six days, which greatly exceeded recommended amounts, but was tolerated by the cells in the system with a single 50% medium change on day 7. Clustering of cells was indicative of their activation in the culture (
The cells from the bioreactor were harvested as the first GNC-activated therapeutic cell composition, which were optionally concentrated using LOVO Automated Cell Processing System (Fresenius Kabi). One sample (Product B) was exposed to 1 nM SI-38E17, which is identical to the first GNC in this case for preparing a second GNC-activated therapeutic cell composition, potential for being used to target treat patients harboring CD19 positive malignancies (
After the second concentration step (100 mL volume) during the processing in the LOVO system, the second GNC-activated therapeutic cells were washed twice before eluting to a final volume of 54 mL in a sterile processing bag. The other sample (Product A) was only exposed to the first GNC protein during the culture phase and not re-exposed during processing in the LOVO system (
Cell viability from the GNC-expanded T cell (GET) culture was >75% and was not affected by exposure to additional GNC reagent (GNC-T, Product B) during processing (
This example illustrates a number of advantages of GNC-T cells in comparison to CAR-T cell preparations. First, the cell composition of the starting material was fresh PBMC from the donor and did not need to be pre-selected for particular subsets of cells or require addition of feeder cells or synthetic beads. The GNC protein was 100% non-nucleotide biological material, and did not require the transfer of RNA or DNA into the cells, or transfection with a viral vector. The GNC-induced expansion yielded a therapeutic dose in 9 days, compared to the average of 40 days for CAR-T cell expansion. The resulting cells were devoid of B cells and highly enriched for activated CD4+ and CD8+ T cells that had potent killing potential against their specific targets. The GNC therapeutic composition was viable and bioactive upon thaw from −80° C. Together these advantages are expected to significantly lower waiting times, costs and issues related to infrastructure and training related to CAR-T cell therapy. Improvements in the purity, safety and quantity of the end product will be of significant benefit to the patient.
Six of the GNC SI-35 class proteins listed in Table 4 were tested for the ability to activate PBMC for redirected T cell cytotoxicity (RTCC) activity against a human ROR1-transduced CHO cell line (
To further demonstrate the killing effects of GNC-labeled PBMC against human tumor cells, a GNC-dose and effector:target ratio escalation experiment was performed using an IncuCyte S3 Live Cell Analysis System (Sartorius/Essen Biosciences) to monitor the cells over time. PBMC from a healthy donor were labeled with GNC protein SI-38E17 at 10-fold serial doses ranging from 0.01 to 100 nM for 30 minutes at 37° C. and then washed prior to culture. The GNC SI-38E17 targets the CD19 antigen expressed on B cell surfaces, and therefore, the Kasumi-2 precursor B cell leukemia line was chosen as a target cell. The Kasumi-2 cell used was transduced to express green fluorescence protein (GFP) and therefore the presence of tumor cells was tracked by measuring the average green fluorescence in 4 images/well collected 9 times over a six-day period. The effector:target (E:T) ratios were escalated by adding GNC-labeled PBMC in a serial 2-fold dilution of 5,000 (1:1) to 160,000 (32:1) cells to duplicate wells. As shown in
As a follow up experiment, three other transformed B cell lines: NALM-6, MEC-1, and Daudi and the acute T cell leukemia line, Jurkat, were used as target cells. These target cells were previously transduced by lentivirus to constitutively express the NucRed 647 molecule. In this assay, PBMC were exposed to 10-fold doses of GNC protein SI-38E17 for 30 minutes at 37° C. and then washed as before. PBMC were plated at 1.2×106 cells/well and 50,000 target tumor cells were added. Cells were placed in IncuCyte S3 set to collect red fluorescence images (4 images/well) collected at 10 time points over a 5.5-day period (
As a different method of quantifying the outcome of cultures of GNC-T cells with tumor cells, we established a limit of quantification (LOQ) curve for detection by flow cytometry. Daudi-Red cells were serially diluted 10-fold in a range from 200,000 to 20 cells and then mixed 1:1 with 1 million PBMC to create samples of 10%, 1.0%, 0.1%, 0.01% and 0.001% tumor cells, which were then analyzed by flow cytometry (
While the present disclosure has been described with reference to particular embodiments or examples, it may be understood that the embodiments are illustrative and that the disclosure scope is not so limited. Alternative embodiments of the present disclosure may become apparent to those having ordinary skill in the art to which the present disclosure pertains. Such alternate embodiments are considered to be encompassed within the scope of the present disclosure. Accordingly, the scope of the present disclosure is defined by the appended claims and is supported by the foregoing description. All references cited or referred to in this disclosure are hereby incorporated by reference in their entireties.
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SGSRYYANWAKGRFTISKDNTKNTVYLQMNSLRAEDTAVYYCARDYMSGSHLWGQGTLVTVSSASTKGPSVFPLAPS
ASSAKGRFTISRDNSKNTLYLQMNSLRAEDTATYFCARLDVGGGGAYIGDIWGQGTLVTVSSGGGGSGGGGSGGGGS
ITSNNIWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDVVMTQSPSTLSASVGDRVTINCQASESISSWLAWYQQKP
GSSAITSNNIWGQGTLVTVSSGGGGSGGGGSDVQLQESGPSLVKPSQSLSLTCTVTGYSITSDFAWNWIRQFPGNKL
AGSAGITYDANWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSAFSFDYAMDLWGQGTLVTVSSGGGGSGGG
DSSSYYMFNLWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSALVMTQSPSTLSASVGDRVTINCQASEDIDTYLAWY
SRISITRDTSKNQFFLQLNSVTIEDTATYYCVTAGRGFPYWGQGTLVTVSAGGGGSGGGGSEVQ
AGITYDANWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSAFSFDYAMDLWGQGTLVTV
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ARGRFTISRPSSKNTVDLQMNSLRAEDTAVYYCARDSGYSDPMWGQGTLVTVSSGGGGSGGGGS
DYASSAKGRFTISRDNSKNTLYLQMNSLRAEDTATYFCARLDVGGGGAYIGDIWGQGTLVTVSS
VITGRDITYYASWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDGGSSAITSNNIWGQG
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SWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDGGSSAITSNNIWGQGTLVTVSTGGGG
ANWAKGRFTISKDNTKNTVYLQMNSLRAEDTAVYYCARDYMSGSHLWGQGTLVTVSSASTKGPS
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FAFGGGTKVEIKGGGGSGGGGSGRSLVESGGGLVQPGGSLRLSCTASGFTISSYHMQWVRQAPG
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SWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDGGSSAITSNNIWGQGTLVTVSTGGGG
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SWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDGGSSAITSNNIWGQGTLVTVSTGGGG
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HLNWYQQKPGKAPKLLIYKASTLASGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQGYSWG
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SWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDGGSSAITSNNIWGQGTLVTVSSGGGG
RYSPSFQGQVTISADKSIRTAYLQWSSLKASDTAMYYCARHVTMIWGVIIDFWGQGTLVTVSSA
LWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSTLSASVGDRVTITCQASQSISSH
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SWYQQKPGKAPKLLIYAAANLASGVPSRFSGSGSGTDFTLTISDLEPGDAATYYCQSTYLGTDY
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AKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDGGSSAITSNNIWGQGTLVTVSSASTKGP
IAAGSAGITYDANWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSAFSFDYAMDLWGQG
This application claims the benefit of filing date of U.S. Provisional Patent Application No. 62/648,888 filed Mar. 27, 2018, and U.S. Provisional Patent Application No. 62/648,880 filed Mar. 27, 2018, the entire disclosures of which are expressly incorporated by reference herein.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2019/024111 | 3/26/2019 | WO | 00 |
