Combination cancer treatment with staggered dosing

Abstract

Methods of enhancing an anticancer therapy in a human cancer patient comprising administering a dose of the anticancer therapy at least 4 days before decreasing soluble CD28 levels in the subject or administering at least 2 doses of the anticancer therapy as a monotherapy before decreasing soluble CD28 levels in the subject are provided. Methods of treating cancer by enhancing an anticancer therapy are also provided.

Description

COMBINATION CANCER TREATMENT WITH STAGGERED DOSINGCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority of U.S. Provisional Patent Application No. 63 / 632,581, filed April 11, 2024, the contents of which are all incorporated herein by reference in their entirety.REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0002] The contents of the electronic sequence listing (BDB-P-022-PCT.xml; Size: 103,673 bytes; and Date of Creation: April 1, 2025) is herein incorporated by reference in its entirety.FIELD OF INVENTION

[0003] The present invention is in the field of combinatorial cancer treatment.BACKGROUND OF THE INVENTION

[0004] The adaptive immune system plays a critical role in the regulation and protection against pathogens and cancer cells, mainly by orchestrating the stimulation of antigen specific helper CD4+ and cytotoxic CD8+ T cells. Durable and persistent activation of T cells by antigen presenting cells (APC), involves i) engagement of the T cell receptor (TCR) with peptides presented by major histocompatibility complexes (MHCs) on APC; and ii) costimulatory CD28 receptors on T cells binding B7-1 (CD80) and B7-2 (CD86) ligands expressed also by the APC. The biological consequences of CD28 co -stimulation are numerous and include control of the T cell cycle, expansion, differentiation, as well as amplification of TCR stimulation by lowering the threshold needed for achieving immune effector function.

[0005] International Patent Applications WO2019175885, WO2020183473,WO2021111441, WO2021181396 and WO2023031943 all describe agents that block proteolytic shedding of membranal CD28 and thus reduce levels of soluble CD28 (sCD28). These agents increase the immune response against cancer and enhance the effectiveness ofPD-1 and PD-L1 based anticancer immunotherapies. Optimized dosing regimens for combined anti-CD28 shedding antibodies and anti-cancer therapies are greatly needed.SUMMARY OF THE INVENTION

[0006] The present invention provides methods of enhancing anticancer therapy in a human cancer patient comprising administering a dose of the anticancer therapy at least 4 days before decreasing soluble CD28 levels in the subject or administering at least 2 doses of the anticancer therapy as a monotherapy before decreasing soluble CD28 levels in the subject. Methods of treating cancer by enhancing an anticancer therapy are also provided.

[0007] According to another aspect, there is provided a method of enhancing an anticancer therapy in a human cancer subject, comprising administering to the subject a dose of the anticancer therapy and at least 4 days after the administering decreasing soluble CD28 (sCD28) levels in the subject, wherein the anticancer therapy is selected from an immune checkpoint inhibitor (ICI) immunotherapy and a chemotherapy, thereby enhancing an anticancer therapy in a human cancer patient.

[0008] According to another aspect, there is provided a method of enhancing an anticancer therapy in a human cancer subject, comprising administering to the subject at least two doses of the anticancer therapy as a monotherapy and after the administering decreasing soluble CD28 (sCD28) levels in the subject, wherein the anticancer therapy is selected from an immune checkpoint inhibitor (ICI) immunotherapy and a chemotherapy, thereby enhancing an anticancer therapy in a human cancer patient.

[0009] According to some embodiments, the anticancer therapy is an anti-programmed death 1 (PD-1) or anti-programmed death ligand 1 (PD-L1) based immunotherapy.

[0010] According to some embodiments, the anti-PD-1 or anti-PD-Ll immunotherapy is a PD-1 blocking antibody or a PD-L1 blocking antibody.[Oi l] According to some embodiments, the PD-1 blocking antibody or a PD-L1 blocking antibody is selected from pembrolizumab, nivolumab, pidilizumab, cemiplimab, atezolizumab, retifanlimab, toripalimab, dostarlimab, avelumab and durvalumab.

[0012] According to some embodiments, the anticancer therapy is anti-PD-1 immunotherapy .

[0013] According to some embodiments, the method comprises continuing to administer the anticancer therapy concomitantly with the decreasing.

[0014] According to some embodiments, at least 4 days is at least 6 days.

[0015] According to some embodiments, at least 4 days is at least 8 days.

[0016] According to some embodiments, the administering a dose of the anticancer therapy is administering at least 2 doses before the decreasing.

[0017] According to some embodiments, at least 2 doses is at least 3 doses.

[0018] According to some embodiments, the at least 4 days is at least 4 days from a first dose of the at least 2 doses.

[0019] According to some embodiments, the decreasing is at least 3 days after a second dose of the at least 2 doses.

[0020] According to some embodiments, the at least 2 doses is at least 3 doses before the decreasing and wherein the decreasing is at least 3 days after a third dose of the at least 3 doses.

[0021] According to some embodiments, the decreasing sCD28 levels in the subject comprises administering an agent that binds to membranal CD28 (mCD28) on the surface of a cell and inhibits proteolytic cleavage of the mCD28.

[0022] According to some embodiments, the agent binds to a stalk region of mCD28 and wherein the stalk region comprises the amino acid sequence GKHLCPSPLFPGPSKP (SEQ ID NO: 5) or KGKHLCPSPLFPGPS (SEQ ID NO: 6) or consists of the amino acid sequence HVKGKHLCPSPLFPGPSKP (SEQ ID NO: 7).

[0023] According to some embodiments, the agent comprises a single domain antibody (sdAb) specific to mCD28.

[0024] According to some embodiments, the sdAb comprises three CDRs wherein CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 8 (IKTMA), CDR2 comprises the amino acid sequence as set forth in SEQ ID NO: 9 (AIASDNRKYYADSVKG), CDR3 comprises the amino acid sequence as set forth in SEQ ID NO: 10 (DVTKEDYWY).

[0025] According to some embodiments, the sdAb is a camelid antibody.

[0026] According to some embodiments, the sdAb comprises the sequence EVQLVESGGGLVQPGGSLRLSCAASGSIASIKTMAWYRQAPGKGRELVTAIASDNRKYYADSVKGRFTISRDNSKTTVYLQMNSLRAEDTAVYYCVVDVTKEDYWYWGQGTLVTVSS (SEQ ID NO: 1).

[0027] According to some embodiments, the agent is a dimeric agent comprising at least two mCD28 binding sdAbs, wherein a first sdAb is linked to a second sdAb by a linker.

[0028] According to some embodiments, the dimeric agent comprises a first polypeptide comprising the first sdAb and a second polypeptide comprising the second sdAb and wherein the linker links the first polypeptide and the second polypeptide.

[0029] According to some embodiments, the first polypeptide comprises a first IgG heavy chain constant region, the second polypeptide comprises a second IgG heavy chain constant region and the linker comprises the first and the second IgG heavy chain constant regions.

[0030] According to some embodiments, the first sdAb is separated from the first IgG heavy chain constant region by an amino acid linker and the second sdAb is separated from the second IgG heavy chain constant region by an amino acid linker.

[0031] According to some embodiments, the amino acid linker comprises a sequence selected from (GGGGS)n, (GS)n, (GGS)n, (GSGGS)n, (EGGGS)n, (EGGS)n and a combination thereof, wherein n is an integer selected from 1, 2, 3, 4, 5, 6, 7, and 8.

[0032] According to some embodiments, the first and the second IgG heavy chain constant region comprise a human IgG4 heavy chain constant region comprising ESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQF NWYVDGVEVHNAKTKPREEQFNSTYRVVSVETVEHQDWENGKEYKCKVSNKGE PSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNG QPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKS LSLSLGK (SEQ ID NO: 11) or a sequence with at least 95% identity thereto.

[0033] According to some embodiments, the first and the second IgG heavy chain constant region compriseESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQF NWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGL PSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNG QPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKS LSLSLGK (SEQ ID NO: 12) or a sequence with at least 95% identity thereto.

[0034] According to some embodiments, the dimeric agent comprises a dimer of the amino acid sequenceEVQLVESGGGLVQPGGSLRLSCAASGSIASIKTMAWYRQAPGKGRELVTAIASDN RKYYADSVKGRFTISRDNSKTTVYLQMNSLRAEDTAVYYCVVDVTKEDYWYWG QGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSESKYGPPCPPCPAPEFEGGPS VFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREE QFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVY TLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF LYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 13).

[0035] According to some embodiments, the method comprises administering a dose of the anticancer therapy concomitantly with each dose of the agent.

[0036] According to some embodiments, the method comprises upon initiating administering the agent administering a combination of the agent and the anticancer therapy every 3 or 4 days.

[0037] According to some embodiments, the cancer is a PD-L1 positive cancer.

[0038] According to some embodiments, the cancer is selected from a breast cancer and a colorectal cancer.

[0039] According to another aspect, there is provided a method of treating cancer in a subject in need thereof, the method comprising administering to the subject a dose of an anticancer therapy and at least 4 days after the administering, administering a dose of an anti-mCD28 cleavage blocking dimeric agent, wherein the anticancer therapy is selected from an immune checkpoint inhibitor (ICI) immunotherapy and a chemotherapy, thereby treating cancer in a subject in need thereof.

[0040] According to another aspect, there is provided a method of treating cancer in a subject in need thereof, the method comprising administering to the subject at least two doses of an anticancer therapy as a monotherapy and after the administering, administering a dose of an anti-mCD28 cleavage blocking dimeric agent, wherein the anticancer therapy is selected from an immune checkpoint inhibitor (ICI) immunotherapy and a chemotherapy, thereby treating cancer in a subject in need thereof.

[0041] According to some embodiments, the anticancer therapy is an anti -programmed death 1 (PD-1) or anti-programmed death ligand 1 (PD-L1) based immunotherapy.

[0042] Further embodiments and the full scope of applicability of the present invention will become apparent from the detailed description given hereinafter. However, it should be understood that the detailed description and specific examples, while indicating preferredembodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figures 1A-1F : (1A) Cartoon of the setup of the initial dosing experiment. Anti-PD- 1 was administered every 4 days starting on day 11 after tumor cell inoculation. VHH18- mlgGl administration began after two doses of anti-PD-1 alone on day 19 and was continued every 4 days in combination with the anti-PD-1. (1B-1C) Line graphs of tumor volume in individual mice after administration of (IB) anti-PD-1 with an isotype control (IC) for VHH18-mIgGlor (IC) administration of anti-PD-1 and VHH18-mIgGl. VHH18- mlgGland the IC dosing was initiated 8 days after the first anti-PD-1 dose. (ID) Bar graph of the percent of mice that developed anti-PD-1 resistant tumors. (IE) Line graph of tumor growth in individual untreated control mice. (IF) Line graph of serum sCD28 levels in mice over the course of the experiment.

[0044] Figures 2A-2F: (2A) A line graph of average MC-38 tumor volume in mice administered anti-PD-1 every 4 days starting on day 13 after tumor cell inoculation and VHH18-mIgGl every 4 days starting on day 25 (3T), anti-PD-1 on day 13 after tumor cell inoculation and VHH18-mIgGl every 4 days starting on day 21 (2T), anti-PD-1 with an isotype control (IC) for VHH18-mIgGl or isotype controls for both drugs. (2B) Dot plot of final tumor volume on day 26 of all individual mice. (2C-2F) Dot plots of immune cell populations in lymph nodes of mice treated with isotype control (IC), anti-PD-1 alone (aPDl) or staggered combination anti-PD-1 and anti-CD28 (aPDl + VHH18-mIgGl) on (2C, 2E and 2F) day 26 and (2D) day 33. The following populations are shown: (2C) CD4 cells as a percent of all T cells, CD8 cells as a percent of all T cells, the ratio of CD8 / CD4 T cells, Ki67+ cells as a percent of all CD4 T cells and Ki67+ cells as a percent of all CD8 T cells; (2D) CD4 cells as a percent of all T cells, CD8 cells as a percent of all T cells, and the ratio of CD8 / CD4 T cells; (2E) CD4 effector memory (EM) cells as a percent of all CD4 T cells and CD8 EM cells as a percent of all CD8 T cells; (2F) CD25+ cells as a percent of all CD4 T cells, CD39+ cells as a percent of all CD4 T cells, CD69+ cells as a percent of all CD8 T cells, CD39+ cells as a percent of all CD8 T cells and CD107a+ cells as a percent of all CD8 T cells.

[0045] Figures 3A-3B: (3A) A line graph of average E0771 tumor volume in mice administered anti-PD-1 every 4 days starting on day 11 after tumor cell inoculation and VHH18-mIgGl every 4 days starting on day 23, anti-PD-1 with an isotype control (IC) for VHH18-mIgGl or isotype controls for both drugs. (3B) Dot plot of final tumor volume on day 33 of all individual mice.

[0046] Figures 4A-4H: (4A) A line graph of average E0771 tumor volume in mice administered anti-PD-1 every 4 days starting on day 7 after tumor cell inoculation and VHH18-mIgGl every 4 days starting on day 19 (3T), anti-PD-1 on day 7 and day 11 after tumor cell inoculation and VHH18-mIgGl in combination with VHH-mlgGl every 4 days starting on day 19 (2T), anti-PD-1 with an isotype control (IC) for VHH18-mIgGl or isotype controls for both drugs. (4B) The line graph of 4A is repeated but without the inferior 2T dosing regimen. (4C) Dot plot of final tumor volume on day 31 of individual mice receiving anti-PD-1 and IC, anti-PD-1 and VHH18-mIgGl with the 3T dosing regimen or only isotype controls. (4D-4H) Dot plots of immune cell populations in lymph nodes of mice treated with isotype control (IC), anti-PD-1 alone (aPDl) or staggered combination anti-PD-1 and anti- CD28 (aPDl + VHH18-mIgGl) on (4D, 4F and 4G) day 31 and (4E, 4F and 4H) day 38. The following populations are shown: (4D) CD4 cells as a percent of all T cells, CD8 cells as a percent of all T cells, the ratio of CD8 / CD4 T cells, Ki67+ cells as a percent of all CD4 T cells and Ki67+ cells as a percent of all CD8 T cells; (4E) CD4 cells as a percent of all T cells, CD8 cells as a percent of all T cells, the ratio of CD8 / CD4 T cells, Ki67+ cells as a percent of all CD4 T cells and Ki67+ cells as a percent of all CD8 T; (4F) CD4 effector memory (EM) cells as a percent of all CD4 T cells, CD4 central memory (CM) cells as a percent of all CD4 cells, CD8 EM cells as a percent of all CD8 T cells and CD8 CM cells as a percent of all CD8 T cells; (4G) CD25+ cells as a percent of all CD4 T cells, CD69+ cells as a percent of all CD4 T cells, CD69+ and Ki67+ cells as a percent of all CD4 T cells, CD69+ cells as a percent of all CD 8 T cells, CD69+ and Ki67+ cells as a percentage of all CD8 T cells and CD107a+ cells as a percent of all CD8 T cells; (4H) CD25+ cells as a percent of all CD4 T cells, CD69+ cells as a percent of all CD4 T cells, CD69+ and Ki67+ cells as a percent of all CD4 T cells, CD69+ cells as a percent of all CD8 T cells, CD69+ and Ki67+ cells as a percentage of all CD8 T cells and CD107a+ cells as a percent of all CD8 T cells .DETAILED DESCRIPTION OF THE INVENTION

[0047] The present invention, in some embodiments, provides methods of enhancing an anticancer therapy. The present invention, further concern methods of enhancing immunotherapy. The present invention further concerns a method of treating cancer by enhancing immunotherapy and methods of treating cancer by enhancing the anticancer therapy.

[0048] By a first aspect, there is provided a method of enhancing an anticancer therapy in a human subject, the method comprising administering to the subject a dose of the anticancer therapy before decreasing soluble CD28 (sCD28) levels in the subject, thereby enhancing the anticancer therapy in the subject.

[0049] By another aspect, there is provided a method of enhancing immunotherapy in a human subject, the method comprising administering to the subject a dose of the immunotherapy before decreasing soluble CD28 (sCD28) levels in the subject, thereby enhancing the immunotherapy in the subject.

[0050] By another aspect, there is provided a method of treating cancer in a subject, the method comprising administering to the subject a dose of an anticancer therapy before reducing sCD28 levels in the subject, thereby treating cancer.

[0051] By another aspect, there is provided a method of treating cancer in a subject, the method comprising administering to the subject a dose of an immunotherapy before reducing sCD28 levels in the subject, thereby treating cancer.

[0052] It will be understood that administering a therapy before reducing sCD28 levels is the same as reducing sCD28 levels after or following administering the therapy. This phrasing may be used interchangeably and so it will be understood that all instances of administering being before reducing sCD28 levels can also be understood as reducing sCD28 levels subsequent to the administering a therapy.

[0053] In some embodiments, the subject is a mammal. In some embodiments, the subject is a human. In some embodiments, the subject is in need of a method of the invention. In some embodiments, the subject is in need of therapy. In some embodiments, therapy is immunotherapy. In some embodiments, the subject suffers from cancer.

[0054] In some embodiments, the cancer can be treated by the anticancer therapy. In some embodiments, the cancer can be treated by immunotherapy. In some embodiments, the cancer is a cancer that can be treated with anti-PD-l / PD-Ll therapy. In some embodiments, the cancer is a cancer that can be treated with anti-CTLA-4 therapy. In some embodiments,the cancer is a cancer that can be treated with chemotherapy. In some embodiments, chemotherapy is chemotherapy alone. In some embodiments, chemotherapy is chemotherapy in combination with anti-PD-l / PD-Ll therapy. In some embodiments, the cancer is refractive to the anticancer therapy. In some embodiments, the cancer is refractive to the immunotherapy therapy. In some embodiments, the cancer is refractive to the chemotherapy. In some embodiments, the subject has undergone PD-1 / PD-L1 therapy. In some embodiments, the subject is a non-responder to PD-1 / PD-L1 therapy. In some embodiments, the subject is naive to PD-1 / PD-L1 therapy. In some embodiments, the subject has undergone CTLA-4 therapy. In some embodiments, the subject is a non-responder to CTLA-4 therapy. In some embodiments, the subject is naive to CTLA-4 therapy. In some embodiments, the cancer is PD-L1 positive cancer. In some embodiments, the cancer is a CTLA-4 cancer. In some embodiments, the subject has undergone chemotherapy. In some embodiments, the subject is a non-responder to chemotherapy. In some embodiments, the subject is naive to chemotherapy. Examples of cancer include, but are not limited to brain cancer, oral cancer, head and neck cancer, esophageal cancer, lung cancer, skin cancer, liver cancer, pancreatic cancer, bladder cancer, renal cancer, blood cancer, bladder cancer, bone cancer, breast cancer, thyroid cancer, cervical cancer, ovarian cancer, testicular cancer, retinoblastoma, gastric cancer, colorectal cancer, and uterine cancer. In some embodiments, the cancer is a solid cancer. In some embodiments, the cancer is a hematopoietic cancer. In some embodiments, the cancer is breast cancer. In some embodiments, the cancer is colorectal cancer. In some embodiments, the colorectal cancer is adenocarcinoma. In some embodiments, the cancer is selected from breast cancer, skin, head and neck, lung, ovarian, kidney, gastric and colorectal cancer. In some embodiments, the skin cancer is melanoma. In some embodiments, lung cancer is non-small cell lung cancer. In some embodiments, the cancer is selected from breast cancer, colon cancer, lung cancer and skin cancer.

[0055] In some embodiments, the cancer is a cancer with elevated sCD28 levels. In some embodiments, the cancer is in a subject with elevated sCD28 levels. In some embodiments, the cancer comprises high sCD28 levels. In some embodiments, the cancer is in a subject with high sCD28 levels. In some embodiments, elevated and / or high sCD28 levels are levels at and / or above 5, 6, 7, 8, 9, 10, 12, 14, 15, 17, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90 or 100 ng / ml. Each possibility represents a separate embodiment of the invention. In some embodiments, the cancer comprises high sCD28 levels. In some embodiments, elevated and / or high sCD28 levels are levels at and / or above 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, or 1000%of the levels in a healthy subject. Each possibility represents a separate embodiment of the invention.

[0056] sCD28 levels can be measured by ELISA for example. In some embodiments, the ELISA is a sandwich ELISA. In some embodiments, the ELISA is a standardized sandwich ELISA. In some embodiments, the ELISA is a Bender MedSystems ELISA. In some embodiments, the ELISA is Bender MedSystems ELISA kit BMS290. In some embodiments, the ELISA is performed with an anti-CD28 antibody such as is described hereinbelow.

[0057] As used herein, the terms “administering,” “administration,” and like terms refer to any method which, in sound medical practice, delivers a composition containing an active agent to a subject in such a manner as to provide a therapeutic effect. One aspect of the present subject matter provides for intravenous administration of a therapeutically effective amount of an agent of the invention to a patient in need thereof. In some embodiments, administering is systemic administering. In some embodiments, administering is intraperitoneal administering. Other suitable routes of administration can include parenteral, subcutaneous, oral, intramuscular, or intratumoral.

[0058] In some embodiments, the immunotherapy is immune checkpoint blockade. In some embodiments, the immunotherapy is immune checkpoint inhibition. In some embodiments, the immunotherapy comprises administering an immunotherapy to the subject. In some embodiments, administering an immunotherapy is administering a dose of immunotherapy. In some embodiments, a dose is at least one dose. In some embodiments, the immunotherapy comprises administering at least one immune checkpoint inhibitor (ICI) to the subject. Examples of immune checkpoint proteins include, but are not limited to programmed death 1 (PD-1), programmed death ligand 1 (PD-L1), Cytotoxic T-lymphocyte associated protein 4 (CTLA4), Lymphocyte Activation Gene 3 (LAG3), Hepatitis A virus cellular receptor 2 (HAVCR2 / TIM3), T cell immunoreceptor with Ig and ITIM domains (TIGIT), B- and T- lymphocyte attenuator (BTLA), and Herpesvirus entry mediator (HVEM).

[0059] In some embodiments, the immunotherapy is PD-1 and / or PD-L1 based immunotherapy. In some embodiments, the immunotherapy is an anti-PD-1 and / or anti-PD- L1 based immunotherapy. In some embodiments, the PD-1 / PD-L1 based immunotherapy comprises administering an anti-PDl or anti-PD-Ll antibody. In some embodiments, the antibody is a blocking antibody. In some embodiment, the antibody is an inhibitory antibody. In some embodiments, the therapy comprises blockade of the PD-1 / PD-L1 checkpoint. Insome embodiments, the immunotherapy comprises inhibiting the PD-1 / PD-L1 axis. In some embodiments, the immunotherapy is an anti-PD-1 immunotherapy. In some embodiments, the immunotherapy is an anti-PD-Ll immunotherapy.

[0060] In some embodiments, the immunotherapy is a CTLA-4 based immunotherapy. In some embodiments, the CTLA-4 based immunotherapy comprises administering an anti- CTLA-4 antibody. In some embodiments, the antibody is a blocking antibody. In some embodiment, the antibody is an inhibitory antibody. In some embodiments, the therapy comprises blockade of the CTLA-4 checkpoint. In some embodiments, the immunotherapy comprises inhibiting the CTLA-4 axis. In some embodiments, the immunotherapy is an anti- CTLA-4 immunotherapy. In some embodiments, the CTLA-4 checkpoint is CTLA-4 binding to CD80 and / or CD86.

[0061] In some embodiments, the immunotherapy is a monotherapy. In some embodiments, the immunotherapy is a combination of immunotherapies. In some embodiments, the immunotherapy is a plurality of immunotherapies. In some embodiments, the immunotherapy is a combination therapy. In some embodiments, the combination is with radiation. In some embodiments, the combination is with chemotherapy. In some embodiments, the immunotherapy is a monotherapy until combined with the decreasing sCD28 levels.

[0062] In some embodiments, the immunotherapy comprises an anti-PD-1 antibody. In some embodiments, the immunotherapy comprises an anti-PD-Ll antibody. In some embodiments, the anti-PD-1 antibody is selected from the group consisting of: pembrolizumab, nivolumab, pidilizumab, cemiplimab, retifanlimab, toripalimab, and dostarlimab. In some embodiments, the anti-PD-Ll antibody is selected from the group consisting of: atezolizumab, avelumab and durvalumab. In some embodiments, the anti-PD- 1 antibody is pembrolizumab. In some embodiments, the anti-PD-1 antibody is the antimouse antibody RMP1-14. In some embodiments, the anti-PD-1 antibody is a humanized RMP1-14 antibody. In some embodiments, the immunotherapy comprises an anti-CTLA-4 antibody. In some embodiments, the anti-CTLA-4 antibody is selected from the group consisting of ipilimumab, botensilimab and tremelimumab. In some embodiments, the immunotherapy comprises an anti-LAG-3 antibody. In some embodiments, the anti-LAG-3 antibody is relatimab or TSR-033.

[0063] In some embodiments, the anticancer therapy is chemotherapy. Chemotherapies are well known in the art and include for example paclitaxel, oxaliplatin, fluorouracil,capecitabine, carboplatin, cisplatin, dacarbazine, gemcitabine, idarubicin, ifosfamide, methotrexate, cyclophosphamide, daunorubicin, doxorubicin, epirubicin, mercaptopurine, pexetrexed, dactinomycin, altretamine, busulfan, cabazitaxel, carmustine, docetaxel, Abraxane, vinorelbine, etoposide, pemetrexed and floxuridine. In some embodiments, the chemotherapy is paclitaxel. In some embodiments, the chemotherapy is oxaliplatin.

[0064] In some embodiments, the chemotherapy is a monotherapy. In some embodiments, the chemotherapy is a combination therapy. In some embodiments, the combination is a combination of at least 2 chemotherapies. In some embodiments, the combination is a combination of the chemotherapy and an immunotherapy. In some embodiments, the combination is a combination of the chemotherapy and a PD-1 and / or PD-L1 based immunotherapy. In some embodiments, the combination is a combination of the chemotherapy and PD-1 / PD-L1 blockade. In some embodiments, the chemotherapy is administered concomitantly with another therapy. In some embodiments, the chemotherapy is administered before the other therapy. In some embodiments, the chemotherapy and the PD-1 / PD-L1 based immunotherapy or blockade are administered before reducing sCD28 levels.

[0065] In some embodiments, enhancing is improving. In some embodiments, enhancing is increasing the efficacy. In some embodiments, enhancing is increasing the anticancer efficacy. In some embodiments, enhancing is not reducing side effects. In some embodiments, enhancing comprising increasing an immune response. In some embodiments, enhancing comprises improving clinical outcome. In some embodiments, enhancing comprises improving treatment of the disease. In some embodiments, enhancing comprises reducing resistance to the anticancer therapy. In some embodiments, enhancing comprises reducing resistance to the immunotherapy. In some embodiments, enhancing comprises reducing resistance to the chemotherapy. In some embodiments, enhancing comprises reducing relapse. In some embodiments, relapse is cancer relapse. In some embodiments, increasing an immune response comprises increasing levels of CD8 positive (CD8+) T cells in the subject. In some embodiments, increasing an immune response comprises increasing the ratio of CD8+ T cells to CD4+ T cells in the subject. In some embodiments, increasing an immune response comprises increasing the activation of T cells in the subject. Activation can be determined by any known activation markers, such as for example, CD69, CD39 and CD 107a. In some embodiments, increasing an immune response comprises increasing active proliferation of T cells in the subject. In some embodiments, increasing an immune response comprises increasing levels of memory T cells in the subject. In some embodiments, memoryT cells are memory CD4+ T cells. In some embodiments, memory T cells are memory CD8 T cells. In some embodiments, memory T cells are effector memory T cells. In some embodiments, effector memory T cells are CD44 positive and CCR7 negative. In some embodiments, memory T cells are central memory T cells. In some embodiments, central memory T cells are CD44 positive and CCR7 positive.

[0066] In some embodiments, enhancing is a statistically significant enhancement. In some embodiments, enhancing is enhancing the antitumor response by the subject. Examples of antitumor response include, but are not limited to tumor regression, tumor shrinkage, tumor necrosis, reduction in tumor burden, an anti-tumor response by the immune system, and preventing or delaying tumor recurrence, tumor growth or tumor metastasis. In some embodiments, enhancing anticancer therapy comprises reducing tumor growth. In some embodiments, enhancing chemotherapy comprises reducing tumor growth. In some embodiments, enhancing immunotherapy comprises reducing tumor growth. In some embodiments, enhancing anticancer therapy comprises reducing tumor size. In some embodiments, enhancing chemotherapy comprises reducing tumor size. In some embodiments, enhancing immunotherapy comprises reducing tumor size. In some embodiments, the reduction is by a statistically significant amount. In some embodiments, the reduction is by at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 92, 95, 97, 99 or 100%. Each possibility represents a separate embodiment of the invention.

[0067] In some embodiments, the anticancer therapy is administered before the reducing. In some embodiments, the immunotherapy is administered before the reducing. In some embodiments, the chemotherapy is administered before the reducing. In some embodiments, after administering the anticancer therapy sCD28 levels are reduced. In some embodiments, after administering the immunotherapy sCD28 levels are reduced. In some embodiments, after administering the chemotherapy sCD28 levels are reduced. . In some embodiments, the method comprises administering the immunotherapy and later reducing sCD28 levels. In some embodiments, the method comprises administering the chemotherapy and later reducing sCD28 levels. In some embodiments, the method comprises administering the anticancer and after reducing sCD28 levels. In some embodiments, the method comprises administering the immunotherapy and after reducing sCD28 levels. In some embodiments, the method comprises administering the chemotherapy and after reducing sCD28 levels.

[0068] In some embodiments, before is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 days before. Each possibility represents a separate embodiment of the invention. In some embodiments, before is at least 3 days before. In some embodiments, before is at least 4 daysbefore. In some embodiments, before is at least 6 days before. In some embodiments, before is at least 8 days before. In some embodiments, a dose of anticancer is administered and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 days after the administering sCD28 levels are reduced in the subject. Each possibility represents a separate embodiment of the invention. In some embodiments, a dose of immunotherapy is administered and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 days after the administering sCD28 levels are reduced in the subject. Each possibility represents a separate embodiment of the invention. In some embodiments, a dose of chemotherapy is administered and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 days after the administering sCD28 levels are reduced in the subject. Each possibility represents a separate embodiment of the invention. In some embodiments, a dose of anticancer therapy is administered and at least 3 days after the administering sCD28 levels are reduced in the subject. In some embodiments, a dose of anticancer therapy is administered and at least 4 days after the administering sCD28 levels are reduced in the subject. In some embodiments, a dose of anticancer therapy is administered and at least 6 days after the administering sCD28 levels are reduced in the subject. In some embodiments, a dose of anticancer therapy is administered and at least 8 days after the administering sCD28 levels are reduced in the subject. In some embodiments, a dose of immunotherapy is administered and at least 3 days after the administering sCD28 levels are reduced in the subject. In some embodiments, a dose of immunotherapy is administered and at least 4 days after the administering sCD28 levels are reduced in the subject. In some embodiments, a dose of immunotherapy is administered and at least 6 days after the administering sCD28 levels are reduced in the subject. In some embodiments, a dose of immunotherapy is administered and at least 8 days after the administering sCD28 levels are reduced in the subject. In some embodiments, a dose of chemotherapy is administered and at least 3 days after the administering sCD28 levels are reduced in the subject. In some embodiments, a dose of chemotherapy is administered and at least 4 days after the administering sCD28 levels are reduced in the subject. In some embodiments, a dose of chemotherapy is administered and at least 6 days after the administering sCD28 levels are reduced in the subject. In some embodiments, a dose of chemotherapy is administered and at least 8 days after the administering sCD28 levels are reduced in the subject.

[0069] In some embodiments, before is at most 6, 8, 11, 14, 15, 18, 20, 21, 24, 25, 28 or 30 days before. Each possibility represents a separate embodiment of the invention. In some embodiments, a dose of anticancer therapy is administered and at most 6, 8, 11, 14, 15, 18,20, 21, 24, 25, 28 or 30 days after the administering sCD28 levels are reduced in the subject. Each possibility represents a separate embodiment of the invention. In some embodiments, a dose of immunotherapy is administered and at most 6, 8, 11, 14, 15, 18, 20, 21, 24, 25, 28 or 30 days after the administering sCD28 levels are reduced in the subject. Each possibility represents a separate embodiment of the invention. In some embodiments, a dose of chemotherapy is administered and at most 6, 8, 11, 14, 15, 18, 20, 21, 24, 25, 28 or 30 days after the administering sCD28 levels are reduced in the subject. Each possibility represents a separate embodiment of the invention. In some embodiments, before is 1 to 15, 1 to 11, 1 to 10, 1 to 8, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 2 to 15, 2 to 11, 2 to 10, 2 to 8, 2 to 6, 2 to 5, 2 to 4, 2 to 3, 3 to 15, 3 to 11, 3 to 10, 3 to 8, 3 to 6, 3 to 5, 3 to 4, 4 to 15, 4 to 11, 4 to 10, 4 to 8, 4 to 6, 4 to 5, 6 to 15, 6 to 11, 6 to 10, 6 to 8, 8 to 15, 8 to 11, or 8 to 10 days before. Each possibility represents a separate embodiment of the invention. In some embodiments, a dose of anticancer therapy is administered and 1 to 15, 1 to 11, 1 to 10, 1 to 8, 1 to 6, 1 to 5, 1 to4, 1 to 3, 2 to 15, 2 to 11, 2 to 10, 2 to 8, 2 to 6, 2 to 5, 2 to 4, 2 to 3, 3 to 15, 3 to 11, 3 to 10, 3 to 8, 3 to 6, 3 to 5, 3 to 4, 4 to 15, 4 to 11, 4 to 10, 4 to 8, 4 to 6, 4 to 5, 6 to 15, 6 to 11, 6 to 10, 6 to 8, 8 to 15, 8 to 11, or 8 to 10 days after the administering sCD28 levels are reduced in the subject. Each possibility represents a separate embodiment of the invention. In some embodiments, a dose of immunotherapy is administered and 1 to 15, 1 to 11, 1 to 10, 1 to 8, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 2 to 15, 2 to 11, 2 to 10, 2 to 8, 2 to 6, 2 to 5, 2 to 4, 2 to 3, 3 to 15, 3 to 11, 3 to 10, 3 to 8, 3 to 6, 3 to 5, 3 to 4, 4 to 15, 4 to 11, 4 to 10, 4 to 8, 4 to 6, 4 to5, 6 to 15, 6 to 11, 6 to 10, 6 to 8, 8 to 15, 8 to 11, or 8 to 10 days after the administering sCD28 levels are reduced in the subject. Each possibility represents a separate embodiment of the invention. In some embodiments, a dose of chemotherapy is administered and 1 to 15, 1 to 11, 1 to 10, 1 to 8, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 2 to 15, 2 to 11, 2 to 10, 2 to 8, 2 to 6, 2 to 5, 2 to 4, 2 to 3, 3 to 15, 3 to 11, 3 to 10, 3 to 8, 3 to 6, 3 to 5, 3 to 4, 4 to 15, 4 to 11, 4 to 10, 4 to 8, 4 to 6, 4 to 5, 6 to 15, 6 to 11, 6 to 10, 6 to 8, 8 to 15, 8 to 11, or 8 to 10 days after the administering sCD28 levels are reduced in the subject. Each possibility represents a separate embodiment of the invention.

[0070] In some embodiments, one dose of anticancer therapy is administered before decreasing sCD28 levels. In some embodiments, two doses of anticancer therapy are administered before decreasing sCD28 levels. In some embodiments, three doses of anticancer therapy are administered before decreasing sCD28 levels. In some embodiments, at least one dose of anticancer therapy is administered before decreasing sCD28 levels. In some embodiments, at least two doses of anticancer therapy are administered beforedecreasing sCD28 levels. In some embodiments, at least three doses of anticancer therapy are administered before decreasing sCD28 levels.

[0071] In some embodiments, one dose of immunotherapy is administered before decreasing sCD28 levels. In some embodiments, two doses of immunotherapy are administered before decreasing sCD28 levels. In some embodiments, three doses of immunotherapy are administered before decreasing sCD28 levels. In some embodiments, at least one dose of immunotherapy is administered before decreasing sCD28 levels. In some embodiments, at least two doses of immunotherapy are administered before decreasing sCD28 levels. In some embodiments, at least three doses of immunotherapy are administered before decreasing sCD28 levels.

[0072] In some embodiments, one dose of chemotherapy is administered before decreasing sCD28 levels. In some embodiments, two doses of chemotherapy are administered before decreasing sCD28 levels. In some embodiments, three doses of chemotherapy are administered before decreasing sCD28 levels. In some embodiments, at least one dose of chemotherapy is administered before decreasing sCD28 levels. In some embodiments, at least two doses of chemotherapy are administered before decreasing sCD28 levels. In some embodiments, at least three doses of chemotherapy are administered before decreasing sCD28 levels.

[0073] In some embodiments, the first dose is before the decreasing. In some embodiments, the second dose is before the decreasing. In some embodiments, the third dose is before the decreasing. In some embodiments, the decreasing is after the first dose. In some embodiments, the decreasing is after the second dose. In some embodiments, the decreasing is after the third dose. In some embodiments, the decreasing is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 days after the first dose. Each possibility represents a separate embodiment of the invention. In some embodiments, the decreasing is at least 3 days after the first dose. In some embodiments, the decreasing is at least 4 days after the first dose. In some embodiments, the decreasing is at least 8 days after the first dose. In some embodiments, the decreasing is at least 12 days after the first dose. In some embodiments, the decreasing is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 days after the second dose. Each possibility represents a separate embodiment of the invention. In some embodiments, the decreasing is at least 3 days after the second dose. In some embodiments, the decreasing is at least 4 days after the second dose. In some embodiments, the decreasing is at least 8 days after the second dose. In some embodiments, the decreasing is at least 12 days after the second dose. In some embodiments, the decreasing is at least 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, or 15 days after the third dose. Each possibility represents a separate embodiment of the invention. In some embodiments, the decreasing is at least 3 days after the third dose. In some embodiments, the decreasing is at least 4 days after the third dose. In some embodiments, the decreasing is at least 8 days after the third dose. In some embodiments, the decreasing is at least 12 days after the third dose.

[0074] In some embodiments, the anticancer therapy is continued during the decreasing. In some embodiments, the method comprises continuing to administer the anticancer therapy during the decreasing. In some embodiments, during is concomitantly to the decreasing. In some embodiments, each decreasing also comprises administering the anticancer therapy. In some embodiments, reducing sCD28 levels is a combination therapy comprising reducing sCD28 levels and administering the anticancer therapy.

[0075] In some embodiments, the immunotherapy is continued during the decreasing. In some embodiments, the method comprises continuing to administer the immunotherapy during the decreasing. In some embodiments, during is concomitantly to the decreasing. In some embodiments, each decreasing also comprises administering the immunotherapy. In some embodiments, reducing sCD28 levels is a combination therapy comprising reducing sCD28 levels and administering the immunotherapy.

[0076] In some embodiments, the chemotherapy is continued during the decreasing. In some embodiments, the method comprises continuing to administer the chemotherapy during the decreasing. In some embodiments, during is concomitantly to the decreasing. In some embodiments, each decreasing also comprises administering the chemotherapy. In some embodiments, reducing sCD28 levels is a combination therapy comprising reducing sCD28 levels and administering the chemotherapy.

[0077] In some embodiments, decreasing sCD28 comprises removing sCD28 from the subject. In some embodiments, decreasing sCD28 comprises sequestering sCD28. In some embodiments, the reducing or decreasing occurs in blood, peripheral blood or the TME of the subject. In some embodiments, the reducing or decreasing occurs in blood. In some embodiments, decreasing sCD28 comprises blocking or inhibiting cleavage of membranal CD28 (mCD28). International Patent Application WO2019175885 discloses that soluble CD28 (sCD28) is produced by proteolytic cleavage of the stalk domain of CD28 and active shedding of the extracellular domain of CD28 from the plasma membrane. The use of antibodies against CD28 which inhibit this shedding and thus enhance the immune response are disclosed therein. The use of these antibodies in enhancing PD-1 / PD-L1 basedimmunotherapy is also disclosed therein. In some embodiments, the decreasing sCD28 does not decrease mCD28 levels. In some embodiments, the decreasing sCD28 increases mCD28 levels.

[0078] In some embodiments, the decreasing comprises administering to the subject an agent that binds to mCD28 on the surface of a cell and inhibits proteolytic cleavage of the mCD28. In some embodiments, the agent is an antibody. In some embodiments, the antibody is an anti-mCD28 antibody. In some embodiments, the anti-mCD28 antibody binds to a stalk region of mCD28. In some embodiments, the stalk region comprises the amino acid sequence GKHLCPSPLFPGPSKP (SEQ ID NO: 5). In some embodiments, the stalk region comprises the amino acid sequence KGKHLCPSPLFPGPS (SEQ ID NO: 6). In some embodiments, the stalk region comprises the amino acid sequence HVKGKHLCPSPLFPGPSKP (SEQ ID NO: 7). In some embodiments, the stalk region consists of SEQ ID NO: 5. In some embodiments, the stalk region consists of SEQ ID NO: 6. In some embodiments, the stalk region consists of SEQ ID NO: 7.

[0079] In some embodiments, the agent is an antibody. In some embodiments, the agent comprises an antibody. In some embodiments, the decreasing comprises administering to the subject at least one anti-CD28 antibody. In some embodiments, the antibody binds sCD28. In some embodiments, the antibody binds mCD28 and inhibits proteolytic cleavage of the mCD28. In some embodiments, the antibody is an anti-shedding antibody. In some embodiments, the antibody binds sCD28 and not mCD28. In some embodiments, the antibody binds mCD28 and not sCD28. In some embodiments, mCD29 is mCD28 on the surface of a cell. In some embodiments, the antibody is mCD28 cleavage blocking antibody. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is specific to CD28. In some embodiments, the antibody is specific to sCD28. In some embodiments, the antibody is specific to mCD28.

[0080] As used herein, the term "antibody" refers to a polypeptide or group of polypeptides that include at least one binding domain that is formed from the folding of polypeptide chains having three-dimensional binding spaces with internal surface shapes and charge distributions complementary to the features of an antigenic determinant of an antigen. An antibody typically has a tetrameric form, comprising two identical pairs of polypeptide chains, each pair having one "light" and one "heavy" chain. The variable regions of each light / heavy chain pair form an antibody binding site. An antibody may be oligoclonal, polyclonal, monoclonal, chimeric, camelised, CDR-grafted, multi- specific, bi-specific, catalytic, humanized, fully human, anti- idiotypic and antibodies that can be labeled insoluble or bound form as well as fragments, including epitope-binding fragments, variants or derivatives thereof, either alone or in combination with other amino acid sequences. An antibody may be from any species. The term antibody also includes binding fragments, including, but not limited to Fv, Fab, Fab', F(ab')2 single stranded antibody (svFC), dimeric variable region (Diabody) and disulphide-linked variable region (dsFv). In particular, antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, i.e., molecules that contain an antigen binding site. Antibody fragments may or may not be fused to another immunoglobulin domain including but not limited to, an Fc region or fragment thereof. The skilled artisan will further appreciate that other fusion products may be generated including but not limited to, scFv- Fc fusions, variable region (e.g., VL and VH)~ Fc fusions and scFv-scFv-Fc fusions.

[0081] Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2) or subclass. In some embodiments, the antibody comprises IgG2 or IgG4. In some embodiments, the antibody comprises IgG2. In some embodiments, the antibody comprises IgG4.

[0082] The basic unit of the naturally occurring antibody structure is a heterotetrameric glycoprotein complex of about 150,000 Daltons, composed of two identical light (L) chains and two identical heavy (H) chains, linked together by both noncovalent associations and by disulfide bonds. Each heavy and light chain also has regularly spaced intra-chain disulfide bridges. Five human antibody classes (IgG, IgA, IgM, IgD and IgE) exist, and within these classes, various subclasses, are recognized based on structural differences, such as the number of immunoglobulin units in a single antibody molecule, the disulfide bridge structure of the individual units, and differences in chain length and sequence. The class and subclass of an antibody is its isotype.

[0083] The amino terminal regions of the heavy and light chains are more diverse in sequence than the carboxy terminal regions, and hence are termed the variable domains. This part of the antibody structure confers the antigen-binding specificity of the antibody. A heavy variable (VH) domain and a light variable (VL) domain together form a single antigenbinding site, thus, the basic immunoglobulin unit has two antigen-binding sites. Particular amino acid residues are believed to form an interface between the light and heavy chain variable domains (Chothia et al., J. Mol. Biol. 186, 651-63 (1985); Novotny and Haber, (1985) Proc. Natl. Acad. Sci. USA 82 4592-4596).

[0084] The carboxy terminal portion of the heavy and light chains form the constant domains i.e. CHI, CH2, CH3, CL. While there is much less diversity in these domains, there are differences from one animal species to another, and further, within the same individual there are several different isotypes of antibody, each having a different function.

[0085] The term “framework region” or “FR” refers to the amino acid residues in the variable domain of an antibody, which are other than the hypervariable region amino acid residues as herein defined. The term “hypervariable region” as used herein refers to the amino acid residues in the variable domain of an antibody, which are responsible for antigen binding. The hypervariable region comprises amino acid residues from a “complementarity determining region” or “CDR”. The CDRs are primarily responsible for binding to an epitope of an antigen. The extent of FRs and CDRs has been precisely defined (see, Kabat et al.).

[0086] Immunoglobulin variable domains can also be analyzed using the IMGT information system (imgt. cines.fr / ) (IMGT® / V-Quest) to identify variable region segments, including CDRs. See, e.g., Brochet, X. et al, Nucl. Acids Res. J6:W503-508 (2008).

[0087] Chothia et al. also defined a numbering system for variable domain sequences that is applicable to any antibody. One of ordinary skill in the art can unambiguously assign this system of "Chothia numbering" to any variable domain sequence, without reliance on any experimental data beyond the sequence itself. As used herein, "Chothia numbering" refers to the numbering system set forth by Chothia et al., Journal of Molecular Biology, "Canonical Structures for the Hypervariable regions of immunoglobulins" (1987) and Chothia et al., Nature, “Conformations of Immunoglobulin Hypervariable Regions” (1989).

[0088] As used herein, the term “humanized antibody” refers to an antibody from a nonhuman species whose protein sequences have been modified to increase similarity to human antibodies. A humanized antibody may be produced by production of recombinant DNA coding for the CDRs of the non-human antibody surrounded by sequences that resemble a human antibody. In some embodiments, the humanized antibody is a chimeric antibody. In some embodiments, humanizing comprises insertion of the CDRs of the invention into a human antibody scaffold or backbone. Humanized antibodies are well known in the art and any method of producing them that retains the CDRs of the invention may be employed.

[0089] The term "monoclonal antibody" or “mAb” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and / or bind the same epitope, except forpossible variants that may arise during production of the monoclonal antibody, such variants generally being present in minor amounts. In contrast to polyclonal antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the monoclonal antibodies are advantageous in that they are uncontaminated by other immunoglobulins. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies and is not to be construed as produced by any specific preparation method. Monoclonal antibodies to be used in accordance with the methods provided herein, may be made by the hybridoma method first described by Kohler et al, Nature 256:495 (1975), or may be made by recombinant DNA methods (see, e.g., U.S. Patent No. 4,816,567). The "monoclonal antibodies" may also be isolated from phage antibody libraries using the techniques described in Clackson et al, Nature 352:624-628 (1991) and Marks et al, J. Mol. Biol. 222:581-597 (1991), for example.

[0090] The mAb of the present invention may be of any immunoglobulin class including IgG, IgM, IgD, IgE or IgA. A hybridoma producing a mAb may be cultivated in vitro or in vivo. High titers of mAbs can be obtained in vivo production where cells from the individual hybridomas are injected intraperitoneally into pristine -primed Balb / c mice to produce ascites fluid containing high concentrations of the desired mAbs. mAbs of isotype IgM or IgG may be purified from such ascites fluids, or from culture supernatants, using column chromatography methods well known to those of skill in the art.

[0091] "Antibody fragments" comprise a portion of an intact antibody, preferably comprising the antigen binding region thereof. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; tandem diabodies (taDb), linear antibodies (e.g., U.S. Patent No. 5,641,870, Example 2; Zapata et al, Protein Eng. 8(10): 1057-1062 (1995)); one-armed antibodies, single variable domain antibodies, minibodies, single-chain antibody molecules; multispecific antibodies formed from antibody fragments (e.g., including but not limited to, Db-Fc, taDb-Fc, taDb-CH3, (scFV)4-Fc, di-scFv, bi-scFv, or tandem (di,tri)- scFv); and Bi-specific T-cell engagers (BiTEs).

[0092] Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each with a single antigen-binding site, and a residual "Fc" fragment, whose name reflects its ability to crystallize readily. Pepsin treatment yields an F(ab')2 fragment that has two antigen-binding sites and is still capable of cross-linking antigen.

[0093] "Fv" is the minimum antibody fragment that contains a complete antigen-recognition and antigen-binding site. This region consists of a dimer of one heavy chain and one light chain variable domain in tight, non-covalent association. It is in this configuration that the three surfaces of the VH-VL dimer. Collectively, the six hypervariable regions confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three hypervariable regions specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.

[0094] The Fab fragment also contains the constant domain of the light chain and the first constant domain (CHI) of the heavy chain. Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CHI domain including one or more cysteines from the antibody hinge region. Fab'-SH is the designation herein for Fab' in which the cysteine residue(s) of the constant domains bear at least one free thiol group. F(ab')2 antibody fragments originally were produced as pairs of Fab' fragments that have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

[0095] The "light chains" of antibodies (immunoglobulins) from any vertebrate species can be assigned to one of two clearly distinct types, called kappa and lambda, based on the amino acid sequences of their constant domains.

[0096] Depending on the amino acid sequence of the constant domain of their heavy chains, antibodies can be assigned to different classes. There are five major classes of intact antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgGl, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy chain constant domains that correspond to the different classes of antibodies are called a, delta, e, gamma, and micro, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.

[0097] "Single-chain Fv" or "scFv" antibody fragments comprise the VH and VL domains of antibody, wherein these domains are present in a single polypeptide chain. In some embodiments, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains that enables the scFv to form the desired structure for antigen binding. For a review of scFv see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer- Verlag, New York, pp. 269-315 (1994).

[0098] The term "diabodies" refers to small antibody fragments with two antigen-binding sites, which fragments comprise a heavy chain variable domain (VH) connected to a lightchain variable domain (VL) in the same polypeptide chain (VH - VL). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigenbinding sites. Diabodies production is known in the art and is described in Natl. Acad. Sci. USA, 90:6444-6448 (1993).

[0099] The term "multispecific antibody" is used in the broadest sense and specifically covers an antibody that has polyepitopic specificity. Such multispecific antibodies include, but are not limited to, an antibody comprising a heavy chain variable domain (VH) and a light chain variable domain (VL), where the VHVL unit has polyepitopic specificity, antibodies having two or more VL and VH domains with each VHVL unit binding to a different epitope, antibodies having two or more single variable domains with each single variable domain binding to a different epitope, full length antibodies, antibody fragments such as Fab, Fv, dsFv, scFv, diabodies, bispecific diabodies, triabodies, tri-functional antibodies, antibody fragments that have been linked covalently or non-covalently. "Polyepitopic specificity" refers to the ability to specifically bind to two or more different epitopes on the same or different target(s).

[0100] The monoclonal antibodies of the invention may be prepared using methods well known in the art. Examples include various techniques, such as those in Kohler, G. and Milstein, C, Nature 256: 495-497 (1975); Kozbor et al, Immunology Today 4: 72 (1983); Cole et al, pg. 77-96 in MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc. (1985).

[0101] Besides the conventional method of raising antibodies in vivo, antibodies can be generated in vitro using phage display technology. Such a production of recombinant antibodies is much faster compared to conventional antibody production and they can be generated against an enormous number of antigens. Furthermore, when using the conventional method, many antigens prove to be non-immunogenic or extremely toxic, and therefore cannot be used to generate antibodies in animals. Moreover, affinity maturation (i.e., increasing the affinity and specificity) of recombinant antibodies is very simple and relatively fast. Finally, large numbers of different antibodies against a specific antigen can be generated in one selection procedure. To generate recombinant monoclonal antibodies, one can use various methods all based on display libraries to generate a large pool of antibodies with different antigen recognition sites. Such a library can be made in several ways: One can generate a synthetic repertoire by cloning synthetic CDR3 regions in a pool of heavy chain germline genes and thus generating a large antibody repertoire, from whichrecombinant antibody fragments with various specificities can be selected. One can use the lymphocyte pool of humans as starting material for the construction of an antibody library. It is possible to construct naive repertoires of human IgM antibodies and thus create a human library of large diversity. This method has been widely used successfully to select a large number of antibodies against different antigens. Protocols for bacteriophage library construction and selection of recombinant antibodies are provided in the well-known reference text Current Protocols in Immunology, Colligan et al (Eds.), John Wiley & Sons, Inc. (1992-2000), Chapter 17, Section 17.1.

[0102] Non-human antibodies may be humanized by any methods known in the art. In one method, the non-human complementarity determining regions (CDRs) are inserted into a human antibody or consensus antibody framework sequence. Further changes can then be introduced into the antibody framework to modulate affinity or immunogenicity.

[0103] In some embodiments, antibodies and portions thereof include: antibodies, fragments of antibodies, Fab and F(ab')2, single-domain antigen -binding recombinant fragments and natural nanobodies. In some embodiments, the antigen binding fragment is selected from the group consisting of a Fv, Fab, F(ab')2, scFV or a scFV2 fragment.

[0104] In some embodiments, the antibody binds to CD28. In some embodiments, the agent binds to CD28. In some embodiments, the CD28 is mammalian CD28. In some embodiments the CD28 is human CD28. In some embodiments, the human CD28 comprises or consists of the amino acid sequence:MLRLLLALNLFPSIQVTGNKILVKQSPMLVAYDNAVNLSCKYSYNLFSREFRASLH KGLDSAVEVCVVYGNYSQQLQVYSKTGFNCDGKLGNESVTFYLQNLYVNQTDIY FCKIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACY SLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO: 25). In some embodiments, mature CD28 lacks a signal peptide and comprises the sequence:NKILVKQSPMLVAYDNAVNLSCKYSYNLFSREFRASLHKGLDSAVEVCVVYGNY SQQEQVYSKTGFNCDGKEGNESVTFYEQNEYVNQTDIYFCKIEVMYPPPYEDNEK SNGTIIHVKGKHECPSPEFPGPSKPFWVEVVVGGVEACYSEEVTVAFIIFWVRSKRS REEHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO: 26). In some embodiments, the CD28 signal peptide comprises or consists of MEREEEAENEFPSIQVTG (SEQ ID NO: 27).

[0105] In some embodiments, the agent binds to a cleavage site in mCD28. In some embodiments, the cleavage site is within the stalk region. In some embodiments, the cleavage site is a cleavage motif. In some embodiments, the MMP-2 cleavage motif is PXX / X, wherein the last X is a hydrophobic residue. In some embodiments, the PXX / X motif in CD28 is PSP / L. In some embodiments, the protease cleavage site is amino acids 142-145 (PSPL) of SEQ ID NO: 25. In some embodiments, the protease cleavage site is amino acids 127-130 (PSPL) of SEQ ID NO: 26. In some embodiments, the protease cleavage site is amino acids 9-12 (PSPL) of SEQ ID NO: 7. In some embodiments, the agent blocks accesses of a protease to a cleavage site. In some embodiments, the agent binds to PSPL in a stalk domain of mCD28. In some embodiments, the protease is matrix metalloproteinase-2 (MMP-2). In some embodiments, the protease is matrix metalloproteinase- 13 (MMP-13).

[0106] In some embodiments, the antibody inhibits proteolytic cleavage of CD28. As used herein, “inhibiting proteolytic cleavage” refers to any reduction in proteolytic cleavage of mCD28. In some embodiments, the inhibition is a reduction in cleavage of at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 97, 99 or 100%. Each possibility represents a separate embodiment of the invention. In some embodiments, inhibiting proteolytic cleavage maintains levels of mCD28 on immune cells. In some embodiments, inhibiting proteolytic cleavage increases levels of mCD28 on immune cells. In some embodiments, inhibiting proteolytic cleavage maintains levels of mCD28 adequate for immune stimulation.

[0107] In some embodiments, the antibody is not an antagonist. In some embodiments, the agent is not an antagonist. In some embodiments, the antibody is not an antagonist of CD28. In some embodiments, an antagonist is a substantial antagonist. In some embodiments, an antagonist is a direct antagonist.

[0108] The term “antagonist” generally refers to a molecule, compound or agent that binds to a receptor at the same site as an agonist or another site, does not activate the receptor and does one or more of the following: interferes with or blocks activation of the receptor by a natural ligand, and interferes with or blocks activation of the receptor by a receptor agonist. In some embodiments, the antibody binds to CD28 and does not block activation of the receptor. In some embodiments, the agent, and / or the antibody does not block activation by CD86. In some embodiments, the antibody does block activation by CD86. In some embodiments, the antibody does not inhibit binding of a ligand to CD28. In some embodiments, inhibit is substantially inhibit. In some embodiments, substantial issignificantly. In some embodiments, substantial antagonism is more than low inhibition. In some embodiments, an agent that is not a substantial antagonist does not inhibit or lowly inhibits. In some embodiments, the antibody lowly inhibits binding of a ligand to CD28. In some embodiments, lowly inhibits comprises less than 50, 45, 40, 35, 30, 25, 20, 15, 10, 7, or 5% inhibition. Each possibility represents a separate embodiment of the invention. In some embodiments, lowly inhibits comprises less than 50% inhibition. In some embodiments, lowly inhibits comprises less than 35% inhibition. In some embodiments, lowly inhibits comprises less than 20% inhibition. In some embodiments, the CD28 ligand is selected from: CD80, CD86 and ICOSL. In some embodiments, the CD28 ligand is CD86. In some embodiments, the CD28 ligand is CD80. In some embodiments, the CD28 ligand is ICOSL. In some embodiments, CD86 is CD86-Fc. In some embodiments, CD80 is CD80- Fc.

[0109] In some embodiments, the sdAb does not bind the ligand binding domain of mCD28. In some embodiments, the sdAb does not obscure or block access to the ligand binding domain. In some embodiments, the sdAb does not bind, obscure or block access to the IgV domain of sCD28. In some embodiments, the IgV domain is the ligand binding domain. In some embodiments, the ligand binding domain comprises amino acids 28-137 of SEQ ID NO: 25. In some embodiments, the ligand binding domain comprises or consists of the amino acid sequenceMLVAYDNAVNLSCKYSYNLFSREFRASLHKGLDSAVEVCVVYGNYSQQLQVYSK TGFNCDGKLGNESVTFYLQNLYVNQTDIYFCKIEVMYPPPYLDNEKSNGTIIHVKG (SEQ ID NO: 28).

[0110] In some embodiments, the antibody is an antibody disclosed in International Patent Application WO2019175885, the contents of which are hereby incorporated herein in their entirety. In some embodiments, the antibody is a single chain antibody. In some embodiments, the antibody is a single domain antibody (sdAb). In some embodiments, the sdAb binds to mCD28. In some embodiments, the sdAb is specific to mCD28. In some embodiments, the antibody is a camelid or shark antibody. In some embodiments, the antibody is a camelid antibody. In some embodiments, the antibody is a VHH. In some embodiments, the antibody is an antibody disclosed in International Patent Application W02020183473, the contents of which are hereby incorporated herein in their entirety. In some embodiments, the antibody is a single chain antibody disclosed in International Patent Application WO2020183473. In some embodiments, the antibody is an antibody disclosed in International Patent Application WO2023031943, the contents of which are herebyincorporated herein in their entirety. In some embodiments, the antibody is a single chain antibody disclosed in International Patent Application WO2023031943.

[0111] In some embodiments, the sdAb binds mCD28, blocks proteolytic cleavage and comprises three CDRs, wherein CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 33 (INAMG), CDR2 comprises the amino acid sequence as set forth in SEQ ID NO: 34 (AISGGGDTYYADSVKG), CDR3 comprises the amino acid sequence as set forth in SEQ ID NO: 31 (DLYGSDYWD). In some embodiments, the sdAb binds mCD28, blocks proteolytic cleavage and comprises three CDRs, wherein CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 17 (INAMA), CDR2 comprises the amino acid sequence as set forth in SEQ ID NO: 18 (AITSSGSTNYANSVKG), CDR3 comprises the amino acid sequence as set forth in SEQ ID NO: 19 (DEYGSDYWI). In some embodiments, the sdAb binds mCD28, blocks proteolytic cleavage and comprises three CDRs, wherein CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 33 (INAMG), CDR2 comprises the amino acid sequence as set forth in SEQ ID NO: 20 (AITSGGSTNYADSVKG), CDR3 comprises the amino acid sequence as set forth in SEQ ID NO: 21 (DLYGEDYWI).

[0112] In some embodiments, the sdAb comprises a sequence comprising and / or consisting ofEVQLVESGGGLVQAGESLRLSCAASGSIASINAMGWYRQAPGSQRELVAAISGGG DTYYADSVKGRFTISRDNAKTTVYLQMNSLRPEDTAVYYCVVDLYGSDYWDWG QGTQVTVSS (SEQ ID NO: 22). In some embodiments, the agent comprises a sequence comprising and / or consisting ofEVQLVESGGGLVQAGGSLRLSCAASGSLFSINAMAWYRQAPGKQRELVAAITSSG STNYANSVKGRFTVSRDNAKNTMYLQMNSLKPEDTAVYYCVVDEYGSDYWIWG QGTQVTVSS (SEQ ID NO: 23). In some embodiments, the agent comprises a sequence comprising and / or consisting ofQVQLVESGGGLVQAGGSLRLSCAASGSIFSINAMGWYRQAPGKQRERVAAITSGG STNYADSVKGRFTISRDNAKNTVYLQMNNLEPRDAGVYYCVVDLYGEDYWIWG QGTQVTVSS (SEQ ID NO: 24).

[0113] In some embodiments, the sdAb comprises three CDRs wherein CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 29 (INSMG), CDR2 comprises the amino acid sequence as set forth in SEQ ID NO: 30 (AINEKLLIYYADSVKG), CDR3 comprises the amino acid sequence as set forth in SEQ ID NO: 31 (DLYGSDYWD). In some embodiments, the sdAb comprises the amino acid sequence EVQLVESGGGLVQAGESLRLSCAASGSIASINSMGWYRQAPGSQRELVAAINEKLLIYYADSVKGRFTISRDNAKTTVYLQMNSLRPEDTAVYYCVVDLYGSDYWDWGQ GTQVTVSS (SEQ ID NO: 32). In some embodiments, the sdAb consists of SEQ ID NO: 32. In some embodiments, the sdAb comprises or consists of a sequence with at least 70, 75, 80, 85, 90, 93, 95, 97, or 99% homology to SEQ ID NO: 32. Each possibility represents a separate embodiment of the invention. In some embodiments, the sequence with homolog comprises the CDRs of SEQ ID NO: 32.

[0114] In some embodiments, the sdAb comprises three CDRs wherein CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 33 (INAMG), CDR2 comprises the amino acid sequence as set forth in SEQ ID NO: 34 (AISGGGDTYYADSVKG), CDR3 comprises the amino acid sequence as set forth in SEQ ID NO: 35 (DMIEQQWWY). In some embodiments, the sdAb comprises the amino acid sequence EVQLVESGGGLVQAGESLRLSCAASGSIASINAMGWYRQAPGSQRELVAAISGGG DTYYADSVKGRFTISRDNAKTTVYLQMNSLRPEDTAVYYCVVDMIEQQWWYWG QGTQVTVSS (SEQ ID NO: 36). In some embodiments, the sdAb consists of SEQ ID NO: 36. In some embodiments, the sdAb comprises or consists of a sequence with at least 70, 75, 80, 85, 90, 93, 95, 97, or 99% homology to SEQ ID NO: 36. Each possibility represents a separate embodiment of the invention. In some embodiments, the sequence with homolog comprises the CDRs of SEQ ID NO: 36.

[0115] In some embodiments, the sdAb comprises three CDRs wherein CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 33 (INAMG), CDR2 comprises the amino acid sequence as set forth in SEQ ID NO: 34 (AISGGGDTYYADSVKG), CDR3 comprises the amino acid sequence as set forth in SEQ ID NO: 37 (DTHRGVYWY). In some embodiments, the sdAb comprises the amino acid sequence EVQLVESGGGLVQAGESLRLSCAASGSIASINAMGWYRQAPGSQRELVAAISGGG DTYYADSVKGRFTISRDNAKTTVYLQMNSLRPEDTAVYYCVVDTHRGVYWYWG QGTQVTVSS (SEQ ID NO: 38). In some embodiments, the sdAb consists of SEQ ID NO: 38. In some embodiments, the sdAb comprises or consists of a sequence with at least 70, 75, 80, 85, 90, 93, 95, 97, or 99% homology to SEQ ID NO: 38. Each possibility represents a separate embodiment of the invention. In some embodiments, the sequence with homolog comprises the CDRs of SEQ ID NO: 38.

[0116] In some embodiments, the sdAb comprises three CDRs wherein CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 8 (IKTMA), CDR2 comprises the amino acid sequence as set forth in SEQ ID NO: 39 (AINYIKEIYYADSVKG), CDR3 comprises the amino acid sequence as set forth in SEQ ID NO: 40 (DVTKEDYWY). In someembodiments, the sdAb comprises the amino acid sequence EVQLVESGGGLVQAGESLRLSCAASGSIASIKTMAWYRQAPGSQRELVAAINYIKE IYYADSVKGRFTISRDNAKTTVYLQMNSLRPEDTAVYYCVVDVTKEDYWYWGQ GTQVTVSS (SEQ ID NO: 41). In some embodiments, the sdAb consists of SEQ ID NO: 41. In some embodiments, the sdAb comprises or consists of a sequence with at least 70, 75, 80, 85, 90, 93, 95, 97, or 99% homology to SEQ ID NO: 41. Each possibility represents a separate embodiment of the invention. In some embodiments, the sequence with homolog comprises the CDRs of SEQ ID NO: 41.

[0117] In some embodiments, the sdAb comprises three CDRs wherein CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 42 (INSMA), CDR2 comprises the amino acid sequence as set forth in SEQ ID NO: 43 (AISNAREVYYADSVKG), CDR3 comprises the amino acid sequence as set forth in SEQ ID NO: 44 (DVYFQEYWY). In some embodiments, the sdAb comprises the amino acid sequence EVQLVESGGGLVQAGESLRLSCAASGSIASINSMAWYRQAPGSQRELVAAISNARE VYYADSVKGRFTISRDNAKTTVYLQMNSLRPEDTAVYYCVVDVYFQEYWYWGQ GTQVTVSS (SEQ ID NO: 45). In some embodiments, the sdAb consists of SEQ ID NO: 45. In some embodiments, the sdAb comprises or consists of a sequence with at least 70, 75, 80, 85, 90, 93, 95, 97, or 99% homology to SEQ ID NO: 45. Each possibility represents a separate embodiment of the invention. In some embodiments, the sequence with homolog comprises the CDRs of SEQ ID NO: 45.

[0118] In some embodiments, the sdAb comprises three CDRs wherein CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 46 (INTMA), CDR2 comprises the amino acid sequence as set forth in SEQ ID NO: 47 (AINSISRTYYADSVKG), CDR3 comprises the amino acid sequence as set forth in SEQ ID NO: 40 (DVTKEDYWY). In some embodiments, the sdAb comprises the amino acid sequence EVQLVESGGGLVQAGESLRLSCAASGSIASINTMAWYRQAPGSQRELVAAINSISR TYYADSVKGRFTISRDNAKTTVYLQMNSLRPEDTAVYYCVVDVTKEDYWYWGQ GTQVTVSS (SEQ ID NO: 48). In some embodiments, the sdAb consists of SEQ ID NO: 48. In some embodiments, the sdAb comprises or consists of a sequence with at least 70, 75, 80, 85, 90, 93, 95, 97, or 99% homology to SEQ ID NO: 48. Each possibility represents a separate embodiment of the invention. In some embodiments, the sequence with homolog comprises the CDRs of SEQ ID NO: 48.

[0119] In some embodiments, the sdAb comprises three CDRs wherein CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 8 (IKTMA), CDR2 comprises the aminoacid sequence as set forth in SEQ ID NO: 9 (AIASDNRKYYADSVKG), CDR3 comprises the amino acid sequence as set forth in SEQ ID NO: 10 (DVTKEDYWY). In some embodiments, the sdAb comprises the amino acid sequence EVQLVESGGGLVQAGESLRLSCAASGSIASIKTMAWYRQAPGSQRELVTAIASDN RKYYADSVKGRFTISRDNAKTTVYLQMNSLRPEDTAVYYCVVDVTKEDYWYWG QGTQVTVSS (SEQ ID NO: 49). In some embodiments, the sdAb consists of SEQ ID NO: 49. In some embodiments, SEQ ID NO: 49is the amino acid sequence of VHH 12A9. In some embodiments, the sdAb comprises or consists of a sequence with at least 70, 75, 80, 85, 90, 93, 95, 97, or 99% homology to SEQ ID NO: 49. Each possibility represents a separate embodiment of the invention. In some embodiments, the sequence with homolog comprises the CDRs of SEQ ID NO: 49.

[0120] In some embodiments, the sdAb comprises the amino acid sequence EVQLVESGGGLVQPGGSLRLSCAASGSIASIKTMAWYRQAPGKQRELVTAIASDN RKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVVDVTKEDYWYWG QGTLVTVSS (SEQ ID NO: 50). In some embodiments, SEQ ID NO: 50 is the amino acid sequence of 12A09_VHH4. In some embodiments, the sdAb consists of SEQ ID NO: 50. In some embodiments, the sdAb comprises or consists of a sequence with at least 70, 75, 80, 85, 90, 93, 95, 97, or 99% homology to SEQ ID NO: 50. Each possibility represents a separate embodiment of the invention. In some embodiments, the sequence with homolog comprises the CDRs of SEQ ID NO: 50.

[0121] In some embodiments, the sdAb comprises the amino acid sequence EVQLVESGGGLVQPGGSLRLSCKASGSIASIKTMAWYRQAPGKGLELVTAIASDN RKYYADSVKGRFTISRDNSKTTVYLQMNSLRAEDTAVYYCVVDVTKEDYWYWG QGTLVTVSS (SEQ ID NO: 51). In some embodiments, SEQ ID NO: 51 is the amino acid sequence of 12A9_VHH12. In some embodiments, the sdAb consists of SEQ ID NO: 51. In some embodiments, the sdAb comprises or consists of a sequence with at least 70, 75, 80, 85, 90, 93, 95, 97, or 99% homology to SEQ ID NO: 51. Each possibility represents a separate embodiment of the invention. In some embodiments, the sequence with homolog comprises the CDRs of SEQ ID NO: 51.

[0122] In some embodiments, the sdAb comprises the amino acid sequence EVQLVESGGGLVQPGGSLRLSCAASGSTASIKTMAWYRQAPGKGLELVTAIASDN RKYYADSVKGRFTISRDNSKTTVYLQMNSLRAEDTAVYYCVVDVTKEDYWYWG QGTLVTVSS (SEQ ID NO: 52). In some embodiments, SEQ ID NO: 52 is the amino acid sequence of 12A09_ VHH16. In some embodiments, the sdAb consists of SEQ ID NO: 52.In some embodiments, the sdAb comprises or consists of a sequence with at least 70, 75, 80, 85, 90, 93, 95, 97, or 99% homology to SEQ ID NO: 52. Each possibility represents a separate embodiment of the invention. In some embodiments, the sequence with homolog comprises the CDRs of SEQ ID NO: 52.

[0123] In some embodiments, the sdAb comprises the amino acid sequence EVQLVESGGGLVQPGGSLRLSCKASGSTASIKTMAWYRQAPGKGLELVTAIASDN RKYYADSVKGRFTISRDNSKTTVYLQMNSLRAEDTAVYYCVVDVTKEDYWYWG QGTLVTVSS (SEQ ID NO: 53). In some embodiments, SEQ ID NO: 53 is the amino acid sequence of 12A9_VHH17. In some embodiments, the sdAb consists of SEQ ID NO: 53. In some embodiments, the sdAb comprises or consists of a sequence with at least 70, 75, 80, 85, 90, 93, 95, 97, or 99% homology to SEQ ID NO: 53. Each possibility represents a separate embodiment of the invention. In some embodiments, the sequence with homolog comprises the CDRs of SEQ ID NO: 53.

[0124] In some embodiments, the sdAb comprises three CDRs wherein CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 8 (IKTMA), CDR2 comprises the amino acid sequence as set forth in SEQ ID NO: 9 (AIASDNRKYYADSVKG), CDR3 comprises the amino acid sequence as set forth in SEQ ID NO: 10 (DVTKEDYWY). In some embodiments, the sdAb comprises the amino acid sequence EVQLVESGGGLVQPGGSLRLSCAASGSIASIKTMAWYRQAPGKGRELVTAIASDN RKYYADSVKGRFTISRDNSKTTVYLQMNSLRAEDTAVYYCVVDVTKEDYWYWG QGTLVTVSS (SEQ ID NO: 1). In some embodiments, SEQ ID NO: 1 is the amino acid sequence of 12A09_ VHH18. In some embodiments, the sdAb consists of SEQ ID NO: 1. In some embodiments, the sdAb comprises or consists of a sequence with at least 70, 75, 80, 85, 90, 93, 95, 97, or 99% homology to SEQ ID NO: 1. Each possibility represents a separate embodiment of the invention. In some embodiments, the sequence with homolog comprises the CDRs of SEQ ID NO: 1.

[0125] In some embodiments, the sdAb comprises three CDRs wherein CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 54 (IRTMA), CDR2 comprises the amino acid sequence as set forth in SEQ ID NO: 55 (AISSGREVYYADSVKG), CDR3 comprises the amino acid sequence as set forth in SEQ ID NO: 56 (DMYWQDYWW). In some embodiments, the sdAb comprises the amino acid sequence EVQLVESGGGLVQAGESLRLSCAASGSIASIRTMAWYRQAPGSQRELVAAISSGRE VYYADSVKGRFTISRDNAKTTVYLQMNSLRPEDTAVYYCVVDMYWQDYWWWG QGTQVTVSS (SEQ ID NO: 57). In some embodiments, the sdAb consists of SEQ ID NO:57. In some embodiments, the sdAb comprises or consists of a sequence with at least 70, 75, 80, 85, 90, 93, 95, 97, or 99% homology to SEQ ID NO: 57. Each possibility represents a separate embodiment of the invention. In some embodiments, the sequence with homolog comprises the CDRs of SEQ ID NO: 57. In some embodiments, the sdAb comprises the amino acid sequenceEVQLVESGGGLVQPGESLRLSCAASGSIASIRTMAWYRQAPGSQRELVAAISSGRE VYYADSVKGRFTISRDNAKTTVYLQMNSLRAEDTAVYYCVVDMYWQDYWWW GQGTQVTVSS (SEQ ID NO: 58). In some embodiments, the sdAb consists of SEQ ID NO: 58. In some embodiments, the sdAb comprises or consists of a sequence with at least 70, 75, 80, 85, 90, 93, 95, 97, or 99% homology to SEQ ID NO: 58. Each possibility represents a separate embodiment of the invention. In some embodiments, the sequence with homolog comprises the CDRs of SEQ ID NO: 58. In some embodiments, the sdAb comprises the amino acid sequenceEVQLVESGGGLVQPGGSLRLSCKASGSIASIRTMAWYRQAPGKGLELVAAISSGRE VYYADSVKGRFTISRDNSKTTVYLQMNSLRAEDTAVYYCVVDMYWQDYWWWG QGTLVTVSS (SEQ ID NO: 59). In some embodiments, the sdAb consists of SEQ ID NO:59. In some embodiments, the sdAb comprises or consists of a sequence with at least 70, 75,80, 85, 90, 93, 95, 97, or 99% homology to SEQ ID NO: 59. Each possibility represents a separate embodiment of the invention. In some embodiments, the sequence with homolog comprises the CDRs of SEQ ID NO: 59. In some embodiments, the sdAb comprises the amino acid sequenceEVQLVESGGGLVQPGGSLRLSCKASGSTASIRTMAWYRQAPGKGLELVSAISSGRE VYYADSVKGRFTISRDNSKTTVYLQMNSLRAEDTAVYYCVVDMYWQDYWWWG QGTLVTVSS (SEQ ID NO: 60). In some embodiments, the sdAb consists of SEQ ID NO:60. In some embodiments, the sdAb comprises or consists of a sequence with at least 70, 75, 80, 85, 90, 93, 95, 97, or 99% homology to SEQ ID NO: 60. Each possibility represents a separate embodiment of the invention. In some embodiments, the sequence with homolog comprises the CDRs of SEQ ID NO: 60.

[0126] In some embodiments, the sdAb comprises the amino acid sequence EVQLVESGGGLVQPGGSLRLSCAASGSIASIRTMAWYRQAPGKGLELVSAISSGRE VYYADSVKGRFTISRDNSKTTVYLQMNSLRAEDTAVYYCVVDMYWQDYWWWG QGTLVTVSS (SEQ ID NO: 61). In some embodiments, the sdAb consists of SEQ ID NO:61. In some embodiments, the sdAb comprises or consists of a sequence with at least 70, 75, 80, 85, 90, 93, 95, 97, or 99% homology to SEQ ID NO: 61. Each possibility represents aseparate embodiment of the invention. In some embodiments, the sequence with homolog comprises the CDRs of SEQ ID NO: 61.

[0127] In some embodiments, the sdAb comprises three CDRs wherein CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 29 (INSMG), CDR2 comprises the amino acid sequence as set forth in SEQ ID NO: 62 (AISDRSEKYYADSVKG), CDR3 comprises the amino acid sequence as set forth in SEQ ID NO: 63 (DHHHSDWWT). In some embodiments, the sdAb comprises the amino acid sequence EVQLVESGGGLVQAGESLRLSCAASGSIASINSMGWYRQAPGSQRELVAAISDRSE KYYADSVKGRFTISRDNAKTTVYLQMNSLRPEDTAVYYCVVDHHHSDWWTWGQ GTQVTVSS (SEQ ID NO: 64). In some embodiments, the sdAb consists of SEQ ID NO: 64. In some embodiments, the sdAb comprises or consists of a sequence with at least 70, 75, 80, 85, 90, 93, 95, 97, or 99% homology to SEQ ID NO: 64. Each possibility represents a separate embodiment of the invention. In some embodiments, the sequence with homolog comprises the CDRs of SEQ ID NO: 64.

[0128] In some embodiments, the sdAb comprises an amino acid sequence selected from SEQ ID NO: 1 and 49-53. In some embodiments, the sdAb consists of an amino acid sequence selected from SEQ ID NO: 1 and 49-53. In some embodiments, the sdAb comprises an amino acid sequence selected from SEQ ID NO: 1 and 50-53. In some embodiments, the sdAb consists of an amino acid sequence selected from SEQ ID NO: 1 and 50-53.

[0129] In some embodiments, the agent is a dimeric agent. In some embodiments, the dimeric agent comprises at least two anti-CD28 antibodies. In some embodiments, the dimeric agent comprises at least anti-mCD28 antibodies. In some embodiments, the dimeric agent comprises at least two mCD28 binding sdAbs. In some embodiments, the dimeric agent comprises at least two mCD28 cleavage blocking sdAbs. In some embodiments, at least two is two. In some embodiments, a first antibody is linked to a second antibody. In some embodiments, a first sdAb is linked to a second sdAb. In some embodiments, the linking is by a linker.

[0130] In some embodiments, the linker is an amino acid linker. In some embodiments, the linker is a flexible linker. In some embodiments, the linker is a GS linker. In some embodiments, a flexible linker comprises or consists of at least one GGGGS (SEQ ID NO: 65) sequence. In some embodiments, a flexible linker comprises or consists of at least one GGGGS sequence. In some embodiments, a flexible linker comprises or consists of at least one GGGGS repeat. In some embodiments, a flexible linker comprises or consists of 1, 3, 5,or 7 GGGGS repeats. Each possibility represents a separate embodiment of the invention. In some embodiments, a flexible linker comprises or consists of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 GGGGS repeats. Each possibility represents a separate embodiment of the invention. In some embodiments, a flexible linker comprises or consists of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 GGGS repeats. Each possibility represents a separate embodiment of the invention. In some embodiments, a flexible linker comprises or consists of 1 GGGGS repeat. In some embodiments, a flexible linker comprises or consists of 3 GGGGS repeats. In some embodiments, a flexible linker comprises or consists of 5 GGGGS repeats. In some embodiments, a flexible linker comprises or consists of 7 GGGGS repeats.

[0131] In some embodiments, the linker is of a length sufficient to allow binding of the first antibody to mCD28. In some embodiments, the linker is of a length sufficient to allow binding of the second antibody to mCD28. In some embodiments, the linker is of a length sufficient to allow binding of the first antibody to the stalk region of mCD28 on a cell. In some embodiments, the linker is of a length sufficient to allow binding of the second antibody to the stalk region of mCD28 on a cell. Thus, the linker must be of a sufficient length to allow a range of movement of the first and second antibodies that allows them to access two stalk domains. In some embodiments, the agent does not induce mCD28 crosslinking. In some embodiments, the agent does not induce mCD28 crosslinking that induces immune activation. In some embodiments, the agent does induce mCD28 crosslinking. In some embodiments, the agent does induce mCD28 crosslinking that induces immune activation.

[0132] As used herein, the term “linked” refers to any method of attachment known in the art by which two moieties are stably connected. In some embodiments, linked is a covalent linkage. In some embodiments, linked is a peptide linkage. In some embodiments, linked is a reversible linkage. In some embodiments, linked is an irreversible linkage. In some embodiments, linked is an amino linkage. In some embodiments, linked is a thiol linkage. In some embodiments, linked is a serine linkage. In some embodiments, the linkage is linkage to a side chain of an amino acid.

[0133] In some embodiments, the dimeric agent comprises a first polypeptide comprising the first antibody. In some embodiments, a polypeptide is a polypeptide chain. In some embodiments, the dimeric agent comprises a second polypeptide comprises the second antibody. In some embodiments, the linker links the first polypeptide to the second polypeptide. In some embodiments, the first polypeptide comprises a first dimerization domain, the second polypeptide comprises a second dimerization domain and the first andsecond dimerization domains dimerize with each other. In some embodiments, dimerize with each other is are configured to dimerize with each other. In some embodiments, the first and second dimerization domains are capable of dimerizing to each other. In some embodiments, the first and second dimerization domains are configured to dimerize with each other.

[0134] As used herein, the term “dimerization domain” refers to an amino acid sequence that upon contacting another amino acid sequence (the other dimerization domain) binds to it to form a dimer. Dimerization domains are well known in the art, as many protein sequences are known to bind to each other. In some embodiments, dimerization comprises formation of a covalent bond between the dimerization domains. In some embodiments, dimerization comprises electrostatic binding. In some embodiments, dimerization does not comprise electrostatic binding. In some embodiments, dimerization is reversible. In some embodiments, dimerization is irreversible. In some embodiments, dimerization comprises a bond forming between the dimerization domains. In some embodiments, the bond is a chemical bond. In some embodiments, the bond is a disulfide bond. In some embodiments, the bond is a peptide bond. Examples of dimerization domain include the hinge domain of antibody heavy chains, the CH1 / CL domains of antibody heavy / light chains, and the ECD domains of TCR alpha / beta to name but a few.

[0135] In some embodiments, the dimerization domain comprises or consists of an immunoglobulin (Ig) hinge domain. In some embodiments, an Ig hinge domain is a heavy chain hinge domain. In some embodiments, the Ig is a human Ig. In some embodiments, the immunoglobulin is elected from IgA, IgD, IgE, IgG and IgM. In some embodiments, the immunoglobulin is IgG. In some embodiments, the IgG is IgGl. In some embodiments, the IgG is IgG2. In some embodiments, the IgG is IgG3In some embodiments, the IgG is IgG4. In some embodiments, the IgG is selected from IgG2 and IgG4. In some embodiments, the first and second dimerization domains are both Ig hinge domains. In some embodiments, the first and second dimerization domains are identical. In some embodiments, the first and second dimerization domains are at least 95% identical. In some embodiments, the first and second dimerization domains are at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 97, 99 or 100% identical. Each possibility represents a separate embodiment of the invention.

[0136] In some embodiments, the hinge domain comprises the amino acid sequence EPKSCDKTHTCPPCPAPELLGG (SEQ ID NO: 66). In some embodiments, the hinge domain consists of the amino acid sequence of SEQ ID NO: 66. In some embodiments, the IgGl hinge comprises or consists of SEQ ID NO: 66. In some embodiments, the hinge domain comprises the amino acid sequence EPKCCVECPPCPAPPAAA (SEQ ID NO: 67).In some embodiments, the hinge domain consists of the amino acid sequence of SEQ ID NO: 67. In some embodiments, the IgG2 hinge comprises or consists of SEQ ID NO: 67. In some embodiments, the hinge domain comprises the amino acid sequence ESKYGPPCPPCPAPEFLGG (SEQ ID NO: 68). In some embodiments, the hinge domain consists of the amino acid sequence of SEQ ID NO: 68. In some embodiments, the IgG4 hinge comprises or consists of SEQ ID NO: 68. In some embodiments, the hinge domain comprises the amino acid sequenceELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPC PRCPAPELLGGP (SEQ ID NO: 69). In some embodiments, the hinge domain consists of the amino acid sequence of SEQ ID NO: 69. In some embodiments, the IgG3 hinge comprises or consists of SEQ ID NO: 69.

[0137] In some embodiments, the dimerization domain comprises or consists of an immunoglobulin (Ig) Fc region. In some embodiments, the first dimerization domain and the second dimerization domain of Ig Fc regions In some embodiments, an Fc region is an Fc domain. In some embodiments, an Fc region is an Fc fragment. In some embodiments, the first polypeptide chain comprises an Fc region. In some embodiments, both the first and second polypeptide chains comprise an Fc region. In some embodiments, the Fc region is an Fc region of an antibody heavy chain. In some embodiments, an Fc region is an antibody heavy chain constant region. In some embodiments, the first and second dimerization domains comprise antibody heavy chain constant regions. In some embodiments, the antibody heavy chain is a human antibody heavy chain. In some embodiments, the heavy chain is an IgG heavy chain. In some embodiments, the IgG is selected from IgGl, IgG2, IgG3 and IgG4. In some embodiments, the IgG is selected from IgG2 and IgG4. In some embodiments, the IgG is IgG2. In some embodiments, the IgG is IgG4.

[0138] In some embodiments, the Fc region comprises an Ig CH2 domain. In some embodiments, the Fc region comprises an Ig heavy chain CH2 domain. In some embodiments, the Fc region comprises an Ig CH3 domain. In some embodiments, the Fc region comprises an Ig heavy chain CH3 domain. In some embodiments, the Fc region comprises or consists of both an Ig CH2 domain and Ig CH3 domain. In some embodiments, the Fc region comprises or consists of both an Ig heavy chain CH2 and an Ig heavy chain CH3 domain. In some embodiments, the first chain comprises a first portion of an Fc region and the second chain comprises a second portion of the Fc region. In some embodiments, the first portion comprises a CH2 domain, a CH3 domain or both. In some embodiments, the second portion comprises a CH2 domain, a CH3 domain or both. In some embodiments,interface of the first portion of an Fc region and the second portion of an Fc region produces a functional Fc region. In some embodiments, interface comprises contact. In some embodiments, interface comprises adjacent positioning. In some embodiments, interface comprises formation of the protein complex of the invention. In some embodiments, interface comprises dimerization of the first and second dimerization domains. In some embodiments, the CH2 domain is an Ig CH2 domain. In some embodiments the CH2 domain is a heavy chain CH2 domain. In some embodiments, the CH3 domain is an Ig CH3 domain. In some embodiments, the CH3 domain is a heavy chain CH3 domain.

[0139] In some embodiments, a CH2 domain comprises the amino acid sequence SVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK (SEQ ID NO: 70). In some embodiments, the CH2 domain consists of SEQ ID NO: 70. In some embodiments, SEQ ID NO: 70 is the IgGl CH2 domain. In some embodiments, a CH2 domain comprises the amino acid sequenceSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPRE EQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTK (SEQ ID NO:71). In some embodiments, the CH2 domain consists of SEQ ID NO: 71. In some embodiments, SEQ ID NO: 71 is the IgG2 CH2 domain. In some embodiments, a CH2 domain comprises the amino acid sequenceSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPRE EQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAK (SEQ ID NO:72). In some embodiments, the CH2 domain consists of SEQ ID NO: 72. In some embodiments, SEQ ID NO: 72 is the IgG4 CH2 domain. In some embodiments, a CH2 domain comprises the amino acid sequenceSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFKWYVDGVEVHNAKTKPRE EQYNSTFRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKTK (SEQ ID NO:73). In some embodiments, the CH2 domain consists of SEQ ID NO: 73. In some embodiments, SEQ ID NO: 73 is the IgG3 CH2 domain.

[0140] In some embodiments, a CH3 domain comprises the amino acid sequence GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 74). In some embodiments, a CH3 domain comprises the amino acid sequence GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQID NO: 75). In some embodiments, the CH3 domain consists of SEQ ID NO: 74. In some embodiments, the CH3 domain consists of SEQ ID NO: 75. In some embodiments, SEQ ID NO: 74 is the IgGl CH3 domain. In some embodiments, SEQ ID NO: 75 is the IgGl CH3 domain. In some embodiments, the SEQ ID NO: 74 sequence is the sequence found predominantly is humans of European and American descent. In some embodiments, SEQ ID NO: 75 is the sequence found predominantly in humans of Asian descent. In some embodiments, a CH3 domain comprises the amino acid sequence GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDISVEWESNGQPENNYKTTPP MLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 76). In some embodiments, the CH3 domain consists of SEQ ID NO: 76. In some embodiments, SEQ ID NO: 76 is the IgG2 CH3 domain. In some embodiments, a CH3 domain comprises the amino acid sequenceGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 77). In some embodiments, the CH3 domain consists of SEQ ID NO: 77. In some embodiments, SEQ ID NO: 77 is the IgG4 CH3 domain. In some embodiments, a CH3 domain comprises the amino acid sequenceGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESSGQPENNYNTTPP MLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSLSLSPGK (SEQ ID NO: 78). In some embodiments, the CH3 domain consists of SEQ ID NO: 78. In some embodiments, SEQ ID NO: 78 is the IgG3 CH3 domain.

[0141] In some embodiments, the dimerization domain comprises a human IgG4 heavy chain constant region. In some embodiments, the human IgG4 heavy chain constant region comprises ESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQF NWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGL PSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNG QPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKS LSLSLGK (SEQ ID NO: 11). In some embodiments, the dimerization domain consists of a human IgG4 heavy chain constant region. In some embodiments, the human IgG4 heavy chain constant region consists of SEQ ID NO: 11. In some embodiments, the first polypeptide comprises a first human IgG4 heavy chain constant region and the second polypeptide comprises a second human IgG4 heavy chain constant region. In some embodiments, the human IgG4 heavy chain constant region comprises at least 70, 75, 80,85, 90, 92, 95, 97, 99 or 100% identity to SEQ ID NO: 11. Each possibility represents a separate embodiment of the invention. In some embodiments, the human IgG4 heavy chain constant region comprises at least 85% identity to SEQ ID NO: 11. In some embodiments, the human IgG4 heavy chain constant region comprises at least 95% identity to SEQ ID NO: 11. In some embodiments, the sequence with identity to SEQ ID NO: 11 retains the ability to homodimerize.

[0142] In some embodiments, the Fc comprises a mutation. In some embodiments, the mutation reduces effector function. In some embodiments, effector function comprises antibody dependent cell cytotoxicity (ADCC), complement dependent cytotoxicity (CDC), antibody-dependent cellular phagocytosis (ADCP) or a combination thereof. In some embodiments, reduced effector function comprises reduced cytotoxicity. In some embodiments, reduces is abolishes. In some embodiments, the Fc is from IgGl or IgG3 and the mutation reduces effector function. In some embodiments, the Fc is from IgGl and comprises at least one mutation that reduces effector function. Mutations that reduce effector function are well known in the art and any such mutation can be used. Examples of such mutations can be found in Saunders, 2019, “Conceptual approaches to modulating antibody effector functions and circulation half-life” Front Immunol., Jun 7; 10: 1296, herein incorporated by reference in its entirety.

[0143] It will be known by a skilled artisan that IgG2 and IgG4 possess greatly reduced effector function and are not generally cytotoxic in nature. Additionally, mutations such as S228P and L235E in IgG4 are known to reduce effector function even more. In some embodiments, the mutation is mutation of serine 228 of the IgG4 human heavy chain to proline (S228P). In some embodiments, the S to P mutation is mutation of S10 of SEQ ID NO: 11 to P. In some embodiments, the mutation is mutation of leucine 235 of the IgG4 human heavy chain to glutamic acid (L235E). In some embodiments, the L to E mutation is mutation of L17 of SEQ ID NO: 11 to E. In some embodiments, IgG4 human heavy chain constant region comprising the S228P and L235E mutations comprises ESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQF NWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGL PSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNG QPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKS LSLSLGK (SEQ ID NO: 12). In some embodiments, the dimerization domain comprises SEQ ID NO: 12. In some embodiments, the IgG4 human heavy chain constant region comprising the S228P and L235E mutations consists of SEQ ID NO: 12. In someembodiments, the dimerization domain consists of SEQ ID NO: 12. In some embodiments, the first polypeptide comprises SEQ ID NO: 12 and the second polypeptide comprises SEQ ID NO: 12.

[0144] Further, mutations that reduce the cytotoxicity / effector function of IgGl and IgG3 are well known in the art. In some embodiments, the IgG comprises at least one mutation. In some embodiments, the mutation is a plurality of mutations. In some embodiments, the mutation decreases cytotoxicity. In some embodiments, the plurality of mutations that decreases cytotoxicity comprise the LALA mutations. In some embodiments, the plurality of mutations that decreases cytotoxicity comprise the PG-LALA mutations. In some embodiments, the dimerization domain comprises a human IgGl heavy chain constant region comprising at least one mutation. In some embodiments, the human IgGl heavy chain constant region comprisesDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPGK (SEQ ID NO: 79). In some embodiments, the dimerization domain consists of a human IgGl heavy chain constant region comprising at least one mutation. In some embodiments, the human IgGl heavy chain constant region consists of SEQ ID NO: 79. In some embodiments, the first polypeptide comprises a first human IgGl heavy chain constant region comprising at least one mutation and the second polypeptide comprises a second human IgG41 heavy chain constant region comprising at least one mutation. In some embodiments, the human IgGl heavy chain constant region comprises at least 70, 75, 80, 85, 90, 92, 95, 97, 99 or 100% identity to SEQ ID NO: 79. Each possibility represents a separate embodiment of the invention. In some embodiments, the human IgGl heavy chain constant region comprises at least 85% identity to SEQ ID NO: 79. In some embodiments, the human IgGl heavy chain constant region comprises at least 95% identity to SEQ ID NO: 79. In some embodiments, the sequence with identity to SEQ ID NO: 79 retains the ability to homodimerize. In some embodiments, the mutation is mutation of proline 329 of the IgGl human heavy chain to glycine (P329G). In some embodiments, the P to G mutation is mutation of P109 of SEQ ID NO: 79 to G. In some embodiments, the mutation is mutation of leucine 234 of the IgGl human heavy chain to alanine (L234A). In some embodiments, the L to A mutation is mutation of L14 of SEQ ID NO: 79 to A. In some embodiments, the mutation is mutation of leucine 235 of the IgGl human heavy chain to alanine (L235A). Insome embodiments, the L to A mutation is mutation of L15 of SEQ ID NO: 79 to A. In some embodiments, the plurality of mutation comprises P109G, L14A and L15A of SEQ ID NO: 79. In some embodiments, the plurality of mutation comprises L14A and L15A of SEQ ID NO: 79. In some embodiments, the plurality of mutation comprises P329G, L234A and L235A of the IgGl human heavy chain. In some embodiments, the plurality of mutation comprises L234A and L235A of the IgGl human heavy chain. It will be understood by a skilled artisan that parallel mutation can also be performed in the IgG3 heavy chain or the heavy chains of non-human IgGls.

[0145] In some embodiments, the first antibody is separated from the first dimerization domain by a linker. In some embodiments, the second antibody is separated from the second dimerization domain by a linker. In some embodiments, the first polypeptide comprises the first antibody, first linker and first dimerization domain. In some embodiments, the second polypeptide comprises the second antibody, second linker and second dimerization domain. In some embodiments, the first antibody is a first sdAb and the second antibody is a second sdAb. In some embodiments, the first antibody is N-terminal to the first dimerization domain. In some embodiments, the first antibody is C-terminal to the first dimerization domain. In some embodiments, the second antibody is N-terminal to the second dimerization domain. In some embodiments, the second antibody is C-terminal to the second dimerization domain.

[0146] In some embodiments, the linker is an amino acid linker. In some embodiments, the linker is a flexible linker. In some embodiments, the linker is a hydrophilic linker. In some embodiments, the linker comprises the amino acid sequence GGGGS (SEQ ID NO: 65). In some embodiments, the linker comprises the amino acid sequence GS. In some embodiments, the linker comprises the amino acid sequence (GGGGS)n wherein n is an integer. In some embodiments, the linker comprises the amino acid sequence AAA(GGGGS)n wherein n is an integer. In some embodiments, the linker comprises the amino acid sequence (GS)n wherein n is an integer. In some embodiments, the linker comprises the amino acid sequence (GGS)n wherein n is an integer. In some embodiments, the linker comprises the amino acid sequence GSGGS (SEQ ID NO: 14). In some embodiments, the linker comprises the amino acid sequence (GSGGS)n wherein n is an integer. In some embodiments, the linker comprises the amino acid sequence EGGGS (SEQ ID NO: 15). In some embodiments, the linker comprises the amino acid sequence (EGGGS)n wherein n is an integer. In some embodiments, the linker comprises the amino acid sequence EGGS (SEQ ID NO: 16). In some embodiments, the linker comprises theamino acid sequence (EGGS)n wherein n is an integer. In some embodiments, n is an integer selected from 1, 2, 3, 4, 5, 6, 7, and 8. In some embodiments, n is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. Each possibility represents a separate embodiment of the invention. In some embodiments, n is 1. In some embodiments, n is 2. In some embodiments, n is 3. In some embodiments, n is 4. In some embodiments, n is 5. In some embodiments, n is 7. In some embodiments, n is 8.

[0147] In some embodiments, the linker comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 amino acids. Each possibility represents a separate embodiment of the invention. In some embodiments, the linker comprises at least 1 amino acid. In some embodiments, the linker comprises at least 5 amino acids. In some embodiments, the linker comprises at least 8 amino acids. In some embodiments, the linker comprises at least 10 amino acids. In some embodiments, the linker comprises at least 13 amino acids. In some embodiments, the linker comprises at least 15 amino acids. In some embodiments, the linker comprises at least 18 amino acids. In some embodiments, the linker comprises at least 25 amino acids. In some embodiments, the linker comprises at least 35 amino acids. In some embodiments, the linker comprises at most 25, 28, 30, 35, 40, 45, 50, 60, 70, 75, 80, 90 or 100 amino acids. Each possibility represents a separate embodiment of the invention. In some embodiments, the linker comprises at most 25 amino acids. In some embodiments, the linker comprises at most 28 amino acids. In some embodiments, the linker comprises at most 35 amino acids. In some embodiments, the linker comprises at most 50 amino acids. In some embodiments, the linker comprises 1-50, 1-28, 1-25, 1-18, 1-15, 1-13, 1-10, 5-50, 5-28, 5-25, 5-18, 5-15, 5-13, 5-10, 10-50, 10-28, 10-25, 10-18, 10-15, 10-13, 15- 50, 15-28, 15-25, 15-18, 25-50 and 28-50 amino acids. Each possibility represents a separate embodiment of the invention. In some embodiments, the linker comprises between 15 and 25 amino acids. In some embodiments, the linker comprises between 15 and 35 amino acids. In some embodiments, the linker comprises between 25 and 35 amino acids. In some embodiments, the linker comprises between 18 and 28 amino acids. In some embodiments, the linker comprises between 10 and 25 amino acids. In some embodiments, the linker comprises between 13 and 28 amino acids. In some embodiments, the linker comprises between 10 and 20 amino acids. In some embodiments, the linker comprises between 13 and 23 amino acids. It will be understood by a skilled artisan that in addition to increasing the number of repeats in the linker, the N- and C- termini can also include additional bases such as additional Gs and / or As.

[0148] In some embodiments, the agent comprises the amino acid sequence EVQLVESGGGLVQPGGSLRLSCAASGSIASIKTMAWYRQAPGKGRELVTAIASDN RKYYADSVKGRFTISRDNSKTTVYLQMNSLRAEDTAVYYCVVDVTKEDYWYWG QGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSESKYGPPCPPCPAPEFEGGPS VFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREE QFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVY TLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF LYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 13). In some embodiments, the agent consists of SEQ ID NO: 13. In some embodiments, the dimeric agent comprises a dimer of SEQ ID NO: 13. In some embodiments, the dimeric agent consists of a dimer of SEQ ID NO: 13. In some embodiments, the agent is a dimer of SEQ ID NO: 13. In some embodiments, the first polypeptide comprises SEQ ID NO: 13 and the second polypeptide comprises SEQ ID NO: 13. In some embodiments, the first polypeptide consists of SEQ ID NO: 13 and the second polypeptide consists of SEQ ID NO: 13.

[0149] In some embodiments, a dose of the immunotherapy is administered with each dose of the agent. In some embodiments, with is concomitantly with. In some embodiments, with is at the same time. In some embodiments, at the same time is within 30 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours or 24 hours of each other. Each possibility represents a separate embodiment of the invention. In some embodiments, upon initiating administration of the agent continuing to administer the agent every 3 or 4 days. In some embodiments, upon initiating administration of the agent continuing to administer the agent every 3 days. In some embodiments, upon initiating administration of the agent continuing to administer the agent every 4 days. In some embodiments, upon initiating administration of the agent continuing to administer the agent in combination with the immunotherapy every 3 to 4 days. In some embodiments, upon initiating administration of the agent continuing to administer the agent in combination with the immunotherapy every 3 days. In some embodiments, upon initiating administration of the agent continuing to administer the agent in combination with the immunotherapy every 4 days.

[0150] In some embodiments, administering the agent is administering a composition comprising the agent. In some embodiments, the composition is a pharmaceutical composition. In some embodiments, the composition comprises a pharmaceutically acceptable carrier, excipient or adjuvant.

[0151] In some embodiments, the composition comprises one dose of the agent. In some embodiments, the composition comprises a therapeutically effective amount of the agent. As used herein, the term "therapeutically effective amount" refers to an amount of the agent effective to treat a disease or disorder in a mammal. The term “a therapeutically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result. The exact dosage form and regimen would be determined by the physician according to the patient's condition. In some embodiments, effective is effective to treat cancer. In some embodiments, effective is effective to enhance the immunotherapy.

[0152] As used herein, the term “carrier,” “excipient,” or “adjuvant” refers to any component of a pharmaceutical composition that is not the active agent. As used herein, the term “pharmaceutically acceptable carrier” refers to non-toxic, inert solid, semi-solid liquid filler, diluent, encapsulating material, formulation auxiliary of any type, or simply a sterile aqueous medium, such as saline. Some examples of the materials that can serve as pharmaceutically acceptable carriers are sugars, such as lactose, glucose and sucrose, starches such as corn starch and potato starch, cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt, gelatin, talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, com oil and soybean oil; glycols, such as propylene glycol, polyols such as glycerin, sorbitol, mannitol and polyethylene glycol; esters such as ethyl oleate and ethyl laurate, agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline, Ringer's solution; ethyl alcohol and phosphate buffer solutions, as well as other non-toxic compatible substances used in pharmaceutical formulations. Some non-limiting examples of substances which can serve as a carrier herein include sugar, starch, cellulose and its derivatives, powered tragacanth, malt, gelatin, talc, stearic acid, magnesium stearate, calcium sulfate, vegetable oils, polyols, alginic acid, pyrogen-free water, isotonic saline, phosphate buffer solutions, cocoa butter (suppository base), emulsifier as well as other non-toxic pharmaceutically compatible substances used in other pharmaceutical formulations. Wetting agents and lubricants such as sodium lauryl sulfate, as well as coloring agents, flavoring agents, excipients, stabilizers, antioxidants, and preservatives may also be present. Any non-toxic, inert, and effective carrier may be used to formulate the compositions contemplated herein. Suitable pharmaceutically acceptable carriers, excipients, and diluents in this regard are well known to those of skill in the art, such as those described in The MerckIndex, Thirteenth Edition, Budavari et ah, Eds., Merck & Co., Inc., Rahway, N.J. (2001); the CTFA (Cosmetic, Toiletry, and Fragrance Association) International Cosmetic Ingredient Dictionary and Handbook, Tenth Edition (2004); and the “Inactive Ingredient Guide,” U.S. Food and Drug Administration (FDA) Center for Drug Evaluation and Research (CDER) Office of Management, the contents of all of which are hereby incorporated by reference in their entirety. Examples of pharmaceutically acceptable excipients, carriers and diluents useful in the present compositions include distilled water, physiological saline, Ringer's solution, dextrose solution, Hank's solution, and DMSO. These additional inactive components, as well as effective formulations and administration procedures, are well known in the art and are described in standard textbooks, such as Goodman and Gillman’s: The Pharmacological Bases of Therapeutics, 8th Ed., Gilman et al. Eds. Pergamon Press (1990); Remington’s Pharmaceutical Sciences, 18th Ed., Mack Publishing Co., Easton, Pa. (1990); and Remington: The Science and Practice of Pharmacy, 21st Ed., Lippincott Williams & Wilkins, Philadelphia, Pa., (2005), each of which is incorporated by reference herein in its entirety. The presently described composition may also be contained in artificially created structures such as liposomes, ISCOMS, slow-releasing particles, and other vehicles which increase the half-life of the peptides or polypeptides in serum. Liposomes include emulsions, foams, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions, lamellar layers and the like. Liposomes for use with the presently described peptides are formed from standard vesicle-forming lipids which generally include neutral and negatively charged phospholipids and a sterol, such as cholesterol. The selection of lipids is generally determined by considerations such as liposome size and stability in the blood. A variety of methods are available for preparing liposomes as reviewed, for example, by Coligan, J. E. et al, Current Protocols in Protein Science, 1999, John Wiley & Sons, Inc., New York, and see also U.S. Pat. Nos. 4,235,871, 4,501,728, 4,837,028, and 5,019,369.

[0153] The carrier may comprise, in total, from about 0.1% to about 99.99999% by weight of the pharmaceutical compositions presented herein.

[0154] In some embodiments, the composition is formulated for administration to a subject. In some embodiments, the composition is formulated for systemic administration. In some embodiments, the composition is formulated for local administration. In some embodiments, local administration is administration to a tumor site. In some embodiments, local administration is intratumoral administration. In some embodiments, systemic administration is intravenous administration. In some embodiments, systemic administration is intraperitoneal administration.

[0155] By another aspect, there is provided an agent for use in a method of the invention.

[0156] By another aspect, there is provided an agent for use in combination with an anticanccr therapy for use in a method of the invention.

[0157] By another aspect, there is provided an agent for use in combination with an immunotherapy for use in a method of the invention.

[0158] By another aspect, there is provided an agent for use in combination with a chemotherapy for use in a method of the invention.

[0159] By another aspect, there is provided an anticancer therapy for use in a method of the invention.

[0160] By another aspect, there is provided an immunotherapy for use in a method of the invention.

[0161] By another aspect, there is provided a chemotherapy for use in a method of the invention.

[0162] By another aspect, there is provided an anticancer therapy for use in combination with an agent for use in a method of the invention.

[0163] By another aspect, there is provided an immunotherapy for use in combination with an agent for use in a method of the invention.

[0164] By another aspect, there is provided a chemotherapy for use in combination with an agent for use in a method of the invention.

[0165] In some embodiments, the method of the invention is treating cancer. In some embodiments, the agent for use is a composition comprising the agent for use. In some embodiments, the use is a staggered dosing regimen of the invention.

[0166] As used herein, the term "about" when combined with a value refers to plus and minus 10% of the reference value. For example, a length of about 1000 nanometers (nm) refers to a length of 1000 nm+- 100 nm.

[0167] It is noted that as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a polynucleotide" includes a plurality of such polynucleotides and reference to "the polypeptide" includes reference to one or more polypeptides and equivalents thereof known to those skilled in the art, and so forth. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intendedto serve as antecedent basis for use of such exclusive terminology as "solely," "only" and the like in connection with the recitation of claim elements, or use of a "negative" limitation.

[0168] In those instances where a convention analogous to "at least one of A, B, and C, etc." is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (e.g., "a system having at least one of A, B, and C" would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). It will be further understood by those within the art that virtually any disjunctive word and / or phrase presenting two or more alternative terms, whether in the description, claims, or drawings, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase "A or B" will be understood to include the possibilities of "A" or "B" or "A and B."

[0169] It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. All combinations of the embodiments pertaining to the invention are specifically embraced by the present invention and are disclosed herein just as if each and every combination was individually and explicitly disclosed. In addition, all subcombinations of the various embodiments and elements thereof are also specifically embraced by the present invention and are disclosed herein just as if each and every such sub-combination was individually and explicitly disclosed herein.

[0170] As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents, unless the context clearly dictates otherwise. The terms “a” (or “an”) as well as the terms “one or more” and “at least one” can be used interchangeably.

[0171] Furthermore, “and / or” is to be taken as specific disclosure of each of the two specified features or components with or without the other. Thus, the term “and / or” as used in a phrase such as “A and / or B” is intended to include A and B, A or B, A (alone), and B (alone). Likewise, the term “and / or” as used in a phrase such as “A, B, and / or C” is intended to include A, B, and C; A, B, or C; A or B; A or C; B or C; A and B; A and C; B and C; A (alone); B (alone); and C (alone).

[0172] Wherever embodiments are described with the language “comprising,” otherwise analogous embodiments described in terms of “consisting of’ and / or “consisting essentially of’ are included.

[0173] Additional objects, advantages, and novel features of the present invention will become apparent to one ordinarily skilled in the art upon examination of the following examples, which are not intended to be limiting. Additionally, each of the various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below finds experimental support in the following examples.

[0174] Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples.EXAMPLES

[0175] Generally, the nomenclature used herein and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological and recombinant DNA techniques. Such techniques are thoroughly explained in the literature. See, for example, "Molecular Cloning: A laboratory Manual" Sambrook et al., (1989); "Current Protocols in Molecular Biology" Volumes I-III Ausubel, R. M., ed. (1994); Ausubel et al., "Current Protocols in Molecular Biology", John Wiley and Sons, Baltimore, Maryland (1989); Perbal, "A Practical Guide to Molecular Cloning", John Wiley & Sons, New York (1988); Watson et al., "Recombinant DNA", Scientific American Books, New York; Birren et al. (eds) "Genome Analysis: A Laboratory Manual Series", Vols. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); methodologies as set forth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; "Cell Biology: A Laboratory Handbook", Volumes I- III Cellis, J. E., ed. (1994); "Culture of Animal Cells - A Manual of Basic Technique" by Freshney, Wiley-Liss, N. Y. (1994), Third Edition; "Current Protocols in Immunology" Volumes I-III Coligan J. E., ed. (1994); Stites et al. (eds), "Basic and Clinical Immunology" (8th Edition), Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (eds), "Strategies for Protein Purification and Characterization - A Laboratory Course Manual" CSHL Press (1996); all of which are incorporated by reference. Other general references are provided throughout this document.Example 1: PD-1 blockade and sCD28 blockade combination with staggered dosing reduces therapy resistance in a colorectal cancer model

[0176] The mouse CD28 stalk region does not contain the cleavage motif PX1X2 / X3 wherein X3 is a hydrophobic residue that exists in human CD28. As such, mice do not naturally produce soluble CD28 (sCD28) in the way that humans do. Therefore, a transgenic mouse expressing human CD28 was used to test CD28 cleavage blockade in vivo. These transgenic animals, developed via knock-in at the mouse CD28 locus, produce a chimeric CD28 protein comprising the human extracellular domain and murine transmembrane and intracellular domains. These mice were used to test combination anti-CD28 shedding and anti-PD-1 therapy.

[0177] In the initial study, mice were injected subcutaneously with 0.5 x 10A6 MC-38 colorectal adenocarcinoma cells and tumors were allowed to form. At day 11 post injection, the mice were administered a PD-1 checkpoint blockade monoclonal antibody that targets murine PD-1 (RMP1-14) at a dose of 2.5 mg / kg body weight by intraperitoneal injections. Some of the mice also received injections with a CD28 cleavage blocking agent intravenously. The agent used is a dimer of a CD28 stalk domain-binding VHH (12A9 VHH18, SEQ ID NO: 1) fused to murine IgGl (mlgGl). Murine IgGl is the equivalent of human IgG4 (hIgG4) as it has no effector function. The mlgGl contained a L234A / L235A / P329G triple mutation that reduces binding to the IgG Fc receptors as well as to complement component Clq (SEQ ID NO: 2). This is similar to the hIgG4 double mutation S228P / L235E which produces reduced Fcgamma receptor binding (SEQ ID NO: X). The VHH was fused to the IgG by a flexible linker (SEQ ID NO: 3). The dimeric agent (SEQ ID NO: 4) was administered at the same time as the anti-PD-1 at a dose of 10 mg / kg body weight. Injections were then continued every 4 days. Both the PD-1 alone and the combination treatment produced an immediate inhibition of tumor growth and indeed produced significant shrinkage by day 15. However, after that point tumors began to grow again. No significant difference in the rate of regrowth (% of cancers developing resistance) was observed between the monotherapy treated and combination therapy treated mice.

[0178] The same experiment was run with E0771 breast cancer cells (0.5xl0A6 cells via injection into the mammary fat pad). Both the monotherapy and the combined treatment produced tumor growth inhibition. Regrowth (resistance) was not observed in these cells.

[0179] To further investigate the combination therapy, the MC-38 test was run again but this time VHH18-mIgG4 administration was only started after mice had already received two doses of anti-PD-1 (Fig. 1A). Mice received a first dose of anti-PD-1 on day 11 (at a dose of 5 mg / kg body weight) and subsequent doses every 4 days. On day 19 (the day of the third anti-PD-1 dose) the mice began receiving the dimeric agent at a dose of 5 mg / kg bodyweight. Anti-CD28 shedding and anti-PD-1 were thus concomitantly administered every 4 days starting on day 19. As before, mice treated with only anti-PD-1 showed an immediate tumor growth retardation after the first dose, but regrowth / relapse following (Fig. IB). Looking at individual mice is especially informative, as 4 of the 5 mice that received only anti-PD-1 showed rapid tumor regrowth / relapse while one mouse showed complete response (CR) and eradication of the tumor (Fig. IB). In contrast, mice that received the staggered dosing with the dimeric agent for the most part did not exhibit tumor regrowth / relapse (Fig. 1C). As before, tumor growth stalled after the first dose of anti-PD-1, but addition of the anti-CD28 shedding dimeric agent blocked regrowth in 6 of the 7 mice tested. Only 1 mouse showed tumor regrowth and it showed slower growth and a smaller tumor volume than each of the 4 anti-PD-1 only therapy regrowth mice at any given time point. Table 1 presents the slope of the tumor growth for each of the 5 resistant / regrowth mice. The one that received the staggered combination therapy had by far the slowest regrowth. Thus, anti-PD- 1 resistant tumors developed in 80% of mice treated with monotherapy, but in only 14% of mice treated with the combination therapy (Fig. ID). The regrowth was also slower in the mouse that received combination therapy. No growth inhibition was observed in mice that received isotype control (IgG4) only (Fig. IE). No improved blocking of regrowth had been observed when anti-CD28 shedding had been administered from the beginning with anti-PD-1.

[0180] Table 1: Slope of tumor regrowth in 5 mice for which regrowth was observed.

[0181] It was hypothesized that T cell activation induced by PD-1 blockade might result in increased mCD28 cleavage and thus increased sCD28 levels. Blood was drawn from the various mice throughout the experiment and serum levels of sCD28 were calculated by a commercial ELISA kit. PD-1 blockade did indeed produce a significant increase in sCD28 levels in the mice as compared to the mice that received isotype control (Fig. IF). This increase was abrogated by dimeric agent staggered administration and indeed sCD28 levels were reduced well below those observed in control mice. Without being bound by any one hypothesis, it may be that initial mCD28 cleavage is beneficial for anti-PD-1 function, but that subsequent reduction of sCD28 enhances therapeutic effect going forward or removes aresistance or control mechanism. In summary, it was apparent that a delayed start to dosing with VHH18-mIgGl was highly effective in treating cancer, reducing the development of resistant tumors and enhancing the overall effect of PD-1 blockade.Example 2: Staggered dosing optimization in colorectal and breast cancer

[0182] A follow up study was conducted to test the ideal dosing regimen for the colorectal cancer cells. Instead of starting anti-PD-1 dosing on day 11, the first dose of 2.5 mg / kg body weight was administered on day 13 when the tumor was larger. Anti-PD-1 was then administered every 3 days. Growth inhibition by anti-PD-1 was again observed, but the level of inhibition was reduced (Fig. 2A). At day 26 when the experiment was terminated a 56% growth inhibition by the monotherapy was observed. Two different setups for the combination therapy were tested. In the first, the dimeric agent was administered at 5 mg / kg body weight concomitantly with the anti-PD-1 starting at the third dose of anti-PD-1 (day 19). In the second, the dimeric agent was administered starting with the fourth dose of anti- PD-1 (day 22). Both regimens produced an improvement as compared to the monotherapy but administering the dimeric agent after 2 initial doses of anti-PD-1 (called 2T) was superior to starting the dimeric agent after 3 initial doses (called 3T) (Fig. 2B). The 2T dosing regimen produced an 83% average reduction in tumor volume which was significantly better than the 56% produced by the monotherapy and slightly better than the 76% produced by the 3T dosing regimen.

[0183] At day 26 and day 33 mice from each arm were sacrificed for an immune cell analysis. Lymph nodes from the mice were drained and processed into a single cell suspension and analyzed by flow cytometry to evaluate various immune cell populations. The combined treatment decreases the CD4+ compartment and increased the CD8+ compartment. This trend was already apparent at day 26 such that the ratio of CD8 to CD4 cells was significantly altered (Fig. 2C) but became even more pronounced and significant at day 33 (Fig. 2D). This is supported by the increase in actively dividing CD8 cells (Ki67+) but not CD4 cells observed on day 26. It was also notable that combination therapy significantly promoted differentiation of both CD4 and CD8 T cells toward the effector memory phenotype (EM, CD44+, CCR7-) (Fig. 2E) and increased T cell activation (Fig. 2F) already at day 26. These changes in T cell populations further speak to the superiority of the staggered combination as compared to anti-PD-1 therapy alone. The provided results were from the 2T staggered combination arm of the study, but similar results are observed for the 3T arm as well.

[0184] The combined treatment with staggered dosing was tested in a mouse model of breast cancer. E0771 breast cancer cells were administered as before and 5 mg / kg body weight anti- PD-1 dosing was started on day 11 and repeated every 4 days. Dimeric agent administration was started after three doses of anti-PD-1 alone, with the first dose administered at 5 mg / kg body weight on day 23 (and repeated with anti-PD-1 every 4 days). The combination therapy was found to be better than anti-PD-1 monotherapy, with the tumor growth inhibition increasing from 45% to 61% (Fig. 3A). Further, when looking at individual mice, it was apparent that 2 mice in the monotherapy arm were resistant to anti-PD-1 therapy, showing identical growth to the isotype control treated mice (Fig. 3B). In contrast, all combination treated mice showed good tumor growth reduction. No difference in regrowth was observed when anti-PD-1 and anti-CD28 shedding had been administered starting at the same time.

[0185] This test was repeated to confirm the superiority of the combination treatment with staggered dosing. E0771 cells were injected, and anti-PD-1 treatment began on day 7, with doses administered every 4 days. Two different combination regimens were tested. In the first, the dimeric agent was administered concomitantly with the anti-PD-1 starting at the third dose of anti-PD-1 (day 15). In the second, the dimeric agent was administered starting with the fourth dose of anti-PD-1 (day 19). Both regimens produced an improvement as compared to the monotherapy but, quite surprisingly, administering the dimeric agent after 3 initial doses of anti-PD-1 (called 3T) was superior to starting after 2 initial doses (called 2T) (Fig. 4A). The 3T dosing regimen produced an 57% average reduction in tumor volume, which was significantly better than the 19% produced by the monotherapy (Fig. 4B-4C).

[0186] Once again immune cell population analysis was performed; this time at days 31 and 38. Insufficient control isotype control mice survived until day 38 and so they are not included in the below analysis. As before, the combined treatment decreased the CD4+ compartment and increased the CD8+ compartment and this trend was already apparent at day 31 (Fig. 4D) but became even more pronounced and significant at day 38 (Fig. 4E). This was again supported by the increase in actively dividing CD8 cells (Ki67+) but not CD4 cells observed on day 31 and day 38 (Fig. 4D-4E). In these mice as well, it was notable that combination therapy significantly promoted differentiation of both CD4 and CD8 T cells toward the memory phenotypes (effector memory-EM and central memory-CM) (Fig. 4F). Increased T cell activation was present already at day 31 (Fig. 4G) and persisted at day 38 (Fig. 4H).

[0187] All of this data taken together indicates that initiating mCD28 cleavage blockade greatly enhances the efficacy of PD-1 / PD-L1 based immunotherapy, decreasing tumorvolume and the prevalence of developing ICI-resistance and relapse, when the mCD28 cleavage blockade is initiated after the PD-1 / PD-L1 therapy has already been going on.Example 3: Staggered with CTLA-4 blockade

[0188] A follow up study is performed to test a second immunotherapy in combination with CD28 cleavage blockade. Specifically, the tumor growth inhibition activity of anti-CTLA- 4, administered either as a monotherapy or in combination with a CD28 cleavage blocking agent, is tested in several syngeneic tumor models.

[0189] To this end, several tumor cell lines, colon adenocarcinoma (MC-38), breast cancer (E0771), Lewis lung carcinoma (LLC1) and / or melanoma (B16-F10), are implanted subcutaneously or orthotopically (0.5-lxl0A6 cells / mouse) into humanized CD28 transgenic C57 / B16 mice (GenOway), and tumors are allowed to form. When tumors reach the desired volume of 50mmA3-120mmA3 (the day depends on the model), mice are administered anti- CTLA-4 antibody (monoclonal Clone 9D9) at a dose of 3-10 mg / Kg, preferably 5 mg / Kg, body weight, intravenously (IV).

[0190] Some mice also receive injections of the dimeric CD28 cleavage blocking agent intravenously, at a dose of 3-10 mg / Kg, preferably 5 mg / Kg, body weight. The anti-CD28 agent is administered either concomitantly with the anti-CTLA-4 or after having already administered two or three doses of anti-CTLA-4. The anti-CD28 shedding agent and the anti-CTLA-4 are administered every 4 days. Anti-CD28 administration is tested concomitantly with anti-CTLA-4, at least 4 days after commencing anti-CTLA-4 treatment or at least 8 days after commencement.

[0191] Tumor volumes are measured three times a week to evaluate the effect of anti-CTLA- 4 treatment as a monotherapy or in combination with the anti-CD28 shedding agent (either concomitantly or with staggered dosing). At the endpoint, tumor samples and draining lymph nodes are extracted and immunophenotyped by flow cytometry for biomarker analysis. To evaluate the anti-cleavage effect of the agent, serum samples are collected from all treated mice once a week and analyzed for soluble CD28 content. A persistent reduction in soluble CD28 levels is observed over time in mice treated with the anti-cleavage agent.

[0192] Addition of CD28 cleavage blockade enhances the effect of the anti-CTLA-4 therapy, however, this enhancement is greatest when the anti-CD28 agent is administered after 2-3 doses of anti-CTLA-4. Thus, the staggered dosing regimen is also superior for the combination of CD28 cleavage blockade with anti-CTLA-4.Example 4: Staggered with chemotherapies

[0193] A follow up study is performed to test chemotherapies in combination with CD28 cleavage blockade. Specifically, the tumor growth inhibition activity of various chemotherapy drugs administered either as a monotherapy, in combination with anti-PDl or in combination with anti-PD-1 and a CD28 cleavage blocking agent, is tested in several syngeneic tumor models.

[0194] To this end, several tumor cell lines, colon adenocarcinoma (MC-38), breast cancer (E0771), Lewis lung carcinoma (LLC1) and / or melanoma (B16-F10), are implanted subcutaneously or orthotopically (0.5-lxl0A6 cells / mouse) into humanized CD28 transgenic C57 / B16 mice (GenOway), and tumors are allowed to form. When tumors reach the desired volume of 50mmA3-120mmA3 (the day depends on the model), mice are administered a chemotherapy at a dose of 3-30 mg / Kg, body weight, intraperitoneally (IP). Paclitaxel and Oxaliplatin are tested along with various other chemotherapies.

[0195] Some mice also receive injections of the dimeric CD28 cleavage blocking agent intravenously, at a dose of 3-10 mg / Kg, preferably 5 mg / Kg, body weight. The anti-CD28 agent is administered either concomitantly with the chemotherapy or after having already administered two or three doses of the chemotherapy. Additionally, an anti-PD-1 antibody is also administered to some mice, such that chemotherapy is tested as a monotherapy, in combination with just anti-PD-1, in combination with just the anti-CD28 and in combination with both anti-PD-1 and anti-CD28. The anti-CD28 shedding agent, the chemotherapy and the anti-PD-1 are administered every 4 days. Anti-CD28 administration is tested concomitantly with the chemotherapy (with or without anti-PD-1), at least 4 days after commencing chemotherapy treatment or at least 8 days after commencement.

[0196] Tumor volumes are measured three times a week to evaluate the effect of chemotherapy as a monotherapy or in combination (either concomitantly or with staggered dosing). At the endpoint, tumor samples and draining lymph nodes are extracted and immunophenotyped by flow cytometry for biomarker analysis. To evaluate the anti-cleavage effect of the agent, serum samples are collected from all treated mice once a week and analyzed for soluble CD28 content. A persistent reduction in soluble CD28 levels is observed over time in mice treated with the anti-cleavage agent.

[0197] Addition of CD28 cleavage blockade enhances the effect of the chemotherapy, especially when combined with anti-PD-1. However, this enhancement is greatest when theanti-CD28 agent is administered after 2-3 doses of chemotherapy. Thus, the staggered dosing regimen is also superior for the combination of CD28 cleavage blockade with chemotherapy.

[0198] Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims.

Claims

CLAIMS:

1. A method of enhancing an anticancer therapy in a human cancer subject, comprising administering to said subject a dose of said anticancer therapy and at least 4 days after said administering decreasing soluble CD28 (sCD28) levels in said subject, wherein said anticancer therapy is selected from an immune checkpoint inhibitor (ICI) immunotherapy and a chemotherapy, thereby enhancing an anticancer therapy in a human cancer patient.

2. A method of enhancing an anticancer therapy in a human cancer subject, comprising administering to said subject at least two doses of said anticancer therapy as a monotherapy and after said administering decreasing soluble CD28 (sCD28) levels in said subject, wherein said anticancer therapy is selected from an immune checkpoint inhibitor (ICI) immunotherapy and a chemotherapy, thereby enhancing an anticancer therapy in a human cancer patient.

3. The method of claim 1 or 2, wherein said anticancer therapy is an anti-programmed death 1 (PD-1) or anti-programmed death ligand 1 (PD-L1) based immunotherapy.

4. The method of claim 3, wherein said anti-PD- 1 or anti-PD-Ll immunotherapy is a PD- 1 blocking antibody or a PD-L1 blocking antibody.

5. The method of claim 4, wherein said PD-1 blocking antibody or a PD-L1 blocking antibody is selected from pembrolizumab, nivolumab, pidilizumab, cemiplimab, atezolizumab, retifanlimab, toripalimab, dostarlimab, avelumab and durvalumab.

6. The method of any one of claims 1 to 5, wherein said anticancer therapy is anti-PD- 1 immunotherapy .

7. The method of any one of claims 1 to 6, comprising continuing to administer said anticancer therapy concomitantly with said decreasing.

8. The method of any one of claims 1 to 7, wherein at least 4 days is at least 6 days.

9. The method of claim 8, wherein at least 4 days is at least 8 days.

10. The method of any one of claims 1 and 3 to 9, wherein said administering a dose of said anticancer therapy is administering at least 2 doses before said decreasing.

11. The method of any one of claims 2 to 10, wherein at least 2 doses is at least 3 doses.

12. The method of claim 10 or 11, wherein said at least 4 days is at least 4 days from a first dose of said at least 2 doses.

13. The method of claim 12, wherein said decreasing is at least 3 days after a second dose of said at least 2 doses.

14. The method of claim 12 or 13, wherein said at least 2 doses is at least 3 doses before said decreasing and wherein said decreasing is at least 3 days after a third dose of said at least 3 doses.

15. The method of any one of claims 1 to 14, wherein said decreasing sCD28 levels in the subject comprises administering an agent that binds to membranal CD28 (mCD28) on the surface of a cell and inhibits proteolytic cleavage of said mCD28.

16. The method of claim 15, wherein said agent binds to a stalk region of mCD28 and wherein said stalk region comprises the amino acid sequence GKHLCPSPLFPGPSKP (SEQ ID NO: 5) or KGKHLCPSPLFPGPS (SEQ ID NO: 6) or consists of the amino acid sequence HVKGKHLCPSPLFPGPSKP (SEQ ID NO: 7).

17. The method of claim 15 or 16, wherein said agent comprises a single domain antibody (sdAb) specific to mCD28.

18. The method of claim 17, wherein said sdAb comprises three CDRs wherein CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 8 (IKTMA), CDR2 comprises the amino acid sequence as set forth in SEQ ID NO: 9 (AIASDNRKYYADSVKG), CDR3 comprises the amino acid sequence as set forth in SEQ ID NO: 10 (DVTKEDYWY).

19. The method of claim 17 or 18, wherein said sdAb is a camelid antibody.

20. The method of claim 19, wherein said sdAb comprises the sequence EVQLVESGGGLVQPGGSLRLSCAASGSIASIKTMAWYRQAPGKGRELVTAIA SDNRKYYADSVKGRFTISRDNSKTTVYLQMNSLRAEDTAVYYCVVDVTKE DYWYWGQGTLVTVSS (SEQ ID NO: 1).

21. The method of any one of claims 17 to 20, wherein said agent is a dimeric agent comprising at least two mCD28 binding sdAbs, wherein a first sdAb is linked to a second sdAb by a linker.

22. The method of claim 21, wherein said dimeric agent comprises a first polypeptide comprising said first sdAb and a second polypeptide comprising said second sdAb and wherein said linker links said first polypeptide and said second polypeptide.

23. The method of claim 22, wherein said first polypeptide comprises a first IgG heavy chain constant region, said second polypeptide comprises a second IgG heavy chain constant region and said linker comprises said first and said second IgG heavy chain constant regions.

24. The method of claim 23, wherein said first sdAb is separated from said first IgG heavy chain constant region by an amino acid linker and said second sdAb is separated from said second IgG heavy chain constant region by an amino acid linker.

25. The method of claim 24, wherein said amino acid linker comprises a sequence selected from (GGGGS)n, (GS)n, (GGS)n, (GSGGS)n, (EGGGS)n, (EGGS)n and a combination thereof, wherein n is an integer selected from 1, 2, 3, 4, 5, 6, 7, and 8.

26. The method of any one of claims 23 to 25, wherein said first and said second IgG heavy chain constant region comprise a human IgG4 heavy chain constant region comprisingESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPE VQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVETVEHQDWENGKEYKC KVSNKGEPSSIEKTISKAKGQPREPQVYTEPPSQEEMTKNQVSETCEVKGFYP SDIAVEWESNGQPENNYKTTPPVEDSDGSFFEYSRETVDKSRWQEGNVFSCS VMHEAEHNHYTQKSESESEGK (SEQ ID NO: 11) or a sequence with at least 95% identity thereto.

27. The method of any one of claims 23 to 26, wherein said first and said second IgG heavy chain constant region compriseESKYGPPCPPCPAPEFEGGPSVFEFPPKPKDTEMISRTPEVTCVVVDVSQEDPE VQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVETVEHQDWENGKEYKC KVSNKGEPSSIEKTISKAKGQPREPQVYTEPPSQEEMTKNQVSETCEVKGFYP SDIAVEWESNGQPENNYKTTPPVEDSDGSFFEYSRETVDKSRWQEGNVFSCSVMHEAEHNHYTQKSESESEGK (SEQ ID NO: 12) or a sequence with at least 95% identity thereto.

28. The method of claim 27, wherein said dimeric agent comprises a dimer of the amino acid sequenceEVQEVESGGGEVQPGGSERESCAASGSIASIKTMAWYRQAPGKGREEVTAIA SDNRKYYADSVKGRFTISRDNSKTTVYLQMNSLRAEDTAVYYCVVDVTKE DYWYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSESKYGPPCPP CPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVD GVEVHNAKTKPREEQFNSTYRVVSVETVEHQDWENGKEYKCKVSNKGEPS SIEKTISKAKGQPREPQVYTEPPSQEEMTKNQVSETCEVKGFYPSDIAVEWES NGQPENNYKTTPPVEDSDGSFFEYSRETVDKSRWQEGNVFSCSVMHEAEHN HYTQKSESESEGK (SEQ ID NO: 13).

29. The method of any one of claims 15 to 28, comprising administering a dose of said anticancer therapy concomitantly with each dose of said agent.

30. The method of claim 29, comprising upon initiating administering said agent administering a combination of said agent and said anticancer therapy every 3 or 4 days.

31. The method of any one of claims 3 to 30, wherein said cancer is a PD-E1 positive cancer.

32. The method of any one of claims 1 to 31, wherein said cancer is selected from a breast cancer and a colorectal cancer.

33. A method of treating cancer in a subject in need thereof, the method comprising administering to said subject a dose of an anticancer therapy and at least 4 days after said administering, administering a dose of an anti-mCD28 cleavage blocking dimeric agent, wherein said anticancer therapy is selected from an immune checkpoint inhibitor (ICI) immunotherapy and a chemotherapy, thereby treating cancer in a subject in need thereof.

34. A method of treating cancer in a subject in need thereof, the method comprising administering to said subject at least two doses of an anticancer therapy as a monotherapy and after said administering, administering a dose of an anti-mCD28cleavage blocking dimeric agent, wherein said anticancer therapy is selected from an immune checkpoint inhibitor (ICI) immunotherapy and a chemotherapy, thereby treating cancer in a subject in need thereof.

35. The method of claim 33 or 34, wherein said anticancer therapy is an anti-programmed death 1 (PD-1) or anti-programmed death ligand 1 (PD-L1) based immunotherapy.

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