BMP1 and PD-1 combined inhibition immunotherapy

Abstract

Methods of improving PD-1 based immunotherapy in a subject suffering from a solid cancer, comprising decreasing bone morphogenetic protein 1 (BMP1) levels or function in T cells or in a tumor microenvironment (TME) or extracellular matrix (ECM) of the solid cancer are provided. Methods of treating a solid cancer comprising administering a PD-1 based immunotherapy and decreasing BMP1 levels or function in T cells or a TME or ECM of the solid cancer are also provided. Antibody drug conjugates comprising an anti-PDl blocking antibody and a BMP1 inhibiting small molecule or antibody or antigen binding fragment thereof small molecule inhibitor and pharmaceutical compositions for use in the methods of the invention are also provided.

Description

BMP1 AND PD-1 COMBINED INHIBITION IMMUNOTHERAPYCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority of U.S. Provisional Patent Application No. 63 / 692,115, filed September 8, 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 (TECH-P-0300-PCT.xml; Size: 44,483 bytes; and Date of Creation: August 29, 2025) is herein incorporated by reference in its entirety.FIELD OF INVENTION

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

[0004] Immunotherapy has been markedly advanced in the last decade by the use of immune checkpoint inhibitors (ICIs). Monoclonal antibodies, targeting mainly CTLA-4 and PD-1- PD-L1 axis check point proteins, are being used in the clinic either as a monotherapy or in combination with other treatment modalities e.g., chemotherapy. ICIs have shown a remarkable therapeutic benefit in several types of cancers, even in patients with advanced metastatic disease who were previously considered non-curable. However, only a small proportion of patients (-20-40%) respond and benefit from this therapy, demonstrating innate resistance. In addition, despite the durable response rates observed with ICIs in responding patients, some of them will face a significant relapse after an initial response, developing acquired resistance to the therapy. Multiple mechanisms have been proposed to elucidate resistance to immunotherapy or relapse, among them are the lack of tumor antigen representation, activation of bypass immune checkpoints, genetic and epigenetic alterations, and tumor microenvironment modification in the form of recruitment of immunosuppressivecells or defect in cytotoxic T cell function. Nevertheless, the precise mechanisms behind tumor-mediated resistance to immunotherapy are still elusive.

[0005] The extracellular matrix (ECM) is composed of different proteins, which are produced intracellularly by cells with the support of enzymes that maintain a specific architecture of the tissue. Among the most abundant ECM components are proteins such as Collagen, Elastin, Fibronectin, and Laminin, each of which contributes to the ECM architecture in the tissue in different ways. Cells residing in the tissue constantly modulate the ECM by the secretion and / or activation of ECM-associated enzymes such as metalloproteinases (MMPs), Lysyl oxidase (LOX), and Cathepsins. These enzymes contribute to the synthesis, degradation and reassembly of the ECM. In cancer, the ECM is usually different by its topography and mechanical behavior than the normal ECM found in the organ. Specifically, collagen is highly enriched in tumors and supports a stiffed architecture. The remodeling of ECM in the growing tumors by the interactions between cancer cells and the environment increases the crosslinking of matrix components therefore making the tumor tissue stiffer. These effects further allow cell signaling pathways supporting tumorigenesis and even metastasis.

[0006] A growing body of evidence suggests that tumor associated ECM and its structure can regulate cancer-immunity. The majority of studies demonstrate that stiffened ECM in tumors reduces the infiltration of anti-tumor immune cells to the tumor site, therefore supporting a ‘cold’ microenvironment which can explain immunotherapy resistance. These effects are not only associated with a physical barrier that inhibits T cell infiltration to tumors but also the fact that changes in tumor ECM support immunosuppression microenvironment mediated through TGF-p. Furthermore, studies demonstrated that TGF-P drives ECM production by cancer-associated fibroblasts, and the activation of several ECM associated enzymes, leading to T lymphocytes exclusion from tumors. However, targeting TGF-P can serve as a double-edge sword, due to its anti- and pro-tumor activities, therefore limiting its therapeutic efficacy. A new combination therapy that targets the ECM and can enhance immune checkpoint inhibitors is therefore greatly needed.SUMMARY OF THE INVENTION

[0007] The present invention provides methods of improving PD-1 based immunotherapy in a subject suffering from a solid cancer, comprising decreasing bone morphogeneticprotein 1 (BMP1) levels or function in T cells or in a tumor microenvironment (TME) or extracellular matrix (ECM) of the solid cancer. Methods of treating a solid cancer comprising administering a PD-1 based immunotherapy and decreasing BMP1 levels or function in T cells or a TME or ECM of the solid cancer are also provided. Antibody drug conjugates comprising an anti-PDl blocking antibody and a BMP1 inhibiting small molecule or antibody or antigen binding fragment thereof small molecule inhibitor and pharmaceutical compositions for use in the methods of the invention are also provided.

[0008] According to a first aspect, there is provided an antibody drug conjugate (ADC) comprising an anti-PDl blocking antibody or antigen binding fragment thereof conjugated to a BMP1 small molecule inhibitor or an anti -BMP 1 inhibiting antibody or antigen binding fragment thereof.

[0009] According to some embodiments, the anti-PDl blocking antibody is selected from pembrolizumab, nivolumab, pidilizumab, cemiplimab, dostarlimab, retifanlimab and toripalimab.

[0010] According to some embodiments, the BMP1 small molecule inhibitor is selected from UK383, 367, S33A, RXP-1001 and FG-2575.

[0011] According to another aspect, there is provided a pharmaceutical composition comprising the ADC of the invention and a pharmaceutically acceptable carrier, excipient or adjuvant.

[0012] According to another aspect, there is provided a method of improving PD-1 based immunotherapy in a subject suffering from a solid cancer, the method comprising decreasing bone morphogenetic protein 1 (BMP1) levels or function in T cells of the subject or in a tumor microenvironment (TME) or extracellular matrix (ECM) of the solid cancer; thereby improving PD-1 based immunotherapy.

[0013] According to another aspect, there is provided a method of treating a solid cancer in a subject in need thereof, the method comprising administering to the subject a PD-1 based immunotherapy and decreasing BMP1 levels or function in T cells of the subject or in a tumor microenvironment (TME) or extracellular matrix (ECM) of the solid cancer, thereby treating cancer.

[0014] According to another aspect, there is provided a method of enhancing cytotoxic immune cell infiltration into a solid cancer in a subject in need thereof, the method comprising decreasing bone morphogenetic protein 1 (BMP1) levels or function in T cellsof the subject or in a tumor microenvironment (TME) or extracellular matrix (ECM) of the solid cancer; thereby enhancing cytotoxic immune cell infiltration into a solid cancer.

[0015] According to another aspect, there is provided a method of decreasing myeloid cell infiltration into a solid cancer in a subject in need thereof, the method comprising decreasing bone morphogenetic protein 1 (BMP1) levels or function in T cells of the subject or in a tumor microenvironment (TME) or extracellular matrix (ECM) of the solid cancer; thereby enhancing cytotoxic immune cell infiltration into a solid cancer.

[0016] According to some embodiments, the decreasing is decreasing BMP1 protein levels or function in a tumor microenvironment (TME) or extracellular matrix (ECM) of the cancer.

[0017] According to some embodiments, the decreasing comprises decreasing secretion of BMP1 from cytotoxic immune cells.

[0018] According to some embodiments, the cytotoxic immune cells are cytotoxic T lymphocytes (CTLs).

[0019] According to some embodiments, the solid cancer is a carcinoma or melanoma.

[0020] According to some embodiments, the cancer is a PD-1 / PD-L1 based immunotherapy resistant cancer, or wherein the subject has previously received PD-1 / PD-L1 based immunotherapy as a monotherapy and has developed resistance.

[0021] According to some embodiments, the cancer is selected from: brain cancer, breast cancer, bone cancer, fat cancer, retinoblastoma, head and neck cancer, tongue cancer, nasopharyngeal cancer, pharyngeal cancer, throat cancer, esophageal cancer, stomach cancer, gastrointestinal cancer, intestinal cancer, lung cancer, colon cancer, colorectal cancer, liver cancer, renal cell carcinoma, pancreatic cancer, gallbladder cancer, penile cancer, thymus cancer, thyroid cancer, urogenital cancer, prostate cancer, kidney cancer, ovarian cancer, cervical cancer, testicular cancer, skin cancer, glioblastoma multiforme (GBM), and uterine cancer.

[0022] According to some embodiments, the cancer is selected from: lung cancer, skin cancer and breast cancer.

[0023] According to some embodiments, the method further comprises administering the PD-1 based immunotherapy to the subject.

[0024] According to some embodiments, the PD-1 based immunotherapy is an immune checkpoint inhibitor of the PD-1 / PD-L1 immune checkpoint.

[0025] According to some embodiments, the PD-1 based immunotherapy is an anti-PDl blocking antibody.

[0026] According to some embodiments, the anti-PDl blocking antibody is selected from pembrolizumab, nivolumab, pidilizumab, cemiplimab, dostarlimab, retifanlimab and toripalimab.

[0027] According to some embodiments, the method comprises administering a BMP1 small molecule inhibitor to the subject.

[0028] According to some embodiments, the BMP1 small molecule inhibitor is selected from UK383, 367, S33A, RXP-1001 and FG-2575.

[0029] According to some embodiments, the method comprises administering an anti -BMP 1 antibody or Sizzled protein to the subject.

[0030] According to some embodiments, the small molecule inhibitor, anti-BMP-1 antibody or Sizzled protein is conjugated to a targeting moiety to T cells, TME or ECM.

[0031] According to some embodiments, the small molecule inhibitor, anti-BMP-1 antibody or Sizzled protein is conjugated to an anti-PDl antibody.

[0032] According to some embodiments, the method comprises administering to the subject a composition comprising an inhibitory nucleic acid molecule comprising a sequence reverse complementary to a sequence of BMP 1, and a T cell targeting moiety.

[0033] According to some embodiments, the small molecule inhibitor, anti-BMP-1 antibody, Sizzled protein or nucleic acid molecule comprising a sequence reverse complementary to a sequence of BMP 1 is conjugated to an anti-PDl antibody.

[0034] According to some embodiments, the T cells are CD8 T cells.

[0035] According to some embodiments, the method comprises adoptive T cell transfer comprising administering to the subject a composition comprising T cells comprising knockdown of endogenous BMP1.

[0036] According to some embodiments, the method comprises administering an antibody drug conjugate (ADC) comprising an anti-PDl blocking antibody conjugated to a BMP1 small molecule inhibitor to the subject.

[0037] According to some embodiments, the anti-PDl blocking antibody is selected from pembrolizumab, nivolumab, pidilizumab, cemiplimab, dostarlimab, retifanlimab andtoripalimab and the BMP1 small molecule inhibitor is selected from UK383,367, S33A, RXP-1001 and FG-2575.

[0038] According to some embodiments, the ADC is an ADC of the invention.

[0039] According to some embodiments, the cytotoxic immune cells comprise CD8 T cells, natural killer cells, chimeric antigen receptor (CAR) T cells or a combination thereof.

[0040] According to some embodiments, the method is a method of enhancing CAR-T therapy and further comprises administering CAR-T cells to the subject.

[0041] According to another aspect, there is provided a pharmaceutical composition comprising a population of T cells comprising an inhibitory nucleic acid molecule comprising a sequence reverse complimentary to a sequence of BMP1 or comprising a genetic knockout of a BMP1 genomic locus and a pharmaceutically acceptable carrier, excipient or adjuvant.

[0042] According to some embodiments, at least 85% of cells in the composition are T cells comprising the inhibitory nucleic acid molecule comprising a sequence reverse complementary to a sequence of BMP1 or comprising a genetic knockout of a BMP1 genomic locus.

[0043] According to some embodiments, the T cells comprise a CRISPR knockout of the BMP1 genomic locus.

[0044] According to some embodiments, the CRISPR knockout was using an sgRNA comprising a forward strand comprising or consisting of SEQ ID NO: 23 and a reverse strand comprising or consisting of SEQ ID NO: 24.

[0045] According to some embodiments, the T cells are CAR-T cells.

[0046] According to some embodiments, the pharmaceutical composition is for use in adoptive cell transfer (ACT).

[0047] According to some embodiments, the pharmaceutical composition is for use in combination with an anti-PD-1 immunotherapy in treating cancer in a subject in need thereof.

[0048] According to some embodiments, the pharmaceutical composition is for use in treating a solid cancer in a subject in need thereof.

[0049] According to another aspect, there is provided a method of producing an antibody drug conjugate (ADC), the method comprising:a. providing an antibody or antigen binding fragment thereof that binds to PD-1 and blocks or inhibits binding to PD-L1; b. providing an agent that binds to BMP1 and inhibits BMP1 protease function; and c. conjugating the provided agent to the provided antibody or antigen binding fragment thereof; thereby producing an ADC.

[0050] According to some embodiments, step (a) comprises providing an antibody or antigen binding fragment thereof that binds to PD-1, measuring the ability of the provided antibody or antigen binding fragment thereof to block or inhibit PD-1 binding to PD-L1 and selecting an antibody or antigen binding fragment thereof that blocks or inhibits the PD-1 binding to PD-L1.

[0051] According to some embodiments, the providing an antibody or antigen binding fragment thereof that binds to PD-1 comprises: a. immunizing an organism with a PD-1 extracellular domain or a fragment thereof and collecting antibodies from the immunized organism; or b. screening a library of antibodies or antigen binding fragments thereof for binding to a PD-1 extracellular domain or a fragment thereof and selecting an agent that binds.

[0052] According to some embodiments, step (b) comprises providing an agent that binds to BMP1, measuring the ability of the provided antibody or antigen binding fragment thereof to inhibit BMP1 protease function and selecting an agent that inhibits BMP1 protease function.

[0053] According to some embodiments, the providing an agent that binds to BMP1 comprises: a. immunizing an organism with BMP1 or a fragment thereof and collecting antibodies from the immunized organism; or b. screening a library of agents for binding to BMP1 or a fragment thereof and selecting an agent that binds.

[0054] According to some embodiments, the method further comprises contacting the provided agent in combination with the provided antibody or antigen binding fragmentthereof with a cancer, and contacting the agent conjugated to the antibody or antigen binding fragment thereof with the cancer and selecting an ADC that produces a superior anticancer effect to the combination.

[0055] According to another aspect, there is provided an ADC produced by a method of the invention.

[0056] 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 preferred embodiments 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

[0057] Figures 1A-1C: ECM content is upregulated in tumors from mice treated with anti-PDl. (1A-1B) C57 / B1 mice bearing LLC tumors were treated with anti-PDl or IgG control. After two weeks, tumors were removed and sectioned. (1A) The tumors were analyzed for ECM content using Sirius red and second harmonic generation (SHG). ECM quantification is shown. As seen in the figure, fibrillar collagen are denser in tumors from anti-PDl treated mice compared to IgG control. (IB) In parallel, tumors were analyzed for ECM content using hydroxyproline and sircol assays as well as prepared for RNA assessment of selected ECM associated genes. Based on the data, tumor ECM is denser and the content is higher in mice treated with anti-PDl compared to control. Statistical significance for was assessed using t test when comparing IgG control and anti-PDl. Significant p values are shown as *p<0.05. (1C) C57 / B1 mice bearing LLC tumors were treated with anti-PD-Ll, anti-CTLA4 or IgG control. After two weeks, tumors were removed and sectioned. The tumors were analyzed for ECM content using Sirius red. The plot shows the area of fibrillar collagen which was unaffected by the two immunotherapy treatments.

[0058] Figures 2A-2B: CD8+ T cells may account for ECM changes following anti-PDl therapy. (2A) SCID mice bearing 4T1 tumors were treated with anti-PDl or IgG control. After 2 weeks, tumors were removed and sectioned for the analysis of ECM content using Sirius red staining. Micrographs and quantification of data are shown. There is no difference in ECM content between IgG and anti-PDl treated mice in these SCID mice. (2B) In a separate experiment, the SCID mice were adoptively transferred with CD8+ T cells isolatedfrom the spleens of BALB / c mice. When tumors reached 50mm3, the mice were adoptively transferred with CD8+ T cells, after which treatment with anti-PDl or IgG control was initiated. After 2 weeks, tumors were removed and analyzed for ECM content using Sirius red staining. Micrographs followed by ECM content quantifications are shown. As can be seen, only in mice adoptively transferred with CD8+ T cells and treated with anti-PDl, tumor ECM displayed higher density. Statistical significance was assessed using one way ANOVA followed by Tukey post-test, when the comparison was carried out between more than two groups, and student t-test when the comparison was carried out by only two groups. Significant p values are shown as **p<0.01 and ***p<0.001.

[0059] Figures 3A-3B: BMP1 is overexpressed in CD8+ T cells from mice treated with anti-PDl. LLC cancer cells were implanted in C57B1 / 6 mice. When tumors reached 50mm3, treatment with anti-PDl or IgG control was initiated. After 2 weeks, spleens were removed and CD8+ T cells were isolated. (3A) mRNA was extracted from the cells, and the expression of selected genes was analyzed by RT-PCR. As shown, levels of BMP1 were significantly upregulated in mice treated with anti-PDl compared to IgG control. (3B) In parallel, CD8+ T cells were cultured in serum-free medium for 24 hours in a concentration of 5xl06 / ml. Conditioned medium was analyzed for the protein levels of BMP1, and similar to RT-PCR levels, it displayed higher levels of BMP1 in anti-PDl group compared to control. Statistical significance for was assessed using t test when comparing IgG control and anti-PDl. Significant p values are shown as *p<0.05.

[0060] Figures 4A-4C: Inhibition of BMP1 in CD8+ T cells resulted in reduced tumor growth. (4A) Mice bearing 4T1 tumors were treated with UK or vehicle control for 2 weeks. Tumor growth was assessed over time. The results demonstrate that tumors from mice treated with UK resulted in accelerated tumor growth compared to control. (4B-4C) 4T1 tumors were implanted in SCID mice. The SCID mice were adoptively transferred with CD8+ T cells that underwent BMP1 knocked down (KD) or control. (4B) Tumor growth was assessed over time. Notably, tumors from mice adoptively transferred with CD8+ T cells that lack the expression of BMP 1 were smaller than those of control mice. After 10 days, tumors were removed and sectioned. Tumor sections were assessed for ECM content using Sirius red staining (PSR) and second harmonic generation (SGH) (4C). The results show no difference in ECM content, at this time point, between the two groups, further suggesting that BMP1 inhibition in non-activated CD8+ T cells did not change ECM content. Statistical significance for was assessed using t test comparing two groups. Significant p values are shown as *p<0.05.

[0061] Figures 5A-5D: The combination of anti-PDl and BMP1 inhibition resulted in improved outcomes in mice resistant to anti-PDl therapy. BALB / c or C57B1 / 6 mice were implanted with (5A) 4T1 breast, (5B) B16 melanoma and (5C) lung carcinoma (LLC) cancer cells. When tumors reached 50mm3, treatment with anti-PDl, UK (BMP1 inhibitor) or their combination, was initiated for 2 weeks. Tumor growth was assessed, and at endpoint, tumors were removed and sectioned. Tumor sections were stained with Sirius red. Micrographs followed by collagen quantification is shown. As can be appreciated from this figure, in all tumor models anti-PDl therapy resulted in no changes in tumor growth, therefore suggesting that these tumors are resistant to anti-PDl therapy. However, in all cases, the combination of UK with anti-PDl resulted in enhanced anti-tumor activity followed reduced tumor ECM content. Notably, UK monotherapy, does not always tend to grow faster than control. Statistical significance was assessed using one way ANOVA followed by Tukey post-test. Significant p values are shown as *p<0.05 and **p<0.01. (5D) Heat map of percentage of different immune cells out of all CD45 cells in the tumor based on FACS analysis.

[0062] Figures 6A-6D: ADC administration results in improved outcomes in mice resistant to anti-PDl therapy. BALB / c or C57B1 / 6 mice were implanted with (6A-B) 4T1 breast, and (6C-D) lung carcinoma (LLC) cancer cells. When tumors reached 50mm3, treatment with anti-PDl, UK (BMP1 inhibitor), their combination, or the ADC of the invention was initiated for 2 weeks. Tumor growth was assessed, and at endpoint, tumors were removed and measured. (6A, 6C) Line graphs plotting tumor volume over time and plots providing the final tumor volume at sacrifice. Statistical significance was assessed using one way ANOVA followed by Tukey post-test. Significant p values are shown as *p<0.05 and **p<0.01. (6B, 6D) Heat maps of percentage of different immune cells out of all CD45 cells in the tumor based on FACS analysis.

[0063] Figure 7: Bar graph of secreted BMP1 in media of PBMCs with and without transfection of an anti-CD19 CAR.DETAILED DESCRIPTION OF THE INVENTION

[0064] The present invention, in some embodiments, provides methods of improving PD-1 based immunotherapy in a subject suffering from a solid cancer, comprising decreasing bone morphogenetic protein 1 (BMP1) levels or function in T cells or in a tumor microenvironment (TME) or extracellular matrix (ECM) of the solid cancer are provided.Methods of treating a solid cancer comprising administering a PD-1 based immunotherapy and decreasing BMP1 levels or function in T cells or a TME or ECM of the solid cancer are also provided. Methods of enhancing cytotoxic immune cell infiltration into a solid cancer, decreasing myeloid cell infiltration into the solid cancer or both in a subject in need thereof, comprising decreasing BMP1 levels or function in T cells or a TME or ECM of the solid cancer are also provided. Antibody drug conjugates (ADCs) comprising an anti-PDl blocking antibody and a BMP1 inhibiting small molecule or antibody or antigen binding fragment thereof small molecule inhibitor are also provided, as are pharmaceutical compositions comprising the ADCs or comprising a population of T cels comprising an inhibitory nucleic acid molecule comprising a sequence reverse complementary to a sequence of BMP 1 or comprising a genetic knockout of a BMP1 genomic locus.

[0065] The invention is based, at least in part, on the surprising finding of changes in the tumor ECM following immunotherapy. It was found that mice treated with anti-PDl immunotherapy showed massive ECM remodeling and ECM dumping in the tumors. These effects were associated with the secretion of BMP 1 from activated cytotoxic T cells upon anti-PDl therapy. Treatment with anti-PDl in mice lacking the lymphoid compartment (SCID mice) resulted in no changes in tumor ECM, unless the mice were previously adoptively transferred with T cells. It was further demonstrated that pharmacological inhibition of BMP 1 with anti-PDl sensitizes tumors otherwise resistant to immunotherapy. Lastly, genetic inhibition of BMP1 solely in cytotoxic T cells, further demonstrates its ECM associated activity following anti-PDl therapy. Further, the combined treatment, especially by way of administering an antibody drug conjugate (ADC) of the anti-PDl antibody and the BMP1 inhibitor, greatly increased the number of cytotoxic immune cells infiltrating the tumor, while simultaneously decreasing the number of myeloid cells infiltrating. Surprisingly, the ADC was found to be superior to just administering each component alone and allowed for a significant decrease in the total inhibitor being administered. These results therefore highlight that combining anti-PDl with BMP1 inhibition specifically in T cells, and especially with the ADC of the invention can improve immunotherapy outcome and sensitize tumors to immunotherapy.

[0066] By a first aspect, there is provided a method of improving PD-1 based immunotherapy in a subject, the method comprising decreasing morphogenetic protein 1 (BMP1) levels or function in the subject, thereby improving PD-1 based immunotherapy.

[0067] By another aspect, there is provided a method of treating cancer in a subject, the method comprising administering to the subject a PD-1 based immunotherapy and decreasing BMP1 levels or function in the subject, thereby treating cancer.

[0068] By another aspect, there is provided a method of enhancing cytotoxic immune cell infiltration into a solid cancer, the method comprising decreasing morphogenetic protein 1 (BMP1) levels or function in the subject, thereby enhancing cytotoxic immune cell infiltration into a solid cancer.

[0069] By another aspect, there is provided a method of decreasing myeloid cell infiltration into a solid cancer, the method comprising decreasing morphogenetic protein 1 (BMP1) levels or function in the subject, thereby decreasing myeloid cell infiltration into a solid cancer.

[0070] In some embodiments, the subject is an animal. As used herein, the term “subject” refers to a full animal and not merely cells or cells in a dish. Thus, administering to a subject refers to providing a therapeutic to a living being and not merely to an experiment in a laboratory such as giving the therapeutic to cells in culture. In some embodiments, the subject is a mammal. In some embodiments, the subject is a human. In some embodiments, the subject suffers from cancer. In some embodiments, the subject is in need of a method of the invention.

[0071] In some embodiments, the cancer is a solid cancer. In some embodiments, the cancer is a tumor. In some embodiments, the cancer comprises a tumor microenvironment (TME). In some embodiments, the cancer comprises extracellular matrix (ECM). Examples of solid cancers include, but are not limited to brain cancer, breast cancer, bone cancer, fat cancer, retinoblastoma, head and neck cancer, tongue cancer, nasopharyngeal cancer, pharyngeal cancer, throat cancer, esophageal cancer, stomach cancer, gastrointestinal cancer, intestinal cancer, lung cancer, colon cancer, colorectal cancer, liver cancer, renal cell carcinoma, pancreatic cancer, gallbladder cancer, penile cancer, thymus cancer, thyroid cancer, urogenital cancer, prostate cancer, kidney cancer, ovarian cancer, cervical cancer, testicular cancer, skin cancer, glioblastoma multiforme (GBM), and uterine cancer. In some embodiments, the cancer is lung cancer. In some embodiments, the cancer is skin cancer. In some embodiments, the cancer is breast cancer. In some embodiments, the cancer is selected from lung cancer, skin cancer and breast cancer. In some embodiments, the cancer is a carcinoma. In some embodiments, the cancer is a melanoma.

[0072] In some embodiments, the cancer is a cancer that can be treated with PD-1 / PD-L1 based immunotherapy. In some embodiments, PD-1 / PD-L1 based immunotherapy is PD-1 based immunotherapy. In some embodiments, the subject has undergone PD-1 / PD-L1 based immunotherapy. In some embodiments, the subject is a non-responder to PD-1 / PD-L1 based immunotherapy. In some embodiments, the cancer is resistant to PD-1 / PD-L1 based immunotherapy. In some embodiments, the cancer is resistant to PD-1 based immunotherapy. In some embodiments, the subject is naive to PD-1 / PD-L1 based immunotherapy. In some embodiments, the methods of the invention are performed together with PD-1 based immunotherapy. In some embodiments, the methods of the invention are performed before PD-1 based immunotherapy. In some embodiments, the method comprises administering adaptive cell transfer (ACT) therapy to the subject. In some embodiments, the method comprises administering CAR-T therapy to the subject.

[0073] In some embodiments, the method comprises administering the PD-1 based immunotherapy to the subject. In some embodiments, a PD-1 based immunotherapy is an anti-PDl immunotherapy. In some embodiments, the PD-1 based immunotherapy comprises administering an anti-PDl antibody. In some embodiments, the antibody is a blocking antibody. In some embodiments, the PD-1 based immunotherapy comprises PD-1 checkpoint blockade. In some embodiments, the therapy comprises blockade of the PD-1 checkpoint. In some embodiments, immune checkpoint blockade comprises administering an immune checkpoint inhibitor. In some embodiments, the subject in need of PD-1 based immunotherapy suffers from cancer. PD-1 based immunotherapies are well known in the art and any such therapy may be used as part of the methods of the invention. In some embodiments, the immune checkpoint inhibitor is an antibody. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the anti-PDl blocking antibody is selected from pembrolizumab, nivolumab, pidilizumab, cemiplimab, dostarlimab, retifanlimab and toripalimab. In some embodiments, the anti-PDl blocking antibody is selected from pembrolizumab, nivolumab, cemiplimab, dostarlimab, retifanlimab and toripalimab. In some embodiments, the anti-PDl blocking antibody is selected from pembrolizumab and nivolumab.

[0074] 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 polypeptidechains, 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 in soluble 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.

[0075] 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 anti-PDl antibody is an IgG4 antibody. In some embodiments, the anti -BMP 1 antibody is an IgGl antibody.

[0076] In some embodiments, the cancer is a cancer that can be treated with adoptive cell transfer (ACT). In some embodiments, the cancer is a cancer that can be treated with chimeric antigen receptor (CAR) T cell therapy. In some embodiments, the adoptive transfer is an adoptive transfer of CAR T cells. In some embodiments, the subject has undergone ACT or CAR-T therapy. In some embodiments, the subject is a non-responder to ACT or CAR-T therapy. In some embodiments, the cancer is resistant to ACT or CAR-T therapy. In some embodiments, the subject is naive to ACT or CAR-T therapy. In some embodiments, the methods of the invention are performed together with ACT or CAR-T therapy. In some embodiments, the methods of the invention are performed before ACT or CAR-T therapy. In some embodiments, enhances is improves. In some embodiments, enhances is increases.

[0077] In some embodiments, the method comprises administering ACT therapy to the subject. In some embodiments, the method comprises administering CAR-T therapy to the subject. In some embodiments, ACT comprises transfer of T cells from a healthy donor to the subject. In some embodiments, ACT comprises transfer of cytotoxic immune cells from a healthy donor to the subject. In some embodiments, the T cells are CAR-T cells. In someembodiments, cytotoxic immune cells are CD8 T cells and natural killer (NK) cells. In some embodiments, cytotoxic immune cells are CD8 T cells. In some embodiments, cytotoxic immune cells are CAR-T cells. In some embodiments, CAR-T therapy comprises transfer of CAR-T cells to the subject. In some embodiments, the T cells are autologous to the subject. In some embodiments, the CAR-T cells are autologous to the subject. In some embodiments, the T cells are syngeneic to the subject. In some embodiments, the CAR-T cells are syngeneic to the subject. In some embodiments, the T cells are allogeneic to the subject. In some embodiments, the CAR-T cells are allogeneic to the subject.

[0078] In some embodiments, CAR-T cells are T cells that are modified ex vivo to express a CAR. CARs are well known in the art. Their structure (extracellular antigen binding domain, transmembrane domain, intracellular domain bearing at least one T cell activating motif, e.g., CD3zeta, ITAM domains) are well-known in the art. A skilled artisan will be familiar with first generation, second generation and third generation CARs and any can be used. The specific CAR to be used to target a specific cancer can be selected by a skilled artisan. CAR-T cells are currently only approved for blood cancers due to their lack of penetrance into solid tumors. The instant methods solve this problem by making the tumor ECM more accessible and increase the cytotoxic activity of T cells through the inhibition of TGF-p. This opens a wide variety of CARs that can now be used that have never been used before. Examples are CARs that can potentially be used against solid tumors but heretofore have not include, but are not limited to anti-CD19, anti-mesothelin CARs, anti-HER2 CARs, anti-EGFR CARs, anti-GD2 CARs, anti-Claudinl8.2 CARs, and anti-HHLA2 CARs, although all CAR-T cells will be enhanced by the method of the invention, since the activity of BMP 1 inhibition is on the T cell and not on the CAR. In some embodiments, the CAR is an anti-CD19 CAR.

[0079] As used herein, the terms “treatment” or “treating” of a disease, disorder, or condition encompasses alleviation of at least one symptom thereof, a reduction in the severity thereof, or inhibition of the progression thereof. Treatment need not mean that the disease, disorder, or condition is totally cured. To be an effective treatment, a useful composition herein needs only to reduce the severity of a disease, disorder, or condition, reduce the severity of symptoms associated therewith, or provide improvement to a patient or subject’s quality of life. In some embodiments, treating cancer comprises shrinking the tumor. In some embodiments, treating cancer comprises killing tumor cells. In some embodiments, treating cancer comprises extending the life of the subject.

[0080] Bone Morphogenetic Protein 1 (BMP1) is a protein that plays a crucial role in the formation of the ECM and the development of various tissues. Unlike other BMPs, which are part of the Transforming Growth Factor-beta (TGF-p) superfamily and are primarily involved in bone and cartilage formation, BMP1 is a metalloprotease. It is not directly involved in the signaling pathways that induce bone and cartilage formation but rather in the processing of converting precursor proteins into their mature, functional forms. BMP1 is known to be involved in the cleavage of procollagen, the precursor of collagen, into its mature form. This process is essential for the proper assembly of collagen fibers, which are critical components of the ECM. BMP1 also processes other ECM proteins, such as laminin and fibronectin, contributing to tissue integrity and repair. BMP1 contains several domains, including a protease domain, a CUB domain, and an EGF-like domain, which are important for its enzymatic activity and interactions with other proteins. BMP1 also has additional signaling roles.

[0081] The human BMP1 gene can be found at Entrez gene# 649. The human BMP1 protein can be found in Uniprot entry Pl 3497. There are several different splice isoforms of the human BMP1 mRNA, and these can be found in RefSeq numbers: NM 001199, NM_001199, NM_006129, NM_033403 and NM_033404. Two protein isoforms of human BMP1 are known and these can be found in RefSeq numbers: NP 001190 and NP 006120.

[0082] In some embodiments, the method comprises decreasing BMP1 levels. In some embodiments, levels are expression. In some embodiments, levels are expression levels. In some embodiments, levels are mRNA levels. In some embodiments, levels are protein levels. In some embodiments, the method comprises decreasing BMP1 section. In some embodiments, the secretion is from cytotoxic immune cells. In some embodiments, the method comprises decreasing BMP1 function. In some embodiments, the function is proteinase activity. In some embodiments, the function is protease activity. In some embodiments, the function is metalloprotease activity. In some embodiments, decreasing function is blocking function. In some embodiments, decreasing function is inhibiting function. In some embodiments, decreasing is inhibiting BMP1 protease activity. In some embodiments, protease activity or function is cleavage. In some embodiments, the cleavage is cleavage of an ECM protein. Examples of ECM proteins cleaved by BMP1 include, but are not limited to collagen, laminin and fibronectin. In some embodiments, the cleavage is cleavage of procollagen to collagen. In some embodiments, the cleavage is laminin processing. Cleavage assays for determining BMP1 function are also well known in the art and generally involve the provision of a substrate protein (which can be labeled or unlabeled)and incubation with BMP1 and the inhibitor. The cleaved form of the protein is then quantified to determine the percent cleavage and thus the functionality of the BMP1 and, reciprocally, the inhibitor. Methods of quantifying the cleavage products include, Western blot, Mass-Spec and fluorescent or colorimetric assays (when the substrate is labeled).

[0083] In some embodiments, the decrease is at least a 50, 60, 70, 75, 80, 85, 90, 92, 95, 97, 99 or 100% decrease. Each possibility represents a separate embodiment of the invention. In some embodiments, the decrease is at least a 50% decrease. In some embodiments, the decrease is at least a 85% decrease. In some embodiments, the decrease is at least a 90% decrease. In some embodiments, the decrease is at least a 95% decrease. In some embodiments, the decrease is at least a 97% decrease. In some embodiments, the decrease is at least a 99% decrease. In some embodiments, the decrease is a 100% decrease.

[0084] In some embodiments, decreasing is to below a predetermined threshold. In some embodiments, decreasing is to below detectable levels. In some embodiments, decreasing is knocking out BMP1 expression. In some embodiments, decreasing is silencing BMP1 expression. In some embodiments, decreasing renders BMP1 expression undetectable. In some embodiments, decreasing renders BMP1 function undetectable. Methods of measuring BMP1 mRNA and protein expression are standard in the art and include for example.

[0085] In some embodiments, decreasing is in immune cells of the subject. In some embodiments, decreasing is decreasing expression of BMP 1 in immune cells of the subject. In some embodiments, decreasing is decreasing secretion of BMP1 from immune cells of the subject. In some embodiments, the immune cells are T cells. In some embodiments, the T cells are CD4 and CD8 T cells. In some embodiments, the T cells are CD8 T cells. In some embodiments, the immune cells are cytotoxic immune cells. In some embodiments, cytotoxic immune cells are selected from T cells and natural killer (NK) cells. In some embodiments, the cytotoxic immune cells are cytotoxic T lymphocytes (CTLs). In some embodiments, the immune cells are tumor resident immune cells. In some embodiments, the immune cells are tumor infiltrating lymphocytes (TILs). In some embodiments, the immune cells are tumor resident T cells.

[0086] In some embodiments, the decreasing is in the tumor microenvironment (TME). In some embodiments, the decreasing is around the tumor. In some embodiments, the decreasing is proximal to the tumor. As used herein, the “TME” refers to the complex and dynamic ecosystem surrounding a tumor, comprising various cellular and non-cellular components that interact with cancer cells. In some embodiments, is immune cells in theTME. In some embodiments, the TME is the extracellular matrix (ECM) in the TME. In some embodiments, the decreasing is in the ECM of the cancer.

[0087] In some embodiments, decreasing comprises administering to the subject an agent that decreases BMP1 levels or function. In some embodiments, decreasing comprises administering to the subject a BMP1 blocking agent. In some embodiments, a BMP1 blocking agent decrease BMP1 levels or function. In some embodiments, decreasing comprises administering to the subject an agent that decreases BMP1 expression or function. In some embodiments, the agent is targeted to immune cells. In some embodiments, the agent is targeted to T cells. In some embodiments, the agent comprises T cell targeting moiety. In some embodiments, the agent is targeted to the TME. In some embodiments, the agent comprises a TME targeting moiety. In some embodiments, the agent is targeted to ECM. In some embodiments, the ECM is ECM of the cancer. In some embodiments, the agent comprises an ECM targeting moiety. In some embodiments, the agent comprises a cancer ECM targeting moiety.

[0088] In some embodiments, the moiety is an antibody. In some embodiments, the antibody is specific to T cells. In some embodiments, the antibody is an anti-CD3 antibody. Anti-CD3 antibodies are well known in the art. A sufficient number of antibodies that bind to CD3 are known in the art such as to be representative of the genus of CD3 binding antibodies. In some embodiments, the antibody is an anti-PD-1 antibody. PD-1 is expressed by cytotoxic immune cells and thus it can be used as a target to bring the agent to T cells. A sufficient number of antibodies that bind to PD-1 are known in the art such as to be representative of the genus of PD-1 binding antibodies. In some embodiments, the antibody is a TME antibody. In some embodiments, the antibody is an ECM antibody. ECM components are well known in the art, as are antibodies to these components. Examples include collagen I, collagen IV, fibronectin, and laminin. The TME is known to be acidic as well as hypoxic, thus targeting moieties specific to acidic or hypoxic conditions can also be used. In some embodiments, the antibody is an anti-cancer antibody. Cancer markers such as EpCAM, HER2, EGFR and PD-L1 can be used to target to the TMEZECM and antibodies to these targets are well known and widely available. In some embodiments, the antibody binds to cancer-associated fibroblasts (CAFs). CAR markers include a-SMA, FAP, PDGFR-B to name but a few and antibodies to these targets are well known.

[0089] In some embodiments, the decreasing comprises administering a BMP1 inhibitor. In some embodiments, the agent is a BMP1 inhibitor. In some embodiments, the inhibitor is a specific inhibitor. In some embodiments, the inhibitor is specific to BMP1. In someembodiments, the inhibitor is a BMP1 selective inhibitor. In some embodiments, a specific inhibitor does not substantially or detectibly inhibit another BMP protein. In some embodiments, a specific inhibitor does not substantially or detectibly inhibit another metalloprotease. In some embodiments, a specific inhibitor does not substantially or detectibly inhibit another tolloid-like proteinase. In some embodiments, the inhibitor comprises a T cell targeting moiety. In some embodiments, the agent is conjugated to a T cell targeting moiety. In some embodiments, the inhibitor comprises a TME targeting moiety. In some embodiments, the agent is conjugated to a TME targeting moiety. In some embodiments, the inhibitor comprises an ECM targeting moiety. In some embodiments, the agent is conjugated to an ECM targeting moiety. In some embodiments, the ECM is cancer ECM.

[0090] In some embodiments, the inhibitor is a small molecule inhibitor. As used herein, the term “small molecule” refers to a molecule with a molecular weight of less than 1 kiloDalton (IkD). In some embodiments, the small molecule comprises a weight of less than 900 Daltons. In some embodiments, the small molecule comprises a weight of less than 500 Daltons. In some embodiments, the small molecule is organic. In some embodiments, the small molecule is not proteinaceous. Examples of BMP1 inhibitors include but are not limited to UK383367 (CAS# 348622-88-8), S33A, RXP-1001 and FG-2575 (FibroGen). BMP1 inhibitors are disclosed in Talantikite et al., “Inhibitors of BMP-l / tolloid-like proteinases: efficacy, selectivity and cellular toxicity”, FEBS Open Bio. 2018 Dec; 8(12): 2011-2021, the contents of which are hereby incorporated by reference in their entirety. In some embodiments, the BMP1 inhibitor is UK383367. In some embodiments, the BMP1 inhibitor is S33A. In some embodiments, the BMP1 inhibitor is RXP-1001. In some embodiments, the BMP1 inhibitor is FG2575. In some embodiments, S331 is. In some embodiments, RXP-1001 is Fmoc-A-Y-Q- ala(PO2-CH2)hPhe-E-P-NH2. In some embodiments, RXP-1001 is. In some embodiments, RXP-1001 is a phosphinic peptide. In some embodiments, the BMP1 inhibitor is conjugated to the anti-PD- 1 antibody or antigen binding fragment thereof. In some embodiments, the BMP1 inhibitor is conjugated to the anti-PD-1 antibody or antigen binding fragment thereof. In some embodiments, the BMP1 small molecule inhibitor is conjugated to the anti-PD-1 antibody or antigen binding fragment thereof. In some embodiments, UK383367 is conjugated to the anti-PD-1 antibody or antigen binding fragment thereof. In some embodiments FG2575 is conjugated to the anti-PD-1 antibody or antigen binding fragment thereof. In some embodiments, RXP-1001 is conjugated to the anti-PD-1 antibody or antigen binding fragment thereof. In some embodiments, S33A is conjugated to the anti-PD-1 antibody or antigen binding fragment thereof.

[0091] In some embodiments, the agent is a proteinaceous agent. In some embodiments, the agent is an antibody or antigen binding fragment thereof. In some embodiments, the antibody is an anti -BMP 1 antibody. In some embodiments, the antibody binds to BMP1. In some embodiments, the antibody binds to the protease domain of BMP 1. In some embodiments, the antibody blocks or occludes the protease domain of BMP 1. In some embodiments, the antibody binds to, blocks or occludes the substrate binding domain of BMP1. In some embodiments, the antibody binds to, blocks or occludes the CUB domain of BMP1. In some embodiments, the antibody binds to, blocks or occludes the EGF-like domain of BMP1. The domains of human BMP1 are delineated in Uniprot entry P13497. Anti-BMPl blocking antibodies are well known in the art and a sufficient number of such antibodies are known and commercially available such as to define the entire genus. In some embodiments, the anti -B MP 1 antibody is an antibody or antigen binding fragment thereof disclosed in International Patent Application W02008011193, the contents of which are hereby incorporated by reference in their entirety. In some embodiments, the anti -B MP 1 antibody is an antibody or antigen binding fragment thereof with the same CDRs as an antibody or antigen binding fragment thereof disclosed in International Patent Application W02008011193.

[0092] In some embodiments, the proteinaceous agent is RXP-1001. In some embodiments, the proteinaceous agent is Sizzled protein. Sizzled is a xenopus protein that is known to bind to and inhibit BMP1. The protein can be found at Uniprot ID 073821 and A0A8J1MPX0. In some embodiments, the amino acid sequence of Sizzled is MSGVFLLLCASMLACTAAFDIGLSTKCVTIPTEMAMCNDVGYSEMRLPNLMGHT NMAEVVPKSAEWQNLLQTGCHPYARTFLCSLFAPVCLDTFIQPCRSMCVAVRDSC APVLACHGHSWPESLDCDRFPAGEDMCLDTLSKEYQYSYKELPKPSCQGCPLIEEF FSHKTVLEAFCDNNFAVKVKLAKKKSASGLYEYETEGPVEFIKQGLLLPYDTRTMI EQWLLINENCAQKLIRTRPTVYVIAGEIHHGKVKVNRIFHWQKKDSQLTLATRRW RHHKC (SEQ ID NO: 25, from 073821). In some embodiments, the amino acid sequence of Sizzled isMSGVFLLLCASMLACTAAFDIGLSTKCVTIPTEMAMCNDVGYSEMRLPNLMGHT NMAEVVPKSAEWQNLLQTGCHPYARTFLCSLFAPVCLDTFIQPCRSMCVAVRDSC APVLACHGHSWPESLDCDRFPAGEDMCLDTLSKEYQYSYKELPKPSCQGCPLIEEF FSHKTVLEAFCDNNFAVKVKLAKKKSASGLYEYETEGPVEFIKQGLLLPYDTRTMI EQWLLINENCAQKLIRTRPTVYVIAGDIHHGKVKVNRIFHWQKKDSQLTLATRRW RHHKC (SEQ ID NO: 26, from A0A8J1MPX0). In some embodiments, the agent is recombinant Sizzled. In some embodiments, Sizzled protein comprises or consists of SEQ ID NO:25 or SEQ ID NO: 26.

[0093] In some embodiments, decreasing comprises administering a nucleic acid molecule specific to BMP1. In some embodiments, decreasing comprises administering a nucleic acid molecule specific to the BMP1 gene. In some embodiments, decreasing comprises administering a nucleic acid molecule specific to a BMP1 mRNA. In some embodiments, the nucleic acid molecule is an inhibitory nucleic acid molecule. Examples of inhibitory nucleic acid molecules include siRNAs, shRNAs and the like. In some embodiments, the inhibitory nucleic acid molecule is comprised within a composition. Gene therapy whereby nucleic acid molecules are administered to a subj ect are well known in the art and the vectors, particles, nanoparticles and compositions for such delivery are well known in the art. In some embodiments, the nucleic acid molecule comprises a sequence reverse complimentary to a sequence of BMP1. In some embodiments, the composition comprising the nucleic acid molecule comprises a T cell targeting moiety. In some embodiments, the composition is a CRISPR-CAS composition. In some embodiments, the nucleic acid molecule is a guide RNA (gRNA).

[0094] In some embodiments, the nucleic acid molecule is reverse complementary to a BMP1 gene. In some embodiments, the nucleic acid molecule is reverse complementary to a BMP1 mRNA. In some embodiments, the nucleic acid molecule comprises at least 10, 15, 20, 22, 25, or 30 nucleotides. Each possibility represents a separate embodiment of the invention. In some embodiments, the nucleotides are reverse complementary to consecutive nucleotides from BMP1. In some embodiments, the sequence of a human BMP1 mRNA is provided in SEQ ID NO: 27. In some embodiments, the sequence of a human BMP1 mRNA is provided in SEQ ID NO: 28. In some embodiments, the sequence of a human BMP1 mRNA is provided in SEQ ID NO: 29. In some embodiments, the sequence of a human BMP1 mRNA is provided in SEQ ID NO: 30.

[0095] In some embodiments, the decreasing comprises administering to the subject at least one agent of the invention. In some embodiments, the decreasing comprises administering to the subject at least one antibody-drug conjugate (ADC) of the invention.

[0096] In some embodiments, the decreasing comprises adoptive cell transfer (ACT). In some embodiments, the treating comprises adoptive cell transfer. In some embodiments, the cells are T cells. In some embodiments, the ACT comprises administering a composition comprising T cells comprising a knockdown or knockout of BMP 1. In some embodiments, BMP1 is endogenous BMP1. In some embodiments, the T cells are CRISPR edited to remove BMP1 expression. In some embodiments, CAR-T cells are CRISPR edited to remove BMP1 expression. In some embodiments, BMP1 is knocked out in the cells. In some embodiments, BMP1 expression is silenced in the cells.

[0097] In some embodiments, the decreasing comprises administering an ADC comprising an anti-PDl blocking antibody conjugated to an anti -BMP 1 agent. In some embodiments, the decreasing comprises administering an ADC comprising an anti-PDl blocking antibody conjugated to a BMP1 small molecule inhibitor. In some embodiments, the decreasing comprises administering an ADC comprising an anti-PDl blocking antibody conjugated to a BMP1 blocking antibody. In some embodiments, blocking is blocking protease function. In some embodiments, the ADC is a bispecific antibody. In some embodiments, the decreasing comprises administering a bispecific antibody that binds to PD-1 and BMP1. In some embodiments, the bispecific antibody blocks PD-1 and BMP1. In some embodiments, blocks is blocks binding of PD-1 to PD-L1, PD-L2 or both. In some embodiments, blocks is blocks BMP1 protease function. In some embodiments, blocks is blocks BMP1 binding to a substrate. In some embodiments, the substrate is selected from procollagen, fibronectin andlaminin. In some embodiments, the substrate is selected from collagen, fibronectin and laminin.

[0098] 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. Other suitable routes of administration can include parenteral, subcutaneous, oral, intramuscular, intratumoral or intraperitoneal. In some embodiments, the administering is systemic administration. In some embodiments, a composition with a targeting moiety is administered and the administration is systemic administration. In some embodiments, the administering is intratumoral administration.

[0099] In some embodiments, a therapeutically effective amount of the PD-1 based immunotherapy is administered and a therapeutically effective amount of the BMP1 blocking agent is administered. The term "therapeutically effective amount" refers to an amount of a drug 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 in treating cancer. In some embodiments, the amount of BMP1 blocking agent is effective in combination with PD-1 based immunotherapy in treating cancer. In some embodiments, a therapeutically effective amount of the combination of the PD-1 based immunotherapy and the BMP1 blocking agent is administered. In some embodiments, effective is effective in combination in treating cancer. In some embodiments, effective is effective in producing a synergistic effect. In some embodiments, synergistic is synergistic in their ability to treat cancer. In some embodiments, synergistic is synergistic in shrinking the cancer.

[0100] By another aspect, there is provided an agent comprising an anti-PDl blocking antibody or antigen binding fragment thereof and a BMP1 blocking agent.

[0101] In some embodiments, the anti-PDl blocking antibody or antigen binding fragment thereof is conjugated to the BMP1 blocking agent. In some embodiments, the anti-PDl blocking antibody or antigen binding fragment thereof is connected to the BMP1 blocking agent. In some embodiments, the agent comprises a first moiety that is the anti-PDl blockingantibody or antigen binding fragment thereof and a second moiety that is the BMP1 blocking agent. In some embodiments, conjugated is linked. In some embodiments, linked is covalently linked. In some embodiments, linked is reversibly linked. In some embodiments, linked is irreversibly linked. In some embodiments, agent is a fusion protein. In some embodiments, the agent is not a fusion protein. In some embodiments, the linking is by a linker. In some embodiments, the linker is a peptide linker. In some embodiments, the linker is an amino acid linker. In some embodiments, the linker is a chemical linker. In some embodiments, the linker is stable in the bloodstream. In some embodiments, the linker is a cleavable linker.

[0102] In some embodiments, the anti-PDl blocking antibody is selected from pembrolizumab, nivolumab, pidilizumab, cemiplimab, dostarlimab, retifanlimab and toripalimab. In some embodiments, the anti-PDl blocking antibody is selected from pembrolizumab, nivolumab, cemiplimab, dostarlimab, retifanlimab and toripalimab. In some embodiments, the anti-PDl blocking antibody is selected from pembrolizumab and nivolumab. In some embodiments, the anti-PDl blocking antibody is an IgG4 antibody.

[0103] In some embodiments, the BMP1 blocking agent blocks BMP1 function. In some embodiments, the BMP1 blocking agent blocks BMP1 binding to its substrate. In some embodiments, the BMP1 blocking agent blocks BMP1 protease function. In some embodiments, the BMP1 blocking agent binds to or occludes the BMP1 protease domain.

[0104] In some embodiments, the BMP1 blocking agent is a small molecule. In some embodiments, the BMP1 blocking agent is an inhibitor. In some embodiments, the BMP1 blocking agent is a small molecule inhibitor. In some embodiments, the BMP1 inhibitor is selected from UK383367, S33A, RXP-1001 and FG-2575. In some embodiments, the BMP1 inhibitor is UK383367. In some embodiments, the BMP1 inhibitor is S33A. In some embodiments, the BMP1 inhibitor is RXP-1001. In some embodiments, the BMP1 inhibitor is FG2575. In some embodiments, the agent is an antibody drug conjugate (ADC). In some embodiments, the ADC comprises an anti-PDl blocking antibody or antigen binding fragment thereof conjugated to a BMP 1 inhibitor. In some embodiments, the ADC comprises an anti-PDl blocking antibody or antigen binding fragment thereof conjugated to a BMP1 small molecule inhibitor.

[0105] Methods of ADC generation and generally of conjugating small molecules to antibodies are well known and routine in the art. Any such method may be used to create the agent of the invention. Conjugation may be carried out by random conjugation (e.g., lysineconjugation, cysteine conjugation) or site-specific conjugation (e.g., using engineered cysteine residues, enzymatic conjugation or click chemistry). In some embodiments, the drug to antibody ratio (DAR) is 1 : 1. In some embodiments, the drug to antibody ratio is 2: 1 (such as when the drug is conjugated to each heavy chain of the antibody). In some embodiments, the DAR is at least 1 :1. In some embodiments, the DAR is at least 2: 1. In some embodiments, the DAR is at least 3: 1. In some embodiments, the DAR is at least 4: 1. In some embodiments, the DAR is between 2: 1 and 8: 1. ADCs that DARs of between 2 and 8 have been clinically approved and it is known that this range is acceptable. The DAR of the ADC of the invention was found to be about 4.3, which falls within this range.

[0106] In some embodiments, the agent of the invention is a fusion protein. In some embodiments, the BMP1 blocking agent is a proteinaceous agent. In some embodiments, the BMP1 blocking agent is a protein or peptide. In some embodiments, the protein is a recombinant protein. In some embodiments, the BMP1 blocking protein is Sizzled. In some embodiments, Sizzled comprises or consists of SEQ ID NO: 25 or SEQ ID NO: 26. These two amino acid sequences differ only in the amino acid at position 248 and both recite a negatively charged amino acid (E or D). In some embodiments, Sizzled is conjugated to the anti-PD-1 antibody or antigen binding fragment thereof. In some embodiments, Sizzled is conjugated by a linker. In some embodiments, the linker is an amino acid linker. In some embodiments, the linker is a chemical linker. In some embodiments, the linker is of a sufficient length such that the antibody still binds to PD-1 and Sizzled still binds to BMP1.

[0107] As used herein, the terms “peptide”, "polypeptide" and "protein" are used interchangeably to refer to a polymer of amino acid residues. In another embodiment, the terms "peptide", "polypeptide" and "protein" as used herein encompass native peptides, peptidomimetics (typically including non-peptide bonds or other synthetic modifications) and the peptide analogues peptoids and semipeptoids or any combination thereof. In another embodiment, the peptides polypeptides and proteins described have modifications rendering them more stable while in the body or more capable of penetrating into cells. In one embodiment, the terms “peptide”, "polypeptide" and "protein" apply to naturally occurring amino acid polymers. In another embodiment, the terms “peptide”, "polypeptide" and "protein" apply to amino acid polymers in which one or more amino acid residue is an artificial chemical analogue of a corresponding naturally occurring amino acid.

[0108] In some embodiments, the BMP1 blocking agent is an antibody or antigen binding fragment thereof. In some embodiments, the antibody is an anti -BMP 1 antibody. In some embodiments, the anti -B MP 1 antibody is an IgG4 antibody. In some embodiments, the anti-BMP1 antibody is an IgGl antibody. In some embodiments, the agent of the invention is a bispecific antibody. In some embodiments, the bispecific antibody is specific to PD-1 and BMP1. In some embodiments, the bispecific antibody binds to PD-1 and binds to BMP1. In some embodiments, the anti-PDl antibody is conjugated to the anti -BMP 1 antibody. In some embodiments, the anti-PDl antibody is linked to the anti -B MP 1 antibody. In some embodiments, the anti -B MP 1 antibody is a BMPl blocking antibody.

[0109] Anti -B MP 1 antibodies are well known in the art and a sufficient number are known and commercially available such as to define the entire genus. Such antibodies can be purchased from ThermoFisher (cat# PA5-82506, MA5-26209, PA5-103660, PA5-37844, PA5-47222, PAI-24843), Fabgennix (cat# BMP-101AP, BMP-112AP), Abnova (cat# H00000649-B01P), GenTex (cat# GTX34060), R&D Systems (cat# AF1927), abeam (cat# ab28953, ab205394), and Sigma-Aldrich (cat# HPA014572) as well as others. Further, therapeutic anti-BMPl antibodies are described in Vukicevic et al., “Bone morphogenetic protein 1.3 inhibition decreases scar formation and supports cardiomyocyte survival after myocardial infarction”, Nat Commun. 2022; 13 : 81 and Bordukal-Niksic and Kufner, “BMP 1.3 protein as potential target in treatment of fibrosis”, RAD CASA - Medical Sciences. 548=56-57 (2021): 56-69, and International Patent Applications W02008 / 011193 and W02022 / 024034.

[0110] Many methods of generating bispecific antibodies are known and routine in the art and any such method may be used. Examples of such technologies include hybrid hybridoma technology, chemical cross-linking, quadroma technology, heterodimer heavy chain formation (e.g., knob-in-holes and similar mutations of the heavy chain), dual-variable domain technology, bispecific engagers (e.g., two scFvs linked by a flexible linker), CrossMab technology, dock-and-lock technology and recombinant DNA engineering. The bispecific antibody can be a full IgG or use only antigen binding domains such as scFvs or single domain antibodies. Regardless, the result is a single agent that binds both PD-1 and BMP1.

[0111] By another aspect, there is provided a composition comprising an agent of the invention.

[0112] In some embodiments, the composition is a pharmaceutical composition and further comprises a pharmaceutically acceptable carrier, excipient or adjuvant. As used herein, the term “carrier,” “adjuvant” or “excipient” refers to any component of a pharmaceutical composition that is not the active agent. As used herein, the term “pharmaceuticallyacceptable 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 Merck Index, Thirteenth Edition, Budavari et al., 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, micelies, insoluble monolayers, liquid crystals, phospholipid dispersions, lamellar layers and the like. Liposomes for use with the presently described peptides / proteins 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.

[0113] The carrier may comprise, in total, from about 0.1% to about 99.99999% by weight of the pharmaceutical compositions presented herein.

[0114] In some embodiments, the composition comprises a therapeutically effective amount of the agent of the invention. In some embodiments, effective is effective in treating cancer. In some embodiments, effective is effective in producing a synergistic effect between the BMP1 blocking agent and the PD-1 based immunotherapy. In some embodiments, synergistic is synergistic in their ability to treat cancer.

[0115] By another aspect, there is provided a composition comprising a population of immune cells comprising an inhibitory nucleic acid molecule comprising a sequence reverse complimentary to a sequence of BMP1 or comprising a genetic knockout of a BMP1 genomic locus.

[0116] In some embodiments, the immune cells are cytotoxic immune cells. In some embodiments, the immune cells are T cells. In some embodiments, the T cells are CD8 T cells. In some embodiments, the immune cells are CD8 and / or CD4 T cells. In some embodiments, at least 10, 20, 25, 30, 40, 50, 60, 70, 75, 80, 85, 90, 92, 95, 97, 99 or 100% of the cells in the composition comprise the inhibitory nucleic acid molecule comprising a sequence reverse complimentary to a sequence of BMP 1 or comprising a genetic knockout of a BMP1 genomic locus. Each possibility represents a separate embodiment of the invention. In some embodiments, at least 20% of the cells in the composition comprise theinhibitory nucleic acid molecule comprising a sequence reverse complimentary to a sequence of BMP1 or comprising a genetic knockout of a BMP1 genomic locus. In some embodiments, at least 85% of the cells in the composition comprise the inhibitory nucleic acid molecule comprising a sequence reverse complimentary to a sequence of BMP 1 or comprising a genetic knockout of a BMP1 genomic locus. In some embodiments, the composition consists of the immune cells. In some embodiments, the composition consists of T cells. In some embodiments, the composition consists of cells comprising a sequence reverse complimentary to a sequence of BMP1 or comprising a genetic knockout of a BMP1 genomic locus.

[0117] In some embodiments, the cells comprise an inhibitory nucleic acid molecule against BMP1. In some embodiments, the inhibitory nucleic acid molecule is a small interfering RNA (siRNA). In some embodiments, the inhibitory nucleic acid molecule is a short hairpin RNA (shRNA). In some embodiments, the cells comprise a CRISPR knockout of the BMP1 genomic locus. In some embodiments, the cells comprise a CRISPR knockout of the BMP1 gene. In some embodiments, the cells comprise a CRISPR knockout of BMP1 mRNA expression. In some embodiments, CRISPR is CRISPR-CAS. In some embodiments, CAS is CAS9. In some embodiments, the knockout was using a guide RNA (gRNA or sgRNA). In some embodiments, the sgRNA comprises a forward strand comprising or consisting of SEQ ID NO: 23. In some embodiments, the guide RNA comprises a reverse strand comprising or consisting of SEQ ID NO: 24.

[0118] In some embodiments, the composition is a pharmaceutical composition. In some embodiments, the composition comprises a pharmaceutically acceptable carrier, excipient or adjuvant. In some embodiments, the composition is for use in adoptive cell transfer (ACT). In some embodiments, the population is an ex vivo population. In some embodiments, the population is an isolated population. In some embodiments, the population is a purified population.

[0119] In some embodiments, the composition is for use in treating cancer. In some embodiments, the composition is for use in combination with an anti-PDl immunotherapy. In some embodiments, the composition is for use in combination with an anti-PDl immunotherapy for treating cancer. In some embodiments, the composition is for use in a method of the invention. In some embodiments, the composition is a therapeutic composition.

[0120] By another aspect, there is provided a method of producing an agent, the method comprising: a. providing an antibody or antigen binding fragment thereof that binds to PD-1 and blocks or inhibits binding to PD-L1; b. providing an agent that binds to BMP1 and inhibits BMP1 function; and c. conjugating the provided agent to the provided antibody or antigen binding fragment thereof; thereby producing an agent.

[0121] In some embodiments, the method is a method of producing an agent of the invention. In some embodiments, the method is a method of producing an anti-cancer agent. In some embodiments, the method is a method of producing an ADC. In some embodiment, the method is a method of producing a bispecific antibody. In some embodiments, the method is a method of designing the agent.

[0122] In some embodiments, step (a) comprises providing an anti-PDl immunotherapy. In some embodiments, step (a) comprises providing a PD-1 based immunotherapy. In some embodiments, step (a) comprises providing an antibody confirmed to bind PD1 and produce checkpoint blockade. In some embodiments, step (a) comprises generating the antibody. In some embodiments, step (a) comprises providing an antibody or antigen binding fragment thereof that binds to PD-1. In some embodiments, generating the antibody comprises immunizing an organism with a PD-1 extracellular domain or a fragment thereof and collecting antibodies from the immunized organism. Human PD-1 protein is provided in Uniprot ID Q15116 and RefSeq entry NP 005009. In some embodiments, human PD-1 protein comprises or consists of the amino acid sequence provided in SEQ ID NO: 33. In some embodiments, the extracellular domain of PD-1 is from amino acids 24-170 of SEQ ID NO: 33. In some embodiments, the organism is immunized with amino acids 24-170 of SEQ ID NO: 33. In some embodiments, the organism is selected from a mouse, a rat, a rabbit, a pig, a camel and a shark. In some embodiments, step (a) comprises screening a library of antibodies or antigen binding fragments thereof for binding to a PD-1 extracellular domain or a fragment thereof and selecting an agent that binds. In some embodiments, the fragment comprises the PD-L1 binding domain. In some embodiments, step (a) further comprises measuring the ability of the provided, generated or selected anti-PDl antibody or antigen fragment thereof to block or inhibit binding of PD-1 to PD-L1, PD-L2 or both. In some embodiments, step (a) further comprises selecting an anti-PDl antibody or antigen bindingfragment thereof that blocks or inhibits PD-1 binding to PD-L1, PD-L2 or both. In some embodiments, the binding is to PD-L1. In some embodiments, the blocking or inhibiting is by at least a threshold amount. In some embodiments, the blocking of inhibiting reduces binding to below a threshold level. In some embodiments, the selected antibody or antigen binding fragment thereof is the provided antibody or antigen binding fragment thereof.

[0123] In some embodiments, step (b) comprises providing an antibody or antigen binding fragment thereof. In some embodiments, step (b) comprises providing a BMPl inhibitor. In some embodiments, step (b) comprises provided a small molecule BMP1 inhibitor. In some embodiments, step (b) comprises providing an agent that binds BMP1. In some embodiments, step (b) comprises providing an antibody confirmed to bind BMP1 and produce checkpoint blockade. In some embodiments, step (b) comprises generating the antibody. In some embodiments, step (b) comprises providing an antibody or antigen binding fragment thereof that binds to BMP1. In some embodiments, generating the antibody comprises immunizing an organism with BMP1 or a fragment thereof and collecting antibodies from the immunized organism. In some embodiments, the organism is selected from a mouse, a rat, a rabbit, a pig, a camel and a shark. In some embodiments, step (b) comprises screening a library of antibodies or antigen binding fragments thereof for binding to BMP1 or a fragment thereof and selecting an agent that binds. In some embodiments, the fragment comprises the protease domain of BMP1. In some embodiments, BMP1 comprises or consists of the amino acid sequence provided in SEQ ID NO: 31. In some embodiments, BMP1 comprises or consists of the amino acid sequence provided in SEQ ID NO: 32. In some embodiments, step (b) further comprises measuring the ability of the provided, generated or selected anti -B MP 1 antibody or antigen fragment thereof to block or inhibit BMP1 function. In some embodiments, the function is protease function. In some embodiments, step (b) further comprises selecting an anti -B MP 1 antibody or antigen binding fragment thereof that blocks or inhibits BMP 1 binding to its substrate. In some embodiments, the binding is to procollagen. In some embodiments, the blocking or inhibiting is by at least a threshold amount. In some embodiments, the blocking of inhibiting reduces binding to below a threshold level. In some embodiments, the blocking of inhibiting reduces BMP1 function to below a threshold level. In some embodiments, the blocking of inhibiting reduces protease activity to below a threshold level. In some embodiments, the threshold is a predetermined threshold. In some embodiments, the selected antibody or antigen binding fragment thereof is the provided antibody or antigen binding fragment thereof.

[0124] In some embodiments, the conjugating comprises linking the provided antibody or antigen binding fragment thereof to the provided agent. In some embodiments, the linking is via a linker. In some embodiments, the linking is chemical linking. In some embodiments, the linker is reversible. In some embodiments, the linking is irreversible. In some embodiments, the conjugating comprises producing a fusion protein of the antibody and the agent. In some embodiments, the agent is an antibody and conjugating comprises producing a bispecific antibody. In some embodiments, the linker is an amino acid linker. In some embodiments, the conjugating is producing an ADC.

[0125] In some embodiments, the method further comprises contacting the provided agent in combination with the provided antibody or antigen binding fragment thereof with a cancer, and contacting the agent conjugated to the antibody or antigen binding fragment thereof with the cancer and selecting an ADC that produces a superior anticancer effect to the combination. In some embodiments, in combination is not as an ADC. In some embodiments, in combination is without conjugation. In some embodiments, in combination comprises the inhibitor and antibody being contacted at the same time. In some embodiments, the contacting is with cancer cells in vitro. In some embodiments, in vitro is in culture. In some embodiments, contacting is with cancer cell in vivo. In some embodiments, in vivo is in a rodent model. In some embodiments, the rodent is a mouse. In some embodiments, contacting is administering to a subject bearing the cancer. In some embodiments, the cancer is a tumor. In some embodiments, the subject is a rodent. In some embodiments, the subject is a mammal. In some embodiments, the subject is a human. It will be understood that an ADC can be based on its producing of a superior anticancer effect as compared to the two agent used together but without conjugation.

[0126] In some embodiments, superior is greater. In some embodiments, greater is significantly greater. In some embodiments, an anticancer effect is shrinking the tumor. In some embodiments, an anticancer effect is slowing growth of the tumor. In some embodiments, an anticancer effect is reducing metastasis. In some embodiments, an anticancer effect is increased immune cell invasion of the tumor. In some embodiments, the immune cell is a cytotoxic immune cell. In some embodiments, the anticancer effect is decreased myeloid cell invasion of the tumor. In some embodiments, decreased and increased are relative as compared to before administration.

[0127] By another aspect, there is provided an agent produced by a method of the invention.

[0128] By another aspect, there is provided a composition comprising an agent produced by a method of the invention.

[0129] 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.

[0130] 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 intended to 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.

[0131] 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."

[0132] 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.

[0133] 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.

[0134] 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

[0135] 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.MATERIALS AND METHODS

[0136] Cell lines: 4T1 murine mammary adenocarcinoma, EMT6 murine breast carcinoma, LLC (murine Lewis lung carcinoma) and B16-F1 (murine melanoma) cell lines were purchased from the American Type Culture Collection (Manassas, VA, USA) and were used within 6 months of thawing. Cells were routinely tested to be mycoplasma-free. All the celllines were maintained under 37°C and 5% CO2 conditions in Dulbecco's modified Eagle's medium (DMEM, Sigma-Aldrich, Rehovot, Israel, Cat# D5796) supplemented with 10% fetal calf serum (FCS, Biological Industries, Israel, Cat# 10270-106), 1% L-glutamine (Cat# 03-020-1B), 1% sodium pyruvate (Cat# S8636), and 1% Pen-Strep-Neomycin (Cat# 03-034- 1B) in solution (Biological Industries, Israel).

[0137] Animal models: The use of animals and experimental protocols were approved by the Animal Care and Use Committee of the Technion. Female BALB / c, C57BL / 6, and severe combined immunodeficient (SCID) mice (8 weeks of age) were purchased from Envigo, Israel. All mice were maintained under specific pathogen-free (SPF) conditions in the animal facility. 4T1 and EMT6 (5xl05 / 50pL in HBSS) were orthotopically injected into the mammary fat pad of 8-10-week-old female BALB / c mice or SCID mice. LLC and Bl 6- F1 (5xl05 / 100pL in HBSS) were subcutaneously injected into the flank of 8-10-week-old female C57BL / 6 mice. Mice were randomly grouped before therapy. Typically, the number of mice per group was set to 5, to reach statistical power, unless indicated otherwise in the text. Tumor size was assessed regularly with Vernier calipers using the formula, width2xlength><0.5. In all experiments, unless otherwise stated, when tumors reached ~50 mm3mice were treated with anti-mouse anti-PD-1 (clone RMP 1-14, BioXCell Cat# BE0146 or ichorbio Cat# ICH1132) antibody. The antibody was given twice a week in a dose of lOOpg / mouse for 2-week period. The control groups were injected with IgG isotype control (BioXCell Cat# BE0089 or ichorbio Cat# ICH2244). In some experiments, mice were treated with the BMP1 inhibitor UK383,367 (UK). UK was dissolved in 15%(wt / vol) hydroxypropyl-P-cyclodextrin to a working concentration of 0.4 pg / pL before injection, and was intraperitoneally injected at 5 mg / kg / day. UK was given every day for 2-week period. The ADC of the invention was given in the same manner as the anti-PDl antibody, that is twice a week in a dose of lOOpg / mouse for 2-week period. This dosing is equivalent to a greatly reduced dose of UK (when part of the conjugate) as compared to UK given freely. Instead of a dose of 5 mg / kg / day (~80 ug / mouse) given every day, the ADC is equivalent to 0.9 ug / mouse given only twice a week.

[0138] For the adoptive transfer experiments of CD8+T cells, single cell suspensions were prepared from spleens harvested from 4T1 tumor bearing BALB / c mice. CD8+T cells were isolated by negative selection (Mojosort™ Mouse CD8a Selection Kit, Biolegend). Five- million cells / mouse were intravenously injected into SCID mice bearing ~50 mm34T1 tumors, and 24 hours later, mice were treated with anti-PDl or IgG control in the same doseand schedule indicated above. CD8+T cells were adoptively transferred once a week, for 2- week period.

[0139] Histology and Immunohistochemistry (IHC): For paraffin-embedded formalin fixed (FFPE) samples, mouse tumors were fixed in 4% neutral buffered formalin, embedded in paraffin, and sectioned at 4pm-thickness. Picrosirius red (PSR) staining for collagen was conducted using 0.1% PSR (Direct Red-80, Sigma- Aldrich) and counterstained with hematoxylin. Images were captured using the Leica DMI6000 B inverted microscope equipped with a Motorized Polarizer (Leica Microsystems, Wetzlar, Germany) at 20x magnification. Slides were scanned using Panoramic 250 Flash III scanner (3DHISTECH, Budapest, Hungary).

[0140] Images were analyzed using Fiji image processing platform. Region of interests (ROIs) / tumor (n=20-30) were segmented by the Trainable Weka Segmentation (TWS) plugin, a machine-learning algorithm. After segmentation, the ROIs were converted to an 8- bit image for calculating the area fraction of each set. A separate classifier was trained for each set. Once trained, the classifier was used on all samples. ROIs excluded regions of adipose tissue, blood vessels and tumor boundaries. The area fraction of each specimen was averaged.

[0141] Second harmonic generation imaging: Frozen blocks of tumors were sliced at a thickness of 100pm in PBS. The slices were using an upright Leica TCS SP8 MP microscope, equipped with external non-descanned detectors (NDD) HyD and acusto optical tunable filter (Leica microsystems CMS GmbH, Germany). Excitation — SHG signal was excited by an 885 nm laser line of a tunable femtosecond laser 680-1080 Coherent vision II (Coherent GmbH USA). Emission signal was collected using an external NDD HyD detector through a long pass filter of 440 nm. Images were acquired using the galvo stage in a format of 1024 * 1024 (XY) through a HC PL APO 40X / 1.10 CS2 objective, and the following parameters: scan speed — 400 Hz; zoom — 0.75; line average — 3; bit depth — 16; FOV- X 0.359 pm, Y 0.359 pm; Z step -0.502 pm; pixel size — 367.25 nm (XY). Z stacks were acquired using the galvo stage, with 0.502 pm intervals. Data were collected (n=3 mice / group). For quantification measurements, images were analyzed using Imaged 1.52V. To avoid edge effects (attenuation of the SHG signal at the top and bottom of the section), only the central image of each z stack was included in the quantification. Mean gray value limited to threshold of each image was calculated for each image and averaged over a set of at least five fields of view.

[0142] Newly synthesized collagen assay: Tumor newly synthesized collagen was quantified using the Sircol collagen assay kit (Biocolor, UK) according to the manufacturer’s instructions. Briefly, tumor samples were extracted into acid-pepsin solution. Then, lOOpL of sample was added to ImL of Sircol dye reagent followed by agitation in a mechanical shaker for 30 min. Then, the mix was centrifuged at 12,000xg for 10 min. The Sirius red dye was released from the pellet with Alkali reagent and light absorption of the samples was measured at 555 nm wavelength using Infinite 200PRO plate reader (Tecan, Switzerland). Collagen tumor content was calculated as a proportion of the tumor tissue.

[0143] Hydroxyproline assay: Total collagen content of tumors tissues was quantified using a hydroxyproline assay kit (Chondrex, Inc., Redmond, WA, USA) according to the manufacturer’s instructions. Briefly, 12N HC1 solution was added to tumor tissue, followed by overnight hydrolysis at 120°C. the processed tissue samples were incubated with chloramine-T solution for 20 min at RT. Each sample was treated with DMAB solution and incubated for 30 min at 60°C for color development. Samples were read at 560 nm wavelength using Infinite 200PRO plate reader.

[0144] Real-time quantitative PCR: RNA was extracted from the tumor, and immune cells, as indicated in the text, using the Total RNA purification kit (Norgen Biotek, Canada) in accordance with the manufacturer’s protocol. Complementary DNA (cDNA) was then synthesized from the mRNA samples using High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, CA). Real-time quantitative PCR (RT-qPCR) reaction was performed using SYBR Green Master Mix and run in CFX Connect Real-Time PCR Detection System (Bio-Rad Laboratories). Analysis was performed using AACt method. Primers are provided in Table 1.

[0145] Table 1 : Primers used for qPCR0146] BMP1 knock-down in CD8+T cells

[0147] Lentiviral plasmid cloning and production: BMP1 sgRNA (forward: 5’CACCGGCCGACTACACCTACGACCT 3’-SEQ ID NO: 23; reverse: 5’AAAC AGGTCGTAGGTGTAGTCGGCC 3’-SEQ ID NO: 24) was cloned into the LentiCRISPR v2 (Addgene Plasmid #52961) vector plasmid (Lenti BMPl) using the golden gate reaction. Sanger sequence was performed to validate correct insertion of the sgRNA. Next, lentiviral particles were generated by co-transfecting HEK-293FT cells with packaging (psPAX2, Addgene Plasmid #12259) and envelope (pMD2.G, Addgene Plasmid #12259) plasmids together with Lenti BMPl vector or an empty vector backbone. After 24hr, fresh RMPL 1640 media was added, and two days later, supernatant was collected and filtered through a 0.45 pm syringe filter.

[0148] Activation and lentiviral transduction of BMP1 knock-down CD8+T cells: Viral transduction of C57BL / 6 derived mouse CD8+T cells was as follows. Briefly, Six-well tissue culture plates were coated for 2hr at 37°C with 2pg / mL anti-CD3s (Clone 145-2C11; BioLegend) in sterile PBS. Isolated CD8+were cultured and activated with the plate bound anti-mouse CD3s and soluble anti-mouse CD28 (2ug / mL; Clone 37.51; BioLegend) in RPMI-1640 media supplemented with 25ng / mL recombinant murine IL-2 (mIL-2) (Peprotech) (T cells media) for 24hr at 37°C. Activated CD8+were then transduced via spinfection using lentiviral supernatant of Lenti BMPl vector or an empty vector backbone (control) containing lOpg / mL polybrene and 25ng / mL mIL-2 for 1.5hr at 2500 RPM. The viral supernatant was gently aspirated and replaced with fresh T cell media and cultured for72hr at 37°C. Subsequently, BMP1 knock-down (KD) CD8+T cells or control (empty vector) CD8+T cells were intravenously injected (5xl06cells / mouse) into SCID mice bearing ~50 mm34T1 tumors, and 24 and 96 hours later, mice were treated with anti-PDl or IgG control. BMP1 knocked down validation was performed using RT-PCR and murine BMP1 protein ELISA kit (Novus Biological).

[0149] Flow cytometry acquisition and analysis: Tumor samples were prepared as single cell suspensions as previously described (Timaner et al., “Analysis of the Stromal Cellular UNIT Components of the Solid Tumor Microenvironment Using Flow Cytometry”, Curr Protoc Cell Biol. 2016 Mar 1 :70: 19.18.1-19.18.1, the contents of which are hereby incorporated by reference in their entirety). Single-cell suspension was incubated with Viability Stain 440UV for 15 min at RT to exclude dead cells. Then, washed and immunostained for the following surface markers CD45, CD3, CD4, CD8, CD25, CD107a, CD44, CD62L, PD1, CTLA-4, CD 19, CD45R / B220, CD49b, CD1 lb, F4 / 80, CD1 lb, Ly6C, Ly6G, CD206, Siglec-F, MHC-II and PD-L1. In addition, immune cells were defined as indicated in Table 2. At least 50,000 events were acquired using a BD FACSymphony™ A5 SE flow cytometer and analyzed with FlowJo V.10 software (FlowJo, Ashland, Oregon, USA).

[0150] Table 2: FACS markers defining immune cell populations.

[0151] Statistical analysis: For adequate statistical power, all experiments were performed with at least 3 biological repeats and 2 technical repeats. In the in vitro and immunostaining studies, analysis was performed on at least three biological repeats and >4 fields / group were assessed. The in vivo experiments were repeated at least twice, with number of mice indicated in the figure legends (usually n=3-5 mice / group). All experiments were performed in a randomized manner. Data are presented as mean ± standard error (SE). Statistically significant differences were assessed by one-way ANOVA, followed by Tukey post-hoc test (when comparing between more than two groups) using GraphPad Prism 5 software (La Jolla, CA). When applicable, estimate of variance was performed and statistical significance comparing only two sets of data was determined by two-tailed Student's t-test. Significance was set at p values <0.05 and designated as follows: *p < 0.05; **p < 0.01; ***p < 0.001.

[0152] ADC Conjugation Process: A lOmM solution of tris(2-carboxyethyl)phosphine (TCEP) was prepared in conjugation buffer consisting of lOOmM phosphate-buffered saline (PBS), 5mM EDTA, pH 7.2. The TCEP solution was added to 10 mg anti -mouse PD1 antibody at a molar ratio of mouse PD1 :TCEP of 1 :5, followed by incubation for 2 hours at 37°C to reduce interchain disulfide bonds.

[0153] Subsequently, lOmM Mal-PEG3-BMPi was added to the reduced anti-mouse PD1 antibody at a molar ratio of mouse PD1 :Mal-PEG3-BMPi of 1 : 10. The reaction mixture was incubated at 4°C for 16-18 hours to generate the antibody-drug conjugate (ADC).

[0154] The resulting ADC was purified using spin desalting columns, and ADC-containing fractions were pooled and concentrated using ultrafiltration tubes. The purified ADC was subjected to further quality control (QC) testing. The drug to antibody ratio (DAR) was calculated and found to be ~4.3.Example 1: ECM undergoes massive remodeling in tumors of mice treated with anti- PD-1 immunotherapy

[0155] To study the effect of immunotherapy on tumor ECM, and ask whether immunotherapy, similar to chemotherapy and surgery, may alter the ECM, LLC tumors were implanted in C57BL / 6 mice. LLC tumors are known to resist immunotherapy that is based on immune checkpoint blockade. When tumors reached 50mm3, treatment with anti-PDl or IgG control was initiated. Treatment was given twice a week for a 2-week period. At the end point tumors were removed and sectioned for analysis of ECM. Figure 1A demonstrates changes in fibrillar collagen using Sirius red staining and two-photo microscopy followed by second harmonic generation. The results show that after anti-PDl therapy, fibrillar collagen is denser and undergoes massive changes when compared to tumors from IgG control. Further analysis of ECM components using hydroxyproline, Sircol, and RT-PCR, demonstrated that there is newly synthesized collagen and that Collagen I, Collagen IV and Fibronectin, but not Laminin are upregulated in the tumors of mice treated with anti-PDl compared to IgG control (Fig. IB). Overall, these results suggest that anti-PDl contributes to tumor ECM remodeling, perhaps through de novo production.

[0156] Two other immunotherapies were also tested for their effect on the tumor ECM. When LLC tumors reached 50mm3, treatment with anti-PD-Ll, anti-CTLA4 or IgG control was initiated. The changes in fibrillar collagen that had been observed when anti-PDl was used were not observed with anti-PD-Ll or anti-CTLA4 (Fig. 1C). This is highly surprising as PD-1 and PD-L1 are part of the same axis and indicates that the effect is likely mediated by the immune cells to which the anti-PDl binds.Example 2: Lymphocytes, and specifically cytotoxic T cells, are associated with ECM remodeling following anti-PDl therapy

[0157] As anti-PDl mostly activates cytotoxic T cells, it was next asked whether mice lacking lymphocytes, such as T cells, display changes in tumor ECM following anti-PDl therapy, therefore indicating the source of cells which accounts for immunotherapy -induced ECM remodeling. To test this, severe-combined immunodeficient mice (SCID) were used, which lack the lymphoid lineage including B and T cells. Breast carcinoma 4T1 tumors were implanted in these mice, and when tumors reached 50mm3treatment with anti-PDl was initiated similar to the schedule described above. After 2 weeks tumors were removed and analyzed for ECM content. Figure 2A demonstrates Sirius red staining of 4T1 tumors implanted in SCID mice. The results show that there was no difference in tumor ECM content between anti-PDl and IgG control treated mice.

[0158] Next, to ask which cell type from the lymphoid compartment is responsible for tumor ECM remodeling following anti-PDl therapy, an adoptive cell transfer experiment was carried out. Specifically, cytotoxic CD8+T cells, known to be activated following anti-PDl therapy were considered the main suspect. These cells were isolated from the spleens of BALB / c mice and adoptively transferred into SCID mice bearing 4T1 tumors. Subsequently, mice were treated with anti-PDl or IgG control, and tumors were removed after 2 weeks to analyze the ECM. The results in Figure 2B show that while in SCID mice lacking T cells there was no change in ECM structure following anti-PDl treatment, mice adoptively transferred with T cells, and subsequently treated with anti-PDl displayed major tumor ECM remodeling. Overall, these results indicate that lymphocytes, and specifically cytotoxic T cells, account for ECM remodeling following anti-PDl therapy.Example 3: BMP1 is upregulated in cytotoxic T cells following anti-PDl therapy

[0159] Next, the potential factors or enzymes which contribute to ECM remodeling following anti-PDl therapy were determined. These factors must be expressed by T cells. To do this, C57BL / 6 mice bearing LLC tumors were treated with anti-PDl or IgG control. After 2 weeks, spleens were removed and CD8+ T cells were isolated. mRNA levels of different ECM associated enzymes were analyzed. It was found that only BMP1 mRNA levels were significantly upregulated in CD8+ T cells isolated from mice treated with anti- PDl compared to all other factors tested (LOX, MMP9, cathepsin D and heparanase) (Fig. 3A). Notably, while LOX was not significantly upregulated it showed elevated levels following anti-PDl therapy. This is not surprising as LOX is known to be downstream to the BMP1 pathway. BMP1 protein levels were also validated in conditioned medium of CD8+ T cells and found to be upregulated in the anti-PDl group as compared to the IgG control group (Fig. 3B). Overall, these results indicate that CD8+ T cells activated by PD-1 blockade highly express BMP1, which can induce tumor ECM remodeling.Example 4: Inhibition of BMP1 solely in CD8+ T cells but not when used systemically decreases tumor growth

[0160] To further analyze the role of BMP 1 in tumor growth and ECM remodeling, two approaches were utilized. First, the effect of systemic inhibition of BMP1 on tumor growth in mice was examined. Secondly, the effect of inhibition of BMP1 solely in CD8+T cells on tumor growth in mice was examined. To do this, UK383,367 (UK), a small molecule drug inhibiting BMP1, was used. The administration of UK to mice bearing 4T1 tumors for two weeks resulted in larger tumors than control (Fig. 4A), similar to other published studies (seeTian C., et al. “Suppression of pancreatic ductal adenocarcinoma growth and metastasis by fibrillar collagens produced selectively by tumor cells”, Nat Commun. 2021;12(l):2328). However, when BMP1 was knocked down solely in CD8+T cells using CRSPR / Cas9 technology followed by CD8+T cell adoptive transfer to SCID mice, this resulted in reduced tumor growth compared to SCID mice adoptively transferred with CD8+T cells in which BMP1 was expressed (Fig. 4B). Notably, no changes in tumor ECM were found between the two groups (Fig. 4C). These results suggest that a local inhibition of BMP 1 specifically in CD8+T cells, decreases tumor growth through means other than ECM remodeling. Of note, in this experiment the effect of BMP 1 in T cells was tested regardless of anti-PDl therapy.Example 5: Inhibition of BMP1 in combination with anti-PDl sensitizes tumors otherwise resistant to immunotherapy.

[0161] As the ECM undergoes massive remodeling following anti-PDl therapy, and the fact that these ECM changes appear to be induced by BMP1, it was further suggested that combining BMP1 inhibition with anti-PDl therapy may support anti-tumor activity, especially in tumors resistant to immunotherapy. To test this, the combination of anti-PDl with UK was assessed in 3 tumor models known to be resistant to immunotherapy: 4T1 (breast cancer), B16 (melanoma), and LLC (lung cancer). Mice bearing such tumors were treated with anti-PDl, UK or their combination, and tumor growth was assessed. At the endpoint, tumors were removed and analyzed for ECM changes. In all tumor models the combination of anti-PDl and UK resulted in decreased tumor growth, while anti-PDl monotherapy or UK monotherapy produced no significant change as compared to control (Fig. 5A-C). The analysis of the ECM revealed that tumor ECM content is reduced in the combination treatment compared to all other treatment groups. Notably, the results of LLC tumors were not found to be statistically significant, nevertheless the same trends as shown in other tumor models were observed (Fig. 5C). Importantly, the reduction in tumor volume produced by the combined treatment was statistically significant even in the LLC model. Further, in all models the combined treatment produced a synergistic effect, as each treatment alone had no effect (UK produced a small not statistically significant tumor volume reduction in the LLC model, but synergism is still clearly present since anti-PDl therapy had no effect whatsoever). Overall, these results indicate that the combination of anti-PDl with BMP1 inhibition produces a synergistic improvement in treatment outcomes in cancers resistant to immunotherapy.

[0162] CD45 positive immune cells were isolated from the extracted tumors of all the mice in all treatment groups, and the infiltration of various immune cell types was quantified. In the control mice (IgG only) a relative reduction in percentage of CD4 T cells, CD8 T cells and NK cells was observed, including few activated and / or cytotoxic CD8 T cells (Fig. 5D). In contrast, myeloid cells were found abundantly in 3 of the 5 tumors and more lowly in another of the 5 tumors. Anti-PDl treatment increased cytotoxic immune cell infiltration and decreased myeloid infiltration and UK treatment had a minor impact on cytotoxic T cell infiltration and myeloid infiltration, however, the combined treatment had a much greater effect. The infiltration of CD8 T cells, activated CD8 T cells, cytotoxic CD8 T cells and NK cells was greatly enhanced by the combined treatment, even over the increase produced by the anti-PDl alone. The reduction in myeloid cell infiltration was comparable to the other two treatments. This demonstrates the synergy of the two treatments, as anti-PDl effectively activates the immune cells and inhibition of BMP1 leaves the TME accessible to those immune cells.Example 6: An antibody-drug conjugate produces superior sensitization of tumors otherwise resistant to immunotherapy.

[0163] In order to create a single therapeutic agent to target both PD-1 and BMP-1 in immune cells (specifically T cells), the BMP-1 inhibitor UK383,367 was conjugated to the anti-PDl antibody (see Materials and Methods). This antibody-drug conjugated (ADC) has the added benefit of targeting the BMP1 inhibitor to T cells. Mice bearing 4T1 and LLC tumors were treated as before, only a fifth group of mice were included who received the ADC. As before the combination of PD-1 blockade and BMP1 inhibition produced a synergistic effect that was significantly greater than either therapy alone. This was observed in both the 4T1 (Fig. 6A) and LLC (Fig. 6C) bearing mice. The ADC of the invention was also significantly superior to either drug alone, but surprisingly in the LLC tumors the ADC was significantly superior to the combination of anti-PD 1 and UK (Fig. 6C). This superiority was observed at all time points after drug administration. The superiority is even more striking when one considers that the ADC amounts to a reduced dosing of UK. Instead of administering 5 mg / kg / day of free UK given every day (~80 ug / mouse every day), the ADC is equivalent to 0.9 ug / mouse given only twice a week. Even with this considerable reduction in dose, the ADC had a superior effect as compared to free UK. It is hypothesized that the targeting of the BMP1 inhibitor to T cells may have produced this superior result, although it is also likely that conjugation to the antibody improved the stability / half-life of UK in the circulation. In 4T1 cells the ADC and combination treatment were not significantly different,though at the last time point measure (18 days) the ADC did appear superior to the combination (Fig. 6A). However, in light of the greatly reduced dose of UK being administered when the ADC is given, even a similar anticancer effect demonstrates the superiority of the ADC over the combination therapy.

[0164] An in-depth look at immune cell infiltration in these tumors showed once again that the combined treatment produced an influx of T cells and NK cells and particularly activated and cytotoxic cells and reduced the infiltration of myeloid cells. (Fig. 6B and 6D). Notably, the ADC produced a greater effect than the two drugs combined, and this difference was more striking in the LLC tumors (Fig. 6D) as compared to the 4T1 tumors (Fig. 6C) which is likely responsible for the stronger effect of the ADC observed in the LLC tumor bearing mice.

[0165] Based on this increased infiltration of cytotoxic immune cells, the combined PD- 1 / BMP1 blocking is tested in further combination with CAR-T cell therapy. The major unmet need in CAR-T therapy of solid tumors is that the engineered T cells do not penetrate the tumor mass and stay at the periphery of the tumor. This is why their therapeutic advantage is highly compromised. The combined therapy overcomes this drawback.

[0166] BPMCs were engineered to express a chimeric antigen receptor (CAR) against CD- 19 and BMP1 levels in the media were measured before and after CAR expression. Secreted BMP1 protein levels were significantly increased after CAR transfection (Fig. 7). This result is confirmed with other CAR constructs against other antigens as well. It is confirmed that this increase in BMP1 is caused by CAR expression but is not specific to any one antigen. Increased BMP1 from CARs would hamper their ability to infiltrate the tumor. Thus, CAR- T therapy is highly suited for combination with BMP1 inhibition and combined PD-1 blockade and BMP1 inhibition.

[0167] Next mice bearing various tumors (breast, lung, skin etc.) are treated with CAR-T cells (with a CAR against a tumor antigen) alone, CAR-T+anti-PDl, CAR-T+UK, CAR- T+anti-PDl+UK, CAR-T+ADC or control. Combining CAR-T with BMP1 inhibition greatly improves CAR-T infiltration into the tumor and retards tumor growth. This effect is enhanced when combined with PD-1 blockade. The ADC of the invention has an even stronger effect that combining PD-1 blockade and BMP1 inhibition.

[0168] 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. An antibody drug conjugate (ADC) comprising an anti-PDl blocking antibody or antigen binding fragment thereof conjugated to a BMP1 small molecule inhibitor or an antiBMP 1 inhibiting antibody or antigen binding fragment thereof.

2. The ADC of claim 1, wherein said anti-PDl blocking antibody is selected from pembrolizumab, nivolumab, pidilizumab, cemiplimab, dostarlimab, retifanlimab and toripalimab.

3. The ADC of claim 1 or 2, wherein said BMP1 small molecule inhibitor is selected from UK383, 367, S33A, RXP-1001 and FG-2575.

4. The ADC of claim 3, wherein said BMP1 small molecule inhibitor is UK383,367.

5. A pharmaceutical composition comprising the ADC of any one of claims 1 to 4 and a pharmaceutically acceptable carrier, excipient or adjuvant.

6. A method of improving PD- 1 based immunotherapy in a subj ect suffering from a solid cancer, the method comprising decreasing bone morphogenetic protein 1 (BMP1) levels or function in T cells of said subject or in a tumor microenvironment (TME) or extracellular matrix (ECM) of said solid cancer; thereby improving PD-1 based immunotherapy.

7. A method of treating a solid cancer in a subj ect in need thereof, the method comprising administering to said subject a PD-1 based immunotherapy and decreasing BMP1 levels or function in T cells of said subject or in a tumor microenvironment (TME) or extracellular matrix (ECM) of said solid cancer, thereby treating cancer.

8. A method of enhancing cytotoxic immune cell infiltration into a solid cancer, decreasing myeloid cell infiltration into said solid cancer or both in a subject in need thereof, the method comprising decreasing bone morphogenetic protein 1 (BMP1) levels or function in T cells of said subject or in a tumor microenvironment (TME) or extracellular matrix (ECM) of said solid cancer; thereby enhancing cytotoxic immune cell infiltration into a solid cancer.

9. The method of any one of claims 6 to 8, wherein said decreasing is decreasing BMP1 protein levels or function in a tumor microenvironment (TME) or extracellular matrix (ECM) of said cancer.

10. The method of any one of claims 6 to 9, wherein said decreasing comprises decreasing secretion of BMP 1 from cytotoxic immune cells.

11. The method of claim 10, wherein said cytotoxic immune cells are cytotoxic T lymphocytes (CTLs).

12. The method of any one of claims 6 to 11, wherein said solid cancer is a carcinoma or melanoma.

13. The method of any one of claims 6 to 12, wherein said cancer is a PD-1 / PD-L1 based immunotherapy resistant cancer, or wherein said subject has previously received PD-1 / PD-L1 based immunotherapy as a monotherapy and has developed resistance.

14. The method of any one of claims 6 to 13, wherein said cancer is selected from: brain cancer, breast cancer, bone cancer, fat cancer, retinoblastoma, head and neck cancer, tongue cancer, nasopharyngeal cancer, pharyngeal cancer, throat cancer, esophageal cancer, stomach cancer, gastrointestinal cancer, intestinal cancer, lung cancer, colon cancer, colorectal cancer, liver cancer, renal cell carcinoma, pancreatic cancer, gallbladder cancer, penile cancer, thymus cancer, thyroid cancer, urogenital cancer, prostate cancer, kidney cancer, ovarian cancer, cervical cancer, testicular cancer, skin cancer, glioblastoma multiforme (GBM), and uterine cancer.

15. The method of claim 14, wherein said cancer is selected from: lung cancer, skin cancer and breast cancer.

16. The method of any one of claims 6 to 15, further comprising administering said PD-1 based immunotherapy to said subject.

17. The method of any one of claims 6 to 16, wherein said PD-1 based immunotherapy is an immune checkpoint inhibitor of the PD-1 / PD-L1 immune checkpoint.

18. The method of claim 17, wherein said PD-1 based immunotherapy is an anti-PDl blocking antibody.

19. The method of claim 18, wherein said anti-PDl blocking antibody is selected from pembrolizumab, nivolumab, pidilizumab, cemiplimab, dostarlimab, retifanlimab and toripalimab.

20. The method of any one of claims 6 to 19, comprising administering a BMP1 small molecule inhibitor to said subject.

21. The method of claim 20, wherein said BMP1 small molecule inhibitor is selected from UK383,367, S33A, RXP-1001 and FG-2575.

22. The method of any one of claims 6 to 19, comprising administering an anti -B MP 1 antibody or Sizzled protein to said subject.

23. The method of any one of claims 20 to 22, wherein said small molecule inhibitor, anti- BMP-1 antibody or Sizzled protein is conjugated to a targeting moiety to T cells, TME or ECM.

24. The method of any one of claims 6 to 19, comprising administering to said subject a composition comprising an inhibitory nucleic acid molecule comprising a sequence reverse complimentary to a sequence of BMP 1, and a T cell targeting moiety.

25. The method of any one of claims 6 to 24, wherein said T cells are CD8 T cells.

26. The method of any one of claims 6 to 19, comprising adoptive T cell transfer comprising administering to said subject a composition comprising T cells comprising knockdown of endogenous BMP1.

27. The method of any one of claims 6 to 19, comprising administering an antibody drug conjugate (ADC) comprising an anti-PDl blocking antibody conjugated to a BMP1 small molecule inhibitor to said subject.

28. The method of claim 27, wherein said anti-PDl blocking antibody is selected from pembrolizumab, nivolumab, pidilizumab, cemiplimab, dostarlimab, retifanlimab and toripalimab and said BMP1 small molecule inhibitor is selected from UK383,367, S33A, RXP-1001 and FG-2575.

29. The method of claim 27 or 28, wherein said ADC is an ADC of any one of claims 1 to 4 or a pharmaceutical composition of claim 5.

30. The method of any one of claims 8 to 29, wherein said cytotoxic immune cells comprise CD8 T cells, natural killer (NK) cells, chimeric antigen receptor (CAR) T cells or a combination thereof.

31. The method of claim 30, being a method of enhancing CAR-T therapy, and further comprising administering CAR-T cells to said subject.

32. A pharmaceutical composition comprising a population of T cells comprising an inhibitory nucleic acid molecule comprising a sequence reverse complimentary to a sequence of BMP1 or comprising a genetic knockout of a BMP1 genomic locus and a pharmaceutically acceptable carrier, excipient or adjuvant.

33. The pharmaceutical composition of claim 32, wherein at least 20% of cells in said composition are T cells comprising said inhibitory nucleic acid molecule comprising a sequence reverse complimentary to a sequence of BMP1 or comprising a genetic knockout of a BMP1 genomic locus.

34. The pharmaceutical composition of claim 32 or 33, wherein said T cells comprise a CRISPR knockout of said BMP1 genomic locus.

35. The pharmaceutical composition of claim 34, wherein said CRISPR knockout was using an sgRNA comprising a forward strand comprising or consisting of SEQ ID NO: 23 and a reverse strand comprising or consisting of SEQ ID NO: 24.

36. The pharmaceutical composition of any one of claims 32 to 35, wherein said T cells are CAR-T cells.

37. The pharmaceutical composition of any one of claims 32 to 36, for use in adoptive cell transfer (ACT).

38. The pharmaceutical composition of any one of claims 32 to 37, for use in combination with an anti-PD-1 immunotherapy in treating cancer in a subject in need thereof.

39. The pharmaceutical composition of any one of claims 5 and 32 to 38, for use in treating a solid cancer in a subject in need thereof.

40. A method of producing an antibody drug conjugate (ADC), the method comprising: a. providing an antibody or antigen binding fragment thereof that binds to PD-1 and blocks or inhibits binding to PD-L1; b. providing an agent that binds to BMP1 and inhibits BMP1 protease function; and c. conjugating said provided agent to said provided antibody or antigen binding fragment thereof; thereby producing an ADC.

41. The method of claim 40, wherein step (a) comprises providing an antibody or antigen binding fragment thereof that binds to PD-1, measuring the ability of said provided antibody or antigen binding fragment thereof to block or inhibit PD-1 binding to PD-L1 and selecting an antibody or antigen binding fragment thereof that blocks or inhibits said PD-1 binding to PD-L1.

42. The method of claim 41, wherein said providing an antibody or antigen binding fragment thereof that binds to PD-1 comprises: a. immunizing an organism with a PD-1 extracellular domain or a fragment thereof and collecting antibodies from the immunized organism; or b. screening a library of antibodies or antigen binding fragments thereof for binding to a PD-1 extracellular domain or a fragment thereof and selecting an agent that binds.

43. The method of any one of claims 40 to 42, wherein step (b) comprises providing an agent that binds to BMP1, measuring the ability of said provided antibody or antigen binding fragment thereof to inhibit BMP1 protease function and selecting an agent that inhibits BMP1 protease function.

44. The method of claim 43, wherein said providing an agent that binds to BMP1 comprises: a. immunizing an organism with BMP1 or a fragment thereof and collecting antibodies from the immunized organism; or b. screening a library of agents for binding to BMP1 or a fragment thereof and selecting an agent that binds.

45. The method of any one of claims 40 to 44, further comprising contacting said provided agent in combination with said provided antibody or antigen binding fragment thereof with a cancer, and contacting said agent conjugated to said antibody or antigen binding fragment thereof with said cancer and selecting an ADC that produces a superior anticancer effect to said combination.

46. An ADC produced by a method of any one of claims 40 to 45.

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