Combination therapy with angiogenesis protein and immune checkpoint inhibitor with / without chemotherapeutics

Abstract

Provided are pharmaceutical compositions for reducing a tumor burden in a subject having a cancer comprising a combination therapy comprising a therapeutically effective dose of a modified CD2 polypeptide derived from human or rat domain one of CD2 polypeptide and a second therapeutic agent comprising an immune checkpoint inhibitor. Also provided are methods of inhibiting or reducing tumor growth and methods of synergistically increasing the activity of an anti-PD-L1 immunotherapeutic in a subject having a cancer comprising administering the pharmaceutical compositions.

Description

COMBINATION THERAPY WITH ANGIOGENESIS PROTEIN AND IMMUNECHECKPOINT INHIBITOR WITH / WITHOUT CHEMOTHERAPEUTICSGOVERNMENT LICENSE RIGHTS

[0001] This invention was made with government support under grant Nos. CA175112, CAI 18113, CA178730, and CA217482 awarded by the National Institute of Health. The government has certain rights in the invention.CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of priority to United States Provisional Application No. 63 / 652,637, filed May 28, 2024, the entire contents of which are incorporated herein by reference.TECHNICAL FIELD

[0003] This disclosure relates in general to compositions and methods to treat a cancer. More particularly, this disclosure relates to compositions and methods comprising a therapeutically effective amount of a modified CD2 polypeptide in combination with an immune checkpoint inhibitor and / or chemotherapeutics.BACKGROUND

[0004] Lung cancer is believed to be more immunogenic often with high levels of PDL-1 expression in tumors (Yu, H., et al., J. Thorac. Oncol. 11(7): 964-75 (2016); Pawelczyk, K., et al., Int. J. Mol. Sci., 20(4) (2019)). Thus, checkpoint inhibitors are regarded as a promising strategy both in single agent and in combination with chemotherapeutics. Due to relatively good clinical outcomes, the check point inhibitor and the combination therapies have been established as standard of care for the treatment of lung cancers (Mountzios, G., et al., Nat. Rev. Clin. Oncol. 20(10): 664-677 (2023); Xiong, W ., et al., Front. Oncol., 11 : 704336 (2021); Gandhi, L., et al., N. Engl. J. Med., 379(11): el8 (2018)). However, despite the success of checkpoint blockade immunotherapies in lung cancer treatment (Pardoll, D. M., Nat. Rev. Cancer., 12(4): 252-64 (2012)), patients have not fully benefitted from the treatments, due to various resistance mechanisms (Creelan, B. C., Cancer Control., 21(1): 80-9 (2014); Onoi, K., et al., J. Clin. Med., 9(5) (2020); Huang, Z., et al., Front. Pharmacol., 11 : 578091 (2020)). It has been observed that many patients do not respond initially to treatment (Catalano, M., Cancers (Basel)., 14(24): 6145 (2022)). It was also noted that even if patients initially responded to the checkpoint blockades treatments, the disease often relapses after a period of response due to development ofimmunotherapy resistance (Mamdani, H., et al., Front Immunol. 13: 823618 (2022); Lahiri, A., Mol Cancer. 22(1): 40 (2023)). It has been gradually realized that tumor immune escape and, also, resistance to immune checkpoint blockade therapies, do not depend solely on the immunogenic potential of cancer cells but rather depend mainly on the tumor stroma. The tumor microenvironment (TME) plays an important role in educating the host immunoresponses in favor of tumor progression. Among all components, tumor angiogenesis and cancer associated fibroblasts (CAF) are particularly important in the tumor’s immunosuppression adaptive program (De Jaeghere, E. A., et al., Trends Cancer., 5(11): 704-723 (2019); Solimando, A. G., et al., Cancers (Basel)., 12(11) (2020); Datta, M., et al., Am Soc Clin Oncol Educ Book., 39: 165-174 (2019)).

[0005] CAFs are the most prominent cell types in TME, which actively engage in crosstalk with the surrounding cells to promote cancer cell proliferation and survival. CAF is a central player for cancer treatment resistance including resistance to immunotherapies (Barrett, R. L., et al., Elife. 9: e57243 (2020); Koppensteiner, L., et al., Front Immunol., 13: 887380 (2022)). The roles played by CAFs in tumor immunity are fourfold; (1) orchestrate the dense fibrotic stroma that forms a physical barrier for immune cell infiltration into tumor, (2) the predominant source of immunosuppressive molecules, such as cytokines / chemokines and growth factors, which suppress immune cell activation, (3) CAF facilitates cancer cells to express high levels of immune checkpoint blockades such as PDL-1, and (4) activated CAFs form a physical trap for checkpoint blockade such as PDL-1 antibody due to high levels expression of PDL-1 on CAF.

[0006] Tumor angiogenic vessels are another critical player in modulating tumor immunity. High tumor microvasculature closely correlates with poor prognosis of cancer patients. Tumor angiogenesis modulates tumor immunity by: (1) Overexpression of several angiogenesis stimulating growth factors, such as vascular growth factors (VEGF) and platelet derived growth factor (PDGF) can directly activate immunosuppressive cells and suppress immune effector cells to shape immunosuppressive microenvironment (Zheng, W ., et al., Front Immunol., 13: 1035323 (2022); Rahma, O. E., et al., Clin. Cancer. Res. 25(18): 5449-5457 (2019); (2) While abnormal vascular structure of tumors prevents immune cell infiltration through a selective immune-cell barrier or inhibits immune cell function by exacerbating hypoxia in the microenvironment (Abou Khouzam, R., et al., Front Immunol., 11 : 613114 (2020)). Meanwhile, activated immunosuppressive cells can further promote abnormal angiogenesis (Noman, M. Z., et al., Am. J. Physiol. Cell. Physiol., 309(9): C569-79 (2015)); (3) Leaky and disorganized tumor vessels often lead to highhypoxia in the tumor. Hypoxia and associated hypoxia factor Hif-la is known to be an important immunosuppressive factor (McGettrick, A. F., et al., Cell Metab., 32(4): 524- 536 (2020); Wu, Q., et al., J. Hematol. Oncol., 15(1): 77 (2022)).

[0007] SUMMARY

[0008] Disclosed herein are compositions and methods to modulate tumor immunity by simultaneously depleting CAFs and tumor angiogenic vessels using a rationally designed protein that induces integrin avbs targeted cell apoptosis. The disclosure offers a unique opportunity for the enhancement of cancer immunotherapies, especially for patients subjects with tumor of dense stroma and high angiogenesis.

[0009] One aspect of the invention provides a pharmaceutical composition for reducing a tumor burden in a subject having a cancer comprising a combination therapy comprising: a first therapeutic agent comprising a therapeutically effective dose of a modified CD2 polypeptide derived from human or rat domain one of CD2 polypeptide, the modified CD2 polypeptide having non-wild type characteristics, the non-wild type characteristics including a P-sheet formed by two segments having at least five amino acids alternating between hydrophilicity and hydrophobicity, an anti-parallel fold, an inwardfacing hydrophobic surface, and an outward-facing hydrophilic surface; a second therapeutic agent comprising an immune checkpoint inhibitor; and a pharmaceutically acceptable excipient; wherein the modified CD2 polypeptide is administered in combination with the immune checkpoint inhibitor and the pharmaceutically acceptable excipient, wherein the modified CD2 polypeptide reduces PD-L1 expression in a tumor’s fibrotic stroma to increase the delivery of the immune checkpoint inhibitor to target cancer cells, wherein PD-L1 expression in a solid tumor’s fibrotic stroma synergistically increases the activity of the immune checkpoint inhibitor.

[0010] In one embodiment, the invention further comprises the combination of one or more additional therapeutic agents, wherein the additional therapeutic agents are anticancer chemotherapy drugs selected from amsacrine, bleomycin, busulfan, capecitabine, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, clofarabine, crisantaspase, cyclophosphamide, cytarabine, dacarbazine, dactinomycin, daunorubicin, docetaxel, doxorubicin, epirubicin, etoposide, fludarabine, fluorouracil, gemcitabine, hydroxycarbamide, idarubicin, ifosfamide, irinotecan, leucovorin, liposomal doxorubicin, liposomal daunorubicin, lomustine, melphalan, mercaptopurine, mesna, methotrexate, mitomycin, mitoxantrone, oxaliplatin, paclitaxel, pemetrexed, pentostatin, procarbazine,raltitrexed, satraplatin, streptozocin, tegafur-uracil, temozolomide, teniposide, thiotepa, tioguanine, topotecan, treosulfan, vinblastine, vincristine, vindesine, vinorelbine, or a combination thereof. Representative pro-apoptotic agents include, but are not limited to fludarabinetaurosporine, cycloheximide, actinomycin D, lactosylceramide, 15d-PGJ(2) and combinations thereof. In some embodiments, the chemotherapeutic is gemcitabine.

[0011] In another embodiment, the modified CD2 polypeptide has at least 99% sequence identity to SEQ ID NO: 1, 2, 3, or 4.

[0012] In yet another embodiment, the immune checkpoint inhibitor is a PD-1 inhibitor. In specific embodiments, the immune checkpoint inhibitor is an anti-PD-1 or anti- PD-L1 antibody. In other embodiments, the immune checkpoint inhibitor is an anti-CTLA- 4 antibody.

[0013] Another aspect of the invention provides a method of inhibiting or reducing tumor growth in a subject having a cancer comprising administering the compositions disclosed herein, wherein the compositions additively or synergistically inhibit or decrease tumor growth in the subject by inducing tumor immunity. In one embodiment, the method further comprises adding one or more therapeutic agents administered to additively or synergistically inhibit or reduce tumor growth. The additional therapeutic agents are anticancer chemotherapy drugs described herein.

[0014] In one embodiment, the subject has a cancer with a solid tumor. In another embodiment the cancer is a lung cancer selected from a non-small cell lung cancer (NSCLC) or a small cell lung cancer (SCLC). In other embodiments, the subject has relapsed from or is refractory to checkpoint inhibitor therapy, prior to treatment with the modified CD2 polypeptide.

[0015] In yet another embodiment, the combination therapy induces integrin avbs targeted cell apoptosis, which induces tumor immunity by simultaneously depleting cancer- associated fibroblasts (CAFs) and tumor angiogenic vessels. The depletion of cancer- associated fibroblasts (CAFs) leads to a depletion of the tumor’s fibrotic stroma. The depletion of the tumor angiogenic vessels decreases tumor angiogenesis, increases tumor perfusion, reduces tumor hypoxia, or the combination thereof.

[0016] An additional aspect of the invention provides method of synergistically increasing the activity of an anti-PD-Ll immunotherapeutic in a subject having a cancer comprising administering the pharmaceutical compositions disclosed herein, wherein the modified CD2 polypeptide reduces PD-L1 expression in a solid tumor’s fibrotic stroma to increase the delivery of the anti-PDL-1 antibody to target cancer cells, and wherein PD-L1expression in the solid tumor’s fibrotic stroma synergistically increases the activity of the anti-PDL-1 antibody. The method further comprising adding one or more additional therapeutic agents administered to additively or synergistically inhibit or reduce tumor growth, wherein the additional therapeutic agents are anti-cancer chemotherapy drugs.

[0017] In one embodiment, the increased delivery of the anti-PDL-1 antibody to target cancer cells synergistically inhibits or reduces tumor growth.

[0018] In another embodiment, the subject has been diagnosed with a lung cancer selected from a non-small cell lung cancer (NSCLC) or a small cell lung cancer (SCLC), wherein the subject has relapsed from or is refractory to checkpoint inhibitor therapy, prior to treatment with the modified CD2 polypeptide.BRIEF DESCRIPTION OF THE FIGURES

[0019] FIGS. 1A-G display that ProAgio depletes CAFs and angiogenic tumor vessels in A549 cell xenograft model.

[0020] FIG. 1A displays quantification of co-IF staining of integrin P3 and a-SMA in cultured normal human lung fibroblasts (NHLF) and commercially available lung CAF (1CAF) isolated from lung cancer patient tumor. The quantity is presented as integrin Ps / a-SMA double positive cell population (%). Error bars represent means ± SEM.

[0021] FIG. IB displays apoptosis of the CAF under treatment of different concentrations (indicated) of ProAgio was measured by an apoptosis kit. Cell apoptosis is presented as the percentage of apoptosis by defining the apoptosis of untreated cells as 0%. Error bars represent means ± SEM.

[0022] FIG. 1C displays mean tumor volume (n = 10) of s.c. xenograft of A549 cells. The tumor bearing mice were treated with indicated agents. The arrow indicates the treatment starting time point. Error bars represent means ± SEM.

[0023] FIG. ID displays quantifications (n = 6) of CD31 staining of A549 tumor sections. Error bars represent means ± SEM.

[0024] FIG. IE displays quantitative analysis of H4C staining of a-SMA. Error bars represent means ± SEM.

[0025] FIG. IF displays quantitative analysis of H4C staining of cirrus red staining. Error bars represent means ± SEM.

[0026] FIG. 1G displays quantitative analysis of hydroxyproline assay in tumor sections / tissues of A549 xenograft mice treated with vehicle or ProAgio. Error bars represent means ± SEM.

[0027] FIGS. 2A-2N display that ProAgio shrank lung tumors by depleting activated CAFs and angiogenic tumor vessels.

[0028] FIG. 2A displays ProAgio or vehicle treatment regimen of GEM-NSCLC mice.

[0029] FIG. 2B displays Kaplan-Meier survival analysis of GEM-NSCLC mice treated with vehicle or 15 mg / kg ProAgio. Treatment was started at indicated time (-179 days of age or 126 days of AdV-Cre delivery). The numbers in parentheses indicate the group size.

[0030] FIG. 2C-E display representative images of lungs of treated mice (treated with indicated agents). FIG. 2C displays lungs before treatment. FIG. 2D displays lungs treated with vehicle. FIG. 2E displays lungs treated with ProAgio.

[0031] FIG. 2F displays quantitative measurements of tumor nodules in lung of the treated mice. Error bars represent means ± SEM.

[0032] FIG. 2G displays quantitative measurements of tumor nodules in lung of the treated mice. Error bars represent means ± SEM.

[0033] FIG. 2H-2I display representative images of Sirius red staining of lung sections from the treated mice. FIG. 2H displays Sirius red staining of lung sections from the mice treated with vehicle. FIG. 21 displays Sirius red staining of lung sections from the mice treated with ProAgio.

[0034] FIG. 2J displays quantifications of Sirius red staining of lung sections from the treated mice. The quantity is presented as fold changes with the Sirius red levels of vehicle treated groups as reference 1. Error bars represent means ± SEM.

[0035] FIG. 2K displays quantifications of IHC of a-SMA staining of lung sections from the treated mice. The quantity is presented as fold changes with the a-SMA levels of vehicle treated groups as reference 1. Error bars represent means ± SEM.

[0036] FIG. 2L-2M display representative images of IHC of CD31 staining of lung sections from the treated mice. The quantity is presented as CD31+pixels per view field. FIG. 2L displays lung sections from mice treated with vehicle. FIG. 2M displays lung sections from mice treated with ProAgio.

[0037] FIG. 2N displays quantifications of IHC of CD31 staining of lung sections from the treated mice. The quantity is presented as CD31+pixels per view field. Error bars represent means ± SEM.

[0038] FIGS. 3A-R display that ProAgio synergistically enhances the effects of immune checkpoint blockade therapies.

[0039] FIG. 3A displays ProAgio, aPDLl, and ProAgio + aPDLl treatment regimen of GEM-NSCLC mice.

[0040] FIG. 3B displays Kaplan-Meier survival analysis of GEM-NSCLC mice treated with indicated agents. Treatment was started at indicated time (-129 days of age or 12 weeks of AdV-Cre delivery). The numbers in parentheses indicate the group size.

[0041] FIG. 3C-3F display representative images of lungs of treated mice. FIG. 3C displays lungs of mice treated with vehicle. FIG. 3D displays lungs of mice treated with aPDLl . FIG. 3E displays lungs of mice treated with ProAgio+ aPDLl. FIG. 3F displays lungs of mice treated with ProAgio.

[0042] FIG. 3G displays quantitative measurements of tumor nodules in lung of the treated mice (treated with indicated agents). Error bars represent means ± SEM.

[0043] FIG. 3H displays quantitative measurements of tumor nodules in lung of the treated mice (treated with indicated agents). Error bars represent means ± SEM.

[0044] FIG. 3F displays representative images of Sirius red staining of lung sections from the treated mice.

[0045] FIG. 3G displays quantifications of Sirius red staining of lung sections from the treated mice. Error bars represent means ± SEM.

[0046] FIG. 3H displays quantifications of IHC of a-SMA staining of lung sections from the treated mice. The quantity in G & H is presented as Sirius red+or a-SMA+area (%). Error bars represent means ± SEM.

[0047] FIG. 3I-3L displays representative images of IHC of CD31 staining of lung sections from the treated mice. The quantity is presented as CD31+area (%).

[0048] FIG. 3M-3N displays quantifications of IHC of CD31 sirius red staining (FIG. 3M) and a-SMA (FIG. 3N) of lung sections from the treated mice. The quantity is presented as CD31+area (%). Error bars represent means ± SEM.

[0049] FIGS. 30-3 S show staining of CD31 demonstrated that ProAgio eliminated angiogenic tumor vessels.

[0050] FIGS. 4A-4L display that ProAgio alters lung tumor immunity.

[0051] FIG. 4A displays ProAgio and vehicle treatment regimen of GEM-NSCLC mice.

[0052] FIG. 4B-4D displays representative images of IHC Hifla staining of lung sections from the treated mice.

[0053] FIG. 4E displays quantifications of IHC Hifla staining of lung sections from the treated mice. Quantifications is presented as Hifla+area (%). Error bars represent means ± SEM.

[0054] FIG. 4F displays FACS analyses of CD4+Treg cells in lung tissues from indicated treatment mice. ETV - early treatment vehicle, ET - early ProAgio treatment, LTV - late treatment vehicle, LT - late ProAgio treatment. Quantities are presented as % of CD4+Treg cells in total lung cells. Error bars represent means ± SEM.

[0055] FIG. 4G displays FACS analyses of MDSC in lung tissues from indicated treatment mice. ETV - early treatment vehicle, ET - early ProAgio treatment, LTV - late treatment vehicle, LT - late ProAgio treatment. Quantities are presented as % of MDSC in total lung cells. Error bars represent means ± SEM.

[0056] FIG. 4H displays FACS analyses of CD8+T cells in lung tissues from indicated treatment mice. ETV - early treatment vehicle, ET - early ProAgio treatment, LTV - late treatment vehicle, LT - late ProAgio treatment. Quantities are presented as % of CD8+T cells in total lung cells. Error bars represent means ± SEM.

[0057] FIG. 41 displays FACS analyses of M1 / M2 macrophage ratio in lung tissues from indicated treatment mice. ETV - early treatment vehicle, ET - early ProAgio treatment, LTV - late treatment vehicle, LT - late ProAgio treatment. M1 / M2 macrophage ratio is presented as fold changes by set reference of ETV M1 / M2 ratio as 1. Error bars represent means ± SEM.

[0058] FIG. 4J displays ELISA analyses of IL6 in lung tissues from indicated treatment mice. The quantities of cytokines / chemokines are presented as ng per ml of lung extracts. Error bars represent means ± SEM.

[0059] FIG. 4K displays ELISA analyses of CXCL2 in lung tissues from indicated treatment mice. The quantities of cytokines / chemokines are presented as ng per ml of lung extracts. Error bars represent means ± SEM.

[0060] FIG. 4L displays ELISA analyses of CDCL12 in lung tissues from indicated treatment mice. The quantities of cytokines / chemokines are presented as ng per ml of lung extracts. Error bars represent means ± SEM.

[0061] FIGS. 5A-K display that ProAgio increases aPDLl delivery to lung cancer nodules.

[0062] FIG. 5A-5C displays representative images of IHC PDL1 staining from the treated mice.

[0063] FIG. 5D displays quantifications of IHC PDL1 staining in lung tissue from the treated mice.

[0064] FIG. 5E displays quantifications of IHC PDL1 staining in tumor nodules from the treated mice.

[0065] FIG. 5F-5H displays representative images of IHC aPDLl staining.

[0066] FIG. 51 displays quantifications of IHC aPDLl staining in lung tissue from the treated mice. Quantifications are presented as aPDLl+area (%).

[0067] FIG. 5 J displays quantifications of IHC PDL1 staining in tumor nodules from the treated mice. Quantifications are presented as aPDLl+area (%).

[0068] FIG. 5K displays the drug actions of ProAgio in lung cancer.

[0069] FIG. 6 displays that ProAgio synergistically enhances the effects of immune checkpoint blockade therapies for melanoma model. The model was generated by orthotopically implant IxlO6B16 cells inoculated into C57BL / 6J mice. Treatment started 13 days post tumor inoculation (tumor size reached -230 mm3), ProAgio 10 mg / kg daily dose for 15 days. Anti-PD-1 antibody (aPD-1) 3 mg / kg two doses a week, total of five doses, and ProAgio + aPD-1. Kaplan-Meier survival analysis of mice of the orthotopic model of B 16 cells treated with indicated agents is shown in the figure. The group size was 12 (n = 12). * P<0.05, *** P<0.005

[0070] FIG. 7 displays that ProAgio synergistically enhances the effects of immune checkpoint blockade therapies for Breast model. The model was generated by orthotopically implant 5xl054T-1 cells into fat pad of mammary gland of female Balb / C mice. Treatment started 15 days post tumor inoculation (tumor size reached -280 mm3), ProAgio 10 mg / kg daily dose for 15 days. Anti-PD-Ll antibody (aPD-Ll) 3 mg / kg one dose a week, total of three doses, and ProAgio + aPD-Ll. Kaplan-Meier survival analysis of mice of the orthotopic model of 4T1 cells treated with indicated agents is shown in the figure. The numbers (n) indicate the group size. ** P<0.01, *** P<0.005

[0071] FIG. 8 displays that ProAgio synergistically enhances the effects of immune checkpoint blockade therapies for pancreatic cancer. Genetically Engineered Mouse (GEM) (KrasG12D / +; Trp53R172H / +; Pdxl-Cre, KPC) pancreatic cancer model was generated by crossbreeding KrasG12D / +with Trp53R172H / +and then crossbreeding with Pdxl-Cre. Treatment started with mice 13 weeks old, ProAgio 10 mg / kg daily dose for 15 days. aPD-LI 4 mg / kg twice a week (5 doses), Gemcitabine 50 mg / kg twice weekly (5 doses), ProAgio + Gemcitabine, ProAgio + Gemcitabine + aPD-Ll. Kaplan-Meier survival analysis of KPC mice treated with indicated agents is shown in the figure. Tumors in 12 out of 14 KPC mice disappeared in the ProAgio + Gemcitabine + aPD-Ll group, ns statistically nonsignificant, ** P<0.01, *** P<0.005.DETAILED DESCRIPTION OF THE INVENTIONI DEFINITIONS

[0072] The following definitions are provided to facilitate understanding of certain terms used throughout this disclosure.

[0073] The term “angiogenesis” refers to the growth, development, and remodeling of the vascular bed, aiming to improve tissue oxygenation and nutrient delivery. This process includes the formation of new capillaries by sprouting from existing blood vessels, as well as the enlargement, maturation, and modification of existing vessels in terms of direction and flow properties, ultimately optimizing blood perfusion of tissues.

[0074] The term “amino acid” refers to naturally occurring and non-natural amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally encoded amino acids are the 20 common amino acids (alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine) and pyrolysine and selenocysteine. Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, by way of example only, an alpha-carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group. Such analogs may have modified R groups (by way of example, norleucine) or may have modified peptide backbones, while still retaining the same basic chemical structure as a naturally occurring amino acid. Non-limiting examples of amino acid analogs include homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium.

[0075] The term “variant” refers to a peptide or polynucleotide that differs from a reference peptide or polynucleotide, but retains essential properties. A typical variant of a peptide differs in amino acid sequence from another, reference peptide. Generally, differences are limited so that the sequences of the reference peptide and the variant are closely similar overall and, in many regions, identical. A variant and reference peptide may differ in amino acid sequence by one or more modifications (e.g., substitutions, additions,and / or deletions). A variant of a peptide includes conservatively modified variants (e.g., conservative variant of about 75%, about 80%, about 85%, about 90%, about 95%, about 98%, and about 99%) of the original sequence. A substituted or inserted amino acid residue may or may not be one encoded by the genetic code. A variant of a peptide may be naturally occurring, such as an allelic variant, or it may be a variant that is not known to occur naturally.

[0076] The term “conservatively modified variants” applies to both natural and nonnatural amino acid and natural and non-natural nucleic acid sequences, and combinations thereof. With respect to particular nucleic acid sequences, “conservatively modified variants” refers to those natural and non-natural nucleic acids which encode identical or essentially identical natural and non-natural amino acid sequences, or where the natural and non-natural nucleic acid does not encode a natural and non-natural amino acid sequence, to essentially identical sequences. By way of example, because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are “silent variations,” which are one species of conservatively modified variations. Thus by way of example every natural or non-natural nucleic acid sequence herein which encodes a natural or non-natural polypeptide also describes every possible silent variation of the natural or non-natural nucleic acid. One of skill will recognize that each codon in a natural or non-natural nucleic acid (except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan) can be modified to yield a functionally identical molecule. Accordingly, each silent variation of a natural and non-natural nucleic acid which encodes a natural and non-natural polypeptide is implicit in each described sequence.

[0077] The term “effective amount,” as used herein, refers to a sufficient amount of an agent or a compound being administered which will relieve to some extent one or more of the symptoms of the disease or condition being treated. The result can be reduction and / or alleviation of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system. By way of example, an agent or a compound being administered includes, but is not limited to, a natural amino acid polypeptide, non-natural amino acid polypeptide, modified natural amino acid polypeptide, or modified non-amino acid polypeptide. Compositions containing such natural amino acid polypeptides, non-natural amino acid polypeptides, modified natural amino acid polypeptides, or modified non-natural amino acid polypeptides can be administered for prophylactic, enhancing, and / or therapeutic treatments. An appropriate “effective” amount in any individual case may be determined using techniques, such as a dose escalation study.

[0078] The term “nucleic acid sequence” as used herein, refers to the order and identity of the nucleotides comprising a nucleic acid.

[0079] The term “nucleic acid” refers to deoxyribonucleotides or ribonucleotides and polymers thereof in single- or double-stranded form. The term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, which have similar binding properties as the reference nucleic acid, and which are metabolized in a manner similar to the reference nucleotides. Examples of such analogs include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs).

[0080] Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed- base and / or deoxyinosine residues. The term nucleic acid is used interchangeably with gene, cDNA, mRNA, oligonucleotide, and polynucleotide.

[0081] The terms “polypeptide,” “peptide” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymer.

[0082] The term “pharmaceutically acceptable”, as used herein, refers to a material, including but not limited, to a salt, carrier or diluent, which does not abrogate the biological activity or properties of the compound, and is relatively nontoxic, i.e., the material may be administered to an individual without causing undesirable biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained.

[0083] The term “prophylactically effective amount,” as used herein, refers that amount of a composition containing at least one non-natural amino acid polypeptide or atleast one modified non-natural amino acid polypeptide prophylactically applied to a patient which will relieve to some extent one or more of the symptoms of a disease, condition or disorder being treated. In such prophylactic applications, such amounts may depend on the patient's state of health, weight, and the like. It is considered well within the skill of the art for one to determine such prophylactically effective amounts by routine experimentation, including, but not limited to, a dose escalation clinical trial.

[0084] The phrase “substantially similar,” in the context of two nucleic acids or polypeptides, refers to two or more sequences or subsequences that have at least 75%, preferably at least 85%, more preferably at least 90%, 95% or higher or any integral value therebetween nucleotide or amino acid residue identity, when compared and aligned for maximum correspondence, as measured using a sequence comparison algorithm such as those described below for example, or by visual inspection. Preferably, the substantial identity exists over a region of the sequences that is at least about 10, preferably about 20, more preferable about 40-60 residues in length or any integral value therebetween, preferably over a longer region than 60-80 residues, more preferably at least about 90-100 residues, and most preferably the sequences are substantially identical over the full length of the sequences being compared, such as the coding region of a nucleotide sequence for example.

[0085] The term “synergistic”, as used herein, refers to a combination of prophylactic or therapeutic effective agents which is more effective than the additive effects of any two or more single agents. A synergistic effect of a combination of prophylactic or therapeutic agents may permit the use of lower dosages of one or more of the agents and / or less frequent administration of the agents to a subject with a specific disease or condition. In some cases, a synergistic effect of a combination of prophylactic or therapeutic agents may be used to avoid or reduce adverse or unwanted side effects associated with the use of any single therapy.

[0086] The term “therapeutically effective amount,” as used herein, refers to the amount of a composition containing at least one non-natural amino acid polypeptide and / or at least one modified non-natural amino acid polypeptide administered to a patient already suffering from a disease, condition or disorder, sufficient to cure or at least partially arrest, or relieve to some extent one or more of the symptoms of the disease, disorder or condition being treated. The effectiveness of such compositions depend on conditions including, but not limited to, the severity and course of the disease, disorder or condition, previous therapy, the patient’s health status and response to the drugs, and the judgment of thetreating physician. By way of example only, therapeutically effective amounts may be determined by routine experimentation, including but not limited to a dose escalation clinical trial.

[0087] The term “subject,” as used herein, refers to, but is not limited to, humans (e.g., a male or female of any age group, e.g., a pediatric subject (e.g., infant, child, adolescent) or adult subject (e.g., young adult, middle-aged adult or senior adult)) and / or other primates (e.g., cynomolgus monkeys, rhesus monkeys); mammals, including commercially relevant mammals such as cattle, pig, horses, sheep, goats, cats, and / or dogs; and / or birds, including commercially relevant birds such as chickens, ducks, geese, quail, and / or turkeys.

[0088] The term “cancer” refers to a condition characterized by unregulated or abnormal cell growth. The terms “cancer cell,” “tumor cell,” and “tumor” refer to abnormal cells or a mass of cells that result from excessive division, which may be malignant or benign, and encompass all pre-cancerous and cancerous cells and tissues.

[0089] The term “modulate” or “modulating” generally refers to the ability to alter, by increasing or decreasing, directly or indirectly promoting / stimulating / up-regulating or interfering with / inhibiting / down-regulating a specific concentration or level, such as acting as an antagonist or agonist.

[0090] The term “fibrotic” diseases, disorders, or conditions include those mentioned herein, and further include acute and chronic, clinical or sub-clinical presentation, in which fibrogenic associated biology or pathology is evident. Fibrotic diseases, disorders, or conditions include diseases, disorders or conditions characterized, in whole or in part, by the excess production of fibrous material, including excess production of fibrotic material within the extracellular matrix, or the replacement of normal tissue elements by abnormal, non-functional, and / or excessive accumulation of matrix-associated components. Fibrotic diseases, disorders, or conditions include, for example, fibrogenic-related biology or pathology characterized by fibrosis.

[0091] The term “fibrotic stroma” as used herein refers to a dense, connective tissue component of the tumor microenvironment (TME) that can negatively impact tumor immunity by creating physical barriers and altering the immune cell landscape. It can lead to immunosuppression, hindering the effectiveness of immunotherapy and promoting tumor growth.

[0092] The term “cell,” “cell population,” or “population of cells” as used herein refers to an isolated cell or plurality of cells excised from a tissue or grown in vitro by tissueculture techniques. Most particularly, a population of cells refers to cells in vivo in a tissue of an animal or human.

[0093] The term “contacting a cell or population of cells” as used herein refers to delivering a peptide or probe according to the present disclosure to an isolated or cultured cell or population of cells or administering the probe in a suitable pharmaceutically acceptable carrier to the target tissue of an animal or human. Administration may be, but is not limited to, intravenous delivery, intraperitoneal delivery, intramuscularly, subcutaneously, or by any other method known in the art. One advantageous method is to deliver directly into a blood vessel leading immediately into a target organ or tissue, thereby reducing dilution of the probe in the general circulatory system.

[0094] The term “pharmaceutically acceptable carrier” as used herein refers to a diluent, adjuvant, excipient, or vehicle with which a heterodimeric probe of the disclosure is administered and which is approved by a regulatory agency of the federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. Such pharmaceutical carriers can be liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. The pharmaceutical carriers can be saline, gum acacia, gelatin, starch paste, talc, keratin, colloidal silica, urea, and the like. When administered to a patient, the heterodimeric probe and pharmaceutically acceptable carriers can be sterile. Water is a useful carrier when the heterodimeric probe is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical carriers also include excipients such as glucose, lactose, sucrose, glycerol monostearate, sodium chloride, glycerol, propylene, glycol, water, ethanol, and the like. The present compositions, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. The present compositions advantageously may take the form of solutions, emulsion, sustained-release formulations, or any other form suitable for use.

[0095] The term “target” as used herein refers to a peptide, cell, tissue, tumor, etc., for which it is desired to detect. The target peptide may be on a cell surface, the cell being isolated from an animal host, a cultured cell, or a cell or population of cells in a tissue of an animal.

[0096] The present disclosure includes peptides which are derivable from the naturally occurring sequence of the peptide. A peptide is said to be “derivable from anaturally occurring amino acid sequence” if it can be obtained by fragmenting a naturally occurring sequence, or if it can be synthesized based upon knowledge of the sequence of the naturally occurring amino acid sequence or of the genetic material (DNA or RNA) that encodes this sequence. Included within the scope of the present disclosure are those molecules which are said to be “derivatives” of a peptide. Such a “derivative” or “variant” shares substantial similarity with the peptide or a similarly sized fragment of the peptide and is capable of functioning with the same biological activity as the peptide.

[0097] The derivatives of the present disclosure include fragments which, in addition to containing a sequence that is substantially similar to that of a naturally occurring peptide may contain one or more additional amino acids at their amino and / or their carboxy termini. Similarly, the disclosure includes peptide fragments which, although containing a sequence that is substantially similar to that of a naturally occurring peptide, may lack one or more additional amino acids at their amino and / or their carboxy termini that are naturally found on the peptide.

[0098] The disclosure also encompasses the obvious or trivial variants of the abovedescribed fragments which have inconsequential amino acid substitutions (and thus have amino acid sequences which differ from that of the natural sequence) provided that such variants have an activity which is substantially identical to that of the above-described derivatives. Examples of obvious or trivial substitutions include the substitution of one basic residue for another (i.e. Arg for Lys), the substitution of one hydrophobic residue for another (i.e. Leu for He), or the substitution of one aromatic residue for another (i.e. Phe for Tyr), etc.II. Compositions

[0099] This disclosure provides, at least in part, compositions and methods comprising a therapeutically effective amount of a modified CD2 polypeptide in combination with an immune checkpoint inhibitor and / or chemotherapeutics.A. Modified CD2 Polypeptide[000100] This disclosure provides compositions for treating a cancer in a mammal, in which the cancer is treated by the administering an anti -angiogenic agent, termed ProAgio to the mammal, in a therapeutically effective amount and frequency to produce a regression or arrest of the cancer without significant toxicity. The cancer may be selected from a group consisting of a solid tumor neoplasm, including lung carcinoma, breast carcinoma, prostate carcinoma, colon carcinoma, prostate carcinoma, ovarian carcinoma, neuroblastoma, central nervous system tumor, neuroblastoma, glioblastoma multiforme or melanoma.While no list can be complete, the anti-angiogenic agent may be used to produce a regression or arrest of most, if not all, solid tumors. The mammal receiving the treatment can be a human.[000101] As used herein, the term “ProAgio” refers to a modified CD2 polypeptide derived from the human or rat domain one of the CD2 polypeptide, possessing non-wild type characteristics. These non-wild type characteristics include a P-sheet formed by two segments with at least five amino acids alternating between hydrophilicity and hydrophobicity, an anti-parallel fold, an inward-facing hydrophobic surface, and an outward-facing hydrophilic surface. Exemplary agents include those disclosed in US Patent No. 9175063.[000102] This disclosure further relates to polypeptide compositions for use in treating cancer. In some embodiments, this disclosure provides polypeptides for use in treating lung cancer. This disclosure also includes an isolated polypeptide that specifically binds to integrin avp3 at PA domain in the region of a2 helix, B-C loop and a2-a3 loop (the “PA groove”). The PA groove is present in nearly all other P integrins and is relatively close to the RGD binding site in the PA domain. Thus, while it is disclosed that ProAgio may target the PA groove of integrin avP3, one skilled in the art should appreciate that ProAgio may be used to target the PA groove of any P integrin containing the PA groove.[000103] An example of such a polypeptide is ProAgio. Exemplary sequences of Pro Agio include an amino acid sequence of a variant domain one of CD2 with the following mutations: E8S, T9V, W10Q, GUM, A12K, D99N, I102V, Q103I, and E104I, and having 85%, 90%, 95%, 99% or 100% sequence identity to: KEITNALSVQMKLGQDINLDIPSFQMSDDIDDIKWEKTSDKKKIAQFRKEKETFKE KDTYELLKNGALKIKHLKTDDQDIYKVSIADTKGKNVLEKIFNLKVII (SEQ ID NO: 1).[000104] Another exemplary sequence of ProAgio includes an amino acid sequence of a variant domain one of CD2 with the M30C mutation, and having 85%, 90%, 95%, 99% or 100% sequence identity to: KEITNALSVQMKLGQDINLDIPSFQCSDDIDDIKWEKTSDKKKIAQFRKEKETFKE KDTYELLKNGALKIKHLKTDDQDIYKVSIADTKGKNVLEKIFNLKVII (SEQ ID NO:2).[000105] Yet another exemplary sequence of Pro Agio includes an amino acid sequence having 85%, 90%, 95%, 99% or 100% sequence identity to:KEITNALETWGALGQDINLDIPSFQMSDDIDDIKWEKTSDKKKIAQFRKEKETFKE KDTYKLFKNGTLKIKHLKTDDQDIYKVSIYDTKGKNVNDVCNFASRQER (SEQ ID N0:3).[000106] One other exemplary sequence of ProAgio includes an amino acid sequence having 85%, 90%, 95%, 99% or 100% sequence identity to:KEITNALETWGALGQDINLDIPSFQMSDDIDDIKWEKTSDKKKIAQFRKEKETFKE KD TYKLFKNGTLKIKHLKTDDQDIYKVSIYDTKGKNVLEKYDYLKIQER (SEQ ID NO: 4).B. Immune Checkpoint Inhibitor[000107] In some embodiments, the disclosure provides a combination therapy comprising of the CD2 polypeptide and / or ProAgio with an immune checkpoint inhibitor for synergistically reducing solid tumors in a subject in need thereof.[000108] Checkpoint blockade immunotherapy can mean a form of cancer treatment that works by targeting specific proteins on the surface of immune cells and cancer cells. These proteins, known as "checkpoints," normally help keep immune responses in check to prevent the immune system from attacking healthy cells in the body. Checkpoint blockade immunotherapy involves using drugs called checkpoint inhibitors to block these checkpoint proteins. By blocking the checkpoints, the drugs allow the immune system to recognize and attack the cancer cells more effectively. i. PD-1 Immune Checkpoint Inhibitor[000109] In particular embodiments, the immune checkpoint inhibitor is a PD-1 antagonist. PD-1 is a member of the CD28 family of receptors that delivers a negative immune response when induced on T cells. Contact between PD-1 and one of its ligands (B7-H1 or B7-DC) induces an inhibitory response that decreases T cell multiplication and / or the strength and / or duration of a T cell response. Suitable PD-1 antagonists are described in U.S. Patent Nos. 8,114,845, 8,609,089, and 8,709,416, which are specifically incorporated by reference herein in their entities, and include compounds or agents that either bind to and block a ligand of PD-1 to interfere with or inhibit the binding of the ligand to the PD-1 receptor, or bind directly to and block the PD-1 receptor without inducing inhibitory signal transduction through the PD-1 receptor.[000110] In some embodiments, the PD-1 receptor antagonist binds directly to the PD- 1 receptor without triggering inhibitory signal transduction and also binds to a ligand of the PD-1 receptor to reduce or inhibit the ligand from triggering signal transduction through the PD-1 receptor. By reducing the number and / or amount of ligands that bind to PD-1receptor and trigger the transduction of an inhibitory signal, fewer cells are attenuated by the negative signal delivered by PD-1 signal transduction and a more robust immune response can be achieved.[000111] It is believed that PD-1 signaling is driven by binding to a PD-1 ligand (such as B7-H1 or B7-DC) in close proximity to a peptide antigen presented by major histocompatibility complex (MHC) (see, for example, Freeman G. J., Proc. Natl. Acad. Sci. U. S. A, 105: 10275-10276 (2008)). Therefore, proteins, antibodies or small molecules that prevent co-ligation of PD-1 and TCR on the T cell membrane are also useful PD-1 antagonists.[000112] In some embodiments, the PD-1 receptor antagonists are small molecule antagonists or antibodies that reduce or interfere with PD-1 receptor signal transduction by binding to ligands of PD-1 or to PD-1 itself, especially where co-ligation of PD-1 with TCR does not follow such binding, thereby not triggering inhibitory signal transduction through the PD-1 receptor.[000113] Other PD-1 antagonists contemplated by the methods of this invention include antibodies that bind to PD-1 or ligands of PD-1, and other antibodies.[000114] Suitable anti -PD-1 antibodies include, but are not limited to, those described in the following US Patent Nos: 7332582, 7488802, 7521051, 7524498, 7563869, 7981416, 8088905, 8287856, 8580247, 8728474, 8779105, 9067999, 9073994, 9084776, 9205148, 9358289, 9387247, 9492539, 9492540, all of which are incorporated by reference in their entireties. See also Berger, R., et al., Clin. Cancer Res., 14: 3044-3051 (2008).[000115] Exemplary anti-PD-Ll antibodies include, but are not limited to, those described in the following US Pat Nos: 8383796, 9102725, 9273135, 9393301, and 9580507 all of which are specifically incorporated by reference herein in their entirety.[000116] Other exemplary PD-1 receptor antagonists include, but are not limited to PD- L2 polypeptides, including homologs and variants of these, as well as active fragments of any of the foregoing, and fusion proteins that incorporate any of these. In some embodiments, the fusion protein includes the soluble portion of B7-DC coupled to the Fc portion of an antibody, such as human IgG, and does not incorporate all or part of the transmembrane portion of human B7-DC.[000117] The PD-1 antagonist can also be a fragment of a mammalian PD-L1, for example from mouse or primate, such as a human, wherein the fragment binds to and blocks PD-1 but does not result in inhibitory signal transduction through PD-1. The fragments can also be part of a fusion protein, for example an Ig fusion protein.[000118] Other useful polypeptides PD-1 antagonists include those that bind to the ligands of the PD-1 receptor. These include the PD-1 receptor protein, or soluble fragments thereof, which can bind to the PD-1 ligands, such as PD-L1 or B7-DC, and prevent binding to the endogenous PD-1 receptor, thereby preventing inhibitory signal transduction. PD- L1 has also been shown to bind the protein B7.1 (Butte, M. J., et al., Immunity, 27(1): 111- 122, (2007)). Such fragments also include the soluble ECD portion of the PD-1 protein that includes mutations, such as the A99L mutation, that increases binding to the natural ligands (Lazar-Molnar, E., et al., Proc. Natl. Acad. Sci. U. S. A., 105(30): 10483-10488 (2008)). B7-1 or soluble fragments thereof, which can bind to the PD-L1 ligand and prevent binding to the endogenous PD-1 receptor, thereby preventing inhibitory signal transduction, are also useful.[000119] PD -1 and PD-L1 anti-sense nucleic acids, both DNA and RNA, as well as siRNA molecules can also be PD-1 antagonists. Such anti-sense molecules prevent expression of PD-1 on T cells as well as production of T cell ligands, such as PD-L1 and / or PD-L2. For example, siRNA (for example, of about 21 nucleotides in length, which is specific for the gene encoding PD-1, or encoding a PD-1 ligand, and which oligonucleotides can be readily purchased commercially) complexed with carriers, such as polyethyleneimine (see Cubillos-Ruiz, J. R., et al., J. Clin. Invest. 119(8): 2231-2244 (2009)), are readily taken up by cells that express PD-1 as well as ligands of PD-1 and reduce expression of these receptors and ligands to achieve a decrease in inhibitory signal transduction in T cells, thereby activating T cells.[000120] PD-1 inhibitors include but are not limited to, e.g., Pembrolizumab (Keytruda), Nivolumab (Opdivo), Cemiplimab (Libtayo); PD-L1 Inhibitors include, e.g., Atezolizumab (Tecentriq), Durvalumab (Imfinzi), and Avelumab (Bavencio). ii. CTLA4 antagonists[000121] Other molecules useful in mediating the effects of T cells in an immune response are also contemplated as additional therapeutic agents. In some embodiments, the molecule is an antagonist of CTLA4, for example an antagonistic anti-CTLA4 antibody. An example of an anti-CTLA4 antibody contemplated for use in the methods of the invention includes an antibody as described in PCT / US2006 / 043690 (Fischkoff et al., WO / 2007 / 056539).[000122] Dosages for anti-PD-1, and anti-CTLA4 antibody, are known in the art and can be in the range of, for example, 0.1 to 100 mg / kg, or with shorter ranges of 1 to 50 mg / kg, or 10 to 20 mg / kg. An appropriate dose for a human subject can be between 5 and15 mg / kg, with 10 mg / kg of antibody (for example, human anti-PD-1 antibody) being a specific embodiment.[000123] Specific examples of an anti-CTLA4 antibody useful in the methods of the invention are Ipilimumab, a human anti-CTLA4 antibody, administered at a dose of, for example, about 10 mg / kg, and Tremelimumab a human anti-CTLA4 antibody, administered at a dose of, for example, about 15 mg / kg. See also Sammartino, C., et al., Clinical Kidney Journal, 3(2): 135-137 (2010).[000124] In other embodiments, the antagonist is a small molecule. A series of small organic compounds have been shown to bind to the B7-1 ligand to prevent binding to CTLA4 (see Erbe, D. V., et al., J. Biol. Chem., 277(9): 7363-7368 (2002)). Such small organics could be administered alone or together with an anti-CTLA4 antibody to reduce inhibitory signal transduction of T cells.C. Chemotherapeutics[000125] In some embodiments, the compositions and methods described herein can include further agents or therapies, including but not limited to, chemotherapeutics, radiation, and surgery.[000126] In additional embodiments, the disclosure provides a combination therapy comprising of the CD2 polypeptide and / or ProAgio with an immune checkpoint inhibitor and / or chemotherapeutics.[000127] Representative chemotherapeutic agents include, but are not limited to amsacrine, bleomycin, busulfan, capecitabine, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, clofarabine, crisantaspase, cyclophosphamide, cytarabine, dacarbazine, dactinomycin, daunorubicin, docetaxel, doxorubicin, epirubicin, etoposide, fludarabine, fluorouracil, gemcitabine, hydroxycarbamide, idarubicin, ifosfamide, irinotecan, leucovorin, liposomal doxorubicin, liposomal daunorubicin, lomustine, melphalan, mercaptopurine, mesna, methotrexate, mitomycin, mitoxantrone, oxaliplatin, paclitaxel, pemetrexed, pentostatin, procarbazine, raltitrexed, satraplatin, streptozocin, tegafur-uracil, temozolomide, teniposide, thiotepa, tioguanine, topotecan, treosulfan, vinblastine, vincristine, vindesine, vinorelbine, or a combination thereof. Representative pro-apoptotic agents include, but are not limited to fludarabinetaurosporine, cycloheximide, actinomycin D, lactosylceramide, 15d-PGJ(2) and combinations thereof.D. Pharmaceutical Compositions[000128] Some embodiments also provide pharmaceutical compositions. Such compositions comprise a therapeutically effective amount of active components (e.g., theCD2 polypeptide plus immune checkpoint inhibitor or the CD2 polypeptide plus anti- immune checkpoint inhibitor plus chemotherapeutic or the CD2 polypeptide plus chemotherapeutic), and a pharmaceutically acceptable carrier. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like. The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Such compositions will contain a therapeutically effective amount of the anti- angiogenic agent together with a suitable amount of carrier so as to provide the form for proper administration to the patient. The formulation should suit the mode of administration.III. Methods of Treatment[000129] In another aspect, featured herein is a method of treating (e.g., inhibiting, reducing, ameliorating, managing, or preventing) a cancer in a subject. The method comprises administering to the subject a CD2 polypeptide and / or ProAgio, or a pharmaceutically acceptable form thereof, in combination with an immune checkpoint inhibitor and / or chemotherapy. In certain embodiments, the method is for use in vivo, e.g., in a subject or as part of a therapeutic protocol.[000130] In another aspect, featured herein is a method of inhibiting the growth or the viability, or both, of a cancer cell. The method includes contacting the cancer cell with a CD2 polypeptide and / or ProAgio, or a pharmaceutically acceptable form thereof, in combination with an immune checkpoint inhibitor and / or chemotherapy.[000131] In some embodiments, the subject is a mammal, e.g., a human. In some embodiments, the subject is at risk or suffers from a cancer, e.g., a cancer described herein.[000132] In some embodiments, the cancer to be treated using the methods or compositions described herein is breast carcinoma, lung carcinoma, non-small cell lung carcinoma, colon carcinoma, prostate carcinoma, ovarian carcinoma, neuroblastoma, central nervous system tumor, glioblastoma multiforme or melanoma.[000133]A. Methods of Modulating Cancer Immunity[000134] The immune cells activated by the disclosed compositions can kill cancer cells and reduce tumor burden in a subject. The term “cancer cell” is meant to encompass both pre-malignant and malignant cancer cells. In some embodiments, cancer refers to a benign tumor, which has remained localized. In other embodiments, cancer refers to a malignant tumor, which has invaded and destroyed neighboring body structures and spread to distant sites. In yet other embodiments, the cancer is associated with a specific cancer antigen (e.g., pan-carcinoma antigen (KS 1 / 4), ovarian carcinoma antigen (CA125), prostate specific antigen (PSA), carcinoembryonic antigen (CEA), CD 19, CD20, HER2 / neu, etc.).[000135] Disclosed herein are methods of modulating tumor immunity by simultaneously depleting CAFs and tumor angiogenic vessels using a rationally designed protein that induces integrin avbs targeted cell apoptosis. The disclosure offers a unique opportunity for the enhancement of cancer immunotherapies, especially for subjects with tumors of dense stroma and high angiogenesis.[000136] Fibrotic stroma and angiogenic tumor vessels play an important role in modulating tumor immunity. A rationally designed protein (ProAgio) targets integrin avPs at a novel site. ProAgio induces apoptosis of cells that express high levels of the integrin. Both activated cancer-associated fibroblasts (CAF) and angiogenic endothelial cells (aEC) in tumor express high levels of integrin avp3. ProAgio simultaneously and specifically induces apoptosis in CAFs and aEC in tumors. Depletion of CAFs and elimination of leaky tumor angiogenic vessels by ProAgio is herein shown to alter tumor immunity. ProAgio reduces CD / Treg and Myeloid-derived suppressor cells (MDSC), increases CDs T-cells, and increases the M1 / M2 macrophage ratio in the tumor. Depletion of dense fibrotic stroma (CAFs) by ProAgio decreases the PDL-1 levels in the stroma areas surrounding the tumors, thus, strongly increasing the delivery of anti -PDL-1 antibody to the target cancer cells. The effects of ProAgio on tumor immunity provide strong synergistic effects of checkpoint inhibitors on lung cancer treatment.B. Method of Treating Lung Cancer[000137] Lung cancers, particularly advanced disease, usually have high levels of activated CAFs and are rich in angiogenic vessels. The highly activated CAFs and angiogenesis orchestrate lung cancer progression and resistance to drug treatment. The cancer promoting CAF and angiogenesis modulate tumor immunity. It is well established that activated CAFs and tumor angiogenesis contribute the checkpoint blockade immunotherapy resistance in lung cancer (De Jaeghere, E. A., et al., Trends Cancer., 5(11): 704-723 (2019); Solimando, A. G., et al., Cancers (Basel)., 12(11) (2020); Datta, M., et al., Am Soc Clin Oncol Educ Book., 39: 165-174 (2019)). Furthermore, lung cancer is often associated with high levels of PDL-1 expression in tumors (Yu, H., et al., J. Thorac. Oncol., 11(7): 964-75 (2016); Pawelczyk, K., et al., Ini. J. Mol. Set., 20(4) (2019)). PDL-1 is not only expressed on lung cancer cells but also on the CAFs in the tumor. The high level of PDL-1 on the CAFs form a molecular trap to prevent PDL-1 blockade from reaching the target cancer cells thus reducing treatment effectiveness of the blockade. Therapeutic strategies targeting tumor fibrotic stroma have been actively explored, particularly in treatment of pancreatic ductal adenocarcinoma (PDAC) due to the unique properties of desmoplasia of PDAC. However, there is a very limited success (Chen, X., et al., Nat. Rev. Drug. Discov., 18(2): 99-115 (2019); Barnett, R. M., et al., J. Natl. Cancer. Inst., 110(1) (2018)). Due to the important role of CAFs and angiogenesis in modulating tumor immunity, targeting CAF and tumor angiogenesis to improve checkpoint blockade immunotherapy in lung cancer treatment has become a promising treatment strategy.[000138] We provide herein an example of simultaneous depletion of lung cancer CAFs and eliminating angiogenic vessels by a novel rationally designed protein drug targeting integrin avbs (Turaga, R. C., et al., Cell Mol Gastroenterol Hepatol., 11(1): 161-179 (2021); Sharma, M., et al., J Exp Med., 218(4) (2021). Depletion of CAFs and elimination of leaky tumor angiogenic vessels by ProAgio alter tumor immunity as revealed by an increase in CD8+T-cells and decreases in CD4+Treg and MDSC cells in tumor. ProAgio also changed tumor immunity by increasing the M1 / M2 macrophage ratio. Due to the effect of ProAgio on tumor immunity, the drug strongly enhanced treatment effectiveness with PDL-1 blockade aPDL-1. The disclosure shows that ProAgio enhances aPDL-1 treatment effectiveness by (Fig. 5G); (1) alteration of tumor immunity due to depletion of CAFs and tumor angiogenesis, (2) increase in tumor blood perfusion and thus increases infiltration of aPDL-1 due to eliminating leaky and disorganized angiogenic tumor vessels, (3) enhancement of anti-tumor immunity due to decrease in tumor hypoxia, and (4) removal of the molecular trap of aPDL-1 due to depletion of activated CAFs. Although checkpointblockade immunotherapy with or without the combination of chemotherapies has become standard of care regimen for lung cancer patients. A large percentage of patients do not respond initially to treatment or relapse after a period of response, mainly due to the immunosuppressive effects of activated CAFs and angiogenesis in the tumors. Thus, (e.g., simultaneous) depletion of angiogenic vessels and activated CAFs by ProAgio will certainly provide very important benefits in lung cancer treatment, especially in combination with checkpoint blockade immunotherapy.[000139] ProAgio decreased PDL-1 in the tumor lung most likely due to the depletion of PDL-1 expressing CAFs and aEC. However, the treatment increased PDL-1 in the tumor nodules, which is consistent with the observation that ProAgio in combination with aPDL- 1 led to such strong enhancement of effectiveness in the treatment. It is well-known that chemotherapies upregulate PDL-1 expression on cancer cells due to induction of apoptosis of cancer cells (Cha, J. H., et al., Mol Cell., 76(3): 359-370 (2019)). Thus, it is plausible that depletion of activated CAFs and angiogenic tumor vessels by ProAgio consequently results in cancer cells apoptosis, thus, increases PDL-1 levels on cancer cells. Increase in PDL-1 levels in lung tumor nodules certainly would increase accumulation of administered aPDL-1. However, the increase in aPDL-1 levels are not proportional to the increase in PDL-1 levels in the tumor nodules. Thus, it is less likely that the increase in aPDL-1 upon ProAgio treatment in the tumor nodules is solely due to increase in PDL-1 levels. Preventing aPDL-1 from trap by activated CAFs and angiogenic vessels play an important role in increase of aPDL-1 in tumor nodules. How ProAgio and aPDL-1 further decreased CAF and angiogenesis in tumor is an open question. A plausible explanation is that aPDL- 1 blocked PDL-1 on CAF and aEC, while ProAgio enhanced inflammation immunity. Thus, the increased infiltration of immune killer cells lead to increased induction of apoptosis of CAFs and aEC.[000140] Both aEC and CAFs in tumor express high levels of integrin avPs (Brooks, P. C., et al., Science., 264(5158): 569-71 (1994); Attieh, Y., et al., J Cell Biol., 216(11): 3509- 3520 (2017)). ProAgio that targets integrin avPs at a novel site and induces apoptosis in the integrin-expressing cells by recruiting caspase 8 at the cytoplasmic domain of P3 (Turaga, R.C., et al., Nat Commun., 7: 11675 (2016)). This disclosure shows that ProAgio induces apoptosis in integrin avp3-expressing CAFs and aEC in lung tumors. Depletion of CAFs by ProAgio decreases intratumoral collagen. Depletion of CAFs by ProAgio consequently decreases tumor immunosuppression effects, e.g. increase in CD8+T-cells and decrease in CD4+Treg cells and MDSC cells in lung tumors. ProAgio also increases the macrophageM1 / M2 ratio in tumor. In addition, the anti-angiogenetic effect of ProAgio eliminates angiogenic leaky tumor vessels, which consequently facilitates immune cell infiltration and decreases hypoxia in murine models of lung cancers. Depletion of CAFs by ProAgio reduces immune checkpoint blockade (PDL-1) levels in tumor stroma regions that are otherwise present due to high levels of expression of PDL-1 on CAFs. In addition, depletion of CAFs by ProAgio prevents checkpoint blockade from being trapped by CAFs as otherwise happens due to high expression of PDL-1 on CAFs. Because of the effects of ProAgio in modulating tumor immunity by simultaneously depleting CAF and eliminating tumor angiogenic vessels, a synergistic treatment effect of ProAgio in combination with check point blockade anti-PDL-1 (aPDL-1) is observed, suggesting an excellent treatment strategy for lung cancer patients, especially for the patients whose tumors exhibit resistance to and have relapsed from checkpoint blockade immunotherapies.IV. Methods of Use[000141] The disclosed compositions can be used to modulate an immune response in a subject in need thereof. One embodiment provides a method of inducing or enhancing an immune response in a subject in need thereof. Typically, the methods include administering a subject an effective amount of the compositions or combination therapies disclosed herein to immunospecifically bind to integrin avp3 at the PA groove and reduce or block the immune suppressive signal of integrin avp3, thus promoting an immune response and enhancing the effectiveness of the PDL-1 antibody. The immune response can be, for example inducing, promoting or enhancing T cell activation, secretion of cytokines by immune cells, T cell proliferation. The disclosed compositions or combination therapies can be administered to a subject in need thereof in an effective amount to overcome T cell suppression. Overcoming T cell suppression can be determined by measuring T cell function using known techniques.A. Methods of Administration[000142] The methods in this application, according to certain embodiments, comprise administering to the subject a therapeutically effective amount of a modified CD2 polypeptide and an immune checkpoint inhibitor. As used herein, the expression “in combination with” means that the checkpoint inhibitor or chemotherapeutic is administered before, after, or concurrent with the modified CD2 polypeptide. The term “in combination with” also includes sequential or concomitant administration of the checkpoint inhibitor and the modified CD2 polypeptide.[000143] In certain embodiments, the methods can comprise administering a modified CD2 polypeptide in combination with a checkpoint inhibitor and / or chemotherapeutics for additive or synergistic activity to treat cancer. In some embodiments, the modified CD2 polypeptide can be administered in combination with chemotherapeutic and other therapeutic agents, as well as radio-therapies. The chemotherapeutic agent may be one or more selected from the group consisting of vinca alkaloid, camptothecan, taxane, or platinum analogue, including vincristine, vinblastine, vinorelbine, vindesine, paclitaxel, docetaxel, 5 FU, cisplatin, carboplatin, irinotecan, topotecan and cyclophosphamide. The chemotherapeutic agent can be administered in a low-dose regimen, in combination with the CD2 polypeptide because of the anti-tumor effect of the anti-angiogenic agent. In some embodiments, the chemotherapeutic agent can be administered at less than the maximum tolerated dose.[000144] For example, when administered “before” the modified CD2 polypeptide, the checkpoint inhibitor may be administered more than 150 hours, about 150 hours, about 100 hours, about 72 hours, about 60 hours, about 48 hours, about 36 hours, about 24 hours, about 12 hours, about 10 hours, about 8 hours, about 6 hours, about 4 hours, about 2 hours, about 1 hour, or about 30 minutes, about 15 minutes, or about 10 minutes prior to the administration of the modified CD2 polypeptide. When administered “after” the modified CD2 polypeptide, the checkpoint inhibitor may be administered about 10 minutes, about 15 minutes, about 30 minutes, about 1 hour, about 2 hours, about 4 hours, about 6 hours, about 8 hours, about 10 hours, about 12 hours, about 24 hours, about 36 hours, about 48 hours, about 60 hours, about 72 hours, or more than 72 hours after the administration of the modified CD2 polypeptide. Administration “concurrent” with the modified CD2 polypeptide means that the checkpoint inhibitor is administered to the subject within less than 10 minutes (before, after, or at the same time) of administration of the modified CD2 polypeptide.[000145] In some embodiments, the subject can be administered one or more doses of the first agent every 1, 2, 3, 4, 5, 6, 7, 14, 21, 28, 35, or 48 days before a first administration of the second or third agent. The modified CD2 polypeptide can be the first, second, or third therapeutic agent.[000146] The disclosed modified CD2 polypeptide and the additional therapeutic agent can be administered as part of a therapeutic regimen. For example, if a first therapeutic agent can be administered to a subject every fourth day, the second therapeutic agent can be administered on the first, second, third, or fourth day, or combinations thereof. The firstT1therapeutic agent or second therapeutic agent may be repeatedly administered throughout the entire treatment regimen.EXAMPLESExample 1: Effects of ProAgio on CAEs isolated from lung cancer tumor[000147] Materials and Methods[000148] Cell line and human CAF: The lung adenocarcinoma cell line A549 was purchased from ATCC. Cells were cultured in DMEM medium under a humidified atmosphere of 5% CO2 at 37 °C, supplemented with 10% fetal bovine serum and 1% penicillin. CAF isolated from patient was purchased from Neuromics. The cells were cultured according to vendor’s suggestion.[000149] Tissue staining: IHC, Sirius red, and Immunofluorescence (IF): IHC, Sirius red and IF staining procedures were similar to those of previous reports (Turaga, R. C., et al., Cell. Mol. Gastroenterol. Hepatol., 11(1): 161-179 (2021); Sharma, M., et al., J. Exp. Med, 218(4) (2021)). Quantification of Sirius red, IHC, and immunofluorescence staining was conducted utilizing Imaged software. The quantities are presented as described in figure legends.[000150] Cell apoptosis assay: The procedures for these assays were similar to our previous reports [36, 37], FACS data were analyzed by FlowJo software (Tree Star).[000151] Results[000152] The effects of ProAgio on the CAFs isolated from lung cancer patient tumor were examined. The lung adenocarcinoma cell line A549 was purchased from ATCC. Cells were cultured in DMEM medium under a humidified atmosphere of 5% CO2 at 37 °C, supplemented with 10% fetal bovine serum and 1% penicillin. CAF isolated from a patient was purchased from Neuromics. The cells were cultured according to the vendor’s suggestion.[000153] Apoptosis of CAF under treatment with concentrations (5 pM, 10 pM, and 15 pM) was measured by an apoptosis kit (FIG. IB). The procedure for this assay was similar to previous reports (Han, H., et al., iScience., 24(10): 103165 (2021); Sharma, M., et al., Journal of Experimental Medicine., 218(4) (2021)). ProAgio induced apoptosis of the CAF.[000154] A s.c. xenograft model of human NSCLC A549 cells was employed to study the effect of ProAgio on tumors. A540 (5xl05) cells were implanted into right flank of nude mice. Tumor growth was measured using caliper ruler. Tumor bearing mice weretreated with vehicle and ProAgio. ProAgio inhibited A549 tumor growth (bottom line, FIG. 1C).[000155] IHC staining of CD31 with A549 tumor sections demonstrated that ProAgio treatment decreased tumor angiogenesis (FIG. ID). Tumor sections / tissues of A549 xenograft mice treated with vehicle or ProAgio underwent IHC of a-SMA (FIG. IE), Sirius red staining (FIG. IF), and hydroxyproline assay (FIG. 1G). The procedure for the hydroxyproline assay was similar to previous reports (Han, H., et al., iScience., 24(10): 103165 (2021); Sharma, M., et al., Journal of Experimental Medicine., 218(4) (2021)). IHC, Sirius red and IF staining procedures were similar to those of previous reports (Turaga, R. C., et al., Cell Mol. Gastroenterol Hepatol., 11(1): 161-179 (2021); Sharma, M., et al., J. Exp. Med., 218(4) (2021)). Quantification of Sirius red, IHC, and immunofluorescence staining was conducted utilizing Imaged software. These assays showed that ProAgio treatment reduced collagen and a-SMA positive cells in the tumors (FIG. 1 E-1G).Example 2: Effects of ProAgio on genetically engineered mouse (GEM) NSCLC model[000156] Materials and Methods[000157] Genetically engineered lung cancer mouse model (GEM-NSCLC) and the treatments: All animal experiments were conducted following NTH guidance and approved by the Institutional Animal Care and Use Committee of Georgia State University, A549 xenograft: A540 (5xl05) cells were implanted into right flank of nude mice. Tumor growth was measured using caliper ruler. For GEM-NSCLC model, 10 to 12-week-old KP mice (LSL-KrasG12D / +; LSL-Trp53R172H / +) were utilized. The breeding offspring mice were genotyped for the correct KrasLSL-G12D and p53 R172H / fl alleles by PCR using following PCR primer pairs following protocols from the Jackson Laboratory website (http: / / web.mit.edu / jacks-lab / protocols_table.html). Genotyping PCR primer pairs P53 forward (5’-AGCCTGCCTAGCTTCCTCAGG-3’SEQ ID NO: 5) and P53 reverse (5’CTTGGAGACATAGCCACACTG-3’ SEQ ID NO: 6), or KRAS reverse (5’- TGTCTTTCCCCAGCACAGT-3’ SEQ ID NO: 7), KRAS forward (5’- GCAGGTCGAGGGACCTAATA-3’ SEQ ID NO: 8) and KRAS WT(5’- CTGCATAGTACGCTATACCCTGT-3’ SEQ ID NO: 9). To induce recombination, a replication-deficient adenovirus expressing Cre (AdV-Cre) from the University of Iowa Gene Transfer Vector Core was delivered via intratracheal intubation. The titer of AdV- Cre is 5.0* 107PFU / ml and the total volume of AdV-Cre is 75pl per mouse. The describedtreatments began 12 weeks after AdV-Cre delivery. Mice were randomly assigned to treatment groups, with early treatment (ET) consisting of 15 mg / kg ProAgio for 4 consecutive days and late treatment (LT) involving the same dosage for 12 days. Survival treatment included 15 mg / kg ProAgio administered for 10 consecutive days, followed by 10 doses every other day, 4 mg / kg / / d / ToMAb anti-mouse PD-L1 (B7-H1) (BE0101, Bioxcell) twice per week for 4 weeks or a combination. At the end of treatments, mice were either sacrificed for analysis or maintained in cages for survival assessment. Organs, tumor tissues, and blood samples were collected for subsequent analyses. Statistical comparisons were made against the control group.[000158] Results[000159] To determine whether ProAgio is effective in lung cancer treatment, a genetically engineered mouse (GEM) NSCLC model (Ref to as GEM-NSCLC) generated from breeding KrasG12Dand P53R172Hmice and intratracheal intubation delivery' of adenoviral coded Cre recombinase (AdV-Cre) was employed

[0031] . For GEM-NSCLC model, 10 to 12-week-old KP mice (LSL-KrasGi2D / +; LSL-Trp53Ri72H / +) were utilized. The breeding offspring mice were genotyped for the correct KrasLSL-G12D and p53 Ri72H / fl alleles by PCR using following PCR primer pairs following protocols from the Jackson Laboratory website. Genotyping PCR primer pairs P53 forward (5’- AGCCTGCCTAGCTTCCTCAGG-3’ SEQ ID NO: 5) and P53 reverse (5’CTTGGAGACATAGCCACACTG-3’ SEQ ID NO: 6), or KRAS reverse (5’- TGTCTTTCCCCAGCACAGT-3’ SEQ ID NO: 7), KRAS forward (5’- GCAGGTCGAGGGACCTAATA-3’ SEQ ID NO: 8) and KRAS WT (5’- CTGCATAGTACGCTATACCCTGT-3’ SEQ ID NO: 9). To induce recombination, a replication-deficient adenovirus expressing Cre (AdV-Cre) from the University of Iowa Gene Transfer Vector Core was delivered via intratracheal intubation. The titer of AdV- Cre is 5. Ox 107PFU / ml and the total volume of AdV-Cre is 75pl per mouse. The described treatments began 12 weeks after AdV-Cre delivery. Mice were randomly assigned to treatment groups, with early treatment (ET) consisting of 15 mg / kg ProAgio for 4 consecutive days and late treatment (LT) involving the same dosage for 12 days. Survival treatment included 15 mg / kg ProAgio administered for 10 consecutive days, followed by 10 doses every other day, 4 mg / kg InVivo MAb anti-mouse PD-L1 (B7-H1) (BE0101, Bioxcell) twice per week for 4 weeks or a combination. At the end of treatments, mice were either sacrificed for analysis or maintained in cages for survival assessment. Organs, tumortissues, and blood samples were collected for subsequent analyses. Statistical comparisons were made against the control group.[000160] The GEM mice were treated with vehicle or ProAgio (15 mg / kg, 15 Q1D doses) 126 days after AdV-Cre delivery' (FIG. 2A). The mice were maintained in their home cages for assessment of survival. ProAgio treatment increased the survival of tumorbearing mice (FIG. 2B). Randomly selected mice (n = 9) were euthanized 10 days after the last dose of treatment.[000161] Examination of lungs of the treated mice revealed that ProAgio reduced the lung tumor nodules both in size and numbers in the GEM mice (FIG. 2C-2G).[000162] Sirius Red and IHC of a-SMA staining demonstrated that ProAgio depletes activated CAFs and reduced tumor collagen levels (FIG. 2H-2K). IHC, Sirius red and IF staining procedures were similar to those of previous reports (Turaga, R. C., et al., Cell Mol. Gastroenterol Hepatol., 11(1): 161-179 (2021); Sharma, M., et al., J. Exp. Med., 218(4) (2021)). Quantification of Sirius red, IHC, and immunofluorescence staining was conducted utilizing ImageJ software.[000163] IHC staining of CD31 with tumor section showed that ProAgio reduced tumor angiogenic vessels (FIG. 2L-2N). These results suggest that ProAgio simultaneously targets CAFs and tumor angiogenesis and is potentially a drug candidate for lung cancer treatment.Example 3: ProAgio enhances checkpoint inhibitor immunotherapy in lung cancer[000164] Due to clinical success, check point inhibitors immunotherapies alone and in combinations with chemotherapies have been established as standard of care treatments for lung cancers (Onoi, K., et al., J. Clin. Med, 9(5) (2020)). ProAgio reduces CD1 lb positive cells in both primary and metastatic tumors of breast cancer models (Sharma, M., et al., J. Exp. Med., 218(4) (2021)). To determine whether ProAgio would enhance checkpoint inhibitor immunotherapy in lung cancer, we employed the GEM-NSCLC model. The GEM-NSCLC model was used as described in Example 2.[000165] The GEM mice were treated with vehicle (bottom line, FIG. 3B), ProAgio (15 mg / kg, 10 Q1D plus 10 Q.O.D. doses, third from bottom line, FIG. 3B), anti-PDL-1 antibody (aPDL-1, 4 mg / kg, twice weekly for four weeks, second from bottom line, FIG. 3B), and ProAgio + aPDLl (top line, FIG. 3B) 12 weeks after delivery of AdV-Cre (FIG. 3A). ProAgio or aPDL-1 alone provided survival advantage, while ProAgio + aPDL-1provided an even greater increase in survival benefits (Tumors in more than half of mice in the ProAgio + aPDL-1 group disappeared) (FIG. 3B).[000166] Examination of lungs of the treated mice revealed that ProAgio reduced the lung tumor nodules both in size and numbers in the GEM mice. ProAgio + aPDLl almost completely eliminated the tumor nodules in the lung of the treated mice, particularly the large size nodules (FIG. 3C-3H).[000167] ProAgio treatment depleted CAF and reduced collagen in tumor. Interestingly, ProAgio + aPDLl further decreased CAFs and collagen in tumor compared to ProAgio alone group (FIG. 3I-3N). This suggests that aPDL-1 might lead to apoptosis of CAFs in the tumor due to block of PDL-1 on the activated CAFs.[000168] Staining of CD31 demonstrated that ProAgio eliminated angiogenic tumor vessels. The ProAgio and aPDL-1 combination led to more reduction in tumor angiogenic vessels (FIG. 3O-3R).Example 4: ProAgio alters lung tumor immunity[000169] Materials and Methods[000170] Immune cell FACS analyses, and hydroxyproline assay: The procedures for these assays were similar to our previous reports (Flan, H., et al., iScience., 24(10): 103165 (2021); Sharma, M., et al., Journal of Experimental Medicine., 218(4) (2021)). FACS data were analyzed by FlowJo software (Tree Star)[000171] Cytokine and chemokine ELISA quantification assay: For quantifying chemokines and cytokines, the lung tissues from tumor bearing mice before and after treatment were collected and analyzed using Elisa Kits according to the manufacturer's instructions. Calibrator blends provided in the kits are added and analyzed together with samples on the same plates. The concentrations of calibrator blends and raw values obtained after reading plates were used to generate standard curves to calculate concentrations of each measurement.[000172] Statistical analyses: Statistical analyses were performed using GraphPad Prism 9.0 software, and each experiment was conducted a minimum of three times. Significance was assessed using either Student's t-test and / or one-way ANOVA for multiple comparisons. In all figures, the statistical significance level was defined as P < 0.05.; n.s. indicates not significant. All data are presented as mean ± sem.[000173] Results[000174] Depletion of CAFs and abrogation of tumor angiogenesis alter tumor immunity. To test whether ProAgio treatment that depletes CAFs and eliminatesangiogenic vessels affects tumor immunity, GEM-NSCLC mice were treated with either 4 daily doses (Referred to as early treatment or ET) or 12 daily doses (Referred to as late treatment or LT) of ProAgio or vehicle 12 weeks after delivery of AdV-Cre, as detailed above in Example 2. Animals were euthanized two days following the last dose (FIG. 4A). Similarly, sirius red and IHC of a-SMA staining indicated reduction of collagen and CAFs in the tumors and surrounding lung tissues in both ET and LT treatment. CD31 staining demonstrated a decrease in tumor angiogenesis. Hypoxia strongly affects immunity (McGettrick, A. F., et al., Cell Metab., 32(4): 524-536 (2020); Wu, Q., et al., J. Hematol. Oncol., 15(1): 77 (20220). Eliminating leaky angiogenic vessels by ProAgio increases blood perfusion into tumor, which consequently reduced tumor hypoxia Hif-la of lung tumor (Turaga, R. C., et al., Cell Mol Gastroenterol Hepatol., 11(1): 161-179 (2021); Sharma, M., et al., J Exp Med., 218(4) (2021)).[000175] IHC staining of lung sections from treated mice suggested that ProAgio treatment decreased Hif-la in lung tumor (FIG. 4B-4E), suggesting that, similar to our observations with pancreatic and breast cancer, ProAgio treatment decreased lung cancer hypoxia due to its anti-angiogenic effects. Immune cells in tumors were subsequently analyzed via FACS. The procedures for this assay was similar to previous reports (Han, H., et al., iScience., 24(10): 103165 (2021); Sharma, M., et al., Journal of Experimental Medicine., 218(4) (2021)). FACS data were analyzed by FlowJo software (Tree Star). ProAgio treatment resulted in a >2-fold and >3-fold increase in CD8+T-cells in ET and LT groups respectively (FIG. 4F). ProAgio treatment led to >3-fold and >10-fold decrease in CD4+Treg cells in ET and LT groups respectively (Fig. 4G). Furthermore, ProAgio treatment also decreased MDSC (FIG. 4H) and increased the Ml to M2 macrophage ratio in tumors (FIG. 41). Overall, ProAgio strongly increased the anti-tumor immunity and reduced cancer immune resistance.[000176] The effects were also reflected by the changes in cytokine / chemokine profiles in the lung of the tumor bearing mice. IL-6, CXCL2, and CXCL12 are cytokine / chemokine that are released by CAFs to regulate tumor immunity and angiogenesis. ProAgio treatment altered IL-6, CXCL2, and CXCL12 levels in the lungs (FIG. 4J-4L).Example 5: Elimination of CAFs and leaky angiogenic vessels by ProAgio increased delivery of aPDL-1 to lung tumors, particularly in the histological carcinoma regions.[000177] Depletion of CAFs and elimination of tumor leaky vessels enhances drug (small and large molecules) delivery to tumors (Turaga, R. C., et al., Cell Mol Gastroenterol Hepatol., 11(1): 161-179 (2021); Sharma, M., et al., J Exp Med., 218(4) (2021)). It is well known that CAFs express high levels of PDL-1 (Kawasaki, K., et al., Cancer Immunol Immunother. 72(11): 3787-3802 (2023)). Thus, dense fibrotic stroma forms both a physical barrier and a molecular trap for the administered aPDL-1, preventing it reaching to its target cancer cells. Depletion of fibrotic stroma and elimination of leaky angiogenic vessels by ProAgio would facilitate the delivery of aPDL-1 to cancer cells in lung tumors. To confirm whether depletion of fibrotic stroma and angiogenic vessels indeed help administered aPDL-1 to reach the target cancer cells, PDL-1 in the lung and lung tumor nodules of GEM-NSCLC mice was analyzed. PDL-1 levels in the lung of tumorbearing mice decreased by approximately 2 folds, while the PDL-1 levels in the tumor nodules increased by approximately 1.4 folds, upon ProAgio treatment (FIG. 5A-5E). The observation suggests that ProAgio depletes high PDL-1 expression activated CAFs in the tumor-containing lung, therefore, leading to a decrease in PDL-1 levels. An increase in PDL-1 levels upon ProAgio treatment would be consistent with the strong synergistic effects of ProAgio in combination with aPDL-1.[000178] The levels of the administered aPDL-1 and the localization of the aPDL-1 relative to a-SMA positive CAF and epithelium carcinoma cancer cells 12 hours after administration were analyzed. The administered aPDL-1 levels were high in the lung tissue surrounding the tumor nodules, while the aPDL-llevels inside the tumor nodules were very low in the vehicle treated group. Conversely, the administered aPDL-1 levels were relatively low in the lung tissue surrounding the tumor nodules, while the aPDL-llevels inside the tumor nodules were very high in the ProAgio treated group (FIG. 5F - 5L). Thus, we conclude that depletion of CAFs by ProAgio in tumor fibrotic stroma reduced PDL-1 levels in the stroma, which consequently prevented the administered aPDL-lfrom being trapped in the tumor microenvironment mainly by CAFs, facilitating accumulation of more administered antibody in the carcinoma areas. This action will enhance the effectiveness of aPDL-1. FIG. 6 and 7 show other examples.

Claims

What is claimed is:

1. A pharmaceutical composition for reducing a tumor burden in a subject having a cancer comprising a combination therapy comprising: a. a first therapeutic agent comprising a therapeutically effective dose of a modified CD2 polypeptide derived from human or rat domain one of CD2 polypeptide, the modified CD2 polypeptide having non-wild type characteristics, the non-wild type characteristics including a P-sheet formed by two segments having at least five amino acids alternating between hydrophilicity and hydrophobicity, an anti-parallel fold, an inward-facing hydrophobic surface, and an outward-facing hydrophilic surface; b. a second therapeutic agent comprising an immune checkpoint inhibitor; and c. a pharmaceutically acceptable excipient; wherein the modified CD2 polypeptide is administered in combination with the immune checkpoint inhibitor and the pharmaceutically acceptable excipient, wherein the modified CD2 polypeptide reduces PD-L1 expression in a tumor’s fibrotic stroma to increase the delivery of the immune checkpoint inhibitor to target cancer cells, and wherein PD-L1 expression in a solid tumor’s fibrotic stroma synergistically increases the activity of the immune checkpoint inhibitor.

2. The pharmaceutical composition according to claim 1, further comprising the combination of one or more additional therapeutic agents, wherein the additional therapeutic agents are anti-cancer chemotherapy drugs selected from gemcitabine, amsacrine, bleomycin, busulfan, capecitabine, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, clofarabine, crisantaspase, cyclophosphamide, cytarabine, dacarbazine, dactinomycin, daunorubicin, docetaxel, doxorubicin, epirubicin, etoposide, fludarabine, fluorouracil, hydroxy carbamide, idarubicin, ifosfamide, irinotecan, leucovorin, liposomal doxorubicin, liposomal daunorubicin, lomustine, melphalan, mercaptopurine, mesna, methotrexate, mitomycin, mitoxantrone, oxaliplatin, paclitaxel, pemetrexed, pentostatin, procarbazine, raltitrexed, satraplatin, streptozocin, tegafur-uracil, temozolomide, teniposide, thiotepa, tioguanine, topotecan, treosulfan, vinblastine, vincristine, vindesine, vinorelbine, or a combination thereof. Representative pro-apoptoticagents include, but are not limited to fludarabinetaurosporine, cycloheximide, actinomycin D, lactosylceramide, 15d-PGJ(2) and combinations thereof.

3. The pharmaceutical composition according to claim 1, wherein the modified CD2 polypeptide has at least 99% sequence identity to SEQ ID NO: 1, 2, 3, or 4.

4. The pharmaceutical composition according to claim 1, wherein the immune checkpoint inhibitor is a PD-1 inhibitor.

5. The pharmaceutical composition according to claim 1, wherein the immune checkpoint inhibitor is an anti-PD-1 or anti-PD-Ll antibody.

6. The pharmaceutical composition according to claim 1, wherein the immune checkpoint inhibitor is an anti-CTLA-4 antibody.

7. A method of inhibiting or reducing tumor growth in a subject having a cancer comprising administering the pharmaceutical composition according to claim 1, wherein the pharmaceutical composition synergistically inhibits or decreases tumor growth in the subject by inducing tumor immunity.

8. The method according to claim 7 further comprising adding one or more additional therapeutic agents administered to additively or synergistically inhibit or reduce tumor growth.

9. The method according to claim 7, wherein the additional therapeutic agents are anti- cancer chemotherapy drugs.

10. The method according to claim 7, wherein the tumor is a solid tumor selected from lung, breast, melanoma, or pancreatic tumors.

11. The method according to claim 7, wherein the cancer is a lung cancer selected from a non-small cell lung cancer (NSCLC) or a small cell lung cancer (SCLC).

12. The method according to claim 7, wherein the subject has relapsed from or is refractory to checkpoint inhibitor therapy, prior to treatment with the modified CD2 polypeptide.

13. The method according to claim 7, wherein the combination therapy induces integrin avbs targeted cell apoptosis.

14. The method according to claim 13, wherein induction of integrin avbs targeted cell apoptosis induces tumor immunity by simultaneously depleting cancer-associated fibroblasts (CAFs) and tumor angiogenic vessels.

15. The method according to claim 14, wherein the depletion of cancer-associated fibroblasts (CAFs) leads to a depletion of the tumor’s fibrotic stroma.

16. The method according to claim 14, wherein the depletion of the tumor angiogenic vessels decreases tumor angiogenesis, increases tumor perfusion, reduces tumor hypoxia, or the combination thereof.

17. A method of synergistically increasing the activity of an anti-PD-Ll immunotherapeutic in a subject having a cancer comprising administering the pharmaceutical composition of claim 1, wherein the modified CD2 polypeptide reduces PD-L1 expression in a solid tumor’ s fibrotic stroma to increase the delivery of the anti-PDL-1 antibody to target cancer cells, and wherein PD-L1 expression in the solid tumor’s fibrotic stroma synergistically increases the activity of the anti-PDL-1 antibody.

18. The method according to claim 17, wherein the increased delivery of the anti-PDL- 1 antibody to target cancer cells synergistically inhibits or reduces tumor growth.

19. The method according to claim 17, further comprising adding one or more additional therapeutic agents administered to additively or synergistically inhibit or reduce tumor growth, wherein the additional therapeutic agents are anti-cancer chemotherapy drugs.

20. The method according to claim 17, wherein the subject has been diagnosed with a lung cancer selected from a non-small cell lung cancer (NSCLC) or a small cell lung cancer (SCLC), wherein the subject has relapsed from or is refractory to checkpoint inhibitor therapy, prior to treatment with the modified CD2 polypeptide.

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