Pan-solid tumor targeting cell therapy

Abstract

The disclosure describes improved chimeric antigen receptors (CARs), improved CAR T cell therapeutics, and their use in treating solid cancers. In particular, the disclosure describes engineered CAR T constructs that employ the integrin-binding peptide 2.5F to target a wide variety of solid tumors.

Description

DESCRIPTION PAN-SOLID TUMOR TARGETING CELL THERAPY PRIORITY CLAIM

[0001] This application claims benefit of priority of U.S. Provisional Application Serial No.63 / 655,736, filed June 4, 2024, the entire content of which are hereby incorporated by reference. REFERENCE TO A SEQUENCE LISTING

[0002] This application contains a Sequence Listing XML, which has been submitted electronically and is hereby incorporated by reference in its entirety. Said Sequence Listing XML, created on May 27, 2025, is named CHOPP0078WO.xml and is 15,948 bytes in size. FIELD OF THE INVENTION

[0003] The present disclosure relates generally to binding agents and methods of use thereof for diagnosing and / or treating cancer. In particular, the disclosure relates to binding agents capable of specifically binding to the engineered knottin peptide 2.5F and their application to chimeric antigen receptor (CAR) construction and use in treating cancer. In addition, systems for delivery agents like CARs and other therapeutic agents to patients using hypoxic responsive elements to drive selective expression are provided. BACKGROUND OF THE INVENTION

[0004] Cancer is the second leading cause of death in the world, accounting for one in six deaths. Current treatment options, including surgery, chemotherapy, radiotherapy, and target therapy, can lead to complete remission. However, most of the patients still did not respond or experienced tumor progression after therapy, calling for new therapeutic strategies in cancer treatment. Adoptive chimeric antigen receptor (CAR) T cell therapy has shown durable complete remissions in leukemia and lymphoma, thus revolutionizing clinical guidelines in hematologic malignancies. However, the clinical outcomes of CAR T therapy in solid tumors have been largely disappointing, caused by loss of the antigen targeted by the CAR due to tumor heterogeneity or tumor adaptation to immunotherapy. While targeting a certain tumor antigen may cause escape of non-targetable tumor cells, CAR-T cells 1 4929-6250-8358, v.2recognizing multiple tumor-associated antigens may also lead to on-target off-tumor toxicity. Improved CAR T cell therapies that overcome one of more of these limitations are therefore greatly in need. 2 4929-6250-8358, v.2SUMMARY

[0005] Thus, in accordance with the present disclosure, there is provided a chimeric antigen receptor (CAR) comprising a knottin 2.5F domain fused to a transmembrane domain, a homodimer forming domain and an intracellular domain. The CAR may further comprise a myc tag immediately downstream of the 2.5F domain. The transmembrane domain may be a human CD8 transmembrane domain, and / or the homodimer forming domain is CD3ζ, and / or the intracellular domain is a CD28, a 41BB, a 28BB, an ICOS or an OX40 intracellular domain. The CAR may further comprising an oxygen-dependent degradation (ODD) domain fused to the C-terminus of the 2.5F domain, such as an ODD domain from HIF-1α.

[0006] In another embodiment, there is provided an expression vector encoding a chimeric antigen receptor (mitCAR) comprising a knottin 2.5F domain fused to a transmembrane domain, a homodimer forming domain and an intracellular domain. The expression vector may further comprise a coding region a myc tag immediately downstream of the coding region for the 2.5F domain. The coding region for the transmembrane domain may be for a human CD8 transmembrane domain, and / or the coding region for the homodimer forming domain may be for a CD3ζ, and / or the intracellular domain is a CD28, a 41BB, a 28BB, an ICOS or an OX40 intracellular domain. The expression vector may further comprise a coding region for an oxygen-dependent degradation (ODD) domain fused to the coding region for the C-terminus of the 2.5F domain, such as an ODD domain from HIF-1α.

[0007] In yet another embodiment, there is provided an engineered cell expressing a CAR as described herein or comprising an expression vector as described herein. The engineered cell may be further engineered to express a cytokine, such as one or more of IL-18, IL12, IL7, IL15, IL10, IL4, IL-9 and / or IL-21. The engineered cell may be an autologous T cell, a macrophage, an NK cell, a monocyte, an NKT cell, a gamma-delta T cell, and allogeneic T cell, or an immortalized cytotoxic cell line, such as TALL104 or NK92. The engineered cell may be further characterized as being a gp100-specific TCR-transgenic T cell.

[0008] In still another embodiment, there is provided a method of killing a cancer cell comprising contacting said cancer cell with an engineered cell as described in the immediately preceding paragraphs, and a method of treating a subject with a solid tumor 3 4929-6250-8358, v.2comprising administering to said subject an engineered cell as described in the immediately preceding paragraphs.

[0009] These methods may be directed to a cancer cell or said solid tumor comprising a lung cancer cell, brain cancer cell, head & neck cancer cell, breast cancer cell, skin cancer cell, liver cancer cell, pancreatic cancer cell, stomach cancer cell, colon cancer cell, rectal cancer cell, uterine cancer cell, cervical cancer cell, ovarian cancer cell, testicular cancer cell, skin cancer cell, or esophageal cancer cell. These methods may further comprise contacting said cancer cell or solid tumor with a second anti-cancer agent or treatment, such as wherein said second anti-cancer agent or treatment is selected from chemotherapy, radiotherapy, immunotherapy, hormonal therapy, or toxin therapy. The second anti-cancer agent or treatment may be given at the same time as said engineered cell, or given before and / or after said engineered cell. The cancer cell or solid tumor may be a metastatic cancer cell / solid tumor, a multiply drug resistant cancer cell / solid tumor or a recurrent cancer cell / solid tumor. The method may further comprise giving at least a second administration of said engineered cell, such as 1, 2, 3, 4, 5, 6, 7, 8, 92010 additional administrations. The administration may be intratumoral, intracranial, into tumor vasculature, local to the tumor, regional to the tumor or systemic. The subject may be a mammal, such as a human, a canine, or a mouse. The tumor may be a solid tumor and said administering said engineered cell results in one or more of cytotoxicity against said solid tumor, and / or tumor regression of said solid tumor.

[0010] In still yet further embodiments, there are provided an mRNA comprising a nucleic acid encoding the CAR as described herein, and a microparticle or nanoparticle comprising such mRNA.

[0011] The use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and / or the specification may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.” The word “about” means plus or minus 5% of the stated number.

[0012] It is contemplated that any method or composition described herein can be implemented with respect to any other method or composition described herein. Other objects, features and advantages of the present disclosure will become apparent from the following detailed description. It should be understood, however, that the detailed 4 4929-6250-8358, v.2description and the specific examples, while indicating specific embodiments of the disclosure, are given by way of illustration only, since various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art from this detailed description. 5 4929-6250-8358, v.2BRIEF DESCRIPTION OF THE DRAWINGS

[0013] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure. The disclosure may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0014] FIGS. 1A-C. Figs. 1 A-C. The development of mitCAR. (Fig.1A) The design of mitCAR. (Fig. 1B) The transduction of efficiency of primary human T cells with mitCAR Fig 1C The level of mitCAR on the surface of transduced primary human T cells.

[0015] Figs. 2 A-C. The specificity of mitCAR. (Fig. 2A) The binding of mitCAR-Fc to A375P, U87 and Jurkat cells. (Fig. 2B) The percentage of CD25+ mitCAR T cells and untransduced (UTD) T cells 72 hours after co-culture with target cells. (Fig. 2C) The level of CD25 on a surface of mitCAR T cells 72 hours after co-culture with target cells.

[0016] Figs. 3A-B. The cytotoxic activity of mitCAR T cells against solid tumor cell lines. (Fig.3A) The cytotoxic activity of mitCAR T cells incubated for 24 hours in different effector-to-target ratios (E:T) with melanoma A375P cell line was assessed by luciferase- based assay. (Fig. 3B) The cytotoxic activity of mitCAR T cells incubated for 24 hours in different effector-to-target ratios (E:T) with glioblastoma U251 cell line was assessed by luciferase-based assay.

[0017] Figs.4A-B. The anti-solid tumor activity of mitCAR T cells. NSG mice engrafted with human melanoma cells, A375P, were injected intratumorally twice with 5 x 10^6 mitCAR T cells or an equal number of control T cells. Tumors were measured daily (Fig. 4A) and survival of tumor-bearing mice were recorded (Fig.4B).

[0018] Fig.5. Binding of mitCAR-Fc to a panel of human solid tumor cell lines.

[0019] Fig. 6. Binding of mitCAR-Fc to primary solid tumor cell lines. Y-axis is normalized binding (from top to bottom) is 100, 80,60,40,20,0. X-axis is log fluorescent intensity. 6 4929-6250-8358, v.2

[0020] Figs. 7A-D. Binding of mitCAR-Fc to primary pediatric and adult tumors. mitCAR-Fc staining of pediatric central nervous system. (Fig. 7A) and neuroblastoma (Fig. 7B) tumor samples as well as glioblastoma organoids (Fig. 7C) and metastatic melanoma tumor sample (Fig. 7D). Y-axis is normalized binding (from top to bottom) is 100, 80,60,40,20,0. X-axis is log fluorescent intensity.

[0021] Fig.8. The cytotoxic activity of mitCAR T cells. The cytotoxic activity of mitCAR T cells was assessed by Incucyte-based real-time assay.

[0022] Fig.9. The cytotoxic activity of mitCAR T cells against primary solid tumor cell lines. The cytotoxic activity of mitCAR T cells incubated for 24 hours in different effector- to-target ratios (E:T) with different primary solid tumor cell line was assessed by Incucyte- based assay.

[0023] Figs. 10A-C. The anti-solid tumor activity of mitCAR T cells against glioblastoma and pancreatic carcinoma xenografts. NSG mice engrafted with human glioblastoma cells, U87MG (Fig. 10A) and U251 (Fig. 10B), or pancreatic carcinoma (Fig. 10C) were injected intratumorally with 5 x 10^6 mitCAR T cells with CD28 or 41BB costimulatory domains (A and B) or CD28 (Fig.10C) or an equal number of control T cells. Tumor size was monitored.

[0024] Figs. 11A-B. The lack of antigen escape in glioblastoma tumors treated with mitCAR T cells. (Fig. 11A) NSG mice engrafted with human glioblastoma cells, U87MG, were injected intratumorally with 5 x 10^6 mitCAR T cells with 41BB domain. Tumor size was monitored. (Fig. 11B) Flow cytometry analysis of mitCAR-Fc binding to tumor cells after progression of mitCAR T or control T cell therapy.

[0025] Figs. 12A-C. Anti-solid tumor activity of IL-18-armored mitCAR cells against glioblastoma and melanoma xenografts. (Fig.12A) Schematics of IL-18-armored mitCAR constructs. (Figs. 12B-C) NSG mince engrafted with human melanoma cells (A375P, Fig. 12B) or glioblastoma cells (U87MG, Fig. 12C) were injected intratumorally with 5 x 10^6 mitCAR T cells, IL-18-armored mitCAR T cells, or an equal number of control T cells. Tumor size was monitored. 7 4929-6250-8358, v.2

[0026] Figs. 13A-B. mRNA based mitCAR T cells exhibit cytotoxicity against solid tumor cell lines. (Fig. 13A) Expression of mitCAR by human T cells after transfection of mRNA-mitCAR. (Fig.13B) The cytotoxic activity of mRNA-mitCAR T cells incubated for 24 hours in different effector-to-target ratios (E:T) with glioblastoma U87MG cell line was assessed by Incucyte-based assay.

[0027] Fig.14. Binding of mitCAR-Fc to a panel of murine solid tumor cell lines.

[0028] Figs. 15A-B. Murine mitCAR T cells. (Fig. 15A) The schematic of murine mitCAR. (Fig.15B) Transduction efficiency of murine mitCAR T cells.

[0029] Fig.16. Murine mitCAR T cells exhibit potent cytotoxic activity against murine solid tumor cell lines. The cytotoxic activity of murine mitCAR T cells incubated for 24 hours in different murine solid tumor cell lines was assessed by Incucyte-based assay.

[0030] Figs. 17A-C. The anti-solid tumor activity of murine mitCAR T cells. C57B / 6 mice engrafted with murine colorectal carcinoma cells (MC38, Fig.17A), glioma cells (CT- 2A, Fig. 17B), or melanoma cells (Yumm1.7, Fig. 17C) and were injected intratumorally with 5 x 10^6 mitCAR T cells, IL-18-armored mitCAR T cells, or an equal number of control T cells. Tumor size was monitored.

[0031] Figs. 18A-B. mitCAR recognizes tumor-associated macrophages and endothelial vessels (ECs) in the tumor microenvironment of murine MC38 tumors. Flow cytometry analysis of mitCAR-Fc binging to myeloid cell populations (Fig.18A) and endothelial cells (Fig.18B) in the tumor microenvironment.

[0032] Fig.19. mitCAR recognizes tumor-associated macrophages in the lymph node of human metastatic melanoma. Y-axis is normalized binding (from top to bottom) is 100, 80,60,40,20,0. X-axis is log fluorescent intensity.

[0033] Figs. 20A-D. mitCAR T cells recognize and target tumor-associated macrophages. (Fig.20A) Flow cytometry analysis of mitCAR-Fc binding to human tumor- associated macrophages (TAMs) differentiated ex vivo. (Fig. 20B) Activation of human mitCAR T cells after co-culture with TAMs assessed by flow cytometry and CD69 staining. (Fig.20C) Degranulation of human mitCAR T cells after co-culture with TAMs assessed by 8 4929-6250-8358, v.2flow cytometry and CD107a staining. (Fig. 20D) Cytotoxic activity in mitCAR T cells against human TAMs assessed by Incucyte imaging.

[0034] Fig. 21. Binding of mitCAR-Fc to canine solid tumor cell lines. Y-axis is normalized binding (from top to bottom) is 100, 80,60,40,20,0. X-axis is log fluorescent intensity.

[0035] Figs. 22A-B. Detrimental effect of mitCAR-targeted integrin loss on tumor cells.

[0036] FIG.23. mitCAR T sequences and component sequences thereof. 9 4929-6250-8358, v.2DETAILED DESCRIPTION

[0086] The curative potential of chimeric antigen receptor (CAR) T cell-based cancer immunotherapies has been established in several cancer types, but solid tumor applications have been limited by a paucity of known tumor-specific membrane proteins. Though membrane proteins represent up to a quarter of the proteome, only a fraction of these are specifically expressed on tumors cells and not on normal tissues, and a smaller proportion are essential to tumor homeostasis.

[0087] Taking advantage of an already developed engineered knottin peptide, 2.5F, which targets five integrins highly expressed in various tumor types, including αvβ1, αvβ3, αvβ5, αvβ6, and α5β1, the inventors engineered T cells with a CAR bearing 2.5F as the targeting domain to simultaneously target all of these integrins. Given the broad-spectrum binding of 2.5F (mitCAR T) to a variety of solid tumor types, the mitCAR T therapy is expected to exert superior antitumor effects against heterogenic solid tumors, compared with traditional CAR T therapy. It can also become an invaluable treatment option for patients bearing rare tumors with no available therapeutics.

[0088] The mitCAR T cell therefore presents the advantage of targeting of both tumor cells and intratumoral myeloid cells, such as tumor-associated macrophages (TAMs). Moreover, since the integrin targeted by mitCAR cells cannot be lost, therefore there no risk of antigen- loss induced tumor escape, as exemplified in slowly dying tumor cells. In sum, such as dual tumor and TAM targeting approach makes this particular CAR the first of its kind and provides a unique advantage in therapeutic settings.

[0089] These and other aspects of the disclosure are discussed in detail below. I. Terminology

[0090] Unless otherwise defined, scientific and technical terms used herein shall have the meaning that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Generally, nomenclatures utilized in connection with, and techniques of, antibodies and related molecules, cell and tissue culture, molecular biology, and protein 10 4929-6250-8358, v.2and oligo- or polynucleotide chemistry and hybridization described herein are those well- known and commonly used in the art.

[0091] Standard techniques are used for recombinant DNA, oligonucleotide synthesis, and cell culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques are performed according to manufacturer’s specifications or as commonly accomplished in the art or as described herein. The foregoing techniques and procedures are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. See, e.g., B. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984); J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbour Laboratory Press (1989); T.A. Brown (editor), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991); D.M. Glover and B.D. Hames (editors), DNA Cloning: A Practical Approach, Volumes 1- 4, IRL Press (1995 and 1996); and F.M. Ausubel et al., (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all updates until present); Ed Harlow and David Lane (editors) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory (1988); and J.E. Coligan et al., (editors) Current Protocols in Immunology, John Wiley & Sons (including all updates until present).

[0092] The description and definitions of variable regions and parts thereof, immunoglobulins, antibodies and fragments thereof herein may be further clarified by the discussion in Kabat Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, Md. (1987 and 1991); Bork et al., J Mol. Biol. 242, 309-320 (1994); Chothia and Lesk J. Mol Biol. 196:901-917 (1987), Chothia et al., Nature 342, 877-883 (1989), and / or or Al-Lazikani et al., J Mol Biol 273, 927-948 (1997).

[0093] As used herein, “antigen-binding site” shall be taken to mean a structure formed by a protein that is capable of binding or specifically binding to an antigen, such as an antibody. The antigen-binding site need not be a series of contiguous amino acids, or even amino acids in a single polypeptide chain. For example, in a Fv comprising two different polypeptide chains from an antibody, the antigen-binding site is made up of a series of amino acids of a VLand a VHthat interact with the antigen and that are generally, however not always in one or more of the CDRs in each variable region. In some embodiments, the antigen-binding 11 4929-6250-8358, v.2site is an antigen-binding site of an antibody. In such embodiments, the antigen-binding site may comprise one or more complementarity-determining regions or “CDRs”. In some embodiments, the antigen-binding site of an antibody comprises at least part of a VHor a VLor a Fv.

[0094] 2.5F is a small, engineered peptide of ∼3.5 kDa, conformationally constrained cystine knot peptide, that binds with high affinity and unique specificity to αvβ1,αvβ3, αvβ5, αvβ6 and α5β1 integrins. The cystine knot structural family, also known as knottins, consists of small polypeptides (30–50 amino acids) linked by at least three interwoven disulfide bonds, creating a rigid molecular “knot” that confers high chemical, thermal, and proteolytic stability. EETI 2.5F, which is based on the Ecballium elaterium trypsin inhibitor-II, was identified via high-throughput screening of yeast-displayed knottin libraries (Kimura et al., Proteins 77(2):359-369, 2009). See also U.S. Patent 9,913,878.

[0095] As used herein the phrase “chimeric antigen receptor (CAR)” refers to a recombinant or synthetic molecule which combines antibody-based specificity for a desired antigen with a T cell receptor-activating intracellular domain to generate a chimeric protein that exhibits cellular immune activity to the specific antigen.

[0096] As used herein the phrase “T Cell Receptor” or “TCR” refers to soluble and non- soluble forms of recombinant T-cell receptor.

[0097] As used herein, a “T-cell receptor (TCR) fusion protein” or “TFP” includes a recombinant polypeptide derived from the various polypeptides comprising the TCR that is generally capable of i) binding to a surface antigen on target cells and ii) interacting with other polypeptide components of the intact TCR complex, typically when co-located in or on the surface of a T-cell.

[0098] As used herein a “T Cell Receptor-like antibody (TCRL” or “peptide-centric CAR (PC-CAR)” refers to an antibody which binds an MHC displaying an HLA-restricted peptide antigen. Binding of the TCRL to its target typically has an MHC-restricted specificity: the TCRL does not bind the MHC in the absence of the complexed peptide, and the TCRL does not bind the peptide in an absence of the MHC. TCRLs are characterized by affinity sufficient to permit specific binding to a tumor antigen even when the TCRL is provided in 12 4929-6250-8358, v.2a soluble, rather than membrane-bound, form. TCRLs are being developed as a new therapeutic class for targeting tumor cells and mediating their specific killing. In addition, TCRLs are valuable research reagents enabling the study of human class I peptide-MHC ligand presentation and TCR-peptide-MHC interactions. In an embodiment, the binding agent of the present disclosure is a TCRL.

[0099] As used herein the phrase “MHC (or HLA)-restricted peptide” refers to a peptide which is potentially presented on an MHC molecule. Such peptides may be identified by laboratory procedures such as Mass-Spectrometry, reverse-immunology or by in-silico analysis. An MHC (or HLA)-presented peptide refers to a peptide which is confirmed in vitro or in vivo as being presented by an MHC molecule.

[0100] The term “cancer” as used herein is defined as a disease characterized by the rapid and uncontrolled growth of aberrant cells. Cancer cells can spread locally or through the bloodstream and lymphatic system to other parts of the body.

[0101] The terms “normoxia” and “hypoxia” are generally known to those of skill in the art. In general, oxygen levels in tumors range from about 0.3 to 4.2% but are mostly below 2%, whereas oxygen levels in normal tissues range from about 3.4 to 6.8%, mostly above 4%. See McKeown S., Br. J. Radiol 87(1035), 2014. For the purposes of this application, normoxia is defined as 3 / 0% or higher oxygen would be lower than 3.0% oxygen.

[0102] A “compound” refers to any molecule including small molecules, polypeptides, and other macromolecules. In some embodiments, a compound is a small molecular weight compound with a molecular weight of less than about 2000 Daltons.

[0103] The term “naturally occurring” (or “native”) as used herein as applied to an object refers to the fact that an object can be found in nature. For example, a polypeptide or polynucleotide sequence that is present in an organism (including viruses) that can be isolated from a source in nature and which has not been intentionally modified by man in the laboratory or otherwise is naturally occurring.

[0104] The term “operably linked” as used herein refers to positions of components so described that are in a relationship permitting them to function in their intended manner. 13 4929-6250-8358, v.2For example, a control sequence “operably linked” to a coding sequence is connected in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences.

[0105] The term “protein” shall be taken to include a single polypeptide chain, i.e., a series of contiguous amino acids linked by peptide bonds or a series of polypeptide chains covalently or non-covalently linked to one another (i.e., a polypeptide complex). For example, the series of polypeptide chains can be covalently linked using a suitable chemical linker or a disulphide bond, for example. Examples of non-covalent bonds include hydrogen bonds, ionic bonds, Van der Waals forces, and hydrophobic interactions.

[0106] The term “polypeptide” or “polypeptide chain” will be understood from the foregoing paragraph to mean a series of contiguous amino acids linked by peptide bonds.

[0107] The term “polynucleotide” as referred to herein means a polymeric form of nucleotides of at least 10 bases in length, either ribonucleotides or deoxynucleotides or a modified form of either type of nucleotide, or RNA-DNA hetero-duplexes. The term includes single and double stranded forms of DNA.

[0108] The term “sequence identity” means that two polynucleotide or amino acid sequences are identical (i.e., on a nucleotide-by-nucleotide or residue-by-residue basis) over the comparison window. The term “percentage of sequence identity” is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, U, or I) or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. The terms “substantial identity” as used herein denotes a characteristic of a polynucleotide or amino acid sequence, wherein the polynucleotide or amino acid comprises a sequence that has at least 85 percent sequence identity, preferably at least 90 to 95 percent sequence identity, more preferably at least 99 percent sequence identity, as compared to a reference sequence over a comparison window of at least 18 nucleotide (6 amino acid) positions, frequently over a window of at least 24-48 nucleotide (8-16 amino acid) positions, wherein the percentage of sequence identity is calculated by comparing the 14 4929-6250-8358, v.2reference sequence to the sequence which may include deletions or additions which total 20 percent or less of the reference sequence over the comparison window. The reference sequence may be a subset of a larger sequence.

[0109] As used herein, the twenty conventional amino acids and their abbreviations follow conventional usage. See Immunology - A Synthesis (2ndEdition, E.S. Golub and D.R. Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)).

[0110] The term “epitope” includes any protein determinant capable of specific binding to an immunoglobulin or T-cell receptor. Epitopic determinants usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and may, but not always, have specific three-dimensional structural characteristics, as well as specific charge characteristics.

[0111] The term “agent” is used herein to denote a chemical compound, a mixture of chemical compounds, a biological macromolecule, or an extract made from biological materials.

[0112] All publications and patents mentioned herein are hereby incorporated by reference in their entirety as if each individual publication or patent was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application, including any definitions herein, will control. However, mention of any reference, article, publication, patent, patent publication, and patent application cited herein is not, and should not be taken as an acknowledgment, or any form of suggestion, that they constitute valid prior art or form part of the common general knowledge in any country in the world.

[0113] In the present description, any concentration range, percentage range, ratio range, or integer range is to be understood to include the value of any integer within the recited range and, when appropriate, fractions thereof (such as one tenth and one hundredth of an integer), unless otherwise indicated. The term “about”, when immediately preceding a number or numeral, means that the number or numeral ranges plus or minus 10%. It should be understood that the terms “a” and “an” as used herein refer to “one or more” of the enumerated components unless otherwise indicated. The use of the alternative (e.g., “or”) 15 4929-6250-8358, v.2should be understood to mean either one, both, or any combination thereof of the alternatives. The term “and / or” should be understood to mean either one, or both of the alternatives. As used herein, the terms “include” and “comprise” are used synonymously.

[0114] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. II. Hypoxia Responsive Control System

[0115] In accordance with the disclosure, there are provided expression control systems that permit regulation of gene expression in hypoxic environments. These systems rely on hypoxia responsive elements (HREs) that are situated upstream and in operational relation to a coding regions, such as CAR, or TCR. In one system, an expression vector comprising an HRE that controls expression of a protein of interest, e.g., a CAR or TCR, is provided. The coding region for the protein of interest may optionally be fused to a coding region for an oxygen-dependent degradation (ODD) domain. Under conditions of hypoxia, expression of the protein of interest is induced, while any “leakage” expression in an oxygen rich environment would results in an oxygen-dependent degradation of the protein of interest. See FIG.8A.

[0116] A variation on this system uses two distinct expression cassettes located in one or two different vectors. The first cassette / vector would comprising a first nucleic acid encoding a transcriptional control factor (TCF), optionally fused a coding region for an oxygen-dependent degradation (ODD) domain under the control of a hypoxia responsive (HRE) promoter element. The second cassette / vector comprises a second nucleic acid encoding a protein of interest, such as a CAR or TCR, under the control a transcription control element bound and activated by the TCF. The same tightly controlled regulation under hypoxic conditions, along with elimination of any “leaky” expression in an oxygen rich environment is provided. See FIG.8B.

[0117] Examples of HREs include those from VEGFA, PGK1, LDHA, EPO, GLUT1, and ALDA genes. Examples of ODDs include those from HIF1a, HIF2a, HIF3a, EPOR, NDRG3, ZHX2 genes. 16 4929-6250-8358, v.2III. Chimeric antigen receptor (CAR) and TCR Fusion Proteins (TFP)

[0118] In an embodiment of the invention, there is provided a T-cell receptor fusion protein (TFP) comprising: a) a T-cell receptor (TCR) subunit comprising: (i) at least a portion of a TCR extracellular domain, and (ii) a TCR intracellular domain comprising a stimulatory domain from an intracellular signaling domain; and b) a binding agent of the disclosure, wherein the TCR subunit and the binding agent are operatively linked, and wherein the TFP incorporates into a TCR when expressed in a T-cell.

[0119] As used herein, the term “at least a portion of a TCR extracellular domain” refers to α and β chains of the TCR comprising variable and constant regions or domains. The term "domain" or "region" can be used interchangeably herein. The variable domain consists of a concatenation of variable region and joining region. The term "TCR alpha variable domain" therefore refers to the concatenation of TRAV and TRAJ regions, and the term TCR alpha constant domain refers to the extracellular TRAC region, or to a C-terminal truncated TRAC sequence. Likewise, the term "TCR beta variable domain" refers to the concatenation of TRBV and TRBD / TRBJ regions, and the term TCR beta constant domain refers to the extracellular TRBV region or to a C-terminal truncated TRBC sequence. The unique sequences defined by the IMGT nomenclature are widely known and accessible to those working in the TCR field. For example, they can be found in the IMGT public database. The "T cell Receptor Factsbook", (2001) LeFranc and LeFranc, Academic Press, ISBN 0-12- 441352-8 also discloses sequences defined by the IMGT nomenclature, but because of its publication date and consequent time-lag, the information therein sometimes needs to be confirmed by reference to the IMGT database. As used herein, the term “wherein the TCR incorporates into a TCR when expressed in a T-cell” refers to the process of a TFP of the invention being expressed and fused to a T-cell receptor of a T-cell.

[0120] Chimeric antigen receptors (CARs) are fusion proteins comprising antigen recognition moieties and T cell-activation domains. Exemplary CARs are provided by US Patent No.8,399,645 and US Patent No.7,638,325. Other exemplary recombinant receptors, including CARs, recombinant T-cell receptors (TCRs), TCR fusion proteins (TFPs), as well 17 4929-6250-8358, v.2as methods for engineering and introducing the receptors into cells, include those described in Int’l Pat. Appl. Nos. WO2017 / 096329, WO2000 / 14257, WO2013 / 126726, WO2012 / 129514, WO2014031687, WO2013 / 166321, and WO2013 / 071154, WO2013 / 123061, and WO / 2014055668; U.S. Pat. App. Nos. US2002131960, US2013287748, and US20130149337; U.S. Pat. Nos. 6,451,995, 7,446,190, 7,638,325, 8,252,592, 8,339,645, 8,398,282, 7,446,179, 6,410,319, 7,070,995, 7,265,209, 7,354,762, 7,446,191, 8,324,353, and 8,479,118; European Pat. App. No. EP2537416; and Sadelain et al. Cancer Discov. April 3(4): 388-398 (2013); Davila et al. PLoS ONE 8(4): e61338 (2013); Turtle et al. Curr. Opin. Immunol. October 24(5): 633-39 (2012); and Wu et al. Cancer, March 18(2): 160-75 (2012). In an embodiment, the binding agent is a TFP as described in U.S. Pat. No.15 / 419,398. IV. Expression Constructs

[0121] According to the disclosure there are provided an engineered vectors that facilitate expression of CAR constructs. Such expression vectors contain elements coding for the non-antigen binding portions of CARs along with cis-acting regulatory elements. The expression vector of some embodiments of the disclosure includes additional sequences which render this vector suitable for replication and integration in prokaryotes, eukaryotes, or preferably both (e.g., as a shuttle vector). In addition, typical cloning vectors may also contain a transcription and translation initiation sequence, transcription and translation terminator and a polyadenylation signal. The nucleic acid construct of some embodiments of the disclosure includes a signal sequence for secretion or presentation of the binding agent from a host cell in which it is placed. Preferably the signal sequence for this purpose is a mammalian signal sequence.

[0122] Eukaryotic promoters typically contain two types of recognition sequences, the TATA box and upstream promoter elements. The TATA box, located 25-30 base pairs upstream of the transcription initiation site, is thought to be involved in directing RNA polymerase to begin RNA synthesis. The other upstream promoter elements determine the rate at which transcription is initiated. Preferably, the promoter utilized by the expression vector is active in the specific cell population transformed. Examples of cell type-specific and / or tissue- specific promoters include promoters such as albumin that is liver specific (Pinkert et al., Genes Dev. 1:268-277 (1987)), lymphoid specific promoters (Calame et al., 18 4929-6250-8358, v.2Adv. Immunol. 43:235-275 (1988)); in particular promoters of T-cell receptors (Winoto et al., EMBO J. 8:729-733 (1989)) and immunoglobulins; (Banerji et al., Cell 33:729-740 (1983)), neuron-specific promoters such as the neurofilament promoter (Byrne et al., Proc. Natl. Acad. Sci. USA 86:5473-5477 (1989)), pancreas-specific promoters (Edlunch et al., Science 230:912-916 (1985)) or mammary gland-specific promoters such as the milk whey promoter (U.S. Pat. No.4,873,316 and European Application Publication No. EP0264166). In the construction of the expression vector, the promoter is preferably positioned approximately the same distance from the heterologous transcription start site as it is from the transcription start site in its natural setting. As is known in the art; however, some variation in this distance can be accommodated without loss of promoter function.

[0123] Enhancer elements can stimulate transcription up to 1,000-fold from linked homologous or heterologous promoters. Enhancers are active when placed downstream or upstream from the transcription initiation site. Many enhancer elements derived from viruses have a broad host range and are active in a variety of tissues. For example, the SV40 early gene enhancer is suitable for many cell types. Other enhancer / promoter combinations that are suitable for some embodiments of the disclosure include those derived from polyoma virus, human or murine cytomegalovirus (CMV), the long-term repeat from various retroviruses such as murine leukemia virus, murine or Rous sarcoma virus and HIV. See, Enhancers and Eukaryotic Expression, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. 1983.

[0124] Polyadenylation sequences can also be added to the expression vector in order to increase the efficiency of TCRL mRNA translation. Two distinct sequence elements are required for accurate and efficient polyadenylation: GU or U rich sequences located downstream from the polyadenylation site and a highly conserved sequence of six nucleotides, AAUAAA, located 11-30 nucleotides upstream. Termination and polyadenylation signals that are suitable for some embodiments of the disclosure include those derived from SV40.

[0125] In addition to the elements already described the expression vector of some embodiments of the disclosure may contain other specialized elements intended to increase the level of expression of cloned nucleic acids or to facilitate the identification of cells that carry the recombinant DNA. For example, a number of animal viruses contain DNA 19 4929-6250-8358, v.2sequences that promote the extra chromosomal replication of the viral genome in permissive cell types. Plasmids bearing these viral replicons are replicated episomally as long as the appropriate factors are provided by genes either carried on the plasmid or with the genome of the host cell.

[0126] The vector may or may not include a eukaryotic replicon. If a eukaryotic replicon is present, then the vector is amplifiable in eukaryotic cells using the appropriate selectable marker. If the vector does not comprise a eukaryotic replicon, no episomal amplification is possible. Instead, the recombinant DNA integrates into the genome of the engineered cell, where the promoter directs expression of the desired nucleic acid.

[0127] Also provided are cells which comprise the polynucleotides / expression vectors as described herein. Such cells are typically selected for high expression of recombinant proteins (e.g., bacterial, plant or eukaryotic cells e.g., CHO, HEK-293 cells), but may also be host cells having a specific immune effector activity (e.g., T cells or NK cells, such as Jurkat cells) when for instance the CDRs of the TCRL are implanted in a T Cell Receptor or CAR transduced in said cells which are used in adoptive cell therapy.

[0128] Next, the CAR library is introduced into mammalian host cells that are then cultured under conditions supporting expression of encoded CARs. The host cells expressing the CAR are then contacted with target antigen positive host cells exhibiting CAR activation are then identified using a variety of different approaches. Once these cells are identified, the binding region can be sequenced and further developed. V. Treatment of Cancers

[0129] In accordance with the present disclosure, there are provided methods of treating cancers, particularly solid cancers. The methods involve the administration of CAR T cells to a subject such that the cells are brought in proximity with a cancer cell / cancer cell environment and their therapeutic effect is delivered. The administration may be performed multiple times (2, 3, 4, 5, 6, 7, 8, 9, 10 or more) to a subject as needed. The subject may be an infant, a pediatric patient, a juvenile, a young adult, and adult or a senior. The subject may be a human, male or female, or may be a non-human mammal. 20 4929-6250-8358, v.2

[0130] In certain aspects the cancer tumor is a renal cell cancer, melanoma, prostate cancer, chronic lymphocytic leukemia, bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestine, gum, head, kidney, liver, lung, nasopharynx, neck, ovary, pancreas, prostate, skin, stomach, testis, tongue, or uterus. In addition, the cancer may specifically be of the following histological type, though it is not limited to these: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposis coli; solid carcinoma; carcinoid tumor, malignant; branchiolo-alveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; acidophil carcinoma; oxyphilic adenocarcinoma; basophil carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; nonencapsulating sclerosing carcinoma; adrenal cortical carcinoma; endometroid carcinoma; skin appendage carcinoma; apocrine adenocarcinoma; sebaceous adenocarcinoma; ceruminous adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; infiltrating duct carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; paget's disease, mammary; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma w / squamous metaplasia; thymoma, malignant; ovarian stromal tumor, malignant; thecoma, malignant; granulosa cell tumor, malignant; androblastoma, malignant; sertoli cell carcinoma; leydig cell tumor, malignant; lipid cell tumor, malignant; paraganglioma, malignant; extra- mammary paraganglioma, malignant; pheochromocytoma; glomangiosarcoma; malignant melanoma; amelanotic melanoma; superficial spreading melanoma; malig melanoma in giant pigmented nevus; epithelioid cell melanoma; blue nevus, malignant; sarcoma; fibrosarcoma; fibrous histiocytoma, malignant; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; mixed tumor, malignant; mullerian mixed tumor; 21 4929-6250-8358, v.2nephroblastoma; hepatoblastoma; carcinosarcoma; mesenchymoma, malignant; brenner tumor, malignant; phyllodes tumor, malignant; synovial sarcoma; mesothelioma, malignant; dysgerminoma; embryonal carcinoma; teratoma, malignant; struma ovarii, malignant; choriocarcinoma; mesonephroma, malignant; hemangiosarcoma; hemangioendothelioma, malignant; kaposi's sarcoma; hemangiopericytoma, malignant; lymphangiosarcoma; osteosarcoma; juxtacortical osteosarcoma; chondrosarcoma; chondroblastoma, malignant; mesenchymal chondrosarcoma; giant cell tumor of bone; ewing's sarcoma; odontogenic tumor, malignant; ameloblastic odontosarcoma; ameloblastoma, malignant; ameloblastic fibrosarcoma; pinealoma, malignant; chordoma; glioma, malignant; ependymoma; astrocytoma; protoplasmic astrocytoma; fibrillary astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroblastoma; primitive neuroectodermal; cerebellar sarcoma; ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumor; meningioma, malignant; neurofibrosarcoma; neurilemmoma, malignant; granular cell tumor, malignant; malignant lymphoma; Hodgkin's disease; Hodgkin's lymphoma; paragranuloma; malignant lymphoma, small lymphocytic; malignant lymphoma, large cell, diffuse; malignant lymphoma, follicular; mycosis fungoides; other specified non- Hodgkin's lymphomas; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small intestinal disease; leukemia; lymphoid leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; myeloid sarcoma; and hairy cell leukemia.

[0131] The term “treatment,” as used herein in the context of treating a condition, pertains generally to treatment and therapy, whether of a human or an animal (e.g., in veterinary applications), in which some desired therapeutic effect is achieved, for example, the inhibition of the progress of the condition, and includes a reduction in the rate of progress, a halt in the rate of progress, regression of the condition, amelioration of the condition, and cure of the condition. Treatment as a prophylactic measure (i.e., prophylaxis, prevention) is also included.

[0132] The term “therapeutically-effective amount,” as used herein, pertains to that amount of binding agent, or a material such as an antibody-drug conjugate, composition or dosage form comprising an active binding agent, which is effective for producing some 22 4929-6250-8358, v.2desired therapeutic effect when administered in accordance with a desired treatment regimen.

[0133] In some embodiments, the treatment reduces or inhibits tumor growth for at least 6, 12, 24, 36, or 48 months. In some embodiments, the treatment enhances an immune response against the tumor.

[0134] The subject / patient may be an animal or any species of mammal, including, without limitation, a horse, a dog, a cat, a pig, or a primate. In a preferred embodiment, the subject / patient is a human. A. Pharmaceutical Formulations and Routes of Administration

[0135] Pharmaceutical compositions provided herein comprise an effective amount of one or more therapeutic compositions and, optionally, an additional agent dissolved or dispersed in a pharmaceutically acceptable carrier. The phrases "pharmaceutical or pharmacologically acceptable" refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to an animal, such as, for example, a human, as appropriate. The preparation of a pharmaceutical composition that contains a therapeutic nucleic acid construct or a therapeutic engineered cell and one or more excipients will be known to those of skill in the art in light of the present disclosure, as exemplified by Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, incorporated herein by reference. Moreover, for animal (e.g., human) administration, it will be understood that preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biological Standards.

[0136] As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, such like materials and combinations thereof, as would be known to one of ordinary skill in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, pp. 1289-1329, incorporated herein by reference). Except insofar as any conventional carrier is incompatible 23 4929-6250-8358, v.2with the active ingredient, its use in therapeutic or pharmaceutical compositions is contemplated.

[0137] In certain embodiments, the pharmaceutical composition may comprise different types of carriers depending on whether it is to be administered in solid, liquid or aerosol form, and whether it need to be sterile for such routes of administration as injection. In certain embodiments, pharmaceutical compositions provided herein can be administered intravenously, intradermally, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostaticaly, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, intrarectally, topically, intratumorally, intramuscularly, intraperitoneally, subcutaneously, subconjunctival, intravesicularlly, mucosally, intrapericardially, intraumbilically, intraocularally, orally, topically, locally, inhalation (e.g. aerosol inhalation), injection, infusion, continuous infusion, localized perfusion bathing target cells directly, via a catheter, via a lavage, in cremes, in lipid compositions (e.g., liposomes), or by other method or any combination of the forgoing as would be known to one of ordinary skill in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, incorporated herein by reference).

[0138] In certain embodiments, the actual dosage amount of a composition administered to a patient can be determined by physical and physiological factors such as body weight, severity of condition, the type of disease being treated, previous or concurrent therapeutic interventions, idiopathy of the patient and on the route of administration. The practitioner responsible for administration will, in any event, determine the concentration of active ingredient(s) in a composition and appropriate dose(s) for the individual subject.

[0139] In certain embodiments, pharmaceutical compositions may comprise, for example, at least about 0.1% of an active compound. In other embodiments, the active compound may comprise between about 2% to about 75% of the weight of the unit, or between about 25% to about 60%, for example, and any range derivable therein. In other non-limiting examples, a dose may also comprise from about 1 microgram / kg / body weight, about 5 microgram / kg / body weight, about 10 microgram / kg / body weight, about 15 microgram / kg / body weight, about 20 microgram / kg / body weight, about 25 microgram / kg / body weight, about 30 microgram / kg / body weight, about 35 microgram / kg / body weight, about 0.04 milligram / kg / body weight, about 0.05 24 4929-6250-8358, v.2milligram / kg / body weight, about 0.06 milligram / kg / body weight, about 0.07 milligram / kg / body weight, about 0.08 milligram / kg / body weight, about 0.09 milligram / kg / body weight, about 0.1 milligram / kg / body weight, about 0.2 milligram / kg / body weight, to about 0.5 mg / kg / body weight or more per administration, and any range derivable therein. In non-limiting examples of a derivable range from the numbers listed herein, a range of about 0.01 mg / kg / body weight to about 0.1 mg / kg / body weight, about 0.04 microgram / kg / body weight to about 0.08 milligram / kg / body weight, etc., can be administered, based on the numbers described above.

[0140] In any case, the composition may comprise various antioxidants to retard oxidation of one or more component. Additionally, the prevention of the action of microorganisms can be brought about by preservatives such as various antibacterial and antifungal agents, including but not limited to parabens (e.g., methylparabens, propylparabens), chlorobutanol, phenol, sorbic acid, thimerosal or combinations thereof.

[0141] In embodiments where the composition is in a liquid form, a carrier can be a solvent or dispersion medium comprising but not limited to, water, ethanol, polyol (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), lipids (e.g., triglycerides, vegetable oils, liposomes) and combinations thereof. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin; by the maintenance of the required particle size by dispersion in carriers such as, for example liquid polyol or lipids; by the use of surfactants such as, for example hydroxypropylcellulose; or combinations thereof such methods. In many cases, it will be preferable to include isotonic agents, such as, for example, sugars, sodium chloride or combinations thereof.

[0142] In other embodiments, one may use eye drops, nasal solutions or sprays, aerosols or inhalants in the present embodiments. Such compositions are generally designed to be compatible with the target tissue type. In a non-limiting example, nasal solutions are usually aqueous solutions designed to be administered to the nasal passages in drops or sprays. Nasal solutions are prepared so that they are similar in many respects to nasal secretions, so that normal ciliary action is maintained. Thus, in preferred embodiments the aqueous nasal solutions usually are isotonic or slightly buffered to maintain a pH of about 5.5 to about 6.5. In addition, antimicrobial preservatives, similar to those used in ophthalmic preparations, drugs, or appropriate drug stabilizers, if required, may be included in the 25 4929-6250-8358, v.2formulation. For example, various commercial nasal preparations are known and include drugs such as antibiotics or antihistamines.

[0143] Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and / or the other ingredients. In the case of sterile powders for the preparation of sterile injectable solutions, suspensions or emulsion, the preferred methods of preparation are vacuum-drying or freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered liquid medium thereof. The liquid medium should be suitably buffered if necessary and the liquid diluent first rendered isotonic prior to injection with sufficient saline or glucose. The preparation of highly concentrated compositions for direct injection is also contemplated, where the use of DMSO as solvent is envisioned to result in extremely rapid penetration, delivering high concentrations of the active agents to a small area.

[0144] The composition must be stable under the conditions of manufacture and storage, and preserved against the contaminating action of microorganisms, such as bacteria and fungi. It will be appreciated that endotoxin contamination should be kept minimally at a safe level, for example, less than 0.5 ng / mg protein.

[0145] In particular embodiments, prolonged absorption of an injectable composition can be brought about by the use in the compositions of agents delaying absorption, such as, for example, aluminum monostearate, gelatin or combinations thereof. B. Combination Therapies

[0146] In order to increase the effectiveness of a nucleic acid, polypeptide or nanoparticle complex of the present embodiments, it may be desirable to combine these compositions with other agents effective in the treatment of the disease of interest. As a non- limiting example, the treatment of cancer may be implemented with a CAR / CAR T cell of the present disclosure along with other anti-cancer agents. An “anti-cancer” agent is capable of negatively affecting cancer in a subject, for example, by killing cancer cells, inducing 26 4929-6250-8358, v.2apoptosis in cancer cells, reducing the growth rate of cancer cells, reducing the incidence or number of metastases, reducing tumor size, inhibiting tumor growth, reducing the blood supply to a tumor or cancer cells, promoting an immune response against cancer cells or a tumor, preventing or inhibiting the progression of cancer, or increasing the lifespan of a subject with cancer. More generally, these other compositions would be provided in a combined amount effective to kill or inhibit proliferation of the cell. This process may involve contacting the cells with the CAR / CAR T cell and the other agent(s) or factor(s) at the same time. This may be achieved by contacting the cell with a single composition or pharmacological formulation that includes both agents, or by contacting the cell with two distinct compositions or formulations, at the same time, wherein one composition includes the CAR / CAR T cell and the other includes the second agent(s).

[0147] Treatment with the CAR / CAR T cell may precede or follow the other agent treatment by intervals ranging from minutes to weeks. In embodiments where the other agent and the anti-cancer peptide or nanoparticle complex are applied separately to the cell, one would generally ensure that a significant period of time did not expire between the time of each delivery, such that the agent and the anti-cancer peptide or nanoparticle complex would still be able to exert an advantageously combined effect on the cell. In such instances, it is contemplated that one may contact the cell with both modalities within about 12-24 hours of each other and, more preferably, within about 6-12 hours of each other. In some situations, it may be desirable to extend the time period for treatment significantly where several days (e.g., 2, 3, 4, 5, 6 or 7 days) to several weeks (e.g., 1, 2, 3, 4, 5, 6, 7 or 8 weeks) lapse between the respective administrations.

[0148] Various combinations may be employed, where the CAR / CAR T cell therapy is “A” and the other agent is “B”: A / B / A B / A / B B / B / A A / A / B A / B / B B / A / A A / B / B / B B / A / B / B B / B / B / A B / B / A / B A / A / B / B A / B / A / B A / B / B / A B / B / A / A B / A / B / A B / A / A / B A / A / A / B B / A / A / A A / B / A / A A / A / B / A In certain embodiments, administration of the CAR / CAR T cell therapy and / or other agent(s) to a patient will follow general protocols for the administration of chemotherapeutics, taking into account the toxicity, if any, of the vector. It is expected that the treatment cycles would be repeated as necessary. It also is contemplated that various standard therapies, as well as 27 4929-6250-8358, v.2surgical intervention, may be applied in combination with the described hyperproliferative cell therapy.

[0149] Chemotherapy. Cancer therapies also include a variety of combination therapies. In some aspects, a TUSC2 therapeutic and / or an immune checkpoint inhibitor of the embodiments is administered (or formulated) in conjunction with a chemotherapeutic agent. For example, in some aspects, the chemotherapeutic agent is a protein kinase inhibitor such as a EGFR, VEGFR, AKT, Erb1, Erb2, ErbB, Syk, Bcr-Abl, JAK, Src, GSK-3, PI3K, Ras, Raf, MAPK, MAPKK, mTOR, c-Kit, eph receptor or BRAF inhibitors. Nonlimiting examples of protein kinase inhibitors include Afatinib, Axitinib, Bevacizumab, Bosutinib, Cetuximab, Crizotinib, Dasatinib, Erlotinib, Fostamatinib, Gefitinib, Imatinib, Lapatinib, Lenvatinib, Mubritinib, Nilotinib, Panitumumab, Pazopanib, Pegaptanib, Ranibizumab, Ruxolitinib, Saracatinib, Sorafenib, Sunitinib, Trastuzumab, Vandetanib, AP23451, Vemurafenib, MK-2206, GSK690693, A-443654, VQD-002, Miltefosine, Perifosine, CAL101, PX-866, LY294002, rapamycin, temsirolimus, everolimus, ridaforolimus, Alvocidib, Genistein, Selumetinib, AZD-6244, Vatalanib, P1446A-05, AG-024322, ZD1839, P276-00, GW572016 or a mixture thereof.

[0150] Yet further combination chemotherapies include, for example, alkylating agents such as thiotepa and cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); a camptothecin (including the synthetic analogue topotecan); bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CB1-TM1); eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as the enediyne antibiotics (e.g., calicheamicin, especially calicheamicin gammalI and 28 4929-6250-8358, v.2calicheamicin omegaI1; dynemicin, including dynemicin A; bisphosphonates, such as clodronate; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromophores, aclacinomysins, actinomycin, authrarnycin, azaserine, bleomycins, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2- pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalarnycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti- adrenals such as mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK polysaccharide complex; razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2”-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside (“Ara-C”); cyclophosphamide; taxoids, e.g., paclitaxel and docetaxel gemcitabine; 6-thioguanine; mercaptopurine; platinum coordination complexes such as cisplatin, oxaliplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (e.g., CPT-11); topoisomerase inhibitor RFS 2000; difluorometlhylornithine (DMFO); retinoids such as retinoic acid; capecitabine; carboplatin, procarbazine, plicomycin, gemcitabien, navelbine, farnesyl-protein tansferase 29 4929-6250-8358, v.2inhibitors, transplatinum, and pharmaceutically acceptable salts, acids or derivatives of any of the above. In certain embodiments, the compositions provided herein may be used in combination with gefitinib. In other embodiments, the present embodiments may be practiced in combination with Gleevac (e.g., from about 400 to about 800 mg / day of Gleevac may be administered to a patient). In certain embodiments, one or more chemotherapeutic may be used in combination with the compositions provided herein.

[0151] Radiotherapy. Other factors that cause DNA damage and have been used extensively include what are commonly known as γ-rays, X-rays, and / or the directed delivery of radioisotopes to tumor cells. Other forms of DNA damaging factors are also contemplated such as microwaves and UV-irradiation. It is most likely that all of these factors effect a broad range of damage on DNA, on the precursors of DNA, on the replication and repair of DNA, and on the assembly and maintenance of chromosomes. Dosage ranges for X-rays range from daily doses of 50 to 200 roentgens for prolonged periods of time (3 to 4 wks), to single doses of 2000 to 6000 roentgens. Dosage ranges for radioisotopes vary widely, and depend on the half-life of the isotope, the strength and type of radiation emitted, and the uptake by the neoplastic cells.

[0152] The terms “contacted” and “exposed,” when applied to a cell, are used herein to describe the process by which a therapeutic composition and a chemotherapeutic or radiotherapeutic agent are delivered to a target cell or are placed in direct juxtaposition with the target cell. To achieve cell killing or stasis, both agents are delivered to a cell in a combined amount effective to kill the cell or prevent it from dividing.

[0153] Immunotherapy. Immunotherapeutics, generally, rely on the use of immune effector cells and molecules to target and destroy cancer cells. The immune effector may be, for example, an antibody specific for some marker on the surface of a tumor cell. The antibody alone may serve as an effector of therapy or it may recruit other cells to actually effect cell killing. The antibody also may be conjugated to a drug or toxin (chemotherapeutic, radionuclide, ricin A chain, cholera toxin, pertussis toxin, etc.) and serve merely as a targeting agent. Alternatively, the effector may be a lymphocyte carrying a 30 4929-6250-8358, v.2surface molecule that interacts, either directly or indirectly, with a tumor cell target. Various effector cells include cytotoxic T cells and NK cells.

[0154] Immunotherapy, thus, could be used as part of a combined therapy, in conjunction with a TUSC2 therapy of the present embodiments. The general approach for combined therapy is discussed below. Generally, the tumor cell must bear some marker that is amenable to targeting, i.e., is not present on the majority of other cells. Many tumor markers exist and any of these may be suitable for targeting in the context of the present embodiments. Common tumor markers include carcinoembryonic antigen, prostate specific antigen, urinary tumor associated antigen, fetal antigen, tyrosinase (p97), gp68, TAG-72, HMFG, Sialyl Lewis Antigen, MucA, MucB, PLAP, estrogen receptor, laminin receptor, erb B and p155.

[0155] Gene Therapy. In yet another embodiment, the secondary treatment is a gene therapy in which a therapeutic polynucleotide is administered before, after, or at the same time as the therapeutic composition. Viral vectors for the expression of a gene product are well known in the art, and include such eukaryotic expression systems as adenoviruses, adeno-associated viruses, retroviruses, herpesviruses, lentiviruses, poxviruses including vaccinia viruses, and papilloma viruses, including SV40. Alternatively, the administration of expression constructs can be accomplished with lipid-based vectors such as liposomes or DOTAP:cholesterol vesicles. All of these methods are well known in the art (see, e.g., Sambrook et al., 1989; Ausubel et al., 1998; Ausubel, 1996).

[0156] Delivery of a vector encoding one of the following gene products will have a combined anti-hyperproliferative effect on target tissues. A variety of proteins are encompassed within the present embodiments, some of which are described below.

[0157] As noted above, the tumor suppressor oncogenes function to inhibit excessive cellular proliferation. The inactivation of these genes destroys their inhibitory activity, resulting in unregulated proliferation.

[0158] Genes that may be employed as secondary treatment in accordance with the present embodiments include p53, p16, Rb, APC, DCC, NF-1, NF-2, WT-1, MEN-I, MEN-II, zac1, p73, VHL, MMAC1 / PTEN, DBCCR-1, FCC, rsk-3, p27, p27 / p16 fusions, 31 4929-6250-8358, v.2p21 / p27 fusions, anti-thrombotic genes (e.g., COX-1, TFPI), PGS, Dp, E2F, ras, myc, neu, raf, erb, fms, trk, ret, gsp, hst, abl, E1A, p300, genes involved in angiogenesis (e.g., VEGF, FGF, thrombospondin, BAI-1, GDAIF, or their receptors), and MCC. Apoptosis, or programmed cell death, is an essential process for normal embryonic development, maintaining homeostasis in adult tissues, and suppressing carcinogenesis (Kerr et al., 1972). The Bcl-2 family of proteins and ICE-like proteases have been demonstrated to be important regulators and effectors of apoptosis in other systems. The Bcl-2 protein, discovered in association with follicular lymphoma, plays a prominent role in controlling apoptosis and enhancing cell survival in response to diverse apoptotic stimuli (Bakhshi et al., 1985; Cleary and Sklar, Proc. Nat’l. Acad. Sci. USA, 82(21):7439-43, 1985; Cleary et al., 1986; Tsujimoto et al., 1985; Tsujimoto and Croce, 1986). The evolutionarily conserved Bcl-2 protein now is recognized to be a member of a family of related proteins, which can be categorized as death agonists or death antagonists.

[0159] Subsequent to its discovery, it was shown that Bcl-2 acts to suppress cell death triggered by a variety of stimuli. Also, it now is apparent that there is a family of Bcl-2 cell death regulatory proteins which share in common structural and sequence homologies. These different family members have been shown to either possess similar functions to Bcl-2 (e.g., BclXL, BclW, BclS, Mcl-1, A1, Bfl-1) or counteract Bcl-2 function and promote cell death (e.g., Bax, Bak, Bik, Bim, Bid, Bad, Harakiri).

[0160] Surgery. Approximately 60% of persons with cancer will undergo surgery of some type, which includes preventative, diagnostic or staging, curative and palliative surgery. Curative surgery is a cancer treatment that may be used in conjunction with other therapies, such as the treatments provided herein, chemotherapy, radiotherapy, hormonal therapy, gene therapy, immunotherapy and / or alternative therapies.

[0161] Curative surgery includes resection in which all or part of cancerous tissue is physically removed, excised, and / or destroyed. Tumor resection refers to physical removal of at least part of a tumor. In addition to tumor resection, treatment by surgery includes laser surgery, cryosurgery, electrosurgery, and microscopically controlled surgery (Mohs’ surgery). It is further contemplated that the present embodiments may be used in 32 4929-6250-8358, v.2conjunction with removal of superficial cancers, precancers, or incidental amounts of normal tissue.

[0162] Upon excision of part of all of cancerous cells, tissue, or tumor, a cavity may be formed in the body. Treatment may be accomplished by perfusion, direct injection or local application of the area with an additional anti-cancer therapy. Such treatment may be repeated, for example, every 1, 2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3, 4, and 5 weeks or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. These treatments may be of varying dosages as well.

[0163] Anti-Inflammatory Agents. In certain aspects TUSC2 therapies and / or an immune checkpoint inhibitor are administered in conjunction with an anti-inflammatory agent. An anti-inflammatory agent is defined herein to refer to an agent that is known or suspected to be of benefit in the treatment or prevention of inflammation in a subject. Corticosteroids are a major class of anti-inflammatory agent. The corticosteroids may be short, medium, or long acting, and may be delivered in a variety of methods. A non-limiting list of corticosteroids contemplated in the present embodiments include the oral corticosteroids such as: cortisone, hydrocortisone, prednisone, and dexamethasone.

[0164] Another major class of anti-inflammatory agents are non-steroidal anti- inflammatory agents. Non-steroidal anti-inflammatory agents include a class of drugs used in the treatment of inflammation and pain. The exact mode of action of this class of drugs is unknown. Examples of members of this class of agents include, but are not limited to, ibuprofen, ketoprofen, flurbiprofen, nabumetone, piroxicam, naproxen, diclofenac, indomethacin, sulindac, tolmetin, etodolac, flufenamic acid, diflunisal, oxaprozin, rofecoxib, and celecoxib. One of ordinary skill in the art would be familiar with these agents. Included in this category are salicylates and derivates of salicylates, such as acetyl salicylic acid, sodium salicylate, choline salicylate, choline magnesium salicylate and diflunisal.

[0165] Other anti-inflammatory agents include anti-rheumatic agents, such as gold salts (e.g., gold sodium thiomalate, aurothioglucose, and auranofin), anti-rheumatic agents (e.g., chloroquine, hydroxychloroquine, and penicillamine), antihistamines (e.g., diphenhydramine, chlorpheniramine, clemastine, hydroxyzine, and triprolidine), and immunosuppressive agents (e.g., methotrexate, mechlorethamine, cyclophosphamide, 33 4929-6250-8358, v.2chlorambucil, cyclosporine, and azathioprine). Other immunosuppressive agents contemplated by the present embodiments is tacrolimus and everolimus. Tacrolimus suppresses interleukin-2 production associated with T-cell activation, inhibits differentiation and proliferation of cytotoxic T cells. Today, it is recognized worldwide as the cornerstone of immunosuppressant therapy. One of ordinary skill in the art would be familiar with these agents, and other members of this class of agents, as well as the mechanism of actions of these agents and indications for use of these agents.

[0166] Other agents. It is contemplated that other agents may be used in combination with the compositions provided herein to improve the therapeutic efficacy of treatment. These additional agents include immunomodulatory agents, agents that affect the upregulation of cell surface receptors and GAP junctions, cytostatic and differentiation agents, inhibitors of cell adhesion, or agents that increase the sensitivity of the hyperproliferative cells to apoptotic inducers. Immunomodulatory agents include tumor necrosis factor; interferon alpha, beta, and gamma; IL-2 and other cytokines; F42K and other cytokine analogs; or MIP-1, MIP-1beta, MCP-1, RANTES, and other chemokines. It is further contemplated that the upregulation of cell surface receptors or their ligands such as Fas / Fas ligand, DR4 or DR5 / TRAIL would potentiate the apoptotic inducing abilities of the compositions provided herein by establishment of an autocrine or paracrine effect on hyperproliferative cells. Increases intercellular signaling by elevating the number of GAP junctions would increase the anti-hyperproliferative effects on the neighboring hyperproliferative cell population. In other embodiments, cytostatic or differentiation agents can be used in combination with the compositions provided herein to improve the anti- hyerproliferative efficacy of the treatments. Inhibitors of cell adhesion are contemplated to improve the efficacy of the present invention. Examples of cell adhesion inhibitors are focal adhesion kinase (FAKs) inhibitors and Lovastatin. It is further contemplated that other agents that increase the sensitivity of a hyperproliferative cell to apoptosis, such as the antibody c225, could be used in combination with the compositions provided herein to improve the treatment efficacy.

[0167] In certain embodiments, hormonal therapy may also be used in conjunction with the present embodiments or in combination with any other cancer therapy previously described. The use of hormones may be employed in the treatment of certain cancers such 34 4929-6250-8358, v.2as breast, prostate, ovarian, or cervical cancer to lower the level or block the effects of certain hormones such as testosterone or estrogen. This treatment is often used in combination with at least one other cancer therapy as a treatment option or to reduce the risk of metastases. VI. Examples

[0168] The following examples are included to demonstrate particular embodiments. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent techniques discovered by the inventor to function well in the practice of embodiments, and thus can be considered to constitute particular modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the disclosure. Example 1 – mitCAR T Cells Expressing the Same

[0169] First, the inventors constructed a lentiviral vector containing a constitutive multi-integrin targeting CAR (mitCAR) by fusing 2.5F (mit) to the human CD8 transmembrane domain, CD3ζ, and CD28 intercellular domain, with a myc tag incorporated immediately downstream of 2.5F detection of CAR expression (FIG. 1A). They then transfected HEK293T cells to package lentivirus. Generated lentivirus was used to transduce primary T cells obtained from healthy donors. Primary T cells were transduced 24 hours after anti-CD3 / CD28 activation in the presence of 5 μg / ml of polybrene. The transduction efficiency based on the expression of blue fluorescence protein (BFP) was assessed by flow cytometry (FIG. 1B). The mean transduction efficiency of primary T cells was >80%. Transduced primary T cells demonstrated high expression of CAR on a surface (FIG.1C).

[0170] To test whether mitCAR T cells can be activated by tumor cells, the inventors co-cultured mitCAR T cells with different cancer cells, including A375P and U87 positive for 2.5F-Fc binding and Jurkat cells that are not recognized by 2.5F (negative control) (FIG.2A).72 hours after co-culture, activation marker CD25 was detected by flow cytometry. The data showed that mitCAR T cells can be activated by A375P and U87 cells 35 4929-6250-8358, v.2and express a higher level of CD25 (FIGS. 2BiC). mitCAR T cells are not activated in the presence of mitCAR-negative Jurkat cells.

[0171] Next, the cytotoxic activity of mitCAR T cells was determined using a luciferase-based killing assay. mitCAR T cells exhibited potent cytotoxic activity against melanoma A375P cells (FIG.3A) and glioblastoma U251 cells (FIG.3B).

[0172] Next, the inventors assessed the anti-solid tumor efficiency of mitCAR T cells in vivo. NSG mice engrafted with human melanoma cells, A375P, were injected intratumorally twice with 5 x 106mitCAR T cells or an equal number of control T cells. The mitCAR T therapy significantly inhibited tumor growth (FIG. 4A) and prolonged survival of tumor-bearing mice (FIG> 4B).

[0173] mitCAR targets all solid tumors and some hematological malignancies. Given the efficacy of mitCAR T in A375 melanoma, the inventors went on and assess if mitCAR T can be used to treat other solid tumors and hematological malignancies. They performed flow cytometry staining of a panel of solid tumor cell lines (including one AML cell line U937). They demonstrated that mitCAR-Fc can bind to all tested human tumor cell lines representing different tumor types, including lung carcinoma, hepatocellular carcinoma, breast cancer, pancreatic cancer, neuroblastoma, colorectal carcinoma, melanoma, glioblastoma, osteosarcoma, Ewing’s sarcoma and AML

[0174] In the next steps, the inventors assessed the binding of mit to primary tumors. They demonstrated that mitCAR can bind to primary pediatric central nervous system (CNS) (FIG. 7A) and neuroblastoma tumor samples (FIG. 7B) as well as to metastatic melanoma tumor samples (FIG.7C).

[0175] Incucyte-based real-time cytotoxicity assay further confirmed that all tested established solid tumor cells recognized by mit can be efficiently killed by mitCAR T (FIG.8).

[0176] Next, the inventors assessed mitCAR T cytotoxic activity against a panel of primary solid tumor cell lines, including melanoma, thyroid carcinoma, glioblastoma, and rhabdomyosarcoma cell lines. These experiments confirmed high cytotoxic activity of 36 4929-6250-8358, v.2mitCAR against different solid tumor cell lines (FIG.9), demonstrating that the mitCAR T can be a pan-tumor targeting T cell therapy.

[0177] mitCAR T cells exhibit potent anti-solid tumor activity in vivo that can be improved by armoring with IL-18. To confirm that mitCAR T cells can target a broad range of tumor types in vivo, the inventors performed studies with NSG mice injected with glioblastoma cell lines (U87MG and U251) and pancreatic carcinoma cells (AsPc-1). NSG mice engrafted with human tumor cells, were injected intratumorally once or twice with 5 x 106mitCAR T cells or an equal number of control T cells. The mitCAR T therapy significantly inhibited the growth of glioblastoma tumors (FIGS. 10A-B) and pancreatic cancer tumors (FIG.10C).

[0178] Next, the inventors determine whether the progression of the tumors after mitCAR T cell therapy could be caused by antigen escape (FIG. 11A). Flow cytometry analysis of mitCAR-Fc binding to tumor cells at the time of progression revealed that disease progression was not caused by the antigen (FIG.11B).

[0179] Analysis of mitCAR-Fc binding of tumors that progressed on mitCAR T cell therapy revealed that disease progression is caused not by antigen escape but rather by CAR T cell failure. Therefore, the inventors aimed to improve CAR T functionality and persistence by armoring it with human IL-18 (FIG. 12A). Armoring mitCAR T cells with IL-18 increased the efficacy of mitCAR T cell therapy of melanoma (FIG. 12B) and glioblastoma tumors (FIG.12C) and provided long-term disease control.

[0180] mRNA-based mitCAR T cells exhibit potent cytotoxicity against solid tumor cell lines. In the next steps, the inventors developed a mRNA version of mitCAR (FIG. 13A). mRNA-mitCAR T cells exhibited cytotoxic activity against glioblastoma U87MG cells in vitro (FIG.13B). These findings confirm the suitability of mRNA-mitCAR.

[0181] Murine mitCAR T cells recognize and target different murine cell lines and have potent anti-solid tumor activity. mitCAR has ability to recognize not only human, but also murine integrins. Therefore, the inventors assessed mitCAR-Fc binding to a panel of murine solid tumor cell lines. All tested murine solid tumor cell lines exhibited strong binding of mitCAR-Fc (FIG.14). 37 4929-6250-8358, v.2

[0182] In the next steps, the inventors developed a retroviral construct encoding murine mitCAR with the same antigen-recognizing domain (2.5F, mit) and murine signaling domains (FIG.15A) and validated it in murine T cells. The mean transduction efficiency of primary murine T cells was >80%. Transduced primary murine T cells demonstrated high expression of CAR on a surface (FIG.15B).

[0183] Further, the inventors assessed the activity of murine mitCAR T cells in vitro. Murine mitCAR T cells exhibited potent cytotoxic activity against a panel of murine solid tumor cell lines (FIG.16).

[0184] Next, the inventors assessed the anti-solid tumor efficiency of murine mitCAR T cells in vivo. C57BL / 6 mice were engrafted with different murine solid tumor cell lines followed by intratumoral injection of 5 x 106mitCAR T cells or an equal number of control T cells. The mitCAR T therapy significantly inhibited tumor growth of colorectal carcinoma MC38 tumors (FIG. 17A), glioma CT-2A tumors (FIG. 17B) and melanoma Yumm1.7 tumors (FIG.17C).

[0185] mitCAR recognizes endothelial cells and tumor-associated macrophages in the tumor microenvironment. The inventors assessed mitCAR-Fc binding to different cell populations in tumor-bearing mice. This analysis revealed that mitCAR-Fc recognizes myeloid cells, especially tumor-associated macrophages (TAMs) in the tumor microenvironment (FIG. 18A) as well as tumor-associated endothelial vessels (ECs) (FIG.18B). These data suggest that mitCAR can be triple-targeting CAR, with tumor- , TAMs-, and ECs-targeting components.

[0186] To confirm these findings in human cells, the inventors analyzed mitCAR- Fc binding to different immune cell populations in the lymph node sample of metastatic melanoma patients. Flow cytometry analyses revealed that mitCAR-Fc recognizes myeloid cells, especially tumor-associated macrophages (TAMs), in human tumors (FIG.19).

[0187] Further, the inventors assessed the mitCAR-Fc binding to tumor- associated macrophages (TAMs) differentiated ex vivo from monocytes obtained from healthy blood donors. TAMs were characterized by a strong mitCAR-Fc binding (FIG. 20A). Human TAMs triggered activation (FIG. 20B) and degranulation (FIG. 20C) of 38 4929-6250-8358, v.2human mitCAR T cells. Moreover, mitCAR-T cells exhibited potent cytotoxicity against human TAMs (FIG. 20D). This data confirms that mitCAR T cells recognize and kill both cancer cells and tumor-associated myeloid cells.

[0188] mitCAR-Fc recognize canine. Dogs that developed spontaneous tumors are often used in the preclinical studies of novel immunotherapies. Motivated by this, the inventors determine whether mitCAR-Fc can bind to canine integrins and recognize canine solid tumor cell lines. Analysis of a panel of canine solid tumor cell lines revealed a strong binding of mitCAR-Fc to canine cells (FIG. 21), suggesting that mitCAR T can be used in preclinical studies with canine tumors.

[0189] mitCAR targets are tumor-essential and partial targeted integrin loss does not prevent mitCAR recognition of tumors. A major barrier for conventional CAR T is that the surface antigen targeted by the CAR is often non-essential for tumor survival. Therefore, the loss of the antigen targeted by the CAR leads to tumor escape. Here, the inventors used CRISPR to knockout integrin αV in U87MG cells, and we found that U87MG cells transduced with CRISPR (mCherry+) gradually died (FIG. 22A). However, because αV-associated integrins only represent part of the integrins targeted by the mitCAR, mitCAR can still efficiently recognize the integrin αV KO U87MG cells (FIG.22B), further reducing the likelihood of tumor escape.

[0190] Discussion. Treatment options for advanced solid tumors have been limited and unsuccessful. As a novel therapeutic approach in cancer treatment, CAR-T therapy has achieved great success in hematological malignancies while solid tumor is still a big challenge. Tumor heterogeneity is considered the major hurdle leading to poor responsiveness. Cellular therapies armed by a single antigen-targeting CAR are highly constrained by tumor antigen heterogeneity and potential antigen-loss-induced tumor escape. Current strategies to overcome this is to develop multi-target CAR-T therapies, still being restrained by lack of ideal tumor targets or high toxicity.

[0191] mitCAR developed by the inventors is a multi-antigen targeting CAR that is characterized by a potent antitumor activity against a broad range of solid tumor types, including rare cancers with no available therapy. They validated the mitCAR expression and cytotoxicity in vitro and in vivo. The data suggest that mitCAR T cell therapy has no risk of 39 4929-6250-8358, v.2antigen escape. Armoring mitCAR T cells with IL-18 provided long-term disease control. Moreover, the inventors developed mRNA-based mitCAR T cells that demonstrated potent cytotoxicity against solid tumor cell lines. Additionally, the inventors demonstrated that mitCAR T cells can recognize and target not only cancer cells but also tumor-associated macrophages and tumor-associated endothelial vessels.

[0192] Given the aberrant expression of 2.5F-targeted integrins on numerous malignant solid tumors, the inventors envision that the mitCAR T developed in this proposal could be a universal T cell therapy for many solid tumors. Patients with rare solid tumors that have no available therapy and patients who failed other therapies. Thus, the targeting of both endothelial cells and tumor-associated macrophages, mitCAR T can be used as a strategy to remodel the immunosuppressive tumor microenvironment and can be combined with other therapeutic strategies. * * * * * * * * * * * * * * * * *

[0193] All of the compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this disclosure have been described in terms of particular embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the disclosure. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the disclosure as defined by the appended claims. 40 4929-6250-8358, v.2

Claims

WHAT IS CLAIMED:

1. A chimeric antigen receptor (mitCAR) comprising a knottin 2.5F domain fused to a transmembrane domain, a homodimer forming domain and an intracellular domain.

2. The CAR of claim 1, further comprising a myc tag immediately downstream of the 2.5F domain.

3. The CAR of claim 1 or claim 2, wherein the transmembrane domain is a human CD8 transmembrane domain, and / or the homodimer forming domain is CD3ζ, and / or the intracellular domain is a CD28, a 41BB, a 28BB, an ICOS or an OX40 intracellular domain.

4. The CAR of any one of claims 1-3, further comprising an oxygen-dependent degradation (ODD) domain fused to the C-terminus of the 2.5F domain, such as an ODD domain from HIF-1α.

5. An expression vector encoding a chimeric antigen receptor (mitCAR) comprising a knottin 2.5F domain fused to a transmembrane domain, a homodimer forming domain and an intracellular domain.

6. The expression vector of claim 5, further comprising a coding region a myc tag immediately downstream of the coding region for the 2.5F domain.

7. The expression vector of claim 5 or claim 6, wherein the coding region for the transmembrane domain is a human CD8 transmembrane domain, and / or the coding region for the homodimer forming domain is CD3ζ, and / or the intracellular domain is a CD28, a 41BB, a 28BB, an ICOS or an OX40 intracellular domain.

8. The expression vector of any one of claims 5-7, further comprising a coding region for an oxygen-dependent degradation (ODD) domain fused to the coding region for the C- terminus of the 2.5F domain, such as an ODD domain from HIF-1α.

9. An engineered cell expressing the CAR of any one of claims 1-4 or comprising the expression vector of any one of claims 5-8. 41 4929-6250-8358, v.

210. The engineered cell of claim 8 or claim 9, wherein the engineered cell is further engineered to express a cytokine.

11. The engineered cell of claim 10, wherein the cytokine is one or more of IL-18, IL12, IL7, IL15, IL10, IL4, IL-9 and / or IL-21.

12. The engineered cell of any one of claims 8-11 wherein the engineered cell is an autologous T cell, a macrophage, an NK cell, a monocyte, an NKT cell, a gamma-delta T cell, and allogeneic T cell, or an immortalized cytotoxic cell line.

13. The engineered cell of claim 12, wherein the immortalized cytotoxic cell line is TALL104 or NK92.

14. The engineered cell of any one of claims 8-13, further characterized as being a gp100- specific TCR-transgenic T cell.

15. A method of killing a cancer cell comprising contacting said cancer cell with an engineered cell of any one of claims 8-14.

16. A method of treating a subject with a solid tumor comprising administering to said subject an engineered cell of any one of claims 8-14.

17. The method of claim 15 or claim 16, wherein said cancer cell is, or said solid tumor comprises, a lung cancer cell, brain cancer cell, head & neck cancer cell, breast cancer cell, skin cancer cell, liver cancer cell, pancreatic cancer cell, stomach cancer cell, colon cancer cell, rectal cancer cell, uterine cancer cell, cervical cancer cell, ovarian cancer cell, testicular cancer cell, skin cancer cell, or esophageal cancer cell.

18. The method of any one of claims 15-17, further comprising contacting said cancer cell or solid tumor with a second anti-cancer agent or treatment, such as wherein said second anti-cancer agent or treatment is selected from chemotherapy, radiotherapy, immunotherapy, hormonal therapy, or toxin therapy.

19. The method of claim 18, wherein said second anti-cancer agent or treatment is given at the same time as said engineered cell. 42 4929-6250-8358, v.

220. The method of claim 18, wherein said second anti-cancer agent or treatment is given before and / or after said engineered cell.

21. The method of any one of claims 15-20, wherein said cancer cell or solid tumor is a metastatic cancer cell / solid tumor, a multiply drug-resistant cancer cell / solid tumor or a recurrent cancer cell / solid tumor.

22. The method of any one of claims 16-21, further comprising giving at least a second administration of said engineered cell, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 20 10 additional administrations.

23. The method of any one of claims 16-21, wherein administration is intratumoral, intracranial, into tumor vasculature, local to the tumor, regional to the tumor or systemic.

24. The method of any one of claims 16-23, wherein the subject is a mammal, such as a human, a canine, or a mouse.

25. The method of any one of claims 16-24, wherein the tumor is a solid tumor and said administering said engineered cell results in one or more of cytotoxicity against said solid tumor, and / or tumor regression of said solid tumor.

26. An mRNA comprising a nucleic acid encoding the CAR of any one of claims 1-4.

27. A microparticle or nanoparticle comprising the mRNA of claim 24. 43 4929-6250-8358, v.2

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