Recombinant constructs, vectors and cells for expressing chimeric antigen receptors, and methods thereof

Abstract

The present invention relates to recombinant constructs encoding chimeric antigen receptors and neutralizing antigen-binding fragments, vectors, engineered chimeric antigen receptors T-cells (CAR-T), and methods for expressing chimeric antigen receptors and neutralizing antigen-binding fragment. The present invention also relates to compositions and methods for enhancing and maintaining chimeric antigen receptor expressing T cells, while reducing cytotoxic effects of CAR-T cell therapies.

Description

RECOMBINANT CONSTRUCTS, VECTORS AND CELLS FOR EXPRESSING CHIMERIC ANTIGEN RECEPTORS, AND METHODS THEREOFFIELD OF THE INVENTION

[0001] The present invention broadly relates to the field of biotechnology and immunology. More particularly, the present invention relates to recombinant constructs and vectors encoding chimeric antigen receptors and cytokine neutralizing antigen-binding fragments, chimeric antigen receptors T-cells (CAR-T) having said recombinant constructs, and methods for producing the same and their use as medicament, particularly for immunotherapy in prostate cancer.BACKGROUND OF THE INVENTION

[0002] Prostate cancer is a common malignancy and ranks the highest in its incidence in men worldwide. Prostate cancer remains the second most frequently diagnosed cancer in men worldwide, with an estimated 110 million new cases per year. In addition, 307,000 deaths are expected, which represent the fifth leading cause of cancer death. Although primary tumors can be successfully treated, there is no curative treatment at the advanced stage. Conventional treatments often involve surgery, radiation therapy, chemotherapy, or androgen deprivation therapy, each associated with significant side effects and limited efficacy, particularly in advanced stages of the disease. Hence, there is an unmet need for more precise and efficient methods of treating the disease.

[0003] Prostate Specific Membrane Antigen (PSMA) is the best characterized antigen in prostate cancer for antibody-based diagnosis and therapeutic intervention. The protein is also known as glutamate carboxypeptidase II (EC 3.4.17.21), N-acetyl linked acidic dipeptidase I (NAALADase) or folate hydrolase. PSMA is a type II membrane glycoprotein consisting of 750 amino acids (aa), with a small intracellular domain of 19aa, a transmembrane domain of 24aa and a large extracellular domain of 707 aa. It shows a high degree of structural similarity and identity to human transferrin receptor. PSMA is highly localized to the surface of prostate cancer cells, is present on cancer cells during all tumor stages, and is enhanced in androgen-independent and metastatic disease. PSMA is not secreted into the extracellular space and undergoes constitutive internalization, which is enhanced by the binding of PSMA-specific antibodies. These characteristics make it an ideal candidate for targeted therapy of advanced prostate cancer. In addition, PSMA was also found to be expressed in neovascular endothelium of virtually allsolid tumor types but not in normal vascular endothelium. Therefore, it is considered to be a unique anti -angiogenic target.

[0004] Interleukin-8 (IL-8) is a cytokine that plays a significant role in inflammation and immune responses. In the context of prostate cancer, IL-8 has been studied for its potential involvement in tumor growth, angiogenesis (the formation of new blood vessels), and metastasis. Prostate cancer cells can produce and secrete IL-8. Elevated levels of IL-8 have been observed in both prostate cancer tissues and in the blood of prostate cancer patients. Given ILS’ s involvement in tumor growth and metastasis, IL-8 has been explored as a potential therapeutic target in prostate cancer and as a means of inhibiting cancer progression. T cell activation is an important step in the protective immunity against pathogenic microorganisms (e.g., viruses, bacteria, and parasites), foreign proteins, and harmful chemicals in the environment, and also as immunity against cancer and other hyperproliferative diseases. T cells express receptors on their surfaces (i.e., T cell receptors) that recognize antigens presented on the surface of cells. During a normal immune response, binding of these antigens to the T cell receptor, in the context of MHC antigen presentation, initiates intracellular changes leading to T cell activation.

[0005] In recent years, adoptive immune cell therapy has been introduced as a novel concept to treat different cancers by redirecting the immune system to eliminate tumor cells. One of the most successful concepts is based on the genetic engineering of T cells to express Chimeric Antigen Receptors (CARs) that bind to tumor antigens or tumor-associated antigens in a Human Leukocyte Antigen (HLA) -independent manner. Chimeric antigen receptors (CARs) are artificial receptors designed to convey antigen specificity to T cells without the requirement for MHC antigen presentation. Chimeric antigen receptor-expressing T cells may be used in various therapies, including cancer therapies. For example, adoptive transfer of T cells expressing CARs is an effective therapy for the treatment of certain hematological malignancies. The use CAR-T therapy is radically changing the area of hematological malignancies, but solid tumors still remain an unchartered field.

[0006] CAR-T cells targeting CD19 have been successfully used to treat B-cell acute lymphoblastic leukemia (B-ALL), with > 90% of patients having complete regression in several clinical trials. Based on this success, over 200 clinical trials have been conducted to treat mainly haematological malignancies. However, to date, CAR-T cell therapy appears to be quite ineffective for solid tumors. The main reason for this failure appears to be the tumor microenvironment (TME), which is the cellular environment in which the tumor resides. Itincludes various immune cells, fibroblasts, extracellular matrix (ECM) and surrounding blood vessels. Several mechanisms have been described that describe the limitation of cytotoxic T- cell activity in TME, including activation of PD-1 based T cell immune checkpoint suppression. Overcoming these limitations in combination with T cell checkpoint antagonists helps to improve antitumor activity in TME.

[0007] The utility of chimeric antigen receptor modified T-cells (CAR-T) to treat cancer has been demonstrated in a number of clinical studies including the approval by the FDA of two of these products for the treatment of pediatric acute lymphocytic leukemia and transformed nonHodgkins Lymphoma. These successful CAR-Ts, tisagenlecleucel and axicabtagene ciloleucel developed by Novartis and Kite Pharmaceuticals, respectively, are autologous products that target the B-cell antigen CD-19. Along with the CD-19 targeting CARs, a number of additional cancer-associated antigens are targeted in ongoing clinical studies by CARs variably employing unique structures, T-cell targets, activation and manufacturing methods and patient pretreatment protocols. The large number of clinical studies employing these differing agents and approaches highlight the complex pharmacology of CAR-Ts. Ma et al (2014)74, pp 286-Santoro et al [Cancer immunol. res. (2015), pp 68-84] describe T cells with chimeric antigen receptors for prostate specific membrane antigen, in which the PSMA binding moiety is derived from antibody J591. Zhong et al [Molecular Therapy (2010), pp.413-420] also discloses chimeric antigen receptors in which the PSMA binding fragment is also derived from antibody J591.

[0008] Over the past few decades, monoclonal antibodies (mabs) that are highly specific and widely used tools for cell targeting, have attracted high interest in medical research and have become the fastest growing class of drugs for the treatment of a variety of human diseases, including cancer. Antibody 7E11 was the first released PSMA-specific mAb and was found to bind to the N-terminus of the intracellular domain of PSMA (MWNLLH). The In-label (Inlabeled) form of 7E11 (ProstaScint, Cytogen, philiadelphia, PA) has been approved by the U.S. Food and Drug Administration (FDA) for the detection and imaging of metastatic prostate cancer in soft tissues. However, 7E11 was unable to bind live cells because the antibody bound an intracellular epitope. EP 1883698 discloses three different mAbs 3 / A12,3ZE7, 3 / F11 which show strong specific binding to the extracellular part of PSMA on the surface of prostate cancer cells and prostate tissue specimens.

[0009] However, the major problems associated with CAR-T therapy in solid tumors are cold tumor microenvironment mainly due to the released cytokines, the infiltration of CAR-T cells into the tumor microenvironment and the low persistence and accelerated exhaustion of CAR-T in tumor microenvironment. Further, some second and third-generation CAR-T cells have been implicated in patient deaths, due to cytokine storm and tumor lysis syndrome caused by highly activated T cells. Thus, to overcome the aforesaid problems in the prior art there exists a need to develop novel chimeric antigen receptors construct, CAR-T cells (CAR-T), methods for producing the same and their use as medicament, particularly for immunotherapy.SUMMARY OF THE INVENTION

[0010] In an aspect of the present disclosure, there is provided a recombinant construct comprising: (a) a first polynucleotide comprising a nucleotide fragment encoding a prostatespecific membrane antigen (PSMA) specific chimeric antigen receptor (CAR); and (b) a second polynucleotide comprising a nucleotide fragment encoding a neutralizing antigen-binding fragment specific for interleukin-8 (IL-8).

[0011] In another aspect of the present disclosure, there is provided a vector comprising the recombinant construct as disclosed herein.

[0012] In an aspect of the present disclosure, there is provided an engineered cell or population thereof, comprising the vector as disclosed herein or the recombinant construct as disclosed herein.

[0013] In an aspect of the present disclosure, there is provided a pharmaceutical composition comprising the engineered cell or population thereof as disclosed herein.

[0014] In another aspect of the present disclosure, there is provided a method for treating cancer in a subject, comprising administering the pharmaceutical composition as disclosed herein, or the engineered cell or population thereof as disclosed herein, to said subject.

[0015] In another aspect of the present disclosure, there is provided a method of killing target cells at a target site, comprising administering the pharmaceutical composition as disclosed herein, or the engineered cell or population thereof as disclosed herein to the target site.

[0016] In yet another aspect of the present disclosure, there is provided a method for increasing cytolysis of target cells at a target site, comprising administering the pharmaceutical composition as disclosed herein, or the engineered cell or population thereof as disclosed herein to the target site.

[0017] These and other features, aspects, and advantages of the present subject matter will be better understood with reference to the following description. This summary is provided tointroduce a selection of concepts in a simplified form. This summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used to limit the scope of the claimed subject matter.BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The following drawings form a part of the present specification and are included to further illustrate aspects of the present disclosure. The disclosure may be better understood by reference to the drawings in combination with the detailed description of the specific embodiments presented herein.

[0019] Figs. 1(a) and 1(b) are schematic representations of recombinant constructs, wherein Fig. 1(a) depicts an exemplary recombinant construct of the present disclosure, for expressing armored PSMA specific CAR, i.e. PSMA specific CAR with IL-8 scFv, and Fig. 1(b) depicts a recombinant construct for expressing unarmored PSMA specific CAR, i.e. PSMA specific CAR without IL-8 scFv, in accordance with the embodiments herein.

[0020] Fig. 2 depicts percentage T cell population (CD4 and CD8 expressing cell) in the cell population expressing unarmored PSMA specific CAR and the armored PSMA specific CAR of the present disclosure, in accordance with the embodiments herein.

[0021] Fig. 3 depicts ELISA results for armored PSMA specific CAR of the present disclosure showing levels of secreted IL-8 scFv (sIL-8 scFv), IL-8 commercial antibody used as control, in accordance with the embodiments herein.

[0022] Fig. 4 depicts the results of BAX expression for Untransduced (UT) T cells, T cells transduced with unarmored PSMA specific CAR and the armored PSMA specific CAR of the present disclosure, respectively, wherein (a) depicts the results of western blotting, and (b) is an ImageJ analysed graph depicting BAX expression, in accordance with the embodiments herein.

[0023] Fig. 5 depicts cytotoxicity at 6 hours for unarmored CAR-T cells and armored CAR-T cells of the present disclosure, wherein a) and b) depict bar and line graph representations respectively, in accordance with the embodiments herein.

[0024] Fig. 6 depicts cytotoxicity at 24 hours for unarmored CAR-T cells and armored CAR-T cells of the present disclosure, wherein a) and b) depict bar and line graph representations respectively, in accordance with the embodiments herein.

[0025] Fig. 7 depicts cytotoxicity at 48 hours for unarmored CAR-T cells and armored CAR-Tcells of the present disclosure, wherein a) and b) depict bar and line graph representations respectively, in accordance with the embodiments herein.

[0026] Fig. 8 depicts cytotoxicity at 72 hours for unarmored CAR-T cells and armored CAR-T cells of the present disclosure, wherein a) and b) depict bar and line graph representations respectively, in accordance with the embodiments herein.

[0027] Figs. 9(a) and 9(b) depicts CAR expression in both armored and unarmored CAR-T cells (stained by protein L staining), wherein Fig. 9(a) depicts CAR expression in unarmored CAR-T cells, and Fig. 9(b) depicts CAR expression in armored CAR-T cells, in accordance with the embodiments herein.

[0028] Figs. 10(a) and 10(b) depicts MitoSOX expression in both armored and unarmored CAR-T cells, wherein Fig. 10(a) depicts MitoSOX expression in unarmored CAR-T cells, and Fig. 10(b) depicts MitoSOX expression in armored CAR-T cells, in accordance with the embodiments herein.

[0029] Figs. 11(a) and 11(b) depicts TMRE expression in both armored and unarmored CAR- T cells, wherein Fig. 11(a) depicts TMRE expression in unarmored CAR-T cells, and Fig. 11(b) depicts TMRE expression in armored CAR-T cells, in accordance with the embodiments herein.DETAILED DESCRIPTION OF THE INVENTION

[0030] At the very outset of the detailed description, it may be understood that the ensuing description only illustrates a particular form of this invention. However, such a particular form is only exemplary embodiment, and without intending to imply any limitation on the scope of this invention. Accordingly, the description is to be understood as an exemplary embodiment and teaching of invention and not intended to be taken restrictively.

[0031] Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skilled in the art to which this invention belongs. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

[0032] A technical problem observed in CAR-T therapy is that CAR-T cells that have application in CAR-T therapy are generally associated with problems such as cold tumor microenvironment (TME), mainly due to the release of cytokines, which leads to poor infiltration of CAR-T cells into the tumor microenvironment and low persistence andaccelerated exhaustion of CAR-T cells in tumor microenvironment.

[0033] A solution provided by the present disclosure is to neutralize the cytokines by providing neutralizing cytokine binding fragments along with the CAR. The problems associated with the existing CAR-T cell therapy is circumvented by the inclusion of a cytokine neutralizing scfv in the CAR construct. Accordingly, embodiments herein provide a recombinant construct for expression in CAR-T cells.

[0034] Further, the present invention relates to engineered cells, particularly chimeric antigen receptors T-cells (CAR-T), methods and use thereof, particularly for immunotherapy. The engineered cells, of the present disclosure, are particularly suitable for allogeneic transplantations, especially because it reduces both the risk of rejection by the host's immune system and the risk of developing graft versus host disease. The present disclosure opens the way to standard and affordable adoptive immunotherapy strategies using T-cells for treating cancer, infections and auto-immune diseases.

[0035] Further, the present invention also provides engineered T cells suitable for immunotherapy. Furthermore, the present invention relates to an engineered T cellexpressing a CAR, comprising an extracellular domain, a transmembrane domain, and an intracellular domain, the extracellular domain comprising an antigen binding domain.Definitions:

[0036] For convenience, before further description of the present disclosure, certain terms employed in the specification, and examples are delineated here. These definitions should be read in the light of the remainder of the disclosure and understood as by a person of skill in the art. The terms used herein have the meanings recognized and known to those of skill in the art, however, for convenience and completeness, particular terms and their meanings are set forth below.

[0037] The articles “a”, “an” and “the” are used to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article.

[0038] The terms “comprise” and “comprising” are used in the inclusive, open sense, meaning that additional elements may be included. It is not intended to be construed as “consists of only”.

[0039] Throughout this specification, unless the context requires otherwise the word “comprise”, and variations such as “comprises” and “comprising”, will be understood to implythe inclusion of a stated element or step or group of elements or steps but not the exclusion of any other element or step or group of elements or steps.

[0040] In an example, “chimeric antigen receptor” or “CAR” is meant, for example, a chimeric polypeptide that comprises a polypeptide sequence that recognizes a target antigen (the terms “antigen-recognition domain”, “antigen recognition region”, “antigen recognition moiety”, or “antigen binding domain” “antigen binding region” are used interchangeably herein), where the polypeptide is further linked to a transmembrane polypeptide and intracellular signaling domain selected to activate T cell and provide specific immunity. An antigen recognition domain may be any polypeptide or fragment thereof, either naturally derived, or synthetic, which can recognize and bind to an antigen. Examples of antigen recognition moieties include, but are not limited to, polypeptides derived from antibodies, such as, for example, single chain variable fragment (scFv), Fab fragment, Fab’ fragment, F(ab‘)2 fragment, and Fv fragment; polypeptides derived from T Cell receptors, such as, for example, TCR variable domains; polypeptides derived from Pattern Recognition Receptors, and any ligand or receptor fragment that binds to the extracellular cognate protein.

[0041] The term "chimeric antigen receptor" (CAR) as used herein refers to a chimeric polypeptide comprising tumor antigen binding domain that is fused to a transmembrane polypeptide and an intracellular signaling domain capable of activating or stimulating T cells. Most commonly, the CAR's extracellular domain is an antigen binding domain composed of a single chain variable fragment (scFv) derived from fusing the variable heavy and light regions of a murine or humanized monoclonal antibody. Alternatively, scFv's may be used that are derived from Fab's (instead of from an antibody, e.g., obtained from Fab libraries). In various embodiments, the scFv is fused to a transmembrane domain and then to intracellular signaling domain. The term “chimeric antigen receptor” may also refer to chimeric receptors that are not derived from antibodies but are chimeric T cell receptors. These chimeric T cell receptors may comprise a polypeptide sequence that recognizes a target antigen, where the recognition sequence may be, for example, but not limited to, the recognition sequence derived from a T cell receptor or an scFv. The intracellular domain polypeptides are those that act to activate the T cell. Chimeric T cell receptors are discussed in, for example, Gross, G., and Eshhar, Z., FASEB Journal 6:3370-3378 (1992), and Zhang, Y., et al., PLOS Pathogens 6: 1-13 (2010).

[0042] As used herein, "first-generation" CARs include those that solely provide CD3 signals upon antigen binding. In the first generation of CAR-T therapies, the intracellular domain consists of CD3 Z- chain or FCSRIY, and it is the primary transmitter of signals from endogenousT cell receptors (TCR). However, these CAR-T cell therapies are not able to produce enough interleukin-2 (IL-2) to kill tumor cells. Most of the studies conducted with first-generation CAR- T cells could not achieve the desired effects because of inadequate proliferation, a short life span in vivo and inadequate secreted cytokine.

[0043] As used herein, "second-generation" CARs include those that provide both costimulation (e.g. CD28 or CD137) and activation (CD3). In the second generation of CAR-T cell therapies, additional signaling domains were added. The intracellular domain consisted of T-cell receptors (TCR), cytokine receptors and costimulatory receptors. Presently, some clinically approved CAR design relies on second-generation CARs which are directed against the CD 19 antigen, a cell surface receptor more abundantly expressed in malignant B cells. These CD 19 directed lentiviral CAR constructs comprise of anti-CD19 scFv derived from a murine FMC63 monoclonal antibody and only one costimulatory domain. Such 2nd generation CARs have shown promising results in patients with refractory and relapsed tumors, where other treatment methods like chemotherapy, radiation therapy and surgery have failed.

[0044] As used herein, "third generation" CARs include those that provide multiple costimulation (e.g. CD28 and CD137) and activation (CD3). In CAR applications to date, the CAR is selected to have high affinity or avidity for the antigen.

[0045] As used herein, the term “extracellular domain,” refers to the part of a CAR that is located outside of the cell membrane and is capable of binding to an antigen, target, or ligand.

[0046] As used herein, the term “transmembrane domain” refers to the portion of a CAR that extends across the cell membrane and anchors the CAR to cell membrane.

[0047] As used herein, the term “intracellular signaling domain” refers to the part of a CAR that is located inside of the cell membrane and is capable of transducing an effector signal.

[0048] As used herein, “T cells” (also referred to as “T lymphocytes”) belong to a group of white blood cells referred to as lymphocytes. Lymphocytes generally are involved in cell- mediated immunity. The “T” in “T cells” refers to cells derived from or whose maturation is influenced by the thymus. T cells can be distinguished from other lymphocyte types such as B cells and Natural Killer (NK) cells by the presence of cell surface proteins known as T cell receptors. The term “activated T cells” as used herein, refers to T cells that have been stimulated to produce an immune response (e.g., clonal expansion of activated T cells) by recognition of an antigenic determinant, such as, for example, presented in the context of a Class II major histo-compatibility (MHC) marker. T cells are activated by the presence of an antigenic determinant, cytokines and / or lymphokines and cluster of differentiation cell surface proteins (e.g., CD3, CD4, CD8, the like and combinations thereof). Cells that express a cluster of differential protein often are said to be “positive” for expression of that protein on the surface of T cells (e.g., cells positive for CD3, CD4, or CD8 expression are referred to as CD3+, CD4+, or CD8+). CD3 and CD4 proteins are cell surface receptors or co-receptors that may be directly and / or indirectly involved in signal transduction in T cells. T cells may either be isolated or obtained from a commercially available source. “T cell” includes all types of immune cells expressing CD3 including T-helper cells (CD4+ cells), cytotoxic T- cells (CD8+ cells), natural killer T-cells , T- regulatory cells (Treg) and gamma-delta T cells. A “cytotoxic cell” includes CD8+ T cells, natural-killer (NK) cells, and neutrophils, which cells are capable of mediating cytotoxicity responses. Non-limiting exemplary sources for such commercially available cell lines include the American Type Culture Collection, or ATCC, (http: / / www.atcc.org / ) and the German Collection of Microorganisms and Cell Cultures (https: / / www.dsmz.de / ).

[0049] T cells express receptors on their surfaces (i.e., T cell receptors) that recognize antigens presented on the surface of cells. During a normal immune response, binding of these antigens to the T cell receptor, in the context of MHC antigen presentation, initiates intracellular changes leading to T cell activation. Chimeric antigen receptors (CARs) are artificial receptors designed to convey antigen specificity to T cells without the requirement for MHC antigen presentation. They include an antigen-specific component, a transmembrane component, and an intracellular component selected to activate the T cell and provide specific immunity. Chimeric antigen receptor-expressing T cells may be used in various therapies, including cancer therapies.

[0050] As used herein, "secreted" refers to a polypeptide that is released from a cell via the secretory pathway through the endoplasmic reticulum, Golgi apparatus, and as a vesicle that transiently fuses at the cell plasma membrane, releasing the proteins outside of the cell.

[0051] The term “chimeric signaling polypeptide” is interchangeable with “chimeric costimulating molecule,” “chimeric co-stimulating polypeptide.”

[0052] As used herein, the terms “chimeric,” “fusion” and “chimeric fusion” are used interchangeably herein with reference to a polypeptide containing two or more proteins (or a portion(s) of one or more of the two or more proteins) that have been joined to create a chimeric polypeptide. The two or more proteins (or portions thereof) may be directly joined to each other, wherein a terminal amino acid residue of one protein (or portion thereof) is directly bonded toa terminal amino acid residue of another protein (or portion thereof), or may be joined through one or more intervening elements (e.g., one or more amino acids that are not part of either protein, such as a linker or adapter, or a non-amino acid polymer). For example, a polypeptide that is produced from nucleic acid encoding a fusion of a multimerizing protein (or portion thereof) and another protein (e.g., a DNA-binding protein, transcription activation protein, pro- apoptotic protein or protein component of an immune cell activation pathway), or portion thereof, may be referred to as a chimeric, fusion or chimeric fusion polypeptide.

[0053] As used herein, the term “expression construct” or “transgene” is defined as a genetic construct having a nucleic acid coding for gene products in which part or all of the nucleic acid encoding sequence is capable of being transcribed can be inserted into the vector.

[0054] As used herein, the term “expression vector” refers to a vector having nucleic acids coding for one or more gene products or part of a gene product, capable of being transcribed. In some cases, RNA molecules are then translated into a protein, polypeptide, or peptide. In other cases, these sequences are not translated, for example, in the production of antisense molecules or ribozymes. Expression vectors can contain a variety of control sequences, which refer to nucleic acid sequences necessary for the transcription and possibly translation of an operatively linked coding sequence in a particular host organism. In addition to control sequences that govern transcription and translation, vectors and expression vectors may contain nucleic acid sequences that serve other functions as well.

[0055] For initial genetic modification of the cells to provide tumor or viral antigen specific cells, a retroviral vector is generally employed for transduction, however any other suitable viral vector or non- viral delivery system can be used. For subsequent genetic modification of the cells to provide cells comprising an antigen presenting complex comprising at least two costimulatory ligands, retroviral gene transfer (transduction) likewise proves effective. Combinations of retroviral vector and an appropriate packaging line are also suitable, where the capsid proteins will be functional for infecting human cells. Various amphotropic virusproducing cell lines are known, including, but not limited to, PA 12 (Miller, et al. (1985) Mol Cell Biol. 5:431-437); PA317 (Miller, et al. (1986) Mol. Cell. Biol. 6:2895-2902); and CRIP (Danos, et al (1988) Proc, Natl. Acad. Sci. USA 85:64606464). Non-amphotropic particles are suitable too, e.g., particles pseudo typed with VSVG, RD1 14 or GALV envelope and any other known art.

[0056] Possible methods of transduction also include direct co-culture of the cells with producercells, e.g., by the method of Bregni, et al. (1992) Blood 80: 1418-1422, or culturing with viral supernatant alone or concentrated vector stocks with or without appropriate growth factors and polycations, e.g., by the method of Xu, et al. (1994) Exp. Hemat. 22:223-230; and Hughes, et al. (1992) J. Clin. Invest. 89: 1817.

[0057] Other transducing viral vectors can be used to express a co-stimulatory ligand of the invention in an immune responsive cell. Preferably, the chosen vector exhibits high efficiency of infection and stable integration and expression (see, e.g., Cayouette et al., Human Gene Therapy 8:423-430, 1997; Kido et al, Current Eye Research 15:833-844, 1996; Bloomer et al, Journal of Virology 71 :6641-6649, 1997; Naldini et al., Science 272:263-267, 1996; and Miyoshi et al., Proc. Natl. Acad. Sci. U.S.A. 94: 10319, 1997). Other viral vectors that can be used include, for example, adenoviral, lentiviral, and adeno-associated viral vectors, vaccinia virus, a bovine papilloma virus, or a herpes virus, such as Epstein-Barr Virus (also see, for example, the vectors of Miller, Human Gene Therapy 15-14, 1990; Friedman, Science 244: 1275-1281, 1989; Eglitis et al., BioTechniques 6:608-614, 1988; Tolstoshev et 1401866742 al., Current Opinion in Biotechnology 1 :55-61, 1990; Sharp, The Lancet 337: 1277- 1278, 1991 ; Cometta et al, Nucleic Acid Research and Molecular Biology 36:31 1- 322, 1987; Anderson, Science 226:401-409, 1984; Moen, Blood Cells 17:407-416, 1991 ; Miller et al., Biotechnology 7:980-990, 1989; LeGal La Salle et al, Science 259:988-990, 1993; and Johnson, Chest 107:77S- 83 S, 1995). Retroviral vectors are particularly well developed and have been used in clinical settings (Rosenberg et al, N. Engl. J. Med 323:370, 1990; Anderson et al., U.S. Pat. No. 5,399,346).

[0058] Non-viral approaches can also be employed for the expression of a protein in cell. For example, a nucleic acid molecule can be introduced into a cell by administering the nucleic acid in the presence of lipofection (Feigner et al., Proc. Natl. Acad. Sci. U.S.A. 84:7413, 1987; Ono et al., Neuroscience Letters 17:259, 1990; Brigham et ah, Am. J. Med. Sci. 298:278, 1989; Staubinger et ah, Methods in Enzymology 101 :512, 1983), asialoorosomucoid- polylysine conjugation (Wu et al., Journal of Biological Chemistry 263: 14621, 1988; Wu et al., Journal of Biological Chemistry 264: 16985, 1989), or by microinjection under surgical conditions (Wolff et al., Science 247: 1465, 1990). Other non-viral means for gene transfer include transfection in vitro using calcium phosphate, DEAE dextran, electroporation, and protoplast fusion. Liposomes can also be potentially beneficial for delivery of DNA into a cell. Transplantation of normal genes into the affected tissues of a subject can also be accomplished by transferring a normal nucleic acid into a cultivatable cell type ex vivo (e.g., an autologous orheterologous primary cell or progeny thereof), after which the cell (or its descendants) are injected into a targeted tissue or are injected systemically. Recombinant receptors can also be derived or obtained using transposases or targeted nucleases (e.g. Zinc finger nucleases, meganucleases, or TALE nucleases). Transient expression may be obtained by RNA electroporation.

[0059] As used herein, the term “gene” is defined as a functional protein-, polypeptide-, or peptide-encoding unit. As will be understood, this functional term includes genomic sequences, cDNA sequences, and smaller engineered gene segments that express, or are adapted to express, proteins, polypeptides, domains, peptides, fusion proteins and / or mutants.

[0060] As used herein, the term “polynucleotide” is defined as a chain of nucleotides. A polynucleotide may comprise one or more nucleotide fragments. Furthermore, nucleic acids are polymers of nucleotides. Thus, nucleic acids and polynucleotides as used herein are interchangeable.

[0061] As used herein, the term “polypeptide” is defined as a chain of amino acid residues, usually having a defined sequence. As used herein the term polypeptide may be interchangeable with the term “proteins”.

[0062] As used herein, “sequence identity” means the extent to which two nucleotide or amino acid sequences are invariant. “Sequence alignment” means the process of lining up two or more sequences to achieve maximal levels of identity (and, in the case of amino acid sequences, conservation) for the purpose of assessing the degree of similarity. Numerous methods for aligning sequences and assessing similarity / identity are known in the art such as, for example, the Cluster Method, wherein similarity is based on the MEGALIGN algorithm, as well as BLASTN, BLASTP, and FASTA. When using any of these programs, the settings may be selected that result in the highest sequence similarity.

[0063] As used herein, the term “promoter” is defined as a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a gene. In some embodiments, the promoter is a developmentally regulated promoter. As used herein, the term “under transcriptional control,” “operably linked,” or “operatively linked” is defined as the promoter is in the correct location and orientation in relation to the nucleic acid to control RNA polymerase initiation and expression of the gene. In some examples, one or more polypeptides are said to be “operatively linked.” In general, the term “operably linked” is meant to indicate that an element, for example: promoter, 2A element,etc. is functionally linked to a coding sequence which may be located downstream to such element in a polynucleotide.

[0064] Promoters may be selected that are appropriate for the vector used to express the CARs and other polypeptides provided herein. Promoters, and other regulatory elements, are selected such that they are functional in the desired cells or tissue. In addition, this list of promoters should not be construed to be exhaustive or limiting; other promoters that are used in conjunction with the promoters and methods disclosed herein.

[0065] As used herein, the terms “pharmaceutically” or “pharmacologically acceptable” refers to molecular entities and compositions that do not produce adverse, allergic, or other untoward reactions when administered to an animal or a human.

[0066] As used herein, “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the vectors or cells presented herein, its use in therapeutic compositions is contemplated. Supplementary active ingredients also can be incorporated into the compositions. In some embodiments, the subject is a mammal.

[0067] As used herein, the term “carrier” refers to any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, oil, lipid, lipid containing vesicle, microsphere, liposomal encapsulation, or other material well known in the art for use in pharmaceutical formulations. It will be understood that the characteristics of the carrier, excipient or diluent will depend on the route of administration for a particular application. As used herein, the term “pharmaceutically acceptable carrier” refers to a non-toxic material that does not interfere with the effectiveness of a composition according to the invention or the biological activity of a composition according to the invention.

[0068] As used herein, the term “cancer” as used herein means any disease, condition, trait, genotype or phenotype characterized by unregulated cell growth or replication as is known in the art. A “cancer cell” is a cell that divides and reproduces abnormally with uncontrolled growth. This cell can break away from the site of its origin (e.g., a tumor) and travel to other parts of the body and set up another site (e.g., another tumor), in a process referred to as metastasis. A “tumor” is an abnormal mass of tissue that results from excessive cell division that is uncontrolled and progressive, and is also referred to as a neoplasm. Tumors can be eitherbenign (not cancerous) or malignant. The compositions and methods described herein are useful for treatment of cancer and tumor cells, i.e., both malignant and benign tumors. Thus, in various embodiments of the methods and compositions described herein, the cancer can include, without limitation, heme cancers, lymphomas, breast cancer, lung cancer, prostate cancer, colorectal cancer, esophageal cancer, stomach cancer, bladder cancer, pancreatic cancer, kidney cancer, cervical cancer, liver cancer, ovarian cancer, and testicular cancer.

[0069] As used herein, the term “subject” refers to an animal, and preferably a mammal. According to particular embodiments, the subject is a mammal including a non-primate (e.g., a camel, donkey, zebra, cow, pig, horse, goat, sheep, cat, dog, rat, rabbit, guinea pig or mouse) or a primate (e.g., a monkey, chimpanzee, or human). In particular embodiments, the subject is a human. In a particular embodiment, the subject is a cancer patient, for eg: prostate cancer patient.

[0070] As used herein, the term “therapeutically effective amount” refers to an amount of an active ingredient or component that elicits the desired biological or medicinal response in a subject. A therapeutically effective amount can be determined empirically and in a routine manner, in relation to the stated purpose.

[0071] As used herein, the terms “treat,” “treating,” and “treatment” are all intended to refer to an amelioration or reversal of at least one measurable physical parameter related to a cancer or autoimmunity, which is not necessarily discernible in the subject, but can be discernible in the subject. The terms “treat,” “treating,” and “treatment,” can also refer to causing regression, preventing the progression, or at least slowing down the progression of the disease, disorder, or condition. In a particular embodiment, “treat,” “treating,” and “treatment” refer to an alleviation, prevention of the development or onset, or reduction in the duration of one or more symptoms associated with the disease, disorder, or condition, such as a tumor or more preferably a cancer. In a particular embodiment, “treat,” “treating,” and “treatment” refer to prevention of the recurrence of the disease, disorder, or condition. In a particular embodiment, “treat,” “treating,” and “treatment” refer to an increase in the survival of a subject having the disease, disorder, or condition. In a particular embodiment, “treat,” “treating,” and “treatment” refer to elimination of the disease, disorder, or condition in the subject.

[0072] Ratios, concentrations, amounts, and other numerical data may be presented herein in a range format. It is to be understood that such range format is used merely for convenience and brevity and should be interpreted flexibly to include not only the numerical values explicitlyrecited as the limits of the range, but also to include all the individual numerical values or subranges encompassed within that range as if each numerical value and sub-range is explicitly recited.

[0073] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the disclosure, the preferred methods, and materials are now described. All publications mentioned herein are incorporated herein by reference.

[0074] The preferred embodiments of the present invention are further described and illustrated in the Figures and Examples of the present application. All aspects disclosed in the Figures or the examples, respectively, relate to the present invention unless expressly excluded. The single features of the present invention as disclosed in the experimental part can be combined unless there are technical reasons which speak against such combination.

[0075] Accordingly, the chimeric antigen receptors (CARs) of the present disclosure have been designed for adoptive immunotherapy by connecting an extracellular antigen-binding domain to a transmembrane domain and an intracellular signaling domain (endodomain). The inventors have designed a novel anti-tumor approach to eradicate tumor cells by adoptive transfer of T cells expressing chimeric antigen receptors to recognize specific antigens presented on tumor cells and activate T cells to specifically lyse these tumor cells. A critical aspect of this CAR strategy is the selection of target epitopes that are specifically or selectively expressed on tumors, are present on tumor cells, and are membrane epitopes not prone to shed or modulate from the cell surface.

[0076] Therefore, the inventors of the present disclosure have developed novel constructs encoding chimeric antigen receptors for expression in T cells, the chimeric antigen receptors comprising an antigen binding domain which binds specifically to prostate cancer antigens. These CAR-T cells are specific to certain tumor antigens and are involved in the treatment of prostate cancer.

[0077] Embodiments herein provide a recombinant construct. The recombinant construct, as disclosed herein, comprises a first polynucleotide and second polynucleotide. In an embodiment, the recombinant construct is a lentiviral construct. In some embodiments, the recombinant construct is a lentiviral construct comprising heterologous polynucleotides having heterologous nucleic acid sequences. In an embodiment, the first polynucleotide and secondpolynucleotide are heterologous polynucleotides.

[0078] In some embodiments, the present disclosure provides a novel genetically modified lentiviral construct comprising heterologous nucleic acid sequence.

[0079] In some embodiments, the heterologous nucleic acid sequence comprises a polynucleotide sequence encoding a 1stpolypeptide sequence of a CAR specific to one or more prostate cancer antigens and / or a 2ndsecond polypeptide sequence of a neutralizing antigenbinding fragment.

[0080] In some embodiments, the first polynucleotide and second polynucleotide may encode a 1stpolypeptide sequence and 2ndpolypeptide sequence, respectively. In various embodiments herein, the first polynucleotide encodes a 1stpolypeptide sequence of a CAR specific to one or more prostate cancer antigens (also referred to herein as PSMA specific CAR); and the second polynucleotide encodes a 2ndpolypeptide sequence of a neutralizing antigen-binding fragment.

[0081] In an embodiment, the recombinant construct comprises: (a) a first polynucleotide comprising a nucleotide fragment encoding a prostate-specific membrane antigen (PSMA) specific CAR; and (b) a second polynucleotide comprises a nucleotide fragment encoding a neutralizing antigen-binding fragment specific for interleukin-8 (IL-8). In an embodiment, the recombinant construct comprises: (a) a first polynucleotide comprising a nucleotide fragment encoding a prostate-specific membrane antigen (PSMA) specific CAR capable of binding to PSMA; and (b) a second polynucleotide comprises a nucleotide fragment encoding a neutralizing antigen-binding fragment capable of binding to interleukin-8 (IL-8). The first polynucleotide, according to embodiments herein, may be positioned upstream or downstream to the second polynucleotide. In an embodiment, the first polynucleotide is positioned upstream to the second polynucleotide. The recombinant construct, according to embodiments herein may encode a first generation, second generation, or third generation CAR. Embodiments herein achieve a fourth generation CAR. In an embodiment, the recombinant construct is for expressing PSMA specific CAR and a neutralizing antigen-binding fragment.

[0082] The term “neutralizing antigen-binding fragment” as used herein refers to a polypeptide capable of binding to an antigen to neutralize the antigen. In an embodiment, the neutralizing antigen-binding fragment is selected from a single chain variable fragment (scFv), Fab fragment , Fab’ fragment, F(ab‘)2 fragment, Fv fragment, or antibody.

[0083] In an embodiment, there is provided a recombinant construct comprising: (a) a firstpolynucleotide encoding a prostate-specific membrane antigen (PSMA) specific CAR; and (b) a second polynucleotide encoding a neutralizing antigen-binding fragment specific for interleukin-8 (IL-8), wherein the PSMA specific CAR comprises an antigen binding domain having at least 90% sequence identity to a sequence as depicted in SEQ ID NO. 1.

[0084] The term “antigen binding domain” as used herein refers to a polypeptide capable of binding to an antigen. In an embodiment, the antigen binding domain is selected from a single chain variable fragment (scFv), Fab fragment , Fab’ fragment, F(ab‘)2 fragment, or Fv fragment. In an embodiment, the antigen binding domain is an scFv fragment. In an embodiment, the antigen binding domain is an scFv fragment of a CAR. In an embodiment, the antigen binding domain is a scFv fragment of a PSMA specific CAR.

[0085] In an embodiment, there is provided a recombinant construct comprising: (a) a first polynucleotide encoding a prostate-specific membrane antigen (PSMA) specific CAR; and (b) a second polynucleotide encoding a neutralizing antigen-binding fragment specific for interleukin-8 (IL-8), wherein the neutralizing antigen-binding fragment comprises a polypeptide having at least 90% sequence identity to a sequence as depicted in SEQ ID NO. 3.

[0086] In some embodiments, the lentiviral vector further comprises a packaging signal (psi or ) or a cPPT / CTS sequence (Central Polypurine Tract / Central Termination sequence). The lentiviral construct may be suitable for insertion into a lentiviral vector.

[0087] In an embodiment, the recombinant construct comprises a nucleotide fragment encoding packaging signal (psi or ) or a cPPT / CTS to facilitate viral particle assembly and transduction efficiency. The nucleotide fragment encoding the packaging signal or the cPPT / CTS, in an embodiment, is positioned upstream to the polynucleotide encoding PSMA specific CAR.

[0088] In some embodiments, the lentiviral vector further comprises one or more restriction sites positioned between elements of said vector.

[0089] In some embodiments, the lentiviral vector further comprising a post- transcriptional regulatory element (PRE) selected from but not limited to a woodchuck hepatitis virus PRE (WPRE) or a hepatitis B virus isolate bba6 PRE (HPRE).

[0090] In some embodiments, the promoter is selected from, but not limited to, EFl alpha promoter, truncated EFl alpha promoter, MNDU promoter, or MNDU3 promoter. In a nonlimiting embodiment, the lentiviral vector comprises an EFla promoter.

[0091] The recombinant construct, according to embodiments herein, may further comprise one or more promoters operably linked to the first polynucleotide, second polynucleotide, or both. In an embodiment, there is provided a recombinant construct comprising a promoter, wherein the promoter is operably linked to the first polynucleotide. In an embodiment, the promoter has a nucleotide sequence selected from SEQ ID NO. 12 or SEQ ID NO. 13. The second polynucleotide, according to some embodiments herein, may be positioned downstream to the first polynucleotide operably linked to the promoter.

[0092] In an embodiment, the recombinant construct may comprise a nucleotide fragment encoding a 2A element. The term “2A element” refers to a small viral peptide that facilitate the production of multiple proteins from a single mRNA molecule by causing the ribosome to skip during translation. In an embodiment, there is provided a recombinant construct, wherein the second polynucleotide comprises a nucleotide fragment encoding a 2A element operably linked upstream to the nucleotide fragment encoding the neutralizing antigen-binding fragment.

[0093] In some embodiments, the recombinant construct may comprise the nucleotide fragment encoding a 2A element positioned between the first polynucleotide and second polynucleotide, to facilitate ribosome skipping and achieve a polycistronic mRNA. The term “polycistronic” mRNA refers to a single messenger RNA that comprises two or more coding sequences (i.e., cistrons) and encodes more than one protein.

[0094] In some embodiments, the recombinant construct may comprise the nucleotide fragment encoding a 2A element positioned upstream to the nucleotide fragment encoding the neutralizing antigen-binding fragment, and downstream to the nucleotide fragment encoding the PSMA specific CAR. Examples of 2A element that may be used in various embodiments herein include, but are not limited to, T2A (Thosea asigna virus 2A) element, P2A (porcine teschovirus-1 2A) element, E2A (equine rhinitis A virus) element, or F2A (foot-and-mouth disease virus) element. In an embodiment, the 2A element is a P2A element. In an embodiment, the nucleotide fragment encoding a P2A is operably linked upstream to the nucleotide fragment encoding the neutralizing antigen-binding fragment, wherein the P2A element has a sequence as set forth in SEQ ID NO. 22 or wherein the nucleotide fragment encoding the P2A element has a sequence as set forth in SEQ ID NO. 23.

[0095] The first polynucleotide, according to some embodiments herein, may be linked to a nucleotide fragment encoding a signal peptide. The second polynucleotide, according to some embodiments herein, may be linked to a nucleotide fragment encoding a signal peptide.Accordingly, the recombinant construct, in some embodiments, may comprise one or more nucleotide fragments encoding a signal or signal peptide. Signal or signal peptide, according to embodiments herein, refers to peptides that direct a nascent protein for folding, processing, and / or subsequent secretion from a cell. In an embodiment, the nucleotide fragment encoding the neutralizing antigen-binding fragment is linked to a nucleotide fragment encoding a signal peptide, wherein the nucleotide fragment encoding a signal peptide has a nucleotide sequence as set forth in SEQ ID NO. 25, and the nucleotide fragment encoding the PSMA specific CAR is linked to a nucleotide fragment encoding a signal peptide, wherein the nucleotide fragment encoding a signal peptide has a nucleotide sequence as set forth in SEQ ID NO. 14.

[0096] In an embodiment, there is provided a recombinant construct comprising a first polynucleotide and a second polynucleotide, wherein the first polynucleotide comprises a nucleotide fragment encoding a signal peptide, and wherein the second polynucleotide comprises a nucleotide fragment encoding a signal peptide.

[0097] In an embodiment, there is provided a recombinant construct, wherein the second polynucleotide comprises a nucleotide fragment encoding a GM-CSF signal operably linked upstream to the nucleotide fragment encoding the neutralizing antigen-binding fragment, wherein the GM-CSF signal has an amino acid sequence as set forth in SEQ ID NO. 24, or wherein the nucleotide fragment encoding the GM-CSF signal has a sequence as set forth in SEQ ID NO. 25.

[0098] In an embodiment, there is provided a recombinant construct, wherein the second polynucleotide comprises a nucleotide fragment encoding a signal sequence and a nucleotide fragment encoding a 2A element operably linked upstream to the second polynucleotide.

[0099] In a non-limiting embodiment, the lentiviral components of said lentiviral vector originate from HIV-1.

[0100] In a non-limiting embodiment, the heterologous nucleic acid sequence is downstream of a Kozak sequence. In an embodiment, the recombinant construct comprises a Kozak sequence such that the first polynucleotide is downstream of a Kozak sequence.

[0101] In some embodiments, the expression of the heterologous sequence is under the control of a subgenomic promoter selected from the group consisting of a CMV promoter, an EFl alpha promoter, and an RSV promoter.

[0102] In some embodiments, said heterologous nucleic acid sequence encodes a 1stpolypeptide sequence encoding a chimeric antigen receptor (CAR) and a 2ndpolypeptide sequence encoding a neutralizing antigen-binding fragment.

[0103] In some embodiments, said CAR comprises, in a N-terminal to C-terminal direction, an antigen binding domain, a transmembrane domain, and one or more signaling domains.

[0104] In some embodiments, said signaling domain comprises one or more primary signaling domains, wherein said signaling domains comprise one or more costimulatory signaling domains.

[0105] In some embodiments, one of said one or more primary signaling domains comprises a CD3-zeta stimulatory domain, wherein one or more of said costimulatory signaling domains comprises an intracellular domain from a protein selected from the group consisting of CD27, CD28, 4-1BB (CD137), 0X40, GITR, CD30, CD40, ICOS, BAFFR, HVEM, ICAM-1, lymphocyte function-associated antigen-1 (LFA-1), CD2, CDS, CD7, CD287, LIGHT, NKG2C, NKG2D, SLAMF7, NKp80, NKp30, NKp44, NKp46, CD 160, B7-H3, and a ligand that specifically binds with CD83.

[0106] In some embodiments, said one or more of said costimulatory signaling domains comprises the 4-lBB(CD137) costimulatory domain, wherein one or more of said costimulatory domains comprises the CD28 costimulatory domain.

[0107] In an embodiment, the CAR is a PSMA specific CAR. In an embodiment, the CAR comprises an antigen binding domain. In a non-limiting embodiment, said antigen binding domain is an scFv.

[0108] In an embodiment, the PSMA specific CAR comprises an antigen binding domain having at least 90% sequence identity to a sequence as set forth in SEQ ID NO. 1. In an embodiment, the PSMA specific CAR comprises a polypeptide having at least 90% sequence identity to a sequence as set forth in SEQ ID NO. 1.

[0109] In some embodiments, said antigen binding domain binds to one or more prostate specific antigens selected from but not limited to PSMA (prostate-specific membrane antigen), PSA (Prostate specific antigen), PAP (prostate acid phosphatase), PSCA (prostate stem cell antigen), EpCaM (epithelial cell adhesion molecule), B7-H3,Muc-l (Mucin 1), CD126, Lewis Y antigen, STEAP1 (six transmembrane epithelial antigen of prostate type 1), STEAP4 (six transmembrane epithelial antigen of prostate type 4).

[0110] In a non-limiting embodiment, the antigen binding domain is PSMA scFv, wherein the antigen binding domain has at least 90% sequence identity to a sequence as set forth in SEQ ID NO. 1. In an embodiment, the CAR is a PSMA specific CAR.

[0111] The PSMA specific CAR, according to embodiments herein, may be selected from a first generation CAR, a second generation CAR, or a third generation. In an embodiment the PSMA specific CAR is a second generation CAR. In another embodiment, the PSMA specific CAR is a third generation CAR. In an embodiment, the PSMA specific CAR comprises a polypeptide having at least 90% sequence identity to a sequence selected from SEQ ID NO. 7, or SEQ ID NO. 5. Accordingly, in an embodiment, there is provided a recombinant construct comprising (a) a first polynucleotide comprising a nucleotide fragment encoding a prostatespecific membrane antigen (PSMA) specific chimeric antigen receptor (CAR); and (b) a second polynucleotide comprising a nucleotide fragment encoding a neutralizing antigen-binding fragment specific for interleukin-8 (IL-8), wherein the PSMA specific CAR comprises a polypeptide having at least 90% sequence identity to a sequence selected from SEQ ID NO. 7, or SEQ ID NO. 5.

[0112] In an embodiment, the PSMA specific CAR comprises a polypeptide having at least 90% sequence identity to a sequence selected from SEQ ID NO. 5, SEQ ID NO. 7, or SEQ ID NO. 9.

[0113] In an embodiment, there is provided a recombinant construct comprising (a) a first polynucleotide comprising a nucleotide fragment encoding a prostate-specific membrane antigen (PSMA) specific chimeric antigen receptor (CAR); and (b) a second polynucleotide comprising a nucleotide fragment encoding a neutralizing antigen-binding fragment specific for interleukin-8 (IL-8), wherein the PSMA specific CAR comprises a hinge and transmembrane domain derived from CD8; a co-stimulatory domain selected from CD-28 co-stimulatory domain, 4 IBB co-stimulatory domain, or combination thereof; and CD3zeta signaling domain.

[0114] In an embodiment, there is provided a recombinant construct comprising (a) a first polynucleotide comprising a nucleotide fragment encoding a prostate-specific membrane antigen (PSMA) specific chimeric antigen receptor (CAR); and (b) a second polynucleotide comprising a nucleotide fragment encoding a neutralizing antigen-binding fragment specific for interleukin-8 (IL-8), wherein the PSMA specific CAR comprises a polypeptide having at least 90% sequence identity to a sequence selected from SEQ ID NO. 1, SEQ ID NO. 5, SEQ ID NO. 7, or SEQ ID NO. 9, or wherein the neutralizing antigen-binding fragment comprises apolypeptide having at least 90% sequence identity to a sequence as set forth in SEQ ID NO. 3, or SEQ ID NO. 20.

[0115] In an embodiment, there is provided a recombinant construct comprising (a) a first polynucleotide comprising a nucleotide fragment encoding a prostate-specific membrane antigen (PSMA) specific chimeric antigen receptor (CAR); and (b) a second polynucleotide comprising a nucleotide fragment encoding a neutralizing antigen-binding fragment specific for interleukin-8 (IL-8), wherein the PSMA specific CAR comprises a polypeptide having at least 90% sequence identity to a sequence selected from SEQ ID NO. 5, SEQ ID NO. 7, or SEQ ID NO. 9, or wherein the neutralizing antigen-binding fragment comprises a polypeptide having at least 90% sequence identity to a sequence as set forth in SEQ ID NO. 20.

[0116] In an embodiment, there is provided a recombinant construct comprising (a) a first polynucleotide comprising a nucleotide fragment encoding a prostate-specific membrane antigen (PSMA) specific chimeric antigen receptor (CAR); and (b) a second polynucleotide comprising a nucleotide fragment encoding a neutralizing antigen-binding fragment specific for interleukin-8 (IL-8), wherein the first polynucleotide comprises a nucleotide fragment having at least 80% sequence identity to a sequence selected from SEQ ID NO. 2, SEQ ID NO. 6, SEQ ID NO. 8, SEQ ID NO. 10, or SEQ ID NO. 11.

[0117] In an embodiment, there is provided a recombinant construct comprising (a) a first polynucleotide comprising a nucleotide fragment encoding a prostate-specific membrane antigen (PSMA) specific chimeric antigen receptor (CAR); and (b) a second polynucleotide comprising a nucleotide fragment encoding a neutralizing antigen-binding fragment specific for interleukin-8 (IL-8), wherein the second polynucleotide comprises a nucleotide fragment having at least 80% sequence identity to a sequence selected from SEQ ID NO. 4, or SEQ ID NO. 21.

[0118] In an embodiment, the recombinant construct comprises a nucleotide fragment having at least 80% sequence identity to a sequence selected from SEQ ID NO. 26. In an embodiment, the recombinant construct encodes a polypeptide having at least 90% sequence identity to a sequence selected from SEQ ID NO. 28.

[0119] Embodiments herein provide a vector. In an embodiment, the vector comprises the recombinant construct as disclosed herein. In an embodiment, there is provided a vector comprising the recombinant construct having (a) a first polynucleotide comprising a nucleotide fragment encoding a prostate-specific membrane antigen (PSMA) specific chimeric antigen receptor (CAR); and (b) a second polynucleotide comprising a nucleotide fragment encoding aneutralizing antigen-binding fragment specific for interleukin-8 (IL-8).

[0120] In an embodiment, the vector is a lentiviral vector. In some embodiments, the lentiviral vector comprises, from 5‘ to 3‘, one or more of the following elements in operable association:(a) a promoter to drive the expression of viral genes,(b) a packaging signal (psi) comprising a major splice donor site (SD),(c) a partial gag sequence consisting of 150-250 nucleotides,(d) a partial env sequence;(e) a Rev-response element (RRE),(f) a partial env sequence comprising splice acceptor site (SA7);(g) a central polypurine tract (cPPT) comprising a splice acceptor site (SAI),(h) a subgenomic promoter,(i) a heterologous nucleic acid sequence encoding a 1stpolypeptide sequence encoding a CAR and a 2ndpolypeptide sequence encoding a neutralizing antigen-binding fragment, and(j) a post-transcriptional regulatory element, which optionally comprises a woodchuck hepatitis virus PRE (WPRE) or a hepatitis B virus isolate bba6 PRE (HPRE).

[0121] In some preferred embodiments, the present disclosure provides a lentiviral vector comprising, from 5‘ to 3‘, one or more of the following elements in operable association:(a) a CMV promoter,(b) an LTR region,(c) an LTR U5 region,(d) a primer binding site (PBS),(e) a packaging signal (psi) comprising a major splice donor site (SD),(f) a partial gag sequence consisting of 150-250 nucleotides,(g) a partial env sequence consisting of a sequence,(h) a Rev-response element (RRE),(i) a partial env sequence comprising splice acceptor site (SA7),(j) a central polypurine tract (cPPT) comprising a splice acceptor site (SAI),(k) an EF 1 a promoter,(l) a heterologous nucleic acid sequence encoding a 1stpolypeptide sequence encoding a CAR and a 2ndpolypeptide sequence encoding a neutralizing antigen-binding fragment,(m) a post-transcriptional regulatory element, which optionally comprises a woodchuck hepatitis virus PRE (WPRE) or a hepatitis B virus isolate bba6 PRE (HPRE),(n) an LTR R region,(o) an LTR U5 region,(p) an SV40 polyA tail,(q) an ampicillin resistance gene (nptll), and(r) a pUC origin of replication.

[0122] In some embodiments, the lentiviral vector comprising a heterologous nucleic acid sequence comprising from 5’ to 3’ direction: i. 1stpolypeptide sequence encoding a CAR comprising the 1stsingle-chain variable fragment (scFv) targeting prostate cancer tumor antigens; and ii. polypeptide sequence encoding a neutralizing antigen-binding fragment.

[0123] In a preferred embodiment, the CAR encoded by the lentiviral construct comprises:(a) an extracellular domain comprising an antigen binding region;(b) a transmembrane domain;(c) one or more co-stimulatory domain; and(d) a CD3 zeta signaling domain.

[0124] In some embodiments, the 1stextracellular domain comprises an antigen-binding domain comprising a polypeptide sequence encoding the variable heavy and light chain regions targeting one or more tumor antigens overexpressed in prostate cancer.

[0125] In some embodiments, the antigen binding domain is specific to PSMA (prostatespecific membrane antigen), PSA (Prostate specific antigen), PAP (prostate acid phosphatase), PSCA (prostate stem cell antigen), EpCaM (epithelial cell adhesion molecule),B7-H3,Muc- l(Mucin 1), CD126, Lewis Y antigen, STEAP1 (six transmembrane epithelial antigen of prostate type 1), STEAP4 (six transmembrane epithelial antigen of prostate type 4).

[0126] In a non-limiting embodiment, the antigen binding domain is specific to PSMA.

[0127] In another non-limiting embodiment, the extracellular antigen-binding domain comprises a linker polypeptide positioned between the VH and VL domains.

[0128] In a non-limiting embodiment, the 1sttransmembrane domain comprises a transmembrane region derived from CD8.

[0129] In a non-limiting embodiment, the co-stimulatory domain comprises of CD-28 signaling domain and a 4 IBB signaling domain or both.

[0130] In some embodiments, the polypeptide comprising an amino acid sequence encoding the variable heavy and light chain regions of neutralizing antigen-binding fragment specific for at least one growth factor, cytokine, or interleukin associated with angiogenesis or vasculogenesis.

[0131] Examples of the neutralizing antigen-binding fragment include, but are not limited to, polypeptides capable of binding to vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), platelet- derived growth factor (PDGF), hepatocyte growth factor (HGF), insulin-like growth factor (IGF), or interleukin-8 (IL-8).

[0132] In a non-limiting embodiment, the neutralizing antigen-binding fragment is specific for binding to IL-8. In an embodiment, the neutralizing antigen-binding fragment is selected from a single chain variable fragment (scFv), Fab fragment, Fab’ fragment, F(ab‘)2 fragment, Fv fragment, or antibody specific for binding to IL-8. In an embodiment, the neutralizing antigenbinding fragment is an scFv fragment specific for IL-8 (also referred to herein as “IL8 scFv” or “IL-8 scFv”).

[0133] In some embodiments, the present disclosure provides a genetically modified lentiviralconstruct comprising a heterologous nucleic acid sequence encoding a polypeptide sequence encoding a CAR comprising from 5’ to 3’ direction: i. an extracellular domain linked, ii. a transmembrane domain, and iii. co-stimulatory domain; and iv. CD3 zeta signaling domain.

[0134] In some embodiments, the extracellular domain is an antigen-binding domain comprising a polypeptide sequence encoding one or more variable heavy and light chain regions targeting one or more prostate cancer antigens, wherein this antigen binding domain is linked via a linker sequence to another polypeptide sequence encoding one or more neutralizing antigen-binding fragments specific for growth factor, cytokine, or interleukin associated with angiogenesis or vasculogenesis.

[0135] In a non-limiting embodiment, the linker sequence is a glycine- serine (GS) sequence.

[0136] In some embodiments, the one or more tumor antigens are PSMA (prostate-specific membrane antigen), PSA (Prostate specific antigen), PAP (prostate acid phosphatase), PSCA (prostate stem cell antigen), EpCaM (epithelial cell adhesion molecule), B7-H3,Muc-l (Mucin 1), CD126, Lewis Y antigen, STEAP1 (six transmembrane epithelial antigen of prostate type 1), STEAP4 (six transmembrane epithelial antigen of prostate type 4).

[0137] In some embodiments, the neutralizing antigen-binding fragment is selected from, but not limited to, a fragment capable of binding to or is specific for vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), platelet- derived growth factor (PDGF), hepatocyte growth factor (HGF), insulin-like growth factor (IGF), or interleukin-8 (IL-8).

[0138] In a non-limiting embodiment, antigen binding domain is specific for PSMA and the neutralizing antigen-binding fragment is specific for IL-8.

[0139] In some embodiments, the extracellular antigen-binding domain comprises a linker polypeptide positioned between the VH and VL domains.

[0140] In some embodiments, the transmembrane domain comprises a transmembrane region derived from CD 8.

[0141] In some embodiments, the co-stimulatory domain comprises a CD-28 signaling domain and a 4 IBB signaling domain.

[0142] In some embodiments, the present disclosure provides a genetically modified lentiviral construct comprising a heterologous nucleic acid sequence encoding a polypeptide sequence comprising from 5’ to 3’ direction: i. a chimeric antigen receptor specific to one or more prostate cancer antigens; and ii. a polypeptide sequence encoding a secretary antibody.

[0143] In some embodiments, the chimeric Antigen Receptor (CAR) construct specific to PSMA comprising a polypeptide sequence encoding a i. an extracellular domain, ii. a transmembrane domain, iii. one or more co-stimulatory domain(s); and iv. CD3 zeta signaling domain.

[0144] In some embodiments, the extracellular domain comprises an antigen-binding domain comprising a polypeptide sequence encoding the variable heavy and light chain regions specific to one or more prostate cancer antigens.

[0145] In some embodiments, the extracellular antigen-binding domain comprises a linker polypeptide positioned between the VH and VL domains.

[0146] In a non-limiting embodiment, the transmembrane domain comprises a transmembrane region derived from CD 8.

[0147] In a non-limiting embodiment, the co-stimulatory domain comprises a CD-28 signaling domain and 4 IBB signaling domain.

[0148] In some embodiments, the neutralizing antigen-binding fragment is specific to vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), platelet- derived growth factor (PDGF), hepatocyte growth factor (HGF), insulin-like growth factor (IGF), or interleukin-8 (IL-8).

[0149] In an embodiment, the neutralizing antigen-binding fragment is an IL-8 scFv. In another embodiment, the neutralizing antigen-binding fragment is a secretory antibody fragment. In a non-limiting embodiment, the secretory antibody is IL-8 antibody.

[0150] In an embodiment, there is provided a vector comprising the recombinant construct having (a) a first polynucleotide comprising a nucleotide fragment encoding a prostate-specificmembrane antigen (PSMA) specific chimeric antigen receptor (CAR); and (b) a second polynucleotide comprising a nucleotide fragment encoding a neutralizing antigen-binding fragment specific for interleukin-8 (IL-8), wherein the PSMA specific CAR comprises a polypeptide having at least 90% sequence identity to a sequence selected from SEQ ID NO. 5, SEQ ID NO. 7, or SEQ ID NO. 9, or wherein the neutralizing antigen-binding fragment comprises a polypeptide having at least 90% sequence identity to a sequence as set forth in SEQ ID NO. 20.

[0151] In an embodiment, there is provided a vector comprising the recombinant construct having (a) a first polynucleotide comprising a nucleotide fragment encoding a prostate-specific membrane antigen (PSMA) specific chimeric antigen receptor (CAR); and (b) a second polynucleotide comprising a nucleotide fragment encoding a neutralizing antigen-binding fragment specific for interleukin-8 (IL-8), wherein the first polynucleotide comprises a nucleotide fragment having at least 80% sequence identity to a sequence selected from SEQ ID NO. 2, SEQ ID NO. 6, SEQ ID NO. 8, SEQ ID NO. 10, or SEQ ID NO. 11, wherein the second polynucleotide comprises a nucleotide fragment having at least 80% sequence identity to a sequence selected from SEQ ID NO. 4, or SEQ ID NO. 21.

[0152] In an embodiment, there is provided a vector, preferably a lentiviral vector, comprising the recombinant construct, wherein the recombinant construct comprises a nucleotide fragment having at least 80% sequence identity to a sequence selected from SEQ ID NO. 26, or wherein the recombinant construct encodes a polypeptide having at least 90% sequence identity to a sequence selected from SEQ ID NO. 28.

[0153] Embodiments herein provide a host cell comprising the vector or the recombinant construct as disclosed herein. In some embodiments, the present disclosure provides a host cell comprising the vector, wherein the host cell is a 293T cell, a Jurkat T cell, or a primary human T cell. In an embodiment, the host cell is an engineered cell.

[0154] Embodiments herein provide an engineered cell or population thereof. In an embodiment, the engineered cell or population thereof comprises the vector or the recombinant construct as disclosed herein. In an engineered cell or population thereof, the engineered cell is selected from an immune cell, natural killer cell, or induced pluripotent cell (iPSC). The recombinant construct, according to embodiments herein, may be transfected into the cell to achieve expression or co-expression of the PSMA specific CAR and the neutralizing antigenbinding fragment. In some embodiments, the PSMA specific CAR and the neutralizing antigen-binding fragment may be secreted by the engineered cell, or population thereof.

[0155] In an embodiment, the immune cell is a T-cell selected from T-helper cells (CD4+ cells), cytotoxic T- cells (CD8+ cells), natural killer T-cells, T-regulatory cells (Treg), and gamma-delta T cells. Accordingly, in an embodiment, there is provided an engineered cell or population thereof, wherein the engineered cell is selected from an immune cell, preferably T- cell; natural killer cell; or induced pluripotent cell (iPSC), wherein the T-cell is selected from T-helper cells (CD4+ cells), cytotoxic T- cells (CD8+ cells), natural killer T-cells, T-regulatory cells (Treg), and gamma-delta T cells.

[0156] In an embodiment, the engineered cell or population thereof comprises the recombinant construct comprising (a) a first polynucleotide comprising a nucleotide fragment encoding a prostate-specific membrane antigen (PSMA) specific chimeric antigen receptor (CAR); and (b) a second polynucleotide comprising a nucleotide fragment encoding a neutralizing antigenbinding fragment specific for interleukin-8 (IL-8), wherein the PSMA specific CAR comprises a polypeptide having at least 90% sequence identity to a sequence selected from SEQ ID NO. 1, SEQ ID NO. 5, SEQ ID NO. 7, or SEQ ID NO. 9, or wherein the neutralizing antigen-binding fragment comprises a polypeptide having at least 90% sequence identity to a sequence selected from SEQ ID NO. 3 or SEQ ID NO. 20.

[0157] In an embodiment, the engineered cell or population thereof expressing the recombinant construct comprising (a) a first polynucleotide comprising a nucleotide fragment encoding a prostate-specific membrane antigen (PSMA) specific chimeric antigen receptor (CAR); and (b) a second polynucleotide comprising a nucleotide fragment encoding a neutralizing antigen-binding fragment specific for interleukin-8 (IL-8), wherein the PSMA specific CAR comprises a polypeptide having at least 90% sequence identity to a sequence selected from SEQ ID NO. 1, SEQ ID NO. 5, SEQ ID NO. 7, or SEQ ID NO. 9, or wherein the neutralizing antigen-binding fragment comprises a polypeptide having at least 90% sequence identity to a sequence selected from SEQ ID NO. 3 or SEQ ID NO. 20.

[0158] In an embodiment, the engineered cell or population thereof expresses the PSMA specific CAR and the neutralizing antigen-binding fragment as described herein.

[0159] In an embodiment, the engineered cell or population thereof comprises the recombinant construct comprising (a) a first polynucleotide comprising a nucleotide fragment encoding a prostate-specific membrane antigen (PSMA) specific chimeric antigen receptor (CAR); and (b) a second polynucleotide comprising a nucleotide fragment encoding a neutralizing antigen-binding fragment specific for interleukin-8 (IL-8), wherein the first polynucleotide comprises a nucleotide fragment having at least 80% sequence identity to a sequence selected from SEQ ID NO. 2, SEQ ID NO. 6, SEQ ID NO. 8, SEQ ID NO. 10, or SEQ ID NO. 11, wherein the second polynucleotide comprises a nucleotide fragment having at least 80% sequence identity to a sequence selected from SEQ ID NO. 4, or SEQ ID NO. 21.

[0160] The engineered cell or population thereof expressing the PSMA specific CAR and the neutralizing antigen-binding fragment is capable of killing target cells or increasing cytolysis of target cells. The term “target cell”, as used herein refers to a cell expressing a tumor or cancer antigen. In an embodiment, the tumor or cancer antigens is selected from PSMA (prostatespecific membrane antigen), PSA (Prostate specific antigen), PAP (prostate acid phosphatase), PSCA (prostate stem cell antigen), EpCaM (epithelial cell adhesion molecule), B7-H3, Muc- l(Mucin 1), CD126, Lewis Y antigen, STEAP1 (six transmembrane epithelial antigen of prostate type 1), and / or STEAP4 (six transmembrane epithelial antigen of prostate type 4). In an embodiment, the target cell is a tumor or cancer cell. In an embodiment, the target cell is a cell expressing PSMA. The engineered cell or population thereof, according to embodiments herein, expressing the PSMA specific CAR and the neutralizing antigen-binding fragment is capable of increasing cytolysis of target cell and neutralizing IL-8. In an embodiment, the engineered cell or population thereof expressing the PSMA specific CAR and the neutralizing antigen-binding fragment is capable of enhancing CAR-T cell function.

[0161] Embodiments herein achieve a pharmaceutical composition comprising the engineered cell or population thereof as disclosed herein. In an embodiment, there is provided a pharmaceutical composition comprising the engineered cell or population thereof, wherein the engineered cell or population thereof comprises the vector having the recombinant construct or the recombinant construct, wherein the recombinant construct comprises (a) a first polynucleotide comprising a nucleotide fragment encoding a prostate-specific membrane antigen (PSMA) specific chimeric antigen receptor (CAR); and (b) a second polynucleotide comprising a nucleotide fragment encoding a neutralizing antigen-binding fragment specific for interleukin-8 (IL-8).

[0162] In some embodiments, the present disclosure provides a method for the preparation of the modified chimeric antigen receptors construct, the method comprising the steps of: a. providing a T cell line; b. generating a polynucleotide encoding the modified CAR targeting the specific tumorantigens; c. introducing a polynucleotide that encodes a CAR into the T cell.

[0163] In some embodiments, the recombinant construct is introduced into natural killer cells (NK), invariant natural killer T-cells (iNKT), diverse natural killer cells (dNKT), cytokine- induced killer cells (CIK) or y-5 T-cells. In some preferred embodiments, allogenic cells may be used.

[0164] In some embodiments, there is provided a method, wherein generating a polynucleotide encoding the modified CAR targeting the specific tumor antigens further comprises introducing a polynucleotide that encodes a CAR into the T cell by electroporation or a viral-based gene transfer system.

[0165] In some embodiments, the viral-based gene transfer system comprises a retroviral vector, adenoviral vector, adeno-associated viral vector, or lentiviral vector.

[0166] In some embodiments, the present disclosure provides an in vitro method for generating a T-cell line that expresses the modified chimeric antigen receptors construct wherein the method comprises the steps: a. providing a T cell line; b. generating a polynucleotide encoding the modified CAR targeting the specific tumor antigens; c. introducing a polynucleotide that encodes a CAR into the T cell.

[0167] In some embodiments, the genetic information coding for the chimeric antigen receptor is introduced with the help of a suitable vector into the target immune cells (e.g. T cell). Such a vector can preferably be a lentivirus vector or a retroviral vector or a transposon or a plasmid. Alternatively, genetic information can be introduced into the genome of the T cell in a targeted fashion with the help of designer nuclease technology, such as the CRISPR / Cas technology or the TALEN technology, as described herein.

[0168] In some embodiments, there is provided a method, wherein generating a polynucleotide encoding the modified CAR targeting the specific tumor antigens further comprises introducing a polynucleotide that encodes a CAR into the T cell by electroporation or a viral-based gene transfer system.

[0169] In some embodiments, the viral-based gene transfer system comprises a retroviral vector, adenoviral vector, adeno-associated viral vector, or lentiviral vector. In some examples, a polynucleotide coding for the chimeric antigen receptor, is included in the same vector, such as, for example, a viral or plasmid vector, as a polynucleotide coding for a second polypeptide. This second polypeptide may be, for example, a chimeric signaling polypeptide, an inducible caspase polypeptide, as discussed herein, or a marker polypeptide. In other examples, the two polypeptides may be expressed separately from the same vector, where each nucleic acid comprising a polynucleotide coding for one of the polypeptides is operably linked to a separate promoter. In yet other examples, one promoter may be operably linked to the two polynucleotides, directing the production of two separate RNA transcripts, and thus two polypeptides; in one example, the promoter may be bi-directional, and the coding regions may be in opposite directions 5‘-3‘. Therefore, the expression construct discussed herein may comprise at least one, or at least two promoters.

[0170] In some embodiments, the present disclosure provides a nucleic acid encoding polynucleotide sequence expressing the chimeric antigen receptor.

[0171] In some embodiments, the nucleic acid is contained within a viral vector selected from the group consisting of but not limited to retroviral vector, adenoviral vector or a lentiviral vector.

[0172] It is understood that in some embodiments, a cell is contacted with the viral vector ex vivo, and in some embodiments, the cell is contacted with the viral vector in vivo. Thus, an expression construct may be inserted into a vector, for example a viral vector or plasmid. The steps of the methods provided may be performed using any suitable method; these methods include, without limitation, methods of transducing, transforming, or otherwise providing nucleic acid to the cell, described herein.

[0173] In some embodiments, the present disclosure provides a method of producing a lentivirus capable of expressing a heterologous nucleic acid sequence, said method comprising:(a) introducing into a cell:(i) the lentiviral vector comprising the heterologous nucleic acid sequence encoding a 1stpolypeptide sequence encoding a CAR and a 2ndpolypeptide sequence encoding a neutralizing antigen-binding fragment, and(ii) one or more lentiviral packaging vectors; and(b) expressing viral proteins encoded by said lentiviral vector and / or said packaging vector in said cell, thereby producing a lentivirus comprising the heterologous nucleic acid sequence of said lentiviral vector, wherein said cell is a 293T cell, a Jurkat T cell, or a primary human T cell.

[0174] In some embodiments, in addition to the CAR construct described herein, another transgene that modulates the immune system, such genes coding for cytokines, chemokine receptors and / or checkpoint inhibitors, may be introduced in the immune cells. In a further preferred embodiment, genome editing is used to disrupt the expression of genes that modulate the immune system, such as genes coding for cytokines, chemokine receptors and / or checkpoint inhibitors.

[0175] In some embodiments, the present disclosure provides a genetically modified T-cell line expressing a genetically modified chimeric antigen receptor construct (CAR-T cells).

[0176] In some embodiments, the modified T cells express a genetically modified lentiviral construct comprising a polypeptide sequence encoding chimeric antigen receptor construct.

[0177] In some embodiments, the present disclosure provides the modified T cells comprising a polypeptide sequence encoding one or more chimeric antigen receptor construct wherein: i. the 1stCAR construct comprises the 1stsingle-chain variable fragment (scFv) targeting prostate cancer tumor antigens and ii. a 2ndCAR construct comprises the 2ndsingle-chain variable fragment (scFv) targeting growth factor, cytokine, or interleukin associated with angiogenesis or vasculogenesis.

[0178] In some embodiments, the 1stCAR construct encoded by the lentiviral construct comprise:(a) the 1stextracellular domain comprising an antigen binding region;(b) the 1sttransmembrane domain;(c) the 1stintracellular signaling domain comprising the co-stimulatory domain and a T cell activation domain.

[0179] In some embodiments, the 1stextracellular domain comprises an antigen-bindingdomain comprising a polypeptide sequence encoding the variable heavy and light chain regions targeting one or more tumor antigens overexpressed in prostate cancer.

[0180] In some embodiments, the antigen binding domain is specific to PSMA (prostatespecific membrane antigen), PSA (Prostate specific antigen), PAP (prostate acid phosphatase), PSCA (prostate stem cell antigen), EpCaM (epithelial cell adhesion molecule),B7-H3,Muc- l(Mucin 1), CD126, Lewis Y antigen, STEAP1 (six transmembrane epithelial antigen of prostate type 1), STEAP4 (six transmembrane epithelial antigen of prostate type 4).

[0181] In a non-limiting embodiment, the antigen binding domain is specific to PSMA.

[0182] In some embodiments, the extracellular antigen-binding domain comprises a linker polypeptide positioned between the VH and VL domains.

[0183] In a non-limiting embodiment, the 1sttransmembrane domain comprises a transmembrane region derived from CD8.

[0184] In a non-limiting embodiment, the 1stco-stimulatory domain comprises a CST-1 signaling domain and a CD-28 signaling domain.

[0185] In some embodiments, the 1stco-stimulatory domain comprises a CST-2 signaling domain and a 4 IBB signaling domain.

[0186] In some embodiments, the 1stT cell activating domain comprises CS and CD3 zeta signaling domain.

[0187] In some embodiments, the 2ndCAR construct encoded by the lentiviral construct comprising:(a) the 2ndextracellular domain comprising an antigen binding region;(b) the 2ndtransmembrane domain;(c) the 2ndintracellular signaling domain(s) comprising the co-stimulatory domain and a T cell activation domain.

[0188] In some embodiments, the 2ndextracellular domain comprises an antigen-binding domain comprising a polypeptide sequence encoding the variable heavy and light chain regions targeting growth factor, cytokine, or interleukin associated with angiogenesis or vasculogenesis.

[0189] In some embodiments, the second antigen is selected from but not limited to vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF), hepatocyte growth factor (HGF), insulin-like growth factor (IGF), or interleukin-8 (IL-8).

[0190] In a non-limiting embodiment, the scFv is specific to second antigen which is IL-8.

[0191] In some embodiments, the extracellular antigen-binding domain comprises a linker polypeptide positioned between the VH and VL domains.

[0192] In a non-limiting embodiment, the 2ndtransmembrane domain comprises a transmembrane region derived from CD8.

[0193] In a non-limiting embodiment, the 2ndco-stimulatory domain comprises a CST-1 signaling domain and a CD-28 signaling domain.

[0194] In a non-limiting embodiment, the 2ndco-stimulatory domain comprises a CST-2 signaling domain and a 4 IBB signaling domain.

[0195] In some embodiments, the 2ndT cell activating domain comprises CS and CD3 zeta signaling domain.

[0196] In some embodiments, the present disclosure provides the modified T cells expressing a genetically modified lentiviral construct comprising a polypeptide sequence encoding a CAR construct comprising: i. an extracellular domain, ii. a transmembrane domain, and iii. an intracellular signaling domain comprising the co-stimulatory domain(s) and a T cell activation domain.

[0197] In some embodiments, the extracellular domain comprises: i. a 1stantigen-binding domain comprising a polypeptide sequence encoding one or more variable heavy and light chain regions targeting one or more tumor antigens overexpressed in prostate cancer; and ii. a 2ndantigen-binding domain comprising a polypeptide sequence encoding one or more variable heavy and light chain regions targeting a second antigen selected from but not limited to growth factor, cytokine, or interleukin associatedwith angiogenesis or vasculogenesis; wherein at least two antigen binding domains are connected via a polypeptide sequence encoding a linker sequence.

[0198] In a non-limiting embodiment, the linker sequence is a glycine- serine (GS) sequence.

[0199] In some embodiments, the one or more tumor antigens are PSMA (prostate-specific membrane antigen), PSA (Prostate specific antigen), PAP (prostate acid phosphatase), PSCA (prostate stem cell antigen), EpCaM (epithelial cell adhesion molecule), B7-H3,Muc-l (Mucin 1), CD126, Lewis Y antigen, STEAP1 (six transmembrane epithelial antigen of prostate type 1), STEAP4 (six transmembrane epithelial antigen of prostate type 4).

[0200] In some embodiments, the second antigen is selected from but not limited to vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF), hepatocyte growth factor (HGF), insulin-like growth factor (IGF), or interleukin-8 (IL-8).

[0201] In a non-limiting embodiment, 1stantigen binding domain is specific for PSMA and the 2ndantigen binding domain is specific to IL-8.

[0202] In some embodiments, the extracellular antigen-binding domain comprises a linker polypeptide positioned between the VH and VL domains.

[0203] In a non-limiting embodiment, the transmembrane domain comprises a transmembrane region derived from CD 8.

[0204] In non-limiting embodiment, the co-stimulatory domain comprises a CST-1 signaling domain and a CD-28 signaling domain.

[0205] In a non-limiting embodiment, the co-stimulatory domain comprises a CST-2 signaling domain and a 4 IBB signaling domain.

[0206] In a non-limiting embodiment, the T cell activating domain comprises CS and CD3 zeta signaling domain.

[0207] In some embodiments, the present disclosure provides the modified T cells expressing a genetically modified lentiviral construct comprising a polypeptide sequence encoding: i. a chimeric antigen receptor specific to tumor antigens overexpressed in prostatecancer; and ii. a polypeptide sequence encoding a secretary antibody.

[0208] In some embodiments, the chimeric Antigen Receptor (CAR) construct specific to prostate cancer tumor antigens comprising a polypeptide sequence encoding a i. an extracellular domain, ii. a transmembrane domain, and iii. an intracellular signaling domain comprising the co-stimulatory domain(s) and a T cell activation domain.

[0209] In some embodiments, the extracellular domain comprises an antigen-binding domain comprising a polypeptide sequence encoding the variable heavy and light chain regions specific one or more prostate cancer tumor antigens.

[0210] In some embodiments, the one or more tumor antigens are PSMA (prostate-specific membrane antigen), PSA (Prostate specific antigen), PAP (prostate acid phosphatase), PSCA (prostate stem cell antigen), EpCaM (epithelial cell adhesion molecule), B7-H3,Muc-l (Mucin 1), CD126, Lewis Y antigen, STEAP1 (six transmembrane epithelial antigen of prostate type 1), STEAP4 (six transmembrane epithelial antigen of prostate type 4).

[0211] In a non-limiting embodiment, the tumor antigen is PSMA.

[0212] In a non-limiting embodiment, the extracellular antigen-binding domain comprises a linker polypeptide positioned between the VH and VL domains.

[0213] In a non-limiting embodiment, the transmembrane domain comprises a transmembrane region derived from CD 8.

[0214] In a non-limiting embodiment, the co-stimulatory domain comprises a CST-1 signaling domain and a CD-28 signaling domain.

[0215] In a non-limiting embodiment, the co-stimulatory domain comprises a CST-2 signaling domain and a 4 IBB signaling domain.

[0216] In a non-limiting embodiment, the T cell activating domain comprises CS and CD3 zeta signaling domain.

[0217] In some embodiments, the present disclosure provides immune cells engineered toexpress chimeric antigen receptors disclosed herein in combination with a therapeutic agent, in particular a cytotoxic agent. In some preferred embodiments, the cytotoxic agents are selected from but not limited to taxol derivatives, 5 -fluorouracil, cyclophosphamide, mitoxantrone, docetaxel, cabazitaxel and etoposide. The following drugs are approved for prostate cancer and preferably used: Abiraterone Acetate, Apalutamide, Bicalutamide, Cabazitaxel, Casodex (Bicalutamide), Degarelix, Docetaxel, Eligard (Leuprolide Acetate), Enzalutamide, Erleada (Apalutamide), Firmagon (Degarelix), Flutamide, Goserelin Acetate, Jevtana (Cabazitaxel), Leuprolide Acetate, Lupron Depot (Leuprolide Acetate), Mitoxantrone Hydrochloride, Nilandron (Nilutamide), Nilutamide, Provenge (Sipuleucel-T), Radium 223 Dichloride, Sipuleucel-T, Taxotere (Docetaxel), Xofigo (Radium 223 Dichloride), Xtandi (Enzalutamide), Zoladex (Gosereliin Acetate), Zytiga (Abiraterone Acetate).

[0218] In some embodiments, the present disclosure provides a pharmaceutical composition, comprising the engineered cells, preferably engineered T-cell, expressing a PSMA specific CAR and neutralizing antigen-binding fragment as described herein; and at least one pharmaceutically acceptable carrier. In an embodiment, pharmaceutically acceptable carrier is selected from diluent or excipients, wherein diluent is selected from water, saline, buffer, or combination thereof; and excipient is selected from physiological saline, glucose, dextrose, parabens, chlorobutanol, phenol, sorbic acid, trehalose, mannitol, sucrose, HEPES, sodium bicarbonate, phosphate buffer, Pluronic F68, ascorbic acid, N-acetylcysteine, or combinations thereof.

[0219] In some embodiments, the pharmaceutical composition may be provided as sterile liquid preparations, e.g., isotonic aqueous solutions, suspensions, emulsions, dispersions, or viscous compositions, which may be buffered to a selected pH, Liquid preparations are normally easier to prepare than gels, other viscous compositions, and solid compositions. Additionally, liquid compositions are somewhat more convenient to administer, especially by injection. Viscous compositions, on the other hand, can be formulated within the appropriate viscosity range to provide longer contact periods with specific tissues. Liquid or viscous compositions can comprise carriers, which can be a solvent or dispersing medium containing, for example, water, saline, phosphate buffered saline, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol, and the like) and suitable mixtures thereof.

[0220] In some embodiments, sterile injectable solutions can be prepared by incorporating the genetically modified immuno-responsive cells utilized in practicing the present invention in the required amount of the appropriate solvent with various amounts of the other ingredients, as desired. Such compositions may be in admixture with a suitable carrier including diluent, orexcipient such as sterile water, physiological saline, glucose, dextrose, or the like. The compositions can also be lyophilized. The pharmaceutical compositions, in some embodiments, may comprise auxiliary substances such as wetting, dispersing, or emulsifying agents (e.g., methylcellulose), pH buffering agents, gelling or viscosity enhancing additives, preservatives, flavoring agents, colors, and the like, depending upon the route of administration and the preparation desired. Standard texts, such as "REMINGTON'S PHARMACEUTICAL SCIENCE", 17th edition, 1985, incorporated herein by reference, may be consulted to prepare suitable preparations, without undue experimentation.

[0221] Various additives which enhance the stability and sterility of the compositions, including antimicrobial preservatives, antioxidants, chelating agents, and buffers, can be added. Prevention of the action of microorganisms can be ensured by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like. Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin. According to the present invention, however, any vehicle, diluent, or additive used would have to be compatible with the genetically modified immune-responsive cells or their progenitors.

[0222] In some embodiments, the present disclosure provides the use of the recombinant construct, vectors, and engineered cell in adoptive immunotherapy. Embodiments herein achieve a method for treating cancer in a subject. In an embodiment, said cancer is prostate cancer. In an embodiment, the method for treating cancer in a subject comprises administering the pharmaceutical composition as disclosed herein, or the engineered cell or population thereof as disclosed herein, to the subject. The present inventors have observed that the engineered cell having the recombinant construct and expressing the PSMA specific CAR and neutralizing antigen-binding fragment, as disclosed herein, are capable of achieving increased cytolysis of PSMA expressing cells.

[0223] Embodiments herein achieve a method for killing target cells. In an embodiment, the method for killing target cells at a target site, comprises administering the pharmaceutical composition as disclosed herein, or the engineered cell or population thereof as disclosed herein, to the target site. The term “target site” as used herein refers to an in vitro or in vivo site having or expected of having target cells. In an embodiment, the target site is a site in a subject. In an embodiment the target site is an in vitro site selected from in vitro tissue culture or cell culture.

[0224] Embodiments herein achieve a method for increasing cytolysis of target cells. In anembodiment, the method for increasing cytolysis of target cells at a target site, comprises administering the pharmaceutical composition as disclosed herein, or the engineered cell or population thereof as disclosed herein, to the target site. In an embodiment, the target cells are cancer cells. In another embodiment, the target cells are PSMA-expressing cancer cells. In another embodiment, the target cells are prostate cancer cells.

[0225] The administration of the pharmaceutical composition as disclosed herein, or the engineered cell or population thereof may be performed using methods known in the field. In an embodiment, administration is by intravenous (IV) or intratumoral (IT) or Intraprostatic or Intra-arterial routes In an embodiment, the subject is human. In another embodiment, the subject is a cancer patient.

[0226] In some embodiments, the present disclosure provides a method of treating prostate cancer in a subject.

[0227] In some embodiments, the chimeric antigen receptor constructs according to the present invention and immune cells containing such constructs can be used for focal therapy with a targeted tumor injection. In this embodiment, which is performed preferably with automated devices that apply the CAR-T cells to certain places in the body of the patient, where local tumor areas are located. With a biopsy needle a sample is then withdrawn whereby a small cavity is formed. In this cavity the transduced or transfected T-cells are introduced and then the needle is withdrawn. This embodiment is particularly advantageous when there are solid tumors which are extremely difficult to treat with regular methods.Kits

[0228] In some embodiments, the present disclosure provides kits for the treatment or prevention of prostate cancer, pathogen infection, immune disorder or allogeneic transplant, in one embodiment, the kit includes a therapeutic or prophylactic composition containing an effective amount of an immuno-responsive cell comprising an activating antigen receptor and a single- chain variable fragment (scFv) that binds an antigen having immunosuppressive activity in unit dosage form. In particular embodiments, the cells further comprise a costimulatory ligand. In some embodiments, the kit comprises a sterile container which contains a therapeutic or prophylactic vaccine; such containers can be boxes, ampules, bottles, vials, tubes, bags, pouches, blister-packs, or other suitable container forms known in the art. Such containers can be made of plastic, glass, laminated paper, metal foil, or other materials suitable for holding medicaments. If desired the immuno-responsive cell is provided together with instructions foradministering the cell to a subject having or at risk of developing a neoplasia, pathogen infection, immune disorder or allogeneic transplant. The instructions will generally include information about the use of the composition for the treatment or prevention of neoplasia, pathogen infection, immune disorder or allogeneic transplant. In other embodiments, the instructions include at least one of the following: description of the therapeutic agent; dosage schedule and administration for treatment or prevention of a neoplasia, pathogen infection, immune disorder or allogeneic transplant or symptoms thereof; precautions; warnings; indications; counter- indications; overdosage information; adverse reactions; animal pharmacology; clinical studies; and / or references. The instructions may be printed directly on the container (when present), or as a label applied to the container, or as a separate sheet, pamphlet, card, or folder supplied in or with the container.

[0229] Table 1 depicts the details of exemplary sequences according to the present disclosure.Table 1:

[0230] Table 2 depicts the exemplary components of the recombinant construct and their function, according to embodiments herein.Table 2:

[0231] The methods and techniques of the present invention 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 unless otherwise indicated. See, e. g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y. (2001); Ausubel et al., Current Protocols in Molecular Biology, J, Greene Publishing Associates (1992, and Supplements to 2002); Handbook of Biochemistry: Section A Proteins, Vol I 1976 CRC Press; Handbook of Biochemistry: Section A Proteins, Vol II 1976 CRC Press. The nomenclatures used in connection with, and the laboratory procedures and techniques of, molecular and cellular biology, protein biochemistry, enzymology and medicinal and pharmaceutical chemistry described herein are those well-known and commonly used in the art.

[0232] Although the subject matter has been described with reference to specific embodiments, this description is not meant to be construed in a limiting sense. Various modifications of the disclosed embodiments, as well as alternate embodiments of the subject matter, will become apparent to persons skilled in the art upon reference to the description of the subject matter. It is therefore contemplated that such modifications can be made without departing from the spirit or scope of the present subject matter as defined.EXAMPLES

[0233] The disclosure will now be illustrated with following examples, which is intended to illustrate the working of disclosure and not intended to take restrictively to imply any limitations on the scope of the present disclosure. Unless defined otherwise, all technical and scientificterms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice of the disclosed methods and compositions, the exemplary methods, devices and materials are described herein. It is to be understood that this disclosure is not limited to particular methods, and experimental conditions described, as such methods and conditions may vary.Materials:

[0234] pMDLg / pRRE (Addgene # 12251); pRSV-Rev (Addgene # 12253), pMD2.G (Addgene # 12259), unarmoured PSMA specific CAR (generated inhouse), armoured PSMA specific CAR (generated inhouse), pCDH-EFla-eFFly-mCherry (Addgene # 104833), LV virus for PSMA expression (generated inhouse); 293T cells (ATCC, CRL-3216), PC3 cells (ATCC, CRL-1435), BD FACS Lyric (R663029000728), Biosafety Cabinet (BSL-2) (Microfilt)( MFI BSC 4x2), Nanodrop Onec(AZY2334563), Victor Nivo Multimode Microplate Reader (HH35L 2023 108), Incubator (CCL-170B-8), Microscope EVOS M5000 (AMF5000). Reagents used are depicted in Table 3:Table 3:Example 1: Production of Lentiviral vector

[0235] Lentiviral vectors and engineered T-cells were prepared using the recombinant construct for expressing PSMA specific CAR and IL-8 scfv of the present disclosure (Fig. 1(a)). Similarly, Lentiviral vectors and engineered T-cells were also prepared using a construct for expressing PSMA specific CAR without IL-8 scFv (Fig. 1(b)).

[0236] The CARs, T cells, constructs, vector, etc prepared using the recombinant construct for PSMA specific CAR and IL-8 scfv of the present disclosure (SEQ ID NO. 26) is referred to herein as “armored” CARs, T cells, constructs, vectors, etc. For eg: “Armored PSMA specific CAR”, “Armored PSMA CAR”, “ IL-8 Armored PSMA CAR”, “Armored CAR-T cells”, “Armored T cells”, “Armored engineered cells”, “Armored CAR”, and so on.

[0237] The CARs, T cells, constructs, vector, etc prepared using the construct for PSMA specific CAR without IL-8 scfv (SEQ ID NO. 27) is referred to herein as “unarmored” CARs, T cells, constructs, vectors, etc. For eg: “Unarmored PSMA specific CAR”, “Unamored PSMA CAR”, “Unarmored CAR-T cells”, “Unarmored T cells”, “Unarmored cells”, “Unarmored CAR”, and so on.

[0238] Armored CAR was expressed using a recombinant construct as depicted in SEQ ID NO. 26, and Unarmored CAR was expressed using a construct as depicted in SEQ ID NO. 27.

[0239] HEK293 T cells (ATCC, CRL-3216) were plated in T75 flasks and grown until a confluency of 70% was achieved. Lentiviral supernatants were collected from HEK293T cells transfected with Lentiviral packaging plasmids (pMDLg / pRRE Addgene # 1225; pRSV-Rev Addgene # 12253; pMD2.G Addgene # 12259) and transfer plasmids with recombinant construct (SEQ ID NO. 26) encoding armored CAR, concentrated by using LV concentrator (Takara, 631232) and resuspended in OptiMEM media (1 / 50111volume of culture- 50 times concentrated ).

[0240] Similarly, transfection was performed using transfer plasmids with construct (SEQ ID NO. 27) encoding unarmored CAR.

[0241] The lentiviral particles obtained were then used to transduce HEK293T cells to obtainviral titre.

[0242] Figs. 1(a) and 1(b) are a schematic representation of recombinant constructs, wherein Fig. 1(a) depicts a recombinant construct for expressing armored PSMA specific CAR, i.e. PSMA specific CAR with IL-8 scFv, and Fig. 1(b) depicts a recombinant construct for expressing unarmored PSMA specific CAR, i.e. PSMA specific CAR without IL-8 scFv, in accordance with the embodiments herein.Example 2: Expressing PSMA specific CAR in engineered T-cells

[0243] HEK293T cells (ATCC, CRL-3216) were plated in a 12-well plate at a density of 0.16 X106 cells / well in DMEM media. LV particles (obtained in Example 1) were added to each of the cells in different volumes from lOpL to 0.078 pL using 2X dilutions in media. The transduced HEK293 T cells (engineered cells) were incubated for 72hrs and surface expression of the PSMA specific CAR (armored and unarmored) was thereafter evaluated using Flow cytometric analysis.

[0244] Flow cytometric analysis: BD FACS lyric flow cytometer was used for data acquisition and FlowJoTM 10 was used for analysis. Cells were stained in IX PBS [Gibco, 18912-014] at 4°C with respective antibodies.

[0245] Fig. 3 depicts percentage T cell population (CD4 and CD8 expressing cell) in engineered cells expressing unarmored CAR and armored CAR of the present disclosure.

[0246] Observation: The distribution of CD4+and CD8+T cell subsets remained comparable between armored and unarmored PSMA specific CAR-T cell populations.Example 3: Evaluation of engineered T-cells for Mitochondrial stress

[0247] Transduced T cells (engineered cells, refer Example 2) were evaluated for Mitochondrial stress by staining with IpM MitoSOX™ Red Mitochondrial superoxide indicator (Cat#2770829, Invitrogen) and lOOnM TMRE-Mitochondrial Membrane Potential Kit (Abeam, Cat#2101039975). The cells were incubated at 37°C for 30 min and washed with PBS (3X).

[0248] Observation: MitoSOX and TMRE expression levels were similar between untransduced, unarmored and armored PSMA specific CAR-T cells, indicating comparable mitochondrial membrane potential.

[0249] Figs. 10(a) and 10(b) depicts MitoSOX expression in both armored and unarmoredCAR-T cells, wherein Fig. 10(a) depicts MitoSOX expression in unarmored CAR-T cells, and Fig. 10(b) depicts MitoSOX expression in armored CAR-T cells, in accordance with the embodiments herein.

[0250] Figs. 11(a) and 11(b) depicts Tetramethylrhodamine ethyl ester (TMRE) expression in both armored and unarmored CAR-T cells, wherein Fig. 11(a) depicts TMRE expression in unarmored CAR-T cells, and Fig. 11(b) depicts TMRE expression in armored CAR-T cells, in accordance with the embodiments herein.Example 4: Evaluation of engineered T-cells for T cell transduction

[0251] The LV particles obatined in Example 1 were titrated and then used to transduce normal donor human T cells at an MOI of 5. T cells were isolated from PBMCs using anti-CD4 and anti-CD8 microbeads (Miltenyi Biotec). Isolated T cells were activated for 24 h in Transact and IL2 containing media (lOOIU / ml). Transduced T cells were expanded for around 7-10 days in TexMACS media (Miltenyi Biotec, 130-097-196) with IL 7 (500IU / ml) IL15 media (84IU / ml). These transduced T cells were evaluated for CAR expression by staining for protein L.

[0252] Observation: Protein L staining revealed that both armored and unarmored CAR constructs display equivalent levels of CAR expression.

[0253] Figs. 9(a) and 9(b) depicts CAR expression in both armored and unarmored CAR-T cells (stained by protein L staining), wherein Fig. 9(a) depicts CAR expression in unarmored CAR-T cells, and Fig. 9(b) depicts CAR expression in armored CAR-T cells, in accordance with the embodiments herein.Example 5: Evaluation of engineered T-cells by ELISA for detection of IL-8 scfv.

[0254] HEK293 T cells were plated at -800% confluency in 100mm dishes and transfected with lOpg of transfer plasmids (having the recombinant construct of SEQ ID NO. 26) in Opti- MEM. Supernatants from transfection were collected at 24hr time point and concentrated using centricon and buffer was exchanged to PBS. The concentrate was then subjected to protein quantification using Pierce BCA protein assay kit (Thermo fischer scientific, 23225). The concentrated protein was then used for ELISA.

[0255] For ELISA, positive control and concentrated harvest were coated onto maxi sorb 96 well plates overnight at 4°C. The following day, the coated plates were washed 3 times with PBS and blocked using 5% skimmed milk. Following another round of washing, IL-8 cytokinewas added to each well and incubated. Post IL-8 cytokine incubation, the plates were washed and biotin labelled IL-8 antibody was added on top. The primary antibody was washed and a streptavidin labelled secondary antibody was thereafter used and detected using TMB. The reaction was quenched using H2SO4. The OD450 was measured on a Victor Nivo Multimode Microplate Reader. This assay was performed in triplicate wells.

[0256] Observation'. Secretion of functional IL-8 scFv by the engineered T-cell having the recombinant construct for PSMA armored CAR was confirmed via ELISA, indicating successful expression and release of the engineered molecule.

[0257] Fig. 2 depicts ELISA results for armored CAR of the present disclosure showing levels of secreted IL-8 scFv (IL-8 commercial antibody used as control), in accordance with the embodiments herein.Example 6: Evaluation by Western blot analysis

[0258] The concentrated protein was collected (refer Example 4), and the protein concentration was measured by BCA protein assay kit (Themofisher scientific). Equal amount of protein was added into SDS-PAGE gel, and then transferred to the poly vinylidene difluoride (PVDF) membrane. The membranes were further blocked in TBST containing 5% BSA for 1 hr, and then were incubated with primary antibody against BAX and GAPDH at 4°C overnight. The membranes were further washed for three times and were incubated with mouse secondary antibodies specific for BAX and GAPDH for 1 h. The bands were scanned and analyzed with chemiluminescence with Clarity Max western ECL substrate (Biorad, Cat#1705062), and images were captured with a ibright Imaging system (Invitrogen, FLI500). GAPDH were used as internal loading control.

[0259] The recombinant construct (armored CAR) according to the present disclosure lowered the apoptosis of the CAR-T cells due to undisrupted metabolism of GLUT1. The lowered Apoptosis is reflected by significant lower expression of BAX (a pro apoptotic protein) compared to third-generation Gen PSMA specific CAR without IL-8 scfv expression and Untransduced control.

[0260] Observation: The engineered CAR-T cells expressing the PSMA armored CAR with IL-8 scFv exhibited reduced expression of pro-apoptotic markers compared to untransduced T- cells and third-generation unarmored CAR-T cells, indicating decreased apoptosis and enhanced T cell fitness.

[0261] Fig. 4 depicts the results of BAX expression for Untransduced T cells, T cells transduced with unarmored and armored CAR respectively, wherein (a) depicts the results of western blotting, and (b) is an imageJ analysed graph depicting BAX expression in both unarmored and armored CAR-T cells compared to un-transduced T-cells and normalised with GAPDH expression, in accordance with the embodiments herein.Example 7: Cytolysis assay

[0262] For co-culture experiments, PC3 cells expressing PSMA and mCherry luciferase were plated at 10 x io3cells per well of 96-well plates in RPMI media. Untransduced T-cells, transduced unarmored T-cells, and transduced armored T-cells (refer Example 2) were then added in the ratios, (i.e. target : effector ratio) of 1 : 1, 1 :2.5, 1 :5 and 1 : 10. Post 6hr, 24hr, 48hr, 72hr of co-culture with PSMA and mCherry luciferase expressing PC3 cells with T cells at said target : effector ratios, coculture assay plates were investigated by addition of D-luciferin to a final concentration of 150pg / mL and luminescence was measured using Victor Nivo Multimode Microplate Reader (HH35L 2023 108). The assay for each time point and each dilution was performed in triplicates.

[0263] Observation: Both, engineered T cells expressing unarmored PSMA specific CAR and engineered T cells expressing armored PSMA specific CAR with sIL-8 scFv effectively mediate cytolysis of PSMA-expressing PC3 cells. However, the engineered T cells expressing armored PSMA specific CAR with IL-8 scFv demonstrate significantly enhanced and more rapid tumor cell killing, even at low effector-to-target ratios. Figs. 5-8 depicts cytotoxicity with both unarmored and armored CAR-T cells compared to basal killing with un-transduced T cells. Fig. 5 depicts cytotoxicity at 6 hours, wherein a) and b) depict bar and line graph representations respectively. Fig. 6 depicts cytotoxicity at 24 hours, wherein a) and b) depict bar and line graph representations respectively. Fig. 7 depicts cytotoxicity at 48 hours, wherein a) and b) depict bar and line graph representations respectively. Fig. 8 depicts cytotoxicity at 72 hours, wherein a) and b) depict bar and line graph representations respectively.

[0264] Armored CAR exhibited increased and accelerated cytolysis evident from the 6hr time point assay. The cytolysis with the armored CAR was higher at even lower concentrations of T cells.

Claims

I / We claim:

1. A recombinant construct comprising: a. a first polynucleotide having a nucleotide fragment encoding a PSMA (prostatespecific membrane antigen) specific CAR (chimeric antigen receptor); and b. a second polynucleotide having a nucleotide fragment encoding a neutralizing antigen-binding fragment specific for interleukin-8 (IL-8).

2. The recombinant construct as claimed in claim 1, wherein the PSMA specific CAR comprises an antigen binding domain having at least 90% sequence identity to a sequence as depicted in SEQ ID NO. 1; or wherein the neutralizing antigen-binding fragment comprises a polypeptide having at least 90% sequence identity to a sequence as depicted in SEQ ID NO. 3.

3. The recombinant construct as claimed in claim 1, wherein the PSMA specific CAR comprises a hinge and transmembrane domain derived from CD8; a co-stimulatory domain selected from CD-28 co-stimulatory domain, 4 IBB co-stimulatory domain, or combination thereof; and CD3zeta signaling domain.

4. The recombinant construct as claimed in claim 1, wherein the recombinant construct comprises a promoter operably linked to said first polynucleotide, wherein the promoter has a sequence selected from SEQ ID NO. 12 or SEQ ID NO. 13.

5. The recombinant construct as claimed in claim 1, wherein the second polynucleotide further comprises a nucleotide fragment encoding a signal and a nucleotide fragment encoding a 2A element, operably linked upstream to the nucleotide fragment encoding the neutralizing antigen-binding fragment.

6. The recombinant construct as claimed in claim 1, wherein the PSMA specific CAR comprises a polypeptide having at least 90% sequence identity to a sequence selected from SEQ ID NO. 5, SEQ ID NO. 7, or SEQ ID NO. 9; or wherein the neutralizing antigen-binding fragment comprises a polypeptide having at least 90% sequence identity to a sequence as set forth in SEQ ID NO. 20.

7. The recombinant construct as claimed in claim 1, wherein the second polynucleotide comprises a nucleotide fragment encoding a P2A element operably linked upstream to the nucleotide fragment encoding the neutralizing antigen-binding fragment, wherein the P2A element has a sequence as set forth in SEQ ID NO. 22 or wherein the nucleotide fragment encoding the P2A element has a sequence as set forth in SEQ ID NO. 23.

8. The recombinant construct as claimed in claim 1, wherein the second polynucleotide comprises a nucleotide fragment encoding a GM-CSF signal operably linked upstream to the nucleotide fragment encoding the neutralizing antigen-binding fragment, wherein theGM-CSF signal has a sequence as set forth in SEQ ID NO. 24, or wherein the nucleotide fragment encoding the GM-SCF signal has a sequence as set forth in SEQ ID NO. 25.

9. The recombinant construct as claimed in claim 1, wherein the first polynucleotide comprises a nucleotide fragment having at least 80% sequence identity to a sequence selected from SEQ ID NO. 2, SEQ ID NO. 6, SEQ ID NO. 8, SEQ ID NO. 10, or SEQ ID NO. 11.

10. The recombinant construct as claimed in claim 1, wherein the second polynucleotide comprises a nucleotide fragment having at least 80% sequence identity to a sequence selected from SEQ ID NO. 4, or SEQ ID NO. 21.

11. The recombinant construct as claimed in claim 1, wherein the recombinant construct has a nucleotide sequence of at least 80% sequence identity to a sequence selected from SEQ ID NO. 26, or wherein the recombinant construct encodes a polypeptide having at least 90% sequence identity to a sequence selected from SEQ ID NO. 28.

12. A vector comprising the recombinant construct as claimed in claim 1.

13. The vector as claimed in claim 12, wherein the vector is a lentiviral vector.

14. An engineered cell or population thereof comprising the vector as claimed in claim 12 or the recombinant construct as claimed in claim 1.

15. The engineered cell or population thereof as claimed in claim 14, wherein the engineered cell is an immune cell, natural killer cell, or induced pluripotent cell (iPSC).

16. A pharmaceutical composition comprising the engineered cell or population thereof as claimed in claim 14.

17. A method for treating cancer in a subject, comprising administering the pharmaceutical composition as claimed in claim 16, or the engineered cell or population thereof as claimed in claim 14, to said subject.

18. A method of killing target cells at a target site, comprising administering the pharmaceutical composition as claimed in claim 16, or the engineered cell or population thereof as claimed in claim 14, to the target site.

19. A method for increasing cytolysis of target cells at a target site, comprising administering the pharmaceutical composition as claimed in claim 16, or the engineered cell or population thereof as claimed in claim 14, to the target site.

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