Uses of immunomodulatory fusion proteins to enhance efficacy of car-expressing immune effector cells

Abstract

The present disclosure provides a population of genetically modified immune cells and uses thereof, the modified immune cells comprising (i) a chimeric antigen receptor (CAR) targeting a target protein on a target cell; (ii) a first immunomodulatory fusion protein comprising an ectodomain derived from a protein of tumor necrosis factor (TNF) superfamily, and an endodomain derived from a costimulatory molecule of a TNF receptor superfamily protein; (iii) a second immunomodulatory fusion protein comprising an ectodomain derived from a protein of TNF receptor superfamily, and an endodomain derived from a common g-chain cytokine receptor; and (iv) a third immunomodulatory fusion protein comprising an ectodomain derived from the same TNF receptor superfamily protein as in the second immunomodulatory fusion protein, and an endodomain derived from an a or b receptor subunit of the common g-chain cytokine receptor family.

Description

USES OF IMMUNOMODULATORY FUSION PROTEINS TO ENHANCE EFFICACY OF CAR-EXPRESSING IMMUNE EFFECTOR CELLSSEQUENCE LISTING STATEMENT

[0001] The instant application contains a Sequence Listing which has been submitted electronically in XML file format and is hereby incorporated by reference in its entirety. Said XML copy, created on July 17, 2024, is named P-636940-USP_07_17_2024_SL.xml and is 84,557 bytes in size.FIELD OF THE DISCLOSURE

[0002] The present disclosure is related in general to the field of immunotherapy by chimeric antigen receptor (CAR)-expressing immune effector cells. In some embodiments, the present disclosure provides immunomodulatory fusion proteins for use in enhancing the efficacy of CAR-expressing immune effector cells.BACKGROUND

[0003] Despite the remarkable success of Adoptive Cell Transfer (ACT) of chimeric antigen receptor (CAR)-T cells in the treatment of hematological malignancies, there is still about 50 % of patients relapse, and efficacy against solid tumors has been limited, in part due to inhibitory tumor microenvironments. Death Receptors (DR), especially Fas cell surface death receptor (Fas, also known as CD95, APO-1 , or TNFRSF6), are expressed in effector immune cells such as T and NK cells. Tumor cells as well as cells of the tumor microenvironment, and immune effector cells, including T and NK cells, express DR ligands (DRL). Therefore, the DR / DRL checkpoint may inhibit cancer immunotherapeutic approaches by limiting the persistence of CAR-expressing cells. Several groups have described strategies to mitigate Fas ligand (FasL)-induced apoptosis in Fas-competent T cells, including genetic engineering immune cells to express non-functional truncated Fas lacking the death domain, which competes with native Fas. Several groups have demonstrated enhanced CAR-T cell efficacy using in vitro and in vivo models when CAR was co-expressed along with immunomodulatory fusion proteins (IFPs) that combine an inhibitory ectodomain of Fas with a costimulatory endodomain of other pro-survival TNF superfamily receptors. There is a need to furtherdevelop immunomodulatory fusion proteins for use in enhancing the efficacy of CAR- expressing immune effector cells.SUMMARY

[0004] In one aspect, the present disclosure provides a population of genetically modified immune cells, wherein the modified immune cells comprising:(i) a chimeric antigen receptor (CAR) targeting a target protein on a target cell;(ii) a first immunomodulatory fusion protein that induces cell death in the target cell, the first fusion protein comprises an ectodomain derived from a protein of tumor necrosis factor (TNF) superfamily, and an endodomain derived from a costimulatory molecule of a TNF receptor superfamily protein;(iii) a second immunomodulatory fusion protein that prevents cell death in the genetically modified immune cell, the second fusion protein comprises an ectodomain derived from a protein of TNF receptor superfamily, and an endodomain derived from a common y- chain cytokine receptor; and(iv) a third immunomodulatory fusion protein comprising an ectodomain derived from the same TNF receptor superfamily protein as in the second immunomodulatory fusion protein, and an endodomain derived from an a or £ receptor subunit of the common y- chain cytokine receptor family.

[0005] In another aspect, the present disclosure provides one or more isolated nucleotide sequences encoding one or more of:(i) a chimeric antigen receptor (CAR) targeting a target protein on a target cell;(ii) a first immunomodulatory fusion protein that induces cell death in the target cell, the first fusion protein comprises an ectodomain derived from a protein of tumor necrosis factor (TNF) superfamily, and an endodomain derived from a costimulatory molecule of a TNF receptor superfamily protein;(iii) a second immunomodulatory fusion protein that prevents cell death in the genetically modified immune cell, the second fusion protein comprises an ectodomain derived from a protein of TNF receptor superfamily, and an endodomain derived from a common y- chain cytokine receptor; and(iv) a third immunomodulatory fusion protein comprising an ectodomain derived from thesame TNF receptor superfamily protein as in the second immunomodulatory fusion protein, and an endodomain derived from an a or 0 receptor subunit of the common y- chain cytokine receptor family.

[0006] In another aspect, the present disclosure provides one or more expression vectors comprising the isolated nucleotide sequences described herein.

[0007] In another aspect, the present disclosure provides a composition comprising the population of genetically modified immune cells disclosed herein and a pharmaceutically acceptable carrier.

[0008] In another aspect, the present disclosure provides a method of treating cancer in a subject in need thereof, comprising administering to the subject the population of genetically modified immune cells described herein.

[0009] These and other aspects of the genetically modified cells and uses thereof will be appreciated from the ensuing descriptions of the figures and detailed description of the invention.BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Some embodiments of the genetically modified cells and uses thereof are herein described, by way of example only, with reference to the accompanying drawings. With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of embodiments of the invention. In this regard, the description taken with the drawings makes apparent to those skilled in the art how embodiments of the invention may be practiced.

[0011] Figure 1 shows the delivery of three signals to immune effector cells through various immunomodulatory fusion proteins expressed by the effector cells to enhance the functions of the effector cells. Signal one (Signal 1 ) is delivered by a chimeric antigen receptor (CAR), for example, an anti-CD19 CAR comprising a CD3z costimulatory domain. Signal two (Signal 2) is delivered by a fusion protein comprising an ectodomain of a tumor necrosis factor (TNF) superfamily protein (or a death receptor ligand, for example, TRAIL or FasL), and an endodomain derived from a costimulatory molecule ofa TNF receptor superfamily protein, for example, CD137 (also known as 4-1 BB), 0X40 (also known as CD134), CD27, CD40, BAFFR (also known as CD268), GITR (also known as CD357), BCMA (also known as CD269), HVEM (also known as CD270), TACI (also known as CD267), CD30, tumor necrosis factor-like weak inducer of apoptosis (TWEAK) receptor, or lymphotoxin [3 receptor (LTbR). Signal three (Signal 3) is delivered by (i) a fusion protein comprising an ectodomain of a TNF receptor superfamily protein (or a death receptor), for example, Fas, and an endodomain derived from a common y-chain cytokine receptor CD132, and (ii) a fusion protein comprising an ectodomain of the same TNF receptor superfamily protein as in (i) (e.g. Fas), and an endodomain derived from an a or p receptor subunit of the common y-chain cytokine receptor family, for example, IL- 2R-p (CD122), IL-7R-a (CD127), IL-9R-a (CD129), or IL-21 R-a (CD360).

[0012] Figure 2 shows one embodiment of expression cassette comprising four coding sequences separated by furin cleavage sites followed by a 2A ribosomal skip peptide. In one embodiment, the sequences encode (i) a chimeric antigen receptor (CAR), (ii) a Fas- CD132 fusion protein, (iii) a Fas-cytokine receptor fusion protein, and (iv) a TRAIL- costimulatory fusion protein.

[0013] Figure 3 shows a schematic of a stress test. Genetically modified immune cells (e.g., CAR T cells) are repeatedly challenged with target cells. Initially, the modified immune cells and the target cells are mixed at a single ratio, e.g., 1 :1 . Growth of the modified immune cells and target cell clearance are measured 3-4 days later. Then the modified immune cells and the target cells are reset at a single ratio, e.g., 1 :1 , and growth of the modified immune cells and target cell clearance are measured 3-4 days later. This growth and challenge cycle is repeated until the modified immune cells can no longer clear the target cells.

[0014] Figure 4 shows a schematic of a single challenge assay. Genetically modified immune cells (e.g., CAR T cells) are mixed with target cells at various effector:target ratios, with constant number of target cells and titrated down number of modified immune cells. Growth of the modified immune cells and target cell killing (cytotoxicity) are measured 4 days later, e.g., by flow cytometry. Target cell killing is normalized to culture with target cells only.

[0015] Figures 5A-5I show representative examples of expression cassettes comprising four coding sequences separated by furin cleavage sites followed by a 2A ribosomal skip peptide (F-2A). Figure 5A represents a sequence encoding 5' to 3' (i) FAS-IL2RP fusion protein, (ii) TRAIL-4-1 BB-costimulatory fusion protein, (iii) FAS-IL2Rg fusion protein, and (iv) a CD19 chimeric antigen receptor (CAR). Figure 5B represents a sequence encoding 5' to 3' (i) FAS-IL7RA fusion protein, (ii) TRAIL-4-1 BB-costimulatory fusion protein, (iii) FAS-IL2Rg fusion protein, and (iv) a CD19 chimeric antigen receptor (CAR). Figure 5C represents a sequence encoding 5' to 3' (i) FAS-IL9Ra fusion protein, (ii) TRAIL-4-1 BB- costimulatory fusion protein, (iii) FAS-IL2Rg fusion protein, and (iv) a CD19 chimeric antigen receptor (CAR). Figure 5D represents a sequence encoding 5' to 3' (i) FAS- IL21 Ro fusion protein, (ii) TRAIL-4-1 BB-costimulatory fusion protein, (iii) FAS-IL2Rg fusion protein, and (iv) a CD19 chimeric antigen receptor (CAR). Figure 5E represents a sequence encoding 5' to 3' (i) FAS-IL2R|3 fusion protein, (ii) TRAIL-OX40-costimulatory fusion protein, (iii) FAS-IL2Rg fusion protein, and (iv) a CD19 chimeric antigen receptor (CAR). Figure 5F represents a sequence encoding 5' to 3' (i) FAS-IL2R|3 fusion protein, (ii) TRAIL-CD27-costimulatory fusion protein, (iii) FAS-IL2Rg fusion protein, and (iv) a CD19 chimeric antigen receptor (CAR). Figure 5G represents a sequence encoding 5' to 3' (i) FAS-IL2R[3 fusion protein, (ii) TRAIL-BAFFR-costimulatory fusion protein, (iii) FAS-IL2Rg fusion protein, and (iv) a CD19 chimeric antigen receptor (CAR). Figure 5H represents a sequence encoding 5' to 3' (i) FAS-IL2R[3 fusion protein, (ii) TRAIL-GITR- costimulatory fusion protein, (iii) FAS-IL2Rg fusion protein, and (iv) a CD19 chimeric antigen receptor (CAR). Figure 51 represents a sequence encoding 5' to 3' (i) FAS- IL2Rp fusion protein, (ii) TRAIL-CD30-costimulatory fusion protein, (iii) FAS-IL2Rg fusion protein, and (iv) a CD19 chimeric antigen receptor (CAR). Abbreviations: F-2A -furin cleavage sites followed by a 2A ribosomal skip peptide; TM - transmembrane region; sp - signal peptide; ICD - intracellular domain.DETAILED DESCRIPTION

[0016] Several studies have shown upregulated expression in immune effector cells of a number of genes that belong to the tumor necrosis factor (TNF) receptor superfamily, both negative and positive regulators of the immune cells. To improve the efficacy and persistent functions of immune effector cells (e.g., CAR-T cell), the present disclosureprovides chimeric receptors (CR) or immunomodulatory fusion proteins that will convert negative, proapoptotic, signal to co-stimulatory, pro-survival signal and, at the same time, induce apoptotic cell death of the targeted cells.

[0017] In some embodiments, the immunomodulatory fusion proteins (IFP) disclosed herein deliver three signals to the effector cells (see Figure 1). As used herein, the terms “immunomodulatory fusion protein” and “fusion protein” may be used interchangeably with all the same qualities and meanings. Signal one is delivered by a chimeric antigen receptor (CAR) targeting a target protein on target cells, for example but not limited to a CD19 target protein. Signal two is delivered by an immunomodulatory fusion protein comprising an ectodomain of a TNF superfamily protein (or a death receptor ligand, for example, TNF-related apoptosis inducing ligand (TRAIL) or FasL, and an endodomain derived from a costimulatory molecule of a TNF receptor superfamily protein, for example, 4-1 BB. This fusion protein would function as a bidirectional receptor, contributing to the apoptotic effect in the target cells by engaging death receptors on the target cells, and simultaneously, providing a costimulatory signal to the immune effector cells. Signal three is delivered by (i) an immunomodulatory fusion protein comprising an ectodomain of a TNF receptor superfamily protein (or a death receptor, for example, Fas), and an endodomain derived from a common y-chain cytokine receptor, and (ii) an immunomodulatory fusion protein comprising an ectodomain of the same TNF receptor superfamily protein (e.g. Fas), and an endodomain derived from an a or [3 receptor subunit of the common y-chain cytokine receptor family, for example, IL-2R-£ and other described herein.

[0018] In some embodiments, when signaling of signal two comprises TRAIL, then signaling of signal three comprises FasL. In some embodiments, when signaling of signal two comprises FasL, then signaling of signal three comprises TRAIL.

[0019] In some embodiments, a target cell comprises a cancer cell.

[0020] In one embodiment, sequences encoding the fusion proteins described herein can be cloned into retroviral or lentiviral constructs to be delivered as individual genes or as a part of a construct separated by self-cleaving peptide sequences (see Figure 2). In another embodiment, sequences encoding the fusion proteins described herein can bedelivered to target cells via other vectors or plasmids generally known in the art by standard techniques such as transduction or transfection etc.

[0021] The genetically modified immune cells disclosed herein can be derived from, for example, NK cells, T cells, NKT-cells, y<5 T cells, mucosal-associated invariant T (MAIT) cells, macrophages, or cytokine-induced killer cells. In one embodiment, these various immune cells are first isolated or generated from human samples using standard procedures known in the art, and then sequences encoding the fusion proteins described herein are delivered to these immune cells using constructs and methodologies as described above to generate the present genetically modified immune cells.

[0022] In some embodiments, an immune effector cell comprises a CAR-T cell. In some embodiments, an immune effector cell comprises a CAR-NK cell. Immune effector cells such as CAR-T or CAR-NK cells can be generated from T or NK cells isolated from human healthy donors PBMCs using standard procedures known in the art. These effector cells as well as others described herein can further be engineered to express the present fusion proteins using standard procedures.

[0023] In one embodiment, the present disclosure provides a population of genetically modified immune cells, wherein the modified immune cells comprise:(i) a chimeric antigen receptor (CAR) targeting a target protein on a target cell;(ii) a first immunomodulatory fusion protein that induces cell death in the target cell, the first fusion protein comprises an ectodomain derived from a protein of tumor necrosis factor (TNF) superfamily, and an endodomain derived from a costimulatory molecule of a TNF receptor superfamily protein;(iii) a second immunomodulatory fusion protein that prevents cell death in the genetically modified immune cell, the second fusion protein comprises an ectodomain derived from a protein of TNF receptor superfamily, and an endodomain derived from a common y- chain cytokine receptor; and(iv) a third immunomodulatory fusion protein comprising an ectodomain derived from the same TNF receptor superfamily protein as in the second immunomodulatory fusion protein, and an endodomain derived from an a or 0 receptor subunit of the common y- chain cytokine receptor family.

[0024] In some embodiments, the ectodomains of the above immunomodulatory fusion proteins (IFPs) comprise signal peptide, extracellular domain, and transmembrane domain. The transmembrane domain is an independent part of the IFP. In some embodiments, the transmembrane domain belongs to the extracellular part of the IFP. In some embodiments, the transmembrane domain belongs to the intracellular part of the IFP.

[0025] In some embodiments, a transmembrane domain comprises a Fas transmembrane domain. In some embodiments, the transmembrane domain comprises a TRAIL transmembrane domain. Since TRAIL protein synthesis begins from endodomain, the order of amino acids was reversed. In the IFPs comprising a TRAIL transmembrane domain, the transmembrane domain is represented by the amino acid sequence FYVYTVAVCLSQLLVTFIVIL in the IFPs set forth in SEQ ID Nos: 3-14.

[0026] A skilled artisan would appreciate that a signal peptide is a peptide segment of about 16-40 amino acids, which is fused to a protein for secretion or targeting to a cell membrane. As used herein, the term “signal peptide” may in some embodiments be used interchangeably with the term “signal sequence”, having all the same meanings and qualities.

[0027] In some embodiments, once an IFP is integrated within the cell membrane, the signal peptide may then be cleaved at its C terminus by a signal peptidase. In some embodiments, the signal peptide is cleaved from the IFP. In some embodiments, the signal peptide is not cleaved from the IFP. The amino acid sequences disclosed herein include the signal peptides.

[0028] Amino acids 1 -21 of SEQ ID NOs: 3-14 and 23-34 (MARPHPWWLCVLGTLVGLS) comprise the signal peptide for the IFPs set forth in SEQ ID NOs: 3-14 and 23-34. Amino acids 1 -25 of SEQ ID NOs: 15-18 (MLGIWTLLPLVLTSVARLSSKSVNA) comprise the signal peptide for the IFPs set forth in SEQ ID NOs: 15-18. Amino acids 1 -23 of SEQ ID NOs: 35-38 (MAPPPARVHLGAFLAVTPNPGSA) comprise the signal peptide for the IFPs set forth in SEQ ID NOs: 35-38. Amino acids 1 -23 of SEQ ID NOs: 39-54 (MAPPPARVHLGAFLAVTPNPGSA) comprise the signal peptides for the IFPs set forth in SEQ ID NOs: 39-54.

[0029] In some embodiments, the CAR disclosed herein is a first generation CAR. In other embodiments, the CAR is a second generation CAR. In other embodiments, the CAR is a third generation CAR. In other embodiments, the CAR described herein encompasses any and all improved versions of CAR known in the art. For example, a first generation anti-CD19 CAR has the amino acid sequence of SEQ ID NO:1 (MALPVTALLLPLALLLHAARPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQ KPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYT FGGGTKLEITGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPD YGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDD TAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSTTTPAPRPPTPAPTIASQPLSLRPEAC RPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCRVKFSRSADAPAYQQG QNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYS EIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPP; CD19 CAR).

[0030] In some embodiments, the genetically modified immune cells can be, but are not limited to, NK cells, T cells, NKT-cells, y<5 T cells, mucosal-associated invariant T (MAIT) cells, macrophages, or cytokine-induced killer cells.

[0031] In some embodiments, the present genetically modified immune cells are targeted to cancer cells expressing the target protein recognized by the present genetically modified immune cells. In some embodiments, the cancer cells are hematopoietic cancer cells. In some embodiments, the cancer cells are cells of solid tumors. In some embodiments, the cancer cells comprise hematopoietic cancer cells or solid cancer cells. In some embodiments, examples of cancer cells include, but are not limited to, B cell acute lymphoblastic leukemia (B-ALL) cells, B cell non-Hodgkin lymphoma (B-NHL) cells, multiple myeloma cells, malignant mesothelioma cells, ovarian cancer cells, breast cancer cells, pancreatic cancer cells, lung cancer cells, gastric cancer cells, endometrial cancer cells, cervical cancer cells, biliary cancer cells, uterine serous carcinoma cells, or cholangiocarcinoma cells.

[0032] In some embodiments, the present genetically modified immune cells are targeted to target proteins expressed on cancer cells. In some embodiments, a target protein is a tumor associated antigen (TAA). Examples of target proteins include, but are not limitedto, target proteins in B-cell malignancies such as CD19, CD20, CD22, and BCMA; target proteins on Acute Myeloid Leukemia such as CD33, CD123, CD70, CLL1 , Flt3, and IL1 Rap; and target proteins on solid tumors such as mesothelin. Further examples of target proteins include G protein-coupled receptor, family C, group 5, member D (GPRC5D), or transmembrane activator and CAML interactor (TACI).

[0033] In some embodiments, a target protein is a protein in the tumor micro-environment. A skilled artisan would appreciate that the terms “tumor microenvironment”, “cancer microenvironment”, “TME”, and “tumor milieu” may be used interchangeably having the same qualities and meanings and encompassing the microenvironment to tumor development. While the normal cellular microenvironment can inhibit malignant cell growth, the modifications that occur in the tumor microenvironment may synergistically support cell proliferation. Tumors shape their microenvironment and support the development of both tumor cells and non-malignant cells. In some instances, the tumor microenvironment affects angiogenesis by interfering with the signaling pathways required for cell recruitment and vascular construction. In addition, proteins secreted by the tumor modify the microenvironment by contributing growth factors and proteases that degrade the extracellular matrix and affect cell motility and adhesion. In some instances, stromal cells secrete extracellular matrix proteins, cytokines, growth factors, proteases, protease inhibitors, and endoglycosidases such as heparanase that would affect tumor growth.

[0034] In some embodiments, the present genetically modified immune cells express CARs targeting the target proteins described herein. Examples of CARs include, but are not limited to, anti-CD19 CAR, anti-CD20 CAR, anti-BCMA CAR, anti-GPRC5D CAR, or anti-TACI CAR. In some embodiments, a CAR is an anti-CD19 CAR. In some embodiments, a CAR is an anti-CD20 CAR. In some embodiments, a CAR is an anti- BCMA CAR. In some embodiments, a CAR is an anti-GPRC5D CAR. In some embodiments, a CAR is an anti-TACI CAR.

[0035] In some embodiments, the first immunomodulatory fusion protein (IFP) described herein comprises an ectodomain derived from a TNF superfamily protein generally known in the art, for example, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL),or Fas ligand (FasL). In some embodiments, an ectodomain of a first IFP comprises a TRAIL. In some embodiments, an ectodomain of a first IFP comprises a FasL. In some embodiments, the first IFP described herein comprises an endodomain derived from a costimulatory molecule of a TNF receptor superfamily protein generally known in the art, for example, CD137 (4-1 BB), 0X40 (CD134), CD27, CD40, BAFFR (CD268), GITR (CD357), BCMA (CD269), HVEM (CD270), TACI (CD267), CD30, tumor necrosis factorlike weak inducer of apoptosis (TWEAK) receptor, or lymphotoxin [3 receptor. In some embodiments, an endodomain of the first IFP is derived from CD137 (4-1 BB). In some embodiments, an endodomain of the first IFP is derived from 0X40 (CD134). In some embodiments, an endodomain of the first IFP is derived from CD27. In some embodiments, an endodomain of the first IFP is derived from CD40. In some embodiments, an endodomain of the first IFP is derived from BAFFR (CD268). In some embodiments, an endodomain of the first IFP is derived from GITR (CD357). In some embodiments, an endodomain of the first IFP is derived from BCMA (CD269). In some embodiments, an endodomain of the first IFP is derived from HVEM (CD270). In some embodiments, an endodomain of the first IFP is derived from TACI (CD267). In some embodiments, an endodomain of the first IFP is derived from CD30. In some embodiments, an endodomain of the first IFP is derived from TWEAK receptor. In some embodiments, an endodomain of the first IFP is derived from lymphotoxin [3 receptor.

[0036] In some embodiments, the present first immunomodulatory fusion protein comprises an ectodomain derived from TRAIL and an endodomain derived from the costimulatory molecule CD137 (4-1 BB) (SEQ ID N0:3). In another embodiment, the present first immunomodulatory fusion protein comprises an ectodomain derived from TRAIL and an endodomain derived from the costimulatory molecule 0X40 (SEQ ID N0:4). In another embodiment, the present first immunomodulatory fusion protein comprises an ectodomain derived from TRAIL and an endodomain derived from the costimulatory molecule CD27 (SEQ ID N0:5). In another embodiment, the present first immunomodulatory fusion protein comprises an ectodomain derived from TRAIL and an endodomain derived from the costimulatory molecule CD40 (SEQ ID N0:6). In another embodiment, the present first immunomodulatory fusion protein comprises an ectodomain derived from TRAIL and an endodomain derived from the costimulatorymolecule BAFFR (SEQ ID NO:7). In another embodiment, the present first immunomodulatory fusion protein comprises an ectodomain derived from TRAIL and an endodomain derived from the costimulatory molecule GITR (SEQ ID NO:8). In another embodiment, the present first immunomodulatory fusion protein comprises an ectodomain derived from TRAIL and an endodomain derived from the costimulatory molecule BCMA (SEQ ID NO:9). In another embodiment, the present first immunomodulatory fusion protein comprises an ectodomain derived from TRAIL and an endodomain derived from the costimulatory molecule HVEM (SEQ ID NQ:10). In another embodiment, the present first immunomodulatory fusion protein comprises an ectodomain derived from TRAIL and an endodomain derived from the costimulatory molecule TACI (SEQ ID NO:11 ). In another embodiment, the present first immunomodulatory fusion protein comprises an ectodomain derived from TRAIL and an endodomain derived from the costimulatory molecule TWEAK receptor (SEQ ID NO:12). In another embodiment, the present first immunomodulatory fusion protein comprises an ectodomain derived from TRAIL and an endodomain derived from the costimulatory molecule CD30 (SEQ ID NO:13). In another embodiment, the present first immunomodulatory fusion protein comprises an ectodomain derived from TRAIL and an endodomain derived from the costimulatory molecule lymphotoxin [3 receptor (SEQ ID NO:14).Table 1 : Representative Amino Acid Sequences of a 1stIFP - SEQ ID NOs:3-14.

[0037] In some embodiments, the present disclosure encompasses sequences that are at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequences disclosed herein, for example but not limited to identity as determined using BlastP software of the National Center of Biotechnology Information (NCBI) using default parameters. A skilled artisan would appreciate that percent identity provides a number that describes how similar the query sequence is to the target sequence (i.e., how many amino acids in each sequence are identical). The higher the percentage identity is, the more significant the match.In one embodiment, the present first immunomodulatory fusion protein comprises an ectodomain derived from FasL and an endodomain derived from the costimulatory molecule CD137 (4-1 BB) (SEQ ID NO:23). In another embodiment, the present first immunomodulatory fusion protein comprises an ectodomain derived from FasL and anendodomain derived from the costimulatory molecule 0X40 (SEQ ID NO:24). In another embodiment, the present first immunomodulatory fusion protein comprises an ectodomain derived from FasL and an endodomain derived from the costimulatory molecule CD27 (SEQ ID NO:25). In another embodiment, the present first immunomodulatory fusion protein comprises an ectodomain derived from FasL and an endodomain derived from the costimulatory molecule CD40 (SEQ ID NO:26). In another embodiment, the present first immunomodulatory fusion protein comprises an ectodomain derived from FasL and an endodomain derived from the costimulatory molecule SAFER (SEQ ID NO:27). In another embodiment, the present first immunomodulatory fusion protein comprises an ectodomain derived from FasL and an endodomain derived from the costimulatory molecule GITR (SEQ ID NO:28). In another embodiment, the present first immunomodulatory fusion protein comprises an ectodomain derived from FasL and an endodomain derived from the costimulatory molecule BCMA (SEQ ID NO:29). In another embodiment, the present first immunomodulatory fusion protein comprises an ectodomain derived from FasL and an endodomain derived from the costimulatory molecule HVEM (SEQ ID NQ:30). In another embodiment, the present first immunomodulatory fusion protein comprises an ectodomain derived from FasL and an endodomain derived from the costimulatory molecule TACI (SEQ ID N0:31 ). In another embodiment, the present first immunomodulatory fusion protein comprises an ectodomain derived from FasL and an endodomain derived from the costimulatory molecule TWEAK receptor (SEQ ID NO:32). In another embodiment, the present first immunomodulatory fusion protein comprises an ectodomain derived from FasL and an endodomain derived from the costimulatory molecule CD30 (SEQ ID NO:33). In another embodiment, the present first immunomodulatory fusion protein comprises an ectodomain derived from FasL and an endodomain derived from the costimulatory molecule lymphotoxin [3 receptor (SEQ ID NO:34).Table 2: Representative Amino Acid Sequences of a 1stIFP - SEQ ID NOs:23-34.

[0038] In some embodiments, the second immunomodulatory fusion protein described herein comprises an ectodomain derived from a TNF receptor superfamily protein generally known in the art, for example, Fas. In another embodiment, the TNF receptor superfamily protein is TRAIL receptor 1 (DR4). In another embodiment, the TNF receptor superfamily protein is TRAIL receptor 2 (DR5). In another embodiment, the TNF receptor superfamily protein is TRAIL receptor 3 (DcR1 ). In another embodiment, the TNF receptor superfamily protein is TRAIL receptor 4 (DcR2). In some embodiments, the present second immunomodulatory fusion protein comprises an endodomain derived from a common y-chain cytokine receptor such as CD132.

[0039] In some embodiments, the present second immunomodulatory fusion protein comprises an ectodomain derived from TRAIL receptor 1 (DR4) and an endodomain derived from the common y-chain cytokine receptor CD132 (SEQ ID NO:35). In another embodiment, the present second immunomodulatory fusion protein comprises anectodomain derived from TRAIL receptor 2 (DR5) and an endodomain derived from the common y -chain cytokine receptor CD132 (SEQ ID NO:36). In another embodiment, the present second immunomodulatory fusion protein comprises an ectodomain derived from TRAIL receptor 3 (DcR1 ) and an endodomain derived from the common y-chain cytokine receptor CD132 (SEQ ID NO:37). In another embodiment, the present second immunomodulatory fusion protein comprises an ectodomain derived from TRAIL receptor 4 (DcR2) and an endodomain derived from the common y-chain cytokine receptor CD132 (SEQ ID NO:38). In another embodiment, the present second immunomodulatory fusion protein comprises a Fas extracellular domain, a Fas transmembrane domain, and an endodomain derived from the common y-chain cytokine receptor CD132 (SEQ ID NO:2) (MLGIWTLLPLVLTSVARLSSKSVNAQVTDINSKGLELRKTVTTVETQNLEGLHHDGQF CHKPCPPGERKARDCTVNGDEPDCVPCQEGKEYTDKAHFSSKCRRCRLCDEGHGLE VEINCTRTQNTKCRCKPNFFCNSTVCEHCDPCTKCEHGIIKECTLTSNTKCKEEGSRS NLGWLCLLLLPIPLIVWVERTMPRIPTLKNLEDLVTEYHGNFSAWSGVSKGLAESLQPD YSERLCLVSEIPPKGGALGEGPGASPCNQHSPYWAPPCYTLKPET; Fas-CD132 fusion protein).

[0040] Table 3: Representative Amino Acid Sequences of a 2ndIFP - SEQ ID NOs:35- 38.

[0041] In some embodiments, the third immunomodulatory fusion protein described herein comprises an ectodomain derived from the same TNF receptor superfamily protein as in the second immunomodulatory fusion protein. In some embodiments, the present third immunomodulatory fusion protein comprises an endodomain derived from an a or p receptor subunit of the common y-chain cytokine receptor family, for example, IL-2R-p (CD122). In another embodiment, the present third immunomodulatory fusion protein comprises an endodomain derived from IL-7R-a (CD127). In another embodiment, the present third immunomodulatory fusion protein comprises an endodomain derived from IL-9R-a (CD129). In another embodiment, the present third immunomodulatory fusion protein comprises an endodomain derived from IL-21 R-a (CD360).

[0042] In some embodiments, the present third immunomodulatory fusion protein comprises a Fas extracellular domain, a Fas transmembrane domain, and an endodomain derived from IL-2R-p (SEQ ID NO:15). In another embodiment, the present third immunomodulatory fusion protein comprises a Fas extracellular domain, a Fas transmembrane domain, and an endodomain derived from IL-7R-a (SEQ ID NO:16). In another embodiment, the present third immunomodulatory fusion protein comprises a Fas extracellular domain, a Fas transmembrane domain, and an endodomain derived from IL-9R-a (SEQ ID NO:17). In another embodiment, the present third immunomodulatory fusion protein comprises a Fas extracellular domain, a Fas transmembrane domain, and an endodomain derived from IL-21 R-a (SEQ ID NO:18).Table 4: Representative Amino Acid Sequences of a 3rdIFP - SEQ ID NOs:15-18.

[0043] In some embodiments, the present third immunomodulatory fusion protein comprises DR4 and an endodomain derived from IL-2R-p (CD122) (SEQ ID NO:39). In another embodiment, the present third immunomodulatory fusion protein comprises DR4 and an endodomain derived from IL-7R-a (CD127) (SEQ ID NO:40). In another embodiment, the present third immunomodulatory fusion protein comprises DR4 and anendodomain derived from IL-9R-0 (CD129) (SEQ ID NO:41 ). In another embodiment, the present third immunomodulatory fusion protein comprises DR4 and an endodomain derived from IL-21 R-a (CD360) (SEQ ID NO:42). In some embodiments, the present third immunomodulatory fusion protein comprises DR5 and an endodomain derived from IL- 2R-|3 (CD122) (SEQ ID NO:43). In another embodiment, the present third immunomodulatory fusion protein comprises DR5 and an endodomain derived from IL- 7R-a (CD127) (SEQ ID NO:44). In another embodiment, the present third immunomodulatory fusion protein comprises DR5 and an endodomain derived from IL- 9R-a (CD129) (SEQ ID NO:45). In another embodiment, the present third immunomodulatory fusion protein comprises DR5 and an endodomain derived from IL- 21 R-a (CD360) (SEQ ID NO:46). In some embodiments, the present third immunomodulatory fusion protein comprises DcR1 and an endodomain derived from IL- 2R-|3 (CD122) (SEQ ID NO:47). In another embodiment, the present third immunomodulatory fusion protein comprises DcR1 and an endodomain derived from IL- 7R-a (CD127) (SEQ ID NO:48). In another embodiment, the present third immunomodulatory fusion protein comprises DcR1 and an endodomain derived from IL- 9R-a (CD129) (SEQ ID NO:49). In another embodiment, the present third immunomodulatory fusion protein comprises DcR1 and an endodomain derived from IL- 21 R-a (CD360) (SEQ ID NQ:50). In some embodiments, the present third immunomodulatory fusion protein comprises DcR2 and an endodomain derived from IL- 2R-|3 (CD122) (SEQ ID NO:51 ). In another embodiment, the present third immunomodulatory fusion protein comprises DcR2 and an endodomain derived from IL- 7R-a (CD127) (SEQ ID NO:52). In another embodiment, the present third immunomodulatory fusion protein comprises DcR2 and an endodomain derived from IL- 9R-a (CD129) (SEQ ID NO:53). In another embodiment, the present third immunomodulatory fusion protein comprises DcR2 and an endodomain derived from IL- 21 R-a (CD360) (SEQ ID NO:54).Table 5: Representative Amino Acid Sequences of a 3rdIFP - SEQ ID NOs:39-54.

[0044] In some embodiments, the present disclosure provides one or more isolated nucleotide sequences encoding one or more of the CAR, first immunomodulatory fusionprotein, second immunomodulatory fusion protein, and third immunomodulatory fusion protein described above. The various components of the various fusion proteins have been described herein. In some embodiments, the nucleotide sequences encode the CAR disclosed herein. In some embodiments, the nucleotide sequences encode the first immunomodulatory fusion protein disclosed herein. In some embodiments, the nucleotide sequences encode the second immunomodulatory fusion protein disclosed herein. In some embodiments, the nucleotide sequences encode the third immunomodulatory fusion protein disclosed herein. In another embodiment, the nucleotide sequences encode more than one of the above four fusion proteins; for example, the nucleotide sequences encode two, three or four of the above four fusion proteins (see Figure 2).

[0045] In some embodiments, the nucleotide sequences encode (1 ) a fusion protein comprising an ectodomain derived from Fas and an endodomain derived from IL-7R-oc, and (2) a fusion protein comprising an ectodomain derived from TRAIL and an endodomain derived from CD137 (4-1 BB) (SEQ ID NO:19). In another embodiment, the nucleotide sequences encode (1 ) a fusion protein comprising an ectodomain derived from Fas and an endodomain derived from IL-7R-a, and (2) a fusion protein comprising an ectodomain derived from TRAIL and an endodomain derived from 0X40 (SEQ ID NO:20). In another embodiment, the nucleotide sequences encode (1 ) a fusion protein comprising an ectodomain derived from Fas and an endodomain derived from IL-9R-a, and (2) a fusion protein comprising an ectodomain derived from TRAIL and an endodomain derived from CD137 (4-1 BB) (SEQ ID N0:21 ). In another embodiment, the nucleotide sequences encode (1 ) a fusion protein comprising an ectodomain derived from Fas and an endodomain derived from IL-9R-oc, and (2) a fusion protein comprising an ectodomain derived from TRAIL and an endodomain derived from 0X40 (SEQ ID NO:22).

[0046] In some embodiments, the isolated nucleotide sequence encoding one or more of the CAR, first immunomodulatory fusion protein, second immunomodulatory fusion protein, and third immunomodulatory fusion protein described above, comprises 1 , 2, 3, or 4 nucleotide sequences. In some embodiments, the isolated nucleotide sequence encoding one or more of the CAR, first immunomodulatory fusion protein, second immunomodulatory fusion protein, and third immunomodulatory fusion protein describedabove, comprises a single nucleotide sequence. In some embodiments, the isolated nucleotide sequence encoding one or more of the CAR, first immunomodulatory fusion protein, second immunomodulatory fusion protein, and third immunomodulatory fusion protein described above, comprises at least 2 nucleotide sequences. In some embodiments, the isolated nucleotide sequence encoding one or more of the CAR, first immunomodulatory fusion protein, second immunomodulatory fusion protein, and third immunomodulatory fusion protein described above, comprises at least 3 nucleotide sequences. In some embodiments, the isolated nucleotide sequence encoding one or more of the CAR, first immunomodulatory fusion protein, second immunomodulatory fusion protein, and third immunomodulatory fusion protein described above, comprises at least 4 nucleotide sequences.

[0047] In some embodiments, a single nucleotide sequence encodes (1 ) a fusion protein comprising an ectodomain derived from Fas and an endodomain derived from IL-7R-oc, and (2) a fusion protein comprising an ectodomain derived from TRAIL and an endodomain derived from CD137 (4-1 BB) (SEQ ID NO:19). In another embodiment, a single nucleotide sequence encodes (1 ) a fusion protein comprising an ectodomain derived from Fas and an endodomain derived from IL-7R-a, and (2) a fusion protein comprising an ectodomain derived from TRAIL and an endodomain derived from 0X40 (SEQ ID NO:20). In another embodiment, a single nucleotide sequence encodes (1 ) a fusion protein comprising an ectodomain derived from Fas and an endodomain derived from IL-9R-a, and (2) a fusion protein comprising an ectodomain derived from TRAIL and an endodomain derived from CD137 (4-1 BB) (SEQ ID NO:21 ). In another embodiment, a single nucleotide sequences encodes (1 ) a fusion protein comprising an ectodomain derived from Fas and an endodomain derived from IL-9R-cc, and (2) a fusion protein comprising an ectodomain derived from TRAIL and an endodomain derived from 0X40 (SEQ ID NO:22).

[0048] In some embodiments, at least 2, 3, or 4 nucleotide sequences encode (1 ) a fusion protein comprising an ectodomain derived from Fas and an endodomain derived from IL- 7R-oc, and (2) a fusion protein comprising an ectodomain derived from TRAIL and an endodomain derived from CD137 (4-1 BB) (SEQ ID NO:19). In another embodiment, atleast 2, 3, or 4 nucleotide sequences encode (1 ) a fusion protein comprising an ectodomain derived from Fas and an endodomain derived from IL-7R-oc, and (2) a fusion protein comprising an ectodomain derived from TRAIL and an endodomain derived from 0X40 (SEQ ID NO:20). In another embodiment, at least 2, 3, or 4 nucleotide sequences encode (1 ) a fusion protein comprising an ectodomain derived from Fas and an endodomain derived from IL-9R-a, and (2) a fusion protein comprising an ectodomain derived from TRAIL and an endodomain derived from CD137 (4-1 BB) (SEQ ID N0:21 ). In another embodiment, at least 2, 3, or 4 nucleotide sequences encode (1 ) a fusion protein comprising an ectodomain derived from Fas and an endodomain derived from IL- 9R-cx, and (2) a fusion protein comprising an ectodomain derived from TRAIL and an endodomain derived from 0X40 (SEQ ID NO:22).Table 6: Representative Nucleotide Sequences Encoding IFPs - SEQ ID NOs:19-22.

[0049] In another embodiment, the present disclosure provides one or more expression vectors comprising the isolated nucleotide sequence(s) described herein. The nucleotide sequence(s) can be put into a single expression vector or two expression vectors or more, as is known in the art, transformed into host cells, where they are expressed to form the IFP of the disclosure. In some embodiments, for example when a single nucleotide sequence is generally used, it can be inserted into an expression vector for transformation into immune cells, wherein the IFP are produced and translocated to the cell membrane. The nucleotide sequence(s) can be put into expression vector(s) that contain the appropriate transcriptional and translational control sequences, including, but not limited to, signal sequences, regulatory sequences, promoters, origins of replication, selection genes, etc.

[0050] In another embodiment, the present disclosure provides a method of treatingcancer in a subject in need thereof, comprising administering to the subject the population of genetically modified immune cells described herein. Examples of cancer to be treated include, but are not limited to, B cell acute lymphoblastic leukemia (B-ALL), B cell nonHodgkin lymphoma (B-NHL), or multiple myeloma. In some embodiments, the population of immune cells is administered to the subject intravenously.

[0051] In some embodiments, the terms “treat”, “treatment”, or “therapy” (as well as different forms thereof) refer to either therapeutic treatment or prophylactic or preventative measures, wherein the object is to prevent or lessen the targeted pathologic condition or disorder as described herein. Thus, in some embodiments, treating may include directly affecting or curing, suppressing, inhibiting, preventing, reducing the severity of, delaying the onset of, reducing symptoms associated with the disease, disorder or condition, or a combination thereof. Thus, in some embodiments, "treating" refers to delaying progression, expediting remission, inducing remission, augmenting remission, speeding recovery, increasing efficacy of or decreasing resistance to alternative therapeutics, or a combination thereof. In some embodiments, "preventing" refers to delaying the onset of symptoms, preventing relapse to a disease, decreasing the number or frequency of relapse episodes, increasing latency between symptomatic episodes, or a combination thereof. In some embodiments, "suppressing" or "inhibiting", refers to reducing the severity of symptoms, reducing the severity of an acute episode, reducing the number of symptoms, reducing the incidence of disease-related symptoms, reducing the latency of symptoms, ameliorating symptoms, reducing secondary symptoms, reducing secondary infections, prolonging patient survival, or a combination thereof.

[0052] The present disclosure also encompasses a composition comprising the population of genetically modified immune cells disclosed herein and a pharmaceutically acceptable carrier. A population of the present genetically modified immune cells can be formulated in a composition with a pharmaceutically acceptable carrier according to methods generally known in the art.

[0053] Compositions suitable for use in the methods disclosed herein include compositions comprising the genetically modified immune cells as described herein are contained in an amount effective to achieve the intended purpose. In some embodiments,a therapeutically effective amount means an amount of the genetically modified immune cells effective to prevent, alleviate or ameliorate symptoms of disease (e.g., cancer) or prolong the survival of the subject being treated. Determination of a therapeutically effective amount is well within the capability of those skilled in the art. The exact formulation, route of administration and dosage can be chosen by one of ordinary skill in the art in view of the patient's condition. The amount of a composition to be administered is dependent on, e.g., the subject being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, etc. Depending on the severity and responsiveness of the condition to be treated, dosing can be of a single or a plurality of administrations, with course of treatment lasting from several days to several weeks or until cure is effected or diminution of the disease state is achieved.

[0054] Administration of a composition comprising the present genetically modified immune cells can be systemic or local. In some embodiments, it would be desirable to administer a composition of the present disclosure locally to the area in need of treatment; this may be achieved by, for example, and not by way of limitation, local infusion, by injection, by means of a catheter, or by means of an implant, said implant being of a porous, non-porous, or gelatinous material.

[0055] Unless otherwise defined, all technical and / or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the invention, exemplary methods and / or materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting. Each literature reference or other citation referred to herein is incorporated herein by reference in its entirety.

[0056] As used herein, the singular form "a", "an" and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a compound" or "at least one compound" may include a plurality of compounds, including mixtures thereof.

[0057] Throughout this application, various embodiments of this invention may bepresented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub ranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed sub ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1 , 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

[0058] Whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range. The phrases “ranging / ranges between” a first indicate number and a second indicate number and “ranging / ranges from” a first indicate number “to” a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals there between.

[0059] In the description presented herein, embodiments of the genetically modified immune cells and methods of making the same have been described. In the description presented herein, each of the steps of the methods of use of the genetically modified immune cells and variations thereof are described. This description is not intended to be limiting and changes in the components, sequence of steps, and other variations would be understood to be within the scope of the present invention.

[0060] It is appreciated that certain features of the genetically modified immune cells and methods of making and using these cells, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the genetically modified immune cells and methods of making and using these cells, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements.

[0061] Various embodiments and aspects of the genetically modified immune cells and methods of making and using these cells as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples.EXAMPLE 1Genetically Modified Immune Cells

[0062] Objective To make and use genetically modified immune cells expressing a chimeric antigen receptor (CAR) and immunomodulatory fusion proteins (IFPs) in order to improve CART efficacy, e.g., efficacy of immunotherapy by the genetically modified immune cells.

[0063] Methods: The genetically modified immune cells disclosed herein can be derived from various kinds of immune cells, including but are not limited to, NK cells, T cells, NKT- cells, yb T cells, mucosal-associated invariant T (MAIT) cells, macrophages, or cytokine- induced killer cells. In some embodiments, these various immune cells (referred as parental cells) are first isolated or generated from human samples using standard procedures known in the art, and then sequences encoding the fusion proteins described herein are delivered to these parental immune cells using constructs and methodologies generally known in the art.

[0064] For illustration purposes, generation of the present genetically modified immune cells from T cells is described below. T cells can be generated by standard methods widely used in the field. See for example, Int. J. Mol. Sci. (2022) 23:3154; STAR Protoc. (2021 ) 2:100920; Methods in Cell Biology (2024) 183:303. Similar methods could be used to generate genetically modified NK cells.

[0065] For example, T cells can be purified from fresh or frozen peripheral blood mononuclear cells using magnetic cell separation tools and reagents, e.g., from Miltenyi Biotec or STEMCELL Technology. Purified T cells can be activated with T cell activation reagents, e.g., CD3 / CD28 T-Activator Dynabeads (Thermo Fisher Scientific, #1 1131 D), or ImmunoCult (STEMCELL Technology, #10971 ), or T Cell TransAct (Miltenyi Biotec, #130-11 1 -160), and IL-7 and IL-15 cytokines in T cell expansion media, e.g. lmmunoCult™-XF T Cell Expansion Medium (STEMCELL, #10981 ), or Xuri T cellexpansion medium (Cytiva, #29185230), or CTS OpTmizer™ T Cell Expansion SFM (GIBCO, #A1048501 ), for 3 days. Then, sequence(s) encoding the CAR and fusion proteins described herein can be delivered to T cells as individual genes or as a part of a construct separated by self-cleaving peptide sequences as described herein, e.g., furin 2A sequences (see Figure 2 and Figures 5A-5I). In some embodiments, the sequences to be delivered include those encoding (i) a chimeric antigen receptor (CAR); (ii) a first immunomodulatory fusion protein comprising an ectodomain derived from a protein of tumor necrosis factor (TNF) superfamily, and an endodomain derived from a costimulatory molecule of TNF receptor superfamily protein; (iii) a second immunomodulatory fusion protein comprising an ectodomain derived from a protein of TNF receptor superfamily, and an endodomain derived from a common y-chain cytokine receptor; and (iv) a third immunomodulatory fusion protein comprising an ectodomain derived from the same TNF receptor superfamily protein as in the second immunomodulatory fusion protein, and an endodomain derived from an a or [3 receptor subunit of the common y-chain cytokine receptor family.

[0066] In some embodiments, the sequences are delivered to the T cells by electroporation using MaxCyte electroporator e.g., ATx, or STx, or Lonza nucleofector e.g., 4D-Nucleofector X (catalog #: AAF-1003X), according to the protocol recommended by manufacturer and expanded for 6 more days in G-Rex vessels. After expansion, T cells can be harvested and analyzed for transduction efficiency, total cell number and viability using flow cytometry.

[0067] Results'. As discussed herein, the present genetically modified immune cells expressing the CAR and the first, second and third immunomodulatory fusion proteins are expected to have enhanced functions against target cells as compared to those cells that only express the CAR. For example, the present genetically modified immune cells would have enhanced anti-tumor activities against the target cells in vitro or in vivo. Enhanced in vitro activities include, but are not limited to, killing of target cells at lower effector-to-target ratio, stronger proliferation of the genetically modified T cells, or higher resistance to exhaustion during repetitive stimulation assay. Enhanced in vivo activities include, but are not limited to, achieving a tumor-free status faster and maintain this condition longer than in control group.

[0068] In one embodiment, the function of the genetically modified immune cells can be examined in an antigen stress test (see Figure 3). Briefly, the genetically modified immune cells (e.g., CAR T cells) are repeatedly challenged with target cells. Initially, the modified immune cells and the target cells are mixed at a single ratio, e.g., 1 :1. Growth of the modified immune cells and target cell clearance are measured 3-4 days later. Then the modified immune cells and the target cells are reset at a single ratio, e.g., 1 :1 , and growth of the modified immune cells and target cell clearance are measured 3-4 days later. This growth and challenge cycle is repeated until the modified immune cells can no longer clear the target cells.

[0069] In another embodiment, the function of the genetically modified immune cells can be examined in a single challenge assay (see Figure 4). In one embodiment, the genetically modified immune cells (e.g., CAR T cells) are mixed with target cells at various effector:target ratios, with constant number of target cells and titrated down number of modified immune cells. Growth of the modified immune cells and targeted cell killing (targeted cytotoxicity) are measured 4 days later, e.g., by flow cytometry. Target cell killing is normalized to culture with target cells only. Methods for measuring cytotoxicity are well known in the art, for example but not limited to methods disclosed in Kiesgen et al., Comparative analysis of assays to measure CAR T-cell-mediated cytotoxicity. Nat Protoc. 2021 Mar;16(3):1331 -1342.

[0070] While certain features of the genetically modified immune cells and methods of making and using these cells have been illustrated and described herein, many modifications, substitutions, changes, and equivalents will now occur to those of ordinary skill in the art. It is, therefore, to be understood that the appended claims are intended to cover all such modifications and changes as fall within the true spirit of these cells and their uses.

Claims

CLAIMSWhat is claimed is:1 . A population of genetically modified immune cells, said modified immune cells comprising(i) a chimeric antigen receptor (CAR) targeting a target protein on a target cell;(ii) a first immunomodulatory fusion protein that induces cell death in the target cell, said first fusion protein comprises an ectodomain derived from a protein of tumor necrosis factor (TNF) superfamily, and an endodomain derived from a costimulatory molecule of a TNF receptor superfamily protein;(iii) a second immunomodulatory fusion protein that prevents cell death in the genetically modified immune cell, said second fusion protein comprises an ectodomain derived from a protein of TNF receptor superfamily, and an endodomain derived from a common y-chain cytokine receptor; and(iv) a third immunomodulatory fusion protein comprising an ectodomain derived from the same TNF receptor superfamily protein as in the second immunomodulatory fusion protein, and an endodomain derived from an a or P receptor subunit of the common y-chain cytokine receptor family.

2. The population of genetically modified immune cells of claim 1 , wherein the immune cell comprises a NK cell, a T cell, a NKT-cell, a yd T cell, a mucosal- associated invariant T (MAIT) cell, a macrophage, or a cytokine-induced killer cell.

3. The population of genetically modified immune cells of claim 1 , wherein the target cell is a cancer cell expressing said target protein on its surface, wherein optionally the target protein comprises CD19, CD20, B cell maturation antigen (BCMA), G protein-coupled receptor, family C, group 5, member D (GPRC5D), or transmembrane activator and CAML interactor (TACI).

4. The population of genetically modified immune cells of claim 1 , wherein the CAR comprises an anti-CD19 CAR, anti-CD20 CAR, anti-BCMA CAR, anti-GPRC5D CAR, or anti-TACI CAR.

5. The population of genetically modified immune cells of claim 1 , wherein the TNF superfamily protein comprises tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) or Fas ligand (FasL).

6. The population of genetically modified immune cells of claim 1 , wherein the protein of TNF receptor superfamily comprises CD137 (4-1 BB), 0X40 (CD134), CD27, CD40, BAFFR (CD268), GITR (CD357), BCMA (CD269), HVEM (CD270), TACI (CD267), CD30, tumor necrosis factor-like weak inducer of apoptosis (TWEAK) receptor, or lymphotoxin [3 receptor (LTbR).

7. The population of genetically modified immune cells of claim 1 , wherein the TNF receptor superfamily protein of the second and third fusion protein comprises TRAIL receptor 1 (DR4), TRAIL receptor 2 (DR5), TRAIL receptor 3 (DcR1 ), TRAIL receptor 4 (DcR2), or Fas (CD95).

8. The population of genetically modified immune cells of claim 1 , wherein(a) the common y-chain cytokine receptor comprises CD132; or(b) the a or p receptor subunit comprises IL-2R-P (CD122), IL-7R-a (CD127), IL-9R-a (CD129), or IL-21 R-a (CD360); or(c) any combination of (a) and (b).

9. The population of genetically modified immune cells of claim 1 , wherein(a) the first immunomodulatory fusion protein has the amino acid sequence of any one of SEQ ID NOs:3-14, or 23-34; or(b) the second immunomodulatory fusion protein has the amino acid sequence of any one of SEQ ID NOs:2, or 35-38; or(c) the third immunomodulatory fusion protein has the amino acid sequenceof any one of SEQ ID NOs:15-18, or 39-54; or(d) any combination of (a), (b), and (c).

10. One or more isolated nucleotide sequences encoding one or more of(i) a chimeric antigen receptor (CAR) targeting a target protein on a target cell;(ii) a first immunomodulatory fusion protein that induces cell death in the target cell, said first fusion protein comprises an ectodomain derived from a protein of tumor necrosis factor (TNF) superfamily, and an endodomain derived from a costimulatory molecule of a TNF receptor superfamily protein;(iii) a second immunomodulatory fusion protein that prevents cell death in the genetically modified immune cell, said second fusion protein comprises an ectodomain derived from a protein of TNF receptor superfamily, and an endodomain derived from a common y-chain cytokine receptor; and(iv) a third immunomodulatory fusion protein comprising an ectodomain derived from the same TNF receptor superfamily protein as in the second immunomodulatory fusion protein, and an endodomain derived from an a or P receptor subunit of the common y-chain cytokine receptor family.1 1. The isolated nucleotide sequence(s) of claim 10, wherein the TNF superfamily protein comprises tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), or Fas ligand (FasL).

12. The isolated nucleotide sequence(s) of claim 10, wherein the protein of TNF receptor superfamily comprises CD137 (4-1 BB), 0X40 (CD134), CD27, CD40, BAFFR (CD268), GITR (CD357), BCMA (CD269), HVEM (CD270), TACI (CD267), CD30, tumor necrosis factor-like weak inducer of apoptosis (TWEAK) receptor, or lymphotoxin p receptor.

13. The isolated nucleotide sequence(s) of claim 10, wherein the TNF receptor superfamily protein of the second and third fusion protein comprises TRAILreceptor 1 (DR4), TRAIL receptor 2 (DR5), TRAIL receptor 3 (DcR1 ), TRAIL receptor 4 (DcR2), or Fas (CD95).

14. The isolated nucleotide sequence(s) of claim 10, wherein(a) the common y-chain cytokine receptor comprises CD132; or(b) the a or p receptor subunit comprises IL-2R- (CD122), IL-7R-a (CD127), IL-9R-a (CD129), or IL-21 R-a (CD360); or(c) any combination of (a) and (b).

15. The isolated nucleotide sequence(s) of claim 10, comprising the sequence of any one of SEQ ID NOs:19-22.

16. One or more expression vectors comprising the isolated nucleotide sequence or sequences of claim 10.

17. A composition comprising the population of genetically modified immune cells of claim 1 and a pharmaceutically acceptable carrier.

18. A composition comprising the nucleotide sequence of claim 10 and a pharmaceutically acceptable carrier.

19. A method of treating cancer in a subject in need thereof, comprising administering to said subject the population of genetically modified immune cells of claim 1 , thereby treating cancer in a subject, wherein optionally the population of immune cells is administered intravenously.

20. The method of claim 19, wherein the cancer is B cell acute lymphoblastic leukemia (B-ALL), B cell non-Hodgkin lymphoma (B-NHL), or multiple myeloma.

Images