Membrane-anchored cytokine fusion proteins and uses thereof

Abstract

Provided are cytokine fusion proteins including a cytokine region, a transmembrane region, and a costimulatory domain. These cytokine fusion proteins, as expressed in immune cells, function in combination with a surface receptor, such as CAR or TCR, and demonstrated superior cytotoxicity, in vivo anti-tumor efficacy, and enhancement of T-cell proliferation capacity. The cytokine fusion proteins of the present disclosure can be suitably used for treating diseases such as cancer.

Description

CYTOKINE FUSION PROTEIN AND USES THEREOFCROSS-REFERENCE TO RELATED APPLICATION(S)

[0001] This application claims the benefit of U. S. Provisional Patent Application No.63 / 749,103, filed on January 24, 2025 and of U. S. Provisional Patent Application No.63 / 885,909, filed on September 22, 2025, both of which applications are incorporated herein by reference in their entirety.INCORPORATION BY REFERENCE OF SEQUENCE LISTING

[0002] The application contains a Sequence Listing which has been submitted electronically in. XML format and is hereby incorporated by reference in its entirety. Said. XML copy, created on January 15, 2026, is named “96SA-401041-WO_SL.xml” and is 122,199 bytes in size. The sequence listing contained in this. XML file is part of the specification and is hereby incorporated by reference herein in its entirety.BACKGROUND

[0003] T cell receptors (TCRs) specifically bind to peptide-MHC complexes on antigen-presenting cells (APCs), a key step for adaptive immunity that requires costimulatory signals, like CD28-B7 interaction, to fully activate T cells. Once activated, T cells proliferate and differentiate into effector or memory T cells, with CD4+ T cells orchestrating immune responses and CD8+ T cells eliminating pathogens or tumor cells, ensuring a balanced defense while preventing autoimmunity.

[0004] Costimulatory signaling is crucial for T cell activation, providing a necessary second signal in addition to TCR engagement. It involves interactions such as CD28 with B7 molecules on APCs, ensuring T cells activate only in response to genuine threats. This mechanism not only prevents autoimmunity but also influences T cell proliferation, survival, differentiation, and memory formation, maintaining a balanced immune response.

[0005] Cytokine signaling, essential for T cell function, involves cytokines acting as intercellular messengers to regulate T cell development, differentiation, and effector functions. Following antigen recognition and costimulatory signaling, T cells produce and respond to cytokines, triggering signaling cascades that guide their proliferation, survival, and differentiation into specific subsets, facilitating a precise immune response and playing roles in autoimmune and inflammatory conditions.

[0006] Native cytokine-based monotherapies face challenges like short half-life, requiring frequent high doses and risking toxicity, including vascular leakage and CNS effects.Systemic administration of cytokines, such as interleukin- 12 (IL- 12) presents significant challenges due to its potential to cause cytokine release syndrome (CRS), a severe inflammatory response characterized by symptoms like fever and, in extreme cases, organ failure. Low biodistribution, narrow therapeutic windows, pathway redundancy, immunosuppressive actions, and pleiotropy complicate treatment outcomes, underscoring the need for improved cytokine therapy strategies for specificity and safety.

[0007] Cytokine fusion proteins emerge as a promising solution to overcome the limitations of native cytokine-based monotherapies and CAR-T or TCR-T therapies in cancer treatment, and offering a safe and tolerable approach to cancer immunotherapy.SUMMARY

[0008] Provided in the present disclosure is a membrane bound cytokine fusion protein comprising a costimulatory domain. Intracellular costimulatory signaling domain can potentially provide cytotoxicity and long-term persistence in modified T cells. The cytokines, such as interleukin, for example IL- 12 or IL- 15, possess potent tumor-suppressor activity including persistent cytotoxic activity of T cells, improve antigen presentation, and reshape endogenous immune inhibitory cells within the tumor microenvironment (TME). The membrane bound cytokine fusion proteins of the present disclosure, with the costimulatory domains described herein, such as the 4- IBB intracellular domain, exhibit potent cytotoxicity and in vivo anti-tumor efficacy, and are safe and tolerable in different animal models of solid tumors.

[0009] In one aspect, provided herein is a fusion protein comprising, from N to C terminus, (a) a cytokine region; (b) a transmembrane region; and (c) a costimulatory domain.

[0010] In certain embodiments, the cytokine region is selected from the group consisting of a polypeptide of interleukin- 12 (IL-12), IL-15, IL-2, IL-4, IL-7, IL-9, IL-18, and IL-21 or a functional portion thereof.

[0011] In certain embodiments, the cytokine region is IL- 12 polypeptide or a functional portion thereof.

[0012] In certain embodiments, wherein the IL- 12 polypeptide or a functional portion thereof comprises a IL-12α subunit and a IL-12β subunit, wherein the IL12α subunit and the IL-12β subunit are connected via a linker and form a heterodimer.

[0013] In certain embodiments, the IL- 12 polypeptide or a functional portion thereof comprises the amino acid sequence of SEQ ID NO: 9 or a peptide having at least 85% sequence identity to SEQ ID NO: 9.

[0014] In certain embodiments, the cytokine region comprises at least a first polypeptide selected from the group consisting of a polypeptide of interleukin- 12 (IL- 12), IL- 15, IL-2, IL-4, IL-7, IL-9, IL- 18, and IL-21 or a functional portion thereof, and a second polypeptide selected from the group consisting of a polypeptide of interleukin- 12 (IL- 12), IL- 15, IL-2, IL-4, IL-7, IL-9, IL-18, and IL-21 or a functional portion thereof.

[0015] In certain embodiments, the cytokine region comprises from N terminus to C terminus the first polypeptide comprising IL- 15 or a functional fragment thereof and the second polypeptide comprising IL- 18 or a functional fragment thereof.

[0016] In certain embodiments, the cytokine region comprises from N terminus to C terminus the first polypeptide comprising IL- 18 or a functional fragment thereof and the second polypeptide comprising IL- 15 or a functional fragment thereof.

[0017] In certain embodiments, the transmembrane region is selected from the group consisting of a transmembrane domain of CD28, CD80, EGFR, 4- IBB, an alpha chain of a T cell receptor, a beta chain of a T cell receptor, a zeta chain of a T cell receptor, CD3 epsilon, CD4, CD5, CD8 alpha, CD9, CD16, CD19, CD22, CD33, CD37, CD45, CD64, CD86, CD 134, CD 137, and CD 154.

[0018] In certain embodiments, the transmembrane region is the CD28 transmembrane domain.

[0019] In certain embodiments, the CD28 transmembrane domain comprises the amino acid sequence of SEQ ID NO: 4 or a peptide having at least 85% sequence identity to SEQ ID NO: 4.

[0020] In certain embodiments, the transmembrane region is the transmembrane domain of CD80.

[0021] In certain embodiments, the CD80 transmembrane domain comprises the amino acid sequence of SEQ ID NO: 3 or a peptide having at least 85% sequence identity to SEQ ID NO: 3.

[0022] In certain embodiments, the costimulatory domain is selected from the group consisting of an intracellular domain (or other suitable portion) of CD28, OX-40, 4-1BB / CD137, CD2, CD7, CD27, CD30, CD40, Programmed Death-1 (PD-1), inducible T cell costimulator (ICOS), lymphocyte function-associated antigen-1 (LFA-1, CDl-la / CD18), CD3 gamma, CD3 delta, CD3 epsilon, CD247, CD276 (B7-H3), LIGHT, (TNFSF14), NKG2C, Ig alpha (CD79a), DAP-10, Fc gamma receptor, MHC class 1 molecule, TNF receptor proteins, an Immunoglobulin protein, cytokine receptor, integrins, Signaling Lymphocytic Activation Molecules (SLAM proteins), activating NK cell receptors, BTLA, a Toll ligand receptor, ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8alpha, CD8beta, IL-2R beta, IL-2R gamma, IL-7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, GDI Id, ITGAE, CD103, ITGAL, GDI la, LFA-1, ITGAM, CD1 lb, ITGAX, GDI 1c, ITGB1, CD29, ITGB2, CD 18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly 108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD 162), LTBR, LAT, GADS, SLP-76, PAG / Cbp, CD 19a, a ligand that specifically binds with CD83, or any combination thereof.

[0023] In certain embodiments, the costimulatory domain is the intracellular domain of CD28. In certain embodiments, the intracellular domain of CD28 comprises the amino acid sequence of SEQ ID NO: 5 or a peptide having at least 85% sequence identity to SEQ ID NO: 5.

[0024] In certain embodiments, the costimulatory domain is the intracellular domain of 4-1BB. In certain embodiments, the intracellular domain of 4-1BB comprises the amino acid sequence of SEQ ID NO: 6 or a peptide having at least 85% sequence identity to SEQ ID NO: 6.

[0025] In certain embodiments, the fusion protein comprising from N to C terminus, (i) the cytokine region comprising the IL- 12 polypeptide or functional portion thereof, the transmembrane region comprising the CD28 transmembrane domain, and the costimulatory domain comprising the intracellular domain of CD28;(ii) the cytokine region comprising the IL-12 polypeptide or functional portion thereof, the transmembrane region comprising the CD28 transmembrane domain, and the costimulatory domain comprising the intracellular domain of 4- IBB;(iii) the cytokine region of IL- 12 polypeptide or functional portion thereof, the transmembrane region comprising the CD80 transmembrane domain, and the costimulatory domain comprising the intracellular domain of 4- IBB; or(iv) the cytokine region of IL- 12 polypeptide or functional portion thereof, the transmembrane region comprising the CD80 transmembrane domain, and the costimulatory domain of the intracellular domain of CD28.

[0026] In certain embodiments, the fusion protein comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 10-14, or a peptide having at least 85%, 90%, 92%, 95%, or 99% sequence identity to an amino acid sequence sequence selected from the group consisting of SEQ ID NOs: 10-14. In certain embodiments, the fusion protein further comprises a hinge region between the cytokine region and the transmembrane region.

[0027] In certain embodiments, the fusion protein comprises from N to C terminus (i) the cytokine region comprising the first polypeptide comprising IL- 15 or a functional fragment thereof and the second polypeptide comprising IL- 18 or a functional fragment thereof, the transmembrane region comprising the CD28 transmembrane domain, and the costimulatory domain comprising the intracellular domain of 4- IBB; (ii) the cytokine region comprising the first polypeptide comprising IL- 15 or a functional fragment thereof and the second polypeptide comprising IL- 18 or a functional fragment thereof, the transmembrane region comprising the CD80 transmembrane domain, and the costimulatory domain comprising the intracellular domain of 4-1BB; (iii) the cytokine region comprising the first polypeptide comprising IL- 15 or a functional fragment thereof and the second polypeptide comprising IL-18 or a functional fragment thereof, the transmembrane region comprising the CD28 transmembrane domain, and the costimulatory domain comprising the intracellular domain ofCD28; (iv) the cytokine region comprising the first polypeptide comprising IL-15 or a functional fragment thereof and the second polypeptide comprising IL- 18 or a functional fragment thereof, the transmembrane region comprising the CD80 transmembrane domain, and the costimulatory domain comprising the intracellular domain of CD28; (v) the cytokine region comprising the first polypeptide comprising IL- 18 or a functional fragment thereof and the second polypeptide comprising IL- 15 or a functional fragment thereof, the transmembrane region comprising the CD28 transmembrane domain, and the costimulatory domain comprising the intracellular domain of 4-1BB; (vi) the cytokine region comprising the first polypeptide comprising IL- 18 or a functional fragment thereof and the second polypeptide comprising IL- 15 or a functional fragment thereof, the transmembrane region comprising the CD80 transmembrane domain, and the costimulatory domain comprising the intracellular domain of 4- IBB; (vii) the cytokine region comprising the first polypeptide comprising IL-18 or a functional fragment thereof and the second polypeptide comprising IL-15 or a functional fragment thereof, the transmembrane region comprising the CD28 transmembrane domain, and the costimulatory domain comprising the intracellular domain of CD28; or (viii) the cytokine region comprising the first polypeptide comprising IL-18 or a functional fragment thereof and the second polypeptide comprising IL- 15 or a functional fragment thereof, the transmembrane region comprising the CD80 transmembrane domain, and the costimulatory domain comprising the intracellular domain of CD28.

[0028] In certain embodiments, the fusion protein comprises an amino acid sequence of SEQ ID NO: 64 or SEQ ID NO: 66, or a peptide having at least 85%, 90%, 92%, 95%, or 99% sequence identity to SEQ ID NO: 64 or SEQ ID NO: 66.

[0029] In certain embodiments, the fusion protein further comprises a hinge region between the cytokine region and the transmembrane region. In certain embodiments, the hinge region is selected from a hinge domain of IgGl, IgG2, IgG3, IgG4, IgA, IgD, IgE, IgM, CD28, or CD8 alpha, any truncation thereof, or any combination thereof.

[0030] In one aspect, provided herein is a fusion protein comprising from N to C terminus, a cytokine region, a transmembrane region, and a costimulatory domain comprising intracellular domain of 4- IBB.

[0031] In certain embodiments, the intracellular domain of 4-1BB comprises the amino acid sequence of SEQ ID NO: 6 or a peptide having at least 85% sequence identity to SEQ ID NO: 6.

[0032] In certain embodiments, the cytokine region is selected from the group consisting of a polypeptide of interleukin- 12 (IL- 12), IL- 15, IL-2, IL-4, IL-7, IL-9, IL- 18, and IL-21 or a functional portion thereof.

[0033] In certain embodiments, the transmembrane region is selected from the group consisting of a transmembrane domain of CD28, CD80, EGFR, 4- IBB, an alpha chain of a T cell receptor, a beta chain of a T cell receptor, a zeta chain of a T cell receptor, CD3 epsilon, CD4, CD5, CD8 alpha, CD9, CD16, CD19, CD22, CD33, CD37, CD45, CD64, CD86, CD134, CD137, and CD154.

[0034] In one aspect, provided herein is a fusion protein comprising, from N to C terminus, (a) a cytokine region; and (b) a transmembrane region.

[0035] In certain embodiments, the cytokine region is selected from the group consisting of a polypeptide of interleukin- 12 (IL- 12), IL- 15, IL-2, IL-4, IL-7, IL-9, IL- 18, and IL-21 or a functional portion thereof. In certain embodiments, the cytokine region is a polypeptide of IL- 12 or a functional portion thereof. In certain embodiments, the polypeptide of IL- 12 or the functional portion thereof comprises a IL-12α subunit and a IL-12β subunit, wherein the IL12α subunit and the IL-12β subunit are connected via a linker and form a heterodimer.

[0036] In certain embodiments, the polypeptide of IL- 12 or the functional portion thereof comprises the amino acid sequence of SEQ ID NO: 9 or a peptide having at least 85% sequence identity to SEQ ID NO: 9. In certain embodiments, the polypeptide of IL- 12 or the functional portion thereof comprises the amino acid sequence of SEQ ID NO: 9.

[0037] In certain embodiments, the transmembrane region is selected from the group consisting of a transmembrane domain of CD28, CD80, EGFR, 4- IBB, an alpha chain of a T cell receptor, a beta chain of a T cell receptor, a zeta chain of a T cell receptor, CD3 epsilon, CD4, CD5, CD8 alpha, CD9, CD16, CD19, CD22, CD33, CD37, CD45, CD64, CD86, CD134, CD137, and CD154.

[0038] In certain embodiments, the transmembrane region is a transmembrane domain of CD28. In certain embodiments, the transmembrane domain of CD28 comprises the amino acid sequence of SEQ ID NO: 4 or a peptide having at least 85% sequence identity to SEQ ID NO: 4.

[0039] In certain embodiments, the transmembrane domain is a transmembrane domain of CD80. In certain embodiments, the transmembrane domain of CD80 comprises the amino acid sequence of SEQ ID NO: 3 or a peptide having at least 85% sequence identity to SEQ ID NO: 3.

[0040] In certain embodiments, (i) the cytokine region comprises a polypeptide of interleukin- 12 (IL- 12) or a functional portion thereof, and the transmembrane region comprises a transmembrane domain of CD28; or (ii) the cytokine region comprises a polypeptide of interleukin- 12 (IL- 12) or a functional portion thereof, and the transmembrane region comprises a transmembrane domain of CD80.

[0041] In certain embodiments, the fusion protein comprises the amino acid sequence of SEQ ID NO: 10 or an amino acid sequence having at least 85%, 90%, 92%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 10. In certain embodiments, the fusion protein comprises the amino acid sequence of SEQ ID NO: 10.

[0042] In certain embodiments, the fusion protein comprises the amino acid sequence of SEQ ID NO: 11 or an amino acid sequence having at least 85%, 90%, 92%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 11. In certain embodiments, the fusion protein comprises the amino acid sequence of SEQ ID NO: 11.

[0043] In one aspect, provided herein is a polynucleotide encoding the fusion protein of any one of the embodiments disclosed herein.

[0044] In another aspect, provided herein is an expression vector comprising an expressible nucleic acid sequence comprising the polynucleotide encoding the fusion protein of any one of the embodiments disclosed herein.

[0045] In certain embodiments, the expression vector further comprises a promoter operably linked to the expressible nucleic acid sequence. In certain embodiments, the promoter is constitutive or inducible.

[0046] In certain embodiments, the constitutive promoter is selected from the group consisting of EF1α, CMV, SV40 or CAG. In certain embodiments, the constitutive promoter is EF1α or CMV.

[0047] In certain embodiments, the inducible promoter is selected from the group consisting of alcohol-regulated promoters, tetracycline-regulated promoters, steroid-regulated promoters, metal-regulated promoters, pathogenesis-regulated promoters, temperature / heat-inducible promoters, and light-regulated promoters. In certain embodiments, the inducible promoter is NF AT.

[0048] In certain embodiments, the expression vector further comprises a woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) and / or a poly A tail 3’ end to the expressible nucleic acid sequence.

[0049] In certain embodiments, the expressible nucleic acid sequence further comprises a second protein encoding sequence.

[0050] In certain embodiments, the fusion protein encoding polynucleotide and the second protein encoding sequence are linked bicistronically.

[0051] In certain embodiments, the fusion protein encoding polynucleotide and the second protein encoding sequence are separated by a self-cleaving peptide or an internal ribosome entry site (IRES).

[0052] In certain embodiments, the self-cleaving protein is a 2A peptide.

[0053] In certain embodiments, the 2A peptide is selected from the group consisting of Foot-and-Mouth Disease Virus 2A (F2A), Thosea asigna virus 2A (T2A), Porcine teschovirus- 1 2A (P2A), Equine rhinitis A virus (E2A), and Thosea asigna Virus Short 2A (S2A).

[0054] In certain embodiments, the expression vector comprises from 5’ to 3’, an EF1α promoter and the expressible nucleic acid sequence comprises a polynucleotide encoding the fusion protein, a T2A peptide, and a woodchuck hepatitis virus posttranscriptional regulatory element (WPRE).

[0055] In certain embodiments, the fusion protein comprises a signal peptide.

[0056] In certain embodiments, the second protein comprises a signal peptide.

[0057] In yet another aspect, the present disclosure provides an isolated immune cell comprising the fusion protein, the fusion protein encoding polynucleotide, or the expression vector of any one of the embodiments disclosed herein.

[0058] In certain embodiments, the immune cell expresses the fusion protein.

[0059] In certain embodiments, the immune cells are selected from the group consisting of T cells, natural killer cells, macrophages, neutrophils, eosinophils, basophils, and dendritic cells.

[0060] In certain embodiments, the T cells are one or more selected from the group consisting of alpha beta (αβ) T cells, and gamma delta (γδ) T cells.

[0061] In certain embodiments, the alpha beta (αβ) T cells are one or more selected from the group consisting of CD4+ / CD8+ double positive T cells, CD8+ cytotoxic T cell, CD4+ helper T cell (such as Th1, Th2, Th17, Th3, or Th9 cells), follicular helper T (Tfh) cells, natural killer T (NKT) cells, tumor infiltrating lymphocytes (TILs), memory T cells (e.g. central memory T cells (Tcm cells), effector memory T cells (Tem cells and TEMRA cells), naive T cells, regulatory T cell ( Treg), CAR-T cells and TCR-T cells..

[0062] In certain embodiments, the TILs comprise CD8+ cytotoxic T cells, CD4+ T cells, and memory T cells.

[0063] In certain embodiments, a chimeric antigen receptor (CAR) is co-expressed.

[0064] In certain embodiments, the intracellular costimulatory domain of the CAR is a CD28 intracellular costimulatory domain, and a costimulatory domain of the fusion protein is an intracellular domain of 4- IBB.

[0065] In certain embodiments, the intracellular costimulatory domain of the CAR is a 4-1BB intracellular costimulatory domain, and a costimulatory domain of the fusion protein is an intracellular domain of 4- IBB.

[0066] In certain embodiments, a T cell receptor (TCR) is co-expressed.

[0067] In certain embodiments, the TCR is an engineered TCR.

[0068] In certain embodiments, the engineered TCR is anti-NY-ESO-1 T cell receptor (1G4).

[0069] In certain embodiments, the CAR or the TCR targets a tumor antigen.

[0070] In certain embodiments, the tumor antigen is selected from the group consisting of CLDN18.2 (claudin 18.2), 5T4 (trophoblast glycoprotein), 707-AP, 9D7, AFP (a-fetoprotein), AlbZIP (androgen-induced bZIP), HPG1 (human prostate specific gene-l), a.5b 1 -Integrity a.5p6-Integrin, a -methylacyl-coenzyme A racemase, ART-4 (ADPribosyltransf erase-4), B7H4 (v-set domain-containing T-cell activation inhibitor 1), B AGE-1 (B melanoma antigen- 1), BCL-2 (B-cell CLL / lymphoma-2), BING-4 (WD repeat domain 46), CA 15-3 / CA 27-29 (mucin 1), CA 19-9 (cancer antigen 19-9), CA 72-4 (cancer antigen 72-4), CAI 25 (cancer antigen 125), calreticulin, CAMEL (CTL-recognized antigen on melanoma), C ASP-8 (caspase 8), cathepsin B, cathepsin L, CD 19 (cluster of differentiation 19), CD20, CD22, CD25, CD30, CD33, CD4, CD52, CD55, CD56, CD80, CEA (carcinoembryonic antigen SG8), CLCA2 (chloride channel accessory 2), CML28 (chronic myelogenous leukemia tumor antigen 28), Coactosin-like protein, Collagen XXIII, COX-2 (cyclooxygenase-2), CT-9 / BRD6 (cancer / testis antigen 9), Cten (c-terminal tensin- like protein), cyclin Bl, cyclin DI, cyp-B, CYPB1 (cytochrome p450 family 1 subfamily b member 1), DAM-10 / MAGE-B1 (melanoma-associated antigen Bl), DAM-6 / MAGE-B2, EGFR / IIerl (epidermal growth factor receptor), EMMPRIN (basigin), EpCam, EphA2 (EPII receptor A2), EphA3, ErbB3 (Erb-B2 receptor tyrosine kinase 3), EZH2 (enhancer of zeste 2 poly comb repressive complex 2 subunit), FGF-5 (fibroblast growth factor 5), FN (fibronectin), Fra-1 (Fosrelated antigen-1), G250 / CAIX (carbonic anhydrase 9), GAGE-1 (G antigen-1), GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE- 7b, GAGE-8, GDEP (gene differentially expressed in prostate), GnT-V (gluconate kinase), gp100 (melanocytes lineage-specific antigen GP100), GPC3 (glypican3), HAGE (helical antigen), HAST-2 (sulfotransferase family 1 A member 1), hepsin, Her2 / neu / ErbB2 (Erb-B2 receptor tyrosine kinase 2), HERV-K-MEL, HNE (medullasin), homeobox NKX 3.1, HOM-TES-14 / SCP-1, HOM-TES-85, HPV-E6, HPVE7, HST-2 (sirtuin-2), hTERT, iCE (caspase 1), IGF-1R (insulin like growth factor- 1 receptor), IL-13Ra2 (interleukin- 13 receptor subunit a 2), IL-2R (interleukin-2 receptor), IL-5 (interleukin-5), immature laminin receptor, kallikrein 2, kallikrein 4, Ki67, KIAA0205 (lysophosphatidylglycerol acyltransf erase 1), KK-LC-1 (kita-kyushu lung cancer antigen- 1), KM-IIN-1, LAGE-1 (L antigen family member- 1), Livin, MAGE- Al, MAGE- A 10, MAGE-A12, MAGEA2, MAGE- A3, MAGE-A4, MAGE-A6, MAGE-A9, MAGE-B1, MAGE-B10. MAGE-B16. MAGEB17, MAGE-B2. MAGE-B3, MAGE-B4, MAGE-B5, MAGE-B6, MAGE-CI, MAGE-C2, MAGE-C3, MAGE-D1,MAGE-D2, MAGE-D4, MAGE-E1, MAGE-E2, MAGE-F1, MAGE-H1. MAGEL2 (melanoma antigen family L2), mammaglobin A, M ART- 1 / Mel an- A (melanoma antigen recognized by T-cells-1), MART-2, matrix protein 22, MC1R (melanocortin 1 receptor), M-CSF (macrophage colony-stimulating factor), Mesothelin, MG50 / PXDN (peroxidasin), MMP 11 (matrix metalloprotease 11), MN / CA IX-antigen (carbonic anhydrase 9), MRP-3 (multidrug resistance-associated protein-3), MUC1 (mucin 1), MUC2, NA88-A (VENT-like homeobox 2 pseudogene 1), N-acetylglucos-aminyltransf erase- V, Neo-PAP (Neo-poly (A) polymerase), NGEP (new gene expressed in prostate), NMP22 (nuclear matrix protein 22), NPM / ALK (nucleophosmin), NSE (neuron-specific enolase), NY-ESO-1, NY-ESO-B, OA1 (osteoarthritis QTL 1), OFA-iLRP (oncofetal antigen immature laminin receptor protein), OGT (O-GlcNAc transferase), OS-9 (endoplasmic reticulum lectin), osteocalcin, osteopontin, p 15 (CDK inhibitor 2B), p53, PAGE-4 (P antigen family member-4), PAI-1 (plasminogen activator inhibitor- 1), PAI-2, PAP (prostatic acid phosphatase), PART-1 (prostate androgen-regulated transcript 1), PATE (prostate and testis expressed 1), PDEF (prostate-derived Ets factor), Pim-l-Kinase (proviral integration site 1), Pinl (Peptidyl-prolyl cis-trans isomerase NIMA-interacting 1), POTE (expressed in prostate, ovary, testis, and placenta), PRAME (preferentially expressed antigen in melanoma), prostein, proteinase-3, PSA (prostate-specific antigen), PSCA (prostate stem cell antigen), PSGR (prostate-specific G-protcin coupled receptor), PSM, PSMA (prostate specific membrane antigen), RAGE-1 (renal tumor carcinoma antigen), RHAMM / CD168, RET1 (renal ubiquitous protein 1), RET2, SAGE (sarcoma antigen), SART-1 (squamous cell carcinoma antigen recognized by T-cells-1), SART-2, SART-3, Spl7 (sperm protein 17), SSX-1 (SSX family member 1), SSX-2 / H0M-MEL-40, SSX-4, STAMP-1 (STEAP2 metalloreductase), STEAP, survivin, survivin-213, TA-90 (tumor associated antigen-90), TAG-72 (tumor associated glycoprotein-72), TARP (TCRy alternate reading frame protein), TGFb (transforming growth factor b), TGFbRl 1 (transforming growth factor b receptor 11), TGM-4 (transglutaminase 4), TRAG-3 (taxol resistance associated gene 3), TRG (T-cell receptor g locus), TRP-1 (transient receptor potential-1), TRP-2 / 6b, TRP-2 / INT2, Trp-p8, Tyrosinase, UPA (U-plasminogen activator), VEGF (vascular endothelial growth factor A), VEGFR-2 / FLK- 1 and WT1 (wilms tumor 1), a-actinin-4 / m, ARTCl / m, bcr / abl, beta-Catenin / m, BRCAl / m, BRCA2 / m, CASP-5 / m, CASP-8 / m, CDC27 / m, CDK4 / m, CDKN2A / m, CML66, COA-l / m, DEK-CAN, EFTUD2 / m, ELF2 / m, ETV6-AML1, FNl / m, GPNMB / m, HLA-A* 0201-R 1701, HLA-A1 1 / m, HLA-A2 / m, HSP70-2M, KIAA0205 / m, K-Ras / m, LDLR-FUT, MART2 / m, MEl / m, MFJM-l / m, MEiM-2 / m, MUM-3 / m, Myosin class 1 / m, neo-PAP / m, NFYC / m, N- Ras / m, OGT / m, OS-9 / m, p53 / m, Pml / RARa, PRDX5 / m, PTPRX / m, RBAF600 / m, SIRT2 / m, SYTSSX-l, SYT-SSX-2, TEL-AML1, TGFbRII and TPI / m.

[0071] In yet another aspect, the present disclosure provides a pharmaceutical composition comprising the isolated immune cell of any one of the embodiments disclosed herein.

[0072] In one aspect, the present disclosure provides a method of producing an isolated immune cell, comprising(i) introducing the fusion protein encoding polynucleotide, or the expression vector into the immune cell; and(ii) maintaining the immune cell under conditions in which the fusion protein encoding polynucleotide or the expression vector is expressed.

[0073] In certain embodiments, the fusion protein encoding polynucleotide is transduced with a virus. In certain embodiments, the virus is a lentivirus or retrovirus.

[0074] In certain embodiments, the expression vector is derived from a viral vector. In certain embodiments, the viral vector is a lentivirus or retrovirus vector.

[0075] In one aspect, the present disclosure provides a method of treating cancer in a subject in need thereof, comprising(i) co-expressing the fusion protein and a CAR or TCR in an immune cell, and administering the immune cell to the subject; or(ii) administering to the subject an immune cell expressing the fusion protein and a CAR or TCR.

[0076] In certain embodiments, wherein the TCR is an engineered TCR.

[0077] In certain embodiments, the cancer is solid tumor.

[0078] In certain embodiments, the solid tumor comprises brain cancer, bladder cancer, liver cancer, colon cancer, rectal cancer, endometrial cancer, pancreatic cancer, small cell lung cancer, non-small cell lung cancer, breast cancer, urethral cancer, head and neck cancer, gastrointestinal cancer, stomach cancer, oesophageal cancer, ovarian cancer, renal cancer, melanoma, cervix cancer, rectum cancer, larynx cancer, prostate cancer and thyroid cancer.

[0079] In certain embodiments, the cancer is hematological malignancy.

[0080] In certain embodiments, the hematological malignancy comprises acute and chronic leukemias (acute myelogenous leukemia (AML), acute lymphoblastic leukemia (ALL), chronic myelogenous leukemia (CML), lymphomas, non-Hodgkin lymphoma (NHL), Hodgkin’s disease, multiple myeloma, and myelodysplastic syndromes.BRIEF DESCRIPTION OF THE DRAWINGS

[0081] FIG. 1 illustrates constructs of the TCR (LI) and cytokine fusion proteins (L2-L6). Figure discloses “G6S” as SEQ ID NO: 8.

[0082] FIGs. 2A and 2B shows the schematic design of transducing the constructs to T cells.

[0083] FIGs. 3A-3D show in vitro cytotoxicity of the hT-222 cells transduced with the constructs of the present disclosure on A375Z cells at different E: T ratios.

[0084] FIGs. 4A-4D show in vitro cytotoxicity of the hT-222 cells transduced with the constructs of the present disclosure on MDA-MB-231Z cells in the presence of NY-ESO peptide (after 4 hours) at different concentrations at the E: T ratio of 1:1 ratio.

[0085] FIGs. 5A-5C show the proliferation of hT-222 cells before freezing (A and B) and after thawing (C).

[0086] FIGs. 6A-6C show the proliferation of hT-222 cells (A), hT-253 cells (B) and hT-254 cells (C) before freezing.

[0087] FIG. 7 shows a schematic design of the stress test to transduced T cells.

[0088] FIGs. 8A-8C show the in vitro cytotoxicity of the hT-222 cells transduced with the constructs of the present disclosure on A375Z cells at different E: T ratios in the repetitive stimulation stress test by adding target cells every 2 days for 5 times.

[0089] FIGs. 9A-9C show the in vitro cytotoxicity of the hT-253 cells transduced with the constructs of the present disclosure on A375Z cells at different E: T ratios in the repetitive stimulation stress test by adding target cells every 3 days for 5 times.

[0090] FIGs. 10A-10C show the in vitro cytotoxicity of the hT-222 cells transduced with the constructs of the present disclosure on A375Z cells in the presence / absence of 300IU / mL IL-2 at different E: T ratios in the repetitive stimulation stress test by adding target cells every 2 days for 4 times.

[0091] FIGs. 11A-11B show the cell number change of T cells in the long-term at an E: T ratio of 2:1, presence of 50IU / mL IL-2, in the repetitive stimulation by adding target cells every 4 days for 3 times.

[0092] FIGs. 12A-12B show the cell number change of T cells in the long-term at an E:T ratio of 2:1, presence of 50IU / mL IL-2, in the repetitive stimulation by adding target cells every 4 days for 4 times.

[0093] FIGs. 13A-13B show the T cell division (%) after 4 day coculture of T cells and A375WT target cells in a E: T ratio of 1:1 (0.5M:0.5M) using Cell Trace Violet.

[0094] FIGs. 14A-14B show the change of CD4 / CD8 proportion before (A) and after (B) the 3rdantigen stimulation in hT-222.

[0095] FIG. 15 shows the schematic design of the in vivo stress test in NSG / NOG mice.

[0096] FIG. 16 shows in vivo imaging of tumor growth in mice demonstrating anti-tumor effects of T cells co-expressing a cytokine fusion protein of the present disclosure.

[0097] FIGs. 17A-17D show the in vivo anti-tumor effects of the T cells transduced with TCR constructs.

[0098] FIG. 18 shows a schematic representation of an in vivo antitumor efficacy experiment in NOG mice.

[0099] FIG. 19 shows in vivo imaging of tumor growth in mice demonstrating anti-tumor effects of T cells co-expressing cytokine fusion proteins of the present disclosure.

[0100] FIGs. 20A-20B show the in vivo anti-tumor effects of T cells co-expressing cytokine fusion proteins of the present disclosure.

[0101] FIG.21 shows a comparison of the in vivo persistence of infused T cells coexpressing cytokine fusion proteins of the present disclosure.

[0102] FIG.22 shows in vivo imaging of tumor growth in mice demonstrating anti-tumor effects of CAR T cells co-expressing a cytokine fusion protein (hyperkine) of the present disclosure at both high and low doses.

[0103] FIGs. 23A-23B show the in vivo anti-tumor effects of CAR T cells co-expressing a cytokine fusion protein (hyperkine) of the present disclosure at both high and low doses.

[0104] FIGs. 24A-24B show the number of CAR T cells in spleen (FIG. 24A) and blood (FIG. 24B) in a tumor mouse model, demonstrating enhanced CAR T cell expansion of CAR T cells that co-express a cytokine fusion protein of the present disclosure.

[0105] FIGs. 25A-25C show the in vitro cytotoxicities of CAR T cells targeting PSMA (FIG. 25A), Claudin 18.2 (FIG. 25B), or GPC3 (FIG. 25C) with and without co-expressed cytokine fusion proteins of the present disclosure, as measured in solid tumor cells.

[0106] FIG.26 shows a schematic depiction of different 28z CAR T cells of the present disclosure and their ranked cytotoxicities as measured in LnCaP-Zsgrccn target cells.

[0107] FIG.27 shows exemplary in vitro cytotoxicity data of different 28z CAR T cells of the present disclosure as measured in LnCaP-Zsgreen target cells.

[0108] FIG.28 shows a schematic depiction of different BBz CAR T cells of the present disclosure and their ranked cytotoxicities as measured in LnCaP-Zsgreen target cells.

[0109] FIG.29 shows exemplary in vitro cytotoxicity data of different BBz CAR T cells of the present disclosure as measured in LnCaP-Zsgreen target cells.

[0110] FIG.30 shows exemplary in vitro cytotoxicity data of different CAR T cells of the present disclosure as measured in LnCaP-Zsgreen target cells.

[0111] FIG.31 shows a schematic depiction of different 1stgeneration PSMA CAR T cells of the present disclosure and their ranked cytotoxicities as measured in LnCzP-Zsgreen target cells.

[0112] FIG. 32 shows exemplary in vitro cytotoxicity data of different 1stgeneration PSMA CAR T cells of the present disclosure as measured in LnCaP-Zsgreen target cells.

[0113] FIG.33 shows a gating strategy used for flow cytometry quantification of hCD3+CAR+T cell populations in an in vivo study evaluating the persistence of CAR T cells expressing membrane-bound cytokine fusion proteins of the present disclosure.

[0114] FIG. 34 shows the number of hCD3+CAR+T cells per milligram of spleen tissue as measured by flow cytometry, where FIG.34A shows results in a mouse prostate cancer model treated with untransduced T cells, PSMA-targeting CAR T cells alone (‘no-armored CAR-T’), PSMA-targeting CAR T cells expressing a membrane-bound IL-12 (‘mbIL-12 armored CAR-T’), and PSMA-targeting CAR T cells expressing a cytokine fusion protein (‘hyperkine armored CAR T’), and FIG. 34B shows results in a mouse adenocarcinoma model treated with untransduced T cells, Claudin (CLDN) 18.2-targeting CAR T cells alone(‘no-armored CAR-T’), CLDN 18.2-targeting CAR T cells expressing a membrane bound IL-12 (‘mbIL-12 armored CAR-T’), and CLDN 18.2-targeting CAR T cells expressing a cytokine fusion protein (‘hyperkine armored CAR T’).

[0115] FIG.35 shows the in vivo anti-tumor effects of Claudin 18.2-targeting CAR T cells co-expressing cytokine fusion proteins of the present disclosure.

[0116] FIG. 36 shows the in vivo persistence in mice of Claudin 18.2-targeting CAR T cells co-expressing a cytokine fusion protein of the present disclosure.

[0117] FIG.37 is a schematic representation of enhancement of T cell functions with a cytokine fusion protein of the present disclosure.

[0118] FIG.38 is a schematic representation of enhancement of T cell functions with a cytokine fusion protein of the present disclosure.

[0119] FIG.39 is a schematic representation of enhancement of CAR T cell functions with a cytokine fusion protein of the present disclosure.

[0120] FIG.40 is a schematic representation of enhancement of CAR T cell functions with a cytokine fusion protein of the present disclosure.

[0121] FIG.41 illustrates lentiviral vector constructs for expression of a negative control, a membrane-bound interleukin- 12 (mbIL-12), and a cytokine fusion protein (hyperkine) in T cells. In all constructs, truncated low-affinity nerve growth factor receptor (LNGFR) (ALNGFR; extracellular and transmembrane domains only) was co-expressed, enabling enrichment of ALNGFR-positive cells.

[0122] FIG.42 illustrates flow cytometry results demonstrating co-expression of ALNGFR and the corresponding transgene (mbIL-12) of the constructs of FIG.41.

[0123] FIG.43 shows a flow cytometry gating strategy used for quantification of T cell proliferation.

[0124] FIGs. 44A-44B show proliferation of T cells expressing the negative control, the mbIL-12, or the cytokine fusion protein (hyperkine constructs) of FIG.41 with single stimulation (FIG. 44A) and repeated stimulation (FIG.44B).

[0125] FIGs. 45A-45B show exemplary in vitro cytotoxicity data of Claudinl8.2-specific CAR T cells co-expressing cytokine fusion protein (hyperkine) or bifunctional cytokine fusion proteins (bifunctional hyperkines) having a membrane -bound interleukin- 15 (mblL-15) domain and a membrane-bound interleukin- 18 (mbIL-18) domain as compared to negative controls at an effector to target (E: T) ratio of 0.5: 1 (FIG. 45A) at an E: T ratio of 0.25:1 (FIG.45B) as measured in Claudinl 8.2-positive tumor cells.

[0126] FIGs.46A-46B show exemplary in vitro cytotoxicity data of PSMA-specific CAR T cells co-expressing cytokine fusion protein (hyperkine) or bifunctional cytokine fusion proteins (bifunctional hyperkines) having a mbIL-15 domain and a mbIL-18 domain as compared to negative controls at an E: T ratio of 0.1:1 (FIG.46A) at an E: T ratio of 0.03:1 (FIG.46B) as measured in PSMA-positive tumor cells.

[0127] FIGs.47A-47B show exemplary in vitro cytotoxicity data of GPC3 -specific CAR T cells co-expressing cytokine fusion protein (hyperkine) or bifunctional cytokine fusion proteins (bifunctional hyperkines) having a mbIL- 15 domain and a mbIL- 18 domain as compared to negative controls at an E: T ratio of 3:1 (FIG.47A) at an E: T ratio of 1:1 (FIG.47B) as measured in GPC3-positive tumor cells.DETAILED DESCRIPTION

[0128] T-cell therapies have achieved remarkable success in blood cancers but remain limited in solid tumors, largely due to the inability to replicate the physiological dynamics of T-cell activation. While vertical strategies — such as incorporating additional costimulatory domains into CARs — have been explored, their benefits in solid tumors have been modest. To overcome this limitation, the present technology provides membrane-anchored cytokine fusion proteins that deliver costimulatory (signal 2) and cytokine (signal 3) inputs in parallel. By mimicking the natural, spatially distinct delivery of activation signals, the cytokine fusion proteins disclosed herein promote a more physiologic and synergistic T-cell activation, offering a promising next-generation approach for treatment of various cancers including solid tumors. Engineered T-cells incorporating the cytokine fusion proteins of the present disclosure exhibit superior cytotoxicity and durable antitumor efficacy in repeated stimulation experiments and in in vivo tumor models. The cytokine fusion proteins of the present disclosure provide a broadly applicable platform to overcome the limitations of current solid tumor-targeted T-cell therapies.Definitions

[0129] It is to be noted that the term “a” or “an” entity refers to one or more of that entity; for example, “an antibody,” is understood to represent one or more antibodies. As such, the terms “a” (or “an”), “one or more,” and “at least one” can be used interchangeably herein.

[0130] The term “about” or “approximately” means within about 20%, such as within about 10%, within about 5%, or within about 1% or less of a given value or range.

[0131] The term “polypeptide” refers to any polymeric chain of amino acids and encompasses native or artificial proteins, polypeptide analogs or variants of a protein sequence, or fragments thereof, unless otherwise contradicted by context. A polypeptide may be monomeric or polymeric; that is, the term encompasses a protein having one or more covalently coupled, or noncovalently coupled, polypeptide chains. A polypeptide fragment comprises at least about 5 contiguous amino acids, at least about 10 contiguous amino acids, at least about 15 contiguous amino acids, or at least about 20 contiguous amino acids, for example.

[0132] “Percent (%) amino acid sequence identity” or “homology” with respect to the polypeptide and antibody sequences identified herein is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the polypeptide being compared, after aligning the sequences considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, Megalign (DNASTAR), or MUSCLE software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full-length of the sequences being compared. For purposes herein, however, % amino acid sequence identity values are generated using the sequence comparison computer program MUSCLE (Edgar, R. C., Nucleic Acids Research 32(5):1792-1797, 2004; Edgar, R. C., BMC Bioinformatics 5(1): 113, 2004).

[0133] “Homologous” refers to the sequence similarity or sequence identity between two polypeptides or between two nucleic acid molecules. When a position in both of the two compared sequences is occupied by the same base or amino acid monomer subunit, e.g., if a position in each of two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The percent of homology between two sequences is a functionof the number of matching or homologous positions shared by the two sequences divided by the number of positions compared times 100. For example, if 6 of 10 of the positions in two sequences are matched or homologous then the two sequences are 60% homologous.Generally, a comparison is made when two sequences are aligned to give maximum homology.

[0134] By “specifically binds” or “has specificity to,” it is generally meant that an antibody binds to an epitope via its antigen-binding domain, and that the binding entails some complementarity between the antigen-binding domain and the epitope. According to this definition, an antibody is said to “specifically bind” to an epitope when it binds to that epitope, via its antigen-binding domain more readily than it would bind to a random, unrelated epitope. The term “specificity” is used herein to qualify the relative affinity by which a certain antibody binds to a certain epitope. For example, antibody “A” may be deemed to have a higher specificity for a given epitope than antibody “B,” or antibody “A” may be said to bind to epitope “C” with a higher specificity than it has for related epitope “D.”

[0135] An “isolated” polypeptide (or construct) or cell is one that has been identified, separated and / or recovered from a component of its production environment (e.g., natural or recombinant). Preferably, the isolated polypeptide or cell is free of association with all other components from its production environment.

[0136] An “isolated” nucleic acid molecule encoding a construct, polypeptide, antibody, or antigen-binding fragment thereof described herein is a nucleic acid molecule that is identified and separated from at least one contaminant nucleic acid molecule with which it is ordinarily associated in the environment in which it was produced. Preferably, the isolated nucleic acid is free of association with all components associated with the production environment. The isolated nucleic acid molecules encoding the polypeptides and antibodies described herein is in a form other than in the form or setting in which it is found in nature. Isolated nucleic acid molecules therefore are distinguished from nucleic acid encoding the polypeptides and antibodies described herein existing naturally in cells. An isolated nucleic acid includes a nucleic acid molecule contained in cells that ordinarily contain the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

[0137] Tumor-infiltrating lymphocyte (TIL), as used herein, refers immune cells, primarily lymphocytes such as T cells and B cells, that have migrated from the bloodstream into the tumor microenvironment. These cells are part of the body's immune response to cancer, and their presence within the tumor tissue is indicative of the immune system's attempt to recognize and attack cancer cells. TILs play a crucial role in anti-tumor immunity, and their abundance and activity are often associated with better clinical outcomes in cancer patients. They are also a focus in immunotherapy strategies, where they can be harvested, expanded, and reinfused into patients to enhance the immune response against tumors.

[0138] Nucleic acid is “operably linked” when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, “operably linked” means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading frame. However, enhancers do not have to be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, the synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice.

[0139] The term “transfected” or “transformed” or “transduced” as used herein refers to a process by which exogenous nucleic acid is transferred or introduced into the host cell. A “transfected” or “transformed” or “transduced” cell is one which has been transfected, transformed or transduced with exogenous nucleic acid. The cell includes the primary subject cell and its progeny.

[0140] A “T cell receptor” or “TCR” is a protein complex located on the surface of T cells which is responsible for recognizing fragments of antigens as peptides bound to major histocompatibility complex (MHC) molecules on the surface of antigen presenting cells (APCs), such as B cells, macrophages, and dendritic cells. Binding of the TCR to the antigen peptide allows the T cells to identify and respond to foreign substances such as pathogens or cancer cells.

[0141] A “chimeric antigen receptor” or “CAR” is an engineered receptor designed to equip an immune cell, particularly T cells, to recognize and attack cancer cells. In certainembodiments, a CAR may include an antigen-binding domain, a transmembrane domain, and one or more signaling domains. A “CAR T cell” is a genetically engineered T cell that expresses a CAR, allowing the T cell to specifically target and kill cancer cells.

[0142] The term “hyperkine” as used herein refers to a fusion protein having one or more cytokine regions such as, but not limited to, interleukins (e.g., IL-12, IL-15, IL-18, etc.) fused to a transmembrane region (e.g., a transmembrane domain of CD28, a transmembrane domain of CD80) and a costimulatory domain (e.g., an intracellular domain of 4-1BB, an intracellular domain of CD28). As used herein, a hyperkine may include a bifunctional hyperkine having two cytokine regions (e.g., two interleukins) fused to a transmembrane region (e.g., a transmembrane domain of CD28, a transmembrane domain of CD80) and a costimulatory domain (e.g., an intracellular domain of 4- IBB, an intracellular domain of CD28), or a multifunctional hyperkine having multiple cytokine regions (e.g., multiple interleukins) fused to a transmembrane region (e.g., a transmembrane domain of CD28, a transmembrane domain of CD80) and a costimulatory domain (e.g., an intracellular domain of 4-1BB, and intracellular domain of CD28). In the present disclosure, a membrane-bound interleukin such as IL- 12 may be abbreviated as “mIL-12” or “mbIL-12”, and the abbreviations “mIL-12” and “mbIL-12” may be used interchangeably. In certain embodiments, the membrane-bound interleukin (e.g., IL-12) is fused to CD28 transmembrane domain and 4- IBB intracellular domain. In certain embodiments, the membrane-bound interleukin (e.g., IL- 12) is fused to CD80 transmembrane domain and 4- IBB intracellular domain. In certain embodiments, the membrane-bound interleukin (e.g., IL- 12) is fused to a CD28 transmembrane domain and CD28 intracellular domain. In certain embodiments, membrane-bound interleukin (e.g., IL- 12) is fused to a CD80 transmembrane domain and a CD28 intracellular domain. In certain non-limiting embodiments, the hyperkine may have an amino acid sequence according to SEQ ID NO: 12 or an amino acid sequence with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 12. In certain non-limiting embodiments, the hyperkine may have an amino acid sequence according to SEQ ID NO: 13 or an amino acid sequence with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 13. In certain non-limiting embodiments, the hyperkine may have an amino acid sequence according to SEQ ID NO: 14 or an amino acid sequence with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 14.

[0143] A “bifunctional hyperkine” as used herein refers to a hyperkine having at least a first cytokine region and a second cytokine region linked together in tandem or with intervening fragments therebetween, wherein the first cytokine region and the second cyokine region are independently selected from cytokines such as, but not limited to, IL-12, IL-15, IL-2, IL-4, IL-7, IL-9, IL-18, and IL-21. The first and second cytokine regions arc fused to a transmembrane region (e.g., a transmembrane domain of CD28, a transmembrane domain of CD80) and a costimulatory domain (e.g., an intracellular domain of 4-1BB, an intracellular domain of CD28). In certain embodiments, the first and second cytokine regions are fused to a CD28 transmembrane domain and a 4- IBB intracellular domain. In certain embodiments, the first and second cytokine regions are fused to a CD80 transmembrane domain and a 4-1BB intracellular domain. In certain embodiments, the first and second cytokine regions are fused to a CD28 transmembrane domain and a CD28 intracellular domain. In certain embodiments, the first and second cytokine regions are fused to a CD80 transmembrane domain and a CD28 intracellular domain. In certain embodiments, the first cytokine region is a membrane-bound IL- 15 (mbIL- 15) and the second cytokine region is a membrane-bound IL- 18 (mbIL-18), and the mbIL-15 and the mbIL-18 are fused to a CD28 transmembrane domain and a 4- IBB intracellular domain. In certain embodiments, the first cytokine region is a mbIL-15 and the second cytokine region is a mbIL-18, and the mbIL-15 and the mbIL-18 are fused to a CD80 transmembrane domain and a 4- IBB intracellular domain. In certain embodiments, the first cytokine region is a mbIL-15 and the second cytokine region is a mbIL-18, and the mbIL-1 and the mbIL-18 are fused to a CD28 transmembrane domain and a CD28 intracellular domain. In certain embodiments, the first cytokine region is a mbIL-15 and the second cytokine region is a mbIL-18, and the mbIL-15 and the mbIL-18 are fused a CD80 transmembrane domain and a CD28 intracellular domain. In certain embodiments, the first cytokine region is a membrane-bound IL- 18 (mbIL-18) and the second cytokine region is a membrane-bound IL- 15 (mbIL-15), and the mbIL-18 and the mbIL-15 are fused to a CD28 transmembrane domain and a 4- IBB intracellular domain. In certain embodiments, the first cytokine region is a mbIL-18 and the second cytokine region is a mbIL-15, and the mbIL-18 and the mbIL-15 are fused to a CD80 transmembrane domain and a 4- IBB intracellular domain. In certain embodiments, the first cytokine region is a mbIL-18 and the second cytokine region is a mbIL-15, and the mbIL-18 and the mbIL-15 are fused to a CD28 transmembrane domain and a CD28 intracellular domain. In certain embodiments, the first cytokine region is a mbIL-18 and the second cytokine region is a mbIL-15, and the mbIL-18 and the mbIL-15 are fused a CD80 transmembrane domain and a CD28 intracellular domain.In certain non- limiting embodiments, the bifunctional hyperkine may have an amino acid sequence according to SEQ ID NO: 64 or an amino acid sequence with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 64. In certain non-limiting embodiments, the bifunctional hyperkine may have an amino acid sequence according to SEQ ID NO: 66 or an amino acid sequence with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 66.

[0144] A “multifunctional hyperkine” as used herein refers to a hyperkine having multiple cytokine regions (e.g., three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, etc.) linked together in tandem or with intervening fragments therebetween, wherein each of the cytokine regions are independently selected from cytokines such as, but not limited to, IL- 12, IL- 15, IL-2, IL-4, IL-7, IL-9, IL- 18, and IL-21 or functional fragments thereof. In certain embodiments, the multiple cytokine regions are fused to a transmembrane domain (e.g., CD28, CD80) and a costimulatory domain (e.g., an intracellular domain of 4- IBB, an intracellular domain of CD28). In certain embodiments, the multiple cytokine regions are fused to a CD28 transmembrane domain and a 4- IBB intracellular domain. In certain embodiments, the multiple cytokine regions are fused to a CD80 transmembrane domain and a 4-1 BB intracellular domain. In certain embodiments, the multiple cytokine regions are fused to a CD28 transmembrane domain and a CD28 intracellular domain. In certain embodiments, the multiple cytokine regions are fused to a CD80 transmembrane domain and a CD28 intracellular domain.

[0145] As used herein, “cis” binding to a receptor refers to binding of an extracellular molecule (such as a cytokine) to a receptor that is expressed on the surface of the same cell (intracellular ligand-receptor binding). In certain embodiments, cis binding refers to binding of a membrane -bound cytokine expressed on the surface of a T cell to a cytokine receptor expressed on the surface of the same T cell. As used herein, “trans” binding to a receptor refers to binding of an extracellular molecule (such as a cytokine) to a receptor that is expressed on the surface of another cell (intercellular ligand-receptor binding). In certain embodiments, trans binding refers to binding of a membrane-bound cytokine expressed on the surface of a T cell to a cytokine receptor expressed on the surface of a different cell.

[0146] As used herein, “signal 1”, “signal 2”, and “signal 3” refer to signals involved in T cell activation and expansion. “Signal 1” is the first stimulatory signal that occurs upon engagement of an antigen binding domain of a TCR or a CAR with its specific antigen. Acostimulatory signal (“signal 2”) may result from binding of a costimulatory receptor, such as CD28, of the T cell to a molecule presented on an APC. A cytokine signal (“signal 3’’) may occur upon release of cytokines from the T cell. In certain embodiments, signal 2 may be triggered via an intracellular signaling domain within the T cell. In certain embodiments, signal 3 may be triggered by cytokine binding to a cytokine receptor.

[0147] As used herein, “vertical’’ augmentation of T cell activation or signaling occurs as a result of the incorporation of one or more additional signaling domains within a TCR or CAR construct, such that the signaling domains are linked to the CAR construct. “Horizontal’’ augmentation of T cell activation, as used herein, occurs by introduction of one or more additional membrane -bound signaling domains within constructs that are not linked to the TCR or CAR construct. In other words, “vertical’’ and “horizontal” augmentation of T cell activation refers to T cell signaling inputs that occur vertically or horizontally with respect to the T cell membrane. In certain embodiments, horizontal augmentation of T cell signaling is provided by a membrane -bound cytokine fusion protein that is co-expressed in the T cell along with a TCR or a CAR. Horizontal augmentation of T cell signaling may deliver spatially discrete costimulatory (signal 2) and cytokine (signal 3) inputs in parallel via a membrane-bound construct that is separate and physically distinct from the TCR or CAR construct. As used herein, “spatially discrete inputs” refers to signaling inputs originating from spatially discrete locations within the cell membrane. In certain embodiments, horizontal augmentation of T cell signaling may provide a more synergistic mode for T cell activation as opposed to vertical augmentation. As used herein, “horizontal” delivery of signal 2 and signal 3 refers to delivery of the signal 2 and signal 3 costimulatory and cytokine signals via a membrane-bound construct that is physically distinct from the membrane -bound TCR or CAR construct.

[0148] As used herein, “spatial dynamics of T cell activation” refers to the dynamics of T cell activation that are related to activation signals originating from different spatial or physical locations within the cell or cell membrane. As used herein, “temporal dynamics of T cell activation” refers to the dynamics of T cell activation that are related to activation signals occurring at different times.

[0149] As used herein, “synergistic” refers to an effect of a combination of elements that is greater than the sum of the individual elements.

[0150] As used herein, an “armored” T cell refers to a T cell which co-expresses a TCR or a CAR construct along with a cytokine fusion protein or hyperkine that delivers horizontal augmentation of the activation and function of the T cell. As used herein, an “unarmored” T cell refers to a T cell that expresses a TCR or a CAR construct and does not co-express a cytokine fusion protein or hyperkine.

[0151] “Chronic antigen stimulation” as used herein refers to the prolonged exposure of immune cells, such as T cells, to specific antigens. Chronic antigen stimulation can lead to various immune responses such as T cell exhaustion.

[0152] ‘ ‘Functional persistence” of a T cell, as used herein, refers to the ability of a modified T cell (e.g., TCR T cell or CAR T cell) to remain alive and active in the body after being infused. High functional persistence allows the T cell to continue identifying and eliminating cancers or pathogens.

[0153] “Post-transfer expansion” as used herein refers to the process of increasing the number of T cell after they have been transferred into a patient for therapeutic purposes.

[0154] As used herein, a “tumor microenvironment” or TME refers to the complex cellular and non-cellular components surrounding a tumor and may include cancer cells, stromal tissue (blood vessels and immune cells), fibroblasts, endothelial cells, the extracellular matrix, and signaling molecules. The TME may play a role in supporting tumor growth and invasion, as well as influence how the tumor responds to treatments.

[0155] As used herein, the terms “treat” or “treatment” refer to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) an undesired physiological change or disorder, such as the progression of cancer. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. “Treatment” can also mean prolonging survival as compared to expected survival if not receiving treatment. Those in need of treatment include those already with the condition or disorder as well as those prone to have the condition or disorder or those in which the condition or disorder is to be prevented.

[0156] By “subject” or “individual” or “animal” or “patient” or “mammal,” is meant any subject, particularly a mammalian subject, for whom diagnosis, prognosis, or therapy isdesired. Mammalian subjects include humans, domestic animals, farm animals, and zoo, sport, or pet animals such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, cows, and so on.

[0157] As used herein, phrases such as “to a patient in need of treatment” or “a subject in need of treatment” includes subjects, such as mammalian subjects, that would benefit from administration of an antibody or composition of the present disclosure used, e.g., for detection, for a diagnostic procedure and / or for treatment.Membrane bound Cytokine Fusion Proteins

[0158] The fusion protein of the present disclosure includes a cytokine region, a transmembrane region and a costimulatory domain. The fusion protein can be expressed on the surface of an immune cell, and thus is bound to the cell membrane.

[0159] Cytokine region

[0160] “Cytokine” is a well-known term of art that refers to any of a class of immunoregulatory proteins (such as interleukin or interferon) that are secreted by cells especially of the immune system and that are modulators of the immune system. Cytokine polypeptides that can be used in the fusion proteins disclosed herein include, but are not limited to transforming growth factors, such as TGF-α and TGF-β (e.g., TGFbetal, TGFbeta2, TGFbeta3); interferons, such as interferon-α, interferon-β, interferon-γ, interferon-kappa and interferon-omega; interleukins, such as IL-1, IL- 1α, IL-2, IL-3, IL- 4, IL- 5, IL-6, IL- 7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-21 and IL-25; tumor necrosis factors, such as tumor necrosis factor alpha and lymphotoxin; chemokines (e.g., C-X-C motif chemokine 10 (CXCF10), CCF19, CCF20, CCF21), and granulocyte macrophage-colony stimulating factor (GM-CS), as well as fragments of such polypeptides that active the cognate receptors for the cytokine (i.e., functional fragments of the foregoing). “Chemokine” is a term of art that refers to any of a family of small cytokines with the ability to induce directed chemo taxis in nearby responsive cells.

[0161] In certain embodiments, the cytokine region is a polypeptide of any cytokine described in the present disclosure, for example, an interleukin, and optionally is selected from the group consisting of a polypeptide of interleukin- 12 (IL-12), IL-15, IL-2, IL-4, IL-7, IL-9, IL- 18, and IL-21 or a functional portion thereof. In certain embodiments, the cytokineregion includes a polypeptide of IL- 12 or a functional portion thereof. In certain embodiments, the cytokine region includes a polypeptide of IL- 15 or a functional portion thereof. In certain embodiments, the cytokine region includes a polypeptide of IL-2 or a functional portion thereof. In certain embodiments, the cytokine region includes a polypeptide of IL-4 or a functional portion thereof. In certain embodiments, the cytokine region includes a polypeptide of IL-7 or a functional portion thereof. In certain embodiments, the cytokine region includes a polypeptide of IL-9 or a functional portion thereof. In certain embodiments, the cytokine region includes a polypeptide of IL- 18 or a functional portion thereof. In certain embodiments, the cytokine region includes a polypeptide of IL-21 or a functional portion thereof.

[0162] In certain embodiments, the cytokine region includes IL- 12 or a functional portion thereof. In certain embodiments, the IL- 12 polypeptide or the functional portion thereof comprises a IL- 12α subunit and a IL- 12β subunit, wherein the IL- 12α subunit and the IL- 12β subunit are connected via a linker and form a heterodimer.

[0163] IL- 12 is a heterodimeric cytokine composed of p35 (35kDa light chain or IL- 12α) and p40 (40kDa heavy chain or IL- 12β) subunits, primarily produced by antigen-presenting cells such as dendritic cells, monocytes, macrophages, and B cells upon toll-like receptor engagement. It plays a critical role in the immune system by promoting the differentiation of naive CD4+ T cells into type 1 T helper (Thl) cells, enhancing proliferation and the cytotoxic functions of natural killer (NK) cells and CD8+ T cells, and stimulating the production of interferon-gamma (IFN-γ). These functions make IL-12 essential for anti-tumor and anti-viral responses, positioning it as a promising candidate in cancer immunotherapy and treatments for chronic infections.

[0164] In certain embodiments, the cytokine region includes IL- 15 or a functional portion thereof. IL- 15 is a cytokine vital for the development, survival, and activation of natural killer (NK) cells and memory CD8+ T cells, playing a significant role in immune responses against tumors and viral infections. It signals through a receptor complex shared with IL-2, but unlike many cytokines, IL- 15 is presented in a cell-bound form for trans -presentation to target cells. Produced by various cells such as macrophages and dendritic cells, IL- 15 enhances cytotoxic functions and promotes cytokine production, contributing to immune response amplification. Its ability to support immune cell activation and proliferation makes IL- 15 a promising candidate for cancer immunotherapy, vaccines, and infectious diseasetreatments, often explored in combination therapies to enhance immune activation with minimized adverse effects.

[0165] In certain embodiments, the cytokine region includes IL- 18 or a functional portion thereof. IL- 18 stimulates various cell types and pleiotropic functions, and plays a role in inducing cell-mediated immunity following infection. IL- 18 is often explored as a therapeutic target for the treatment of inflammatory and autoimmune diseases.

[0166] In certain embodiments, the cytokine region includes one or more polypeptides (e.g., two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, etc.) linked together in tandem or with intervening fragments and / or linkers therebetween, wherein each of the polypeptides are selected from any one of the cytokines or functional portions thereof described in the present disclosure. In certain embodiments, the cytokine region includes one or more polypeptides (e.g., two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, etc.) linked together in tandem or with intervening fragments and / or linkers therebetween, wherein each of the polypeptides are selected from the group consisting of IL-12, IL-15, IL-2, IL-4, IL-7, IL-9, IL-18, and IL-21 or functional portions thereof.

[0167] In some embodiments, the cytokine region comprises at least a first polypeptide selected from the group consisting of a polypeptide of interleukin- 12 (IL- 12), IL- 15, IL-2, IL-4, IL-7, IL-9, IL- 18, and IL-21 or functional portions thereof, and a second polypeptide selected from the group consisting of a polypeptide of interleukin- 12 (IL-12), IL-15, IL-2, IL-4, IL-7, IL-9, IL-18, and IL-21 or functional portions thereof. In some embodiments, the cytokine region comprises, from N terminus to C terminus, a first polypeptide comprising IL-15 or a functional fragment thereof and a second polypeptide comprising IL- 18 or a functional fragment thereof. In some embodiments, the cytokine region comprises, from N terminus to C terminus, a first polypeptide comprising IL- 18 or a functional fragment thereof and a second polypeptide comprising IL- 15 or a functional fragment thereof.

[0168] Linkers within the scope of the present disclosure are characterized in terms of amino acid content, length, rigidity and secondary structure. Linkers within the scope of the present disclosure separate a functional polypeptide and another functional polypeptide and allow proper folding and functioning of each domain. In this manner, a linker can be tailored to the particular functional polypeptide and the other functional polypeptide. According to oneaspect, functional independence of the structural and fused (heterologous) domains is maximized by a suitable linker to limit steric interference between domains during the export and assembly processes of the bacterial cell. According to an additional aspect, cell stress is minimized by limiting the overall length of the fusion protein. Longer linker sequences and higher induction levels stress the biosynthetic machinery of the cells, inhibiting cell growth and leading to cell lysis in extreme cases.

[0169] Linkers within the scope of the present disclosure include amino acid residues. The amino acid residues may be any of the naturally occurring amino acid residues. Amino acid residues may also be synthetic amino acids known to those of skill in the art. Representative amino acids which may be used in linkers include Glycine, Alanine, Valine, Leucine, Isoleucine, Serine, Cysteine, Selenocysteine, Threonine, Methionine, Proline, Phenylalanine, Tyrosine, Tryptophan, Histidine, Lysine, Arginine, Aspartate, Glutamate, Asparagine, and Glutamine.

[0170] According to one aspect, the linker length can be any length which may be expressed from a cell, such as a bacterial cell when linking the antibody domains. In some embodiments, the linker has a length of about four to about fifty amino acids. According to one aspect, a peptide linker sequence is a polypeptide sequence of at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 24, 48 or more amino acids.

[0171] In some embodiments, the peptide linker sequence comprises a flexible polypeptide, e.g., a polypeptide not having a rigid secondary and / or tertiary structure. In some embodiments, the linker sequence comprises glycine and serine residues. Tn some embodiments at least 50% of the amino acids comprised by the linker sequence are glycine or serine residues, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or more are glycine or serine residues. In some embodiments, the linker sequence consists of glycine and serine residues.

[0172] In some embodiments, the linker is (GS)n (SEQ ID NO: 68), (G3S)n (SEQ ID NO: 69), (G4S)n (SEQ ID NO: 70), or (G6S)n (SEQ ID NO: 71), wherein n is any integer between 1-20. In some embodiments, the linker includes an amino acid sequence of GGGGGGS (SEQ ID NO: 8).

[0173] In certain embodiments, the IL- 12 polypeptide or a functional portion thereof includes the amino acid sequence of SEQ ID NO: 9 or a peptide having at least 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 9. In certain embodiments, the IL- 12 polypeptideor a functional portion thereof includes a peptide having at least 85% sequence identity to SEQ ID NO: 9. In certain embodiments, the IL- 12 polypeptide or a functional portion thereof includes a peptide having at least 86% sequence identity to SEQ ID NO: 9. In certain embodiments, the IL- 12 polypeptide or a functional portion thereof includes a peptide having at least 87% sequence identity to SEQ ID NO: 9. In certain embodiments, the IL-12 polypeptide or a functional portion thereof includes a peptide having at least 88% sequence identity to SEQ ID NO: 9. In certain embodiments, the IL-12 polypeptide or a functional portion thereof includes a peptide having at least 89% sequence identity to SEQ ID NO: 9. In certain embodiments, the IL- 12 polypeptide or a functional portion thereof includes a peptide having at least 90% sequence identity to SEQ ID NO: 9. In certain embodiments, the IL-12 polypeptide or a functional portion thereof includes a peptide having at least 91% sequence identity to SEQ ID NO: 9. In certain embodiments, the IL- 12 polypeptide or a functional portion thereof includes a peptide having at least 92% sequence identity to SEQ ID NO: 9. In certain embodiments, the IL- 12 polypeptide or a functional portion thereof includes a peptide having at least 93% sequence identity to SEQ ID NO: 9. In certain embodiments, the IL-12 polypeptide or a functional portion thereof includes a peptide having at least 94% sequence identity to SEQ ID NO: 9. In certain embodiments, the IL- 12 polypeptide or a functional portion thereof includes a peptide having at least 95% sequence identity to SEQ ID NO: 9. In certain embodiments, the IL- 12 polypeptide or a functional portion thereof includes a peptide having at least 96% sequence identity to SEQ ID NO: 9. In certain embodiments, the IL-12 polypeptide or a functional portion thereof includes a peptide having at least 97% sequence identity to SEQ ID NO: 9. In certain embodiments, the IL- 12 polypeptide or a functional portion thereof includes a peptide having at least 98% sequence identity to SEQ ID NO: 9. In certain embodiments, the IL- 12 polypeptide or a functional portion thereof includes a peptide having at least 99% sequence identity to SEQ ID NO: 9. In certain embodiments, the IL-12 polypeptide or a functional portion thereof comprises the amino acid sequence of SEQ ID NO: 9. In certain embodiments, the IL- 12 polypeptide or a functional portion thereof consists of the amino acid sequence of SEQ ID NO: 9.

[0174] In certain embodiments, the IL- 15 polypeptide or a functional portion thereof includes the amino acid sequence of SEQ ID NO: 17 or a peptide having at least 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 17. In certain embodiments, the IL-15 polypeptide or a functional portion thereof includes a peptide having at least 85% sequence identity to SEQ ID NO: 17. In certain embodiments, the IL-15 polypeptide or a functionalportion thereof includes a peptide having at least 86% sequence identity to SEQ ID NO: 17. In certain embodiments, the IL- 15 polypeptide or a functional portion thereof includes a peptide having at least 87% sequence identity to SEQ ID NO: 17. In certain embodiments, the IL- 15 polypeptide or a functional portion thereof includes a peptide having at least 88% sequence identity to SEQ ID NO: 17. In certain embodiments, the IL- 15 polypeptide or a functional portion thereof includes a peptide having at least 89% sequence identity to SEQ ID NO: 17. In certain embodiments, the IL- 15 polypeptide or a functional portion thereof includes a peptide having at least 90% sequence identity to SEQ ID NO: 17. In certain embodiments, the IL- 15 polypeptide or a functional portion thereof includes a peptide having at least 91% sequence identity to SEQ ID NO: 17. In certain embodiments, the IL-15 polypeptide or a functional portion thereof includes a peptide having at least 92% sequence identity to SEQ ID NO: 17. In certain embodiments, the IL- 15 polypeptide or a functional portion thereof includes a peptide having at least 93% sequence identity to SEQ ID NO: 17. In certain embodiments, the IL- 15 polypeptide or a functional portion includes thereof a peptide having at least 94% sequence identity to SEQ ID NO: 17. In certain embodiments, the IL- 15 polypeptide or a functional portion thereof includes a peptide having at least 95% sequence identity to SEQ ID NO: 17. In certain embodiments, the IL-15 polypeptide or a functional portion thereof includes a peptide having at least 96% sequence identity to SEQ ID NO: 17. In certain embodiments, the IL-15 polypeptide or a functional portion thereof includes a peptide having at least 97% sequence identity to SEQ ID NO: 17. In certain embodiments, the IL- 15 polypeptide or a functional portion thereof includes a peptide having at least 98% sequence identity to SEQ ID NO: 17. In certain embodiments, the IL-15 polypeptide or a functional portion thereof includes a peptide having at least 99% sequence identity to SEQ ID NO: 17. In certain embodiments, the IL-15 polypeptide or a functional portion thereof comprises the amino acid sequence of SEQ ID NO: 17. In certain embodiments, the IL- 15 polypeptide or a functional portion thereof consists of the amino acid sequence of SEQ ID NO: 17.

[0175] In certain embodiments, the IL- 18 polypeptide or a functional portion thereof includes the amino acid sequence of SEQ ID NO: 19 or a peptide having at least 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 19. In certain embodiments, the IL- 18 polypeptide or a functional portion thereof includes a peptide having at least 85% sequence identity to SEQ ID NO: 19. In certain embodiments, the IL- 18 polypeptide or a functional portion thereof includes a peptide having at least 86% sequence identity to SEQ ID NO: 19.In certain embodiments, the IL- 18 polypeptide or a functional portion thereof includes a peptide having at least 87% sequence identity to SEQ ID NO: 19. In certain embodiments, the IL- 18 polypeptide or a functional portion thereof includes a peptide having at least 88% sequence identity to SEQ ID NO: 19. In certain embodiments, the IL-18 polypeptide or a functional portion thereof includes a peptide having at least 89% sequence identity to SEQ ID NO: 19. In certain embodiments, the IL- 18 polypeptide or a functional portion thereof includes a peptide having at least 90% sequence identity to SEQ ID NO: 19. In certain embodiments, the IL- 18 polypeptide or a functional portion thereof includes a peptide having at least 91% sequence identity to SEQ ID NO: 19. In certain embodiments, the IL-18 polypeptide or a functional portion thereof includes a peptide having at least 92% sequence identity to SEQ ID NO: 19. In certain embodiments, the IL- 18 polypeptide or a functional portion thereof includes a peptide having at least 93% sequence identity to SEQ ID NO: 19. In certain embodiments, the IL- 18 polypeptide or a functional portion thereof includes a peptide having at least 94% sequence identity to SEQ ID NO: 19. In certain embodiments, the IL- 18 polypeptide or a functional portion thereof includes a peptide having at least 95% sequence identity to SEQ ID NO: 19. In certain embodiments, the IL-18 polypeptide or a functional portion thereof includes a peptide having at least 96% sequence identity to SEQ ID NO: 19. In certain embodiments, the IL-18 polypeptide or a functional portion thereof includes a peptide having at least 97% sequence identity to SEQ ID NO: 19. In certain embodiments, the IL- 18 polypeptide or a functional portion thereof includes a peptide having at least 98% sequence identity to SEQ ID NO: 19. In certain embodiments, the IL-18 polypeptide or a functional portion thereof includes a peptide having at least 99% sequence identity to SEQ ID NO: 19. In certain embodiments, the IL- 18 polypeptide or a functional portion thereof comprises the amino acid sequence of SEQ ID NO: 19. In certain embodiments, the IL- 18 polypeptide or a functional portion thereof consists of the amino acid sequence of SEQ ID NO: 19.

[0176] Transmembrane Region

[0177] The fusion protein of the present disclosure can be designed with a transmembrane region that is fused to the cytokine region. In some instances, the transmembrane region can be selected or modified by amino acid substitution. The transmembrane region may be derived either from a natural or from a synthetic source. Where the source is natural, the region may be derived from any membrane-bound or transmembrane protein. As used herein, a ‘transmembrane region’ includes the transmembrane domain and may or may notinclude additional intracellular and / or extracellular fragments. As used herein, a ‘transmembrane domain’ refers to a segment of a protein that spans the cell membrane. As used herein, ‘TM’ refers to the transmembrane region of the fusion protein.

[0178] Non-limiting examples of such proteins include CD28, CD8alpha, CD8beta, 4-1BB, B7-H3, BAFFR, BLAME, BTLA, CD100, CD103, CD11a, CD11b, CD11c, CD11d, CD160, CD18, CD19, CD19a, CD2, CD247, CD27, CD276, CD29, CD3 delta, CD3 epsilon, CD3 gamma, CD30, CD4, CD40, CD49a, CD49D, CD49f, CD69, CD7, CD79a, CD84, CD96, CDS, CEACAM1, CRTAM, DAP-10, DNAM1, Fc gamma receptor, GADS, GITR, HVEM, IA4, ICAM-1, IGOS, IL-2R beta, IL-2R gamma, IL-7R alpha, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB1, ITGB2, ITGB7, KIRDS2, LAT, LFA-1, LIGHT, LTBR, Ly9, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80, OX-40, PAG, PD-1, PSGL1, SELPLG, SLAM, SLAMF4, SLAMF6, SLAMF7, SLP-76, TNFR2, TNFSF14, TRANCE, VLA1, VLA-6, a cytokine receptor, a MHC class 1 molecule, a SLAM protein, a TNF receptor protein, a Toll ligand receptor, an activating NK cell receptor, an immunoglobulin protein, and an integrin.

[0179] In certain embodiments, the transmembrane region comprises any transmembrane domain described in the present disclosure, and optionally is selected from the group consisting of a transmembrane domain of CD28, CD80, EGFR, 4- IBB, an alpha chain of a T cell receptor, a beta chain of a T cell receptor, a zeta chain of a T cell receptor, CD3 epsilon, CD4, CD5, CD8 alpha, CD9, CD16, CD19, CD22, CD33, CD37, CD45, CD64, CD86, CD134, CD137, and CD154. In some embodiments, the transmembrane domain comprises a transmembrane domain of CD28. In some embodiments, the transmembrane domain comprises a transmembrane domain of CD80. In some embodiments, the transmembrane domain comprises a transmembrane domain of EGFR. In some embodiments, the transmembrane domain comprises a transmembrane domain of 4- IBB. In some embodiments, the transmembrane domain comprises a transmembrane domain of an alpha chain of a T cell receptor. In some embodiments, the transmembrane domain comprises a transmembrane domain of a beta chain of a T cell receptor. In some embodiments, the transmembrane domain comprises a transmembrane domain of a zeta chain of a T cell receptor. In some embodiments, the transmembrane domain comprises a transmembrane domain of CD3 epsilon. In some embodiments, the transmembrane domain comprises a transmembrane domain of CD4. In some embodiments, the transmembrane domain comprises a transmembrane domain of CD5. In some embodiments, the transmembranedomain comprises a transmembrane domain of CD8 alpha. In some embodiments, the transmembrane domain comprises a transmembrane domain of CD9. In some embodiments, the transmembrane domain comprises a transmembrane domain of CD 16. In some embodiments, the transmembrane domain comprises a transmembrane domain of CD 19. In some embodiments, the transmembrane domain comprises a transmembrane domain of CD22. In some embodiments, the transmembrane domain comprises a transmembrane domain of CD33. In some embodiments, the transmembrane domain comprises a transmembrane domain of CD37. In some embodiments, the transmembrane domain comprises a transmembrane domain of CD45. In some embodiments, the transmembrane domain comprises a transmembrane domain of CD64. In some embodiments, the transmembrane domain comprises a transmembrane domain of CD86. In some embodiments, the transmembrane domain comprises a transmembrane domain of CD 134. In some embodiments, the transmembrane domain comprises a transmembrane domain of CD137. In some embodiments, the transmembrane domain comprises a transmembrane domain of CD 154.

[0180] In certain embodiments, the transmembrane region includes the CD28 transmembrane domain. In certain embodiments, the CD28 transmembrane domain includes the amino acid sequence of SEQ ID NO: 4 or a peptide having at least 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 4. In certain embodiments, the CD28 transmembrane domain comprises a peptide having at least 85% sequence identity to SEQ ID NO: 4. In certain embodiments, the CD28 transmembrane domain comprises a peptide having at least 86% sequence identity to SEQ ID NO: 4. In certain embodiments, the CD28 transmembrane domain comprises a peptide having at least 87% sequence identity to SEQ ID NO: 4. In certain embodiments, the CD28 transmembrane domain comprises a peptide having at least 88% sequence identity to SEQ ID NO: 4. In certain embodiments, the CD28 transmembrane domain comprises a peptide having at least 89% sequence identity to SEQ ID NO: 4. In certain embodiments, the CD28 transmembrane domain comprises a peptide having at least 90% sequence identity to SEQ ID NO: 4. In certain embodiments, the CD28 transmembrane domain comprises a peptide having at least 91% sequence identity to SEQ ID NO: 4. In certain embodiments, the CD28 transmembrane domain comprises a peptide having at least 92% sequence identity to SEQ ID NO: 4. In certain embodiments, the CD28 transmembrane domain comprises a peptide having at least 93% sequence identity to SEQ ID NO: 4. In certain embodiments, the CD28 transmembrane domain comprises a peptide having at least94% sequence identity to SEQ ID NO: 4. In certain embodiments, the CD28 transmembrane domain comprises a peptide having at least 95% sequence identity to SEQ ID NO: 4. In certain embodiments, the CD28 transmembrane domain comprises a peptide having at least 96% sequence identity to SEQ ID NO: 4. In certain embodiments, the CD28 transmembrane domain comprises a peptide having at least 97% sequence identity to SEQ ID NO: 4. In certain embodiments, the CD28 transmembrane domain comprises a peptide having at least 98% sequence identity to SEQ ID NO: 4. In certain embodiments, the CD28 transmembrane domain comprises a peptide having at least 99% sequence identity to SEQ ID NO: 4. In certain embodiments, the CD28 transmembrane domain comprises the amino acid sequence of SEQ ID NO: 4. In certain embodiments, the CD28 transmembrane domain consists of the amino acid sequence of SEQ ID NO: 4.

[0181] In certain embodiments, the transmembrane region includes the CD80 transmembrane domain. In certain embodiments, the CD80 transmembrane domain includes the amino acid sequence of SEQ ID NO: 3 or a peptide having at least 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 3. In certain embodiments, the CD80 transmembrane domain comprises a peptide having at least 85% sequence identity to SEQ ID NO: 3. In certain embodiments, the CD80 transmembrane domain comprises a peptide having at least 86% sequence identity to SEQ ID NO: 3. In certain embodiments, the CD80 transmembrane domain comprises a peptide having at least 87% sequence identity to SEQ ID NO: 3. In certain embodiments, the CD80 transmembrane domain comprises a peptide having at least 88% sequence identity to SEQ ID NO: 3. In certain embodiments, the CD80 transmembrane domain comprises a peptide having at least 89% sequence identity to SEQ ID NO: 3. In certain embodiments, the CD80 transmembrane domain comprises a peptide having at least 90% sequence identity to SEQ ID NO: 3. In certain embodiments, the CD80 transmembrane domain comprises a peptide having at least 91% sequence identity to SEQ ID NO: 3. In certain embodiments, the CD80 transmembrane domain comprises a peptide having at least 92% sequence identity to SEQ ID NO: 3. In certain embodiments, the CD80 transmembrane domain comprises a peptide having at least 93% sequence identity to SEQ ID NO: 3. In certain embodiments, the CD80 transmembrane domain comprises a peptide having at least 94% sequence identity to SEQ ID NO: 3. In certain embodiments, the CD80 transmembrane domain comprises a peptide having at least 95% sequence identity to SEQ ID NO: 3. In certain embodiments, the CD80 transmembrane domain comprises a peptide having at least 96% sequence identity to SEQ ID NO: 3. In certain embodiments, the CD80 transmembranedomain comprises a peptide having at least 97% sequence identity to SEQ ID NO: 3. In certain embodiments, the CD80 transmembrane domain comprises a peptide having at least 98% sequence identity to SEQ ID NO: 3. In certain embodiments, the CD80 transmembrane domain comprises a peptide having at least 99% sequence identity to SEQ ID NO: 3. In certain embodiments, the CD80 transmembrane domain comprises the amino acid sequence of SEQ ID NO: 3. In certain embodiments, the CD80 transmembrane domain consists of the amino acid sequence of SEQ ID NO: 3.

[0182] Costimulatory Domain

[0183] A “costimulatory domain” as used herein refers to a molecule that provides a signal which mediates a T cell response, including, but not limited to, proliferation, activation, differentiation, and the like. It functions as a secondary signal, working in conjunction with the primary signal provided by the T-cell receptor (TCR) or chimeric antigen receptor (CAR) to ensure proper immune response. A “costimulatory domain” of the present disclosure may include all or a portion of a protein’s intracellular domain (ICD), wherein an “intracellular domain” or “ICD” refers to the intracellular portion of a membrane -bound protein. A “costimulatory domain” and an “intracellular costimulatory domain” are used interchangeably herein.

[0184] The costimulatory domain is typically found on antigen-presenting cells (APCs), such as dendritic cells and macrophages. When an APC encounters a pathogen, it processes the pathogen's antigens and presents them to T cells via the major histocompatibility complex (MHC) molecules. This interaction between the TCR on the T cell and the MHC-antigen complex on the APC provides the primary signal for T-cell activation.

[0185] However, this primary signal alone is often not sufficient to fully activate T cells. The costimulatory domain on the APC provides the necessary secondary signal, which can either enhance or inhibit the T-cell response. The most well-known costimulatory molecules are CD80 (B7-1) and CD86 (B7-2), which interact with CD28 on the T cell to deliver a positive costimulatory signal, promoting T-cell activation, proliferation, and survival.

[0186] Non-limiting examples of the costimulatory domains include the intracellular domain (or other suitable portion) derived from the costimulatory receptors selected from the group consisting of CD28, OX-40, 4-1BB / CD137, CD2, CD7, CD27, CD30, CD40, Programmed Death- 1 (PD-1), inducible T cell costimulator (ICOS), lymphocyte function- associated antigen-1 (LFA-1, CDl-la / CD18), CD3 gamma, CD3 delta, CD3 epsilon, CD247, CD276(B7-H3), LIGHT, (TNFSF14), NKG2C, Ig alpha (CD79a), DAP-10, Fc gamma receptor, MHC class 1 molecule, TNF receptor proteins, an Immunoglobulin protein, cytokine receptor, integrins, Signaling Lymphocytic Activation Molecules (SLAM proteins), activating NK cell receptors, BTLA, a Toll ligand receptor, ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD 19, CD4, CD8alpha, CD8beta, IL-2R beta, IL-2R gamma, IL-7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG / Cbp, CD19a, a ligand that specifically binds with CD83, or any combination thereof.

[0187] CD28 (Uniprot Pl 0747 for human protein) is a protein found on the surface of T cells, belonging to the immunoglobulin superfamily, and serves as a crucial costimulatory receptor for T cell activation. It binds to the ligands CD80 and CD86 on antigen-presenting cells, providing necessary secondary signals that complement the primary antigen recognition signal from the T cell receptor (TCR). The intracellular domain of CD28 plays a vital role in enhancing T cell activation, proliferation, and survival by transmitting signals that increase cytokine production and support sustained immune responses. In cancer immunotherapy, this domain is frequently used in chimeric antigen receptor (CAR) T cell constructs to boost their efficacy in targeting and eliminating cancer cells by ensuring optimal T cell activation and function.

[0188] 4 -IBB, also known as CD137 (Uniprot Q07011 for human protein), is a protein expressed on activated T cells and other immune cells, belonging to the tumor necrosis factor receptor family. It functions as a costimulatory molecule that enhances T cell activation, proliferation, and survival, playing a critical role in immune response regulation. The intracellular domain of 4-1BB, upon binding with its ligand, initiates a signaling cascade that boosts T cell activation, increases their survival and persistence, and enhances their cytotoxicity. This makes 4- IBB particularly valuable in cancer immunotherapy, where its intracellular domain is used as a costimulatory element in chimeric antigen receptor (CAR) T cell or TCR-T cells constructs. This application improves the efficacy of CAR T cells orTCR-T cells in targeting and eliminating cancer cells by strengthening their activation and persistence.

[0189] In certain embodiments, the costimulatory domain includes the intracellular domain of CD28. In certain embodiments, the costimulatory domain includes the amino acid sequence of SEQ ID NO: 5 or a peptide having at least 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 5. In certain embodiments, the costimulatory domain comprises a peptide having at least 85% sequence identity to SEQ ID NO: 5. In certain embodiments, the costimulatory domain comprises a peptide having at least 86% sequence identity to SEQ ID NO: 5. In certain embodiments, the costimulatory domain comprises a peptide having at least 87% sequence identity to SEQ ID NO: 5. In certain embodiments, the costimulatory domain comprises a peptide having at least 88% sequence identity to SEQ ID NO: 5. In certain embodiments, the costimulatory domain comprises a peptide having at least 89% sequence identity to SEQ ID NO: 5. In certain embodiments, the costimulatory domain comprises a peptide having at least 90% sequence identity to SEQ ID NO: 5. In certain embodiments, the costimulatory domain comprises a peptide having at least 91% sequence identity to SEQ ID NO: 5. In certain embodiments, the costimulatory domain comprises a peptide having at least 92% sequence identity to SEQ ID NO: 5. In certain embodiments, the costimulatory domain comprises a peptide having at least 93% sequence identity to SEQ ID NO: 5. In certain embodiments, the costimulatory domain comprises a peptide having at least 94% sequence identity to SEQ ID NO: 5. In certain embodiments, the costimulatory domain comprises a peptide having at least 95% sequence identity to SEQ ID NO: 5. In certain embodiments, the costimulatory domain comprises a peptide having at least 96% sequence identity to SEQ ID NO: 5. In certain embodiments, the costimulatory domain comprises a peptide having at least 97% sequence identity to SEQ ID NO: 5. In certain embodiments, the costimulatory domain comprises a peptide having at least 98% sequence identity to SEQ ID NO: 5. In certain embodiments, the costimulatory domain comprises a peptide having at least 99% sequence identity to SEQ ID NO: 5. In certain embodiments, the costimulatory domain comprises the amino acid sequence of SEQ ID NO: 5. In certain embodiments, the costimulatory domain consists of the amino acid sequence of SEQ ID NO: 5.

[0190] In certain embodiments, the costimulatory domain includes the intracellular domain of 4-1BB. In certain embodiments, the intracellular domain of 4-1BB comprises the amino acid sequence of SEQ ID NO: 6 or a peptide having at least 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 6. In certain embodiments, the costimulatory domaincomprises a peptide having at least 85% sequence identity to SEQ ID NO: 6. In certain embodiments, the costimulatory domain comprises a peptide having at least 86% sequence identity to SEQ ID NO: 6. In certain embodiments, the costimulatory domain comprises a peptide having at least 87% sequence identity to SEQ ID NO: 6. In certain embodiments, the costimulatory domain comprises a peptide having at least 88% sequence identity to SEQ ID NO: 6. In certain embodiments, the costimulatory domain comprises a peptide having at least 89% sequence identity to SEQ ID NO: 6. In certain embodiments, the costimulatory domain comprises a peptide having at least 90% sequence identity to SEQ ID NO: 6. In certain embodiments, the costimulatory domain comprises a peptide having at least 91% sequence identity to SEQ ID NO: 6. In certain embodiments, the costimulatory domain comprises a peptide having at least 92% sequence identity to SEQ ID NO: 6. In certain embodiments, the costimulatory domain comprises a peptide having at least 93% sequence identity to SEQ ID NO: 6. In certain embodiments, the costimulatory domain comprises a peptide having at least 94% sequence identity to SEQ ID NO: 6. In certain embodiments, the costimulatory domain comprises a peptide having at least 95% sequence identity to SEQ ID NO: 6. In certain embodiments, the costimulatory domain comprises a peptide having at least 96% sequence identity to SEQ ID NO: 6. In certain embodiments, the costimulatory domain comprises a peptide having at least 97% sequence identity to SEQ ID NO: 6. In certain embodiments, the costimulatory domain comprises a peptide having at least 98% sequence identity to SEQ ID NO: 6. In certain embodiments, the costimulatory domain comprises a peptide having at least 99% sequence identity to SEQ ID NO: 6. In certain embodiments, the costimulatory domain comprises the amino acid sequence of SEQ ID NO: 6. In certain embodiments, the costimulatory domain consists of the amino acid sequence of SEQ ID NO: 6.

[0191] In one aspect, the fusion protein of the present disclosure includes from N to C terminus, a cytokine region, a transmembrane region, and a costimulatory domain comprising the intracellular domain of CD28.

[0192] In one aspect, the fusion protein of the present disclosure includes from N to C terminus, a cytokine region, a transmembrane region, and a costimulatory domain comprising the intracellular domain of 4- IBB.

[0193] Exemplary Fusion Proteins

[0194] In certain embodiments, the fusion protein of the present disclosure includes from N to C terminus, the cytokine region of IL- 12 polypeptide or a functional portion thereof andthe CD80 transmembrane domain. In certain embodiments, the fusion protein of the present disclosure includes from N terminus to C terminus, IL-12 β subunit p40, a linker, IL-12 α subunit p35, and the CD80 transmembrane domain. In certain embodiments, the fusion protein of the present disclosure includes the amino acid sequence of SEQ ID NO: 10 or a peptide having at least 85%, 90%, 92%, 95%, 98% or 99% sequence identity to SEQ ID NO: 10. In certain embodiments, the fusion protein of the present disclosure includes a peptide having at least 85% sequence identity to SEQ ID NO: 10. In certain embodiments, the fusion protein of the present disclosure includes a peptide having at least 86% sequence identity to SEQ ID NO: 10. In certain embodiments, the fusion protein of the present disclosure includes a peptide having at least 87% sequence identity to SEQ ID NO: 10. In certain embodiments, the fusion protein of the present disclosure includes a peptide having at least 88% sequence identity to SEQ ID NO: 10. In certain embodiments, the fusion protein of the present disclosure includes a peptide having at least 89% sequence identity to SEQ ID NO: 10. In certain embodiments, the fusion protein of the present disclosure includes a peptide having at least 90% sequence identity to SEQ ID NO: 10. In certain embodiments, the fusion protein of the present disclosure includes a peptide having at least 91 % sequence identity to SEQ ID NO: 10. In certain embodiments, the fusion protein of the present disclosure includes a peptide having at least 92% sequence identity to SEQ ID NO: 10. In certain embodiments, the fusion protein of the present disclosure includes a peptide having at least 93% sequence identity to SEQ ID NO: 10. In certain embodiments, the fusion protein of the present disclosure includes a peptide having at least 94% sequence identity to SEQ ID NO: 10. In certain embodiments, the fusion protein of the present disclosure includes a peptide having at least 95% sequence identity to SEQ ID NO: 10. In certain embodiments, the fusion protein of the present disclosure includes a peptide having at least 96% sequence identity to SEQ ID NO: 10. In certain embodiments, the fusion protein of the present disclosure includes a peptide having at least 97% sequence identity to SEQ ID NO: 10. In certain embodiments, the fusion protein of the present disclosure includes a peptide having at least 98% sequence identity to SEQ ID NO: 10. In certain embodiments, the fusion protein of the present disclosure includes a peptide having at least 99% sequence identity to SEQ ID NO: 10. In certain embodiments, the fusion protein of the present disclosure comprises the amino acid sequence of SEQ ID NO: 10. In certain embodiments, the fusion protein of the present disclosure consists of the amino acid sequence of SEQ ID NO: 10.

[0195] In certain embodiments, the fusion protein of the present disclosure includes from N to C terminus, the cytokine region of IL- 12 polypeptide or a functional portion thereof and the CD28 transmembrane domain. In certain embodiments, the fusion protein of the present disclosure includes from N terminus to C terminus, IL-12 β subunit p40, a linker, IL-12 α subunit p35, and the CD28 transmembrane domain. In certain embodiments, the fusion protein of the present disclosure includes the amino acid sequence of SEQ ID NO: 11 or a peptide having at least 85%, 90%, 92%, 95%, 98% or 99% sequence identity to SEQ ID NO: 11. In certain embodiments, the fusion protein of the present disclosure includes a peptide having at least 85% sequence identity to SEQ ID NO: 11. In certain embodiments, the fusion protein of the present disclosure includes a peptide having at least 86% sequence identity to SEQ ID NO: 11. In certain embodiments, the fusion protein of the present disclosure includes a peptide having at least 87% sequence identity to SEQ ID NO: 11. In certain embodiments, the fusion protein of the present disclosure includes a peptide having at least 88% sequence identity to SEQ ID NO: 11. In certain embodiments, the fusion protein of the present disclosure includes a peptide having at least 89% sequence identity to SEQ ID NO: 11. In certain embodiments, the fusion protein of the present disclosure includes a peptide having at least 90% sequence identity to SEQ ID NO: 11. In certain embodiments, the fusion protein of the present disclosure includes a peptide having at least 91% sequence identity to SEQ ID NO: 11. In certain embodiments, the fusion protein of the present disclosure includes a peptide having at least 92% sequence identity to SEQ ID NO: 11. In certain embodiments, the fusion protein of the present disclosure includes a peptide having at least 93% sequence identity to SEQ ID NO: 11. In certain embodiments, the fusion protein of the present disclosure includes a peptide having at least 94% sequence identity to SEQ ID NO: 11. In certain embodiments, the fusion protein of the present disclosure includes a peptide having at least 95% sequence identity to SEQ ID NO: 11. In certain embodiments, the fusion protein of the present disclosure includes a peptide having at least 96% sequence identity to SEQ ID NO: 11. In certain embodiments, the fusion protein of the present disclosure includes a peptide having at least 97% sequence identity to SEQ ID NO: 11. In certain embodiments, the fusion protein of the present disclosure includes a peptide having at least 98% sequence identity to SEQ ID NO: 11. In certain embodiments, the fusion protein of the present disclosure includes a peptide having at least 98% sequence identity to SEQ ID NO: 11. In certain embodiments, the fusion protein of the present disclosure comprises the aminoacid sequence of SEQ ID NO: 11. In certain embodiments, the fusion protein of the present disclosure consists of the amino acid sequence of SEQ ID NO: 11.

[0196] In certain embodiments, the fusion protein of the present disclosure includes from N to C terminus, the cytokine region of IL- 12 polypeptide or functional portion thereof, the CD80 transmembrane domain, and the costimulatory domain of the intracellular domain of 4-1BB. In certain embodiments, the fusion protein of the present disclosure includes from N terminus to C terminus, IL-12 β subunit p40, a linker, IL-12 α subunit p35, the CD80 transmembrane domain, and the 4- IBB intracellular domain. In certain embodiments, the fusion protein of the present disclosure includes the amino acid sequence of SEQ ID NO: 12 or a peptide having at least 85%, 90%, 92%, 95%, 98% or 99% sequence identity to SEQ ID NO: 12. In certain embodiments, the fusion protein includes an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 12. In certain embodiments, the fusion protein includes an amino acid sequence having at least 86% sequence identity to SEQ ID NO: 12. In certain embodiments, the fusion protein includes an amino acid sequence having at least 87% sequence identity to SEQ ID NO: 12. In certain embodiments, the fusion protein includes an amino acid sequence having at least 88% sequence identity to SEQ ID NO: 12. In certain embodiments, the fusion protein includes an amino acid sequence having at least 89% sequence identity to SEQ ID NO: 12. In certain embodiments, the fusion protein includes an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 12. In certain embodiments, the fusion protein includes an amino acid sequence having at least 91% sequence identity to SEQ ID NO: 12. In certain embodiments, the fusion protein includes an amino acid sequence having at least 92% sequence identity to SEQ ID NO: 12. In certain embodiments, the fusion protein includes an amino acid sequence having at least 93% sequence identity to SEQ ID NO: 12. In certain embodiments, the fusion protein includes an amino acid sequence having at least 94% sequence identity to SEQ ID NO: 12. In certain embodiments, the fusion protein includes an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 12. In certain embodiments, the fusion protein includes an amino acid sequence having at least 96% sequence identity to SEQ ID NO: 12. In certain embodiments, the fusion protein includes an amino acid sequence having at least 97% sequence identity to SEQ ID NO: 12. In certain embodiments, the fusion protein includes an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 12. In certain embodiments, the fusion protein includes an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 12. In certain embodiments, the fusion protein of thepresent disclosure comprises the amino acid sequence of SEQ ID NO: 12. In certain embodiments, the fusion protein of the present disclosure consists of the amino acid sequence of SEQ ID NO: 12.

[0197] In certain embodiments, the fusion protein of the present disclosure includes from N to C terminus, the cytokine region of IL- 12 polypeptide or functional portion thereof, the CD28 transmembrane domain, and the costimulatory domain of the intracellular domain of CD28. In certain embodiments, the fusion protein of the present disclosure includes from N terminus to C terminus, IL-12 β subunit p40, a linker, IL-12 α subunit p35, the CD28 transmembrane domain, and the CD28 intracellular domain. In certain embodiments, the fusion protein of the present disclosure includes the amino acid sequence of SEQ ID NO: 13. In certain embodiments, the fusion protein of the present disclosure includes the amino acid sequence of SEQ ID NO: 13 or a peptide having at least 85%, 90%, 92%, 95%, 98% or 99% sequence identity to SEQ ID NO: 13. In certain embodiments, the fusion protein includes an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 13. In certain embodiments, the fusion protein includes an amino acid sequence having at least 86% sequence identity to SEQ ID NO: 13. In certain embodiments, the fusion protein includes an amino acid sequence having at least 87% sequence identity to SEQ ID NO: 13. In certain embodiments, the fusion protein includes an amino acid sequence having at least 88% sequence identity to SEQ ID NO: 13. In certain embodiments, the fusion protein includes an amino acid sequence having at least 89% sequence identity to SEQ ID NO: 13. In certain embodiments, the fusion protein includes an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 13. In certain embodiments, the fusion protein includes an amino acid sequence having at least 91% sequence identity to SEQ ID NO: 13. In certain embodiments, the fusion protein includes an amino acid sequence having at least 92% sequence identity to SEQ ID NO: 13. In certain embodiments, the fusion protein includes an amino acid sequence having at least 93% sequence identity to SEQ ID NO: 13. In certain embodiments, the fusion protein includes an amino acid sequence having at least 94% sequence identity to SEQ ID NO: 13. In certain embodiments, the fusion protein includes an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 13. In certain embodiments, the fusion protein includes an amino acid sequence having at least 96% sequence identity to SEQ ID NO: 13. In certain embodiments, the fusion protein includes an amino acid sequence having at least 97% sequence identity to SEQ ID NO: 13. In certain embodiments, the fusion protein includes an amino acid sequence having at least 98%sequence identity to SEQ ID NO: 13. In certain embodiments, the fusion protein includes an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 13. In certain embodiments, the fusion protein of the present disclosure comprises the amino acid sequence of SEQ ID NO: 13. In certain embodiments, the fusion protein of the present disclosure consists of the amino acid sequence of SEQ ID NO: 13.

[0198] In certain embodiments, the fusion protein of the present disclosure includes from N to C terminus, the cytokine region including the IL- 12 polypeptide or functional portion thereof, the CD28 transmembrane domain, and the costimulatory domain including the 4-1BB intracellular domain. In certain embodiments, the fusion protein of the present disclosure includes from N terminus to C terminus, IL-12 β subunit p40, a linker, IL-12 α subunit p35, the CD28 transmembrane domain and the 4-1BB intracellular domain. In certain embodiments, the fusion protein of the present disclosure includes the amino acid sequence of SEQ ID NO: 14 or a peptide having at least 85%, 90%, 92%, 95%, 98% or 99% sequence identity to SEQ ID NO: 14. In certain embodiments, the fusion protein of the present disclosure comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 14. In certain embodiments, the fusion protein of the present disclosure comprises an amino acid sequence having at least 86% sequence identity to SEQ ID NO: 14. In certain embodiments, the fusion protein of the present disclosure comprises an amino acid sequence having at least 87% sequence identity to SEQ ID NO: 14. In certain embodiments, the fusion protein of the present disclosure comprises an amino acid sequence having at least 88% sequence identity to SEQ ID NO: 14. In certain embodiments, the fusion protein of the present disclosure comprises an amino acid sequence having at least 89% sequence identity to SEQ ID NO: 14. In certain embodiments, the fusion protein of the present disclosure comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 14. In certain embodiments, the fusion protein of the present disclosure comprises an amino acid sequence having at least 91% sequence identity to SEQ ID NO: 14. In certain embodiments, the fusion protein of the present disclosure comprises an amino acid sequence having at least 92% sequence identity to SEQ ID NO: 14. In certain embodiments, the fusion protein of the present disclosure comprises an amino acid sequence having at least 93% sequence identity to SEQ ID NO: 14. In certain embodiments, the fusion protein of the present disclosure comprises an amino acid sequence having at least 94% sequence identity to SEQ ID NO: 14. In certain embodiments, the fusion protein of the present disclosure comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 14. In certain embodiments,the fusion protein of the present disclosure comprises an amino acid sequence having at least 96% sequence identity to SEQ ID NO: 14. In certain embodiments, the fusion protein of the present disclosure comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO: 14. In certain embodiments, the fusion protein of the present disclosure comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 14. In certain embodiments, the fusion protein of the present disclosure comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 14. In certain embodiments, the fusion protein of the present disclosure comprises the amino acid sequence of SEQ ID NO: 14. In certain embodiments, the fusion protein of the present disclosure consists of the amino acid sequence of SEQ ID NO: 14.

[0199] In certain embodiments, the cytokine fusion protein of the present disclosure includes one or more cytokine regions such as, but not limited to, interleukins or fragments thereof (e.g., IL-12, IL-15, IL-2, IL-4, IL-7, IL-9, IL-18, IL-21, etc.) fused to a transmembrane domain (e.g., CD28, CD80) and a costimulatory domain (e.g., an intracellular domain of 4-1BB, an intracellular domain of CD28). In certain embodiments, the cytokine fusion protein may include two cytokine regions (e.g., two interleukins or functional fragments thereof) fused to a transmembrane domain (e.g., CD28, CD80) and a costimulatory domain (e.g., an intracellular domain of 4-1 BB, an intracellular domain of CD28). In certain embodiments, the cytokine fusion protein includes multiple cytokine regions (e.g., multiple interleukins or functional fragments thereof) fused to a transmembrane domain (e.g.. CD28, CD80) and a costimulatory domain (e.g., an intracellular domain of 4- IBB, an intracellular domain of CD28).

[0200] Exemplary Bifunctional / Multifunctional Fusion Proteins

[0201] In certain embodiments, the cytokine region of the fusion protein of the present disclosure comprises at least a first polypeptide comprising a first cytokine or functional fragment thereof, and a second polypeptide comprising a second cytokine or functional fragment thereof. The first cytokine or functional fragment thereof and the second cytokine or functional fragment thereof may be directly connected in tandem or connected via an intervening fragment and / or linker. In certain embodiments, the cytokine region comprises at least a first polypeptide selected from the group consisting of a polypeptide of interleukin- 12 (IL-12), IL-15, IL-2, IL-4, IL-7, IL-9, IL-18, and IL-21 or a functional portion thereof, and a second polypeptide selected from the group consisting of a polypeptide of interleukin- 12 (IL-12), IL- 15, IL-2, IL-4, IL-7, IL-9, IL- 18, and IL-21 or a functional portion thereof. In certainembodiments, the first polypeptide and the second polypeptide are fused to a transmembrane domain (e.g., CD28, CD80) and a costimulatory domain (e.g., an intracellular domain of 4-1BB, an intracellular domain of CD28). In certain embodiments, the first polypeptide and the second polypeptide are fused to a CD28 transmembrane domain and a 4- IBB intracellular domain. In certain embodiments, the first polypeptide and the second polypeptide are fused to a CD80 transmembrane domain and a 4- IBB intracellular domain. In certain embodiments, the first polypeptide and the second polypeptide are fused to a CD28 transmembrane domain and a CD28 intracellular domain. In certain embodiments, the first polypeptide and the second polypeptide are fused to a CD80 transmembrane domain and a CD28 intracellular domain.

[0202] In certain embodiments, the cytokine region comprises from N terminus to C terminus the first polypeptide comprising IL- 15 or a functional fragment thereof and the second polypeptide comprising IL- 18 or a functional fragment thereof. In certain embodiments, the cytokine region comprises from N terminus to C terminus the first polypeptide comprising IL- 18 or a functional fragment thereof and the second polypeptide comprising IL- 15 or a functional fragment thereof._In certain embodiments, the first polypeptide is IL- 15 or a functional fragment thereof and the second polypeptide is IL- 18 or a functional fragment thereof, and the IL-15 and the IL- 18 (or functional fragments thereof) are fused to a CD28 transmembrane domain and a 4- IBB intracellular domain. In certain embodiments, the first polypeptide is IL- 15 or a functional fragment thereof and the second polypeptide is IL- 18 or a functional fragment thereof, and the IL- 15 and the IL- 18 (or functional fragments thereof) are fused to a CD80 transmembrane domain and a 4-1BB intracellular domain. In certain embodiments, the first polypeptide is IL-15 or a functional fragment thereof and the second polypeptide is IL- 18 or a functional fragment thereof, and the IL- 15 and the IL- 18 (or functional fragments thereof) are fused to a CD28 transmembrane domain and a CD28 intracellular domain. In certain embodiments, the first polypeptide is IL- 15 or a functional fragment thereof and the second polypeptide is IL- 18 or a functional fragment thereof, and the IL- 15 or the IL- 18 (or functional fragments thereof) are fused a CD80 transmembrane domain and a CD28 intracellular domain.

[0203] In certain embodiments, the first polypeptide is IL- 18 or a functional fragment thereof and the second polypeptide is IL- 15 or a functional fragment thereof, and the IL- 18 and the IL- 15 (or functional fragments thereof) are fused to a CD28 transmembrane domain and a 4-1BB intracellular domain. In certain embodiments, the first polypeptide is IL-18 or afunctional fragment thereof and the second polypeptide is IL- 15 or a functional fragment thereof, and the IL- 18 and the IL- 15 (or functional fragments thereof) are fused to a CD80 transmembrane domain and a 4- IBB intracellular domain. In certain embodiments, the first polypeptide is a IL- 18 or a functional fragment thereof and the second polypeptide is IL- 15 or a functional fragment thereof, and the IL- 18 and the IL- 15 (or functional fragments thereof) are fused to a CD28 transmembrane domain and a CD28 intracellular domain. In certain embodiments, the first polypeptide is IL- 18 or a functional fragment thereof and the second polypeptide is IL- 15 or a functional fragment thereof, and the IL- 18 and the IL- 15 (or functional fragments thereof) are fused a CD80 transmembrane domain and a CD28 intracellular domain.

[0204] In certain embodiments, the cytokine region comprises the amino acid sequence of SEQ ID NO: 64 or an amino acid sequence having at least 85%, 90%, 92%, 95%, or 99% sequence identity to SEQ ID NO: 64. In certain embodiments, the cytokine region comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 64. In certain embodiments, the cytokine region comprises an amino acid sequence having at least 86% sequence identity to SEQ ID NO: 64. In certain embodiments, the cytokine region comprises an amino acid sequence having at least 87% sequence identity to SEQ ID NO: 64. In certain embodiments, the cytokine region comprises an amino acid sequence having at least 88% sequence identity to SEQ ID NO: 64. In certain embodiments, the cytokine region comprises an amino acid sequence having at least 89% sequence identity to SEQ ID NO: 64. In certain embodiments, the cytokine region comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 64. In certain embodiments, the cytokine region comprises an amino acid sequence having at least 91 % sequence identity to SEQ ID NO: 64. In certain embodiments, the cytokine region comprises an amino acid sequence having at least 92% sequence identity to SEQ ID NO: 64. In certain embodiments, the cytokine region comprises an amino acid sequence having at least 93% sequence identity to SEQ ID NO: 64. In certain embodiments, the cytokine region comprises an amino acid sequence having at least 94% sequence identity to SEQ ID NO: 64. In certain embodiments, the cytokine region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 64. In certain embodiments, the cytokine region comprises an amino acid sequence having at least 96% sequence identity to SEQ ID NO: 64. In certain embodiments, the cytokine region comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO: 64. In certain embodiments, the cytokine region comprises an amino acid sequence having at least 98%sequence identity to SEQ ID NO: 64. In certain embodiments, the cytokine region comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 64. In certain embodiments, the cytokine region comprises the amino acid sequence of SEQ ID NO: 64. In certain embodiments, the cytokine region consists of the amino acid sequence of SEQ ID NO: 64.

[0205] In certain embodiments, the cytokine region comprises the amino acid sequence of SEQ ID NO: 66 or an amino acid sequence having at least 85%, 90%, 92%, 95%, or 99% sequence identity to SEQ ID NO: 66. In certain embodiments, the cytokine region comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 66. In certain embodiments, the cytokine region comprises an amino acid sequence having at least 86% sequence identity to SEQ ID NO: 66. In certain embodiments, the cytokine region comprises an amino acid sequence having at least 87% sequence identity to SEQ ID NO: 66. In certain embodiments, the cytokine region comprises an amino acid sequence having at least 88% sequence identity to SEQ ID NO: 66. In certain embodiments, the cytokine region comprises an amino acid sequence having at least 89% sequence identity to SEQ ID NO: 66. In certain embodiments, the cytokine region comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 66. In certain embodiments, the cytokine region comprises an amino acid sequence having at least 91 % sequence identity to SEQ ID NO: 66. In certain embodiments, the cytokine region comprises an amino acid sequence having at least 92% sequence identity to SEQ ID NO: 66. In certain embodiments, the cytokine region comprises an amino acid sequence having at least 93% sequence identity to SEQ ID NO: 66. In certain embodiments, the cytokine region comprises an amino acid sequence having at least 94% sequence identity to SEQ ID NO: 66. In certain embodiments, the cytokine region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 66. In certain embodiments, the cytokine region comprises an amino acid sequence having at least 96% sequence identity to SEQ ID NO: 66. In certain embodiments, the cytokine region comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO: 66. In certain embodiments, the cytokine region comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 66. In certain embodiments, the cytokine region comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 66. In certain embodiments, the cytokine region comprises the amino acid sequence of SEQ ID NO: 66. In certain embodiments, the cytokine region consists of the amino acid sequence of SEQ ID NO: 66.

[0206] In certain embodiments, the cytokine region of the fusion protein of the present disclosure comprises multiple polypeptides (e.g., three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, etc.) each comprising a cytokine region or functional fragment thereof, wherein each of the cytokine regions are independently selected from cytokines such as, but not limited to, IL- 12, IL- 15, IL-2, IL-4, IL-7, IL-9, IL- 18, and IL-21 or functional fragments thereof. In certain embodiments, the multiple cytokine regions are fused to a transmembrane domain (e.g., CD28, CD80) and a costimulatory domain (e.g., an intracellular domain of 4-1BB, an intracellular domain of CD28). In certain embodiments, the multiple cytokine regions are fused to a CD28 transmembrane domain and a 4- IBB intracellular domain. In certain embodiments, the multiple cytokine regions are fused to a CD80 transmembrane domain and a 4- IBB intracellular domain. In certain embodiments, the multiple cytokine regions are fused to a CD28 transmembrane domain and a CD28 intracellular domain. In certain embodiments, the multiple cytokine regions are fused to a CD80 transmembrane domain and a CD28 intracellular domain.

[0207] Hinge region

[0208] The fusion protein of the present disclosure may also include a hinge region. The hinge region may be located between the cytokine region and the transmembrane region.

[0209] A variety of hinges can be employed, including portions or derivatives of the molecules of all or some of CD28, OX-40, 4-1BB / CD137, CD2, CD7, CD27, CD30, CD40, programmed death-1 (PD-1), inducible T cell costimulator (ICOS), lymphocyte function-associated antigen- 1 (LFA-1, CDl-la / CD18), CD3 gamma, CD3 delta, CD3 epsilon, CD247, CD276 (B7-H3), LIGHT, (TNFSF14), NKG2C, Ig alpha (CD79a), DAP-10, Fc gamma receptor, MHC class 1 molecule, TNF receptor proteins, an Immunoglobulin protein, IgGl, IgG2, IgG3, IgG4, IgA, IgD, IgE, IgM, cytokine receptor, integrins, Signaling Lymphocytic Activation Molecules (SLAM proteins), activating NK cell receptors, BTLA, a Toll ligand receptor, ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8alpha, CD8beta, IL-2R beta, IL-2R gamma, IL-7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100(SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD 150, IPO-3), BLAME (SLAMF8), SELPLG (CD 162), LTBR, LAT, GADS, SLP-76, PAG / Cbp, CD 19a, a ligand that specifically binds with CD83, or any combination thereof. The extracellular domain may be derived either from a natural or from a synthetic source.

[0210] In certain embodiments, the hinge region is a hinge domain of IgGl, IgG2, IgG3, IgG4, IgA, IgD, IgE, IgM, CD28, or CD8 alpha, any truncation thereof, or any combination thereof.

[0211] It is appreciated that the amino acid sequences of the fusion proteins described herein can be modified to include those having one, two or three amino acid addition, deletion and / or substitutions. In some embodiments, the substitutions can be conservative substitutions.

[0212] A “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a nonessential amino acid residue in an immunoglobulin polypeptide is preferably replaced with another amino acid residue from the same side chain family. In another embodiment, a string of amino acids can be replaced with a structurally similar string that differs in order and / or composition of side chain family members.

[0213] Non-limiting examples of conservative amino acid substitutions are provided in the table below, where a similarity score of 0 or higher indicates conservative substitution between the two amino acids.Table A. Amino Acid Similarity MatrixC G P S A T D E N Q H K R V M 1 L F Y w w -8 -7 -6 -2 -6 -5 -7 -7 -4 -5 -3 -3 2 -6 -4 -5 -2 0 0 17 Y 0 -5 -5 -3 -3 -3 -4 -4 -2 -4 0 -4 -5 -2 -2 -1 -1 7 10 F -4 -5 -5 -3 -4 -3 -6 -5 -4 -5 -2 -5 -4 -1 0 1 2 9 L -6 -4 -3 -3 -2 -2 -4 -3 -3 -2 -2 -3 -3 2 4 2 61 -2 -3 -2 -1 -1 0 -2 -2 -2 -2 -2 -2 -2 4 2 5M -5 -3 -2 -2 -1 -1 -3 -2 0 -1 -2 0 0 2 6V -2 -1 -1 -1 0 0 -2 -2 -2 -2 -2 -2 -2 4R -4 -3 0 0 -2 -1 -1 -1 0 1 2 3 6K -5 -2 -1 0 -1 0 0 0 1 1 0 5H -3 -2 0 -1 -1 -1 1 1 2 3 6Q -5 -1 0 -1 0 -1 2 2 1 4N -4 0 -1 1 0 0 2 1 2E -5 0 -1 0 0 0 3 4D -5 1 -1 0 0 0 4T -2 0 0 1 1 3A -2 1 1 1 2S 0 1 1 1P -3 -1 6G -3 5C 12Table B. Conservative Amino Acid SubstitutionsFor Amino Acid Substitution WithAlanine D-Ala, Gly, Aib, [3-Ala, L-Cys, D-CysArginine D-Arg, Lys, D-Lys, Orn D-OrnAsparagine D-Asn, Asp, D-Asp, Glu, D-Glu Gin, D-GInAspartic Acid D-Asp, D-Asn, Asn, Glu, D-Glu, Gin, D-GInCysteine D-Cys, S-Me-Cys, Met, D-Met, Thr, D-Thr, L-Ser, D-Ser Glutamine D-GIn, Asn, D-Asn, Glu, D-Glu, Asp, D-AspGlutamic Acid D-Glu, D-Asp, Asp, Asn, D-Asn, Gin, D-GInGlycine Ala, D-Ala, Pro, D-Pro, Aib, P-AlaIsoleucine D-lle, Vai, D-Val, Leu, D-Leu, Met, D-MetLeucine Vai, D-Val, Met, D-Met, D-lle, D-Leu, HeLysine D-Lys, Arg, D-Arg, Orn, D-OrnMethionine D-Met, S-Me-Cys, lie, D-lle, Leu, D-Leu, Vai, D-Val Phenylalanine D-Phe, Tyr, D-Tyr, His, D-His, Trp, D-TrpProline D-ProSerine D-Ser, Thr, D-Thr, allo-Thr, L-Cys, D-CysThreonine D-Thr, Ser, D-Ser, allo-Thr, Met, D-Met, Vai, D-Val Tyrosine D-Tyr, Phe, D-Phe, His, D-His, Trp, D-TrpValine D-Val, Leu, D-Leu, He, D-lle, Met, D-Met

[0214] It will also be understood by one of ordinary skill in the art that fusion proteins as disclosed herein may be modified such that they vary in amino acid sequence from which they were derived. For example, a polypeptide or amino acid sequence derived from a designated protein may be similar, e.g., have a certain percent identity to the starting sequence, e.g., it may be 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identical to the starting sequence.Polynucleotides Encoding the Fusion Proteins

[0215] The present disclosure also provides isolated polynucleotides or nucleic acid molecules encoding the fusion proteins, variants or derivatives thereof of the disclosure.

[0216] The present disclosure also provides polynucleotides encoding the fusion proteins, recombinant expression vectors and isolated cells producing the fusion proteins, and pharmaceutical compositions comprising the fusion proteins disclosed herein. Methods of using the fusion proteins of the present disclosure to treat diseases are also provided.

[0217] Nucleic acid sequences encoding the fusion proteins disclosed herein are typically inserted in an expression vector for introduction into cells to express the desired fusion proteins. Accordingly, in certain aspects, the invention provides expression vectors including expressible nucleic acid sequence that entails the fusion protein encoding polynucleotides disclosed herein and cells expressing or comprising these vectors and nucleic acid sequences.

[0218] Vectors

[0219] The term “vector,” as used herein, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes the vector as a selfreplicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as “expression vectors.”

[0220] Such vectors may easily be selected from the group consisting of plasmids, phages, and viruses (e.g., baculoviruses, vaccinia viruses, lentiviruses, adenoviruses, adeno-associated viruses, and retroviruses).

[0221] Numerous expression vector systems may be employed. For example, one class of vector utilizes DNA elements which are derived from animal viruses such as bovine papilloma virus, polyoma virus, adenovirus, vaccinia virus, baculovirus, retroviruses (RSV,MMTV or MOMLV), or SV40 virus. Others involve the use of polycistronic systems with internal ribosome binding sites.

[0222] Promoters

[0223] In certain embodiments, the expression vector includes a promoter. A “promoter” refers to a control region of a nucleic acid at which initiation and rate of transcription of the remainder of a nucleic acid sequence are controlled. A promoter drives transcription or of the nucleic acid sequence that it regulates, thus, it is typically located at or near the transcriptional start site of a gene. A promoter, in some embodiments, is 100 to 1000 nucleotides in length. A promoter may also contain sub-regions at which regulatory proteins and other molecules may bind, such as RNA polymerase and other transcription factors. Promoters may be constitutive (e.g., CAG promoter, cytomegalovirus (CMV) promoter), inducible (also referred to as activatable), repressible, tissue-specific, developmental stagespecific or any combination of two or more of the foregoing.

[0224] A promoter is considered to be “operably linked” when it is in a correct functional location and orientation relative to a sequence of nucleic acid that it regulates (e.g., to control (“drive”) transcriptional initiation and / or expression of that sequence).

[0225] A promoter, in some embodiments, is naturally associated with a nucleic acid and may be obtained by isolating the 5’ non-coding sequence(s) located upstream of the coding region of the given nucleic acid. Such a promoter is referred to as an “endogenous ” promoter. A promoter, in some embodiments, is not naturally associated with a nucleic acid. Such a promoter is referred to as a “heterologous” promoter and includes, for example, promoters that regulate other nucleic acids and promoters obtained from other cells. A heterologous promoter may be synthetic or recombinant. Synthetic heterologous promoters, in some embodiments, contain various elements obtained from known transcriptional regulatory regions. Synthetic heterologous promoters, in some embodiments, contain mutations that alter expression through methods of genetic engineering that are known in the art. Recombinant heterologous promoters, in some embodiments, are produced by recombinant cloning, nucleic acid amplification (e.g., polymerase chain reaction (PCR)), or a combination of recombinant cloning and nucleic acid amplification (see U. S. Pat. Nos. 4,683,202 and 5,928,906). Other methods of producing synthetic and recombinant heterologous promoters are contemplated herein.

[0226] In certain embodiments, the promoters are constitutive. Constitutive promoters are active under all conditions and drive continuous gene expression at a relatively constant rate. They are not regulated by specific external signals or environmental factors. Genes under the control of constitutive promoters are typically expressed at a basal level in all cells or tissues of an organism. Examples of constitutive promoters include the EF1α (Elongation Factor 1 Alpha) promoter, the PGK (Phosphoglycerate Kinase) promoter, the GAPDH (Glyceraldehyde 3-Phosphate Dehydrogenase) promoter, Cytomegalovirus (CMV) Promoter, Simian Virus 40 (SV40) Promoter, and Chicken Beta-Actin Promoter with a CMV Enhancer (CAG).

[0227] In certain embodiments, the constitutive promoter is EF1α or CMV.

[0228] In certain embodiments, the promoter is EF1α. In certain embodiments, the EF1α includes a nucleic acid sequence of SEQ ID NO: 15.

[0229] A promoter, in some embodiments, is an inducible promoter. An “inducible promoter” regulates (e.g., activates or inactivates) transcriptional activity of a nucleic acid to which it is operably linked when the promoter is influenced by or contacted by a corresponding regulator}' protein.

[0230] Examples of effector substances that regulate inducible promoters (e.g., via regulation of a regulatory protein) include, without limitation, physiological conditions, such as changes in light, pH, temperature, radiation, osmotic pressure, saline gradients and cell surface binding. Inducible promoters may also be regulated by varying the concentration of extrinsic or intrinsic effector substances. Examples of extrinsic effector substances include, without limitation, amino acids and amino acid analogs, saccharides and polysaccharides, nucleic acids, protein transcriptional activators and repressors, cytokines, toxins, petroleum-based compounds, metal (e.g., copper) containing compounds, salts, ions, enzyme substrate analogs, hormones or combinations of any two or more of the foregoing. Other effector substances are known in the art and may be used in accordance with the present disclosure.

[0231] Examples of inducible promoters include, without limitation, chemically- or biochemically-regulated and physically-regulated promoters, such as alcohol-regulated promoters, tetracycline-regulated promoters (e.g., anhydrotetracycline (aTc)-responsive promoters and other tetracycline-responsive promoter systems, which include a tetracycline repressor protein (tetR), a tetracycline operator sequence (tetO) and a tetracycline transactivator fusion protein (tTA)), steroid-regulated promoters (e.g., promoters based on therat glucocorticoid receptor, human estrogen receptor, moth ecdysone receptors, and promoters from the steroid / retinoid / thyroid receptor superfamily), metal-regulated promoters (e.g., promoters derived from metallothionein (proteins that bind and sequester metal ions) genes from yeast, mouse and human), pathogenesis-regulated promoters (e.g., induced by salicylic acid, ethylene or benzothiadiazole (BTH)), temperature / heat-inducible promoters (e.g., heat shock promoters), and light-regulated promoters (e.g., light responsive promoters from plant cells). Other inducible promoters are known in the art and may be used in accordance with the present disclosure.

[0232] In certain embodiments, the inducible promoter is nuclear factor of activated t-cells (NF AT) promoter.

[0233] Enhancers

[0234] Engineered nucleic acids, in some embodiments, comprise enhancers. An “enhancer” is a cis-acting regulatory sequence of nucleotides involved in the transcriptional activation of a nucleic acid sequence operably linked to a promoter. The enhancer may be located at any functional location upstream or downstream from the promoter.

[0235] Terminators

[0236] Engineered nucleic acids, in some embodiments, comprise terminators. A “terminator” is a sequence of nucleotides that causes transcription to stop. A terminator may be unidirectional or bidirectional. A terminator comprises a DNA sequence involved in specific termination of an RNA transcript by an RNA polymerase and prevents transcriptional activation of downstream nucleic acid sequences by upstream promoters.

[0237] The most commonly used type of terminator is a forward terminator. When placed downstream of a nucleic acid sequence that is usually transcribed, a forward transcriptional terminator will cause transcription to abort. In some embodiments, bidirectional transcriptional terminators are used, which usually cause transcription to terminate on both the forward and reverse strand. In some embodiments, reverse transcriptional terminators are provided, which usually terminate transcription on the reverse strand only.

[0238] Examples of terminators for use in accordance with the present disclosure include, without limitation, termination sequences of genes such as, for example, the bovine growth hormone terminator, and viral termination sequences such as, for example, the TO terminator, the TE terminator, Lambda T1 and the T1T2 terminator found in bacterial systems. In someembodiments, the termination signal may be a sequence that cannot be transcribed or translated, such as those resulting from a sequence truncation.

[0239] Selectable Marker

[0240] The expression vector, in some embodiments, comprise a nucleic acid encoding a selectable marker protein to facilitate cloning of the desired gene and the ability of the vector to enter and / or replicate in eukaryotic or prokaryotic cells. A selectable marker is a gene introduced into a cell that confers a trait suitable for artificial selection. A selectable marker may be, for example, an antibiotic resistance gene. Non-limiting examples of antibiotic resistance genes include gene encoding resistance to ampicillin, chloroamphenicol, tetracycline or kanamycin. For example, beta-lactamase confers ampicillin resistance to bacterial hosts, the neo gene obtained from Tn5, confers resistance to kanamycin in bacteria and geneticin in eukaryotic cells, the mutant FabI gene (mFabl) obtained from the Escherichia coli genome confers triclosan resistance to the host, and URA3, an orotidine-5' phosphate decarboxylase obtained from yeast is a positive and negative selectable marker.

[0241] Additional elements may also be needed for optimal synthesis of mRNA. These elements may include signal sequences, splice signals, as well as transcriptional promoters, enhancers, and termination signals.

[0242] In other embodiments, the fusion proteins as described herein may be expressed using polycistronic constructs. In such expression systems, multiple gene products of interest such as two or more proteins may be produced from a single polycistronic construct. These systems advantageously use an internal ribosome entry site (IRES) to provide relatively high levels of polypeptides in eukaryotic host cells. Compatible IRES sequences are disclosed in U. S. Pat. No. 6,193,980.

[0243] In certain embodiments, the expression vector is a bicistronic or multi-cistronic construct.

[0244] In certain embodiments, expression of the fusion protein and the one or more proteins are driven by the same promoter. The one or more protein encoding sequence are operably linked to the fusion protein encoding polynucleotides within a single polycistronic construct, such that the fusion protein and the one or more proteins can be co-expressed.

[0245] In certain embodiments, the fusion protein encoding polynucleotides and the one or more protein encoding sequences are separated by IRES or self-cleaving peptide, such as the 2A peptide, such that they are translated separately.

[0246] The 2A peptide also known as self-cleaving peptides or 2A sequences, are small peptide sequences that mediate the co-translational cleavage of polyproteins into individual proteins. These peptides are commonly used in molecular biology and genetic engineering to express multiple proteins from a single mRNA transcript. The 2A peptides may include foot-and-mouth disease virus 2A (F2A), thosea asigna virus 2A (T2A), porcine tescho virus- 1 2A (P2A), equine rhinitis A virus (E2A), and thosea asigna virus short 2A (S2A). In certain embodiments, the 2 A peptide is T2A.

[0247] In certain embodiments, the one or more protein can be low-affinity nerve growth factor receptor (LNGFR) or a functional portion thereof. In certain embodiments, the LNGFR includes a functional portion without a cytoplasmic domain. Such functional portion is used for purification of cells wherein the fusion protein encoding a polynucleotide described herein is co-expressed.

[0248] The expression vector of the present disclosure may also include one or more enhancer elements to improve expression of the protein of interest. The enhancer element may be positioned 5’ or 3’ to the expressible nucleic acid sequence used to express the protein of interest.

[0249] In certain embodiments, the enhancer element includes a woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) 5’ or 3’ end to the expressible nucleic acid sequence.

[0250] In certain embodiments, the WPRE includes a nucleic acid sequence of SEQ ID NO: 16.

[0251] In certain embodiments, the enhancer element includes a poly A tail 3 ’ end to the expressible nucleic acid sequence.

[0252] In certain embodiments, the enhancer element includes a woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) 3’ or 5’ end to the expressible nucleic acid sequence.

[0253] In certain embodiments, the enhancer element includes a WPRE 5’ end to the expressible nucleic acid sequence, and a poly A tail 3’ end to the expressible nucleic acid sequence.

[0254] In certain embodiments, the enhancer element includes a WPRE and a poly A tail both 3’ end to the expressible nucleic acid sequence.

[0255] In certain embodiments, the expression vector of the present disclosure includes from 5’ to 3’, the EF1α promoter, the fusion protein encoding polynucleotide, the 2A encoding sequence, the one or more protein encoding sequences, and the WPRE.

[0256] In certain embodiments, the one or more protein further comprises a signal peptide.Immune Cells

[0257] Immune cells enclosing the fusion proteins of the present disclosure or one or more polynucleotides encoding the fusion proteins are also provided.

[0258] A membrane bound cytokine fusion protein having a costimulatory domain can be inserted into and expressed by an immune cell on its surface, such as T cell. The immune cells express a CAR or TCR that can recognize a specific antigen and, when bound to that antigen, activate the immune cell to attack and destroy the cell bearing that antigen. The costimulatory domain in the fusion protein can potentially improve T cell cytotoxicity, proliferation and long-term persistence upon antigen encounter in the context of solid tumors, where there is a tumor microenvironment.

[0259] The “immune cell”, also known as leukocytes or white blood cells, are the key components of the immune system responsible for defending the body against infections and foreign substances. They are broadly classified into two categories: innate and adaptive immune cells. Innate immune cells, which provide the first line of defense, include neutrophils, eosinophils, basophils, monocytes (which differentiate into macrophages), dendritic cells, and natural killer (NK) cells. Adaptive immune cells, which provide a more specific and long-lasting immune response, consist of lymphocytes: B cells, which produce antibodies, and T cells, which arc further divided into subtypes such as CD4+ T helper cells (Thl, Th2, Thl7, and regulatory T cells), CD8+ cytotoxic T cells, memory T cells, follicular helper T (Tfh) cells, and natural killer T (NKT) cells. Each subtype of immune cell has distinct functions and mechanisms that contribute to immune surveillance, pathogen elimination, and immune regulation.

[0260] As used herein, the terms “T lymphocyte” and “T cell” are used interchangeably and refer to a principal type of white blood cell that completes maturation in the thymus and that has various roles in the immune system, including the identification of specific foreign antigens in the body and the activation and deactivation of other immune cells. A T cell can be any T cell, such as a cultured T cell, e.g., a primary T cell, or a T cell from a cultured T cell line, e.g., Jurkat, SupTl, etc., or a T cell obtained from a mammal. The T cell can be CD3+ cells. The T cell can be alpha beta (a, J3) T cells, or gamma delta (y6) T cells. The alpha beta (a|3) T cell can be any type of T cell and can be of any developmental stage, including but not limited to, CD4+ / CD8+ double positive T cells, CD4+ helper T cells (e.g., Thl, Th2 and Thl7 cells), CD8+ T cells (e.g., cytotoxic T cells), tumor infiltrating lymphocytes (TILs), memory T cells, naive T cells, regulator T cells, and the like. Additional types of helper T cells include cells such as Th3 (Treg), Th9, or Tfh cells. Additional types of memory T cells include cells such as central memory T cells (Tcm cells), effector memory T cells (Tem cells and TEMRA cells). The T cell can also refer to a genetically engineered T cell, such as a T cell modified to express a T cell receptor (TCR) or a chimeric antigen receptor (CAR). The T cell can also be differentiated from a stem cell or progenitor cell.

[0261] As used herein, the term “NK cell” or “Natural Killer cell” refer to a subset of peripheral blood lymphocytes defined by the expression of CD56 or CD16 and the absence of the T cell receptor (CD3). As used herein, the terms “adaptive NK cell” and “memory NK cell” are interchangeable and refer to a subset of NK cells that are phenotypically CD3- and CD56+, expressing at least one of NKG2C and CD57, and optionally, CD 16, but lack expression of one or more of the following: PLZF, SYK, FceRy, and EAT-2. In some embodiments, isolated subpopulations of CD56+ NK cells comprise expression of CD16, NKG2C, CD57, NKG2D, NCR ligands, NKp30, NKp40, NKp46, activating and inhibitory KIRs, NKG2A and / or DNAM-1. CD56+ can be dim or bright expression.

[0262] The alpha beta (aP) T cells as used herein are characterized by their T cell receptors composed of alpha (a) and beta (P) chains. These receptors are highly diverse, allowing ap T cells to recognize a wide variety of peptide antigens presented by major histocompatibility complex (MHC) molecules on other cells. The main subtypes of aP T cells include CD4+ T helper cells, which are further divided into Thl, Th2, Thl7, and regulatory T cells (Treg) that coordinate various immune responses and maintain immune tolerance; CD8+ cytotoxic T cells, which directly kill infected or cancerous cells; memory T cells, which provide longterm immunity; follicular helper T cells (Tfh), which assist B cells in antibody production;and natural killer T (NKT) cells, which bridge innate and adaptive immunity by recognizing lipid antigens. The αβ T cells can also refer to a genetically engineered T cell, such as an αβ T cell modified to express a T cell receptor (TCR) or a chimeric antigen receptor (CAR).

[0263] Gamma Delta (y8) T cells are a distinct subset of T cells, characterized by their T cell receptors composed of gamma (γ) and delta (δ) chains, which are less diverse compared to αβ T cell receptors. Unlike αβ T cells, γδ T cells can recognize a broad range of antigens without the need for presentation by major histocompatibility complex (MHC) molecules, allowing them to directly identify and respond to antigens. They are primarily involved in bridging innate and adaptive immunity, contributing to early immune responses, and are abundant in epithelial tissues. The main subtypes of γδ T cells are classified based on the types of TCR chains they express, such as Vγ9Vδ2 and Vγ4Vδ1, with each subtype having distinct roles in immune surveillance, tumor immunity, and response to infections. These cells are known for their rapid response to pathogens, potential cytotoxicity, and production of cytokines, playing crucial roles in immune regulation and protection against a variety of diseases.

[0264] “ CD4+ T cells” refers to a subset of T cells that express CD4 on their surface and are associated with cell-mediated immune response. They are characterized by the secretion profiles following stimulation, which may include secretion of cytokines such as IFN-gamma, TNF-alpha, IL2, IL4 and IL10.“CD4” are 55-kD glycoproteins originally defined as differentiation antigens on T-lymphocytes, but also found on other cells including monocytes / macrophages. CD4 antigens are members of the immunoglobulin supergene family and are implicated as associative recognition elements in MHC (major histocompatibility complex) class Il-restricted immune responses. On T-lymphocytes they define the helper / inducer subset.

[0265] ‘ ‘CD8+ T cells” refers to a subset of T cells which express CD8 on their surface, are MHC class I-restricted, and function as cytotoxic T cells. “CD8” molecules are differentiation antigens found on thymocytes and on cytotoxic and suppressor T-lymphocytes. CD8 antigens are members of the immunoglobulin supergene family and are associative recognition elements in major histocompatibility complex class I-restricted interactions.

[0266] Memory T cells are a subset of T cells that remain in the body after an initial infection has been cleared. They are long-lived and can rapidly expand to large numbers upon reexposure to their specific antigen. This allows for a quicker and more effective immuneresponse during subsequent encounters with the same pathogen, forming the basis of immunological memory. Subtypes of the memory T cells include central memory T cells (TCM), effector memory T cells (TEM) and tissue-resident memory T cells (TRM).

[0267] Naive T cells are mature T cells that have left the thymus but have not yet encountered their specific antigen. They circulate through the peripheral lymphoid organs, such as lymph nodes and the spleen, where they can encounter antigens presented by antigen-presenting cells. Upon activation by their specific antigen, naive T cells proliferate and differentiate into effector T cells or memory T cells, depending on the immune response required. Subtypes of the effector T cells include CD4+ naive T cells and CD8+ naive T cells.

[0268] Regulatory T cells are often referred to as Tregs, are a specialized subset of T cells that help maintain immune system homeostasis by suppressing excessive immune responses and preventing autoimmune diseases. They play a crucial role in maintaining tolerance to self-antigens and preventing the immune system from attacking the body's own tissues. Tregs can inhibit the activity of other immune cells, thereby modulating the immune response to prevent tissue damage and maintain balance within the immune system. The regulatory T cells include natural Tregs (nTregs) and induced Tregs (iTregs).

[0269] By “tumor infiltrating lymphocytes” or “TILs” herein is meant a population of cells originally obtained as white blood cells that have left the bloodstream of a subject and migrated into a tumor. TILs as described herein include, but are not limited to, CD8+ cytotoxic T cells, CD4+ T cells (e.g. Thl and Thl7 cells), and memory T cells. TILs include both primary and secondary TILs. “Primary TILs” are those that are obtained from patient tissue samples as outlined herein (sometimes referred to as “freshly harvested”), and “secondary TILs” are any TIL cell populations that have been expanded or proliferated as discussed herein, including, but not limited to bulk TILs and expanded TILs (“REP TILs” or “post-REP TILs”).

[0270] Cell surface receptors, such as CAR or TCR can be co-expressed with the membrane bound cytokine fusion protein described herein.

[0271] TCRs

[0272] The term “T-cell receptor (TCR)” as used herein refers to a protein receptor on T cells that is composed of a heterodimer of an alpha (α) and beta (β) chain, although in some cells the TCR consists of gamma and delta (y8) chains.

[0273] In certain embodiments, the TCR includes an alpha (α) and beta (β) chain.

[0274] In certain embodiments, the TCR may be modified on any cell including a TCR, including αβ (alpha beta) T cells (such as a helper T cell, a cytotoxic T cell, a memory T cell, regulatory T cell, natural killer T cell), natural killing cells and gamma delta T cell, for example.

[0275] In certain embodiments, the T cells that include a TCR described herein is αβ (alpha beta) T cells.

[0276] In certain embodiments, the TCR can be genetically modified to specifically target a particular antigen, such that it becomes an antigen-specific TCR or engineered TCR. The engineered TCR described herein may include a chain selected from among a, b, g and d chains, or chimeric scFv, which are linked to the intracellular signaling domains of FcR-gamma or CD3 zeta to trigger T-cell effector function.

[0277] In some embodiments, the TCR is anti-NY-ESO-1 T cell receptor (1G4).

[0278] CARs

[0279] A chimeric antigen receptor (CAR) is generally a set of polypeptides which, when existing on an immune cell, causes the immune cell to have specificity to a target cell (normally a cancer cell) while causing signal transduction in the cell. A CAR at minimum comprises an extracellular antigen recognition domain which recognizes the target antigen to be described below, a transmembrane domain, and an intracellular signal transduction domain, wherein the intracellular signal transduction domain is derived from the promoting molecules or costimulatory molecules.

[0280] The structure of CARs commonly used today for clinical applications comprises a single chain variable fragment domain (hereinafter scFv) which gives specificity to an antigen, a spacer domain to regulate the distance between the scFv and the cell membrane, a transmembrane domain, and an intracellular signaling domain (hereinafter ISD). The ISD in turn comprises a costimulatory domain (CD28, CD 137 or 0X40) which contributes to in vivo proliferation and long life of one or multiple T cells, and a TCR signaling domain (CD3 zeta) which contributes to T cell activation. T-cells modified to express CAR that have been prepared in this manner can be activated by recognizing cancer cells which express the target antigen with high specificity, effectively induce the death of such cancer cells, simultaneously proliferate exponentially in the body, and remain alive for a long time. For example, when CAR-T cells (CART- 19) prepared to target CD 19, a B cell-specific antigen, were administered to a B-cell leukemia patient, it was reported that the cells proliferated to1,000 to 10,000 times and remained alive in the body for several years. As a result, CART-19 exhibited 90% complete response in a clinical trial carried out on terminal acute lymphoblastic leukemia (B-ALL) patients on whom conventional chemotherapy, etc., had not been effective, leading to a rare case of licensing to a global pharmaceuticals company in the early investigator-initiated clinical trial phase. It became the first CAR-T cell therapy agent to receive U. S. FDA approval in 2017, and thereafter, a second CAR-T was also approved.

[0281] In some embodiments, the target of the CAR or engineered TCR may be a human tumor antigen whose expression is increased in a cancer which is to be treated. For example, the target can be selected from CLDN18.2 (claudin 18.2), 5T4 (trophoblast glycoprotein), 707-AP, 9D7, AFP (a-fetoprotein), AlbZIP (androgen-induced bZIP), HPG1 (human prostate specific gene-1), a.5b 1 -Integrity a.5p6-Integrin, a-methylacyl-coenzyme A racemase, ART-4 (ADPribosyltransferase-4), B7H4 (v-set domain-containing T-cell activation inhibitor 1), BAGE-1 (B melanoma antigen- 1), BCL-2 (B-cell CLL / lymphoma-2), BING-4 (WD repeat domain 46), CA 15-3 / CA 27-29 (mucin 1), CA 19-9 (cancer antigen 19-9), CA 72-4 (cancer antigen 72-4), CA125 (cancer antigen 125), calreticulin, CAMEL (CTL-recognized antigen on melanoma), C ASP-8 (caspase 8), cathepsin B, cathepsin L, CD 19 (cluster of differentiation 19), CD20, CD22, CD25, CD30, CD33, CD4, CD52, CD55, CD56, CD80, CEA (carcinoembryonic antigen SG8), CLCA2 (chloride channel accessory 2), CML28 (chronic myelogenous leukemia tumor antigen 28), Coactosin-like protein, Collagen XXIII, COX-2 (cyclooxygenase-2), CT-9 / BRD6 (cancer / testis antigen 9), Cten (c-terminal tensin-like protein), cyclin Bl, cyclin DI, cyp-B, CYPB1 (cytochrome p450 family 1 subfamily b member 1), DAM-10 / MAGE-B1 (melanoma-associated antigen Bl), DAM-6 / MAGE-B2, EGFR / Herl (epidermal growth factor receptor), EMMPRIN (basigin), EpCam, EphA2 (EPH receptor A2), EphA3, ErbB3 (Erb-B2 receptor tyrosine kinase 3), EZH2 (enhancer of zeste 2 poly comb repressive complex 2 subunit), FGF-5 (fibroblast growth factor 5), FN (fibronectin), Fra-1 (Fosrelated antigen-1), G250 / CAIX (carbonic anhydrase 9), GAGE-1 (G antigen-1), GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7b, GAGE-8, GDEP (gene differentially expressed in prostate), GnT-V (gluconate kinase), gp100 (melanocytes lineage-specific antigen GP100), GPC3 (glypican3), HAGE (helical antigen), HAST-2 (sulfotransferase family 1 A member 1), hepsin, Her2 / neu / ErbB2 (Erb-B2 receptor tyrosine kinase 2), HERV-K-MEL, HNE (medullasin), homeobox NKX 3.1, HOM-TES-14 / SCP-1, HOM-TES-85, HPV-E6, HPVE7, HST-2 (sirtuin-2), hTERT, iCE (caspase 1), IGF-1R (insulin like growth factor- 1 receptor), IL-13Ra2 (interleukin- 13 receptor subunit a 2), IL-2R(interleukin-2 receptor), IL-5 (interleukin-5), immature laminin receptor, kallikrein 2, kallikrein 4, Ki67, KIAA0205 (lysophosphatidylglycerol acyltransferase 1), KK-LC-1 (kita-kyushu lung cancer antigen- 1), KM-HN-1, LAGE-1 (L antigen family member- 1), Livin, MAGE-A1, MAGE- A 10, MAGE-A12, MAGEA2, MAGE- A3, MAGE-A4, MAGE-A6, MAGE-A9, MAGE-B1, MAGE-BIO, MAGE-B16, MAGEB17, MAGE-B2, MAGE-B3, MAGE-B4, MAGE-B5, MAGE-B6, MAGE-CI, MAGE-C2, MAGE-C3, MAGE-D1, MAGE-D2, MAGE-D4, MAGE-E1, MAGE-E2, MAGE-F1, MAGE-H1, MAGEL2 (melanoma antigen family L2), mammaglobin A, M ART- 1 / Mel an- A (melanoma antigen recognized by T-cells-1), MART-2, matrix protein 22, MC1R (melanocortin 1 receptor), M-CSF (macrophage colony-stimulating factor), Mesothelin, MG50 / PXDN (peroxidasin), MMP 11 (matrix metalloprotease 11), MN / CA IX-antigen (carbonic anhydrase 9), MRP-3 (multidrug resistance-associated protein-3), MUC1 (mucin 1), MUC2, NA88-A (VENT-like homeobox 2 pseudogene 1), N-acetylglucos-aminyltransferase- V, Neo-PAP (Neo-poly (A) polymerase), NGEP (new gene expressed in prostate), NMP22 (nuclear matrix protein 22), NPM / ALK (nucleophosmin), NSE (neuron-specific enolase), NY-ESO-1, NY-ESO-B, OA1 (osteoarthritis QTL 1), OFA-iLRP (oncofetal antigen immature laminin receptor protein), OGT (O-GlcNAc transferase), OS-9 (endoplasmic reticulum lectin), osteocalcin, osteopontin, p 15 (CDK inhibitor 2B), p53, PAGE-4 (P antigen family member-4), PAI-1 (plasminogen activator inhibitor- 1), PAL2, PAP (prostatic acid phosphatase), PART-1 (prostate androgen-regulated transcript 1), PATE (prostate and testis expressed 1), PDEF (prostate-derived Ets factor), Pim-l-Kinase (pro viral integration site 1), Pinl (Peptidyl -prolyl cis-trans isomerase NIMA-interacting 1), POTE (expressed in prostate, ovary, testis, and placenta), PRAME (preferentially expressed antigen in melanoma), prostein, proteinase-3, PSA (prostate-specific antigen), PSCA (prostate stem cell antigen), PSGR (prostate-specific G-protein coupled receptor), PSM, PS MA (prostate specific membrane antigen), RAGE-1 (renal tumor carcinoma antigen), RHAMM / CD168, RET1 (renal ubiquitous protein 1), RET2, SAGE (sarcoma antigen), SART-1 (squamous cell carcinoma antigen recognized by T-cells-1), SART-2, SART-3, Spl7 (sperm protein 17), SSX-1 (SSX family member 1), SSX-2 / HOM-MEL-40, SSX-4, STAMP-1 (STEAP2 metalloreductase), STEAP, survivin, survivin-213, TA-90 (tumor associated antigen-90), TAG-72 (tumor associated glycoprotein-72), TARP (TCRy alternate reading frame protein), TGFb (transforming growth factor b), TGFbRl 1 (transforming growth factorb receptor 11), TGM-4 (transglutaminase 4), TRAG-3 (taxol resistance associated gene 3), TRG (T-cell receptor g locus), TRP-1 (transient receptor potential-1), TRP-2 / 6b, TRP-2 / INT2, Trp-p8, Tyrosinase, UPA (U-plasminogen activator),VEGF (vascular endothelial growth factor A), VEGFR-2 / FLK- 1 and WT1 (wilms tumor 1), or may be a mutated form of human tumor antigen discovered in the cancer to be treated, selected from among a-actinin-4 / m, ARTCl / m, bcr / abl, beta-Catenin / m, BRCAl / m, BRCA2 / m, CASP-5 / m, CASP-8 / m, CDC27 / m, CDK4 / m, CDKN2A / m, CML66, COA-l / m, DEK-CAN, EFTUD2 / m, ELF2 / m, ETV6-AML1, FNl / m, GPNMB / m, HLA-A* 0201-R 1701, HLA-A1 1 / m, HLA-A2 / m, HSP70-2M, KIAA0205 / m, K-Ras / m, LDLR-FUT, MART2 / m, MEl / m, MFJM-l / m, MEiM-2 / m, MUM-3 / m, Myosin class 1 / m, neo-PAP / m, NFYC / m, N- Ras / m, OGT / m, OS-9 / m, p53 / m, Pml / RARa, PRDX5 / m, PTPRX / m, RBAF600 / m, SIRT2 / m, SYTSSX-l, SYT-SSX-2, TEL-AML1, TGFbRII and TPl / m. For example, the target antigen may be selected between CD 19 or CD22.

[0282] Methods of Producing an Immune Cell

[0283] In one aspect, present disclosure also provides a method of producing an immune cell expressing the fusion protein, or entailing the fusion protein encoding polynucleotide or the expression vector as described herein. The method includes (i) introducing the fusion protein encoding polynucleotide, or the expression vector including the fusion protein encoding polynucleotide into the immune cell; and (ii) maintaining the immune cell under conditions in which the fusion protein encoding polynucleotide is expressed.

[0284] A variety of methods for introducing nucleic acid (e.g., transfection, transduction, and / or transposon system) encoding the cytokine fusion protein as a transmembrane polypeptide into an immune cell can be used. Examples of such methods include chemicalbased methods (e.g., involving the use of calcium phosphate; highly branched organic compounds (e.g., dendrimers); liposomes (lipofection); and / or cationic polymers (e.g., DEAE dextran; polyethylenimine)), non-chemical-based methods (e.g., electroporation; cell squeezing; sonoporation; optical transfection; impalefection; hydrodynamic delivery), particle -based methods (e.g., gene gun; magnetofection; particle bombardment), vectorbased methods (e.g., vectors including viral vectors such as retroviral vector, lentiviral vectors, adenoviral vectors, etc.), nucleotransfection, transposon-based methods (e.g., Sleeping Beauty, PiggyBAC, etc.) and / or RNA transfection.

[0285] In certain embodiments, the fusion protein encoding polynucleotide is transduced with a virus. In certain embodiments, the expression vector including the fusion protein encoding polynucleotide is derived from a viral vector. In certain embodiments, the virus is a lenti virus or retrovirus.

[0286] Also there are a variety of methods of maintaining the immune cells under conditions in which (i) the cytokine fusion protein is expressed as a membrane-bound polypeptide and / or (ii) the immune cells comprising membrane-bound cytokine fusion protein proliferate can be used. For example, immune cells can be grown and / or maintained at an appropriate temperature and gas mixture (e.g., about 25°C to about 37°C, about 5% CO2in a cell incubator). Culture conditions can vary widely, and variation of conditions for a particular cell type can result in different phenotypes. In addition to temperature and gas mixture, a commonly varied factor in culture systems is the cell growth medium. Recipes for growth media can vary in pH, glucose concentration, growth factors, and the presence of other nutrients. The growth factors used to supplement media are often derived from the serum of animal blood, such as fetal bovine serum (FBS), bovine calf serum, equine serum, porcine serum and / or human platelet lysate (hPL). Other factors considered for maintaining cells include plating density (number of cells per volume of culture medium) and growth of the cells in suspension or adherent cultures.

[0287] The methods can further comprise isolating or separating the one or more immune cells produced by the methods provided herein, In addition, the methods can further comprises culturing the one or more immune cells. In some aspects, an immune cell line is produced.Compositions and Methods of Treatments

[0288] The present disclosure also provides pharmaceutical compositions. In one aspect, provided herein are compositions comprising an immune cell described herein and a pharmaceutically acceptable carrier. In another aspect, provided herein are pharmaceutical compositions for immune therapy of human patients comprising the immune cells described above. In some embodiments, the immune cell is originally derived from the patient. In some embodiments, the patient has a tumor or cancer in which an increase or variation in levels of cancer antigen targeted by the CAR or engineered TCR expressed in the cell is detected.

[0289] In a specific embodiment, the term “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U. S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. Further, a “pharmaceutically acceptable carrier” will generally be a non- toxic solid, semisolid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.

[0290] “Effective amount” means the amount of active pharmaceutical agent (e.g., an FcRn antagonist of the present disclosure) sufficient to effectuate a desired physiological outcome in an individual in need of the agent. The effective amount may vary among individuals depending on the health and physical condition of the individual to be treated, the formulation of the composition, assessment of the individual's medical condition, and other relevant factors.

[0291] The term “carrier” refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or Ph buffering agents such as acetates, citrates or phosphates. Antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; and agents for the adjustment of tonicity such as sodium chloride or dextrose are also envisioned. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like. The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in Remington’s Pharmaceutical Sciences by E. W. Martin, incorporated herein by reference. Such compositions will contain a therapeutically effective amount of the antigen-binding polypeptide, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient. The formulation should suit the mode of administration. The parental preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

[0292] In some embodiments, the immune cells can be used as immune cell therapeutic agents; such immune cells are normally used for the treatment of cancers but are not limited thereto. In further embodiments, to make these immune cells recognize cancers, they are modified to express cell surface receptors which target cancer antigens.

[0293] Using the vector according to one embodiment, immune cells can be produced, wherein the immune cells express the membrane bound cytokine fusion protein as described herein and CAR or engineered TCR, specific to target molecules. Said immune cells can be used to provide a pharmaceutical composition for immune therapy.

[0294] In some embodiment, provided are methods of treatment comprising administering to a subject having a disease or a condition the immune cell described above or the composition described above. In some embodiments, the genetically engineered antigen receptor specifically binds to an antigen associated with the disease or the condition.

[0295] In another aspect, provided herein are immune cells and compositions. In some embodiments, provided are immune cells and compositions described above for use in treating a disease or a condition.

[0296] In another aspect, provided herein is use of the immune cells or compositions. In some embodiments, provided is use of the immune cells or compositions described above in the manufacture of a medicament for use in a method for treating a disease or a condition. In some embodiments, the surface antigen receptor specifically binds to an antigen associated with the disease or the condition.

[0297] In yet another aspect, provided herein is a method of treating cancer in a subject in need thereof, including (i) co-expressing the fusion protein and a CAR or TCR in an immune cell as described herein, and administering the immune cell to the subject; or (ii) administering to the subject an immune cell expressing the fusion protein and a CAR or TCR as described herein.

[0298] In certain embodiments, the TCR is an engineered TCR, such as an antigen-specific TCR.

[0299] In certain embodiments, the TCR is anti-NY-ESO-1 T cell receptor (1G4).

[0300] A variety of diseases may be ameliorated by introducing immune cells as described herein to a subject suitable for adoptive immune therapy. In some embodiments, the produced CAR-T cells or engineered TCR-T cells as provided is for allogeneic adoptive cell therapies.Additionally provided herein are therapeutic use of the compositions described herein, comprising introducing the composition to a subject suitable for adoptive cell therapy, wherein the subject has an autoimmune disorder; a cancer including a hematological malignancy or a solid tumor; or an infection associated with HIV, RSV, EB V, CMV, adenovirus, or BK polyomavirus.

[0301] In some embodiments, the disease or the condition is cancer, such as hematological malignancies or solid tumor. Examples of hematological malignancies include, but are not limited to, acute and chronic leukemias (acute myelogenous leukemia (AML), acute lymphoblastic leukemia (ALL), chronic myelogenous leukemia (CML), lymphomas, nonHodgkin lymphoma (NHL), Hodgkin’s disease, multiple myeloma, and myelodysplastic syndromes. Examples of solid cancers include, but are not limited to, brain cancer, bladder cancer, liver cancer, colon cancer, rectal cancer, endometrial cancer, pancreatic cancer, small cell lung cancer, non-small cell lung cancer, breast cancer, urethral cancer, head and neck cancer, gastrointestinal cancer, stomach cancer, oesophageal cancer, ovarian cancer, renal cancer, melanoma, cervix cancer, rectum cancer, larynx cancer, prostate cancer and thyroid cancer.

[0302] Examples of various autoimmune disorders include, but are not limited to, alopecia areata, autoimmune hemolytic anemia, autoimmune hepatitis, dermatomyositis, diabetes (type 1), some forms of juvenile idiopathic arthritis, glomerulonephritis, Graves’ disease, Guillain-Barre syndrome, idiopathic thrombocytopenic purpura, myasthenia gravis, some forms of myocarditis, multiple sclerosis, pemphigus / pemphigoid, pernicious anemia, polyarteritis nodosa, polymyositis, primary biliary cirrhosis, psoriasis, rheumatoid arthritis, scleroderma / systemic sclerosis, Sjogren’s syndrome, systemic lupus, erythematosus, some forms of thyroiditis, some forms of uveitis, vitiligo, granulomatosis with polyangiitis (Wegener’s).

[0303] Examples of viral infections include, but are not limited to, HIV- (human immunodeficiency virus), HSV- (herpes simplex virus), KSHV- (Kaposi's sarcoma-associated herpesvirus), RSV- (Respiratory Syncytial Virus), EBV- (Epstein-Barr virus), CMV- (cytomegalovirus), VZV (Varicella zoster virus), adenovirus-, a lentivims-, a BK polyomavirus- associated disorders.

[0304] In one aspect, a composition described herein can be provided in unit dosage form wherein each dosage unit, e.g., an injection, contains a predetermined amount of thecomposition, alone or in appropriate combination with other active agents. The term unit dosage form as used herein refers to physically discrete units suitable as unitary dosages for human and animal subjects, each unit containing a predetermined quantity of a composition described herein, alone or in combination with other active agents, calculated in an amount sufficient to produce the desired effect, in association with a pharmaceutically acceptable diluent, carrier, or vehicle, where appropriate. The specifications for the novel unit dosage forms of cells or compositions described herein depend on the particular pharmacodynamics associated with the pharmaceutical composition in the particular subject.

[0305] In some embodiments, the preferred pharmaceutical dosage form for the cells or compositions described herein may be determined based on the content of the present disclosure and general knowledge of formulation techniques and according to the intended administration pathway, method of delivery and the target dose. The method of administration notwithstanding, the effective dose may be calculated in according to the patient’s body weight, surface area or organ size. Calculations to determine the appropriate administration doses for therapy using the respective dosage forms stated in the present specification, as well as additional purification, are carried out on a daily basis in the art, and are included within the scope of work carried out on a daily basis in the art. The appropriate administration doses may be identified through use of appropriate dose-response data.

[0306] Pharmaceutical compositions described herein can be used alone or in combination with other known agents useful for treating cancer. Whether delivered alone or in combination with other agents, pharmaceutical compositions described herein can be delivered via various routes and to various sites in a mammalian, particularly human, body to achieve a particular effect. One skilled in the art will recognize that, although more than one route can be used for administration, a particular route can provide a more immediate and more effective reaction than another route. For example, intradermal delivery may be advantageously used over inhalation for the treatment of melanoma. Local or systemic delivery can be accomplished by administration comprising application or instillation of the formulation into body cavities, inhalation or insufflation of an aerosol, or by parenteral introduction, comprising intramuscular, intravenous, intraportal, intrahepatic, peritoneal, subcutaneous, or intradermal administration. Exemplary route of administration to a subject includes intravenous (IV) injection, and regional (intratumoral, intraperitoneal) administration. In some embodiment, the pharmaceutical composition can be administered via infusion into a solid tumor.

[0307] In some embodiments, the composition further includes a second therapeutic agent. The combined administration of a second therapeutic agent includes co-administration (concurrent administration), using separate formulations or a single pharmaceutical formulation, and consecutive administration in either order, wherein preferably there is a time period while both (or all) therapeutic agents (medicaments) simultaneously exert their biological activities.

[0308] In some embodiments, the second therapeutic agent comprising an antibody, or an antibody fragment that targets an antigen associated with a condition, a disease, or an indication may be used with these effector cells in a combinational therapy. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is a humanized antibody, a humanized monoclonal antibody, or a chimeric antibody. In some embodiments, the antibody, or antibody fragment, specifically binds to a viral antigen. In other embodiments, the antibody, or antibody fragment, specifically binds to a tumor antigen. In some embodiments, the antibodies suitable for combinational treatment as an additional therapeutic agent to the administered genomically engineered immune cells include, but are not limited to, anti-CD20 (rituximab, veltuzumab, ofatumumab, ublituximab, ocaratuzumab, obinutuzumab), anti-HER2 (trastuzumab, pertuzumab), anti-CD52 (alemtuzumab), anti-EGFR (certuximab), anti-GD2 (dinutuximab), anti-PDEl (avelumab), anti-CD38 (daratumumab, isatuximab, MOR202), anti-CD123 (7G3, CSL362), anti-SLAMF7 (elotuzumab); and their humanized or Fc modified variants or fragments, or their functional equivalents and biosimilars.

[0309] Desirably an effective amount or sufficient number of the isolated transduced T cells is present in the composition and introduced into the subject such that long-term, specific, anti-tumor responses are established to reduce the size of a tumor or eliminate tumor growth or regrowth than would otherwise result in the absence of such treatment. Desirably, the amount of transduced T cells reintroduced into the subject causes a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 100% decrease in tumor size when compared to otherwise same conditions wherein the transduced T cells are not present.

[0310] Accordingly, the amount of transduced T cells administered should take into account the route of administration and should be such that a sufficient number of the transduced T cells will be introduced so as to achieve the desired therapeutic response.

[0311] Furthermore, the amounts of each active agent included in the compositions described herein (e.g., the amount per each cell to be contacted or the amount per certain body weight) can vary in different applications. In general, the concentration of transduced T cells desirably should be sufficient to provide in the subject being treated at least from about 1 x 106to about 1 x 109transduced T cells, even more desirably, from about 1 x 107to about 5 x 108transduced T cells, although any suitable amount can be utilized either above, e.g., greater than 5 x 10scells, or below, e.g., less than 1 x 107cells. The dosing schedule can be based on well-established cell-based therapies (see, e.g., Topalian and Rosenberg, 1987; U. S. Pat. No. 4,690,915), or an alternate continuous infusion strategy can be employed.

[0312] These values provide general guidance of the range of transduced T cells to be utilized by the practitioner upon optimizing the methods described herein. The recitation herein of such ranges by no means precludes the use of a higher or lower amount of a component, as might be warranted in a particular application. For example, the actual dose and schedule can vary depending on whether the compositions are administered in combination with other pharmaceutical compositions, or depending on interindividual differences in pharmacokinetics, drug disposition, and metabolism. One skilled in the art readily can make any necessary adjustments in accordance with the exigencies of the particular situation.Kits

[0313] Any of the compositions described herein may be comprised in a kit. In some embodiments, the CAR T-cells or engineered TCR-T cells are provided in the kit, which also may include reagents suitable for expanding the cells, such as media, aAPCs, growth factors, antibodies (e.g., for sorting or characterizing CAR T-cells or TCR-T cells) and / or plasmids encoding CARs or TCRs or transposase.

[0314] In a non-limiting example, the kit includes a CAR or engineered TCR expression construct, one or more reagents to generate a CAR or engineered TCR expression construct, cells for transfection of the expression construct, and / or one or more instruments to obtain allogeneic cells for transfection of the expression construct (such an instrument may be a syringe, pipette, forceps, and / or any such medically approved apparatus).

[0315] In some embodiments, an expression construct for eliminating endogenous TCR a / b expression, one or more reagents to generate the construct, and / or CAR+ or engineered TCRT cells are provided in the kit. In some aspects, the kit comprises reagents or apparatuses for electroporation of cells.

[0316] The kits may comprise one or more suitably aliquoted compositions described herein or reagents for generating compositions as described herein. The components of the kits may be packaged either in aqueous media or in lyophilized form. The container means of the kits may include at least one vial, test tube, flask, bottle, syringe, or other container means, into which a component may be placed, and in certain embodiments, suitably aliquoted. Where there is more than one component in the kit, the kit also will generally contain a second, third, or other additional container into which the additional components may be separately placed. However, various combinations of components may be comprised in a vial. The kits described herein also will typically include a means for containing the chimeric receptor construct and any other reagent containers in close confinement for commercial sale. Such containers may include injection or blow molded plastic containers into which the desired vials are retained, for example.Table 1. Amino Acid and Nucleic Acid SequencesName Sequence SEQ ID NO: IL-12 p40 MCHQQLVISWFSLVFLASPLVAIWELKKDVYVVELDWYPD 1 subunit (Uniprot APGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEF P29460) GDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEP KNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDP QGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEES LPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKN SRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDR VFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCS IL-12 p40 ATGTGTCACCAGCAGTTGGTCATCTCTTGGTTTTCCCTGG 52 subunit (Uniprot TTTTTCTGGCATCTCCCCTCGTGGCCATATGGGAACTGAA P29460) GAAAGATGTTTATGTCGTAGAAlTGGATrGGTATCCGGAT GCCCCTGGAGAAATGGTGGTCCTCACCTGTGACACCCCTGNucleic acid AAGAAGATGGTATCACCTGGACCTTGGACCAGAGCAGTG sequence AGGTCTTAGGCTCTGGCAAAACCCTGACCATCCAAGTCA(5’— >3’) AAGAGTTTGGAGATGCTGGCCAGTACACCTGTCACAAAG GAGGCGAGGTTCTAAGCCATTCGCTCCTGCTGCTTCACAA AAAGGAAGATGGAATTTGGTCCACTGATATTTTAAAGGA CCAGAAAGAACCCAAAAATAAGACCTTTCTAAGATGCGA GGCCAAGAATTATTCTGGACGTTTCACCTGCTGGTGGCTG ACGACAATCAGTACTGATTTGACATTCAGTGTCAAAAGC AGCAGAGGCTCTTCTGACCCCCAAGGGGTGACGTGCGGA GCTGCTACACTCTCTGCAGAGAGAGTCAGAGGGGACAAC AAGGAGTATGAGTACTCAGTGGAGTGCCAGGAGGACAGT GCCTGCCCAGCTGCTGAGGAGAGTCTGCCCATTGAGGTC ATGGTGGATGCCGTTCACAAGCTCAAGTATGAAAACTAC ACCAGCAGCTTCTTCATCAGGGACATCATCAAACCTGACCCACCCAAGAACTTGCAGCTGAAGCCATTAAAGAATTCTCGGCAGGTGGAGGTCAGCTGGGAGTACCCTGACACCTGGA GTACTCCACATTCCTACTTCTCCCTGACA1TCTGCGTTCAG GTCCAGGGCAAGAGCAAGAGAGAAAAGAAAGATAGAGT CTTCACGGACAAGACCTCAGCCACGGTCATCTGCCGCAA AAATGCCAGCATTAGCGTGCGGGCCCAGGACCGCTACTA TAGCTCATCTTGGAGCGAATGGGCATCTGTGCCCTGCAGT IL-12 p35 RNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYP 2 subunit (Uniprot CTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITN P29459) GSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLM DPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFY KTKIKLCILLHAFRIRAVTIDRVMSYLNAS IL-12 p35 AGAAACCTCCCCGTGGCCACTCCAGACCCAGGAATGTTC 53 subunit (Uniprot CCATGCCTTCACCACTCCCAAAACCTGCTGAGGGCCGTCA P29459) GCAACATGCTCCAGAAGGCCAGACAAACTCTAGAATTTT Nucleic acid ACCCTTGCACTTCTGAAGAGATTGATCATGAAGATATCAC sequence AAAAGATAAAACCAGCACAGTGGAGGCCTGTTTACCATT (5’— >3’) GGAATTAACCAAGAATGAGAGTTGCCTAAATTCCAGAGA GACCTCTTTCATAACTAATGGGAGTTGCCTGGCCTCCAGA AAGACCTCTTTTATGATGGCCCTGTGCCTTAGTAGTATTT ATGAAGACTTGAAGATGTACCAGGTGGAGTTCAAGACCA TGAATGCAAAGCTTCTGATGGATCCTAAGAGGCAGATCT TTCTAGATCAAAACATGCTGGCAGTTATTGATGAGCTGAT GCAGGCCCTGAATTTCAACAGTGAGACTGTGCCACAAAA ATCCTCCCTTGAAGAACCGGATTTTTATAAAACTAAAATC AAGCTCTGCATACTTCTTCATGCTTTCAGAATTCGGGCAG TGACTATTGATAGAGTGATGAGCTATCTGAATGCTTCC CD80 LLPSWAITLLSVNGIFVICCL 3 transmembranedomain (UniprotP33681)CD80 CTGCTCCCATCCTGGGCCATTACCTTAATCTCAGTAAATG 54 transmembrane GAATTTTTGTGATATGCTGCCTGdomain (UniprotP33681)Nucleic acidsequence(5’— >3’)CD28 FWVLVVVGGVLACYSLLVTVAFIIFWV 4 transmembranedomain (UniprotP10747)CD28 TTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCT 55 transmembrane ATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGT domain (Uniprot GPl 0747)Nucleic acidsequence(5’— >3’)CD28 RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS 5 intracellulardomain (UniprotPl 0747)CD28 AGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATG 56 intracellular AACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCAT domain (Uniprot TACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATC P10747) GCTCCNucleic acidsequence(5’— >3’)41BB KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL 6 intracellulardomain (UniprotQ07011)41BB AAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAA 57 intracellular CCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGAT domain (Uniprot GGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGA Q07011) TGTGAACTGNucleic acidsequence(5’— >3’)Tanker GGGGGGS 8 IL- 12 MCIIQQLVISWFSLVFLASPLVAIWELKKDVYVVELDWYPD 9 polypeptide APGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLT1QVKEF GDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEP KNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDP QGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEES LPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKN SRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDR VFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCSG GGGGGSRNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKAR QTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNS RETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKT MNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQK SSLEEPDFYKTK1KLCILLHAFRIRAVTIDRVMSYLNASIL- 12 ATGTGTCACCAGCAGTTGGTCATCTCTTGGTTTTCCCTGG 58 polypeptide TTTTTCTGGCATCTCCCCTCGTGGCCATATGGGAACTGAA GAAAGATGTTTATGTCGTAGAATTGGATTGGTATCCGGATNucleic acid GCCCCTGGAGAAATGGTGGTCCTCACCTGTGACACCCCTG sequence AAGAAGATGGTATCACCTGGACCTTGGACCAGAGCAGTG (5’— >3’) AGGTCTTAGGCTCTGGCAAAACCCTGACCATCCAAGTCA AAGAGTTTGGAGATGCTGGCCAGTACACCTGTCACAAAG GAGGCGAGGTTCTAAGCCATTCGCTCCTGCTGCTTCACAA AAAGGAAGATGGAATTTGGTCCACTGATATTTTAAAGGA CCAGAAAGAACCCAAAAATAAGACCTTTCTAAGATGCGA GGCCAAGAATTATTCTGGACGTTTCACCTGCTGGTGGCTG ACGACAATCAGTACTGATTTGACATTCAGTGTCAAAAGC AGCAGAGGCTCTTCTGACCCCCAAGGGGTGACGTGCGGA GCTGCTACACTCTCTGCAGAGAGAGTCAGAGGGGACAAC AAGGAGTATGAGTACTCAGTGGAGTGCCAGGAGGACAGT GCCTGCCCAGCTGCTGAGGAGAGTCTGCCCATTGAGGTC ATGGTGGATGCCGTTCACAAGCTCAAGTATGAAAACTAC ACCAGCAGCTTCTTCATCAGGGACATCATCAAACCTGACC CACCCAAGAACTTGCAGCTGAAGCCATTAAAGAATTCTCGGCAGGTGGAGGTCAGCTGGGAGTACCCTGACACCTGGAGTACTCCACATTCCTACTTCTCCCTGACATTCTGCGTTCAG GTCCAGGGCAAGAGCAAGAGAGAAAAGAAAGATAGAGT CTTCACGGACAAGACCTCAGCCACGGTCATCTGCCGCAA AAATGCCAGCATTAGCGTGCGGGCCCAGGACCGCTACTA TAGCTCATCTTGGAGCGAATGGGCATCTGTGCCCTGCAGT GGTGGCGGTGGCGGCGGATCTAGAAACCTCCCCGTGGCC ACTCCAGACCCAGGAATGTTCCCATGCCTTCACCACTCCC AAAACCTGCTGAGGGCCGTCAGCAACATGCTCCAGAAGG CCAGACAAACTCTAGAATTTTACCCTTGCACTTCTGAAGA GATTGATCATGAAGATATCACAAAAGATAAAACCAGCAC AGTGGAGGCCTGTTTACCATTGGAATTAACCAAGAATGA GAGTTGCCTAAATTCCAGAGAGACCTCTTTCATAACTAAT GGGAGTTGCCTGGCCTCCAGAAAGACCTCTTTTATGATGG CCCTGTGCCTTAGTAGTATTTATGAAGACTTGAAGATGTA CCAGGTGGAGTTCAAGACCATGAATGCAAAGCTTCTGAT GGATCCTAAGAGGCAGATCTTTCTAGATCAAAACATGCT GGCAGTTATTGATGAGCTGATGCAGGCCCTGAATTTCAAC AGTGAGACTGTGCCACAAAAATCCTCCCTTGAAGAACCG GATTTTTATAAAACTAAAATCAAGCTCTGCATACTTCTTC ATGCTTTCAGAATTCGGGCAGTGACTATTGATAGAGTGAT GAGCTATCTGAATGCTTCCL2: IL- 12- MCHQQLVISWFSLVFLASPLVAIWELKKDVYVVELDWYPD 10 CD80TM APGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEF GDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEP KNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDP QGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEES LPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKN SRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDR VFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCSG GGGGGSRNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKAR QTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNS RETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKT MNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQK SSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNASLLPS W A1TL1S VNGIF VICCLL2: IL- 12- ATGTGTCACCAGCAGTTGGTCATCTCTTGGTTTTCCCTGG 59 CD80TM TTTTTCTGGCATCTCCCCTCGTGGCCATATGGGAACTGAA GAAAGATGTTTATGTCGTAGAATTGGATTGGTATCCGGATNucleic acid GCCCCTGGAGAAATGGTGGTCCTCACCTGTGACACCCCTG sequence AAGAAGATGGTATCACCTGGACCTTGGACCAGAGCAGTG (5’— >3’) AGGTCTTAGGCTCTGGCAAAACCCTGACCATCCAAGTCA AAGAGTTTGGAGATGCTGGCCAGTACACCTGTCACAAAG GAGGCGAGGTTCTAAGCCATTCGCTCCTGCTGCTTCACAA AAAGGAAGATGGAATTTGGTCCACTGATATTTTAAAGGA CCAGAAAGAACCCAAAAATAAGACCTTTCTAAGATGCGA GGCCAAGAATTATTCTGGACGTTTCACCTGCTGGTGGCTG ACGACAATCAGTACTGATTTGACATTCAGTGTCAAAAGC AGCAGAGGCTCTTCTGACCCCCAAGGGGTGACGTGCGGA GCTGCTACACTCTCTGCAGAGAGAGTCAGAGGGGACAAC AAGGAGTATGAGTACTCAGTGGAGTGCCAGGAGGACAGT GCCTGCCCAGCTGCTGAGGAGAGTCTGCCCATTGAGGTC ATGGTGGATGCCGTTCACAAGCTCAAGTATGAAAACTAC ACCAGCAGCTTCTTCATCAGGGACATCATCAAACCTGACC CACCCAAGAACTTGCAGCTGAAGCCATTAAAGAATTCTCGGCAGGTGGAGGTCAGCTGGGAGTACCCTGACACCTGGAGTACTCCACATTCCTACTTCTCCCTGACATTCTGCGTTCAG GTCCAGGGCAAGAGCAAGAGAGAAAAGAAAGATAGAGT CTTCACGGACAAGACCTCAGCCACGGTCATCTGCCGCAA AAATGCCAGCATTAGCGTGCGGGCCCAGGACCGCTACTA TAGCTCATCTTGGAGCGAATGGGCATCTGTGCCCTGCAGT GGTGGCGGTGGCGGCGGATCTAGAAACCTCCCCGTGGCC ACTCCAGACCCAGGAATGTTCCCATGCCTTCACCACTCCC AAAACCTGCTGAGGGCCGTCAGCAACATGCTCCAGAAGG CCAGACAAACTCTAGAATTTTACCCTTGCACTTCTGAAGA GATTGATCATGAAGATATCACAAAAGATAAAACCAGCAC AGTGGAGGCCTGTTTACCATTGGAATTAACCAAGAATGA GAGTTGCCTAAATTCCAGAGAGACCTCTTTCATAACTAAT GGGAGTTGCCTGGCCTCCAGAAAGACCTCTTTTATGATGG CCCTGTGCCTTAGTAGTATTTATGAAGACTTGAAGATGTA CCAGGTGGAGTTCAAGACCATGAATGCAAAGCTTCTGAT GGATCCTAAGAGGCAGATCTTTCTAGATCAAAACATGCT GGCAGTTATTGATGAGCTGATGCAGGCCCTGAATTTCAAC AGTGAGACTGTGCCACAAAAATCCTCCCTTGAAGAACCG GATTTTTATAAAACTAAAATCAAGCTCTGCATACTTCTTC ATGCTTTCAGAATTCGGGCAGTGACTATTGATAGAGTGAT GAGCTATCTGAATGCTTCCCTGCTCCCATCCTGGGCCATT ACCTTAATCTCAGTAAATGGAATTTTTGTGATATGCTGCC TGL3: IL-12- MCHQQLVISWFSLVFLASPLVAIWELKKDVYVVELDWYPD 11 CD28TM APGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEF GDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEP KNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDP QGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEES LPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKN SRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDR VFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCSG GGGGGSRNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKAR QTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNS RETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKT MNAKLLMDPKRQ1FLDQNMLAV1DELMQALNFNSETVPQK SSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNASFWVL VVVGGVLACYSLLVTVAFIIFWVL3: IL- 12- ATGTGTCACCAGCAGTTGGTCATCTCTTGGTTTTCCCTGG 60 CD28TM TTTTTCTGGCATCTCCCCTCGTGGCCATATGGGAACTGAA GAAAGATGTTTATGTCGTAGAATTGGATTGGTATCCGGATNucleic acid GCCCCTGGAGAAATGGTGGTCCTCACCTGTGACACCCCTG sequence AAGAAGATGGTATCACCTGGACCTTGGACCAGAGCAGTG (5’— >3’) AGGTCTTAGGCTCTGGCAAAACCCTGACCATCCAAGTCA AAGAGTTTGGAGATGCTGGCCAGTACACCTGTCACAAAG GAGGCGAGGTTCTAAGCCATTCGCTCCTGCTGCTTCACAA AAAGGAAGATGGAATTTGGTCCACTGATATTTTAAAGGA CCAGAAAGAACCCAAAAATAAGACCTTTCTAAGATGCGA GGCCAAGAATTATTCTGGACGTTTCACCTGCTGGTGGCTG ACGACAATCAGTACTGATTTGACATTCAGTGTCAAAAGC AGCAGAGGCTCTTCTGACCCCCAAGGGGTGACGTGCGGA GCTGCTACACTCTCTGCAGAGAGAGTCAGAGGGGACAAC AAGGAGTATGAGTACTCAGTGGAGTGCCAGGAGGACAGT GCCTGCCCAGCTGCTGAGGAGAGTCTGCCCATTGAGGTC ATGGTGGATGCCGTTCACAAGCTCAAGTATGAAAACTACACCAGCAGCTTCTTCATCAGGGACATCATCAAACCTGACCCACCCAAGAACTTGCAGCTGAAGCCATTAAAGAATTCTC GGCAGGTGGAGGTCAGCTGGGAGTACCCTGACACCTGGA GTACTCCACATTCCTACTTCTCCCTGACATTCTGCGTTCAG GTCCAGGGCAAGAGCAAGAGAGAAAAGAAAGATAGAGT CTTCACGGACAAGACCTCAGCCACGGTCATCTGCCGCAA AAATGCCAGCATTAGCGTGCGGGCCCAGGACCGCTACTA TAGCTCATCTTGGAGCGAATGGGCATCTGTGCCCTGCAGT GGTGGCGGTGGCGGCGGATCTAGAAACCTCCCCGTGGCC ACTCCAGACCCAGGAATGTTCCCATGCCTTCACCACTCCC AAAACCTGCTGAGGGCCGTCAGCAACATGCTCCAGAAGG CCAGACAAACTCTAGAATTTTACCCTTGCACTTCTGAAGA GATTGATCATGAAGATATCACAAAAGATAAAACCAGCAC AGTGGAGGCCTGTTTACCATTGGAATTAACCAAGAATGA GAGTTGCCTAAATTCCAGAGAGACCTCTTTCATAACTAAT GGGAGTTGCCTGGCCTCCAGAAAGACCTCTTTTATGATGG CCCTGTGCCTTAGTAGTATTTATGAAGACTTGAAGATGTA CCAGGTGGAGTTCAAGACCATGAATGCAAAGCTTCTGAT GGATCCTAAGAGGCAGATCTTTCTAGATCAAAACATGCT GGCAGTTATTGATGAGCTGATGCAGGCCCTGAATTTCAAC AGTGAGACTGTGCCACAAAAATCCTCCCTTGAAGAACCG GATTTTTATAAAACTAAAATCAAGCTCTGCATACTTCTTC ATGCTTTCAGAATTCGGGCAGTGACTATTGATAGAGTGAT GAGCTATCTGAATGCTTCCTTTTGGGTGCTGGTGGTGGTT GGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGG CCTTTATTATTTTCTGGGTGL4: IL-12- MCHQQLVISWFSLVFLASPLVAIWELKKDVYVVELDWYPD 12 CD80TM-4-1BB APGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEF ICD GDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEP KNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDP QGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEES LPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKN SRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDR VFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCSG GGGGGSRNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKAR QTLEFYPCTSEE1DHEDITKDKTSTVEACLPLELTKNESCLNS RETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKT MNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQK SSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNASLLPS WAITLISVNGIFVICCLKRGRKKLLYIFKQPFMRPVQTTQEED GCSCRFPEEEEGGCELL4: IL- 12- ATGTGTCACCAGCAGTTGGTCATCTCTTGGTTTTCCCTGG 61 CD80TM-4-1BB TTTTTCTGGCATCTCCCCTCGTGGCCATATGGGAACTGAA ICD GAAAGATGTTTATGTCGTAGAATTGGATTGGTATCCGGAT GCCCCTGGAGAAATGGTGGTCCTCACCTGTGACACCCCTGNucleic acid AAGAAGATGGTATCACCTGGACCTTGGACCAGAGCAGTG sequence AGGTCTTAGGCTCTGGCAAAACCCTGACCATCCAAGTCA (5’^3’) AAGAGTTTGGAGATGCTGGCCAGTACACCTGTCACAAAG GAGGCGAGGTTCTAAGCCATTCGCTCCTGCTGCTTCACAA AAAGGAAGATGGAATTTGGTCCACTGATATTTTAAAGGA CCAGAAAGAACCCAAAAATAAGACCTTTCTAAGATGCGA GGCCAAGAATTATTCTGGACGTTTCACCTGCTGGTGGCTG ACGACAATCAGTACTGATTTGACATTCAGTGTCAAAAGC AGCAGAGGCTCTTCTGACCCCCAAGGGGTGACGTGCGGA GCTGCTACACTCTCTGCAGAGAGAGTCAGAGGGGACAACAAGGAGTATGAGTACTCAGTGGAGTGCCAGGAGGACAGTGCCTGCCCAGCTGCTGAGGAGAGTCTGCCCATTGAGGTC ATGGTGGATGCCGTTCACAAGCTCAAGTATGAAAACTAC ACCAGCAGCTTCTTCATCAGGGACATCATCAAACCTGACC CACCCAAGAACTTGCAGCTGAAGCCATTAAAGAATTCTC GGCAGGTGGAGGTCAGCTGGGAGTACCCTGACACCTGGA GTACTCCACATTCCTACTTCTCCCTGACATTCTGCGTTCAG GTCCAGGGCAAGAGCAAGAGAGAAAAGAAAGATAGAGT CTTCACGGACAAGACCTCAGCCACGGTCATCTGCCGCAA AAATGCCAGCATTAGCGTGCGGGCCCAGGACCGCTACTA TAGCTCATCTTGGAGCGAATGGGCATCTGTGCCCTGCAGT GGTGGCGGTGGCGGCGGATCTAGAAACCTCCCCGTGGCC ACTCCAGACCCAGGAATGTTCCCATGCCTTCACCACTCCC AAAACCTGCTGAGGGCCGTCAGCAACATGCTCCAGAAGG CCAGACAAACTCTAGAATTTTACCCTTGCACTTCTGAAGA GATTGATCATGAAGATATCACAAAAGATAAAACCAGCAC AGTGGAGGCCTGTTTACCATTGGAATTAACCAAGAATGA GAGTTGCCTAAATTCCAGAGAGACCTCTTTCATAACTAAT GGGAGTTGCCTGGCCTCCAGAAAGACCTCTTTTATGATGG CCCTGTGCCTTAGTAGTATTTATGAAGACTTGAAGATGTA CCAGGTGGAGTTCAAGACCATGAATGCAAAGCTTCTGAT GGATCCTAAGAGGCAGATCTTTCTAGATCAAAACATGCT GGCAGTTATTGATGAGCTGATGCAGGCCCTGAATTTCAAC AGTGAGACTGTGCCACAAAAATCCTCCCTTGAAGAACCG GATTTTTATAAAACTAAAATCAAGCTCTGCATACTTCTTC ATGCTTTCAGAATTCGGGCAGTGACTATTGATAGAGTGAT GAGCTATCTGAATGCTTCCCTGCTCCCATCCTGGGCCATT ACCTTAATCTCAGTAAATGGAATTTTTGTGATATGCTGCC TGAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAAC AACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAG ATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAG GATGTGAACTGL5: IL- 12- MCHQQLVISWFSLVFLASPLVAIWELKKDVYVVELDWYPD 13 CD28TM-CD28 APGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEF ICD GDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEP KNKTFLRCEAKNYSGRFTCWWLTT1STDLTFSVKSSRGSSDP QGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEES LPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKN SRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDR VFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCSG GGGGGSRNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKAR QTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNS RETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKT MNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQK SSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNASFWVL VVVGGVLACYSLLVTVAFIIFWVRSKRSRLLIISDYMNMTPR RPGPTRKHYQPYAPPRDFAAYRSL5: IL- 12- ATGTGTCACCAGCAGTTGGTCATCTCTTGGTTTTCCCTGG 62 CD28TM-CD28 TTTTTCTGGCATCTCCCCTCGTGGCCATATGGGAACTGAA ICD GAAAGATGTTTATGTCGTAGAATTGGATTGGTATCCGGAT GCCCCTGGAGAAATGGTGGTCCTCACCTGTGACACCCCTGNucleic acid AAGAAGATGGTATCACCTGGACCTTGGACCAGAGCAGTG sequence AGGTCTTAGGCTCTGGCAAAACCCTGACCATCCAAGTCA (5’— >3’) AAGAGTTTGGAGATGCTGGCCAGTACACCTGTCACAAAGGAGGCGAGGTTCTAAGCCATTCGCTCCTGCTGCTTCACAAAAAGGAAGATGGAATTTGGTCCACTGATATTTTAAAGGACCAGAAAGAACCCAAAAATAAGACCTTTCTAAGATGCGA GGCCAAGAATTATTCTGGACGTTTCACCTGCTGGTGGCTG ACGACAATCAGTACTGATTTGACATTCAGTGTCAAAAGC AGCAGAGGCTCTTCTGACCCCCAAGGGGTGACGTGCGGA GCTGCTACACTCTCTGCAGAGAGAGTCAGAGGGGACAAC AAGGAGTATGAGTACTCAGTGGAGTGCCAGGAGGACAGT GCCTGCCCAGCTGCTGAGGAGAGTCTGCCCATTGAGGTC ATGGTGGATGCCGTTCACAAGCTCAAGTATGAAAACTAC ACCAGCAGCTTCTTCATCAGGGACATCATCAAACCTGACC CACCCAAGAACTTGCAGCTGAAGCCATTAAAGAATTCTC GGCAGGTGGAGGTCAGCTGGGAGTACCCTGACACCTGGA GTACTCCACATTCCTACTTCTCCCTGACATTCTGCGTTCAG GTCCAGGGCAAGAGCAAGAGAGAAAAGAAAGATAGAGT CTTCACGGACAAGACCTCAGCCACGGTCATCTGCCGCAA AAATGCCAGCATTAGCGTGCGGGCCCAGGACCGCTACTA TAGCTCATCTTGGAGCGAATGGGCATCTGTGCCCTGCAGT GGTGGCGGTGGCGGCGGATCTAGAAACCTCCCCGTGGCC ACTCCAGACCCAGGAATGTTCCCATGCCTTCACCACTCCC AAAACCTGCTGAGGGCCGTCAGCAACATGCTCCAGAAGG CCAGACAAACTCTAGAATTTTACCCTTGCACTTCTGAAGA GATTGATCATGAAGATATCACAAAAGATAAAACCAGCAC AGTGGAGGCCTGTTTACCATTGGAATTAACCAAGAATGA GAGTTGCCTAAATTCCAGAGAGACCTCTTTCATAACTAAT GGGAGTTGCCTGGCCTCCAGAAAGACCTCTTTTATGATGG CCCTGTGCCTTAGTAGTATTTATGAAGACTTGAAGATGTA CCAGGTGGAGTTCAAGACCATGAATGCAAAGCTTCTGAT GGATCCTAAGAGGCAGATCTTTCTAGATCAAAACATGCT GGCAGTTATTGATGAGCTGATGCAGGCCCTGAATTTCAAC AGTGAGACTGTGCCACAAAAATCCTCCCTTGAAGAACCG GATTTTTATAAAACTAAAATCAAGCTCTGCATACTTCTTC ATGCTTTCAGAATTCGGGCAGTGACTATTGATAGAGTGAT GAGCTATCTGAATGCTTCCTTTTGGGTGCTGGTGGTGGTT GGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGG CCTTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCT CCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCC GGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCA CGCGACTTCGCAGCCTATCGCTCCL6: IL-12- MCHQQLVISWFSLVFLASPLVAIWELKKDVYVVELDWYPD 14 CD28TM-4-1BB APGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEF ICD GDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEP KNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDP QGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEES LPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKN SRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDR VFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCSG GGGGGSRNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKAR QTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNS RETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKT MNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQK SSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNASFWVL VVVGGVLACYSLLVTVAFIIFWVKRGRKKLLYIFKQPFMRP VQTTQEEDGCSCRFPEEEEGGCELL6: IL- 12- ATGTGTCACCAGCAGTTGGTCATCTCTTGGTTTTCCCTGG 63 CD28TM-4-1BB TTTTTCTGGCATCTCCCCTCGTGGCCATATGGGAACTGAAICD GAAAGATGTTTATGTCGTAGAATTGGATTGGTATCCGGATGCCCCTGGAGAAATGGTGGTCCTCACCTGTGACACCCCTG Nucleic acid AAGAAGATGGTATCACCTGGACC1TGGACCAGAGCAGTG sequence AGGTCTTAGGCTCTGGCAAAACCCTGACCATCCAAGTCA (5’— >3’) AAGAGTTTGGAGATGCTGGCCAGTACACCTGTCACAAAG GAGGCGAGGTTCTAAGCCATTCGCTCCTGCTGCTTCACAA AAAGGAAGATGGAATTTGGTCCACTGATATTTTAAAGGA CCAGAAAGAACCCAAAAATAAGACCTTTCTAAGATGCGA GGCCAAGAATTATTCTGGACGTTTCACCTGCTGGTGGCTG ACGACAATCAGTACTGATTTGACATTCAGTGTCAAAAGC AGCAGAGGCTCTTCTGACCCCCAAGGGGTGACGTGCGGA GCTGCTACACTCTCTGCAGAGAGAGTCAGAGGGGACAAC AAGGAGTATGAGTACTCAGTGGAGTGCCAGGAGGACAGT GCCTGCCCAGCTGCTGAGGAGAGTCTGCCCATTGAGGTC ATGGTGGATGCCGTTCACAAGCTCAAGTATGAAAACTAC ACCAGCAGCTTCTTCATCAGGGACATCATCAAACCTGACC CACCCAAGAACTTGCAGCTGAAGCCATTAAAGAATTCTC GGCAGGTGGAGGTCAGCTGGGAGTACCCTGACACCTGGA GTACTCCACATTCCTACTTCTCCCTGACATTCTGCGTTCAG GTCCAGGGCAAGAGCAAGAGAGAAAAGAAAGATAGAGT CTTCACGGACAAGACCTCAGCCACGGTCATCTGCCGCAA AAATGCCAGCATTAGCGTGCGGGCCCAGGACCGCTACTA TAGCTCATCTTGGAGCGAATGGGCATCTGTGCCCTGCAGT GGTGGCGGTGGCGGCGGATCTAGAAACCTCCCCGTGGCC ACTCCAGACCCAGGAATGTTCCCATGCCTTCACCACTCCC AAAACCTGCTGAGGGCCGTCAGCAACATGCTCCAGAAGG CCAGACAAACTCTAGAATTTTACCCTTGCACTTCTGAAGA GATTGATCATGAAGATATCACAAAAGATAAAACCAGCAC AGTGGAGGCCTGTTTACCATTGGAATTAACCAAGAATGA GAGTTGCCTAAATTCCAGAGAGACCTCTTTCATAACTAAT GGGAGTTGCCTGGCCTCCAGAAAGACCTCTTTTATGATGG CCCTGTGCCTTAGTAGTATTTATGAAGACTTGAAGATGTA CCAGGTGGAGTTCAAGACCATGAATGCAAAGCTTCTGAT GGATCCTAAGAGGCAGATCTTTCTAGATCAAAACATGCT GGCAGTTATTGATGAGCTGATGCAGGCCCTGAATTTCAAC AGTGAGACTGTGCCACAAAAATCCTCCCTTGAAGAACCG GATTTTTATAAAACTAAAATCAAGCTCTGCATACTTCTTC ATGCTTTCAGAATTCGGGCAGTGACTATTGATAGAGTGAT GAGCTATCTGAATGCTTCCTTTTGGGTGCTGGTGGTGGTT GGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGG CCTTTATTATTTTCTGGGTGAAACGGGGCAGAAAGAAACT CCTGTATATATTCAAACAACCATTTATGAGACCAGTACAA ACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCA GAAGAAGAAGAAGGAGGATGTGAACTG IL- 15 NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMK 17CFLLELQVISLESGDASIIIDTVENLIILANNSLSSNGNVTESG CKECEELEEKNIKEELQSFVHIVQMFINTSIL- 15 AACTGGGTCAACGTGATCAGCGACCTGAAGAAGATCGAG 18 nucleic acid GACCTGATCCAGAGCATGCACATCGACGCCACACTGTAC sequence (5’— > ACCGAGTCCGACGTGCACCCCAGCTGCAAGGTGACCGCC 3’) ATGAAGTGCTTCCTGCTGGAGCTGCAGGTGATCAGCCTG GAGAGCGGCGACGCCAGCATCCACGACACCGTGGAGAAC CTGATCATCCTGGCCAACAACAGCCTGTCCAGCAACGGC AACGTGACCGAGAGCGGCTGCAAGGAGTGCGAGGAGCT GGAGGAGAAGAACATCAAGGAGTTCCTGCAGAGCTTCGTCCACATCGTGCAGATGTTCATCAACACGAGCIL- 18 YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDN 19 APRTIFIISMYKDSQPRGMAVTISVKCEKISTLSCENKIISFKE MNPPDNIKDTKSDIIFFQRSVPGHDNKMQFESSSYEGYFLAC EKERDLFKLILKKEDELGDRSIMFTVQNED IL- 18 TACTTCGGCAAGCTTGAGAGCAAGCTGAGCGTCATCAGA 20 Nucleic acid AACCTGAACGACCAGGTTCTCTTCATTGACCAGGGCAAC sequence (5’— > AGGCCCCTGTTCGAGGACATGACCGACAGCGACTGCAGA 3’) GACAACGCACCCAGGACCATCTTCATTATCAGTATGTATA AGGACAGCCAGCCCAGAGGCATGGCCGTAACCATCAGCG TGAAGTGCGAGAAGATTAGCACCCTCTCCTGCGAGAACA AGATTATTTCCTTCAAGGAGATGAACCCCCCCGACAACAT CAAGGACACAAAGAGTGACATCATCTTCTTCCAGAGAAG TGTCCCCGGCCATGACAACAAGATGCAGTTCGAGAGCAG CAGCTACGAGGGCTACTTCCTCGCCTGCGAGAAGGAGAG AGACCTTTTCAAGCTCATTCTGAAGAAGGAGGACGAGCT GGGGGACAGAAGCATCATGTTCACCGTTCAGAACGAGGA CBifunctional MDWTWILFLVAAATRVHSNWVNVISDLKKIEDLIQSMHIDA 64 hyperkine (mblL- TLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVEN 15 - mbIL-18) LIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQ MFINTSGGGGSYFGKLESKLSVIRNLNDQVLFIDQGNRPLFE DMTDSDCRDNAPRTIFIISMYKDSQPRGMAVTISVKCEKIST LSCENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHDNKMQFE SSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNEDS GGGGSGGGGSGGGGSGGGGSGGGSLQFWVLVVVGGVLAC YSLLVTVAFIIFWVKRGRKKLLYIFKQPFMRPVQTTQEEDGC SCRFPEEEEGGCELBifunctional ATGGACTGGACCTGGATACTCTTCCTGGTCGCCGCCGCCA 65 hyperkine (mblL- CAAGGGTGCACAGCAACTGGGTCAACGTGATCAGCGACC 15 -mbIL-18) TGAAGAAGATCGAGGACCTGATCCAGAGCATGCACATCG ACGCCACACTGTACACCGAGTCCGACGTGCACCCCAGCTNucleic acid GCAAGGTGACCGCCATGAAGTGCTTCCTGCTGGAGCTGC sequence AGGTGATCAGCCTGGAGAGCGGCGACGCCAGCATCCACG (5’— >3’) ACACCGTGGAGAACCTGATCATCCTGGCCAACAACAGCC TGTCCAGCAACGGCAACGTGACCGAGAGCGGCTGCAAGG AGTGCGAGGAGCTGGAGGAGAAGAACATCAAGGAGTTC CTGCAGAGCTTCGTCCACATCGTGCAGATGTTCATCAACA CGAGCGGTGGGGGAGGAAGCTACTTCGGCAAGCTTGAGA GCAAGCTGAGCGTCATCAGAAACCTGAACGACCAGGTTC TCTTCATTGACCAGGGCAACAGGCCCCTGTTCGAGGACAT GACCGACAGCGACTGCAGAGACAACGCACCCAGGACCAT CTTCATTATCAGTATGTATAAGGACAGCCAGCCCAGAGG CATGGCCGTAACCATCAGCGTGAAGTGCGAGAAGATTAG CACCCTCTCCTGCGAGAACAAGATTATTTCCTTCAAGGAG ATGAACCCCCCCGACAACATCAAGGACACAAAGAGTGAC ATCATCTTCTTCCAGAGAAGTGTCCCCGGCCATGACAACA AGATGCAGTTCGAGAGCAGCAGCTACGAGGGCTACTTCC TCGCCTGCGAGAAGGAGAGAGACCTTTTCAAGCTCATTCT GAAGAAGGAGGACGAGCTGGGGGACAGAAGCATCATGT TCACCGTTCAGAACGAGGACAGCGGGGGCGGTGGAAGCG GAGGAGGCGGAAGTGGCGGCGGAGGCTCAGGCGGCGGA GGTTCCGGCGGAGGAAGCCTTCAGTTTTGGGTGCTGGTG GTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCA GTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGA TTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGBifunctional MDWTWILFLVAAATRVHSYFGKLESKLSVIRNLNDQVLFID 66 hyperkine (mblL- QGNRPLFEDMTDSDCRDNAPRTIFIISMYKDSQPRGMAVTIS 18-mbIL-15) VKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGH DNKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMF TVQNEDGGGGSNWVNVISDLKKIEDLIQSMHIDATLYTESD VHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNS LSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSSG GGGSGGGGSGGGGSGGGGSGGGSLQFWVLVVVGGVLACY SLLVTVAFIIFWVKRGRKKLLYIFKQPFMRPVQTTQEEDGCS CRFPEEEEGGCELBifunctional ATGGACTGGACCTGGATACTCTTCCTGGTCGCCGCCGCCA 67 hyperkine (mblL- CAAGGGTGCACAGCTACTTCGGCAAGCTTGAGAGCAAGC18 -mbIL-15) TGAGCGTCATCAGAAACCTGAACGACCAGGTTCTCTTCAT TGACCAGGGCAACAGGCCCCTGTTCGAGGACATGACCGANucleic acid CAGCGACTGCAGAGACAACGCACCCAGGACCATCTTCAT sequence TATCAGTATGTATAAGGACAGCCAGCCCAGAGGCATGGC(5’— >3’) CGTAACCATCAGCGTGAAGTGCGAGAAGATTAGCACCCT CTCCTGCGAGAACAAGATTATTTCCTTCAAGGAGATGAA CCCCCCCGACAACATCAAGGACACAAAGAGTGACATCAT CTTCTTCCAGAGAAGTGTCCCCGGCCATGACAACAAGAT GCAGTTCGAGAGCAGCAGCTACGAGGGCTACTTCCTCGC CTGCGAGAAGGAGAGAGACCTTTTCAAGCTCATTCTGAA GAAGGAGGACGAGCTGGGGGACAGAAGCATCATGTTCAC CGTTCAGAACGAGGACGGTGGGGGAGGAAGCAACTGGG TCAACGTGATCAGCGACCTGAAGAAGATCGAGGACCTGA TCCAGAGCATGCACATCGACGCCACACTGTACACCGAGT CCGACGTGCACCCCAGCTGCAAGGTGACCGCCATGAAGT GCTTCCTGCTGGAGCTGCAGGTGATCAGCCTGGAGAGCG GCGACGCCAGCATCCACGACACCGTGGAGAACCTGATCA TCCTGGCCAACAACAGCCTGTCCAGCAACGGCAACGTGA CCGAGAGCGGCTGCAAGGAGTGCGAGGAGCTGGAGGAG AAGAACATCAAGGAGTTCCTGCAGAGCTTCGTCCACATC GTGCAGATGTTCATCAACACGAGCAGCGGGGGCGGTGGA AGCGGAGGAGGCGGAAGTGGCGGCGGAGGCTCAGGCGG CGGAGGTTCCGGCGGAGGAAGCCTTCAGTTTTGGGTGCT GGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTA GTAACAGTGGCCTTTATTATTTTCTGGGTGAAACGGGGCA GAAAGAAACTCCTGTATATATTCAAACAACCATTTATGA GACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTG EXAMPLESExample 1. Design of Constructs and Expression on Human Primary T Cells

[0317] Gene constructs and cloning

[0318] Sequences of membrane-bound IL-12 (mbIL12) fusion proteins and NY-ESO-1-specific TCR-engineered T cells (1G4) are cloned and the constructs of the fusion proteinsare designed as illustrated in FIG. 1. Protein and / or nucleic acid sequences of the constructs and construct components of FIG. 1 are provided in Table 2. The NY-ES 0-1 -specific TCR sequence corresponds to the LI sequence of Table 2. T2A is a 2A peptide-based coexpression system that enables the independent expression of multiple proteins from a single open reading frame (ORF). ALNGFR is a truncated version of low-affinity nerve growth factor receptor (LNGFR) that lacks the intracellular domain. ALNGFR was used as a selection marker to monitor gene transduction and to enrich CAR T-cells. All constructs were cloned under a EF1α promoter using a lentivirus vector backbone. The amino acid sequences were obtained from Uniprot. Human IL- 12 is composed by p40 and p35 subunits. IL- 12 p40 subunit was linked to IL-12 p35 subunit by a Gly6Ser linker (SEQ ID NO: 8). The signal peptide of p40 subunit was maintained and the signal peptide of p35 was deleted. Additionally to the IL- 12, CD80TM or CD28TM was used to anchor the cytokine to the cell membrane. In the novel IL- 12 based fusion proteins, downstream of the transmembrane domain, the intracellular domain of a costimulatory receptor (CD28 or 41 BB) is added. Table 2. Amino Acid and Nucleic Acid Sequences11Construct Sequence SEQ ID NO: L1.1G4 WT MSIGLLCCAALSLLWAGPVNAGVTQTPKFQVLKTGQSMTL 23 QCAQDMNHEYMSWYRQDPGMGLRLIHYSVGAGITDQGEV PNGYNVSRSTTEDFPLRLLSAAPSQTSVYFCASSYVGNTGEL FFGEGSRLTVLEDLKNVFPPEVAVFEPSEAE1SHTQKATLVC LATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALND SRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQ DRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILL GKATLYAVLVSALVLMAMVKRKDSRGRAKRSGSGATNFSL LKQAGDVEENPGPMETLLGLLILWLQLQWVSSKQEVTQIPA ALSVPEGENLVLNCSFTDSAIYNLQWFRQDPGKGLTSLLLIQ SSQREQTSGRLNASLDKSSGRSTLYIAASQPGDSATYLCAVR PLYGGSYIPTFGRGTSLIVHPYIQNPDPAVYQLRDSKSSDKSV CLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAV AWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFET DTNLNFQNLSVIGFR1LLLKVAGFNLLMTLRLWSS* L1.1G4 WT ATGAGCATCGGCCTCCTGTGCTGTGCAGCCTTGTCTCTCC 24 TGTGGGCAGGTCCAGTGAATGCTGGTGTCACTCAGACCCnucleic acid CAAAATTCCAGGTCCTGAAGACAGGACAGAGCATGACAC(5’— > 3’) TGCAGTGTGCCCAGGATATGAACCATGAATACATGTCCT GGTATCGACAAGACCCAGGCATGGGGCTGAGGCTGATTC ATTACTCAGTTGGTGCTGGTATCACTGACCAAGGAGAAG TCCCCAATGGCTACAATGTCTCCAGATCAACCACAGAGG ATTTCCCGCTCAGGCTGCTGTCGGCTGCTCCCTCCCAGAC ATCTGTGTACTTCTGTGCCAGCAGTTACGTCGGGAACACC GGGGAGCTGTTTTTTGGAGAAGGCTCTAGGCTGACCGTA CTGGAGGACCTGAAAAACGTGTTCCCACCCGAGGTCGCTGTGTTTGAGCCATCAGAAGCAGAGATCTCCCACACCCAAAAGGCCACACTGGTATGCCTGGCCACAGGCTTCTACCCC GACCACGTGGAGCTGAGCTGGTGGGTGAATGGGAAGGAG GTGCACAGTGGGGTCAGCACAGACCCGCAGCCCCTCAAG GAGCAGCCCGCCCTCAATGACTCCAGATACTGCCTGAGC AGCCGCCTGAGGGTCTCGGCCACCTTCTGGCAGAACCCC CGCAACCACTTCCGCTGTCAAGTCCAGTTCTACGGGCTCT CGGAGAATGACGAGTGGACCCAGGATAGGGCCAAACCC GTCACCCAGATCGTCAGCGCCGAGGCCTGGGGTAGAGCA GACTGTGGCTTCACCTCCGAGTCTTACCAGCAAGGGGTCC TGTCTGCCACCATCCTCTATGAGATCTTGCTAGGGAAGGC CACCTTGTATGCCGTGCTGGTCAGTGCCCTCGTGCTGATG GCTATGGTCAAGAGAAAGGATTCCAGAGGCCGGGCCAAG CGGTCCGGATCCGGAGCCACCAACTTCAGCCTGCTGAAG CAGGCCGGCGACGTGGAGGAGAACCCCGGCCCCATGGAG ACCCTCTTGGGCCTGCTTATCCTTTGGCTGCAGCTGCAAT GGGTGAGCAGCAAACAGGAGGTGACGCAGATTCCTGCAG CTCTGAGTGTCCCAGAAGGAGAAAACTTGGTTCTCAACT GCAGTTTCACTGATAGCGCTATTTACAACCTCCAGTGGTT TAGGCAGGACCCTGGGAAAGGTCTCACATCTCTGTTGCTT ATTCAGTCAAGTCAGAGAGAGCAAACAAGTGGAAGACTT AATGCCTCGCTGGATAAATCATCAGGACGTAGTACTTTAT ACATTGCAGCTTCTCAGCCTGGTGACTCAGCCACCTACCT CTGTGCTGTGAGGCCCCTGTACGGAGGAAGCTACATACC TACATTTGGAAGAGGAACCAGCCTTATTGTTCATCCGTAT ATCCAGAACCCTGACCCTGCGGTGTACCAGCTGAGAGAC TCTAAATCCAGTGACAAGTCTGTCTGCCTATTCACCGATT TTGATTCTCAAACAAATGTGTCACAAAGTAAGGATTCTGA TGTGTATATCACAGACAAAACTGTGCTAGACATGAGGTC TATGGACTTCAAGAGCAACAGTGCTGTGGCCTGGAGCAA CAAATCTGACTTTGCATGTGCAAACGCCTTCAACAACAGC ATTATTCCAGAAGACACCTTCTTCCCCAGCCCAGAAAGTT CCTGTGATGTCAAGCTGGTCGAGAAAAGCTTTGAAACAG ATACGAACCTAAACTTTCAAAACCTGTCAGTGATTGGGTT CCGAATCCTCCTCCTGAAAGTGGCCGGGTTTAATCTGCTC ATGACGCTGCGGCTGTGGTCCAGC7AGL2. mbIL12 MCHQQLVISWFSLVFLASPLVAIWELKKDVYVVELDWYPD 25 (CD80TM)- APGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEF LNGFR GDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEP KNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDP QGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEES mbIL12- LPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKN CD80TM-72A-A SRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDR VFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCSG LNGFR GGGGGSRNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKAR QTLEFYPCTSEEIDIIEDITKDKTSTVEACLPLELTKNESCLNS RETSFITNGSCLASRKTSFMMALCLSS1YEDLKMYQVEFKT MNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQK SSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNASLLPS WMTLISVNGIFVICCLRAKRSGSGEGRGSLLTCGDVEENPGP MGAGATGRAMDGPRLLLLLLLGVSLGGAKEACPTGLY THSGECCKACNLGEGVAQPCGANQTVCEPCLDSVTFSD VVSATEPCKPCTECVGLQSMSAPCVEADDAVCRCAYGY YQDETTGRCEACRVCEAGSGLVFSCQDKQNTVCEECPD GTYSDEANHVDPCLPCTVCEDTERQLRECTRWADAECEEIPGRWITRSTPPEGSDSTAPSTQEPEAPPEQDLIASTVAGVVTTVMGSSQPVVTRGTTDNLIPVYCSILAAVVVGLVAYI AFKRW*L2. mbIL12 ATGTGTCACCAGCAGTTGGTCATCTCTTGGTTTTCCCTGGT 26 (CD80TM)- TTTTCTGGCATCTCCCCTCGTGGCCATATGGGAACTGAAG LNGFR AAAGATGTTTATGTCGTAGAATTGGATTGGTATCCGGATG CCCCTGGAGAAATGGTGGTCCTCACCTGTGACACCCCTGA nucleic acid AGAAGATGGTATCACCTGGACCTTGGACCAGAGCAGTGA (5’— > 3’) GGTCTTAGGCTCTGGCAAAACCCTGACCATCCAAGTCAA AGAGTTTGGAGATGCTGGCCAGTACACCTGTCACAAAGG mbIL12- AGGCGAGGTTCTAAGCCATTCGCTCCTGCTGCTTCACAAA CD80TM-r2A- AAGGAAGATGGAATTTGGTCCACTGATATTTTAAAGGAC ALNGFR CAGAAAGAACCCAAAAATAAGACCTTTCTAAGATGCGAG GCCAAGAATTATTCTGGACGTTTCACCTGCTGGTGGCTGA CGACAATCAGTACTGATTTGACATTCAGTGTCAAAAGCA GCAGAGGCTCTTCTGACCCCCAAGGGGTGACGTGCGGAG CTGCTACACTCTCTGCAGAGAGAGTCAGAGGGGACAACA AGGAGTATGAGTACTCAGTGGAGTGCCAGGAGGACAGTG CCTGCCCAGCTGCTGAGGAGAGTCTGCCCATTGAGGTCAT GGTGGATGCCGTTCACAAGCTCAAGTATGAAAACTACAC CAGCAGCTTCTTCATCAGGGACATCATCAAACCTGACCCA CCCAAGAACTTGCAGCTGAAGCCATTAAAGAATTCTCGG CAGGTGGAGGTCAGCTGGGAGTACCCTGACACCTGGAGT ACTCCACATTCCTACTTCTCCCTGACATTCTGCGTTCAGGT CCAGGGCAAGAGCAAGAGAGAAAAGAAAGATAGAGTCT TCACGGACAAGACCTCAGCCACGGTCATCTGCCGCAAAA ATGCCAGCATTAGCGTGCGGGCCCAGGACCGCTACTATA GCTCATCTTGGAGCGAATGGGCATCTGTGCCCTGCAGTGG TGGCGGTGGCGGCGGATCTAGAAACCTCCCCGTGGCCAC TCCAGACCCAGGAATGTTCCCATGCCTTCACCACTCCCAA AACCTGCTGAGGGCCGTCAGCAACATGCTCCAGAAGGCC AGACAAACTCTAGAATTTTACCCTTGCACTTCTGAAGAGA TTGATCATGAAGATATCACAAAAGATAAAACCAGCACAG TGGAGGCCTGTTTACCATTGGAATTAACCAAGAATGAGA GTTGCCTAAATTCCAGAGAGACCTCTTTCATAACTAATGG GAGTTGCCTGGCCTCCAGAAAGACCTCTTTTATGATGGCC CTGTGCCTTAGTAGTATTTATGAAGACTTGAAGATGTACC AGGTGGAGTTCAAGACCATGAATGCAAAGCTTCTGATGG ATCCTAAGAGGCAGATCTTTCTAGATCAAAACATGCTGG CAGTTATTGATGAGCTGATGCAGGCCCTGAATTTCAACAG TGAGACTGTGCCACAAAAATCCTCCCTTGAAGAACCGGA TTTTTATAAAACTAAAATCAAGCTCTGCATACTTCTTCAT GCTTTCAGAATTCGGGCAGTGACTATTGATAGAGTGATG AGCTATCTGAATGCTTCCCTGCTCCCATCCTGGGCCATTA CCTTAATCTCAGTAAATGGAATTTTTGTGATATGCTGCCT GCGGGCCAAGCGGTCCGGATCCGGAGAGGGCAGAGGAAGT CTGCTAACATGCGGTGACGTCGAGGAGAATCCTGGCCCAAT GGGGGCAGGTGCCACCGGCCGCGCCATGGACGGGCC GCGCCTGCTGCTGTTGCTGCTTCTGGGGGTGTCCCTT GGAGGTGCCAAGGAGGCATGCCCCACAGGCCTGTACA CACACAGCGGTGAGTGCTGCAAAGCCTGCAACCTGGG CGAGGGTGTGGCCCAGCCTTGTGGAGCCAACCAGACC GTGTGTGAGCCCTGCCTGGACAGCGTGACGTTCTCCG ACGTGGTGAGCGCGACCGAGCCGTGCAAGCCGTGCA CCGAGTGCGTGGGGCTCCAGAGCATGTCGGCGCCGTGCGTGGAGGCCGACGACGCCGTGTGCCGCTGCGCCTACGGCTACTACCAGGATGAGACGACTGGGCGCTGCGA GGCGTGCCGCGTGTGCGAGGCGGGCTCGGGCCTCGT GTTCTCCTGCCAGGACAAGCAGAACACCGTGTGCGAG GAGTGCCCCGACGGCACGTATTCCGACGAGGCCAACC ACGTGGACCCGTGCCTGCCCTGCACCGTGTGCGAGGA CACCGAGCGCCAGCTCCGCGAGTGCACACGCTGGGCC GACGCCGAGTGCGAGGAGATCCCTGGCCGTTGGATTA CACGGTCCACACCCCCAGAGGGCTCGGACAGCACAGC CCCCAGCACCCAGGAGCCTGAGGCACCTCCAGAACAA GACCTCATAGCCAGCACGGTGGCAGGTGTGGTGACCA CAGTGATGGGCAGCTCCCAGCCCGTGGTGACCCGAGG CACCACCGACAACCTCATCCCTGTCTATTGCTCCATCC TGGCTGCTGTGGTTGTGGGTCTTGTGGCCTACATAGC CTTCAAGAGGTGGTAGL3.mbIL12- MCHQQLVISWFSLVFLASPLVAIWELKKDVYVVELDWYPD 27 (CD28TM)- APGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEF LNGFR GDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEP KNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDP QGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEES mbIL12- LPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKN CD28TM-F2A-A SRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDR LNGFR VFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCSG GGGGGSRNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKAR QTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNS RETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKT MNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQK SSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNASFWVL VVVGGVLACYSLLVTVAFIIFWVRAKRSGSGEGRGSLLTCGD VEEAPGPMGAGATGRAMDGPRLLLLLLLGVSLGGAKEA CPTGLYTHSGECCKACNLGEGVAQPCGANQTVCEPCLD SVTFSDVVSATEPCKPCTECVGLQSMSAPCVEADDAVCR CAYGYYQDETTGRCEACRVCEAGSGLVFSCQDKQNTVC EECPDGTYSDEANHVDPCLPCTVCEDTERQLRECTRWA DAECEEIPGRWITRSTPPEGSDSTAPSTQEPEAPPEQDLIA STVAGVVTTVMGSSQPVVTRGTTDNLIPVYCSILAAVVV GLVAYIAFKRW*L3.mbIL12- ATGTGTCACCAGCAGTTGGTCATCTCTTGGTTTTCCCTGGT 28 (CD28TM)- TTTTCTGGCATCTCCCCTCGTGGCCATATGGGAACTGAAG LNGFR AAAGATGTTTATGTCGTAGAATTGGATTGGTATCCGGATG CCCCTGGAGAAATGGTGGTCCTCACCTGTGACACCCCTGA nucleic acid AGAAGATGGTATCACCTGGACCTTGGACCAGAGCAGTGA (5’— > 3’) GGTCTTAGGCTCTGGCAAAACCCTGACCATCCAAGTCAA AGAGTTTGGAGATGCTGGCCAGTACACCTGTCACAAAGG mbIL12- AGGCGAGGTTCTAAGCCATTCGCTCCTGCTGCTTCACAAA CD28TM-F2A- AAGGAAGATGGAATTTGGTCCACTGATATTTTAAAGGAC ALNGFR CAGAAAGAACCCAAAAATAAGACCTTTCTAAGATGCGAG GCCAAGAATTATTCTGGACGTTTCACCTGCTGGTGGCTGA CGACAATCAGTACTGATTTGACATTCAGTGTCAAAAGCA GCAGAGGCTCTTCTGACCCCCAAGGGGTGACGTGCGGAG CTGCTACACTCTCTGCAGAGAGAGTCAGAGGGGACAACA AGGAGTATGAGTACTCAGTGGAGTGCCAGGAGGACAGTG CCTGCCCAGCTGCTGAGGAGAGTCTGCCCATTGAGGTCAT GGTGGATGCCGTTCACAAGCTCAAGTATGAAAACTACAC CAGCAGCTTCTTCATCAGGGACATCATCAAACCTGACCCACCCAAGAACTTGCAGCTGAAGCCATTAAAGAATTCTCGGCAGGTGGAGGTCAGCTGGGAGTACCCTGACACCTGGAGT ACTCCACATTCCTACTTCTCCCTGACATTCTGCGTTCAGGT CCAGGGCAAGAGCAAGAGAGAAAAGAAAGATAGAGTCT TCACGGACAAGACCTCAGCCACGGTCATCTGCCGCAAAA ATGCCAGCATTAGCGTGCGGGCCCAGGACCGCTACTATA GCTCATCTTGGAGCGAATGGGCATCTGTGCCCTGCAGTGG TGGCGGTGGCGGCGGATCTAGAAACCTCCCCGTGGCCAC TCCAGACCCAGGAATGTTCCCATGCCTTCACCACTCCCAA AACCTGCTGAGGGCCGTCAGCAACATGCTCCAGAAGGCC AGACAAACTCTAGAATTTTACCCTTGCACTTCTGAAGAGA TTGATCATGAAGATATCACAAAAGATAAAACCAGCACAG TGGAGGCCTGTTTACCATTGGAATTAACCAAGAATGAGA GTTGCCTAAATTCCAGAGAGACCTCTTTCATAACTAATGG GAGTTGCCTGGCCTCCAGAAAGACCTCTTTTATGATGGCC CTGTGCCTTAGTAGTATTTATGAAGACTTGAAGATGTACC AGGTGGAGTTCAAGACCATGAATGCAAAGCTTCTGATGG ATCCTAAGAGGCAGATCTTTCTAGATCAAAACATGCTGG CAGTTATTGATGAGCTGATGCAGGCCCTGAATTTCAACAG TGAGACTGTGCCACAAAAATCCTCCCTTGAAGAACCGGA TTTTTATAAAACTAAAATCAAGCTCTGCATACTTCTTCAT GCTTTCAGAATTCGGGCAGTGACTATTGATAGAGTGATG AGCTATCTGAATGCTTCCTTTTGGGTGCTGGTGGTGGTTG GTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGC CTTTATTATTTTCTGGGTGCGGGCCAAGCGGTCCGGATCCGGAGAGGGCAGAGGAAGTCTGCTAACATGCGGTGACGTCGAGGAGAATCCTGGCCCAATGGGGGCAGGTGCCACCGGCCG CGCCATGGACGGGCCGCGCCTGCTGCTGTTGCTGCTT CTGGGGGTGTCCCTTGGAGGTGCCAAGGAGGCATGCC CCACAGGCCTGTACACACACAGCGGTGAGTGCTGCAA AGCCTGCAACCTGGGCGAGGGTGTGGCCCAGCCTTGT GGAGCCAACCAGACCGTGTGTGAGCCCTGCCTGGACA GCGTGACGTTCTCCGACGTGGTGAGCGCGACCGAGCC GTGCAAGCCGTGCACCGAGTGCGTGGGGCTCCAGAG CATGTCGGCGCCGTGCGTGGAGGCCGACGACGCCGT GTGCCGCTGCGCCTACGGCTACTACCAGGATGAGACG ACTGGGCGCTGCGAGGCGTGCCGCGTGTGCGAGGCG GGCTCGGGCCTCGTGTTCTCCTGCCAGGACAAGCAGA ACACCGTGTGCGAGGAGTGCCCCGACGGCACGTATTC CGACGAGGCCAACCACGTGGACCCGTGCCTGCCCTGC ACCGTGTGCGAGGACACCGAGCGCCAGCTCCGCGAGT GCACACGCTGGGCCGACGCCGAGTGCGAGGAGATCC CTGGCCGTTGGATTACACGGTCCACACCCCCAGAGGG CTCGGACAGCACAGCCCCCAGCACCCAGGAGCCTGAG GCACCTCCAGAACAAGACCTCATAGCCAGCACGGTGG CAGGTGTGGTGACCACAGTGATGGGCAGCTCCCAGCC CGTGGTGACCCGAGGCACCACCGACAACCTCATCCCT GTCTATTGCTCCATCCTGGCTGCTGTGGTTGTGGGTC TTGTGGCCTACATAGCCTTCAAGAGGTGGTAGL4.mbIL12 MCHQQLVISWFSLVFLASPLVAIWELKKDVYVVELDWYPD 29 (CD80TM)- APGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEF 41BBICD - GDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEP LNGFR KNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDP QGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEES LPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRmbIL12- VFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCSG CD80TM-4- GGGGGSRNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKAR 1BBICD- T2A- QTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNS ALNGFR RETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKT MNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQK SSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNASLLPSWAITLISVNGIFVICCLKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRAKRSGSGEGRGSLLTCGDVEEN PGPMGAGATGRAMDGPRLLLLLLLGVSLGGAKEACPTG LYTHSGECCKACNLGEGVAQPCGANQTVCEPCLDSVTF SDVVSATEPCKPCTECVGLQSMSAPCVEADDAVCRCAY GYYQDETTGRCEACRVCEAGSGLVFSCQDKQNTVCEEC PDGTYSDEANHVDPCLPCTVCEDTERQLRECTRWADAE CEEIPGRWITRSTPPEGSDSTAPSTQEPEAPPEQDLIASTV AGVVTTVMGSSQPVVTRGTTDNLIPVYCSILAAVVVGLV AYIAFKRW*L4.mbIL12 ATGTGTCACCAGCAGTTGGTCATCTCTTGGTTTTCCCTGGT 30 (CD80TM)- TTTTCTGGCATCTCCCCTCGTGGCCATATGGGAACTGAAG 41BBICD - AAAGATGTTTATGTCGTAGAATTGGATTGGTATCCGGATG LNGFR CCCCTGGAGAAATGGTGGTCCTCACCTGTGACACCCCTGA AGAAGATGGTATCACCTGGACCTTGGACCAGAGCAGTGA nucleic acid GGTCTTAGGCTCTGGCAAAACCCTGACCATCCAAGTCAA (5’-> 3’) AGAGTTTGGAGATGCTGGCCAGTACACCTGTCACAAAGG AGGCGAGGTTCTAAGCCATTCGCTCCTGCTGCTTCACAAA mbIL12- AAGGAAGATGGAATTTGGTCCACTGATATTTTAAAGGAC CD80TM-4- CAGAAAGAACCCAAAAATAAGACCTTTCTAAGATGCGAG 1BBICD- T2A- GCCAAGAATTATTCTGGACGTTTCACCTGCTGGTGGCTGA ALNGFR CGACAATCAGTACTGATTTGACATTCAGTGTCAAAAGCA GCAGAGGCTCTTCTGACCCCCAAGGGGTGACGTGCGGAG CTGCTACACTCTCTGCAGAGAGAGTCAGAGGGGACAACA AGGAGTATGAGTACTCAGTGGAGTGCCAGGAGGACAGTG CCTGCCCAGCTGCTGAGGAGAGTCTGCCCATTGAGGTCAT GGTGGATGCCGTTCACAAGCTCAAGTATGAAAACTACAC CAGCAGCTTCTTCATCAGGGACATCATCAAACCTGACCCA CCCAAGAACTTGCAGCTGAAGCCATTAAAGAATTCTCGG CAGGTGGAGGTCAGCTGGGAGTACCCTGACACCTGGAGT ACTCCACATTCCTACTTCTCCCTGACATTCTGCGTTCAGGT CCAGGGCAAGAGCAAGAGAGAAAAGAAAGATAGAGTCT TCACGGACAAGACCTCAGCCACGGTCATCTGCCGCAAAA ATGCCAGCATTAGCGTGCGGGCCCAGGACCGCTACTATA GCTCATCTTGGAGCGAATGGGCATCTGTGCCCTGCAGTGG TGGCGGTGGCGGCGGATCTAGAAACCTCCCCGTGGCCAC TCCAGACCCAGGAATGTTCCCATGCCTTCACCACTCCCAA AACCTGCTGAGGGCCGTCAGCAACATGCTCCAGAAGGCC AGACAAACTCTAGAATTTTACCCTTGCACTTCTGAAGAGA TTGATCATGAAGATATCACAAAAGATAAAACCAGCACAG TGGAGGCCTGTTTACCATTGGAATTAACCAAGAATGAGA GTTGCCTAAATTCCAGAGAGACCTCTTTCATAACTAATGG GAGTTGCCTGGCCTCCAGAAAGACCTCTTTTATGATGGCC CTGTGCCTTAGTAGTATTTATGAAGACTTGAAGATGTACC AGGTGGAGTTCAAGACCATGAATGCAAAGCTTCTGATGG ATCCTAAGAGGCAGATCTTTCTAGATCAAAACATGCTGG CAGTTATTGATGAGCTGATGCAGGCCCTGAATTTCAACAG TGAGACTGTGCCACAAAAATCCTCCCTTGAAGAACCGGATTTTTATAAAACTAAAATCAAGCTCTGCATACTTCTTCATGCTTTCAGAATTCGGGCAGTGACTATTGATAGAGTGATG AGCTATCTGAATGCTTCCCTGCTCCCATCCTGGGCCATTA CCTTAATCTCAGTAAATGGAATTTTTGTGATATGCTGCCT GAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAA CAACCATTTATGAGACCAGTACAAACTACTCAAGAGG AAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGA AGGAGGATGTGAACTGCGGGCCA4GCGGPCCGGAPCCG GAGAGGGCAGAGGAAGTCTGCTAACATGCGGTGACGTCGAG GAGAATCCTGGCCCAATGGGGGCAGGTGCCACCGGCCG CGCCATGGACGGGCCGCGCCTGCTGCTGTTGCTGCTT CTGGGGGTGTCCCTTGGAGGTGCCAAGGAGGCATGCC CCACAGGCCTGTACACACACAGCGGTGAGTGCTGCAA AGCCTGCAACCTGGGCGAGGGTGTGGCCCAGCCTTGT GGAGCCAACCAGACCGTGTGTGAGCCCTGCCTGGACA GCGTGACGTTCTCCGACGTGGTGAGCGCGACCGAGCC GTGCAAGCCGTGCACCGAGTGCGTGGGGCTCCAGAG CATGTCGGCGCCGTGCGTGGAGGCCGACGACGCCGT GTGCCGCTGCGCCTACGGCTACTACCAGGATGAGACG ACTGGGCGCTGCGAGGCGTGCCGCGTGTGCGAGGCG GGCTCGGGCCTCGTGTTCTCCTGCCAGGACAAGCAGA ACACCGTGTGCGAGGAGTGCCCCGACGGCACGTATTC CGACGAGGCCAACCACGTGGACCCGTGCCTGCCCTGC ACCGTGTGCGAGGACACCGAGCGCCAGCTCCGCGAGT GCACACGCTGGGCCGACGCCGAGTGCGAGGAGATCC CTGGCCGTTGGATTACACGGTCCACACCCCCAGAGGG CTCGGACAGCACAGCCCCCAGCACCCAGGAGCCTGAG GCACCTCCAGAACAAGACCTCATAGCCAGCACGGTGG CAGGTGTGGTGACCACAGTGATGGGCAGCTCCCAGCC CGTGGTGACCCGAGGCACCACCGACAACCTCATCCCT GTCTATTGCTCCATCCTGGCTGCTGTGGTTGTGGGTC TTGTGGCCTACATAGCCTTCAAGAGGTGGTAGL5.mbIL12 MCHQQLVISWFSLVFLASPLVAIWELKKDVYVVELDWYPD 31 (CD28TM)- APGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEF CD28ICD - GDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEP LNGFR KNKTFLRCEAKNYSGRFTCWWLTT1STDLTFSVKSSRGSSDP QGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEES mbIL12- LPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKN CD28TM- SRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDR CD28ICD -T2A- VFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCSG ALNGFR GGGGGSRNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKAR QTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNS RETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKT MNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQK SSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNASFWVL VVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTP RRPGPTRKHYOPYAPPRDFAAYRSPAATP. SG. SGEGPG. S'LLTC’ GDVEEAPGPMGAGATGRAMDGPRLLLLLLLGVSLGGAK EACPTGLYTHSGECCKACNLGEGVAQPCGANQTVCEPC LDSVTFSDVVSATEPCKPCTECVGLQSMSAPCVEADDAV CRCAYGYYQDETTGRCEACRVCEAGSGLVFSCQDKQNT VCEECPDGTYSDEANHVDPCLPCTVCEDTERQLRECTR WADAECEEIPGRWITRSTPPEGSDSTAPSTQEPEAPPEQD LIASTVAGVVTTVMGSSQPVVTRGTTDNLIPVYCSILAAVVVGLVAYIAFKRW*L5.mbIL12 ATGTGTCACCAGCAGTTGGTCATCTCTTGGTTTTCCCTGGT 32 (CD28TM)- TTTTCTGGCATCTCCCCTCGTGGCCATATGGGAACTGAAG CD28ICD - AAAGATGTTTATGTCGTAGAATTGGATTGGTATCCGGATG LNGFR CCCCTGGAGAAATGGTGGTCCTCACCTGTGACACCCCTGA AGAAGATGGTATCACCTGGACCTTGGACCAGAGCAGTGA nucleic acid GGTCTTAGGCTCTGGCAAAACCCTGACCATCCAAGTCAA (5’— > 3’) AGAGTTTGGAGATGCTGGCCAGTACACCTGTCACAAAGG AGGCGAGGTTCTAAGCCATTCGCTCCTGCTGCTTCACAAA mbIL12- AAGGAAGATGGAATTTGGTCCACTGATATTTTAAAGGAC CD28TM- CAGAAAGAACCCAAAAATAAGACCTTTCTAAGATGCGAG CD28ICD-r2A- GCCAAGAATTATTCTGGACGTTTCACCTGCTGGTGGCTGA ALNGFR CGACAATCAGTACTGATTTGACATTCAGTGTCAAAAGCA GCAGAGGCTCTTCTGACCCCCAAGGGGTGACGTGCGGAG CTGCTACACTCTCTGCAGAGAGAGTCAGAGGGGACAACA AGGAGTATGAGTACTCAGTGGAGTGCCAGGAGGACAGTG CCTGCCCAGCTGCTGAGGAGAGTCTGCCCATTGAGGTCAT GGTGGATGCCGTTCACAAGCTCAAGTATGAAAACTACAC CAGCAGCTTCTTCATCAGGGACATCATCAAACCTGACCCA CCCAAGAACTTGCAGCTGAAGCCATTAAAGAATTCTCGG CAGGTGGAGGTCAGCTGGGAGTACCCTGACACCTGGAGT ACTCCACATTCCTACTTCTCCCTGACATTCTGCGTTCAGGT CCAGGGCAAGAGCAAGAGAGAAAAGAAAGATAGAGTCT TCACGGACAAGACCTCAGCCACGGTCATCTGCCGCAAAA ATGCCAGCATTAGCGTGCGGGCCCAGGACCGCTACTATA GCTCATCTTGGAGCGAATGGGCATCTGTGCCCTGCAGTGG TGGCGGTGGCGGCGGATCTAGAAACCTCCCCGTGGCCAC TCCAGACCCAGGAATGTTCCCATGCCTTCACCACTCCCAA AACCTGCTGAGGGCCGTCAGCAACATGCTCCAGAAGGCC AGACAAACTCTAGAATTTTACCCTTGCACTTCTGAAGAGA TTGATCATGAAGATATCACAAAAGATAAAACCAGCACAG TGGAGGCCTGTTTACCATTGGAATTAACCAAGAATGAGA GTTGCCTAAATTCCAGAGAGACCTCTTTCATAACTAATGG GAGTTGCCTGGCCTCCAGAAAGACCTCTTTTATGATGGCC CTGTGCCTTAGTAGTATTTATGAAGACTTGAAGATGTACC AGGTGGAGTTCAAGACCATGAATGCAAAGCTTCTGATGG ATCCTAAGAGGCAGATCTTTCTAGATCAAAACATGCTGG CAGTTATTGATGAGCTGATGCAGGCCCTGAATTTCAACAG TGAGACTGTGCCACAAAAATCCTCCCTTGAAGAACCGGA TTTTTATAAAACTAAAATCAAGCTCTGCATACTTCTTCAT GCTTTCAGAATTCGGGCAGTGACTATTGATAGAGTGATG AGCTATCTGAATGCTTCCTTTTGGGTGCTGGTGGTGGTTG GTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGC CTTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCT CCTGCACAGTGACTACATGAACATGACTCCCCGCCGC CCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCC CACCACGCGACTTCGCAGCCTATCGCTCCCGGGCCAAG CGGTCCGGA TCCGGA GA GGGCAGA GGAA GTCTGCTAA CA TG CGGTGACGTCGAGGAGAATCCTGGCCCAATGGGGGCAGG TGCCACCGGCCGCGCCATGGACGGGCCGCGCCTGCT GCTGTTGCTGCTTCTGGGGGTGTCCCTTGGAGGTGCC AAGGAGGCATGCCCCACAGGCCTGTACACACACAGCG GTGAGTGCTGCAAAGCCTGCAACCTGGGCGAGGGTGT GGCCCAGCCTTGTGGAGCCAACCAGACCGTGTGTGAG CCCTGCCTGGACAGCGTGACGTTCTCCGACGTGGTGAGCGCGACCGAGCCGTGCAAGCCGTGCACCGAGTGCGTGGGGCTCCAGAGCATGTCGGCGCCGTGCGTGGAGG CCGACGACGCCGTGTGCCGCTGCGCCTACGGCTACTA CCAGGATGAGACGACTGGGCGCTGCGAGGCGTGCCG CGTGTGCGAGGCGGGCTCGGGCCTCGTGTTCTCCTGC CAGGACAAGCAGAACACCGTGTGCGAGGAGTGCCCC GACGGCACGTATTCCGACGAGGCCAACCACGTGGACC CGTGCCTGCCCTGCACCGTGTGCGAGGACACCGAGCG CCAGCTCCGCGAGTGCACACGCTGGGCCGACGCCGA GTGCGAGGAGATCCCTGGCCGTTGGATTACACGGTCC ACACCCCCAGAGGGCTCGGACAGCACAGCCCCCAGCA CCCAGGAGCCTGAGGCACCTCCAGAACAAGACCTCAT AGCCAGCACGGTGGCAGGTGTGGTGACCACAGTGATG GGCAGCTCCCAGCCCGTGGTGACCCGAGGCACCACCG ACAACCTCATCCCTGTCTATTGCTCCATCCTGGCTGCT GTGGTTGTGGGTCTTGTGGCCTACATAGCCTTCAAGA GGTGG / 1GL6.mbIL12 MCHQQLVISWFSLVFLASPLVAIWELKKDVYVVELDWYPD 33 (CD28TM)- APGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEF 41BB-ICD - GDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEP LNGFR KNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDP QGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEES mbIL12- LPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKN CD28TM-4- SRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDR 1BBICD-T2A- VFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCSG ALNGFR GGGGGSRNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKAR QTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNS RETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKT MNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQK SSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNASFWVL VVVGGVLACYSLLVTVAFIIFWVKRGRKKLLYIFKOPFMR PVOTTOEEDGCSCRFPEEEEGGCELRAATRSGSGEGRGSLLr CGDVESVPGPMGAGATGRAMDGPRLLLLLLLGVSLGGA KEACPTGLYTHSGECCKACNLGEGVAQPCGANQTVCEP CLDSVTFSDVVSATEPCKPCTECVGLQSMSAPCVEADDA VCRCAYGYYQDETTGRCEACRVCEAGSGLVFSCQDKQN TVCEECPDGTYSDEANHVDPCLPCTVCEDTERQLRECTR WADAECEEIPGRWITRSTPPEGSDSTAPSTQEPEAPPEQD LIASTVAGVVTTVMGSSQPVVTRGTTDNLIPVYCSILAAV VVGLVAYIAFKRW*L6.mbIL12 ATGTGTCACCAGCAGTTGGTCATCTCTTGGTTTTCCCTGGT 34 (CD28TM)- TTTTCTGGCATCTCCCCTCGTGGCCATATGGGAACTGAAG 41BB4CD - AAAGATGTTTATGTCGTAGAATTGGATTGGTATCCGGATG LNGFR CCCCTGGAGAAATGGTGGTCCTCACCTGTGACACCCCTGA AGAAGATGGTATCACCTGGACCTTGGACCAGAGCAGTGA nucleic acid GGTCTTAGGCTCTGGCAAAACCCTGACCATCCAAGTCAA (5’— > 3’) AGAGTTTGGAGATGCTGGCCAGTACACCTGTCACAAAGG AGGCGAGGTTCTAAGCCATTCGCTCCTGCTGCTTCACAAA mbIL12- AAGGAAGATGGAATTTGGTCCACTGATATTTTAAAGGAC CD28TM-4- CAGAAAGAACCCAAAAATAAGACCTTTCTAAGATGCGAG 1BBICD-T2A- GCCAAGAATTATTCTGGACGTTTCACCTGCTGGTGGCTGA ALNGFR CGACAATCAGTACTGATTTGACATTCAGTGTCAAAAGCA GCAGAGGCTCTTCTGACCCCCAAGGGGTGACGTGCGGAG CTGCTACACTCTCTGCAGAGAGAGTCAGAGGGGACAACA AGGAGTATGAGTACTCAGTGGAGTGCCAGGAGGACAGTGCCTGCCCAGCTGCTGAGGAGAGTCTGCCCATTGAGGTCATGGTGGATGCCGTTCACAAGCTCAAGTATGAAAACTACAC CAGCAGCTTCTTCATCAGGGACATCATCAAACCTGACCCA CCCAAGAACTTGCAGCTGAAGCCATTAAAGAATTCTCGG CAGGTGGAGGTCAGCTGGGAGTACCCTGACACCTGGAGT ACTCCACATTCCTACTTCTCCCTGACATTCTGCGTTCAGGT CCAGGGCAAGAGCAAGAGAGAAAAGAAAGATAGAGTCT TCACGGACAAGACCTCAGCCACGGTCATCTGCCGCAAAA ATGCCAGCATTAGCGTGCGGGCCCAGGACCGCTACTATA GCTCATCTTGGAGCGAATGGGCATCTGTGCCCTGCAGTGG TGGCGGTGGCGGCGGATCTAGAAACCTCCCCGTGGCCAC TCCAGACCCAGGAATGTTCCCATGCCTTCACCACTCCCAA AACCTGCTGAGGGCCGTCAGCAACATGCTCCAGAAGGCC AGACAAACTCTAGAATTTTACCCTTGCACTTCTGAAGAGA TTGATCATGAAGATATCACAAAAGATAAAACCAGCACAG TGGAGGCCTGTTTACCATTGGAATTAACCAAGAATGAGA GTTGCCTAAATTCCAGAGAGACCTCTTTCATAACTAATGG GAGTTGCCTGGCCTCCAGAAAGACCTCTTTTATGATGGCC CTGTGCCTTAGTAGTATTTATGAAGACTTGAAGATGTACC AGGTGGAGTTCAAGACCATGAATGCAAAGCTTCTGATGG ATCCTAAGAGGCAGATCTTTCTAGATCAAAACATGCTGG CAGTTATTGATGAGCTGATGCAGGCCCTGAATTTCAACAG TGAGACTGTGCCACAAAAATCCTCCCTTGAAGAACCGGA TTTTTATAAAACTAAAATCAAGCTCTGCATACTTCTTCAT GCTTTCAGAATTCGGGCAGTGACTATTGATAGAGTGATG AGCTATCTGAATGCTTCCTTTTGGGTGCTGGTGGTGGTTG GTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGC CTTTATTATTTTCTGGGTGAAACGGGGCAGAAAGAAACT CCTGTATATATTCAAACAACCATTTATGAGACCAGTAC AAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATT TCCAGAAGAAGAAGAAGGAGGATGTGAACTGCGGGCC AAGCGGTCCGGATCCGGAGAGGGCAGAGGAAGTCTGCTAAC ATGCGGTGACGTCGAGGAGAATCCTGGCCCAATGGGGGCA GGTGCCACCGGCCGCGCCATGGACGGGCCGCGCCTG CTGCTGTTGCTGCTTCTGGGGGTGTCCCTTGGAGGTG CCAAGGAGGCATGCCCCACAGGCCTGTACACACACAG CGGTGAGTGCTGCAAAGCCTGCAACCTGGGCGAGGGT GTGGCCCAGCCTTGTGGAGCCAACCAGACCGTGTGTG AGCCCTGCCTGGACAGCGTGACGTTCTCCGACGTGGT GAGCGCGACCGAGCCGTGCAAGCCGTGCACCGAGTG CGTGGGGCTCCAGAGCATGTCGGCGCCGTGCGTGGA GGCCGACGACGCCGTGTGCCGCTGCGCCTACGGCTAC TACCAGGATGAGACGACTGGGCGCTGCGAGGCGTGC CGCGTGTGCGAGGCGGGCTCGGGCCTCGTGTTCTCCT GCCAGGACAAGCAGAACACCGTGTGCGAGGAGTGCC CCGACGGCACGTATTCCGACGAGGCCAACCACGTGGA CCCGTGCCTGCCCTGCACCGTGTGCGAGGACACCGAG CGCCAGCTCCGCGAGTGCACACGCTGGGCCGACGCC GAGTGCGAGGAGATCCCTGGCCGTTGGATTACACGGT CCACACCCCCAGAGGGCTCGGACAGCACAGCCCCCAG CACCCAGGAGCCTGAGGCACCTCCAGAACAAGACCTC ATAGCCAGCACGGTGGCAGGTGTGGTGACCACAGTGA TGGGCAGCTCCCAGCCCGTGGTGACCCGAGGCACCAC CGACAACCTCATCCCTGTCTATTGCTCCATCCTGGCTG CTGTGGTTGTGGGTCTTGTGGCCTACATAGCCTTCAAGAGGTGG7AGEfl a promoter AGCTTTGCAAAGATGGATAAAGTTTTAAACAGAGAGGAA 15 (1264bp) TCTTTGCAGCTAATGGACCTTCTAGGTCTTGAAAGGAGTG GGAATTGGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACA nucleic acid TCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGC sequence AATTGAACCGGTGCCTAGAGAAGGTGGCGCGGGGTAAAC (5’— >3’) TGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCG AGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGCCG TGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACA GGTAAGTGCCGTGTGTGGTTCCCGCGGGCCTGGCCTCTTT ACGGGTTATGGCCCTTGCGTGCCTTGAATTACTTCCACCT GGCTGCAGTACGTGATTCTTGATCCCGAGCTTCGGGTTGG AAGTGGGTGGGAGAGTTCGAGGCCTTGCGCTTAAGGAGC CCCTTCGCCTCGTGCTTGAGTTGAGGCCTGGCCTGGGCGC TGGGGCCGCCGCGTGCGAATCTGGTGGCACCTTCGCGCCT GTCTCGCTGCTTTCGATAAGTCTCTAGCCATTTAAAATTTT TGATGACCTGCTGCGACGCTTTTTTTCTGGCAAGATAGTC TTGTAAATGCGGGCCAAGATCTGCACACTGGTATTTCGGT TTTTGGGGCCGCGGGCGGCGACGGGGCCCGTGCGTCCCA GCGCACATGTTCGGCGAGGCGGGGCCTGCGAGCGCGGCC ACCGAGAATCGGACGGGGGTAGTCTCAAGCTGGCCGGCC TGCTCTGGTGCCTGGCCTCGCGCCGCCGTGTATCGCCCCG CCCTGGGCGGCAAGGCTGGCCCGGTCGGCACCAGTTGCG TGAGCGGAAAGATGGCCGCTTCCCGGCCCTGCTGCAGGG AGCTCAAAATGGAGGACGCGGCGCTCGGGAGAGCGGGC GGGTGAGTCACCCACACAAAGGAAAAGGGCCTTTCCGTC CTCAGCCGTCGCTTCATGTGACTCCACGGAGTACCGGGCG CCGTCCAGGCACCTCGATTAGTTCTCGAGCTTTTGGAGTA CGTCGTCTTTAGGTTGGGGGGAGGGGTTTTATGCGATGGA GTTTCCCCACACTGAGTGGGTGGAGACTGAAGTTAGGCC AGCTTGGCACTTGATGTAATTCTCCTTGGAATTTGCCCTTT TTGAGTTTGGATCTTGGTTCATTCTCAAGCCTCAGACAGT GGTTCAAAGTTTTTTTCTTCCATTTCAGGTGTCGTGA WPRE (610bp) AATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTG 16GTATTCTTAACTATGTTGCTCCTTTTACGCTATGTGGATAC nucleic acid GCTGcrTrAArGCC'rrrG'rATCArGcrAn'GC'rTCCCGTAr sequence GGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTGCTGT (5’— >3’) CTCTTTATGAGGAGTTGTGGCCCGTTGTCAGGCAACGTGG CGTGGTGTGCACTGTGTTTGCTGACGCAACCCCCACTGGT TGGGGCATTGCCACCACCTGTCAGCTCCTTTCCGGGACTT TCGCTTTCCCCCTCCCTATTGCCACGGCGGAACTCATCGC CGCCTGCCTTGCCCGCTGCTGGACAGGGGCTCGGCTGTTG GGCACTGACAATTCCGTGGTGTTGTCGGGGAAGCTGACG TCCTTTCCATGGCTGCTCGCCTGTGTTGCCACCTGGATTCT GCGCGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTCAAT CCAGCGGACCTTCCTTCCCGCGGCCTGCTGCCGGCTCTGC GGCCTCTTCCGCGTCTTCGCCTTCGCCCTCAGACGAGTCG GATCTCCCTTTGGGCCGCCTCCCCGCCTGGAATTCGAGCT CGGTACC ALNGFR MGAGATGRAMDGPRLLLLLLLGVSLGGAKEACPTGLYTHS 21GECCKACNLGEGVAQPCGANQTVCEPCLDSVTFSDVVSAT EPCKPCTECVGLQSMSAPCVEADDAVCRCAYGYYQDETTG RCEACRVCEAGSGLVFSCQDKQNTVCEECPDGTYSDEANH VDPCLPCTVCEDTERQLRECTRWADAECEEIPGRWITRSTPP EGSDSTAPSTQEPEAPPEQDLIASTVAGVVTTVMGSSQPVVTRGTTDNLIPVYCSILAAVVVGLVAYIAFKR*ALNGFR ATGGGGGCAGGTGCCACCGGCCGCGCCATGGACGGGCCG 22 CGCCTGCTGCTGTTGCTGCTTCTGGGGGTGTCCCTTGGAGNucleic acid GTGCCAAGGAGGCATGCCCCACAGGCCTGTACACACACA sequence (5’— > GCGGTGAGTGCTGCAAAGCCTGCAACCTGGGCGAGGGTG3’) TGGCCCAGCCTTGTGGAGCCAACCAGACCGTGTGTGAGC CCTGCCTGGACAGCGTGACGTTCTCCGACGTGGTGAGCG CGACCGAGCCGTGCAAGCCGTGCACCGAGTGCGTGGGGC TCCAGAGCATGTCGGCGCCGTGCGTGGAGGCCGACGACG CCGTGTGCCGCTGCGCCTACGGCTACTACCAGGATGAGA CGACTGGGCGCTGCGAGGCGTGCCGCGTGTGCGAGGCGG GCTCGGGCCTCGTGTTCTCCTGCCAGGACAAGCAGAACA CCGTGTGCGAGGAGTGCCCCGACGGCACGTATTCCGACG AGGCCAACCACGTGGACCCGTGCCTGCCCTGCACCGTGT GCGAGGACACCGAGCGCCAGCTCCGCGAGTGCACACGCT GGGCCGACGCCGAGTGCGAGGAGATCCCTGGCCGTTGGA TTACACGGTCCACACCCCCAGAGGGCTCGGACAGCACAG CCCCCAGCACCCAGGAGCCTGAGGCACCTCCAGAACAAG ACCTCATAGCCAGCACGGTGGCAGGTGTGGTGACCACAG TGATGGGCAGCTCCCAGCCCGTGGTGACCCGAGGCACCA CCGACAACCTCATCCCTGTCTATTGCTCCATCCTGGCTGC TGTGGTTGTGGGTCTTGTGGCCTACATAGCCTTCAAGAGG TGAaAmino acid and / or nucleic acid residues that are bold, italic, and underlined at the start and stop of the sequences indicate start or stop codons. An asterisk (*) indicates a stop codon.

[0319] Lentivirus production

[0320] Lenti-X 293T cells were transiently transfected with a lentiviral backbone construct encoding the NY ESO specific TCR (1G4 TCR) or IL-12 fusion proteins and three packaging plasmids (pMD2. G (#12259; Addgene), pMDLg / pRRE (#12251; Addgene), and pRSV Rev (#12253: Addgene) using OPTI MEM and lipof ectamine 2000 (Thermofisher). The mixture was incubated for 15 min at room temperature and dropwise into Lenti-X cells. After 42h of incubation at 37°C, virus supernatant was collected, centrifuged and filtered to eliminate remaining cells. Virus supernatant was used for further transduction into human primary T cells.

[0321] T cell activation and co-transduction

[0322] As shown in FIG. 2B, whole peripheral blood from healthy donors was obtained from ASAN Medical Hospital (Seoul, South Korea: isolated human T cells are subsequently denoted as “hT-xxx” based on the patient sample numbers). Peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation using Lymphoprep and SepMate PBMC Isolation Tubes (Stemcell Technologies), and T cells were subsequently isolated using a Pan-T cell isolation kit (Miltenyi Biotec). T cells were activated using Dynabeads CD3 / CD28 activation beads (Thermofisher) at a 1:1 bead: T cell ratio and cultured for 24h in a 37°C incubator with 5% CO2. 24h after T cell activation, the firsttransduction was performed using a NY-ESO specific TCR (1G4 TCR) lentivirus supernatant or T cell media in the case of Non-Transduced T cells (NTD) for negative control. For transduction, 1 M T cells were combined with 2mL of virus and protamine sulfate in presence of 300IU / mL IL-2 and spinfected at 1000 RCF for Ih and thirty minutes. 24h after first transduction, second transduction using the IL- 12 fusion proteins lentivirus supernatant was performed same as described before. 24h after second transduction, dynabeads were removed and T cells were subcultured every two days. Transduction efficiency was checked five days after second transduction. Seven days after second transduction, T cells were sorted by dLNGFR using CD271 Microbeads (Milteny Biotec) and subcultured for two days more. T cells were stocked using Bambanker solution (Nippon Genetics) and stored at -80°C for further analysis. The T cell media consists on RPMI-1640 supplemented with 10% FBS, 1% penincilin / streptadivin, 55uM 2-mercaptoethanol, 1% Glutamax, 1% Sodium pymvate, 1% MEM Non-essential amino acid solution and 1%HEPES. T cells were cultured at a concentration of IM cells / mL and supplemented with 300units / mL interleukin 2 (IL- 2).

[0323] Flow cytometry

[0324] 1G4 TCR expression was measured using VP13.1 antibody conjugated with APC or FITC fluorochrome (Biolegend). Membrane bound IL-12 was measured by cell surface staining using an antihuman IL- 12 (p40 / p70) antibody (BD Biosciences). dLNGFR was measured using an anti -human CD271 antibody conjugated with APC or FITC (Milteny Biotec). All flow cytometry data were acquired with a BD LSRFortessa X-20 Cell Analyzer (BD Biosciences) and analyzed using the FlowJo software (BD Biosciences).

[0325] Transfection efficiency

[0326] All the constructs in T cells (hT-222, hT-253 or hT-254, where “hT” represents human T cells) isolated from patient blood donated are expressed at high transfection efficiency as measured by anti-LNGFR antibody.

[0327] Transduction efficiency

[0328] Transduction efficiency assesses the expression levels of fusion proteins on the surface of human primary T cells (hT-253). Constructs co-expressing a IL-12 fusion protein containing the CD80TM (L2: 1G4+ mbIL12-CD80TM and L4: IG4+ mbIL12-CD80TM-41BB) show low expression (4-10%) of the fusion proteins-data from hT-253 and hT-254 donors- compared to those that expressed a CD28TM (L3:lG4+mbIL12-CD28TM, L5: 1G4+ mbIL12-CD28TM-CD28 and L6: lG4+mbIL12-CD28TM-41BB) (17-23%). These resultssuggest that the choice of transmembrane domains are relevant and in the context of membrane-bound IL- 12 fusion proteins, being CD28TM a better choice than CD80TM.

[0329] Sorting efficiency

[0330] For hT-253 and hT-222 (in vitro) and hT-254 in vivo) donors, TCR-T cells expressing the IL- 12 based fusion proteins were sorted by the LNGFR tag using CD271 microbeads. After sorting, the sorting efficiencies of constructs containing a CD28TM (L3:lG4+mbIL12-CD28TM, L5: 1G4+ mbIL12-CD28TM-CD28 and L6: lG4+mbIL12-CD28TM-41BB) was over 80%, with the sorting efficiencies of L3 and L6 being over 90%. Meanwhile, the sorting efficiencies of the CD80-based fusion proteins (L2:1G4+ mbIL12-CD80TM and L4: 1G4+ mbIL12-CD80TM-41BB) was much lower (15-40%).Example 2. In Vitro Cytotoxicity

[0331] 5 x 103A375-Zsgreen target cells or 5 x 103MDAMB231-Zs cells were seeded per well one day before coculture in a 96-well flat-bottom plate in lOOpl of media. 1G4 TCR-T cells alone or co-transduced constructs co-expressing a IL- 12 fusion protein were cocultured with target cells at different effector-to-target (E: T) ratios from 0.1:1 to 1: 1 in 200ul of media. Triplicates were plated for group. The GFP fluorescence intensity of target cells was measured every 2 hours using the Incucyte S3 Live-Cell Analysis System. Integrated total GFP intensity per well was used as a quantitative measure of the amount of viable target cells.

[0332] For the A375-Zsgreen target cells, as can be seen from FIGs. 3A-3D, at an E: T ratio of 1:1, both 1G4WT alone or with fusion proteins is able to kill all cancer cells (FIG. 3A), while at low E: T ratios, in general, all groups show significantly higher cytotoxicity compared to 1G4WT (FIGs. 3B-3D).

[0333] For the MDAMB231-Zs cells, NY-ESO1 peptide (SLLMWITQV; SEQ ID NO: 35) was added and incubated for 4 hours and the cell killing effects were evaluated at an E: T ratio of 1:1. Cell killing was observed in the presence of the antigen NY-ESO1 peptide (FIGs. 4A-4C), and there was no non-specific killing in the absence of the antigen NY-ESO1 peptide (FIG.4D), indicating that the presence of mbIL12 alone or with costimulatory receptors does not induce non-specific killing of cells.

[0334] The T cell proliferation was tested before and after thawing. Homeostatic proliferation of the fusion protein-expressing T cells hT-222 showed similar trend before freezing (FIGs.5A-5B), and showed different viability (FIG. 5C).

[0335] The T cell homeostatic proliferation between different T cells transduced with the constructs before freezing was also tested (FIGs. 6A-6C).Example 3. In vitro Stress Test and T-cell Long-Term Proliferation Assay

[0336] Cytotoxicity in the stress test

[0337] The cell killing effect of the T cells can also be evaluated under stress conditions, such as by repetitively addition of target cells (FIG. 7). 5 x 103A375-Zsgreen target cells were seeded in 100 pl medium per well on Day-1 before coculture with lOOpl T cells in a 96-well flat-bottom plate Day 0. 50 pl of 1G4 TCR-T cells (hT-222) alone or in combination with a IL- 12 fusion protein were cocultured with target cells at different effector- to-target (E: T) ratios from 0.1:1 to 0.5:1 in a total of lOOpl of media. Every two days, 1 x 104A375-Zsgreen cells were added to the previous coculture in a 30pl of volume. A total of 5 stimulations for a period of 11 days was performed. Triplicates were plated for group. The GFP fluorescence intensity of target cells was measured every 2 hours using the Incucyte S3 Live-Cell Analysis System. Integrated total GFP intensity per well was used as a quantitative measure of the amount of viable target cells (Method 1).

[0338] Using Method 1, there are no significant differences in the 0.5:1 E: T ratio group (initial ratio) between different groups incorporating a mbIL12 alone or mbIL12+ costimulatory receptor (FIG. 8C). In the 0.25:1 E: T ratio group, L4 kills significantly better as compared to L3 after 5threp. stimulation (FIG.8B). In the 0.1:1 ratio group, fusion proteins incorporating a costimulatory receptor kills significantly better than mbIL12 alone after 5thstimulation (FIG. 8A).

[0339] In a further stress test using Method 1, the T cells (hT-253) were stimulated for 5 times by target cells every 3 days during 15 days at different E: T ratios of 0.1:1, 0.25:1 and 0.5:1. As shown in FIGs. 9A-9C, 1G4 T cells transduced with L6 showed much higher cytotoxicity than L4 in all the E: T ratios.

[0340] An alternation can be performed following the same protocol on Day - 1 and Day 0 to obtain a cell coculture as in Method 1. On Day +1, another 5xl03 / well A375Z cells were seeded in a new plate in lOOpl of medium and were added with lOOpl of the previous cellcoculture (hT-222) on Day +2 at the indicated E: T ratio. Every two days (e.g. Day 4 and 6), 100 pl of medium from the preceding culture (mainly T cells, e.g. hT-222) was collected and added to a new plate containing IxlO4A375 target cells in lOOpl (seeded the day before). IL2 is added when T cells are added for coculture at a concentration of 300IU / mL. A total of 4 stimulations for a period of 8 days was performed (Method 2, FIG. 7).

[0341] In this experiment (FIGs. 10A-10C), after the third restimulation, 1G4WT cannot control the tumor growth and its effect is similar to NTD and no T cells. L2, L3 and L5 are able to control the tumor until the third restimulation but loss of control is observed after the fourth restimulation. In presence of IL2, no significant differences was observed between different groups either with mbIL12 alone or with a costimulatory receptor.

[0342] In Method 2, if lOO l of T cells from a previous coculture are transferred to the new plate, the number of T cells from different groups might be different due to the different proliferation. It might be possible that in L4 group, the number of T cells present are higher and induce the cytotoxicity faster, showing a significance after 4 stimulations.

[0343] T-cell long-term proliferation assay

[0344] A375 cancer cells were used as targets for repetitive stimulation assays. One day before the coculture, 2 x 105A375 target cells were plated per well in ImL of media in a 24-well plate. The next day, these target cells were treated with mitomycin C (Sigma Aldrich) at a concentration of lOpg / mL to obtain a layer of feeder cells. After incubation at 37°C for 3h, A375 cells were washed with DPBS three times. For co-culture, 1 x 1061G4 TCR-T cells alone or cotransduced constructs co-expressing a IL- 12 fusion protein were added into mitomycin C treated A375 cells (E: T ratio at 2: 1) in the presence of 50IU / mL IL-2. Four days later, T cells were counted using a Countess II Automated Cell Counter (Thermo Fisher Scientific) and re-challenged with fresh mitomycin C-pretreated A375 cells. A total of 3 stimulations at an interval of 4 days were performed for a total period of 12 days (FIG. 11A), or a total of 4 stimulations at an interval of 4 days were performed for a total period of 16 days (FIG. 12A).

[0345] On day 12, the T cell immunophenotype including memory T cell markers (CD45RA+ and CD62L+ or CCR7 / CD45RO) and exhaustion markers (TIM3+, PD-1+, LAG3+) were measured. As shown in FIG. 11B, after 3 stimulations, T cells (hT-222) with L4 and L5 having the costimulatory domain showed significantly higher cell growth.

[0346] As shown in FIG. 12B, T cells (hT-253) showed a mild cell growth after 4 stimulations.

[0347] The T cell division was measured by cell trace violet, in which the 1G4 TCR combined with membrane- bound IL 12 fusion proteins proliferate much better as compared to 1G4 TCR alone (FIGs. 13A-13B).

[0348] CD4 / CD8 proportion change in T cells

[0349] The CD4 / CD8 proportion of T cells (hT-222) before and after the repetitive third stimulation of the T-cell long-term proliferation assay shown in FIG. 11 were also measured. The CD4 / CD8 proportion is 50 / 50% before the stimulation (FIG. 14A). However, after the third stimulation, all groups containing mbIL12 fusion proteins reprogram all T cells to CD8 phenotype (FIG. 14B).Example 4. In Vivo Anti-Tumor Effect (In Vivo Stress Test) with TCR T cells

[0350] In vivo xenograft mouse model

[0351] Tumor was induced in 6-8 weeks old female NOD. Cg-PrkdcscldIL2rgtm1Sug / ShiJic (NOG) mice by 1 x 106A375-luciferase-Zsgreen target cells, which were injected subcutaneously in the flank of the mouse in presence of Matrigel (Corning). After 6 days, tumor randomization was conducted and the next day 5 x 105T cells transduced with 1G4 TCR-T cells alone or in combination with IL- 12 fusion proteins were infused intravenously. Tumor growth was monitored twice a week using a caliper digital system and weekly using an In vivo Imaging System (IVIS) (FIG. 15).

[0352] FIG. 16 shows in vivo imaging of tumor sizes over the course of 28 days in mice infused with untransduced T cells, IG4 TCR T cells, and IG4 TCR T cells in combination with a membrane cytokine fusion protein L3 (IL-12-CD28TM) or L6 (IL-12-CD28TM-41BB ICD; “hyperkine”). FIG. 16 shows a clear reduction in tumor size in mice in the L6 group over the course of the study.

[0353] As shown in FIGs. 17A-17B, in the condition of an E: T ratio of 0.5:1 (in vivo stress conditions), the L6 group (lG4+mbIL12-CD28TM-41BB) was able to eliminate all the tumor after 21 days of tumor induction compared to the other constructs (only 1G4 TCR or lG4TCR+mbIL-12). The L4 and L5 groups (which also incorporates the 3 signals for T cell activation; L5 (in which a CD28 ICD is incorporated in the fusion protein) and L4 (in whichthe CD80TM is present instead))- also showed significantly better tumor weight reduction compared to 1G4 WT but to a lesser extent than L6 (FIG. 17C).

[0354] Additionally, when TCR-T cells were assessed in circulation on day 21, only the L6 group had a considerable amount of T cells in circulation, suggesting persistence of these TCR-T cells in the long term (FIG. 17D).

[0355] Some mice died due to graft- versus-host disease (GvHD) as it is a humanized mouse model.Example 5. In Vivo Antitumor Efficacy of NY-ESO-1 Specific TCR T Cells Expressing Cytokine Fusion Proteins

[0356] An in vivo study was conducted to evaluate the antitumor efficacy of NY-ESO-1 -specific TCR-T cells engineered to express a cytokine fusion protein (L6) in a NY-ESO-1-positive melanoma tumor model.

[0357] Human peripheral blood-derived T lymphocytes were isolated and transduced with lentiviral vectors encoding various gene constructs to establish four experimental CAR-T cell groups, as follows:Group 1 (Gl, negative control): Untransduced T cells lacking NY-ESO-1 -specific TCR; Group 2 (G2, No-armored TCR-T): TCR-T cells expressing a NY-ESO-1 -specific TCR (LI, Table 2);Group 3 (G3, mbIL-12 armored TCR-T): TCR-T cells co-expressing a membrane-bound-IL-12 (mbIL-12) along with the NY-ESO-1 -specific TCR (LI, Table 2);Group 4 (G4, Hyperkine #1 (L4, Table 2) armored TCR-T): TCR-T cells co-expressing Hyperkine, comprising mbIL-12 and an additional costimulatory domain (CD80 transmembrane-41BB intracellular domain), along with the NY-ESO-l-specific TCR (LI, Table 2): andGroup 5 (G5, Hyperkine #2 (L6, Table 2) armored TCR-T): TCR-T cells co-expressing Hyperkine, comprising mbIL-12 and an additional costimulatory domain (CD28 transmembrane-4- IBB intracellular domain), along with the NY-ESO-l-specific TCR (LI, Table 2).

[0358] To establish the NY-ESO-1 -positive melanoma model, 5-week-old NOG mice (NOD.Cg-PrkdcscidIL2γgtm1Sug / JicKoat) were subcutaneously injected with IxlO6A375melanoma cells engineered to express EGFP and firefly luciferase (A375-GFP-Fluc). On day 6 after tumor implantation, tumor engraftment was confirmed by two complementary methods: (i) bioluminescent imaging using IVIS® Spectrum system and (ii) caliper measurement of tumor dimensions (see FIG. 18). Mice with detectable bioluminescent signals and measurable tumors within a comparable size range were randomized into experimental groups.

[0359] On day 7 after tumor implantation, cultured NY-ESO-1 -specific TCR-T cells were harvested and prepared for infusion. The cells were centrifuged at 600 ×g for 3 minutes, the supernatant was removed, and the cells were resuspended in fresh culture medium.Following cell counting, the suspension was centrifuged again, washed with Dulbecco's phosphate-buffered saline (D-PBS), and adjusted to a concentration of 0.5xl06NY-ESO-1 TCR-positive T cells per 200 pL. Each mouse received an intravenous injection of 200 pL of the prepared cell suspension.

[0360] After TCR-T cell administration, tumor burden was assessed weekly using two complementary methods: (i) bioluminescent imaging using IVIS® Spectrum system, in which photon flux (photons / sec) from tumor cells was quantified within a defined region of interest (ROI) using Live Image® software, and (ii) caliper measurement of tumor dimensions, with tumor volume calculated according to the formula (length x width2) / 2.

[0361] To evaluate in vivo persistence of the infused TCR-T cells, spleens were harvested on day 28 after TCR-T cell administration. Spleens were processed into single-cell suspensions, and red blood cells were lysed and removed. The splenocytes were stained with antibodies against human CD3 (hCD3), and flow cytometry was performed to determine the percentage of hCD3+cells. Persistence of TCR-T cells was calculated based on the total splenocyte count and the proportion of hCD3+cells.

[0362] Referring to FIGs. 19-20, in the untransduced T cell group (Gl) and no-armored TCR-T group (G2), tumor progression was evident, as demonstrated by continuous increase in bioluminescent photon flux and tumor volume measurements over time. The mbIL-12-armored TCR-T group (G3) exhibited partial tumor control, with reduced but still detectable tumor burden relative to Gl and G2. By contrast, both Hyperkine- armored TCR-T group (G4 and G5) demonstrated marked antitumor activity. In particular, the G5 group (Hyperkine #2 (L6)), incorporating CD28 transmembrane-4- IBB intracellular domain) demonstrated the strongest and most sustained tumor suppression, with significant reductions in bothbioluminescent signal intensity and tumor volume compared to all other groups (*p<0.05, **p<0.01, ***p<0.001).

[0363] Evaluation of in vivo persistence at day 28 of groups G1-G3 showed only minimal numbers of hCD3+T cells, with average counts of 109±128 (G1), 5±3 (G2), 6±5 (G3), and 7±4 (G4), respectively. By contrast, the Hyperkine #2 (L6) TCR-T group (G5) exhibited an average of 28,887±24,501 hCD3+T cells, representing more than a 288-fold increase in persistence compared to the other groups (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001) (see FIG.21).

[0364] These findings demonstrate that application of the cytokine fusion protein L6 comprising the mbIL-12 - CD28 transmembrane - 4-1BB intracellular domain structure markedly enhanced in vivo persistence and resulted in durable tumor regression. The data confirms that this configuration represents an optimal design for promoting long-term survival and expansion of TCR-T cells, thereby providing a useful armoring strategy to improve the therapeutic efficacy on next-generation T cell therapies.Example 6. In Vivo Anti-Tumor Effect and Expansion of CAR T cells with a Coexpressed Cytokine Fusion Protein

[0365] The anti-tumor effect of CAR T cells alone or in combination with a co-expressed IL-12 fusion protein was investigated in an in vivo xenograft mouse model using a method similar to that described in Example 4. Tumor was induced in 6-8 weeks old female NOD.Cg-PrkdcscidIL2rgtm1Sug / ShiJic (NOG) mice by 1 x 106A375-luciferase-Zsgreen target cells, which were injected subcutaneously in the flank of the mouse in presence of Matrigel (Corning). Tumor randomization was conducted and the next day untransduced αβ T Cells, high dose (4xl06) CAR T cells alone (unarmored) or co-expressing the L6 cytokine fusion protein (IL-12-CD28TM-41BB ICD; “hyperkine”; see Table 2), and low dose (2xl06) CAR T cells alone (unarmored) or co-expressing the L6 cytokine fusion protein (IL-12-CD28TM-41 BB ICD; “hyperkine”) were infused intravenously. Tumor growth was monitored using a caliper digital system and using an In vivo Imaging System (IVIS) over the course of 30 days before and after CAR T cell treatment.

[0366] FIG.22 shows in vivo imaging of tumor sizes in mice infused with untransduced ap T cells, a high dose (4x106) of Claudinl8.2-targeting CAR T cells alone (unarmored) or coexpressing the L6 cytokine fusion protein (IL-12-CD28TM-41BB ICD; “hyperkine”), and a low dose (2xl06) of Claudinl8.2-targeting CAR T cells alone (unarmored) or co-expressingthe L6 cytokine fusion protein (IL-12-CD28TM-41BB ICD; “hyperkine”). The Claudinl8.2-targeting CAR sequence is provided in Table 3. As can be seen, co-expression of the L6 fusion protein clearly improved the anti-tumor effects of the CART T cells.

[0367] FIGs. 23A-23B show the anti-tumor effects (as measured by photons / second) as a function of day after CAR T cell infusion in the different treatment groups (where mock = untransduced αβ T cells) at both the high and low doses of CAR T cells. At both doses, the CAR T cells co-expressing L6 (hyperkine) demonstrated enhanced anti-tumor effects in the mice compared with the unarmored CAR T cells.

[0368] FIGs. 24A-24B show the number of CAR T cells in spleen (FIG. 24A) and in blood (FIG. 24B) in the different treatment groups after CAR T cell infusion. The number of CAR T cells in the L6 (hyperkine) group was more than two orders of magnitude higher than the unarmored and mock treatment groups in the spleen measurements (FIG. 24A). In blood, the L6 (hyperkine) treatment group showed high levels of CAR T cells in circulation, and was the only treatment group showing any detectable levels of CAR T cells (FIG. 24B). These results demonstrate that the co-expressed cytokine fusion protein promotes T cell expansion and persistence in vivo.Example 7. In vitro Cytotoxicity of CAR T Cells Expressing Cytokine Fusion Proteins

[0369] An in vitro repeated stimulation killing assay was performed to evaluate the cytotoxicity of CAR-T cells under sustained stress conditions. The assay was designed to stimulate continuous antigenic pressure by periodically adding fresh target cells over multiple rounds of stimulation. Three tumor cell lines expressing target antigen PSMA, Claudinl8.2, or GPC3 were employed. The target cells were engineered to stably express a fluorescent reporter (Zsgreen or GFP) to enable real-time viability assessment. Four parallel CAR-T cell treatment groups were tested for each target cell model:Group 1 (Gl, negative control): Target cell only;Group 2 (G2, negative control): T cells with no CAR (Untransduced T cells);Group 3 (G3, No-armored CAR-T): CAR-T cells expressing a tumor antigen-specific CAR targeting PSMA, Claudinl8.2, or GPC3 (Table 3);Group 4 (G4, mbIL-12 armored CAR-T): CAR-T cells co-expressing a mbIL-12 (L3, Table 2) along with a CAR targeting PSMA, Claudin, or GPC3 (Table 3); andGroup 5 (G5, Hyperkine armored CAR-T): CAR-T cells co-expressing Hyperkine (L6, Table 2), comprising mbIL-12 and an additional costimulatory domain, along with a CAR targeting PSMA, Claudin, or GPC3 (Table 3).

[0370] The CAR sequences targeting PSMA, CLDN 18.2, and GPC3 used in the experiment are provided in Table 3.Table 3. CAR SequencesaAntigen Target / CAR Sequence SEQ Construct ID NO: DescriptionPSMA / MALPVTALLLPLALLLHAARPELVLTQPPSASGTPGQRVTI 36 SCSGSNSNVGRDTVNWYQQLPGTAPKLLMYSDNQRPSGVPLeader seq- DRFSGSKSGTSASLAISGLQSEDEADYYCAAWDDSLNGWVF PSMA scFv- GGGTKLTVLGGSSRSSSSGGGGSGGGGEVQLVESGGGVVQMyc - CD28hinge- PGRSLRLSCAASGFTFNSYAMHWVRQAPGKGLEWVAVTSF CD28TM- DGSNKYYADSVKGRFTISRDNSNNTLYLQMSSLRSGDSAVY CD28ICD-CDJY YCAKDGGPYYDGTFYVSGMDVWGQGTTVTVSPASPTSPKV TSEQKLISEEDLIEVMYPPPYLDNEKSNGTIIIHVKGKHLCPSP LFPGPSKPFWLVWGGVLACYSLLVTVAFIIIFWVRSKRSRLLH SDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRS ADAPAYOOGONOLYNELNLGRREEYDVLDKRRGRDPEMGGKP RRKNPOEGLYNELOKDKMAEAYSEIGMKGERRRGKGHDGLYQ GLSTATKDTYDALHMOALPPR*PSMA / ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGG 37CCTTGCTGCTCCACGCCGCCAGGCCGGAGCTCGTGTTGnucleic acid ACGCAGCCGCCCTCAGCGTCTGGGACCCCCGGGCAGAGG(5’— >3’) GTCACCATCTCTTGTTCTGGAAGCAACTCCAACGTGGGAC GTGACACTGTAAACTGGTATCAGCAACTCCCGGGGACGGLeader seq- CCCCCAAACTCCTCATGTATAGTGATAATCAACGGCCCTC PSMA scFv- AGGGGTTCCTGACCGATTCTCTGGCTCCAAGTCTGGCACCMy c -CD28hinge- TCAGCCTCCCTGGCCATCAGTGGGCTCCAGTCTGAGGATG CD28TM- AGGCTGATTATTACTGTGCAGCATGGGATGACAGCCTGA CD28ICD-CDA' ATGGTTGGGTGTTCGGCGGAGGCACCAAGCTGACCGTCC TAGGTGGTTCCTCTAGATCTTCCTCCTCTGGTGGCGGTGG CTCGGGCGGTGGTGGGGAGGTGCAGCTGGTGGAGTCTGG GGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTC CTGTGCAGCCTCTGGATTCACCTTCAATAGTTATGCTATG CACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTAGAGTGGGTGGCAGTCACTTCATTTGATGGAAGTAATAAATATTATG CAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACA ATTCCAACAACACACTGTATCTGCAAATGAGCAGCCTGA GATCTGGGGACTCGGCTGTCTATTACTGTGCGAAAGACG GGGGACCTTATTATGACGGAACCTTCTACGTGTCCGGTAT GGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCCC AGCATCCCCGACCAGCCCCAAGGTCACTAGTGAGCAGAA ACTTATATCAGAGGAAGAT’CTTATTGAAGTTATGTATCCTC CTCCTTACCTAGACAATGAGAAGAGCAATGGAACCATTA TCCATGTGAAAGGGAAACACCTTTGTCCAAGTCCCCTATTT'CCCGGACCTTCT'AAGCCCTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCrGGGTGAGGAGTAAGAGGAGCAGGCTCC TGCACAGTGACTACATGAACATGACTCCCCGCCGCCC CGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCA CCACGCGACTTCGCAGCCTATCGCTCCAGAGTGA4G7TC AGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGA ACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGT ACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATG GGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTA CAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGA GATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCAC GATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACC TACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCTAA CLDN18.2 / MALPVTALLLPLALLLHAARPEVQLVESGGGLVQPGGSLR 38LSCAASGSIFSNTYMGWYRQAPGKQRELVSRISGGGSVHYLLeader seq- DSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCYAHYW CLDN18.2 scFv- MDYWGOGTLVTVSSEOALZ. SEEDLIEVMYPPPYLDNEKSNG My c -CD28hinge- TIIHVKGKHLCPSPLFPGPSKPFWLVWGGVLACySLLVTVAF / CD28TM- ZFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRD CD28ICD-CD3C VKKNR' SRVKFSRSADAPAYOOGONQLYNELNLGRREEYDVLD KRRGRDPEMGGKPRRKNPOEGLYNELQKDKMAEAYSEIGMK GERRRGKGHDGLYOGLSTATKDTYDALHMQALPPR* CLDN18.2 / A7GGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGG 39CCTTGCTGCTCCACGCCGCCAGGCCGGAGGTGCAACTnucleic acid GGTCGAGAGCGGAGGCGGCCTGGTGCAGCCCGGCGGCAG (5’^3’) CCTGAGACTGAGCTGTGCTGCTTCTGGCAGCATCTTCAGC AATACCTACATGGGCTGGTATAGACAGGCCCCTGGCAAALeader seq- CAGCGGGAACTGGTGTCCAGAATCAGCGGCGGAGGATCT CLDN18.2 scFv- GTGCACTACCTCGACAGCGTGAAGGGCAGATTCACCATC Mvc-CD28hinge- TCCCGCGACAACGCCAAGAACACCCTGTACCTGCAGATG CD28TM- AACAGCCTGCGGGCCGAAGATACAGCCGTGTACTACTGC TACGCCCACTACTGGATGGACTACTGGGGCCAGGGCACA CD28ICD-CD3ζ CTGGTGACCGTGTCTAGCGAGCAGAAACrrATArCAGAGG 4AGATC7TATTGAAGTTATGTATCCTCCTCCTTACCTAGAC AATGAGAAGAGCAATGGAACCATTATCCATGTGAAAGGG AAACACCTTTGTCCAAGTCCCCTATTTCCCGGACCTTCTA AGCCCTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCT TGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGG TGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTA CATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGC AAGCATTACCAGCCCTATGCCCCACCACGCGACTTCG CAGCCTATCGCTCCAGAGTGAAGTTCAGCAGGAGCGCAGA CGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACG AGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACA AGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAG AAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAA AGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAG GCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCA GGGTCTCAGTACAGCCA CCAAGGACA CCTA CGA CGCCCTTCA CATGCAGGCCCTGCCCCCTCGCTAA GPC3 / MALPVTALLLPLALLLHAARPQVKLEESGGGSVQAGESLT 40LSCAAAGLTFNDYAMVWFRQAPGKEREFVAGISRSGGTIRYLeader seq- EDSVKGRFTISRDNARNAVYLQMNSLKPEDTAVYYCNRLH GPC3 scFv-Myc- SYEYADWGQGTQVTVSSEGALZSEEDLIEVMYPPPYLDNEK_CD28hinge- SNGTIIHVKGKHLCPSPLFPGPSKPFWLVWGGVLACYSLLVTCD28TM- VAFIIFWRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPCD28ICD-CD3ζ PRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR* GPC3 / A7GGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGG 41CCTTGCTGCTCCACGCCGCCAGGCCGCAAGTGAAGCTnucleic acid GGAAGAATCTGGAGGCGGCTCTGTGCAGGCCGGCGAGAG(5’— >3’) CCTGACCCTGAGCTGTGCCGCTGCTGGCCTGACCTTCAAC GACTACGCCATGGTGTGGTTCAGACAGGCTCCTGGCAAALeader seq- GAGAGAGAGTTCGTGGCCGGCATCTCCCGGTCTGGCGGAGPC3 scFv-Afyc- ACAATCAGATACGAGGACTCCGTCAAGGGCAGATTTACC CD28hinge- ATCAGCCGGGACAACGCCAGAAATGCCGTGTACCTGCAG CD28TM- ATGAACTCCCTGAAGCCCGAAGATACAGCCGTGTACTAC CD28ICD- CD3C TGCAACCGGCTGCACAGCTACGAGTACGCCGACTGGGGC CAGGGCACCCAGGTGACCGTGTCCTCTGAGCAGAAAC77ATA TCAGAGGAAGA TCTT’ATTGAAGTTATGT ATCCTCCTCCT TACCTAGACAATGAGAAGAGCAATGGAACCATTATCCAT GTGAAAGGGAAACACCTTTGTCCAAGTCCCCTATTTCCCG GACCTYCYAAGCCCTTTTGGGTGCTGGTGGTGGTTGGTGGA GTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTA TTZTCrGGGrGAGGAGTAAGAGGAGCAGGCTCCTGCAC AGTGACTACATGAACATGACTCCCCGCCGCCCCGGGC CCACCCGCAAGCATTACCAGCCCTATGCCCCACCACG CGACTTCGCAGCCTATCGCTCCAGAGTGAAG77CAGCAG GAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAG CTCTATAACGAGCTCAA TCTAGGA CGAAGAGAGGAGTA CGA T GTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGG AAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATG AACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTG GGA TGAAA GGCGA GCGCCGGAGGGGCAA GGGGCA CGA TGG CCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCA CA TGCAGGCCCTGCCCCCTCGCTAA“Amino acid and / or nucleic acid residues that are bold, italic, and underlined at the start and stop of the sequences indicate start or stop codons. An asterisk (*) indicates a stop codon.

[0371] For all assays, the integrated total fluorescence intensity per well was recorded as a quantitative measure of viable target cells, with measurements taken every 4 or 8 hours using the Incucyte® S3 Live-Cell Analysis System. Each experimental condition was set up in triplicate to ensure robustness of the data.

[0372] For the PSMA-targeted cytotoxicity assay, LNCaP-Zsgreen prostate cancer cells (PSMA-positive) were used as target cells. On Day -1, IxlO4LNCaP-Zsgreen cells in 50 pL of complete growth medium were seeded into each well of a 96-well plate. On Day 0, CAR-T cells (from Group 1-5) were added to the wells in an additional 50 pL of medium at an effector-to-target (E: T) ratio of 0.03:1 (resulting in a total volume of 100 pL per well). Every two days, IxlO4fresh LNCaP-Zsgreen cells in 30 pL of medium were added to each well to stimulate continuous antigenic stimulation, for a total of six stimulations over a 14-dayperiod. Throughout the assay, GFP intensity was monitored every 4 hours. Integrated total GFP intensity per well served as the quantitative readout of viable LNCaP-Zsgreen cells.

[0373] For the Claudinl8.2-targeted cytotoxicity assay, AsPC-l-Claudinl8.2-GFP-Fluc pancreatic cancer cells were used as targets. On Day -1, IxlO4AsPC-l-Claudinl8.2-GFP-Fluc cells in 50 pL of medium were plated per well in a 96-well plate. On Day 0, CAR-T cells (from Group 1-5) were added in 50 pL of medium at an E: T ratio of 0.01:1 (total 100 pL per well). Every three days, IxlO4fresh AsPC-l-Claudinl8.2-GFP-Fluc cells in 30 pL of medium were added to each well. GFP fluorescence was recorded every 8 hours, and integrated total GFP intensity was used as the measure of viable AsPC-l-Claudinl8.2 cells throughout the 14-day repeated stimulation period.

[0374] For the GPC3-targeted cytotoxicity assay, HepG2-Zsgreen hepatocellular carcinoma cells (GPC3-positive) served as targets. On Day -1, each well was seeded with 1×104HepG2-Zsgreen cells in 50 pL of medium. On Day 0, CAR-T cells (from Group 1-5) were co-cultured with the target cells in 50 pL of medium at an E: T ratio of 3: 1 (total 100 pL per well). Every three days, IxlO4fresh HepG2-Zsgreen cells in 30 pL of medium were added to each well. As with the other assay, GFP fluorescence was measured every 8 hours, and the integrated total GFP intensity per well used to quantify the amount of viable HepG2-Zsgreen target cells throughout the assay.

[0375] Across all three tumor models (PSMA, Claudinl8.2, and GPC3), the untransduced T cell group (G2) and the no-armored CAR-T group (G3) showed limited and transient cytotoxic activity, with progressive increases in GFP fluorescence intensity over repeated stimulation cycles, indicating continued tumor cell survival and regrowth. The mbIL-12-armored CAR-T cells (G4) demonstrated improved cytotoxic activity relative to no-armored CAR-T cells, as reflected by reduced GFP intensity values across stimulation rounds.Notably, the Hyperkine-expressing CAR-T cells (G5) exhibited the most pronounced and sustained cytotoxicity effect, maintaining significantly lower GFP intensity throughout the course of repeated stimulations compared to other groups (see FIGs. 25A-25C). Statistical analysis confirmed that the suppression of tumor cell growth by Hyperkine-expressing CAR-T cells was significant in all tested models (*p<0.05, **p<0.01. ***p<0.001. and ****p<0.0001).

[0376] These results confirm that CAR-T cells engineered to express Hyperkine display superior cytotoxic durability under conditions of continuous antigenic stimulation. In each ofthe PSMA-, Claudinl8.2-, and GPC3-positive tumor models, Hyperkine-armored CAR-T cells achieved significantly enhanced tumor cell killing relative to no-armored CAR-T or mbIL-12-armored CAR-T cells. These data indicate that Hyperkine provides a distinct functional advantage in sustaining CAR-T cells effector activity under repeated stimulation stress, thereby representing a valuable armoring strategy to improve the therapeutic durability of CAR-T cell therapy against solid tumors.

[0377] As shown herein, engineered T cells expressing cytokine fusion proteins exhibit superior cytotoxicity and durable antitumor efficacy in repeated stimulation assays.Example 8. In vitro Cytotoxicity of Hyperkine-Expressing CAR T Cells

[0378] An in vitro study was conducted to evaluate the cytotoxicities of CAR T cells engineered to express a membrane-bound interleukin- 12 fusion protein (mIL-12 / mbIL-12) with or without a 4- IBB intracellular costimulatory domain. The CAR and mIL-12 fusion protein sequences of the evaluated CAR T cells are provided in Table 4. Schematic depiction of the different CAR T cell groups tested are also provided in FIGs. 26, 28, and 31. Table 4. CAR T cellsCAR T cell CAR mIL-12 description mIL-12 Description (target antigen-costimulatory SEQ ID domain-intracellular signaling NO domain)28z PSMA-CD28-CD3C none NA 28z-L3 PSMA-CD28-CD3C L3 (IL-12-CD28TM) 27 28z-L6 PSMA-CD28-CD3ζ L6 (IL-12-CD28TM-4- 331BB ICD (hyperkine))BBz PSMA-4-lBB-CD3£ none NA BBz-L3 PSMA-4-1BB-CD3C L3 (IL-12-CD28TM) 27 BBz-L6 PSMA-4-1BB-CD3ζ L6 (IL-12-CD28TM-4- 331BB ICD (hyperkine))1stPSMA PSMA- CD3C none NA 1stPSMA+L3 PSMA- CD3C L3 (IL-12-CD28TM) 27 1stPSMA+L6 PSMA- CD3ζ L6 (IL-12-CD28TM-4- 331BB ICD (hyperkine))

[0379] Table 5 provides the amino acid and coding nucleic acid sequences of the CAR constructs used in the experiments.Table 5. CAR SequencesaCAR CAR Sequence SEQ IDNO:PSMA-CD28- MALPVTALLLPLALLLHAARPELVLTQPPSASGTPGQRVTI 36 CD3ζ SCSGSNSNVGRDTVNWYQQLPGTAPKLLMYSDNQRPSGVP DRFSGSKSGTSASLAISGLQSEDEADYYCAAWDDSLNGWVFLeader seq- GGGTKLTVLGGSSRSSSSGGGGSGGGGEVQLVESGGGVVQ PSMA scFv- PGRSLRLSCAASGFTFNSYAMHWVRQAPGKGLEWVAVTSF My c - CD28hinge- DGSNKYYADSVKGRFTISRDNSNNTLYLQMSSLRSGDSAVY CD28TM- YCAKDGGPYYDGTFYVSGMDVWGQGTTVTVSPASPTSPKV CD28ICD-CDJY TSEEQKLISEEDLIEVMYPPPY LDNEKSNGTIIHVKGKHLCPSP LFPGPSKPFVVVLVWGGVIACYSLLVTVAFIFWVRSKRSRLLH SDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSREKFSRS ADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR*PSMA-CD28- ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGG 43 CD3^ CCTTGCTGCTCCACGCCGCCAGGCCGGAGCTCGTGTTG ACGCAGCCGCCCTCAGCGTCTGGGACCCCCGGGCAGAGG nucleic acid GTCACCATCTCTTGTTCTGGAAGCAACTCCAACGTGGGAC (5’— >3’) GTGACACTGTAAACTGGTATCAGCAACTCCCGGGGACGG CCCCCAAACTCCTCATGTATAGTGATAATCAACGGCCCTCLeader seq- AGGGGTTCCTGACCGATTCTCTGGCTCCAAGTCTGGCACC PSMA scFv- TCAGCCTCCCTGGCCATCAGTGGGCTCCAGTCTGAGGATG My c -CD28hinge- AGGCTGATTATTACTGTGCAGCATGGGATGACAGCCTGA CD28TM- ATGGTTGGGTGTTCGGCGGAGGCACCAAGCTGACCGTCC CD28ICI)-C7 / ?c' TAGGTGGTTCCTCTAGATCTTCCTCCTCTGGTGGCGGTGG CTCGGGCGGTGGTGGGGAGGTGCAGCTGGTGGAGTCTGG GGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTC CTGTGCAGCCTCTGGATTCACCTTCAATAGTTATGCTATG CACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTAGAGTGG GTGGCAGTCACTTCATTTGATGGAAGTAATAAATATTATG CAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACA ATTCCAACAACACACTGTATCTGCAAATGAGCAGCCTGA GATCTGGGGACTCGGCTGTCTATTACTGTGCGAAAGACG GGGGACCTTATTATGACGGAACCTTCTACGTGTCCGGTAT GGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCCC AGCATCCCCGACCAGCCCCAAGGTCACTAGTGAGCAGAA ACTTATA TCAGAGGAAGATC77ATTGAAGTTATGTATCCTC CTCCTTACCTAGACAATGAGAAGAGCAATGGAACCATTA TCCATGTGAAAGGGAAACACCTTTGTCCAAGTCCCCTATT TCCCGGACCTTTCTAAGCCCTTTTGGGTGCTGGTGGTGGTTG GTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCT TTATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCC TGCACAGTGACTACATGAACATGACTCCCCGCCGCCC CGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCA CCACGCGACTTCGCAGCCTATCGCTCCAGAGAGAAGTTC AGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGA ACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGT ACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATG GGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTA CAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGA GATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCAC GATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACC TACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCTAA PSMA-4-1BB- MALPVTALLLPLALLLHAARPELVLTQPPSASGTPGQRVTI 44 CD3ζ SCSGSNSNVGRDTVNWYQQLPGTAPKLLMYSDNQRPSGVPDRFSGSKSGTSASLAISGLQSEDEADYYCAAWDDSLNGWVFGGGTKLTVLGGSSRSSSSGGGGSGGGGEVQLVESGGGVVQ Leader seq- PGRSLRLSCAASGFTFNSYAMHWVRQAPGKGLEWVAVTSF PSMA scFv- DGSNKYYADSVKGRFTISRDNSNNTLYLQMSSLRSGDSAVY Mvc-CD8hinge- YCAKDGGPYYDGTFYVSGMDVWGQGTTVTVSPASPTSPKV CD8TM- TSEGKLLS'EEDLTTTPAPRPPTPAPTIASQPLSLRPEACRPAAG 41BBICI) C / Z’< GAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKK LLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVK FSRSADAPAYOOGONOLYNELNLGRREEYDVLDKRRGRDPEM GGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGH DGLYQGLSTATKDTYDALHMQALPPR*PSMA-4-1BB- ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGG 45 CD3ζ CCTTGCTGCTCCACGCCGCCAGGCCGGAGCTCGTGTTG ACGCAGCCGCCCTCAGCGTCTGGGACCCCCGGGCAGAGG nucleic acid GTCACCATCTCTTGTTCTGGAAGCAACTCCAACGTGGGAC (5’— >3’) GTGACACTGTAAACTGGTATCAGCAACTCCCGGGGACGG CCCCCAAACTCCTCATGTATAGTGATAATCAACGGCCCTCLeader seq- AGGGGTTCCTGACCGATTCTCTGGCTCCAAGTCTGGCACC PSMA scFv- TCAGCCTCCCTGGCCATCAGTGGGCTCCAGTCTGAGGATG Mvc-CD8hinge- AGGCTGATTATTACTGTGCAGCATGGGATGACAGCCTGA CD8TM- ATGGTTGGGTGTTCGGCGGAGGCACCAAGCTGACCGTCC 41BBICD-CD3ζ TAGGTGGTTCCTCTAGATCTTCCTCCTCTGGTGGCGGTGG CTCGGGCGGTGGTGGGGAGGTGCAGCTGGTGGAGTCTGG GGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTC CTGTGCAGCCTCTGGATTCACCTTCAATAGTTATGCTATG CACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTAGAGTGG GTGGCAGTCACTTCATTTGATGGAAGTAATAAATATTATG CAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACA ATTCCAACAACACACTGTATCTGCAAATGAGCAGCCTGA GATCTGGGGACTCGGCTGTCTATTACTGTGCGAAAGACG GGGGACCTTATTATGACGGAACCTTCTACGTGTCCGGTAT GGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCCC AGCATCCCCGACCAGCCCCAAGGTCACTAGTGAGCAGAA ACTTATATCAGAGGAAGATCTTACCACGACGCCAGCGCCG CGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCC CTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGG GGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGAT ATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTT CTCCTGTCACTGGTTATCACCCTTTACTGCAAAGGGGGCAG AAAGAAACTCCTGTATATATTCAAACAACCATTTATGA GACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAG CTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAA CTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTA CCAGCAGGGCCAGAA CCAGCTCTATAA CGAGCTCAA TCTAGG ACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCC GGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCT CAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCG GAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAG GGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAG CCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTG CCCCCTCGCTAA PSMA- CD3ζ MALPVTALLLPLALLLHAARPELVLTQPPSASGTPGQRVTI 46SCSGSNSNVGRDTVNWYQQLPGTAPKLLMYSDNQRPSGVPLeader seq- DRFSGSKSGTSASLAISGLQSEDEADYYCAAWDDSLNGWVFGGGTKLTVLGGSSRSSSSGGGGSGGGGEVQLVESGGGVVQPGRSLRLSCAASGFTFNSYAMHWVRQAPGKGLEWVAVTSFPSMA scFv- DGSNKYYADSVKGRFTISRDNSNNTLYLQMSSLRSGDSAVYMyc -CD8hinge- YCAKDGGPYYDGTFYVSGMDVWGQGTTVTVSPASPTSPKV CD8TM-CD3ζTSEEQKLISEEDLTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACVIYIWAPLAGTCGVLLLSLVITLYCRVKFSRS ADAPAYOOGONOLYNELNLGRREEYDVLDKRRGRDPEMGGKP RRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR*PSMA- CD3ζ ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGG 47CCTTGCTGCTCCACGCCGCCAGGCCGGAGCTCGTGTTGnucleic acid ACGCAGCCGCCCTCAGCGTCTGGGACCCCCGGGCAGAGG(5’— >3’) GTCACCATCTCTTGTTCTGGAAGCAACTCCAACGTGGGAC GTGACACTGTAAACTGGTATCAGCAACTCCCGGGGACGGLeader seq- CCCCCAAACTCCTCATGTATAGTGATAATCAACGGCCCTC PSMA scFv- AGGGGTTCCTGACCGATTCTCTGGCTCCAAGTCTGGCACCMyc-CD8hinge- TCAGCCTCCCTGGCCATCAGTGGGCTCCAGTCTGAGGATG CD8TM-CD3ζ AGGCTGATTATTACTGTGCAGCATGGGATGACAGCCTGA ATGGTTGGGTGTTCGGCGGAGGCACCAAGCTGACCGTCC TAGGTGGTTCCTCTAGATCTTCCTCCTCTGGTGGCGGTGG CTCGGGCGGTGGTGGGGAGGTGCAGCTGGTGGAGTCTGG GGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTC CTGTGCAGCCTCTGGATTCACCTTCAATAGTTATGCTATG CACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTAGAGTGG GTGGCAGTCACTTCATTTGATGGAAGTAATAAATATTATG CAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACA ATTCCAACAACACACTGTATCTGCAAATGAGCAGCCTGA GATCTGGGGACTCGGCTGTCTATTACTGTGCGAAAGACG GGGGACCTTATTATGACGGAACCTTCTACGTGTCCGGTAT GGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCCC AGCATCCCCGACCAGCCCCAAGGTCACTAGTGAGCAGAA ACTTATATCAGAGGAAGATCTTACCACGACGCCAGCGCCG CGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCC CTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGG GGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGAT ATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTT CTCCTGTCACTGGTTATCACCCTTTACTGCAGAGTGAAGTTCA GCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAA CCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTA CGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGG GGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTAC AATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAG ATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACG ATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCTAA“Amino acid and / or nucleic acid residues that are bold, italic, and underlined at the start and stop of the sequences indicate start or stop codons. An asterisk (*) indicates a stop codon.

[0380] 5 x 103LnCaP-Zsgreen target cells were seeded per well one day before coculture in a 96-well flat-bottom plate in 100 pl of media. CAR T cells alone or CAR T cells expressing mIL-12 fusion proteins (see Table 4) were cocultured with the target cells at different effector-to-target ratios from 0.1: 1 to 1: 1 in 200 pl of media. Triplicates were plated for each group. Rcstimulation with the target cells was performed every 3 days according to Method1 described above. The GFP fluorescence intensity of the target cells was measured every 2 hours using the Incucyte S3 Live-Cell Analysis System. Integrated total GFP intensity per well was used as a quantitative measure of the amount of viable target cells. The amounts of viable target cells (as measured by green fluorescence intensity) as a function of time for the 28z, BBz, and 1stPSMA CAR T cell groups are shown in FIG. 27, FIG. 29, and FIG.30, respectively. The data plots include data sets having non-transduced cells (NTD) and low-affinity nerve growth factor receptor (LNGFR).

[0381] As can be seen from FIGs. 26-27, the cytotoxicities of the 28z CAR T cells follows the order of 28z < 28z-L3 < 28z-L6. Accordingly, the highest cytotoxicity was observed with the hyperkine-expressing CAR T cells (28z-L6). In contrast, for the BBz CAR T cells, cytotoxicity follows the order to BBz < BBz-L3 = BBz-L6 (see FIGs. 28-29). The hyperkine-expressing BBz CAR T cells (BBz-L6) showed the same cytotoxicity as the mlL-12-expressing CAR T cells lacking the 4-1BB intracellular domain (BBz-L3). Additionally, comparison of the 28z CAR T cell data (FIGs. 26-27) with the BBz CAR T cell data (FIGs.28-29) suggests a possible cytotoxic benefit for pairing a CD28 costimulatory domain in the CAR with a 4-1BB domain in the hyperkine (as in 28z-L6) as opposed to pairing a 4-1BB costimulatory domain in the CAR with a 4- IBB domain in the hyperkine (as in BBz-L6) (also see FIG. 30).

[0382] Referring to FIGs.31-32, the cytotoxicities of the 1stPSMA CAR T cells followed the order of 1stPSMA < 1stPSMA+L3 < 1stPSMA+L6. Thus, as with the 28z CAR T cells, the highest cytotoxicity was observed with the hyperkine-expressing CAR T cell (1stPSMA+L6). In all data sets, co-expression of the cytokine fusion protein (L3 or L6) showed enhanced cytotoxicity over the CAR T cell alone.

[0383] It is further noted that in all data sets, the CAR T cells co-expressing an mIL-12 fusion protein, even in the absence of a costimulatory domain on the fusion protein, exhibited higher cytotoxicities than the CAR T cells alone (compare, for example, 28z and 28z-L3, BBz and BBz-L3, and 1stPSMA and 1stPSMA+L3).Example 9. In vivo Persistence with Hyperkine-Expressing CAR T Cells

[0384] An in vivo study was conducted to evaluate persistence of CAR T cells engineered to express hyperkine (L6).

[0385] Human peripheral blood-derived T lymphocytes were isolated and transduced with lentiviral vectors encoding various gene constructs to establish four experimental CAR-T cell groups, as follows:

[0386] Group 1 (Gl, negative control): T cells with no CAR (untransduced T cells);

[0387] Group 2 (G2, No-armored CAR-T): CAR-T cells expressing a tumor antigenspecific CAR (targeting either PSMA or Claudin 18.2; see Table 3);

[0388] Group 3 (G3, mbIL-12 armored CAR-T): CAR-T cells co-expressing a mbIL-12 (L3, Table 2) along with CAR; and

[0389] Group 4 (G4, hyperkine armored CAR-T): CAR-T cells co-expressing hyperkine (L6, Table 2) along with CAR.

[0390] In the prostate cancer model, CAR constructs targeted PSMA. In the adenocarcinoma model, CAR constructs targeted Claudin (CLDN) 18.2. The mbIL-12 and hyperkine constructs were identical between models, with Hyperkine incorporating an additional costimulatory domain, 41-BB.

[0391] To establish a PSMA-positive tumor model, NOG (NOD. Cg-Prkdcscid IL2ygtmlSug / JicKoat) mice were subcutaneously inoculated with 4xl06PC3-PSMA-GFP-Fluc human prostate cancer cells (engineered to express PSMA, EGFP, and firefly luciferase) in 100 pL of volume. Eight days post-inoculation, tumor engraftment was confirmed using bioluminescence imaging (IVIS® Spectrum system). Successfully engrafted mice were randomized into groups Gl through G4. On day 9 post-tumor implantation, CAR-T cells were harvested and prepared. For each mouse, 4xl06PSMA-specific CAR-T cells in 200 pL of sterile PBS were intravenously administered via tail vein injection. Group 1 mice received an equivalent dose of untransduced T cells.

[0392] Similarly, for the Claudin 18.2-positive model, 5-week-old NOG mice were subcutaneously inoculated with 3x106AsPC-1 -Claudin 18.2-GFP-Fluc cells. On day 13 postimplantation, tumor formation was confirmed, and mice were randomly allocated into Gl through G4. On day 14 post-implantation, mice received 2x106Claudin 18.2-specific CAR-T cells (or control T cells for Gl) via tail vein injection.

[0393] Following CAR-T cell administration, tumor progression or regression was monitored weekly by IVIS bioluminescence imaging. Photon flux (photons / sec) from tumor cells was quantified using Living Image® software through ROI-based analysis.

[0394] To assess in vivo persistence of CAR-T cells, spleens were harvested at endpoint: day 25 for PSMA CAR-T models and day 31 for Claudin 18.2 CAR-T models. Harvested spleens were processed into single-cell suspensions, and red blood cells were lysed. Splenocytes were stained with antibodies against human CD3 (hCD3) and a CAR detection reagent (e.g., Myc tag). Flow cytometric analysis was performed using a gating strategy (sec FIG.33) to quantify hCD3+CAR+cell populations. CAR-T cell persistence was calculated as the number of hCD3+CAR+cells per mg of spleen tissue, based on total cell count and spleen mass.

[0395] The data revealed clear differences in CAR T cell persistence across the treatment groups (see FIG.34). As expected, Group 1 exhibited negligible levels of human CAR+T cells. Group 2 showed detectable CAR-T cells but at relatively low levels. Group 3 showed an increased level of CAR T cell persistence in vivo compared to Group 2, consistent with the known proliferation effects of IL- 12 on T cells. Notably, Group 4 demonstrated the highest levels of CAR-T cell persistence in both tumor models (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).

[0396] In the PSMA-positive tumor model (see FIG.34A), mice treated with hyperkine (L6)-expressing CAR T cells (G4) had on average 412,000 ± 2,828 CAR+T cells per mg of spleen, whereas the mbIL-12 CAR T group (G3) had only 18,633 ± 1,250 CAR+T cells / mg. This data represents roughly a 22-fold increase in persisting CAR T cell numbers for hyperkine (G4) compared to the mbIL-12 CAR T cells (G3) in the prostate tumor setting. A similar trend was observed in the Claudin 18.2 tumor model: Group 4 mice had 74,533 ± 17,602 CAR+T cells per mg spleen versus 14,692 ± 5,625 cells / mg in Group 3, approximately a 5-fold higher persistence with Hyperkine CAR-T (see FIG.34B).

[0397] Taken together, these data demonstrate that CAR-T cells engineered to express hyperkine (L6) exhibit significantly enhanced in vivo persistence, as evidenced by substantially increased numbers of CAR+T cells in spleen tissue at endpoint. These findings suggest that incorporation of hyperkine provides a distinct advantage in promoting CAR-T cell survival and expansion in vivo. Accordingly, hyperkine represents a refined armoring strategy for improving the persistence and therapeutic durability of CAR-T cell therapies in solid tumor contexts.Example 10. In Vivo Antitumor Efficacy an Persistence of CAR T Cells Expressing Cytokine Fusion Proteins

[0398] This example describes an in vivo evaluation of the antitumor efficacy of Claudinl8.2-specific CAR-T cells expressing cytokine fusion proteins in a Claudinl8.2-positive adenocarcinoma tumor model.

[0399] Human peripheral blood-derived T lymphocytes were isolated and transduced with lentiviral vectors encoding various gene constructs to establish four experimental CAR-T cell groups, as follows:Group 1 (Gl, negative control): T cells with no CAR (Untransduced T cells);Group 2 (G2, No-armored CAR-T): CAR-T cells expressing a tumor antigen-specific CAR (targeting Claudinl8.2, Table 3); andGroup 3 (G3, Hyperkine (L6, Table 2) armored CAR-T): CAR-T cells co-expressing Hyperkine, comprising mbIL-12 and an additional costimulatory domain, along with CAR.

[0400] A Claudinl8.2-positive adenocarcinoma model was established in 5-week-old-NOG (NOD.Cg-PrkdcscidIL2γgtm1Sug / JicKoat) mice by subcutaneous injection of 3xl06AsPC-1-Claudinl8.2-GFP-Fluc tumor cells in 100 pL of medium. The tumor cells were engineered to express enhanced green fluorescent protein (EGFP) and firefly luciferase (Flue). On day 13 after tumor implantation, tumor engraftment was confirmed by bioluminescent imaging using IVIS® Spectrum system. Mice with detectable bioluminescent signal were randomly allocated into four experimental group as follows:

[0401] On day 14 after tumor implantation, cultured Claudinl8.2-specific CAR-T cells (Group 1-3) were harvested, centrifuged at 600 ×g for 3 minutes, and resuspended in fresh culture medium. The cells were counted, centrifuged again, washed with D-PBS, and adjusted to a final concentration of 0.5xl06CAR-T cells in 200 pL. Each mouse received an intravenous injection of 200 pL of the prepared suspension containing 0.5xl06CAR-T cells.

[0402] Following CAR-T cell infusion, tumor progression was monitored weekly by bioluminescent imaging using the IVIS® Spectrum system. The photon flux (photons / sec) emitted by the tumor cells was quantified in a defined region of interest (ROI) using Live Image® software.

[0403] On day 31 after CAR-T cell administration, spleens were harvested and processed into single-cell suspensions. Red blood cells were lysed and splenocytes were stained with antibodies against human CD3 (hCD3) and a CAR detection tag (e.g., Myc tag). Flow cytometry was performed to determine the frequency of hCD3+CAR+cells. The total numberof hCD3+CAR+T cells per spleen was calculated based on the overall splenocyte count and the percentage of hCD3+CAR+cells.

[0404] In the control group (Gl) receiving untransduced T cells, bioluminescent tumor signals increased progressively. In contrast, in the group receiving Hyperkine (L6)- armored CAR-T cells (G3), tumor growth was significantly suppressed compared to no-armored CAR-T group (G2) (*p<0.05, **p<0.01, and ***p<0.001) (see FIG. 35).

[0405] No human CAR+T cells were detected in the untransduced T cell group (Gl). The No-armored CAR-T group (G2) exhibited an average of 339,663±426,189 CAR+T cells, whereas the Hyperkine armored CAR-T group (G3) exhibited 11,451, 194±3, 506, 412 CAR+T cells, corresponding to approximately 33-fold increase in persistence (FIG. 36).

[0406] The results confirmed that CAR-T cells armored with cytokine fusion proteins of the present disclosure demonstrated markedly enhanced antitumor efficacy and superior in vivo persistence. This finding indicates that the cytokine fusion protein promotes CAR T cell survival and expansion, thereby representing a useful armoring strategy to improve therapeutic efficacy.Example 11. Cytokine Fusion Protein to Enhance T Cell Function

[0407] Referring to FIGs.37-38, schematic representations of enhancement of T cell function with a cytokine fusion protein of the present disclosure are shown. The T cell is engineered to co-express a cytokine fusion protein of the present disclosure. Engagement of a TCR of the T cell with an antigen peptide presented by the MHC complex of an APC provides a first signal (signal 1) for stimulating activation of the T cell. A second costimulatory signal (signal 2) for activation of the T cell occurs upon binding of a costimulatory receptor, such as CD28, to a molecule presented on the APC, and / or via costimulatory domains within the cytokine fusion protein. Cytokine signaling triggered by binding of the cytokine fusion protein (or hyperkine) to a receptor provides a third signal (signal 3) for activation of the T cell. The cytokine fusion protein may bind to a receptor on the surface of the same T cell (cis binding) or on the surface of a different T cell (trans binding). The cytokine fusion protein of the present disclosure may deliver spatially discrete costimulatory (signal 2) and cytokine (signal 3) inputs in parallel which enables a more physiologic and synergistic mode of T cell activation. As demonstrated herein horizontal signal 2 and signal 3 augmentation by membrane-bound cytokine fusion proteins enhances effector function while sustaining proliferative potential within the tumor microenvironment.Example 12. Evolution of CAR T Cell Design with Cytokine Fusion Protein

[0408] A potential problem associated with current CAR T cell designs is limited signaling enhancement which results from suboptimal engagement of proximal signaling components. This may lead to suppressed CAR T cell functions. FIGs.39-40 show schematic representations of enhancement of CAR T cell functions (2ndgeneration CAR T Cell) with a cytokine fusion protein of the present disclosure. The CAR T cell is engineered to co-express a cytokine fusion protein of the present disclosure. Binding of the antigen binding domain of the CAR to its antigen target provides signal 1 for T cell activation. Costimulatory and cytokine signal inputs (signals 2 and 3) are delivered via the costimulatory domain of the fusion protein and upon cis or trans binding of the cytokine portion of the cytokine fusion protein to its cytokine receptor. Unlike “vertical” augmentation of costimulatory signals -such as via the incorporation of additional signaling domains into the CAR construct - the separate membrane-bound cytokine fusion protein (e.g., hyperkine) provides “horizontal” augmentation of CAR T cell activation by providing horizontal delivery of costimulatory (signal 2) and cytokine (signal 3) inputs in parallel. As demonstrated herein horizontal signal 2 and signal 3 augmentation by membrane-bound cytokine fusion proteins enhances effector function while sustaining proliferative potential within the tumor microenvironment.Example 13. Membrane- Anchored Cytokine Fusion Protein Enhances the Functional Persistence of Tumor-Specific TCR T Cells and CAR T Cells

[0409] Vertical augmentation of costimulatory signals — such as the incorporation of additional signaling domains into CAR constructs — has been extensively investigated to enhance the efficacy of engineered T cells against solid tumors. However, these approaches often fail to recapitulate the spatial and temporal dynamics of physiologic T cell activation, limiting their therapeutic benefit. To overcome this limitation, a hyperkine (L6) has been developed. The hyperkine (L6) is a membrane-anchored costimulatory-cytokine receptor designed to enable horizontal augmentation of TCR and CAR signaling. The hyperkine delivers spatially discrete costimulatory (signal 2) and cytokine (signal 3) inputs in parallel, enabling a more physiologic and synergistic mode of T cell activation (see FIGs.37-40). Structurally, the hyperkine consists of membrane-bound interleukin- 12 (mbIL-12) fused to a CD28 transmembrane domain and a 4- IBB intracellular domain. As demonstrated herein, human T cells expressing either an NY-ESO-l-specific TCR or CARs targeting PSMA, Claudinl8.2, or GPC3 were further engineered to co-express the hyperkine. In vitro, hyperkine- armored T cells exhibited markedly enhanced cytotoxicity under chronic antigenstimulation, outperforming both unarmored T cells and those equipped with conventional mbIL-12 armoring. In multiple xenograft models, both TCR-T and CAR-T cells modified with Hyperkine demonstrated superior anti-tumor efficacy. This enhanced in vivo performance was closely linked to robust post-transfer expansion of Hyperkine-expressing CAR-T cells, suggesting that the coordinated horizontal delivery of signal 2 and 3 not only boosts effector function but also sustains persistence and proliferation within the tumor microenvironment. Together, these findings establish that horizontal integration of costimulatory and cytokine signaling through Hyperkine provides a physiologically aligned and synergistic activation framework — representing a next-generation armoring strategy to improve the potency and durability of engineered T cell therapies against solid tumors. Example 14. In vitro Proliferation Assay of Hyperkine-Expressing T Cells Following TCR Stimulation

[0410] An in vitro study was performed to evaluate the proliferative capacity of T cells engineered to express Hyperkine, a membrane -bound IL- 12 (mbIL-12) construct incorporating an additional costimulatory domain, in comparison to mbIL-12-expressing T cells. The assay was designed to assess whether Hyperkine (L6) enhances T cell proliferation under conditions of TCR stimulation.

[0411] Human peripheral blood-derived T cells were isolated and transduced with lentiviral vectors encoding the respective gene construct. In all groups, truncated low-affinity nerve growth factor receptor (LNGFR) (ALNGFR; extracellular and transmembrane domains only) was co-expressed, enabling enrichment of ALNGFR-positive cells prior to proliferation assay. The constructs used for each group are schematically illustrated in FIG.41. Following ALNGFR-based enrichment, co-expression of ALNGFR and the corresponding transgene (mbIL-12) was confirmed by flow cytometry, as shown in FIG.42.

[0412] The experimental groups were as follows:

[0413] Group 1 (Gl, negative control): T cells transduced with ALNGFR (SEQ ID NO: 21, Table 1) only (negative control construct of FIG.41).

[0414] Group 2 (G2, mbIL-12 armored T cells): T cells co-expressing ALNGFR and mblL-12 (mbIL-12 armored construct of FIG.41; L3, Table 2).

[0415] Group 3 (G3, Hyperkine armored T cells): T cells co-expressing ALNGFR and Hyperkine, comprising mbIL-12 with an additional costimulatory domain (hyperkine armored construct of FIG.41; L6, Table 2).

[0416] Following enrichment, each group of T cells was subjected to TCR stimulation, and proliferation was assessed over time. TCR stimulation was performed using an anti-CD3 antibody (OKT3 clone) in the absence of exogenous recombinant IL-2. Both singlestimulation and chronic- stimulation conditions were tested. Quantitative assessment of T cell proliferation was performed using CountBright™ Absolute Counting Beads, and the number of ALNGFR+live cells was determined by flow cytometry. Fold change in proliferation was calculated relative to the initial cell count.

[0417] For single stimulation, non-treated 12-well plates were coated with anti-CD3 antibody (OKT3 clone) at 10 pg / mL in D-PBS (1 mL per well) and incubated overnight at 4°C, followed by washing once with D-PBS to remove unbound antibody. T cells were harvested, centrifuged at 600 ×g for 3 minutes, and resuspended in fresh assay medium. Based on the proportion of ALNGFR+cells, the suspension was adjusted to 3xl05cells / 3 mL, and cells were seeded into antibody-coated wells. Proliferation was evaluated on day 3, 6, and 9 poststimulation by quantifying the number of ALNGFR+live cells.

[0418] For chronic stimulation, plates were coated with anti-CD3 antibody as described above. T cells were prepared in fresh assay medium and adjusted to 5xl05ALNGFR+cells / 3 mL prior to seeding. On day 3 after the first stimulation, cells were harvested, counted using CountBright™ Absolute Counting Beads, and re-seeded at the same density (5x l()' ALNGFR+cells / 3 mL) onto newly coated plates for re-stimulation. This process was repeated at 3-day intervals, and proliferation was evaluated up to day 9. Cumulative proliferation capacity was determined by sequentially multiplying the fold change values obtained at each stimulation step.

[0419] Quantification of T cell proliferation was performed by flow cytometry using a gating strategy (see FIG.43) to identify ALNGFR+live cells. The absolute number of cells was calculated using the following formula:(cellsx Absolute count - I uL ) (Cell count x Counting beads volumex (beadsx = - - -; - - -; - X Counting beads concentration -\ Counting bead count x cell volume ) \ uL /

[0420] In both single and chronic stimulation conditions, Hyperkine- armored T cells (G3) exhibited significantly greater proliferation compared to mbIL-12 armored T cells (G2) and LNGFR-only control T cells (Gl) (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).Whereas proliferation in Gl and G2 groups declined or plateaued with successivestimulations, Hyperkine armored T cells continued to expand over time as shown in FIGs. 44A -44B.

[0421] These results demonstrate that T cells engineered to express Hyperkine maintained superior proliferative capacity under prolonged TCR stimulation compared to conventional mbIL-12 armored T cells. Importantly, the presence of Hyperkine provided a proliferative advantage to T cells and enabled sustained proliferation under chronic stimulation conditions. This finding indicates that Hyperkine provides a robust armoring strategy to overcome proliferative limitations observed in conventional T cell therapies targeting solid tumors and may be broadly applicable to various T cell modalities including TCR-T, CAR-T, and TIL therapies.Example 15: In vitro Antitumor Activity of Hyperkine- and Bifunctional Hyperkine- Armored CAR-T Cells Under Repeated Stimulation

[0422] An in vitro repeated stimulation killing assay was performed to evaluate the cytotoxic activity of CAR-T cells under sustained antigenic stress conditions against Claudinl8.2, PSMA, GPC3-positive tumor cells. Five parallel CAR-T cell treatment groups were tested:Group 1 (Gl, negative control): Target cell only.Group 2 (G2, negative control): T cells with no CAR (Untransduccd T cells).Group 3 (G3, Hyperkine armored CAR-T): CAR-T cells co-expressing Hyperkine, comprising mbIL-12 and an additional costimulatory domain ...

Claims

CLAIMSWhat is claimed is:

1. A fusion protein comprising, from N to C terminus,(a) a cytokine region;(b) a transmembrane region; and(c) a costimulatory domain.

2. The fusion protein of claim 1, wherein the cytokine region is selected from the group consisting of a polypeptide of interleukin- 12 (IL- 12), IL- 15, IL-2, IL-4, IL-7, IL-9, IL- 18, and IL- 21 or a functional portion thereof.

3. The fusion protein of claim 2, wherein the cytokine region is IL- 12 polypeptide or a functional portion thereof.

4. The fusion protein of claim 3, wherein the IL- 12 polypeptide or a functional portion thereof comprises a IL- 12a subunit and a IL- 120 subunit, wherein the IL12a subunit and the IL- 120 subunit are connected via a linker and form a heterodimer.

5. The fusion protein of any one of claims 3-4, wherein the IL- 12 polypeptide or a functional portion thereof comprises the amino acid sequence of SEQ ID NO: 9 or a peptide having at least 85% sequence identity to SEQ ID NO: 9.

6. The fusion protein of claim 1, wherein the cytokine region includes one or more polypeptides each selected from the group consisting of a polypeptide of interleukin- 12 (IL-12), IL-15, IL-2, IL-4, IL-7, IL-9, IL-18, and IL-21 or a functional portion thereof.

7. The fusion protein of claim 1, wherein the cytokine region comprises at least:a first polypeptide selected from the group consisting of a polypeptide of interleukin- 12 (IL-12), IL-15, IL-2, IL-4, IL-7, IL-9, IL-18, and IL-21 or a functional portion thereof; anda second polypeptide selected from the group consisting of a polypeptide of interleukin- 12 (IL- 12), IL- 15, IL-2, IL-4, IL-7, IL-9, IL- 18, and IL-21 or a functional portion thereof.

8. The fusion protein of claim 7, wherein the cytokine region comprises from N terminus to C terminus the first polypeptide comprising IL- 15 or a functional fragment thereof and the second polypeptide comprising IL- 18 or a functional fragment thereof.

9. The fusion protein of claim 7, wherein the cytokine region comprises from N terminus to C terminus the first polypeptide comprising IL- 18 or a functional fragment thereof and the second polypeptide comprising IL- 15 or a functional fragment thereof.

10. The fusion protein of any one of claims 1-9, wherein the transmembrane region is selected from the group consisting of a transmembrane domain of CD28, CD80, EGFR, 4-1BB, an alpha chain of a T cell receptor, a beta chain of a T cell receptor, a zeta chain of a T cell receptor, CD3 epsilon, CD4, CD5, CD8 alpha, CD9, CD16, CD19, CD22, CD33, CD37, CD45, CD64, CD86, CD 134, CD 137, and CD 154.

11. The fusion protein of any one of claims 1-10, wherein the transmembrane region is the CD28 transmembrane domain.

12. The fusion protein of claim 11, wherein the CD28 transmembrane domain comprises the amino acid sequence of SEQ ID NO: 4 or a peptide having at least 85% sequence identity to SEQ ID NO: 4.

13. The fusion protein of any one of claims 1-10, wherein the transmembrane region is the transmembrane domain of CD80.

14. The fusion protein of claim 13, wherein the CD80 transmembrane domain comprises the amino acid sequence of SEQ ID NO: 3 or a peptide having at least 85% sequence identity to SEQ ID NO: 3.

15. The fusion protein of any one of claims 1-14, wherein the costimulatory domain is selected from the group consisting of an intracellular domain (or other suitable portion) of CD28, OX-40, 4-1BB / CD137, CD2, CD7, CD27, CD30, CD40, Programmed Death- 1 (PD-1), inducible T cell costimulator (ICOS), lymphocyte function-associated antigen- 1 (LFA-1, CDl-la / CD18), CD3 gamma, CD3 delta, CD3 epsilon, CD247, CD276 (B7-H3), LIGHT, (TNFSF14), NKG2C, Ig alpha (CD79a), DAP-10, Fc gamma receptor, MHC class 1 molecule, I NF receptor proteins, an Immunoglobulin protein, cytokine receptor, integrins, Signaling Lymphocytic Activation Molecules (SLAM proteins), activating NK cell receptors, BTLA, a Toll ligand receptor, ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8alpha, CD8bcta, IL-2R beta, IL-2R gamma, IL-7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, GDI Id, ITGAE, CD103, ITGAL, GDI la, LFA-1, ITGAM, GDI lb. ITGAX, GDI 1c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD 162), LTBR, LAT, GADS, SLP-76, PAG / Cbp, CD 19a, a ligand that specifically binds with CD83, or any combination thereof.

16. The fusion protein of claim 15, wherein the costimulatory domain is the intracellular domain of CD28.

17. The fusion protein of claim 16, wherein the intracellular domain of CD28 comprises the amino acid sequence of SEQ ID NO: 5 or a peptide having at least 85% sequence identity to SEQ ID NO: 5.

18. The fusion protein of claim 15, wherein the costimulatory domain is the intracellular domain of 4- IBB.

19. The fusion protein of claim 18, wherein the intracellular domain of 4- IBB comprises the amino acid sequence of SEQ ID NO: 6 or a peptide having at least 85% sequence identity to SEQ ID NO: 6.

20. The fusion protein of any one of claims 1-5 and 10-18, comprising from N to C terminus,(i) the cytokine region comprising the IL- 12 polypeptide or functional portion thereof, the transmembrane region comprising the CD28 transmembrane domain, and the costimulatory domain comprising the intracellular domain of CD28;(ii) the cytokine region comprising the IL- 12 polypeptide or functional portion thereof, the transmembrane region comprising the CD28 transmembrane domain, and the costimulatory domain comprising the intracellular domain of 4- IBB;(iii) the cytokine region of IL- 12 polypeptide or functional portion thereof, the transmembrane region comprising the CD80 transmembrane domain, and the costimulatory domain comprising the intracellular domain of 4- IBB; or(iv) the cytokine region of IL- 12 polypeptide or functional portion thereof, the transmembrane region comprising the CD80 transmembrane domain, and the costimulatory domain comprising the CD28 intracellular domain.

21. The fusion protein of claim 20, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 10-14, or a peptide having at least 85%, 90%, 92%, 95%, or 99% sequence identity to any sequence selected from the group consisting of SEQ ID NOs: 10-14.

22. The fusion protein of any one of claims 6-18, comprising from N to C terminus, (i) the cytokine region comprising the first polypeptide comprising IL- 15 or a functional fragment thereof and the second polypeptide comprising IL- 18 or a functional fragment thereof, the transmembrane region comprising the CD28 transmembrane domain, and the costimulatory domain comprising the intracellular domain of 4- IBB;(ii) the cytokine region comprising the first polypeptide comprising IL- 15 or a functional fragment thereof and the second polypeptide comprising IL- 18 or a functional fragment thereof, the transmembrane region comprising the CD80 transmembrane domain, and the costimulatory domain comprising the intracellular domain of 4- IBB;(iii) the cytokine region comprising the first polypeptide comprising IL- 15 or a functional fragment thereof and the second polypeptide comprising IL- 18 or a functional fragment thereof, the transmembrane region comprising the CD28 transmembrane domain, and the costimulatory domain comprising the intracellular domain of CD28;(iv) the cytokine region comprising the first polypeptide comprising IL- 15 or a functional fragment thereof and the second polypeptide comprising IL- 18 or a functional fragment thereof, the transmembrane region comprising the CD80 transmembrane domain, and the costimulatory domain comprising the intracellular domain of CD28;(v) the cytokine region comprising the first polypeptide comprising IL- 18 or a functional fragment thereof and the second polypeptide comprising IL- 15 or a functional fragment thereof, the transmembrane region comprising the CD28 transmembrane domain, and the costimulatory domain comprising the intracellular domain of 4-1BB;(vi) the cytokine region comprising the first polypeptide comprising IL- 18 or a functional fragment thereof and the second polypeptide comprising IL- 15 or a functional fragment thereof, the transmembrane region comprising the CD80 transmembrane domain, and the costimulatory domain comprising the intracellular domain of 4- IBB;(vii) the cytokine region comprising the first polypeptide comprising IL- 18 or a functional fragment thereof and the second polypeptide comprising IL- 15 or a functional fragment thereof, the transmembrane region comprising the CD28 transmembrane domain, and the costimulatory domain comprising the intracellular domain of CD28; or(viii) the cytokine region comprising the first polypeptide comprising IL- 18 or a functional fragment thereof and the second polypeptide comprising IL- 15 or a functional fragment thereof, the transmembrane region comprising the CD80 transmembrane domain, and the costimulatory domain comprising the intracellular domain of CD28.

23. The fusion protein of any one of claims 6-9 or 22, comprising an amino acid sequence of SEQ ID NO: 64 or SEQ ID NO: 66, or a peptide having at least 85%, 90%, 92%, 95%, or 99% sequence identity to SEQ ID NO: 64 or SEQ ID NO: 66.

24. The fusion protein of any one of claims 1-23, further comprising a hinge region between the cytokine region and the transmembrane region.

25. The fusion protein of claim 24, wherein the hinge region is selected from a hinge domain of IgGl, IgG2, IgG3, IgG4, IgA, IgD, IgE, IgM, CD28, or CD8 alpha, any truncation thereof, or any combination thereof.

26. A fusion protein comprising from N to C terminus, a cytokine region, a transmembrane region, and a costimulatory domain comprising intracellular domain of 4-1BB.

27. The fusion protein of claim 26, wherein the intracellular domain of 4- IBB comprises the amino acid sequence of SEQ ID NO: 6 or a peptide having at least 85% sequence identity to SEQ ID NO: 6.

28. The fusion protein of claim 26 or 27, wherein the cytokine region is selected from the group consisting of a polypeptide of interleukin- 12 (IL- 12), IL- 15, IL-2, IL-4, IL-7, IL-9, IL-18, and IL-21 or a functional portion thereof.

29. The fusion protein of any one of claims 26-28, wherein the transmembrane region is selected from the group consisting of a transmembrane domain of CD28, CD80, EGFR, 4-1BB, an alpha chain of a T cell receptor, a beta chain of a T cell receptor, a zeta chain of a T cell receptor, CD3 epsilon. CD4, CD5, CD8 alpha, CD9, CD16, CD19, CD22, CD33, CD37, CD45, CD64, CD86, CD134, CD137, and CD154.

30. A fusion protein comprising, from N to C terminus,(a) a cytokine region: and(b) a transmembrane region.

31. The fusion protein of claim 30, wherein the cytokine region is selected from the group consisting of a polypeptide of interleukin- 12 (IL-12), IL-15, IL-2, IL-4, IL-7, IL-9, IL-18, and IL-21 or a functional portion thereof.

32. The fusion protein of claims 30 or 31, wherein the cytokine region is a polypeptide of IL- 12 or a functional portion thereof.

33. The fusion protein of claim 32, wherein the polypeptide of IL- 12 or the functional portion thereof comprises a IL- 12α subunit and a IL- 12β subunit, wherein the IL 12α subunit and the IL- 12β subunit are connected via a linker and form a heterodimer.

34. The fusion protein of claim 32 or 33, wherein the polypeptide of IL-12 or the functional portion thereof comprises the amino acid sequence of SEQ ID NO: 9 or a peptide having at least 85% sequence identity to SEQ ID NO: 9.

35. The fusion protein of claim 34, wherein the polypeptide of IL- 12 or the functional portion thereof comprises the amino acid sequence of SEQ ID NO: 9.

36. The fusion protein of any one of claims 30-35, wherein the transmembrane region is selected from the group consisting of a transmembrane domain of CD28, CD80, EGFR, 4- 1 BB, an alpha chain of a T cell receptor, a beta chain of a T cell receptor, a zeta chain of a T cell receptor, CD3 epsilon, CD4, CD5, CD8 alpha, CD9, CD16, CD19, CD22, CD33, CD37, CD45, CD64, CD86, CD134, CD137, and CD154.

37. The fusion protein of any one of claims 30-36, wherein the transmembrane region is a transmembrane domain of CD28.

38. The fusion protein of claim 37, wherein the transmembrane domain of CD28 comprises the amino acid sequence of SEQ ID NO: 4 or a peptide having at least 85% sequence identity to SEQ ID NO: 4.

39. The fusion protein of any one of claims 30-36, wherein the transmembrane domain is a transmembrane domain of CD80.

40. The fusion protein of claim 39, wherein the transmembrane domain of CD80 comprises the amino acid sequence of SEQ ID NO: 3 or a peptide having at least 85% sequence identity to SEQ ID NO: 3.

41. The fusion protein of claim 30, wherein:(i) the cytokine region comprises a polypeptide of interleukin- 12 (IL- 12) or a functional portion thereof, and the transmembrane region comprises a transmembrane domain of CD28: or(ii) the cytokine region comprises a polypeptide of interleukin- 12 (IL- 12) or a functional portion thereof, and the transmembrane region comprises a transmembrane domain of CD80.

42. The fusion protein of claim 30, wherein the fusion protein comprises the amino acid sequence of SEQ ID NO: 10 or an amino acid sequence having at least 85%, 90%, 92%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 10.

43. The fusion protein of claim 30, wherein the fusion protein comprises the amino acid sequence of SEQ ID NO: 10.

44. The fusion protein of claim 30, wherein the fusion protein comprises the amino acid sequence of SEQ ID NO: 11 or an amino acid sequence having at least 85%, 90%, 92%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 11.

45. The fusion protein of claim 30, wherein the fusion protein comprises the amino acid sequence of SEQ ID NO: 11.

46. A polynucleotide encoding the fusion protein of any one of claims 1-45.

47. An expression vector comprising an expressible nucleic acid sequence comprising a polynucleotide encoding the fusion protein of any one of claims 1-45.

48. The expression vector of claim 47, further comprising a promoter operably linked to the expressible nucleic acid sequence.

49. The expression vector of claim 48, wherein the promoter is constitutive or inducible.

50. The expression vector of claim 49, wherein the constitutive promoter is selected from the group consisting of EF1α, CMV, SV40 or CAG.

51. The expression vector of claim 50, wherein the constitutive promoter is EF1α or CMV.

52. The expression vector of claim 49, wherein the inducible promoter is selected from the group consisting of alcohol-regulated promoters, tetracycline-regulated promoters, steroid-regulated promoters, metal-regulated promoters, pathogenesis-regulated promoters, temperature / heat-inducible promoters, and light-regulated promoters.

53. The expression vector of claim 49, wherein the inducible promoter is NFAT.

54. The expression vector of any one of claims 47-53, further comprising a woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) and / or a poly A tail 3’ end to the expressible nucleic acid sequence.

55. The expression vector of any one of claims 47-54, wherein the expressible nucleic acid sequence further comprises a second protein encoding sequence.

56. The expression vector of claim 55, wherein the fusion protein encoding polynucleotide and the second protein encoding sequence are linked bicistronically.

57. The expression vector of any one of claims 55-56, wherein the fusion protein encoding polynucleotide and the second protein encoding sequence are separated by a selfcleaving peptide or an internal ribosome entry site (IRES).

58. The expression vector of claim 57, wherein the self-cleaving protein is a 2A peptide.

59. The expression vector of claim 58, wherein the 2A peptide is selected from the group consisting of Foot-and-Mouth Disease Virus 2A (F2A), Thosea asigna virus 2A (T2A), Porcine tescho virus- 1 2A (P2A), Equine rhinitis A virus (E2A), and Thosea asigna Virus Short 2A (S2A).

60. The expression vector of any one of claims 47-59, comprising from 5’ to 3’, an EF1α promoter and the expressible nucleic acid sequence comprising a polynucleotide encoding the fusion protein, a T2A peptide, and a woodchuck hepatitis virus posttranscriptional regulatory element (WPRE).

61. The expression vector of any one of claims 47-60, wherein the fusion protein comprises a signal peptide.

62. The expression vector of any one of claims 55-61, wherein the second protein comprises a signal peptide.

63. An isolated immune cell comprising the fusion protein of any one of claims 1-45, the fusion protein encoding polynucleotide of claim 45 or the expression vector of any one of claims 46-61.

64. The isolated immune cell of claim 63, which expresses the fusion protein.

65. The isolated immune cell of claim 63 or 64, which is selected from the group consisting of T cells, B cell, natural killer cells, macrophages, neutrophils, eosinophils, basophils, and dendritic cells.

66. The isolated immune cell of any one of claims 63-65, wherein the T cells are one or more selected from the group consisting of alpha beta (a ) T cells, and gamma delta (y5) T cells.

67. The isolated immune cell of claim 66, wherein the alpha beta (a ) T cells are one or more selected from the group consisting of CD4+ / CD8+ double positive T cells, CD8+ cytotoxic T cell, CD4+ helper T cell (such as Thl, Th2, Thl7, Th3, or Th9 cells), follicular helper T (Tfh) cells, natural killer T (NKT) cells, tumor infiltrating lymphocytes (TILs), memory T cells (e.g. central memory T cells (Tcm cells), effector memory T cells (Tem cells and TEMRA cells), naive T cells, regulatory T cell (Treg), CAR-T cells and TCR-T cells.

68. The isolated immune cell of claim 67, wherein the TILs comprise CD8+ cytotoxic T cells, CD4+ T cells, and memory T cells.

69. The isolated immune cell of any one of claims 63-68, wherein a chimeric antigen receptor (CAR) is co-expressed.

70. The isolated immune cell of claim 69, wherein the CAR comprises an antigen binding domain, a transmembrane domain, and an intracellular costimulatory domain.

71. The isolated immune cell of claim 70, wherein the intracellular costimulatory domain of the CAR is a CD28 intracellular costimulatory domain, and wherein a costimulatory domain of the fusion protein is an intracellular domain of 4- IBB.

72. The isolated immune cell of claim 70, wherein the intracellular costimulatory domain of the CAR is a 4- IBB intracellular costimulatory domain, and wherein a costimulatory domain of the fusion protein is an intracellular domain of 4- IBB.

73. The isolated immune cell of any one of claims 63-72, wherein a T cell receptor (TCR) is co-expressed.

74. The isolated immune cell of claim 73, wherein the TCR is an engineered TCR.

75. The isolated immune cell of claim 74, wherein the engineered TCR is anti-NY-ESO-1 T cell receptor (1G4).

76. The isolated immune cell of any one of claims 69-75, wherein the CAR or the TCR targets a tumor antigen.

77. The isolated immune cell of claim 76, wherein the tumor antigen is selected from the group consisting of CLDN18.2 (claudin 18.2), 5T4 (trophoblast glycoprotein), 707-AP, 9D7, AFP (a-fetoprotein), AlbZIP (androgen-induced bZIP), HPG1 (human prostate specific gene-l), a.5b 1 -Integrity a.5p6-Integrin, a -methylacyl-coenzyme A racemase, ART-4 (ADPribosyltransf erase-4), B7H4 (v-set domain-containing T-cell activation inhibitor 1), BAGE-1 (B melanoma antigen- 1), BCL-2 (B-cell CLL / lymphoma-2), BING-4 (WD repeat domain 46), CA E5-3 / CA 27-29 (mucin 1), CA 19-9 (cancer antigen 19-9), CA 72-4 (cancer antigen 72-4), CAI 25 (cancer antigen 125), calreticulin, CAMEL (CTL-recognized antigen on melanoma), C ASP-8 (caspase 8), cathepsin B, cathepsin L, CD 1 (cluster of differentiation 19), CD20, CD22, CD25, CD30, CD33, CD4, CD52, CD55, CD56, CD80, CEA (carcinoembryonic antigen SG8), CLCA2 (chloride channel accessory 2), CML28 (chronic myelogenous leukemia tumor antigen 28), Coactosin-like protein, Collagen XXIII, COX-2 (cyclooxygenase-2), CT-9 / BRD6 (cancer / testis antigen 9), Cten (c-terminal tensin-like protein), cyclin Bl, cyclin DI, cyp-B, CYPB1 (cytochrome p450 family 1 subfamily b member 1), DAM-10 / MAGE-B1 (melanoma-associated antigen Bl), DAM-6 / MAGE-B2, EGFR / Herl (epidermal growth factor receptor), EMMPRIN (basigin), EpCam, EphA2 (EPH receptor A2), EphA3, ErbB3 (Erb-B2 receptor tyrosine kinase 3), EZH2 (enhancer of zeste 2 poly comb repressive complex 2 subunit), FGF-5 (fibroblast growth factor 5), FN (fibronectin), Fra-1 (Fosrelated antigen-1), G250 / CAIX (carbonic anhydrase 9), GAGE-1 (G antigen-1), GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7b, GAGE-8, GDEP (gene differentially expressed in prostate), Gn T-V (gluconate kinase), gp100 (melanocytes lineage-specific antigen GP100), GPC3 (glypican3), HAGE (helical antigen), HAST-2 (sulfotransferase family 1 A member 1), hepsin, Her2 / neu / ErbB2 (Erb-B2 receptor tyrosinekinase 2), HERV-K-MEL, HNE (medullasin), homeobox NKX 3.1, HOM-TES-14 / SCP-1, HOM-TES-85, HPV-E6, HPVE7, HST-2 (sirtuin-2), hTERT, iCE (caspase 1), IGF-1R (insulin like growth factor- 1 receptor), IL-13Ra2 (interleukin- 13 receptor subunit a 2), IL-2R (interleukin-2 receptor), IL-5 (interleukin-5), immature laminin receptor, kallikrein 2, kallikrein 4, Ki67, KIAA0205 (lysophosphatidylglycerol acyltransf erase 1), KK-LC-1 (kita-kyushu lung cancer antigen- 1), KM-HN-1, LAGE-1 (L antigen family member- 1), Livin, MAGE-A1, MAGE- A 10, MAGE-A12, MAGEA2, MAGE- A3, MAGE-A4, MAGE-A6, MAGE-A9, MAGE-B1, MAGE-BIO, MAGE-B16, MAGEB17, MAGE-B2, MAGE-B3, MAGE-B4, MAGE-B5, MAGE-B6, MAGE-CI, MAGE-C2, MAGE-C3, MAGE-D1, MAGE-D2, MAGE-D4, MAGE-E1, MAGE-E2, MAGE-F1, MAGE-H1, MAGEL2 (melanoma antigen family L2), mammaglobin A, M ART- 1 / Mel an- A (melanoma antigen recognized by T-cells-1), MART-2, matrix protein 22, MC1R (melanocortin 1 receptor), M-CSF (macrophage colony-stimulating factor), Mesothelin, MG50 / PXDN (peroxidasin), MMP 11 (matrix metalloprotease 11), MN / CA IX-antigen (carbonic anhydrase 9), MRP-3 (multidrug resistance-associated protein-3), MUC1 (mucin 1), MUC2, NA88-A (VENT-like homeobox 2 pseudogene 1), N-acetylglucos-aminyltransf erase- V, Neo-PAP (Neo-poly (A) polymerase), NGEP (new gene expressed in prostate), NMP22 (nuclear matrix protein 22), NPM / ALK (nucleophosmin), NSE (neuron-specific enolase), NY-ESO-1, NY-ESO-B, OA1 (osteoarthritis QTL 1), OFA-iLRP (oncofetal antigen immature laminin receptor protein), OGT (O-GlcNAc transferase), OS-9 (endoplasmic reticulum lectin), osteocalcin, osteopontin, p 15 (CDK inhibitor 2B), p53, PAGE-4 (P antigen family member-4), PAI-1 (plasminogen activator inhibitor- 1), PAL2, PAP (prostatic acid phosphatase), PART-1 (prostate androgen-regulated transcript 1 ), PATE (prostate and testis expressed 1 ), PDEF (prostate-derived Ets factor), Pim-l-Kinase (proviral integration site 1), Pinl (Peptidyl-prolyl cis-trans isomerase NIMA-interacting 1), POTE (expressed in prostate, ovary, testis, and placenta), PRAME (preferentially expressed antigen in melanoma), prostein, proteinase-3, PSA (prostate-specific antigen), PSCA (prostate stem cell antigen), PSGR (prostate-specific G-protein coupled receptor), PSM, PSMA (prostate specific membrane antigen), RAGE-1 (renal tumorcarcinoma antigen), RHAMM / CD168, RET1 (renal ubiquitous protein 1), RET2, SAGE (sarcoma antigen), SART-1 (squamous cell carcinoma antigen recognized by T-cells-1), SART-2, SART-3, Spl7 (sperm protein 17), SSX-1 (SSX family member 1), SSX-2 / H0M-MEL-40, SSX-4, STAMP4 (STEAP2 metalloreductase), STEAP, survivin, survivin-213, TA-90 (tumor associated antigen-90), TAG-72 (tumor associated glycoprotein-72), TARP (TCRy alternate reading frame protein), TGFb (transforming growth factor b), TGFbRl 1 (transforming growth factor b receptor 11), TGM-4 (transglutaminase 4), TRAG-3 (taxol resistance associated gene 3), TRG (T-cell receptor g locus), TRP-1 (transient receptor potential-1), TRP-2 / 6b, TRP-2 / INT2, Trp-p8, Tyrosinase, UPA (U-plasminogen activator), VEGF (vascular endothelial growth factor A), VEGFR-2 / FLK- 1 and WT1 (wilms tumor 1), a-actinin-4 / m, ARTCl / m, bcr / abl, bcta-Catcnin / m, BRCAl / m, BRCA2 / m, CASP-5 / m, CASP-8 / m, CDC27 / m, CDK4 / m, CDKN2A / m, CME66, COA-l / m, DEK-CAN, EFTUD2 / m, ELF2 / m, ETV6-AML1, FNl / m, GPNMB / m, HLA-A* 0201-R 1701, HLA-A1 1 / m, HLA-A2 / m, HSP70-2M, KIAA0205 / m, K-Ras / m, LDLR-FUT, MART2 / m, MEl / m, METM-l / m, MEiM-2 / m, MUM-3 / m, Myosin class 1 / m, neo-PAP / m, NFYC / m, N- Ras / m, OGT / m, OS-9 / m, p53 / m, Pml / RARa, PRDX5 / m, PTPRX / m, RBAF600 / m, SIRT2 / m, SYTSSX-l, SYT-SSX-2, TEL-AML1, TGFbRII and TPI / m.

78. A pharmaceutical composition comprising the isolated immune cell of any one of claims 63-77.

79. A method of producing an isolated immune cell of any one of claims 63-77, comprising(i) introducing the fusion protein encoding polynucleotide of claim 46, or the expression vector of any one of claims 47-62 into the immune cell; and(ii) maintaining the immune cell under conditions in which the fusion protein encoding polynucleotide or the expression vector is expressed.

80. The method of claim 79, wherein the fusion protein encoding polynucleotide is transduced with a virus.

81. The method of claim 80, wherein the virus is a lentivirus or retrovirus.

82. The method of claim 79, wherein the expression vector is derived from a viral vector.

83. The method of claim 82, wherein the viral vector is a lentivirus or retrovirus vector.

84. A method of treating cancer in a subject in need thereof, comprising(i) co-expressing the fusion protein of any one of claims 1-45 and a CAR or TCR in an immune cell, and administering the immune cell to the subject; or(ii) administering to the subject an immune cell expressing the fusion protein of any one of claims 1-45 and a CAR or TCR.

85. The method of claim 84, wherein the TCR is an engineered TCR.

86. The method of claim 84 or 85, wherein the cancer is solid tumor.

87. The method of claim 86, wherein the solid tumor comprises brain cancer, bladder cancer, liver cancer, colon cancer, rectal cancer, endometrial cancer, pancreatic cancer, small cell lung cancer, non-small cell lung cancer, breast cancer, urethral cancer, head and neck cancer, gastrointestinal cancer, stomach cancer, oesophageal cancer, ovarian cancer, renal cancer, melanoma, cervix cancer, rectum cancer, larynx cancer, prostate cancer and thyroid cancer.

88. The method of claim 84 or 85, wherein the cancer is hematological malignancy.

89. The method of claim 88, wherein the hematological malignancy comprises acute and chronic leukemias (acute myelogenous leukemia (AML), acute lymphoblastic leukemia (ALL), chronic myelogenous leukemia (CML), lymphomas, non-Hodgkin lymphoma (NHL), Hodgkin’s disease, multiple myeloma, and myelodysplastic syndromes.

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