Fusion protein and method of use thereof

Abstract

This disclosure relates to a fusion protein (e.g., a reverse cytokine receptor) comprising the extracellular domain of an immunosuppressive cytokine receptor (e.g., IL-4R) and the intracellular domain of an immunostimulatory receptor (e.g., an IL-23 receptor complex). This fusion protein (e.g., a reverse cytokine receptor) binds to inhibitory / suppressive cytokines and converts their intracellular signals into immunostimulatory / activating signals. This disclosure further relates to polynucleotides encoding such fusion proteins, modified cells expressing such fusion proteins, therapeutic uses of such fusion proteins, and pharmaceutical compositions comprising such fusion proteins.

Description

[Technical Field]

[0001] cross reference This application claims priority to International Patent Application No. PCT / CN2023 / 107742, filed on 17 July 2023, which is incorporated herein by reference in whole.

[0002] Sequence List This application refers by reference to the sequence listing titled "IEC240173PCT SEQUENCE LISTING.xml", which was submitted with this application as an XML file, created on July 16, 2024, and has a size of 51,855 bytes.

[0003] Technical field This disclosure relates to a fusion protein (e.g., a reverse cytokine receptor) comprising the extracellular domain of an immunosuppressive cytokine receptor (e.g., IL-4R) and the intracellular domain of an immunostimulatory receptor (e.g., an IL-23 receptor complex). [Background technology]

[0004] background Adoptive transfer of T cells genetically modified by chimeric antigen receptors (CARs) is considered highly promising as a treatment for hematological malignancies, but the application of CAR-T cells to the treatment of solid tumors has achieved only limited success. Existing theories suggest that the immunosuppressive microenvironment of solid tumors, characterized by limited nutrients and oxygen, accumulation of inhibitory cells and cytokines, vascular disorder, and endothelial dysfunction, poses a major obstacle to cancer immunotherapy, including CAR-T cell therapy.

[0005] To increase immune cell proliferation and antitumor efficacy in an inhibitory microenvironment, it is necessary to modify CAR-expressing immune cells to be inherently resistant to anti-inflammatory cytokines. Reverse cytokine receptors (ICRs) can be used to increase the potency of immune cells (e.g., T cells). [Overview of the project]

[0006] overview This disclosure relates to a fusion protein (e.g., a reverse cytokine receptor (ICR)) comprising the extracellular domain of an immunosuppressive cytokine receptor (e.g., IL-4R) and the intracellular domain of an immunostimulatory receptor (e.g., an IL-23 receptor complex). This fusion protein binds to inhibitory / suppressive cytokines and converts their intracellular signals into immunostimulatory / activating signals. This disclosure further provides nucleic acids encoding such fusion proteins, polypeptides containing such fusion proteins, modified cells expressing such fusion proteins, therapeutic uses of such fusion proteins, and pharmaceutical compositions containing such fusion proteins.

[0007] In one embodiment, the present disclosure relates to a fusion protein comprising (1) an extracellular domain that specifically binds to interleukin-4 (IL-4), and (2) an intracellular domain; the fusion protein transmits interleukin-23 (IL-23) pathway signals upon binding to IL-4.

[0008] In some embodiments, the fusion protein comprises a first polypeptide chain and a second polypeptide chain.

[0009] In some embodiments, the first polypeptide chain comprises (1) a first extracellular domain including an interleukin-4 receptor alpha (IL-4Rα) extracellular domain, and (2) a first intracellular domain including an interleukin-12 receptor beta-1 subunit (IL-12Rβ1) intracellular domain or an interleukin-23 receptor (IL-23R) intracellular domain.

[0010] In some embodiments, the second polypeptide chain comprises (1) a second extracellular domain including an interleukin-2 receptor gamma (IL-2Rγ) extracellular domain, and (2) a second intracellular domain including an IL-12Rβ1 intracellular domain or an IL-23R intracellular domain.

[0011] In some embodiments, the first polypeptide chain comprises (1) a first extracellular domain containing an IL-4Rα extracellular domain and (2) a first intracellular domain containing an IL-12Rβ1 intracellular domain, and the second polypeptide chain comprises (1) a second extracellular domain containing an IL-2Rγ extracellular domain and (2) a second intracellular domain containing an IL-23R intracellular domain.

[0012] In some embodiments, the first polypeptide chain comprises (1) a first extracellular domain containing an IL-4Rα extracellular domain and (2) a first intracellular domain containing an IL-23R intracellular domain, and the second polypeptide chain comprises (1) a second extracellular domain containing an IL-2Rγ extracellular domain and (2) a second intracellular domain containing an IL-12Rβ1 intracellular domain.

[0013] In one embodiment, this disclosure, (a) (1) The first extracellular domain containing the IL-4Rα extracellular domain, and (2) First intracellular domain containing the IL-12Rβ1 intracellular domain A first polypeptide chain including, (1) A second extracellular domain including the IL-2Rγ extracellular domain, and (2) Second intracellular domain containing the IL-23R intracellular domain A second polypeptide chain including including; or (b) (1) The first extracellular domain containing the IL-4Rα extracellular domain, and (2) First intracellular domain containing the IL-23R intracellular domain A first polypeptide chain including, (1) A second extracellular domain including the IL-2Rγ extracellular domain, and (2) Second intracellular domain containing the IL-12Rβ1 intracellular domain A second polypeptide chain containing and Regarding fusion proteins, including those mentioned above.

[0014] In some embodiments, the first polypeptide chain and the second polypeptide chain are linked to each other by a 2A cleavage - capable linker.

[0015] In some embodiments, the first extracellular domain and the second extracellular domain form a binding site for IL - 4, the first intracellular domain and the second intracellular domain form an IL - 23 receptor complex, and when the fusion protein binds to IL - 4, signal transduction is transmitted through the IL - 23 receptor complex.

[0016] In some embodiments, the IL - 4Rα extracellular domain comprises the amino acid sequence shown in SEQ ID NO: 1, or an amino acid sequence having at least 90%, 95%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 1.

[0017] In some embodiments, the IL - 2Rγ extracellular domain comprises the amino acid sequence shown in SEQ ID NO: 5, or an amino acid sequence having at least 90%, 95%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 5.

[0018] In some embodiments, the IL - 12Rβ1 intracellular domain comprises the amino acid sequence shown in SEQ ID NO: 4, or an amino acid sequence having at least 90%, 95%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 4.

[0019] In some embodiments, the IL - 23R intracellular domain comprises the amino acid sequence shown in SEQ ID NO: 8, or an amino acid sequence having at least 90%, 95%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 8.

[0020] In some embodiments, the fusion protein further comprises a transmembrane domain.

[0021] In some embodiments, the transmembrane domain is selected from the group consisting of the transmembrane domain of IL-4Rα, the transmembrane domain of IL-2Rγ, the transmembrane domain of IL-12Rβ1, and the transmembrane domain of IL-23R.

[0022] In some embodiments, the transmembrane domain includes the amino acid sequence shown in SEQ ID NO: 3 or 7, or an amino acid sequence having at least 90%, 95%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 3 or 7.

[0023] In some embodiments, the fusion protein comprises a first transmembrane domain in the first polypeptide chain and a second transmembrane domain in the second polypeptide chain; optionally, the first and second transmembrane domains may be dimerized.

[0024] In some embodiments, the fusion protein further includes a connecting sequence located between the C-terminus of the extracellular domain and the N-terminus of the transmembrane domain.

[0025] In some embodiments, the connecting sequence includes the amino acid sequence shown in SEQ ID NO: 2 or 6, or an amino acid sequence having at least 90%, 95%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 2 or 6.

[0026] In some embodiments, the fusion protein comprises a first and / or second polypeptide chain having an amino acid sequence that is at least 90%, 95%, or 99% identical to the amino acid sequence shown in any of SEQ ID NOs: 9 to 14.

[0027] In some embodiments, the fusion protein is a reverse cytokine receptor (ICR).

[0028] In one embodiment, the present disclosure relates to nucleic acids comprising one or more nucleic acid sequences encoding a fusion protein or a portion thereof as described herein.

[0029] In some embodiments, the nucleic acid further comprises a second nucleic acid sequence encoding a modified receptor, the modified receptor comprising an extracellular antigen-binding domain or a ligand-binding domain and optionally an intracellular signaling domain.

[0030] In some embodiments, the nucleic acid sequence encoding the modified receptor is located upstream or downstream of at least one of the one or more nucleic acid sequences encoding the fusion protein, and optionally the modified receptor nucleic acid sequence and the fusion protein nucleic acid sequence are separated by a linker nucleic acid sequence encoding a 2A cleavable linker.

[0031] In some embodiments, the 2A cleavable linker includes the amino acid sequence shown in any one of SEQ ID NOs: 31-33, or a functional variant having at least about 90% sequence identity thereto.

[0032] In some embodiments, the modified receptor is selected from the group consisting of a modified T cell receptor (TCR), a chimeric antigen receptor (CAR), a T cell antigen coupling compound (TAC), or a portion thereof.

[0033] In some embodiments, the modified receptor is a CAR.

[0034] In some embodiments, CAR comprises an extracellular antigen-binding domain that specifically binds to an antigen, and the antigens include BCMA, CLL1, CD4, GPC3, GPRC5D, GU2CYC, CD19, MUC16, MUC1, CAIX, CEA, CD8, CD7, CD10, CD20, CD22, CD30, CD33, CD34, CD38, CD41, CD44, CD49f, CD56, CD74, CD133, CD138, EGP-2, EGP-40, EpCAM, ERBB2 (HER-2), ERBB3, ERBB4, FBP, fetal acetylcholine receptor, folate receptor-α, GD2, GD3, hTERT, IL-13R-α2, κ- The tumor antigen is selected from the group consisting of light chain, KDR, LeY, L1 cell adhesion molecule, MAGE-A1, mesothelin, MAGEA3, p53, MART1, GP100, proteinase-3 (PR3), tyrosinase, survivorbin, hTERT, EphA2, NY-ESO-1, h5T4, PSCA, PSMA, ROR1, TAG-72, VEGF-R2, WT-1, CD123, CD44V6, NKCS1, IGF1R, EGFR, EGFR-VIII, claudin 18.2, claudin 6, NKG2D, delta-like 3 (DLL3), CD70, CS-1, c-Met, glycolipid F77, PD-L1, and PD-L2.

[0035] In some embodiments, the tumor antigen is GPC3.

[0036] In some embodiments, the CAR includes a primary intracellular signaling domain in immune cells and / or a co-stimulatory signaling domain.

[0037] In some embodiments, the primary intracellular signaling domain is derived from CD3ζ, and the co-stimulatory signaling domain is derived from a co-stimulatory molecule selected from the group consisting of ligands for CD27, CD28, CD137, OX40, CD30, CD40, CD3, LFA-1, ICOS, CD2, CD7, LIGHT, NKG2C, B7-H3, CD83, and combinations thereof.

[0038] In some embodiments, the CAR comprises a transmembrane domain derived from a molecule selected from the group consisting of CD8α, CD4, CD28, CD137, CD80, CD86, CD152, and PD1.

[0039] In some embodiments, the CAR further includes a hinge domain located between the C-terminus of the extracellular antigen-binding domain and the N-terminus of the transmembrane domain.

[0040] In some embodiments, the CAR further comprises a signal peptide located at the N-terminus of the extracellular antigen-binding domain.

[0041] In some embodiments, the nucleic acid encodes the amino acid sequence shown in any one of SEQ ID NOs: 9-14 and 20-22, or a functional variant having at least about 90% sequence identity thereto.

[0042] In one embodiment, this disclosure relates to a vector comprising nucleic acid as described herein.

[0043] In one embodiment, this disclosure relates to modified cells comprising a fusion protein, a nucleic acid, and / or a vector as described herein.

[0044] In some embodiments, the modified cells further include a modified receptor, which comprises an extracellular antigen-binding domain or a ligand-binding domain and optionally an intracellular signaling domain.

[0045] In some embodiments, the modified receptor is selected from the group consisting of a modified T cell receptor (TCR), a chimeric antigen receptor (CAR), a T cell antigen coupling compound (TAC), or a portion thereof.

[0046] In some embodiments, the modified receptor specifically binds to the antigen.

[0047] In some embodiments, the modified cells include a polypeptide encoding a fusion protein and / or a polypeptide encoding a CAR, wherein the polypeptide includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 9-14 and 20-22, or a functional variant having at least about 90% sequence identity thereto.

[0048] In some embodiments, the modified cells are immune cells.

[0049] In some embodiments, immune cells are selected from the group consisting of T cells, NK cells, peripheral blood mononuclear cells (PBMCs), hematopoietic stem cells, pluripotent stem cells, embryonic stem cells, and combinations thereof.

[0050] In some embodiments, the immune cells are T cells, and optionally, the immune cells are αβT cells or γδT cells.

[0051] In one embodiment, this disclosure relates to a pharmaceutical composition comprising modified cells as described herein and a pharmaceutically acceptable carrier.

[0052] In one embodiment, the present disclosure relates to a method for producing modified cells, the method comprising introducing a vector described herein into cells.

[0053] In one embodiment, the present disclosure relates to a method for treating a disease or disorder in a subject, comprising administering to the subject in need a therapeutically effective amount of the modified cells described herein or the pharmaceutical composition described herein.

[0054] In some embodiments, the disease or disorder is cancer, autoimmune disease, or tumor.

[0055] In some embodiments, the disease or disorder is breast cancer, lung cancer, pancreatic cancer, melanoma, oral cancer, mesothelioma, ovarian cancer, colorectal cancer, gastric cancer, cervical cancer, brain cancer, skin cancer, multiple myeloma, lymphoma, epithelial neoplasm, soft tissue sarcoma, esophageal cancer, or CNS tumor. [Brief explanation of the drawing]

[0056] [Figure 1] Figures 1a and 1b show the design schemes for two IL4R-IL23R reverse cytokine receptors (ICRs). The IL4R-IL23R ICR consists of two fusion polypeptide chains, which are formed by cleavage of a self-cleaving 2A linker after expression of the IL4R-IL23R ICR nucleic acid sequence. The two polypeptide chains can form a dimeric fusion protein. As shown in Figure 1a, the nucleic acid sequence of IL4R-IL23R#01 includes, from the 5' end to the 3' end, the IL-4Rα extracellular domain (ECD), as well as the IL-12Rβ1 transmembrane domain (TM) and intracellular domain (ICD), a 2A linker, the IL-2Rγ extracellular domain (ECD), and the IL-23R transmembrane domain (TM) and intracellular domain (ICD). As shown in Figure 1b, the nucleic acid sequence of IL4R-IL23R#02 contains the IL-2Rγ extracellular domain (ECD), the IL-12Rβ1 intracellular domain (ICD) 2A linker, the IL-4Rα extracellular domain (ECD), and the IL-23R intracellular domain (ICD) from the 5' to the 3' end. This ICR sequence was then ligated to the CAR encoding sequence using a self-cleaving 2A (e.g., P2A) linker. [Figure 2] Figures 2a-2h show the activation of the pSTAT signaling pathway in CAR-T cells after in vitro treatment with IL-4. Figures 2a-2f show histograms of phosphorylated STAT3 and STAT4 expression by flow cytometry under different concentrations of IL-4. Figures 2g-2h show the median fluorescence intensity (median) of phosphorylated STAT3 and STAT4 expression under IL-4. The results show that after IL-4 treatment, the expression levels of pSTAT3 and pSTAT4 in H93-IL4R-IL23R#01 and H93-IL4R-IL23R#02 CAR-T cells increased significantly, while there was no clear change in the expression levels of pSTAT3 and pSTAT4 in H93 CAR-T cells. [Figure 3]Figures 3a–3c show the expansion capacity of HCC CAR-T cells after treatment with five stimulation rounds in a rechallenge assay. CAR positivity (Figure 3a), CAR-T cell amplification factor (Figure 3b), and CAR-T cell viability were detected (Figure 3c). After sequential antigen stimulation, H93-IL4R-IL23R#01 CAR-T cells showed superior expansion capacity. R0, R1, R2, R3, R4, and R5 refer to rounds 1, 2, 3, 4, and 5, respectively. [Figure 4] Figures 4a and 4b show the results of in vitro long-term cytotoxicity assays of GPC3 CAR-T cells against HCC-positive cell lines (Hep3B2.1-7) with and without 10 ng / mL of IL-4, respectively. These results indicate that the long-term cytotoxicity of H93-IL4R-IL23R#01 CAR-T cells was significantly enhanced in the presence of 10 ng / mL of IL-4. [Figure 5] Figures 5a and 5b show the IFNγ and TNFα release of GPC3 CAR-T cells co-cultured with target cells in long-term cytotoxicity assays, with and without 10 ng / mL of IL-4, respectively. In the presence of 10 ng / mL of IL-4, IFNγ and TNFα release from H93-IL4R-IL23R#01 CAR-T cells was upregulated, while IFNγ and TNFα release from H93 CAR-T cells was significantly reduced. [Figure 6-1] Figure 6 shows the sequence described in this application. [Figure 6-2] See the explanation in Figure 6-1. [Figure 6-3] See the explanation in Figure 6-1. [Figure 6-4] See the explanation in Figure 6-1. [Figure 6-5] See the explanation in Figure 6-1. [Figure 6-6] See the explanation in Figure 6-1. [Modes for carrying out the invention]

[0057] Detailed explanation Cytokines are a broad category of small proteins crucial for cellular signaling. Cytokines are peptides and cannot cross the cellular lipid bilayer into the cytoplasm. Cytokines have been shown to act as immunomodulators in autocrine, paracrine, and endocrine signaling. Among these cytokines, interleukins (ILs) are particularly important, typically expressed and secreted by leukocytes (white blood cells) and other somatic cells. Each cytokine has a distinct function and can elicit different responses depending on its target, cellular source, and different stages of the immune response. It is well recognized that cytokines, chemokines, and their receptors play a critical role in regulating the functional and phenotypic characteristics of CAR-expressing cells (e.g., CAR-T cells); influencing parameters such as persistence, transport, memory cell formation, and proliferation.

[0058] Immunosuppressive cytokines, including IL-10, TGFβ, and IL-4, are key components of the tumor microenvironment (TME) that contribute to CAR-T cell dysfunction. These cytokines induce immunosuppression through several mechanisms, including the recruitment and activation of regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs), and the inhibition of effector function in CAR-T cells. In addition, these cytokines can inhibit the activity of several endogenous antitumor immune cells, such as T cells, NK cells, dendritic cells, and M1 macrophages.

[0059] To enhance the efficacy of immune cells in an inhibitory microenvironment, CAR-expressing immune cells can be modified to become inherently resistant to anti-inflammatory cytokines. Reverse cytokine receptors (ICRs) are modified cytokine receptors designed to enhance immune cell activity by overcoming cytokine-induced immunosuppression.

[0060] This disclosure provides a fusion protein comprising (1) an extracellular domain that specifically binds to interleukin-4 (IL-4) and (2) an intracellular domain; the fusion protein, upon binding to IL-4, transmits interleukin-23 (IL-23) pathway signals. In some embodiments, the fusion protein is a reverse cytokine receptor (ICR). The ICR may comprise an extracellular domain of an immunosuppressive cytokine receptor (e.g., IL-4R) and an intracellular domain of an immunostimulatory receptor (e.g., IL-23R).

[0061] As used herein, the term “reverse cytokine receptor” or “ICR” generally refers to a chimeric protein comprising the extracellular domain of one cytokine receptor and the intracellular domain of another cytokine receptor. This chimeric protein can be designed to act as a “switch receptor” capable of counteracting the effects of immunosuppressive cytokines in certain diseases, particularly cancer. ICRs can be created by replacing the extracellular domain of a cytokine receptor that recognizes immunosuppressive cytokines with the extracellular domain of a cytokine receptor that recognizes cytokines involved in immune activation. When an ICR encounters an immunosuppressive cytokine, it can trigger downstream signaling pathways of the activated cytokine receptor, thus potentially helping to restore or enhance immune function.

[0062] As used herein, the term "IL-4Rα" refers to a polypeptide derived from wild-type IL-4 receptor alpha or a functional variant thereof. IL-4Rα may be wild-type IL-4Rα (e.g., human IL-4Rα). IL-4Rα may have one or more mutations (e.g., insertions, deletions, or substitutions). IL-4Rα may be human IL-4Rα. The extracellular domain of IL-4Rα may encompass extracellular domains derived from wild-type IL-4Rα or a functional variant thereof, or a portion thereof.

[0063] As used herein, the term "IL-23R" refers to polypeptides derived from wild-type IL-23R or its functional variants. IL-23R may be wild-type IL-23R (e.g., human IL-23R). IL-23R may have one or more mutations (e.g., insertions, deletions, or substitutions). IL-23R may be human IL-23R.

[0064] As used herein, the term "IL-2Rβ" refers to polypeptides derived from wild-type IL-2Rβ or its functional variants. IL-2Rβ may be wild-type IL-2Rβ (e.g., human IL-2Rβ). IL-2Rβ may have one or more mutations (e.g., insertions, deletions, or substitutions). IL-2Rβ may be human IL-2Rβ.

[0065] As used herein, the term "IL-2Rγ" refers to a polypeptide derived from wild-type IL-2Rγ or a functional variant thereof. IL-2Rγ may be wild-type IL-2Rγ (e.g., human IL-2Rγ). IL-2Rγ may have one or more mutations (e.g., insertions, deletions, or substitutions). IL-2Rγ may be human IL-2Rγ.

[0066] As used herein, the term "IL-12Rβ1" refers to a polypeptide derived from wild-type IL-12Rβ1 or a functional variant thereof. IL-12Rβ1 may be wild-type IL-12Rβ1 (e.g., human IL-12Rβ1). IL-12Rβ1 may have one or more mutations (e.g., insertions, deletions, or substitutions). IL-12Rβ1 may be human IL-12Rβ1.

[0067] As used herein, the terms “extracellular domain” or “extracellular region” are used synonymously to refer to the portion of the receptor located outside the cell membrane. The extracellular domain may be the entire portion of the receptor located outside the cell membrane, or only a portion thereof (for example, at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the entire portion). The extracellular domain may be derived from the extracellular domain of the wild-type receptor or a functional variant thereof. The extracellular domain may have one or more mutations, including, for example, insertions, deletions, or substitutions. The IL-4Rα extracellular domain may be derived from the extracellular domain of wild-type IL-4Rα or a functional variant thereof, or a portion thereof.

[0068] As used herein, the terms “intracellular domain,” “intracellular region,” or “cytoplasmic region” are used synonymously to refer to the portion of a receptor located inside the cell. The intracellular domain may be the entire portion of the receptor located inside the cell, or only a portion thereof (for example, at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the entire portion). The intracellular domain may originate from the intracellular domain of the wild-type receptor or a functional variant thereof. The intracellular domain may have one or more mutations, including, for example, insertions, deletions, or substitutions. The IL-23R intracellular domain may be an intracellular domain derived from wild-type IL-23R or a functional variant thereof, or a portion thereof. The IL-12Rβ1 intracellular domain may be an intracellular domain derived from wild-type IL-12Rβ1 or a functional variant thereof, or a portion thereof.

[0069] As used herein, the terms “transmembrane domain” or “transmembrane region” are used synonymously to refer to the portion of the receptor embedded in the cell membrane. The transmembrane domain may be the entire portion of the receptor embedded in the cell membrane, or only a portion thereof (e.g., at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the entire portion). The transmembrane domain may originate from the transmembrane domain of the wild-type receptor or a functional variant thereof. The transmembrane domain may have one or more mutations, including, for example, insertions, deletions, or substitutions. The transmembrane domain may be the transmembrane domain of an ICR. The transmembrane domain may be the transmembrane domain of a CAR.

[0070] As used herein, the terms “hinge domain” or “hinge region” are used synonymously to refer to the portion of a receptor that connects the transmarine domain to the extracellular domain. The hinge region may be part of the extracellular domain. The hinge region may originate from the hinge region of the wild-type receptor or a functional variant thereof. The hinge region may have one or more mutations, including, for example, insertions, deletions, or substitutions. The hinge region may be the hinge region of the ICR. The hinge region may be the hinge region of the CAR.

[0071] As used herein, “vector” is any construct having the ability to deliver one or more polynucleotides of interest to a host cell once the vector has been introduced into the host cell. “Expression vector” has the ability to deliver and express one or more polynucleotides of interest as encoded polypeptides within the host cell into which the expression vector has been introduced. Thus, in an expression vector, the polynucleotides of interest are positioned for expression in the vector by being operably ligated to, near, or adjacent to, the integration site of the polynucleotides of interest either within the vector or in the genome of the host cell, with regulatory elements such as promoters, enhancers, and / or poly-A tails, so that the polynucleotides of interest are translated in the host cell into which the expression vector has been introduced.

[0072] As used herein, the term “chimeric antigen receptor” or “CAR” refers to a genetically modified receptor that can be used to graft one or more antigen specificities onto immune effector cells such as T cells. Some CARs are also known as “artificial T cell receptors,” “chimeric T cell receptors,” or “chimeric immune receptors.” A CAR may comprise an extracellular ligand-binding domain or extracellular antigen-binding domain specific to one or more antigens (such as tumor antigens), a transmembrane domain, and an intracellular signaling domain for T cells and / or other receptors. “CAR-T cells” refers to T cells that express a CAR.

[0073] As used herein, the terms “T cell receptor” or “TCR” refer to an endogenous or modified T cell receptor comprising an extracellular antigen-binding domain that binds to a specific antigen peptide bound to an MHC molecule. A TCR may comprise a TCRα polypeptide chain and a TCRβ polypeptide chain. A TCR may comprise a TCRγ polypeptide chain and a TCRδ polypeptide chain. A TCR may bind specifically to a tumor antigen. “TCR-T” refers to a T cell expressing a recombinant TCR. When a heterologous antigen receptor, such as a heterologous TCR or CAR, is expressed, the immunogenicity specificity of T cells may be altered, so that T cells recognize or exhibit improved recognition of one or more tumor antigens present on the surface of cancer cells in an individual with cancer.

[0074] As used herein, the term “fusion protein” refers to a protein complex having one or more polypeptides with a desired function. This construct may or may consist of one polypeptide. A fusion protein can be a fusion polypeptide.

[0075] As used herein, the term “cancer” refers to cells that have the capacity for autonomous proliferation. Examples of such cells include cells exhibiting an abnormal state or condition characterized by rapidly growing cell growth. The term means that, regardless of histopathological type or invasive stage, it includes cancerous growth, e.g., tumors; carcinogenic processes, metastatic tissues, and malignant transformed cells, tissues, or organs. It also includes malignant tumors of various organ systems, such as the respiratory, cardiovascular, renal, reproductive, hematological, nervous, hepatic, gastrointestinal, and endocrine systems; as well as adenocarcinomas, including many colon cancers, renal cell carcinomas, prostate cancers and / or testicular tumors, non-lung small cell carcinomas, and small intestine cancers. "Spontaneous" cancers include any cancer that is not experimentally induced by transplantation of cancer cells into a subject, and include, for example, spontaneously occurring cancers, cancers caused by exposure of a patient to one or more carcinogens, cancers resulting from transgenic oncogene insertions or tumor suppressor gene knockouts, and cancers caused by infections, such as viral infections. The term "carcinoma" is recognized in the art and refers to malignant tumors of epithelial or endocrine tissue. This term also includes carcinosarcoma, which includes malignant tumors composed of carcinomatous and sarcomatous tissues. "Adenocarcinoma" refers to carcinomas originating from glandular tissue, or carcinomas in which tumor cells form recognizable glandular structures. The term "sarcoma" is recognized in the art and refers to malignant tumors of mesenchymal origin. The term "hematopoietic neoplasm" includes diseases involving proliferative / neoplastic cells of hematopoietic origin. Hematopoietic neoplasms may originate from myeloid, lymphoid, or erythroid cell lineages, or their progenitor cells.

[0076] When used herein, the terms “subject” and “patient” are used synonymously throughout this specification and refer to the animal, human or non-human to which the treatment relating to the method of this disclosure is to be provided. This disclosure is intended for veterinary and non-veterinary applications. Human patients may be adult humans or young humans (e.g., humans under 18 years of age). In addition to humans, patients include, but are not limited to, mice, rats, hamsters, guinea pigs, rabbits, ferrets, cats, dogs and primates. For example, non-human primates (e.g., monkeys, chimpanzees, gorillas, etc.), rodents (e.g., rats, mice, gerbils, hamsters, ferrets, rabbits), rabbits, pigs (e.g., pigs, miniature pigs), equids, canids, felines, bovines and other domestic animals, livestock and zoo animals.

[0077] Unless otherwise defined, all scientific and technical terms used herein have the same meaning as those generally understood by those skilled in the art to which this disclosure pertains. This disclosure describes methods and materials for use in this disclosure; other preferred methods and materials known in the art may also be used. Materials, methods, and examples are illustrative and not intended to limit the scope of use. All publications, patent applications, patents, sequences, database entries, and other references referred to herein are incorporated by reference in whole. In case of any conflict, including definitions, this disclosure shall prevail.

[0078] Fusion proteins (e.g., reverse cytokine receptors) In one embodiment, the present disclosure provides a fusion protein comprising (1) an extracellular domain that specifically binds to interleukin-4 (IL-4) and (2) an intracellular domain; the fusion protein, upon binding to IL-4, transmits interleukin-23 (IL-23) pathway signals.

[0079] In one embodiment, the disclosure provides a fusion protein comprising (a) a first polypeptide chain comprising (1) a first extracellular domain comprising the extracellular domain of a cytokine receptor and (2) a first intracellular domain comprising the intracellular domain of a cytokine receptor, and (b) a second polypeptide chain comprising (3) a second extracellular domain comprising the extracellular domain of a cytokine receptor and (4) a second intracellular domain comprising the intracellular domain of a cytokine receptor, wherein the first extracellular domain and the second extracellular domain form a binding site for IL-4, the first intracellular domain and the second intracellular domain form an IL-23 receptor complex, and when the fusion protein binds to IL-4, signal transduction is transmitted through the IL-23 receptor complex.

[0080] The first and second extracellular domains may be derived from cytokine receptors for immunosuppressive cytokines, such as TGFβ, IL-10, IL-4, IL-13, IL-6, IL-8, IL-5, VEGF, IL-22, IL-1, IL-1β, IL-35, TNF, GM-CSF, M-CSF, or G-CSF. The first and second intracellular domains may be derived from cytokine receptors for immunostimulatory cytokines, such as TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, CD28, OX-40, 4-1BB, CD80, CD86, ICOS, CD40, CD27, CD30, CD226, IL-7, IL-2, IL-15, IL-21, IL-12, IL-18, IL-9, IL-23, or IFN-γ.

[0081] In some embodiments, the fusion protein comprises a first polypeptide chain comprising (1) a first extracellular domain containing an interleukin-4 receptor alpha (IL-4Rα) extracellular domain, and (2) a first intracellular domain containing an interleukin-12 receptor beta-1 subunit (IL-12Rβ1) intracellular domain or an interleukin-23 receptor (IL-23R) intracellular domain. In some embodiments, the fusion protein comprises a second polypeptide chain comprising (1) a second extracellular domain containing an interleukin-2 receptor gamma (IL-2Rγ) extracellular domain, and (2) a second intracellular domain containing an interleukin-12 receptor beta-1 subunit (IL-12Rβ1) intracellular domain or an IL-23R intracellular domain.

[0082] In particular, this disclosure provides a fusion protein comprising a first extracellular domain and a second extracellular domain derived from an IL-4 receptor for IL-4 (e.g., IL-4Rα or IL-2Rγ) and a first intracellular domain and a second intracellular domain derived from an IL-23 receptor complex (e.g., IL-12Rβ1 or IL-23R). In some embodiments, the fusion protein is (a) (1) The first extracellular domain containing the IL-4Rα extracellular domain, and (2) First intracellular domain containing the IL-12Rβ1 intracellular domain A first polypeptide chain, and (3) A second extracellular domain including the IL-2Rγ extracellular domain, and (4) Second intracellular domain containing the IL-23R intracellular domain A second polypeptide chain including; or (b) (1) The first extracellular domain containing the IL-4Rα extracellular domain, and (2) First intracellular domain containing the IL-23R intracellular domain A first polypeptide chain containing, and (3) A second extracellular domain including the IL-2Rγ extracellular domain, and (4) Second intracellular domain containing the IL-12Rβ1 intracellular domain The second polypeptide chain, which includes Includes.

[0083] In some embodiments, the fusion protein is a reverse cytokine receptor (ICR).

[0084] Reverse cytokine receptors (ICRs) are fusion proteins created by fusing one or more extracellular domains of one or more cytokine receptors with one or more intracellular domains of different cytokine receptors. These fusion proteins can be designed to act as "switch receptors" that counteract the effects of immunosuppressive cytokines in certain diseases, particularly cancer. ICRs can be created by replacing the extracellular domain of a cytokine receptor that recognizes immunosuppressive cytokines with the extracellular domain of a cytokine receptor that recognizes cytokines involved in immune activation. When an ICR encounters an immunosuppressive cytokine, it can trigger downstream signaling pathways of the activating cytokine receptor, potentially helping to restore or enhance immune function.

[0085] IL-4 is primarily immunosuppressive. IL-4 is involved in the differentiation of naive CD4 T cells into IL-4-producing Th2 cells, and Th2 cells are associated with lower antitumor activity compared to IFNγ-producing CD4 T cells (Th1). Adoptive transfer of tumor-specific IL-4-producing cytotoxic CD8 T cells (Tc2) was also less effective in controlling tumor growth compared to IFNγ-producing CD8 T cells (Tc1). In addition, several cancer types express IL-4 and IL-4R, suggesting a role in tumor progression. The IL-4Rα chain (CD124) is a 140 kDa heterodimer complex that acts as a common monomer in both type 1 and type 2 receptor complexes. IL-4Rα interacts with the gamma chain of the IL-2 receptor (also called IL-2Rγ, γc, common gamma chain, or CD132) to form type 1 IL-4 receptors, and can also interact with the 65-70 kDa IL-13 binding receptor alpha-1 (IL-13Rα1) chain to form type 2 IL-4 / IL-13 receptors.

[0086] IL-2 regulates the activity of white blood cells (leukocytes, mostly lymphocytes) involved in immunity. IL-2 is part of the body's natural response to microbial infections, in distinguishing between foreign ("non-self") and "self." IL-2 mediates its effects by binding to IL-2 receptors expressed on lymphocytes. The main sources of IL-2 are activated CD4+ T cells and activated CD8+ T cells. IL-2 binds to IL-2 receptors, which have three forms, often referred to as "chains," resulting from different combinations of three different proteins: α (alpha) (also called IL-2Rα, CD25, or Tac antigen), β (beta) (also called IL-2Rβ, or CD122), and γ (gamma) (also called IL-2Rγ, γc, common gamma chain, or CD132). These subunits are also part of the receptors for other cytokines. The three IL-2R forms include α, βγ, and αβγ. The α chain binds to IL-2 with low affinity. The β and γ combination forms a βγ complex that binds to IL-2 with intermediate affinity, mainly on memory T cells and NK cells. All three receptor chains form an αβγ complex, which binds to IL-2 with high affinity (Kd approximately 10) on activated T cells and regulatory T cells. -11 They bind at M). The α chain is not involved in signal transduction, but the β chain complexes with an enzyme called Janus kinase 1 (JAK1), which has the ability to add phosphate groups to molecules. Similarly, the γ chain complexes with another tyrosine kinase called JAK3. These enzymes are activated when IL-2 binds to the external domain of IL-2R. As a result, three intracellular signal transduction pathways are triggered: the MAP kinase pathway, the phosphoinositide 3-kinase (PI3K) pathway, and the JAK-STAT pathway.

[0087] IL-4 binds to a cell surface receptor complex consisting of IL-4Rα and IL-2Rγ. The receptors for IL-4 and IL-2 share several common characteristics; both use IL-2Rγ as a receptor component, and both activate Janus kinases JAK-1 and JAK-3. Despite these similarities, IL-4 elicits specific responses, including tyrosine phosphorylation of 4PS / IRS-2 and induction of CD23.

[0088] IL-23 is a STAT3-activating cytokine consisting of the IL-23α p19 and IL-12β p40 subunits, both of which are expressed in activated macrophages and dendritic cells. IL-23 is part of the IL-12 cytokine family. IL-23 is known to promote the proliferation of memory T cells, particularly T helper type 17 (TH17) cells expressing the IL-23 receptor (IL-23R). IL-23 is primarily expressed in macrophages and dendritic cells (DCs). IL-23R is found in memory T cells, NKT cells, macrophages, DCs, and naive T cells activated by TGF-β and IL-6. The primary biological effects of IL-23 initially identified include stimulation of antigen presentation by DCs, T cell differentiation into Th17 cells, and interferon-γ (IFN-γ) production. IL-23 also acts as a terminal effector cytokine by directly acting on macrophages. This can be interpreted, in part, as an autocrine loop of IL-23 on macrophages. In addition, intraperitoneal administration of recombinant IL-23 in mice induces the expression of mRNA encoding IL-1 and TNF-α in peritoneal macrophages. Incorporation of p40 into CAR-modified or TCR-modified T cells enhanced their antitumor activity in xenograft and syngeneic mouse models.

[0089] The functional receptor for IL-23 (IL-23 receptor complex) consists of a heterodimer between the interleukin-12 receptor β1 subunit (IL-12Rβ1) and IL-23R. In the complex with IL-12Rβ1, IL-23R is activated by the cytokine IL-23. IL-23R is a type I permeable protein and contains a signal peptide and an intracellular portion with an N-terminal fibronectin III-like domain and three possible tyrosine phosphorylation domains.

[0090] In one embodiment, the disclosure provides a fusion protein (e.g., ICR) comprising an extracellular domain derived from the IL-4 receptor (e.g., IL-4Rα). The extracellular domain (i.e., IL-4Rα, or IL-2Rγ) can specifically bind to IL-4. Upon binding to IL-4, the fusion protein (e.g., ICR) can be activated by IL-4 (e.g., human IL-4). Binding of the IL-4R extracellular domain to IL-4 may result in the fusion protein (e.g., ICR) activating signaling of the IL-23R intracellular signaling domain, which, through transmission by native cellular elements, may provide biological activity that mimics the native response of IL-23R (e.g., phosphorylation of STAT3 and / or STAT4).

[0091] An ICR may be a fusion protein comprising two polypeptide chains. An ICR may comprise a first polypeptide chain and a second polypeptide chain. The first polypeptide chain may comprise a first extracellular domain derived from IL-4R (e.g., IL-4Rα) and a first intracellular domain derived from IL-12Rβ1. The second polypeptide chain may comprise a second extracellular domain derived from IL-2R (e.g., IL-2Rγ) and a second intracellular domain derived from IL-23R. Alternatively, the first polypeptide chain may comprise a first extracellular domain derived from IL-2R (e.g., IL-2Rγ) and a first intracellular domain derived from IL-12Rβ1. The second polypeptide chain may comprise a second extracellular domain derived from IL-4R (e.g., IL-4Rα) and a second intracellular domain derived from IL-23R. The ICRs described herein may have the structures and / or sequences shown in Table 1.

[0092] Each of the first and second polypeptide chains may further include a transmembrane domain between the extracellular and intracellular domains. In some embodiments, the fusion protein includes a first transmembrane domain in the first polypeptide chain and a second transmembrane domain in the second polypeptide chain; optionally, the first and second transmembrane domains may be dimerized. The first polypeptide chain may include, from the N-terminus to the C-terminus, a first extracellular domain derived from IL-4R (e.g., IL-4Rα), a first transmembrane domain (e.g., a transmembrane domain derived from IL-12Rβ1), and a first intracellular domain derived from IL-12Rβ1. The second polypeptide chain may include, from the N-terminus to the C-terminus, a second extracellular domain derived from IL-2R (e.g., IL-2Rγ), a second transmembrane domain (e.g., a transmembrane domain derived from IL-23R), and a second intracellular domain derived from IL-23R. Alternatively, the first polypeptide chain may include, from the N-terminus to the C-terminus, a first extracellular domain derived from IL-2R (e.g., IL-2Rγ), a first transmembrane domain (e.g., a transmembrane domain derived from IL-12Rβ1), and a first intracellular domain derived from IL-12Rβ1. The second polypeptide chain may include, from the N-terminus to the C-terminus, a second extracellular domain derived from IL-4R (e.g., IL-4Rα), a second transmembrane domain (e.g., a transmembrane domain derived from IL-23R), and a second intracellular domain derived from IL-23R.

[0093] The fusion protein (e.g., ICR) may contain an IL-4Rα extracellular domain (ECD). The IL-4Rα extracellular domain may contain an amino acid sequence that is at least 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 1. The IL-4Rα extracellular domain (ECD) may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 mutations (e.g., compared to the entire SEQ ID NO: 1 or wild-type IL-4Rα extracellular domain). These mutations may be amino acid insertions, deletions, or substitutions. Insertions, deletions, and substitutions may occur within the IL-4Rα extracellular domain, or at one or both ends of the IL-4Rα extracellular domain.

[0094] The fusion protein (e.g., ICR) may contain an IL-2Rγ extracellular domain (ECD). The IL-2Rγ extracellular domain may contain an amino acid sequence that is at least 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 5. The IL-2Rγ extracellular domain (ECD) may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 mutations (e.g., compared to the entire SEQ ID NO: 5 or wild-type IL-2Rγ extracellular domain). These mutations may be amino acid insertions, deletions, or substitutions. Insertions, deletions, and substitutions may occur within the IL-2Rγ extracellular domain, or at one or both ends of the IL-2Rγ extracellular domain.

[0095] A fusion protein (e.g., ICR) may contain an IL-23R intracellular domain (ICD). The IL-23R intracellular domain may contain an amino acid sequence that is at least 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of Sequence ID No. 8. The IL-23R ICD may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 mutations (e.g., compared to Sequence ID No. 8 or the entire wild-type IL-23R ICD). These mutations may be amino acid insertions, deletions, or substitutions. Insertions, deletions, and substitutions may occur within the range of the IL-23R ICD, or at one or both ends of the IL-23R ICD.

[0096] A fusion protein (e.g., ICR) may contain an IL-12Rβ1 intracellular domain (ICD). The IL-12Rβ1 intracellular domain may contain an amino acid sequence that is at least 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 4. The IL-12Rβ1 ICD may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 mutations (e.g., compared to the entire SEQ ID NO: 4 or wild-type IL-12Rβ1 ICD). These mutations may be amino acid insertions, deletions, or substitutions. Insertions, deletions, and substitutions may occur within the range of the IL-12Rβ1 ICD, or at one or both ends of the IL-12Rβ1 ICD.

[0097] A fusion protein (e.g., ICR) may contain an IL-2Rβ intracellular domain (ICD). The IL-2Rβ intracellular domain may contain an amino acid sequence that is at least 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 16 or SEQ ID NO: 17. The IL-2Rβ ICD may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 mutations (compared to, for example, SEQ ID NO: 16, SEQ ID NO: 17, or the entire wild-type IL-2Rβ ICD). These mutations may be amino acid insertions, deletions, or substitutions. Insertions, deletions, and substitutions may occur within the range of the IL-2Rβ ICD, or at one or both ends of the IL-2Rβ ICD.

[0098] A fusion protein (e.g., ICR) may contain an IL-23R transmembrane domain (TM). The IL-23R transmembrane domain may be derived from wild-type IL-23R or a functional variant thereof, or a portion thereof. The IL-23R transmembrane domain may contain an amino acid sequence that is at least 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 7. IL-23R TM may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 mutations (e.g., compared to SEQ ID NO: 7 or wild-type IL-23R TM as a whole). These mutations may be amino acid insertions, deletions, or substitutions. Insertions, deletions, and substitutions may occur within the range of IL-23R™ or at one or both ends of IL-23R™.

[0099] A fusion protein (e.g., ICR) may contain an IL-12Rβ1 transmembrane domain (TM). The IL-12Rβ1 transmembrane domain may be derived from wild-type IL-12Rβ1 or a functional variant thereof, or a portion thereof. The IL-12Rβ1 transmembrane domain may contain an amino acid sequence that is at least 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 3. IL-12Rβ1 TM may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 mutations (e.g., compared to SEQ ID NO: 3 or wild-type IL-12Rβ1 TM as a whole). These mutations may be amino acid insertions, deletions, or substitutions. Insertions, deletions, and substitutions may be within the range of IL-12Rβ1™ or at one or both ends of IL-12Rβ1™.

[0100] A fusion protein (e.g., ICR) may contain an IL-2Rβ transmembrane domain (TM). The IL-2Rβ transmembrane domain may be derived from wild-type IL-2Rβ or a functional variant thereof, or a portion thereof. The IL-2Rβ transmembrane domain may contain an amino acid sequence that is at least 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 15. The IL-2Rβ TM may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 mutations (e.g., compared to SEQ ID NO: 15 or the entire wild-type IL-2Rβ TM). These mutations may be amino acid insertions, deletions, or substitutions. Insertions, deletions, and substitutions may be within the range of IL-2Rβ™ or at one or both ends of IL-2Rβ™.

[0101] The fusion protein (e.g., ICR) may further include a conjugate sequence. The conjugate sequence may be selected from the sequence of the IL-23R extracellular domain located near the membrane (named the IL-23R conjugate sequence) or the sequence of the IL-12Rβ1 extracellular domain located near the membrane (named the IL-12Rβ1 conjugate sequence). The fusion protein (e.g., ICR) may include the IL-23R conjugate sequence. The IL-23R conjugate sequence may contain an amino acid sequence that is at least 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 6. The IL-23R linker may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 mutations (for example, compared to SEQ ID NO: 6). These mutations may be amino acid insertions, deletions, or substitutions. Insertions, deletions, and substitutions may be within the range of the IL-23R linker, or at one or both ends of the IL-23R linker. The fusion protein (e.g., ICR) may contain an IL-12Rβ1 linker. The IL-12Rβ1 linker may contain an amino acid sequence that is at least 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 2. The IL-12Rβ1 linker sequence may contain one, two, or three mutations (for example, compared to sequence number 2). These mutations may be amino acid insertions, deletions, or substitutions. Insertions, deletions, and substitutions may be within the range of the IL-12Rβ1 linker sequence, or at one or both ends of the IL-12Rβ1 linker sequence.

[0102] A fusion protein (e.g., ICR) may comprise two polypeptide chains. The first polypeptide chain may comprise an IL-4Rα extracellular domain (e.g., SEQ ID NO: 1), an IL-12Rβ1 transmembrane domain (e.g., SEQ ID NO: 3), and an IL-12Rβ1 intracellular domain (e.g., SEQ ID NO: 4), from the N-terminus to the C-terminus. The first polypeptide chain may comprise at least 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical amino acid sequence to SEQ ID NO: 9. The first polypeptide chain may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations (when compared to, for example, SEQ ID NO: 9). These mutations may be amino acid insertions, deletions, or substitutions. Insertions, deletions, and substitutions may be within the scope of the first polypeptide chain, or at one end or both ends of the first polypeptide chain.

[0103] A fusion protein (e.g., ICR) may contain two polypeptide chains. The second polypeptide chain may contain, from the N-terminus to the C-terminus, an IL-2Rγ extracellular domain (e.g., SEQ ID NO: 5), an IL-23R transmembrane domain (e.g., SEQ ID NO: 7), and an IL-23R intracellular domain (e.g., SEQ ID NO: 8). The second polypeptide chain may contain an amino acid sequence that is at least 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 10. The second polypeptide chain may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more mutations (for example, when compared to SEQ ID NO: 10). These mutations may be amino acid insertions, deletions, or substitutions. Insertions, deletions, and substitutions may be within the scope of the second polypeptide chain, or at one end or both ends of the second polypeptide chain.

[0104] A fusion protein (e.g., ICR) may comprise two polypeptide chains. The first polypeptide chain may comprise an IL-2Rγ extracellular domain (e.g., SEQ ID NO: 5), an IL-12Rβ1 transmembrane domain (e.g., SEQ ID NO: 3), and an IL-12Rβ1 intracellular domain (e.g., SEQ ID NO: 4), from the N-terminus to the C-terminus. The first polypeptide chain may comprise at least 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical amino acid sequence to SEQ ID NO: 11. The first polypeptide chain may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations (when compared to, for example, SEQ ID NO: 11). These mutations may be amino acid insertions, deletions, or substitutions. Insertions, deletions, and substitutions may be within the scope of the first polypeptide chain, or at one end or both ends of the first polypeptide chain.

[0105] A fusion protein (e.g., ICR) may contain two polypeptide chains. The second polypeptide chain may contain, from N-terminus to C-terminus, an IL-4Rα extracellular domain (e.g., SEQ ID NO: 1), an IL-23R transmembrane domain (e.g., SEQ ID NO: 7), and an IL-23R intracellular domain (e.g., SEQ ID NO: 8). The second polypeptide chain may contain at least 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical amino acid sequence to SEQ ID NO: 12. The second polypeptide chain may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations (e.g., compared to SEQ ID NO: 12). These mutations may be amino acid insertions, deletions, or substitutions. Insertions, deletions, and substitutions may be within the scope of the second polypeptide chain, or at one end or both ends of the second polypeptide chain.

[0106] A fusion protein (e.g., ICR) may comprise two polypeptide chains. In some embodiments, the first and second polypeptide chains are linked together by a 2A cleavable linker, such as a P2A or T2A linker. A fusion protein (e.g., ICR) may comprise a first polypeptide chain containing the amino acid sequence of SEQ ID NO: 9 and a second polypeptide chain containing the amino acid sequence of SEQ ID NO: 10. A fusion protein (e.g., ICR) may comprise a first polypeptide chain containing the amino acid sequence of SEQ ID NO: 11 and a second polypeptide chain containing the amino acid sequence of SEQ ID NO: 12. Before cleavage by the 2A linker, a fusion protein (e.g., ICR) may comprise a polypeptide containing one of the amino acid sequences of SEQ ID NOs: 13-14. In some embodiments, a fusion protein (e.g., ICR) comprises first and / or second polypeptide chains containing the amino acid sequence shown in any of SEQ ID NOs: 9-14, or an amino acid sequence having at least 90%, 95%, or 99% identity with the amino acid sequence shown in any of SEQ ID NOs: 9-14.

[0107] Modified receptors (e.g., CAR and TCR) One aspect of this disclosure provides cells (e.g., immune cells) expressing the reverse cytokine receptor described herein and also expressing a modified receptor. The modified receptor may comprise an extracellular ligand-binding domain or an extracellular antigen-binding domain and optionally an intracellular signaling domain. Exemplary modified receptors include, but are not limited to, chimeric antigen receptors (CARs), modified T cell receptors (TCRs), and T cell antigen-coupling (TAC) receptors. The modified receptor may comprise an extracellular antigen-binding domain that specifically binds to an antigen (e.g., a tumor antigen), a transmembrane domain, and an intracellular signaling domain. The intracellular signaling domain may comprise a primary intracellular signaling domain and / or a co-stimulatory signaling domain. The intracellular signaling domain may comprise the intracellular signaling domain of a TCR co-receptor. The modified receptor may be encoded by heterologous polynucleotides operably linked to a promoter (such as a constitutive promoter or an inductive promoter).

[0108] The modified receptor may comprise one or more specific binding domains that target at least one tumor antigen, and one or more intracellular effector domains, such as one or more primary intracellular signaling domains and / or costimulatory signaling domains.

[0109] The modified receptor may be a chimeric antigen receptor (CAR). Many chimeric antigen receptors are known in the art and may be suitable for the modified cells described herein. CARs specific to any cell surface marker can also be constructed, for example, by utilizing antigen-binding fragments or antibody variable domains of antibody molecules.

[0110] The CAR of this disclosure may include an extracellular domain containing at least one antigen-binding domain that specifically binds to at least one tumor antigen, a transmembrane domain, and an intracellular signaling domain.

[0111] Intracellular signaling domains can generate signals that promote the immune effector function of CAR-containing cells, such as CAR-T cells. Immune effector function or immune effector response refers to the function or response of immune effector cells, such as immune effector cells, that enhance or promote the immune attack of target cells. For example, immune effector function or response may refer to the properties of T cells or NK cells that promote the killing of target cells or the inhibition of their growth or proliferation. Examples of immune effector function in CAR-T cells include cytolytic activity (antibody-dependent cell-mediated cytotoxicity, or ADCC, etc.) and helper activity (cytokine secretion, etc.). Intracellular signaling domains can generate signals that promote the proliferation and / or survival of CAR-containing cells. CARs may contain one or more intracellular signaling domains selected from the signaling domains of CD28, CD137, CD3, CD27, CD40, ICOS, GITR, and OX40. Signaling domains of naturally occurring molecules may include the entire intracellular or cytoplasmic portion of the molecule, or the entire native intracellular signaling domain, or fragments or derivatives thereof.

[0112] The intracellular signaling domain of CAR may include a primary intracellular signaling domain. A "primary intracellular signaling domain" refers to a cytoplasmic signaling sequence that acts to induce immune effector function in a stimulating manner. The primary intracellular signaling domain may contain an immune receptor tyrosine activation motif or a signaling motif known as ITAM. The primary intracellular signaling domain may include a functional signaling domain of a protein selected from the group consisting of CD3 zeta, CD3 gamma, CD3 delta, CD3 epsilon, common FcR gamma (FCER1G), FcR beta (Fc epsilon Rib), CD79a, CD79b, Fc gamma R IIa, DAP10, and DAP12. The primary intracellular signaling domain may include a non-functional or attenuated signaling domain of a protein selected from the group consisting of CD3 zeta, CD3 gamma, CD3 delta, CD3 epsilon, common FcR gamma (FCER1G), FcR beta (Fc epsilon Rib), CD79a, CD79b, Fc gamma R IIa, DAP10, and DAP12. A non-functional or attenuated signaling domain may be a mutant signaling domain having a point mutation, insertion, or deletion that attenuates or eliminates one or more immunoeffector functions, such as cytolytic activity or helper activity, including antibody-dependent cell-mediated cytotoxicity (ADCC). The CAR may include a non-functional or attenuated CD3 zeta (i.e., CD3ζ or CD3z) signaling domain. The intracellular signaling domain does not necessarily include the primary intracellular signaling domain. Attenuated primary intracellular signaling domains, while having the same construct, may induce immunoeffector function (such as cytolytic function against target cells) at or below approximately 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, or 10% compared to CARs with a wild-type primary intracellular signaling domain.

[0113] The intracellular signaling domain of a CAR may contain one or more (one, two, three, or more) co-stimulatory signaling domains. A "co-stimulatory signaling domain" may be a cellular portion of a co-stimulatory molecule. The term "co-stimulatory molecule" refers to a cognitive-binding partner on an immune cell (such as a T cell) that specifically binds to a co-stimulatory ligand and thereby mediates a co-stimulatory response by the immune cell, including, but not limited to, proliferation and survival. Co-stimulatory molecules are cell surface molecules other than antigen receptors or their ligands that contribute to an efficient immune response. Co-stimulatory molecules can be represented by the following protein families: TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocyte activators (SLAM proteins), and activated NK cell receptors. Examples of co-stimulatory molecules, though not limited to them, include MHC class I molecules, BTLA and Toll ligand receptors, as well as OX40, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), and 4-1BB (CD137).Further examples of such co-stimulatory molecules include CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, CD4, CD8 alpha, CD8 beta, 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, Ligands that specifically bind to LFA-1, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CDIOO (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a, and CD83 are examples.

[0114] A CAR may contain a single co-stimulatory signaling domain. A CAR may contain two or more co-stimulatory signaling domains. The intracellular signaling domain may contain a functional primary intracellular signaling domain and one or more co-stimulatory signaling domains. A CAR does not have to contain a functional primary intracellular signaling domain (such as CD3ζ). A CAR may consist of one or more co-stimulatory signaling domains, or contain an intracellular signaling domain that is essentially derived therefrom. A CAR may consist of a non-functional or attenuated primary intracellular signaling domain (such as mutant CD3ζ) and one or more co-stimulatory signaling domains, or contain an intracellular signaling domain that is essentially derived therefrom. When the antigen-binding domain binds to a tumor antigen, the co-stimulatory signaling domain of the CAR may transmit signals for enhanced proliferation, survival, and differentiation of CAR-containing modified immune cells (such as T cells), and for inhibition of activation-induced cell death. One or more co-stimulatory signaling domains may originate from one or more molecules selected from the group consisting of ligands that specifically bind to CD27, CD28, 4-1BB (i.e., CD137), OX40, CD30, CD40, CD3, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and CD83.

[0115] The intracellular signaling domain of CAR may include a co-stimulatory signaling domain derived from CD28. The intracellular signaling domain may include a primary intracellular signaling domain of CD3ζ and a co-stimulatory signaling domain of CD28. The intracellular signaling domain in the chimeric receptor of the present application may include a co-stimulatory signaling domain derived from 4-1BB (i.e., CD137). The intracellular signaling domain may include a primary intracellular signaling domain of CD3ζ and a co-stimulatory signaling domain of 4-1BB. The intracellular signaling domain may include a polypeptide containing a co-stimulatory signaling domain of 4-1BB and a primary intracellular signaling domain of CD3ζ from the N-terminus to the C-terminus.

[0116] The intracellular signaling domain of CAR may include a CD28 co-stimulatory signaling domain and a 4-1BB co-stimulatory signaling domain. The intracellular signaling domain may include a CD3ζ primary intracellular signaling domain, a CD28 co-stimulatory signaling domain, and a 4-1BB co-stimulatory signaling domain. The intracellular signaling domain may include a polypeptide containing a CD28 co-stimulatory signaling domain, a 4-1BB co-stimulatory signaling domain, and a CD3ζ primary intracellular signaling domain from the N-terminus to the C-terminus.

[0117] The antigen-binding domain of CARs can be scFv, Fv, Fab, (Fab')2, minibody, diabody, single-domain antibody (sdAb), or V. H The CAR may contain one or more antibodies or antibody fragments (such as one, two, three, four, five, or six) that can be selected from the H domain. The antigen-binding domain of the CAR may contain an extracellular portion of a ligand or receptor that specifically binds to a tumor antigen. The CAR may be monospecific, bispecific, or multispecific. The antigen-binding domain of the CAR may specifically bind to a single tumor antigen. The antigen-binding domain of the CAR may bind to two or more tumor antigens. Modified receptors (e.g., CARs) can redirect the specificity of modified cells through the expression of chimeric antigen receptors (CARs) or TCRs on these cells.

[0118] Antigens include BCMA, CLL1, CD4, GPC3, GPRC5D, GU2CYC, CD19, MUC16, MUC1, CAIX, CEA, CD8, CD7, CD10, CD20, CD22, CD30, CD33, CD34, CD38, CD41, CD44, CD49f, CD56, CD74, CD133, CD138, EGP-2, EGP-40, EpCAM, ERBB2, ERBB3, ERBB4, FBP, fetal acetylcholine receptor, folate receptor-α, GD2, GD3, hTERT, IL-13Rα2, κ-light chain, KDR, LeY, L1 cell adhesion molecule, MAGE-A1, mesothelin, and MAGE-A3. The tumor antigen may be selected from a group consisting of p53, MART1, GP100, proteinase-3 (PR3), tyrosinase, survivorbin, hTERT, EphA2, NY-ESO-1, h5T4, PSCA, PSMA, ROR1, TAG-72, VEGF-R2, WT-1, CD123, CD44V6, NKCS1, IGF1R, EGFR, EGFR-VIII, claudin 18.2, claudin 6, NKG2D, delta-like 3 (DLL3), CD70, CS-1, c-Met, glycolipid F77, PD-L1, PD-L2, and other clinically significant tumor antigens, and combinations thereof. The antigen may be GPC3.

[0119] Tumor antigens may originate from intracellular proteins of tumor cells. Tumor antigens may be expressed on the surface of tumor cells. Many tumor antigen-specific TCRs (including tumor-associated antigens) have been described, including, for example, TCRs for tumor antigens in NY-ESO-1 cancer-testis antigen, p53 tumor suppressor antigen, melanoma (e.g., MARTI, gp100), leukemia (e.g., WT1, minor histocompatibility antigens), and breast cancer (e.g., HER2, NY-BR1).

[0120] The transmembrane domain of CARs includes the alpha, beta, or zeta chains of T cell receptors, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, OX40, CD2, CD27, LFA-1 (CD11a, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD160, and CD1 9, IL-2R beta, IL-2R gamma, IL-7Ra, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, IT GAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM1, CRT The transmembrane domains of AM, Ly9 (CD229), CD160 (BY55), PSGL1, CDIOO (SEMA4D), SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, and / or NKG2C can be selected. The transmembrane domain of CAR may be a CD4, CD3, CD8α, or CD28 transmembrane domain. The transmembrane domain of CAR may include a CD8α transmembrane domain. The transmembrane domain may originate from a molecule selected from the group consisting of CD8α, CD4, CD28, CD137, CD80, CD86, CD152, and PD1.

[0121] The extracellular domain may be connected to the transmembrane domain by a hinge domain. The hinge domain may be the hinge domain of CD8α.

[0122] CAR may also include signal peptides (SPs), such as the CD8α signal peptide. The signal peptide may contain a sequence that is at least 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 25.

[0123] In the art, many CARs targeting various different tumor antigens, such as CD19 CARs or BCMA CARs, have been widely disclosed. The extracellular antigen-binding domain of a CD19 CAR may be, or may be, a CD19 binding fragment (e.g., FMC63, SJ25C1, or those disclosed in various different patents such as International Publication 2022 / 012683). BCMA CARs have also been well described, and relevant patents include, but are not limited to, International Publication 2016 / 014789, International Publication 2016 / 014565, International Publication 2013 / 154760, and International Publication 2018 / 028647. The extracellular antigen-binding domain of a BCMA CAR may be, or may be, a BCMA binding fragment. The BCMA binding fragment can bind to one or more epitopes on BCMA. A BCMA CAR may be a bivalent CAR containing two anti-BCMA sdAbs targeting the same or different BCMA epitopes.

[0124] The CAR may be a GPC3 CAR ("H93" or "H93 CAR"). The GPC3 CAR may be monospecific. The GPC3 CAR may be bispecific or bivalent. The extracellular antigen-binding domain of the GPC3 CAR may be, or may be, one or more GPC3-binding moieties. The GPC3-binding moieties may bind to one or more epitopes on GPC3. The GPC3 CAR may be a bivalent CAR containing one or more anti-GPC3 scFv targeting the same or different GPC3 epitopes.

[0125] The GPC3 CAR may contain, from the N-terminus to the C-terminus, a CD8α signaling peptide, an extracellular antigen-binding domain containing anti-GPC3 scFv, a CD8α hinge domain, a CD8α transmembrane domain, a 4-1BB costimulatory signaling domain, and a CD3ζ primary intracellular signaling domain. The anti-GPC3 scFv may contain a sequence that is at least 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 30.

[0126] The CD8α hinge domain may contain a sequence that is at least 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of Sequence ID No. 26.

[0127] The CD8α transmembrane domain may contain a sequence that is at least 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of Sequence ID No. 27.

[0128] The 4-1BB costimulatory signaling domain may contain a sequence that is at least 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of Sequence ID No. 28.

[0129] The CD3ζ primary intracellular signaling domain may contain a sequence that is at least 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of Sequence ID No. 29.

[0130] GPC3 CARs may contain sequences that are at least 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of Sequence ID No. 20. GPC3 CARs may specifically bind to GPC3-positive tumor cells (e.g., PLCPRF5 cells).

[0131] Modified receptors may be modified T cell receptors or modified T cell receptors. Modified TCRs may be specific to tumor antigens. Tumor antigens include BCMA, CLL1, CD4, GPC3, GPRC5D, GU2CYC, CD19, MUC16, MUC1, CAIX, CEA, CD8, CD7, CD10, CD20, CD22, CD30, CD33, CD34, CD38, CD41, CD44, CD49f, CD56, CD74, CD133, CD138, EGP-2, EGP-40, EpCAM, ERBB2, ERBB3, ERBB4, FBP, fetal acetylcholine receptor, folate receptor-α, GD2, GD3, HER-2, hTERT, IL-13R-α2, κ-light chain, KDR, LeY, L1 cell adhesion molecule, MAGE-A1, mesothelin, The following can be selected from a group consisting of MAGEA3, p53, MART1, GP100, proteinase-3 (PR3), tyrosinase, survivorbin, hTERT, EphA2, NY-ESO-1, h5T4, PSCA, PSMA, ROR1, TAG-72, VEGF-R2, WT-1, CD123, CD44V6, NKCS1, IGF1R, EGFR, EGFR-VIII, claudin 18.2, claudin 6, NKG2D, delta-like 3 (DLL3), CD70, CS-1, c-Met, glycolipid F77, PD-L1, PD-L2, and other clinically significant tumor antigens, as well as combinations thereof. Tumor antigens may originate from intracellular proteins of tumor cells. Tumor antigens may be expressed on the surface of tumor cells. Many TCRs specific to tumor antigens (including tumor-associated antigens) have been described, including, for example, TCRs for tumor antigens in NY-ESO-1 cancer-testis antigen, p53 tumor suppressor antigen, melanoma (e.g., MARTI, gp100), leukemia (e.g., WT1, minor histocompatibility antigens), and breast cancer (e.g., HER2, NY-BR1). Any TCR known in the art may be used. TCRs may exhibit enhanced affinity for tumor antigens.Exemplary TCRs and methods for introducing TCRs into immune cells are described, for example, in U.S. Patent No. 5,830,755 and Kessels et al. Immunotherapy through TCR gene transfer. Nat. Immunol. 2,957-961 (2001) (these are incorporated herein by reference in whole).

[0132] The TCR receptor complex is an octameric complex formed by the variable TCR receptor α and β chains (or γ and δ chains in the case of γδ T cells) and three dimeric signaling modules CD3δ / ε, CD3γ / ε, and CD247 (T cell surface glycoprotein CD3 zeta chain) ζ / ζ or ζ / η. Ionizable residues in the transmembrane domains of each subunit form a polar interaction network that holds the complex together. The TCR complex has the function of activating the signaling cascade in T cells.

[0133] A modified receptor may be a modified TCR comprising one or more T cell receptor (TCR) fusion proteins (TFPs). Exemplary TFPs are described, for example, in U.S. Patent Application Publication No. 20170166622A1, which is incorporated herein by reference in whole. A TFP may include an extracellular domain of a TCR subunit comprising a TCR alpha chain, a TCR beta chain, a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, a CD3 delta TCR subunit, functional fragments thereof, and an extracellular domain or portion thereof of a protein selected from the group consisting of at least one, but not more than 20, amino acid sequences thereof. A TFP may also include a transmembrane domain comprising a transmembrane domain of a protein selected from the group consisting of a TCR alpha chain, a TCR beta chain, a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, a CD3 delta TCR subunit, functional fragments thereof, and an extracellular domain of a protein selected from the group consisting of at least one, but not more than 20, amino acid sequences thereof. TFP may include a transmembrane domain containing a protein transmembrane domain selected from the group consisting of TCR alpha chain, TCR beta chain, TCR zeta chain, CD3 epsilon TCR subunit, CD3 gamma TCR subunit, CD3 delta TCR subunit, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD137, CD154, functional fragments thereof, and a transmembrane domain of a protein having at least one, but not more than 20, modifications.

[0134] TFP may include a TCR subunit comprising at least a portion of the extracellular domain of the TCR and an intracellular domain of the TCR comprising a stimulating domain from the intracellular signaling domain of CD3 epsilon; and an antigen-binding domain, the TCR subunit and the antigen-binding domain being operably linked, and when expressed in T cells, TFP is taken up by the TCR.

[0135] The modified receptor may be a T cell antigen-coupling (TAC) receptor. An exemplary TAC receptor is described, for example, in U.S. Patent Application Publication No. 20160368964A1, which is incorporated herein by reference. The TAC may include an antigen-binding domain, a TCR-binding domain that specifically binds to proteins associated with the TCR complex, and a T cell receptor signaling domain. The antigen-binding domain may be an antibody fragment that specifically binds to a tumor antigen, such as scFv or VHH. The antigen-binding domain may be a designed ankyrin repeat (DARPin) polypeptide. Tumor antigens include BCMA, CLL1, CD4, GPC3, GPRC5D, GU2CYC, CD19, MUC16, MUC1, CAIX, CEA, CD8, CD7, CD10, CD20, CD22, CD30, CD33, CD34, CD38, CD41, CD44, CD49f, CD56, CD74, CD133, CD138, EGP-2, EGP-40, EpCAM, ERBB2, ERBB3, ERBB4, FBP, fetal acetylcholine receptor, folate receptor-α, GD2, GD3, HER-2, hTERT, IL-13R-α2, κ-light chain, KDR, LeY, L1 cell adhesion molecule, MAGE-A1, mesothelin. The following can be selected from the group consisting of MAGEA3, p53, MART1, GP100, proteinase-3 (PR3), tyrosinase, survivin, hTERT, EphA2, NY-ESO-1, h5T4, PSCA, PSMA, ROR1, TAG-72, VEGF-R2, WT-1, CD123, CD44V6, NKCS1, IGF1R, EGFR, EGFR-VIII, claudin 18.2, claudin 6, NKG2D, delta-like 3 (DLL3), CD70, CS-1, c-Met, glycolipid F77, PD-L1, PD-L2, and other clinically significant tumor antigens, and combinations thereof. Tumor antigens may be derived from intracellular proteins of tumor cells. Tumor antigens may be expressed on the surface of tumor cells. Proteins associated with the TCR complex may be CD3, such as CD3E. The TCR-binding domain may be a single-chain antibody, such as scFv, or V HIt may also be H. The TCR-binding domain may be derived from UCHT1. The TAC receptor may include a cytoplasmic domain and a transmembrane domain. The T cell receptor signaling domain may include a cytoplasmic domain derived from the TCR co-receptor. Exemplary TCR co-receptors include, but are not limited to, CD4, CD8, CD28, CD45, CD4, CD5, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154. The TAC receptor may include a transmembrane domain and a cytoplasmic domain derived from CD4. The TAC receptor may include a transmembrane domain and a cytoplasmic domain derived from CD8 (e.g., CD8α).

[0136] T cell coreceptors are expressed as membrane proteins on T cells. This can lead to the stabilization of the TCR:peptide:MEC complex, facilitating signal transduction. Two subtypes of T cell coreceptors, CD4 and CD8, exhibit strong specificity for specific MEC classes. The CD4 coreceptor can stabilize only the TCR:MEC II complex, while the CD8 coreceptor can stabilize only the TCR:MEC I complex. Differences in CD4 and CD8 expression in different T cell types result in characteristically distinct T cell functional subpopulations. CD8+ T cells are cytotoxic T cells.

[0137] Modified receptors (such as CARs, TCRs, or TACs) can target one or more tumor antigens. Tumor antigens are proteins produced by tumor cells that can elicit an immune response, particularly a T-cell-mediated immune response. The choice of antigen to target will depend on the specific type of cancer being treated. Examples of tumor antigens include glioma-associated antigen, carcinoembryonic antigen (CEA), β-human chorionic gonadotropin, α-fetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CAIX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxylesterase, mut hsp70-2, M-CSF, prostase, prostate-specific antigen (PSA), PAP, NY-ESO-1, LAGE-LA, p53, prostain, PSMA, HER2 / neu, survivorbin and telomerase, prostate cancer tumor antigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrin B2, CD22, insulin growth factor (IGF)-I, IGF-II, IGF-I receptor and mesothelin.

[0138] Tumor antigens may include one or more antigenic cancer epitopes associated with malignant tumors. Malignant tumors express several proteins that can act as target antigens for immune attack. These molecules include, but are not limited to, tissue-specific antigens such as MART-1, tyrosinase, and gp100 in melanoma, and prostatic acid phosphatase (PAP) and prostate-specific antigen (PSA) in prostate cancer. Other target molecules belong to the group of transformation-related molecules, such as the oncogene HER2 / Neu / ErbB-2. Yet another group of target antigens are carcinoembryonic antigens, such as carcinoembryonic antigens (CEAs). In B-cell lymphomas, tumor-specific idiotype immunoglobulins constitute truly tumor-specific immunoglobulin antigens unique to individual tumors. B-cell differentiation antigens such as CD19, CD20, and CD37 are other candidate target antigens in B-cell lymphomas.

[0139] Tumor antigens can be tumor-specific antigens (TSAs) or tumor-associated antigens (TAAs). TSAs are specific to tumor cells and are not present in other cells of the body. TAA-associated antigens are not specific to tumor cells; instead, they are expressed on normal cells under conditions that impair immune tolerance to the antigen. Antigen expression on tumors can occur under conditions that enable the immune system to respond to the antigen. TAAs may be antigens expressed on normal cells during fetal development when the immune system is immature and unable to respond to the antigen, or TAAs may be antigens that are normally present at very low levels on normal cells but expressed at much higher levels on tumor cells.

[0140] Non-limiting examples of TSA or TAA antigens include, namely, differentiation antigens such as MART-1 / Melan A (MART-I), gp 100 (Pmel 17), tyrosinase, TRP-1, TRP-2 and tumor-specific multisystem antigens such as MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, and p15; overexpressed embryonic antigens such as CEA; overexpressed oncogenes and mutated tumor suppressor genes such as p53, Ras, and HER2 / neu; unique tumor antigens resulting from chromosomal translocations, such as BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, and MYL-RAR; and viral antigens such as Epstein-Barr virus antigen (EBVA) and human papillomavirus (HPV) antigens E6 and E7. Other large protein-based antigens include TSP-180, MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO, p185erbB2, p180erbB-3, c-met, nm-23HI, PSA, TAG-72, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, beta-catenin, CDK4, Mum-1, p 15, p 16, 43-9F, 5T4, 791Tgp72, alpha-fetoprotein, beta-HCG, BCA225, BTAA, CA 125, CA 15-3, CA 27.29, BCAA, CA 195, CA Examples include 242, CA-50, CAM43, CD68\P1, CO-029, FGF-5, G250, Ga733\EpCAM, HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB / 70K, NY-CO-1, RCAS 1, SDCCAG16, TA-90\Mac-2 binding protein\cyclophyllin C-related protein, TAAL6, TAG72, TLP, and TPS.

[0141] nucleic acid This disclosure provides (i) nucleic acids encoding fusion proteins (e.g., ICR) as described herein, and (ii) nucleic acids encoding modified receptors (e.g., CAR, TAC, or TCR). The nucleic acids of this disclosure may comprise a nucleic acid sequence encoding any one of the ICR, CAR, and / or TCR disclosed herein. The nucleic acid may encode both (1) CAR and (2) fusion proteins (e.g., ICR). The nucleic acid may contain one, two, three, four, five or more copies of the coding sequence for the fusion protein (e.g., ICR). The expression of the fusion protein (e.g., ICR) may be controlled by non-native regulatory elements.

[0142] The nucleic acids of this disclosure may comprise a first nucleic acid sequence and a second nucleic acid sequence. The first nucleic acid may be upstream of the second nucleic acid, or the first nucleic acid may be downstream of the second nucleic acid. The first and second nucleic acid sequences may be separated by a linker. The linker used in this disclosure allows multiple proteins to be encoded by the same nucleic acid sequence (e.g., a polycistronic or bicistronic sequence), and the multiple proteins are translated as a polyprotein, which is then dissociated into separate protein components. The nucleic acid may comprise, from the 5' end to the 3' end, a first nucleic acid sequence, a linker, and a second nucleic acid sequence. The nucleic acid may comprise, from the 5' end to the 3' end, a second nucleic acid sequence, a linker, and a first nucleic acid sequence. The first nucleic acid sequence may encode a CAR as described herein, and the second nucleic acid sequence may encode a fusion protein (e.g., an ICR) as described herein.

[0143] Linkers may include nucleic acid sequences encoding intrasequence ribosome entry sites (IRESs). As used herein, “intrasequence ribosome entry site” or “IRES” refers to an element, such as ATG, that facilitates direct intrasequence ribosome entry into the start codon of a protein-coding region, thereby resulting in cap-independent translation of a gene. Various intrasequence ribosome entry sites are known to those skilled in the art and include, without limitation, IRESs available from viral or cellular mRNA sources, such as immunoglobulin heavy chain binding protein (BiP); vascular endothelial growth factor (VEGF); fibroblast growth factor 2; insulin-like growth factor; translation initiation factor eIF4G; yeast transcription factors TFIID and HAP4; and IRESs available from, for example, cardioviruses, rhinoviruses, aftviruses, HCV, friend mouse leukemia virus (FrMLV), and Moloney mouse leukemia virus (MoMLV). Those skilled in the art will be able to select an appropriate IRES.

[0144] A linker may contain a nucleic acid sequence encoding a self-cleaving peptide. As used herein, “self-cleaving peptide” or “2A linker” refers to an oligopeptide that enables multiple proteins to be encoded as a polyprotein, which then dissociates into component proteins during translation. The use of the term “self-cleaving” is not intended to imply a proteolytic cleavage reaction. Various self-cleaving or 2A linkers are known to those skilled in the art and include, without limitation, members of the Picornaviridae family, e.g., foot-and-mouth disease virus (FMDV), equine rhinitis A virus (ERAV0), Thosea assigna (Thosea Examples include the asigna virus (TaV) and porcine scutellaria virus-1 (PTV-1); as well as those found in cardioviruses such as tylovirus and encephalomyocarditis virus. 2A linkers derived from FMDV, ERAV, PTV-1, and TaV are referred to herein as "F2A," "E2A," "P2A," and "T2A," respectively. The P2A linker has at least 70% or 80% of the amino acid sequence of SEQ ID NO: 31 or SEQ ID NO: 32. The T2A linker may have a sequence that is 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical. The T2A linker may have a sequence that is at least 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 33.

[0145] Various linker sequences are known in the art and include, without limitation, glycineserine (GS) spacers (also known as GS linkers), such as (GS)n, (SG)n, (GSGGS)n, and (GGGS)n (wherein n represents an integer of at least 1). Exemplary linker sequences may include amino acid sequences that are at least 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, or SEQ ID NO: 39.

[0146] The nucleic acids of this disclosure may include restriction enzyme site sequences. Exemplary restriction enzyme site sequences may include nucleic acid sequences that are at least 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleic acid sequence of GTTAAC or ACTAGT.

[0147] The nucleic acids of this disclosure may be operably coupled to transcriptional regulatory elements, such as promoters and enhancers.

[0148] The promoter may be a CD8 cell-specific promoter, a CD4 cell-specific promoter, a neutrophil-specific promoter, or an NK-specific promoter. For example, the CD4 gene promoter can be used; see, e.g., Salmon et al. Proc.Natl.Acad.Sci.USA(1993)90:7739; and Marodon et al.(2003)Blood 101:3416. Another example is the use of the CD8 gene promoter. NK cell-specific expression can be achieved using the NcrI(p46) promoter; see, e.g., Eckelhart et al.Blood(2011)117:1565.

[0149] Other examples of suitable promoters include the pre-early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a potent constitutive promoter sequence capable of driving high levels of expression of any nucleic acid sequence operably ligated to it. Other constitutive promoter sequences can also be used, but are not limited to, the Simian virus 40 (SV40) early promoter, the mouse mammary cancer virus (MMTV) or human immunodeficiency virus (HIV) long-terminal repeat (LTR) promoter, the MoMuLV promoter, the avian leukemia virus promoter, the Epstein-Barr virus pre-early promoter, the Roussarcoma virus promoter, the EF-1α promoter, and human gene promoters, including, but are not limited to, actin promoters, myosin promoters, hemoglobin promoters, and creatine kinase promoters. Furthermore, this disclosure should not be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of this disclosure. Using an inducible promoter provides a molecular switch capable of turning on the expression of the nucleic acid sequence to which it is operably ligated when such expression is desired, or turning off the expression when such expression is not desired. Examples of inductive promoters include, but are not limited to, the metallothionine promoter, the glucocorticoid promoter, the progesterone promoter, and the tetracycline promoter.

[0150] The nucleic acids of this disclosure may be provided for (i) the production of fusion proteins (e.g., ICRs) described herein, and / or (ii) the production of CARs described herein (e.g., in mammalian cells). The nucleic acids of this disclosure may be provided for nucleic acid amplification.

[0151] A vector, such as an expression vector (e.g., a lentiviral vector), can be used to introduce the nucleic acids described herein into immune cells (e.g., T cells) or their precursors. The vectors of this disclosure (e.g., lentiviral vectors) may contain one or more nucleic acids encoding the fusion proteins described herein (e.g., ICR). The vectors (e.g., lentiviral vectors) may contain additional elements that would be useful for the functional expression of the fusion proteins described herein (e.g., ICR) and / or the CARs described herein. The expression vectors may contain mammalian promoters. The vectors may contain an elongation factor 1-α promoter (EF-1α promoter). The use of the EF-1α promoter may improve the expression efficiency of downstream transgenes (e.g., nucleic acids encoding CARs). Physiological promoters (e.g., EF-1α promoters) may be less likely to induce integration-mediated genotoxicity and may negate the retroviral vector's ability to transform stem cells. Other physiological promoters suitable for use in vectors (e.g., lentiviral vectors) may also be incorporated into the vectors of this disclosure. Vectors (e.g., lentiviral vectors) may contain non-essential cis-acting sequences to improve titer and gene expression.

[0152] Nucleic acids may encode naked CARs. Nucleic acids may contain, from the 5' end to the 3' end, a CD8α signal peptide, an extracellular antigen-binding domain, a CD8α hinge domain, a CD8α transmembrane domain, a CD137 costimulatory signaling domain, and a CD3ζ primary intracellular signaling domain. Nucleic acids may encode an amino acid sequence that is at least 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence shown in Sequence ID No. 20.

[0153] A fusion protein (e.g., ICR) may contain two polypeptide chains. A nucleic acid may encode (1) a first polypeptide chain and / or (2) a second polypeptide chain. The nucleic acid may contain the coding sequence for the first polypeptide chain from the 5' end to the 3' end. The nucleic acid may contain the coding sequence for the second polypeptide chain from the 5' end to the 3' end. The nucleic acid may encode an amino acid sequence that is at least 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence shown in any one of sequence numbers 9-12. The nucleic acid may comprise, from its 5' end to its 3' end, a coding sequence for a first polypeptide chain, a 2A cleavable linker, and a second polypeptide chain. The nucleic acid may encode an amino acid sequence that is at least 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO: 13 or SEQ ID NO: 14. Exemplary structures of the nucleic acids described herein are shown in Figures 1a and 1b. The first polypeptide chain may comprise a first extracellular domain derived from IL-4Rα and a first intracellular domain derived from IL-12Rβ1. The second polypeptide chain may comprise a second extracellular domain derived from IL-2Rγ and a second intracellular domain derived from IL-23R. The nucleic acid may encode an amino acid sequence that is at least 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence shown in Sequence ID No. 13. The first polypeptide chain may include a first extracellular domain derived from IL-2Rγ and a first intracellular domain derived from IL-12Rβ1. The second polypeptide chain may include a second extracellular domain derived from IL-4Rα and a second intracellular domain derived from IL-23R.Nucleic acids may encode an amino acid sequence that is at least 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence shown in Sequence ID No. 14.

[0154] Nucleic acids may encode (1) CAR and (2) ICR. Nucleic acids may contain, from the 5' end to the 3' end, a coding sequence for CAR, a 2A cleavable linker, and an ICR. An ICR may be a fusion protein containing two polypeptide chains. An ICR may contain a first polypeptide chain and a second polypeptide chain. Nucleic acids may contain, from the 5' end to the 3' end, a coding sequence for CAR, a 2A cleavable linker, a first polypeptide chain, a 2A cleavable linker, and a second polypeptide chain. The nucleic acid may encode an amino acid sequence that is at least 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO: 21 or SEQ ID NO: 22. The first polypeptide chain may include a first extracellular domain derived from IL-4Rα and a first intracellular domain derived from IL-12Rβ1. The second polypeptide chain may include a second extracellular domain derived from IL-2Rγ and a second intracellular domain derived from IL-23R. The nucleic acid may encode an amino acid sequence that is at least 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO: 21. The first polypeptide chain may include a first extracellular domain derived from IL-2Rγ and a second intracellular domain derived from IL-12Rβ1. The second polypeptide chain may include a second extracellular domain derived from IL-4Rα and a second intracellular domain derived from IL-23R. Nucleic acids may encode an amino acid sequence that is at least 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence shown in Sequence ID No. 22.

[0155] To determine the percentage identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison (for example, gaps can be introduced in one or both of the first and second amino acid or nucleic acid sequences for optimal alignment, and non-homologous sequences can be ignored for comparison purposes). The length of the reference sequence aligned for comparison purposes is at least 80% of the reference sequence length, and may be at least 90%, 95%, or 100%. Next, amino acid residues or nucleotides at corresponding amino acid or nucleotide positions are compared. The molecules are identical at a given position when the position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence. The percentage identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps that need to be introduced for optimal alignment of the two sequences and the length of each gap. For example, the comparison of sequences and the determination of percentage identity between two sequences can be achieved using a Blossum 62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5.

[0156] Modified cells One aspect of this disclosure provides modified cells comprising a fusion protein (e.g., ICR) as described herein. Modified cells comprising a fusion protein (e.g., ICR) as described herein may further comprise a modified receptor (e.g., CAR). Modified cells comprising an ICR as described herein may further comprise a CAR (a CAR equipped with an ICR).

[0157] Modified receptors (e.g., CARs) can redirect the specificity of modified cells through the expression of chimeric antigen receptors (CARs) or TCRs on those cells. CAR expression can be induced via electroporation of modified cells for insertion of genetic material, or by infecting such cells with a viral vector, such as a lentivirus or retrovirus, containing the desired genetic material. Such gene editing can improve the efficacy of modified cells by improving homing, cytokine production, euthanasia and reuse, and / or engraftment.

[0158] Modified cells containing the fusion protein described herein (e.g., ICR) may express two or more polypeptide chains. Modified cells containing the fusion protein described herein (e.g., ICR) may express two or more modified receptors, such as any combination of CAR, TCR, or TAC receptors. Modified cells containing the fusion protein described herein (e.g., ICR) may be used for the treatment of cancer.

[0159] Compared to cells without the fusion protein described herein (e.g., ICR), modified cells containing the fusion protein described herein (e.g., ICR) may exhibit higher cytotoxicity against tumor cells. Compared to cells without the ICR described herein, modified cells containing the ICR described herein may exhibit higher persistence and / or proliferation in the tumor microenvironment.

[0160] In one embodiment, the Disclosure provides a modified cell comprising (i) a fusion protein described herein (e.g., ICR) and (ii) a modified receptor described herein (e.g., CAR). The modified cell may be an immune cell. The modified cell may comprise one or more nucleic acids encoding (i) a fusion protein described herein (e.g., ICR) and (ii) a modified receptor described herein (e.g., CAR). Thus, such a modified cell may possess specificity directed by the modified receptor (e.g., CAR) expressed therein. For example, a modified cell of the Disclosure comprising one or more CARs may possess specificity for one or more antigens on a target cell (e.g., one or more tumor antigens on a cancer cell).

[0161] Modified cells may be modified immune cells. Modified cells may be selected from the group consisting of T cells, αβT cells, γδT cells, NK cells, peripheral blood mononuclear cells (PBMCs), hematopoietic stem cells, pluripotent stem cells, embryonic stem cells, and combinations thereof. Modified cells may be T cells. Modified cells may be NK cells. Modified cells may be αβT cells. Modified cells may be γδT cells. Modified cells may be Vδ1 T cells.

[0162] Modified cells may be autologous, syngeneic, allogeneic, or heterogeneous cells to the recipient individual. Modified cells may be modified by altering the major histocompatibility complex (MHC) profile, by inactivating β2-microglobulin to prevent the formation of functional class I MHC molecules, or by inactivating class II MHC molecules. Modified cells may be autologous cells obtained from the recipient human subject. Modified cells may be autologous T cells obtained from the recipient human subject.

[0163] Modified cells as described herein may include eukaryotic cells, such as mammalian cells. Modified cells may also be human cells. Modified cells may also be equine, cattle, rodent, sheep, canine, or feline cells.

[0164] In one embodiment, the disclosure provides modified cells expressing a fusion protein (e.g., ICR) comprising an extracellular domain derived from the IL-4 receptor (e.g., IL-4Rα) and an intracellular domain derived from the IL-23 receptor complex. The fusion protein (e.g., ICR) containing the extracellular domain of IL-4R (i.e., IL-4Rα) on the modified cells can specifically bind to IL-4. Upon binding to IL-4, the fusion protein (e.g., ICR) activates signaling in the IL-23R intracellular signaling domain, which can then be transmitted through native cellular elements to provide biological activity that mimics the native response of IL-23R.

[0165] A fusion protein (e.g., ICR) may comprise a first polypeptide chain and a second polypeptide chain. The first polypeptide chain may comprise a first extracellular domain derived from IL-4Rα and a first intracellular domain derived from IL-12Rβ1. The second polypeptide chain may comprise a second extracellular domain derived from IL-2Rγ and a second intracellular domain derived from IL-23R. The first polypeptide chain may comprise a first extracellular domain derived from IL-2Rγ and a first intracellular domain derived from IL-12Rβ1. The second polypeptide chain may comprise a second extracellular domain derived from IL-4Rα and a second intracellular domain derived from IL-23R.

[0166] The modified cells may contain polypeptides encoding a fusion protein (e.g., ICR), the fusion protein (e.g., ICR) comprising first and / or second polypeptide chains containing an amino acid sequence shown in any of SEQ ID NOs: 9-14, or an amino acid sequence having at least 90%, 95%, or 99% identity with the amino acid sequence shown in any of SEQ ID NOs: 9-14. The fusion protein (e.g., ICR) may comprise a first polypeptide chain containing the amino acid sequence of SEQ ID NO: 9 and a second polypeptide chain containing the amino acid sequence of SEQ ID NO: 10. The fusion protein (e.g., ICR) may comprise a first polypeptide chain containing the amino acid sequence of SEQ ID NO: 11 and a second polypeptide chain containing the amino acid sequence of SEQ ID NO: 12. Before cleavage by a 2A linker (e.g., a P2A linker or a T2A linker), the fusion protein (e.g., ICR) may contain a polypeptide containing one of the amino acid sequences of SEQ ID NOs: 13-14.

[0167] Compared to cells without ICR, modified cells containing ICR may show enhanced proliferation when exposed to immunosuppressive cytokines in the tumor microenvironment (TME). Modified cells containing ICR may experience reduced or cessation of proliferation when one or more immunosuppressive cytokines are reduced or eliminated from the TME (e.g., by killing tumor cells). Compared to cells without ICR, modified cells containing ICR may show enhanced cytotoxicity when exposed to immunosuppressive cytokines in the TME.

[0168] The modified receptor may also be a CAR. A CAR may contain a polypeptide comprising, from the N-terminus to the C-terminus, a CD8α signaling peptide, an extracellular antigen-binding domain, a CD8α hinge domain, a CD8α transmembrane domain, a CD137 costimulatory signaling domain, and a CD3ζ primary intracellular signaling domain. A CAR may be a GPC3 CAR ("H93" or "H93 CAR"). A CAR may contain a polypeptide having at least 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical amino acid sequence to SEQ ID NO: 20.

[0169] The modified cell may comprise (i) a fusion protein described herein (e.g., ICR) and (ii) a modified receptor described herein (e.g., CAR), wherein the fusion protein described herein (e.g., ICR) and (ii) the modified receptor (e.g., CAR) are linked to each other by a 2A cleavable linker. In some embodiments, the modified cell comprises a polypeptide encoding the fusion protein and / or a polypeptide encoding the CAR, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 9-14 and 20-22, or a functional variant having at least about 90% sequence identity thereto.

[0170] Modified cells may include both ICRs and CARs (CARs with ICRs). Modified cells may be modified CAR-T cells (CAR-T cells with ICRs). The expression of CARs and ICRs in modified cells can be determined by flow cytometry (FACS). Modified cells (e.g., H93-IL4R-IL23R cells) may have CAR positivity rates of over 5%, over 10%, over 15%, over 20%, over 25%, over 30%, over 35%, over 40%, over 45%, over 50%, over 55%, over 60%, over 65%, over 70%, over 75%, over 80%, over 90%, or over 95%. Modified cells may have CAR positivity rates of less than 5%, less than 10%, less than 15%, less than 20%, less than 25%, less than 30%, less than 35%, less than 40%, less than 45%, less than 50%, less than 55%, less than 60%, less than 65%, less than 70%, less than 75%, less than 80%, less than 90%, or less than 95%. Modified cells may have CAR positivity rates of 10% to 100%, 20% to 100%, 30% to 100%, 40% to 100%, or 30% to 95%.

[0171] IL-4 (e.g., 20 ng / ml IL-4) may activate STAT signaling in modified cells (e.g., phosphorylated STAT3 or phosphorylated STAT4). IL-4 (e.g., 20 ng / ml IL-4) can increase the amount of phosphorylated STAT3 in modified cells by more than 5%, more than 10%, more than 15%, more than 20%, more than 30%, more than 40%, more than 50%, more than 60%, more than 70%, more than 80%, more than 90%, more than 100%, more than 150%, more than 200%, more than 250%, more than 300%, more than 400%, more than 500%, more than 600%, more than 700%, more than 800%, more than 900%, or more than 1000%. IL-4 (e.g., 20 ng / ml IL-4) can increase the amount of phosphorylated STAT4 in modified cells by more than 5%, more than 10%, more than 15%, more than 20%, more than 30%, more than 40%, more than 50%, more than 60%, more than 70%, more than 80%, more than 90%, more than 100%, more than 150%, more than 200%, more than 250%, more than 300%, more than 400%, more than 500%, more than 600%, more than 700%, more than 800%, more than 900%, or more than 1000%. Modified cells containing ICR (e.g., H93-IL4R-IL23R cells) may show increased responsiveness to IL-4 stimulation compared to cells without ICR (e.g., H93 cells). Compared to cells without ICR, STAT3 and / or STAT4 phosphorylation in modified cells containing ICR in response to IL-4 stimulation may be more than 5%, more than 10%, more than 15%, more than 20%, more than 30%, more than 40%, more than 50%, more than 60%, more than 70%, more than 80%, more than 90%, more than 100%, more than 150%, more than 200%, more than 250%, more than 300%, more than 400%, more than 500%, more than 600%, more than 700%, more than 800%, more than 900%, or more than 1000% higher.

[0172] To evaluate the CAR positivity, amplification, and viability of modified cells in vitro, modified cells are repeatedly stimulated with tumor cells (e.g., Hep3B2.1-7 cells) over several rounds in a rechallenge assay with or without IL-4 (e.g., 10 ng / mL of IL-4). The effector cell:target cell (E:T) ratio may be 0.5:1, 1:1, 2:1, 2.5:1, 5:1, or 10:1. Modified cells may be amplified more than 1x, 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, 10x, 15x, 20x, 25x, 30x, 35x, 40x, 45x, 50x, 60x, 70x, 8x, 30x, or 400x after 1, 2, 3, 4, or 5 rounds of stimulation in a rechallenge assay. Each stimulation round lasts 1, 2, 3, 4, or 5 days. Compared to cells without ICR (e.g., H93 cells), the amplification of modified cells containing ICR (e.g., H93-IL4R-IL23R cells) may increase by more than 5%, more than 10%, more than 15%, more than 20%, more than 30%, more than 40%, more than 50%, more than 60%, more than 70%, more than 80%, more than 90%, more than 100%, more than 150%, more than 200%, more than 250%, more than 300%, more than 400%, more than 500%, more than 600%, more than 700%, more than 800%, more than 900%, or more than 1000%.

[0173] Modified cells may have CAR positivity rates of more than 5%, more than 10%, more than 15%, more than 20%, more than 25%, more than 30%, more than 35%, more than 40%, more than 45%, more than 50%, more than 55%, more than 60%, more than 65%, more than 70%, more than 75%, more than 80%, or more than 90% after 1, 2, 3, 4, or 5 rounds of stimulation in a rechallenge assay. Modified cells may have CAR positivity rates of less than 5%, less than 10%, less than 15%, less than 20%, less than 25%, less than 30%, less than 35%, less than 40%, less than 45%, less than 50%, less than 55%, less than 60%, less than 65%, less than 70%, less than 75%, less than 80%, or less than 90% after 1, 2, 3, 4, or 5 rounds of stimulation in a rechallenge assay. Compared to cells without ICR (e.g., H93 cells), modified cells containing ICR (e.g., H93-IL4R-IL23R cells) exhibit similar CAR positivity rates after 1, 2, 3, 4, or 5 rounds of stimulation in a rechallenge assay.

[0174] Modified cells may have survival rates of more than 5%, more than 10%, more than 15%, more than 20%, more than 25%, more than 30%, more than 35%, more than 40%, more than 45%, more than 50%, more than 55%, more than 60%, more than 65%, more than 70%, more than 75%, more than 80%, or more than 90% after 1, 2, 3, 4, or 5 rounds of stimulation in a rechallenge assay. Modified cells may have survival rates of less than 5%, less than 10%, less than 15%, less than 20%, less than 25%, less than 30%, less than 35%, less than 40%, less than 45%, less than 50%, less than 55%, less than 60%, less than 65%, less than 70%, less than 75%, less than 80%, or less than 90% after 1, 2, 3, 4, or 5 rounds of stimulation in a rechallenge assay. Compared to cells without ICR (e.g., H93 cells), modified cells containing ICR (e.g., H93-IL4R-IL23R cells) may exhibit similar survival rates after 1, 2, 3, 4, or 5 rounds of stimulation in a rechallenge assay.

[0175] The cytotoxicity of modified cells against tumor cells may be evaluated by a long-term cytotoxicity assay, in which case the modified cells are co-cultured with tumor cells (e.g., Hep3B2.1-7 cells) with or without IL-4 (e.g., 10 ng / ml IL-4). The effector cell:target cell (E:T) ratio may be 1:40, 0.5:1, 1:1, 2:1, 2.5:1, 5:1, or 10:1. Long-term cytotoxicity can be measured by a normalized cell index (real-time cell index / cell index before adding effector cells). Modified cells may have a normalized cell index greater than 1, greater than 2, greater than 3, greater than 4, greater than 5, or greater than 6 after co-culturing with tumor cells (e.g., Hep3B2.1-7 cells) with or without IL-4 (e.g., 10 ng / ml IL-4) for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 days. Compared to cells without ICR (e.g., H93 cells), the normalized cell index of modified cells containing ICR (e.g., H93-IL4R-IL9R cells) is greater when co-cultured with tumor cells (e.g., Hep3B2.1-7 cells) with or without IL-4 (e.g., 10 ng / ml IL-4). After co-culturing for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 days with or without IL-4, the levels may be lower by more than 5%, more than 10%, more than 15%, more than 20%, more than 30%, more than 40%, more than 50%, more than 60%, more than 70%, more than 80%, more than 90%, more than 100%, more than 150%, more than 200%, more than 250%, more than 300%, more than 400%, more than 500%, more than 600%, more than 700%, more than 800%, more than 900%, or more than 1000%.

[0176] Modified cells were able to secrete cytokines (e.g., IFN-γ and / or TNF-α) in long-term cytokine release assays, where modified cells were co-cultured with tumor cells (e.g., Hep3B2.1-7 cells) with or without IL-4 (e.g., 10 ng / ml IL-4). The effector cell:target cell (E:T) ratio could be 1:40, 0.5:1, 1:1, 2:1, 2.5:1, 5:1, or 10:1. Modified cells were co-cultured with tumor cells (e.g., Hep3B2.1-7 cells) with IL-4 (e.g., 10 ng / ml IL-4). After co-culturing for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 days with or without IL-4, the following concentrations were observed: over 50 pg / ml, over 100 pg / ml, over 200 pg / ml, over 300 pg / ml, over 400 pg / ml, over 500 pg / ml, over 1000 pg / ml, over 1500 pg / ml, and 2 Modified cells can secrete IFNγ in amounts greater than 000 pg / ml, greater than 2500 pg / ml, greater than 3000 pg / ml, greater than 4000 pg / ml, greater than 5000 pg / ml, greater than 6000 pg / ml, greater than 7000 pg / ml, greater than 8000 pg / ml, greater than 10000 pg / ml, greater than 15000 pg / ml, greater than 20000 pg / ml, or greater than 25000 pg / ml. Modified cells can secrete IL-4 (e.g., 10 ng / ml) along with tumor cells (e.g., Hep3B2.1-7 cells). After co-culturing for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 days with or without IL-4, the following concentrations were determined: less than 50 pg / ml, less than 100 pg / ml, less than 200 pg / ml, less than 300 pg / ml, less than 400 pg / ml, less than 500 pg / ml, less than 1000 pg / ml, less than 1500 pg / ml, and 20 It secretes IFNγ in amounts less than 00 pg / ml, less than 2500 pg / ml, or less than 3000 pg / ml, less than 4000 pg / ml, less than 5000 pg / ml, less than 6000 pg / ml, less than 7000 pg / ml, less than 8000 pg / ml, less than 10000 pg / ml, less than 15000 pg / ml, less than 20000 pg / ml, or less than 25000 pg / ml.Modified cells were co-cultured with tumor cells (e.g., Hep3B2.1-7 cells) with or without IL-4 (e.g., 10 ng / ml IL-4) for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 days, after which IL-4 concentrations were adjusted to 500-5000 pg / ml, 1000-4000 pg / ml, 1000-3000 pg / ml, 1500-3000 pg / ml, 50-500 pg / ml, 50-400 pg / ml, 100-1000 pg / ml, and 100 IFNγ can be secreted in amounts of ~800 pg / ml, 100~600 pg / ml, 100~400 pg / ml, 200~400 pg / ml, 200~300 pg / ml, 200~1000 pg / ml, 200~800 pg / ml, 1500~3000 pg / ml, 1000~15000 pg / ml, 5000~15000 pg / ml, or 10000~20000 pg / ml. Compared to cells without ICR (e.g., H93 cells), modified cells containing ICR (e.g., H93-IL4R-IL9R cells) secrete IFNγ, along with IL-4 (e.g., 10 ng / ml), as do tumor cells (e.g., Hep3B2.1-7 cells). After co-culturing with IL-4 for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 days, the amount may increase by more than 5%, more than 10%, more than 15%, more than 20%, more than 30%, more than 40%, more than 50%, more than 60%, more than 70%, more than 80%, more than 90%, more than 100%, more than 150%, more than 200%, more than 250%, more than 300%, more than 400%, more than 500%, more than 600%, more than 700%, more than 800%, more than 900%, or more than 10.00%.

[0177] Modified cells, along with tumor cells (e.g., Hep3B2.1-7 cells), contain IL-4 (e.g., 10 ng / ml). After co-culturing for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 days with or without IL-4, TNFα can be secreted in amounts greater than 10 pg / ml, 20 pg / ml, 30 pg / ml, 40 pg / ml, 50 pg / ml, 60 pg / ml, 70 pg / ml, 80 pg / ml, 90 pg / ml, 100 pg / ml, 200 pg / ml, 300 pg / ml, 400 pg / ml, 500 pg / ml, 600 pg / ml, 700 pg / ml, 800 pg / ml, or 900 pg / ml. Modified cells can secrete TNFα in amounts of less than 10 pg / ml, less than 20 pg / ml, less than 30 pg / ml, less than 40 pg / ml, less than 50 pg / ml, less than 60 pg / ml, less than 70 pg / ml, less than 80 pg / ml, less than 900 pg / ml, or less than 900 pg / ml after co-culturing with tumor cells (e.g., Hep3B2.1-7 cells) with or without IL-4 (e.g., 10 ng / ml IL-4) for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 days.Modified cells are co-cultured with tumor cells (e.g., Hep3B2.1-7 cells) with or without IL-4 (e.g., 10 ng / ml IL-4) for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 days, after which the IL-4 levels are adjusted to 10-1000 pg / ml, 10-800 pg / ml, 10-500 pg / ml, 10-400 pg / ml, 10-300 pg / ml, 10-200 pg / ml, 20-200 pg / ml, 20-300 pg / ml, and 20- It can secrete TNFα in amounts of 400 pg / ml, 50-1000 pg / ml, 50-800 pg / ml, 50-400 pg / ml, 50-200 pg / ml, 50-150 pg / ml, 100-800 pg / ml, 200-800 pg / ml, 200-600 pg / ml, 200-500 pg / ml, 200-400 pg / ml, 300-800 pg / ml, 400-800 pg / ml, 400-900 pg / ml, 400-600 pg / ml, 300-600 pg / ml, or 350-600 pg / ml. Compared to cells without ICR (e.g., H93 cells), modified cells containing ICR (e.g., H93-IL4R-IL9R cells) secrete TNFα, along with IL-4 (e.g., 10 ng / ml), compared to tumor cells (e.g., Hep3B2.1-7 cells). After co-culturing with IL-4 for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 days, the amount may increase by more than 5%, more than 10%, more than 15%, more than 20%, more than 30%, more than 40%, more than 50%, more than 60%, more than 70%, more than 80%, more than 90%, more than 100%, more than 150%, more than 200%, more than 250%, more than 300%, more than 400%, more than 500%, more than 600%, more than 700%, more than 800%, more than 900%, or more than 1000%.

[0178] When modified cells are exposed to tumor cells (e.g., Hep3B2.1-7 cells), the modified cells can secrete more cytokines (e.g., IFNγ and / or TNFα). The effector cell:target cell (E:T) ratio can be 1:40, 0.5:1, 1:1, 2:1, 2.5:1, 5:1, or 10:1. When exposed to tumor cells (e.g., Hep3B2.1-7 cells), IFNγ secretion by modified cells may increase by more than 5%, more than 10%, more than 15%, more than 20%, more than 30%, more than 40%, more than 50%, more than 60%, more than 70%, more than 80%, more than 90%, more than 100%, more than 150%, more than 200%, more than 250%, more than 300%, more than 400%, more than 500%, more than 600%, more than 700%, more than 800%, more than 900%, or more than 1000%. When exposed to tumor cells (e.g., Hep3B2.1-7 cells), TNFα secretion by modified cells may increase by more than 5%, more than 10%, more than 15%, more than 20%, more than 30%, more than 40%, more than 50%, more than 60%, more than 70%, more than 80%, more than 90%, more than 100%, more than 150%, more than 200%, more than 250%, more than 300%, more than 400%, more than 500%, more than 600%, more than 700%, more than 800%, more than 900%, or more than 1000%.

[0179] Treatment method The ICRs, polynucleotides, and modified cells described herein can be used in a variety of experimental, therapeutic, and commercial applications.

[0180] In one embodiment, the present disclosure provides a pharmaceutical composition comprising modified cells as described herein and a pharmaceutically acceptable carrier.

[0181] In one embodiment, the Disclosure provides a method for treating a disease or disorder in a subject (e.g., a human subject), comprising administering to the subject an effective amount of modified cells described herein or a pharmaceutical composition described herein. The disease or disorder may be cancer, an autoimmune disease, a tumor, or an infection.

[0182] A disease or disorder may be a solid tumor. A "solid tumor" usually refers to an abnormal mass of tissue that does not contain cysts or fluid areas. Solid tumors can be benign (non-cancerous) or malignant (cancerous). Different types of solid tumors are named after the type of cells that form them. Examples of solid tumors include sarcomas, carcinomas, and lymphomas. Leukemia (a cancer of the blood) generally does not form solid tumors.

[0183] In one embodiment, the present disclosure provides a method for modulating an immune response, comprising administering an effective amount of the modified cells described herein to a subject in need thereof.

[0184] The term "effective dose," as used herein, means the amount of medication that is effective in terms of the dosage and duration required to achieve the desired result.

[0185] In another aspect, the Disclosure provides a method for treating cancer, comprising administering an effective amount of the modified cells described herein to a subject in need. Examples of cancers that can be treated include, but are not limited to, small cell lung cancer (SCLC), large cell neuroendocrine carcinoma (LCNC), neuroendocrine prostate cancer (NEPC), pancreatic neuroendocrine tumors (PNET), gastrointestinal neuroendocrine carcinoma, chronic lymphocytic leukemia, chronic myeloid leukemia, acute myeloid leukemia, acute lymphoblastic leukemia, and leukemias including T-cell and B-cell leukemia, lymphomas (Hodgkin and non-Hodgkin), lymphoproliferative disorders, plasmacytoma, histiocytoma, melanoma, adenoma, sarcoma, solid tissue carcinoma, hypoxic tumor, squamous cell carcinoma, genitourinary cancers such as cervical cancer and bladder cancer, hematopoietic cancer, head and neck cancer, and nervous system cancer. Cancer may be breast cancer, lung cancer, pancreatic cancer, melanoma, oral cancer, mesothelioma, ovarian cancer, colorectal cancer, gastric cancer, cervical cancer, brain cancer, skin cancer, multiple myeloma, lymphoma, epithelial neoplasm, soft tissue sarcoma, esophageal cancer, or CNS tumor.

[0186] This disclosure further includes the use of modified cells described herein in the manufacture of pharmaceuticals or pharmaceutical compositions for modulating immune responses, treating infectious diseases, or treating cancer, as described above.

[0187] Modified cells can also be used in experimental models, for example, to further study and elucidate cell function.

[0188] One or more of the modified cells described herein may be administered to a subject in a single, integrated form, such as intravenous injection, or in multiple forms, for example, by multiple intravenous infusions or injections, or by subcutaneous injection. The modified cells may expand in vivo within the subject's body after administration. The modified cells may be frozen, and the same cell preparation may be used to provide cells for multiple treatments. The modified cells of this disclosure, and the pharmaceutical compositions containing them, may be packaged as kits. The kits may include instructions (e.g., written instructions) on the use of the modified cells and the compositions containing them.

[0189] The treatment method may include administering a therapeutically effective dose of modified cells to the subject. The therapeutically effective dose of modified cells may be administered over a period of at least one week, two weeks, three weeks, one month, two months, three months, four months, five months, six months, or one year. The therapeutically effective dose of modified cells may also be administered over a period of at least one week. The therapeutically effective dose of modified cells may also be administered over a period of at least two weeks.

[0190] The modified cells described herein can be administered before, during, or after the onset of a disease or condition, and the timing of administration of the modified cells can vary. For example, the modified cells can be used as a prophylactic agent and can be administered sequentially to subjects susceptible to a condition or disease to reduce the likelihood of the disease or condition developing. The modified cells can be administered to subjects at the time of symptom onset or as soon as possible after the onset. Administration of the modified cells can be initiated immediately after the onset of symptoms, within the first three hours, within the first six hours, within the first 24 hours, within 48 hours, or within any time after the onset of symptoms. The initial administration can be carried out using any formulation described herein by any practical route (e.g., intravenous infusion or injection), including any route described herein. In some examples, the administration of the modified cells in this disclosure is intravenous. One or more doses of modified cells can be administered as soon as feasible after the onset of cancer or infectious disease, over a period of time necessary to treat the disease, such as approximately 24 to 48 hours, approximately 48 hours to approximately 1 week, approximately 1 week to approximately 2 weeks, approximately 2 weeks to approximately 1 month, or approximately 1 month to approximately 3 months. For the treatment of cancer, one or more doses of modified cells can be administered several years after the onset of cancer, before or after other treatments. In some cases, modified cells can be administered for at least approximately 10 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, 24 hours, at least 48 hours, at least 72 hours, at least 96 hours, at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 1 year, at least 2 years, at least 3 years, at least 4 years, or at least 5 years. The duration of treatment may vary depending on the subject.

[0191] Methods for administering modified cells for adoptive cell therapy are known and can be used in connection with the provided methods and compositions. For example, for adoptive T cell therapy, see, for example, U.S. Patent Publication No. 2003 / 0170238 to Gruenberg et al.; U.S. Patent No. 4,690,915 to Rosenberg; Rosenberg (2011) Nat Rev Clin Oncol. 8(10):577-85). See, for example, Themeli et al. (2013) Nat Biotechnol. 31(10):928-933; Tsukahara et al. (2013) Biochem Biophys Res Commun 438(1):84-9; and Davila et al. (2013) PLoS ONE 8(4):e61338. Cell therapy, such as adoptive T-cell therapy, may be performed by autologous transplantation, in which cells are isolated from and / or prepared by other means from a subject to receive cell therapy, or prepared from a sample derived from such subject. Thus, the cells originate from the subject in need of treatment and cells, e.g., a patient, and are administered to the same subject after isolation and processing.

[0192] Cell therapy (e.g., adoptive T-cell therapy) can be performed by allogeneic transplantation, in which cells are isolated and / or prepared by other means from a subject other than the subject that will receive or will ultimately receive cell therapy, e.g., a first subject. In such embodiments, the cells are then administered to a different subject of the same species, e.g., a second subject. The first and second subjects may be genetically identical. The first and second subjects may be genetically similar. The second subject may express the same HLA class or supertype as the first subject.

[0193] The subject (e.g., a human subject) may have been treated with a therapeutic agent targeting a disease or condition, such as a tumor, prior to administration of cells or a cell-containing composition. The subject may be refractory or unresponsive to other therapeutic agents. The subject may have a persistent or recurrent disease after treatment with another therapeutic intervention, such as chemotherapy, radiation therapy, and / or hematopoietic stem cell transplantation (HSCT), including, for example, allogeneic HSCT. This administration may effectively treat the subject even if the subject has become resistant to other therapies.

[0194] The subjects may respond to other therapeutic agents, and treatment with those agents reduces the disease burden. The subjects may initially respond to therapeutic agents, but exhibit a relapse of the disease or condition over time. The subjects do not need to show a relapse. The subjects may be determined to be at high risk of relapse, and therefore, cells are administered prophylactically, for example, to reduce the likelihood of relapse or to prevent relapse. The subjects do not need to have received prior treatment with other therapeutic agents.

[0195] The subjects may have persistent or relapsing disease following treatment with other therapeutic interventions, such as chemotherapy, radiation therapy, and / or hematopoietic stem cell transplantation (HSCT), including allogeneic HSCT. This treatment may effectively treat the subjects even if they have become resistant to other therapies.

[0196] The modified cells described herein may be administered to animals, such as mammals, and more specifically to humans, for the treatment of cancer. In addition, the modified cells may be used to treat any cancer-related condition, particularly cell-mediated immune responses to one or more tumor cells, where it is desirable to treat or alleviate the disease.

[0197] Modified cells described herein (e.g., immune cells, T cells, or NK cells) may be included in compositions for immunotherapy. The composition comprises a pharmaceutical composition and may further comprise a pharmaceutically acceptable carrier. A therapeutically effective dose of the pharmaceutical composition containing the modified cells can be administered.

[0198] Modified cells can be used immediately in the therapeutic, experimental, or commercial applications described above, or the cells can be cryopreserved for later use. The pharmaceutical composition can be contained in a container, pack, or dispenser together with instructions for administration.

[0199] The modified cells disclosed herein can be formulated into unit dosage forms suitable for single-dose administration of precise dosages. The unit dosage forms may contain additional lymphocytes. In the unit dosage forms, the formulation is divided into unit doses containing appropriate amounts of one or more compounds. The unit doses may be in the form of packages containing individual doses of the formulation. Non-limiting examples include packaged tablets or capsules, and powders in vials or ampoules. Aqueous suspension compositions can be packaged in non-resealable containers for single doses. Resealable containers for multiple doses can be used, for example, in combination with or without preservatives. Pharmaceutical compositions may be preservative-free. Formulations for parenteral injection may be in the form of unit dosage forms in ampoules, or in multi-dose containers with preservatives. [Examples]

[0200] This disclosure is further illustrated by the following examples, which do not limit the scope of this disclosure as set forth in the claims.

[0201] Example 1. Creation of GPC3 CAR-T cells expressing IL-4Rα reverse cytokine receptor (ICR) A CAR backbone sequence encoding a GPC3 CAR (H93 CAR, SEQ ID NO: 20) backbone polypeptide was chemically synthesized. This sequence included an antigen-binding domain containing a CD8α signal peptide (SEQ ID NO: 25), an anti-GPC3 scFv (SEQ ID NO: 30), a CD8α hinge domain (SEQ ID NO: 26), a CD8α transmembrane domain (SEQ ID NO: 27), a CD137 costimulatory signaling domain (SEQ ID NO: 28), and a CD3ζ primary intracellular signaling domain (SEQ ID NO: 29) from the N-terminus to the C-terminus. This sequence was then cloned into a pre-modified lentiviral vector (pLSINK-BBzBB) by operably ligating it downstream of a constitutive hEF1α promoter for in vitro transcription. To co-express armor elements, IL4R-IL23R, ICR5, and ICR6 sequences were also chemically synthesized and ligated to the C-terminus of the CD3ζ primary intracellular signaling domain, respectively, using the cloneEZ method. As shown in Figures 1a and 1b, this ICR was linked to the coding sequence of the CAR by a self-cleaving 2A (e.g., P2A, SEQ ID NO: 31) linker. Lentiviral expression vectors were prepared that co-express IL4R-IL23R#01 (SEQ ID NO: 13) and H93 CAR (SEQ ID NO: 20) (collectively, H93-IL4R-IL23R#01, SEQ ID NO: 21), or IL4R-IL23R#02 (SEQ ID NO: 14) and H93 CAR (SEQ ID NO: 20) (collectively, H93-IL4R-IL23R#02, SEQ ID NO: 22). The ICR structures and sequences of the IL-4 reverse cytokine receptors described herein are shown in Table 1.

[0202] [Table 1]

[0203] A lentiviral packaging plasmid mixture containing pMDLg.pRRE (Addgene#12251), pRSV-REV (Addgene#12253), and pMD2.G (Addgene#12259) was pre-mixed with polyetherimide (PEI) in a pre-optimized ratio along with a vector expressing the CAR construct, and then incubated at 25°C for 5 minutes. Next, the transfection mixture was added to HEK293 cells. The cells were then incubated overnight at 37°C in a 5% CO2 cell incubator. After centrifugation at 4°C and 3000g for 15 minutes, the supernatant was collected, filtered through a 0.45 μm PES filter, and then the lentivirus was concentrated by ultracentrifugation. The supernatant was then carefully discarded, and the virus pellet was carefully rinsed with pre-cooled DPBS. The virus was properly resuspended and stored at -80°C. Viral titer was determined by transduction titer measurement of CHO (Chinese hamster ovary) cell lines.

[0204] Human T cells were purified from commercially available PBMCs using the Miltenyi Pan T cell isolation kit (catalog number 130-096-535) according to the manufacturer's protocol as described below. First, the cell count was determined, and the cell suspension was centrifuged at 300 g for 10 minutes. Next, the supernatant was completely discarded, and the cell pellet was 10 7 Each total cell was resuspended in 40 μL of MACS buffer (DPBS supplemented with 8 μM EDTA + 0.5% FBS). 10 7 Add 10 μL of Pan T cell biotin-antibody cocktail per total number of cells, mix thoroughly, and incubate in a refrigerator (2-8°C) for approximately 5 minutes. Then 10 7 30 μL of MACS buffer was added per cell. 10 720 μL of Pan T cell microbead cocktail was added per cell. This cell suspension mixture was thoroughly mixed and incubated in a refrigerator (2-8°C) for a further 10 minutes. A minimum of 500 μL was required for magnetic separation. For magnetic separation, an LS column was placed in the magnetic field of a suitable MACS separator. The column was prepared by rinsing with 3 mL of buffer. The cell suspension was then applied to the column, and the flow-through containing unlabeled cells was collected, which corresponded to the concentrated T cell fraction. Further T cells were recovered by washing the column with 3 mL of buffer and collecting the unlabeled cells that passed through. These unlabeled cells also corresponded to concentrated T cells and were combined with the flow-through from the previous step. Next, the pooled concentrated T cells were centrifuged and resuspended in 1 L of TexMACS GMP Medium (Miltenyi #170-076-309) containing 300 IU / mL IL-2.

[0205] The prepared T cells were then pre-activated for 48-96 hours using the Human T Cell Activation / Expansion Kit (Miltenyi #130-091-441) with anti-CD3 / CD28 MACSiBead particles added in a 1:2 bead-to-cell ratio according to the manufacturer's protocol.

[0206] Pre-activated T cells were transduced using the lentivirus stock by directly adding it to culture medium (TexMACS GMP medium supplemented with 300 IU / mL of IL-2). Next, the transduced cells were transferred to a cell culture incubator at 37°C with 5% CO2 for transgene expression.

[0207] On day 7, CAR expression levels were determined by flow cytometry. In short, 3 × 10⁶ samples were taken from each group. 5Individual T cells were harvested and then incubated with FITC-GPC3 protein-Fc tag (Acrobiosystems#GP3-HF258-200UG) and PE-anti-IL4Rα antibody (Biolegend#355004), followed by incubation at 4°C for 30 minutes. As shown in the table below, the CAR positivity rates for UnT, H93, H93-IL4R-IL23R#01, H93-IL4R-IL23R#02, H93-ICR5, and H93-ICR6 CAR-T cells were 0.30%, 60.09%, 45.52%, 33.32%, 61.48%, and 61.96%, respectively. The IL4Rα expression levels in UnT, H93, H93-IL4R-IL23R#01, H93-IL4R-IL23R#02, H93-ICR5, and H93-ICR6 CAR-T cells were 0.56%, 0.32%, 47.14%, 32.50%, 51.89%, and 49.11%, respectively.

[0208] [Table 2]

[0209] Example 2. Expression levels of pSTAT3 and pATAT4 in IL-4-treated IL4R-IL23R-expressing CAR-T cells. To study intracellular signaling in CAR-T cells in vitro in the presence of IL-4, CAR-T cells were treated with 20 ng / mL recombinant human IL-4 (ACRO#IL4-H4218) for 15 minutes, and then measured for pSTAT3 and pSTAT4 by FACS. CAR-T cells were fixed with paraformaldehyde and permeabilized with Tween-20 for 10 minutes. Cells were resuspended in 100 μL of DPBS containing FITC-GPC3 protein-Fc tag (Acrobiosystems#GP3-HF258-200UG) and pSTAT3 antibody (Biolegend#651004) at 4°C for 30 minutes. Cells were washed with DPBS, resuspended in 100 μL of DPBS, and then detected by FACS. As shown in Figures 2a to 2c, STAT3 phosphorylation in H93-IL4R-IL23R#01 and H93-IL4R-IL23R#02 CAR-T cells was significantly increased compared to H93 CAR-T cells after treatment with 20 ng / mL of IL-4. As shown in Figure 2g, the median fluorescence intensity (MFI) of pSTAT3 in IL-4-free H93-IL4R-IL23R#01, H93-IL4R-IL23R#02, and H93 CAR-T cells was 1267, 952, and 1792, respectively. However, the MFI of pSTAT3 in H93-IL4R-IL23R#01, H93-IL4R-IL23R#02, and H93 CAR-T cells treated with 20 ng / mL of IL-4 was 8867, 8000, and 3644, respectively.

[0210] After 15 minutes of treatment with 20 ng / mL of IL-4, cells were measured by FACS using a FITC-GPC3 protein-Fc tag (Acrobiosystems#GP3-HF258-200UG) and a phospho-STAT4 (Tyr693) antibody (Invitrogen #17-9044-42). As shown in Figures 2d-2f, STAT4 phosphorylation in H93-IL4R-IL23R#01 and H93-IL4R-IL23R#02 CAR-T cells was significantly increased after treatment with 20 ng / mL of IL-4 compared to H93 CAR-T cells. The MFI of pSTAT4 in IL-4-free H93-IL4R-IL23R#01, H93-IL4R-IL23R#02, and H93 CAR-T cells was 349, 346, and 363, respectively. However, the MFI values ​​of pSTAT4 in H93-IL4R-IL23R#01, H93-IL4R-IL23R#02, and H93 CAR-T cells treated with 20 ng / mL of IL-4 were 1224, 1087, and 477, respectively.

[0211] These results demonstrate that H93-IL4R-IL23R#01 and H93-IL4R-IL23R#02 CAR-T cells co-expressing GPC3 CAR and IL4R-IL23R fusion proteins can promote STAT3 and STAT4 phosphorylation in response to IL-4 stimulation, suggesting that H93-IL4R-IL23R#01 and H93-IL4R-IL23R#02 CAR-T cells can convert IL-4R signaling to IL23R signaling, thereby inducing a downstream response. In summary, these results firmly demonstrate that the IL4R-IL23R construct converts IL-4-mediated inhibitory signals to IL-23R-mediated signals in CAR-T cells, thereby conferring greater efficacy to CAR-T cells in killing target cells (e.g., tumor cells) even in the presence of IL-4.

[0212] Example 3. A rechallenge model in GPC3 CAR-T cells expressing IL4R-IL23R. To evaluate the persistence of CAR-T cells in vitro, a repeated challenge model of CAR-T cells was set up. The rechallenged assay included two treatment groups: the H93 CAR-T group (initial CAR-T cells were H93 CAR-T cells) and the H93-IL4R-IL23R#01 CAR-T group (initial CAR-T cells were H93-IL4R-IL23R#01 CAR-T cells). In the initial round (Round 1), CAR-T cells were co-cultured overnight with GPC3-expressing PLCPRF5 cells (ATCC#CRL-8024) in an E / T ratio of 2:1. After centrifugation at 300g for 10 minutes, the CAR-T cells were resuspended in fresh medium (RPMI 1640 medium, 10% FBS and 300 IU / mL IL2) and cultured for a further 2 days. Cell viability, count, and CAR positivity were analyzed. The cells were reused for four more rounds using the same treatment as in Round 1. After each target cell stimulation round, a new set of PLCPRF5 cells was added in each round according to the number of CAR-T cells.

[0213] At the end of each stimulation round, the percentage of CAR-positive T cells after co-culture with PLCPRF5 cells was detected by FACS (Figure 3a). In the rechallenge model assay, without external IL4 addition, the percentage of CAR-positive T cells increased almost identically between the H93 CAR-T group and the H93-IL4R-IL23R#01 CAR-T group, from 35% to 98.58% and 99.12%, respectively.

[0214] At the end of each round, the count and viability of CAR-T cells after co-culture with PLCPRF5 cells were detected by trypan blue staining using a T4 cell counter. 20 μL of cell suspension was mixed with 20 μL of trypan blue, pipetted into a disposable hemocytometer, and analyzed using a Cellometer T4. The amplification factor of CAR-T cells was calculated according to the total number of T cells.

[0215] As shown in FIGS. 3b to 3c, in the rechallenge model assay, the amplification fold of H93 CAR-T cells increased from 1 to 18.42, while that of H93-IL4R-IL23R#01 CAR-T cells increased from 1 to 387.85. These results indicate that co-expression with the IL4R-IL23R construct results in high potency in CAR-T cells compared to the expression of CAR alone, due to the sharp increase in the currently low concentration of IL4 released by T cells and target cells themselves.

[0216] Example 4. Real-time cell analysis (RTCA) assay and cytokine release in CAR-T cells expressing ICR The real-time cytotoxic efficacy of CAR-T cells co-cultured with Hep3B2.1-7 cells was measured by RTCA assay. Hep3B2.1-7 cells (6×10 3 / well) were seeded in 96-well E-plates and detected overnight by an RTCA analyzer. Next, the target cells were incubated with CAR-T or UnT cells at an E / T ratio of 1:40 with or without 10 ng / mL of IL-4, and the cytotoxic efficacy of T cells during that time was detected in real-time. The cell index is an indicator of activity, adhesion, and the number of target cells (normalized cell index = real-time cell index / cell index at the time point before adding effector cells).

[0217] During the co-culture process, monitoring was stopped every three days, 90 μL of culture supernatant was collected from each well to determine cytokines, and then 90 μL of fresh medium was added to continue monitoring. For the cytokine release to be analyzed, the concentration of produced IFN-γ in the culture supernatant was measured using an HTRF kit (Cisbio, catalog number 62HIFNGPEG), and TNF-α was measured using an HTRF kit (Cisbio, catalog number 62HTNFAPEG). In short, the HTRF reagents were warmed to room temperature for at least 30 minutes before the assay. 16 μL / well supernatant from the co-culture assay was transferred to a 384-well assay plate (Greiner Bio-One, #784075), and then added along with 4 μL / well of pre-mixed HTRF reagent prepared according to the kit manual. The plate was then sealed with Parafilm, and the IFN-γ test was incubated overnight at room temperature, or the TNF-α test was incubated for 2 hours at room temperature. The plates were read using an HTRF-compatible Tecan Spark 10M reader. IFN-γ and TNF-α concentrations were calculated by referencing the signals obtained using the calibration curve provided by the kit.

[0218] As shown in Figures 4a and 4b, the cell index of Hep3B2.1-7 cells co-cultured with UnT cells increased significantly over time, with normalized cell indices of 4.13 and 3.76 at the end of the experiment with and without 10 ng / mL IL-4. H93, H93-IL4R-IL23R#01, H93-ICR5, and H93-ICR6 CAR-T cells showed varying degrees of cytotoxicity against Hep3B2.1-7 cells over time. As shown in Figure 4a, the normalized cell indices of Hep3B2.1-7 cells co-cultured with H93, H93-IL4R-IL23R#01, H93-ICR5, and H93-ICR6 CAR-T cells without 10 ng / mL IL-4 were 1.96, 1.85, 2.43, and 1.52, respectively. As shown in Figure 4b, the normalized cell indices of H93, H93-IL4R-IL23R#01, H93-ICR5, and H93-ICR6 CAR-T cells in the presence of 10 ng / mL of IL-4 were 2.26, 0.03, 1.03, and 0.17, respectively. These results indicate that the long-term cytotoxicity of CAR-T cells co-expressing IL4R-IL23R or IL4R-IL2R constructs is enhanced in the presence of IL-4 (10 ng / mL), and H93 CAR-T cells co-expressing IL4R-IL23R showed significantly stronger long-term cytotoxicity compared to H93 CAR-T cells co-expressing IL4R-IL2R (H93-ICR5 or H93-ICR6).

[0219] As shown in Figure 5a, IFN-γ release from H93 CAR-T cells with and without 10 ng / mL of IL-4 was 125 pg / mL and 21511 pg / mL, respectively. On day 12, IFN-γ release from H93-IL4R-IL23R#01, H93-ICR5, and H93-ICR6 CAR-T cells without 10 ng / mL of IL-4 was 1410 pg / mL, 353 pg / mL, and 438 pg / mL, respectively. On day 12, IFN-γ release from H93-IL4R-IL23R#01, H93-ICR5, and H93-ICR6 CAR-T cells with 10 ng / mL of IL-4 was 16382 pg / mL, 193 pg / mL, and 7379 pg / mL, respectively. As shown in Figure 5b, TNF-α release from H93 CAR-T cells with and without 10 ng / mL of IL-4 on day 12 was 50 pg / mL and 226 pg / mL, respectively. TNF-α release from H93-IL4R-IL23R#01, H93-ICR5, and H93-ICR6 CAR-T cells without 10 ng / mL of IL-4 on day 12 was 64 pg / mL, 57 pg / mL, and 57 pg / mL, respectively. TNF-α release from H93-IL4R-IL23R#01, H93-ICR5, and H93-ICR6 CAR-T cells with 10 ng / mL of IL-4 on day 12 was 858 pg / mL, 62 pg / mL, and 363 pg / mL, respectively. These results suggest that IL-4 can significantly inhibit H93 cytokine release, and that cytokine release from H93-IL4R-IL23R#01 cells is higher than that from H93 CAR-T cells (H93-ICR5 or H93-ICR6) that co-express IL4R-IL2R.

[0220] Other Embodiments This disclosure is provided in conjunction with a detailed description thereof, but it should be understood that the foregoing description is intended to be illustrative and not to limit the scope of this disclosure as defined by the attached claims. Other embodiments, advantages, and variations are within the following claims.

Claims

1. A fusion protein comprising (1) an extracellular domain that specifically binds to interleukin-4 (IL-4), and (2) an intracellular domain; the fusion protein transmits an interleukin-23 (IL-23) pathway signal when it binds to IL-4.

2. The fusion protein according to claim 1, comprising a first polypeptide chain and a second polypeptide chain.

3. The first polypeptide chain is (1) A first extracellular domain containing the interleukin-4 receptor alpha (IL-4Rα) extracellular domain, (2) A first intracellular domain comprising an interleukin-12 receptor beta-1 subunit (IL-12Rβ1) intracellular domain or an interleukin-23 receptor (IL-23R) intracellular domain and The fusion protein according to claim 2, comprising:

4. The second polypeptide chain is (1) A second extracellular domain containing the interleukin-2 receptor gamma (IL-2Rγ) extracellular domain, (2) A second intracellular domain comprising an IL-12Rβ1 intracellular domain or an IL-23R intracellular domain A fusion protein according to claim 2 or claim 3, comprising:

5. (a) The first polypeptide chain comprises (1) a first extracellular domain containing an IL-4Rα extracellular domain and (2) a first intracellular domain containing an IL-12Rβ1 intracellular domain, and the second polypeptide chain comprises (1) a second extracellular domain containing an IL-2Rγ extracellular domain and (2) a second intracellular domain containing an IL-23R intracellular domain; or (b) The first polypeptide chain comprises (1) a first extracellular domain containing an IL-4Rα extracellular domain and (2) a first intracellular domain containing an IL-23R intracellular domain, and the second polypeptide chain comprises (1) a second extracellular domain containing an IL-2Rγ extracellular domain and (2) a second intracellular domain containing an IL-12Rβ1 intracellular domain, A fusion protein according to any one of claims 2 to 4.

6. (a) (1) A first extracellular domain containing the IL-4Rα extracellular domain, (2) The first intracellular domain including the IL-12Rβ1 intracellular domain and A first polypeptide chain, and (1) A second extracellular domain containing the IL-2Rγ extracellular domain, (2) A second intracellular domain including the IL-23R intracellular domain and A second polypeptide chain including; or (b) (1) A first extracellular domain containing the IL-4Rα extracellular domain, (2) The first intracellular domain including the IL-23R intracellular domain and A first polypeptide chain, and (1) A second extracellular domain containing the IL-2Rγ extracellular domain, (2) A second intracellular domain including the IL-12Rβ1 intracellular domain and A second polypeptide chain containing A fusion protein containing [the specified ingredient].

7. The fusion protein according to any one of claims 2 to 6, wherein the first polypeptide chain and the second polypeptide chain are linked to each other by a 2A cleavable linker.

8. The first extracellular domain and the second extracellular domain form a binding site for IL-4. The first intracellular domain and the second intracellular domain form an IL-23 receptor complex, and When the fusion protein binds to IL-4, signal transduction is transmitted through the IL-23 receptor complex. A fusion protein according to any one of claims 2 to 7.

9. The fusion protein according to any one of claims 3 to 8, wherein the IL-4Rα extracellular domain comprises the amino acid sequence shown in SEQ ID NO: 1, or an amino acid sequence having at least 90%, 95%, or 99% identity with the amino acid sequence shown in SEQ ID NO:

1.

10. The fusion protein according to any one of claims 4 to 8, wherein the IL-2Rγ extracellular domain includes the amino acid sequence shown in SEQ ID NO: 5, or an amino acid sequence having at least 90%, 95%, or 99% identity with the amino acid sequence shown in SEQ ID NO:

5.

11. The fusion protein according to any one of claims 3 to 10, wherein the IL-12Rβ1 intracellular domain includes the amino acid sequence shown in SEQ ID NO: 4, or an amino acid sequence having at least 90%, 95%, or 99% identity with the amino acid sequence shown in SEQ ID NO:

4.

12. The fusion protein according to any one of claims 3 to 11, wherein the IL-23R intracellular domain includes the amino acid sequence shown in SEQ ID NO: 8, or an amino acid sequence having at least 90%, 95%, or 99% identity with the amino acid sequence shown in SEQ ID NO:

8.

13. A fusion protein according to any one of claims 1 to 12, further comprising a transmembrane domain.

14. The fusion protein according to claim 13, wherein the transmembrane domain is selected from the group consisting of the transmembrane domain of IL-4Rα, the transmembrane domain of IL-2Rγ, the transmembrane domain of IL-12Rβ1, and the transmembrane domain of IL-23R.

15. The fusion protein according to claim 13 or claim 14, wherein the transmembrane domain comprises the amino acid sequence shown in SEQ ID NO: 3 or 7, or an amino acid sequence having at least 90%, 95%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 3 or 7.

16. The fusion protein according to any one of claims 13 to 15, wherein the fusion protein comprises a first transmembrane domain in the first polypeptide chain and a second transmembrane domain in the second polypeptide chain; optionally, the first transmembrane domain and the second transmembrane domain may be dimerized.

17. The fusion protein according to any one of claims 13 to 16, further comprising a connecting sequence located between the C-terminus of the extracellular domain and the N-terminus of the transmembrane domain.

18. The fusion protein according to claim 17, wherein the connecting sequence includes the amino acid sequence shown in SEQ ID NO: 2 or 6, or an amino acid sequence having at least 90%, 95%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 2 or 6.

19. A fusion protein according to any one of claims 1 to 18, comprising a first and / or second polypeptide chain having at least 90%, 95%, or 99% identity with the amino acid sequence shown in any of SEQ ID NOs: 9 to 14, or with the amino acid sequence shown in any of SEQ ID NOs: 9 to 14.

20. A fusion protein according to any one of claims 1 to 19, which is an inverse cytokine receptor (ICR).

21. A nucleic acid comprising one or more nucleic acid sequences encoding a fusion protein or a portion thereof according to any one of claims 1 to 20.

22. The nucleic acid according to claim 21, wherein the nucleic acid further comprises a second nucleic acid sequence encoding a modified receptor, and the modified receptor comprises an extracellular antigen-binding domain or a ligand-binding domain and optionally an intracellular signaling domain.

23. The nucleic acid according to claim 22, wherein the nucleic acid sequence encoding the modified receptor is located upstream or downstream of at least one of the one or more nucleic acid sequences encoding the fusion protein, and optionally the modified receptor nucleic acid sequence and the fusion protein nucleic acid sequence are separated by a linker nucleic acid sequence encoding a 2A cleavable linker.

24. The nucleic acid according to claim 23, wherein the 2A cleavable linker comprises the amino acid sequence shown in any one of SEQ ID NOs: 31 to 33, or a functional variant having at least about 90% sequence identity thereto.

25. The nucleic acid according to any one of claims 22 to 24, wherein the modified receptor is selected from the group consisting of a modified T cell receptor (TCR), a chimeric antigen receptor (CAR), a T cell antigen coupling compound (TAC), or a portion thereof.

26. The nucleic acid according to claim 25, wherein the modified receptor is CAR.

27. The CAR comprises an extracellular antigen-binding domain that specifically binds to an antigen, and the antigen is BCMA, CLL1, CD4, GPC3, GPRC5D, GU2CYC, CD19, MUC16, MUC1, CAIX, CEA, CD8, CD7, CD10, CD20, CD22, CD30, CD33, CD34, CD38, CD41, CD44, CD49f, CD56, CD74, CD133, CD138, EGP-2, EGP-40, EpCAM, ERBB2 (HER-2), ERBB3, ERBB4, FBP, fetal acetylcholine receptor, folate receptor-α, GD2, GD3, hTERT, IL-13R-α2, κ-light chain, KDR, LeY The nucleic acid according to claim 26, which is a tumor antigen selected from the group consisting of L1 cell adhesion molecule, MAGE-A1, mesothelin, MAGEA3, p53, MART1, GP100, proteinase-3 (PR3), tyrosinase, survivorbin, hTERT, EphA2, NY-ESO-1, h5T4, PSCA, PSMA, ROR1, TAG-72, VEGF-R2, WT-1, CD123, CD44V6, NKCS1, IGF1R, EGFR, EGFR-VII, claudin 18.2, claudin 6, NKG2D, delta-like 3 (DLL3), CD70, CS-1, c-Met, glycolipid F77, PD-L1, and PD-L2.

28. The nucleic acid according to claim 27, wherein the tumor antigen is GPC3.

29. The nucleic acid according to any one of claims 26 to 28, wherein the CAR comprises a primary intracellular signaling domain of an immune cell and / or a co-stimulatory signaling domain.

30. The nucleic acid according to claim 29, wherein the primary intracellular signaling domain is derived from CD3ζ, and the co-stimulatory signaling domain is derived from a co-stimulatory molecule selected from the group consisting of ligands and combinations thereof of CD27, CD28, CD137, OX40, CD30, CD40, CD3, LFA-1, ICOS, CD2, CD7, LIGHT, NKG2C, B7-H3, CD83.

31. The nucleic acid according to any one of claims 26 to 30, wherein the CAR comprises a transmembrane domain derived from a molecule selected from the group consisting of CD8α, CD4, CD28, CD137, CD80, CD86, CD152, and PD1.

32. The nucleic acid according to any one of claims 26 to 31, wherein the CAR further comprises a hinge domain located between the C-terminus of the extracellular antigen-binding domain and the N-terminus of the transmembrane domain.

33. The nucleic acid according to any one of claims 26 to 32, wherein the CAR further comprises a signal peptide located at the N-terminus of the extracellular antigen-binding domain.

34. The nucleic acid according to any one of claims 21 to 33, encoding the amino acid sequence shown in any one of SEQ ID NOs: 9-14 and 20-22, or a functional variant having at least about 90% sequence identity thereto.

35. A vector comprising the nucleic acid described in any one of claims 21 to 34.

36. A modified cell comprising a fusion protein according to any one of claims 1 to 20, a nucleic acid according to any one of claims 21 to 34, and / or a vector according to claim 35.

37. Further containing modified receptors, The modified receptor comprises an extracellular antigen-binding domain or a ligand-binding domain and optionally an intracellular signaling domain. The modified cell according to claim 36.

38. The modified cell according to claim 37, wherein the modified receptor is selected from the group consisting of a modified T cell receptor (TCR), a chimeric antigen receptor (CAR), a T cell antigen coupling compound (TAC), or a portion thereof.

39. The modified cell according to claim 37 or claim 38, wherein the modified receptor specifically binds to the antigen.

40. It comprises a polypeptide encoding a fusion protein and / or a polypeptide encoding a CAR, and The polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 9-14 and 20-22, or a functional variant having at least about 90% sequence identity thereto. Modified cells according to any one of claims 36 to 39.

41. A modified cell according to any one of claims 36 to 40, which is an immune cell.

42. The modified cell according to claim 41, wherein the immune cells are selected from the group consisting of T cells, NK cells, peripheral blood mononuclear cells (PBMCs), hematopoietic stem cells, pluripotent stem cells, embryonic stem cells, and combinations thereof.

43. The modified cell according to claim 42, wherein the immune cell is a T cell, and optionally the immune cell is an αβT cell or a γδT cell.

44. A pharmaceutical composition comprising modified cells according to any one of claims 36 to 43 and a pharmaceutically acceptable carrier.

45. A method for producing modified cells, comprising introducing the vector described in claim 35 into cells.

46. A method for treating a disease or disorder in a subject, comprising administering to the subject in need of such treatment a therapeutically effective amount of a modified cell according to any one of claims 36 to 43 or a pharmaceutical composition according to claim 44.

47. The method according to claim 46, wherein the disease or disorder is cancer, an autoimmune disease, or a tumor.

48. The method according to claim 46 or claim 47, wherein the disease or disorder is breast cancer, lung cancer, pancreatic cancer, melanoma, oral cancer, mesothelioma, ovarian cancer, colorectal cancer, gastric cancer, cervical cancer, brain cancer, skin cancer, multiple myeloma, lymphoma, epithelial neoplasm, soft tissue sarcoma, esophageal cancer, or CNS tumor.

Images