Fusion proteins and methods of use thereof

Abstract

The present application relates to fusion proteins (e.g., reverse cytokine receptors) comprising an extracellular domain of an immunosuppressive cytokine receptor (e.g., IL-4R) and an intracellular domain of an immunostimulatory receptor (e.g., IL-23 receptor complex). A fusion protein (e.g., a reverse cytokine receptor) binds to an inhibitory cytokine and converts its intracellular signal to an immunostimulatory / activatable signal. The application further relates to polynucleotides encoding such fusion proteins, engineered cells expressing such fusion proteins, therapeutic uses of such fusion proteins, and pharmaceutical compositions comprising such fusion proteins.

Description

[0001] Cross-references

[0002] This application claims priority to International Patent Application No. PCT / CN2023 / 107742, filed on July 17, 2023, the contents of which are incorporated herein by reference in their entirety.

[0003] sequence list

[0004] This application incorporates by reference a sequence list submitted with this application, which is an XML file titled "IEC240173PCT SEQUENCE LISTING.xml", created on July 16, 2024, and is 51,855 bytes in size. Technical Field

[0005] This disclosure relates to fusion proteins (e.g., reverse cytokine receptors) that comprise an extracellular domain of an immunosuppressive cytokine receptor (e.g., IL-4R) and an intracellular domain of an immunostimulatory receptor (e.g., the IL-23 receptor complex). Background Technology

[0006] While adoptive transfer of chimeric antigen receptor (CAR) genetically engineered T cells has shown great promise as a treatment for hematologic malignancies, the application of CAR-T cells to treat solid tumors has had limited success. Current theory suggests that the immunosuppressive microenvironment of solid tumors, characterized by nutrient and oxygen restriction, accumulation of suppressor cells and cytokines, vascular disturbances, and endothelial dysfunction, constitutes a major obstacle to cancer immunotherapy (including CAR-T cell therapy).

[0007] To enhance the proliferation and antitumor efficacy of immune cells in an inhibitory microenvironment, CAR-expressing immune cells need to be modified to possess inherent resistance to anti-inflammatory cytokines. Inverse cytokine receptors (ICRs) can be used to increase the potency of immune cells (e.g., T cells). Summary of the Invention

[0008] This disclosure relates to fusion proteins (e.g., inverse cytokine receptors (ICRs)) comprising an extracellular domain of an immunosuppressive cytokine receptor (e.g., IL-4R) and an intracellular domain of an immunostimulatory receptor (e.g., the IL-23 receptor complex). The fusion protein binds to an inhibitory / suppressive cytokine and converts its intracellular signal into an immunostimulatory / activating signal. This disclosure further provides nucleic acids encoding such fusion proteins, polypeptides containing such fusion proteins, engineered cells expressing such fusion proteins, therapeutic uses of such fusion proteins, and pharmaceutical compositions comprising such fusion proteins.

[0009] In one respect, this disclosure relates to a fusion protein comprising: (1) an extracellular domain that specifically binds to interleukin-4 (IL-4), and (2) an intracellular domain; wherein, upon binding to IL-4, the fusion protein transmits interleukin-23 (IL-23) pathway signals.

[0010] In some embodiments, the fusion protein comprises a first polypeptide chain and a second polypeptide chain.

[0011] In some embodiments, the first polypeptide chain comprises (1) a first extracellular domain comprising an extracellular domain of an interleukin-4 receptor α (IL-4Rα) and (2) a first intracellular domain comprising an intracellular domain of an interleukin-12 receptor β1 subunit (IL-12Rβ1) or an intracellular domain of an interleukin-23 receptor (IL-23R).

[0012] In some embodiments, the second polypeptide chain comprises (1) a second extracellular domain comprising an extracellular domain of interleukin-2 receptor γ (IL-2Rγ), and (2) a second intracellular domain comprising an intracellular domain of IL-12Rβ1 or an intracellular domain of IL-23R.

[0013] In some embodiments, the first polypeptide chain comprises (1) a first extracellular domain comprising an IL-4Rα extracellular domain and (2) a first intracellular domain comprising an IL-12Rβ1 intracellular domain, and the second polypeptide chain comprises (1) a second extracellular domain comprising an IL-2Rγ extracellular domain and (2) a second intracellular domain comprising an IL-23R intracellular domain.

[0014] In some embodiments, the first polypeptide chain comprises (1) a first extracellular domain comprising an IL-4Rα extracellular domain and (2) a first intracellular domain comprising an IL-23R intracellular domain, and the second polypeptide chain comprises (1) a second extracellular domain comprising an IL-2Rγ extracellular domain and (2) a second intracellular domain comprising an IL-12Rβ1 intracellular domain.

[0015] In one respect, this disclosure relates to a fusion protein comprising:

[0016] (a) A first polypeptide chain containing the following:

[0017] (1) The first extracellular domain containing the IL-4Rα extracellular domain, and

[0018] (2) The first intracellular domain containing the IL-12Rβ1 intracellular domain, and

[0019] A second polypeptide chain containing the following items:

[0020] (1) A second extracellular domain containing the IL-2Rγ extracellular domain, and

[0021] (2) A second intracellular domain containing the intracellular domain of IL-23R; or

[0022] (b) A first polypeptide chain containing the following:

[0023] (1) The first extracellular domain containing the IL-4Rα extracellular domain, and

[0024] (2) The first intracellular domain containing the IL-23R intracellular domain, and

[0025] A second polypeptide chain containing the following items:

[0026] (1) A second extracellular domain containing the IL-2Rγ extracellular domain, and

[0027] (2) A second intracellular domain containing the intracellular domain of IL-12Rβ1.

[0028] In some embodiments, the first polypeptide chain and the second polypeptide chain are connected to each other via a cleavable 2A connector.

[0029] In some embodiments, the first extracellular domain and the second extracellular domain form the binding site for IL-4, wherein the first intracellular domain and the second intracellular domain form the IL-23 receptor complex, and wherein signal transduction is transmitted through the IL-23 receptor complex after the fusion protein binds to IL-4.

[0030] In some embodiments, the extracellular domain of IL-4Rα comprises the amino acid sequence shown in SEQ ID NO: 1 or has an amino acid sequence that is at least 90%, 95%, or 99% identical to the amino acid sequence shown in SEQ ID NO: 1.

[0031] In some embodiments, the extracellular domain of IL-2Rγ comprises the amino acid sequence shown in SEQ ID NO: 5 or has at least 90%, 95% or 99% identical amino acid sequence to the amino acid sequence shown in SEQ ID NO: 5.

[0032] In some embodiments, the intracellular domain of IL-12Rβ1 contains the amino acid sequence shown in SEQ ID NO: 4 or has an amino acid sequence that is at least 90%, 95%, or 99% identical to the amino acid sequence shown in SEQ ID NO: 4.

[0033] In some embodiments, the intracellular domain of IL-23R comprises the amino acid sequence shown in SEQ ID NO: 8 or has an amino acid sequence that is at least 90%, 95%, or 99% identical to the amino acid sequence shown in SEQ ID NO: 8.

[0034] In some embodiments, the fusion protein further includes a transmembrane domain.

[0035] 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.

[0036] In some embodiments, the transmembrane domain comprises the amino acid sequence shown in SEQ ID NO: 3 or 7, or an amino acid sequence that is at least 90%, 95%, or 99% identical to the amino acid sequence shown in SEQ ID NO: 3 or 7.

[0037] In some embodiments, the fusion protein includes a first transmembrane domain in a first polypeptide chain and a second transmembrane domain in a second polypeptide chain; optionally, the first transmembrane domain and the second transmembrane domain may dimerize.

[0038] In some embodiments, the fusion protein further includes a linker sequence located between the C-terminus of the extracellular domain and the N-terminus of the transmembrane domain.

[0039] In some embodiments, the linker sequence comprises the amino acid sequence shown in SEQ ID NO: 2 or 6, or an amino acid sequence that is at least 90%, 95%, or 99% identical to the amino acid sequence shown in SEQ ID NO: 2 or 6.

[0040] In some embodiments, the fusion protein comprises a first and / or a second polypeptide chain, the first and / or the second polypeptide chain comprising an amino acid sequence shown in any one of SEQ ID NO: 9-14 or having an amino acid sequence that is at least 90%, 95% or 99% identical to an amino acid sequence shown in any one of SEQ ID NO: 9-14.

[0041] In some embodiments, the fusion protein is an inverse cytokine receptor (ICR).

[0042] In one respect, this disclosure relates to a nucleic acid comprising one or more nucleic acid sequences encoding the fusion protein described herein or a portion thereof.

[0043] In some embodiments, the nucleic acid further comprises a second nucleic acid sequence encoding an engineered receptor, wherein the engineered receptor comprises an extracellular antigen-binding domain or a ligand-binding domain, and optionally an intracellular signal transduction domain.

[0044] In some embodiments, the nucleic acid sequence encoding the engineered receptor is upstream or downstream of at least one of one or more nucleic acid sequences encoding the fusion protein, and optionally the engineered receptor nucleic acid sequence and the fusion protein nucleic acid sequence are separated by a adapter nucleic acid sequence encoding a 2A cleavable adapter.

[0045] In some embodiments, the 2A cleavable adapter comprises an amino acid sequence shown in any one of SEQ ID NO: 31-33 or a functional variant having at least about 90% sequence identity with it.

[0046] In some embodiments, the engineered receptor is selected from the group consisting of engineered T-cell receptors (TCRs), chimeric antigen receptors (CARs), T-cell antigen conjugates (TACs), or a subset thereof.

[0047] In some embodiments, the engineered receptor is a CAR.

[0048] In some embodiments, the CAR includes an extracellular antigen-binding domain that specifically binds to an antigen, wherein the antigen is a tumor antigen selected from the group consisting of: 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 receptors. The following are listed: folate receptor-α, GD2, GD3, hTERT, IL-13R-α2, κ-light chain, KDR, LeY, L1 cell adhesion molecule, MAGE-A1, mesothelin, MAGEA3, p53, MART1, GP100, protease-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, duracin 18.2, duracin 6, NKG2D, delta-like ligand 3 (DLL3), CD70, CS-1, c-Met, glycolipid F77, PD-L1, and PD-L2.

[0049] In some embodiments, the tumor antigen is GPC3.

[0050] In some embodiments, the CAR includes primary intracellular signal transduction domains of immune cells and / or co-stimulatory signal transduction domains.

[0051] In some embodiments, the primary intracellular signal transduction domain is derived from CD3ζ, and the co-stimulatory signal transduction domain is derived from ligands of co-stimulatory molecules selected from the group consisting of: CD27, CD28, CD137, OX40, CD30, CD40, CD3, LFA-1, ICOS, CD2, CD7, LIGHT, NKG2C, B7-H3, CD83, and combinations thereof.

[0052] In some embodiments, the CAR includes a transmembrane domain derived from molecules selected from the group consisting of: CD8α, CD4, CD28, CD137, CD80, CD86, CD152, and PD1.

[0053] 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.

[0054] In some embodiments, the CAR further comprises a signal peptide located at the N-terminus of the extracellular antigen-binding domain.

[0055] In some embodiments, the nucleic acid encodes the amino acid sequence shown in any one of SEQ ID NO: 9-14 and 20-22 or a functional variant having at least about 90% sequence identity with it.

[0056] In one respect, this disclosure relates to a vector containing the nucleic acid described herein.

[0057] In one respect, this disclosure relates to an engineered cell comprising the fusion protein, nucleic acid, and / or vector described herein.

[0058] In some embodiments, the engineered cell further includes an engineered receptor, wherein the engineered receptor includes an extracellular antigen-binding domain or a ligand-binding domain, and optionally an intracellular signal transduction domain.

[0059] In some embodiments, the engineered receptor is selected from the group consisting of engineered T-cell receptors (TCRs), chimeric antigen receptors (CARs), T-cell antigen conjugates (TACs), or a subset thereof.

[0060] In some embodiments, engineered receptors bind specifically to antigens.

[0061] In some embodiments, the engineered cells comprise a polypeptide encoding a fusion protein and / or a polypeptide encoding a CAR, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 9-14 and 20-22 or a functional variant having at least about 90% sequence identity with it.

[0062] In some embodiments, the engineered cells are immune cells.

[0063] In some embodiments, 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.

[0064] In some embodiments, the immune cells are T cells, optionally αβT cells or γδT cells.

[0065] In one respect, this disclosure relates to a pharmaceutical composition comprising the engineered cells and pharmaceutically acceptable carriers described herein.

[0066] In one respect, this disclosure relates to a method for producing engineered cells, the method comprising introducing the vector described herein into the cells.

[0067] In one aspect, this disclosure relates to a method of treating a subject’s disease or disorder, the method comprising administering to the subject in need a therapeutically effective amount of the engineered cells or pharmaceutical composition described herein.

[0068] In some embodiments, the disease or disorder is cancer, an autoimmune disease, or a tumor.

[0069] In some embodiments, the disease or disorder is breast cancer, lung cancer, pancreatic cancer, melanoma, oral cancer, mesothelioma, ovarian cancer, colorectal cancer, stomach cancer, cervical cancer, brain cancer, skin cancer, multiple myeloma, lymphoma, epithelial tumor, soft tissue sarcoma, esophageal cancer, or CNS tumor. Attached Figure Description

[0070] Figures 1a-1b Two design schemes for IL4R-IL23R reverse cytokine receptors (ICRs) are shown. The IL4R-IL23R ICR consists of two fusion polypeptide chains, which are cleaved via a self-cleaving 2A linker after expression of the IL4R-IL23R ICR nucleic acid sequence. The two polypeptide chains can form a dimer fusion protein. Figure 1aThe nucleic acid sequence of IL4R-IL23R#01, as shown, includes the extracellular domain (ECD) of IL-4Rα and the transmembrane domain (TM) and intracellular domain (ICD) of IL-12Rβ1 from the 5' end to the 3' end, a 2A linker, the extracellular domain (ECD) of IL-2Rγ, and the transmembrane domain (TM) and intracellular domain (ICD) of IL-23R. Figure 1b As shown, the nucleic acid sequence of IL4R-IL23R#02 contains the IL-2Rγ extracellular domain (ECD) and IL-12Rβ1 intracellular domain (ICD), 2A adapter, IL-4Rα extracellular domain (ECD), and IL-23R intracellular domain (ICD) from the 5' to the 3' end. The ICR sequence is then linked to the sequence encoding the CAR via a self-cleaving 2A (e.g., P2A) adapter.

[0071] Figures 2a-2h The pSTAT signaling pathway in CAR-T cells was activated after in vitro treatment with IL-4. Figures 2a-2f Histograms of phosphorylated STAT3 and STAT4 expression obtained by flow cytometry under different concentrations of IL-4 are shown. Figure 2g-2h The median fluorescence intensity (median) of phosphorylated STAT3 and STAT4 expression under the influence of IL-4 is shown. The results indicate that IL-4 treatment significantly increased the expression levels of pSTAT3 and pSTAT4 in H93-IL4R-IL23R#01 and H93-IL4R-IL23R#02 CAR-T cells, while no significant change was observed in the expression levels of pSTAT3 and pSTAT4 in H93 CAR-T cells.

[0072] Figures 3a-3c This demonstrates the expansion capacity of HCC CAR-T cells after five rounds of stimulation in a re-stimulation assay. The percentage of CAR-positive cells was also measured. Figure 3a ), CAR-T cell expansion fold ( Figure 3b ) and CAR-T cell viability ( Figure 3c Following continuous antigen stimulation, H93-IL4R-IL23R#01 CAR-T cells exhibited excellent proliferative capacity. R0, R1, R2, R3, R4, and R5 represent rounds 1, 2, 3, 4, and 5, respectively.

[0073] Figures 4a-4bThe results of in vitro long-term cytotoxicity assays of GPC3 CAR-T cells against the HCC-positive cell line (Hep3B2.1-7) are presented with and without 10 ng / mL IL-4. The results showed that the long-term cytotoxicity of H93-IL4R-IL23R#01 CAR-T cells was significantly enhanced in the presence of 10 ng / mL IL-4.

[0074] Figures 5a-5b The release of IFNγ and TNFα from GPC3 CAR-T cells co-cultured with target cells in a long-term cytotoxicity assay was shown in the presence and absence of 10 ng / mL IL-4. In the presence of 10 ng / mL IL-4, the release of IFNγ and TNFα from H93-IL4R-IL23R#01 CAR-T cells was upregulated, while the release of IFNγ and TNFα from H93 CAR-T cells was significantly reduced.

[0075] Figure 6 The sequences listed in this application are shown. Detailed Implementation

[0076] Cytokines are a large class of small proteins that play a crucial role in cell signaling. Cytokines are peptides and cannot cross the lipid bilayer into the cytoplasm. They have been shown to participate in autocrine, paracrine, and endocrine signaling as immunomodulators. Among these cytokines, interleukins (ILs) are typically expressed and secreted by leukocytes, as well as some other somatic cells. Each cytokine has a different function and can elicit different responses depending on the target, cell origin, and stage of the immune response. It is generally accepted that cytokines, chemokines, and their receptors play a key role in regulating the functional and phenotypic characteristics of CAR-expressing cells (e.g., CAR-T cells) and influencing parameters such as persistence, transport, memory cell formation, and proliferation.

[0077] Immunosuppressive cytokines (including IL-10, TGFβ, and IL-4) are key components of the tumor microenvironment (TME) and can lead to CAR-T cell dysfunction. These cytokines induce immunosuppression through several mechanisms, such as recruiting and activating regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs), and inhibiting the effector functions of CAR-T cells. Furthermore, they can suppress the activity of several endogenous anti-tumor immune cells, such as T cells, NK cells, dendritic cells, and M1 macrophages.

[0078] To enhance the efficacy of immune cells in an inhibitory microenvironment, CAR-expressing immune cells can be modified to possess inherent resistance to anti-inflammatory cytokines. Inverse cytokine receptors (ICRs) are modified forms of cytokine receptors designed to enhance immune cell activity by overcoming cytokine-induced immunosuppression.

[0079] This disclosure provides a fusion protein comprising: (1) an extracellular domain that specifically binds to interleukin-4 (IL-4), and (2) an intracellular domain; wherein, upon binding to IL-4, the fusion protein transmits interleukin-23 (IL-23) pathway signaling. In some embodiments, the fusion protein is an inverse 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).

[0080] As used herein, the term "reverse cytokine receptor" or "ICR" generally refers to a chimeric protein containing an extracellular domain of one cytokine receptor and an intracellular domain of a different cytokine receptor. This chimeric protein can be engineered to act as a "switch receptor," counteracting the effects of immunosuppressive cytokines in certain diseases, particularly cancer. An ICR is established by exchanging 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, thereby helping to restore or enhance immune function.

[0081] As used herein, the term "IL-4Rα" refers to a polypeptide derived from wild-type IL-4 receptor α or a functional variant thereof. IL-4Rα can be wild-type IL-4Rα (e.g., human IL-4Rα). IL-4Rα can have one or more mutations (e.g., insertion, deletion, or substitution). IL-4Rα can be human IL-4Rα. The extracellular domain of IL-4Rα can encompass extracellular domains derived from wild-type IL-4Rα or a functional variant thereof, or a portion thereof.

[0082] As used herein, the term "IL-23R" refers to a polypeptide derived from wild-type IL-23R or a functional variant thereof. IL-23R can be wild-type IL-23R (e.g., human IL-23R). IL-23R can have one or more mutations (e.g., insertion, deletion, or substitution). IL-23R can be human IL-23R.

[0083] As used herein, the term "IL-2Rβ" refers to a polypeptide derived from wild-type IL-2Rβ or a functional variant thereof. IL-2Rβ can be wild-type IL-2Rβ (e.g., human IL-2Rβ). IL-2Rβ can have one or more mutations (e.g., insertion, deletion, or substitution). IL-2Rβ can be human IL-2Rβ.

[0084] As used herein, the term "IL-2Rγ" refers to a polypeptide derived from wild-type IL-2Rγ or a functional variant thereof. IL-2Rγ can be wild-type IL-2Rγ (e.g., human IL-2Rγ). IL-2Rγ can have one or more mutations (e.g., insertion, deletion, or substitution). IL-2Rγ can be human IL-2Rγ.

[0085] As used herein, the term "IL-12Rβ1" refers to a polypeptide derived from wild-type IL-12Rβ1 or a functional variant thereof. IL-12Rβ1 can be wild-type IL-12Rβ1 (e.g., human IL-12Rβ1). IL-12Rβ1 can have one or more mutations (e.g., insertion, deletion, or substitution). IL-12Rβ1 can be human IL-12Rβ1.

[0086] As used herein, the terms “extracellular domain” or “extracellular region” are used interchangeably to refer to the portion of the receptor outside the cell membrane. An extracellular domain may be the entire portion of the receptor outside 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). An extracellular domain may be derived from the wild-type receptor or an extracellular domain of a functional variant thereof. An extracellular domain may have one or more mutations, including, for example, insertions, deletions, or substitutions. The extracellular domain of IL-4Rα may be an extracellular domain derived from wild-type IL-4Rα or an extracellular domain of a functional variant thereof or a portion thereof.

[0087] As used herein, the terms "intracellular domain" and "intracellular region / cytoplasmic region" are used interchangeably to refer to a portion of the receptor within the cell. An intracellular domain can be the entire intracellular portion of the receptor 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). An intracellular domain can be derived from the wild-type receptor or an intracellular domain of a functional variant thereof. An intracellular domain can have one or more mutations, including, for example, insertions, deletions, or substitutions. The IL-23R intracellular domain can be an intracellular domain derived from wild-type IL-23R or a functional variant thereof, or a portion thereof. The IL-12Rβ1 intracellular domain can be an intracellular domain derived from wild-type IL-12Rβ1 or a functional variant thereof, or a portion thereof.

[0088] As used herein, the terms “transmembrane domain” or “transmembrane region” are used interchangeably to refer to the portion of the receptor embedded in the cell membrane. A 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). A transmembrane domain may be derived from a wild-type receptor or a transmembrane domain of a functional variant thereof. A transmembrane domain may have one or more mutations, including, for example, insertions, deletions, or substitutions. A transmembrane domain may be the transmembrane domain of an ICR. A transmembrane domain may be the transmembrane domain of a CAR.

[0089] As used herein, the terms "hinge domain" or "hinge region" are used interchangeably to refer to the portion of the receptor that connects the transmembrane region and the extracellular domain. The hinge region may be part of the extracellular domain. The hinge region may be derived from the hinge region of a 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 an ICR. The hinge region may be the hinge region of a CAR.

[0090] As used herein, a “vector” is any construct capable of delivering one or more polynucleotides of interest to a host cell when the vector is introduced into the host cell. An “expression vector” is capable of delivering one or more polynucleotides of interest to a host cell in which the expression vector has been introduced and expressing those polynucleotides as encoded polypeptides. Thus, in an expression vector, the polynucleotide of interest is positioned for expression in the vector by operatively linking to regulatory elements such as promoters, enhancers, and / or poly-A tails, which are located within the vector or in the genome of the host cell at or near the integration site of the polynucleotide of interest, such that the polynucleotide of interest will be translated in the host cell in which the expression vector has been introduced.

[0091] As used herein, the term "chimeric antigen receptor" or "CAR" refers to a genetically engineered receptor that can be used to specifically transplant one or more antigens onto immune effector cells, such as T cells. Some CARs are also referred to as "artificial T cell receptors," "chimeric T cell receptors," or "chimeric immune receptors." CARs may contain 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 cell receptors and / or other receptors. "CAR-T cell" refers to a T cell expressing a CAR.

[0092] As used herein, the term "T cell receptor" or "TCR" refers to an endogenous or modified T cell receptor that contains an extracellular antigen-binding domain that binds to a specific antigenic peptide bound to an MHC molecule. A TCR may contain a TCRα polypeptide chain and a TCRβ polypeptide chain. A TCR may contain a TCRγ polypeptide chain and a TCRδ polypeptide chain. A TCR can specifically bind to tumor antigens. "TCR-T" refers to a T cell expressing a recombinant TCR. Expression of heterologous antigen receptors (such as heterologous TCRs or CARs) can alter the immunogenicity specificity of T cells, enabling them to recognize one or more tumor antigens present on the surface of cancer cells in an individual with cancer, or exhibiting improved recognition of one or more tumor antigens present on the surface of cancer cells in an individual with cancer.

[0093] As used herein, the term "fusion protein" refers to a protein complex having one or more polypeptides that perform the desired function. An construct may contain or consist of a single polypeptide. A fusion protein may be a fusion polypeptide.

[0094] As used herein, the term “cancer” refers to cells capable of autonomous growth. Examples of such cells include cells exhibiting an abnormal state or condition characterized by rapid proliferative cell growth. The term is intended to include cancerous growth, such as tumors; carcinogenic processes, metastatic tissues, and malignantly transformed cells, tissues, or organs, regardless of histopathological type or stage of invasiveness. It also includes malignant tumors of various organ systems, such as the respiratory, cardiovascular, renal, reproductive, hematopoietic, nervous, hepatic, gastrointestinal, and endocrine systems; and adenocarcinomas, including most colon cancers, renal cell carcinomas, prostate and / or testicular tumors, non-small cell lung cancer, and small bowel cancer. “Naturally occurring” cancer includes any cancer not experimentally induced by implanting cancer cells into a subject, and includes, for example, spontaneously occurring cancer, cancer caused by patient exposure to one or more carcinogens, cancer caused by insertion of a transgenic oncogene or knockout of a tumor suppressor gene, and cancer caused by infection (e.g., viral infection). The term “cancer” is generally accepted in the field as referring to a malignant tumor of epithelial or endocrine tissue. The term also includes carcinosarcoma, which comprises malignant tumors composed of both carcinomatous and sarcomatous tissue. "Adenocarcinoma" refers to cancer derived from glandular tissue or in which tumor cells form identifiable glandular structures. The term "sarcoma" is generally accepted in the field as referring to a malignant tumor derived from mesenchyme. The term "hematopoietic neoplastic disorder" includes diseases involving proliferative / tumor cells of hematopoietic origin. Hematopoietic neoplastic disorders can arise from myeloid, lymphoid, or erythroid lineages or their precursor cells.

[0095] As used herein, the terms “subject” and “patient” are used interchangeably throughout the specification and describe an animal, human, or non-human, to which treatment is administered according to the methods disclosed herein. This disclosure contemplates both veterinary and non-veterinary applications. Human patients may be adults or adolescents (e.g., humans under the age of 18). In addition to humans, patients include, but are not limited to, mice, rats, hamsters, guinea pigs, rabbits, ferrets, cats, dogs, and primates. For example, this includes non-human primates (e.g., monkeys, chimpanzees, gorillas, etc.), rodents (e.g., rats, mice, gerbils, hamsters, ferrets, rabbits), lagomorphs, pigs (e.g., pigs, miniature pigs), horses, dogs, cats, cattle, and other domesticated, farm, and zoo animals.

[0096] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. This document describes the methods and materials used in this disclosure; other suitable methods and materials known in the art may also be used. Materials, methods, and examples are illustrative only and are not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated herein by reference in their entirety. In case of conflict, this specification, including the definitions, shall prevail.

[0097] Fusion proteins (e.g., reverse cytokine receptors)

[0098] In one aspect, this disclosure provides a fusion protein comprising: (1) an extracellular domain that specifically binds to interleukin-4 (IL-4), and (2) an intracellular domain; wherein, upon binding to IL-4, the fusion protein transmits interleukin-23 (IL-23) pathway signals.

[0099] In one aspect, this disclosure provides a fusion protein comprising: (a) a first polypeptide chain comprising: (1) a first extracellular domain comprising an extracellular domain of a cytokine receptor and (2) a first intracellular domain comprising an intracellular domain of a cytokine receptor, and (b) a second polypeptide chain comprising: (3) a second extracellular domain comprising an extracellular domain of a cytokine receptor and (4) a second intracellular domain comprising an intracellular domain of a cytokine receptor, wherein the first extracellular domain and the second extracellular domain form a binding site for IL-4, and the first intracellular domain and the second intracellular domain form an IL-23 receptor complex, and wherein, upon binding of the fusion protein to IL-4, signal transduction is conducted via the IL-23 receptor complex.

[0100] The first and second extracellular domains can be derived from cytokine receptors of immunosuppressive cytokines (e.g., 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 can be derived from cytokine receptors of immunostimulatory cytokines (e.g., 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).

[0101] In some embodiments, the fusion protein comprises a first polypeptide chain comprising (1) a first extracellular domain comprising an extracellular domain of an interleukin-4 receptor α (IL-4Rα) and (2) a first intracellular domain comprising an intracellular domain of an interleukin-12 receptor β1 subunit (IL-12Rβ1) or an intracellular domain of an interleukin-23 receptor (IL-23R). In some embodiments, the fusion protein comprises a second polypeptide chain comprising (1) a second extracellular domain comprising an extracellular domain of an interleukin-2 receptor γ (IL-2Rγ) and (2) a second intracellular domain comprising an intracellular domain of an interleukin-12 receptor β1 subunit (IL-12Rβ1) or an intracellular domain of an IL-23R.

[0102] Specifically, this disclosure provides a fusion protein comprising a first extracellular domain and a second extracellular domain of an IL-4 receptor (e.g., IL-4Rα or IL-2Rγ) derived from IL-4, and a first intracellular domain and a second intracellular domain of an IL-23 receptor complex (e.g., IL-12Rβ1 or IL-23R). In some embodiments, the fusion protein comprises:

[0103] (a) A first polypeptide chain containing the following:

[0104] (1) The first extracellular domain containing the IL-4Rα extracellular domain, and

[0105] (2) A first intracellular domain containing an IL-12Rβ1 intracellular domain, and a second polypeptide chain containing the following:

[0106] (3) A second extracellular domain containing the IL-2Rγ extracellular domain, and

[0107] (4) A second intracellular domain containing the intracellular domain of IL-23R; or

[0108] (b) A first polypeptide chain containing the following:

[0109] (1) The first extracellular domain containing the IL-4Rα extracellular domain, and

[0110] (2) A first intracellular domain containing an IL-23R intracellular domain, and a second polypeptide chain containing the following:

[0111] (3) A second extracellular domain containing the IL-2Rγ extracellular domain, and

[0112] (4) A second intracellular domain containing the intracellular domain of IL-12Rβ1.

[0113] In some embodiments, the fusion protein is an inverse cytokine receptor (ICR).

[0114] Inverse 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 a different cytokine receptor. These fusion proteins can be engineered to act as "switch receptors," counteracting the effects of immunosuppressive cytokines in certain diseases, particularly cancer. ICRs are established by exchanging 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, thereby helping to restore or enhance immune function.

[0115] IL-4 is primarily immunosuppressive. It participates in the differentiation of naive CD4 T cells into IL-4-producing Th2 cells, which exhibit lower antitumor activity compared to IFNγ-producing CD4 T cells (Th1). Adoptive transfer of tumor-specific IL-4-producing cytotoxic CD8 T cells (Tc2) is also less effective in controlling tumor growth compared to IFNγ-producing CD8 T cells (Tc1). Furthermore, several cancer types express IL-4 and IL-4R, suggesting their role in tumor progression. The IL-4Rα chain (CD124) is a 140 kDa heterodimeric complex that acts as a common monomer in both type 1 and type 2 receptor complexes. IL-4Rα interacts with the γ chain of the IL-2 receptor (also known as IL-2Rγ, γc, common γ chain, or CD132) to form the type 1 IL-4 receptor, and can also interact with the 65-70 kDa IL-13 binding receptor α1 (IL-13Rα1) chain to form the type 2 IL-4 / IL-13 receptor.

[0116] IL-2 regulates the activity of white blood cells (usually lymphocytes) responsible for immunity. IL-2 is part of the body's natural response to microbial infection and is involved in distinguishing foreign ("non-self") from "self." IL-2 mediates its action by binding to the IL-2 receptor expressed by lymphocytes. The primary sources of IL-2 are activated CD4+ T cells and activated CD8+ T cells. IL-2 binds to the IL-2 receptor, which has three forms, generated by different combinations of three different proteins (often referred to as "chains"): α (alpha) (also known as IL-2Rα, CD25, or Tac antigen), β (beta) (also known as IL-2Rβ or CD122), and γ (gamma) (also known as IL-2Rγ, γc, shared γ chain, or CD132). These subunits are also part of receptors for other cytokines. The three IL-2R forms include α, βγ, and αβγ. The α chain binds IL-2 with low affinity. β and γ combine to form the βγ complex, which binds to IL-2 with moderate affinity (primarily on memory T cells and NK cells). All three receptor chains form the αβγ complex, which binds to activated T cells and regulatory T cells with high affinity (approximately 10 Kd). -11 The α chain binds to IL-2. The β chain does not participate in signal transduction, while the β chain complexes with an enzyme called Janus kinase 1 (JAK1), which adds a phosphate group to the molecule. Similarly, the γ chain complexes with another tyrosine kinase called JAK3. These enzymes are activated by the binding of IL-2 to the outer domain of IL-2R. Thus, three intracellular signaling pathways are initiated: the MAP kinase pathway, the phosphatidylinositol 3-kinase (PI3K) pathway, and the JAK-STAT pathway.

[0117] IL-4 binds to a surface receptor complex consisting of IL-4Rα and IL-2Rγ. The receptors for IL-4 and IL-2 share several common features; 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.

[0118] IL-23 is one of the STAT3-activating cytokines and is composed of the IL-23α p19 and IL-12β p40 subunits, both of which are expressed by activated macrophages and dendritic cells. IL-23 belongs to the IL-12 cytokine family. IL-23 is known to promote the proliferation of memory T cells, particularly helper T cell type 17 (TH17 cells) cells that express the IL-23 receptor (IL-23R). IL-23 is primarily expressed by macrophages and dendritic cells (DCs). Upon activation by TGF-β and IL-6, IL-23R is found on memory T cells, NKT cells, macrophages, DCs, and naive T cells. The major biological effects of IL-23 initially identified consist of stimulating antigen presentation by DCs, T cell differentiation into Th17 cells, and the production of interferon-γ (IFN-γ). IL-23 also acts as a terminal effector cytokine through direct action on macrophages. This can be partially understood as an autocrine loop of IL-23 on macrophages. Furthermore, intraperitoneal administration of recombinant IL-23 in mice induced the expression of mRNAs encoding IL-1 and TNF-α in peritoneal macrophages. Incorporation of p40 into CAR- or TCR-engineered T cells enhanced their antitumor activity in xenograft and syngeneic mouse models.

[0119] The functional receptor for IL-23 (the IL-23 receptor complex) is composed of a heterodimer between the interleukin-12 receptor β1 subunit (IL-12Rβ1) and IL-23R. Upon complexation with IL-12Rβ1, IL-23R is activated by the cytokine IL-23. IL-23R is a type I penetrating protein and contains a signal peptide, an N-terminal fibronectin III-like domain, and an intracellular portion containing three potentially tyrosine phosphorylated domains.

[0120] In one aspect, this disclosure provides a fusion protein (e.g., an ICR) comprising an extracellular domain derived from an 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., the ICR) can be activated by IL-4 (e.g., human IL-4). Following binding of the IL-4R extracellular domain to IL-4, the fusion protein (e.g., the ICR) can activate signal transduction of the IL-23R intracellular signal transduction domain, which is transduced via native cellular elements to provide biological activity mimicking the native responses of IL-23R (e.g., STAT3 and / or STAT4 phosphorylation).

[0121] An ICR can be a fusion protein comprising two polypeptide chains. An ICR can comprise a first polypeptide chain and a second polypeptide chain. The first polypeptide chain can 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 can 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 can 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 can 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 can have the structures and / or sequences described in Table 1.

[0122] Each of the first and second polypeptide chains may further include a transmembrane domain between an extracellular domain and an intracellular domain. 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 dimerize. The first polypeptide chain may include, from its N-terminus to its 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 its N-terminus to its 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 its N-terminus to its 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 its N-terminus to its 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.

[0123] Fusion proteins (e.g., ICRs) may contain an IL-4Rα extracellular domain (ECD). The IL-4Rα extracellular domain 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 sequences 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 mutations (e.g., compared to SEQ ID NO: 1 or the entire 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.

[0124] Fusion proteins (e.g., ICRs) may contain an IL-2Rγ extracellular domain (ECD). The IL-2Rγ extracellular domain 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 sequences 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 mutations (e.g., compared to SEQ ID NO: 5 or the entire 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.

[0125] Fusion proteins (e.g., ICRs) may contain an intracellular domain (ICD) of IL-23R. The IL-23R intracellular domain 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 the amino acid sequence of SEQ ID NO: 8. The IL-23R ICD may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations (e.g., compared to SEQ 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 be within the IL-23R ICD or at one or both ends of the IL-23R ICD.

[0126] Fusion proteins (e.g., ICRs) may contain an intracellular domain (ICD) of IL-12Rβ1. The IL-12Rβ1 intracellular domain 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 sequences 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 mutations (e.g., compared to SEQ ID NO: 4 or the entire wild-type IL-12Rβ1 ICD). These mutations may be amino acid insertions, deletions, or substitutions. Insertions, deletions, and substitutions may be within the IL-12Rβ1 ICD or at one or both ends of the IL-12Rβ1 ICD.

[0127] Fusion proteins (e.g., ICRs) may contain an intracellular domain (ICD) of IL-2Rβ. The IL-2Rβ intracellular domain 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 sequences to those 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 (e.g., compared to 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 be within the IL-2Rβ ICD or at one or both ends of the IL-2Rβ ICD.

[0128] Fusion proteins (e.g., ICRs) may contain an IL-23R transmembrane domain (TM). The IL-23R transmembrane domain may be a transmembrane domain 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. The 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 the entire wild-type IL-23RTM). These mutations may be amino acid insertions, deletions, or substitutions. Insertion, deletion, and substitution can occur within IL-23R™ or at one or both ends of IL-23R™.

[0129] Fusion proteins (e.g., ICRs) may contain an IL-12Rβ1 transmembrane domain (TM). The IL-12Rβ1 transmembrane domain may be a transmembrane domain 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. The 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 the entire wild-type IL-12Rβ1 TM). These mutations may be amino acid insertions, deletions, or substitutions. Insertion, deletion, and substitution can occur within IL-12Rβ1™, or at one or both ends of IL-12Rβ1™.

[0130] Fusion proteins (e.g., ICRs) may contain an IL-2Rβ transmembrane domain (TM). The IL-2Rβ transmembrane domain may be a transmembrane domain 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. Insertion, deletion, and substitution can occur within IL-2Rβ™, or at one or both ends of IL-2Rβ™.

[0131] Fusion proteins (e.g., ICRs) may further include a linker sequence. The linker sequence may be selected from a membrane-proximal sequence within the extracellular domain of IL-23R (designated as the IL-23R linker sequence) or a membrane-proximal sequence within the extracellular domain of IL-12Rβ1 (designated as the IL-12Rβ1 linker sequence). Fusion proteins (e.g., ICRs) may include an IL-23R linker sequence. The IL-23R linker sequence 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 sequences to the amino acid sequence of SEQ ID NO: 6. The IL-23R linker sequence may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations (e.g., compared to SEQ ID NO: 6). These mutations may be amino acid insertions, deletions, or substitutions. Insertions, deletions, and substitutions may occur within the IL-23R linker sequence or at one or both ends of the IL-23R linker sequence. Fusion proteins (e.g., ICRs) may contain an IL-12Rβ1 linker sequence. The IL-12Rβ1 linker 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: 2. The IL-12Rβ1 linker sequence may have one, two, or three mutations (e.g., compared to SEQ ID NO: 2). These mutations may be amino acid insertions, deletions, or substitutions. Insertions, deletions, and substitutions may occur within the IL-12Rβ1 linker sequence or at one or both ends of the IL-12Rβ1 linker sequence.

[0132] The fusion protein (e.g., ICR) may comprise two polypeptide chains. The first polypeptide chain may comprise, from the N-terminus to the C-terminus, 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). The first 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 sequences to the sequence in SEQ ID NO: 9. The first polypeptide chain may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations (e.g., compared to SEQ ID NO: 9). These mutations may be amino acid insertions, deletions, or substitutions. Insertion, deletion, and substitution can occur within the first polypeptide chain, or at one or both ends of the first polypeptide chain.

[0133] The fusion protein (e.g., ICR) may comprise two polypeptide chains. The second polypeptide chain may comprise, 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 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 sequences to the sequence in SEQ ID NO: 10. 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: 10). These mutations may be amino acid insertions, deletions, or substitutions. Insertions, deletions, and substitutions may occur within the second polypeptide chain or at one or both ends of the second polypeptide chain.

[0134] The fusion protein (e.g., ICR) may comprise two polypeptide chains. The first polypeptide chain may comprise, from the N-terminus to the C-terminus, an extracellular domain of IL-2Rγ (e.g., SEQ ID NO: 5), a transmembrane domain of IL-12Rβ1 (e.g., SEQ ID NO: 3), and an intracellular domain of IL-12Rβ1 (e.g., SEQ ID NO: 4). The first 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 sequences to those of SEQ ID NO: 11. The first polypeptide chain may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations (e.g., compared to SEQ ID NO: 11). These mutations may be amino acid insertions, deletions, or substitutions. Insertion, deletion, and substitution can occur within the first polypeptide chain, or at one or both ends of the first polypeptide chain.

[0135] The fusion protein (e.g., ICR) may comprise two polypeptide chains. The second polypeptide chain may comprise, from the N-terminus to the 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 sequences to those of 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. Insertion, deletion, and substitution can occur within the second polypeptide chain, or at one or both ends of the second polypeptide chain.

[0136] A fusion protein (e.g., an ICR) may comprise two polypeptide chains. In some embodiments, the first and second polypeptide chains are linked together via a 2A cleavable linker (such as a P2A or T2A linker). A fusion protein (e.g., an 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., an 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 being cleaved by a 2A linker, a fusion protein (e.g., an ICR) may comprise a polypeptide containing the amino acid sequence of any one of SEQ ID NO: 13-14. In some embodiments, a fusion protein (e.g., an ICR) comprises a first and / or a second polypeptide chain containing the amino acid sequence shown in any one of SEQ ID NO: 9-14 or having at least 90%, 95%, or 99% identical amino acid sequences to any one of SEQ ID NO: 9-14.

[0137] Engineered receptors (e.g., CAR and TCR)

[0138] One aspect of this disclosure provides cells (e.g., immune cells) that express the reversed cytokine receptor described herein and engineered receptors. Engineered receptors may include an extracellular ligand-binding domain or an extracellular antigen-binding domain, and optionally an intracellular signaling domain. Exemplary engineered receptors include, but are not limited to, chimeric antigen receptors (CARs), engineered T-cell receptors (TCRs), and T-cell antigen-coupled device (TAC) receptors. Engineered receptors may include an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain that specifically binds to an antigen (e.g., a tumor antigen). The intracellular signaling domain may include a primary intracellular signaling domain and / or a co-stimulatory signaling domain. The intracellular signaling domain may include the intracellular signaling domain of a TCR co-receptor. Engineered receptors may be encoded by heteropolynucleotides operatively linked to a promoter, such as a constitutive or inducible promoter.

[0139] Engineered receptors may include 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 co-stimulatory signaling domains.

[0140] Engineered receptors can be chimeric antigen receptors (CARs). Many chimeric antigen receptors are known in the art and can be adapted to the engineered cells described herein. CARs can also be constructed to be specific for any cell surface marker by using, for example, antigen-binding fragments of antibody molecules or variable domains of antibodies.

[0141] The CAR disclosed herein may include an extracellular domain, a transmembrane domain, and an intracellular signal transduction domain, wherein the extracellular domain includes at least one antigen-binding domain that specifically binds at least one tumor antigen.

[0142] Intracellular signaling domains can generate signals that promote immune effector functions in CAR-containing cells (e.g., CAR-T cells). Immune effector functions or immune effector responses refer to functions or responses of immune effector cells that enhance or promote immune attack on target cells. For example, immune effector functions or responses can refer to the properties of T or NK cells that promote the killing of target cells or inhibit the growth or proliferation of target cells. Examples of immune effector functions, such as in CAR-T cells, include cytolytic activities (such as antibody-dependent cytotoxicity or ADCC) and helper activities (such as the secretion of cytokines). 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 CD28, CD137, CD3, CD27, CD40, ICOS, GITR, and OX40. Signaling domains of naturally occurring molecules may comprise the entire intracellular (or cytoplasmic) portion of the molecule or the entire native intracellular signaling domain, or fragments or derivatives thereof.

[0143] The intracellular signaling domains of CARs can include primary intracellular signaling domains. A "primary intracellular signaling domain" refers to a cytoplasmic signaling sequence that acts in a stimulatory manner to induce immune effector function. Primary intracellular signaling domains can contain signaling motifs known as immune receptor tyrosine-based activation motifs or ITAMs. Primary intracellular signaling domains can contain functional signaling domains of proteins selected from the following groups: CD3ζ, CD3γ, CD3δ, CD3ε, shared FcRγ (FCER1G), FcRβ (FcεRib), CD79a, CD79b, FcγR IIa, DAP10, and DAP12. Primary intracellular signal transduction domains may contain nonfunctional or attenuated signal transduction domains of proteins selected from the group consisting of: CD3ζ, CD3γ, CD3δ, CD3ε, shared FcRγ (FCER1G), FcRβ (FcεRib), CD79a, CD79b, FcγR IIa, DAP10, and DAP12. Nonfunctional or attenuated signal transduction domains may be mutant signal transduction domains with point mutations, insertions, or deletions that attenuate or eliminate one or more immune effector functions, such as cytolytic or accessory activities, including antibody-dependent cytotoxicity (ADCC). CARs may contain a nonfunctional or attenuated CD3ζ (i.e., CD3ζ or CD3z) signal transduction domain. Intracellular signal transduction domains may not contain primary intracellular signal transduction domains. Compared to CARs with the same construct but with wild-type primary intracellular signaling domains, the weakened primary intracellular signaling domains can induce immune effector functions (such as cell lysis against target cells) of no more than about 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10% or less.

[0144] The intracellular signaling domain of a CAR may contain one or more (such as any one of 1, 2, 3 or more) co-stimulatory signaling domains. A “co-stimulatory signaling domain” can be the intracellular portion of a co-stimulatory molecule. The term “co-stimulatory molecule” refers to a homologous binding partner on an immune cell (such as a T cell) that specifically binds to a co-stimulatory ligand, thereby mediating a co-stimulatory response of the immune cell, such as, 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 activating molecules (SLAM proteins), and activating NK cell receptors. Co-stimulatory molecules include, but are not limited to, MHC class I molecules, BTLA and Toll ligand receptors, and OX40, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), and 4-1BB (CD137). Other examples of such co-stimulatory molecules include CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, CD4, CD8α, CD8β, IL-2Rβ, IL-2Rγ, IL-7Rα, 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, L... FA-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 ligands that specifically bind to CD83.

[0145] CARs may contain a single costimulatory signaling domain. CARs may contain two or more costimulatory signaling domains. Intracellular signaling domains may contain a functional primary intracellular signaling domain and one or more costimulatory signaling domains. CARs may not contain a functional primary intracellular signaling domain (such as CD3ζ). CARs may contain an intracellular signaling domain consisting of one or more costimulatory signaling domains, or substantially consisting of one or more costimulatory signaling domains. CARs may contain an intracellular signaling domain consisting of a non-functional or attenuated primary intracellular signaling domain (such as mutant CD3ζ) and one or more costimulatory signaling domains, or substantially consisting of a non-functional or attenuated primary intracellular signaling domain (such as mutant CD3ζ) and one or more costimulatory signaling domains. After the antigen-binding domain binds to a tumor antigen, the costimulatory signaling domains of the CAR can transduce signals to enhance the proliferation, survival, and differentiation of CAR-equipped modified immune cells (such as T cells) and inhibit activation-induced cell death. One or more co-stimulatory signal transduction domains can be derived from one or more molecules selected from the group consisting of: 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 ligands that specifically bind to CD83.

[0146] The intracellular signal transduction domain of a CAR may include a co-stimulatory signal transduction domain derived from CD28. The intracellular signal transduction domain may include a primary intracellular signal transduction domain of CD3ζ and a co-stimulatory signal transduction domain of CD28. The intracellular signal transduction domain in the chimeric receptor of this application may include a co-stimulatory signal transduction domain derived from 4-1BB (i.e., CD137). The intracellular signal transduction domain may include a primary intracellular signal transduction domain of CD3ζ and a co-stimulatory signal transduction domain of 4-1BB. The intracellular signal transduction domain may comprise a polypeptide containing, from the N-terminus to the C-terminus, a co-stimulatory signal transduction domain of 4-1BB and a primary intracellular signal transduction domain of CD3ζ.

[0147] The intracellular signal transduction domains of CARs may include a co-stimulatory signal transduction domain of CD28 and a co-stimulatory signal transduction domain of 4-1BB. The intracellular signal transduction domains may also include a primary intracellular signal transduction domain of CD3ζ, a co-stimulatory signal transduction domain of CD28, and a co-stimulatory signal transduction domain of 4-1BB. Alternatively, the intracellular signal transduction domains may comprise a polypeptide containing, from N-terminus to C-terminus, a co-stimulatory signal transduction domain of CD28, a co-stimulatory signal transduction domain of 4-1BB, and a primary intracellular signal transduction domain of CD3ζ.

[0148] The antigen-binding domain of a CAR may contain one or more (such as 1, 2, 3, 4, 5, 6 or more) antibodies or antibody fragments, which may be selected from scFv, Fv, Fab, (Fab′)2, mini antibodies, biantibodies, single-domain antibodies (sdAb), or V. H The H domain. The antigen-binding domain of a CAR may contain an extracellular portion of a ligand or receptor that specifically binds to a tumor antigen. CARs can be monospecific, bispecific, or multispecific. The antigen-binding domain of a CAR can specifically bind to a single tumor antigen. The antigen-binding domain of a CAR can bind to two or more tumor antigens. Engineered receptors (e.g., CARs) can redirect the specificity of engineered cells by expressing chimeric antigen receptors (CARs) or TCRs on these cells.

[0149] Antigens can be tumor antigens selected from the following groups: 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, MAGEA3, p53, MART1, GP100, protease-3 (PR3), tyrosinase, survival protein, hTERT, EphA2, NY-ESO-1, h5T4, PSCA, PSMA, ROR1, TAG-72, VEGF-R2, WT-1, CD123, CD44V6, NKCS1, IGF1R, EGFR, EGFR-VIII, dentin 18.2, dentin 6, NKG2D, delta-like ligand 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.

[0150] Tumor antigens can be derived from intracellular proteins of tumor cells. Tumor antigens can be expressed on the surface of tumor cells. Many TCRs specific to tumor antigens, including tumor-associated antigens, have been described, including, for example, the NY-ESO-1 cancer-testis antigen, the p53 tumor suppressor antigen, and TCRs targeting tumor antigens in melanoma (e.g., MARTI, gp 100), leukemia (e.g., WT1, minor histocompatibility antigen), and breast cancer (e.g., HER2, NY-BR1).

[0151] The transmembrane domain of a CAR can be selected from the following transmembrane domains: α, β, or ζ chains of the T cell receptor; CD28, CD3ε, 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; CD 19. IL-2Rβ, IL-2Rγ, IL-7Ra, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGA M, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT 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. The transmembrane domain of a CAR can be a CD4, CD3, CD8α, or CD28 transmembrane domain. The transmembrane domain of a CAR can include a CD8α transmembrane domain. Transmembrane domains can be derived from molecules selected from the group consisting of: CD8α, CD4, CD28, CD137, CD80, CD86, CD152, and PD1.

[0152] Extracellular domains can connect to transmembrane domains via hinge domains. The hinge domain can be a CD8α hinge domain.

[0153] The CAR may also contain a signal peptide (SP), such as the CD8α signal peptide. The signal peptide 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 to the amino acid sequence of SEQ ID NO: 25.

[0154] Many CARs targeting different tumor antigens have been widely disclosed in the art, such as CD19 CARs or BCMACARs. The extracellular antigen-binding domain of a CD19 CAR may be or include a CD19-binding fragment (e.g., FMC63, SJ25C1, or those disclosed in various patents such as WO 2022 / 012683). BCMA CARs have also been well described, with relevant patents including, but not limited to, WO 2016 / 014789, WO 2016 / 014565, WO 2013 / 154760, and WO 2018 / 028647. The extracellular antigen-binding domain of a BCMACAR may be or include a BCMA-binding fragment. The BCMA-binding fragment may bind to one or more epitopes on BCMA. A BCMA CAR may be a bivalent CAR comprising two anti-BCMA sdAbs targeting the same or different BCMA epitopes.

[0155] The CAR can be a GPC3 CAR (“H93” or “H93 CAR”). A GPC3 CAR can be monospecific. A GPC3 CAR can be bispecific or bivalent. The extracellular antigen-binding domain of the GPC3 CAR can be or include one or more GPC3-binding moieties. The GPC3-binding moieties can bind to one or more epitopes on GPC3. A GPC3 CAR can be a bivalent CAR containing one or more anti-GPC3 scFvs targeting the same or different GPC3 epitopes.

[0156] The GPC3 CAR may comprise, from the N-terminus to the C-terminus, the following: a CD8α signal peptide, an extracellular antigen-binding domain containing anti-GPC3 scFv, a CD8α hinge domain, a CD8α transmembrane domain, a 4-1BB co-stimulatory signal transduction domain, and a CD3ζ primary intracellular signal transduction domain. The anti-GPC3 scFv 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 that of SEQ ID NO: 30.

[0157] The CD8α hinge domain 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% of the amino acid sequence of SEQ ID NO: 26.

[0158] The CD8α transmembrane domain 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% of the amino acid sequence of SEQ ID NO: 27.

[0159] The 4-1BB co-stimulatory signal transduction domain 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: 28.

[0160] The CD3ζ primary intracellular signal transduction domain 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% of the amino acid sequence of SEQ ID NO: 29.

[0161] The GPC3 CAR 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: 20. The GPC3 CAR can specifically bind to GPC3-positive tumor cells (e.g., PLCPRF5 cells).

[0162] Engineered receptors can be modified T-cell receptors or engineered T-cell receptors. Engineered TCRs can be specific for tumor antigens. Tumor antigens can be selected from the following groups: 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, Le Y, L1 cell adhesion molecules, MAGE-A1, mesothelin, MAGEA3, p53, MART1, GP100, protease-3 (PR3), tyrosinase, survival proteins, hTERT, EphA2, NY-ESO-1, h5T4, PSCA, PSMA, ROR1, TAG-72, VEGF-R2, WT-1, CD123, CD44V6, NKCS1, IGF1R, EGFR, EGFR-VIII, dentin 18.2, dentin 6, NKG2D, delta-like ligand 3 (DLL3), CD70, CS-1, c-Met, glycolipid F77, PD-L1, PD-L2, and other clinically significant tumor antigens, and combinations thereof. Tumor antigens can be derived from intracellular proteins of tumor cells. Tumor antigens can be expressed on the surface of tumor cells. Many TCRs specific to tumor antigens, including tumor-associated antigens, have been described, including, for example, the NY-ESO-1 cancer-testis antigen, the p53 tumor suppressor antigen, and TCRs targeting tumor antigens in melanoma (e.g., MARTI, gp 100), leukemia (e.g., WT1, minor histocompatibility antigen), and breast cancer (e.g., HER2, NY-BR1). Any TCR known in the art can be used. TCRs can have enhanced affinity for tumor antigens. Exemplary TCRs and methods for introducing TCRs into immune cells have been 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), which are incorporated herein by reference in their entirety.

[0163] The TCR receptor complex is an octamer formed by the variable TCR receptor α and β chains (or γ and δ chains in the case of γδ T cells) with three dimeric signaling modules CD3δ / ε, CD3γ / ε, and CD247 (the CD3ζ chain of the T cell surface glycoprotein) ζ / ζ or ζ / η. Ionizable residues in the transmembrane domains of each subunit form an interacting polar network that holds the complex together. The TCR complex has the function of activating the signaling cascade in T cells.

[0164] Engineered receptors can be engineered TCRs comprising one or more T-cell receptor (TCR) fusion proteins (TFPs). Exemplary TFPs have been described, for example, in US 20170166622A1, which is incorporated herein by reference in its entirety. A TFP may comprise an extracellular domain of a TCR subunit comprising an extracellular domain of a protein selected from the group consisting of: a TCRα chain, a TCRβ chain, a CD3ε TCR subunit, a CD3γ TCR subunit, a CD3δ TCR subunit, a functional fragment thereof, and an amino acid sequence having at least one but no more than 20 modified amino acids. A TFP may comprise a transmembrane domain comprising a transmembrane domain of a protein selected from the group consisting of: a TCRα chain, a TCRβ chain, a CD3ε TCR subunit, a CD3γ TCR subunit, a CD3δ TCR subunit, a functional fragment thereof, and an amino acid sequence having at least one but no more than 20 modified amino acids. TFP may contain a transmembrane domain comprising a transmembrane domain of a protein selected from the group consisting of: TCRα chain, TCRβ chain, TCRζ chain, CD3εTCR subunit, CD3γTCR subunit, CD3δTCR subunit, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD137, CD154, their functional fragments, and their amino acid sequences having at least one but no more than 20 modifications.

[0165] TFP may comprise a TCR subunit and an antigen-binding domain, the TCR subunit comprising at least a portion of the extracellular domain of the TCR and an intracellular domain of the TCR containing a stimulatory domain from the intracellular signaling domain of CD3ε; wherein the TCR subunit is operatively linked to the antigen-binding domain, and wherein the TFP is incorporated into the TCR when expressed in T cells.

[0166] Engineered receptors can be T-cell antigen-coupled receptors (TACs). For example, an exemplary TAC receptor has been described in US20160368964A1, which is incorporated herein by reference. A TAC may comprise an antigen-binding domain, a TCR-binding domain that specifically binds to a protein 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 engineered ankyrin repeat (DARPin) polypeptide. Tumor antigens can be selected from the following groups: 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, Le Y, L1 cell adhesion molecules, MAGE-A1, mesothelin, MAGEA3, p53, MART1, GP100, protease-3 (PR3), tyrosinase, survival proteins, hTERT, EphA2, NY-ESO-1, h5T4, PSCA, PSMA, ROR1, TAG-72, VEGF-R2, WT-1, CD123, CD44V6, NKCS1, IGF1R, EGFR, EGFR-VIII, dentin 18.2, dentin 6, NKG2D, delta-like ligand 3 (DLL3), CD70, CS-1, c-Met, glycolipid F77, PD-L1, PD-L2, and other clinically significant tumor antigens, and combinations thereof. Tumor antigens can be derived from intracellular proteins of tumor cells. Tumor antigens can be expressed on the surface of tumor cells. Proteins associated with the TCR complex can be CD3, such as CD3E. The TCR-binding domain can be a single-chain antibody, such as scFv or V. HH. 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 (such as CD8α).

[0167] T cell co-receptors are expressed as membrane proteins on T cells. They can stabilize the TCR:peptide:MEC complex and promote signal transduction. Two subtypes of T cell co-receptors, CD4 and CD8, exhibit strong specificity for specific MEC classes. The CD4 co-receptor stabilizes only the TCR:MEC II complex, while the CD8 co-receptor stabilizes only the TCR:MEC I complex. Differential expression of CD4 and CD8 across different T cell types leads to distinct functional subsets of T cells. CD8+ T cells are cytotoxic T cells.

[0168] Engineered receptors (such as CARs, TCRs, or TACs) can target one or more tumor antigens. Tumor antigens are proteins produced by tumor cells that can trigger an immune response, particularly a T-cell-mediated immune response. The choice of target antigen will depend on the specific type of cancer to be treated. Exemplary tumor antigens include, for example, glioma-associated antigen, carcinoembryonic antigen (CEA), β-human chorionic gonadotropin, alpha-fetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CAIX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxylesterase, muta-hsp70-2, M-CSF, prostate enzymes, prostate-specific antigen (PSA), PAP, NY-ESO-1, LAGE-1a, p53, prostaglandins, PSMA, HER2 / neu, survival proteins and telomerase, prostate cancer tumor antigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, liver glycoside B2 (ephrinB2), CD22, insulin-like growth factor (IGF)-I, IGF-II, IGF-I receptor, and mesothelin.

[0169] Tumor antigens can contain one or more antigenic cancer epitopes associated with malignant tumors. Malignant tumors express a number of proteins that can be used 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 prostate acid phosphatase (PAP) and prostate-specific antigen (PSA) in prostate cancer. Other target molecules belong to the transformation-related molecular group, such as the oncogene HER2 / Neu / ErbB-2. Another group of target antigens is oncoemulsification antigens, such as carcinoembryonic antigen (CEA). In B-cell lymphomas, tumor-specific idiotype immunoglobulins constitute the true tumor-specific immunoglobulin antigens unique to the individual tumor. B-cell differentiation antigens (such as CD19, CD20, and CD37) are other candidates for target antigens in B-cell lymphomas.

[0170] Tumor antigens can be tumor-specific antigens (TSA) or tumor-associated antigens (TAAs). TSAs are specific to tumor cells and are not present on other cells in the body. TAA-associated antigens are not specific to tumor cells; instead, they are also expressed on normal cells under conditions that do not induce immune tolerance to the antigen. Antigen expression on tumors can occur under conditions that enable the immune system to respond to the antigen. TAAs can be antigens expressed on normal cells during embryonic development (when the immune system is immature and unable to respond), or they can be antigens that are normally present at very low levels on normal cells but expressed at much higher levels on tumor cells.

[0171] Non-limiting examples of TSA or TAA antigens include the following: differentiation antigens, such as MART-1 / MelanA (MART-I), gp 100 (Pmel 17), tyrosinase, TRP-1, TRP-2, and tumor-specific multi-lineage antigens, such as MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, p15; overexpressed embryonic antigens, such as CEA; overexpressed oncogenes and mutated tumor suppressor genes, such as p53, Ras, HER2 / neu; unique tumor antigens resulting from chromosomal translocations, such as BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, 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, CA72-4, CAM 17.1, NuMa, K-ras, β-catenin, CDK4, Mum-1, p15, p16, 43-9F, 5T4, 791Tgp72, alpha-fetoprotein, β-HCG, BCA225, BTAA, CA 125, CA 15-3, CA 27.29, BCAA, CA 195, CA 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, cyclophilin C-related protein, TAAL6, TAG72, TLP, and TPS.

[0172] Nucleic acid

[0173] This disclosure provides (i) nucleic acids encoding fusion proteins (e.g., ICRs) described herein, and (ii) nucleic acids encoding engineered receptors (e.g., CARs, TACs, or TCRs). The nucleic acids disclosed herein may contain nucleic acid sequences encoding any one of the ICRs, CARs, and / or TCRs disclosed herein. The nucleic acids may simultaneously encode (1) a CAR and (2) a fusion protein (e.g., an ICR). The nucleic acids may contain one, two, three, four, five, or more copies of the coding sequence for the fusion protein (e.g., an ICR). Expression of the fusion protein (e.g., an ICR) may be controlled by non-natural regulatory elements.

[0174] The nucleic acid disclosed herein may comprise a first nucleic acid sequence and a second nucleic acid sequence. The first nucleic acid may be upstream of or 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 encoded by the same nucleic acid sequence (e.g., a polycistronic or bicistronic sequence), which are translated into a polymer that dissociates into individual protein components. The nucleic acid may comprise a first nucleic acid sequence, a linker, and a second nucleic acid sequence from its 5' to 3' end. Alternatively, the nucleic acid may comprise a second nucleic acid sequence, a linker, and a first nucleic acid sequence from its 5' to 3' end. The first nucleic acid sequence may encode the CAR described herein, and the second nucleic acid sequence may encode the fusion protein described herein (e.g., an ICR).

[0175] The adapter may contain a nucleic acid sequence encoding an internal ribosome entry site (IRES). As used herein, “internal ribosome entry site” or “IRES” refers to an element that facilitates direct entry of an internal ribosome into a protein-coding region of a start codon (such as ATG), thereby leading to cap-independent translation of the gene. Various internal ribosome entry sites are known to those skilled in the art, including but not limited to IRES derived 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 IRES derived from, for example, heart virus, rhinovirus, foot-and-mouth disease virus, 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.

[0176] Linkers may contain nucleic acid sequences encoding self-cleaving peptides. As used herein, a “self-cleaving peptide” or “2A linker” refers to an oligopeptide that allows multiple proteins to be encoded as polyproteins that dissociate into component proteins upon 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, including but not limited to those found in members of the Picornaviridae virus family, such as foot-and-mouth disease virus (FMDV), equine rhinitis A virus (ERAV0), Thosea asignavirus (TaV), and porcine chexenvirus-1 (PTV-1); and cardiogenic viruses, such as Theylvirus 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 adapter may have 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: 32. The T2A adapter may have 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: 33.

[0177] Various adapter sequences are known in the art, including but not limited to glycine-serine (GS) spacers (also known as GS adapters), such as (GS)n, (SG)n, (GSGGS)n, and (GGGS)n, where n represents an integer of at least 1. Exemplary adapter sequences 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 sequences to those in 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.

[0178] The nucleic acid disclosed herein may contain a restriction enzyme site sequence. Exemplary restriction enzyme site sequences may contain a nucleic 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 nucleic acid sequence of GTTAAC or ACTAGT.

[0179] The nucleic acids disclosed herein can be operatively linked to transcriptional control elements, such as promoters and enhancers.

[0180] The promoter can be a CD8 cell-specific promoter, a CD4 cell-specific promoter, a neutrophil-specific promoter, or an NK cell-specific promoter. For example, the CD4 gene promoter can be used; see, for example, Salmon et al., Proc. Natl. Acad. Sci. USA (1993) 90:7739; and Marodon et al., (2003) Blood 101:3416. As another example, the CD8 gene promoter can be used. NK cell-specific expression can be achieved by using the NcrI (p46) promoter; see, for example, Eckelhart et al., Blood (2011) 117:1565.

[0181] Other examples of suitable promoters include the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strongly constitutive promoter sequence capable of driving high-level expression of any nucleic acid sequence operatively linked to it. Other constitutive promoter sequences may also be used, including, but not limited to, the simian virus 40 (SV40) early promoter, the mouse mammary tumor virus (MMTV) or human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, the MoMuLV promoter, the avian leukosis virus promoter, the Epstein-Barr virus immediate early promoter, the Raoult sarcoma virus promoter, the EF-1α promoter, and human gene promoters (such as, but not limited to, the actin promoter, myosin promoter, hemoglobin promoter, and creatine kinase promoter). Furthermore, this disclosure is not limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of this disclosure. The use of inducible promoters provides a molecular switch capable of turning on the expression of a nucleic acid sequence operatively linked to it when such expression is desired, or turning off such expression when it is not desired. Examples of inducible promoters include, but are not limited to, metallothionein promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters.

[0182] The nucleic acids disclosed herein may be provided for the generation of (i) the fusion proteins described herein (e.g., ICRs), and / or (ii) the CARs described herein (e.g., in mammalian cells). The nucleic acids disclosed herein may be provided for the amplification of these nucleic acids.

[0183] Nucleic acids described herein can be introduced into immune cells (e.g., T cells) or their precursors using vectors (e.g., expression vectors, such as lentiviral vectors). Vectors disclosed herein (e.g., lentiviral vectors) may contain one or more nucleic acids encoding fusion proteins (e.g., ICRs) described herein. Vectors (e.g., lentiviral vectors) may contain additional elements that contribute to the functional expression of fusion proteins (e.g., ICRs) and / or CARs described herein. Expression vectors may contain mammalian promoters. Vectors may contain elongation factor-1-α promoters (EF-1α promoters). Using EF-1α promoters can increase the expression efficiency of downstream transgenes (e.g., nucleic acids encoding CARs). Physiological promoters (e.g., EF-1α promoters) are unlikely to induce integration-mediated genotoxicity and may eliminate the ability of retroviral vectors to transform stem cells. Other physiological promoters suitable for use in vectors (e.g., lentiviral vectors) may be incorporated into the vectors disclosed herein. Vectors (e.g., lentiviral vectors) may contain non-essential cis-acting sequences that can improve titers and gene expression.

[0184] The nucleic acid can encode a naked CAR. From the 5' to the 3' end, the nucleic acid may contain a CD8α signal peptide, an extracellular antigen-binding domain, a CD8α hinge domain, a CD8α transmembrane domain, a CD137 co-stimulatory signal transduction domain, and a CD3ζ primary intracellular signal transduction domain. The nucleic acid can 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: 20.

[0185] Fusion proteins (e.g., ICRs) may comprise two polypeptide chains. The 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 its 5' end to its 3' end. The nucleic acid may contain the coding sequence for the second polypeptide chain from its 5' end to its 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 SEQ ID NO: 9-12. The nucleic acid may contain the coding sequences for the first polypeptide chain, a 2A cleavable linker, and the second polypeptide chain from its 5' end to its 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 SEQ ID NO: 13 or SEQ ID NO: 14. Exemplary structures of the nucleic acids described herein are shown in […]. Figures 1a-1b The first polypeptide chain may contain a first extracellular domain derived from IL-4Rα and a first intracellular domain derived from IL-12Rβ1. The second polypeptide chain may contain 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: 13. The first polypeptide chain may contain a first extracellular domain derived from IL-2Rγ and a first intracellular domain derived from IL-12Rβ1. The second polypeptide chain may contain a second extracellular domain derived from IL-4Rα 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: 14.

[0186] The nucleic acid may encode (1) a CAR and (2) an ICR. The nucleic acid may contain coding sequences for the CAR, the 2A cleavable adapter, and the ICR from the 5' to the 3' end. The ICR may be a fusion protein comprising two polypeptide chains. The ICR may comprise a first polypeptide chain and a second polypeptide chain. The nucleic acid may contain coding sequences for the CAR, the 2A cleavable adapter, the first polypeptide chain, the 2A cleavable adapter, and the second polypeptide chain from the 5' 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 SEQ ID NO: 21 or SEQ ID NO: 22. 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 contain 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 contain a first extracellular domain derived from IL-2Rγ and a second intracellular domain derived from IL-12Rβ1. The second polypeptide chain may contain a second extracellular domain derived from IL-4Rα 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: 22.

[0187] To determine the percentage of identity between two amino acid sequences or two nucleic acid sequences, these sequences are aligned for optimal comparison (e.g., vacancies may be introduced in one or both of the first and second amino acid or nucleic acid sequences for optimal alignment, and non-homologous sequences may 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%. The amino acid residues or nucleotides at corresponding amino acid or nucleotide positions are then compared. The molecules are identical at that position when a 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 of identity between two sequences is a function of the number of identical positions shared by the two sequences, taking into account the number of vacancies that need to be introduced to achieve optimal alignment and the length of each vacancies. For example, a Blossum 62 scoring matrix with a vacancy penalty of 12, a vacancy extension penalty of 4, and a frameshift vacancy penalty of 5 can be used to perform sequence comparison and determine the percentage of identity between two sequences.

[0188] engineered cells

[0189] One aspect of this disclosure provides engineered cells comprising the fusion protein (e.g., ICR) described herein. Engineered cells comprising the fusion protein (e.g., ICR) described herein may further comprise an engineered receptor (e.g., CAR). Engineered cells comprising the ICR described herein may further comprise a CAR (a CAR armored with an ICR).

[0190] Engineered receptors (e.g., CARs) can redirect the specificity of engineered cells by expressing chimeric antigen receptors (CARs) or TCRs on these cells. CAR expression can be induced by electroporating engineered cells to insert genetic material or by infecting these cells with viral vectors, such as lentiviruses or retroviruses containing the desired genetic material. Such gene editing can improve the potency of engineered cells by enhancing homing, cytokine production, recirculating killing, and / or improving implantation.

[0191] Engineered cells containing the fusion proteins described herein (e.g., ICR) can express more than one polypeptide chain. Engineered cells containing the fusion proteins described herein (e.g., ICR) can express more than one engineered receptor (such as any combination of CAR, TCR, or TAC receptors). Modified cells containing the fusion proteins described herein (e.g., ICR) can be used to treat cancer.

[0192] Compared to cells that do not contain the fusion protein (e.g., ICR) described herein, modified cells containing the fusion protein (e.g., ICR) described herein may exhibit higher cytotoxicity against tumor cells. Compared to cells that do not contain the ICR described herein, modified cells containing the ICR described herein may exhibit higher persistence and / or proliferation in the tumor microenvironment.

[0193] In one aspect, this disclosure provides an engineered cell comprising (i) the fusion protein (e.g., ICR) described herein and (ii) the engineered receptor (e.g., CAR) described herein. The modified cell may be an immune cell. The modified cell may contain one or more nucleic acids encoding (i) the fusion protein (e.g., ICR) described herein and (ii) the engineered receptor (e.g., CAR) described herein. Therefore, such an engineered cell may have specificity determined by the engineered receptor (e.g., CAR) expressed therein. For example, the modified cell of this disclosure comprising one or more CARs may be specific for one or more antigens on target cells (e.g., one or more tumor antigens on cancer cells).

[0194] Engineered cells can be modified immune cells. Engineered cells can be selected from the following groups: T cells, αβ T cells, γδ T cells, NK cells, peripheral blood mononuclear cells (PBMCs), hematopoietic stem cells, pluripotent stem cells, embryonic stem cells, and combinations thereof. Engineered cells can be T cells. Engineered cells can be NK cells. Engineered cells can be αβ T cells. Engineered cells can be γδ T cells. Engineered cells can be Vδ1 T cells.

[0195] For the individual receiving them, engineered cells can be autologous cells, syngeneic cells, allogeneic cells, or xenogeneic cells. Engineered cells can 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. Engineered cells can be autologous cells obtained from the recipient subject. Engineered cells can also be autologous T cells obtained from the recipient subject.

[0196] The engineered cells described herein may include eukaryotic cells, such as mammalian cells. Engineered cells may be human cells. Engineered cells may be horse, cow, mouse, sheep, dog, or cat cells.

[0197] In one aspect, this disclosure provides modified cells expressing a fusion protein (e.g., ICR) comprising an extracellular domain derived from an IL-4 receptor (e.g., IL-4Rα) and an intracellular domain derived from an IL-23 receptor complex. In engineered cells, the fusion protein (e.g., ICR) comprising the extracellular domain of IL-4R (i.e., IL-4Rα) can specifically bind to IL-4. Upon binding to IL-4, the fusion protein (e.g., ICR) can activate signal transduction of the IL-23R intracellular signaling domain, which is transduced via native cellular elements to provide biological activity mimicking the natural response of IL-23R.

[0198] Fusion proteins (e.g., ICRs) 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.

[0199] Engineered cells may contain polypeptides encoding fusion proteins (e.g., ICR), wherein the fusion protein (e.g., ICR) comprises a first and / or a second polypeptide chain containing an amino acid sequence shown in any one of SEQ ID NO: 9-14 or an amino acid sequence having at least 90%, 95%, or 99% identical to any one of the amino acid sequences shown in SEQ ID NO: 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 being cleaved by a 2A adapter (e.g., a P2A adapter or a T2A adapter), the fusion protein (e.g., ICR) may comprise a polypeptide containing an amino acid sequence of any one of SEQ ID NO: 13-14.

[0200] Compared to cells without ICRs, engineered cells containing ICRs can exhibit enhanced proliferation upon exposure to immunosuppressive cytokines in the tumor microenvironment (TME). When one or more immunosuppressive cytokines in the TME are reduced or eliminated (e.g., after tumor cell killing), engineered cells containing ICRs may exhibit reduced proliferation or no proliferation. Compared to cells without ICRs, engineered cells containing ICRs can exhibit enhanced cytotoxicity upon exposure to immunosuppressive cytokines in the TME.

[0201] The engineered receptor can be a CAR. A CAR can comprise a polypeptide comprising, from its N-terminus to its C-terminus: a CD8α signal peptide, an extracellular antigen-binding domain, a CD8α hinge domain, a CD8α transmembrane domain, a CD137 co-stimulatory signal transduction domain, and a CD3ζ primary intracellular signal transduction domain. The CAR can be a GPC3 CAR (“H93” or “H93 CAR”). A CAR can comprise a polypeptide containing 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 that of SEQ ID NO: 20.

[0202] Engineered cells may comprise (i) a fusion protein (e.g., ICR) as described herein and (ii) an engineered receptor (e.g., CAR) as described herein, wherein the fusion protein (e.g., ICR) and (ii) the engineered receptor (e.g., CAR) are linked to each other via a 2A cleavable linker. In some embodiments, the engineered cells comprise a polypeptide encoding the fusion protein and / or a polypeptide encoding the CAR, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 9-14 and 20-22 or a functional variant having at least about 90% sequence identity with it.

[0203] Engineered cells can contain both ICR and CAR (CAR armored with ICR). Engineered cells can be modified CAR-T cells (CAR-T cells armored with ICR). Flow cytometry (FACS) can be used to determine the expression of CAR and ICR in engineered cells. Engineered cells (e.g., H93-IL4R-IL23R cells) can have CAR positivity rates greater than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, or 95%. Engineered cells can also have CAR positivity rates less than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, or 95%. Engineered cells can have CAR positivity rates of 10%-100%, 20%-100%, 30%-100%, 40%-100%, or 30%-95%.

[0204] IL-4 (e.g., 20 ng / ml IL-4) can activate STAT signaling (e.g., STAT3 phosphorylation or STAT4 phosphorylation) in engineered cells. IL-4 (e.g., 20 ng / ml IL-4) can increase the amount of phosphorylated STAT3 in engineered 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 engineered 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%. Engineered cells containing ICRs (e.g., H93-IL4R-IL23R cells) may respond more strongly to IL-4 stimulation compared to cells without ICRs (e.g., H93 cells). Following IL-4 stimulation, compared to cells without ICR, engineered cells containing ICR showed significantly higher phosphorylation of STAT3 and / or STAT4 by more than 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, or 1000%.

[0205] To assess the CAR positivity, expansion, and viability of engineered cells in vitro, in the re-stimulation assay, engineered cells were repeatedly stimulated 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 0.5:1, 1:1, 2:1, 2.5:1, 5:1, or 10:1. In re-stimulation assays, after 1, 2, 3, 4, or 5 rounds of stimulation, engineered cells could expand by more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 150, 200, 300, or 400 times. Each round of stimulation lasted 1, 2, 3, 4, or 5 days. Compared to cells without ICRs (e.g., H93 cells), the expansion of engineered cells containing ICRs (e.g., H93-IL4R-IL23R cells) can be increased 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%.

[0206] In re-stimulation assays, after 1, 2, 3, 4, or 5 rounds of stimulation, engineered cells could exhibit CAR positivity rates greater than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 90%. Conversely, in re-stimulation assays, after 1, 2, 3, 4, or 5 rounds of stimulation, engineered cells could exhibit CAR positivity rates less than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 90%. In re-stimulation assays, engineered cells containing ICRs (e.g., H93-IL4R-IL23R cells) showed similar CAR positivity rates to cells without ICRs (e.g., H93 cells) after 1, 2, 3, 4, or 5 rounds of stimulation.

[0207] In the re-excitation assay, after 1, 2, 3, 4, or 5 rounds of stimulation, engineered cells could exhibit viability greater than 5%, greater than 10%, greater than 15%, greater than 20%, greater than 25%, greater than 30%, greater than 35%, greater than 40%, greater than 45%, greater than 50%, greater than 55%, greater than 60%, greater than 65%, greater than 70%, greater than 75%, greater than 80%, or greater than 90%. Conversely, in the re-excitation assay, after 1, 2, 3, 4, or 5 rounds of stimulation, engineered cells could exhibit viability 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%. In re-stimulation assays, engineered cells containing ICRs (e.g., H93-IL4R-IL23R cells) showed similar viability to cells without ICRs (e.g., H93 cells) after 1, 2, 3, 4, or 5 rounds of stimulation.

[0208] Cytotoxicity of engineered cells against tumor cells can be assessed in long-term cytotoxicity assays, in which engineered 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 can 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 normalized cell index (real-time cell index / cell index at the time point before the addition of effector cells). After co-culturing with tumor cells (e.g., Hep3B2.1-7 cells) for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 days with or without IL-4 (e.g., 10 ng / ml IL-4), engineered cells can have normalized cell indices higher than 1, 2, 3, 4, 5, or 6, respectively. After co-culturing with tumor cells (e.g., Hep3B2.1-7 cells) for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 days with or without IL-4 (e.g., 10 ng / ml IL-4), the normalized cell index of engineered cells containing ICR (e.g., H93-IL4R-IL9R cells) could decrease 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%.

[0209] In long-term cytokine release assays, engineered cells can secrete cytokines (e.g., IFN-γ and / or TNF-α), wherein engineered 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 can be 1:40, 0.5:1, 1:1, 2:1, 2.5:1, 5:1, or 10:1. After co-culturing with tumor cells (e.g., Hep3B2.1-7 cells) for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 days with or without IL-4 (e.g., 10 ng / ml IL-4), engineered cells were able to secrete greater than 50 pg / ml, greater than 100 pg / ml, greater than 200 pg / ml, greater than 300 pg / ml, greater than 4000 pg / ml, greater than 500 pg / ml, greater than 1000 pg / ml, greater than 1500 pg / ml, greater than 2000 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, and greater than 10000 pg / ml. IFNγ at concentrations of pg / ml, greater than 15000 pg / ml, greater than 20000 pg / ml, or greater than 25000 pg / ml. After co-culturing with tumor cells (e.g., Hep3B2.1-7 cells) for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 days with or without IL-4 (e.g., 10 ng / ml IL-4), engineered cells secreted less than 50 pg / ml, less than 100 pg / ml, less than 200 pg / ml, less than 300 pg / ml, less than 4000 pg / ml, less than 500 pg / ml, less than 1000 pg / ml, less than 1500 pg / ml, less than 2000 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, or less than 10000 pg / ml. IFNγ in amounts of pg / ml, less than 15000 pg / ml, less than 20000 pg / ml, or less than 25000 pg / ml.After co-culturing with tumor cells (e.g., Hep3B2.1-7 cells) for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 days with or without IL-4 (e.g., 10 ng / ml IL-4), engineered cells can secrete 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, 100-800 pg / ml, 100-600 pg / ml, 100-400 pg / ml, 200-400 pg / ml, and 200-300 pg / ml, respectively. IFNγ in an amount of 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. After co-culturing with tumor cells (e.g., Hep3B2.1-7 cells) in the presence of IL-4 (e.g., 10 ng / ml IL-4) for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 days, engineered cells containing ICR (e.g., H93-IL4R-IL9R cells) showed significantly higher IFNγ secretion 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% compared to cells without ICR (e.g., H93 cells).

[0210] After co-culturing with tumor cells (e.g., Hep3B2.1-7 cells) for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 days with or without IL-4 (e.g., 10 ng / ml IL-4), engineered cells can secrete amounts of TNFα greater than 10 pg / ml, greater than 20 pg / ml, greater than 30 pg / ml, greater than 40 pg / ml, greater than 50 pg / ml, greater than 60 pg / ml, greater than 70 pg / ml, greater than 80 pg / ml, greater than 90 pg / ml, greater than 100 pg / ml, greater than 200 pg / ml, greater than 300 pg / ml, greater than 400 pg / ml, greater than 500 pg / ml, greater than 600 pg / ml, greater than 700 pg / ml, greater than 800 pg / ml, or greater than 900 pg / ml. After co-culturing with tumor cells (e.g., Hep3B2.1-7 cells) for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 days with or without IL-4 (e.g., 10 ng / ml IL-4), engineered cells can secrete amounts of TNFα 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 90 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 600 pg / ml, less than 700 pg / ml, less than 800 pg / ml, or less than 900 pg / ml.After co-culturing with tumor cells (e.g., Hep3B2.1-7 cells) for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 days with or without IL-4 (e.g., 10 ng / ml IL-4), engineered cells can secrete 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, 20-400 pg / ml, 50-1000 pg / ml, 50-800 pg / ml, 50-400 pg / ml, 50-200 pg / ml, and 50-150 pg / ml, respectively. 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 of TNFα. After co-culturing with tumor cells (e.g., Hep3B2.1-7 cells) in the presence of IL-4 (e.g., 10 ng / ml IL-4) for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 days, engineered cells containing ICRs (e.g., H93-IL4R-IL9R cells) showed significantly higher TNFα secretion compared to cells without ICRs (e.g., H93 cells), exceeding 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, or 1000%.

[0211] When engineered cells are exposed to tumor cells (e.g., Hep3B2.1-7 cells), the engineered 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. Exposure to tumor cells (e.g., Hep3B2.1-7 cells) can increase IFNγ secretion from engineered 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%. Exposure to tumor cells (e.g., Hep3B2.1-7 cells) can increase TNFα secretion in engineered 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%.

[0212] Treatment

[0213] The ICRs, polynucleotides, and engineered cells described herein can be used in a variety of experimental, therapeutic, and commercial applications.

[0214] In one respect, this disclosure provides a pharmaceutical composition comprising the engineered cells and pharmaceutically acceptable carriers described herein.

[0215] In one aspect, this disclosure provides a method for treating a disease or disorder in a subject (e.g., a human subject), the method comprising administering to the subject an effective amount of the engineered cells or pharmaceutical composition described herein. The disease or disorder may be cancer, an autoimmune disease, a tumor, or an infection.

[0216] Diseases or disorders can be solid tumors. A "solid tumor" is an abnormal mass of tissue that does not typically contain cysts or fluid-filled areas. Solid tumors can be benign (non-cancerous) or malignant (cancerous). Different types of solid tumors are named after the types of cells that form them. Examples of solid tumors are sarcomas, carcinomas, and lymphomas. Leukemia (blood cancers) typically does not form solid tumors.

[0217] In one respect, this disclosure provides a method for modulating an immune response, the method comprising administering an effective amount of the engineered cells described herein to a subject in need.

[0218] As used herein, the term "effective dose" means the amount that is effective at the necessary dosage and duration to achieve the desired result.

[0219] On the other hand, this disclosure provides a method for treating cancer, comprising administering an effective amount of the engineered 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 tumor (PNET), gastrointestinal neuroendocrine carcinoma, leukemia (including chronic lymphocytic leukemia, chronic myeloid leukemia, acute myeloid leukemia, acute lymphoblastic leukemia, and T-cell and B-cell leukemia), lymphoma (Hodgkin's and non-Hodgkin's), lymphoproliferative disorders, plasmacytoma, histiocytoma, melanoma, adenoma, sarcoma, solid tissue cancer, hypoxic tumors, squamous cell carcinoma, genitourinary cancers (such as cervical cancer and bladder cancer), hematopoietic system cancers, head and neck cancers, and nervous system cancers. Cancer can be breast cancer, lung cancer, pancreatic cancer, melanoma, oral cancer, mesothelioma, ovarian cancer, colorectal cancer, stomach cancer, cervical cancer, brain cancer, skin cancer, multiple myeloma, lymphoma, epithelial tumors, soft tissue sarcomas, esophageal cancer, or CNS tumors.

[0220] This disclosure further includes the use of the engineered cells described herein in the manufacture of medicaments or pharmaceutical compositions for modulating immune responses, treating infections, or treating cancers as described above.

[0221] Engineered cells can also be used in experimental models, for example, to further study and elucidate cell function.

[0222] One or more of the engineered cells described herein can be administered to a subject in a single, uniform form (such as intravenous injection) or in multiple forms (e.g., as multiple intravenous infusions or injections, or subcutaneous injections). Following administration to a subject, the engineered cells can be expanded in the subject's body. The engineered cells can be frozen to provide cells for multiple treatments with the same cell preparation. The engineered cells disclosed herein and pharmaceutical compositions containing the engineered cells disclosed herein can be packaged as kits. Kits may include instructions for use of the engineered cells and compositions containing the engineered cells (e.g., written instructions).

[0223] Treatment methods may include administering a therapeutically effective amount of engineered cells to the subject. The therapeutically effective amount of engineered cells may be administered for at least 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, or 1 year. The therapeutically effective amount of engineered cells may be administered for at least one week. The therapeutically effective amount of engineered cells may be administered for at least two weeks.

[0224] The engineered cells described herein can be administered before, during, or after the onset of a disease or condition, and the timing of administration can vary. For example, engineered cells can be used as a preventative agent and can be administered continuously to subjects with a condition or predisposition to reduce the likelihood of developing the disease or condition. Engineered cells can be administered to subjects during or as soon as possible after the onset of symptoms. Administration of engineered cells can begin immediately upon the onset of symptoms, within 3 hours before the onset of symptoms, within 6 hours before the onset of symptoms, within 24 hours before the onset of symptoms, within 48 hours before the onset of symptoms, or at any time after the onset of symptoms. The initial administration can be via any practical route (e.g., intravenous infusion or injection), such as any of the routes described herein using any of the formulations described herein. In some instances, the administration of the engineered cells disclosed herein is intravenous. After the onset of cancer or an infectious disease, one or more doses of engineered cells can be administered as soon as practicable, and the duration of treatment can be as long as necessary, such as approximately 24 hours to approximately 48 hours, approximately 48 hours to approximately 1 week, approximately 1 week to approximately 2 weeks, approximately 2 weeks to approximately 1 month, and approximately 1 month to approximately 3 months. For cancer treatment, one or more doses of engineered cells can be administered several years after the onset of cancer and before or after other treatments. In some instances, engineered cells can be administered for at least about 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 can vary for each subject.

[0225] Methods for administering engineered cells for adoptive cell therapy are known and can be used in conjunction with the provided methods and compositions. For example, adoptive T-cell therapy methods are described in, for example, U.S. Patent Application Publication No. 2003 / 0170238 by Gruenberg et al.; U.S. Patent No. 4,690,915 by Rosenberg; Rosenberg (2011) Nat RevClin 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; Davila et al., (2013) PLoS ONE 8(4): e61338. Cell therapy (e.g., adoptive T-cell therapy) can be performed via autologous transfer, wherein cells are isolated from and / or otherwise prepared from a subject to receive cell therapy or from a sample derived from such a subject. Therefore, cells can be derived from subjects who require treatment (e.g., patients), and after separation and processing, these cells can be applied to the same subject.

[0226] Cell therapy (e.g., adoptive T-cell therapy) can be performed via allogeneic transfer, where cells are isolated and / or otherwise prepared from a subject other than the one to be or ultimately receive the 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.

[0227] The subject (e.g., a human subject) may have been treated with a therapeutic agent targeting a disease or condition (e.g., a tumor) prior to administration of the cells or a composition containing cells. The subject may be refractory or unresponsive to other therapeutic agents. The subject may have a persistent or relapsing disease, for example, after treatment with another therapeutic intervention, including chemotherapy, radiation, and / or hematopoietic stem cell transplantation (HSCT), such as allogeneic HSCT. Even if the subject has become resistant to another therapy, administration may still be effective in treating the subject.

[0228] The subject may respond to another treatment agent, and treatment with that agent reduces the disease burden. The subject may initially respond to the treatment agent but exhibit a relapse of the disease or condition over time. The subject may not experience a relapse. The subject may be identified as being at risk of relapse, such as being at high risk of relapse, and therefore prophylactically administered cells, for example, to reduce the likelihood of relapse or to prevent relapse. The subject may not have previously received treatment with another treatment agent.

[0229] Subjects may have persistent or recurrent diseases, for example, after treatment with another therapeutic intervention, including chemotherapy, radiation, and / or hematopoietic stem cell transplantation (HSCT), such as allogeneic HSCT. Even if the subject has become resistant to another therapy, administration can still effectively treat the subject.

[0230] The engineered cells described in this article can be administered to animals (such as mammals, and even humans) to treat cancer. Additionally, engineered cells can be used to treat any condition associated with cancer, particularly cell-mediated immune responses against one or more tumor cells, where treatment or mitigation of the disease is desired.

[0231] The engineered cells described herein (e.g., immune cells, T cells, or NK cells) may be included in a composition for immunotherapy. The composition may include a pharmaceutical composition and further include a pharmaceutically acceptable carrier. A therapeutically effective amount of the pharmaceutical composition containing engineered cells may be administered.

[0232] The engineered cells can be used immediately for the aforementioned therapeutic, experimental, or commercial applications, or they can be cryopreserved for later use. The pharmaceutical composition may be included in a container, package, or dispenser along with the instructions for use.

[0233] The engineered cells disclosed herein can be formulated into unit dosage forms suitable for precise single-dose administration. The unit dosage form may contain additional lymphocytes. In the unit dosage form, 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 discrete amounts of the formulation. Non-limiting examples are packaged tablets or capsules, and powders in vials or ampoules. Aqueous suspension compositions may be packaged in single-dose, non-resealable containers. Multi-dose, resealable containers may be used, for example, with or without preservatives. Pharmaceutical compositions may not contain preservatives. Formulations for parenteral injection may be presented in unit dosage forms, such as in ampoules or in multi-dose containers with preservatives.

[0234] Example

[0235] The disclosure is further described in the following examples, which do not limit the scope of the disclosure as set forth in the claims.

[0236] Example 1. Generation of GPC3 CAR-T cells expressing IL-4Rα inverse cytokine receptor (ICR)

[0237] A chemically synthesized CAR backbone sequence encoding the GPC3 CAR (H93 CAR, SEQ ID NO: 20) backbone polypeptide was cloned into a pre-modified lentiviral vector (pLSINK-BBzBB), which was located downstream of and operatively linked to the constitutive hEF1α promoter for in vitro transcription. The CAR backbone sequence, from the N-terminus to the C-terminus, contains a CD8α signal peptide (SEQ ID NO: 25), an antigen-binding domain with 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 co-stimulatory signal transduction domain (SEQ ID NO: 28), and a CD3ζ primary intracellular signal transduction domain (SEQ ID NO: 29). To co-express armor elements, IL4R-IL23R, ICR5, and ICR6 sequences were chemically synthesized and ligated to the C-terminus of the CD3ζ primary intracellular signal transduction domain using the cloneEZ method. Figures 1a-1b As shown, the ICR is linked to the CAR-encoding sequence via a self-cleaving 2A (e.g., P2A, SEQ ID NO: 31) linker. Lentiviral expression vectors co-expressing the following were prepared: IL4R-IL23R#01 (SEQ ID NO: 13) and H93 CAR (SEQ ID NO: 20) (collectively referred to as H93-IL4R-IL23R#01, SEQ ID NO: 21); or IL4R-IL23R#02 (SEQ ID NO: 14) and H93 CAR (SEQ ID NO: 20) (collectively referred to as H93-IL4R-IL23R#02, SEQ ID NO: 22). The ICR structure and sequence of the IL-4 inverse cytokine receptor described herein are shown in Table 1.

[0238] Table 1. List of structures of IL4R-IL23R inverse cytokine receptors (ICRs)

[0239]

[0240] A mixture of lentiviral packaging plasmids containing pMDLg.pRRE (Addgene#12251), pRSV-REV (Addgene#12253), and pMD2.G (Addgene#12259) was premixed with a vector expressing the CAR construct at a pre-optimized ratio with polyetherimide (PEI) and incubated at 25°C for 5 min. The transfection mixture was then added to HEK293 cells. Cells were then incubated overnight in a cell culture incubator at 37°C and 5% CO2. The supernatant was collected after centrifugation at 4°C and 3000 g for 15 min and filtered through a 0.45 μm PES filter, followed by ultracentrifugation for lentiviral concentration. The supernatant was then carefully discarded, and viral particles were carefully washed with pre-chilled DPBS. The virus was resuspended appropriately and stored at -80°C. Viral titers were determined by titration using a transduced CHO (Chinese hamster ovary) cell line.

[0241] Human T cells were purified from commercial 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 number was determined, and the cell suspension was centrifuged at 300 g for 10 min. The supernatant was then completely discarded, and the cells were centrifuged every 10... 7 Each total cell count was resuspended in 40 μL of MACS buffer (DPBS supplemented with 8 μM EDTA + 0.5% FBS). Every 10 cells... 7 Add 10 μL of Pan T cell biotin-antibody mixture to each total cell, mix thoroughly, and incubate in a refrigerator (2°C–8°C) for approximately 5 min. Then add every 10 cells... 7 Add 30 μL of MACS buffer to each cell. Add 10 μL of MACS buffer per cell. 7 Add 20 μL of the Pan T cell microbead mixture to each cell. Thoroughly mix the cell suspension mixture and incubate for another 10 min in a refrigerator (2°C–8°C). Magnetic separation requires at least 500 μL. For magnetic separation, place the LS column in the magnetic field of a suitable MACS separator. This column is prepared by rinsing with 3 mL of buffer. The cell suspension is then applied to the column, and the effluent containing unlabeled cells is collected, representing the enriched T cell fraction. Additional T cells are collected by washing the column with 3 mL of buffer and collecting the passing unlabeled cells. These unlabeled cells again represent the enriched T cells and are combined with the effluent from the previous step. The combined enriched T cells are then centrifuged and resuspended in 1 L of TexMACS GMP Medium (Miltenyi #170-076-309) containing 300 IU / mL IL-2.

[0242] Subsequently, according to the manufacturer's protocol, the prepared T cells were pre-activated for 48-96 h using a human T cell activation / expansion kit (Miltenyi #130-091-441), in which anti-CD3 / CD28 MACSiBead particles were added at a bead-to-cell ratio of 1:2.

[0243] Preactivated T cells were transduced with lentiviral stock solution by adding it directly to the culture medium (TexMACS GMP medium supplemented with 300 IU / mL IL-2). The transduced cells were then transferred to a cell culture incubator at 37°C and 5% CO2 for transgene expression.

[0244] On day 7, CAR expression levels were assessed by flow cytometry. In short, 3 × 10⁶ cells were collected from each group. 5 T cells were collected and then incubated with FITC-GPC3 protein-Fc tag (Acrobiosystems#GP3-HF258-200UG) and PE-anti-IL4Rα antibody (Biolegend#355004) at 4°C for 30 min. As shown in the table below, the CAR positivity rates of 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 of 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.

[0245] Table 2. CAR and ICR expression

[0246]

[0247] Example 2. Expression levels of pSTAT3 and pATAT4 in CAR-T cells expressing IL4R-IL23R after IL-4 treatment.

[0248] To investigate intracellular signal transduction in CAR-T cells in the presence of IL-4, CAR-T cells were treated with 20 ng / mL recombinant human IL-4 (ACRO#IL4-H4218) for 15 min, and then pSTAT3 and pSTAT4 levels were measured by FACS. CAR-T cells were fixed with paraformaldehyde and permeabilized with Tween-20 for 10 min. At 4°C, cells were resuspended in 100 μL of DPBS containing the FITC-GPC3 protein-Fc tag (Acrobiosystems#GP3-HF258-200UG) and pSTAT3 antibody (Biolegend#651004) for 30 min. Cells were washed with DPBS and resuspended in 100 μL of DPBS, and then analyzed by FACS. Figures 2a-2c As shown, after treatment with 20 ng / mL IL-4, STAT3 phosphorylation was significantly increased in H93-IL4R-IL23R#01 and H93-IL4R-IL23R#02 CAR-T cells compared to H93 CAR-T cells. Figure 2g The median fluorescence intensity (MFI) of pSTAT3 in H93-IL4R-IL23R#01, H93-IL4R-IL23R#02, and H93 CAR-T cells was 1267, 952, and 1792, respectively, in the absence of IL-4. However, after treatment with 20 ng / mL IL-4, the MFI of pSTAT3 in H93-IL4R-IL23R#01, H93-IL4R-IL23R#02, and H93 CAR-T cells was 8867, 8000, and 3644, respectively.

[0249] After treatment with 20 ng / mL IL-4 for 15 min, 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). Figure 2d-2fAs shown, after treatment with 20 ng / mL IL-4, STAT4 phosphorylation was significantly increased in H93-IL4R-IL23R#01 and H93-IL4R-IL23R#02 CAR-T cells compared to H93 CAR-T cells. In the absence of IL-4, the MFI of pSTAT4 in H93-IL4R-IL23R#01, H93-IL4R-IL23R#02, and H93 CAR-T cells were 349, 346, and 363, respectively. However, after treatment with 20 ng / mL IL-4, the MFI of pSTAT4 in H93-IL4R-IL23R#01, H93-IL4R-IL23R#02, and H93 CAR-T cells were 1224, 1087, and 477, respectively.

[0250] These results demonstrate that, upon IL-4 stimulation, H93-IL4R-IL23R#01 and H93-IL4R-IL23R#02 CAR-T cells co-expressing the GPC3 CAR and IL4R-IL23R fusion proteins promote STAT3 and STAT4 phosphorylation, indicating that H93-IL4R-IL23R#01 and H93-IL4R-IL23R#02 CAR-T cells can convert IL-4R signaling into IL23R signaling and elicit downstream responses. In summary, these results strongly suggest that the IL4R-IL23R construct in CAR-T cells converts IL-4-mediated inhibitory signaling into IL-23R-mediated signaling, conferring greater efficacy against target cells (e.g., tumor cells) even in the presence of IL-4.

[0251] Example 3. Re-excitation model in GPC3 CAR-T cells expressing IL4R-IL23R

[0252] To assess the persistence of CAR-T cells in vitro, a CAR-T cell re-challenge model was established. Two treatment groups were used in the re-challenge assay: the H93 CAR-T group (the initial CAR-T cells were H93 CAR-T cells) and the H93-IL4R-IL23R#01 CAR-T group (the initial CAR-T cells were H93-IL4R-IL23R#01 CAR-T cells). As the first round (Round 1), CAR-T cells were co-cultured overnight with GPC3-expressing PLCPRF5 cells (ATCC#CRL-8024) at a 2:1 E / T ratio. After centrifugation at 300 g for 10 min, the CAR-T cells were resuspended in fresh medium (RPMI 1640 medium, 10% FBS, and 300 IU / mL IL2) and cultured for another two days. Cell viability, cell count, and CAR positivity were analyzed. These cells were used in the same treatment as in Round 1 for four rounds. The number of newly added PLCPRF5 cells in each round is adjusted based on the number of CAR-T cells after each round of target cell stimulation.

[0253] At the end of each round of stimulation, the percentage of CAR-positive T cells co-cultured with PLCPRF5 cells was detected by FACS. Figure 3a During the re-excitation model assay, in the absence of exogenous IL4 addition, the increase in the percentage of CAR-positive T cells in the H93 CAR-T group was almost similar to that in the H93-IL4R-IL23R#01 CAR-T group, increasing from 35% to 98.58% and 99.12%, respectively.

[0254] At the end of each round, the count and viability of CAR-T cells co-cultured with PLCPRF5 cells were detected using a T4 cell counter and trypan blue staining. 20 μL of cell suspension was mixed with 20 μL of trypan blue, pipetted into a disposable counting chamber, and analyzed using a Cellometer T4 analyzer. The fold increase of CAR-T cells was calculated based on the total number of T cells.

[0255] like Figures 3b-3c As shown, during the re-excitation model assay, the fold increase of H93 CAR-T cells increased from 1-fold to 18.42-fold, while that of H93-IL4R-IL23R#01 CAR-T cells increased from 1-fold to 387.85-fold. These results indicate that co-expression with the IL4R-IL23R construct provides CAR-T cells with greater proliferative potency at low concentrations of IL4 released by T cells and target cells themselves, compared to CAR expression alone.

[0256] Example 4. Real-time cell analysis (RTCA) assay and cytokine release in CAR-T cells expressing ICR

[0257] Real-time cytotoxic potency in CAR-T cells co-cultured with Hep3B2.1-7 cells was measured using RTCA assay. Hep3B2.1-7 cells (6 × 10⁶ cells per well) were cultured in the same wells. 3 Target cells were seeded in 96-well E-plates and incubated overnight using an RTCA analyzer. Then, with or without 10 ng / mL IL-4, target cells were incubated with CAR-T or UnT cells at an E / T ratio of 1:40, with T cell cytotoxicity assessed in real-time during this period. Cell indexes indicated the activity, adhesion, and number of target cells (normalized cell index = real-time cell index / cell index at the time point before the addition of effector cells).

[0258] During co-culture, monitoring was paused every 3 days, and 90 μL of culture supernatant was collected from each well for cytokine assays. Then, 90 μL of fresh culture medium was added to continue monitoring. To analyze cytokine release, the concentration of IFN-γ produced 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). Briefly, the HTRF reagents were warmed to room temperature for at least 30 min before assays. 16 μL / well of supernatant from the co-culture assay was transferred to a 384-well assay plate (Greiner Bio-One, #784075), followed by the addition of 4 μL / well of premixed HTRF reagent prepared according to the kit manual. The plate was then sealed with a paraffin membrane and incubated overnight at room temperature for IFN-γ assays or for 2 hours at room temperature for TNF-α assays. The plate was read on an HTRF-compatible Tecan Spark 10M reader. The concentrations of IFN-γ and TNF-α were calculated based on the signals obtained from the standard curve provided in the kit.

[0259] like Figures 4a-4b The cell index of Hep3B2.1-7 cells co-cultured with UnT cells showed a significant increase over time, with normalized cell indices of 4.13 and 3.76 at the end of the experiment, with and without 10 ng / mL IL4. H93, H93-IL4R-IL23R#01, H93-ICR5, and H93-ICR6 CAR-T cells exhibited varying degrees of cytotoxicity against Hep3B2.1-7 cells over time. Figure 4aAs shown, in the absence of 10 ng / mL IL-4, 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 were 1.96, 1.85, 2.43, and 1.52, respectively. Figure 4b As shown, in the presence of 10 ng / mL IL-4, the normalized cell indexes of H93, H93-IL4R-IL23R#01, H93-ICR5, and H93-ICR6 CAR-T cells were 2.26, 0.03, 1.03, and 0.17, respectively. These results indicate that CAR-T cells co-expressing IL4R-IL23R or IL4R-IL2R constructs exhibit enhanced long-term cytotoxicity in the presence of IL-4 (10 ng / mL), and H93 CAR-T cells co-expressing IL4R-IL23R showed significantly stronger long-term cytotoxicity than H93 CAR-T cells co-expressing IL4R-IL2R (H93-ICR5 or H93-ICR6).

[0260] like Figure 5a The results showed that, with and without 10 ng / mL IL-4, IFN-γ release from H93 CAR-T cells was 125 pg / mL and 21511 pg / mL, respectively. On day 12, without 10 ng / mL IL-4, IFN-γ release from H93-IL4R-IL23R#01, H93-ICR5, and H93-ICR6 CAR-T cells was 1410 pg / mL, 353 pg / mL, and 438 pg / mL, respectively. On day 12, with 10 ng / mL IL-4, IFN-γ release from H93-IL4R-IL23R#01, H93-ICR5, and H93-ICR6 CAR-T cells was 16382 pg / mL, 193 pg / mL, and 7379 pg / mL, respectively. Figure 5bAs shown, on day 12, TNF-α release from H93 CAR-T cells was 50 pg / mL with and 226 pg / mL without 10 ng / mL IL-4. On day 12, without 10 ng / mL IL-4, TNF-α release from H93-IL4R-IL23R#01, H93-ICR5, and H93-ICR6 CAR-T cells was 64 pg / mL, 57 pg / mL, and 57 pg / mL, respectively. Furthermore, on day 12, with 10 ng / mL IL-4, TNF-α release from H93-IL4R-IL23R#01, H93-ICR5, and H93-ICR6 CAR-T cells was 858 pg / mL, 62 pg / mL, and 363 pg / mL, respectively. These results indicate that IL-4 can significantly inhibit the release of H93 cytokines, and that the release of cytokines by H93-IL4R-IL23R#01 cells is higher than that by H93 CAR-T cells (H93-ICR5 or H93-ICR6) that co-express IL4R-IL2R.

[0261] Other embodiments

[0262] It should be understood that although this disclosure has been described in conjunction with its detailed description, the foregoing description is intended to illustrate and not limit the scope of this disclosure, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of 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; wherein, upon binding to IL-4, the fusion protein transmits interleukin-23 (IL-23) pathway signals.

2. The fusion protein of claim 1, wherein the fusion protein comprises a first polypeptide chain and a second polypeptide chain.

3. The fusion protein of claim 2, wherein the first polypeptide chain comprises (1) a first extracellular domain comprising an extracellular domain of an interleukin-4 receptor α (IL-4Rα) and (2) a first intracellular domain comprising an intracellular domain of an interleukin-12 receptor β1 subunit (IL-12Rβ1) or an intracellular domain of an interleukin-23 receptor (IL-23R).

4. The fusion protein of claim 2 or claim 3, wherein the second polypeptide chain comprises (1) a second extracellular domain comprising an extracellular domain of interleukin-2 receptor γ (IL-2Rγ), and (2) a second intracellular domain comprising an intracellular domain of IL-12Rβ1 or IL-23R.

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

6. A fusion protein, said fusion protein comprising: (a) A first polypeptide chain containing the following: (1) The first extracellular domain containing the IL-4Rα extracellular domain, and (2) A first intracellular domain containing an IL-12Rβ1 intracellular domain, and a second polypeptide chain containing the following: (1) A second extracellular domain containing the IL-2Rγ extracellular domain, and (2) A second intracellular domain containing the intracellular domain of IL-23R; or (b) A first polypeptide chain containing the following: (1) The first extracellular domain containing the IL-4Rα extracellular domain, and (2) The first intracellular domain containing the IL-23R intracellular domain, and A second polypeptide chain containing the following items: (1) A second extracellular domain containing the IL-2Rγ extracellular domain, and (2) A second intracellular domain containing the intracellular domain of IL-12Rβ1.

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

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

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

1.

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

5.

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

4.

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

8.

13. The fusion protein of any one of claims 1-12, wherein the fusion protein further comprises a transmembrane domain.

14. The fusion protein of 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 of claim 13 or claim 14, wherein the transmembrane domain comprises the amino acid sequence shown in SEQ ID NO: 3 or 7 or has at least 90%, 95% or 99% identical amino acid sequence to the amino acid sequence shown in SEQ ID NO: 3 or 7.

16. The fusion protein of any one of claims 13-15, 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 dimerize.

17. The fusion protein of any one of claims 13-16, wherein the fusion protein further comprises a linker sequence located between the C-terminus of the extracellular domain and the N-terminus of the transmembrane domain.

18. The fusion protein of claim 17, wherein the linker sequence comprises the amino acid sequence shown in SEQ ID NO: 2 or 6, or has at least 90%, 95%, or 99% identical amino acid sequence to the amino acid sequence shown in SEQ ID NO: 2 or 6.

19. The fusion protein of any one of claims 1-18, wherein the fusion protein comprises a first and / or a second polypeptide chain, the first and / or the second polypeptide chain comprising an amino acid sequence shown in any one of SEQ ID NO: 9-14 or having at least 90%, 95% or 99% identical amino acid sequences to any one of SEQ ID NO: 9-14.

20. The fusion protein of any one of claims 1-19, wherein the fusion protein 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 as described in any one of claims 1-20.

22. The nucleic acid of claim 21, wherein the nucleic acid further comprises a second nucleic acid sequence encoding an engineered receptor, wherein the engineered receptor comprises an extracellular antigen-binding domain or a ligand-binding domain, and optionally an intracellular signal transduction domain.

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

24. The nucleic acid of claim 23, wherein the 2A cleavable adapter comprises an amino acid sequence shown in any one of SEQ ID NO: 31-33 or a functional variant having at least about 90% sequence identity with it.

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

26. The nucleic acid of claim 25, wherein the engineered receptor is a CAR.

27. The nucleic acid of claim 26, wherein the CAR comprises an extracellular antigen-binding domain that specifically binds to an antigen, wherein the antigen is a tumor antigen selected from the group consisting of: 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, L1 cell adhesion molecule, MAGE-A1, mesothelin, MAGEA3, p53, MART1, GP100, protease-3 (PR3), tyrosinase, survival protein, hTERT, EphA2, NY-ESO-1, h5T4, PSCA, PSMA, ROR1, TAG-72, VEGF-R2, WT-1, CD123, CD44V6, NKCS1, IGF1R, EGFR, EGFR-VIII, duracin 18.2, duracin 6, NKG2D, delta-like ligand 3 (DLL3), CD70, CS-1, c-Met, glycolipid F77, PD-L1 and PD-L2.

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

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

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

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

32. The nucleic acid of any one of claims 26-31, 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 of any one of claims 26-32, 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 21-33, wherein the nucleic acid encodes an amino acid sequence shown in any one of SEQ ID NO: 9-14 and 20-22 or a functional variant having at least about 90% sequence identity with it.

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

36. An engineered cell comprising a fusion protein as described in any one of claims 1-20, a nucleic acid as described in claims 21-34, and / or a vector as described in claim 35.

37. The engineered cell of claim 36, wherein the engineered cell further comprises an engineered receptor, wherein the engineered receptor comprises an extracellular antigen-binding domain or a ligand-binding domain, and optionally an intracellular signal transduction domain.

38. The engineered cell of claim 37, wherein the engineered receptor is selected from the group consisting of engineered T-cell receptors (TCR), chimeric antigen receptors (CAR), T-cell antigen conjugates (TAC), or a subset thereof.

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

40. The engineered cell of any one of claims 36-39, wherein the engineered cell comprises a polypeptide encoding a fusion protein and / or a polypeptide encoding a CAR, and wherein the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 9-14 and 20-22 or a functional variant having at least about 90% sequence identity with it.

41. The engineered cell according to any one of claims 36-40, wherein the engineered cell is an immune cell.

42. The engineered cell of claim 41, wherein the immune cell is 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 engineered cell of claim 42, wherein the immune cell is a T cell, optionally an αβ T cell or a γδ T cell.

44. A pharmaceutical composition comprising engineered cells and a pharmaceutically acceptable carrier as described in any one of claims 36-43.

45. A method for producing engineered cells, the method comprising introducing the carrier of claim 35 into the cells.

46. ​​A method of treating a disease or disorder in a subject, the method comprising administering to a subject in need a therapeutically effective amount of engineered cells as described in any one of claims 36-43 or a pharmaceutical composition as described in claim 44.

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

48. The method of 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 tumor, soft tissue sarcoma, esophageal cancer, or CNS tumor.

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