Recombinant polypeptides, recombinant nucleic acids encoding them, and their use in the treatment of cancer

Abstract

Recombinant polypeptides and recombinant nucleic acids encoding them are disclosed herein. According to some embodiments of this disclosure, a recombinant polypeptide comprises a first bifunctional domain and a first single-chain variable region fragment (scFv) or peptide ligated to the N-terminus of the first bifunctional domain. Optionally, the recombinant polypeptide further comprises a second scFv ligated to the N-terminus of the first scFv or peptide. Methods for treating cancer using immune cells expressing recombinant polypeptides are also disclosed herein.

Description

[Technical Field]

[0001] This disclosure generally relates to the field of disease treatment. More specifically, this disclosure relates to the treatment of cancer with novel cell therapies. [Background technology]

[0002] Cancer is a leading cause of death worldwide, accounting for approximately 10 million deaths in 2020, representing roughly one in six deaths. One of the defining characteristics of cancer is that abnormal cells grow rapidly and uncontrollably, potentially invading or metastasizing to any part of the body. The most common cancers include breast cancer, lung cancer, colorectal cancer, prostate cancer, skin cancer, and stomach cancer. Signs and symptoms associated with cancer usually vary depending on which part of the body is affected. Common signs and symptoms of cancer include fatigue, lumps, weight changes (such as unintentional loss or gain), changes in bowel or bladder habits, difficulty swallowing, nausea, vomiting, fever, night sweats, bleeding, bruising, cough, and / or difficulty breathing. Symptoms of advanced cancer further include pain, decreased physical strength, and paralysis that affect the cancer patient's movement, resulting in depression, loss of independence, and social isolation. In addition to the patient themselves, cancer also affects the patient's family and friends. Many caregivers experience physical, mental, and emotional struggles. Several reports suggest that a cancer diagnosis can also negatively impact the health of caregivers. For example, they may have a higher rate of depression, weakened immune responses, and cardiovascular disease. [Overview of the project] [Problems that the invention aims to solve]

[0003] While various treatment approaches are currently available, including chemotherapy, surgery, radiation therapy, hormone therapy, targeted therapy, and most recently, immunotherapy and cell-based therapies, none of these treatments have yielded satisfactory results. Cancer continues to pose significant health risks and increases personal, familial, social, and economic burdens. For these reasons, there is ongoing interest in developing novel drugs and methods to treat cancer.

Means for Solving the Problem

[0004] The following presents a simplified summary of the present disclosure to provide a basic understanding to the reader. This summary is not an extensive overview of the present disclosure, nor does it identify the main / determinative elements of the invention, nor define the scope of the invention. Its sole purpose is to present, in a simplified form, some of the concepts disclosed herein as a prelude to the more detailed description presented later.

[0005] As embodied and broadly described herein, a first aspect of the present disclosure relates to a recombinant polypeptide comprising a bifunctional domain and a single-chain variable region fragment (scFv) or a peptide linked to the N-terminus of the bifunctional domain. According to an embodiment of the present disclosure, the bifunctional domain can penetrate the cell membrane (i.e., function as a transmembrane domain) and mediate signal transduction within the cell (i.e., function as an intracellular domain).

[0006] According to some embodiments of the present disclosure, the bifunctional domain comprises, in this order from its N-terminus to its C-terminus, intracellular loops 1 (ICL1), ICL2, ICL3, and the C-terminal region of a G protein-coupled receptor (GPCR), and does not have the extracellular domain and transmembrane domain of the GPCR. According to some preferred embodiments, the bifunctional domain consists of ICL1, ICL2, ICL3, and the C-terminal region of the GPCR.

[0007] The GPCR is preferably a class A GPCR. According to some exemplary embodiments, the GPCR is a cannabinoid receptor 2 (CNR2), a hydroxycarboxylic acid receptor 2 (HCAR2), a G protein-coupled receptor 84 (GPR84), or a P2Y purinergic receptor 14 (P2Y14).

[0008] According to one embodiment, the bifunctional domain includes ICL1, ICL2, ICL3 and the C-terminal region of CNR2. In this embodiment, the bifunctional domain derived from CNR2 includes the amino acid sequence of Sequence ID No: 2.

[0009] According to another embodiment, the bifunctional domain includes ICL1, ICL2, ICL3 and the C-terminal region of HCAR2. In this embodiment, the bifunctional domain derived from HCAR2 includes the amino acid sequence of SEQ ID NO: 4.

[0010] According to another embodiment, the bifunctional domain comprises ICL1, ICL2, ICL3 and the C-terminal region of GPR84. In this embodiment, the bifunctional domain derived from GPR84 comprises the amino acid sequence of SEQ ID NO: 6.

[0011] In yet another embodiment, the bifunctional domain includes ICL1, ICL2, ICL3 and the C-terminal region of P2Y14. In this embodiment, the bifunctional domain derived from P2Y14 includes the amino acid sequence of SEQ ID NO: 8.

[0012] According to certain embodiments of this disclosure, the scFv is specific to CD3 or NKp46.

[0013] According to some preferred embodiments, the recombinant polypeptide further comprises an scFv specific to a tumor-associated antigen (TAA).

[0014] According to alternative embodiments of the present disclosure, the recombinant polypeptide comprises two bifunctional domains (i.e., a first and a second bifunctional domain), the second bifunctional domain being located at the C-terminus of and ligated to the first bifunctional domain. In this embodiment, the first bifunctional domain comprises ICL1, ICL2, ICL3 and the C-terminal region of the first GPCR in this order from its N-terminus to its C-terminus; and the second bifunctional domain comprises ICL1, ICL2, ICL3 and the C-terminal region of the second GPCR in this order from its N-terminus to its C-terminus. The first and second bifunctional domains do not contain the extracellular and transmembrane domains of the GPCRs (i.e., the first and second GPCRs). According to some preferred embodiments, the first bifunctional domain consists of ICL1, ICL2, ICL3 and the C-terminal region of the first GPCR, and the second bifunctional domain consists of ICL1, ICL2, ICL3 and the C-terminal region of the second GPCR. Preferably, the second GPCR is different from the first GPCR.

[0015] In a preferred embodiment, the first and second GPCRs are each a class A GPCR. According to some embodiments, the first and second GPCRs are independently selected from the group consisting of CNR2, HCAR2, GPR84, and P2Y14.

[0016] According to one embodiment, the first bifunctional domain includes ICL1, ICL2, ICL3 and the C-terminal region of CNR2, and the second bifunctional domain includes ICL1, ICL2, ICL3 and the C-terminal region of P2Y14. In this embodiment, the bifunctional domain derived from CNR2 includes the amino acid sequence of SEQ ID NO: 2, and the bifunctional domain derived from P2Y14 includes the amino acid sequence of SEQ ID NO: 8.

[0017] According to another embodiment, the first bifunctional domain includes ICL1, ICL2, ICL3 and the C-terminal region of CNR2, and the second bifunctional domain includes ICL1, ICL2, ICL3 and the C-terminal region of HCAR2. In this embodiment, the bifunctional domain derived from CNR2 includes the amino acid sequence of SEQ ID NO: 2, and the bifunctional domain derived from HCAR2 includes the amino acid sequence of SEQ ID NO: 4.

[0018] According to alternative embodiments of the present disclosure, the recombinant polypeptide comprises three bifunctional domains (i.e., first, second, and third bifunctional domains), wherein the second bifunctional domain is located at the C-terminus of and ligated to the first bifunctional domain, and the third bifunctional domain is located at the C-terminus of and ligated to the second bifunctional domain. In these embodiments, the first bifunctional domain comprises ICL1, ICL2, ICL3 and the C-terminal region of the first GPCR in this order from its N-terminus to its C-terminus, the second bifunctional domain comprises ICL1, ICL2, ICL3 and the C-terminal region of the second GPCR in this order from its N-terminus to its C-terminus, and the third bifunctional domain comprises ICL1, ICL2, ICL3 and the C-terminal region of the third GPCR in this order from its N-terminus to its C-terminus. None of the first to third bifunctional domains described above contain the extracellular domain and transmembrane domain of the GPCR (i.e., the first, second, and third GPCRs). According to some preferred embodiments, the first bifunctional domain consists of ICL1, ICL2, ICL3 and the C-terminal region of the first GPCR, the second bifunctional domain consists of ICL1, ICL2, ICL3 and the C-terminal region of the second GPCR, and the third bifunctional domain consists of ICL1, ICL2, ICL3 and the C-terminal region of the third GPCR. Preferably, the first, second, and third GPCRs are different from each other.

[0019] In a preferred embodiment, the first, second, and third GPCRs are each a class A GPCR. According to a particular embodiment, the first, second, and third GPCRs are independently selected from the group consisting of CNR2, HCAR2, GPR84, and P2Y14.

[0020] According to one embodiment, the first bifunctional domain includes ICL1, ICL2, ICL3 and the C-terminal region of CNR2, the second bifunctional domain includes ICL1, ICL2, ICL3 and the C-terminal region of P2Y14, and the third bifunctional domain includes ICL1, ICL2, ICL3 and the C-terminal region of HCAR2. In this embodiment, the bifunctional domain derived from CNR2 includes the amino acid sequence of SEQ ID NO: 2, the bifunctional domain derived from P2Y14 includes the amino acid sequence of SEQ ID NO: 8, and the bifunctional domain derived from HCAR2 includes the amino acid sequence of SEQ ID NO: 4.

[0021] Also disclosed herein are recombinant nucleic acids encoding the recombinant polypeptides of this disclosure, and immune cells expressing the recombinant polypeptides.

[0022] According to certain embodiments of the present disclosure, the recombinant nucleic acid comprises a promoter and first and second coding sequences operably linked to the promoter, wherein the first coding sequence codes for the scFv or peptide, and the second coding sequence is located downstream of the first coding sequence and codes for one or more bifunctional domains.

[0023] In some embodiments, the second coding sequence encodes a bifunctional domain derived from one of the following: for example, CNR2-, HCAR2-, GPR84-, or P2Y14-. According to one exemplary embodiment, the second coding sequence encoding the bifunctional domain derived from CNR2 (SEQ ID NO: 2) includes the nucleotide sequence of SEQ ID NO: 1. According to one exemplary embodiment, the second coding sequence encoding the bifunctional domain derived from HCAR2 (SEQ ID NO: 4) includes the nucleotide sequence of SEQ ID NO: 3. According to another exemplary embodiment, the second coding sequence encoding the bifunctional domain derived from GPR84 (SEQ ID NO: 6) includes the nucleotide sequence of SEQ ID NO: 5. According to yet another exemplary embodiment, the second coding sequence encoding the bifunctional domain derived from P2Y14 (SEQ ID NO: 8) includes the nucleotide sequence of SEQ ID NO: 7.

[0024] In some embodiments, the second coding sequence codes for two bifunctional domains (i.e., a first and a second bifunctional domain), the second bifunctional domain being located at the C-terminus of and ligated to the first bifunctional domain. According to one exemplary embodiment, the second coding sequence encoding a bifunctional domain derived from CNR2 (SEQ ID NO: 2) and a bifunctional domain derived from P2Y14 (SEQ ID NO: 8) includes the nucleotide sequence of SEQ ID NO: 29. According to another exemplary embodiment, the second coding sequence encoding a bifunctional domain derived from CNR2 (SEQ ID NO: 2) and a bifunctional domain derived from HCAR2 (SEQ ID NO: 4) includes the nucleotide sequence of SEQ ID NO: 31.

[0025] In a particular embodiment, the second coding sequence codes for three bifunctional domains (i.e., first, second, and third bifunctional domains), the second bifunctional domain being located at the C-terminus of and ligated to the first bifunctional domain, and the third bifunctional domain being located at the C-terminus of and ligated to the second bifunctional domain. According to one exemplary embodiment, the second coding sequence encoding the bifunctional domain derived from CNR2 (SEQ ID NO: 2), the bifunctional domain derived from P2Y14 (SEQ ID NO: 8), and the bifunctional domain derived from HCAR2 (SEQ ID NO: 4) includes the nucleotide sequence of SEQ ID NO: 27.

[0026] Depending on the desired purpose, the immune cells expressing the recombinant polypeptide may be T cells, natural killer (NK) cells, B cells, basophils, eosinophils, dendritic cells (DCs), or neutrophils. According to one exemplary embodiment, the immune cells are T cells. According to another exemplary embodiment, the immune cells are NK cells.

[0027] Another aspect of the present disclosure relates to a method for treating cancer in a subject. The method includes administering an effective amount of the immune cells of the present disclosure to the subject.

[0028] Examples of cancers treatable with the immune cells and / or methods of this disclosure include, but are not limited to, breast cancer, gastric cancer, colorectal cancer, gallbladder cancer, prostate cancer, cervical cancer, ovarian cancer, chronic or acute lymphoblastic leukemia, bladder cancer, kidney cancer, hepatocellular carcinoma, head and neck squamous cell carcinoma, glioblastoma, esophageal cancer, pancreatic cancer, oral cancer, lung cancer, melanoma, and lymphoma.

[0029] In all embodiments of this disclosure, the subject is a mammal, preferably a human.

[0030] Many of the features and benefits associated with this disclosure will be better understood by referring to the following detailed description, which is considered in conjunction with the attached drawings.

[0031] This description will be better understood in conjunction with the following detailed explanation, which should be considered in conjunction with the attached drawings. [Brief explanation of the drawing]

[0032] [Figure 1] Figure 1 is a schematic diagram of a BE-TM / IC polypeptide according to one embodiment of the present disclosure. [Figure 2] Figure 2 is a schematic diagram of a 2xBE-TM / IC polypeptide according to another embodiment of the present disclosure. [Figure 3A] Figure 3A is a schematic diagram of a 2xBE-multiTM / IC polypeptide according to one embodiment of the present disclosure. [Figure 3B] Figure 3B is a schematic diagram of a 2xBE-multiTM / IC polypeptide according to another embodiment of the present disclosure. [Figure 4] Figure 4 shows the results of a tumor killing assay demonstrating the cytotoxic effect of engineered T cells expressing FRa_CAR or CAR-delITAM-CPH according to Example 1.2 of this disclosure. [Figure 5] Figure 5 shows the results of a tumor killing assay demonstrating the cytotoxic effect of modified T cells expressing FRa_CAR or FRa-CPH™ / IC according to Example 1.2 of this disclosure. [Figure 6] Figure 6 shows the results of a tumor killing assay demonstrating the cytotoxic effects of modified T cells expressing HER2_TAC (Her2_TAC-T), HER2-CD3-syn GPR84 (Her2_syn GPR84), or HER2-CD3-syn CPH (Her2_syn CPH) according to Example 1.3 of this disclosure. [Figure 7A] Figure 7A shows the results of an analysis demonstrating the cytotoxic effect of specific modified T cells on cancer cells according to Example 1.3 of this disclosure. FRa_syn CPH: Modified T cells expressing FRa_CD3_syn CPH. FRa_TAC-T: Modified T cells expressing FRa_TAC. [Figure 7B]Figure 7B shows the results of an analysis demonstrating the cytotoxic effect of specific modified T cells on cancer cells according to Example 1.3 of this disclosure. FRa_syn CPH: Modified T cells expressing FRa_CD3_syn CPH. FRa_CAR-T: Modified T cells expressing FRa_CAR. [Figure 8A] Figure 8A is a histogram showing the expression levels of interleukin-2 (IL-2; Figure 8A) in the supernatant of cultured cells according to Example 1.3 of this disclosure. Cancer cells were co-cultured with specific modified T cells for 48 hours, and then cytokine assays were analyzed. FRa_syn CPH: Modified T cells expressing FRa_CD3_syn CPH. FRa_TAC-T: Modified T cells expressing FRa_TAC. FRa_CAR-T: Modified T cells expressing FRa_CAR. **p<0.01;***p<0.001;ns (no significant difference): p≧0.05. [Figure 8B] Figure 8B is a histogram showing the expression levels of interferon-gamma (IFN-γ; Figure 8B) in the supernatant of cultured cells according to Example 1.3 of this disclosure. Cancer cells were co-cultured with specific modified T cells for 48 hours, and then cytokine assays were analyzed. FRa_syn CPH: Modified T cells expressing FRa_CD3_syn CPH. FRa_TAC-T: Modified T cells expressing FRa_TAC. FRa_CAR-T: Modified T cells expressing FRa_CAR. **p<0.01;***p<0.001;ns (no significant difference): p≧0.05. [Figure 8C] Figure 8C is a histogram showing the expression level of tumor necrosis factor-alpha (TNF-α; Figure 8C) in the supernatant of cultured cells according to Example 1.3 of this disclosure. Cancer cells were co-cultured with specific modified T cells for 48 hours, and then cytokine assays were analyzed. FRa_syn CPH: Modified T cells expressing FRa_CD3_syn CPH. FRa_TAC-T: Modified T cells expressing FRa_TAC. FRa_CAR-T: Modified T cells expressing FRa_CAR. **p<0.01;***p<0.001;ns (no significant difference): p≧0.05. [Figure 9]Figure 9 is a histogram showing the cytotoxic effect of specific modified T cells according to Example 1.3 of this disclosure. FRa_CPH: Modified T cells expressing FRa-CD3-syn CPH. FRa_CH: Modified T cells expressing FRa-CD3-syn CH. FRa_CP: Modified T cells expressing FRa-CD3-syn CP. FRa_C: Modified T cells expressing FRa_CD3_syn CNR2. FRa_H: Modified T cells expressing FRa-CD3-syn HCAR2. FRa_P: Modified T cells expressing FRa-CD3-syn P2Y14. [Figure 10] Figure 10 is a line graph showing the tumor volume of mice administered with untransduced T cells, modified T cells expressing FRa_TAC (FRa_TAC-T), or modified T cells expressing FRa_CD3_syn CPH (FRa_syn CPH) according to Example 2 of this disclosure. *p<0.05;**p<0.01. [Figure 11] Figure 11 is a line graph showing the survival probability of mice administered with non-transduced T cells, modified T cells expressing FRa_TAC (FRa_TAC-T), or modified T cells expressing FRa_CD3_syn CPH (FRa_syn CPH) according to Example 2 of this disclosure. [Modes for carrying out the invention]

[0033] Following common practice, the components or elements shown in each figure are not depicted to scale, but are illustrated in a way that most clearly illustrates the specific features or elements relevant to the present invention. Furthermore, the reference numerals and numerals in each figure indicate components or parts.

[0034] The detailed description below, with reference to the accompanying drawings, is intended to describe embodiments of the present disclosure and does not represent the only possible forms in which these embodiments can be constructed or utilized. This description illustrates the function and construction and operation procedures of these embodiments. However, the same or equivalent functions and procedures may be achieved by other embodiments.

[0035] I. Definition For convenience, the specific terms used in the specification, examples, and appended claims are summarized here. Unless otherwise defined herein, the scientific and technical terms used in this disclosure have the meanings commonly understood and used by those skilled in the art. Unless otherwise required by context, singular terms are to be interpreted as including the plural form of the same term, and plural terms are to be interpreted as including the singular form. In particular, as used herein and in the claims, the singular “a” and “an” include a plural reference unless explicitly indicated otherwise by context. Also, as used herein and in the claims, the terms “at least one” and “one or more” have the same meaning and include one, two, three, or more.

[0036] Although the numerical ranges and parameters defining the broad scope of the present invention are approximate, the numerical values ​​described in specific examples are reported as accurately as possible. However, any numerical value inherently includes a certain degree of error that inevitably arises from the standard deviation observed in each test measurement. Furthermore, the term “about” as used herein generally means within 10%, 5%, 1%, or 0.5% of a given value or range. Alternatively, the term “about” means within the acceptable standard error of the mean, as considered by those skilled in the art. Unless otherwise expressly specified in the examples / operations, all numerical ranges, quantities, values, and percentages disclosed herein, such as quantities of material, durations of time, temperatures, operating conditions, and ratios of quantities, should be understood in all cases as being modified by the term “about.” Accordingly, unless otherwise indicated, the numerical parameters described in this disclosure and the appended claims are approximate values ​​that may vary as desired. At a minimum, each numerical parameter should be interpreted in light of the reported number of significant figures and by applying the usual rounding techniques.

[0037] As used herein, the term “recombinant nucleic acid” refers to a nucleic acid produced by recombinant DNA technology, wherein the nucleotide sequence of the nucleic acid is not identical to any sequence found in nature. The term “recombinant polypeptide” refers to a polypeptide expressed and isolated from cells or cell lines transfected with an expression vector containing a polypeptide coding sequence (e.g., the recombinant nucleic acid of this disclosure), wherein the coding sequence is not associated with cells or nature.

[0038] As used herein, the term “operably linked” refers to both an expression regulatory sequence that is continuous with the gene of interest (e.g., the first and second coding sequences of this disclosure) and an expression regulatory sequence that acts in trans or at a distant location to control the gene of interest (e.g., a promoter of this disclosure).

[0039] As discussed herein, minor variations in the amino acid sequence of a polypeptide or the nucleotide sequence of a nucleic acid are intended to be included within the concept of the invention as described herein and claimed, provided that the variations in the amino acid / nucleotide sequence maintain at least 85% sequence identity (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity). The polypeptides or nucleic acids of this disclosure may be specifically modified to alter the properties of the polypeptide or nucleic acid, regardless of their physiological activity. For example, due to codon degeneracy, specific nucleotides can be modified without affecting the physiological activity of the encoded polypeptide (i.e., multiple codons can encode the same amino acid residue during protein synthesis; for example, a leucine residue can be encoded by the codons "UUA", "UUG", "CUA", "CUG", "CUU", or "CUC", and a serine residue can be encoded by the codons "UCA", "UCG", "UCC", "UCU", "AGU", or "AGC"). Furthermore, specific amino acids can be modified and / or deleted without affecting the physiological activity of the polypeptide in this study. In particular, conservative amino acid substitutions are intended. Conservative substitutions are those performed within a family of related amino acids in the side chain. Genetically encoded amino acids are generally classified into families: (1) acidic = aspartic acid, glutamic acid; (2) basic = lysine, arginine, histidine; (3) nonpolar = alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) uncharged polar = glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. More preferred families are the aliphatic hydroxy family (serine and threonine), the amide-containing family (asparagine and glutamine), the aliphatic family (alanine, valine, leucine, and isoleucine), and the aromatic family (phenylalanine, tryptophan, and tyrosine).For example, substituting leucine with isoleucine or valine individually, aspartic acid with glutamic acid, threonine with serine, or substitutions between structurally similar amino acids is reasonably expected to have no significant effect on the binding or properties of the resulting molecule. This is especially true when the substitution does not involve amino acids within the framework site.

[0040] "Percentage (%) sequence identity" is defined as the percentage of amino acid residues / nucleotides in a candidate sequence that are identical to the amino acid residues / nucleotides of a given polypeptide / nucleic acid after aligning sequences and introducing gaps as necessary to achieve maximum percentage sequence identity, with conservative substitutions not considered as part of sequence identity. Alignment for determining percentage sequence identity can be achieved in various ways within the scope of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithm necessary to achieve maximum alignment over the entire length of the sequences being compared. For the purposes of this specification, sequence comparisons between two amino acid sequences / nucleic acids were performed using the computer programs Blasp (protein-protein BLAST) / Blastn (nucleic acid-nucleic acid BLAST), which are provided online by the National Center for Biotechnology Information (NCBI). The percentage of amino acid sequence / nucleic acid identity between a given amino acid sequence / nucleic acid A and a given amino acid sequence / nucleic acid B (which can be expressed as a certain percentage of amino acid sequence / nucleic acid identity between a given amino acid sequence / nucleic acid A and a given amino acid sequence / nucleic acid B) is calculated by the following formula. JPEG2026518063000002.jpg1526 Here, X is the number of amino acid residues / nucleic acids that the sequence alignment program BLAST scored as identical in the alignment of programs A and B, and Y is the total number of amino acid residues / nucleic acids of the shorter of A or B.

[0041] As used herein, the terms “link” and “connect” are interchangeable to refer to any means of connecting two components, either directly or indirectly.

[0042] The terms “administered,” “administering,” and “administration” are used interchangeably herein and refer to modes of delivery including, but not limited to, the delivery of the active substance of the present invention (e.g., immune cells) into a tumor, intravenous, intraarterial, or peritoneal cavity.

[0043] As used herein, the terms “treat,” “treating,” and “treatment” are interchangeable and encompass the partial or complete prevention, improvement, mitigation, and / or management of cancer-related symptoms, secondary disorders, or conditions. As used herein, “treating” means applying or administering the immune cells of the present invention to a subject having cancer-related symptoms, secondary disorders, or conditions for the purpose of partially or completely reducing, improving, mitigating, delaying the onset, inhibiting progression, reducing severity, and / or reducing the incidence of one or more cancer-related symptoms, secondary disorders, or characteristics. Cancer-related symptoms, secondary disorders, and / or conditions include, but are not limited to, fatigue, lumps, weight changes (e.g., unintentional loss or increase), changes in bowel or bladder habits, dysphagia, nausea, vomiting, fever, night sweats, bleeding, bruising, cough, shortness of breath, pain, decreased physical strength, and paralysis. Treatment may be administered to subjects exhibiting only the initial signs of cancer-related symptoms, disorders, and / or conditions, with the aim of reducing the risk of developing such symptoms, disorders, and / or conditions. Treatment is generally considered “effective” if one or more symptoms or clinical markers are reduced, as defined herein. Alternatively, treatment is considered “effective” if the progression of symptoms, disorders, or conditions is reduced or halted.

[0044] As used herein, the term “effective dose” specifies the amount of a component sufficient to produce a desired response. For therapeutic purposes, the effective dose is also the amount in which the therapeutically beneficial effects of the component outweigh the toxic or adverse effects. The effective dose of a drug will provide treatment for a disease or condition, such that it does not need to cure the disease or condition, but the onset of the disease or condition is delayed, inhibited, or prevented, or the symptoms of the disease or condition are improved. The effective dose may be divided into one, two, or more doses in a form suitable for administration over a specified period of time. A particular effective or sufficient dose will vary depending on factors such as the specific condition being treated, the patient’s physical condition (e.g., size, age, or sex), the type of mammal or animal being treated, the duration of treatment, the nature of any concomitant therapy, and the specific formulation being employed. The effective dose may be expressed, for example, by cell count per unit body weight (cells / kg) or cell count per unit body surface area (cells / m²). 2 ), or may be expressed as the number of cells per subject (number of cells / subject). A person skilled in the art can calculate the human equivalent dose (HED) of a drug (e.g., the immune cells) based on the dose determined from an animal model. For example, when estimating the maximum safe dose for use in human subjects, one can follow the industry guidance published by the U.S. Food and Drug Administration (FDA) titled "Estimating the Maximum Safe Starting Dose in Initial Clinical Trials for Therapeutics in Adult Healthy Volunteers."

[0045] The term "subject" refers to animals, including human species, that can be treated by the immune cells and / or methods of the present invention. The term "subject" is intended to refer to both males and females unless one sex is specifically indicated.

[0046] II. Description of the Invention GPCRs, also known as "seven-transmembrane domain receptors," are membrane-bound receptors coupled to heterotrimeric G proteins that regulate responses to sensory stimuli such as light, odor, hormones, cytokines, or neurotransmitters. Structurally, GPCRs consist of seven transmembrane alpha helices (H1-H7) linked by three extracellular loops (ECL1, ECL2, ECL3) and three intracellular loops (ICL1, ICL2, ICL3), and possess an extracellular N-terminal region (also known as the "N-terminal tail") and an intracellular C-terminal region (also known as the "C-terminal tail"). GPCRs are classified into six classes based on sequence homology and functional similarity, including Class A: rhodopsin-like receptors, Class B: secretin family, Class C: metabotropic glutamate receptors, Class D: fungal conjugation pheromone receptors, Class E: cAMP receptors, and Class F: frizzled (FZD) and smoothed (SMO) receptors.

[0047] This disclosure is at least in part based on the unexpected discovery that a reconstituted peptide containing the ICL1, ICL2, ICL3, and C-terminal regions of a class A GPCR can function as a bifunctional domain (hereinafter referred to as the "TM / IC domain") that can span / traverse the cell membrane (i.e., function as a transmembrane domain) and mediate intracellular signaling (i.e., function as an intracellular domain). This novel TM / IC domain is useful for coupling with one or more extracellular proteins (e.g., one or more scFv or peptides) to express extracellular proteins on the surface of cells (e.g., immune cells). According to some exemplary embodiments of this disclosure, this novel TM / IC domain is ligated to two extracellular proteins (e.g., two scFv, two peptides, or one scFv and one peptide), which function as binding elements (BEs) and are specific to TAA and immune cell-associated molecules (IAMs, i.e., molecules expressed on the surface of immune cells and preferably associated with immune cell activation), respectively. The binding elements (BEs) thus formed redirect the specificity of immune cells to tumor cells expressing TAA via TAA-binding scFv / peptide, and then mediate the activation of immune cells via IAM-binding scFv / peptide and the TM / IC domain, which is useful in initiating the effector functions of immune cells (e.g., proliferation, cytokine secretion, and / or cell lysis). Therefore, this novel TM / IC domain provides a novel strategy for constructing different types of chimeric immune cell receptors (e.g., chimeric T cell receptors or chimeric NK cell receptors) for use in cell therapy.

[0048] Accordingly, this disclosure provides several recombinant polypeptides, including BE-TM / IC polypeptides, 2xBE-TM / IC polypeptides, and 2xBE-multiTM / IC polypeptides, each comprising one or two TM / IC domains that function as bifunctional domains for immobilizing the polypeptide to a cell membrane, and one or more BEs that function as targeting domains for interacting with target molecules. Also disclosed herein are nucleic acids for expressing recombinant polypeptides, immune cells on which the recombinant polypeptides are expressed, and methods for treating diseases (e.g., cancer) using immune cells.

[0049] (i) BE-TM / IC polypeptide and the nucleic acid encoding it A first aspect of this disclosure relates to BE-TM / IC polypeptides and nucleic acids encoding BE-TM / IC polypeptides.

[0050] Herein, we refer to Figure 1, which shows a schematic diagram of a BE-TM / IC polypeptide according to one embodiment of the present disclosure. In the structure, the BE-TM / IC polypeptide comprises one TM / IC domain and one binding element (BE) ligated to the N-terminus of the TM / IC domain, where the TM / IC domain contains the ICL1, ICL2, ICL3 and C-terminal region (C) of a GPCR in this order from its N-terminus to its C-terminus. Depending on the desired purpose, the BE may be an scFv or a peptide, where the BE is an scFv, which has a common structure known in the art and includes a heavy chain variable domain (VH) and a light chain variable domain (VL). After expression in a cell (e.g., an immune cell), the BE (i.e., scFv or peptide) functions as an extracellular domain that recognizes and targets the corresponding molecule, and the TM / IC domain functions as a bifunctional domain that fixes the BE to the cell membrane and mediates the signal transmitted therefrom.

[0051] According to several embodiments of this disclosure, BE is an scFv specific to a receptor, co-receptor, costimulatory molecule, or cell adhesion molecule of immune cells (e.g., T cells, NK cells, B cells, basophils, eosinophils, DCs, or neutrophils). In one embodiment, BE is an scFv specific to CD3, a protein complex and T cell co-receptor involved in T cell activation. According to one example of this disclosure, the anti-CD3 scFv contains the amino acid sequence of SEQ ID NO: 10. In another embodiment, BE is an scFv specific to NKp46, a major NK cell activation receptor. According to one example of this disclosure, the anti-NKp46 scFv contains the amino acid sequence of SEQ ID NO: 26. In yet another embodiment, BE is an scFv specific to CD2, a cell adhesion molecule that modulates T cell and NK cell activation. According to one example of this disclosure, the anti-CD2 scFv contains the amino acid sequence of SEQ ID NO: 53 or 55. In yet another embodiment, BE is an scFv specific to an inducible costimulatory molecule (ICOS), a costimulatory molecule associated with T cell activation. According to one example of this disclosure, anti-ICOS scFv contains the amino acid sequence of SEQ ID NO: 57.

[0052] According to certain embodiments of this disclosure, BE is a peptide recognized by an immunomodulatory receptor or molecule. In one embodiment, BE is an inducible costimulatory molecule ligand (ICOS-L). According to one example of this disclosure, the ICOS-L peptide comprises the amino acid sequence of SEQ ID NO: 59. In another embodiment, BE is cell adhesion molecule 1 (CAMD1). According to one example of this disclosure, the CAMD1 peptide comprises the amino acid sequence of SEQ ID NO: 61. In another embodiment, BE is an IL-2 binder. According to one example of this disclosure, the IL-2 binder comprises the amino acid sequence of SEQ ID NO: 63. As can be understood, BE may vary depending on the desired purpose, e.g., the type of immune cell to be activated. Those skilled in the art can select an appropriate BE for use in the present invention according to the intended purpose.

[0053] The TM / IC domain of the BE-TM / IC polypeptide is characterized by having the intracellular domain of the GPCR (including ICL1, ICL2, ICL3, and the C-terminal region) but without the extracellular domain and transmembrane domain of the GPCR.

[0054] Depending on the desired purpose, the ICL1, ICL2, ICL3 and C-terminal regions of the TM / IC domain may be linked with or without a linker sequence. For example, the TM / IC domain of this disclosure may be in the form of "ICL1-linker-ICL2-linker-ICL3-linker-C-terminal region," where ICL1, ICL2, ICL3 and the C-terminal region are linked via a suitable linker sequence. Alternatively, the TM / IC domain of this disclosure may be in the form of "ICL1-ICL2-ICL3-C-terminal region," where ICL1, ICL2, ICL3 and the C-terminal region are linked without a linker sequence. According to some preferred embodiments, the TM / IC domain of this disclosure consists of the ICL1, ICL2, ICL3 and C-terminal region of a GPCR.

[0055] Preferably, the GPCR is a class A GPCR. For example, CNR2, HCAR2, GPR84, or P2Y14.

[0056] According to some embodiments, the TM / IC domain referred to as “syn CNR2” includes ICL1 (aa 60-71), ICL2 (aa 130-149), ICL3 (aa 215-246), and the C-terminal region (aa 302-360) of CNR2. In one exemplary embodiment, syn CNR2 is in the form of “ICL1-ICL2-ICL3-C-terminal region,” "SSHQLRRKPSYLDRYLCLRYPPSYKALLTRGRKAHQHVASLSGHQDRQVPGMARMRLDVRLAKTRSGEIRSSAHHCLAHWKKCVRGLGSEAKEEAPRSSVTETEADGKITPWPDSRDLDLSDC" It contains the amino acid sequence (SEQ ID NO: 2).

[0057] According to some embodiments, the TM / IC domain referred to as “syn HCAR2” includes ICL1 (aa 55-63), ICL2 (aa 124-142), ICL3 (aa 214-229), and the C-terminal region (aa 295-363) of HCAR2. In one exemplary embodiment, syn HCAR2 is in the form of “ICL1-ICL2-ICL3-C-terminal region,” "HLKSWKSSRDRYFRVVHPHHALNKISNRSLRQRQMDRHAKIKRAYFSSPSFPNFFSTLINRCLQRKMTGEPDNNRSTSVELTGDPNKTRGAPEALMANSGEPWSPSYLGPTSP" It contains the amino acid sequence (SEQ ID NO: 4).

[0058] According to certain embodiments, the TM / IC domain referred to as “syn GPR84” includes ICL1 (aa 48-57), ICL2 (aa 116-144), ICL3 (aa 202-320), and the C-terminal region (aa 374-396) of GPR84. In one exemplary embodiment, syn GPR84 is in the form of “ICL1-ICL2-ICL3-C-terminal region,” "QPKLRTRFNLLGRYLLIAHPKLFPQVFSAKGIVLALVSTHRQVKRAAQALDQYKLRQASIHSNHVARTDEAMPGRFQELDSRLASGGPSEGISSEPVSAATTQTLEGDSSEVGDQINSKRAKQMAEKSPPEASAKAQPIKGARRAPDSSSEFGKVTRMNRQFRQAYGSILKRGPRSFHRLH" It contains the amino acid sequence (SEQ ID NO: 6).

[0059] According to certain embodiments, the TM / IC domain referred to as “syn P2Y14” includes ICL1 (aa 51-55), ICL2 (aa 118-139), ICL3 (aa 210-234), and the C-terminal region (aa 300-338) of P2Y14. In one exemplary embodiment, syn P2Y14 is in the form of “ICL1-ICL2-ICL3-C-terminal region,” "PSSKSDRYYKIVKPLWTSFIQSVSYSKTKKIFKSHLKSSRNSTSVKKKSSRNQPFREILCKKLHIPLKAQNDLDISRIKRGNTTLESTDTL" It contains the amino acid sequence (SEQ ID NO: 8).

[0060] According to some exemplary embodiments, for the purpose of activating T cells, the BE-TM / IC polypeptide of this disclosure comprises an anti-CD3 scFv (e.g., the anti-CD3 scFv of SEQ ID NO: 10) and a TM / IC domain (e.g., any of syn CNR2, syn HCAR2, syn GPR84, and syn P2Y14). Depending on the desired purpose, the anti-CD3 scFv may alternatively comprise a Y177T mutation, e.g., the anti-CD3 scFv (huUCHT1 Y177T) described in U.S. Patent Application No. US 2020 / 0392247. Alternatively, for the purpose of activating NK cells, the BE-TM / IC polypeptide of this disclosure may comprise an anti-NKp46 scFv (e.g., the anti-NKp46 scFv of SEQ ID NO: 26) and a TM / IC domain (e.g., any of syn CNR2, syn HCAR2, syn GPR84, and syn P2Y14).

[0061] Preferably, the BE is linked to the TM / IC domain via a linker. According to some preferred embodiments, the linker comprises an amino acid residue independently selected from the group consisting of glycine (G) and serine (S) residues. In one exemplary embodiment, the linker comprises the amino acid sequence "GGGGS" (SEQ ID NO: 51). The linker may be any linker known to link two peptides. Those skilled in the art can select a suitable linker for linking the BE and TM / IC domain of this disclosure according to the intended purpose.

[0062] As an example, the amino acid sequence of a BE-TM / IC polypeptide called "CD3-syn HCAR2" containing anti-CD3 scFv (SEQ ID NO: 10) and syn HCAR2 (SEQ ID NO: 4) is provided below, where anti-CD3 scFv is marked in italics, syn HCAR2 is in bold, and the linker sequence is placed between them.

[0063] Sequence ID 16 (amino acid sequence of CD3-syn HCAR2) JPEG2026518063000003.jpg49166

[0064] Also disclosed herein is a nucleic acid encoding the BE-TM / IC polypeptide of this disclosure. The nucleic acid comprises a promoter, a BE coding sequence (first coding sequence), and a TM / IC coding sequence (second coding sequence), wherein the BE coding sequence and the TM / IC coding sequence are operably linked to the promoter, and the TM / IC coding sequence is located downstream of the BE coding sequence.

[0065] The promoter may be an inductive promoter or a constitutive promoter. As is known in the art, the term “inductive promoter” refers to a promoter whose performance is conditioned not on endogenous factors, but on artificially controllable environmental conditions and external stimuli. Examples of inductive promoters suitable for driving the expression of the first and second coding sequences of nucleic acids of this disclosure include, but are not limited to, heat shock-inducible promoters, metallothionein promoters, ecdysone-inducible promoters, FKBP dimerization-inducible promoters, Gal4-estrogen receptor fusion protein regulatory promoters, steroid-inducible promoters, streptogramin-responsive promoters, and tetracycline-regulating promoters. With respect to constitutive promoters, these are promoters whose activity is maintained at a relatively constant level in all cells of an organism, with little or no regard to the cellular environmental conditions (e.g., substrate concentration). Non-limiting examples of constitutive promoters suitable for use in nucleic acids of this disclosure include the cytomegalovirus (CMV) promoter, the Roussarcoma virus (RSV) promoter, the Simian virus (SV40) promoter, the mouse mammary tumor virus (MMTV) promoter, the phosphoglycerate kinase (PGK) promoter, the chicken beta-actin promoter, the elongation factor 1-alpha (EF1-α) promoter, the human H1 promoter, and the U6 promoter. According to one exemplary embodiment, the promoter is the EF1-α promoter.

[0066] In some embodiments, the BE coding sequence codes for anti-CD3 scFv, for example, the anti-CD3 scFv of SEQ ID NO: 10, where the anti-CD3 scFv coding sequence contains a nucleotide sequence that is at least 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 9. Preferably, it is at least 90% identical to SEQ ID NO: 9, and more preferably, at least 95% identical to SEQ ID NO: 9. In one example of this disclosure, the anti-CD3 scFv coding sequence contains the nucleotide sequence of SEQ ID NO: 9, i.e., a nucleotide sequence that is 100% identical to SEQ ID NO: 9.

[0067] In some embodiments, the BE coding sequence codes for anti-NKp46 scFv, for example, anti-NKp46 scFv of SEQ ID NO: 26, where the anti-NKp46 scFv coding sequence contains a nucleotide sequence that is at least 85% identical to SEQ ID NO: 25. Preferably, it is at least 90% identical to SEQ ID NO: 25, and more preferably, at least 95% identical to SEQ ID NO: 25. In one example of this disclosure, the anti-NKp46 scFv coding sequence contains the nucleotide sequence of SEQ ID NO: 25.

[0068] In certain embodiments, the BE coding sequence codes for anti-CD2 scFv, for example, the anti-CD2 scFv of SEQ ID NO: 53 or 55, where the anti-CD2 scFv coding sequence includes a nucleotide sequence that is at least 85% identical to SEQ ID NO: 52 or 54. Preferably, it is at least 90% identical to SEQ ID NO: 52 or 54, and more preferably, at least 95% identical to SEQ ID NO: 52 or 54. In one example of the present disclosure, the BE coding sequence encoding the anti-CD2 scFv of SEQ ID NO: 53 includes the nucleotide sequence of SEQ ID NO: 52. In another example of the present disclosure, the BE coding sequence encoding the anti-CD2 scFv of SEQ ID NO: 55 includes the nucleotide sequence of SEQ ID NO: 54.

[0069] In certain embodiments, the BE coding sequence codes for anti-ICOS scFv, for example, the anti-ICOS scFv of SEQ ID NO: 57, where the anti-ICOS scFv coding sequence includes a nucleotide sequence that is at least 85% identical to SEQ ID NO: 56. Preferably, it is at least 90% identical to SEQ ID NO: 56, and more preferably, at least 95% identical to SEQ ID NO: 56. In one example of this disclosure, the BE coding sequence encoding the anti-ICOS scFv of SEQ ID NO: 57 includes the nucleotide sequence of SEQ ID NO: 56.

[0070] In various embodiments, the BE coding sequence codes for the ICOS-L peptide, for example, the ICOS-L peptide of SEQ ID NO: 59, where the ICOS-L coding sequence includes a nucleotide sequence that is at least 85% identical to that of SEQ ID NO: 58. Preferably, it is at least 90% identical to that of SEQ ID NO: 58, and more preferably, at least 95% identical to that of SEQ ID NO: 58. In one example of this disclosure, the BE coding sequence encoding ICOS-L of SEQ ID NO: 59 includes the nucleotide sequence of SEQ ID NO: 58.

[0071] In some embodiments, the BE coding sequence codes for the CAMD1 peptide, for example, the CAMD1 peptide of SEQ ID NO: 61, where the CAMD1 coding sequence includes a nucleotide sequence that is at least 85% identical to SEQ ID NO: 60. Preferably, it is at least 90% identical to SEQ ID NO: 60, and more preferably, at least 95% identical to SEQ ID NO: 60. In one example of this disclosure, the BE coding sequence encoding the CAMD1 peptide of SEQ ID NO: 61 includes the nucleotide sequence of SEQ ID NO: 60.

[0072] In an alternative embodiment, the BE coding sequence codes for an IL-2 binder, for example, the IL-2 binder of SEQ ID NO: 63, where the IL-2 binder coding sequence includes a nucleotide sequence that is at least 85% identical to SEQ ID NO: 62. Preferably, it is at least 90% identical to SEQ ID NO: 62, and more preferably, at least 95% identical to SEQ ID NO: 62. In an example of this disclosure, the BE coding sequence encoding the IL-2 binder of SEQ ID NO: 63 includes the nucleotide sequence of SEQ ID NO: 62.

[0073] The TM / IC coding sequence encodes the TM / IC domain of the present disclosure. According to a particular embodiment, the TM / IC coding sequence encodes syn CNR2 (SEQ ID NO: 2), where the syn CNR2 coding sequence includes a nucleotide sequence that is at least 85% identical to SEQ ID NO: 1. Preferably, it is at least 90% identical to SEQ ID NO: 1, and more preferably, at least 95% identical to SEQ ID NO: 1. In an example of the present disclosure, the syn CNR2 coding sequence includes the nucleotide sequence of SEQ ID NO: 1.

[0074] According to a particular embodiment, the TM / IC coding sequence codes for syn HCAR2 (SEQ ID NO: 4), where the syn HCAR2 coding sequence includes a nucleotide sequence that is at least 85% identical to SEQ ID NO: 3. Preferably, it is at least 90% identical to SEQ ID NO: 3, and more preferably, at least 95% identical to SEQ ID NO: 3. In an example of this disclosure, the syn HCAR2 coding sequence includes the nucleotide sequence of SEQ ID NO: 3.

[0075] According to some embodiments, the TM / IC coding sequence codes for syn GPR84 (SEQ ID NO: 6), where the syn GPR84 coding sequence contains a nucleotide sequence that is at least 85% identical to SEQ ID NO: 5. Preferably, it is at least 90% identical to SEQ ID NO: 5, and more preferably, at least 95% identical to SEQ ID NO: 5. In one example of this disclosure, the syn GPR84 coding sequence contains the nucleotide sequence of SEQ ID NO: 5.

[0076] According to some embodiments, the TM / IC coding sequence codes for syn P2Y14 (SEQ ID NO: 8), where the syn P2Y14 coding sequence includes a nucleotide sequence that is at least 85% identical to SEQ ID NO: 7. Preferably, it is at least 90% identical to SEQ ID NO: 7, and more preferably, at least 95% identical to SEQ ID NO: 7. In one example of this disclosure, the syn P2Y14 coding sequence includes the nucleotide sequence of SEQ ID NO: 7.

[0077] As an example, the nucleotide sequence of the nucleic acid encoding the CD3-syn HCAR2 (SEQ ID NO: 16) of this disclosure is provided below, where the anti-CD3 scFv coding sequence (SEQ ID NO: 9) is marked in italics, the syn HCAR2 coding sequence (SEQ ID NO: 3) is marked in bold, and the linker sequence is placed between them.

[0078] Sequence ID 15 (nucleotide sequence of the nucleic acid encoding CD3-syn HCAR2) JPEG2026518063000004.jpg136166

[0079] The sequences of the BEs, TM / IC domains, and BE-TM / IC constructs of this disclosure described in Section (i) are summarized in Table 1.

[0080] Table 1. Sequence summaries of BEs, TM / IC domains and BE-TM / IC constructs of this disclosure. [Table 1]

[0081] (ii) 2xBE-TM / IC polypeptide and the nucleic acid encoding it For the purpose of inducing an antigen-targeting effect, the BE-TM / IC polypeptide described in Section (i) may further contain a second BE (e.g., an anti-antigen scFv), i.e., in the form of a 2xBE-TM / IC domain. Accordingly, a second aspect of the present disclosure relates to the 2xBE-TM / IC polypeptide and the nucleic acids encoding the 2xBE-TM / IC polypeptide.

[0082] Herein, we refer to Figure 2, which shows a schematic diagram of a 2xBE-TM / IC polypeptide according to one embodiment of the present disclosure.Structurally, the 2xBE-TM / IC polypeptide comprises a first BE (BE1), a second BE (BE2) ligated to the C-terminus of the first BE (BE1), and a TM / IC domain ligated to the C-terminus of the second BE (BE2). Preferably, the first BE is an anti-antigen scFv (first scFv, e.g., anti-TAA scFv), and the second BE is a cell-targeting scFv (second scFv, e.g., anti-CD3 scFv, anti-NKp46 scFv, anti-CD2 scFv, or anti-ICOS scFv) or a cell-targeting peptide (e.g., ICOS-L, CAMD1, or IL-2 binder). As described in Section (i) above, the anti-antigen scFv and the cell-targeting scFv each contain a heavy chain variable domain (VH) and a light chain variable domain (VL), respectively, and the TM / IC domain contains the ICL1, ICL2, ICL3 and C-terminal region (C) of the GPCR in that order from the N-terminus to the C-terminus.

[0083] Optionally, the anti-antigen scFv, cell-targeting scFv / peptide, and TM / IC domain may be linked with or without a linker sequence. Preferably, the anti-antigen scFv and cell-targeting scFv / peptide are linked via a first linker sequence, and the cell-targeting scFv / peptide and TM / IC domain are linked via a second linker sequence. As understood, the first and second linker sequences may be the same or different.

[0084] According to some preferred embodiments of this disclosure, the anti-antigen scFv is anti-TAA scFv. Depending on the intended purpose, TAA may be any antigen overexpressed or associated with tumor / cancer cells, such as alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), glycan antigen 19-9 (CA 19-9), epidermal growth factor receptor (HER1), HER2, HER3, HER4, epithelial tumor antigen (ETA), folate receptor alpha (FRa), ganglioside GD2, Globo H, melanoma-associated antigen (MAGE), mucin 1 (MUC 1), mucin 16 (MUC 16, also known as "ovarian cancer-associated tumor marker CA125"), mesothelin, prostate-specific membrane antigen (PSMA), prostate stem cell antigen (PSCA), receptor tyrosine kinase-like orphan receptor 1 (ROR1), glypican 3 (GPC3), tumor-associated glycoprotein 72 (TAG72), or claudin-18 isoform 2 (CLDN18.2). In one embodiment, the anti-antigen scFv is anti-FRa scFv. According to one example of this disclosure, anti-FRa scFv includes the amino acid sequence of SEQ ID NO: 12. In another embodiment, the anti-antigen scFv is anti-HER2 scFv. According to one example of this disclosure, anti-HER2 scFv includes the amino acid sequence of SEQ ID NO: 14.

[0085] According to certain preferred embodiments, the cell-targeting scFv / peptides are specific to receptors, co-receptors, costimulatory molecules, or cell adhesion molecules of immune cells (e.g., T cells, NK cells, B cells, basophils, eosinophils, DCs, or neutrophils). Examples include anti-CD3 scFv, anti-NKp46 scFv, anti-CD2 scFv, anti-ICOS scFv, ICOS-L peptide, CAMD1 peptide, or IL-2 binder. Those skilled in the art can select an appropriate scFv / peptide for use in the present invention according to the intended purpose, e.g., the immune cells to be activated.

[0086] As described above, the TM / IC domain is characterized by having the intracellular domain of the GPCR (including ICL1, ICL2, ICL3 and the C-terminal region) but not the extracellular domain and transmembrane domain of the GPCR, where the ICL1, ICL2, ICL3 and the C-terminal region of the TM / IC domain may or may not be linked with a linker sequence. Preferably, the TM / IC domain of this disclosure consists of the ICL1, ICL2, ICL3 and the C-terminal region of the GPCR. The GPCR is preferably a class A GPCR, such as CNR2, HCAR2, GPR84, or P2Y14.

[0087] According to some embodiments of the present disclosure, the TM / IC domain may be any of syn CNR2 (SEQ ID NO: 2), syn HCAR2 (SEQ ID NO: 4), syn GPR84 (SEQ ID NO: 6), or syn P2Y14 (SEQ ID NO: 8), as described in Section (i) of the present disclosure.

[0088] According to some embodiments, the 2BE-TM / IC polypeptide includes the form of a 2xscFv-TM / IC polypeptide, i.e., two scFvs linked to the N-terminus of the TM / IC domain. According to an exemplary embodiment of the present disclosure, the 2xscFv-TM / IC polypeptide referred to as "FRa-CD3-syn HCAR2" includes, in this order from the N-terminus to the C-terminus, an anti-FRa scFv (e.g., the anti-FRa scFv of SEQ ID NO: 12), an anti-CD3 scFv (e.g., the anti-CD3 scFv of SEQ ID NO: 10), and the syn HCAR2 of the present disclosure. In one exemplary embodiment, the FRa-CD3-syn HCAR2 polypeptide includes the amino acid sequence of SEQ ID NO: 18.

[0089] According to some embodiments, the 2xscFv-TM / IC polypeptide referred to as “FRa-CD3-syn CNR2” comprises, in this order from the N-terminus to the C-terminus, anti-FRa scFv (e.g., anti-FRa scFv of SEQ ID NO: 12), anti-CD3 scFv (e.g., anti-CD3 scFv of SEQ ID NO: 10), and syn CNR2 of the present disclosure. In one exemplary embodiment, the FRa-CD3-syn CNR2 polypeptide comprises the amino acid sequence of SEQ ID NO: 20.

[0090] According to certain embodiments, the 2xscFv-TM / IC polypeptide referred to as "FRa-CD3-syn P2Y14" comprises, in this order from the N-terminus to the C-terminus, anti-FRa scFv (e.g., anti-FRa scFv of SEQ ID NO: 12), anti-CD3 scFv (e.g., anti-CD3 scFv of SEQ ID NO: 10), and syn P2Y14 of the present disclosure. In one exemplary embodiment, the FRa-CD3-syn P2Y14 polypeptide comprises the amino acid sequence of SEQ ID NO: 22.

[0091] According to alternative embodiments, the 2xscFv-TM / IC polypeptide referred to as "HER2-CD3-syn GPR84" comprises, in this order from the N-terminus to the C-terminus, anti-HER scFv (e.g., anti-HER2 scFv of SEQ ID NO: 14), anti-CD3 scFv (e.g., anti-CD3 scFv of SEQ ID NO: 10), and syn GPR84 of the present disclosure. In one exemplary embodiment, the HER2-CD3-syn GPR84 polypeptide comprises the amino acid sequence of SEQ ID NO: 24.

[0092] Also disclosed herein are nucleic acids encoding the 2xBE-TM / IC polypeptide of the present disclosure. The nucleic acid comprises a promoter, a first coding sequence encoding a first BE (i.e., an anti-antigen scFv, e.g., anti-TAA scFv), a second coding sequence encoding a second BE (i.e., a cell-targeting scFv / peptide, e.g., CD3 scFv or anti-NKp46 scFv), and a third coding sequence encoding the TM / IC domain of the present disclosure, wherein the first, second, and third coding sequences are operably linked to the promoter, the second coding sequence is located downstream of the first coding sequence, and the third coding sequence is located downstream of the second coding sequence.

[0093] As described above, the promoter may be an inductive promoter (e.g., a heat shock-inducible promoter, a metallothionein promoter, an ecdysone-inducible promoter, an FKBP dimerization-inducible promoter, a Gal4-estrogen receptor fusion protein regulatory promoter, a steroid-inducible promoter, a streptogramin-responsive promoter, or a tetracycline-regulating promoter) or a constitutive promoter (e.g., a CMV promoter, an RSV promoter, an SV40 promoter, an MMTV promoter, a PGK promoter, an EF1-α promoter, a human H1 promoter, or a U6 promoter). According to one exemplary embodiment, the promoter is the EF1-α promoter.

[0094] According to some embodiments, the anti-antigen scFv is anti-FRa scFv, where the anti-FRa scFv coding sequence includes a nucleotide sequence that is at least 85% identical to SEQ ID NO: 11. Preferably, it is at least 90% identical to SEQ ID NO: 11, and more preferably, at least 95% identical to SEQ ID NO: 11. In one particular example, the anti-FRa scFv coding sequence includes the nucleotide sequence of SEQ ID NO: 11.

[0095] According to a particular embodiment, the anti-antigen scFv is anti-HER2 scFv, where the anti-HER2 scFv coding sequence includes a nucleotide sequence that is at least 85% identical to SEQ ID NO: 13. Preferably, it is at least 90% identical to SEQ ID NO: 13, and more preferably, at least 95% identical to SEQ ID NO: 13. In one particular example, the anti-HER2 scFv coding sequence includes the nucleotide sequence of SEQ ID NO: 13.

[0096] According to some embodiments, the nucleic acid encodes FRa-CD3-syn HCAR2 (SEQ ID NO: 18) and contains a nucleotide sequence that is at least 85% identical to SEQ ID NO: 17. Preferably, it is at least 90% identical to SEQ ID NO: 17, and more preferably, at least 95% identical to SEQ ID NO: 17. According to one exemplary embodiment, the nucleic acid encoding FRa-CD3-syn HCAR2 contains the nucleotide sequence of SEQ ID NO: 17.

[0097] According to some embodiments, the nucleic acid encodes FRa-CD3-syn CNR2 (SEQ ID NO: 20) and contains a nucleotide sequence that is at least 85% identical to SEQ ID NO: 19. Preferably, it is at least 90% identical to SEQ ID NO: 19, and more preferably, at least 95% identical to SEQ ID NO: 19. According to one exemplary embodiment, the nucleic acid encoding FRa-CD3-syn CNR2 contains the nucleotide sequence of SEQ ID NO: 19.

[0098] According to a particular embodiment, the nucleic acid encodes FRa-CD3-syn P2Y14 (SEQ ID NO: 22) and contains a nucleotide sequence that is at least 85% identical to SEQ ID NO: 21. Preferably, it is at least 90% identical to SEQ ID NO: 21, and more preferably, at least 95% identical to SEQ ID NO: 21. According to one exemplary embodiment, the nucleic acid encoding FRa-CD3-syn P2Y14 contains the nucleotide sequence of SEQ ID NO: 21.

[0099] According to another embodiment, the nucleic acid encodes HER2-CD3-syn GPR84 (SEQ ID NO: 24) and contains a nucleotide sequence that is at least 85% identical to SEQ ID NO: 23. Preferably, it is at least 90% identical to SEQ ID NO: 23, and more preferably, at least 95% identical to SEQ ID NO: 23. According to one exemplary embodiment, the nucleic acid encoding HER2-CD3-syn GPR84 contains the nucleotide sequence of SEQ ID NO: 23.

[0100] The sequences of the 2xBE-TM / IC constructs of this disclosure described in Section (ii) are summarized in Table 2.

[0101] Table 2. Summary of the arrangement of the 2xBE-TM / IC constructs in this disclosure. [Table 2]

[0102] (iii) 2xBE-multiTM / IC polypeptide and nucleic acid encoding it In addition to the TM / IC domains described in sections (i) and (ii) above, which include ICL1, ICL2, ICL3, and the C-terminal region of a single GPCR, the inventors of the present disclosure have further discovered that combinations of two or three TM / IC domains can achieve the same bifunctional effect, i.e., TM / IC domains can cooperate to fix polypeptides to the cell membrane and act as bifunctional domains that mediate intracellular signaling. Accordingly, a third aspect of the present disclosure relates to 2xBE-multiTM / IC polypeptides and nucleic acids encoding 2xBE-multiTM / IC polypeptides.

[0103] The structure of the 2xBE-multiTM / IC polypeptide is very similar to that of the 2xBE-TM / IC polypeptide described in Section (ii) above, except that instead of one TM / IC domain, two or three TM / IC domains work together to function as a bifunctional domain.

[0104] Figure 3A provides a schematic diagram of a 2xBE-multiTM / IC according to one embodiment of the present disclosure. As shown in Figure 3A, the 2xscFv-multiTM / IC comprises two TM / IC domains, where the first TM / IC domain (1 ST The TM / IC domain includes ICL1(I1), ICL2(I2), ICL3(I3), and the C-terminal region (C) of the first GPCR, and the second TM / IC domain (2 nd The TM / IC domain includes ICL1 (I1), ICL2 (I2), ICL3 (I3), and the C-terminal region (C) of the second GPCR. Preferably, the first and second GPCRs are class A GPCRs, for example, CNR2, HCAR2, GPR84, and P2Y14, and the second GPCR is different from the first GPCR. Neither the first nor the second TM / IC domain contains the extracellular and transmembrane domains of the GPCRs (i.e., the first and second GPCRs). As described above, the ICL1, ICL2, ICL3, and C-terminal regions of the first and second TM / IC domains may be linked with or without a linker sequence. Preferably, the first TM / IC domain consists of ICL1, ICL2, ICL3 and the C-terminal region of the first GPCR, and the second TM / IC domain consists of ICL1, ICL2, ICL3 and the C-terminal region of the second GPCR.

[0105] In certain preferred embodiments, the first and second TM / IC domains are linked to each other without a linker sequence. Alternatively, the first and second TM / IC domains may be linked via a suitable linker sequence.

[0106] According to one embodiment, the 2xBE-multiTM / IC polypeptide comprises syn CNR2 and syn P2Y14 (in the form of the 2xTM / IC domain). In this embodiment, the 2xTM / IC domain is referred to as "syn CP" and contains the following amino acid sequence. JPEG2026518063000007.jpg27166 (Sequence ID 30) Here, the amino acid sequence of syn CNR2 (sequence number 2) is marked in italics, and the amino acid sequence of syn P2Y14 (sequence number 8) is marked in bold.

[0107] According to another embodiment, the 2xBE-multiTM / IC polypeptide comprises syn CNR2 and syn HCAR2 (in the form of the 2xTM / IC domain). In this embodiment, the 2xTM / IC domain is referred to as "syn CH" and contains the following amino acid sequence. JPEG2026518063000008.jpg27166 (Sequence ID 32) Here, syn CNR2 (sequence number 2) is marked in italics, and syn HCAR2 (sequence number 4) is marked in bold.

[0108] Figure 3B provides a schematic diagram of a 2xBE-multiTM / IC according to another embodiment of the present disclosure. As shown in Figure 3B, the 2xBE-multiTM / IC comprises three TM / IC domains, where the first TM / IC domain (1st TM / IC domain) comprises ICL1(I1), ICL2(I2), ICL3(I3), and the C-terminal region (C) of the first GPCR; the second TM / IC domain (2nd TM / IC domain) comprises ICL1(I1), ICL2(I2), ICL3(I3), and the C-terminal region (C) of the second GPCR; and the third TM / IC domain (3rd TM / IC domain) comprises ICL1(I1), ICL2(I2), ICL3(I3), and the C-terminal region (C) of the third GPCR. Preferably, the first, second, and third GPCRs are each a class A GPCR, for example, CNR2, HCAR2, GPR84, and P2Y14, and the first to third GPCRs are distinct from each other. None of the first to third TM / IC domains contain the extracellular domain and transmembrane domain of the GPCR. As described above, the ICL1, ICL2, ICL3, and C-terminal regions of the first, second, and third TM / IC domains may or may not be linked with a linker sequence. Preferably, the first TM / IC domain consists of the ICL1, ICL2, ICL3, and C-terminal region of the first GPCR, the second TM / IC domain consists of the ICL1, ICL2, ICL3, and C-terminal region of the second GPCR, and the third TM / IC domain consists of the ICL1, ICL2, ICL3, and C-terminal region of the third GPCR.

[0109] In certain preferred embodiments, the first to third TM / IC domains are linked without a linker sequence (i.e., "1 st TM / IC Domain-2 nd TM / IC Domain-3 rd (In the form of a "TM / IC domain"). Alternatively, the first to third TM / IC domains may be linked via an appropriate linker sequence, for example, "1 st TM / IC Domain-Linker-2 nd TM / IC Domain-Linker-3rd "TM / IC domain", "1" st TM / IC domain - 2 nd TM / IC domain - linker - 3 rd "TM / IC domain" or "1" st TM / IC domain - linker - 2 nd TM / IC domain - 3 rd It is in the form of "TM / IC domain".

[0110] According to one embodiment, the 2xBE - multiTM / IC polypeptide comprises syn CNR2, syn P2Y14, and syn HCAR2 (in the form of a 3xTM / IC domain). This 3xTM / IC domain is referred to as "syn CPH" and comprises the following amino acid sequence. JPEG2026518063000009.jpg41166 (SEQ ID NO: 28) Here, the syn CNR2 sequence (SEQ ID NO: 2) is marked in italics, the syn HCAR2 sequence (SEQ ID NO: 4) is marked in bold, and the syn P2Y14 sequence (SEQ ID NO: 8) is placed between the syn CNR2 and syn HCAR2 sequences.

[0111] Depending on the desired purpose, the 2xTM / IC domain or 3xTM / IC domain may be linked to one or two scFv / peptides (e.g., an anti - antigen scFv and / or a cell - targeting scFv / peptide) as described above, thereby forming an scFv / peptide - 2xTM / IC polypeptide (comprising one scFv / peptide and two TM / IC domains), a 2xscFv / peptide - 2xTM / IC polypeptide (comprising two scFv / peptides and two TM / IC domains), an scFv / peptide - 3xTM / IC polypeptide (comprising one scFv / peptide and three TM / IC domains), or a 2xscFv / peptide - 3xTM / IC polypeptide (comprising two scFv / peptides and three TM / IC domains).

[0112] In one embodiment, an scFv-3x™ / IC polypeptide referred to as "HER2-CD3-syn CPH" is provided, which comprises anti-HER2 scFv, anti-CD3 scFv, and syn CPH in this order from the N-terminus to the C-terminus. According to this embodiment, the HER2-CD3-syn CPH polypeptide comprises the amino acid sequence of SEQ ID NO: 42.

[0113] In another embodiment, an scFv-3x™ / IC polypeptide referred to as "FRa-CD3-syn CPH" is provided, which comprises, in this order from the N-terminus to the C-terminus, anti-FRa scFv, anti-CD3 scFv, and syn CPH. According to this embodiment, the FRa-CD3-syn CPH polypeptide comprises the amino acid sequence of SEQ ID NO: 44.

[0114] In yet another embodiment, an scFv-2x™ / IC polypeptide referred to as "FRa-CD3-syn CP" is provided, which comprises, in this order from the N-terminus to the C-terminus, anti-FRa scFv, anti-CD3 scFv, and syn CP. According to this embodiment, the FRa-CD3-syn CP polypeptide comprises the amino acid sequence of SEQ ID NO: 48.

[0115] In yet another embodiment, an scFv-2x™ / IC polypeptide referred to as "FRa-CD3-syn CH" is provided, which comprises, in this order from the N-terminus to the C-terminus, anti-FRa scFv, anti-CD3 scFv, and syn CH. According to this embodiment, the FRa-CD3-syn CH polypeptide comprises the amino acid sequence of SEQ ID NO: 50.

[0116] The Disclosure also provides nucleic acids encoding 2xBE-multiTM / IC polypeptides. According to certain embodiments of the Disclosure, the nucleic acid comprises a promoter, a first coding sequence encoding a first BE (i.e., an anti-antigen scFv; e.g., anti-TAA scFv), a second coding sequence encoding a second BE (i.e., a cell-targeting scFv / peptide; e.g., anti-CD3 scFv or anti-NKp46 scFv), and a third coding sequence encoding a multiTM / IC domain, wherein the first, second, and third coding sequences are operably linked to the promoter, the second coding sequence is located downstream of the first coding sequence, and the third coding sequence is located downstream of the second coding sequence.

[0117] The promoter, the first code sequence, and the second code sequence are very similar to those of the 2xBE-TM / IC construct described in Section (ii) of this disclosure. Therefore, for brevity, a detailed description is omitted.

[0118] The difference between the third coding sequence of a 2xBE-multiTM / IC construct and the third coding sequence of a 2xBE-TM / IC construct is that the third coding sequence of a 2xBE-multiTM / IC construct includes multiple (two or three) coding segments for expressing multiple (two or three) TM / IC domains. For example, the third coding sequence may include two coding segments expressing syn CNR2 and syn P2T14 (i.e., syn CP), or two coding segments expressing syn CNR2 and syn HCAR2 (i.e., syn CH), respectively. Alternatively, the third coding sequence may include three coding segments expressing syn CNR2, syn P2T14, and syn HCAR2 (i.e., syn CPH), respectively.

[0119] According to some embodiments, the third coding sequence codes for syn CP (SEQ ID NO: 30) and includes a nucleotide sequence that is at least 85% identical to SEQ ID NO: 29. Preferably, it is 90% identical to SEQ ID NO: 29, more preferably 100% identical to SEQ ID NO: 29, and most preferably 100% identical to SEQ ID NO: 29. According to some embodiments, the third coding sequence codes for syn CH (SEQ ID NO: 32) and includes a nucleotide sequence that is at least 85% identical to SEQ ID NO: 31. Preferably, it is 90% identical to SEQ ID NO: 31, more preferably 100% identical to SEQ ID NO: 31, and most preferably 100% identical to SEQ ID NO: 31. According to another embodiment, the third coding sequence codes for syn CPH (SEQ ID NO: 28) and includes a nucleotide sequence that is at least 85% identical to SEQ ID NO: 27. Preferably, it is 90% identical to SEQ ID NO: 27, more preferably 100% identical to SEQ ID NO: 27, and most preferably 100% identical to SEQ ID NO: 27.

[0120] In one example, the nucleic acid encoding the HER2-CD3-syn CPH polypeptide (SEQ ID NO: 42) contains the nucleotide sequence of SEQ ID NO: 41. In another example, the nucleic acid encoding the FRa-CD3-syn CPH polypeptide (SEQ ID NO: 44) contains the nucleotide sequence of SEQ ID NO: 43. In yet another example, the nucleic acid encoding the FRa-CD3-syn CP polypeptide (SEQ ID NO: 48) contains the nucleotide sequence of SEQ ID NO: 47. In one particular example, the nucleic acid encoding the FRa-CD3-syn CH polypeptide (SEQ ID NO: 50) contains the nucleotide sequence of SEQ ID NO: 49.

[0121] The sequences of the 2xTM / IC domains, 3xTM / ID domains, 2xBE-2xTM / IC constructs, and 2xBE-3xTM / IC constructs described in Section (iii) are summarized in Table 3.

[0122] Table 3. Sequence summaries of the 2xTM / IC domains, 2xBE-2xTM / IC constructs, and 2xBE-3xTM / IC constructs of this disclosure. [Table 3]

[0123] (iv) Recombinant polypeptide-expressing immune cells and their use in the treatment of cancer A fourth aspect of the present disclosure relates to engineered immune cells expressing any recombinant polypeptide of the present disclosure (i.e., any of the BE-TM / IC polypeptide, 2xBE-TM / IC polypeptide, and 2xBE-multiTM / IC polypeptide described in sections (i) to (iii) of the present disclosure, respectively).

[0124] According to certain embodiments, modified immune cells of the Disclosure are prepared by introducing the nucleic acid of the Disclosure into immune cells via a suitable method, and subsequently culturing the introduced immune cells under suitable conditions (e.g., 37°C) for a certain period (e.g., 48-72 hours or longer), thereby enabling the introduced immune cells to express the polypeptide of the Disclosure on their surface. Methods for introducing nucleic acids into cells for expression purposes are known to those skilled in the art, and include, for example, viral or non-viral methods. Specifically, in viral methods, the nucleic acid encoding the protein of interest (e.g., the nucleic acid encoding the BE-TM / IC polypeptide, 2xBE-TM / IC polypeptide, or 2xBE-multiTM / IC polypeptide of the Disclosure) is introduced into immune cells via a viral vector. Exemplary viral vectors suitable for this purpose include, but are not limited to, Sendai virus, adenovirus, adeno-associated virus (AAV), retrovirus, lentivirus, and Sindbisvirus. In nonviral methods, nucleic acids encoding the target protein are introduced into immune cells via nonviral techniques such as mRNA transfection, miRNA transfection or infection, transposons (e.g., PiggyBac and Sleeping Beauty), plasmid transfection, liposome magnetfection, minicircle transfection, electroporation, or other known methods for introducing nucleic acids into host cells.

[0125] The immune cells may be T cells, NK cells, B cells, basophils, eosinophils, DCs, or neutrophils, depending on their intended purpose. To be clear, the cell-targeting scFv of the recombinant polypeptides of this disclosure may vary depending on the type of immune cell. For example, if the immune cell is a T cell, the cell-targeting scFv is anti-CD3 scFv. Or, if the immune cell is an NK cell, the cell-targeting scFv is anti-NKp46 scFv.

[0126] According to certain embodiments of this disclosure, modified immune cells expressing anti-TAA scFv and cell-targeting scFv / peptides (e.g., anti-CD3 scFv, anti-NKp46 scFv, anti-CD2 scFv, anti-ICOS scFv, ICOS-L peptide, CAMD1 peptide, or IL-2 binder) can target and kill cancer cells expressing TAA.

[0127] In some exemplary embodiments, the modified immune cells are modified T cells. Compared to modified T cells using a chimeric antigen receptor (CAR-T) or a T cell antigen coupler (TAC-T), the modified immune cells of this disclosure are characterized by lacking any of the following components: (1) a CAR-T costimulatory domain (e.g., CD28 and 4-1BB), (2) a CAR-T activation domain (e.g., CD3-zeta ITAM), (3) a CAR-T or TAC-T transmembrane domain (e.g., a transmembrane domain of CD28, CD8, or CD4), and (4) an intracellular CD4 or CD8 domain for delivering signals to the CD3 complex as TAC-T does.

[0128] Another aspect of this disclosure provides a method for treating cancer in a subject. This method includes administering an effective amount of the modified immune cells of this disclosure to the subject.

[0129] According to certain exemplary embodiments of this disclosure, the effective amount of the modified immune cells of this disclosure is approximately 1 × 10⁶ per single dose. 5 ~1 × 10 10These are individual cells, for example, 1 × 10⁶ 5 ,2×10 5 ,3×10 5 ,4×10 5 ,5×10 5 ,6×10 5 ,7×10 5 ,8×10 5 ,9×10 5 ,1×10 6 ,2×10 6 ,3×10 6 ,4×10 6 ,5×10 6 ,6×10 6 ,7×10 6 ,8×10 6 ,9×10 6 ,1×10 7 ,2×10 7 ,3×10 7 ,4×10 7 ,5×10 7 ,6×10 7 ,7×10 7 ,8×10 7 ,9×10 7 ,1×10 8 ,2×10 8 ,3×10 8 ,4×10 8 ,5×10 8 ,6×10 8 ,7×10 8 ,8×10 8 ,9×10 8 ,1×10 9 ,2×10 9 ,3×10 9 ,4×10 9 ,5×10 9 ,6×10 9 ,7×10 9 ,8×10 9 ,9×10 9 , or 1 × 10 10 This is the dose per cell.

[0130] Depending on the intended purpose, immune cells and modified immune cells derived therefrom may be derived from the subject being treated / administered (i.e., autotransplantation), from another subject of the same species (i.e., allogeneic transplantation), or from a subject of a different species (i.e., xenotransplantation). Preferably, the transplantation is autotransplantation or allogeneic transplantation. If the transplantation is allogeneic transplantation, the method may further include a step of administering immunosuppressive therapy to the subject prior to, simultaneously with, or after the administration of the modified immune cells of this disclosure in order to suppress the subject's immune response to the allogeneic modified immune cells. Immunosuppression may be achieved by any agent and / or method known to those skilled in the art for preventing rejection, for example, by gamma irradiation or administration of immunosuppressants.

[0131] Non-limiting examples of cancers treatable by the immune cells and / or methods of this disclosure include breast cancer, gastric cancer, colorectal cancer, gallbladder cancer, prostate cancer, cervical cancer, ovarian cancer, chronic or acute lymphoblastic leukemia, bladder cancer, kidney cancer, hepatocellular carcinoma, head and neck squamous cell carcinoma, glioblastoma, esophageal cancer, pancreatic cancer, oral cancer, lung cancer, melanoma, and lymphoma.

[0132] The immune cells of this disclosure may be administered to a subject via an appropriate route, such as intratumor, intravenous, intra-arterial, or intraperitoneal injection.

[0133] In all embodiments of this disclosure, the subject is a mammal, such as a human, mouse, rat, rabbit, or monkey. Preferably, the subject is a human.

[0134] The following examples are provided to illustrate specific aspects of the present invention and to assist those skilled in the art in carrying it out. These examples should not be considered to limit the scope of the present invention in any way. Without further detail, those skilled in the art will be able to make the most of the present invention based on the description herein. All publications cited herein are incorporated herein by reference in their entirety. [Examples]

[0135] Materials and methods Nucleic acid construction

[0136] In this disclosure, one scFv-TM / IC construct (referred to as the "CD3-syn HCAR2 construct"), four 2xscFv-TM / IC constructs (referred to as the "FRa-CD3-syn HCAR2 construct", the "FRa-CD3-syn CNR2 construct", the "HER2-CD3-syn GPR84 construct", and the "FRa-CD3-syn P2Y14 construct", respectively), and four 2xscFv-multiTM / IC constructs (referred to as the "HER2-CD3-syn CPH construct", the "FRa-CD3-syn CPH construct", the "FRa-CD3-syn CP construct", and the "FRa-CD3-syn CH construct", respectively) are provided.

[0137] In the preparation of scFv-TM / IC constructs, each scFv DNA sequence, including the selected TM / IC DNA sequence, was optimized based on the use of human DNA codons. The scFv-TM / IC DNA sequences were designed entirely de novo and subsequently synthesized in vitro. Third-generation lentiviral backbones were digested using desired restriction enzymes and used as vectors. The synthetic DNA was amplified by polymerase chain reaction (PCR) to produce inserts, which were incorporated into the vector using the Gibson assembly® method. The DNA was transformed into Stbl3 competent E. coli, cultured at 30°C, and the resulting plasmids were validated using Sanger sequencing.

[0138] In the preparation of the 2xscFv-TM / IC construct, two selected scFv sequences were cloned into the 2xscFv construct via de novo ligation, employing optimized DNA codons based on the use of human DNA codons. The de novo-designed 2xscFv construct was subsequently synthesized in vitro. The desired scFv-TM / IC lentiviral backbone construct was selectively cleaved using the desired restriction enzyme, and the scFv region was removed to use as a vector. The 2xscFv insert DNA was PCR-amplified as an insert from the synthetic DNA. The resulting PCR-amplified insert was incorporated into the vector using the Gibson assembly® method. The DNA was transformed into Stbl3 competent E. coli and cultured at 30°C, and the resulting plasmid was confirmed using Sanger sequencing.

[0139] To prepare the 2xscFv-multiTM / IC construct, selected TM / IC sequences were cloned into the multiTM / IC construct via de novo ligation, incorporating optimized DNA codons based on human DNA codon usage. The de novo-designed multiTM / IC construct was subsequently synthesized in vitro. The third-generation lentiviral backbone was selectively cleaved using desired restriction enzymes and used as a vector. The 2xscFv insert DNA was amplified from the 2xscFv-TM / IC construct via PCR, and the multiTM / IC DNA was similarly amplified by PCR. The resulting PCR-amplified insert was incorporated into the vector using the Gibson assembly® method. The DNA was transformed into Stbl3 competent E. coli and cultured at 30°C, and the resulting plasmid was confirmed using Sanger sequencing.

[0140] cell culture Human T-cell lymphoblastic lymphoma cell line SupT1 was cultured in RPMI-1640 basal medium supplemented with 10% fetal bovine serum (FBS) and 1x penicillin-streptomycin. Human primary T cells were cultured in RPMI-1640 basal medium supplemented with 10% fetal bovine serum (FBS), 1x beta-mercaptoethanol, 1x sodium pyruvate, and 1x non-essential amino acids (NEAA). Cells were cultured at 37°C in a 5% CO2 humidified incubator.

[0141] Human high-grade serous ovarian adenocarcinoma cell lines Ovcar3 or Ovcar4 were cultured in RPMI-1640 basal medium supplemented with 20% (Ovcar3) or 10% (Ovcar4) FBS and 1x penicillin-streptomycin. Cells were cultured at 37°C in a 5% CO2 humidified incubator.

[0142] Preparation of lentiviruses The scFv-TM / IC, 2xscFv-TM / IC, and 2xscFv-multiTM / IC constructs were each prepared as lentiviral particles and transfected into human SupT1 cells and human primary T cells. Briefly, 12 million 293 T cells (ATCC,CRL-3216) were seeded into 15 cm plates. Overnight, packaging plasmids (containing plasmids encoding Rev response element (RRE; 9.375 μg), REV (9.375 μg), and vesicular stomatitis virus glycoprotein (VSV-G; 3.75 μg), respectively) and lentiviral backbone plasmids (i.e., scFv-TM / IC, 2xscFv-TM / IC, or 2xscFv-multiTM / IC constructs; 7.5 μg) were co-transfected into 293 T cells with Lipofectamine® 3000 according to the manufacturer's protocol. The supernatant was collected after 48 hours and filtered through a 0.45 μm filter. The resulting product was concentrated, and lentiviral titer was measured by evaluating transfection efficiency in SupT1 cells. Briefly, lentiviral particles were added to SupT1 cells at a concentration of 20,000 cells per well, following a 3-fold serial dilution protocol from 1:27 to 1:2187 dilutions. Protein L expression was examined 3 days after viral introduction. Cells were incubated at 37°C in a 5% CO2 environment.

[0143] Preparation of modified T cells Human primary T cells were cultured in RPMI-1640 basal medium supplemented with 10% FBS, 1x beta-mercaptoethanol, 1x NEAA, 1x Glutamax®, 1x sodium pyruvate, and 10 mM N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES). Human primary T cells were activated on thawing day (day 0) using Dynabeads® Human T-Activator CD3 / CD28. On day 1, viruses (MOI = 1-7.5 based on SupT1 titer) were added along with lenti-boost® according to the manufacturer's protocol. Protein L expression was examined on day 7, and cells were harvested on day 9. Subsequently, the cells were cultured at 37°C in a 5% CO2 incubator throughout the entire process and then stored in liquid nitrogen.

[0144] In this study, three modified T cell groups were used as a control group. These included: (a) FRa_CAR-T: A modified T cell expressing the FRa chimeric antigen receptor (FRa_CAR), whose structure includes an anti-FRa scFv, a hinge domain, a transmembrane domain, a CD28 costimulatory domain, and a CD3 zeta ITAM motif (from N-terminus to C-terminus), the FRa chimeric antigen receptor containing the amino acid sequence of SEQ ID NO: 34, and encoded by the nucleotide sequence of SEQ ID NO: 33. (b) HER2_TAC-T: Modified T cells expressing a TAC containing anti-HER2 scFv, anti-CD3 scFv, and CD4 intracellular domains, where the TAC contains the amino acid sequence of SEQ ID NO: 40 and is encoded by the nucleotide sequence of SEQ ID NO: 39. (c)FRa-TAC-T: Modified T cells expressing a TAC containing anti-FRa scFv, anti-CD3 scFv, and CD4 intracellular domains, where the TAC contains the amino acid sequence of SEQ ID NO: 46 and is encoded by the nucleotide sequence of SEQ ID NO: 45.

[0145] Flow cytometry Expression levels of the scFv-TM / IC, 2xscFv-TM / IC, and 2xscFv-multiTM / IC polypeptides of this disclosure on the cell membrane of human SupT1 cells and primary T cells were determined by flow cytometry. Briefly, modified SupT1 / primary human cells were washed twice with phosphate-buffered saline (PBS) and then stained with a viability dye at 4°C for 30 minutes. Subsequently, the cells were washed twice with PBS and then stained with phycoerythrin (PE)-conjugated Protein L antibody at 4°C for 45 minutes. The cells were then washed twice with PBS and resuspended in 200 μl of PBS before evaluation by flow cytometry. Data were analyzed using software. Construct surface expression was determined by gating the PE-positive population within a live cell gate.

[0146] Animal experiments NCG mice were used in this study to evaluate the antitumor effects of the modified T cells of this disclosure. Briefly, 5 × 10¹⁶ cells in 100 μl of PBS. 6 OVCAR-3 cells were subcutaneously inoculated into the flank of each NCG mouse. Tumor size was recorded twice a week. The volume of tumors transplanted subcutaneously in vivo was determined using a digital caliper. Tumor volume was calculated using the modified ellipsoid formula, 1 / 2 × (length × width × width). The average tumor size was approximately 100 mm. 3 When this is reached, modified T cells expressing FRa_CD3_syn CPH and FRa-TAC respectively (0.3 × 10⁻¹⁰ 6 or 1.5 × 10 6 The drug was administered intravenously (iv) to mice.

[0147] Example 1: Characterization of modified T cells according to the present disclosure 1.1 Expression of the scFv-TM / IC, 2xscFv-TM / IC, and 2xscFv-multiTM / IC constructs of this disclosure

[0148] In this example, we investigated the expression of the scFv-TM / IC construct of this disclosure (i.e., the CD3-syn HCAR2 construct), the 2xscFv-TM / IC construct (including the FRa-CD3-syn HCAR2, FRa-CD3-syn CNR2, HER2-CD3-syn GPR84, and FRa-CD3-syn P2Y14 constructs), and the 2xscFv-multiTM / IC construct (including the HER2-CD3-syn CPH, FRa-CD3-syn CPH, FRa-CD3-syn CP, and FRa-CD3-syn CH constructs) in modified T cells. As described in the "Materials and Methods" section of this disclosure, human T cell lymphoblastic lymphoma cell line SupT1 and human primary T cells were transfected with lentiviruses expressing the scFv-TM / IC, 2xscFv-TM / IC, and 2xscFv-multiTM / IC polypeptides of this disclosure, respectively. After culturing at 37°C for 7 days, the expression of the polypeptides of this disclosure was determined by flow cytometry. The data are summarized in Table 4.

[0149] Table 4. Expression levels of specific constructs on the cell membrane of human SupT1 cells and primary T cells. [Table 4]

[0150] The data in Table 4 demonstrate that the scFv-TM / IC, 2xscFv-TM / IC, and 2xscFv-multiTM / IC polypeptides of this disclosure were expressed on the cell membranes of human SupT1 cells and primary T cells.

[0151] In FRa-CD3-syn CPH, the anti-CD3 scFv was further substituted with anti-CD2 scFv (SEQ ID NO: 53), anti-CD2 scFv (SEQ ID NO: 55), anti-ICOS scFv (SEQ ID NO: 57), anti-ICOS-L (SEQ ID NO: 59), CAMD1 (SEQ ID NO: 61), or IL-2 binder (SEQ ID NO: 63). The resulting constructs were named "FRa-CD2(1)-syn CPH," "FRa-CD2(2)-syn CPH," "FRa-ICOS-syn CPH," "FRa-ICOS-L-syn CPH," "FRa-CAMD1-syn CPH," and "FRa-IL-2 binder-syn CPH," respectively. After being prepared as lentiviral particles, they were transfected into human primary T cells. After culturing at 37°C for 7 days, the expression of the polypeptides disclosed herein was determined by flow cytometry. The data are summarized in Table 5.

[0152] Table 5. Expression levels of specific constructs on the cell membrane of human primary T cells [Table 5]

[0153] The data in Table 5 confirm that the constructs of this disclosure (in the form of 2xscFv-multiTM / IC or scFv-peptide-multiTM / IC) are membrane-expressed on T cells.

[0154] The data from this embodiment demonstrate that the TM / IC, 2xTM / IC, and 3xTM / IC domains of this disclosure are suitable for use as transmembrane domains for immobilizing one or more targeting elements (e.g., scFv and / or peptides) on the surface of immune cells (e.g., T cells), respectively.

[0155] 1.2 Influence of CD3-mediated pathways on the tumor-killing activity of modified T cells In this example, the ITAM domain of FRa_CAR was replaced with the syn CPH of this disclosure. The resulting polypeptide was named "CAR-delITAM-CPH," and it contained, in this order from the N-terminus to the C-terminus, an anti-FRa scFv, a hinge domain, a transmembrane domain, a CD28 costimulatory domain, and the syn CPH of this disclosure. According to sequencing results, CAR-delITAM-CPH (encoded by the nucleotide sequence of SEQ ID NO: 35) contained the amino acid sequence of SEQ ID NO: 36. Flow cytometry data showed that both FRa_CAR and CAR-delITAM-CPH were expressed in human primary T cells (data not shown). However, modified T cells expressing CAR-delITAM-CPH did not show a killing effect against Ovcar4 cancer cells (Figure 4).

[0156] Next, the syn CPH of this disclosure was ligated to anti-FRa scFv. The resulting FRa-CPH TM / IC was encoded by the nucleotide sequence of SEQ ID NO: 37 and contained the amino acid sequence of SEQ ID NO: 38. According to flow cytometry results, FRa-CPH TM / IC could be expressed in human primary T cells (data not shown). Nevertheless, modified T cells expressing FRa-CPH TM / IC did not show a killing effect against Ovcar4 cancer cells compared to FRa-CAR modified T cells (Figure 5).

[0157] The data in Figures 4 and 5 suggest that the CD3-mediated signaling pathway plays a crucial role in T cell activation and subsequent tumor-killing activity.

[0158] 1.3 Tumor-killing activity of modified T cells In this study, we investigated the tumor-killing activity of two exemplary HER2-expressing modified T cells against Ovcar4 cancer cells: modified T cells expressing HER2-CD3-syn GPR84 (HER2-CD3-syn GPR84-T cells) and modified T cells expressing HER2-CD3-syn CPH (HER2-CD3-syn CPH-T cells). HER2_TAC-T was used as the control group in this study. As shown in the data in Table 6, polypeptides expressed by each of the HER2-CD3-based constructs were detected on the surface of human primary T cells.

[0159] Table 6. Expression levels of specific HER2-CD3-based constructs on the cell membrane of human primary T cells. [Table 6]

[0160] In tumor killing assays, modified T cells expressing a HER2-CD3-based construct were co-cultured with Ovcar4 ovarian cancer cells at effector-to-target ratios (E:T) of 4:1, 2:1, 1:1, or 1:2. After 48 hours, the T cells were harvested, and the survival rate of the cancer cells remaining in the wells was evaluated as the killing result. Meanwhile, the T cells were transferred to wells of another cancer cell line seeded as part of a repeated killing procedure. The data in Figure 6 (killing results in a total of 5 repeats) and Table 7 demonstrate that the modified T cells described herein exhibited repeated cytotoxic effects against ovarian cancer cells in long-term killing assays.

[0161] Table 7. Cytotoxic effects of specific modified T cells on cancer cells. [Table 7]

[0162] The effect of modified T cells expressing FRa_CD3_syn CPH (SEQ ID NO: 44, i.e., FRa_CD3_syn CPH-T cells) on Ovcar4 cancer cells was also determined. FRa-TAC-T and FRa_CAR-T cells were used as control groups in this study. As shown in the data in Table 8, polypeptides expressed by each of the FRa-based constructs were detected on the surface of human primary T cells.

[0163] Table 8. Expression levels of specific FRa-based constructs on the cell membrane of human primary T cells [Table 8]

[0164] To perform tumor killing assays, modified T cells expressing the FRa-based construct were co-cultured with Ovcar4 ovarian cancer cells at different E:T ratios. After 48 hours, the T cells were harvested, and the viability of the remaining cancer cells in the well was evaluated as the killing result. Meanwhile, the T cells were transferred to wells of another cancer cell seeded as a repeat killing procedure. The data in Figures 7A, 7B, and Table 9 demonstrate that the modified T cells of this disclosure exhibited cytotoxic effects against ovarian cancer cells in the long-term killing assay, with E:T ratios ranging from 0.0625:1 to 8:1.

[0165] Table 9. Cytotoxic effects of specific modified T cells on cancer cells. [Table 9]

[0166] In addition to Ovcar4 cancer cells, this study also determined the antitumor effect of modified T cells expressing FRa_CD3_syn CPH (i.e., FRa_CD3_syn CPH-T cells) against OVCAR-3 cancer cells, a highly malignant serous ovarian adenocarcinoma cell line isolated from malignant ascites fluid of ovarian patients. FRa-TAC-T and FRa_CAR-T were used as control groups in this study. Modified T cells were co-cultured with OVCAR-3 cancer cells at an E:T ratio of 1:1. After 72 hours, the T cells were transferred to wells of another cancer cell line seeded in a repeated killing procedure. After the second round of killing assays, the supernatant was collected and subjected to a cytokine assay. The percentage of viable cells (%) treated with the FRa_CD3_syn CPH-T, FRa-TAC-T, and FRa_CAR-T cells of this disclosure were approximately 1% (standard error: 0%), 2% (standard error: 1%), and 2% (standard error: 0%), respectively (data not shown). This demonstrates the tumor-killing activity of the tested FRa-targeted modified T cells. According to the cytokine assay results, treatment with the FRa_CD3_syn CPH-T cells of this disclosure induced significantly higher expression levels of pro-inflammatory Th1 cytokines, including IL-2 (Figure 8A), IFN-γ (Figure 8B), and TNF-α (Figure 8C), compared to positive controls (i.e., FRa-TAC-T and FRa_CAR-T).

[0167] On the other hand, the data in Figure 9 confirms that each modified T cell expressing FRa-CD3-syn CPH (FRa_CPH), FRa-CD3-syn CH (FRa_CH), FRa-CD3-syn CP (FRa_CP), FRa_CD3_syn CNR2 (FRa_C), FRa-CD3-syn HCAR2 (FRa_H), and FRa-CD3-syn P2Y14 (FRa_P), respectively, showed cytotoxic effects (over 80% killing activity) against Ovcar4 cancer cells.

[0168] Example 2: In vivo activity In this example, the antitumor effect of the modified T cells of this disclosure was evaluated in an animal model. OVCAR-3 cells were subcutaneously inoculated into the flanks of mice as described in the "Materials and Methods" section of this disclosure. The average tumor size was approximately 100 mm.3 When this is reached, FRa_CD3_syn CPH-T or FRa-TAC-T cells (0.3 × 10) 6 or 1.5 × 10 6 The drug was administered intravenously (iv) to tumor-carrying mice.

[0169] The data in Figures 10 and 11 demonstrate that administration of the FRa_CD3_syn CPH-T cells of this disclosure significantly inhibited tumor growth (Figure 10) and extended the survival rate of tumor-carrying mice (Figure 11), compared to positive control FRa-TAC-T cells.

[0170] In conclusion, this disclosure provides several TM / IC domains, each of which binds to one or more scFvs / peptides and is useful for expressing scFv / peptides on the cell membrane of cells (e.g., T cells). Modified T cells expressing TM / IC-based polypeptides according to the embodiments of this disclosure exhibit targeting and cytotoxic effects against cancer cells and thus provide a potential means for treating cancer.

[0171] The above description of embodiments is given for illustrative purposes only, and it will be understood that various modifications can be made by those skilled in the art. The above specification, examples, and data provide a complete description of the structure and use of exemplary embodiments of the present invention. Although various embodiments of the present invention have been described above with some specificity or by reference to one or more individual embodiments, those skilled in the art will be able to make numerous modifications to the disclosed embodiments without departing from the spirit or scope of the invention.

Claims

1. It comprises a first bifunctional domain and a first single-chain variable region fragment (scFv) or peptide linked to the N-terminus of the first bifunctional domain, A recombinant polypeptide characterized in that the first bifunctional domain comprises the intracellular loop 1 (ICL1), ICL2, ICL3, and C-terminal region of a first G protein-coupled receptor (GPCR) in that order, and lacks the extracellular domain and transmembrane domain of the first GPCR.

2. The recombinant polypeptide according to claim 1, wherein the first bifunctional domain comprises ICL1, ICL2, ICL3 and the C-terminal region of the first GPCR.

3. The recombinant polypeptide according to claim 1, wherein the first GPCR is a class A GPCR.

4. The recombinant polypeptide according to claim 3, wherein the first GPCR is cannabinoid receptor 2 (CNR2), hydroxycarboxylic acid receptor 2 (HCAR2), G protein-coupled receptor 84 (GPR84), or P2Y purine receptor 14 (P2Y14).

5. The recombinant polypeptide according to claim 4, wherein the first GPCR is CNR2 and the first bifunctional domain comprises the amino acid sequence of SEQ ID NO:

2.

6. The recombinant polypeptide according to claim 4, wherein the first GPCR is HCAR2, and the first bifunctional domain comprises the amino acid sequence of SEQ ID NO:

4.

7. The recombinant polypeptide according to claim 4, wherein the first GPCR is GPR84, and the first bifunctional domain comprises the amino acid sequence of SEQ ID NO:

6.

8. The recombinant polypeptide according to claim 4, wherein the first GPCR is P2Y14, and the first bifunctional domain comprises the amino acid sequence of SEQ ID NO:

8.

9. The recombinant polypeptide according to claim 1, wherein the first scFv is specific to CD3 or NKp46.

10. The recombinant polypeptide according to claim 1, further comprising a first scFv or a second scFv ligated to the N-terminus of a peptide, wherein the second scFv is specific to a tumor-associated antigen (TAA).

11. It further includes a second bifunctional domain located at the C-terminus of the first bifunctional domain and ligated thereto, The second bifunctional domain includes ICL1, ICL2, ICL3 and the C-terminal region of the second GPCR in this order. The second bifunctional domain is characterized by lacking the extracellular domain and transmembrane domain of the second GPCR. The recombinant polypeptide according to claim 1, wherein the second GPCR is different from the first GPCR.

12. The recombinant polypeptide according to claim 11, wherein the second bifunctional domain comprises ICL1, ICL2, ICL3 and the C-terminal region of the second GPCR.

13. The recombinant polypeptide according to claim 11, wherein the first GPCR and the second GPCR are each a class A GPCR.

14. The recombinant polypeptide according to claim 13, wherein the first GPCR and the second GPCR are independently selected from the group consisting of CNR2, HCAR2, GPR84, and P2Y14.

15. The first GPCR and the second GPCR are CNR2 and P2Y14, respectively. The recombinant polypeptide according to claim 14, wherein the first and second bifunctional domains each contain the amino acid sequences of SEQ ID NO: 2 and SEQ ID NO: 8, respectively.

16. The first GPCR and the second GPCR are CNR2 and HCAR2, respectively. The recombinant polypeptide according to claim 14, wherein the first and second bifunctional domains each contain the amino acid sequences of SEQ ID NO: 2 and SEQ ID NO: 4, respectively.

17. It further includes a third bifunctional domain located at the C-terminus of the second bifunctional domain and ligated thereto, The third bifunctional domain includes ICL1, ICL2, ICL3 and the C-terminal region of the third GPCR in this order. The third bifunctional domain is characterized by lacking the extracellular domain and transmembrane domain of the third GPCR. The recombinant polypeptide according to claim 11, wherein the first GPCR, the second GPCR, and the third GPCR are different from each other.

18. The recombinant polypeptide according to claim 17, wherein the third bifunctional domain comprises ICL1, ICL2, ICL3 and the C-terminal region of the third GPCR.

19. The recombinant polypeptide according to claim 17, wherein the first GPCR, the second GPCR, and the third GPCR are each a class A GPCR.

20. The recombinant polypeptide according to claim 17, wherein the first GPCR, the second GPCR, and the third GPCR are independently selected from the group consisting of CNR2, HCAR2, GPR84, and P2Y14.

21. The first GPCR, the second GPCR, and the third GPCR are CNR2, P2Y14, and HCAR2, respectively. The recombinant polypeptide according to claim 20, wherein the first, second, and third bifunctional domains each contain the amino acid sequences of SEQ ID NO: 2, SEQ ID NO: 8, and SEQ ID NO: 4, respectively.

22. A recombinant nucleic acid encoding a recombinant polypeptide according to claim 1, comprising a promoter and a first coding sequence and a second coding sequence operably linked to the promoter, wherein the first coding sequence encodes the first single-chain variable region fragment (scFv) or the peptide, and the second coding sequence is located downstream of the first coding sequence and encodes the first bifunctional domain.

23. The recombinant nucleic acid according to claim 22, further comprising a third coding sequence operably coupled to the promoter and located upstream of the first coding sequence, wherein the third coding sequence codes for a second scFv specific to a tumor-associated antigen (TAA).

24. The aforementioned second coding sequence is located at the C-terminus of the first bifunctional domain and further codes for a second bifunctional domain ligated thereto. The second bifunctional domain includes ICL1, ICL2, ICL3 and the C-terminal region of the second GPCR in this order. The second bifunctional domain is characterized by lacking the extracellular domain and transmembrane domain of the second GPCR. The recombinant nucleic acid according to claim 22, wherein the second GPCR is different from the first GPCR.

25. The aforementioned second coding sequence is located at the C-terminus of the aforementioned second bifunctional domain and further codes for a third bifunctional domain ligated thereto. The third bifunctional domain includes ICL1, ICL2, ICL3 and the C-terminal region of the third GPCR in this order. The third bifunctional domain is characterized by lacking the extracellular domain and transmembrane domain of the third GPCR. The recombinant nucleic acid according to claim 24, wherein the first GPCR, the second GPCR, and the third GPCR are different from each other.

26. A method for treating cancer in a subject, comprising administering to the subject an effective amount of immune cells expressing the recombinant polypeptide described in claim 1.

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