CD24-binding protein and its use

Abstract

A group of CD24-binding proteins and their uses are provided. Specifically, a group of antigen-binding proteins is provided in which cells expressing the antigen-binding protein may have cytotoxicity and safety. The use of antigen-binding proteins in the preparation of pharmaceuticals is also provided.

Description

Technical Field

[0001] This application relates to the field of biomedicine, and particularly to CD24 binding proteins and their use. [[ID=б]]

Background Art

[0002] Chimeric antigen receptors (CARs) specifically recognize antigens expressed on the surface of tumor cells and induce CAR-expressing immune cells to eliminate tumors. For example, when there are antigen-positive cells targeted by the CAR, cells expressing the CAR can recognize and kill these antigen-positive cells.

[0003] However, existing CARs in the art still have problems such as low activity, weak cytotoxic efficacy of CAR-expressing cells, and problematic in vivo safety. Therefore, it is desirable to further optimize CARs in the art to achieve the effect of increasing the cytotoxic efficacy of CAR-expressing cells and improving safety.

Summary of the Invention

[0004] This application provides an antigen-binding protein having an improved structure, and the antigen-binding protein can be in the form of a chimeric antigen receptor (CAR). The antigen-binding protein can have one or more effects selected from the group consisting of: (1) excellent expression efficiency; (2) significant anti-tumor activity of cells expressing the antigen-binding protein; (3) sustained anti-tumor activity of cells expressing the antigen-binding protein; (4) significant cell activation ability of cells expressing the antigen-binding protein; and (5) appropriate cytokine secretion ability of cells expressing the antigen-binding protein.

[0005] In one aspect, this application provides an antigen-binding protein that binds to CD24, and then cells expressing the antigen-binding protein have the ability to express cytokines at a low level. For example, the antigen-binding fragment includes a light chain variable region and a heavy chain variable region, has a linker between the light chain variable region and the heavy chain variable region, and the linker has a length of 10 amino acids or less.

[0006] In another embodiment, the present application provides a polypeptide comprising the antigen-binding protein of the present application.

[0007] In another embodiment, the present application provides nucleic acids encoding antigen-binding proteins and / or polypeptides of the present application.

[0008] In another embodiment, the present application provides a vector comprising the nucleic acid of the present application.

[0009] In another embodiment, the present application provides cells comprising the antigen-binding protein of the present application, the polypeptide of the present application, the nucleic acid of the present application and / or the vector of the present application.

[0010] In another embodiment, the present application provides a method for preparing the antigen-binding protein and / or polypeptide of the present application, comprising culturing cells of the present application under conditions that enable the expression of the antigen-binding protein and / or polypeptide.

[0011] In another embodiment, the present application provides a composition comprising the antigen-binding protein of the present application, the polypeptide of the present application, the nucleic acid of the present application, the vector of the present application and / or the cell of the present application, and optionally, a pharmaceutically acceptable carrier.

[0012] In another embodiment, the present application provides a kit comprising the antigen-binding protein of the present application, the polypeptide of the present application, the nucleic acid of the present application, the vector of the present application, the cells of the present application and / or the composition of the present application.

[0013] In another embodiment, the present application provides a method for stimulating an immune response, comprising administering the antigen-binding protein of the present application, the polypeptide of the present application, the nucleic acid of the present application, the vector of the present application, the cells of the present application, the composition of the present application, and / or the kit of the present application.

[0014] In another embodiment, the present application provides the use of the antigen-binding proteins, polypeptides, nucleic acids, vectors, cells, compositions, and / or kits of the present application in the preparation of a pharmaceutical product to be used to prevent, alleviate, and / or treat tumors.

[0015] In another embodiment, the present application provides a method for preventing, mitigating and / or treating a tumor, comprising administering the antigen-binding protein of the present application, the polypeptide of the present application, the nucleic acid of the present application, the vector of the present application, the cells of the present application, the composition of the present application and / or the kit of the present application.

[0016] In another embodiment, the present application provides the use of the antigen-binding proteins, polypeptides, nucleic acids, vectors, cells, compositions, and / or kits of the present application for the prevention, mitigation, and / or treatment of tumors.

[0017] Other aspects and advantages of this application may be readily apparent to those skilled in the art from the following detailed description. The following detailed description is merely illustrative and descriptive of exemplary embodiments of this application. As will be understood to those skilled in the art, the content of this application enables those skilled in the art to modify specific embodiments disclosed without departing from the spirit and scope of the invention contained herein. Accordingly, the accompanying drawings and the description in the specification of this application are illustrative and not limiting.

[0018] Specific features of the invention included in this application are enumerated in the appended claims. The features and advantages of the invention included in this application can be better understood by referring to the exemplary embodiments and accompanying drawings detailed below. The accompanying drawings are briefly illustrated below. [Brief explanation of the drawing]

[0019] [Figure 1]Shows the antitumor activities and cytokine expression levels of H3L3-CAR and PP6373-CAR. By modifying the sequences of H3L3-CAR, C1 was induced, and by modifying the sequences of PP6373-CAR, C2 was induced. The modified C1 and C2 showed even higher antitumor activities and cytokine secretion levels. [Figure 2] Shows the expression of CAR structures such as C3, C5, C7, C9, C11, C13, and C15 induced by adjusting the hinge region, transmembrane region, and co-stimulatory domain of the CAR structure based on C1. [Figure 3] Shows the antitumor activities of CAR structures such as C1, C3, C5, C7, C9, C11, C13, and C15 in this application. [Figure 4] Shows the cytokine secretion of CAR structures such as C1, C3, C5, C7, C9, C11, C13, and C15 in this application. [Figure 5] Shows the expression of CAR structures such as C4, C6, C8, C10, C12, C14, and C16 induced by adjusting the hinge region, transmembrane region, and co-stimulatory domain of the CAR structure based on C2. [Figure 6] Shows the antitumor activities of CAR structures such as C2, C4, C6, C8, C10, C12, C14, and C16 in this application. [Figure 7] Shows the cytokine secretion of CAR structures such as C2, C4, C6, C8, C10, C12, C14, and C16 in this application. [Figure 8] Shows the expression of C23, C27, and C28 induced by adjusting the length of the linker or the type of the transmembrane region in this application. [Figure 9] Shows the in vitro cytotoxic efficacy of C23, C27, and C28 induced by adjusting the length of the linker or the type of the transmembrane region in this application. [Figure 10] Shows the cytokine secretion of C23, C27, and C28 induced by adjusting the length of the linker or the type of the transmembrane region in this application. [Figure 11]Shows the expression of C28, C29 and C30 induced by adjusting the linker length of the present application. [Figure 12] Shows the in vitro cytotoxic efficacy and cytokine secretion of C28, C29 and C30 induced by adjusting the linker length of the present application. [Figure 13] Shows the expression of C31 induced by adding the chemokine receptor CXCR2 of the present application. [Figure 14] Shows the in vitro killing efficacy of C31 induced by adding the chemokine receptor CXCR2 of the present application. [Figure 15] Shows the cytokine secretion of C31 induced by adding the chemokine receptor CXCR2 of the present application. [Figure 16] Shows the in vitro cytotoxic efficacy and cytokine secretion of C32 induced by adding the cytokine IL-12β(p40) of the present application. [Figure 17] Shows the ability of the CAR structures of C8 and C28 of the present application to remove tumors and their safety results. [Figure 18] Shows the ability of the CAR structures of C28 and C31 of the present application to reach the tumor site. [Figure 19] Shows the ability of the CAR structures of C28 and C31 of the present application to remove tumors. [Figure 20] Shows the ability of the CAR structures of C28, C31 and C32 of the present application to remove tumors and the statistics of their safety results.

Mode for Carrying Out the Invention

[0020] Hereinafter, embodiments of the invention of the present application will be exemplified by specific examples. Those skilled in this technology can easily understand other advantages and effects of the invention of the present application from the content disclosed in this specification.

[0021] In this application, the term "CD24" generally refers to the CD24 protein or the gene encoding it. CD24 can be found under UniProt number P25063. This term includes its variants, homologs, and functionally active fragments.

[0022] In this application, the term “antigen-binding protein” generally refers to a protein capable of binding to one or more antigens or functionally active fragments thereof. For example, exemplary antigen-binding proteins include chimeric antigen receptors and various other molecules known in the art that contain domains capable of recognizing antigens.

[0023] In this application, the term “chimeric antigen receptor (CAR)” generally refers to a fusion protein comprising an extracellular domain capable of binding to an antigen and at least one transmembrane domain. A CAR may be a core component of a CAR cell and may include an antigen (e.g., tumor-specific antigen and / or tumor-associated antigen) binding domain, a hinge domain, a transmembrane domain, and an intracellular domain.

[0024] In this application, the term "signal peptide" generally refers to a propeptide that exists as the N-terminal peptide on a precursor protein. The signal peptide functions to facilitate the translocation of an expression polypeptide attached to the endoplasmic reticulum, thereby enabling the expression of the target protein on the cell membrane, during which time the signal peptide can generally be cleaved. For example, without the signal peptide, the biological activity of the antigen-binding protein in this application would also be unaffected. In organisms producing polypeptides, the signal peptide can be heterologous or homologous.

[0025] In this application, the term "antigen-binding domain" generally refers to (specifically) binding to a given target epitope or site on a target molecule (antigen), interacting with a given target epitope or site, or identifying a domain of a given target epitope or site.

[0026] In this application, the term “specific binding” generally refers to measurable and / or reproducible interactions, such as binding between a target and an antibody, where the presence of the target may be determined within a heterogeneous population of molecules (including biomolecules). For example, an antibody that specifically binds to a target (which may be an epitope) is an antibody that binds to the target with higher affinity, binding activity, ease and / or duration than it would to other targets. In some embodiments, the antibody specifically binds to an epitope on a protein, and the epitopes are conserved across different species of proteins. In other embodiments, specific binding may include, but is not necessarily, exclusive binding.

[0027] In this application, the term “antibody” generally refers to immunoglobulins or their fragments or derivatives, and includes any polypeptide containing an antigen-binding site, whether in vitro or in vivo. This term includes, but is not limited to, polyclonal antibodies, monoclonal antibodies, monospecific antibodies, multispecific antibodies, nonspecific antibodies, humanized antibodies, single-chain antibodies, chimeric antibodies, synthetic antibodies, recombinant antibodies, hybrid antibodies, mutant antibodies, and transplanted antibodies. The term “antibody” may also include antibody fragments such as Fab, F(ab')2, Fv, scFv, Fd, dAb, and other antibody fragments that maintain antigen-binding function.

[0028] In this application, the term "single-chain antibody (scFv)" may refer to an antibody formed by linking the heavy chain variable region and the light chain variable region of an antibody via a linker.

[0029] In this application, the term "hinge domain" generally refers to the junction region between the antigen-binding domain and the transmembrane domain. Generally, the hinge domain has a certain degree of flexibility, and CAR molecules may or may not contain a hinge domain.

[0030] In this application, the term “transmembrane domain” generally refers to a domain within a CAR that crosses the cell membrane, connects to intracellular signaling domains, and helps transmit signals.

[0031] In this application, the term "CD8a" generally refers to the T cell surface glycoprotein CD8 alpha chain. CD8a can be found under UniProt number P01732. This term includes its variants, homologs, and functionally active fragments. In this application, the term "CD28" generally refers to the T cell-specific surface glycoprotein CD28. CD28 can be found under UniProt number P10747. This term includes its variants, homologs, and functionally active fragments.

[0032] In this application, the term "intracellular domain" generally refers to the intracellular fraction of a molecule. The intracellular domain generates signals that promote the immune effector function of CAR-containing cells, for example, involving cytolytic activity and co-activation. For example, the intracellular domain may consist only of cleaved fractions that preserve intracellular signaling function.

[0033] In this application, the term “costimulatory signaling domain” generally refers to an intracellular domain capable of providing immune costimulatory molecules. In this application, the term “costimulation” generally refers to a source of lymphocyte activation signals typically generated by the interaction of costimulatory molecules on the surface of immune cells involved in adaptive immunity with their receptors (between T cells and B cells, or between antigen-presenting cells and T cells).

[0034] In this application, the term "signaling domain" generally refers to a domain located within a cell that is capable of conducting signals. In this application, an intracellular signaling domain is capable of transmitting signals to a cell.

[0035] In this application, the term "CD3ζ" generally refers to the CD247 or T cell surface glycoprotein CD3 zeta chain. CD3ζ can be found under UniProt number P20963. This term includes its variants, homologs, and functionally active fragments.

[0036] For example, the sequences referred to in this application include sequences that are at least about 95% homologous to this sequence. For example, the sequences referred to in this application include sequences that are at least about 95%, 96%, 97%, 98%, or 99% homologous to this sequence.

[0037] In this application, the term “immune cells” generally refers to cells involved in the immune response, such as cells that promote immune effector responses. Examples of immune cells include, but are not limited to, T cells, B cells, natural killer (NK) cells, NKT cells, mast cells, granulocytes, monocytes, lymphocytes, and macrophages. The term also includes engineered immune cells, such as immune cells that have been genetically modified by adding exogenous genetic material in the form of DNA or RNA to the entire genetic material of the cell.

[0038] In this application, the term “cytotoxic efficacy” refers to the killing of cells by exposing them to an effective amount of antibody, immunoconjugate, bispecific / multispecific molecule, or a combination thereof. The method may optionally include killing antigen-positive cells in the presence of effector cells, for example, by CDC, apoptosis, ADCC, ADCP, phagocytosis, or a combination of two or more of these mechanisms.

[0039] In this application, the term "directly or indirectly linked" refers to direct linkage by peptide bonds, or indirect linkage by linkers or non-peptide linkages.

[0040] In this application, the term “proliferation” refers to an increase in cell division (symmetric or asymmetric division of cells). “Proliferation” may refer to symmetric or asymmetric division of T cells. “Increased proliferation” occurs when the number of cells in a treated sample increases compared to the number of cells in an untreated sample.

[0041] In this application, the term “subject” includes any human or non-human animal. The term “non-human animal” includes all vertebrates, e.g., mammals and non-mammals, e.g., non-human primates, sheep, dogs, cats, cattle, horses, chickens, amphibians and reptiles. Non-human animals may be non-human primates, sheep, dogs, cats, cattle and horses, and other mammals.

[0042] In this application, the term “therapeutic effective dose” refers to a dose of the antibody of this application that is sufficient to prevent or alleviate symptoms associated with a disease or disorder (e.g., cancer). The therapeutic effective dose is relevant to the disease being treated, and a person skilled in the art can readily determine the actual effective dose.

[0043] In this application, the term "pharmaceutical" generally refers to a chemical compound or composition that can induce a desired therapeutic effect when administered correctly to a patient.

[0044] In this application, the term “composition” refers to a mixture comprising one or more of the compounds of this application, or a physiologically / pharmaceutically acceptable salt or prodrug thereof, with other chemical components, such as physiologically / pharmaceutically acceptable carriers and excipients. Pharmaceutical compositions are intended to facilitate administration to a living organism and enhance the absorption of the active ingredient, thereby exerting biological activity. Therapeutic compositions are generally sterile and should remain stable under manufacturing and storage conditions. Compositions may be formulated as solutions, microemulsions, dispersants, liposomes, or other ordered structures suitable for high antibody concentrations. Sterile injections may be prepared by incorporating the required amount of the active compound (i.e., antibody or antibody portion) along with one or a combination of the components listed above into a suitable solvent, followed by filtration and sterilization as necessary.

[0045] In this application, the term “vector” generally refers to a nucleic acid molecule to which another nucleic acid it is bound can be transferred. One type of vector is a “plasmid,” which refers to a circular double-stranded DNA loop to which other DNA segments can be ligated. Another type of vector is a viral vector, in which other DNA segments can be ligated to a viral genome. Some vectors can replicate autonomously within the host cell into which they are introduced (e.g., bacterial vectors with bacterial origins of replication, and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors), once introduced into a host cell, can be integrated into the host cell's genome and thus replicate together with the host genome. These vectors include, for example, naked RNA polynucleotides, naked DNA polynucleotides, polynucleotides composed of DNA and RNA of the same strand, polylysine conjugate DNA or polylysine conjugate RNA, peptide conjugate DNA or peptide conjugate RNA, liposome conjugate DNA, etc., which cannot replicate autonomously. Furthermore, some vectors can lead to the expression of the gene to which these vectors are effectively ligated. Such vectors are referred to in this application as “recombinant expression vectors” (or simply “expression vectors”). Generally, expression vectors used in recombinant DNA technology are typically in plasmid form. In this specification, the terms “plasmid” and “vector” are used interchangeably, since plasmids are the most commonly used form of vectors.

[0046] In this application, the term “adjuvant” generally refers to any substance that assists or modulates the action of a pharmaceutical product. This includes, but is not limited to, immunoadjuvants that enhance or diversify the immune response to an antigen.

[0047] In this application, the terms “tumor” or “tumor cell” generally refer to or represent a physiological condition in mammals typically characterized by unregulated cell proliferation. Examples of tumors include, but are not limited to, carcinomas, lymphomas, blastomas (including medulloblastomas and retinoblastomas), sarcomas (including liposarcomas and synovial cell sarcomas), neuroendocrine tumors (including carcinoid tumors, gastrinomas and islet cell carcinomas), mesotheliomas, schwannomas (including acoustic neuromas), meningiomas, adenocarcinomas, and melanomas. "Tumor cells" may further include "solid tumors," which refer to tumors selected from the group consisting of gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, bladder cancer, hepatocellular carcinoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial cancer or uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, hepatocellular carcinoma, anal cancer, penile cancer, testicular cancer, esophageal cancer, bile duct tumors, and head and neck cancers.

[0048] In this application, the term “antigen-positive tumor” refers to tumor cells that express an antigen protein on their surface. To determine whether cells express an antigen protein on their surface, antigen mRNA expression is considered to be related to antigen protein expression on the cell surface, and antigen mRNA expression can be determined by a method selected from in situ hybridization and RT-PCR (including quantitative RT-PCR). Alternatively, antigen protein expression on the cell surface may be determined by methods such as immunohistochemical testing and FACS, for example, using an antibody against the antigen protein. For example, an antigen-positive tumor may be a transplant tumor in a mammal. In this application, the term “antigen-positive tumor cell” refers to a cell that expresses an antigen protein on its surface.

[0049] Detailed description of the invention In one embodiment, the present application provides a group of improved antigen-binding protein G1s in which G1 binds to CD24, and the enhanced effect of cells expressing the G1 on the ability to kill CD24-positive tumor cells, or on the ability of cells expressing the G1 to express cytokines after stimulation by CD24-positive tumor cells, is higher than that of cells expressing existing CD24 antigen-binding proteins. For example, about 5%, 10%, 20%, 30%, 40%, 50%, 70%, 100%, 150%, 200%, 300%, 400%, 500%, 4000%, 5000%, or 5000% higher than that of cells expressing CD24 antigen-binding proteins disclosed in the Art.

[0050] In one embodiment, the present application provides a group of G1-based, further improved antigen-binding protein G2s, wherein G2 binds to CD24, and G2-expressing cells maintain the ability to kill CD24-positive tumor cells and express cytokines after stimulation by CD24-positive tumor cells at least about 50% lower than that of cells expressing G1. For example, at least about 5%, 10%, 20%, 30%, 40%, or 50% lower than that of G1-expressing cells.

[0051] In one embodiment, the present application provides a group of G3 antigen-binding proteins based on G1 or G2, which are further improved, for use in intracellular expression, wherein G3-expressing cells also express chemokine receptors. For example, the chemokine receptor may be a chemokine receptor known in the art. For example, the chemokine receptor may include CXCR2 or a functional fragment thereof. For example, CXCR2 may include the amino acid sequence described in SEQ ID NO: 53.

[0052] In one embodiment, the present application provides a group of G3 antigen-binding proteins based on G1 or G2, which are further improved, for use in intracellular expression, and G3-expressing cells also express cytokines. For example, the cytokines may include IL-12β or a functional fragment thereof. For example, IL-12β may include the amino acid sequence described in SEQ ID NO: 50. In one embodiment, the present application provides an antigen-binding protein which binds to CD24, and subsequently, cells expressing the antigen-binding protein have the ability to express cytokines at low levels.

[0053] For example, cytokines include, but are not limited to, IFN-γ, IL-6, TNF-α, and / or IL-2.

[0054] For example, with respect to the antigen-binding protein described, compared to corresponding cells that do not express the antigen-binding protein of this application, cells expressing the antigen-binding protein have cytokine expression levels of approximately 2000 pg / ml or less in the co-culture supernatant of these cells and tumor cells over approximately 24 hours. For example, compared to corresponding cells that do not express the antigen-binding protein of this application, cells expressing the antigen-binding protein have cytokine expression levels of approximately 2000 pg / ml or less, approximately 1000 pg / ml or less, approximately 500 pg / ml or less, approximately 200 pg / ml or less, approximately 100 pg / ml or less, or approximately 50 pg / ml or less in the co-culture supernatant of these cells and tumor cells over approximately 24 hours.

[0055] For example, an antigen-binding protein contains an antigen-binding domain. For example, the antigen-binding domain can recognize an antibody or its antigen-binding fragment against an antigen. For example, the antigen-binding fragment contains a single-chain antibody. For example, the antigen-binding fragment contains a heavy chain variable region. For example, the heavy chain variable region contains the sequence described in SEQ ID NO: 23 or SEQ ID NO: 24. For example, the heavy chain variable region contains HCDR 1-3. For example, the heavy chain variable region contains HCDR 1-3 described in SEQ ID NO: 42-44. For example, an antigen-binding fragment contains a light chain variable region. For example, the light chain variable region contains the sequence described in SEQ ID NO: 25 or SEQ ID NO: 26. For example, the light chain variable region contains LCDR 1-3. For example, the light chain variable region contains LCDR 1-3 described in SEQ ID NO: 45-47.

[0056] For example, the antigen-binding fragment includes a light chain variable region and a heavy chain variable region. For example, the light chain variable region includes the sequence described in SEQ ID NO: 25 or SEQ ID NO: 26, and the heavy chain variable region includes the sequence described in SEQ ID NO: 23 or SEQ ID NO: 24. For example, a linker is contained between the light chain variable region and the heavy chain variable region. For example, the linker has a length of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids. For example, the linker includes an alanine-serine polymer or other flexible peptide sequence known in the art. For example, the linker includes amino acid substitutions. For example, a linker may include one or more GGGGS (as described in SEQ ID NO: 27), for example, a sequence of 1, 2, 3, 4, or 5 GGGGS; for example, a linker may include one or more G (as described in SEQ ID NO: 28), for example, a sequence of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 G; for example, a linker may include one or more GS (as described in SEQ ID NO: 29), for example, a sequence of 1, 2, 3, 4, or 5 GS; for example, a linker may include one or more GGGS (as described in SEQ ID NO: 30), for example, a sequence of 1, 2, 3, 4, or 5 GGGS; for example, a linker may include one or more GSGGS (as described in SEQ ID NO: 31), for example, a sequence of 1 or 2 GSGGS.

[0057] For example, the antigen-binding protein further comprises a hinge domain directly or indirectly linked to the antigen-binding domain. For example, the hinge domain includes hinge domain structures known in the art for chimeric antigen receptors. For example, the hinge domain includes, but is not limited to, a hinge domain of CD8 or CD28, or a functional fragment thereof. For example, the hinge domain includes the sequence described in SEQ ID NO: 32 or 33.

[0058] For example, the antigen-binding protein further comprises a transmembrane domain directly or indirectly linked to the hinge domain. For example, the transmembrane domain of this application includes intracellular domain structures known in the art for chimeric antigen receptors. For example, the transmembrane domain includes, but is not limited to, a transmembrane domain of CD8 or CD28, or a functional fragment thereof. For example, the transmembrane domain includes the sequence described in SEQ ID NO: 34 or 35.

[0059] For example, the antigen-binding protein further comprises an intracellular domain directly or indirectly linked to the transmembrane domain. For example, the intracellular domain of this application comprises an intracellular domain structure known in the art for chimeric antigen receptors. For example, the antigen-binding protein comprises one or more intracellular domains. For example, the antigen-binding protein comprises 1, 2, 3, 4, or 5 additional intracellular domains.

[0060] For example, the intracellular domain may include a co-stimulatory signaling domain and / or a signal transduction domain. For example, the intracellular domain may include one, two, three, four, or five co-stimulatory signaling domains. For example, the intracellular domain may include one co-stimulatory signaling domain. For example, the co-stimulatory domain includes, but is not limited to, the co-stimulatory domain of 41-BB, CD28, or OX40 or a functional fragment thereof. For example, the co-stimulatory domain includes the sequence described in SEQ ID NO: 36, SEQ ID NO: 37, or SEQ ID NO: 38. For example, the co-stimulatory domain includes the co-stimulatory domain of 41-BB, CD28, or a functional fragment thereof. For example, the co-stimulatory domain includes the sequences described in SEQ ID NO: 36 and SEQ ID NO: 37. For example, the co-stimulatory domain includes the co-stimulatory domain of 41-BB or a functional fragment thereof. For example, the co-stimulatory domain includes the sequence described in SEQ ID NO: 36. For example, the co-stimulatory domain includes the co-stimulatory domain of CD28 or a functional fragment thereof. For example, the co-stimulatory domain includes the sequence described in SEQ ID NO: 37. For example, the co-stimulatory domain includes the co-stimulatory domain of OX40 or a functional fragment thereof. For example, the co-stimulatory domain includes the sequence described in SEQ ID NO: 38. For example, the intracellular domain may include 1, 2, 3, 4, or 5 signaling domains. For example, the signaling domain includes, but is not limited to, the signaling domain of CD3ζ or a functional fragment thereof. For example, the signaling domain includes the sequence described in SEQ ID NO: 39.

[0061] For example, antigen-binding proteins include chimeric antigen receptors.

[0062] For example, a chimeric antigen receptor may include, in order from the N-terminus to the C-terminus, an antigen-binding domain, a hinge domain, a transmembrane domain, and / or an intracellular domain.

[0063] For example, the chimeric antigen receptor of this application includes the H3L3 heavy chain variable region described in SEQ ID NO: 23, the H3L3 light chain variable region described in SEQ ID NO: 25, the CD28 hinge domain described in SEQ ID NO: 32, the CD28 transmembrane domain described in SEQ ID NO: 34, the CD28 intracellular costimulatory domain described in SEQ ID NO: 37, the 41-BB intracellular costimulatory domain described in SEQ ID NO: 36, and the CD3ζ intracellular signaling domain described in SEQ ID NO: 39.

[0064] For example, the chimeric antigen receptor of this application includes the H3L3 heavy chain variable region described in SEQ ID NO: 23, the H3L3 light chain variable region described in SEQ ID NO: 25, the CD8 hinge domain described in SEQ ID NO: 33, the CD28 transmembrane domain described in SEQ ID NO: 34, the CD28 intracellular costimulatory domain described in SEQ ID NO: 37, the 41-BB intracellular costimulatory domain described in SEQ ID NO: 36, and the CD3ζ intracellular signaling domain described in SEQ ID NO: 39.

[0065] For example, the chimeric antigen receptor of this application includes the H3L3 heavy chain variable region described in SEQ ID NO: 23, the H3L3 light chain variable region described in SEQ ID NO: 25, the CD28 hinge domain described in SEQ ID NO: 32, the CD28 transmembrane domain described in SEQ ID NO: 34, the CD28 intracellular costimulatory domain described in SEQ ID NO: 37, and the CD3ζ intracellular signaling domain described in SEQ ID NO: 39.

[0066] For example, the chimeric antigen receptor of this application includes the H3L3 heavy chain variable region described in SEQ ID NO: 23, the H3L3 light chain variable region described in SEQ ID NO: 25, the CD8 hinge domain described in SEQ ID NO: 33, the CD28 transmembrane domain described in SEQ ID NO: 34, the CD28 intracellular costimulatory domain described in SEQ ID NO: 37, and the CD3ζ intracellular signaling domain described in SEQ ID NO: 39.

[0067] For example, the chimeric antigen receptor of this application includes the H3L3 heavy chain variable region described in SEQ ID NO: 23, the H3L3 light chain variable region described in SEQ ID NO: 25, the CD28 hinge domain described in SEQ ID NO: 32, the CD28 transmembrane domain described in SEQ ID NO: 34, the 41BB intracellular costimulatory domain described in SEQ ID NO: 36, and the CD3ζ intracellular signaling domain described in SEQ ID NO: 39.

[0068] For example, the chimeric antigen receptor of this application includes the H3L3 heavy chain variable region described in SEQ ID NO: 23, the H3L3 light chain variable region described in SEQ ID NO: 25, the CD8 hinge domain described in SEQ ID NO: 33, the CD28 transmembrane domain described in SEQ ID NO: 34, the 41BB intracellular costimulatory domain described in SEQ ID NO: 36, and the CD3ζ intracellular signaling domain described in SEQ ID NO: 39.

[0069] For example, the chimeric antigen receptor of this application includes the H3L3 heavy chain variable region described in SEQ ID NO: 23, the H3L3 light chain variable region described in SEQ ID NO: 25, the CD28 hinge domain described in SEQ ID NO: 32, the CD28 transmembrane domain described in SEQ ID NO: 34, the OX40 intracellular costimulatory domain described in SEQ ID NO: 38, and the CD3ζ intracellular signaling domain described in SEQ ID NO: 39.

[0070] For example, the chimeric antigen receptor of this application includes the H3L3 heavy chain variable region described in SEQ ID NO: 23, the H3L3 light chain variable region described in SEQ ID NO: 25, the CD8 hinge domain described in SEQ ID NO: 33, the CD28 transmembrane domain described in SEQ ID NO: 34, the OX40 intracellular costimulatory domain described in SEQ ID NO: 38, and the CD3ζ intracellular signaling domain described in SEQ ID NO: 39.

[0071] For example, the chimeric antigen receptor of this application includes the PP6373 heavy chain variable region described in SEQ ID NO: 24, the PP6373 light chain variable region described in SEQ ID NO: 26, the CD28 hinge domain described in SEQ ID NO: 32, the CD28 transmembrane domain described in SEQ ID NO: 34, the CD28 intracellular costimulatory domain described in SEQ ID NO: 37, the 41-BB intracellular costimulatory domain described in SEQ ID NO: 36, and the CD3ζ intracellular signaling domain described in SEQ ID NO: 39.

[0072] For example, the chimeric antigen receptor of this application includes the PP6373 heavy chain variable region described in SEQ ID NO: 24, the PP6373 light chain variable region described in SEQ ID NO: 26, the CD8 hinge domain described in SEQ ID NO: 33, the CD28 transmembrane domain described in SEQ ID NO: 34, the CD28 intracellular costimulatory domain described in SEQ ID NO: 37, the 41-BB intracellular costimulatory domain described in SEQ ID NO: 36, and the CD3ζ intracellular signaling domain described in SEQ ID NO: 39.

[0073] For example, the chimeric antigen receptor of this application includes the PP6373 heavy chain variable region described in SEQ ID NO: 24, the PP6373 light chain variable region described in SEQ ID NO: 26, the CD28 hinge domain described in SEQ ID NO: 32, the CD28 transmembrane domain described in SEQ ID NO: 34, the CD28 intracellular costimulatory domain described in SEQ ID NO: 37, and the CD3ζ intracellular signaling domain described in SEQ ID NO: 39.

[0074] For example, the chimeric antigen receptor of this application includes the PP6373 heavy chain variable region described in SEQ ID NO: 24, the PP6373 light chain variable region described in SEQ ID NO: 26, the CD8 hinge domain described in SEQ ID NO: 33, the CD28 transmembrane domain described in SEQ ID NO: 34, the CD28 intracellular costimulatory domain described in SEQ ID NO: 37, and the CD3ζ intracellular signaling domain described in SEQ ID NO: 39.

[0075] For example, the chimeric antigen receptor of this application includes the PP6373 heavy chain variable region described in SEQ ID NO: 24, the PP6373 light chain variable region described in SEQ ID NO: 26, the CD28 hinge domain described in SEQ ID NO: 32, the CD28 transmembrane domain described in SEQ ID NO: 34, the 41-BB intracellular costimulatory domain described in SEQ ID NO: 36, and the CD3ζ intracellular signaling domain described in SEQ ID NO: 39.

[0076] For example, the chimeric antigen receptor of this application includes the PP6373 heavy chain variable region described in SEQ ID NO: 24, the PP6373 light chain variable region described in SEQ ID NO: 26, the CD8 hinge domain described in SEQ ID NO: 33, the CD28 transmembrane domain described in SEQ ID NO: 34, the 41-BB intracellular costimulatory domain described in SEQ ID NO: 36, and the CD3ζ intracellular signaling domain described in SEQ ID NO: 39.

[0077] For example, the chimeric antigen receptor of this application includes the PP6373 heavy chain variable region described in SEQ ID NO: 24, the PP6373 light chain variable region described in SEQ ID NO: 26, the CD28 hinge domain described in SEQ ID NO: 32, the CD28 transmembrane domain described in SEQ ID NO: 34, the OX40 intracellular costimulatory domain described in SEQ ID NO: 38, and the CD3ζ intracellular signaling domain described in SEQ ID NO: 39.

[0078] For example, the chimeric antigen receptor of this application includes the PP6373 heavy chain variable region described in SEQ ID NO: 24, the PP6373 light chain variable region described in SEQ ID NO: 26, the CD8 hinge domain described in SEQ ID NO: 33, the CD28 transmembrane domain described in SEQ ID NO: 34, the OX40 intracellular costimulatory domain described in SEQ ID NO: 38, and the CD3ζ intracellular signaling domain described in SEQ ID NO: 39.

[0079] For example, the chimeric antigen receptor of this application may also be linked to a chemokine receptor via a self-cleaving peptide at the C-terminus of the CD3ζ intracellular signaling domain. In another example, the chimeric antigen receptor of this application may also be linked to a cytokine via a self-cleaving peptide at the C-terminus of the CD3ζ intracellular signaling domain. For example, linkage may be downward via a 2A peptide. An exemplary 2A peptide may be P2A or T2A having the sequences described in SEQ ID NOs. 51 and 52.

[0080] For example, a chemokine receptor may be CXCR2 or a functional fragment thereof. An exemplary amino acid sequence of CXCR2 may be as described in SEQ ID NO: 53.

[0081] For example, the cytokine may be IL-12β or a functional fragment thereof. An exemplary amino acid sequence of IL-12β may be as described in SEQ ID NO: 50.

[0082] For example, the improved antigen-binding protein of this application comprises the amino acid sequence described in any one of SEQ ID NOs: 1-21, 48, and 49.

[0083] For example, the improved antigen-binding protein of this application includes the amino acid sequence described in SEQ ID NO: 1.

[0084] For example, the improved antigen-binding protein of this application includes the amino acid sequence described in SEQ ID NO: 2.

[0085] For example, the improved antigen-binding protein of this application includes the amino acid sequence described in SEQ ID NO: 3.

[0086] For example, the improved antigen-binding protein of this application includes the amino acid sequence described in SEQ ID NO: 4.

[0087] For example, the improved antigen-binding protein of this application includes the amino acid sequence described in SEQ ID NO: 5.

[0088] For example, the improved antigen-binding protein of this application includes the amino acid sequence described in SEQ ID NO: 6.

[0089] For example, the improved antigen-binding protein of this application includes the amino acid sequence described in SEQ ID NO: 7.

[0090] For example, the improved antigen-binding protein of this application includes the amino acid sequence described in SEQ ID NO: 8.

[0091] For example, the improved antigen-binding protein of this application includes the amino acid sequence described in SEQ ID NO: 9.

[0092] For example, the improved antigen-binding protein of this application includes the amino acid sequence described in SEQ ID NO: 10.

[0093] For example, the improved antigen-binding protein of this application includes the amino acid sequence described in SEQ ID NO: 11.

[0094] For example, the improved antigen-binding protein of this application includes the amino acid sequence described in SEQ ID NO: 12.

[0095] For example, the improved antigen-binding protein of this application includes the amino acid sequence described in SEQ ID NO: 13.

[0096] For example, the improved antigen-binding protein of this application includes the amino acid sequence described in SEQ ID NO: 14.

[0097] For example, the improved antigen-binding protein of this application includes the amino acid sequence described in SEQ ID NO: 15.

[0098] For example, the improved antigen-binding protein of this application includes the amino acid sequence described in SEQ ID NO: 16.

[0099] For example, the improved antigen-binding protein of this application includes the amino acid sequence described in SEQ ID NO: 17.

[0100] For example, the improved antigen-binding protein of this application includes the amino acid sequence described in SEQ ID NO: 18.

[0101] For example, the improved antigen-binding protein of this application includes the amino acid sequence described in SEQ ID NO: 19.

[0102] For example, the improved antigen-binding protein of this application includes the amino acid sequence described in SEQ ID NO: 20.

[0103] For example, the improved antigen-binding protein of this application includes the amino acid sequence described in SEQ ID NO: 21.

[0104] For example, the improved antigen-binding protein of this application includes the amino acid sequence described in SEQ ID NO: 48.

[0105] For example, the improved antigen-binding protein of this application includes the amino acid sequence described in SEQ ID NO: 49.

[0106] For example, antigen-binding proteins include chimeric antigen receptors.

[0107] For example, the chimeric antigen receptor of this application may be expressed on the surface of immune cells. For example, the immune cells of this application may include immune effector cells. For example, the immune cells of this application may include T cells, NK cells, and mixtures of the above cells. For example, the immune cells of this application may include one or more chimeric antigen receptors of this application.

[0108] In one embodiment, the present application provides a polypeptide that may contain the antigen-binding protein of the present application. For example, the polypeptide of the present application may further contain one or more full-length cytokines or fragments thereof.

[0109] In one embodiment, the present application provides nucleic acids that can encode the antigen-binding protein and / or polypeptide of the present application.

[0110] In one embodiment, the present application provides a vector that may contain the nucleic acid of the present application.

[0111] In one embodiment, the present application provides a cell which may include the antigen-binding protein of the present application, the polypeptide of the present application, the nucleic acid of the present application and / or the vector of the present application. For example, the cell may include immune cells. For example, the cell may include NK cells and / or T cells.

[0112] In one embodiment, the present application provides a method for preparing the antigen-binding protein and / or polypeptide of the present application, which may include culturing cells of the present application under conditions that enable the expression of the antigen-binding protein and / or polypeptide.

[0113] In one embodiment, the present application provides a composition that may comprise the antigen-binding protein of the present application, the polypeptide of the present application, the nucleic acid of the present application, the vector of the present application, and / or the cell of the present application, and optionally, a pharmaceutically acceptable carrier. For example, the pharmaceutically acceptable carrier may comprise any or all of a solvent, dispersion medium, isotonic agent, and absorption retarder that are compatible with immunoeffector cells. Such carriers are generally safe and nontoxic.

[0114] In one embodiment, the present application provides a kit that may comprise the antigen-binding protein of the present application, the polypeptide of the present application, the nucleic acid of the present application, the vector of the present application, the cells of the present application, and / or the pharmaceutical composition of the present application.

[0115] In one embodiment, the present application provides a method for stimulating an immune response, which may include administering an antigen-binding protein, a polypeptide, a nucleic acid, a vector, a cell, a pharmaceutical composition, and / or a kit of the present application. For example, a method for stimulating an immune response may be in vitro and / or ex vivo. For example, a method for stimulating an immune response may be a method for non-therapeutic purposes. In one embodiment, stimulating an immune response may involve increasing the ability of target cells to kill, decreasing their ability to release cytokines, and / or increasing the activation level of cells.

[0116] In one embodiment, the present application provides the use of the antigen-binding protein, polypeptide, nucleic acid, vector, cell, pharmaceutical composition, and / or kit of the present application in the preparation of a pharmaceutical, the pharmaceutical used to prevent, alleviate, and / or treat a tumor. For example, the tumor may include solid tumors and / or hematological malignancies. For example, the tumor may be selected from the group consisting of gastric cancer, liver cancer, and leukemia. For example, the tumor of the present application may be selected from all cancer types, for example, the tumor may include tumors that are positive for tumor antigens or have high expression of tumor antigens. For example, the tumor may be selected from the group consisting of lung cancer, breast cancer, liver cancer, brain cancer, cervical cancer, ovarian cancer, kidney cancer, testicular cancer, prostate cancer, and neuroblastoma.

[0117] In one embodiment, the present application provides antigen-binding proteins, polypeptides, nucleic acids, vectors, cells, pharmaceutical compositions, and / or kits that can be used to prevent, mitigate, and / or treat tumors. For example, tumors may include solid tumors and / or hematological malignancies. For example, tumors may be selected from the group consisting of gastric cancer, liver cancer, and leukemia. For example, tumors of the present application may be selected from all cancer types, for example, tumors may include tumors that are positive for tumor antigens or have high expression of tumor antigens. For example, tumors may be selected from the group consisting of lung cancer, breast cancer, liver cancer, brain cancer, cervical cancer, ovarian cancer, kidney cancer, testicular cancer, prostate cancer, and neuroblastoma.

[0118] In one embodiment, the present application provides a method for preventing, mitigating and / or treating a tumor, comprising administering the antigen-binding protein of the present application, the polypeptide of the present application, the nucleic acid of the present application, the vector of the present application, the cells of the present application, the pharmaceutical composition of the present application and / or the kit of the present application. For example, the tumor may include solid tumors and / or hematological malignancies. For example, the tumor may be selected from the group consisting of gastric cancer, liver cancer and leukemia. For example, the tumor of the present application may be selected from all cancer types, for example, the tumor may include tumors that are positive for tumor antigens or have high expression of tumor antigens. For example, the tumor may be selected from the group consisting of lung cancer, breast cancer, liver cancer, brain cancer, cervical cancer, ovarian cancer, kidney cancer, testicular cancer, prostate cancer and neuroblastoma.

[0119] In another aspect, the present application further provides the following embodiments.

[0120] 1. A group of improved antigen-binding protein G1s in which G1 can bind to CD24, and the enhanced effect of cells expressing said G1 on the ability to kill CD24-positive tumor cells, or on the ability of cells expressing said G1 to express cytokines after stimulation by CD24-positive tumor cells, is higher than that of cells expressing existing CD24 antigen-binding proteins.

[0121] 2. The antigen-binding protein G1 of Embodiment 1, wherein the cytokine comprises IFN-γ and / or IL-2.

[0122] 3. A group of further improved antigen-binding protein G2s based on any one of the G1s in Embodiments 1-2, wherein G2 can bind to CD24, and G2-expressing cells maintain the ability to kill CD24-positive tumor cells and express cytokines after stimulation by CD24-positive tumor cells at least about 50% lower than that of cells expressing G1.

[0123] 4. A group of improved antigen-binding protein G3s for use in intracellular expression, based on any one of the G1s of Embodiments 1-2 or the G2 of Embodiment 3, wherein the G3-expressing cells co-express a chemokine receptor, preferably the chemokine receptor comprising CXCR2.

[0124] 5. A group of G3 antigen-binding proteins for use in intracellular expression, wherein G3-expressing cells co-express cytokines, preferably including IL-12β, based on any one of the G1s of Embodiments 1-2, or a further improved antigen-binding protein based on the G2 of Embodiment 3.

[0125] 6. An antigen-binding protein according to any one of Embodiments 1 to 5, wherein the antigen-binding fragment comprises a light chain variable region and a heavy chain variable region, and has a linker between the light chain variable region and the heavy chain variable region, and the linker has a length of 10 amino acids or less.

[0126] 7. The antigen-binding protein of Embodiment 6, wherein the linker has a length of 5 amino acids or less.

[0127] 8. Any one of Embodiments 1 to 7, wherein the heavy chain variable region described in SEQ ID NO: 23 or 24 contains H-CDRs 1 to 3, and the light chain variable region described in SEQ ID NO: 25 or 26 contains L-CDRs 1 to 3.

[0128] 9. An antigen-binding protein according to any one of Embodiments 1 to 8, wherein the heavy chain variable region contains H-CDR 1 to 3 as described in SEQ ID NOs. 42 to 44, and the light chain variable region contains L-CDR 1 to 3 as described in SEQ ID NOs. 45 to 47.

[0129] 10. An antigen-binding protein according to any one of Embodiments 1 to 9, wherein the signal peptide comprises the sequence described in SEQ ID NO: 22.

[0130] 11. Any one of the antigen-binding proteins from Embodiments 1 to 10, further comprising a hinge domain directly or indirectly linked to the antigen-binding domain.

[0131] 12. The antigen-binding protein of Embodiment 11, wherein the hinge domain comprises a hinge domain of CD8 or CD28, or a functional fragment thereof.

[0132] 13. An antigen-binding protein from any one of embodiments 11 to 12, wherein the hinge domain contains the sequence described in SEQ ID NO: 32 or 33.

[0133] 14. Any one of the antigen-binding proteins from Embodiments 1 to 13, further comprising a transmembrane domain directly or indirectly linked to a hinge domain.

[0134] 15. The antigen-binding protein of Embodiment 14, wherein the transmembrane domain comprises a transmembrane domain of CD8 or CD28, or a functional fragment thereof.

[0135] 16. An antigen-binding protein according to any one of embodiments 14 to 15, wherein the transmembrane domain comprises the sequence described in SEQ ID NO: 34 or 35.

[0136] 17. Any one of embodiments 1 to 16 further comprising an intracellular domain directly or indirectly linked to a transmembrane domain.

[0137] 18. The antigen-binding protein of Embodiment 17, wherein the intracellular domain comprises a costimulatory domain containing a costimulatory domain of 41-BB, CD28, or OX40, or a functional fragment thereof.

[0138] 19. The antigen-binding protein of Embodiment 18, comprising one or more of the co-stimulatory domains.

[0139] 20. An antigen-binding protein from any one of embodiments 18 to 19, wherein the co-stimulatory domain comprises the sequence described in SEQ ID NO: 36, 37, or 38.

[0140] 21. Any one of the antigen-binding proteins of Embodiments 1 to 20, wherein the intracellular domain comprises a signaling domain including a signaling domain of CD3ζ or a functional segment thereof.

[0141] 22. The antigen-binding protein of Embodiment 21, wherein the intracellular signaling domain includes the sequence described in Sequence ID No. 39.

[0142] 23. An antigen-binding protein comprising a chimeric antigen receptor, one of any one of embodiments 1 to 22.

[0143] 24. Antigen-binding proteins of embodiments 1 to 23, selected from any one of the sequences described in SEQ ID NOs: 1 to 21, 48, and 49.

[0144] 25. A polypeptide comprising one antigen-binding protein from any one of Embodiments 1 to 24.

[0145] 26. A polypeptide of Embodiment 25, further comprising a tagged protein containing the sequence described in Sequence ID No. 40.

[0146] 27. A polypeptide of Embodiment 26, wherein a polypeptide tagged protein and an antigen-binding protein are linked via the sequence described in SEQ ID NO: 41.

[0147] 28. A nucleic acid encoding any one antigen-binding protein from Embodiments 1 to 24 and / or any one polypeptide from Embodiments 25 to 27.

[0148] 29. A vector containing the nucleic acid of Embodiment 28.

[0149] 30. A cell comprising one antigen-binding protein from any of Embodiments 1 to 24, one polypeptide from any of Embodiments 25 to 27, the nucleic acid of Embodiment 28, and / or the vector of Embodiment 29.

[0150] 31. Cells of Embodiment 30, including immune cells.

[0151] 32. Any one of the cells from Embodiments 30 to 31, comprising NK cells and / or T cells.

[0152] 33. A method for preparing an antigen-binding protein from any one of Embodiments 1 to 24 and / or a polypeptide from any one of Embodiments 25 to 27, comprising culturing cells from any one of Embodiments 30 to 32 under conditions that enable the expression of the antigen-binding protein and / or polypeptide.

[0153] 34. A composition comprising an antigen-binding protein from any one of Embodiments 1 to 24, a polypeptide from any one of Embodiments 25 to 27, a nucleic acid from Embodiment 28, a vector from Embodiment 29, and / or a cell from any one of Embodiments 30 to 32, and optionally a pharmaceutically acceptable carrier.

[0154] 35. A kit comprising one antigen-binding protein from any of Embodiments 1 to 24, one polypeptide from any of Embodiments 25 to 27, a nucleic acid from Embodiment 28, a vector from Embodiment 29, one cell from any of Embodiments 30 to 32, and / or a composition from Embodiment 34.

[0155] 36. A method for stimulating an immune response, comprising administering an antigen-binding protein from any one of Embodiments 1 to 24, a polypeptide from any one of Embodiments 25 to 27, a nucleic acid from Embodiment 28, a vector from Embodiment 29, a cell from any one of Embodiments 30 to 32, a composition from Embodiment 34, and / or a kit from Embodiment 35.

[0156] 37. Use of any one antigen-binding protein of Embodiments 1 to 24, any one polypeptide of Embodiments 25 to 27, a nucleic acid of Embodiment 28, a vector of Embodiment 29, any one cell of Embodiments 30 to 32, a composition of Embodiment 34, and / or a kit of Embodiment 35 in the preparation of a pharmaceutical product, the pharmaceutical product being used to prevent, alleviate, and / or treat tumors.

[0157] 38. Use of Embodiment 37, wherein the tumor includes solid tumors and / or hematological malignancies.

[0158] 39. Use of any one of Embodiments 37 to 38, wherein the tumor is selected from the group consisting of lung cancer, breast cancer, liver cancer, brain cancer, cervical cancer, ovarian cancer, kidney cancer, testicular cancer, prostate cancer, and neuroblastoma.

[0159] While we do not wish to be bound by any theory, the following examples are merely illustrative of the proteins, preparation methods, and uses of this application, and are not intended to limit the scope of the invention. [Examples]

[0160] [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7] [Table 1-8]

[0161] Example 1 Expression of H3L3-CAR and PP6373-CAR Anti-CD24 antibodies can be used to react with a wide range of cancer cells and produce chimeric antigen receptors to confer anticancer activity to T cells. In one embodiment, an antigen-binding fragment of CD24 was inserted into a CAR-T vector known in the art. These antigen-binding fragments include, but are not limited to, a PP6373 / H3L3 single-stranded Fv sequence (where the heavy chain variable region may be as described in SEQ ID NO: 24 and the light chain variable region as described in SEQ ID NO: 26, or where the heavy chain variable region may be as described in SEQ ID NO: 23 and the light chain variable region as described in SEQ ID NO: 25) or another single-stranded anti-CD24 mAb (alpha-CD24SC). The construct was then inserted into a gene vector known in the art, including those derived from retroviruses, lentiviruses, adeno-associated viruses, or adenovirus vectors.

[0162] (I) Preparation of T lymphocytes PBMCs (Day 0) from healthy donors were stimulated for 24 hours with anti-human CD3, anti-human CD28, and recombinant human fibronectin (RetroNectin, RN). Activated T cells were either mocked (control T cells) or infected with a lentivirus containing CD24-CAR (Day 1). On Days 3 and 5, the culture medium was replenished and the cells were expanded. On Day 7, the cells were stained with CD24-FITC, and the expression of antigen-binding regions on the surface of T cells was identified by flow cytometry to detect FITC and RFP signals.

[0163] (II) In vitro cytotoxicity test To test the antitumor activity of CAR-T cells, control T cells or CD24 CAR-T cells were co-cultured overnight with tumor cells that highly expressed the target molecule CD24 and possessed luciferase activity. Tumor cell lysis was measured using a luciferase detection kit (Yeasen) and calculated using the following formula: Lysis % = 100% - co-culture signal / tumor cell signal.

[0164] (III) Cytokine assay To evaluate the safety of CAR-T cells, the expression levels of interferon-gamma (IFN-γ) and interleukin-2 (IL-2) in the supernatant of co-cultures of CAR-T cells and tumor cells were measured using an ELISA kit (Invitrogen).

[0165] First, the structures of H3L3-CAR and PP6373-CAR were optimized by removing the five amino acids preceding 4-1BB, respectively, to obtain C1 and C2. As shown in Figure 1, the binding ability of the antigen-binding domains of C1 and C2 on the surface of T cells was significantly increased. C1 and C2 also exhibited increased antitumor activity and cytokine expression levels. The above results indicate that, compared to the previous H3L3-CAR and PP6373-CAR, the structurally optimized C1 and C2 can express the CAR structure more efficiently, resulting in stronger antitumor activity and higher cytokine secretion levels.

[0166] Example 2 Structural optimization of the CD24-CAR By modifying the hinge region, transmembrane region, and co-stimulatory domain of the CAR structure based on C1, CAR structures such as C3, C5, C7, C9, C11, C13, and C15 were induced (Figure 2). Accordingly, by modifying the hinge region, transmembrane region, and co-stimulatory domain of the CAR structure based on C2, CAR structures such as C4, C6, C8, C10, C12, C14, and C16 were induced (Figure 5). Co-culture results showed that C5, C8, C9, C10, and C11 possessed excellent antitumor activity (Figures 3 and 6).

[0167] Based on further detection of cytokine secretion, it was found that C7 / C8 produces low levels of IFN-γ and IL-2 (Figures 4 and 7), demonstrating that the C7 / C8 CAR structure (hinge region: CD8, transmembrane region: CD28™, and costimulatory domain: CD28) possesses excellent antitumor activity and superior safety. Furthermore, it was shown that the second-generation CAR structure of CD24-CAR can achieve greater in vitro cytotoxicity than the third generation.

[0168] Example 3 Optimization of the linker in the CD24-CAR structure To further optimize the CAR structure, the linkers between the heavy and light chains of the C6 and C8 antibodies were modified, shortening the amino acid sequence from (GGGGS)3 to GGGGS to obtain C27 and C28, and changing the C7 transmembrane region CD28™ to CD8™ to obtain C23 (Figure 8). By comparing the in vitro cytotoxic efficacy and cytokine secretion levels of these CAR structures, it was found that there was little difference in in vitro cytotoxic efficacy in CAR-T cells (Figure 9), but C28 with the shortened linker significantly reduced cytokine secretion levels (Figure 10).

[0169] To further investigate the effect of linker length on CAR function, the linker of C7 was extended from (GGGGS)3 to (GGGGS)4 to obtain C29, while the linker of C8 was shortened from (GGGGS)3 to (GGGGS)2 to obtain C30 (Figure 11). Based on in vitro experiments, C29 with the extended linker was found to have a significant increase in cytotoxic efficacy and IFN-γ secretion levels compared to C7 (Figure 12). By comparing C8, C28, and C30, a positive correlation was also found between linker length and IFN-γ secretion levels. By comparing C8, C28, and C30, a positive correlation was found between linker length and IFN-γ and IL-2 secretion levels, indicating that shortening the linker length had little effect on the cytotoxic efficacy of CAR-T. The results above demonstrate that the inventors can optimize the antitumor effect and cytokine secretion levels of CAR-T cells by modifying linker length, and that, in the case of high antigen-target affinity, shortening the linker can maintain the cytotoxic efficacy of CAR-T cells while significantly reducing cytokine production. Therefore, C28 was even more effective in avoiding the risk of cytokine storms during treatment.

[0170] Example 4 Functional optimization of the CD24-CAR To enhance the chemotactic ability of CAR-T cells and the persistence of cytokine IFN-γ secretion, the chemokine receptor CXCR2 and the cytokine IL-12β were linked to the CD3ζ 3' terminus of the C28 CAR sequence via a P2A linker to obtain C31 and C32 (Figure 13). Based on in vitro experiments, C31 expressing the chemokine receptor CXCR2 showed significantly increased cytotoxicity against SK-OV-3 target cells compared to C28, and the concentrations of IFN-γ, TNF-α, IL-2, and other cytokines produced by killing target cells SK-OV-3 and NIH-OVCAR3 were also significantly increased (Figures 14 and 15). By comparing C28 and C32, it was found that there was no significant difference in in vitro cytotoxicity between C32 and C28 expressing IL-12β, but the secretion levels of cytokines such as IFN-γ, TNF-α, and IL-2 were significantly increased (Figure 16). These results suggest that the expression of the chemokine receptor CXCR2 helped improve the antitumor effect and cytokine secretion levels of C28, while IL-12β can significantly enhance cytokine release from C28.

[0171] Example 5 Animal model testing (I) Verification of in vivo functionality for C8 and C28 Purchased 4-5 week old NCG immunodeficient mice were reared in SPF-grade animal rooms for one week. Then, each mouse was given 2 x 10⁶ doses. 6 SK-OV-3-luc cells were injected intraperitoneally. Nineteen days after abdominal tumor formation, intraperitoneal tumors were detected in mice by invivoymeger. Mice were grouped according to tumor size, with 6 × 10⁶ cells in each case. 6Individual C8, C28, and NC (i.e., mock T) cells, along with saline as a blank control, were injected into the tail vein. The body weight of the mice was measured weekly, and the distribution and size of tumors in these mice were monitored using an in vivo measure. As shown in Figure 17, C8 and C28 substantially eliminated tumor cells two weeks after CAR-T reinfusion. The results showed no significant difference between C8 and C28 in terms of antitumor capacity, indicating that C28 has superior in vivo safety.

[0172] (II) Verification of the in vivo migratory ability of C28 and C31 Purchased 4-5 week old NCG immunodeficient mice were reared in SPF-grade animal rooms for one week. Then, 2 x 10 units were placed on the back of each mouse. 6 SK-OV-3-luc cells were subcutaneously injected. Thirteen days after tumor formation, tumors were detected on the backs of mice by in vitro measuring. Mice were grouped by tumor size, and each case was tagged with the fluorescent dye DiD (1 × 10⁶). 7 Individual C28, C31, and NC (i.e., mock T) cells, along with saline as a blank control, were injected into the tail vein. CAR-T and tumor distribution and size were monitored in these mice using an in vivo measure. As shown in Figure 18, the DiD signal of C31 cells was significantly higher than that of C28 and control cells within 6 days after CAR-T reinjection, indicating that C31 cells possessed stronger in vivo migration and viability.

[0173] (III) Comparison of in vivo functionality of C28 and C31 Purchased 4-5 week old NCG immunodeficient mice were reared in SPF-grade animal rooms for one week. Then, 2 x 10 units were placed on the back of each mouse. 6 SK-OV-3-luc cells were subcutaneously injected. Fourteen days after abdominal tumor formation, intraperitoneal tumors were detected in mice by invivoymeger. Mice were grouped according to tumor size, with 1 × 10⁶ cells in each case. 7Individual C28, C31, and NC (i.e., mock T) cells, along with saline as a blank control, were injected into the tail vein. The body weight of the mice was measured weekly, and the distribution and size of tumors in these mice were monitored using an in vivo measurer. As shown in Figure 19, both C28 and C31 were able to eliminate tumor cells after reinjection; C31 completely eliminated tumor cells in the first week after CAR-T reinjection, and C28 was able to eliminate tumor cells in the second week after reinjection. There were no mouse deaths during the experiment, demonstrating the excellent safety of both C28 and C31.

[0174] (IV) Comparison of in vivo functions of C28, C31, and C32 Purchased 4-5 week old NCG immunodeficient mice were reared in SPF-grade animal rooms for one week. Then, each mouse was given 2 x 10⁶ doses. 6 SK-OV-3-luc cells were injected intraperitoneally. Eighteen days after abdominal tumor formation, intraperitoneal tumors were detected in mice by invivoymeger. Mice were grouped according to tumor size, with 1 × 10⁶ cells in each case. 7 Individual C28, C31, C32, and NC (i.e., mock T) cells, along with saline as a blank control, were injected into the tail vein. Mice were weighed weekly, and tumor distribution and size were monitored using an in vivo measure. As shown in Figure 20, both C31 and C32 substantially eliminated tumor cells in the first week after CAR-T injection and completely eliminated the tumor in the second week after re-injection. However, the death of mice four weeks after T-cell re-injection with C32 is thought to be due to C32's superior amplification ability, leading to a large number of T cells appearing in the peripheral blood of NCG mice, resulting in GVHD and ultimately causing mouse death. Furthermore, C28 was able to substantially eliminate tumor cells in the third week after re-injection, demonstrating a good tumor-removing effect of C28. These results indicate that C31 possesses superior tumor-removing ability and even greater in vivo safety.

[0175] In summary, this application exemplifies the significant potential of the antibody CD24 CAR-T based on this application in cancer treatment. It demonstrated high safety and efficacy in both in vivo and in vitro studies without the risk of off-target effects such as killing normal PBMCs.

[0176] The detailed description provided herein is provided by description and examples and is not intended to limit the scope of the appended claims. Various modifications of the embodiments enumerated in this application to date will be obvious to those skilled in the art and should remain within the scope of the appended claims and their equivalents.

Claims

1. A group of improved antigen-binding protein G1s, wherein G1 can bind to CD24, and the enhanced effect of cells expressing G1 on the ability to kill CD24-positive tumor cells, or on the ability of cells expressing G1 to express cytokines after stimulation by CD24-positive tumor cells, is higher than that of cells expressing existing CD24 antigen-binding proteins.

2. The antigen-binding protein G1 according to claim 1, wherein the cytokine comprises IFN-γ and / or IL-2.

3. A group of further improved antigen-binding protein G2s based on G1 according to any one of claims 1 to 2, wherein G2 can bind to CD24, G2-expressing cells maintain the ability to kill CD24-positive tumor cells, and the ability to express cytokines after stimulation by said CD24-positive tumor cells is at least about 50% lower than that of cells expressing G1.

4. A group of improved antigen-binding protein G3s based on G1 according to any one of claims 1 to 2, or based on G2 according to claim 3, for use in intracellular expression, wherein G3-expressing cells also express chemokine receptors, preferably the chemokine receptors comprising CXCR2.

5. A group of improved antigen-binding protein G3s based on G1 according to any one of claims 1 to 2, or based on G2 according to claim 3, for use in intracellular expression, wherein G3-expressing cells also express cytokines, preferably the cytokines containing IL-12β.

6. The antigen-binding protein according to any one of claims 1 to 5, wherein the antigen-binding fragment comprises a light chain variable region and a heavy chain variable region, and has a linker between the light chain variable region and the heavy chain variable region, and the linker has a length of 10 amino acids or less.

7. The antigen-binding protein according to claim 6, wherein the linker has a length of 5 amino acids or less.

8. The antigen-binding protein according to any one of claims 1 to 7, wherein the heavy chain variable region described in SEQ ID NO: 23 or 24 contains H-CDR 1 to 3, and the light chain variable region described in SEQ ID NO: 25 or 26 contains L-CDR 1 to 3.

9. The antigen-binding protein according to any one of claims 1 to 8, wherein the heavy chain variable region contains H-CDR 1 to 3 as described in SEQ ID NOs. 42 to 44, and the light chain variable region contains L-CDR 1 to 3 as described in SEQ ID NOs. 45 to 47.

10. The antigen-binding protein according to any one of claims 1 to 9, wherein the signal peptide comprises the sequence described in SEQ ID NO:

22.

11. The antigen-binding protein according to any one of claims 1 to 10, further comprising a hinge domain directly or indirectly linked to the antigen-binding domain.

12. The antigen-binding protein according to claim 11, wherein the hinge domain comprises a hinge domain of CD8 or CD28, or a functional fragment thereof.

13. The antigen-binding protein according to any one of claims 11 to 12, wherein the hinge domain comprises the sequence described in SEQ ID NO: 32 or 33.

14. The antigen-binding protein according to any one of claims 1 to 13, further comprising a transmembrane domain directly or indirectly linked to the hinge domain.

15. The antigen-binding protein according to claim 14, wherein the transmembrane domain comprises a transmembrane domain of CD8 or CD28, or a functional fragment thereof.

16. The antigen-binding protein according to any one of claims 14 to 15, wherein the transmembrane domain comprises the sequence described in SEQ ID NO: 34 or 35.

17. The antigen-binding protein according to any one of claims 1 to 16, further comprising an intracellular domain directly or indirectly linked to the transmembrane domain.

18. The antigen-binding protein according to claim 17, wherein the intracellular domain comprises a costimulatory domain containing a costimulatory domain of 41-BB, CD28, or OX40, or a functional fragment thereof.

19. The antigen-binding protein according to claim 18, comprising one or more of the aforementioned costimulatory domains.

20. The antigen-binding protein according to any one of claims 18 to 19, wherein the co-stimulatory domain comprises the sequence described in SEQ ID NO: 36, 37, or 38.

21. The antigen-binding protein according to any one of claims 1 to 20, wherein the intracellular domain comprises a signal transduction domain including a signal transduction domain of CD3ζ or a functional segment thereof.

22. The antigen-binding protein according to claim 21, wherein the intracellular signaling domain comprises the sequence described in SEQ ID NO:

39.

23. An antigen-binding protein according to any one of claims 1 to 22, comprising a chimeric antigen receptor.

24. An antigen-binding protein according to claims 1 to 23, selected from any one of the sequences described in SEQ ID NOs: 1 to 21, 48, and 49.

25. A polypeptide comprising an antigen-binding protein according to any one of claims 1 to 24.

26. The polypeptide according to claim 25, further comprising a tagged protein containing the sequence described in SEQ ID NO:

40.

27. The polypeptide according to claim 26, wherein the tagged protein and the antigen-binding protein of the polypeptide are linked via the sequence described in SEQ ID NO:

41.

28. A nucleic acid encoding an antigen-binding protein according to any one of claims 1 to 24 and / or a polypeptide according to any one of claims 25 to 27.

29. A vector comprising the nucleic acid described in claim 28.

30. A cell comprising an antigen-binding protein according to any one of claims 1 to 24, a polypeptide according to any one of claims 25 to 27, a nucleic acid according to claim 28, and / or a vector according to claim 29.

31. The cells according to claim 30, including immune cells.

32. The cells according to any one of claims 30 to 31, comprising NK cells and / or T cells.

33. A method for preparing an antigen-binding protein and / or a polypeptide according to any one of claims 1 to 24, comprising culturing cells according to any one of claims 30 to 32 under conditions that enable the expression of the antigen-binding protein and / or the polypeptide.

34. A composition comprising an antigen-binding protein according to any one of claims 1 to 24, a polypeptide according to any one of claims 25 to 27, a nucleic acid according to claim 28, a vector according to claim 29 and / or a cell according to any one of claims 30 to 32, and optionally a pharmaceutically acceptable carrier.

35. A kit comprising an antigen-binding protein according to any one of claims 1 to 24, a polypeptide according to any one of claims 25 to 27, a nucleic acid according to claim 28, a vector according to claim 29, a cell according to any one of claims 30 to 32, and / or a composition according to claim 34.

36. A method for stimulating an immune response, comprising administering an antigen-binding protein according to any one of claims 1 to 24, a polypeptide according to any one of claims 25 to 27, a nucleic acid according to claim 28, a vector according to claim 29, a cell according to any one of claims 30 to 32, a composition according to claim 34, and / or a kit according to claim 35.

37. Use in the preparation of a pharmaceutical product, wherein the pharmaceutical product is used to prevent, alleviate and / or treat a tumor.

38. The use according to claim 37, wherein the tumor includes solid tumors and / or hematological malignancies.

39. The use according to any one of claims 37 to 38, wherein the tumor is selected from the group consisting of lung cancer, breast cancer, liver cancer, brain cancer, cervical cancer, ovarian cancer, kidney cancer, testicular cancer, prostate cancer, and neuroblastoma.

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