Antibody that specifically binds to claudin 18.2, method for preparing the same, and its application

Abstract

The present invention provides anti-claudin 18.2 nanobodies or their binding fragments. The antibody exhibits good specificity, and immune effector cells targeting claudin 18.2 prepared using the antibody show good therapeutic efficacy in treating or improving diseases with positive claudin 18.2 expression.

Description

[Technical Field]

[0001] The present invention relates to the field of engineered immunotherapy, and more particularly to antibodies that specifically bind to claudin 18.2, methods for preparing the same, and applications thereof.

[0002] Conventional technology Claudin family proteins are widely distributed in the tight junctions of epithelial cells. Claudin 18.2 is a membrane protein with four transmembrane domains and two extracellular loop domains, with its N-terminus and C-terminus located in the cytoplasm. The two extracellular domains can function as specific targets for targeted therapy. In normal tissues, claudin 18.2 is specifically expressed in highly differentiated gastric epithelial cells. In addition to gastric cancer, claudin 18.2 is also expressed at high rates in tumor cells of other cancers, including bile duct cancer, ovarian cancer, lung cancer, esophageal cancer, and pancreatic cancer.

[0003] Chimeric antigen receptor T cells (CAR T cells) are a novel immunotherapy that targets specific antigens on the surface of tumor cells. They are currently being applied to the development of cell therapies for solid tumors.

[0004] The advantages of camel-derived antibodies compared to conventional antibodies include their stable properties, good water solubility, low immunogenicity, stronger affinity, low molecular weight, ability to penetrate into antigens, and stronger tissue penetration. Camel-derived antibodies can be used as a diagnostic tool, and can also be used for CAR-T delivery and drug targeting, thereby enabling the development of CAR-Ts with higher affinity, stronger specificity, and lower immunogenicity. [Overview of the project]

[0005] The object of the present invention is to provide an antibody that has high affinity and high biological activity and can specifically recognize claudin 18.1 and / or claudin 18.2 antigens, and applications thereof.

[0006] In a first aspect of the present invention, a nanobody that specifically binds to claudin 18.2 is provided. The complementarity-determining region of the VHH chain of the nanobody, i.e., the CDR region, (1) CDR1 shown in Sequence ID No. 36, CDR2 shown in Sequence ID No. 37, and CDR3 shown in Sequence ID No. 38, (2) CDR1 shown in Sequence ID 39, CDR2 shown in Sequence ID 40, and CDR3 shown in Sequence ID 41, (3) CDR1 shown in Sequence ID 42, CDR2 shown in Sequence ID 43, and CDR3 shown in Sequence ID 44, (4) CDR1 shown in Sequence ID 45, CDR2 shown in Sequence ID 46, and CDR3 shown in Sequence ID 47, (5) CDR1 shown in Sequence ID No. 48, CDR2 shown in Sequence ID No. 49, and CDR3 shown in Sequence ID No. 50, (6) CDR1 shown in Sequence ID 51, CDR2 shown in Sequence ID 52, and CDR3 shown in Sequence ID 53, (7) CDR1 shown in Sequence ID 39, CDR2 shown in Sequence ID 54, and CDR3 shown in Sequence ID 41, (8) CDR1 shown in Sequence ID 39, CDR2 shown in Sequence ID 55, and CDR3 shown in Sequence ID 41, (9) CDR1 shown in Sequence ID No. 67, CDR2 shown in Sequence ID No. 68, and CDR3 shown in Sequence ID No. 41, (10) CDR1 shown in Sequence ID No. 39, CDR2 shown in Sequence ID No. 69, and CDR3 shown in Sequence ID No. 41, and / or (11) Selected from the group consisting of CDR1 shown in Sequence ID No. 36, CDR2 shown in Sequence ID No. 65, and CDR3 shown in Sequence ID No. 66.

[0007] In another preferred embodiment, CDR1, CDR2, and CDR3 are separated by framework regions FR1, FR2, FR3, and FR4 of the VHH chain.

[0008] In another preferred embodiment, the nanobodies that specifically bind to claudin 18.2 include humanized antibodies, camel-derived antibodies, and chimeric antibodies.

[0009] In another preferred embodiment, the amino acid sequence of the nanobody that specifically binds to claudin 18.2 is represented by any one of SEQ ID NOs: 1 to 26.

[0010] In another preferred embodiment, the amino acid sequence of the VHH chain of the nanobody is selected from the group consisting of SEQ ID NOs: 1 to 26, or combinations thereof.

[0011] In another preferred embodiment, the CDR region of the nanobody's VHH chain contains an amino acid sequence having at least 80%, preferably at least 90%, more preferably at least 95%, or even more preferably at least 99% sequence identity with any one of SEQ ID NOs: 1 to 26. In another preferred embodiment, the amino acid sequence of the CDR region of the nanobody's VHH chain contains one or more amino acid substitutions, preferably conservative amino acid substitutions, compared to any one of SEQ ID NOs: 1 to 26.

[0012] In another preferred embodiment, any of the above amino acid sequences further comprises a derivative sequence that optionally includes the addition, deletion, modification and / or substitution of at least one (e.g., 1 to 3, preferably 1 to 2, more preferably 1) amino acid and retains specific binding ability to claudin 18.2.

[0013] In another preferred embodiment, claudin 18.2 is human or non-human mammalian claudin 18.2.

[0014] In another preferred embodiment, claudin 18.2 is human, mouse, rat, or non-human primate (such as monkey) claudin 18.2.

[0015] In a second aspect of the present invention, there is provided a multivalent antibody or multispecific antibody that specifically binds to Claudin 18.2, comprising at least one antibody component targeting a Claudin 18.2 epitope, wherein the antibody component is an anti-Claudin 18.2 nanobody described in the first aspect of the present invention.

[0016] In another preferred embodiment, the anti-Claudin 18.2 multivalent antibody or multispecific antibody comprises one or more anti-Claudin 18.2 nanobodies.

[0017] In another preferred embodiment, the anti-Claudin 18.2 antibody comprises a monobody, a diabody (bivalent antibody), a tetrabody (tetravalent antibody), and / or a multibody (multivalent antibody).

[0018] In another preferred embodiment, the anti-Claudin 18.2 antibody comprises one or more VHH chains having an amino acid sequence shown in any one of SEQ ID NOs: 1 to 26.

[0019] In another preferred embodiment, the anti-Claudin 18.2 antibody comprises two VHH chains having an amino acid sequence shown in any one of SEQ ID NOs: 1 to 26.

[0020] In another preferred embodiment, the antibody is selected from animal-derived antibodies, chimeric antibodies, humanized antibodies, or combinations thereof.

[0021] In a third aspect of the present invention, a recombinant protein is provided, (i) a nanobody that specifically binds to Claudin 18.2 described in the first aspect of the present invention, or a multivalent antibody or multispecific antibody that specifically binds to Claudin 18.2 described in the second aspect of the present invention, and (ii) optionally, a tag sequence that aids in expression and / or purification.

[0022] In another preferred embodiment, the antibody is a multivalent antibody.

[0023] In another preferred embodiment, the tag array includes an Fc tag, an HA tag, a flag tag, and a 6His tag.

[0024] In another preferred embodiment, the Fc tag includes hIgG1Fc and mIgG1Fc.

[0025] In another preferred embodiment, the recombinant protein specifically binds to claudin 18.1 and / or claudin 18.2 proteins.

[0026] In a fourth aspect of the present invention, a chimeric antigen receptor (CAR) fusion protein is provided, wherein the chimeric antigen receptor (CAR) fusion protein extends from the N-terminus to the C-terminus. (i) an antigen-binding domain that specifically binds to claudin 18.2, comprising an antigen-binding domain containing a nanobody as described in the first aspect of the present invention, (ii) Transmembrane domain, (iii) at least one co-stimulatory domain, and (iv) Contains an activation domain

[0027] In another preferred embodiment, the antigen-binding domain is monovalent or polyvalent.

[0028] In another preferred embodiment, the antigen-binding domain is derived from the nanobody described in the first aspect of the present invention or the recombinant protein described in the third aspect of the present invention.

[0029] In another preferred embodiment, the CAR has a structure represented by formula Ia. L-VHH-H-TM-C-CD3ζ (Ia) During the ceremony, Each "-" independently represents a linker peptide or peptide bond. L is a signal peptide sequence, VHH is an antigen-binding domain that specifically binds to claudin 18.2. H is the hinge region, TM is a transmembrane domain, C is a co-stimulatory signaling domain, and CD3ζ is a cytoplasmic signaling sequence derived from CD3ζ (including the wild type or its variants / modified versions).

[0030] In another preferred embodiment, L is a signal peptide of a protein selected from the group consisting of CD28, 4-1BB, GM-CSF, CD3, CD8a, or a combination thereof.

[0031] In another preferred embodiment, L is a signal peptide derived from CD8.

[0032] In another preferred embodiment, L comprises the amino acid sequence shown in SEQ ID NO: 27.

[0033] In another preferred embodiment, the amino acid sequence of VHH is shown in SEQ ID NOs: 2, 5, or 18.

[0034] In another preferred embodiment, H comprises the amino acid sequence shown in SEQ ID NO: 28.

[0035] In another preferred embodiment, TM includes a transmembrane region derived from CD28.

[0036] In another preferred embodiment, C is a transmembrane region of a protein selected from the group consisting of CD28, 4-1BB, CD8a, or a combination thereof.

[0037] In another preferred embodiment, C comprises a co-stimulatory signaling molecule derived from 4-1BB.

[0038] In another preferred embodiment, the CAR fusion protein has the amino acid sequence shown in SEQ ID NOs. 33-35.

[0039] In a fifth aspect of the present invention, an antibody-drug conjugate is provided, the antibody-drug conjugate is, (a) a nanobody according to the first aspect of the present invention, a polyvalent antibody or multispecific antibody according to the second aspect of the present invention, or a recombinant protein according to the third aspect of the present invention, (b) A conjugate portion conjugated to an antibody portion, the conjugate portion comprising a conjugate portion selected from the group consisting of a detectable label, a drug, a toxin, a cytokine, a radionuclide, an enzyme, or a combination thereof.

[0040] In another preferred embodiment, the antibody portion is conjugated to the conjugate portion via a chemical bond or linker.

[0041] A sixth aspect of the present invention provides a polynucleotide encoding a protein selected from the group consisting of a nanobody described in the first aspect of the present invention, a multivalent antibody or multispecific antibody described in the second aspect of the present invention, a recombinant protein described in the third aspect of the present invention, or a CAR fusion protein described in the fourth aspect of the present invention.

[0042] A seventh aspect of the present invention provides an expression vector comprising the polynucleotide described in the sixth aspect of the present invention.

[0043] In another preferred embodiment, the expression vector is selected from the group consisting of DNA, RNA, viral vectors, plasmids, transposons, other gene transfer systems, or combinations thereof.

[0044] In another preferred embodiment, the expression vector is a lentiviral vector.

[0045] An eighth aspect of the present invention provides a host cell that expresses an expression vector described in the seventh aspect of the present invention, or a polynucleotide described in the sixth aspect of the present invention incorporated into its genome, or a nanobody described in the first aspect of the present invention, a multivalent antibody or multispecific antibody described in the second aspect of the present invention, a recombinant protein described in the third aspect of the present invention, or a CAR fusion protein described in the fourth aspect of the present invention.

[0046] In another preferred embodiment, the cells are isolated cells and / or the cells are genetically engineered cells.

[0047] In another preferred embodiment, the cells are mammalian cells.

[0048] In another preferred embodiment, the cells are T cells.

[0049] In another preferred embodiment, the host cells are engineered immune cells.

[0050] In another preferred embodiment, the manipulated immune cells are (i) Chimeric antigen receptor αβ T cells (CAR-T cells), (ii) Chimeric antigen receptor γδ T cells (CAR-T cells), (iii) Chimeric antigen receptor NKT cells (CAR-NKT cells), (iv) Chimeric antigen receptor NK cells (CAR-NK cells), and (v) Selected from a group consisting of chimeric antigen receptor macrophages.

[0051] A ninth aspect of the present invention provides a method for preparing engineered immune cells, comprising the step of introducing a nucleic acid molecule described in the sixth aspect of the present invention or a vector described in the seventh aspect of the present invention into T cells or NK cells to obtain engineered immune cells expressing a CAR fusion protein described in the fourth aspect of the present invention.

[0052] In another preferred embodiment, the method further includes the step of determining the function and effectiveness of the resulting manipulated immune cells.

[0053] A tenth aspect of the present invention provides a formulation comprising a nanobody described in the first aspect of the present invention, a multivalent or multispecific antibody described in the second aspect of the present invention, a recombinant protein described in the third aspect of the present invention, a CAR fusion protein described in the fourth aspect of the present invention, a vector described in the seventh aspect of the present invention, or a host cell described in the eighth aspect of the present invention, and a pharmaceutically acceptable carrier, diluent, or excipient.

[0054] In another preferred embodiment, the host cells are engineered immune cells.

[0055] In an eleventh aspect of the present invention, a pharmaceutical composition is provided, (i) an immune cell expressing a nanobody according to the first aspect of the present invention, a polyvalent antibody or multispecific antibody according to the second aspect of the present invention, a recombinant protein according to the third aspect of the present invention, or a CAR fusion protein according to the fourth aspect of the present invention, and (ii) A pharmaceutical composition is provided comprising a pharmaceutically acceptable carrier.

[0056] In another preferred embodiment, the conjugated portion of the immunoconjugate is a drug, toxin, and / or therapeutic isotope.

[0057] In another preferred embodiment, the drug composition further comprises other drugs for treating an immune system disorder or a tumor disorder.

[0058] In another preferred embodiment, the drug composition is used to prepare a drug for preventing and / or treating a disease or condition associated with Claudine 18.2.

[0059] In another preferred embodiment, the disease or condition associated with Claudine 18.2 is selected from the group consisting of breast cancer, gastric cancer, colorectal cancer, pancreatic cancer, prostate cancer, cervical cancer, head and neck cancer, lung cancer, esophageal cancer, kidney cancer, bladder cancer, uterine cancer, ovarian cancer, or a combination thereof.

[0060] A twelfth aspect of the present invention provides a kit comprising a nucleic acid molecule described in the sixth aspect of the present invention, a recombinant protein described in the third aspect of the present invention, or a vector described in the seventh aspect of the present invention.

[0061] In another preferred embodiment, the kit is used to prepare immune cells expressing the receptor CAR fusion protein described in the fourth aspect of the present invention.

[0062] A thirteenth aspect of the present invention provides the use of immune cells expressing the nanobody described in the first aspect of the present invention, the multivalent or multispecific antibody described in the second aspect of the present invention, the recombinant protein described in the third aspect of the present invention, or the CAR fusion protein described in the fourth aspect of the present invention, in the preparation of drugs for the prevention and / or treatment of cancer or tumors related to claudin 18.2.

[0063] A fourteenth aspect of the present invention provides a method for treating a disease, comprising administering immune cells expressing a nanobody described in the first aspect of the present invention, a multivalent or multispecific antibody described in the second aspect of the present invention, a recombinant protein described in the third aspect of the present invention, or a CAR fusion protein described in the fourth aspect of the present invention to a target in need thereof.

[0064] In another preferred embodiment, the disease is a cancer or tumor having positive expression of claudin 18.2.

[0065] Within the scope of the present invention, it should be understood that the above-described technical features of the present invention and the technical features specifically described below (as in the examples) can be combined to form new or preferred technical solutions. Due to space limitations, such technical solutions are not detailed herein. [Brief explanation of the drawing]

[0066] [Figure 1] This shows the binding efficiency of the anti-claudin 18.2 recombinant antibody to 293FT-CLDN18.1 and 293FT-CLDN18.2 cells. [Figure 2] Exemplary designs of single-CAR and dual-epitope CARs for chimeric antigen receptors targeting claudin 18.2 are shown. [Figure 3] This shows the CAR expression levels of T cells targeting claudin 18.2. [Figure 4] This study demonstrates the memory phenotype of CAR-T cells targeting claudin 18.2 under continuous in vitro culture conditions. [Figure 5] This study demonstrates the transient killing effect of CAR-T cells, and the cytotoxicity of CAR-T cells targeting 293FT-claudin 18.2, 293FT-claudin 18.1, and claudin 18.2 for targeting 293FT cells. [Figure 6] This study demonstrates the in vitro cytotoxicity of CAR-T cells targeting claudin 18.2, specifically for use as a cellular AGS-claudin 18.2 target. [Figure 7] This shows the level of cytokine release when CAR-T cells targeting claudin 18.2 are co-cultured with target cells. [Figure 8] This paper compares the in vitro cytotoxicity of humanized claudin 18.2 in CAR-T cells and maternal CAR-T cells. [Figure 9] This shows the binding affinity of the anti-claudin 18.2 recombinant antibody to the claudin 18.2 VLP antigen, as determined by ELISA. [Figure 10] This study demonstrates the in vivo efficacy of CAR-T cells targeting claudin 18.2. [Modes for carrying out the invention]

[0067] Following extensive and thorough research, the inventors unexpectedly obtained, for the first time, an anti-CLDN18.2 antibody with high affinity and excellent antitumor activity. Specifically, after extensive screening, the inventors obtained, for the first time, multiple nanobodies that specifically bind to CLDN18.2. Based on these nanobodies, the inventors further prepared recombinant antibodies, humanized antibodies, and chimeric antigen receptors targeting claudin 18.2. Based on alpaca heavy chain antibodies that specifically bind to claudin 18.2, the inventors provide CAR-T cells with higher affinity, stronger specificity to the target antigen, and lower immunogenicity compared to conventional antibodies. The CAR-T cells of the inventors exhibit excellent target cell-killing activity and can successfully infiltrate solid tumors, thereby demonstrating excellent antitumor effects in animals. The inventors then completed the invention.

[0068] term To facilitate understanding of this disclosure, certain terms are defined first. Where used in this application, unless expressly otherwise specified herein, the following terms shall have the meanings set forth below. Further definitions are provided throughout this application.

[0069] The term “approximately” may mean that a value or composition is within a specific tolerance range for that value or composition as determined by those skilled in the art, which depends in part on how the value or composition is measured or determined. For example, as used herein, the expression “approximately 100” includes 99 and 101 and all values ​​in between (e.g., 99.1, 99.2, 99.3, 99.4).

[0070] As used herein, the terms “comprise” or “contain” may be open-ended, semi-closed-ended, or closed-ended. In other words, the terms also include “substantially composed of” or “composed of.”

[0071] Sequence identity is determined by comparing two aligned sequences along a predetermined comparison window (which may be 50%, 60%, 70%, 80%, 90%, 95%, or 100% of the length of the reference nucleotide sequence or protein) and determining the number of positions in which the same residues appear. Typically, sequence identity is expressed as a percentage. Methods for measuring sequence identity of nucleotide sequences are well known to those skilled in the art.

[0072] As used herein, the terms “heavy chain variable region” and “VH” may be used interchangeably.

[0073] As used herein, the terms “variable region” and “complementarity-determining region (CDR)” can be used interchangeably.

[0074] In the present invention, the terms "antibody of the present invention," "protein of the present invention," or "polypeptide of the present invention" are interchangeable and all refer to antibodies that specifically bind to CLDN18.2, such as proteins or polypeptides having a heavy chain variable region (such as the amino acid sequences described in SEQ ID NOs. 1 to 14). They may or may not contain starting methionine.

[0075] CLDN18.2 Full-length human CLDN18 is a tetratransmembrane protein containing 261 amino acids and possessing four transmembrane hydrophobic regions and two extracellular loop structures. Loop 1 is formed by the bending and connection of transmembrane region 1 and transmembrane region 2, while loop 2 is formed by the bending and connection of transmembrane region 3 and transmembrane region 4. Human CLDN18.1 and CLDN18.2 differ by 8 amino acids in the amino acid composition of the N-terminus, transmembrane region 1, and extracellular loop 1, but are otherwise identical. They have 92% sequence identity.

[0076] antibody As used herein, the term “antibody” refers to immunoglobulin, which is a tetrapeptide chain structure formed from two identical heavy chains and two identical light chains linked by interchain disulfide bonds.

[0077] Existing antibody numbering schemes include the following: 1. The Kabat scheme (Kabat et al., 1991) is based on the location of regions with high sequence variation between sequences of the same domain type. Antibody heavy chain (VH) and light chain (Vλ and Vκ) variable domains are numbered differently. 2. Chothia's scheme (Al-Lazikani, 1997) is identical to Kabat's scheme, but modifies it so that insertions correspond to structural loops when they are annotated around the first VH complementarity determination region (CDR). Similarly, the Enhanced Chothia scheme (Abhinandan and Martin, 2008) performs further structural correction of indel locations. 3. In contrast to these Kabat-like schemes, IMGT (Lefranc, 2003) and AHo (Honegger and Pluckthun, 2001) both define unique schemes for antibody and T cell receptor (TCR) (Vα and Vβ) variable domains. Therefore, equivalent residue positions can be easily compared between domain types. IMGT and AHo differ in the number of positions they annotate (128 and 149, respectively) and the locations where indels are thought to arise.

[0078] Immunoglobulins differ in antigenicity due to differences in the amino acid composition and sequence of the heavy chain constant region. Therefore, immunoglobulins can be classified into five classes or immunoglobulin isotypes, namely IgM, IgD, IgG, IgA, and IgE, with their corresponding heavy chains being μ, δ, γ, α, and ε, respectively. Each class of Ig can be subdivided into different subclasses according to differences in the amino acid composition of the hinge region and the number and position of heavy chain disulfide bonds. For example, IgG can be classified into IgG1, IgG2, IgG3, and IgG4. The light chain can be a κ or λ chain depending on its constant region. Each of the five classes of Ig may have a κ or λ chain. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known to those skilled in the art.

[0079] The light chain of the antibody according to the present invention may further include a light chain constant region, the light chain constant region including a human or mouse κ chain, λ chain, or a variant thereof.

[0080] In the present invention, the heavy chain of the antibody according to the present invention may further include a heavy chain constant region, which includes human or mouse IgG1, IgG2, IgG3, IgG4 or their variants. The sequence of approximately 110 amino acids toward the N-terminus of the antibody's heavy and light chains changes significantly and constitutes a variable region (Fv region). The sequence of other amino acids toward the C-terminus is relatively stable and constitutes a constant region. The variable region includes three hypervariable regions (HVRs) and four framework regions (FRs) having relatively conserved sequences. The three hypervariable regions determine the specificity of the antibody and are also called complementarity-determining regions (CDRs). Each light chain variable region (LCVR) and each heavy chain variable region (HCVR) consists of three CDR regions and four FR regions, which are arranged in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 toward the carboxyl terminus. The three CDR regions of the light chain are named LCDR1, LCDR2, and LCDR3, and the three CDR regions of the heavy chain are named LHCDR1, HCDR2, and HCDR3. In Example 13 of the present invention, the six CDRs of the ch782 antibody were defined with reference to the Kabat method and the Chothia method.

[0081] In the present invention, the term "mouse antibody" refers to an anti-CLDN18.2 monoclonal antibody prepared in accordance with the knowledge and art of the art. During preparation, a test subject is injected with the CLDN18.2 antigen, and hybridomas expressing an antibody having a desired sequence or functional feature are isolated. In preferred embodiments of the present invention, the mouse CLDN18.2 antibody or its antigen-binding fragment may further comprise the light chain constant region of the mouse κ chain, λ chain, or their variants, or the heavy chain constant region of mouse IgG1, IgG2, IgG3, or their variants.

[0082] The term "chimeric antibody" refers to an antibody formed by fusing the variable region of a mouse antibody with the constant region of a human antibody. Chimeric antibodies can mitigate the immune response induced by mouse antibodies.

[0083] The term "humanized antibody," also known as CDR transplantation antibody, refers to antibodies produced by transplanting mouse CDR sequences into a human antibody variable region framework, i.e., different types of human germline antibody framework sequences. Humanized antibodies can overcome heterogeneous reactions induced by chimeric antibodies containing large amounts of mouse protein components. Such framework sequences can be obtained from publicly available DNA databases containing germline antibody gene sequences or from published references. To avoid a decrease in activity due to reduced immunogenicity, Human antibody variable region framework sequences can be subjected to minimal reverse mutations or reverse mutations while maintaining activity.

[0084] The term “antibody antigen-binding fragment” (or “antibody fragment” for short) refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., CLDN18.2). It has been shown that the antigen-binding function of an antibody can be performed using fragments of a full-length antibody. Examples of binding fragments included in the term “antibody antigen-binding fragment” include: (i) Fab fragment (a monovalent fragment consisting of VL, VH, CL, and CH1 domains), (ii) F(ab')2 fragment (a divalent fragment consisting of two Fab fragments connected by disulfide bonds in the hinge region), (iii) Fd fragment (consisting of VH domain and CH1 domain), (iv) Fv fragment (consisting of the VH domain and VL domain of one arm of the antibody).

[0085] Fv antibodies contain heavy chain variable regions and light chain variable regions, but do not have a constant region. Fv antibodies are the smallest antibody fragments that possess all antigen-binding sites. Generally, Fv antibodies can further contain a polypeptide linker between the VH domain and the VL domain to form the structure necessary for antigen binding.

[0086] The term "CDR" refers to one of the six hypervariable regions within the variable domain of an antibody that primarily contribute to antigen binding. One of the most commonly used definitions of the six CDRs is provided by Kabat EA et al., (1991) Sequences of protein of immunological interest. NIH Publication, No. 91-3242.

[0087] The term “epitope” or “antigenic determinant” refers to a site on an antigen to which an immunoglobulin or antibody specifically binds (for example, a specific site on the CLDN18.2 molecule). An epitope typically contains at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 consecutive or discontinuous amino acids in a unique spatial configuration.

[0088] The terms "specific binding," "selective binding," "to bind selectively," and "to bind specifically" refer to the binding of an antibody to a specific epitope on an antigen. Typically, an antibody binds to approximately 10 -7 Less than M, for example, approximately 10 -8 Less than M, 10 -9 Less than M, or 10 10 It binds with an affinity (KD) of less than M.

[0089] The term "competitive binding" refers to an antibody that recognizes the same epitope (also called an antigenic determinant) or a portion of the same epitope on the extracellular domain of CLDN18.2 and binds to the antigen in such a way that the monoclonal antibody of the present invention binds to it. An antibody that binds to the same epitope as the monoclonal antibody of the present invention refers to an antibody that recognizes and binds to the amino acid sequence of CLDN18.2 recognized by the monoclonal antibody of the present invention.

[0090] The term "KD" or "Kd" refers to the dissociation equilibrium constant of a particular antibody-antigen interaction. Typically, the antibodies of the present invention have a dissociation equilibrium constant of about 10. -7 Less than M, for example, about 10 -8 M, 10 -9 M, or 10 -10It binds to CLDN18.2 with a dissociation equilibrium constant (KD) less than or equal to M.

[0091] As used herein, the term “antigenic determinant” refers to a discontinuous three-dimensional spatial site on an antigen that is recognized by the antibody or antigen-binding fragment of the present invention.

[0092] The present invention includes not only the whole antibody, but also fragments of the antibody or fusion proteins formed by the antibody and other sequences, the fragments or fusion proteins having immunoactivity. Therefore, the present invention also includes antibody fragments, derivatives, and analogs.

[0093] In the present invention, the antibody includes mouse antibodies, chimeric antibodies, humanized antibodies, or fully human antibodies prepared using techniques well known to those skilled in the art. Recombinant antibodies, such as chimeric and humanized monoclonal antibodies, contain both human and non-human portions and can be prepared using DNA recombination techniques well known in the art.

[0094] As used herein, the term “monoclonal antibody” refers to an antibody secreted by a clone derived from a single cell. Monoclonal antibodies are highly specific and target a single antigenic epitope. The cell may be a eukaryotic cell line, a prokaryotic cell line, or a phage clone cell line.

[0095] In the present invention, the antibody may be monospecific, bispecific, triplicate, or more multispecific.

[0096] In the present invention, the antibody of the present invention further comprises its conserved variants. A conserved variant refers to a polypeptide formed by replacing up to 10 amino acids, preferably up to 8, more preferably up to 5, and most preferably 3, amino acids with similar or close properties, compared to the amino acid sequence of the antibody of the present invention. These conserved variant polypeptides are preferably produced by performing amino acid substitutions according to Table A below.

[0097] [Table 1]

[0098] Humanized anti-CLDN18.2 antibody The present invention provides a humanized anti-CLDN18.2 antibody (hereinafter referred to as CLDN18.2 antibody). Specifically, the present invention provides a humanized antibody having high specificity and high affinity for CLDN18.2. The humanized antibody comprises a heavy chain and a light chain, the heavy chain comprising the amino acid sequence of the heavy chain variable region (VH), and the light chain comprising the amino acid sequence of the light chain variable region (VL).

[0099] Generally, there are two types of human framework regions that can be selected for antibody humanization: one derived from well-known mature antibodies, and the other from human germline sequences. Framework regions from well-known mature antibodies typically contain somatic mutation sites, which can lead to potential immunogenicity. Compared to mature antibodies, framework regions from human germline sequences theoretically have lower immunogenicity. Furthermore, framework regions have a more flexible structure and strong plasticity, and are more readily able to accept different CDR regions. There is a bias in the frequency of use of human antibody germline genes in the human body. Antibodies humanized by selecting and using frequently used germline anti-framework regions have advantages such as lower immunogenicity, higher expression levels, and structural stability.

[0100] In a preferred embodiment of the present invention, when humanization is performed, the germline sequence having the highest similarity to the mouse antibody is not selected. Instead, both similarity and frequency of use in the human body are considered, and after extensive experimental screening, a framework region with a preferred sequence is selected and humanization is performed. The human antibody germline framework region is selected and used for CDR transplantation, and the humanized antibody thus constructed has a more stable structure, higher expression levels, lower immunogenicity, and higher druggability.

[0101] In another preferred embodiment, the heavy chain steady region and / or light chain steady region may be a humanized heavy chain steady region or a humanized light chain steady region. More preferably, the humanized heavy chain steady region or light chain steady region is a heavy chain steady region such as human IgG1, IgG2, or a human κ or λ light chain steady region.

[0102] In another preferred embodiment, the sequence formed by adding, deleting, modifying and / or substituting at least one amino acid sequence is preferably an amino acid sequence having at least 80%, preferably at least 85%, more preferably at least 90%, and most preferably at least 95% homology.

[0103] The antibody of the present invention may be a double-chain antibody or a single-chain antibody, and preferably a fully humanized antibody.

[0104] The antibody derivatives according to the present invention may be single-chain antibodies and / or antibody fragments such as Fab, Fab', (Fab')2, or other antibody derivatives known in the art, as well as any one or more of IgA, IgD, IgE, IgG, and IgM antibodies or other subtypes.

[0105] The antibody of the present invention may be a humanized antibody or a CDR-implanted and / or modified antibody targeting CLDN18.2.

[0106] In the present invention described above, the number of amino acids added, deleted, modified and / or substituted is preferably 40% or less of the total number of amino acids in the initial amino acid sequence, more preferably 35% or less, more preferably 1% to 33%, more preferably 5% to 30%, more preferably 10% to 25%, and more preferably 15% to 20%.

[0107] Antibody preparation The CLDN18.2 antibody of the present invention can be produced using any method suitable for producing monoclonal antibodies. For example, animals can be immunized with linked or naturally occurring CLDN18.2 proteins or fragments thereof. Appropriate immunization methods, including adjuvants, immunostimulants, and repeated booster immunizations, can be used, and one or more of these methods can be used.

[0108] Any suitable form of CLDN18.2 may serve as an immunogen (antigen) for producing CLDN18.2-specific non-human antibodies, which are then screened for biological activity. The immunogen may be used alone or in combination with one or more immunogenicity enhancers known in the art. The immunogen may be purified from natural sources or produced in genetically modified cells. The DNA encoding the immunogen may be either genomic or non-genomic (e.g., cDNA) with respect to the source. The DNA encoding the immunogen can be expressed using a suitable gene vector. Vectors include, but are not limited to, adenovirus vectors, baculovirus vectors, plasmids, and non-viral vectors.

[0109] Humanized antibodies can be selected from any type of immunoglobulin, including IgM, IgD, IgG, IgA, and IgE. Similarly, any type of light chain can be used in the compounds and methods herein. Specifically, κ chains or λ chains or their variants can be used in the compounds and methods of the present invention.

[0110] The DNA molecule sequences of the antibodies or fragments thereof of the present invention can be obtained using conventional techniques such as PCR amplification or genome library screening, among other methods. Furthermore, the light chain and heavy chain coding sequences can be fused together to form a single-chain antibody.

[0111] After obtaining the relevant sequence, it can be obtained in large quantities using recombination. This typically involves cloning the relevant sequence into a vector, then introducing the vector into cells, and then isolating the relevant sequence from host cells grown by conventional methods to obtain the relevant sequence.

[0112] Furthermore, especially when the fragment length is short, the relevant sequence can be artificially synthesized. Generally, fragments with very long sequences can be obtained by first synthesizing several smaller fragments and then ligating them together. The DNA sequence can then be introduced into various existing DNA molecules (or vectors, etc.) and cells that are well known in the field.

[0113] The term "nucleic acid molecule" refers to DNA molecules and RNA molecules. Nucleic acid molecules can be single-stranded or double-stranded, but are preferably double-stranded DNA. When a nucleic acid is functionally related to another nucleic acid sequence, the nucleic acid is "operably ligated." For example, if a promoter or enhancer affects the transcription of a coding sequence, the promoter or enhancer is operably ligated to the coding sequence.

[0114] The term "vector" refers to a nucleic acid molecule that can transport another nucleic acid to which it is ligated. In one embodiment, the vector is a "plasmid," which refers to a circular double-stranded DNA loop to which additional DNA segments can be ligated.

[0115] The present invention further relates to vectors comprising the above-mentioned appropriate DNA sequence and appropriate promoter or regulatory sequence. These vectors can be used to transform appropriate host cells, enabling the host cells to express proteins.

[0116] The term "host cell" refers to the cell into which the expression vector has been introduced. The host cell may be a prokaryotic cell such as a bacterial cell, a lower eukaryotic cell such as a yeast cell, or a higher eukaryotic cell such as a plant or animal cell (e.g., a mammalian cell).

[0117] The step of transforming host cells with recombinant DNA according to the present invention can be carried out using techniques well known in the art. The resulting transformants express the polypeptide encoded by the gene of the present invention by culturing them according to conventional methods. Culturing is carried out in a normal culture medium under appropriate conditions, depending on the host cells used.

[0118] Generally, host cells obtained from transformation are cultured under conditions suitable for the expression of the antibody of the present invention. Then, a conventional immunoglobulin purification process is carried out by conventional isolation and purification methods well known to those skilled in the art, such as protein A-Sepharose chromatography, hydroxyapatite chromatography, gel electrophoresis, dialysis, ion exchange chromatography, hydrophobic chromatography, molecular sieve chromatography, or affinity chromatography.

[0119] The resulting monoclonal antibodies can be identified by conventional methods. For example, the binding specificity of monoclonal antibodies can be determined using immunoprecipitation or in vitro binding assays (e.g., radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA)).

[0120] Antibody preparations Antibodies exhibit different stabilities in different formulation buffers, as evidenced by changes such as charge heterogeneity, antibody molecule degradation, and polymerization. These changes in quality characteristics are related to the physical and chemical properties of the antibody itself. Therefore, in antibody drug development, it is necessary to screen formulation buffers suitable for different antibodies according to their physical and chemical properties. Currently, commonly used antibody buffer systems include phosphate buffer, citrate buffer, and histidine buffer. Simultaneously, to maintain antibody stability, different concentrations of salt ions or excipients such as sorbitol, trehalose, and sucrose, as well as appropriate amounts of surfactants such as Tween, may be added depending on the antibody's characteristics.

[0121] Pharmaceutical composition The present invention further provides compositions. In preferred embodiments, the composition is a pharmaceutical composition comprising the aforementioned antibody or its active fragment, fusion protein or ADC, or corresponding CAR-T cell, and a pharmaceutically acceptable carrier. Generally, these substances may be formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous carrier medium, where the pH is typically about 5 to 8, and preferably about 6 to 8, but the pH value may vary depending on the properties of the formulated substance and the disease being treated. The formulated pharmaceutical composition may be administered by conventional means including, but not limited to, intratumoral, intraperitoneal, intravenous, or topical administration.

[0122] The antibodies of the present invention can also be expressed intracellularly by nucleotide sequence for use in cell therapies such as chimeric antigen receptor T cell (CAR-T) immunotherapy.

[0123] The pharmaceutical composition of the present invention can be used directly to bind to the CLDN18.2 protein molecule and therefore can be used for the prevention and treatment of CLDN18.2-related diseases. Furthermore, other therapeutic agents can be used simultaneously.

[0124] The pharmaceutical composition of the present invention comprises a safe and effective amount (e.g., 0.001 wt% to 99 wt%, preferably 0.01 wt% to 90 wt%, more preferably 0.1 wt% to 80 wt%) of the monoclonal antibody (or its conjugate) of the present invention, and a pharmaceutically acceptable carrier or excipient. Such carriers include, but are not limited to, physiological saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical formulation should be adapted to the mode of administration. The pharmaceutical composition of the present invention can be formulated, for example, in the form of an injectable preparation prepared by conventional methods using physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as injectable preparations and liquid preparations are preferably manufactured under sterile conditions. The dose of the active ingredient is a therapeutically effective dose, such as about 1 μg / kg body weight to about 5 mg / kg body weight per day. Furthermore, the polypeptide of the present invention may also be used in combination with other therapeutic agents.

[0125] When using a pharmaceutical composition, administer a safe and effective amount of the pharmaceutical composition to the mammal. A safe and effective amount is typically at least about 10 mg / kg body weight and, in most cases, does not exceed about 50 mg / kg body weight. Preferably, the dose is about 10 mg / kg body weight to about 20 mg / kg body weight. Of course, factors such as the route of administration and the patient's health condition should also be considered for a specific dose, and these are all within the scope of the skill of an experienced physician.

[0126] Detection applications and kits The antibody of the present invention can be used for detection purposes, such as detecting a sample, in order to provide diagnostic information.

[0127] In the present invention, the sample (collected material) can be a cell, tissue sample, biopsy specimen, etc. The term "biopsy" as used in the present invention should include all types of biopsies well known to those skilled in the art. Therefore, the biopsy used in the present invention may include, for example, a tissue sample prepared by endoscopy, organ puncture, or needle biopsy.

[0128] The samples used in this invention include fixed or preserved cell or tissue samples.

[0129] The present invention further provides a kit containing the antibody (or a fragment thereof) of the present invention. In a preferred embodiment of the present invention, the kit further includes a container, a user manual, a buffer, and the like. In a preferred embodiment, the antibody of the present invention may be immobilized on a detection plate.

[0130] The main advantages of this invention are as follows: 1. The single-domain antibody (VHH domain) against Cladin 18.2 of the present invention has high specificity and affinity. 2. The affinity of the anti-Cladin 18.2 antibody of the present invention for CLDN18.2 is at least 100 times higher than its affinity for CLDN18.1 (cell-based). 3. The Cladin 18.2 CART cells of the present invention exhibit specific in vitro cytotoxicity against Cladin 18.2-positive target cells, which is significantly higher than the in vitro killing efficiency against Cladin 18.1 target cells. 4. The cradin-18.2 CART cells of the present invention can effectively inhibit the growth of cradin-18.2-positive tumors in vitro and exhibit long-term antitumor effects. 5. The Cladin 18.2 CAR05 T cells of the present invention exhibit a stronger immunomemory phenotype during continuous in vitro culture. 6. The humanized cradin 18.2 CART cells of the present invention do not show a significant decrease in cytotoxicity to target cells or a significant decrease in affinity to target cells compared to their maternal CAR-T cells. 7. The antibodies of the present invention can be used for the specific detection of claudin 18.2 protein and / or claudin 18.1 protein.

[0131] The present invention will be further described below with reference to specific examples. It should be understood that these examples are used solely to illustrate the present invention and not to limit its scope. Experimental methods without specific conditions shown in the following examples typically follow conventional conditions, e.g., those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or conditions recommended by the manufacturer. Unless otherwise specified, percentages and parts refer to weight percentages and parts by weight.

[0132] Unless otherwise specified, the materials and reagents used in the examples of this invention are all commercially available products.

[0133] Example 1 1. Screening of alpaca antibodies The phage antibody library was screened for antibodies. The process for establishing the alpaca antibody library is briefly described below.

[0134] Antigen Claudin 18.2 VLP (0.5 mg) and 293-Claudin 18.2 cells (2e7) were injected into the neck of an alpaca by multi-site injection. After multiple immunizations, 50 ml of peripheral blood was collected for antibody titer detection and nanobody library construction.

[0135] 1.1 Construction of the 1.1 VHH phage display library After multiple immunizations of alpacas, their serum titers met the criteria for library construction, i.e., 1) ELISA titer > 32000 and 2) FACS titer > 200.

[0136] PBMCs were separated, total RNA of PBMCs was extracted and reverse transcribed into cDNA. VHH was amplified by nested PCR and the VHH fragment was inserted into the phagemid. The phagemid was amplified and recovered to obtain an antibody library.

[0137] The VHH3 antibody library was determined to have a library capacity of 5.2×10 9 and a recombination efficiency of 90% (number of clonal PCR positive clones / total number of clones), meeting the relevant indicators of a high-quality immune library.

[0138] 1.2 Screening of Claudin 18.2 antibodies by cell panning This process is briefly described as follows. 1.2.1 Pre-treatment: Claudin 18.1 overexpressing cells (as negative cells) were used and added to the antibody library. 1.2.2 Binding step: Step 1 was centrifuged to obtain the supernatant, and the pre-treated antibody library was used to bind to Claudin 18.1 and Claudin 18.2 overexpressing cells respectively for screening in the liquid phase. 1.2.3 Washing: Washing was performed 6 - 8 times. 1.2.4 Amplification: NEBalpha5F' cells were infected, helper phages were added, and the cells were cultured overnight. 1.2.5 Plating was performed to determine the degree of enrichment (enrichment was considered successful if the number of positive cell enriched clones / the number of negative cell enriched clones was greater than 10). 1.2.6 Phages from round R1 were collected and enriched for screening in the next round.

[0139] 1.3 Identification of clones obtained by cell panning-phage flow cytometry (phage-based FACS) 1.3.1 Identification of clones obtained by cell panning: Clones that bind to claudin 18.2 overexpressing cells were randomly selected, and phages were obtained by expanding culture. The binding affinity to claudin 18.1 and claudin 18.2 overexpressing cells was then identified. 1.3.1 Cell preparation: 2 × 10⁶ cells overexpressing claudin 18.1 and 18.2 5 The cells / groups were centrifuged and washed three times with DPBS. 1.3.2 Sample Incubation The supernatant of phages obtained from the growth cultures was added to each sample, and the samples were incubated at 4°C for 2 hours. 1.3.3 Incubation with secondary antibody PE-labeled anti-M13 antibody (concentration 5 ug / ml, 100 ul) was added and incubated at 4°C for 45 minutes. 1.3.4 Washing with DPBS The samples were washed three times with DPBS, and then resuspended in 300 µl of DPBS. 1.3.5 Flow Cytometry Detection The resuspended cells were detected using a flow cytometer. Fluorescence values ​​of the cells revealed that all 14 randomly selected clones bound to claudin 18.2-overexpressing cells, but not to claudin 18.1-overexpressing cells.

[0140] 1.4 Sequencing of positive clones Fourteen positive clones (from 1.3) were selected for sequencing. The clone numbers and VHH domain sequence IDs are shown in the table below. In the table, CDR1, CDR2, and CDR3 are defined based on Kabat numbering (see Deschacht et al., 2010. Journal of Immunology, 184:5696-704). Clones 7, 8, and 9 are identical to clones 12 and 13 with respect to CDR 1, 2, and 3, the only difference being the FR region, as shown in Table 1 below.

[0141] [Table 2]

[0142] 1.5 NGS sequencing of phages obtained by cell panning and enrichment. Using cells overexpressing 293FT-claudin 18.2, the phage pool obtained by panning and enrichment was subjected to PCR to amplify VHH3 gene fragments, and 30 VHH3 gene sequences were obtained by NGS sequencing.

[0143] Thirty VHH3 genes were further tested for the binding performance of their expressed VHH domains to claudin 18.2, yielding six nanobodies with optimal performance. The sequences of the VHH domains of these nanobodies, as well as the sequences of CDR1, CDR2, and CDR3, are shown in Table 2.

[0144] [Table 3]

[0145] 1.6 Determination of affinity for preferred sequences - ELISA The affinity of the recombinant antibody was determined by ELISA. Four sequences (clones 1, 5, 8, and 10) were selected from 14 clones to synthesize VHH-hIgG1Fc recombinant antibodies, and the affinity of the antibodies to the antigens was determined. The antigens were claudin 18.2 VLP and claudin 18.2 recombinant protein (His-tagged), respectively. The ELISA EC50 data are shown in Table 2.

[0146] The measurement method is as follows: 1.5.1 Coating: On day 1, the antigen (200 ng / well) was coated onto the ELISA plate overnight at 4°C. 1.5.2 Blocking: On day 2, the ELISA plates were blocked with 0.5% BSA at room temperature for 2 hours. 1.5.3 Binding: On day 2, seven concentrations of VHH hIgG1 Fc recombinant antibody were obtained by diluting them 1:3, starting with 10 μg / mL. The diluted solutions were added to ELISA plates at 100 μL / well and left at room temperature for 2 hours. 1.5.4 The ELISA plate was washed with PBS-Tween, 100 μL of anti-human IgG1Fc HRP antibody was added, and the plate was left at room temperature for 1 hour. 1.5.5 Chromogenicity: The ELISA plate was washed with PBS-Tween, then washed with PBS, and 100 μL of TMB was added. The plate was then left at 37°C for 5-10 minutes. 1.5.6 Termination: 50 μL of 1 M phosphoric acid was added to stop the reaction. 1.5.7 Measurement: Absorbance values ​​at 450 nm were measured using an ELISA reader, and the results are shown in Table 3.

[0147] [Table 4]

[0148] As shown in Table 3, all recombinant antibodies in this disclosure bind to claudin 18.2 VLP or claudin 18.2-his antigen in a dose-dependent manner, with binding efficacy ranging from 4 nM to 200 nM (EC50 value).

[0149] 1.7 Determination of affinity for preferred sequences - FACS The ability of recombinant antibodies to bind to 293FT-h claudin 18.1 cells (negative cells) and 293FT-h claudin 18.2 cells (positive cells) was determined using cell-based flow cytometry. 293FT-claudin 18.1 and 293FT-h claudin 18.2 cells were incubated with four preferred recombinant antibody concentrations (5 ug / mL and 10 ug / mL) at 4°C for 30 minutes, followed by washing with DPBS. After washing, the cell precipitate was resuspended in a secondary antibody (1:100, goat anti-human IgG (H+L), FITC), incubated in the dark for 15 minutes, and then washed with DPBS. The cells were subjected to flow cytometry to determine the binding levels of recombinant antibodies to 293FT-h claudin 18.1 and 293FT-h claudin 18.2 cells. Each recombinant antibody was labeled with an Fc tag and named 1-hIgG1Fc, 5-hIgG1Fc, 8-hIgG1Fc, and 10-hIgG1Fc, respectively. The recombinant antibodies were used at a concentration of 10 ug / mL. The binding ability of each recombinant antibody was expressed as a binding percentage, which was calculated as (cells with antibody binding / total cells) × 100%.

[0150] The results are shown in Figure 1. The binding rates of recombinant antibodies to claudin 18.1 cells were 2.75%, 2.06%, 2.26%, and 2.06%, respectively, while the binding rates of recombinant antibodies to claudin 18.2 cells were 88.94%, 95.96%, 93.86%, and 96.1%, respectively, demonstrating the binding specificity of recombinant antibodies to human claudin 18.2.

[0151] 1.8 Humanized anti-claudin 18.2 antibody The humanized anti-claudin 18.2 antibody was obtained based on a humanized reconstituted claudin 18.2 VHH antibody, according to literature such as Vincke et al., Journal of Biochemistry, 284(5):3273-3284 (2009). The framework region of the humanized VHH domain disclosed in this application was reconstituted based on it to reduce antigenicity. In the design of the humanized single-domain antibody derived from camelid species, the numbering of characteristic residues in their VHH was based on Kabat, such as residues 11, 37, 44, 45, and 47 (Muyldermans, Molecular Biotechnology Review, 74:277-302 (2001)). The humanized sequences are shown in Table 4.

[0152] [Table 5]

[0153] 1.9 Exemplary Designs of Single-CAR and Dual-Epitope CARs for Chimeric Antigen Receptors Targeting Claudin 18.2 Exemplary structures of claudin 18.2 single-CAR and double-CAR structures were designed as shown in Figure 2. Exemplary amino acid sequences of the CAR constructs are listed in Table 5.

[0154] Example 2 Preparation of CAR-T targeting Claudine 18.2 2.1 Preparation of CAR-T cells targeting Claudine 18.2 General materials and methods: Peripheral blood mononuclear cells (PBMCs) were isolated from donor blood, and T cells were proliferated. Peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation using Ficoll. Then, T cells were enriched, activated with magnetic beads conjugated with anti-CD3 / anti-CD28, cultured, and proliferated.

[0155] Target cell lines, PBMCs, and CAR-T cultures: 1. Cell lines 293FT-Luc, 293FT-CLDN18.1-luc, 293FT-CLN18.2-Luc, AGS-CLDN18.2-Luc, AGS-CLDN18.1(KO CLDN18.2)-Luc (KO CLDN18.2 refers to the knockout of endogenous claudin 18.2), Aspc-1-CLDN18.2-Luc, and 293FT series cells were cultured in DMEM medium. All cells labeled with the Luc tag stably expressed firefly luciferase. AGS-CLDN18.2-Luc and AGS-CLDN18.1(KO CLDN18.2)-Luc series cells were cultured in F12 medium, and Aspc-1-CLDN18.2-Luc cells were cultured in RPMI1640 medium. RPMI1640, F12, and DMEM media were all supplemented with 10% (v / v) fetal bovine serum, 100 U / ml penicillin and streptomycin, 2 mM glutamine, and 1 mM sodium pyruvate. All cells were cultured in a 37C, 5% CO2 incubator. 2. CAR-T cells were cultured in X-vivo15 medium (containing 5% FBS, 2 mM L-glutamine, 1 mM sodium pyruvate, and 300 IU / ml rhIL-2). rhIL-2 was added to the CAR-T cell culture medium. 300 IU / ml of IL-2 was added every two days. All cells were cultured at 37°C under 5% CO2.

[0156] CAR-T preparation The VHH coding sequences of the selected antibodies were optimized using gene codon optimization. Nucleotide molecules encoding chimeric antigen receptor (CAR) backbone polypeptides were synthesized and cloned into lentiviral vectors (pCDH backbone vectors with GFP expression sequences). A flag tag was inserted at the N-terminus of the hinge region. The VHH coding sequences were selected from Tables 1, 2, and 4 and corresponded to their CAR-T numbering, as specifically shown in Table 5. The control antibody (IMAB) had the IMAB362 sequence, including its heavy and light chain variable region fragments.

[0157] A mixture containing a lentiviral packaging plasmid and a vector expressing a CAR construct was pre-mixed with polyetherimide (PEI) in a pre-optimized ratio and incubated at room temperature for 15–20 minutes. The transfection mixture was then added to HEK293 cells, and the cells were cultured for 48–72 hours. The supernatant was collected and the lentivirus was concentrated.

[0158] Primary T cells activated by Dynabeads CD3 / CD28 were transfected with lentivirus. Three days after transfection, the percentage of CAR-positive cells was determined using an anti-flag tag antibody, and the FITC channel was simultaneously identified (indicating GFP expression). Three batches of CAR-T cells were produced separately, and their CAR positivity was determined to be in the range of 30% to 70%. The flag and GFP were shown to be expressed in a near 1:1 ratio, demonstrating successful co-expression of CAR and GFP, as seen in Figure 3.

[0159] [Table 6]

[0160] Example 3: In vitro function of CAR-T cells targeting claudin 18.2 3.1 Analysis of CAR-T cell memory phenotypes CAR-T cells were cultured continuously for 8-10 days, and the cell phenotype was determined using a flow cytometer. Anti-fragment antibodies were used to differentiate between CAR-positive and CAR-negative cells. Simultaneously, T cell memory phenotypes were determined using CD45RA, CD45RO, and CCR7 antibodies. CAR-positive cells were gated to distinguish between naive and T cells. CM , T EM , and T EMRA The cell population was analyzed, as shown in Figure 4. CD45RA of CAR15, CAR16, and CAR05. + vs CCR7 + (Naive cell ratio was higher than in the other groups: 20%, 23%, and 13% vs. approximately 2%), CD45RA +The ratio to CD45RO cells was also higher than that of the other groups, indicating a higher level of cell youth compared to other CAR-T cells.

[0161] 3.2 Cytotoxicity of CAR-T cells targeting claudin 18.2 Target cells expressing luciferase containing 293FT-Luc, 293FT-claudin 18.1-Luc, and 293FT-claudin 18.2-Luc were constructed. For measurement, luciferin is added as a substrate, after which luciferase reacts with luciferin and emits fluorescence. By measuring the fluorescence intensity, luciferase activity can be measured, allowing for the determination of the number of viable cells among the target cells and the evaluation of CAR-T cell cytotoxicity. Specific cytotoxicity is calculated using the following formula: Specific cytotoxicity % = 100% × (RLU) のみ -RLU 試料 / RLU のみ ). RLU 試料 RLU refers to the luciferase activity measured in wells containing CAR T cells that target claudin 18.2. のみ This refers to the luciferase activity measured in the well containing only the target cells.

[0162] Luciferase-expressing target cells 293FT-Luc, 293FT-claudin 18.1-Luc, and 293FT-claudin 18.2-Luc were incubated with CAR-T for 6 hours in an effector-to-target ratio (E:T = 1:1), and their cytotoxicity was compared.

[0163] The results are shown in Figure 5. The three data bars, from left to right, represent the lysis rates of target cells 293FT-Luc, 293FT-claudin 18.1-Luc, and 293FT-claudin 18.2-Luc by CAR T cells, respectively.

[0164] The results showed that IMAB, CAR02, CAR04-CAR13, and CAR18 T cells did not exhibit significant cytotoxicity against 293FT-Luc cells and 293FT-Luc cells that stably express human claudin 18.1. CAR01 and CAR17 CAR-T cells showed significant cytotoxicity against 293FT-Luc cells that stably express human claudin 18.1 and claudin 18.2, while CAR14 T cells showed significant cytotoxicity against 293FT-Luc, 293FT-claudin 18.1-Luc, and 293FT-claudin 18.2-Luc cells. Furthermore, CAR02, CAR05, and CAR18 T cells showed better killing specificity (they had the highest ratio of their lysis rate against 293FT-claudin 18.2-Luc cells to their lysis rate against 293FT-claudin 18.1-Luc cells).

[0165] The results of real-time cell analysis (RTCA) are shown in Figure 6. At an effector-to-target ratio (1:1), CAR16 showed no significant cytotoxicity against AGS cells stably expressing human claudin 18.2, while CAR15 showed lower cytotoxicity against AGS cells stably expressing human claudin 18.2 compared to other CAR-T cells. With the exception of CAR15 and CAR16, all CAR-T cells showed significant cytotoxicity when co-incubated with target cells AGS-claudin 18.2 for 10 hours.

[0166] 3.3 Release of cytokines CAR-T cells and AGS-claudin 18.2 cells were mixed in RPMI medium in a 1:1 ratio at 100 µl each. The density was 1 × 10⁶ cells per CAR-T cell. 5 The concentration was / ml. Cells were cultured overnight in a 96-well plate. The culture medium was then collected and centrifuged. The supernatant was taken, and the cytokines IFN-γ, IL-2, IL-4, IL-6, IL-10, and TNF released into the cell culture medium were measured using a cytokine detection kit (CBA kit, BD).

[0167] The detection results are shown in Figure 7. Large amounts of IL-2, TNFα, and IFNγ were detected in the co-culture supernatants of CAR 17-CAR 20, CAR02-CAR06, and CAR10, indicating the specific release of cytokines, including IL-2, TNFα, and IFNγ, during the action of claudin 18.2 CAR-T cells against CLDN18.2-positive target cells.

[0168] Advantageously, these CAR T cells of the present invention do not induce significant IL-6 release, suggesting a low risk of inducing a cytokine storm.

[0169] Furthermore, CAR05 T cells unexpectedly elicited high releases of IL-2, TNFα, and IFNγ (significantly higher than other CAR T cells).

[0170] Example 4: In vitro data of humanized VHH domain CAR-T cells Using the same method as in Example 3 to determine the in vitro function of CAR-T cells targeting claudin 18.2, the in vitro function of humanized VHH domain CAR-T cells was determined, as shown in Figure 8.

[0171] The CAR-T phenotype was analyzed after 8 days of culture. The results showed a significant difference in phenotype between CAR21 and CAR05, as well as a significantly lower CD45RA+CCR7+ (naive) cell ratio in CAR21 compared to CAR05, CAR22, and CAR23 (27.5% vs. 2.4%), indicating a lower T phenotype in CAR21. EMRA The cell ratio was significantly higher than that of CAR05 (61.4% vs. 43.9%). This is consistent with the immediate killing results of CAR21, and the immediate killing rate was higher than that of CAR05.

[0172] The results are shown in Figure 8. Humanized domain CAR-T cells were compared to maternal CAR-T cells in terms of immediate killing efficacy. Among the humanized domain CAR-T cells, CAR21 (humanized) showed higher immediate killing efficacy against 293FT-CLDN18.2-Luc (80% vs. 50%) and Nugc4-CLDN18.2-Luc (70% vs. 45%) compared to CAR05 (non-humanized), while CAR22, CAR23 (humanized), and CAR05 (non-humanized) showed similar immediate killing efficacy. CAR25 (humanized) and CAR18 (non-humanized) showed immediate lysis rates of 75% vs. 60% against 293FT-CLDN18.2-Luc, while CAR26 (humanized) and CAR02 (non-humanized) showed immediate lysis rates of 70% vs. 60% against 293FT-CLDN18.2-Luc. The results showed that humanized CAR25 and CAR26 exhibited higher immediate killing efficiency than maternal CAR-T.

[0173] Example 5: Determination of affinity 5.1 Determining the binding curve of recombinant antibodies against claudin 18.2 VLP antigen using ELISA. The recombinant antibodies listed in the table below were synthesized, and their binding to the claudin 18.2-VLP protein was determined using ELISA. The specific procedure for the ELISA experiment is as follows.

[0174] Claudin 18.2-VLP protein was added to ELISA plates at 200 ng / well, and the plates were coated overnight at 4°C. The plates were washed three times with PBS, and 200 μL / well of 1% BSA / PBS was added, followed by blocking at 37°C for 1 hour. After washing the plates with 100 μL of PBS, the gradient dilution of the aforementioned fusion antibody was added, and binding was allowed to proceed at 37°C for 1 hour. The plates were rinsed three times with PBST. 100 μL of HRP-goat anti-mouse IgG Fc, diluted 1:5000, was added, and binding was allowed to proceed at 37°C for 1 hour. The plates were rinsed three times with PBST. 100 μL / well of TMB chromogenic solution was added, and color development was allowed to proceed at 37°C for 10 minutes. The reaction was stopped by adding 50 μL of 1 M phosphate, and the absorbance at 450 nm was measured using an ELISA reader.

[0175] Figure 9 shows the results of an experiment testing the antigen-binding ability of VHH using ELISA technology. The EC of the nanobody of the present invention tested is shown below. 50 All values ​​were superior to those of the positive control antibody IMAB.

[0176] EC 50 Based on the values, affinity capabilities were ranked from highest to lowest as follows: 5H3 > 5 > 2, 18, 5H2 > 5H1 > IMAB.

[0177] 5.2 Determine the binding capacity of recombinant antibodies to 293FT-CLDN18.1-Luc or 293FT-CLDN18.2-Luc cells using flow cytometry. Negative 293FT-claudin 18.1 cells and positive 293FT-claudin 18.2 cells were each co-incubated with the fusion antibody anti-claudin 18.2VHH-mlg2aFc at 4°C for 1 hour. The cells were washed three times with PBS, and a secondary antibody (1:200 APC goat anti-mouse IgGFc) was added, followed by incubation at 4°C for 30 minutes. The cells were washed once with PBS and subjected to flow cytometry. The results of VHH affinity measurement using FACS are shown in Table 6.

[0178] [Table 7]

[0179] 5.3 Use of SPR technology to determine the affinity of recombinant antibodies against human claudin 18.2 protein Six antibodies were diluted to 5 μg / mL with the working reagent. Approximately 200 RU of the diluted solution was injected into the protein A capture experimental channel (Fc2) at a flow rate of 10 μL / min. Ligand capture did not need to be performed in the reference channel (Fc1).

[0180] Human claudin 18.2 protein was diluted 2-fold with the working reagent (Table 5). The diluted human claudin 18.2 protein was sequentially injected into the experimental channel (Fc2) and the reference channel (Fc1) at a flow rate of 30 μL / min, allowing binding and dissociation to proceed at respective times. Both binding and dissociation steps were performed in the working reagent. After analysis for each concentration, the tip had to be regenerated by using glycine hydrochloride with a pH of 1.5 at a flow rate of 20 μL / min for 30 seconds to wash away the ligand and undissociated analytes. When analyzing for the next concentration, the experimental channel (Fc2) had to recapture the same amount of ligand.

[0181] The KD value for each sample was calculated using Biocore 8K analysis software, Biocore Insight Evaluation Software. A reference channel (Fc1) was used for background subtraction.

[0182] The lower the dissociation constant KD value (ratio of dissociation rate Kd to binding rate Ka), the higher the affinity of the antibody. The KD value varies depending on the differences in the antibody-antigen complex and depends on Ka and Kd.

[0183] The results are shown in Table 7. The KD values ​​for H1-mIgG Fc, IMAB-mIgG Fc, 5H2-mIgG2a Fc, 5-mIgG2a Fc, and 2-mIgG2a Fc were 7.21 nM, 5.65 nM, 3.32 nM, 3.02 nM, and 1.1 nM, respectively. Ranking of antibody affinity (from highest to lowest): 2-mIgG2a Fc, 5-mIgG2a Fc, 5H2-mIgG2a Fc, IMAB-mIgG Fc, and 5H1-mIgG Fc.

[0184] Ka ranking (highest to lowest): 2-mIgG2a Fc, IMAB-mIgG Fc, 5H2-mIgG2a Fc, 5-mIgG2a Fc, and 5H1-mIgG Fc.

[0185] Kd ranking (from highest to lowest): IMAB-mIgG Fc, 5H1-mIgG Fc, 2-mIgG2a Fc, 5H2-mIgG2a Fc, and 5-mIgG2a Fc.

[0186] [Table 8]

[0187] 5.4 Studies on in vivo antitumor effects The in vivo antitumor effect of claudin 18.2 CAR-T cells was evaluated in the Aspc-1 18.2-Luc pancreatic cancer NOG-DKO mouse model (NOD.Cg-B2mem1Tac Prkdcscid H2-Ab1tm1Doi Il2rgtm1Sug / JicCrl).

[0188] 2E6 AsPC-1 CLDN18.2-Luc cells (CLDN18.2+MSLN+) were subcutaneously injected. When the tumor volume reached approximately 200 mm3, the mice were divided into groups of 3-4 mice each. One day after group division, 200 μL of CLDN18.2 CAR-T cells, MSLN CAR-T cells, or NT cells were injected via the tail vein (5E6 CAR-T / mouse). One day after CAR-T injection, a small amount of mouse blood was collected to determine the number of CAR-T cells surviving in vivo. Subsequently, blood was collected weekly to check for various CAR-T cell phenotypes, and the size of the subcutaneous tumors and the body weight of the mice were measured twice a week.

[0189] The results are shown in Figure 10. Claudin 18.2 CAR-T cells and MSLN CAR-T cells showed significant in vivo antitumor effects against transplanted tumor cells. VHH candidate CAR-T cells, including CAR02, 05, and 18, showed similar antitumor effects. Mice remained alive without tumor recurrence at 80 and 90 days after CAR-T cell reinjection. When control MSLN CAR-T cells targeting MSLN were used, tumor cells could be killed, but tumor recurrence began at 80 days after CAR-T cell reinjection and continued by 90 days, with tumor volume approximately 200 mm². 3 It reached its goal.

[0190] In vivo experimental results showed that CAR-T (CAR02, 05, 18) cells targeting claudin 18.2 possessed strong and persistent antitumor activity.

[0191] [Table 9]

[0192] [Table 10]

[0193] All documents referenced herein are incorporated by reference as each document is incorporated by reference individually. Furthermore, those skilled in the art, having read the above teachings of the invention, should understand that various changes or modifications can be made to the invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. The complementarity-determining region of the VHH chain of the nanobody, i.e., the CDR region, (1) CDR1 shown in Sequence ID 36, CDR2 shown in Sequence ID 37, and CDR3 shown in Sequence ID 38, (2) CDR1 shown in Sequence ID No. 39, CDR2 shown in Sequence ID No. 40, and CDR3 shown in Sequence ID No. 41, (3) CDR1 shown in Sequence ID No. 42, CDR2 shown in Sequence ID No. 43, and CDR3 shown in Sequence ID No. 44 (4) CDR1 shown in Sequence ID No. 45, CDR2 shown in Sequence ID No. 46, and CDR3 shown in Sequence ID No. 47, (5) CDR1 shown in Sequence ID No. 48, CDR2 shown in Sequence ID No. 49, and CDR3 shown in Sequence ID No. 50, (6) CDR1 shown in Sequence ID 51, CDR2 shown in Sequence ID 52, and CDR3 shown in Sequence ID 53, (7) CDR1 shown in Sequence ID 39, CDR2 shown in Sequence ID 54, and CDR3 shown in Sequence ID 41, (8) CDR1 shown in Sequence ID 39, CDR2 shown in Sequence ID 55, and CDR3 shown in Sequence ID 41, (9) CDR1 shown in Sequence ID 67, CDR2 shown in Sequence ID 68, and CDR3 shown in Sequence ID 41, (10) CDR1 shown in Sequence ID No. 39, CDR2 shown in Sequence ID No. 69, and CDR3 shown in Sequence ID No. 41, and / or (11) A nanobody selected from the group consisting of CDR1 shown in SEQ ID NO: 36, CDR2 shown in SEQ ID NO: 65, and CDR3 shown in SEQ ID NO: 66, which specifically binds to claudin 18.

2.

2. The nanobody according to claim 1, wherein CDR1, CDR2, and CDR3 are separated by framework regions FR1, FR2, FR3, and FR4 of the VHH chain.

3. The nanobody according to claim 1, wherein the amino acid sequence of the VHH chain of the nanobody is selected from the group consisting of SEQ ID NOs: 1 to 26, or combinations thereof.

4. A polyvalent antibody or multispecific antibody that specifically binds to claudin 18.2, wherein the polyvalent antibody or multispecific antibody comprises at least one antibody component that targets the claudin 18.2 epitope, and the antibody component is a nanobody as described in claim 1.

5. The anti-claudin 18.2 antibody comprises one or more VHH chains having the amino acid sequence shown in any one of SEQ ID NOs: 1 to 26, according to claim 4, a multivalent antibody or a multispecific antibody.

6. Recombinant protein, wherein the recombinant protein is (i) a nanobody that specifically binds to claudin 18.2 as described in claim 1, or a polyvalent antibody or multispecific antibody that specifically binds to claudin 18.2 as described in claim 4, (ii) Recombinant protein comprising, optionally, a tag sequence that aids in expression and / or purification.

7. The aforementioned chimeric antigen receptor (CAR) fusion protein extends from the N-terminus to the C-terminus. (i) an antigen-binding domain that specifically binds to claudin 18.2, comprising the nanobody described in claim 1, (ii) Transmembrane domain, (iii) at least one co-stimulatory domain, and (iv) A chimeric antigen receptor (CAR) fusion protein containing an activating domain.

8. The CAR has the structure shown in formula Ia, L-VHH-H-TM-C-CD3ζ (Ia) During the ceremony, Each "-" independently represents a linker peptide or peptide bond. L is the signal peptide sequence, VHH is an antigen-binding domain that specifically binds to claudin 18.

2. H is the hinge region, TM is a transmembrane domain, C is a co-stimulatory signaling domain, and The chimeric antigen receptor (CAR) fusion protein according to claim 7, wherein CD3ζ is a cytoplasmic signaling sequence derived from CD3ζ (including wild-type or its variants / modified forms).

9. The chimeric antigen receptor (CAR) fusion protein according to claim 8, wherein the amino acid sequence of VHH is as shown in SEQ ID NO: 2, 5, or 18.

10. The antibody-drug conjugate is (a) a nanobody according to claim 1, a polyvalent antibody or multispecific antibody according to claim 4, or a recombinant protein according to claim 6, (b) An antibody-drug conjugate comprising a conjugate portion conjugated to the antibody portion, the conjugate portion being selected from the group consisting of a detectable label, a drug, a toxin, a cytokine, a radionuclide, an enzyme, or a combination thereof.

11. The polynucleotide is a polynucleotide that encodes a protein selected from the group consisting of the nanobody described in claim 1, the multivalent antibody or multispecific antibody described in claim 4, the recombinant protein described in claim 6, or the CAR fusion protein described in claim 7.

12. An expression vector, wherein the expression vector comprises the polynucleotide described in claim 11.

13. A host cell, wherein the host cell contains the expression vector described in claim 12, or has the polynucleotide described in claim 11 incorporated into its genome, or expresses a nanobody described in claim 1, a multivalent antibody or multispecific antibody described in claim 4, a recombinant protein described in claim 6, or a CAR fusion protein described in claim 7.

14. A pharmaceutical composition, wherein the pharmaceutical composition is i) Immune cells expressing the nanobody described in claim 1, the multivalent antibody or multispecific antibody described in claim 4, the recombinant protein described in claim 6, or the CAR fusion protein described in claim 7, and (ii) A pharmaceutical composition comprising a pharmaceutically acceptable carrier.

15. Use of immune cells expressing the nanobody described in claim 1, the multivalent antibody or multispecific antibody described in claim 4, the recombinant protein described in claim 6, or the CAR fusion protein described in claim 7, wherein the use is in the preparation of a drug for the prevention and / or treatment of cancer or tumors related to Claudin 18.2.

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