Antibody specifically binding to claudin18.2 and preparation method therefor and use thereof

Abstract

Provided is an anti-Claudin18.2 nanobody or a binding fragment thereof. The antibody has good specificity, and immune effector cells targeting Claudin18.2 which are prepared from the antibody show a good therapeutic effect in treating or ameliorating diseases having positive expression of Claudin18.2.

Description

Antibodies that specifically bind to Claudin18.2, their preparation methods, and applications. Technical Field

[0001] This invention relates to the field of engineered immunotherapy, and more specifically to antibodies that specifically bind to Claudin18.2, their preparation methods, and applications. Background Technology

[0002] Claudin family proteins are widely distributed in the tight junction structures of epithelial cells. Claudin 18.2 is a membrane protein with four transmembrane regions: the N-terminus and C-terminus are located in the cytoplasm, while two extracellular loop regions can serve as specific targets for targeted therapy. In normal tissues, Claudin 18.2 is specifically expressed in highly differentiated epithelial cells of the stomach. Besides gastric cancer, Claudin 18.2 is also expressed in a high proportion of tumor cells in cholangiocarcinoma, ovarian cancer, lung cancer, esophageal cancer, and pancreatic cancer.

[0003] Chimeric antigen receptor T cell (CAR T) is a novel immunotherapy method targeting specific antigens on the surface of tumor cells. Currently, it is being used to develop cell therapy for solid tumors.

[0004] Camel-derived antibodies have the advantages of being stable, water-soluble, low-immunogenic, highly affinity, and small in molecular weight. They can penetrate deep into the antigen and have stronger tissue penetration than traditional antibodies. They can be used as diagnostic tools and for the delivery of CAR-T and targeted drugs to develop a CAR-T with higher affinity, stronger specificity, and lower immunogenicity.

[0005] Summary of the Invention

[0006] The purpose of this invention is to provide an antibody with high affinity and high biological activity that can specifically recognize Claudin 18.1 and / or Claudin 18.2 antigens and its application.

[0007] In a first aspect of the invention, a nanobody that specifically binds to Claudin18.2 is provided, wherein the complementarity-determining region (CDR) of the VHH chain in the nanobody is selected from the group consisting of:

[0008] (1) CDR1 shown in SEQ ID NO:36, CDR2 shown in SEQ ID NO:37, and CDR3 shown in SEQ ID NO:38;

[0009] (2) CDR1 shown in SEQ ID NO:39, CDR2 shown in SEQ ID NO:40, and CDR3 shown in SEQ ID NO:41;

[0010] (3) CDR1 shown in SEQ ID NO:42, CDR2 shown in SEQ ID NO:43, and CDR3 shown in SEQ ID NO:44;

[0011] (4) CDR1 shown in SEQ ID NO:45, CDR2 shown in SEQ ID NO:46, and CDR3 shown in SEQ ID NO:47;

[0012] (5) CDR1 shown in SEQ ID NO:48, CDR2 shown in SEQ ID NO:49, and CDR3 shown in SEQ ID NO:50;

[0013] (6) CDR1 shown in SEQ ID NO:51, CDR2 shown in SEQ ID NO:52, and CDR3 shown in SEQ ID NO:53;

[0014] (7) CDR1 shown in SEQ ID NO:39, CDR2 shown in SEQ ID NO:54, and CDR3 shown in SEQ ID NO:41;

[0015] (8) CDR1 shown in SEQ ID NO:39, CDR2 shown in SEQ ID NO:55, and CDR3 shown in SEQ ID NO:41;

[0016] (9) CDR1 shown in SEQ ID NO:67, CDR2 shown in SEQ ID NO:68, and CDR3 shown in SEQ ID NO:41;

[0017] (10) CDR1 shown in SEQ ID NO:39, CDR2 shown in SEQ ID NO:69, and CDR3 shown in SEQ ID NO:41; and / or

[0018] (11) CDR1 shown in SEQ ID NO:36, CDR2 shown in SEQ ID NO:65, and CDR3 shown in SEQ ID NO:66.

[0019] In another preferred embodiment, CDR1, CDR2 and CDR3 are separated by the frame regions FR1, FR2, FR3 and FR4 of the VHH chain.

[0020] In another preferred embodiment, the nanobody that specifically binds to Claudin18.2 includes humanized antibodies, camel-derived antibodies, and chimeric antibodies.

[0021] In another preferred embodiment, the amino acid sequence of the nanobody that specifically binds to Claudin18.2 is shown in any of SEQ ID NO:1 to 26.

[0022] In another preferred embodiment, the amino acid sequence of the VHH chain of the nanobody is selected from the group consisting of SEQ ID NO:1 to 26, or combinations thereof.

[0023] In another preferred embodiment, the CDR region of the nanobody VHH chain contains an amino acid sequence having at least 80%, preferably at least 90%, more preferably at least 95%, and even more preferably at least 99% sequence similarity to any one of SEQ ID NO:1 to 26.

[0024] In another preferred embodiment, the amino acid sequence of the CDR region of the nanobody VHH chain contains one or more amino acid substitutions compared to any of SEQ ID NO:1 to 26, preferably conservative amino acid substitutions.

[0025] In another preferred embodiment, any of the amino acid sequences described above further includes a derived sequence which has optionally been added, deleted, modified and / or substituted at least one (e.g., 1-3, preferably 1-2, more preferably 1) amino acid and retains the ability to specifically bind to Claudin18.2.

[0026] In another preferred embodiment, the Claudin18.2 is Claudin18.2 of a human or non-human mammal.

[0027] In another preferred embodiment, the Claudin18.2 is Claudin18.2 of a human, mouse, rat, or non-human primate (such as a monkey).

[0028] In a second aspect of the invention, a multivalent antibody or multispecific antibody that specifically binds to Claudin18.2 is provided, the multivalent antibody or multispecific antibody comprising at least one antibody element targeting the Claudin18.2 epitope, the antibody element being the anti-Claudin18.2 nanobody described in the first aspect of the invention.

[0029] In another preferred embodiment, the anti-Claudin18.2 multivalent antibody or multispecific antibody comprises one or more anti-Claudin18.2 nanobodies.

[0030] In another preferred embodiment, the anti-Claudin18.2 antibody comprises a monomer, a bivalent (bivalent antibody), a tetravalent (tetravalent antibody), and / or a multivalent (multivalent antibody).

[0031] In another preferred embodiment, the anti-Claudin18.2 antibody comprises one or more VHH chains having an amino acid sequence as shown in any of SEQ ID NO:1 to 26.

[0032] In another preferred embodiment, the anti-Claudin18.2 antibody comprises two VHH chains having amino acid sequences as shown in SEQ ID NO:1-26.

[0033] In another preferred embodiment, the antibody is selected from: animal-derived antibodies, chimeric antibodies, humanized antibodies, or combinations thereof.

[0034] In a third aspect of the invention, a recombinant protein is provided, said recombinant protein having:

[0035] (i) the Claudin18.2 nanobody specifically binding as described in the first aspect of the present invention, or the Claudin18.2 specific multivalent antibody or multispecific antibody specifically binding as described in the second aspect of the present invention; and

[0036] (ii) Optional tag sequences to assist in expression and / or purification.

[0037] In another preferred embodiment, the antibody is a multivalent antibody.

[0038] In another preferred embodiment, the tag sequence includes an Fc tag, an HA tag, a Flag tag, and a 6His tag.

[0039] In another preferred embodiment, the Fc tag comprises hIgG1Fc and mIgG1Fc.

[0040] In another preferred embodiment, the recombinant protein specifically binds to Claudin18.1 and / or Claudin18.2 proteins.

[0041] In a fourth aspect of the invention, a chimeric antigen receptor (CAR) fusion protein is provided, characterized in that the chimeric antigen receptor (CAR) fusion protein comprises, from the N-terminus to the C-terminus:

[0042] (i) Specifically binds to the antigen-binding domain of Claudin18.2, wherein the antigen-binding domain contains the nanobody described in the first aspect of the present invention;

[0043] (ii) Transmembrane structural domains;

[0044] (iii) at least one costimulatory domain; and

[0045] (iv) Activate the structural domain.

[0046] In another preferred embodiment, the antigen-binding domain is monovalent or polyvalent.

[0047] In another preferred embodiment, the antigen-binding domain is derived from the nanobody described in the first aspect of the invention or the recombinant protein described in the third aspect of the invention.

[0048] In another preferred embodiment, the CAR has the structure shown in Formula Ia:

[0049] L-VHH-H-TM-C-CD3ζ(Ia)

[0050] In the formula,

[0051] Each "-" independently represents a linking peptide or peptide bond;

[0052] L represents the signal peptide sequence;

[0053] VHH is the antigen-binding domain that specifically binds to Claudin 18.2;

[0054] H represents the hinge area;

[0055] TM represents a transmembrane domain;

[0056] C is the co-stimulation signal structure domain;

[0057] CD3ζ is a cytoplasmic signaling sequence derived from CD3ζ (including wild type or its mutants / modifiers).

[0058] 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.

[0059] In another preferred embodiment, L is a signal peptide derived from CD8.

[0060] In another preferred embodiment, the L comprises an amino acid sequence as shown in SEQ ID NO:27.

[0061] In another preferred embodiment, the amino acid sequence of the VHH is shown in SEQ ID NO:2, 5, 18.

[0062] In another preferred embodiment, H comprises an amino acid sequence as shown in SEQ ID NO:28.

[0063] In another preferred embodiment, the TM includes a transmembrane region derived from CD28.

[0064] 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.

[0065] In another preferred embodiment, the C comprises a co-stimulatory signaling molecule derived from 4-1BB.

[0066] In another preferred embodiment, the CAR fusion protein has the amino acid sequence shown in SEQ ID NO: 33-35.

[0067] In a fifth aspect of the invention, an antibody-drug conjugate is provided, the antibody-drug conjugate comprising:

[0068] (a) the nanobody described in the first aspect of the present invention, the multivalent antibody or multispecific antibody described in the second aspect of the present invention, and the recombinant protein described in the third aspect of the present invention; and

[0069] (b) A conjugation portion conjugated to the antibody portion, the conjugation portion being selected from the group consisting of: detectable markers, drugs, toxins, cytokines, radionuclides, enzymes, or combinations thereof.

[0070] In another preferred embodiment, the antibody portion is coupled to the coupling portion via a chemical bond or a linker.

[0071] In a sixth aspect of the invention, a polynucleotide is provided, the polynucleotide encoding a protein selected from the group consisting of: nanobodies described in the first aspect of the invention, or multivalent or multispecific antibodies described in the second aspect of the invention, recombinant proteins described in the third aspect of the invention, or CAR fusion proteins described in the fourth aspect of the invention.

[0072] In a seventh aspect of the invention, an expression vector is provided, the expression vector containing the polynucleotide described in the sixth aspect of the invention.

[0073] 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.

[0074] In another preferred embodiment, the expression vector is a lentiviral vector.

[0075] In an eighth aspect of the invention, a host cell is provided, the host cell containing the expression vector described in the seventh aspect of the invention, or having integrated the polynucleotide described in the sixth aspect of the invention into its genome, or expressing the nanobody described in the first aspect of the invention, the multivalent antibody or multispecific antibody described in the second aspect of the invention, the recombinant protein described in the third aspect of the invention, or the CAR fusion protein described in the fourth aspect of the invention.

[0076] In another preferred embodiment, the cells are isolated cells, and / or the cells are genetically engineered cells.

[0077] In another preferred embodiment, the cell is a mammalian cell.

[0078] In another preferred embodiment, the cell is a T cell.

[0079] In another preferred embodiment, the host cell is an engineered immune cell.

[0080] In another preferred embodiment, the engineered immune cells are selected from the group consisting of:

[0081] (i) Chimeric antigen receptor αβ T cells (CAR-T cells);

[0082] (ii) Chimeric antigen receptor γδ T cells (CAR-T cells);

[0083] (iii) Chimeric antigen receptor NKT cells (CAR-NKT cells);

[0084] (iv) Chimeric antigen receptor NK cells (CAR-NK cells);

[0085] (v) Chimeric antigen receptor macrophages.

[0086] In a ninth aspect of the present invention, a method for preparing engineered immune cells is provided, comprising the following steps: transducing a nucleic acid molecule as described in the sixth aspect of the present invention or a vector as described in the seventh aspect of the present invention into T cells or NK cells, thereby obtaining the engineered immune cells, wherein the engineered immune cells express the CAR fusion protein as described in the fourth aspect of the present invention.

[0087] In another preferred embodiment, the method further includes the step of: testing the function and effectiveness of the obtained engineered immune cells.

[0088] In a tenth aspect of the invention, a formulation is provided comprising the nanobody described in the first aspect of the invention, the multivalent antibody or multispecific antibody described in the second aspect of the invention, the recombinant protein described in the third aspect of the invention, the CAR fusion protein described in the fourth aspect of the invention, or the carrier described in the seventh aspect of the invention, or the host cell described in the eighth aspect of the invention, and pharmaceutically acceptable carriers, diluents or excipients.

[0089] In another preferred embodiment, the host cell is an engineered immune cell.

[0090] In an eleventh aspect of the present invention, a pharmaceutical composition is provided, the pharmaceutical composition comprising:

[0091] (i) the nanobody described in the first aspect of the present invention, or the multivalent antibody or multispecific antibody described in the second aspect of the present invention, or the recombinant protein described in the third aspect of the present invention, or immune cells expressing the CAR fusion protein described in the fourth aspect of the present invention; and

[0092] (ii) Pharmaceutically acceptable carriers.

[0093] In another preferred embodiment, the conjugation portion of the immunoconjugate is a drug, a toxin, and / or a therapeutic isotope.

[0094] In another preferred embodiment, the pharmaceutical composition further contains other drugs for treating immune system diseases or tumor diseases.

[0095] In another preferred embodiment, the pharmaceutical composition is used to prepare a medicine for the prevention and / or treatment of diseases or conditions related to Claudin 18.2.

[0096] In another preferred embodiment, the disease or condition associated with Claudin 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 combinations thereof.

[0097] In a twelfth aspect of the invention, a kit is provided containing the nucleic acid molecule described in the sixth aspect of the invention, the recombinant protein described in the third aspect of the invention, or the vector described in the seventh aspect of the invention.

[0098] 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.

[0099] In a thirteenth aspect of the invention, the use of the nanobody described in the first aspect of the invention, or the multivalent antibody or multispecific antibody described in the second aspect of the invention, or the recombinant protein described in the third aspect of the invention, or the immune cells expressing the CAR fusion protein described in the fourth aspect of the invention, is provided for the preparation of a medicament for the prevention and / or treatment of cancers or tumors associated with Claudin18.2.

[0100] In a fourteenth aspect of the invention, a method for treating a disease is provided, comprising administering to a subject requiring treatment: a nanobody as described in the first aspect of the invention, or a multivalent or multispecific antibody as described in the second aspect of the invention, or a recombinant protein as described in the third aspect of the invention, or immune cells expressing a CAR fusion protein as described in the fourth aspect of the invention.

[0101] In another preferred embodiment, the disease is a cancer or tumor that is positive for Claudin18.2 expression.

[0102] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0103] Figure 1 shows the binding efficiency of the anti-Claudin 18.2 recombinant antibody to 293FT-CLDN18.1 and 293FT-CLDN18.2 cells.

[0104] Figure 2 shows exemplary designs of single-CAR and dual-epitope CARs targeting the chimeric antigen receptor of Claudin 18.2.

[0105] Figure 3 shows the CAR expression level targeting Claudin 18.2T cells.

[0106] Figure 4 shows the memory phenotype of Claudin 18.2 CAR-T cells cultured in vitro.

[0107] Figure 5 shows the cytotoxicity of CAR-T transient killing results targeting Claudin 18.2 CAR-T cells to target cells 293FT-Claudin18.2, 293FT-Claudin18.1, and 293FT cells.

[0108] Figure 6 shows the in vitro cytotoxicity of Claudin 18.2-targeted CAR-T cells to AGS-Claudin18.2 target cells.

[0109] Figure 7 shows the cytokine release levels when Claudin18.2 CAR-T cells are co-cultured with target cells.

[0110] Figure 8 shows a comparison of the in vitro cytotoxicity of humanized Claudin18.2 CAR-T cells with that of maternal CAR-T cells.

[0111] Figure 9 shows the affinity of the anti-Claudin 18.2 recombinant antibody for binding to the Claudin 18.2 VLP antigen as determined by ELISA.

[0112] Figure 10 shows the in vivo efficacy of targeting Claudin18.2 CAR-T cells. Detailed Implementation

[0113] Through extensive and in-depth research, the inventors have unexpectedly obtained, for the first time, an anti-CLDN18.2 antibody with excellent high affinity and high anti-tumor activity. Specifically, after extensive screening, the inventors obtained, for the first time, multiple nanobodies that specifically bind to CLDN18.2. Based on the nanobodies, this invention further prepared recombinant antibodies and humanized antibodies, as well as a chimeric antigen receptor targeting Claudin 18.2. Based on an alpaca heavy chain antibody that specifically binds to Claudin18.2, this invention provides CAR-T cells with higher affinity, stronger specificity to the target antigen, and lower immunogenicity compared to traditional antibodies. The CAR-T cells of this invention exhibit excellent target cell killing activity, can effectively infiltrate solid tumors, and exert excellent tumor-suppressing effects in animals. This invention was completed based on these findings.

[0114] the term

[0115] To facilitate a clearer understanding of this disclosure, certain terms are first defined. As used herein, unless otherwise expressly specified herein, each of the following terms shall have the meaning given below. Other definitions are set forth throughout the application.

[0116] The term “about” can refer to a value or composition within an acceptable margin of error for a particular value or composition as determined by a person skilled in the art, depending in part on how the value or composition is measured or determined. For example, as used herein, the expression “about 100” includes all values ​​between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0117] As used herein, the terms “containing” or “including (comprise)” can be open-ended, semi-closed, or closed. In other words, the terms also include “consistently made of” or “composed of”.

[0118] 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 a reference nucleotide sequence or a protein) and determining the number of positions where identical residues occur. This is typically expressed as a percentage. The measurement of sequence identity of nucleotide sequences is a method well known to those skilled in the art.

[0119] As used in this article, the terms "heavy chain variable region" and "V" are used interchangeably. H "They can be used interchangeably."

[0120] As used in this article, the terms “variable region” and “complementarity determining region (CDR)” are used interchangeably.

[0121] In this invention, the terms "antibody of the invention," "protein of the invention," or "peptide of the invention" are used interchangeably and all refer to antibodies that specifically bind to CLDN18.2, such as proteins or peptides having a heavy chain variable region (such as the amino acid sequence shown in SEQ ID NO:1-14). They may or may not contain an initiating methionine.

[0122] CLDN18.2

[0123] Human CLDN18 contains 261 amino acids in its full length. It is a four-transmembrane protein with four transmembrane hydrophobic regions and two extracellular loop structures. Loop 1 is formed by transmembrane regions 1 and 2, while loop 2 is formed by transmembrane regions 3 and 4. Human CLDN18.1 and CLDN18.2 differ in 8 amino acids at the N-terminus, transmembrane region 1, and extracellular loop 1; the remaining parts are identical, with a sequence similarity of 92%.

[0124] Antibody

[0125] As used in this article, the term "antibody" refers to an immunoglobulin, which is a tetrapeptide chain structure consisting of two identical heavy chains and two identical light chains linked by interchain disulfide bonds.

[0126] Existing antibody numbering schemes include:

[0127] 1. The Kabat scheme (Kabat et al., 1991) is based on the location of highly variable regions between sequences of the same domain type, with different numbering for antibody heavy (VH) and light (Vλ and Vκ) variable domains.

[0128] 2. Chothia's scheme (Al-Lazikani, 1997) is similar to Kabat's scheme, but corrects the placement of the annotations around the first VH complementarity determination region (CDR) to correspond to the structural loop. Similarly, the enhanced Chothia scheme (Abhinandan and Martin, 2008) further structurally modifies the insertion position.

[0129] 3. In contrast to these Kabat-like schemes, IMGT (Lefranc, 2003) and AHo (Honegger and Plückthun, 2001) have defined unique schemes for variable domains of antibodies and T-cell receptors (TCRs) (Vα and Vβ). Therefore, equivalent residue positions can be readily compared between domain types. IMGT and AHo differ in the number of positions they annotated (128 and 149, respectively) and in the locations where they believe indels occur.

[0130] Immunoglobulins differ in the amino acid composition and sequence of their heavy chain constant regions, thus exhibiting different antigenicities. Based on this, immunoglobulins can be classified into five classes, or isotypes, namely IgM, IgD, IgG, IgA, and IgE, with their corresponding heavy chains being μ, δ, γ, α, and ε chains, respectively. Within the same class of Ig, differences in the amino acid composition of the heavy chain region and the number and position of disulfide bonds in the heavy chain can further lead to different subclasses; for example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4. Light chains are classified as κ or λ chains based on their constant regions. Each of the five classes of Ig can possess either 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.

[0131] The antibody light chain of the present invention may further include a light chain constant region, wherein the light chain constant region comprises a human or mouse κ, λ chain or a variant thereof.

[0132] In this invention, the antibody heavy chain may further include a heavy chain constant region, which contains human or mouse IgG1, IgG2, IgG3, IgG4, or variants thereof. The sequence of approximately 110 amino acids near the N-terminus of the antibody heavy and light chains varies considerably and is called the variable region (Fv region); the remaining amino acid sequence near the C-terminus is relatively stable and is called the constant region. The variable region includes three hypervariable regions (HVR) and four relatively conserved backbone regions (FR). The three hypervariable regions determine the antibody's specificity and are also called complementarity-determining regions (CDR). Each light chain variable region (LCVR) and heavy chain variable region (HCVR) consists of three CDR regions and four FR regions, arranged in the following order from the amino terminus to the terminal terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The three CDR regions of the light chain refer to LCDR1, LCDR2, and LCDR3; the three CDR regions of the heavy chain refer to HCDR1, HCDR2, and HCDR3. In Example 13 of this invention, the six CDRs of the ch782 antibody are divided using the Kabat and Chothia methods.

[0133] In this invention, the term "mouse antibody" refers to a monoclonal antibody against CLDN18.2 prepared according to the knowledge and skills in the art. Preparation involves injecting the test subject with the CLDN18.2 antigen, followed by isolating hybridomas expressing antibodies with the desired sequence or functional characteristics. In a preferred embodiment of the invention, the mouse CLDN18.2 antibody or its antigen-binding fragment may further comprise a light chain constant region of a mouse κ, λ chain or a variant thereof, or further comprise a heavy chain constant region of mouse IgG1, IgG2, IgG3 or a variant thereof.

[0134] The term "chimeric antibody" refers to an antibody formed by fusing the variable region of a murine antibody with the constant region of a human antibody, which can reduce the immune response induced by murine antibodies.

[0135] The term "humanized antibody," also known as a CDR-grafted antibody, refers to an antibody generated by grafting a mouse CDR sequence into the variable region framework of a human antibody, i.e., a different type of human germline antibody framework sequence. Humanized antibodies can overcome the heterologous response induced by chimeric antibodies due to the large amount of mouse protein components they carry. Such framework sequences can be obtained from public DNA databases containing germline antibody gene sequences or from publicly available references. To avoid a decrease in activity along with a decrease in immunogenicity, minimal reverse or reversion mutations can be performed on the human antibody variable region framework sequence to maintain activity.

[0136] The term "antigen-binding fragment of an antibody" (or simply "antibody fragment") refers to one or more fragments of an antibody that maintain the ability to specifically bind to an antigen (e.g., CLDN18.2). It has been shown that fragments of full-length antibodies can be used for antigen-binding function. Examples of binding fragments included in the term "antigen-binding fragment of an antibody" include...

[0137] (i)Fab fragment, a monovalent fragment composed of VL, VH, CL and CH1 domains;

[0138] (ii) F(ab')2 fragment, which is a divalent fragment containing two Fab fragments connected by a disulfide bridge on the chain region;

[0139] (iii) Fd fragments composed of VH and CH1 domains;

[0140] (iv) The Fv fragment consisting of the VH and VL domains of the single arm of the antibody.

[0141] Fv antibodies contain variable regions on the heavy and light chains, but no constant regions, and are the smallest antibody fragments with all antigen-binding sites. Typically, Fv antibodies also contain a polypeptide linker between the VH and VL domains and can form the structure required for antigen binding.

[0142] The term "CDR" refers to one of the six hypervariable regions within the variable domain of an antibody that primarily facilitate antigen binding. One of the most commonly used definitions of these six CDRs is provided by Kabat EA et al., (1991) Sequences of proteins of immunological interest. NIH Publication, No. 91-3242.

[0143] The term "epitope" or "antigenic determinant" refers to a site on an antigen where an immunoglobulin or antibody specifically binds (e.g., a specific site on the CLDN18.2 molecule). Epitopes typically consist of 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 conformation.

[0144] The terms "specific binding," "selective binding," "selective binding," and "specific binding" refer to the binding of an antibody to a pre-defined epitope on an antigen. Typically, antibodies bind at a concentration of approximately less than 10... -7 M, for example, approximately less than 10 -8 M, 10 -9 M or 10 10 M or lower affinity (KD) binding.

[0145] The term "competitive binding" refers to an antibody that recognizes or binds to the same epitope (also called an antigenic determinant) or a portion of the same epitope on the extracellular region of CLDN18.2 as the monoclonal antibody of the present invention. 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.

[0146] The term "KD" or "Kd" refers to the dissociation equilibrium constant of a specific antibody-antigen interaction. Typically, the antibodies of this invention have a dissociation equilibrium constant of less than approximately 10. -7 M, for example, less than approximately 10 -8 M, 10 -9 M or 10 -10 M or a smaller dissociation equilibrium constant (KD) is combined with CLDN18.2.

[0147] As used herein, the term "antigen 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.

[0148] This invention includes not only complete antibodies, but also fragments of immunologically active antibodies or fusion proteins formed by antibodies and other sequences. Therefore, this invention also includes fragments, derivatives, and analogs of said antibodies.

[0149] In this invention, antibodies include mouse, chimeric, humanized, or fully human antibodies prepared using techniques well known to those skilled in the art. Recombinant antibodies, such as chimeric and humanized monoclonal antibodies, including both human and non-human portions, can be prepared using DNA recombination techniques well known in the art.

[0150] 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, targeting a single antigenic epitope. The cell may be a eukaryotic, prokaryotic, or phage clone.

[0151] In this invention, the antibody can be monospecific, bispecific, trispecific, or more multiple specific.

[0152] In this invention, the antibody also includes its conserved variants, which are polypeptides formed by replacing up to 10, preferably up to 8, more preferably up to 5, and most preferably up to 3 amino acids with amino acids of similar or analogous properties compared to the amino acid sequence of the antibody of this invention. These conserved variant polypeptides are preferably generated by amino acid substitutions according to Table A below.

[0153] Table A

[0154] Anti-CLDN18.2 humanized antibody

[0155] This invention provides a humanized antibody against CLDN18.2 (hereinafter referred to as CLDN 18.2 antibody). Specifically, this invention provides a humanized antibody with high specificity and high affinity against CLDN18.2, comprising a heavy chain and a light chain, wherein the heavy chain contains a heavy chain variable region (VH) amino acid sequence and the light chain contains a light chain variable region (VL) amino acid sequence.

[0156] In antibody humanization, there are generally two choices for the human backbone region: known mature antibodies and human Germline sequences. Known mature antibody backbone regions often contain somatic mutation sites, potentially introducing immunogenicity. Compared to mature antibodies, human Germline sequence backbone regions theoretically have lower immunogenicity, are more structurally flexible and malleable, and readily accept different CDR regions. The frequency of use of human antibody Germline genes in the human body exhibits a certain bias; antibodies humanized from frequently used Germline backbone regions have advantages such as low immunogenicity, high expression levels, and structural stability.

[0157] In a preferred embodiment of the present invention, the Germline sequence with the highest similarity to the mouse antibody was not selected during humanization. Instead, considering both similarity and frequency of human use, a preferred sequence backbone region was selected for humanization after extensive experimental screening. Using the human antibody Germline backbone region for CDR transplantation results in a more stable humanized antibody structure with higher expression levels, lower immunogenicity, and higher drugability.

[0158] In another preferred embodiment, the heavy chain constant region and / or light chain constant region may be a humanized heavy chain constant region or a light chain constant region. More preferably, the humanized heavy chain constant region or light chain constant region is a heavy chain constant region of human IgG1, IgG2, etc., or a light chain constant region of human kappa, Lambda.

[0159] In another preferred embodiment, the sequence formed by adding, deleting, modifying and / or substituting at least one amino acid sequence preferably has a homology of at least 80%, more preferably at least 85%, more preferably at least 90%, and most preferably at least 95% of the amino acid sequence.

[0160] The antibodies of the present invention can be double-chain or single-chain antibodies, and are preferably fully humanized antibodies.

[0161] The antibody derivatives described in this invention may be single-chain antibodies and / or antibody fragments, such as Fab, Fab', (Fab')2, or other known antibody derivatives in the field, as well as any one or more of IgA, IgD, IgE, IgG, and IgM antibodies or other subtypes of antibodies.

[0162] The antibody of this invention may be a humanized antibody targeting CLDN18.2, a CDR grafted and / or modified antibody.

[0163] In the above-described content of the present invention, the number of added, deleted, modified and / or substituted amino acids is preferably no more than 40% of the total number of amino acids in the initial amino acid sequence, more preferably no more than 35%, more preferably 1-33%, more preferably 5-30%, more preferably 10-25%, and more preferably 15-20%.

[0164] Antibody preparation

[0165] Any method suitable for producing monoclonal antibodies can be used to produce the CLDN18.2 antibody of the present invention. For example, animals can be immunized with linked or naturally occurring CLDN18.2 protein or fragments thereof. Suitable immunization methods can be used, including adjuvants, immunostimulants, repeated booster immunizations, or one or more routes.

[0166] Any suitable form of CLDN18.2 can be used as an immunogen (antigen) to generate non-human antibodies specific to CLDN18.2 and to screen for the biological activity of said antibodies. The immunogen can be used alone or in combination with one or more immunogenic enhancers known in the art. The immunogen can be purified from a natural source or produced in genetically modified cells. The DNA encoding the immunogen can be genomic or non-genomic (e.g., cDNA). The DNA encoding the immunogen can be expressed using suitable genetic vectors, including but not limited to adenovirus vectors, baculovirus vectors, plasmids, and nonviral vectors.

[0167] Humanized antibodies can be selected from any type of immunoglobulin, including IgM, IgD, IgG, IgA, and IgE. Similarly, any class of light chains can be used in the compounds and methods described herein. Specifically, κ, λ chains, or variants thereof, can be used in the compounds and methods of this invention.

[0168] The DNA sequences of the antibodies or fragments thereof of this invention can be obtained using conventional techniques, such as PCR amplification or genomic library screening. Furthermore, the coding sequences of the light and heavy chains can be fused together to form single-chain antibodies.

[0169] Once the relevant sequence is obtained, it can be obtained in large quantities using recombination methods. This typically involves cloning it into a vector, transferring it into cells, and then isolating the sequence from the proliferated host cells using conventional methods.

[0170] In addition, sequences can be synthesized artificially, especially when the fragment length is short. Typically, long sequences are obtained by first synthesizing multiple small fragments and then ligating them. This DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art.

[0171] The term "nucleic acid molecule" refers to both DNA and RNA molecules. Nucleic acid molecules can be single-stranded or double-stranded, but double-stranded DNA is preferred. Nucleic acids are "effectively linked" when placed in a functional relationship with another nucleic acid sequence. For example, if a promoter or enhancer affects the transcription of a coding sequence, then the promoter or enhancer is effectively linked to said coding sequence.

[0172] The term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. In one implementation, the vector is a "plasmid," which refers to a circular double-stranded DNA loop to which an additional DNA segment can be linked.

[0173] The present invention also relates to vectors comprising the aforementioned suitable DNA sequences and suitable promoters or control sequences. These vectors can be used to transform suitable host cells to enable them to express proteins.

[0174] The term "host cell" refers to a cell into which an expression vector has been introduced. Host cells can be prokaryotic cells, such as bacterial cells; lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as plant or animal cells (such as mammalian cells).

[0175] The steps of transforming host cells with recombinant DNA as described in this invention can be performed using techniques well known in the art. The obtained transformants can be cultured using conventional methods, and the transformants express the polypeptide encoded by the gene of this invention. Depending on the host cell used, the cells are cultured in a conventional culture medium under suitable conditions.

[0176] Typically, host cells transformed with the antibody are cultured under conditions suitable for antibody expression according to the present invention. The antibody of the present invention is then purified using conventional immunoglobulin purification steps, such as protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, ion exchange chromatography, hydrophobic chromatography, molecular sieve chromatography, or affinity chromatography, which are well known to those skilled in the art.

[0177] The obtained monoclonal antibodies can be identified using conventional methods. For example, the binding specificity of monoclonal antibodies can be determined by immunoprecipitation or in vitro binding assays (such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA)).

[0178] antibody preparations

[0179] Antibodies exhibit varying degrees of stability in different formulation buffers, manifesting as changes in charge heterogeneity, antibody molecule degradation, and polymerization. These changes in quality properties are related to the antibody's inherent physicochemical properties. Therefore, in antibody drug development, it is necessary to screen suitable formulation buffers based on the physicochemical properties of different antibodies. Currently, commonly used antibody formulation buffer systems include phosphate buffer, citrate buffer, and histidine buffer. Depending on the antibody's properties, different concentrations of salt ions or excipients such as sorbitol, trehalose, and sucrose, as well as appropriate amounts of surfactants such as Tween, are added to maintain antibody stability.

[0180] Pharmaceutical Composition

[0181] The present invention also provides a composition. In a preferred embodiment, the composition is a pharmaceutical composition comprising the aforementioned antibody or its active fragment or fusion protein or its ADC or corresponding CAR-T cell, and a pharmaceutically acceptable carrier. Typically, these substances are formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous carrier medium, wherein the pH is typically about 5-8, preferably about 6-8, although the pH may vary depending on the nature of the formulated substance and the condition to be treated. The formulated pharmaceutical composition can be administered via conventional routes, including (but not limited to): intratumoral, intraperitoneal, intravenous, or local administration.

[0182] The antibody described in this invention can also be expressed in cells by a nucleotide sequence for cell therapy, such as for chimeric antigen receptor T-cell immunotherapy (CAR-T).

[0183] The pharmaceutical compositions of the present invention can be directly used 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 concurrently.

[0184] The pharmaceutical compositions of the present invention contain a safe and effective amount (e.g., 0.001-99 wt%, preferably 0.01-90 wt%, more preferably 0.1-80 wt%) of the above-described monoclonal antibody (or conjugate thereof) of the present invention, and a pharmaceutically acceptable carrier or excipient. Such carriers include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical formulation should be matched to the route of administration. The pharmaceutical compositions of the present invention can be formulated into injectable forms, for example, prepared by conventional methods using physiological saline or an aqueous solution containing glucose and other excipients. Pharmaceutical compositions such as injections and solutions are preferably manufactured under sterile conditions. The dosage of the active ingredient is a therapeutically effective amount, for example, about 1 microgram / kg body weight to about 5 milligrams / kg body weight per day. Furthermore, the peptides of the present invention can also be used with other therapeutic agents.

[0185] When using a pharmaceutical composition, a safe and effective amount of the composition is administered to a mammal, wherein the safe and effective amount is generally at least about 10 micrograms per kilogram of body weight, and in most cases does not exceed about 50 milligrams per kilogram of body weight, preferably about 10 micrograms per kilogram of body weight to about 20 milligrams per kilogram of body weight. Of course, the specific dosage should also take into account factors such as the route of administration and the patient's health condition, which are all within the scope of the skill of a skilled physician.

[0186] Detection uses and kits

[0187] The antibodies of this invention can be used in detection applications, such as for testing samples, to provide diagnostic information.

[0188] In this invention, the samples used include cells, tissue samples, and biopsy specimens. The term "biopsy" as used in this invention should include all types of biopsies known to those skilled in the art. Therefore, biopsies used in this invention can include tissue samples prepared, for example, by endoscopic methods or by puncture or needle biopsy of organs.

[0189] The samples used in this invention include fixed or preserved cell or tissue samples.

[0190] The present invention also provides a kit containing the antibody (or fragment thereof) of the present invention. In a preferred embodiment of the present invention, the kit further includes a container, instructions for use, a buffer, etc. In a preferred embodiment, the antibody of the present invention can be immobilized on a detection plate.

[0191] The main advantages of this invention include:

[0192] 1. The single-domain antibody (VHH domain) of Claudin 18.2 of this invention has high specificity and affinity.

[0193] 2. The anti-Claudin 18.2 antibody of the present invention has an affinity for CLDN18.2 that is at least 100 times greater than its affinity for CLDN18.1 (cell based).

[0194] 3. The Claudin 18.2 CAR T cells of the present invention exhibit specific in vitro cytotoxicity against Claudin 18.2 positive target cells, which is significantly higher than the in vitro killing efficiency of Claudin 18.1 target cells.

[0195] 4. The Claudin 18.2CAR T cells of the present invention can effectively inhibit the growth of Claudin18.2 positive tumors in vivo and show a long-lasting anti-tumor effect.

[0196] 5. The Claudin 18.2CAR05 T cells of the present invention exhibit a stronger immune memory phenotype during continuous in vitro culture.

[0197] 6. Compared with its parent CAR-T cells, the humanized Claudin 18.2CAR T cells of the present invention do not show a significant reduction in toxicity to target cells, nor a significant reduction in affinity.

[0198] 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.

[0199] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0200] Unless otherwise specified, all materials and reagents used in the embodiments of this invention are commercially available products.

[0201] Example 1

[0202] 1. Alpaca antibody screening

[0203] Antibodies were screened from a phage antibody library. The process of establishing an alpaca antibody library is briefly described below:

[0204] The antigen Claudin 18.2VLP (0.5 mg) and 293-Claudin 18.2 cells (2e7) were injected at multiple points under the neck of alpacas. After multiple rounds of immunization, 50 ml of peripheral blood was collected for antibody titer detection and nanobody library construction.

[0205] 1.1 Construction of VHH phage display library

[0206] After multiple rounds of immunization, the serum titers of alpacas met the following criteria: 1) ELISA titer > 32000, 2) FACS titer > 200, thus meeting the conditions for library construction.

[0207] PBMCs were isolated, total RNA was extracted from PBMCs, and then reverse transcribed into cDNA. VHH fragments were amplified by nested PCR, and then the VHH fragments were inserted into phage particles. The phages were amplified and harvested to obtain an antibody library.

[0208] The VHH3 antibody library was found to have a capacity of 5.2 × 10⁻⁶. 9 The recombination efficiency (number of clonal PCR positive clones / total number of clones) was 90%, which meets the relevant indicators for a high-quality immune library.

[0209] 1.2 Screening for Claudin18.2 antibodies via cell panning

[0210] The process can be briefly described as follows:

[0211] 1.2.1 Pretreatment: Claudin 18.1 overexpressing cells (as negative cells) were added to the antibody library;

[0212] 1.2.2 Combining the steps, centrifuge the supernatant from step 1, and use the pretreated antibody library to bind to Claudin18.1 and Claudin18.2 overexpressing cells respectively, and screen by liquid chromatography;

[0213] 1.2.3 Clean, clean 6-8 times;

[0214] 1.2.4 Amplification: NEBalpha5F' cells were infected, helper phages were added, and the cells were cultured overnight.

[0215] 1.2.5 Plate smear detection enrichment (enrichment is considered successful if the number of positive cell clones / the number of negative cell clones > 10);

[0216] 1.2.6 Harvest R1 round phages and enrich them for the next round of screening;

[0217] 1.3 Identification of clones obtained from cell screening - phage-based FACS

[0218] 1.3.1 Cell screening and clone identification: Clones that bind to Claudin18.2 overexpressing cells were randomly selected, expanded cultured to obtain phages, and their binding affinity to Claudin 18.1 and Claudin 18.2 overexpressing cells was identified.

[0219] 1.3.1 Cell preparation:

[0220] Claudin 18.1 and 18.2 overexpressing cells at 2 × 10 5 1 cell / group, centrifuged, washed 3 times with DPBS;

[0221] 1.3.2 Sample incubation

[0222] Add the supernatant of the expanded phage culture separately and incubate at 4°C for 2 hours;

[0223] 1.3.3 Secondary Antibody Incubation

[0224] Add PE-labeled anti-M13 antibody (concentration 5ug / ml, 100ul) and incubate at 4℃ for 45min;

[0225] 1.3.4 Washing with DPBS

[0226] Wash three times with DPBS and resuspend in 300 μL of DPBS.

[0227] 1.3.5 Flow Cytometry

[0228] Fluorescence analysis of the resuspended cells using flow cytometry showed that all 14 random clones bound to cells overexpressing Claudin 18.2, but not to cells overexpressing Claudin 18.1.

[0229] 1.4 Sequencing of positive clones

[0230] Fourteen positive clones (from 1.3) were sequenced. The clone numbers and VHH domain sequence IDs are shown in the table below. CDR1, CDR2, and CDR3 were defined according to the Kabat numbering system, referencing Deschacht et al., 2010, *Journal of Immunology*, 184:5696-704. Clones 7, 8, 9 and 12, 13 shared the same CDR1, 2, and 3, differing only in the FR region, as shown in Table 1 below.

[0231] Table 1

[0232] 1.5 Cell screening, phage enrichment, and NGS sequencing

[0233] Phage pools enriched by screening cells overexpressing 293FT-Claudin 18.2 were amplified with VHH3 gene fragments by PCR and NGS sequencing to obtain 30 VHH3 gene sequences.

[0234] For these 30 VHH3 genes, the binding performance of the expressed VHH domain with Claudin 18.2 was further tested, and 6 nanobodies with the best performance were obtained. The sequences of their VHH domains, as well as the sequences of CDR1, CDR2 and CDR3, are shown in Table 2.

[0235] Table 2: NGS sequencing analysis of phages enriched by cell screening

[0236] 1.6 Affinity Detection of Optimized Sequences - ELISA

[0237] The affinity of recombinant antibodies was detected using the ELISA method. Four sequences (clones 1, 5, 8, and 10) were selected from 14 clones to synthesize recombinant antibody VHH-hIgG1Fc. Its affinity with the antigen was detected. The antigens were Claudin 18.2VLP and Claudin 18.2 recombinant protein (His tag), respectively. The ELISA EC50 data are shown in Table 2.

[0238] The detection method is as follows:

[0239] 1.5.1 Coating: On day 1, the antigen (200 ng / well) was coated onto the ELISA plate and incubated overnight at 4°C;

[0240] 1.5.2 Blocking: On the second day, block the ELISA plate with 0.5% BSA at room temperature for 2 hours;

[0241] 1.5.3 Binding: On the second day, VHH-hIgG1 Fc was added to ELISA plates at a concentration of 10 μg / mL, with 7 concentrations prepared at a 1:3 ratio. The plates were incubated at room temperature for 2 hours.

[0242] 1.5.4 Washing: Wash the ELISA plate with PBS-Tween, add 100 μL of anti-human IgG1Fc HRP antibody, and incubate at room temperature for 1 h;

[0243] 1.5.5 Color development: Wash the ELISA plate with PBS-Tween, then wash with PBS, add 100 μL TMB, and incubate at 37°C for 5-10 min;

[0244] 1.5.6 Termination: The reaction was terminated by adding 50 μL of 1M phosphoric acid;

[0245] 1.5.7 Measurement values: The absorbance at 450 nm was measured using an ELISA reader, and the results are shown in Table 3.

[0246] Table 3

[0247] As shown in Table 3, the recombinant antibodies disclosed herein bind to Claudin18.2 VLP or Claudin18.2-his antigen in a dose-dependent manner, with binding potency in the range of 4 nM-200 nM (EC50 value).

[0248] 1.7 Affinity Detection of Optimized Sequences - FACS

[0249] Cell-based flow cytometry was used to detect the binding ability of recombinant antibodies to 293FT-hClaudin18.1 cells (negative cells) and 293FT-hClaudin18.2 cells (positive cells). 293FT-Claudin 18.1 and 293FT-Claudin 18.2 cells were incubated with recombinant antibodies containing four preferred sequences (5 μg / mL and 10 μg / mL), respectively, at 4°C for 30 min. The cells were then washed with DPBS, and the cell pellet was resuspended in secondary antibody (1:100, Goat anti-human IgG (H+L), FITC), and incubated in the dark for another 15 min. After washing with DPBS, the cells were used for flow cytometry to detect the binding level of recombinant antibodies to 293FT-hClaudin18.1 and 293FT-hClaudin18.2 cells. The recombinant antibodies were labeled with Fc tags, namely 1-hIgG1Fc, 5-hIgG1Fc, 8-hIgG1Fc, and 10-hIgG1Fc, and the concentration of the recombinant antibodies used was 10 μg / mL. The binding ability of each tested recombinant antibody was described as a binding percentage, calculated as (cells with antibody binding / total cells) × 100%.

[0250] The results are shown in Figure 1. The binding percentages of the recombinant antibody to Claudin 18.1 cells were 2.75%, 2.06%, 2.26%, and 2.06%, respectively, while the binding percentages to Claudin 18.2 cells were 88.94%, 95.96%, 93.86%, and 96.1%, respectively. This indicates that the recombinant antibody has specific binding to human Claudin 18.2 cells.

[0251] 1.8 Humanized anti-Claudin 18.2 antibody

[0252] The humanized anti-Claudin18.2 antibody is based on the Claudin 18.2 VHH antibody. The antibody was humanized 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 modified based on this, with the aim of reducing antigenicity. The design of the humanized single-domain antibody from camelid species uses Kabat as the marker residue numbering in the VHH domain, for example, residues 11, 37, 44, 45, and 47 (Muyldermans, *Review of Molecular Biotechnology*, 74:277 302 (2001)). The humanized sequence is shown in Table 4.

[0253] Table 4

[0254] 1.9 Exemplary designs of single-CAR and dual-epitope CARs targeting chimeric antigen receptors of Claudin 18.2

[0255] Example structures of Claudin 18.2 single and double CAR are shown in Figure 2, and exemplary amino acid sequences of CAR constructs are listed in Table 5.

[0256] Example 2: Preparation of Claudin18.2-targeted CAR-T

[0257] 2.1 Preparation of Claudin18.2-targeted CAR-T

[0258] General materials and methods:

[0259] Peripheral blood mononuclear cells (PBMCs) were isolated from donor blood and T cells were expanded using Ficoll density gradient centrifugation. The T cells were then enriched, activated by magnetic beads conjugated with anti-CD3 / anti-CD28, cultured, and expanded.

[0260] Target cell lines, PBMCs, CAR-T culture:

[0261] 1. Cell lines 293FT-Luc, 293FT-CLDN18.1-luc, 293FT-CLDN18.2-Luc, AGS-CLDN18.2-Luc, AGS-CLDN18.1(KO CLDN18.2)-Luc (KO CLDN18.2 refers to the knockout of endogenous Claudin18.2), Aspc-1-CLDN 18.2-Luc, and the 293FT series were cultured in DMEM medium. All Luc-tagged cells stably expressed firefly luciferase. AGS-CLDN 18.2-Luc and AGS-CLDN 18.1(KO CLDN18.2)-Luc series cells were cultured in F12 medium, and Aspc-1-CLDN 18.2-Luc cells were cultured in RPMI 1640 medium. RPMI 1640, F12, and DMEM media were all supplemented with 10% (v / v) fetal bovine serum and 100 U / ml penicillin and streptomycin, 2 mM glutamine, and 1 mM sodium pyruvate. All cells were cultured in an incubator at 37°C and 5% CO2.

[0262] 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). The CAR-T cell culture medium was supplemented with rhIL-2, with 300 IU / ml IL-2 added every two days. All cells were cultured at 37°C and 5% CO2.

[0263] CAR-T preparation

[0264] Gene codon optimization was used to optimize the VHH coding sequence of the selected antibody, synthesize a nucleotide molecule encoding a chimeric antigen receptor (CAR) backbone polypeptide, clone it into a lentiviral vector (pCDH backbone vector with GFP expression sequence), insert a flag tag into the N-terminus of the hinge region, and select the VHH coding sequence from Tables 1, 2 and 4 corresponding to their CAR-T numbers, as detailed in Table 5. The control antibody (IMAB) is a fragment with the IMAB362 sequence including its heavy chain and light chain variable regions.

[0265] The mixture containing the lentiviral packaging plasmid and the vector expressing the CAR construct were premixed 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 cultured for 48-72 hours. The supernatant was collected and the lentivirus was concentrated.

[0266] Dynabeads CD3 / CD28-activated primary T cells were transfected with lentivirus. Three days post-transfection, the percentage of CAR-positive cells was detected using an anti-flag tag antibody, and FITC channels (showing GFP expression) were also detected. Three batches of CAR-T cells were produced, with CAR positivity rates ranging from 30% to 70%. Flag and GFP expression were nearly 1:1, indicating successful co-expression of CAR and GFP (see Figure 3). Table 5 shows the correspondence between the CAR VHH amino acid sequences and corresponding VHH domains used to prepare Claudin18.2-targeting CAR-T cells.

[0267] Example 3: In vitro function of Claudin18.2 CAR-T cells

[0268] 3.1 CAR-T cell memory phenotype analysis

[0269] After CAR-T cells were cultured for 8-10 days, cell phenotype was detected using flow cytometry. Anti-flag antibodies were used to distinguish between CAR-positive and CAR-negative cells. Simultaneously, T cell memory phenotype was detected using CD45RA, CD45RO, and CCR7 antibodies. Cells were then enclosed in CAR-positive cells for analysis. T CM T EM T EMRA The cell community is shown in Figure 4, with CD45RA cells from CAR15, CAR16, and CAR05. + CCR7 + ( The proportion of CD45RA cells was higher than in other groups (20%, 23%, 13% vs. approximately 2%), and CD45RA... + CD45RO - The proportion of cells was also greater than in other groups, indicating that its cells were more youthful than other CAR-T cells.

[0270] 3.2 Cytotoxicity of Claudin18.2-targeted CAR-T cells

[0271] Target cells expressing luciferase were constructed including 293FT-Luc, 293FT-Claudin18.1-Luc, and 293FT-Claudin18.2-Luc. Upon addition of the luciferase substrate, luciferase reacts with luciferin to produce fluorescence. By detecting the fluorescence intensity, the activity of luciferase can be measured, and the number of viable target cells can be determined, thereby assessing the cytotoxicity of CAR-T cells. Specific cytotoxicity was calculated using the following formula: Specific cytotoxicity % = 100% × (RLU) only -RLU 样品 / RLU only RLU 样品 This indicates the luciferase activity measured in wells containing Claudin18.2 CAR T cells, and RLU... only This refers to the luciferase activity measured in the target cell-only wells.

[0272] 293FT-Luc, 293FT-Claudin18.1-Luc, and 293FT-Claudin18.2-Luc target cells expressing luciferase were co-incubated with CAR-T at an effector:target ratio (E:T = 1:1) for 6 hours, and cytotoxicity was compared.

[0273] The results are shown in Figure 5. From left to right, the three data bars represent the lysis rates of CAR T cells on target cells 293FT-Luc, 293FT-Claudin18.1-Luc, and 293FT-Claudin18.2-Luc, respectively.

[0274] The results showed that IMAB, CAR02, CAR04 to CAR13, and CAR18 T cells did not exhibit significant cytotoxicity against 293FT-Luc cells and 293FT-Luc cells stably expressing human Claudin18.1. CAR01 and CAR17 CAR-T cells showed significant cytotoxicity against 293FT-Luc cells stably expressing human Claudin18.1 and Claudin18.2, while CAR14 T cells showed significant cytotoxicity against 293FT-Luc, 293FT-Claudin18.1-Luc, and 293FT-Claudin18.2-Luc cells. Furthermore, CAR02, CAR05, and CAR18 T cells exhibited superior killing specificity (their ratio of lysis rate against 293FT-Claudin18.2-Luc cells to lysis rate against 293FT-Claudin18.1-Luc cells was the highest).

[0275] The results of real-time cell analysis (RTCA) are shown in Figure 6. At an effector-to-target ratio of 1:1, except for CAR16, no significant cytotoxicity was observed in AGS cells stably expressing human Claudin18.2. CAR15 showed lower cytotoxicity to AGS cells stably expressing human Claudin18.2 than other CAR-T cells. Except for CAR15 and CAR16, all other CAR-T cells showed significant cytotoxicity when co-incubated with the target cells AGS-Claudin18.2 for 10 hours.

[0276] 3.3 Release of cytokines

[0277] 100 μL each of CAR-T cells and AGS-Claudin18.2 cells were mixed in RPMI medium at a 1:1 ratio, with a density of 1 × 10⁻⁶ cells / mL. 5 / ml per CAR-T cell, cultured overnight in 96-well plates. The culture medium was then collected and centrifuged, and the supernatant was used to detect the release of cytokines IFN-γ, IL-2, IL-4, IL-6, IL-10 and TNF into the cell culture medium using a cytokine assay kit (CBA kit, BD).

[0278] As shown in Figure 7, a large amount of IL-2, TNFα and IFNγ were detected in the co-culture supernatant of CAR17-CAR20, CAR02 to CAR06 and CAR10, indicating that Claudin18.2 CAR-T cells specifically released cytokines including IL-2, TNFα and IFNγ during the action of CLDN18.2 positive target cells.

[0279] Advantageously, none of these CAR T cells of the present invention lead to a large release of IL-6, suggesting a low risk of causing a cytokine storm.

[0280] In addition, CAR05 T cells unexpectedly led to a high release of IL-2, TNFα, and IFNγ (significantly higher than other CAR T cells).

[0281] Example 4: In vitro data of humanized VHH domain CAR-T cells

[0282] Using the same in vitro functional method for targeting Claudin18.2 CAR-T cells as in Example 3, the in vitro function of humanized VHH domain CAR-T cells was detected as shown in Figure 8.

[0283] Analysis of CAR-T phenotypes after 8 days of culture showed that, apart from significant differences between CAR21 and CAR05 phenotypes, CAR21 also exhibited... The proportion of CAR21 T cells was significantly lower than that of CAR05, CAR22, and CAR23 (27.5% vs. 2.4%). EMRA The proportion of cells was significantly higher than that of CAR05 (61.4% vs. 43.9%), consistent with its transient killing results, and the transient killing rate was greater than that of CAR05.

[0284] The results, as shown in Figure 8, compared the transient killing efficacy of humanized CAR-T cells with maternal CAR-T cells, showed that CAR21 (humanized) had higher transient killing efficacy against 293FT-CLDN18.2-Luc (80% vs. 50%) and Nugc4-CLDN18.2-Luc (70% vs. 45%) than CAR05 (unhumanized). The transient killing efficiencies of CAR22, CAR23 (humanized) and CAR05 (unhumanized) were similar. The transient fragmentation rates of CAR25 (humanized) and CAR18 (unhumanized) against 293FT-CLDN18.2-Luc were 75% vs. 60%, and the transient fragmentation rates of CAR26 (humanized) and CAR02 (unhumanized) against 293FT-CLDN18.2-Luc were 70% vs. 60%. The results show that humanized CAR25 and CAR26 exhibit higher transient killing efficiency than the parent CAR-T.

[0285] Example 5 Affinity Detection

[0286] 5.1 ELISA was used to detect the binding curve of the recombinant antibody to the Claudin18.2 VLP antigen.

[0287] The recombinant antibodies listed in the table below were synthesized, and ELISA was used to detect the binding of Claudin18.2-VLP protein to these recombinant antibodies. The specific procedures for the ELISA experiment are as follows:

[0288] Add 200 ng Claudin 18.2-VLP protein to each well of the ELISA plate and incubate overnight at 4°C. Wash three times with PBS, add 200 μL of 1% BSA / PBS to each well, and block at 37°C for 1 hour. Wash the plate with 100 μL of PBS, add serially diluted fusion antibody, and incubate at 37°C for 1 hour. Wash three times with PBST, add 100 μL of 1:5000 diluted HRP-goat anti-mouse IgG Fc, and incubate at 37°C for 1 hour. Wash three times with PBST, add 100 μL of TMB chromogenic buffer per well, incubate at 37°C for 10 minutes, stop the reaction with 50 μL of 1M phosphate, and measure the absorbance at 450 nm using an ELISA reader.

[0289] The experimental results of ELISA detection of VHH binding antigen ability are shown in Figure 9. Several ELISA results of the nanobodies of this invention were tested. 50 The values ​​were all superior to those of the positive control antibody IMAB.

[0290] Based on EC 50 The affinity values, ranked from highest to lowest, are 5H3>5>2,18,5H2>5H1>IMAB.

[0291] 5.2 Flow cytometry was used to detect the binding ability of the recombinant antibody to 293FT-CLDN18.1-Luc or 293FT-CLDN18.2-Luc cells.

[0292] 293FT-Claudin 18.1 cells were used as negative cells and 293FT-Claudin 18.2 cells were used as positive cells. They were incubated with serially diluted anti-Claudin18.2 VHH-mIgG2aFc fusion antibody at 4°C for 1 hour. After washing three times with PBS, secondary antibody (1:200 APC goat anti-mouse IgG Fc) was added and incubated at 4°C for 30 minutes. After washing once with PBS, the cells were analyzed by flow cytometry. The results of VHH affinity detection by FACS are shown in Table 6.

[0293] Table 6

[0294] 5.3 Detection of the affinity between the recombinant antibody and human Claudin18.2 protein using SPR technology.

[0295] Six antibodies were diluted to 5 μg / mL with the running reagent and injected into approximately 200 RU of the Protein A capture assay channel (Fc2) at a flow rate of 10 μL / min. The reference channel (Fc1) did not require ligand capture.

[0296] Human Claudin-18.2 protein was serially diluted 2-fold using the running 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, with binding and dissociation times as specified. Both binding and dissociation steps were performed using the running reagent. After each concentration analysis, the chip was regenerated with glycine hydrochloride at pH 1.5 at a flow rate of 20 μL / min for 30 s to wash away ligands and undissociated analytes. For the next concentration analysis, the experimental channel (Fc2) needed to recapture the same amount of ligands.

[0297] The KD value for each sample was calculated using Biacore Insight Evaluation Software (8K analysis software). The reference channel (Fc1) was used for background subtraction.

[0298] A lower dissociation constant KD (the ratio of dissociation rate Kd to binding rate Ka) indicates higher antibody affinity. The KD value varies depending on the different complexes of the antibody and antigen, and depends on both Ka and Kd.

[0299] The results are shown in Table 7. The KD values ​​of 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. The antibody affinity ranking (from highest to lowest) was: 2-mIgG2a Fc, 5-mIgG2a Fc, 5H2-mIgG2a Fc, IMAB-mIgG Fc, and 5H1-mIgG Fc.

[0300] Ka sorting (from largest to smallest): 2-mIgG2a Fc, IMAB-mIgG Fc, 5H2-mIgG2a Fc, 5-mIgG2a Fc, 5H1-mIgG Fc.

[0301] Kd sorting (from largest to smallest): IMAB-mIgG Fc, 5H1-mIgG Fc, 2-mIgG2a Fc, 5H2-mIgG2a Fc, 5-mIgG2a Fc.

[0302] Table 7. Affinity test results of four antibodies with human Claudin-18.2 protein.

[0303] 5.4 In vivo antitumor efficacy study

[0304] The in vivo antitumor efficacy of Claudin18.2 CAR-T cells was evaluated in an Aspc-1 18.2-Luc pancreatic cancer NOG-DKO mouse model (NOD.Cg-B2mem1Tac Prkdcscid H2-Ab1tm1Doi Il2rgtm1Sug / JicCrl).

[0305] 2E6 AsPC-1CLDN18.2-Luc cells (CLDN18.2+MSLN+) were subcutaneously injected. When the tumor burden reached approximately 200 mm3, mice were divided into groups of 3-4. One day after grouping, 200 μL of CLDN18.2 CAR-T cells, MSLN CAR-T cells, or NT cells were injected via the tail vein, or 5E6 CAR-T cells per mouse. On the first day after CAR-T injection, a small amount of blood was collected from the mice to detect the number of CAR-T cells surviving in vivo. Subsequently, blood samples were collected weekly to detect various CAR-T cell phenotypes, and subcutaneous tumor size and mouse weight were measured twice weekly.

[0306] As shown in Figure 10, Claudin18.2 CAR-T cells and MSLN CAR-T cells exhibited significant anti-tumor activity against transplanted tumor cells in vivo. VHH candidate CAR-T cells, including CAR02, 05, and 18, showed similar tumor-suppressing effects. Mice remained alive without tumor recurrence at Day 80 and Day 90 after CAR-T cell infusion. While control MSLN CAR-T cells targeting MSLN also killed tumor cells, tumor recurrence began at Day 80 after CAR-T cell infusion, reaching a tumor volume of approximately 200 mm² by Day 90. 3 .

[0307] In vivo experimental results showed that targeting Claudin18.2 CAR-T (CAR02, 05, 18) cells had strong and persistent anti-tumor activity.

[0308] Table 8

[0309] Table 9 CAR Composition Sequence

[0310] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A nanobody that specifically binds to Claudin18.2, characterized in that, The complementarity-determining region (CDR) of the VHH chain in the nanobody is selected from the following group: (1) CDR1 shown in SEQ ID NO:36, CDR2 shown in SEQ ID NO:37, and CDR3 shown in SEQ ID NO:38; (2) CDR1 shown in SEQ ID NO:39, CDR2 shown in SEQ ID NO:40, and CDR3 shown in SEQ ID NO:41; (3) CDR1 shown in SEQ ID NO:42, CDR2 shown in SEQ ID NO:43, and CDR3 shown in SEQ ID NO:44; (4) CDR1 shown in SEQ ID NO:45, CDR2 shown in SEQ ID NO:46, and CDR3 shown in SEQ ID NO:47; (5) CDR1 shown in SEQ ID NO:48, CDR2 shown in SEQ ID NO:49, and CDR3 shown in SEQ ID NO:50; (6) CDR1 shown in SEQ ID NO:51, CDR2 shown in SEQ ID NO:52, and CDR3 shown in SEQ ID NO:53; (7) CDR1 shown in SEQ ID NO:39, CDR2 shown in SEQ ID NO:54, and CDR3 shown in SEQ ID NO:41; (8) CDR1 shown in SEQ ID NO:39, CDR2 shown in SEQ ID NO:55, and CDR3 shown in SEQ ID NO:41; (9) CDR1 shown in SEQ ID NO:67, CDR2 shown in SEQ ID NO:68, and CDR3 shown in SEQ ID NO:41; (10) CDR1 shown in SEQ ID NO:39, CDR2 shown in SEQ ID NO:69, and CDR3 shown in SEQ ID NO:41; and / or (11) CDR1 shown in SEQ ID NO:36, CDR2 shown in SEQ ID NO:65, and CDR3 shown in SEQ ID NO:

66.

2. The nanobody as described in claim 1, characterized in that, The CDR1, CDR2, and CDR3 are separated by the frame regions FR1, FR2, FR3, and FR4 of the VHH chain.

3. The nanobody as described in claim 1, characterized in that, The amino acid sequence of the VHH chain of the nanobody is selected from the following group: SEQ ID NO:1~26, or a combination thereof.

4. A multivalent or multispecific antibody that specifically binds to Claudin18.2, characterized in that, The multivalent or multispecific antibody comprises at least one antibody element targeting the Claudin18.2 epitope, wherein the antibody element is the nanobody of claim 1.

5. The multivalent or multispecific antibody as described in claim 4, characterized in that, The anti-Claudin18.2 antibody comprises one or more VHH chains having an amino acid sequence as shown in any of SEQ ID NO:1 to 26.

6. A recombinant protein, characterized in that, The recombinant protein has the following characteristics: (i) the nanobody that specifically binds to Claudin18.2 as described in claim 1, or the multivalent antibody or multispecific antibody that specifically binds to Claudin18.2 as described in claim 4; and (ii) Optional tag sequences to assist in expression and / or purification.

7. A chimeric antigen receptor (CAR) fusion protein, characterized in that, The chimeric antigen receptor (CAR) fusion protein comprises, from the N-terminus to the C-terminus: (i) Specifically binds to the antigen-binding domain of Claudin18.2, wherein the antigen-binding domain contains the nanobody of claim 1; (ii) Transmembrane structural domains; (iii) at least one costimulatory domain; and (iv) Activate the structural domain.

8. The chimeric antigen receptor (CAR) fusion protein as described in claim 7, characterized in that, The CAR described above has the structure shown in Formula Ia: L-VHH-H-TM-C-CD3ζ (Ia) In the formula, Each "-" independently represents a linking peptide or peptide bond; L represents the signal peptide sequence; VHH is the antigen-binding domain that specifically binds to Claudin 18.2; H represents the hinge area; TM represents a transmembrane domain; C is the co-stimulation signal structure domain; CD3ζ is a cytoplasmic signaling sequence derived from CD3ζ (including wild type or its mutants / modifiers).

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

10. An antibody-drug conjugate, characterized in that, The antibody-drug conjugate contains: (a) The nanobody as claimed in claim 1, the multivalent antibody or multispecific antibody as claimed in claim 4, and the recombinant protein as claimed in claim 6; as well as (b) A conjugation portion conjugated to the antibody portion, the conjugation portion being selected from the group consisting of: detectable markers, drugs, toxins, cytokines, radionuclides, enzymes, or combinations thereof.

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

12. An expression carrier, characterized in that, The expression vector contains the polynucleotide of claim 11.

13. A host cell, characterized in that, The host cell contains the expression vector of claim 12, or its genome integrates the polynucleotide of claim 11, or expresses the nanobody of claim 1, the multivalent antibody or multispecific antibody of claim 4, the recombinant protein of claim 6, or the CAR fusion protein of claim 7.

14. A pharmaceutical composition, characterized in that, The pharmaceutical composition contains: (i) the nanobody of claim 1, or the multivalent or multispecific antibody of claim 4, or the recombinant protein of claim 6, or immune cells expressing the CAR fusion protein of claim 7; and (ii) Pharmaceutically acceptable carriers.

15. The use of the nanobody of claim 1, or the multivalent antibody or multispecific antibody of claim 4, or the recombinant protein of claim 6, or immune cells expressing the CAR fusion protein of claim 7, characterized in that, Used to prepare medicines for the prevention and / or treatment of cancers or tumors associated with Claudin 18.2.

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