WO2024131683A1

WO2024131683A1 - Antibody binding to cldn18.2, antibody-drug conjugate and use thereof

Info

Publication number: WO2024131683A1
Application number: PCT/CN2023/139288
Authority: WO
Inventors: 周祥山; 殷惠军; 张苗; 孙乐桥; 吴坤宝; 曹峰琦; 李娴; 于朋飞
Original assignee: 华润生物医药有限公司
Priority date: 2022-12-19
Filing date: 2023-12-15
Publication date: 2024-06-27

Abstract

An antibody specifically binding to CLDN18.2, an antigen-binding fragment thereof or a variant thereof, and an antibody-drug conjugate. The antibody-drug conjugate comprises a drug-conjugated antibody specifically binding to CLDN18.2, and an antigen-binding fragment thereof, or a variant thereof, and the antibody, the antigen-binding fragment thereof, or the variant thereof comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1 as shown in SEQ ID NO. 1, HCDR2 as shown in SEQ ID NO. 2, and HCDR3 as shown in SEQ ID NO. 3; and the light chain variable region comprises LCDR1 as shown in SEQ ID NO. 4, LCDR2 as shown in SEQ ID NO. 5, and LCDR3 as shown in SEQ ID NO. 6. The antibody is a fully human monoclonal antibody binding to a claudin 18.2 protein (such as human claudin 18.2, mouse claudin 18.2 and monkey claudin 18.2), has high ADCC activity, CDC activity and/or claudin 18.2 protein binding stability, and has high endocytosis activity.

Description

Antibodies and antibody-drug conjugates binding to CLDN18.2 and their uses

Reference to a sequence listing

This application contains a Sequence Listing in computer readable form, which is incorporated herein by reference.

Priority information

This application claims priority and benefits of patent application No. 202211632003.8 filed with the State Intellectual Property Office of China on December 19, 2022, and the entire text of which is incorporated herein by reference.

This application claims priority and benefits of patent application No. 202211631999.0 filed with the State Intellectual Property Office of China on December 19, 2022, and the entire text of which is incorporated herein by reference.

Technical Field

The present invention relates to the field of biomedicine, and in particular to an antibody binding to CLDN18.2 and an application thereof, and an antibody-drug conjugate targeting CLDN18.2 and an application thereof.

Background technique

Tumors have become a serious threat to human life and health. Targeted therapy can specifically target abnormal cells in tumors. Targeted drugs are developed for abnormal tumor molecules and genes. They use abnormal tumor cells or genes as targets to prevent the growth of tumor cells and kill them. Therefore, targeted therapy will not accidentally harm normal cells, and all the drug effects are used on tumor cells. The drug effect is stronger than conventional chemotherapy drugs, but the side effects are much smaller than conventional chemotherapy.

CLDN18.2 is expressed in patients with primary pancreatic ductal adenocarcinoma and non-small cell lung cancer (NSCLC), but not in the corresponding normal tissues; it is expressed more in advanced Siberian cell carcinoma (SRCC) and damaged squamous epithelium of Barrett's esophagus. In addition, the expression of CLDN18.2 is not limited to primary lesions, but is also highly expressed in metastatic lesions, which may be involved in the proliferation and chemotaxis of malignant tumor cells.

Obtaining drugs targeting CLDN18.2 can meet the needs of treating a variety of tumors.

Antibody-drug conjugate (ADC) is a small molecule drug with biological activity connected to a monoclonal antibody through a chemical chain. The monoclonal antibody acts as a carrier to transport the small molecule drug to the target cell, and can be used as a tumor-targeted drug. Targeted drugs use abnormal tumor cells or genes as targets to prevent the growth of tumor cells and kill them, so targeted therapy will not accidentally harm normal cells, and all the drug effects are used on tumor cells. The drug effect is stronger than conventional chemotherapy drugs, but the side effects are much smaller than conventional chemotherapy.

Therefore, ADC drugs containing CLDN18.2 antigen targeting may be widely used in clinical anti-tumor treatment.

Summary of the invention

In response to the technical problems existing in the prior art, the present invention proposes an antibody, an antigen-binding fragment or a variant thereof that specifically binds to CLDN18.2, which includes a heavy chain variable region and a light chain variable region, wherein: the heavy chain variable region comprises HCDR1 as shown in SEQ ID NO.1, HCDR2 as shown in SEQ ID NO.2, and HCDR3 as shown in SEQ ID NO.3; and the light chain variable region comprises LCDR1 as shown in SEQ ID NO.4, LCDR2 as shown in SEQ ID NO.5, and LCDR3 as shown in SEQ ID NO.6.

In some embodiments, the heavy chain comprises the variable region shown in SEQ ID NO.7; and the light chain comprises the variable region shown in SEQ ID NO.8.

In some embodiments, the antibody or antigen-binding portion thereof is selected from the group consisting of a whole antibody, a bispecific antibody, a monoclonal antibody, a chimeric antibody, a humanized antibody, and a fully human antibody.

In some embodiments, the above-mentioned antibodies, antigen-binding fragments thereof or variants thereof further include a heavy chain constant region and a light chain constant region, wherein: the antibody heavy chain constant region is selected from the IgG series antibodies; the light chain constant region is selected from κ or λ chain.

In some embodiments, the IgG series antibody is selected from one or more of IgG1, IgG2, and IgG4.

In some embodiments, the antigen binding fragment is selected from the group consisting of a Fab fragment, a Fab' fragment, a F(ab) ₂ fragment, a Fv fragment, and a ScFv.

In some embodiments, the CLDN18.2 is selected from the group consisting of human CLDN18.2, mouse CLDN18.2, and monkey CLDN18.2.

A fusion protein comprising any of the above antibodies, antigen-binding fragments thereof or variants thereof.

One or more isolated nucleic acid molecules encoding an antibody, antigen-binding fragment thereof or variant thereof as described above, or a fusion protein as described above.

One or more vectors comprising one or more isolated nucleic acid molecules as above.

A cell comprising one or more isolated nucleic acid molecules as above or one or more vectors as above.

In some embodiments, the cell is further a CAR-T or CAR-NK cell comprising one or more isolated nucleic acid molecules as above or one or more vectors as above.

A method for producing any of the above antibodies, antigen-binding fragments thereof, or variants thereof, or fusion proteins as above, comprising culturing the above cells under conditions that allow the above antibodies, antigen-binding fragments thereof, or variants thereof, or fusion proteins as above to be expressed.

A composition comprising any of the above antibodies, antigen-binding fragments thereof or variants thereof, the above fusion proteins, the above one or more isolated nucleic acid molecules, the above one or more vectors and/or the above cells, and optionally a pharmaceutically acceptable excipient.

Use of any of the above antibodies, antigen-binding fragments thereof or variants thereof, the above fusion proteins, the above one or more isolated nucleic acid molecules, the above one or more vectors and/or the above cells in the preparation of a medicament for preventing and/or treating cancer or tumors.

Furthermore, in some embodiments, the drug is a drug for cell therapy.

In some embodiments, the cancer or tumor is a cancer or tumor that is positive for CLDN18.2 expression.

Preferably, the cancer or tumor is selected from bladder cancer, ovarian cancer, lung cancer, adenocarcinoma, gastric cancer, breast cancer, liver cancer, pancreatic cancer, skin cancer, malignant melanoma, head and neck cancer, sarcoma, bile duct cancer, kidney cancer, colon cancer, small intestine cancer, testicular embryonal carcinoma, placental choriocarcinoma, cervical cancer, testicular cancer, uterine cancer, esophageal cancer and gallbladder cancer cells.

Use of the antibody, antigen-binding fragment or variant thereof, or fusion protein thereof as described above in the preparation of a reagent for determining the presence and/or amount of CLDN18.2 in a sample.

A pharmaceutical composition comprising: any of the above antibodies, antigen-binding fragments thereof or variants thereof, the above fusion proteins, the above one or more isolated nucleic acid molecules, or the above one or more vectors and/or the above cells.

An antibody-drug conjugate, comprising the antibody, antigen-binding fragment or variant thereof as described above covalently bound to a therapeutic agent.

In some embodiments, the therapeutic agent is a cytotoxic agent or a cell proliferation inhibitor selected from auristatin, DM1, MMAE (Monomethy lauristatin E), MMAF (Monomethy lauristatin F), PDB, dukamycin, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunomycin ... One or more of abacterial agents, such as aunorubicin, dihydroxy anthracin, maytansinoids (such as DM-1 and DM-4), diketones, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, epirubicin and cyclophosphamide and their analogs. The present invention provides an antibody, a fully human monoclonal antibody that binds to claudin18.2 (such as human claudin18.2, mouse claudin18.2, monkey claudin18.2), which has higher ADCC activity, CDC activity and/or claudin18.2 binding stability compared to the antibody molecules in the prior art, and has higher endocytosis activity.

The present invention also proposes an antibody-drug conjugate, which comprises an antibody, an antigen-binding fragment thereof or a variant thereof that specifically binds to CLDN18.2 and is conjugated to a drug, wherein the antibody, the antigen-binding fragment thereof or a variant thereof comprises a heavy chain variable region and a light chain variable region, wherein: the heavy chain variable region comprises HCDR1 as shown in SEQ ID NO.1, HCDR2 as shown in SEQ ID NO.2, and HCDR3 as shown in SEQ ID NO.3; and the light chain variable region comprises LCDR1 as shown in SEQ ID NO.4, LCDR2 as shown in SEQ ID NO.5, and LCDR3 as shown in SEQ ID NO.6.

In some embodiments, the antibody, its antigen-binding fragment or its variant further includes: a heavy chain variable region shown in SEQ ID NO.7; and a light chain variable region shown in SEQ ID NO.8.

In some embodiments, the antibody, antigen-binding fragment thereof or variant thereof further comprises a heavy chain constant region and a light chain constant region, wherein: the antibody heavy chain constant region is selected from the IgG series of antibodies; and the light chain constant region is selected from κ or λ chain.

In some embodiments, the IgG series antibody is selected from one or more of IgG1, IgG2 and IgG4.

In some embodiments, the antigen binding fragment is selected from the group consisting of a Fab fragment, a Fab' fragment, a F(ab) ₂ fragment, a Fv fragment and a ScFv.

In some embodiments, the antibody-drug conjugate as described in any of the above items, the molecular formula of the antibody-drug conjugate is: Ab-[L-D]n, wherein Ab represents an anti-CLDN18.2 antibody, an antigen-binding fragment thereof or a variant thereof, L represents a linker, D represents a drug, and n represents the average number of drug connections relative to each molecule of Ab.

In some embodiments, the drug D is a cytotoxic agent or a cell proliferation inhibitor.

In some embodiments, the drug is selected from one or more of calicheamicins, duocarmycins, anthramycin derivatives PBD, camptothecin derivatives, dolastatin and auristatins, maytansine and its derivatives.

In some embodiments, further, the drug is selected from MMAF, MMAE, MMAD, PBD, dukamycin, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxy anthracin, maytansinoids (such as DM-1 and DM-4), diketone, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, epirubicin and cyclophosphamide and one or more of their analogs.

In some embodiments, the linker L is a cysteine coupling linker, a lysine coupling linker, a valine-citrulline (Val-Cit, vc) linker, an SPDB linker, an SMCC linker, or a SMAC (sortasemediated antibody conjugation technology) linker.

A method for preparing an antibody-drug conjugate, comprising: preparing an antibody, an antigen-binding fragment thereof or a variant thereof that specifically binds to CLDN18.2, which comprises a heavy chain variable region and a light chain variable region, wherein: the heavy chain variable region comprises HCDR1 as shown in SEQ ID NO.1, HCDR2 as shown in SEQ ID NO.2, and HCDR3 as shown in SEQ ID NO.3; and the light chain variable region comprises LCDR1 as shown in SEQ ID NO.4, LCDR2 as shown in SEQ ID NO.5, and LCDR3 as shown in SEQ ID NO.6; coupling the antibody, the antigen-binding fragment thereof or a variant thereof to MMAC using a valine-citrulline (Val-Cit, vc) linker; and obtaining an anti-CLDN18.2 antibody-drug conjugate.

A composition, such as any of the above antibody-drug conjugates, or the antibody-drug conjugate prepared by the above method, and optionally a pharmaceutically acceptable excipient.

Use of any of the above antibody-drug conjugates, or the antibody-drug conjugate prepared by the above method, in the preparation of a drug for preventing and/or treating cancer or tumors.

In some embodiments, the drug is a cell therapy drug.

In some embodiments, the cancer or tumor is selected from bladder cancer, ovarian cancer, lung cancer, adenocarcinoma, gastric cancer, breast cancer, liver cancer, pancreatic cancer, skin cancer, malignant melanoma, head and neck cancer, sarcoma, bile duct cancer, kidney cancer, colon cancer, small intestine cancer, testicular embryonal carcinoma, placental choriocarcinoma, cervical cancer, testicular cancer, uterine cancer, esophageal cancer and gallbladder cancer cells.

Use of the antibody drug conjugate as described above in the preparation of a reagent for determining the presence and/or amount of CLDN18.2 in a sample.

A pharmaceutical composition comprising: the antibody-drug conjugate as described in any one of the above items or the antibody-drug conjugate prepared by the method described above.

The present invention provides an antibody, a fully human monoclonal antibody that binds to claudin18.2 (eg, human claudin18.2, mouse claudin18.2, monkey claudin18.2), which has higher ADCC activity, CDC activity and/or claudin18.2 binding stability compared to antibody molecules in the prior art.

BRIEF DESCRIPTION OF THE DRAWINGS

The preferred embodiments of the present invention will be further described in detail below with reference to the accompanying drawings, wherein:

FIG1 is a diagram showing specific detection of human CLDN18.2 and CLDN18.1 according to an embodiment of the present invention; wherein the horizontal axis is the fluorescence intensity of Anti-hIgG-Fc-AF647, which detects the binding strength of the antibody to CLDN18.1 and CLDN18.2; the vertical axis SSC-H is the biased dispersion of the cells, which detects the complexity of the cells; P1026 is the screened CLDN18.2 specific binding antibody; NC (Negative control) is a negative control, and the specific antibody is Human IgG1, kappa Isotype control, brand: CrownBio, item number: C0001-4, batch number AB190016, which is a negative control antibody that does not bind to CLDN18.1 and CLDN18.2; IMAB362 is a positive control, which specifically binds to human CLDN18.2, according to Clone: 175D10 (Heavy Chain: SEQ ID NO: 103; Light Chain: SEQ ID NO: 104) of the patent CN101312989A of the IMAB362 antibody. ID NO: 110), synthesizing the light and heavy chain sequences respectively, and transiently expressing them through the ExpiCHO ^™ expression system to prepare IMAB362, which is a positive control antibody that does not bind to CLDN18.1 but binds to CLDN18.2;

Fig. 2 is a result of antibody species cross-detection according to an embodiment of the present invention, wherein the horizontal axis is the fluorescence intensity of Anti-hIgG-Fc-AF647, detecting the binding strength of the antibody and CLDN18.2; the vertical axis SSC-H is the biased dispersion of the cells, detecting the complexity of the cells; P1026 is the screened CLDN18.2 specific binding antibody; NC (Negative control) is a negative control, and the specific antibody is Human IgG1, kappa Isotype control, brand: CrownBio, item number: C0001-4, batch number AB190016, which is a negative control antibody that does not bind to CLDN18.2; IMAB362 is a positive control, specifically binding to human CLDN18.2, according to Clone: 175D10 (Heavy Chain: SEQ ID NO: 103; Light Chain: SEQ ID NO: 110) of the patent CN101312989A of the IMAB362 antibody, the light and heavy chain sequences were synthesized respectively, and the light and heavy chain sequences were synthesized by ExpiCHO ^TM expression system, transient expression to prepare IMAB362, a positive control antibody that does not bind to CLDN18.1 but binds to CLDN18.2;

FIG3 is a diagram showing antibody binding ability detection according to an embodiment of the present invention; wherein the abscissa represents antibody concentration and the ordinate represents mean fluorescence intensity;

FIG4 shows the ADCC activity of CLDN18.2 antibody against MC38 cells overexpressing human claudin18.2 according to one embodiment of the present invention;

FIG5 shows the CDC activity of CLDN18.2 antibody on MC38 cells overexpressing human claudin18.2 according to one embodiment of the present invention;

FIG6 shows the endocytic activity of the CLDN18.2 antibody according to one embodiment of the present invention;

FIG. 7A shows the anti-tumor efficacy of a CLDN18.2 antibody-drug conjugate in a GSU gastric cancer model according to an embodiment of the present invention; and

FIG. 7B shows the anti-tumor efficacy of the CLDN18.2 antibody-drug conjugate in a gastric cancer PDX model according to an embodiment of the present invention.

Detailed ways

In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, but not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

In the following detailed description, reference may be made to the various specification drawings that are part of the present application and are used to illustrate specific embodiments of the present application. In the accompanying drawings, similar reference numerals describe substantially similar components in different figures. The various specific embodiments of the present application are described below in sufficient detail so that a person of ordinary skill in the art with relevant knowledge and skills in the art can implement the technical solutions of the present application. It should be understood that other embodiments may also be used or structural, logical or electrical changes may be made to the embodiments of the present application.

Antibody therapy has been approved for the treatment of various cancers worldwide, and has significantly improved patient prognosis and overall survival. Various antibody molecules can specifically bind to tumor surface antigens, and antibodies trigger antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) through Fc fragments, ultimately causing tumor cell death.

As used herein, the term "antibody" generally refers to an immunoglobulin molecule composed of two identical pairs of polypeptide chains, each pair of polypeptide chains having one "light" (L) chain and one "heavy" (H) chain. The light chains of antibodies can be divided into kappa and lambda light chains. The heavy chains can be classified as μ, δ, γ, α or ε, and the isotypes of antibodies are defined as IgM, IgD, IgG, IgA and IgE, respectively. Within the light and heavy chains, the variable and constant regions are connected by a "J" region of about 12 or more amino acids, and the heavy chain also includes a "D" region of about 3 or more amino acids. Each heavy chain consists of a heavy chain variable region ( _VH ) and a heavy chain constant region ( _CH ). The heavy chain constant region consists of three domains (CH1, CH2 and CH3). Each light chain consists of a light chain variable region ( _VL ) and a light chain constant region ( _CL ). The light chain constant region consists of one domain, _CL . The constant region of an antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (such as effector cells) and the first component (Clq) of the classical complement system. _{The VH} and _VL regions can also be subdivided into highly variable regions called complementarity determining regions (CDRs), which are interspersed between more conserved regions called framework regions (FRs). Each _VH and _VL consists of 3 CDRs and 4 FRs arranged in the following order from N-terminus to C-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions ( _VH and _VL ) of each heavy/light chain pair form an antibody binding site, respectively. The distribution of amino acids into regions or domains follows the definition of Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)) or Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883.

The present antibody is not limited by any antibody production method. For example, it includes recombinant antibodies, monoclonal antibodies and polyclonal antibodies. The antibody can be an antibody of different types, for example, IgG (e.g., IgG 1, IgG2, IgG3 or IgG4 subtype), IgA1, IgA2, IgD, IgE or IgM antibody.

As used herein, the term "antigen-binding fragment" generally refers to one or more fragments of a full-length antibody that retain the ability to bind to the same antigen (e.g., CLDN18.2) to which the antibody binds and competes with the intact antibody for antigen-specific binding. Antigen-binding fragments can be produced by recombinant DNA technology or by enzymatic or chemical cleavage of intact antibodies. In some cases, antigen-binding sites include Fab, Fab', F(ab') ₂ , F(ab) ₂ , Fd, Fv, dAb and complementary determining region (CDR) fragments, single-chain antibodies (e.g., scFv), chimeric antibodies, diabodies, and polypeptides, which contain at least a portion of an antibody sufficient to confer specific antigen-binding ability to the polypeptide.

In some embodiments, the amino acid sequence included in the antibody or antigen-binding fragment has one or more modification groups. For example, the antibody or antigen-binding fragment disclosed in the present invention may include a flexible linker sequence, or may be modified to add a functional group (e.g., PEG, a drug, a toxin, or a label).

The antibodies and antigen binding fragments disclosed in the present invention include modified derivatives, i.e., modified by covalent attachment of any type of molecule to the antibody or antigen binding fragment, wherein the covalent attachment does not prevent the antibody or antigen binding fragment from binding to the epitope. Including but not limited to the following examples, the antibody or antigen binding fragment can be glycosylated, acetylated, pegylated, phosphorylated, amidated, derivatized by known protecting/blocking groups, proteolytically cleaved, attached to cell ligands or other proteins, etc. Any of the numerous chemical modifications can be performed by existing techniques, including but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc.

In some embodiments, the antibody or antigen-binding fragment may be conjugated to a therapeutic agent, a prodrug, a peptide, a protein, an enzyme, a virus, a lipid, a biological response modifier, a pharmaceutical agent, or PEG.

The antibody or antigen binding fragment can be detectably labeled by coupling it to a chemiluminescent compound. The presence of the chemiluminescent labeled antibody or antigen binding fragment is then determined by detecting the luminescence that occurs during the chemical reaction. Examples of chemiluminescent labeling compounds include luminol, isoluminol, aromatic acridinium esters, imidazoles, acridinium salts, and oxalate esters.

As used herein, the term "whole antibody" refers to an antibody having the full-length sequence of an antibody. In some embodiments, the whole antibody may be a chimeric antibody, a humanized antibody, or a fully human antibody. In other embodiments, the whole antibody may be a monoclonal antibody or a polyclonal antibody. In one embodiment, the whole antibody is an immunogenic fragment that binds to the Claudin18.2 antigen. Further, the whole antibody is a CLDN18.2 antibody that contains the full-length sequence of the CLDN18.2 antibody.

As used herein, the term "monoclonal antibody" generally refers to antibodies produced by the same immune cells, which are clones of unique parental cells. Monoclonal antibodies can have monovalent affinity because they bind to the same epitope (the part of the antigen recognized by the antibody). It has become an important tool in biochemistry, molecular biology and medicine. In recent years, a variety of monoclonal antibody technologies have been developed, such as phage display, single B cell culture, single cell expansion from various B cell populations, and single plasma cell interrogation technologies. The present application provides a separated monoclonal antibody, for example, a fully human monoclonal antibody that binds to claudin18.2 (human claudin18.2, mouse claudin18.2, monkey claudin18.2), which has higher ADCC activity, CDC activity and/or claudin18.2 binding stability compared to existing anti-CLDN18.2 antibody molecules.

As used herein, "chimeric antibodies" have different portions derived from different animal species, such as those having variable regions derived from mouse antibodies and human immunoglobulin constant regions. Chimerization of antibodies is achieved by joining the variable regions of mouse antibody heavy and light chains to the constant regions of human heavy and light chains (e.g., as described by Kraus et al. in Methods in Molecular Biology series, Recombinant antibodies for cancer therapy ISBN-089603-918-8).

As used in this article, "CAR-T" means: CAR-T therapy is chimeric antigen receptor T cell immunotherapy, which is a new type of precision targeted therapy for treating tumors. It has achieved good results in clinical tumor treatment and is a new type of tumor immunotherapy that is accurate, fast, efficient, and has the potential to cure cancer.

As used herein, "CAR-NK" or "CAR-NK cells" refers to CAR-NK cell therapy, CAR-NK cells, which are composed of extracellular signaling domains, transmembrane regions, and intracellular domains that recognize tumor-specific antigens, and they can establish new activation pathways to enhance the lysis of target cells. CAR-NK cells specifically recognize antigen-expressing tumors through CAR, and eliminate tumors through the NK cell receptors themselves. Among them, CAR is a chimeric antigen receptor, and NK cells are natural killer cells, which are immune cells of large granular lymphocytes different from T and B lymphocytes. The activity of NK cells depends on the balance of stimulatory and inhibitory signals, rather than antigen specificity.

As used herein, "humanization" means that the antibody interacts with the target antigen primarily through amino acid residues located in the six heavy and light chain complementary determining regions (CDRs). For this reason, the amino acid sequences inside the CDRs between individual antibodies are more diverse than the sequences outside the CDRs. Because the CDR sequences are responsible for most of the antibody-antigen interactions, it is possible to express recombinant antibodies that mimic the properties of specific naturally occurring antibodies by constructing expression vectors, which include CDR sequences of specific naturally occurring antibodies grafted to framework sequences from different antibodies with different properties (see, e.g., Riechmann, L. et al. (1998) Nature 332: 323-327; Jones, P. et al. (1986) Nature 321: 522-525 and Queen, C. et al. (1989) Proc. Natl. Acad. Sci. U.S.A. 86: 10029-10033). Such framework sequences can be obtained from public DNA databases including germline antibody gene sequences. These germline sequences will differ from mature antibody gene sequences in that they do not include the fully assembled variable genes, which are formed by V(D)J joining during B cell maturation.

"Fully human" or "completely human" means that all human antibody genes are transferred to genetically engineered antibody gene-deficient animals through transgenic or transchromosome technology, so that the animals express human antibodies and achieve the purpose of fully humanized antibodies. It usually refers to antibodies with antibody regional therapeutic agents derived from fully human amino acid sequences, in which antigen specificity has been selected in vivo by using genetically modified mice or by antibody engineering methods combined with screening. Compared with humanized antibodies, fully human antibodies have a lower risk of inducing immune responses in humans, stronger specific immune effects, and higher ADCC activity, CDC activity and/or claudin18.2 binding stability.

As used herein, the term "bispecific antibody" generally refers to an artificial protein that can bind to two different types of antigens at the same time. The main types of manufacturing methods are quadromas, chemical conjugation, and genetic recombination. The IgG-like form retains the conventional monoclonal antibody (mAb) structure of two Fab arms and one Fc region, except that two Fab sites bind to different antigens. Each heavy chain and light chain pair comes from a unique mAb. The Fc region made of two heavy chains forms the third binding site. Non-IgG-like forms include chemically connected Fabs, which are composed only of Fab regions, and various types of divalent and trivalent single-chain variable fragments (scFvs). There are also fusion proteins that simulate two antibody variable domains. Bispecific antibodies have higher cytotoxic potential and bind to antigens that are relatively weakly expressed at lower effective doses. In addition, targeting more than one molecule can be used to circumvent the regulation of parallel pathways and avoid resistance to treatment.

Bispecific antibodies include cross-linked or "heteroconjugate" antibodies. For example, one of the antibodies in the heteroconjugate can be coupled to avidin and the other antibody to biotin. Heteroconjugate antibodies can also be produced using any convenient cross-linking method. Suitable cross-linking agents and cross-linking techniques are well known in the art and are disclosed in U.S. Pat. No. 4,676,980, which is incorporated herein by reference in its entirety.

As used herein, the term "Fab fragment" generally refers to a portion of an immunoglobulin molecule (such as an antigen binding fragment). A Fab fragment may comprise a portion of a light chain and a heavy chain, and have a single antigen binding site. Fab fragments may be obtained by digesting immunoglobulin molecules with papain. For example, a Fab fragment may consist of a constant domain and a variable domain of each heavy chain and light chain. The variable domain may contain a paratope (antigen binding site) comprising a set of complementary determining regions at the amino terminus of the immunoglobulin molecule. Papain may be used to cleave an immunoglobulin molecule into two Fab fragments and one Fc fragment. Pepsin cleaves below the hinge region, thereby forming a F(ab') ₂ fragment and a pFc' fragment. A divalent F(ab) ₂ or F(ab') ₂ fragment has two antigen binding regions connected by disulfide bonds. Reduction of the F(ab) ₂ or F(ab') ₂ fragment produces two monovalent Fab or Fab' fragments, which have free sulfhydryl groups that can be used for conjugation with other molecules.

The term "Fv fragment" as used herein generally refers to the smallest fragment made by enzymatic cleavage of IgG and IgM class antibodies. Fv fragments have an antigen binding site made of VH and VL regions, but they lack CH1 and CL regions. VH and VL chains are bound together in Fv fragments by non-covalent interactions.

As used herein, the term "ScFv" generally refers to a single-chain antibody fragment. ScFv may refer to a recombinant single-chain polypeptide molecule in which the light chain and heavy chain variable regions of an antibody are connected by a peptide linker. Single-chain antibodies (ScFv) generally do not include portions of the Fc region of an antibody that are involved in effector functions and are therefore naked antibodies, although methods are known to add such regions to known ScFv molecules if desired. See Helfrich et al., A rapid and versatile method for harnessing ScFv antibody fragments with various biological functions. J Immunol Methods 237:131-145 (2000) and de Haard et al., Creating and engineering human antibodies for immunotherapy. Advanced Drug Delivery Reviews 31:5-31 (1998).

As used herein, the term "IgG" generally refers to an antibody of a subtype. Each IgG has two antigen binding sites. Representing approximately 75% of human serum antibodies, IgG is the most common antibody type found in circulation. Recognized immunoglobulin genes include kappa, lambda, alpha, gamma (IgG1, IgG2, IgG3, IgG4).

The teachings given herein with respect to a specific amino acid sequence (such as those shown in the sequence listing) should be understood to also relate to variants of the specific sequence, thereby generating a sequence that is functionally equivalent to the specific sequence, such as an amino acid sequence that exhibits properties that are equivalent to or similar to those of the specific amino acid sequence. An important property is to retain the antibody binding to its target or to maintain the effector function of the antibody. Preferably, when a sequence that is a variant relative to a specific sequence replaces a specific sequence in an antibody, the sequence retains the antibody binding to CLDN18.2, and preferably retains the function of the antibody as described herein, such as CDC-mediated cleavage or ADCC-mediated cleavage.

It will be appreciated by those skilled in the art that, in particular, the sequences of the CDRs, hypervariable regions and variable regions can be modified without losing the ability to bind to CLDN18.2. For example, the CDR regions are identical or highly homologous to the antibody regions specified herein. It is expected that "highly homologous" can be 1 to 5, preferably 1 to 4, such as 1 to 3 or 1 or 2 substitutions can be made in the CDRs. In addition, the hypervariable regions and variable regions can be modified so that they show substantial homology to the antibody regions explicitly disclosed herein. As used herein, the CDRs may be CDR regions of the heavy chain variable region, or CDR regions of the light chain region. Further, the CDR regions may refer to CDR1, CDR2 and/or CDR3 of the heavy chain and/or light chain. Further, HCDR refers to the complementarity determining region of the heavy chain variable region, specifically including HCDR1, HCDR2 and HCDR3; LCDR refers to the complementarity determining region of the light chain variable region, specifically including LCDR1, LCDR2 and LCDR3.

As used herein, the term "variant" generally refers to a protein that differs from the parent molecule by at least one amino acid. A variant may refer to the molecule itself, a composition comprising the molecule. When such a molecule is a polypeptide or protein, it may also refer to the amino acid sequence of the molecule. In some cases, a variant differs from its parent molecule (e.g., a protein) in one or more amino acids, such as 1-50, 1-40, 1-30, 1-20, 1-15, 1-14, 1-13, 1-12, 1-11, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3 or 1-2 amino acids in addition, deletion or substitution. In some cases, a variant may have at least about 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more) sequence homology to the amino acid sequence of its parent molecule.

In some embodiments, the antibodies or antigen-binding fragments described herein can be coupled to a drug. Antibody drug conjugates comprising antibodies or antigen-binding fragments thereof can be covalently or non-covalently bound to a drug. In certain embodiments, the drug is a cytotoxic agent or a cell proliferation inhibitor.

In some embodiments, the molecular formula of the antibody-drug conjugate is: Ab-[L-D]n, wherein Ab represents an anti-CLDN18.2 antibody, an antigen-binding fragment thereof, or a variant thereof, L represents a linker, D represents a drug, and n represents the average number of drug connections relative to each molecule of Ab. In some embodiments, the linker L is a cysteine-coupled linker, a lysine-coupled linker, a valine-citrulline (Val-Cit, vc) linker, an SPDB linker, an SMCC linker, or a SMAC (sortasemediated antibody conjugation technology) linker.

In some embodiments, the drug is selected from one or more of calicheamicins, duocarmycins, anthramycin derivatives PBD, camptothecin derivatives, dolastatin and auristatins, maytansine and its derivatives. Furthermore, the drug is selected from MMAF, MMAE, MMAD, PBD, dukamycin, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxy anthracin, maytansinoids (such as DM-1 and DM-4), diketone, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, epirubicin and cyclophosphamide and one or more of their analogs.

"Sequence similarity" indicates the percentage of amino acids that are identical or that represent conservative amino acid substitutions. "Sequence identity" between two amino acid sequences indicates the percentage of identical amino acids between the sequences.

As used herein, the term "binding specificity" generally refers to the ability of a substance to specifically bind to another substance and not to randomly bind to any other substance. For example, a protein can specifically bind to another protein due to its specific structure. For example, a targeting moiety can exhibit binding specificity to a corresponding tumor antigen.

As used herein, the term "claudin18.2" is interchangeable with "CLDN18.2". The claudin18.2 protein contains four transmembrane domains and two extracellular loops that can be used for monoclonal antibody binding, and claudin18.2 antibodies have been used in cancer treatment research. For example, claudiximab (IMAB362), a human-mouse chimeric antibody developed by Astellas, has shown positive data in clinical trials for the treatment of gastric and gastroesophageal junction cancer. Claudin 18.2 is present in large quantities in a large proportion of primary gastric cancer and its metastatic cancer, and plays an important role in its malignant transformation. For example, frequent ectopic activation of Claudin 18.2 has been found in pancreatic, esophageal, ovarian, and lung tumors (Niimi et al., (2001) Mol Cell Biol 21(21): 7380-7390; Tanaka et al. (2011) J Histochem Cytochem 59(10): 942-952; Micke et al., (2014) Int J Cancer 135(9): 2206-2214; Shimobaba et al. (2016) Biochim Biophys Acta 18 CLDN18.2 expression can be detected in a variety of cancers or tumors such as bladder cancer, ovarian cancer, lung cancer, adenocarcinoma, gastric cancer, breast cancer, liver cancer, pancreatic cancer, skin cancer, malignant melanoma, head and neck cancer, sarcoma, bile duct cancer, kidney cancer, colon cancer, small intestine cancer, testicular embryonal carcinoma, placental choriocarcinoma, cervical cancer, testicular cancer, uterine cancer, esophageal cancer and gallbladder cancer cells.

As used herein, the terms "claudin18.2 antibody", "CLDN18.2 antibody", "anti-claudin18.2 antibody" and "anti-CLDN18.2 antibody" are interchangeable. In some embodiments, the antibody or antigen-binding fragment thereof of the present invention is capable of specifically binding to CLDN18.2 (claudin18.2). In some embodiments, the antibody or antigen-binding fragment thereof is a murine antibody, a chimeric antibody, a humanized antibody or a fully human antibody. In some embodiments, the antibody or antigen-binding fragment thereof of the present invention has the following properties: a) specific binding to CLDN18.2; b) high affinity; c) strong ADCC activity; d) strong CDC activity. In some embodiments, anti-CLDN18.2 antibodies can impair the growth of tumors or cancer cells that express positive CLDN18.2. Because Claudin 18.2 is present in large quantities in a large proportion of primary gastric cancers and their metastatic cancers, and plays an important role in their malignant transformation. Anti-CLDN18.2 antibodies can damage, for example, tumors in the pancreas, esophagus, ovary and lung. Furthermore, in some embodiments, anti-CLDN18.2 antibodies can also have a therapeutic effect on other cancers or tumors, including but not limited to bladder cancer, ovarian cancer, lung cancer, adenocarcinoma, gastric cancer, breast cancer, liver cancer, pancreatic cancer, skin cancer, malignant melanoma, head and neck cancer, sarcoma, bile duct cancer, kidney cancer, colon cancer, small intestine cancer, testicular embryonal carcinoma, placental choriocarcinoma, cervical cancer, testicular cancer, uterine cancer, esophageal cancer and gallbladder cancer cells.

In some embodiments, the antibody capable of binding to CLDN18.2 preferably comprises one or more complementary determining regions (HCDRs), such as HCDR1, HCDR2 and HCDR3, of the heavy chain variable region ( _VH ) of an anti-CLDN18.2 monoclonal antibody (preferably a monoclonal antibody against CLDN18.2 described herein), and preferably comprises one or more complementary determining regions (LCDRs), such as LCDR1, LCDR2 and LCDR3, of the light chain variable region ( _VL ) described herein. In one embodiment, the one or more complementary determining regions (CDRs) are selected from a set of complementary determining regions CDR1, CDR2 and CDR3 described herein. In a particularly preferred embodiment, the antibody capable of binding to CLDN18.2 preferably comprises the complementarity determining regions CDR1, CDR2 and CDR3 of the heavy chain variable region ( _VH ) and/or light chain variable region ( _VL ) of the anti-CLDN18.2 monoclonal antibody, and preferably comprises the complementarity determining regions CDR1, CDR2 and CDR3 of the heavy chain variable region ( _VH ) and/or light chain variable region ( _VL ) described herein.

In one embodiment, the amino acid sequence of the complementarity determining region HCDR1 of the heavy chain variable region ( _VH ) of the monoclonal antibody against CLDN18.2 in the antibody capable of binding to CLDN18.2 is as shown in SEQ ID NO.1, the amino acid sequence of HCDR2 is as shown in SEQ ID NO.2, and the amino acid sequence of HCDR3 is as shown in SEQ ID NO.3. Among them, SEQ ID NO.1 is NYEMN; SEQ ID NO.2 is YITGSGRTIYYADSVKG; and SEQ ID NO.3 is YDYGDFDF.

In one embodiment, the amino acid sequence of the complementary determining region LCDR1 of the light chain variable region (V _L ) of the monoclonal antibody against CLDN18.2 in the antibody capable of binding to CLDN18.2 is shown in SEQ ID NO. 4, the amino acid sequence of LCDR2 is shown in SEQ ID NO. 5, and the amino acid sequence of LCDR3 is shown in SEQ ID NO. 6. Among them, SEQ ID NO. 4 is RASQGISSWLA; SEQ ID NO. 5 is AASSLQS; and SEQ ID NO. 6 is QQANSFPLT.

In one embodiment, an antibody comprising one or more CDRs, a set of CDRs or a combination of CDR sets as described herein comprises said CDRs together with their intervening framework regions.

In one embodiment, an antibody comprising one or more CDRs, a set of CDRs or a combination of sets of CDRs as described herein comprises said CDRs in a human antibody framework.

In one embodiment, the amino acid sequence of the heavy chain variable region ( _VH ) of the monoclonal antibody against CLDN18.2 in the antibody capable of binding to CLDN18.2 is as shown in SEQ ID NO. 7, and the amino acid sequence of the light chain variable region ( _VL ) of the monoclonal antibody against CLDN18.2 in the antibody capable of binding to CLDN18.2 is as shown in SEQ ID NO. 8. Wherein, SEQ ID NO. 7 is QVQLVESGGGLVQPGGSLRLSCAASGFTFSNYEMNWVRQAPGKGLEWVAYITGSGRTIYYADSVKGRFTISRDNAKKSLYLQMNSLRSEDTAVYYCAIYDYGDFDFWGQGTLVTVSS; SEQ ID NO. 8 is DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFPLTFGGGTKVEIK.

The antibody mentioned herein comprising a specific chain relative to its heavy chain, or a specific region or sequence preferably relates to a situation in which all heavy chains of the antibody comprise the specific chain, region or sequence. This applies correspondingly to the light chain of the antibody.

As used herein, the term "nucleic acid" or "nucleotide" is intended to include DNA and RNA. The nucleic acid may be single-stranded or double-stranded, but is preferably double-stranded DNA.

According to the present invention, the term "expression" is used in its most general sense and includes the production of RNA or RNA and protein/peptide. It also includes partial expression of nucleic acid. In addition, expression can be transient or stable.

The term "transgenic animal" refers to an animal having a genome comprising one or more transgenes, preferably a heavy chain transgene and/or a light chain transgene, or a transchromosome (integrated or not integrated into the native genomic DNA of the animal), and preferably an animal capable of expressing the transgene. For example, a transgenic mouse may have a human light chain transgene and a human heavy chain transgene or a human heavy chain transchromosome, such that when immunized with a CLDN18.2 antigen and/or a cell expressing CLDN18.2, the mouse produces human anti-CLDN18.2 antibodies. The human heavy chain transgene may be integrated into the chromosomal DNA of the mouse, such as in transgenic mice, such as HuMAb mice, such as HCo7 or HCol2 mice, or the human heavy chain transgene may be maintained extrachromosomally, such as in the case of transchromosomal (e.g., KM) mice described in WO 02/43478. Such transgenic and transchromosomal mice are capable of producing multiple isotypes (e.g., IgG, IgA and/or IgE) of human monoclonal antibodies to CLDN18.2 by undergoing V-D-J recombination and isotype switching.

As used herein, the term "substantially not" generally refers to little or almost no binding to a specific substance. For example, very little or almost no (e.g., less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.5%, less than 0.1% or less than 0.01%) antibodies, antigen-binding fragments thereof or variants thereof according to the present application bind to CLDN18.1.

As used herein, "reduce", "lower" or "inhibit" means an overall decrease in level (such as expression level or cell proliferation level) or is capable of causing an overall decrease in level (such as expression level or cell proliferation level), preferably 5% or more, 10% or more, 20% or more, more preferably 50% or more, and most preferably 75% or more.

Terms such as "increase" or "enhance" preferably relate to an increase or enhancement of about at least 10%, preferably at least 20%, preferably at least 30%, more preferably at least 40%, more preferably at least 50%, even more preferably at least 80% and most preferably at least 100%, at least 200%, at least 500%, at least 1000%, at least 10000% or even more.

While the following provides considerations regarding potential mechanisms of therapeutic efficacy for the antibodies of the invention, they should not be construed as limiting the invention in any way.

The antibodies described herein preferably interact with components of the immune system, preferably through ADCC or CDC. The antibodies described herein may also be used to target payloads (e.g., radioisotopes, drugs, or toxins) to directly kill tumor cells or may be used in conjunction with traditional biological or chemotherapeutic agents to attack tumors through complementary mechanisms of action, which may include anti-tumor immune responses that may have been impaired due to cytotoxic side effects of chemotherapeutic agents on T lymphocytes. However, the antibodies described herein may also work only by binding to CLDN18.2 on the cell surface, thereby blocking cell proliferation.

"ADCC" and "antibody-dependent cell-mediated cytotoxicity" as used herein refer to Antibody-Dependent Cell-mediated Cytotoxicity, which describes the cell-killing ability of effector cells, which preferably requires target cells labeled by antibodies. When the Fab fragment of the antibody binds to the antigen epitope on the tumor cell and the Fc domain of the antibody engages the Fc receptor (FcR) on the surface of the immune effector cell (such as NK cells, macrophages, neutrophils, etc.), ADCC preferably occurs, that is, ADCC mediates the direct killing of target cells by immune effector cells.

Several families of Fc receptors have been identified, and specific cell populations characteristically express defined Fc receptors. ADCC can be considered a mechanism for directly inducing variable degrees of immediate tumor destruction, which results in antigen presentation and induction of tumor-directed T-cell responses. Preferably, in vivo induction of ADCC will result in tumor-directed T-cell responses and host-derived antibody responses.

The "CDC" and "complement-dependent cytotoxicity" mentioned in this article refer to Complement Dependent Cytotoxicity, which is another cell killing method that can be directed by antibodies. It is a cytotoxic effect involving complement, that is, specific antibodies bind to corresponding antigens on the cell membrane surface to form a complex and activate the classical complement pathway. The membrane-attacking complex formed exerts a lytic effect on target cells.

IgM is the most effective isotype for complement activation. IgG1 and IgG3 are also very effective in directional CDC via the classical complement-activation pathway. Preferably, in this cascade, the formation of the antigen-antibody complex leads to the exposure of multiple C1q binding sites (C1q is one of the three subcomponents of complement C1) that are closely adjacent to the CH2 domain of the antibody molecule involved in the IgG molecule. Preferably, these exposed C1q binding sites convert the previously low-affinity C1q-IgG interaction into one of the high-affinity ones, which triggers a cascade involving a series of other complement protein events and leads to the proteolytic release of effector-cell chemokines/activators C3a and C5a. Preferably, the complement cascade terminates when the membrane attack complex is formed, which creates holes in the cell membrane that facilitate the free entry and exit of water and solutes into and out of the cell.

In the present application, antibodies can be produced by a variety of techniques, including conventional monoclonal antibody methodology, such as the standard somatic cell hybridization technique of Kohler and Milstein (Nature 256:495, 1975). Although the somatic cell hybridization procedure is preferred, in principle, other techniques for producing monoclonal antibodies can be used, such as viral or oncogenic transformation of B-lymphocytes or phage display technology using antibody gene libraries.

The preferred animal system for preparing hybridomas secreting monoclonal antibodies is the murine system, more preferably the mouse system.Immunization protocols and techniques for isolating immune cells for fusion are known in the art.

Other preferred animal systems for preparing hybridomas secreting monoclonal antibodies are the rat and rabbit systems (eg, as described in Spieker-Polet et al., Proc. Natl. Acad. Sci. USA 92:9348 (1995); see also Rossi et al., Am. J. Clin. Pathol. 124:295 (2005)).

In another preferred embodiment, human monoclonal antibodies can be generated using transgenic or transchromosomal mice that carry parts of the human immune system rather than the mouse system. These transgenic and transchromosomal mice include mice known as HuMAb mice and KM mice, respectively, and are collectively referred to herein as "transgenic mice." The production of human antibodies in such transgenic mice can be performed as described in detail for CD20 in WO2004035607.

However, another strategy for generating monoclonal antibodies involves isolating the gene encoding the antibody directly from lymphocytes producing antibodies of defined specificities, as described by Babcock et al. (1996; A novel strategy for generating monoclonal antibodies from single, isolated lymphocytes producing antibodies of defined specificities). Details of recombinant antibody engineering can also be found in Welschof and Kraus (Recombinant antibodes for cancer therapy ISBN-0-89603-918-8) and Benny K.C.Lo Antibody Engineering ISBN 1-58829-092-1.

To generate antibodies, as described above, a concentrated preparation of a carrier-coupled peptide derived from an antigen sequence (i.e., a sequence for an antibody to be directed), a recombinantly expressed antigen or its fragment and/or an antigen-expressing cell can be used to immunize mice. Alternatively, a DNA encoding an antigen or its fragment can be used to immunize mice. In the case where antibodies are not produced by immunization with purified or concentrated antigen preparations, cells expressing the antigen (e.g., cell lines) can also be used to immunize mice to promote an immune response.

To generate hybridomas that produce monoclonal antibodies, spleen cells and lymph node cells can be isolated from immunized mice and fused to appropriate immortalized cell lines, such as mouse myeloma cell lines. The resulting hybridomas can then be screened for the production of antigen-specific antibodies. The individual wells can then be screened for hybridomas that secrete antibodies by ELISA. Using cells that express the antigen, antibodies specific to the antigen can be identified by immunofluorescence and FACS analysis. The hybridomas that secrete antibodies can be re-inoculated and screened again, and if they are still positive for the monoclonal antibody, they can be subcloned by limiting dilution. Stable subclones can then be cultured in vitro to generate antibodies for characterization in tissue culture medium.

Antibodies can also be produced in host cell transfectomas using, for example, a combination of recombinant DNA technology and gene transfection methods as are well known in the art (Morrison, S. (1985) Science 229:1202).

For example, in one embodiment, the target gene (e.g., antibody gene) can be connected to an expression vector, such as a eukaryotic expression plasmid used in the GS gene expression system disclosed in WO 87/04462, WO 89/01036 and EP 338841, or other expression systems well known in the art. Purified plasmids with cloned antibody genes can be introduced into eukaryotic host cells, such as CHO cells, NS/0 cells, HEK293T cells or HEK293 cells or other eukaryotic cells, such as plant-derived cells, fungal cells or yeast cells. The method for introducing these genes can be the method described in the field, such as electroporation, liposomes (lipofectine/lipofectamine) or other methods. After these antibody genes are introduced into host cells, cells expressing antibodies can be identified and selected. These cells represent transfectomas, and the expression level of the transfectomas can then be amplified and scaled up to produce antibodies. Recombinant antibodies can be isolated and purified from these culture supernatants and/or cells.

Alternatively, the cloned antibody genes can be expressed in other expression systems, including prokaryotic cells, such as microorganisms, such as E. coli. In addition, antibodies can be produced in transgenic non-human animals (such as milk of sheep and rabbits or eggs of hens) or in transgenic plants; see, e.g., Verma, R., et al.

(1998) J.Immunol.Meth.216:165-181; Pollock, et al.

(1999) J. Immunol. Meth. 231: 147-157; and Fischer, R., et al.

(1999) Biol. Chem. 380:825-839.

As used herein, the term "isolated nucleic acid molecule(s)" generally refers to a polymeric form of nucleotides of any length, whether deoxyribonucleotides or ribonucleotides or their analogs, separated from their natural environment or artificially synthesized.

As used herein, the term "fusion protein" may generally refer to a polypeptide comprising an amino acid sequence of a polypeptide fused directly or indirectly (e.g., via a linker) to an amino acid sequence of a heterologous polypeptide (i.e., a polypeptide unrelated to the previous polypeptide or its domain). Or consisting of the same.

As used herein, the term "one or more vectors" generally refers to a nucleic acid vector into which a polynucleotide encoding a protein can be inserted and expressed. The genetic material elements carried in the vector can be expressed in a host cell by transforming, transducing or transfecting the host cell with a vector. Embodiments of the vector include: plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs) or P1-derived artificial chromosomes (PACs); bacteriophages such as lambda phages or M13 phages and animal viruses. Animal viruses used as vectors include retroviruses (including slow viruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex viruses), oncolytic viruses, icteroviruses, baculoviruses, papilloma viruses, papovaviruses (such as SV40 viruses). The vector may include a variety of elements that control expression, including promoter sequences, transcription start sequences, enhancer sequences, selection elements and reporter genes. In addition, the vector may also include a replication origin. The vector may also include components that help it enter the cell, such as viral particles, liposomes or protein shells, but not just these substances. In some embodiments, the antibodies or antigen-binding portions thereof of the invention may also be encoded by or carried by an oncolytic virus.

The ability of an antibody to bind antigen can be determined using standard binding assays (eg, ELISA, Western blot, immunofluorescence, and flow cytometric analysis).

To purify the antibodies, the selected hybridomas can be grown in spinner bottles for monoclonal antibody purification. Alternatively, the antibodies can be produced in a dialysis-based bioreactor. The supernatant can be filtered and, if necessary, concentrated before affinity chromatography with protein G-agarose or protein A-agarose. The eluted IgG can be detected by gel electrophoresis and high performance liquid chromatography to ensure purity. The buffer can be exchanged for PBS and the concentration can be determined by OD ₂₈₀ using an extinction coefficient of 1.43. The monoclonal antibodies can be aliquoted and stored at -80°C.

To determine whether a selected monoclonal antibody binds to a unique epitope, site-directed mutagenesis or multiple site-directed mutagenesis can be used.

To determine the isotype of the antibody, an isotype ELISA (e.g., Zymed, Roche Diagnostics) with different commercial kits can be performed. The wells of the microtiter plate can be coated with anti-mouse Ig. After blocking, the plate and the isotype control of the monoclonal antibody or purification are reacted at ambient temperature for 2 hours. Then, the wells can be reacted with a probe coupled with mouse IgG1, IgG2a, IgG2b or IgG3, IgA or mouse IgM-specific peroxidase. After washing, the plate can be developed with ABTS substrate (1 mg/ml) and analyzed at OD _405-650 . Alternatively, the IsoStrip mouse monoclonal antibody typing kit can be used as described by the manufacturer.

To demonstrate the presence of antibodies in the sera of immunized mice or that the monoclonal antibodies bind to live cells expressing the antigen, flow cytometry can be used. Cell lines expressing the antigen naturally or after transfection and negative controls lacking antigen expression (grown under standard growth conditions) can be mixed with varying concentrations of monoclonal antibodies in hybridoma supernatants or in PBS containing 1% FBS and can be incubated at 4°C for 30 min. After washing, APC- or Alexa Flour647- (AF647)-labeled anti-IgG antibodies can bind to the antigen-bound monoclonal antibodies under the same conditions as the primary antibody staining. Samples can be analyzed by flow cytometry using a FACS instrument that gates single live cells using the light and side scatter properties. To distinguish antigen-specific monoclonal antibodies from nonspecific binders in a single measurement, a co-transfection approach can be used. Cells transiently transfected with plasmids encoding the antigen and a fluorescent marker can be stained as described above. Transfected cells can be detected in a different fluorescent channel than antibody-stained cells. Because most transfected cells express both transgenes, antigen-specific monoclonal antibodies preferentially bind to cells expressing the fluorescent marker, while non-specific antibodies bind to untransfected cells at comparable rates. Alternative assays to fluorescence microscopy can be used in addition to or in place of flow cytometry. As described above, cells can be stained and examined by fluorescence microscopy.

To demonstrate the presence of antibodies in the sera of immunized mice or that the monoclonal antibodies bind to living cells expressing the antigen, immunofluorescence microscopy analysis can be used.

Cell extracts from cells expressing the antigen and appropriate negative controls can be prepared and subjected to sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. After electrophoresis, the separated antigens are transferred to nitrocellulose membranes, blocked and probed with the monoclonal antibodies to be tested. IgG binding can be detected using anti-mouse IgG peroxidase and developed with ECL substrates.

The reactivity of the antibody with the antigen can be further tested by immunohistochemistry in a manner well known to the skilled artisan, such as using paraformaldehyde- or acetone-fixed frozen sections or paraffin-embedded tissue sections fixed with paraformaldehyde from non-cancerous or cancerous tissue samples obtained from patients during routine surgical procedures or from mice bearing xenograft tumors inoculated with a cell line expressing the antigen either spontaneously or after transfection.

The antibodies can be tested for their ability to mediate phagocytosis and to kill cells expressing CLDN18.2. Testing of monoclonal antibody activity in vitro will provide an initial screen prior to testing in vivo models.

ADCC Assay: Polymorphonuclear cells (PMN), NK cells, monocytes, mononuclear cells or other effector cells from healthy donors can be purified by Ficoll Hypaque density centrifugation, followed by lysis of contaminating erythrocytes. Washed effector cells can be suspended in RPMI supplemented with 10% heat-inactivated fetal bovine serum or 5% heat-inactivated human serum and mixed with 51Cr-labeled target cells expressing CLDN18.2 at different ratios of effector cells to target cells. Alternatively, target cells can be labeled with a fluorescence enhancing ligand (BATDA). Highly fluorescent chelates of europium with enhancing ligands released from dead cells can be measured by a fluorimeter. Another alternative technique can utilize target cell transfection with luciferase. The added lucifer yellow can then be oxidized only by live cells. Purified anti-CLDN18.2 IgG can then be added at different concentrations. An irrelevant human IgG can be used as a negative control. The assay can be performed at 37°C for 4 to 20 hours, depending on the type of effector cells used. By measuring 51Cr release or the presence of EuTDA chelates in the culture supernatant, cell lysis of the sample can be determined. Alternatively, luminescence caused by oxidation of Lucifer Yellow can be a measure of viable cells. Anti-CLDN18.2 monoclonal antibodies can also be tested in different combinations to determine whether multiple monoclonal antibodies enhance cell lysis.

CDC test: The ability of monoclonal anti-CLDN18.2 antibodies to mediate CDC can be tested using a variety of known techniques. For example, serum complement can be obtained from blood in a manner known to the skilled person. To determine the CDC activity of mAb, different methods can be used. For example, 51Cr release can be measured or increased membrane permeability can be assessed using propidium iodide (PI) exclusion assays. In short, target cells can be washed and 5x 10 ⁵ /ml can be incubated with different concentrations of mAb at room temperature or at 37°C for 10-30min. Then, serum or plasma can be added to a final concentration of 20% (v/v) and the cells can be incubated at 37°C for 20-30min. All cells from each sample can be added to the PI solution in a FACS tube. Then, using FACSArray, the mixture can be immediately analyzed by flow cytometry analysis.

In an alternative assay, the induction of CDC can be determined on adherent cells. In one embodiment of the assay, cells were inoculated at a density of 3x 10 ⁴ cells/well in a tissue-culture flat-bottom microtiter plate 24 h before the assay. The next day, the growth medium was removed and the cells were incubated with antibodies in triplicate. Control cells were incubated with growth medium or growth medium containing 0.2% saponin for determining background lysis and maximum lysis, respectively. After incubation at room temperature for 20 min, the supernatant was removed and DMEM (pre-warmed to 37° C.) containing 20% (v/v) human plasma or serum was added to the cells and incubated for another 20 min at 37° C. All cells from each sample were added to a propidium iodide solution (10 μg/ml). Then, the supernatant was replaced with PBS containing 2.5 μg/ml ethidium bromide, and the fluorescence emission after excitation at 520 nm was measured at 600 nm using a Tecan Safire. The percentage of specific lysis was calculated as follows: % specific lysis = (fluorescence sample - fluorescence background) / (fluorescence maximum lysis - fluorescence background) x 100.

Monoclonal antibody-induced apoptosis and cell proliferation inhibition: To test the ability to induce apoptosis, monoclonal anti-CLDN18.2 antibodies, for example, can be incubated with CLDN18.2-positive tumor cells (such as SNU-16, DAN-G, KATO-III or CLDN18.2-transfected tumor cells) at 37°C for about 20 hours. Cells can be harvested, washed in Annexin-V binding buffer (BD biosciences), and incubated with Annexin-V-conjugated FITC or APC (BD biosciences) in the dark for 15 min. All cells from each sample can be added to a PI solution (10 μg/ml in PBS) in a FACS tube and immediately evaluated by flow cytometry (as above). Alternatively, commercially available kits can be used to detect overall inhibition of cell proliferation by monoclonal antibodies. The DELFIA Cell Proliferation Kit (Perkin-Elmer, Cat. No. AD0200) is a non-isotopic immunoassay based on the measurement of 5-bromo-2'-deoxyuridine (BrdU) incorporation during DNA synthesis of proliferating cells in microplates. Incorporated BrdU is detected using a europium-labeled monoclonal antibody. To allow antibody detection, cells are fixed and DNA is denatured using Fix solution. Unbound antibody is washed off and DELFIA Inducer is added to dissociate europium ions from the labeled antibody into solution where they form highly fluorescent chelates with components of the DELFIA Inducer. The measured fluorescence - using time-resolved fluorimetry in the assay - is proportional to DNA synthesis in the cells of each well.

CHO stable cells (Cell Pool) were used to detect cross-reaction with 18.1 by flow cytometry. The antibody CLDN-BC-P1026-hH1 is a CLDN18.2-specific antibody and does not react with CLDN18.1.

The relevant information about P1026 antibody is as follows:

Cell line CLDN-BC-P1026-hH1;

Source: Obtained by antigen immunization of fully human mice.

As used herein, the term "cell" generally refers to a cell into which a vector is introduced, including many cell types, such as prokaryotic cells such as Escherichia coli and Bacillus subtilis, fungal cells such as yeast cells or Aspergillus cells, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK293 cells, MC38 cells or human cells.

As used herein, the term "conditions enabling expression" generally refers to conditions enabling expression of the antibody, its Fab or its variant of the present application. In some embodiments, the conditions enabling expression include but are not limited to incubation time, temperature and culture medium, and may depend on the cell type, and may be easily determined by one of ordinary skill in the art. In some embodiments, in the process of producing the antibody, its Fab or its variant of the present application, the cells are grown in culture and grown in any device (including fermentation tank) that can be used to grow the culture. The cells may be grown into a monolayer or attached to a surface. Alternatively, the cells may be grown in suspension. The cells may be grown in serum-free medium.

As used herein, the term "cancer" generally refers to a group of diseases involving abnormal cell growth, which has the potential to invade or spread to other parts of the body. Cancer is fundamentally a disease of tissue growth regulation. In order for normal cells to be transformed into cancer cells, genes that regulate cell growth and differentiation must be changed. The affected genes are divided into two major categories. Oncogenes are genes that promote cell growth and reproduction. Tumor suppressor genes are genes that inhibit cell division and survival. Malignant transformation can occur through the formation of new oncogenes, inappropriate overexpression of normal oncogenes, or insufficient expression or inactivation of tumor suppressor genes. Generally, to transform normal cells into cancer cells, changes in multiple genes are required. Cancer is classified by cell type, including carcinomas, sarcomas, lymphomas and leukemias, germ cell tumors and blastomas.

As used herein, the term "T cell" generally refers to a type of lymphocyte (a subtype of white blood cell) that plays a central role in cell-mediated immunity. T cells can be distinguished from other lymphocytes (such as B cells and natural killer cells) by the presence of T cell receptors on the cell surface. They are called T cells because they mature from thymocytes in the thymus. Most human T cells rearrange their alpha and beta chains on the cell receptor and are called alpha beta T cells (αβT cells), which are part of the adaptive immune system. Specialized γδT cells (a small subset of T cells in humans, more common in ruminants) have an invariant T cell receptor with limited diversity, are able to effectively present antigens to other T cells, and are considered part of the innate immune system.

As used herein, the term "MC38" cells refers to mouse colon cancer cells. MC38 mouse colorectal cancer cells overexpress human claudin18.2, simulating colorectal cancer with high expression of claudin18.2. Mouse colorectal cancer cells are used to simulate the human body as much as possible in the context of immune-competent mice.

As used herein, the term "pharmaceutically acceptable excipient" generally refers to any and all solvents, dispersion media, coatings, isotonic agents, and absorption delaying agents that are compatible with drug administration. In some embodiments, the excipient of CLDN18.2 stabilizes its expression or increases its expression. In some embodiments, the CLDN18.2 expression stabilizer or expression increaser is oxaliplatin and/or 5-FU. In some embodiments, preferably, when CLDN18.2 is administered, it is expressed on the cell surface of cancer cells. Further, the cancer cells are cancer cells associated with claudin18.2, such as solid tumor cells, such as gastric cancer cells, pancreatic cancer cells, esophageal cancer cells, intestinal cancer cells, liver cancer cells, lung cancer cells, etc. The CLDN18.2 antibody or its antigen-binding portion of the present application has an in vivo anti-tumor effect.

As used herein, the term "about" generally refers to an approximate value of a given value that can be reasonably inferred based on ordinary skills in the art, including equivalent values and approximate values due to the experimental and/or measurement conditions of the given value. For example, it can refer to a value that is no more than 10% higher or lower than the value modified by the term. For example, the term "about 5 μg/kg" refers to a range of 4.5 μg/kg to 5.5 μg/kg. As another example, "about 1 hour" means a range of 48 minutes to 72 minutes.

As used herein, the term "effective amount" generally refers to a dose sufficient to provide a sufficiently high concentration to confer a beneficial effect on its recipient. The specific therapeutically effective dose level for any particular subject will depend on a variety of factors, including the condition being treated, the severity of the condition, the activity of the specific component, the route of administration, the clearance rate, the duration of treatment, the age, weight, sex, diet and general health of the subject, and other relevant factors.

As used herein, the term "binding specificity" generally refers to the ability to specifically bind (e.g., immunoreact with) a given target (while not binding or substantially not binding to non-targets). The antibodies (or antigen-binding fragments or variants thereof) of the present application may be monospecific and include one or more binding sites that specifically bind to a target, or may be multispecific (e.g., bispecific or trispecific) and include two or more binding sites that specifically bind to the same or different targets.

As used herein, the term "modification" generally refers to any manipulation of the peptide backbone (e.g., amino acid sequence) of a polypeptide or any post-translational modification (e.g., glycosylation). For example, the modification is compared to the sequence of the corresponding wild-type polypeptide. The modification can be a substitution, addition, and/or deletion of one or more amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more).

As used herein, the term "amino acid substitution" generally refers to the replacement of one amino acid at a specific position in a polypeptide with another amino acid.

As used herein, the term "isolated polynucleotide" generally refers to a polymeric form of nucleotides of any length, whether deoxyribonucleotides or ribonucleotides, or analogs thereof, separated from its natural environment or artificially synthesized.

In one aspect, the present application provides an antibody, an antigen-binding fragment thereof, or a variant thereof that can bind to CLDN18.2. The antibody, the antigen-binding fragment thereof, or a variant thereof can specifically bind to CLDN18.2 and substantially not bind to CLDN18.1.

In the present application, it is preferred that the antibody, antigen-binding fragment thereof or variant thereof remains in the form of a monomer (eg, rather than a dimer, trimer or other multimer).

The antibody according to the present application may be selected from the group consisting of: a monoclonal antibody, a chimeric antibody, a humanized antibody, a fully human antibody and a bispecific antibody.

The antigen-binding fragment according to the present application may be selected from the group consisting of: a Fab fragment, a Fab' fragment, a F(ab) ₂ fragment, a F(ab') ₂ fragment, a Fv fragment and a ScFv.

In some cases, the variant may be a polypeptide that differs from the antibody or antigen-binding fragment thereof according to the present application in the addition, deletion or substitution of one or more amino acids (such as 1-50, 1-40, 1-30, 1-20, 1-15, 1-14, 1-13, 1-12, 1-11, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3 or 1-2 amino acids).

In some cases, the variant can be a polypeptide having at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more) sequence identity with an antibody or antigen-binding fragment thereof according to the present application.

In some cases, the reference antibody comprises light chain LCDR1-3 and heavy chain HCDR1-3, the heavy chain HCDR1 may comprise the amino acid sequence shown in SEQ ID NO:1, the heavy chain HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:2, the heavy chain HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:3, the light chain LCDR1 may comprise the amino acid sequence shown in SEQ ID NO:4, the light chain LCDR2 may comprise the amino acid sequence shown in SEQ ID NO:5, and the light chain LCDR3 may comprise the amino acid sequence shown in SEQ ID NO:6.

In some embodiments, the preparation method of anti-CLDN18.2 antibodies is a conventional method in the art. In certain embodiments, the prepared antibodies do not cause harmful immune responses in animals (e.g., humans) to be treated. In some embodiments, the antibodies, antigen-binding fragments, or derivatives disclosed in the present invention are modified using techniques recognized in the art to reduce their immunogenicity. For example, antibodies can be humanized, primatized, deimmunized, or chimeric antibodies can be prepared. In some embodiments, the antibodies of the present application are fully human antibodies.

The preparation of scFv can refer to the technology for producing single-chain units (U.S. Patent 4,694,778; Bird, Science 242: 423-442 (1988), Huston et al., Proc. Natl. Acad. Sci. USA 55: 5879-5883 (1988) and Ward et al., Nature 334: 544-554 (1989) and Nie et al., Antibody Therapeutics 3 (1): 18-62 (2020)). The heavy chain and light chain fragments of the Fv region are bridged by amino acids to form a single-chain unit to produce a single-chain fusion peptide. The technology for assembling functional Fv fragments in Escherichia coli can also be used (Skerra et al., Science 242: 1038-1041 (1988)).

Examples of techniques that can be used to produce single-chain Fvs (scFvs) and antibodies include those described in U.S. Pat. Nos. 4,946,778 and 5,258,498, as well as Huston et al., Methods in Enzymology 203:46-88 (1991), Shu et al., Proc. Natl. Sci. USA 90:1995-1999 (1993), and Skerra et al., Science 240:1038-1040 (1988). For certain uses including the use of antibodies in humans and in vitro detection experiments, chimeric antibodies, humanized antibodies, or fully human antibodies can be used. Chimeric antibodies are a class of molecules in which different portions of an antibody are derived from different animal species, such as an antibody having the variable regions of a mouse monoclonal antibody and the constant regions of a human immunoglobulin. Methods for producing chimeric antibodies are known in the art, see Morrison, Science 229:1202 (1985); Oi et al., BioTechniques 4:214 (1986); Gillies et al., J. Immunol. Methods 125:191-202 (1989); Neuberger et al., Nature 372:604-608 (1984); Takeda et al., Nature 314:452-454 (1985); and U.S. Patents 5,807,715, 4,816,567 and 4,816,397, the entire contents of which are incorporated herein by reference.

In some embodiments, hybridoma technology is used to prepare to produce antibodies of the present invention. Monoclonal antibodies are prepared using, for example, the hybridoma method, such as described by Kohler and Milstein, Nature, 256:495 (1975). In the hybridoma method, mice, hamsters or other suitable host animals are usually immunized with an immunizing agent to induce lymphocytes to produce or be able to produce antibodies that specifically bind to the immunizing agent.

The immunizing agent will generally include a protein antigen, a fragment thereof, or a fusion protein thereof. Typically, if human cells are desired, peripheral blood lymphocytes are used; if non-human mammalian sources are desired, spleen lymphocytes or lymph node cells are used. In some embodiments, spleen lymphocytes are used; then the lymphocytes are fused with immortalized cell lines using a suitable fusion agent, such as polyethylene glycol, to form hybridoma cells (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) pages 59-103). Immortalized cell lines are generally transformed mammalian cells, particularly myeloma cells of rodent, bovine and human origin. Typically, rat or mouse myeloma cell lines are used. In some embodiments, spleen lymphocytes and mouse myeloma cells are used for fusion. Hybridoma cells can be cultured in an appropriate culture medium, and in some embodiments the culture medium contains one or more substances that inhibit the growth or survival of unfused immortalized cells. For example, if the parental cells lack hypoxanthine-guanine phosphoribosyltransferase (HGPRT or HPRT), the culture medium for the hybridoma typically includes hypoxanthine, aminopterin and thymidine ("HAT medium"), which prevents the growth of HGPRT-deficient cells. In some embodiments, the binding specificity of the monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by in vitro binding assays, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). Such techniques and assays are known in the art. The binding affinity of the monoclonal antibodies can be determined, for example, by the Scatchard analysis of Munson and Pollard, Anal. Biochem., 107: 220 (1980). In addition, in the therapeutic application of monoclonal antibodies, it is important to identify antibodies with high specificity and high binding affinity to the target antigen.

After the desired hybridoma cells are identified, the clones can be subcloned using limiting dilution procedures and grown using standard methods (see Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) pp. 59-103). Suitable culture media for this purpose include, for example, Dulbecco's modified Eagle's medium and RPMI-1640 medium.

In some embodiments, the monoclonal antibodies secreted by the subclones can be isolated or purified by conventional techniques, such as protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

Monoclonal antibodies can also be prepared by recombinant DNA methods, such as described in U.S. Patent No. 4,816,567. The DNA encoding the monoclonal antibodies described herein can be separated and sequenced using conventional methods (e.g., by using oligonucleotide probes that can specifically bind to the genes of the heavy and light chains of the antibody). The DNA encoding the antibodies described herein can also be synthesized according to the antibody sequence design according to conventional methods. The isolated or synthesized DNA is inserted into an expression vector, which is then transfected into a host cell such as a Chinese hamster ovary (CHO) cell, a human embryonic kidney (HEK) 293 cell, ape COS cell, PER.NS0 cell, SP2/0, YB2/0 or myeloma cell that does not produce immunoglobulin in addition, thereby obtaining the synthesized monoclonal antibody in a recombinant host cell.

In some embodiments, hybridoma technology is used to prepare and produce antibodies or antigen-binding fragments of the present invention, and mice are immunized with a protein preparation containing CLDN18.2, and spleen lymphocytes of the mice are fused with myeloma cells after immunization, and hybridomas are screened by CLDN18.2 protein, and positive hybridomas are limitedly diluted, further subcloned, and the binding ability of hybridoma strains to CLDN18.2 protein is identified again to prepare anti-CLDN18.2 antibodies. In some embodiments, the mouse is a female Balb/c mouse aged 6-8 weeks.

The binding specificity of the antibodies or antigen-binding fragments disclosed in the present invention can be detected by in vitro assays, such as co-immunoprecipitation, radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).

For certain uses including the use of antibodies in humans and in vitro detection experiments, chimeric antibodies, humanized antibodies, or fully human antibodies may be used in some embodiments. Chimeric antibodies are a class of molecules in which different parts of an antibody are derived from different animal species, such as antibodies having the variable region of a mouse monoclonal antibody and the constant region of a human immunoglobulin. Methods for producing chimeric antibodies are known in the art, see Morrison, Science 229:1202 (1985); Oi et al., BioTechniques 4:214 (1986), Gillies et al., J. Immunol. Methods 125:191-202 (1989), and U.S. Patents 5,807,715, 4,816,567, and 4,816397, the entire contents of which are incorporated herein by reference.

Transgenic mice can also be used to produce fully human antibodies, which cannot express functional endogenous immunoglobulins but can express human immunoglobulin genes. For example, human heavy chain and light chain immunoglobulin gene complexes can be introduced randomly or by homologous recombination into mouse embryonic stem cells. Alternatively, in addition to human heavy chain and light chain genes, human variable regions, constant regions, and diversity regions can also be introduced into mouse embryonic stem cells. Mouse heavy chain and light chain immunoglobulin genes can be introduced into human immunoglobulin loci by homologous recombination and lose function. In particular, homozygous deletions in the JH region can prevent the production of endogenous antibodies. Modified embryonic stem cells are amplified and microinjected into blastocysts to produce chimeric mice. Chimeric mice are then cultivated to produce homozygous offspring expressing human antibodies. Transgenic mice are immunized in a conventional manner with selected antigens, such as all or part of the desired polypeptide targets. Monoclonal antibodies targeting antigens can be obtained from immunized transgenic mice using conventional hybridoma technology. The human immunoglobulin transgenes carried by transgenic mice are rearranged during B cell differentiation, followed by class switching and somatic mutations. Therefore, using this technology, IgG, IgA, IgM and IgE antibodies that can be used for treatment can be produced.In the present application, the anti-CLDN18.2 antibody can be a fully human antibody.

In other embodiments, DNA encoding the desired monoclonal antibody can be easily isolated and sequenced using conventional methods (e.g., using oligonucleotide probes that can specifically bind to genes encoding mouse antibody heavy and light chains). Once isolated, the DNA can be placed in an expression vector and then transfected into a prokaryotic or eukaryotic host cell such as an E. coli cell, a simian COS cell, a Chinese hamster ovary (CHO) cell, or a myeloma cell that does not otherwise produce immunoglobulins. The isolated DNA (which may be synthetic as described herein) can also be used to prepare sequences of constant and variable regions of antibodies, as described in U.S. Patent No. 5,658,570, the entire contents of which are incorporated herein by reference. The method extracts RNA from selected cells and converts it into cDNA, which is then amplified by PCR technology using Ig-specific primers. Suitable probes for this purpose are also mentioned in U.S. Patent No. 5,658,570.

In addition, using conventional recombinant DNA technology, one or more CDRs of the antibody or antigen-binding fragment of the present invention can be inserted into the framework region, for example, into the human framework region to construct a humanized non-fully human antibody. The framework region can be a naturally occurring or shared framework region, preferably a human framework region (see Chothia et al., J. Mol. Biol. 278: 457-479 (1998), which lists a series of human framework regions). Some polynucleotides can encode antibodies that specifically bind to at least one epitope of the target antigen produced by the combination of framework regions and CDRs. One or more amino acid substitutions are made in the framework region, and amino acid substitutions that can improve the binding of the antibody to its antigen can be selected. In addition, this method can be used to replace or delete cysteine residues in one or more variable regions involved in the formation of interchain disulfide bonds, thereby producing antibody molecules lacking one or more interchain disulfide bonds. Other changes to polynucleotides within the technical scope of the art are also covered in the present invention.

In addition, another efficient method for producing recombinant antibodies is disclosed in Newman, Biotechnology 10:1455-1460 (1992), in particular, the technology can produce primate antibodies containing monkey variable region and human constant region sequences, the entire content of which is incorporated herein by reference. In addition, the technology is also mentioned in commonly assigned U.S. Patents 5,658,570, 5,693,780 and 5,756,096, the entire content of each patent is incorporated herein by reference.

Antibodies can be prepared using conventional recombinant DNA techniques. Antibody-producing vectors and cell lines can be selected, constructed, and cultured using techniques known to those skilled in the art. These techniques are described in various laboratory manuals and major publications, such as Recombinant DNA Technology for Production of Protein Therapeutics in Cultured Mammalian Cells, D.L. Hacker, F.M. Wurm, in Reference Module in Life Sciences, 2017, the entire contents of which, including supplementary content, are incorporated herein by reference.

In some embodiments, the DNA encoding the antibody can be designed and synthesized according to the antibody amino acid sequence described herein in a conventional manner, placed in an expression vector, and then transfected into a host cell, and the transfected host cell is cultured in a culture medium to produce a monoclonal antibody. In some embodiments, the antibody expression vector includes at least one promoter element, an antibody coding sequence, a transcription termination signal, and a polyA tail. Other elements include enhancers, Kozak sequences, and donor and acceptor sites for RNA splicing on both sides of the insertion sequence. Efficient transcription can be obtained by the early and late promoters of SV40, the long terminal repeats from retroviruses such as RSV, HTLV1, HIVI, and the early promoters of cytomegalovirus, and promoters of other cells such as the actin promoter can also be used. Suitable expression vectors may include pIRES1neo, pRetro-Off, pRetro-On, PLXSN, or Plncx, pcDNA3.1 (+/-), pcDNA/Zeo (+/-), pcDNA3.1/Hygro (+/-), PSVL, PMSG, pRSVcat, pSV2dhfr, pBC12MI and pCS2, etc. Commonly used mammalian host cells include 293 cells, Cos1 cells, Cos7 cells, CV1 cells, mouse L cells and CHO cells, etc.

In some embodiments, the inserted gene fragment needs to contain a screening marker, and common screening markers include dihydrofolate reductase, glutamine synthetase, neomycin resistance, hygromycin resistance and other screening genes, so as to facilitate the screening and separation of successfully transfected cells. The constructed plasmid is transfected into host cells without the above genes, and after culturing in a selective medium, the successfully transfected cells grow in large quantities and produce the desired target protein.

In addition, mutations can be introduced into the nucleotide sequence encoding the antibody of the present invention using standard techniques known to those skilled in the art, including but not limited to site-directed mutagenesis and PCR-mediated mutations that result in amino acid substitutions. Variants (including derivatives) encode less than 50 amino acid substitutions, less than 40 amino acid replacements, less than 30 amino acid replacements, less than 25 amino acid replacements, less than 20 amino acid replacements, less than 15 amino acid replacements, less than 10 amino acid replacements, less than 5 amino acid replacements, less than 4 amino acid replacements, less than 3 amino acid replacements, or less than 2 amino acid replacements relative to the original heavy chain variable region and light chain variable region. Alternatively, mutations can be introduced randomly along all or part of the coding sequence, such as by saturation mutations, and the biological activity of the resulting mutants can be screened to identify mutants that retain activity.

The present invention also provides the use and administration method of a drug or composition with an anti-CLDN18.2 antibody or antigen-binding fragment as the main component. In some embodiments, a method for treating or improving various types of cancers or tumors and other related diseases is provided, the method comprising administering an effective dose of an anti-CLDN18.2 antibody or antigen-binding fragment to a patient in need. In some embodiments, the use of an anti-CLDN18.2 antibody or antigen-binding fragment for treating or improving cancer or tumors and other related diseases is provided. In some embodiments, the use of the anti-CLDN18.2 antibody or antigen-binding fragment in the preparation of a drug for treating or improving cancer or tumors and other related diseases is provided.

The specific dosage and treatment regimen for any particular patient will depend on various factors, including the specific antibody, antigen binding fragment or derivative used, the patient's age and weight, general health, sex and diet, as well as the time of administration, frequency of excretion, drug combination, and the severity of the specific disease being treated. These factors are judged by medical care personnel included in the scope of ordinary technicians in the field. The dosage will also depend on the individual patient to be treated, the route of administration, the type of formulation, the characteristics of the compound used, the severity of the disease, and the desired effect. The dosage used can be determined by pharmacological and pharmacokinetic principles well known in the art. For example, in some embodiments, the dosage of the antibody of the present invention administered to the patient is 0.01 mg/kg to 100 mg/kg of patient body weight each time; in some embodiments, the dosage is administered once every 1 week, 2 weeks, 3 weeks, or monthly.

The methods of administration of antibodies and Fabs thereof include, but are not limited to, intradermal, intramuscular, peritoneal, intravenous, subcutaneous, nasal, epidural injection and oral administration. Antibodies, Fabs or compositions can be administered by any convenient route, such as by infusion or push injection, absorbed by epithelial or mucocutaneous membranes (e.g., oral mucosa, rectal and intestinal mucosa, etc.), and can be co-administered with other bioactive agents. Therefore, pharmaceutical compositions containing antibodies and Fabs of the present invention can be administered orally, rectally, parenterally, intracisternal, intravaginal, intraperitoneal, externally (e.g., by powder, ointment, drops or transdermal patch), orally or by oral or nasal spray.

The term "parenteral" as used herein refers to modes of administration including intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion.

The administration can be systemic or local.

The antibodies, antigen-binding fragments or pharmaceutical compositions of the invention can be applied topically to the area in need of treatment; this can be achieved by, but not limited to, local infusion during surgery, such as topical application in conjunction with postoperative wound dressings, by injection, by catheter, by suppository or by implant, which is a porous, non-porous or gel-like material, including membranes (e.g., silicone rubber membranes) or fibers. In some embodiments, when administering proteins (including antibodies) of the invention, care must be taken to use materials that do not absorb proteins.

In some embodiments, the compositions of the invention comprise nucleic acids or polynucleotides encoding antibodies, which can be administered in vivo to promote expression of the protein encoded by them by constructing them as part of an appropriate nucleic acid expression vector, and then administering the above-mentioned part of the vector to make it intracellular, for example, by using retroviral vectors (see U.S. Patent 4,980,286), or by direct injection, or by using microparticle bombardment (e.g., gene gun; Biolistic, Dupont), or by coating with lipids or cell surface receptors or transfection agents, or by linking to homeobox peptides known to enter the cell nucleus (see, e.g., Joliot et al., 1991, Proc. Natl. Acad. Sci. USA 88: 1864-1868), etc. Alternatively, the nucleic acid can be introduced into the cell by homologous recombination and integrated into the host cell DNA for expression.

Typically, a method for treating a disease is performed in vitro, including administering the antibody, antigen-binding fragment or derivative of the present invention, then testing the desired therapeutic or preventive activity in vivo in an acceptable animal model, and finally administering to the human body. Suitable animal models (including transgenic animals) are well known to those of ordinary skill in the art. For example, in vitro assays for demonstrating the therapeutic use of the antibody or antigen-binding fragment of the present invention include the effects of the antibody or antigen-binding fragment on a cell line or a patient's tissue sample. The effects of the antibody or antigen-binding fragment on a cell line and/or a tissue sample can be detected using techniques known to those skilled in the art, such as techniques disclosed in other parts of the present invention. According to the present invention, in vitro assays that can be used to determine whether to administer a specific antibody or antigen-binding fragment include in vitro cell culture experiments, in which a patient's tissue sample is cultured in culture, and exposed to or otherwise administered with a compound, and the effects of such a compound on the tissue sample are observed.

Various known delivery systems can be used to administer the antibodies of the present invention or polynucleotides encoding the antibodies of the present invention, such as encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the compound, receptor-mediated endocytosis (see, for example, Wu and Wu, 1987, J. Biol. Chem. 262: 4429-4432), construction of nucleic acids as part of a retrovirus or other vector, etc.

In some embodiments, in some embodiments, the antibodies or antigen-binding fragments of the present application are used in combination with other treatment methods. The anti-CLDN18.2 antibodies or antigen-binding fragments of the present invention can be combined with other treatment or prevention schemes, including the administration of one or more antibodies or antigen-binding fragments of the present invention and one or more other therapeutic agents or methods together or in combination. In some embodiments, other treatment schemes include but are not limited to radiotherapy, chemotherapy, hormone therapy, etc. For combined treatment, antibodies can be administered simultaneously or separately with other therapeutic agents. When administered separately, the antibodies of the present invention can be administered before or after the administration of another other therapeutic agent.

In some embodiments, the antibodies of the invention are administered in combination with a chemotherapeutic agent. In some embodiments, chemotherapeutic agents that can be administered with the antibodies of the invention include, but are not limited to, carboplatin (Baldin), cisplatin (Cisplatin, Cisplatin-AQ), cyclophosphamide (Cytoxan, Cytoxan), docetaxel (Taxotere), doxorubicin (Adriamycin), erlotinib (Tarceva), etoposide (Vanbis), fluorouracil (5-FU), gemcitabine (Gemzar), imatinib mesylate (Gleevec), irinotecan (Irinotecan), methotrexate (Diazo Tablets, Methotrexate Sodium, Methotrexate), paclitaxel (Taxol, Abraxane), sorafenib (Nexavar), sunitinib (Sutent), topotecan (Hycamtin), vincristine (Oncovin, Vincasar PFS) and vinblastine (Velban).

In some embodiments, the antibodies of the present invention are administered in combination with cytokines, chemokines or co-stimulatory molecules. In some embodiments, cytokines, chemokines or co-stimulatory molecules that can be administered with the antibodies of the present invention include but are not limited to CCR7, CCL19, CCL21, CCL2, CCL3, CCL5, CCL16, CXCR4, CXCR7, CXCL12, interleukins (e.g., IL-1-IL17), interferons (e.g., IFNα1, IFNα8, IFNα10, IFNα13, IFNα14, IFNα16, IFNα17, IFNα21, IFNβ1, IFNW, IFNE1 and IFNK), hematopoietic factors, TGFs (e.g., TGF-α, TGF-β, and other members of the TGF family), 4-1BB, 4-1BB-L , CD137, CD137L, CTLA-4GITR, GITRL, Fas, Fas-L, TNFR1, TRAIL-R1, TRAIL-R2, p75NGF-R, DR6, RANK, EDAR1, XEDAR, Fn114, Troy/Trade, TAJ, TNFRII, HVEM, CD27, CD30, CD40, 4-1BB, OX40, GITR, GITRL, TACI, BAFF-R, BCMA, RELT, CD95 (Fas/APO-1), glucocorticoid-induced TNFR-related protein, TNF receptor-associated apoptosis-mediating protein (TRAMP) and death receptor-6 (DR6), etc.

In some embodiments, the antibodies of the present application are administered in combination with immunotherapeutics. In some embodiments, immunotherapeutics that can be administered with the antibodies of the present invention include, but are not limited to, abavoizumab (CA-125), abciximab (CD41), adelimumab (EpCAM), afutumumab (CD20), pegol (VEGFR2), atumomab pentetate (CEA), Amatuxi monoclonal antibody (MORAb-009), maanatumomab (TAG-72), abortuzumab (HLA-DR), acitumomab (CEA), baviximab (phosphatidylserine), betumomab (CD22), belimumab (BAFF), bevacizumab (VEGF-A), bivacizumab (CD44v 6), Rantumomab (CD19), Brentuximab vedotin (CD30TNFRSF8), Mecanizumab (Mucin CanAg), Cantuzumab bravtansine (MUC1), Capromab pentotide (prostate cancer cells), Carlu monoclonal antibody (CNTO888), Catumaxomab (EpCAM, CD3), Cetuximab (EGFR), Pocitaluzumab (EpCAM), Citutumumab (IGF-1 receptor), Cliximab (tight junction protein), Clivatuzumab tetraxetan (MUC1), Canatumumab (TR AIL-R2), Daclizumab (CD40), Dalotuzumab (insulin-like growth factor I receptor), Denosumab (RANKL), Demostatumab (B lymphoma cells), Daclizumab (DR5), Emetaximab (GD3 ganglioside), Efloriximab (EpCAM), Elotuzumab (SLAMF7), Enavatuzumab (PDL192), Ensituximab (NPC-1C), Epratuzumab (CD22), Ermazosumab (HER2/neu, CD3), Irrezizumab (integrin αvβ3), Farletuzumab (folate receptor 1 ), FBTA05 (CD20), Ficlatuzu (SCH900105), Fentumumab (IGF-1 receptor), Flanvotu (glycoprotein 75), Fusutumumab (TGF-β), Galiximab (CD80), Ganitu (IGF-I), Gemtuzumab (CD33), Gevokizu (IL-1β), Girentuxi (carbonic anhydrase 9 (CA-IX)), Ibritumomab (CD20), Icrucumab (VEGFR-1), Igovotumab (CA-125), Indatuximab ( SDC1), Intetumumab (CD51), Inotuzumab ozogamicin (CD22), Ipilimumab (CD152), Itumumab (CD30), Labetuzumab (CEA), Lexatumumab (TRAIL-R2), Rivimab (HBsAg), Lintuzumab (CD33), Lorvotuzumab mertansine (CD56), Rucamumab (CD40), Rucirumab (CD23), Mapatumumab (TRAIL-R1), Matuzumab (EGFR), Mepolizumab (IL-5), Milatuzumab (CD74), Mitumumab (GD3 neurotransmitter). gangliosides), Mogamulizu mAb (CCR4), Moxetumo mAb pasudotox (CD22), Tadalafil (C242 antigen), Tadalafil (5T4), Narnatu mAb (RON), Necitumumu mAb (EGFR), Nimotuzumab (EGFR), Nivolu mAb (IgG4), Ofatumumab (CD20), Olaratu mAb (PDGF-Rα), Onartuzu mAb (human discrete factor receptor kinase), Mogamulizu mAb (EpCAM), Ogovotumab (CA-125), Oxelu mAb (OX-40), Panitumumab ( EGFR), Patritumumab (HER3), Pemtumo (MUC1), Pertuzumab (HER2/neu), Pintumomab (adenocarcinoma antigen), Protumumab (vimentin), Racotumo (N-glycolylneuraminic acid), Radretu (fibronectin extradomain-B), Ravelumab (rabies virus glycoprotein), Ramucirumab (VEGFR2), Rilotumumab (HGF), Rituximab (CD20), Rotrulumumab (IGF-1 receptor), Samalizu (CD200), Ciroizumab (FAP), Siltuxi (IL-6), Tabalumab (BAFF), Talizumab (α-fetoprotein), Patamumomab (CD19), Tetumomab (cerebrospinal fluid C), Teprotumumab (CD221), Tesitumumab (CTLA-4), Tegazumab (TRAIL-R2), TNX-650 (IL-13), Tositumomab (CD20), Trastuzumab (HER2/neu), TRBS07 (GD2), Tremelimumab (CTLA-4), Simoleukin (EpCAM), Ublituximab (MS4A1), Urelu (4-1BB), Volociximab (integrin α5β1).

In some embodiments, the present application relates to an antibody-drug conjugate, which comprises an antibody, an antigen-binding fragment thereof, or a variant thereof that specifically binds to CLDN18.2 and is conjugated to a drug, wherein the antibody, the antigen-binding fragment thereof, or a variant thereof comprises a heavy chain variable region and a light chain variable region, wherein: the heavy chain variable region comprises HCDR1 as shown in SEQ ID NO.1, HCDR2 as shown in SEQ ID NO.2, and HCDR3 as shown in SEQ ID NO.3; and the light chain variable region comprises LCDR1 as shown in SEQ ID NO.4, LCDR2 as shown in SEQ ID NO.5, and LCDR3 as shown in SEQ ID NO.6.

The antibody, antigen-binding fragment or variant thereof further comprises: a heavy chain variable region as shown in SEQ ID NO.7; and a light chain variable region as shown in SEQ ID NO.8. The antibody or its antigen-binding portion is selected from the following groups: a whole antibody, a bispecific antibody, a monoclonal antibody, a chimeric antibody, a humanized antibody and a fully human antibody. The antibody, antigen-binding fragment or variant thereof further comprises a heavy chain constant region and a light chain constant region, wherein: the antibody heavy chain constant region is selected from an IgG series antibody; and the light chain constant region is selected from a κ or λ chain.

In some embodiments, the molecular formula of the antibody drug conjugate is: Ab-[L-D]n, wherein Ab represents an anti-CLDN18.2 antibody, an antigen-binding fragment thereof, or a variant thereof, L represents a linker, D represents a drug, and n represents the average number of drug connections relative to each molecule of Ab. The drug D is a cytotoxic agent or a cell proliferation inhibitor. The drug is selected from one or more of calicheamicins, duocarmycins, anthramycin derivatives PBD, camptothecin derivatives, dolastatin, auristatins, maytansine, and derivatives thereof.

The immune antigen and anti-CLDN18.2 antibody of the present application can be obtained according to the sequence table disclosed in the present application and conventional technical means in the art, which will not be described in detail here. The antibody or antigen-binding fragment of the present application will be described below through specific examples.

Example 1 Preparation of anti-CLDN18.2 antibodies

The anti-CLDN18.2 antibody of the present application is codon optimized by CHO cell preference, and the light and heavy chain sequences are synthesized respectively, and the heavy chain is connected to the PEE12.4 expression vector, and the light chain is connected to the PEE6.4 expression vector. After the sequencing is correct, the plasmid is extracted, and the antibody is transiently transfected and purified using the Expi CHO-S ^TM expression system. Among them, the sequence of the anti-CLDN18.2 antibody is shown in Table 1:

Table 1 Amino acid sequence of anti-CLDN18.2 antibody P1026

The method for producing antibodies will not be described in detail in this application.

Example 2 Detection of anti-CLDN18.2 antibodies

Flow cytometry was used to detect the ability of anti-CLDN18.2 antibodies to bind to cells expressing CLDN18.2, and further to detect their specificity of not binding to CLDN18.1. Furthermore, species cross-detection was performed on anti-CLDN18.2 antibodies.

The experimental steps are outlined as follows:

1. Affinity detection

The positive control antibody and the antibody to be tested were diluted to 20 μg/mL respectively, and appropriate CLDN18.2 and CLDN18.1 overexpressing cells were taken and distributed to 96-well plates. An appropriate amount of antibody was added to each well to make the final concentration of 10 μg/mL. After incubation at 4°C for 30 minutes, PBS was washed twice, and an appropriate amount of anti-human IgG Fc AF647 fluorescent secondary antibody diluted at a ratio of 1:5000 was added. After incubation at 4°C for 30 minutes, PBS was washed twice, and an appropriate amount of PBS was added. The binding signal was detected by flow cytometry. At the same time, CHO-S and NC blank controls were set up in the experiment. FlowJo was used to analyze the activity of each antibody binding to CLDN18.2 and CLDN18.1, and the antibody specifically binding to CLDN18.2 was selected to enter the next EC50 detection, species cross detection and affinity detection.

Figure 1 is a specific detection of human CLDN18.2 and CLDN18.1 according to an embodiment of the present invention; wherein the horizontal axis is the fluorescence intensity of Anti-hIgG-Fc-AF647, detecting the binding strength of the antibody to CLDN18.1 and CLDN18.2; the vertical axis SSC-H is the biased dispersion of the cell, detecting the complexity of the cell. As shown in Figure 1A, the fully human antibody P1026 (CLDN18.2 specific binding antibody) only binds to CLDN18.2 and does not bind to CLDN18.1.

2. Activity detection

The positive control antibody and the antibody to be tested were diluted to 20 μg/mL respectively. Appropriate amounts of human, mouse and monkey CLDN18.2 overexpressing cells and human CLDN18.1 overexpressing cells were taken and distributed to 96-well plates. Appropriate amounts of antibodies were added to each well to make the final concentration 10 μg/mL. After incubation at 4°C for 30 minutes, the plates were washed twice with PBS, and appropriate amounts of anti-human IgG Fc AF647 fluorescent secondary antibodies diluted at a ratio of 1:5000 were added. After incubation at 4°C for 30 minutes, the plates were washed twice with PBS, and appropriate amounts of PBS were added. The binding signals were detected by flow cytometry. At the same time, CHO-S and NC blank controls were set up in the experiment. FlowJo was used to analyze the activity of each antibody in binding to human, mouse and monkey CLDN18.2 and CLDN18.1.

Figure 2 is a result of antibody species cross-detection according to an embodiment of the present invention, wherein the horizontal axis is the fluorescence intensity of Anti-hIgG-Fc-AF647, detecting the binding strength of the antibody and CLDN18.2; the vertical axis SSC-H is the biased dispersion of the cell, detecting the complexity of the cell. As shown in Figure 2, the fully human antibody P1026 (CLDN18.2 specific binding antibody) only binds to CLDN18.2 and does not bind to CLDN18.1; the fully human antibody P1026 can bind to CLDN18.2 of humans, mice and cynomolgus monkeys, and its binding effect is comparable to that of the known CLDN18.2 inhibitor Zolbetuximab (IMAB362).

3. Binding strength test

The positive control antibody and the antibody to be tested were diluted in a gradient dilution; an appropriate amount of CLDN18.2 overexpressing cells were taken and distributed into 96-well plates, and an appropriate amount of antibody was added to each well. After incubation at 4°C for 30 minutes, the cells were washed twice with PBS, and an appropriate amount of anti-human IgG Fc AF647 fluorescent secondary antibody diluted at a ratio of 1:5000 was added. After incubation at 4°C for 30 minutes, the cells were washed twice with PBS, and an appropriate amount of PBS was added. The binding signal was detected by flow cytometry. At the same time, CHO-S and NC blank controls were set up in the experiment. FlowJo was used to analyze the MFI of each antibody binding to CLDN18.2, and the MFI was imported into GraphPad Prism for analysis to obtain the EC ₅₀ value of each antibody binding to CLDN18.2.

Figure 3 is an antibody binding ability test according to an embodiment of the present invention; wherein the abscissa is the antibody concentration and the ordinate is the average fluorescence intensity; Table 2 is the antibody binding and affinity test data according to an embodiment of the present application. As shown in Figure 3 and Table 2, compared with the known CLDN18.2 inhibitor IMAB362, the fully human anti-CLDN18.2 antibody P1026 of the present application has stronger binding and affinity.

Table 2 Binding and affinity test data of fully human anti-CLDN18.2 antibodies

Example 3 ADCC activity of anti-CLDN18.2 antibodies (antibody dependent cell-mediated cytotoxicity)

In this experiment, ADCC Reporter Bioassay was selected to evaluate the ADCC activity of CLDN18.2 antibody. This is a bioluminescent reporter gene detection system that uses artificially constructed effector cells to replace NK cells and is combined with highly sensitive detection reagents to quantify the biological activity of therapeutic antibody drugs based on the ADCC mechanism of action in their activation pathways. It is a mechanism of action detection method.

Take the target cells MC38-hCLDN18.2 and effector cells FcR-TANK in the logarithmic growth phase for cell counting, adjust the cell density to 4×10 ⁵ /mL and 1.6×10 ⁶ /mL, add 50 μL of each cell to a 96-well plate (effector-target ratio is 4:1). Dilute the antibodies to be tested into different concentrations and add them to each well, with a volume of 50 μL per well. Effector cells, target cells and antibodies are mixed in a ratio of 1:1:1 and incubated at 37°C for 4 hours. LDH method is used to detect cell killing.

45 minutes before the end of incubation, add 1× lysis solution to the target cell maximum release well, mix well, and continue to culture for 45 minutes. After the incubation, remove the culture plate, aspirate 50 μL of supernatant into a new ELISA plate, add 50 μL of LDH detection solution to each well, tap the plate, mix well, and incubate at room temperature in the dark for 10-30 minutes. Add 50 μL of stop solution to each well and tap the plate to mix well. Read the absorbance values of OD ₄₉₀ and OD ₆₈₀ respectively with an ELISA reader and calculate the increment. Calculate cytotoxicity (Cytotoxicity)% according to the following formula:

in,

Experimental value: Experimental well increment value – culture medium control well increment value

Target spontaneous value: target cell spontaneous well increment value – medium control well increment value

Effector Spontaneous: Effector spontaneous release well increment value – Medium control well increment value

Target Maximum: Target cell maximum release well increment value – equal volume control well increment value

Graphpadprism was used to draw a curve and perform nonlinear fitting of the data using the fitting method [Agonist] vs. response--Variable slope (four parameters) to obtain the EC ₅₀ value of the test article. Human IgG1 was used as a negative control and CLN-Zolbetuximab-IgG1 was used as a positive antibody control.

Figure 4 shows the ADCC activity of the CLDN18.2 antibody on MC38 cells overexpressing human claudin18.2 according to one embodiment of the present application. Table 3 shows the ADCC activity of the CLDN18.2 antibody on MC38 cells overexpressing human claudin18.2. Among them, IMAB362-IgG1 is used as an antibody positive control. As shown in Figure 4 and Table 3, antibody P1026 has a strong ADCC effect, and antibody P1026 has a stronger ADCC effect than the positive antibody CLN-zolbetuximab-IgG1.

Table 3 ADCC activity of CLDN18.2 antibody against MC38 cells overexpressing human claudin18.2

Example 4 CDC activity of anti-CLDN18.2 antibodies (complement dependent cytoxicity)

Take 5×10 ⁴ /well of target cells in logarithmic growth phase and plate them in 96-well plates, 90μL of cell suspension in each well, and incubate overnight. Dilute the antibody to be tested into different concentrations and add them to each well. After incubating the target cells with antibodies for 30 minutes at 37℃, 5% CO ₂ , add complement stock solution to all wells at 12.5μL/well, mix well, and incubate in a 37℃, 5% CO ₂ incubator for 2 hours. Use Presto Blue to detect cell killing.

After the incubation, the culture plate was removed and the fluorescence value of each well was measured by ELISA at 560/590 nm. The cytotoxicity (Cytotoxicity) % was calculated according to the following formula:

in,

Flu s: is the fluorescence value of the experimental well (i.e., wells with cells and test products of different concentrations);

Flu c: the average fluorescence value of the experimental control wells (i.e., wells with cells and culture medium but no test product);

Flu b: is the average fluorescence value of the blank wells (i.e., the wells with only culture medium added, without cells and test products).

Graphpadprism was used to draw a curve and perform nonlinear fitting of the data using the fitting method [Agonist] vs. response--Variable slope (four parameters) to obtain the EC ₅₀ value of the test article.

IMAB362-IgG1 served as a positive antibody control.

Figure 5 shows the CDC activity of CLDN18.2 antibody on MC38 cells overexpressing human claudin18.2 according to one embodiment of the present application. Table 4 shows the CDC activity of CLDN18.2 antibody on MC38 cells overexpressing human claudin18.2. As shown in Figure 5 and Table 4, antibody P1026 has a significant CDC effect in the presence of complement.

Table 4 CDC activity of CLDN18.2 antibody against MC38 cells overexpressing human claudin18.2

Example 5 Endocytic activity of anti-CLDN18.2 antibodies

CHO-S-hCLDN18.2 cells were mixed with the CDLN18.2 antibody to be tested at a final concentration of 10 nM, the negative control hIgG1 at 10 nM, and the control IMAB362 antibody (all the above antibodies were labeled with pHAb dye), and incubated at 37°C for 0 hr, 1 hr, 4 hr, and 8 hr, respectively. The flow cytometer collected the cell endocytosis data after different treatment times. FlowJo X10.0.7 was used to analyze and export the ratio of CLDN18.2 antibody and hIgG1 to CLDN18.2 expressed on the surface of CHO-S-hCLDN18.2 cells and then internalized into the cell to emit fluorescence under different treatment time. GraphPad Prism 7.00 was used to process and analyze the data and draw the endocytosis curve.

Figure 6 is an endocytic activity curve of the anti-CLDN18.2 antibody P1026 according to one embodiment of the present application. As shown in Figure 6, compared with IMAB362, P1026 can show endocytic activity more quickly, and the endocytic activity within 1 hour is close to 40, which is much higher than IMAB362 and the negative control (NC).

Example 6 In vivo anti-tumor effect of anti-CLDN18.2 antibody drug conjugate

In this example, P1026 samples were prepared by transient expression, coupled with toxin using VC-MMAE linker, and in vivo efficacy evaluation was performed.

Method for preparing antibody drug conjugate

(1) dissolving the linker-cytotoxic drug (VC-MMAE) in dimethyl sulfoxide to obtain a linker-cytotoxic drug stock solution;

(2) Dissolve the reducing agent in a buffer solution to prepare a reducing agent stock solution.

(3) subjecting the naked antibody sample of P1026 to a reduction reaction with the reducing agent buffer in step (2) for 1-2 hours to obtain an antibody-reduced solution;

(4) adding the linker-cytotoxic drug stock solution in step (1) dropwise to the antibody reduced solution, and performing addition reaction for 1-2 hours to obtain an antibody-conjugated drug.

1. GSU Gastric Cancer Model

GSU human gastric cancer tumor cells were obtained from ATCC. The tumor cells were cultured in RPMI-1640 medium containing inactivated 10% fetal bovine serum, 100U/ml penicillin and 2mM glutamine in an incubator at 37°C and 5% _CO2. The cells were subcultured every 3 to 4 days after they were fully grown, and the tumor cells in the logarithmic growth phase were used for inoculation of tumors in vivo.

GSU tumor cells were adjusted to a concentration of 5×10 ⁷ /ml with PBS and inoculated subcutaneously in the right flank of experimental mice, 5×10 ⁶ /mouse. When the average tumor volume reached about 40-60 mm ³ , the animals were divided into 3 groups, each with 6 animals. The drugs were administered on the day of grouping. The dosage of IMAB362 was 10 mg/kg, the administration route was ip (intraperitoneal injection), and the administration frequency was tiw (administration three times a week). The dosage of P1026ADC was 3 mg/kg, the administration route was ip (intraperitoneal injection), and the administration frequency was tiw (administration three times a week).

After grouping, the tumor volume was measured twice a week using a vernier caliper. The tumor volume was measured before euthanasia. The long diameter and short diameter of the tumor were measured. The volume calculation formula was: tumor volume = 0.5 × long diameter × short diameter 2. After the last administration, the weight of the experimental animals and the tumor growth status were observed for 7 days. During the observation period, the tumor volume and animal weight were measured twice a week, and the measured values were recorded. After the observation was completed, the experiment was terminated.

Figure 7A shows the anti-tumor effect of the antibody drug conjugate P1026-ADC in the GSU gastric cancer model according to one embodiment of the present application. Table 5-1 shows the anti-tumor effect of the antibody drug conjugate P1026-ADC in the GSU gastric cancer model according to one embodiment of the present application. As shown in Figure 7A and Table 5-1, 3 mg/kg of the antibody drug conjugate of the present application can reduce the tumor volume to disappear, which is much better than the existing anti-gastric cancer tumor drug IMAB362.

Table 5-1 Antitumor effect of P1026-ADC in GSU gastric cancer model

GAS076 is a patient tissue xenograft model (PDX) of human gastric cancer. Its tumor tissue is passaged in mice. When the subcutaneous tumor volume grows to ^500-1000mm3 , the tumor tissue is removed under sterile conditions, cut into small pieces with a diameter of about 2-3mm, and inoculated subcutaneously in the right flank of the experimental mouse with a trocar. One piece of tumor tissue is inoculated in each mouse.

When the average tumor volume reached ^about 120-130 mm3, the animals were divided into 5 groups, with 6 animals in each group. Dosing began on the day of grouping. The dose of IMAB362 was 10 mg/kg, and the dosing frequency was biw (twice a week). The doses of P1026ADC were 1, 3, and 10 mg/kg, respectively, and the dosing frequency was biw (twice a week).

FIG. 7B shows the anti-tumor effect of the gastric cancer PDX model of the antibody drug conjugate P1026-ADC according to one embodiment of the present application. Table 5-2 shows the anti-tumor effect of the gastric cancer PDX model of the antibody drug conjugate P1026-ADC according to one embodiment of the present application. As shown in FIG. 7B and Table 5-2, the anti-tumor effect of P1026-ADC is much better than the existing anti-gastric cancer tumor drug IMAB362. Even when P1026-ADC is used at 10 mg/kg, the tumor volume can be reduced to disappearance.

Table 5-2 Anti-tumor effect of ADC in gastric cancer PDX model

The above embodiments are only used to illustrate the present invention, but not to limit the present invention. Ordinary technicians in the relevant technical field can make various changes and modifications without departing from the scope of the present invention. Therefore, all equivalent technical solutions should also fall within the scope of the present invention.

Claims

An antibody, an antigen-binding fragment or a variant thereof that specifically binds to CLDN18.2, comprising a heavy chain variable region and a light chain variable region, wherein: the heavy chain variable region comprises HCDR1 as shown in SEQ ID NO.1, HCDR2 as shown in SEQ ID NO.2, and HCDR3 as shown in SEQ ID NO.3; and the light chain variable region comprises LCDR1 as shown in SEQ ID NO.4, LCDR2 as shown in SEQ ID NO.5, and LCDR3 as shown in SEQ ID NO.6.
An antibody, an antigen-binding fragment or a variant thereof according to claim 1, wherein: the heavy chain comprises the variable region shown in SEQ ID NO.7; and the light chain comprises the variable region shown in SEQ ID NO.8.
The antibody, antigen-binding fragment or variant thereof according to claim 1 or 2, wherein the antibody or antigen-binding portion thereof is selected from the following group: a whole antibody, a bispecific antibody, a monoclonal antibody, a chimeric antibody, a humanized antibody and a fully human antibody.
The antibody, antigen-binding fragment or variant thereof according to claim 1, further comprising a heavy chain constant region and a light chain constant region, wherein: the antibody heavy chain constant region is selected from the IgG series of antibodies; and the light chain constant region is selected from κ or λ chain.
The antibody, antigen-binding fragment or variant thereof according to claim 4, wherein the IgG series antibody is selected from one or more of IgG1, IgG2 and IgG4.
The antibody, antigen-binding fragment or variant thereof according to claim 1, wherein the antigen-binding fragment is selected from the following group: a Fab fragment, a Fab' fragment, a F(ab) 2 fragment, a Fv fragment and a ScFv.
The antibody, antigen-binding fragment or variant thereof according to claim 1, wherein the CLDN18.2 is selected from the following group: human CLDN18.2, mouse CLDN18.2 and monkey CLDN18.2.
A fusion protein comprising the antibody, antigen-binding fragment or variant thereof according to any one of claims 1 to 7.
One or more isolated nucleic acid molecules encoding an antibody, antigen-binding fragment or variant thereof according to any one of claims 1 to 7, or a fusion protein according to claim 8.
One or more vectors comprising one or more isolated nucleic acid molecules according to claim 9.
A cell comprising one or more isolated nucleic acid molecules according to claim 9 or one or more vectors according to claim 10.
The cell according to claim 11, which is further a CAR-T or CAR-NK cell comprising one or more isolated nucleic acid molecules according to claim 9 or one or more vectors according to claim 10.
A method for producing an antibody, an antigen-binding fragment thereof or a variant thereof according to any one of claims 1 to 7, or a fusion protein according to claim 8, comprising culturing a cell according to claim 11 or 12 under conditions that allow the expression of the antibody, an antigen-binding fragment thereof or a variant thereof according to any one of claims 1 to 7, or a fusion protein according to claim 8.
A composition comprising an antibody, an antigen-binding fragment thereof or a variant thereof according to any one of claims 1 to 7, a fusion protein according to claim 8, one or more isolated nucleic acid molecules according to claim 9, one or more vectors according to claim 10 and/or a cell according to claim 11 or 12, and optionally a pharmaceutically acceptable excipient.
Use of an antibody, an antigen-binding fragment or a variant thereof according to any one of claims 1 to 7, a fusion protein according to claim 8, one or more isolated nucleic acid molecules according to claim 9, one or more vectors according to claim 10 and/or a cell according to claim 11 or 12 in the preparation of a medicament for preventing and/or treating cancer or tumors.
The use according to claim 15, wherein the drug is a drug for cell therapy.
The use according to claim 15, wherein the cancer or tumor is a cancer or tumor that is positive for CLDN18.2 expression.
The use according to claim 17, wherein the cancer or tumor is selected from bladder cancer, ovarian cancer, lung cancer, adenocarcinoma, gastric cancer, breast cancer, liver cancer, pancreatic cancer, skin cancer, malignant melanoma, head and neck cancer, sarcoma, bile duct cancer, kidney cancer, colon cancer, small intestine cancer, testicular embryonal carcinoma, placental choriocarcinoma, cervical cancer, testicular cancer, uterine cancer, esophageal cancer and gallbladder cancer cells.
Use of the antibody, antigen-binding fragment or variant thereof according to any one of claims 1 to 7, or the fusion protein according to claim 8 in the preparation of a reagent for determining the presence and/or amount of CLDN18.2 in a sample.
A pharmaceutical composition comprising: an antibody, an antigen-binding fragment thereof or a variant thereof according to any one of claims 1 to 7, a fusion protein according to claim 8, one or more isolated nucleic acid molecules according to claim 9 or one or more vectors according to claim 10 and/or a cell according to claim 11 or 12.
An antibody-drug conjugate, comprising an antibody, an antigen-binding fragment thereof or a variant thereof that specifically binds to CLDN18.2 and is conjugated to a drug, wherein the antibody, the antigen-binding fragment thereof or a variant thereof comprises a heavy chain variable region and a light chain variable region, wherein: the heavy chain variable region comprises HCDR1 as shown in SEQ ID NO.1, HCDR2 as shown in SEQ ID NO.2, and HCDR3 as shown in SEQ ID NO.3; and the light chain variable region comprises LCDR1 as shown in SEQ ID NO.4, LCDR2 as shown in SEQ ID NO.5, and LCDR3 as shown in SEQ ID NO.6.
The antibody-drug conjugate according to claim 21, wherein the antibody, its antigen-binding fragment or its variant further comprises: a heavy chain variable region shown in SEQ ID NO.7; and a light chain variable region shown in SEQ ID NO.8.
The antibody-drug conjugate according to claim 21 or 22, wherein the antibody or antigen-binding portion thereof is selected from the group consisting of a whole antibody, a bispecific antibody, a monoclonal antibody, a chimeric antibody, a humanized antibody and a fully human antibody.
The antibody-drug conjugate according to claim 21, wherein the antibody, antigen-binding fragment thereof or variant thereof further comprises a heavy chain constant region and a light chain constant region, wherein: the antibody heavy chain constant region is selected from the IgG series of antibodies; and the light chain constant region is selected from κ or λ chain.
According to the antibody-drug conjugate of claim 24, the IgG series antibody is selected from one or more of IgG1, IgG2 and IgG4.
The antibody drug conjugate according to claim 21, wherein the antigen binding fragment is selected from the following group: a Fab fragment, a Fab' fragment, a F(ab) 2 fragment, a Fv fragment and a ScFv.
The antibody drug conjugate according to claim 21, wherein the CLDN18.2 is selected from the following group: human CLDN18.2, mouse CLDN18.2 and monkey CLDN18.2.
The antibody-drug conjugate according to any one of claims 21 to 27, wherein the molecular formula of the antibody-drug conjugate is: Ab-[L-D]n, wherein Ab represents an anti-CLDN18.2 antibody, an antigen-binding fragment thereof or a variant thereof, L represents a linker, D represents a drug, and n represents the average number of drug connections per Ab molecule.
The antibody-drug conjugate according to any one of claims 21 to 28, wherein the drug D is a cytotoxic agent or a cell proliferation inhibitor.
The antibody-drug conjugate according to claim 29, wherein the drug is selected from one or more of calicheamicins, duocarmycins, anthramycin derivatives PBD, camptothecin derivatives, dolastatin and auristatins, maytansine and its derivatives.
According to the drug conjugate of claim 29, further, the drug is selected from MMAF, MMAE, MMAD, PBD, dukamicin, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxy anthracin, maytansinoids (such as DM-1 and DM-4), diketone, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, epirubicin and cyclophosphamide and one or more thereof.
According to the drug conjugate of claim 28, the linker L is a cysteine conjugation linker, a lysine conjugation linker, a valine-citrulline (Val-Cit, vc) linker, an SPDB linker, an SMCC linker, or a SMAC (sortasemediated antibody conjugation technology) linker.
A method for preparing an antibody-drug conjugate, comprising:

An antibody, an antigen-binding fragment thereof, or a variant thereof that specifically binds to CLDN18.2 is prepared, which comprises a heavy chain variable region and a light chain variable region, wherein: the heavy chain variable region comprises HCDR1 as shown in SEQ ID NO.1, HCDR2 as shown in SEQ ID NO.2, and HCDR3 as shown in SEQ ID NO.3; and the light chain variable region comprises LCDR1 as shown in SEQ ID NO.4, LCDR2 as shown in SEQ ID NO.5, and LCDR3 as shown in SEQ ID NO.6;

The antibody, antigen-binding fragment or variant thereof is coupled to MMAE using a valine-citrulline (Val-Cit, vc) linker;

Obtain anti-CLDN18.2 antibody-drug conjugate.
A composition comprising the antibody-drug conjugate according to any one of claims 21-22, or the antibody-drug conjugate prepared according to the method of claim 33, and optionally a pharmaceutically acceptable excipient.
Use of the antibody-drug conjugate according to any one of claims 21 to 32, or the antibody-drug conjugate prepared according to the method of claim 33, in the preparation of a medicament for preventing and/or treating cancer or tumors.
The use according to claim 35, wherein the drug is a drug for cell therapy.
The use according to claim 35, wherein the cancer or tumor is a cancer or tumor that is positive for CLDN18.2 expression.
The use according to claim 37, wherein the cancer or tumor is selected from bladder cancer, ovarian cancer, lung cancer, adenocarcinoma, gastric cancer, breast cancer, liver cancer, pancreatic cancer, skin cancer, malignant melanoma, head and neck cancer, sarcoma, bile duct cancer, kidney cancer, colon cancer, small intestine cancer, testicular embryonal carcinoma, placental choriocarcinoma, cervical cancer, testicular cancer, uterine cancer, esophageal cancer and gallbladder cancer cells.
Use of an antibody drug conjugate according to any one of claims 21 to 27 in the preparation of a reagent for determining the presence and/or amount of CLDN18.2 in a sample.
A pharmaceutical composition comprising: an antibody-drug conjugate according to any one of claims 21-22 or an antibody-drug conjugate prepared according to the method of claim 33.