WO2023025306A1 - Bispecific antibody targeting pd-l1 and cldn18.2, and preparation method therefor and use thereof - Google Patents

Abstract

A bispecific antibody targeting PD-L1 and CLDN18.2, and a preparation method therefor and the use thereof. The bispecific antibody or an antigen-binding portion thereof comprises a first antigen-binding domain targeting PD-L1 and a second antigen-binding domain targeting CLDN18.2, wherein the first antigen-binding domain comprises CDR1-3 in a sequence represented by SEQ ID NO: 3, and the second antigen-binding domain comprises CDR1-3 in a sequence represented by SEQ ID NO: 4. The SEC-HPLC monomer purity of the bispecific antibody is greater than 90% and Tm is greater than 60°Ϲ, and the specificity is equivalent to those of control antibodies (an anti-PD-L1 antibody and an anti-CLDN18.2 antibody).

Description

Bispecific antibody targeting PD-L1 and CLDN18.2 and its preparation method and application

This application claims the priority of Chinese patent application 2021109973035 with a filing date of 2021/8/27. This application cites the full text of the above-mentioned Chinese patent application.

technical field

The invention belongs to the field of biomedicine, and in particular relates to a bispecific antibody targeting PD-L1 and CLDN18.2 and its preparation method and application.

Background technique

CLDN18 is a member of the Claudin protein family and was discovered by Shoichiro Tsukita et al. in 1998. It is an important molecule that constitutes the tight junction of epithelial cells, determines the permeability of epithelial cells, and also plays a role in blocking the diffusion of proteins and lipids on the cell membrane surface (Gunzel , D. and A.S. Yu(2013). "Claudins and the modulation of tight junction permeability" Physiol Rev 93(2):525-569). The human CLDN18 gene has two different exon 1, which can be alternatively spliced after transcription to generate two protein subtypes CLDN18.1 and CLDN18.2 with different sequences only at the N-terminus. Both CLDN18 subtype proteins consist of 261 amino acids and have four transmembrane domains, but they are distributed in different tissues. CLDN18.1 is mainly expressed in lung tissue, and CLDN18.2 is only expressed in differentiated stomach tissue. On mucosal epidermal cells, not on gastric stem cells (Sahin, Ugur, et al."Claudin-18 splice variant 2 is a pan-cancer target suitable for therapeutic antibody development"Clinical Cancer Research14.23(2008):7624-7634 ). CLDN 18.2 is highly expressed in a variety of tumor tissues, such as non-small cell lung cancer (25%), gastric cancer (70%), pancreatic cancer (50%) and esophageal cancer (30%), but almost no expression in normal tissues ( Kumar, V., et al. (2018). "Emerging Therapies in the Management of Advanced-Stage Gastric Cancer" Front Pharmacol 9:404), due to its specific expression in tumor cells and normal tissues, it has become a very A potential target for antitumor drugs. Up to now, the fastest progress is IMAB362 developed by German company Ganymed. In the phase II trial of gastric cancer, IMAB362 significantly prolongs the survival time (13.2 vs. 8.4 months) compared with standard chemotherapy, and the advantage is more obvious in patients with high expression of CLDN18.2.

PD-1 (programmed death 1, programmed death receptor 1) is an important immunosuppressive molecule, which was originally cloned from the apoptotic mouse T cell hybridoma 2B4.11. Immunomodulation targeting PD-1 is of great significance in anti-tumor, anti-infection, anti-autoimmune diseases and organ transplant survival. The main ligands of PD-1 are PD-L1 and PD-L2, among which PD-L1 plays an important role in mediating the escape of tumor cells. It is highly expressed in tumor cells and some antigen-presenting cells, and the expression level can be Induced by various cytokines such as IFN-γ, TGFβ, etc. In the tumor microenvironment, the upregulation of PD-L1 expression can directly inhibit the anti-tumor response of T cells and mediate the immune escape of tumor cells through the PD-1 signaling pathway. Blocking the interaction between PD-1 and PD-L1 can effectively restore the tumor-killing function of T cells. In recent years, antibody drugs that specifically block PD-1/PD-L1 have achieved shocking therapeutic effects in the medical community, and have the potential to treat various types of tumors.

However, patients may develop drug resistance or non-response after receiving monoclonal antibody therapy, and some diseases are affected by multiple factors in the body, including different signaling pathways, different cytokines and receptor regulation mechanisms, etc., a single target Immunotherapy does not appear to be sufficient to destroy cancer cells. As a result, researchers have begun to investigate new immunotherapy strategies, such as drug combinations or designing bispecific or multispecific antibodies to improve efficacy.

Contents of the invention

The technical problem to be solved by the present invention is to provide a bispecific antibody targeting PD-L1 and CLDN18.2 in order to overcome the lack of effective bispecific antibodies targeting PD-L1 and CLDN18.2 in the prior art And its preparation method and application.

The present invention provides a bispecific antibody or an antigen-binding part thereof, which comprises a first antigen-binding domain targeting PD-L1 and a second antigen-binding domain targeting CLDN18.2, wherein:

The first antigen-binding domain comprises: CDR1-3 in the sequence shown in SEQ ID NO:3;

The second antigen-binding domain comprises: CDR1-3 in the sequence shown in SEQ ID NO:4;

The CDRs are defined using the AbM antibody numbering system.

In the above-mentioned bispecific antibody or its antigen-binding portion, the first antigen-binding domain or the second antigen-binding domain comprises VHH; or the first antigen-binding domain and the second antigen-binding domain The two antigen-binding domains also contain VHH;

The VHH of the first antigen-binding domain comprises: HCDR1 as shown in SEQ ID NO:28, HCDR2 as shown in SEQ ID NO:29 and HCDR3 as shown in SEQ ID NO:30;

The VHH of the second antigen-binding domain comprises: HCDR1 as shown in SEQ ID NO:31, HCDR2 as shown in SEQ ID NO:32 and HCDR3 as shown in SEQ ID NO:33.

Preferably: the first antigen-binding domain comprises at least one VHH ^PD-L1 , and the VHH ^PD-L1 comprises the amino acid sequence shown in SEQ ID NO: 3 or at least 80%, 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%, or at least 99% sequence identity and retains specificity for PD-L1 Homologous sequences for sexual binding affinity; and,

The second antigen-binding domain comprises at least one VHH ^CLDN18.2 , and the VHH ^CLDN18.2 comprises the amino acid sequence shown in SEQ ID NO: 4 or has at least 80%, 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%, or at least 99% sequence identity and retain specific binding affinity for CLDN18.2 Homologous sequences;

Wherein, the number of the VHH ^CLDN18.2 and the VHH ^PD-L1 is preferably 1-2 respectively; when the antigen-binding domain contains two VHHs, the two VHHs are connected by a linker; the linker is preferably The peptide sequence, more preferably comprises or consists of (G4S)n, where n=1-10, optionally the linker comprises the sequence shown in SEQ ID NO:5.

The bispecific antibody or antigen-binding portion thereof as described above preferably has the following structure (from N-terminus to C-terminus):

The first antigen-binding domain, the constant region, and the second antigen-binding domain are operably linked to each other in any order; for example, the first antigen-binding domain is operably linked to the constant region and the constant region is operably linked to the second antigen-binding domain. two antigen binding domains, or vice versa;

Alternatively, the second antigen binding domain is operably linked to the first antigen binding domain, which is operably linked to the constant region, or vice versa;

Alternatively, both the first antigen-binding domain and the second antigen-binding domain are operably linked to the N-terminus of the constant region.

Wherein the constant region may be a complete human IgG constant region, such as a kappa light chain constant region, a human IgG1 heavy chain constant region or a human IgG1 Fc region, wherein:

The human IgG1 Fc region preferably has an amino acid sequence as shown in SEQ ID NO: 8;

The human kappa light chain constant region preferably has an amino acid sequence as shown in SEQ ID NO:7;

The human IgG1 heavy chain constant region preferably has the amino acid sequence shown in SEQ ID NO: 6, its CH1 includes the amino acid sequence shown in SEQ ID NO: 10, and CH2 includes the amino acid sequence shown in SEQ ID NO: 11 , CH3 comprises the amino acid sequence shown in SEQ ID NO:12.

In a preferred embodiment of the present invention, the C-terminal of the first antigen-binding domain is connected to the N-terminal of the human IgG Fc region, and the N-terminal of the second antigen-binding domain is connected to the C-terminal of the human IgG Fc region. end.

In another preferred embodiment of the present invention, the C-terminus of the first antigen-binding domain is connected to the N-terminus of the light chain constant region of the complete human IgG constant region, and the C-terminus of the second antigen-binding domain is Linked to the N-terminus of the heavy chain constant region of an intact human IgG constant region. Wherein, the antigen-binding domain and the constant region may be connected directly or through a linker.

In another preferred embodiment of the present invention, the C-terminal of the first antigen-binding domain is linked to the N-terminal of the human IgG Fc region, and the N-terminal of the second antigen-binding domain is linked to the first antigen Binding to the N-terminus of the domain. Wherein, the antigen-binding domain and the Fc region may be connected directly or through a linker.

In a specific embodiment of the present invention, the bispecific antibody or its antigen-binding portion contains two first polypeptide chains, and the first polypeptide chains are represented by the following formula:

VHH ^PD-L1 -hinge region-CH2-CH3-linker-VHH ^CLDN18.2 , preferably comprising the sequence shown in SEQ ID NO:13;

Alternatively, VHH ^CLDN18.2 -linker-VHH ^PD-L1 -hinge region-CH2-CH3 preferably comprises the sequence shown in SEQ ID NO:18;

Alternatively, VHH ^PD-L1 -linker-VHH ^CLDN18.2 -hinge region-CH2-CH3 preferably comprises the sequence shown in SEQ ID NO:19.

As described above, when the constant region is an intact human IgG constant region, the first antigen-binding domain and the second antigen-binding domain are operably linked to a light chain constant region and a heavy chain constant region, respectively The operative connection is preferably carried out by a linker; the linker is preferably a peptide sequence, more preferably comprising or consisting of (G4S)n, wherein n=1-10, optionally together comprising such as SEQ ID NO: The sequence shown in 5.

Preferably, the bispecific antibody or its antigen-binding portion comprises two first polypeptide chains and two second polypeptide chains: the first polypeptide chain has the formula VHH ^CLDN18.2 -CH1-hinge region-CH2-CH3, and the second polypeptide chain is shown in the formula VHH ^PD-L1 -CL; or the first polypeptide chain is shown in the formula VHH ^CLDN18.2 -linker-CH1-hinge region- Shown in CH2-CH3, and the second polypeptide chain is shown in the formula VHH ^PD-L1 -Linker-CL; preferably: the first polypeptide chain includes amino acids shown in SEQ ID NO:14 sequence, and the second polypeptide chain comprises the amino acid sequence shown in SEQ ID NO: 15; or the first polypeptide chain comprises the amino acid sequence shown in SEQ ID NO: 16, and the second multiple The peptide chain comprises the amino acid sequence shown in SEQ ID NO:17.

Alternatively, a first polypeptide chain and a second polypeptide chain are included: the first polypeptide chain is shown in the formula VHH ^PD-L1 -hinge region-CH2-CH3, and the second polypeptide chain is shown in the formula VHH ^CLDN18.2 -hinge region-CH2-CH3; or, the first polypeptide chain is shown in the formula VHH ^PD-L1 -hinge region-CH2-CH3, and the second polypeptide chain is shown in the formula VHH ^CLDN18.2 -linker-VHH ^CLDN18.2 -hinge region-CH2-CH3; alternatively, the first polypeptide chain is such as VHH ^{PD- L1} -linker-VHH ^PD-L1 -hinge region-CH2-CH3 shown, and the second polypeptide chain is shown as VHH ^CLDN18.2 -hinge region-CH2-CH3; or, the first polypeptide chain is shown as VHH ^PD-L1 -linker-VHH ^PD-L1 -hinge Region-CH2-CH3, and the second polypeptide chain is shown as VHH ^CLDN18.2 -linker-VHH ^CLDN18.2 -hinge region-CH2-CH3; preferably, the first polypeptide chain The amino acid sequence of the polypeptide chain is as shown in SEQ ID NO:20, and the amino acid sequence of the second polypeptide chain is as shown in SEQ ID NO:21; or the amino acid sequence of the first polypeptide chain is as shown in SEQ ID NO:20 shown, and the amino acid sequence of the second polypeptide chain is shown in SEQ ID NO: 22; or the amino acid sequence of the first polypeptide chain is shown in SEQ ID NO: 23, and the amino acid sequence of the second polypeptide chain The sequence is shown in SEQ ID NO:21; or, the amino acid sequence of the first polypeptide chain is shown in SEQ ID NO:23, and the amino acid sequence of the second polypeptide chain is shown in SEQ ID NO:22.

The hinge region described in the present invention preferably contains the amino acid sequence shown in SEQ ID NO:9.

The invention also provides an isolated nucleic acid encoding the bispecific antibody or antigen-binding portion thereof as described above.

The present invention also provides an expression vector comprising the isolated nucleic acid as described above.

The present invention also provides a transformant comprising the above-mentioned isolated nucleic acid or the above-mentioned recombinant expression vector;

Preferably, the host cells of the transformant are prokaryotic cells or eukaryotic cells, the prokaryotic cells are preferably E. coli cells such as TG1, BL21, and the eukaryotic cells are preferably HEK293 cells or CHO cells.

The present invention also provides a method for preparing a bispecific antibody, which comprises culturing the transformant as described above, and obtaining the bispecific antibody from the culture.

The present invention also provides a pharmaceutical composition, which comprises the above-mentioned bispecific antibody or its antigen-binding portion, and a pharmaceutically acceptable carrier;

Preferably, the pharmaceutical composition further includes other anti-tumor antibodies as active ingredients.

The present invention also provides an antibody-drug conjugate, which comprises a cytotoxic agent, and the above-mentioned bispecific antibody or an antigen-binding part thereof;

Preferably, the cytotoxic agent is MMAF or MMAE.

The present invention also provides the above-mentioned bispecific antibody or its antigen-binding part, the above-mentioned pharmaceutical composition, and the above-mentioned antibody-drug conjugate in the preparation of drugs for diagnosis, prevention and/or treatment of tumors Applications.

The present invention also provides a kit, which includes the above-mentioned bispecific antibody or its antigen-binding part, the above-mentioned pharmaceutical composition, and the above-mentioned antibody drug conjugate;

Preferably, the kit further includes (i) a device for administering the bispecific antibody or antibody drug conjugate or pharmaceutical composition; and/or (ii) instructions for use.

The present invention also provides a kit of medicines, which includes a medicine box A and a medicine box B, wherein:

The kit A contains the above-mentioned bispecific antibody or its antigen-binding part, the above-mentioned pharmaceutical composition, and the above-mentioned antibody drug conjugate;

The kit B contains other anti-tumor antibodies or pharmaceutical compositions containing the other anti-tumor antibodies, and/or consists of hormone preparations, targeted small molecule preparations, proteasome inhibitors, imaging agents, diagnostic agents, chemotherapeutic agents, One or more of the group consisting of oncolytic drugs, cytotoxic agents, cytokines, activators of co-stimulatory molecules, inhibitors of inhibitory molecules, and vaccines.

The present invention also provides a non-diagnostic method of detecting a specific antigen in vitro or in vivo, which comprises the use of a bispecific antibody or an antigen-binding portion thereof as described above for detection.

On the basis of conforming to common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain preferred examples of the present invention.

The reagents and raw materials used in the present invention are all commercially available.

The positive and progressive effects of the present invention are: 1. The inventors listed various configurations of bispecific antibodies that can be formed by VHH ^CLDN18.2 and VHH ^PD-L1 , and found the following preferred structures: Figure 1A-Figure 1D, corresponding to The bispecific antibodies are BsAb1, BsAb8, BsAb10, BsAb17 and BsAb18; 2. The SDS-PAGE purity and SEC-HPLC monomer purity of the preferred bispecific antibodies BsAb1, BsAb8, BsAb10, BsAb17 and BsAb18 are all greater than 90% and the Tm 3. Preferred bispecific antibodies BsAb1, BsAb8, BsAb10, BsAb17, and BsAb18 bind to CLDN18.2 or bind to PD-L1 and control antibodies (anti-PD-L1 L1 antibody and anti-CLDN18.2 antibody) are comparable.

Description of drawings

Figures 1A-1G show schematic structures of bispecific antibodies described in the present application.

2A-2B are gel electrophoresis images. The samples in Fig. 2A are respectively BsAb1, BsAb8, BsAb10, D21-4, NA3SH1-T4-hVH6, BsAb17 and IPI, and the samples in Fig. 2B are respectively BsAb18, BsAb19, BsAb20, BsAb21, BsAb22, and IPI, wherein BsAb1, BsAb8, BsAb10, BsAb17, BsAb18, BsAb19, BsAb20, BsAb21, BsAb22 are purified candidate bispecific antibodies, the reference product IPI is Ipilimumab, and lane M is the protein indicator (Marker).

Figures 3A-3I show the SEC-HPLC monomer detection profiles of candidate bispecific antibodies.

Figures 4A-4E show the binding activity of candidate bispecific antibodies to human recombinant protein PD-L1-His.

Figures 5A-5B show the binding activity of candidate bispecific antibodies to huPD-L1-CHO-S cells.

Figures 6A-6B show the binding activity of candidate bispecific antibodies to huCLDN18.2-HEK293 cells.

7A-7B show the binding activity of candidate bispecific antibodies to huCLDN18.1-HEK293 cells.

Figures 8A-8B show the detection of the blocking activity of candidate bispecific antibodies against PD-1/PD-L1 by FACS method.

Figures 9A-9B show the antibody-dependent cell-mediated cytotoxicity (ADCC) of candidate bispecific antibodies on huCLDN18.2-PANC-1 cells.

Figure 10 shows the detection of the blocking activity of candidate bispecific antibodies against PD-1/PD-L1 by luciferase reporter gene method.

Figure 11 shows the in vivo tumor inhibition results of the candidate bispecific antibody CLDN18.2-NUGC4.

Figure 12 shows the in vivo tumor inhibition results of the candidate bispecific antibody CLDN18.2-MC38.

Detailed ways

The present invention is further illustrated below by means of examples, but the present invention is not limited to the scope of the examples. For the experimental methods that do not specify specific conditions in the following examples, select according to conventional methods and conditions, or according to the product instructions.

The following examples are intended to illustrate the invention only and therefore should not be construed as limiting the invention in any way.

After the present invention combines CLDN18.2/PD-L1 dual targets with bispecific antibodies, it can simultaneously target PD-L1 and CLND18.2 overexpression cancers, such as gastric cancer and gastroesophageal cancer. Compared with single target antibodies, it is extremely Greatly increased the number of adapted patients.

Herein, unless the context clearly dictates otherwise, when the term "antibody" is referred to, it includes not only whole antibodies but also antigen-binding fragments of antibodies.

As used herein, the term "antibody" is used in the broadest sense herein to refer to a protein comprising an antigen binding site, encompassing natural and artificial antibodies of various structures, including but not limited to monoclonal antibodies, polyclonal Antibodies, multispecific antibodies (eg, bispecific antibodies), single chain antibodies, single domain antibodies, whole antibodies and antibody fragments. Preferably, the antibodies of the invention are single domain antibodies or heavy chain antibodies.

As used herein, the term "single domain antibody" generally refers to an antibody in which a single variable domain (e.g., a heavy chain variable domain (VH) or a light chain variable domain (VL), derived Antigen binding can be conferred from heavy chain variable domains of camelid heavy chain antibodies, VH-like single domains derived from fish IgNARs (v-NARs). That is, the single variable domain does not need to interact with another variable domain in order to recognize the target antigen. Examples of single domain antibodies include those derived from camelids (llamas and camels) and cartilaginous fish (eg nurse sharks) (WO2005/035572). A single domain antibody derived from Camelidae, also referred to in this application as VHH, consists of only one heavy chain variable region, consisting of only one chain from C-terminus to N-terminus FR4-CDR3-FR3-CDR2-FR2- Antibodies to CDR1-FR1 are also referred to as "nanobodies". Single-domain antibodies are currently known as the smallest unit that can bind to a target antigen.

The term "complementarity determining region" or "CDR region" or "CDR" is an antibody variable domain that is hypervariable in sequence and forms a structurally defined loop ("hypervariable loop") and/or contains antigen-contacting residues ("antigen contact point"). The CDR is mainly responsible for binding to the antigenic epitope, and the sequential numbering from the N-terminus includes CDR1, CDR2 and CDR3. In a given heavy chain variable region amino acid sequence, the precise amino acid sequence boundaries of each CDR can be determined using any one or combination of a number of well known antibody CDR assignment systems. It is well known to those skilled in the art that the CDR of an antibody can be defined by various methods in the art, such as Chothia (Chothia et al. (1989) Nature 342:877-883.A1 based on the three-dimensional structure of the antibody and the topology of the CDR loop -Lazikani et al., Journal of Molecular Biology, 273, 927-948 (1997)), Kabat based on antibody sequence variability (Kabat et al., U.S. Department of Health and Human Services, National Institutes of Health (1987)), AbM (University of Bath), Contact (University College London), the International ImMunoGeneTics database (IMGT) (World Wide Web imgt.cines.fr/), and North CDR definitions based on affinity propagation clustering utilizing a large number of crystal structures. It will be understood by those skilled in the art that, unless otherwise specified, the terms "CDR" and "complementarity determining region" of a given antibody or region thereof (e.g., variable region) are to be understood as encompassing the above-mentioned existing ones as described by the present invention. Complementarity-determining regions defined in any one of the known schemes.

As used herein, the term "antigen-binding fragment" of an antibody refers to a polypeptide comprising a fragment of a full-length antibody that retains the ability to specifically bind to the same antigen to which the full-length antibody binds, and/or competes with the full-length antibody for Specific binding to an antigen, which is also referred to as an "antigen-binding moiety". See generally Fundamental Immunology, Ch.7 (Paul, W., ed., 2nd ed., Raven Press, NY (1989), which is incorporated herein by reference in its entirety for all purposes. Recombinant DNA techniques or Antigen-binding fragments of antibodies are produced by enzymatic or chemical cleavage of intact antibodies. In some cases, antigen-binding fragments include Fab, Fab', F(ab') ₂ , Fd, Fv, dAb, and complementarity determining region (CDR) fragments , single chain antibodies (eg, scFv), single domain antibodies (eg, VHH), chimeric antibodies, diabodies (diabodies), and polypeptides comprising at least a portion of an antibody sufficient to confer specific antigen-binding ability to the polypeptide.

As used herein, the term "isolated" means obtained from the natural state by artificial means. If an "isolated" substance or component occurs in nature, it may be that its natural environment has been altered, the substance has been isolated from its natural environment, or both. For example, a certain unisolated polynucleotide or polypeptide naturally exists in a living animal, and the high-purity identical polynucleotide or polypeptide isolated from this natural state is called "" Detached". The term "isolated" does not exclude the admixture of artificial or synthetic substances, nor the presence of other impure substances which do not affect the activity of the substance.

As used herein, the term "host cell" refers to cells that can be used to introduce vectors, including but not limited to, prokaryotic cells such as Escherichia coli, fungal cells such as yeast cells, such as S2 Drosophila cells or Sf9 etc., or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK293 cells or human cells.

As used herein, the term "KD" refers to the dissociation equilibrium constant for a particular antibody-antigen interaction, which is used to describe the binding affinity between an antibody and an antigen. The smaller the equilibrium dissociation constant, the tighter the antibody-antigen binding, and the higher the affinity between the antibody and the antigen. ^Typically , the antibody binds with an equilibrium dissociation constant (KD) of less than about ^10-5 M, such as less than about ^10-6 M, ^10-7 M, ^10-8 M, ^10-9 M, or ^10-10 M or less Antigens, eg, as determined in a BIACORE instrument using surface plasmon resonance (SPR).

"Pharmaceutically acceptable carrier" or "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, isotonic and absorption delaying agents, and the like that are physiologically compatible. Pharmaceutical carriers suitable for use in the present invention can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable excipients include starch, dextrose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, dried skim milk, glycerin , propylene, glycol, water, ethanol, etc. For the use of excipients and their uses, see also "Handbook of Pharmaceutical Excipients", Fifth Edition, R.C. Rowe, P.J. Seskey and S.C. Owen, Pharmaceutical Press, London, Chicago. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions may take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations and the like. It can be prepared by mixing an antibody or antigen-binding fragment thereof of the invention having the desired purity with one or more optional pharmaceutical excipients (Remington's Pharmaceutical Sciences, 16th Ed., Osol, A. Ed. (1980)) The pharmaceutical preparation or composition according to the present invention is preferably in the form of a freeze-dried preparation or an aqueous solution. The pharmaceutical compositions or formulations of the invention may also contain more than one active ingredient as required for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. For example, it is desirable to also provide other anti-infective active ingredients, such as other antibodies, anti-infective active agents, small molecule drugs or immunomodulators and the like. The active ingredients are suitably present in combination in amounts effective for the intended use. Sustained release formulations can be prepared. Suitable examples of sustained release formulations include semipermeable matrices of solid hydrophobic polymers containing the antibodies or antigen-binding fragments thereof of the invention in the form of shaped articles, eg films or microcapsules.

Embodiment 1 raw material preparation

1.1 Antigen preparation

The DNA sequences of human recombinant protein PD-L1 (UniProtKB-Q9NZQ7) and human recombinant protein PD-1 (UniProtKB-Q15116) were synthesized by General Biosystems (Anhui) Co., Ltd. for the target fragment gene. Each fragment of interest was amplified by PCR, and a His tag was introduced at the C-terminus by primers, and then constructed into the eukaryotic expression vector pcDNA3.4 (Invitrogen) by homologous recombination. The constructed recombinant protein expression vectors were respectively transformed into Escherichia coli DH5α, cultured overnight at 37°C, and then plasmids were extracted using an endotoxin-free plasmid extraction kit (OMEGA, D6950-01) to obtain endotoxin-free plasmids and For eukaryotic expression.

Both human recombinant protein PD-L1-His and human recombinant protein PD-1-His were expressed through the Expi293 transient expression system (ThermoFisher, A14635). For the transient method, see Expi293 ^TM Expression System USER GUIDE. After 5-7 days of transfection, the cell expression supernatant was centrifuged at 15,000 g for 10 min at high speed, and the obtained His-tagged protein expression supernatant was affinity-purified with Ni Smart Beads 6FF (Changzhou Tiandi Renhe Biotechnology Co., Ltd., SA036050), and then Gradient concentrations of imidazole elute the target protein. The eluted proteins were replaced into PBS buffer through ultrafiltration concentrator tubes (Millipore, UFC901096), and frozen at -80°C after being identified by SDS-PAGE and qualified for activity identification.

1.2 Preparation of control antibody

In this example, the anti-human CLDN18.2 antibody NA3SH1-T4-hVH6 (VHH ^CLDN18.2 -huFc, amino acid sequence shown in SEQ ID NO: 1) is a self-developed anti-human CLDN18.2 antibody, anti-human PD- L1 antibody D21-4 (VHH ^{PD- L1} -huFc, amino acid sequence shown in SEQ ID NO: 2) is derived from patent application PCT/CN2020/125301.

NA3SH1-T4-hVH6 and D21-4 are antibodies obtained through alpaca immunization, phage display library construction, screening, humanization and functional identification. The control antibodies D21-4 and NA3SH1-T4-hVH6 were expressed using a transient system (ExpiCHO). For the transient method, see ExpiCHO ^™ Expression System Kit User Guide. The expressed cell suspension was subjected to high-speed centrifugation and the supernatant was taken, and the obtained supernatant was filtered through a 0.22 μm filter membrane, and then purified by using a Protein A/G affinity chromatography column affinity method. After purification, the target protein was eluted with 100 mM glycine salt (pH 3.0), then concentrated and replaced, and then frozen at -80°C after being identified by SDS-PAGE and qualified for activity identification.

Amino acid sequence of NA3SH1-T4-hVH6 (SEQ ID NO: 1):

Amino acid sequence (SEQ ID NO:2) of D21-4:

1.3 Cell line construction

In this example, the HEK293 cell line overexpressing human CLDN18.2 (huCLDN18.2-HEK293 cell line), the HEK293 cell line overexpressing human CLDN18.1 (huCLDN18.1-HEK293 cell line) and the overexpression CHO-S cell line of human PD-L1 (huPD-L1-CHO-S cell line). The cell line construction method is as follows:

1.3.1 Construction of huCLDN18.2-HEK293 cell line and huCLDN18.1-HEK293 cell line

DNA fragments of human CLDN18.2 (UniProtKB-P56856-2) and human CLDN18.1 (UniProtKB-P56856-1) were synthesized by gene synthesis technology, and cloned into the expression vector pLVX-puro (Clontech, Cat#632164) . Introduce the E. coli into E. coli through the transformation method, pick out the E. coli single clone and sequence to obtain the correct plasmid clone, carry out the plasmid extraction and re-sequencing confirmation.

Recovery and cultivation of HEK293 (

CRL-1573 ^TM ) cells were continuously passaged for 2-3 times. The day before transfection, the cells were inoculated into the cell culture dish at a density of 3×10 ⁵ cells/mL, and the next day the cells reached about 70% confluence. use. Digest the cells with Trypsin (Gibco, 25200-072) containing 0.25% EDTA by volume for 2 minutes, collect the cells, centrifuge the cells at 100 g for 5 minutes at room temperature, discard the supernatant, add 1×DPBS (Yuancui, B210) to resuspend the cells and For counting, 5×10 ⁶ cells were centrifuged and collected, and the cells were resuspended with 250 μL Buffer R (Invitrogen, Neon ^TM Kit, PK10096) buffer, and 25 μg of the target plasmid was added to it, and mixed gently with a pipette. Afterwards, the suspension was placed in an electroporation apparatus (Invitrogen, Neon ^TM Transfection System, MP922947) for electrotransformation, and the reaction conditions were set at 1100V/20ms/2 times for electroporation. After electroporation, the resulting cells were transferred to DMEM medium (Gibco, 11995065) containing 10% by volume of FBS (Gibco, 15140-141) without antibiotics, and then the cells were seeded into 10cm×10cm cells Culture in the culture dish for 48 h, then divide the cells into 96-well cell culture plates at an average density of 0.5 cells/well, add puromycin (Gibco, A111138-03) at a final concentration of 2 μg/mL as a screening pressure, 2 Observe the growth of the cell line clones in about a week, pick the single cell clones grown in the 96-well plate, transfer to the 24-well culture plate to continue to expand the culture, and then successfully obtained huCLDN18.2-HEK293 and huCLDN18.1 after identification by FACS -A stable transfected cell line of HEK293.

1.3.2 Construction of huPD-L1-CHO-S cell line

A DNA fragment of human PD-L1 (UniProtKB-Q9NZQ7) was synthesized by gene synthesis technology and cloned into an expression vector. Introduce Escherichia coli through the method of transformation, pick a single clone of Escherichia coli and sequence to obtain the correct plasmid clone, carry out plasmid extraction and re-sequence confirmation.

CHO-S (Thermo, A1461801) cells were cultured using Gibco's CD-CHO serum-free medium (Catalog No.: 10743029). The day before electroporation, the cells were subcultured to 5×10 ⁶ cells/mL, and the next day, the constructed plasmid was introduced into CHO-S cells using Invitrogen’s electroporation kit (Catalog No.: MPK10096) and electroporation instrument (Cat. No.: MP922947). The cells after electroporation were transferred to CD-CHO medium and placed in a cell culture incubator at 37°C for 48 hours. Spread 2,000 cells/well of electroporated CHO-S cells into a 96-well plate, add MSX (Millipore, GSS-1015-F) and GS supplement (Sigma, 58672C-100ml) at a final concentration of 30 μM, and place in 37°C carbon dioxide They were cultured in an incubator, and after 10 days, supplemented with a medium containing 30 μM MSX and 1×GS supplement. Single-cell clones grown in 96-well plates were picked and transferred to 24-well culture plates for further expansion. After identification by FACS, a stable transgenic cell line of huPD-L1-CHO-S was successfully obtained.

Example 2 Construction of anti-CLDN18.2&PD-L1 bispecific antibody

This example describes the structure of an exemplary anti-CLDN18.2 & PD-L1 bispecific antibody (BsAb) and the construction of an expression vector. Nine constructs were designed and constructed: the amino acid sequence of VHH ^PD-L1 is from D21-4, and its variable region amino acid sequence is shown in SEQ ID NO:3; the amino acid sequence of VHH ^CLDN18.2 is from NA3SH1-T4-hVH6, which can The amino acid sequence of the variable region is shown in SEQ ID NO: 4; the amino acid sequence of the linker is GGGGSGGGGSGGGGS (SEQ ID NO: 5). Exemplary BsAb constructs are shown in Table 1 and the corresponding amino acid sequences are provided in Table 2.

Construct BsAb1: Contains two identical polypeptide chains from N-terminus to C-terminus comprising VHH domain of anti-PD-L1 Nanobody, IgG1 heavy chain hinge region, IgG1 heavy chain Fc, linker and anti-CLDN18.2 VHH domains of Nanobodies. BsAb1 has the format of Figure 1A.

Construct BsAb8: Contains two identical first polypeptide chains, comprising VHH domain of anti-CLDN18.2 Nanobody from N-terminus to C-terminus, IgG1 heavy chain constant region domain (including IgG1 heavy chain CH1, IgG1 heavy chain Hinge region, IgG1 heavy chain Fc); contains two identical second polypeptide chains, from N-terminus to C-terminus, including the VHH domain of anti-PD-L1 Nanobody and the CL domain of antibody kappa light chain. BsAb8 has the format of Figure 1B.

Construct BsAb10: Contains two identical first polypeptide chains, comprising from N-terminus to C-terminus the VHH domain of anti-CLDN18.2 Nanobody, linker, IgG1 heavy chain constant region domain (including IgG1 heavy chain CH1, IgG1 heavy chain hinge region, IgG1 heavy chain Fc); Contains two identical second polypeptide chains, from N-terminus to C-terminus containing VHH domain of anti-PD-L1 Nanobody, linker and CL structure of antibody kappa light chain area. BsAb10 has the format of Figure 1C.

Construct BsAb17: Contains two identical polypeptide chains from N-terminus to C-terminus comprising VHH domain of anti-CLDN18.2 Nanobody, linker, VHH domain of anti-PD-L1 Nanobody, IgG1 heavy chain hinge region and IgG1 heavy chain Fc. BsAb17 has the format of Figure 1D.

Construct BsAb18: Contains two identical polypeptide chains from N-terminus to C-terminus comprising VHH domain of anti-PD-L1 Nanobody, linker, VHH domain of anti-CLDN18.2 Nanobody, IgG1 heavy chain hinge region and IgG1 heavy chain Fc. BsAbl8 has the format of Figure 1D.

Construct BsAb19: Contains two different polypeptide chains, the first polypeptide chain contains the VHH domain of anti-PD-L1 Nanobody, IgG1 heavy chain hinge region and IgG1 heavy chain Fc (knob) from N-terminal to C-terminal, the second The polypeptide chain comprises the VHH domain of the anti-CLDN18.2 Nanobody, the IgG1 heavy chain hinge region and the IgG1 heavy chain Fc (hole) from the N-terminus to the C-terminus. BsAb19 has the format of Figure 1E.

Construct BsAb20: Contains two different polypeptide chains, the first polypeptide chain contains the VHH domain of anti-PD-L1 Nanobody, IgG1 heavy chain hinge region and IgG1 heavy chain Fc (knob) from N-terminal to C-terminal, the second The polypeptide chain comprises the VHH domain of the anti-CLDN18.2 Nanobody, the linker, the VHH domain of the anti-CLDN18.2 Nanobody, the IgG1 heavy chain hinge region and the IgG1 heavy chain Fc (hole) from the N-terminal to the C-terminal. BsAb20 has the format of Figure IF.

Construct BsAb21: Contains two different polypeptide chains, the first polypeptide chain contains VHH domain of anti-PD-L1 nanobody from N-terminus to C-terminus, linker, VHH domain of anti-PD-L1 nanobody, IgG heavy chain hinge region and IgG1 heavy chain Fc (knob), the second polypeptide chain from N-terminal to C-terminal contains VHH domain of anti-CLDN18.2 Nanobody, IgG1 heavy chain hinge region and IgG1 heavy chain Fc (hole). BsAb21 has the format of Figure IF.

Construct BsAb22: Contains two different polypeptide chains, the first polypeptide chain contains VHH domain of anti-PD-L1 Nanobody from N-terminus to C-terminus, linker, VHH domain of anti-PD-L1 Nanobody, IgG1 heavy chain hinge region and IgG1 heavy chain Fc (knob), the second polypeptide contains VHH domain of anti-CLDN18.2 Nanobody, linker, VHH domain of CLDN18.2 Nanobody, IgG1 heavy chain from N-terminus to C-terminus Hinge region and IgG1 heavy chain Fc (hole). BsAb22 has the format of Figure 1G.

According to the structure of the construct, fragments of the variable region and constant region of each antibody are amplified by PCR method, and the fragments are connected by overlap extension PCR method, and then constructed into modified eukaryotic expression vectors by homologous recombination method On the plasmid pcDNA3.4 (Invitrogen), the full-length gene of the polypeptide chain constituting the complete construct. The constructed vectors containing the full-length gene of the polypeptide chain of the construct were respectively transformed into Escherichia coli DH5α, and cultured overnight at 37°C. The endotoxin-free plasmid extraction kit (OMEGA, D6950-01) was used for plasmid extraction to obtain an endotoxin-free construct polypeptide chain plasmid for eukaryotic expression.

Table 1 Constructs of anti-CLDN18.2&PD-L1 bispecific antibodies

Wherein, Hinge is the hinge region.

The amino acid sequence of table 2 construct

VHH ^PD-L1 amino acid sequence (SEQ ID NO: 3)

VHH ^CLDN18.2 amino acid sequence (SEQ ID NO: 4)

IgG1 heavy chain constant region amino acid sequence (SEQ ID NO: 6)

Amino acid sequence of κ light chain constant region (SEQ ID NO: 7)

Amino acid sequence of IgG1 Fc region (SEQ ID NO: 8)

IgG1 hinge region amino acid sequence (SEQ ID NO: 9)

IgG1 CH1 amino acid sequence (SEQ ID NO: 10)

IgG1 CH2 amino acid sequence (SEQ ID NO: 11)

IgG1 CH3 amino acid sequence (SEQ ID NO: 12)

Example 3 Expression, purification, and analysis of physicochemical properties of anti-CLDN18.2&PD-L1 bispecific antibody

3.1 Expression and purification of anti-CLDN18.2&PD-L1 bispecific antibody

The construct in Example 2 was expressed through the ExpiCHO transient expression system (Thermo Fisher, A29133), and the specific method was as follows: on the day of transfection, confirm that the cell density was about 7×10 ⁶ to 1×10 ⁷ viable cells/mL, The cell survival rate is >98%. At this point, adjust the cells to a final concentration of 6×10 ⁶ cells/mL with fresh ExpiCHO expression medium pre-warmed at 37°C. Dilute the target plasmid with 4°C pre-cooled OptiPRO ^TM SFM (add 1 μg of plasmid to 1 mL of the medium), and at the same time, dilute ExpiFectamine ^TM CHO with OptiPRO ^TM SFM, then mix the two in equal volumes and gently blow and mix to prepare Form ExpiFectamine ^TM CHO/plasmid DNA mixture, incubate at room temperature for 1-5min, slowly add to the prepared cell suspension and shake gently at the same time, and finally place in a cell culture shaker at 37°C, 8% CO ₂ conditions under cultivation. 18-22 hours after transfection, add ExpiCHO ^™ Enhancer and ExpiCHO ^™ Feed to the culture medium, and place the shaker flask in a shaker at 32°C and 5% CO ₂ to continue culturing. On day 5 after transfection, the same volume of ExpiCHO ^TM Feed was added, and the cell suspension was mixed gently while adding slowly. After 7-15 days of transfection, the cell culture supernatant expressing the target protein was centrifuged at 15,000 g for 10 min at high speed, and the resulting supernatant was affinity purified with MabSelect SuRe LX (GE, 17547403), and then purified with 100 mM sodium acetate (pH 3.0 ) to elute the target protein, then neutralize it with 1M Tris-HCl, and finally replace the obtained protein into PBS buffer through an ultrafiltration concentrator tube (Millipore, UFC901096).

3.2 Determination of the concentration of anti-CLDN18.2&PD-L1 bispecific antibody

The concentration of the purified bispecific antibody in Example 3.1 was measured with a verified ultra-micro spectrophotometer (Hangzhou Aosheng Instrument Co., Ltd., Nano-300), and the measured A280 value was divided by the theoretical extinction coefficient of the antibody. The value is used as the antibody concentration value for follow-up research. After passing the quality inspection, it is aliquoted and stored at -80°C.

3.3 SDS-PAGE identification of anti-CLDN18.2&PD-L1 bispecific antibody

Non-reducing solution preparation: candidate bispecific antibody, control antibody D21-4 and NA3SH1-T4-hVH6, and reference product IPI (the IPI is the abbreviation of Ipilimumab, prepared by the method in Example 3.1 ) 1 μg was added with 5×SDS loading buffer and 40 mM iodoacetamide, heated in a dry bath at 75° C. for 10 min, cooled to room temperature, centrifuged at 12,000 rpm for 5 min, and the supernatant was taken. Preparation of reducing solution: add 2 μg of candidate bispecific antibody, control antibody D21-4 and NA3SH1-T4-hVH6, and reference product IPI to 5×SDS loading buffer and 5mM DTT, heat in a dry bath at 100°C for 10 minutes, and cool to room temperature. Centrifuge at 12000rpm for 5min, and take the supernatant. Add the supernatant to Bis-tris 4-15% gradient gel (purchased from GenScript), electrophoresis at a constant voltage of 110V, stop running when the Coomassie brilliant blue migrates to the bottom of the gel, take out the gel slice and place it in the Coomassie brilliant blue staining solution Incubate for 1-2 hours, discard the staining solution, add destaining solution, replace the destaining solution 2-3 times as needed, decolorize until the gel background is transparent, and store in deionized water. After decolorization, it was scanned with EPSON V550 color scanner, and the purity of reduced and non-reduced bands was calculated by ImageJ according to the peak area normalization method.

The results are shown in Figures 2A-2B: the bands of the candidate bispecific antibody and the reference product IPI reduced gel and non-reduced gel all conformed to the expected size, and the purity was about 90%.

3.4 SEC-HPLC monomer purity identification of anti-CLDN18.2&PD-L1 bispecific antibody

Material preparation: 1. Mobile phase: 150 mmol/L phosphate buffer, pH 7.4; 2. Sample preparation: Candidate bispecific antibodies were diluted to 0.5 mg/mL with mobile phase solution. Agilent HPLC 1100 chromatographic column (XBridge BEH SEC 3.5μm, 7.8mm I.D.×30cm, Waters) flow rate was set to 0.8mL/min, injection volume was 20μL, VWD detector wavelength was 280nm and 214nm.

The SEC-HPLC results of candidate bispecific antibodies are as follows: the percentages of high molecular polymers, antibody monomers and low molecular substances in the sample were calculated according to the area normalization method, and the results are shown in Figures 3A-3I and Table 3.

3.5 Study on thermal stability of anti-CLDN18.2&PD-L1 bispecific antibody

Differential scanning fluorometry (DSF) can provide information about protein structure stability based on the fluorescence change process in protein maps, detect protein configuration changes, and obtain protein melting temperature (Tm). In this example, the Tm value of the trifunctional antibody was detected by the DSF method.

The candidate bispecific antibody solution was prepared at 0.2 mg/mL, and each test product was added to a 96-well plate (Nunc) at 19 μL/well, three parallel wells were set, and PBS and IPI (Ipilimlumab) were used as references, and then Add 1 μL of SYPRO orange dye at a concentration of 100× to each well, mix well by blowing with a pipette gun, and prepare for use on the machine. The sample thermal stability test adopts ABI 7500FAST RT-PCR instrument, the test type selects melting curve, adopts continuous mode, scans the temperature range from 25 to 95 ° C, the heating rate is 1%, 25 ° C for 5 minutes, collects data during the heating process, and reports Select "ROX" for the group, select "None" for the quenching group, and use a reaction volume of 20 μL. The temperature corresponding to the first peak and valley of the first derivative of the melting curve is determined as the melting temperature Tm of the antibody.

The experimental results are shown in Table 3, and the results show that the Tm of the candidate bispecific antibodies in this batch of samples are all greater than 60°C, so they have good thermal stability.

Table 3 Preparation and physicochemical data of anti-CLDN18.2&PD-L1 bispecific antibody

Example 4 Affinity Activity Analysis of Anti-CLDN18.2&PD-L1 Bispecific Antibody

4.1 ELISA method to detect the binding ability of candidate bispecific antibodies to human recombinant protein PD-L1-His

Coat human recombinant protein PD-L1-His on a 96-well ELISA plate overnight at 4°C. The next day, the well plate was washed 3 times with PBST and blocked with 5% skimmed milk for 2 h. After the plate was washed 3 times with PBST, different concentrations of candidate bispecific antibody and control antibody D21-4 were added and incubated for 1 h. Afterwards, after washing with PBST for 3 times, the secondary antibody Anti-human-IgG-Fc-HRP (abcam, ab79225) was added and incubated for 1 h. After incubation, the plate was washed six times with PBST, and TMB (SurModics, TMBS-1000-01) was added to develop the color. According to the color development results, 2M HCl was added to terminate the reaction, and the plate was read at OD450 by a microplate reader (Molecular Devices, SpecterMax 190). Data were analyzed using PRISM ^™ (GraphPad Software, San Diego, CA), and _EC50 values were calculated.

The results of the ELISA binding assay are shown in Figures 4A-4E and Table 4, the candidate bispecific antibody exhibited comparable binding ability to PD-L1 as the control antibody D21-4.

4.2 FACS-based detection of the binding ability of candidate bispecific antibodies to huPD-L1-CHO-S

Collect the huPD-L1-CHO-S cells in the exponential growth phase, centrifuge at 300g to remove the supernatant, resuspend the cells in FACS buffer (PBS containing 1% BSA), count and adjust the density of the cell suspension to 2×10 ⁶ /mL. Subsequently, huPD-L1-CHO-S cells were added to a 96-well round bottom plate at 100 μL per well, and centrifuged at 300 g to remove the supernatant. Add different concentrations of candidate bispecific antibody, control antibody D21-4 dilution and human IgG1 isotype antibody (as isotype control) to the corresponding wells, resuspend the cells and place them at 4°C for 30 min. After the incubated cell mixture was washed 3 times, a PE-labeled anti-human-IgG-Fc flow antibody (Abcam, 98596) was added, resuspended and incubated at 4°C for 30 min. After the incubated cell mixture was washed three times, 200 μL of FACS buffer was added to resuspend the cells, and the cells were detected and analyzed by flow cytometry (Beckman, CytoFLEX AOO-1-1102). Data were analyzed using PRISM ^™ (GraphPad Software, San Diego, CA), and _EC50 values were calculated.

The results of FACS binding assays are shown in Figures 5A-5B and Table 4, the candidate bispecific antibody exhibited comparable binding ability to PD-L1 as the control antibody D21-4.

4.3 FACS-based detection of the binding ability of candidate antibodies to huCLDN18.2-HEK293 cells

Collect the huCLDN18.2-HEK293 cells in the exponential growth phase, centrifuge at 300g to remove the supernatant, resuspend the cells in FACS buffer (PBS containing 1% BSA), count and adjust the density of the cell suspension to 2×10 ⁶ /mL . Subsequently, huCLDN18.2-HEK293 cells were added to a 96-well round bottom plate at 100 μL per well, and centrifuged at 300 g to remove the supernatant. Different concentrations of candidate bispecific antibodies, control antibody NA3SH1-T4-hVH6 and human IgG1 isotype antibody (as isotype control) were added to the corresponding wells, and the cells were resuspended and incubated at 4°C for 30 min. After the incubated cell mixture was washed 3 times, a PE-labeled anti-human-IgG-Fc flow antibody (Abcam, 98596) was added, resuspended and incubated at 4°C for 30 min. After the incubated cell mixture was washed three times, 200 μL of FACS buffer was added to resuspend the cells, and finally analyzed by flow cytometry (Beckman, CytoFLEX AOO-1-1102). Data were analyzed using PRISM™ (GraphPad Software, San Diego, CA), and _EC50 values were calculated.

FACS binding assay results are shown in Figures 6A-6B and Table 4, some candidate bispecific antibodies BsAb1, BsAb8, BsAb10, BsAb17 and BsAb18 showed comparable binding ability to CLDN18.2 as the control antibody NA3SH1-T4-hVH6.

Table 4 Antigen binding data of anti-CLDN18.2&PD-L1 bispecific antibody

4.4 FACS-based detection of the affinity of candidate antibodies to huCLDN18.1-HEK293 cells

Collect the huCLDN18.1-HEK293 cells in the exponential growth phase, centrifuge at 300g to remove the supernatant, resuspend the cells with the prepared FACS buffer (PBS containing 1% BSA), count and adjust the density of the cell suspension to 2×10 ⁶ /mL. Subsequently, huCLDN18.1-HEK293 cells were added to a 96-well round bottom plate at 100 μL per well, and centrifuged at 300 g to remove the supernatant. Different concentrations of candidate bispecific antibodies and human IgG1 isotype antibodies (as isotype controls) were added to corresponding wells, resuspended and placed at 4°C for 30 min incubation. After the incubated cell mixture was washed 3 times, a PE-labeled anti-human-IgG-Fc flow antibody (Abcam, 98596) was added, resuspended and incubated at 4°C for 30 min. After the incubated cell mixture was washed three times, 200 μL of FACS buffer was added to resuspend the cells, and finally analyzed by flow cytometry (Beckman, CytoFLEX AOO-1-1102).

The results are shown in Figures 7A-7B, none of the candidate bispecific antibodies binds to huCLDN18.1-HEK293, indicating that the candidate bispecific antibodies specifically bind to CLDN18.2 but not to CLDN18.1.

4.5 Detection of the affinity of candidate bispecific antibodies to PD-L1 based on Fortebio

In this example, the affinity of the candidate bispecific antibody to human recombinant protein PD-L1-His was tested using Fortebio BLItz instrument.

Weigh 10g of BSA, measure 5mL of Tween 20, add to 1000mL of 10×PBS, mix well to make 10×KB buffer, filter and store in separate packages. Take 0.1mL of 0.1M glycine solution with pH=2.0 and add it to 0.9mL of ultrapure water, mix well to make sensor regeneration buffer. Human recombinant protein PD-L1-His as an antigen was diluted to 10 μg/mL with 10×KB buffer, and the candidate bispecific antibody and control antibody D21-4 were serially diluted 2 times with 10×KB buffer, in order of 100, 50, 25, 0nM. Under dark conditions, use 10×KB buffer to pre-wet the sensor (Anti-Penta-HIS, HIS1K, Fortebio, CA), start testing the sample plate (GreinierBio, PN655209) after at least 10 minutes, and follow the preset procedure after the test is correct. First, the antigen human recombinant protein PD-L1-His was used for binding for 300s. After the binding was completed, continue to equilibrate in 10×KB buffer for 30s. Then, the sensor bound to the antigen was transferred to different concentrations of antibody diluents for binding for 300s. After the signal was stable , and then transferred to 10×KB buffer, the dissociation time was 900s, and finally KD, Kon and Koff were obtained by fitting the binding and dissociation data of different concentrations of antibodies. The results are shown in Table 5, and the candidate bispecific antibodies all showed The affinity for PD-L1 was comparable to that of the control antibody D21-4.

Table 5 BLI determination of the affinity of anti-CLDN18.2&PD-L1 bispecific antibodies to PD-L1

Example 5 Analysis of blocking activity of anti-CLDN18.2&PD-L1 bispecific antibody

In this example, the FACS method is used to detect the blocking activity of candidate bispecific antibodies against PD-1/PD-L1. The specific method is as follows: collect the cultured huPD-L1-CHO-S cells, centrifuge at 300g to remove the supernatant, and separate the cells Resuspend with the prepared FACS buffer, count and adjust the cell suspension density to 2×10 ⁶ /mL. Add 100 μL of huPD-L1-CHO-S cells to a 96-well round-bottom plate per well, centrifuge at 300 g to remove the supernatant, and then add different concentrations of candidate bispecific antibody, control antibody D21-4, and human IgG1 isotype antibody ( Isotype control), resuspend the cells and incubate at 4°C for 30 minutes. After the incubated cell mixture was washed 3 times, 100 μL of biotin-labeled PD-1-His protein dilution (1 μg/mL) was added, and incubated at 4° C. for 30 minutes. After washing 3 times, add PE-labeled streptavidin (eBioscience, 12-4317-87), incubate at 4°C for 30 minutes, wash the incubated cell mixture 3 times, add 200 μL of FACS buffer to resuspend the cells, and finally pass the flow cytometry Cytometer (Beckman, CytoFLEX AOO-1-1102) was tested on the machine. Data were analyzed using PRISM ^™ (GraphPad Software, San Diego, CA), and _IC50 values were calculated.

The results of FACS binding assays are shown in Figures 8A-8B and Table 6. Some candidate bispecific antibodies BsAb1, BsAb8, BsAb10, BsAb17, and BsAb18 exhibited comparable resistance to PD-1/PD-L1 as the control antibody D21-4. break activity.

Table 6 Blocking activity of anti-CLDN18.2&PD-L1 bispecific antibodies

Antibody name	IC 50 (nM)
BsAb1	6.136
BsAb8	6.414
BsAb10	6.760
BsAb17	7.430
BsAb18	5.937
BsAb19	12.16
BsAb20	10.85
BsAb21	10.87
BsAb22	8.388

D21-4

5.399

Example 6 ADCC activity analysis of anti-CLDN18.2&PD-L1 bispecific antibody

After binding the Fc end of the bispecific antibody of the present invention to CD16a (V158) and the VHH end to target cells, the expression of NF-AT protein in Jurkat cells will be activated, and the combination of NF-AT and its response elements will trigger its downstream fluorescence If the enzyme is expressed and stimulated with antibodies of different concentration gradients, a fluorescence reading curve with antibody concentration dependence will be obtained, so that the ADCC activity of the antibody can be evaluated.

In a 96-well cell culture plate, add a total volume of 1× ¹⁰⁴ cells/well of huCLDN18.2-PANC-1 cells (PANC stably transfected with full-length human CLDN18.2 (UniProtKB-P56856-2)) in a total volume of 50 μL per well. -1 cell (

CRL-1469 ^TM )) and 1×10 ⁵ /well Jurkat-NFAT-Luciferase-CD16a (V158) cells (stable transfection CD16a (V158) sequence (UniProtKB-P08637) and containing NF-AT-re nucleic acid sequence pGL4.30 plasmid (promega, #E8481) to Jurkat cells (

TIB-152) obtained stable transfected cell line), adding 50 μL of serially diluted candidate bispecific antibody and control bispecific antibody QP3711461 (prepared according to WO2020259566A1, the amino acid sequences of heavy chain and light chain are as shown in SEQ ID NO: 24 and shown in SEQ ID NO:25), incubated in a 37°C incubator for 6h. Add 50 μL Bright-Lite (vazyme, product number: DD1204-03) to each well, incubate in the dark for 10 min, and detect the fluorescent signal.

The ADCC detection results are as follows: As shown in Figure 9A-9B, both bispecific antibodies BsAb8 and BsAb17 exhibited ADCC activity, and BsAb8 was superior to the control bispecific antibody QP3711461.

QP3711461 heavy chain amino acid sequence (SEQ ID NO: 24)

QP3711461 light chain amino acid sequence (SEQ ID NO: 25)

Example 7 Analysis of blocking activity of anti-CLDN18.2&PD-L1 bispecific antibody on PD-1/PD-L1

This example established the Jurkat-PD-1-NFAT cell line (stable expression of PD-1 and luciferase) as effector cells and CHO-PD-L1-CD3L (stable expression of PD-L1 and Anti-CD3-scFv), The combination of PD-1 and PD-L1 can block the downstream signal transduction of CD3 to inhibit the expression of luciferase. When the anti-PD-L1 antibody is added, the blocking effect is reversed, and the luciferase is expressed. Antibodies with different concentration gradients are used After stimulation, a fluorescence readout curve with the concentration dependence of the antibody will be obtained, so that the blocking activity of the antibody can be evaluated.

Add 2× ¹⁰⁴ CHO-PD-L1-CD3L cells (stable expression of PD-L1 (UniProtKB-Q9NZQ7) and anti- CD3-scFv) and 1×10 ⁵ /well Jurkat-PD-1-NFAT cells (Jurkat cells (

TIB-152) stably expresses PD-1 (UniProtKB-Q15116) and luciferase). Add 50 μL of serially diluted candidate bispecific antibody and control bispecific antibody QP3711461, and incubate in a 37°C incubator for 6 hours. Add 50 μL Bright-Lite (vazyme, product number: DD1204-03) to each well, incubate in the dark for 10 min, and detect the fluorescent signal.

The blocking activity test results are as follows: As shown in Figure 10, both bispecific antibodies BsAb8 and BsAb17 exhibited PD-L1 blocking activity, and BsAb17 was superior to the control bispecific antibody QP3711461.

Example 8 Anti-CLDN18.2 & PD-L1 bispecific antibody CLDN18.2-NUGC4 in vivo tumor inhibition experiment

Through the humanized model of the immune system, the in vivo tumor synergistic inhibition experiment of CLDN18.2 and PD-L1 was simulated. 6-8 weeks old female NCG mice (purchased from Jiangsu Jicui Yaokang, strain: NOD/ShiLtJGpt-Prkdcem26Cd52Il2rgem26Cd22/Gpt) were used, and the experimental mice were kept in an independent ventilation box with constant temperature and humidity, and the temperature of the feeding room was 21-24°C. Humidity 30-53%. 2×10 ⁶ CLDN18.2-NUGC4 cells (identified by FACS to overexpress human CLDN18.2) were subcutaneously injected into the right back of each mouse (day 0), and then randomized into groups (8 mice per group) Rats): respectively PBS treatment group, PBS group+PBMC group, BsAb8 administration group, BsAb17 administration group, QP3711461 administration group, each administration group was set up as a high-dose group. On the second day after bearing the tumor, inject PBMC (C2106025) through the tail vein, inject 5×10 ⁶ PBMC cells per mouse, and give the first administration 2 hours later, twice a week, intraperitoneal injection (ip) medicine for a total of 3 weeks.

Observe and record the tumor length (mm) and width (mm) at any time, calculate its tumor volume (V), the calculation method is V=(length×width 2)/2, tumor inhibition rate TGI (%)=(1-administration The average tumor volume of the group/the average tumor volume of the PBS-treated group)×100%. The tumor suppression results are shown in Figure 11 and Table 7. It can be seen from the results that all drug groups exhibited tumor growth inhibition relative to the PBS treatment group; BsAb17 had the best tumor growth inhibition at a dose of 10mpk, better than BsAb8 and the control bispecific antibody QP3711461, and both showed partial tumor remission trend.

Table 7 The tumor inhibition rate TGI (%) of different administration groups of anti-CLDN18.2&PD-L1 bispecific antibody

Antibody name	dose (mpk)	TGI(%)
BsAb17	10	30.3
BsAb8	10	10.9
QP3711461	10	19.0

Example 9 Anti-CLDN18.2 & PD-L1 bispecific antibody CLDN18.2-MC38 in vivo tumor inhibition experiment

D21-4 has only weak mouse cross activity, so the MC38-CLDN18.2 model can only be used to evaluate the tumor killing effect of ADCC and CDC at the 18.2 end of BsAb8 and BsAb17 molecules. Use 6-8 weeks of female C57 mice (purchased from Weitong Lihua, strain: C57BL/6N, the experimental mice are raised in an independent ventilation box with constant temperature and humidity, the temperature of the feeding room is 21-24 °C, and the humidity is 30-53% .2×10 ⁶ CLDN18.2-MC38 cells (identified by FACS overexpressing human CLDN18.2)/each mouse were subcutaneously injected on the right back (day 0), the tumor grew to 100 mm ³ , and then randomized Grouping (each group, 6 mice): PBS treatment group, BsAb8 administration group, BsAb17 administration group, IMAB362 prepared according to patent US20180127489A1, the amino acid sequence of its heavy chain is shown in SEQ ID NO: 32, the light chain The amino acid sequence is as shown in SEQ ID NO: 33) administration group, and each administration group is provided with a middle dosage group. The drugs were administered immediately after grouping, and were administered twice a week by intraperitoneal injection (ip) for a total of 3 weeks.

Observe and record the tumor length (mm) and width (mm) at any time, calculate its tumor volume (V), the calculation method is V=(length×width 2)/2, tumor inhibition rate TGI (%)=(1-administration The average tumor volume of the group/the average tumor volume of the PBS-treated group)×100%. The tumor suppression results are shown in Figure 12 and Table 8. As can be seen from the results: all the administration groups showed tumor growth inhibition relative to the PBS treatment group; BsAb17 had the best tumor growth inhibition at a dose of 10mpk, with a TGI of 71%, which was significantly better than BsAb8 and IMAB362, both of which exhibited relatively Strong tumor response trend.

Table 8 The tumor inhibition rate TGI (%) of different administration groups of anti-CLDN18.2&PD-L1 bispecific antibody

Antibody name	dose (mpk)	TGI(%)
BsAb17	10	71.0
BsAb8	10	42.3
IMAB362	10	42.3

IMAB362 heavy chain amino acid sequence (SEQ ID NO: 26)

IMAB362 light chain amino acid sequence (SEQ ID NO: 27)

Although the specific implementations of the present invention have been described above, those skilled in the art should understand that these are only examples, and various changes or changes can be made to these implementations without departing from the principle and essence of the present invention. Revise. Accordingly, the protection scope of the present invention is defined by the appended claims.

Claims (18)

1. A bispecific antibody or an antigen-binding portion thereof, comprising a first antigen-binding domain targeting PD-L1 and a second antigen-binding domain targeting CLDN18.2, wherein:

   The first antigen-binding domain comprises: CDR1-3 in the sequence shown in SEQ ID NO:3;

   The second antigen-binding domain comprises: CDR1-3 in the sequence shown in SEQ ID NO:4;

   The CDRs are defined using the AbM numbering system.
2. The bispecific antibody or antigen-binding portion thereof according to claim 1, the first antigen-binding domain, and/or the second antigen-binding domain comprising VHH;

   The VHH of the first antigen-binding domain comprises: HCDR1 as shown in SEQ ID NO:28, HCDR2 as shown in SEQ ID NO:29 and HCDR3 as shown in SEQ ID NO:30;

   The VHH of the second antigen-binding domain comprises: HCDR1 as shown in SEQ ID NO:31, HCDR2 as shown in SEQ ID NO:32 and HCDR3 as shown in SEQ ID NO:33.
3. The bispecific antibody or antigen-binding portion thereof of claim 1 or 2, wherein:

   The first antigen-binding domain comprises at least one VHH ^{PD-L1 comprising} the amino acid sequence shown in SEQ ID NO: 3 or having at least 80% sequence identity therewith and retaining the affinity for PD-L1 Homologous sequences of specific binding affinity; and/or,

   The second antigen binding domain comprises at least one VHH ^CLDN18.2 comprising the amino acid sequence shown in SEQ ID NO: 4 or having at least 80% sequence identity thereto and retaining ^the alignment to CLDN18.2 Homologous sequences of specific binding affinity;

   Wherein, the number of the VHH ^CLDN18.2 and the VHH ^PD-L1 is preferably 1-2 respectively; when the antigen-binding domain contains two VHHs, the two VHHs are connected by a linker; the linker is preferably The peptide sequence more preferably comprises or consists of (G4S)n, wherein n=1-10, n is a natural number; optionally the linker comprises the sequence shown in SEQ ID NO:5.
4. The bispecific antibody or antigen-binding portion thereof according to any one of claims 1 to 3, wherein, from the N-terminus to the C-terminus, the first antigen-binding domain, the constant region and the second antigen-binding domain are in any order, can be are operatively interconnected; for example:

   The first antigen binding domain is operably linked to a constant region and the constant region is operably linked to a second antigen binding domain, or vice versa;

   Alternatively, the second antigen binding domain is operably linked to the first antigen binding domain, which is operably linked to the constant region, or vice versa;

   Alternatively, both the first antigen-binding domain and the second antigen-binding domain are operably linked to the N-terminus of the constant region.
5. The bispecific antibody or antigen-binding portion thereof according to claim 4, wherein the constant region is a human IgG constant region, such as a human kappa light chain constant region, a human IgG1 heavy chain constant region or a human IgG1 Fc region, wherein:

   The human IgG1 Fc region preferably has an amino acid sequence as shown in SEQ ID NO: 8;

   The human kappa light chain constant region preferably has an amino acid sequence as shown in SEQ ID NO:7;

   The human IgG1 heavy chain constant region preferably has the amino acid sequence shown in SEQ ID NO:6.
6. The bispecific antibody or its antigen-binding portion according to claim 5, which contains two first polypeptide chains, and the first polypeptide chains are represented by the following formula:

   VHH ^PD-L1 -hinge region-CH2-CH3-linker-VHH ^CLDN18.2 , preferably comprising the sequence shown in SEQ ID NO:13;

   Alternatively, VHH ^CLDN18.2 -linker-VHH ^PD-L1 -hinge region-CH2-CH3 preferably comprises the sequence shown in SEQ ID NO:18;

   Alternatively, VHH ^PD-L1 -linker-VHH ^CLDN18.2 -hinge region-CH2-CH3 preferably comprises the sequence shown in SEQ ID NO:19.
7. The bispecific antibody or its antigen-binding part according to claim 5, when the constant region is a complete human IgG constant region, the first antigen-binding domain and the second antigen-binding domain can be respectively Operably linked to the light chain constant region and the heavy chain constant region; preferably said operably linked via a linker; said linker preferably comprises a peptide sequence, more preferably comprises or consists of (G4S)n, where n=1 -10, n is a natural number; Optionally, the sequence shown in SEQ ID NO:5 is included.
8. The bispecific antibody or antigen-binding portion thereof according to claim 7, comprising two first polypeptide chains and two second polypeptide chains: said first polypeptide chain is of the formula VHH ^CLDN18.2 -CH1 -Hinge region-shown in CH2-CH3, and the second polypeptide chain is shown in the formula VHH ^PD-L1 -CL; or the first polypeptide chain is shown in the formula VHH ^CLDN18.2 -Linker-CH1-hinge region-CH2-CH3, and the second polypeptide chain is shown in the formula VHH ^PD-L1 -linker-CL; preferably: the first polypeptide chain comprises the formula shown in SEQ ID NO:14 amino acid sequence, and the second polypeptide chain comprises the amino acid sequence shown in SEQ ID NO:15; or the first polypeptide chain comprises the amino acid sequence shown in SEQ ID NO:16, and the second polypeptide chain comprises the amino acid sequence shown in SEQ ID NO:16, and the second polypeptide chain comprises the amino acid sequence shown in SEQ ID NO:15; The two polypeptide chains comprise the amino acid sequence shown in SEQ ID NO: 17;

   Wherein, the hinge region preferably contains the amino acid sequence shown in SEQ ID NO:9.
9. An isolated nucleic acid encoding the bispecific antibody or antigen-binding portion thereof according to any one of claims 1-8.
10. A recombinant expression vector comprising the isolated nucleic acid of claim 9.
11. A transformant comprising the isolated nucleic acid as claimed in claim 9 or the recombinant expression vector as claimed in claim 10;

    Preferably, the host cells of the transformant are prokaryotic cells or eukaryotic cells, the prokaryotic cells are preferably E. coli cells such as TG1, BL21, and the eukaryotic cells are preferably HEK293 cells or CHO cells.
12. A method for preparing a bispecific antibody, comprising culturing the transformant as claimed in claim 11, and obtaining the bispecific antibody from the culture.
13. A pharmaceutical composition, characterized in that the pharmaceutical composition comprises the bispecific antibody or antigen-binding portion thereof according to any one of claims 1-8, and a pharmaceutically acceptable carrier;

    Preferably, the pharmaceutical composition further includes other anti-tumor antibodies as active ingredients.
14. An antibody-drug conjugate comprising a cytotoxic agent, and the bispecific antibody or antigen-binding portion thereof according to any one of claims 1-8;

    Preferably, the cytotoxic agent is MMAF or MMAE.
15. A bispecific antibody or antigen-binding portion thereof according to any one of claims 1 to 8, a pharmaceutical composition according to claim 13, or an antibody-drug conjugate according to claim 14 in the preparation of a diagnostic , The application in the medicine of preventing and/or treating tumor.
16. A kit comprising the bispecific antibody or antigen-binding portion thereof according to any one of claims 1 to 8, the pharmaceutical composition according to claim 13, the antibody drug conjugate according to claim 14 Union;

    Preferably, the kit further includes (i) a device for administering the bispecific antibody or antibody drug conjugate or pharmaceutical composition; and/or (ii) instructions for use.
17. A kind of set medicine box, it comprises medicine box A and medicine box B, wherein:

    The kit A contains the bispecific antibody or its antigen-binding portion according to any one of claims 1-8, the pharmaceutical composition according to claim 13, the antibody-drug conjugate according to claim 14 thing;

    The kit B contains other anti-tumor antibodies or pharmaceutical compositions containing the other anti-tumor antibodies, and/or consists of hormone preparations, targeted small molecule preparations, proteasome inhibitors, imaging agents, diagnostic agents, chemotherapeutic agents, One or more of the group consisting of oncolytic drugs, cytotoxic agents, cytokines, activators of co-stimulatory molecules, inhibitors of inhibitory molecules, and vaccines.
18. A non-diagnostic method for detecting a specific antigen in vitro or in vivo, comprising using the bispecific antibody or antigen-binding portion thereof according to any one of claims 1-8 for detection.

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