WO2023193767A1

WO2023193767A1 - Antigen binding protein and application thereof

Info

Publication number: WO2023193767A1
Application number: PCT/CN2023/086611
Authority: WO
Inventors: 邓俗俊; 刘培培; 顾春银; 王宗达; 曹晓丹; 杨欣秀
Original assignee: 上海济煜医药科技有限公司
Priority date: 2022-04-08
Filing date: 2023-04-06
Publication date: 2023-10-12
Also published as: TW202405013A

Abstract

The present application relates to an antigen binding protein, and specifically relates to a PD-L1 binding protein and/or relates to a bispecific binding protein capable of binding to PD-L1 and PD-1. The present application further provides an application of the antigen binding protein in treatment of diseases or disorders.

Description

An antigen-binding protein and its application

Technical field

This application relates to the field of biomedicine, specifically to an antigen-binding protein and its application.

Background technique

Programmed death 1 (programmed death 1), referred to as PD-1, is widely expressed in immune cells and is an important immunosuppressive molecule. The main ligand of PD-1 is PD-L1, and PD-L1 is mainly expressed on the surface of tumor cells. After the ligand PD-L1 binds to the receptor PD-1, it inhibits T cell activation in the tumor microenvironment, causing the immune system such as T cells to be unable to kill tumor cells normally, thereby achieving immune escape.

A number of therapeutic antibodies targeting this signaling pathway have been developed, such as Pembrolizumab and Nivolumab. However, the response rate is generally low during the treatment process and drug resistance is easy to develop. sexual phenomena. Therefore, there is an urgent need for anti-tumor PD-L1 antibodies with stable structure, good efficacy, and suitable for large-scale industrial production, as well as multispecific antibodies containing PD-L1 binding domains.

Contents of the invention

The present application provides an antigen-binding protein, which may have one or more of the following properties: (1) capable of binding to primate-derived _PD- L1 protein; and (2) can stimulate immune cells to secrete cytokines, for example, stimulate lymphocytes to secrete IL-2.

In one aspect, the present application provides an antigen-binding protein comprising an antigen-binding fragment capable of binding PD-L1. For example, the antigen-binding protein includes an antibody heavy chain variable region VH, the VH includes HCDR1, HCDR2 and HCDR3, the HCDR3 includes the amino acid sequence shown in SEQ ID NO: 6, and the HCDR2 includes SEQ ID NO: 5 The amino acid sequence shown is, and the HCDR1 includes the amino acid sequence shown in SEQ ID NO: 1. For example, the antigen-binding protein includes an antibody light chain variable region VL, the VL includes LCDR1, LCDR2 and LCDR3, the LCDR3 includes the amino acid sequence shown in SEQ ID NO: 25, and the LCDR2 includes SEQ ID NO: 24 The amino acid sequence shown is, and the LCDR1 includes the amino acid sequence shown in SEQ ID NO: 23.

In another aspect, the present application provides a polypeptide, the antigen-binding protein comprising a first targeting moiety capable of binding to PD-L1 and a second targeting moiety capable of binding to PD-1. For example, the first targeting moiety includes the PD-L1 antigen-binding protein described in any one of the present applications. For example, the second targeting portion includes the antibody heavy chain variable region VH, the VH of the second targeting portion includes HCDR1, HCDR2 and HCDR3, and the HCDR3 of the second targeting portion includes SEQ ID NO: 14 The amino acid sequence shown in SEQ ID NO: 13, HCDR2 of the second targeting part includes the amino acid sequence shown in SEQ ID NO: 13 acid sequence, and the HCDR1 of the second targeting portion includes the amino acid sequence shown in SEQ ID NO: 12. For example, the second targeting portion includes an antibody light chain variable region VL, the VL of the second targeting portion includes LCDR1, LCDR2, and LCDR3, and the LCDR3 of the second targeting portion includes SEQ ID NO: 25. The amino acid sequence shown is, the LCDR2 of the second targeting portion includes the amino acid sequence shown in SEQ ID NO: 24, and the LCDR1 of the second targeting portion includes the amino acid sequence shown in SEQ ID NO: 23. For example, the polypeptide is a multispecific antibody with a common light chain, such as a bispecific antibody.

In another aspect, the present application provides a nucleic acid encoding the antigen-binding protein of the present application and/or the polypeptide of the present application.

In another aspect, the present application provides a vector comprising the nucleic acid of the present application.

In another aspect, the present application provides a cell comprising the antigen-binding protein of the present application, the polypeptide of the present application, and/or the nucleic acid of the present application.

On the other hand, the present application provides a method for preparing the antigen-binding protein of the present application and/or the polypeptide of the present application, which method includes culturing under conditions that allow the expression of the antigen-binding protein and/or the polypeptide. cells according to the present application.

In another aspect, the present application provides a conjugate comprising an antigen-binding protein of the present application and/or a polypeptide of the present application.

On the other hand, the present application provides a pharmaceutical composition comprising the antigen-binding protein of the present application, the polypeptide of the present application, the nucleic acid of the present application, the vector of the present application, the cell of the present application, and/or The conjugates of the present application, and optionally a pharmaceutically acceptable carrier.

On the other hand, the present application provides a kit, which includes the antigen-binding protein of the present application, the polypeptide of the present application, the nucleic acid of the present application, the vector of the present application, the cells of the present application, and the conjugation of the present application. substances, and/or pharmaceutical compositions of the present application.

On the other hand, the present application provides a method for influencing cells to produce cytokines, the method comprising administering the antigen-binding protein of the present application, the polypeptide of the present application, the nucleic acid of the present application, the vector of the present application, the cells of the present application, The conjugate of the present application, the pharmaceutical composition of the present application, and/or the kit of the present application.

On the other hand, the present application provides a method for promoting cell bridging, the method comprising administering the polypeptide of the present application, the nucleic acid of the present application, the vector of the present application, the cell of the present application, the conjugate of the present application, the The pharmaceutical composition, and/or the kit of the present application.

On the other hand, the present application provides the antigen-binding protein of the present application, the polypeptide of the present application, the nucleic acid of the present application, the vector of the present application, the cell of the present application, the conjugate of the present application, the pharmaceutical composition of the present application, and /or the kit of this application Use in the preparation of medicaments for preventing, alleviating and/or treating tumors.

On the other hand, the present application provides the antigen-binding protein of the present application, the polypeptide of the present application, the nucleic acid of the present application, the vector of the present application, the cell of the present application, the conjugate of the present application, the pharmaceutical composition of the present application, and / Or the kit of the present application, which is used to prevent, alleviate and / or treat tumors.

On the other hand, the present application provides a method for preventing, alleviating and/or treating tumors, comprising administering the antigen-binding protein of the present application, the polypeptide of the present application, the nucleic acid of the present application, the vector of the present application, the cells of the present application, The conjugate of the present application, the pharmaceutical composition of the present application, and/or the kit of the present application.

Those skilled in the art will readily appreciate other aspects and advantages of the present application from the detailed description below. Only exemplary embodiments of the present application are shown and described in the following detailed description. As those skilled in the art will realize, the contents of this application enable those skilled in the art to make changes to the specific embodiments disclosed without departing from the spirit and scope of the invention covered by this application. Accordingly, the drawings and descriptions of the present application are illustrative only and not restrictive.

Description of the drawings

The specific features of the invention to which this application relates are set forth in the appended claims. The features and advantages of the invention to which this application relates can be better understood by reference to the exemplary embodiments described in detail below and the accompanying drawings. A brief description of the drawings is as follows:

Figure 1 shows the activation results of the antibody of the present application on the release of IL-2 from lymphocytes.

Figure 2 shows the activation results of the antibody of the present application on the release of IL-2 from lymphocytes.

Figure 3 shows the activation results of another antibody of the present application on the release of IL-2 from lymphocytes.

Figure 4 shows an exemplary bispecific antibody construction method of the present application.

Figure 5 shows the activation results of the bispecific antibody of the present application on the release of IL-2 from lymphocytes.

Figure 6 shows the results of the cell bridging function of the bispecific antibody of the present application.

Figure 7 shows the inhibitory effect of the bispecific antibody of the present application on tumor growth.

Detailed ways

The implementation of the invention of the present application will be described below with specific examples. Those familiar with this technology can easily understand other advantages and effects of the invention of the present application from the content disclosed in this specification.

Definition of Terms

In this application, the term "PD-L1" generally refers to the programmed death ligand 1 protein. PD-L1 is also known as cluster of differentiation 274 (CD274) or B7 homolog 1 (B7-H1), and is the protein encoded by the CD274 gene (in humans). PD-L1 can bind to its receptors, such as programmed death 1 (PD-1). The complexation of PD-L1 and PD-1 exerts an immunosuppressive effect by inhibiting T cell proliferation and producing the cytokines IL-2 and IFN-γ. The term "PD-L1" encompasses any native PD-L1 or modified PD-L1 from any vertebrate source, including mammals, such as primates (e.g., humans or monkeys) and rodents ( e.g. mouse or rat). The term encompasses "full-length", unprocessed PD-L1 as well as any form of PD-L1 resulting from processing in the cell. PD-L1 can exist as a transmembrane protein or as a soluble protein. The term also encompasses naturally occurring variants of PD-L1, such as splice variants or allelic variants. The basic structure of PD-L1 includes four domains: extracellular Ig-like V-type domain and Ig-like C2-type domain, transmembrane domain and cytoplasmic domain. PD-L1 sequences are known in the art. Information on the human PD-L1 gene, including the genomic DNA sequence, can be found, for example, under NCBI Gene ID No. 29126. The amino acid sequence of an exemplary full-length human PD-L1 protein can be found under UniProt accession number Q9NZQ7.

In this application, the term "PD-1" generally refers to the programmed death 1 receptor, which may also be referred to as "programmed death 1", "CD279", "cluster of differentiation 279", "PD1", "PDCD1" or "CD297". PD-1 proteins usually include an extracellular IgV domain, a transmembrane region and an intracellular tail. PD-1 is commonly expressed on T cells, B cells, natural killer T cells, activated monocytes, and dendritic cells (DC). PD-1 can bind to its ligands PD-L1 and PD-L2. The term "PD-1" encompasses any native PD-1 or modified PD-1 from any vertebrate source, including mammals, such as primates (e.g., humans or monkeys) and rodents ( e.g. mouse or rat). The term encompasses "full-length", unprocessed PD-1 as well as any form of PD-1 resulting from processing in the cell. PD-1 can exist as a transmembrane protein or as a soluble protein. "PD-1" includes complete PD-1 and its fragments, and also includes functional variants, isoforms, species homologs, derivatives, analogs of PD-1, and those that have at least one property in common with PD-1 Analogues of epitopes. PD-1 sequences are known in the art. For example, an exemplary full-length human PD-1 protein sequence can be found under NCBI accession number NP_005009.2, and an exemplary full-length cynomolgus monkey PD-1 protein sequence can be found under NCBI accession number NP_001271065 or UniProt accession number BOLAJ3 turn up.

As used herein, the term "antigen-binding protein" generally refers to a protein comprising an antigen-binding portion, and optionally a scaffold or backbone portion that allows the antigen-binding portion to adopt a conformation that promotes binding of the antigen-binding protein to the antigen. Antigen binding proteins may typically comprise an antibody light chain variable region (VL), an antibody heavy chain variable region (VH), or both, and functional fragments thereof. The variable regions of the heavy and light chains contain binding domains that interact with the antigen. Examples of antigen-binding proteins include, but are not limited to, antibodies, antigen-binding fragments, immunoconjugates, multispecific antibodies (e.g., bispecific antibodies), antibody fragments, antibody derivatives, antibody analogs, or fusion proteins, etc., as long as they display The required antigen-binding activity can be obtained.

In this application, the term "antibody" generally refers to an immunoglobulin reactive to a specified protein or peptide or fragment thereof. The antibodies may be from any class, including, but not limited to, IgG, IgA, IgM, IgD, and IgE, and from any subclass (eg, IgGl, IgG2, IgG3, and IgG4). The antibody may have a heavy chain constant region selected from, for example, IgGl, IgG2, IgG3, or IgG4. The antibody may also have a light chain selected from, for example, kappa (κ) or lambda (λ). Antibodies of the present application can be derived from any species.

As used herein, the term "antigen-binding fragment" generally refers to a portion of an antibody molecule that contains the amino acid residues that interact with the antigen and confer specificity and affinity to the antibody for the antigen. Examples of antigen-binding fragments may include, but are not limited to, Fab, Fab', F(ab) ₂ , Fv fragment, F(ab') ₂ , scFv, di-scFv and/or dAb. In this application, the term "Fab" generally refers to a fragment containing the variable domain of the heavy chain and the variable domain of the light chain, and also containing the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. ; The term "Fab'" generally refers to a fragment that is different from Fab by adding a small number of residues (including one or more cysteines from the antibody hinge region) to the carboxyl terminus of the heavy chain CH1 domain; the term "F(ab ') ₂ " usually refers to a dimer of Fab', an antibody fragment containing two Fab fragments connected by a disulfide bridge on the hinge region. The term "Fv" generally refers to the smallest antibody fragment containing intact antigen recognition and binding sites. In some cases, the fragment may consist of a heavy chain variable domain and a light chain variable domain as a dimer in tight non-covalent association; the term "dsFv" generally refers to a disulfide-stabilized Fv fragment, The bond between its single light chain variable domain and its single heavy chain variable domain is a disulfide bond. The term "dAb fragment" generally refers to an antibody fragment consisting of a VH domain. In this application, the term "scFv" generally refers to a monovalent molecule formed by covalently connecting a heavy chain variable domain and a light chain variable domain of an antibody through a flexible peptide linker; such scFv molecules may have general Structure: NH ₂ -VL-linker-VH-COOH or NH ₂ -VH-linker-VL-COOH.

In this application, the term "variable region" or "variable domain" generally refers to the domain of an antibody heavy or light chain that is involved in binding of the antibody to an antigen. In this application, the term "variable" generally means that certain portions of the sequence of the variable domain of an antibody vary strongly, resulting in the binding and specificity of various specific antibodies for their specific antigens. Variability is not evenly distributed throughout the variable regions of an antibody. It is concentrated in three segments in the light chain variable region and heavy chain variable region, known as the complementarity determining region (CDR) or hypervariable region (HVR), which are LCDR1, LCDR2, LCDR3, HCDR1, HCDR2 and HCDR3. The more highly conserved portions of the variable domains are called framework regions (FR). The variable domains of the native heavy and light chains each contain four FR regions (H-FR1, H-FR2, H-FR3, H-FR4, L-FR1, L-FR2, L-FR3, L-FR4) , most adopt β-sheet configuration and are connected through three CDR structural loop regions. The CDRs in each chain are held closely together by the FR region and, together with the CDRs from the other chain, form the antigen-binding site of the antibody.

In the art, the variable regions of an antibody can be encoded or the CDRs of an antibody can be delineated by a variety of methods, such as the Kabat numbering scheme and definition rules based on sequence variability (see, Kabat et al., Protein Sequences in Immunology, 5 ed., National Institutes of Health, Bethesda, MD (1991)), based on the location of structural loop regions. Chothia numbering scheme and definition rules (see, Al-Lazikani et al., J Mol Biol 273:927-48, 1997), efranc et al.'s IMGT numbering scheme and definition rules based on amino acid sequence alignment of germline V genes, and Honneger's numbering scheme (AHo's), Martin numbering scheme, Gelfand numbering scheme, etc. (See Mathieu Dondelinger et al., Understanding the Significance and Implications of Antibody Numbering and Antigen-Binding Surface/Residue Definition, Front. Immunol., 16 October 2018.) . In this application, the antibody variable regions are numbered according to Chothia rules, and the antibody CDR regions are defined according to Chothia.

In this application, the term "monoclonal antibody" generally refers to an antibody obtained from a population of antibodies that are essentially homogeneous, i.e., the individual antibodies making up the population are identical except for possible naturally occurring mutations that may be present in minimal amounts and/or In addition to post-translational modifications (such as isomerization, amidation). Monoclonal antibodies are highly specific and target a single antigenic site.

In this application, the term "chimeric antibody" generally refers to an antibody in which the variable regions are derived from one species and the constant regions are derived from another species. Typically, the variable regions are derived from an antibody from a laboratory animal, such as a rodent (a "parent antibody"), and the constant regions are derived from a human antibody, such that the resulting chimeric antibody is more effective in a human individual than the parent (eg, mouse-derived) antibody. Less likely to trigger an adverse immune response.

In this application, the term "humanized antibody" generally refers to an antibody in which some or all of the amino acids outside the CDR region of a non-human antibody (eg, a mouse antibody) are replaced with corresponding amino acids derived from human immunoglobulins. Additions, deletions, insertions, substitutions or modifications of amino acids in the CDR regions may also be allowed as long as they retain the ability of the antibody to bind a specific antigen. The humanized antibody may optionally comprise at least a portion of a human immunoglobulin constant region. "Humanized antibodies" retain antigen specificity similar to the original antibody. "Humanized" forms of non-human (eg, murine) antibodies may minimally comprise chimeric antibodies derived from sequences derived from non-human immunoglobulins. In some cases, CDR region residues in a human immunoglobulin (recipient antibody) can be used with a non-human species (donor antibody) having the desired properties, affinity, and/or ability (such as mouse, rat , rabbit or non-human primate) CDR region residue substitution. In some cases, FR region residues of a human immunoglobulin can be replaced with corresponding non-human residues. Furthermore, humanized antibodies may contain amino acid modifications that are not present in the recipient antibody or in the donor antibody.

In this application, the term "fully human antibody" generally refers to an antibody in which all parts (including the variable and constant regions of the antibody) are encoded by genes of human origin. Methods for obtaining fully human antibodies in this field include phage display technology, transgenic mouse technology, ribosome display technology, and RNA-polypeptide technology.

In this application, the terms "binding,""specificbinding," or "specific for" generally refer to a measurable and reproducible interaction, such as binding between an antigen and an antibody, which can be determined in the presence of a molecule The presence of a target in a heterogeneous population (including biological molecules). For example, an antibody binds to an epitope through its antigen-binding domain, and this binding requires some complementarity between the antigen-binding domain and the epitope. For example, an antibody that specifically binds a target (which may be an epitope) is An antibody that binds this target with greater affinity, avidity, more readily, and/or for a greater duration than it binds other targets. An antibody is said to "specifically bind" an antigen when it binds to an epitope more readily through its antigen-binding domain than it would to a random, unrelated epitope.

In this application, the terms "KD" and " _KD " are used interchangeably and generally refer to the equilibrium dissociation constant. "KD" is the dissociation rate constant (kdis, also known as "off-rate". )(koff)" or "kd") to the binding rate constant (kon, also known as "binding rate (kon)" or "ka"). The binding affinity of an antigen-binding protein (eg, an antibody) for an antigen can be expressed using the association rate constant (kon), the dissociation rate constant (kdis), and the equilibrium dissociation constant (KD). Methods for determining association and dissociation rate constants are well known in the art, including but not limited to biofilm interference technique (BLI), radioimmunoassay (RIA), equilibrium dialysis, surface plasmon resonance (SPR), fluorescence resonance energy transfer (FRET) , co-immunoprecipitation (Co-IP) and protein chip technology. The measured affinity for a particular protein-protein interaction can differ if measured under different conditions (eg, salt concentration, pH).

In this application, the term "primate" generally refers to aye-aye and ape species, and includes monkey species, such as those from the genus Macaca (e.g., Macaca fascicularis and/or rhesus monkeys (Macaca mulatta)) monkeys and baboons (Papio ursinus), as well as marmosets (species from the genus Callithrix), squirrel monkeys (species from the genus Saimiri) and tamarins (from the genus Tamarinus) Saguinus), as well as ape species such as chimpanzees (Pan troglodytes), and also includes Homo sapiens.

In this application, the terms "polypeptide" or "protein" are used interchangeably and generally refer to a polymer of amino acid residues. The term also applies to amino acid polymers in which one or more amino acid residues are analogs or mimetics of the corresponding naturally occurring amino acids, as well as naturally occurring amino acid polymers. The term may also include modified amino acid polymers, for example, by the addition of sugar residues to form glycoproteins or by phosphorylation. Polypeptides and proteins may be produced from naturally occurring and non-recombinant cells or from genetically engineered or recombinant cells, and may comprise molecules having the amino acid sequence of the native protein, or having the deletion, addition, or deletion of one or more amino acids of the native sequence. and/or substituted molecules. The terms "polypeptide" and "protein" particularly include sequences in which one or more amino acids of the antigen-binding proteins described herein are deleted, added and/or substituted. For example, a polypeptide of the present application may comprise an antigen-binding protein. For example, a polypeptide of the present application may comprise a multispecific antigen-binding protein, such as a bispecific antibody.

In this application, the term "isolated" generally refers to biological material (eg, viruses, nucleic acids, or proteins) that is substantially free of components that normally accompany or interact with it in its naturally occurring environment. The isolated biological material optionally contains additional materials that the biological material is not found to have in its natural environment (eg, nucleic acids or proteins). In this application, when referring to a protein, "isolated" generally refers to the removal of the molecule from the entire organism in which it is found naturally occurring. Isolation and separation, or the substantial absence of other biological macromolecules of the same type. When it comes to a nucleic acid molecule, it is completely or partially separated from the sequence to which it is naturally associated, or the nucleic acid has heterologous sequences to which it is associated, or the nucleic acid is separated from the chromosome.

In this application, the term "immunoconjugate" generally refers to a substance formed by connecting an antigen-binding protein to other active agents. The other active agents can be small molecule active agents, such as chemotherapeutic agents, toxins, immunotherapeutic agents, and imaging probes. or spectroscopic probes.

In this application, the term "nucleic acid" molecule generally refers to an isolated form of nucleotides, deoxyribonucleotides or ribonucleotides or analogs thereof of any length, isolated from their natural environment or artificially synthesized.

In this application, the term "vector" generally refers to a nucleic acid molecule capable of self-replication in a suitable host, which transfers the inserted nucleic acid molecule into and/or between host cells. The vectors may include vectors primarily used for insertion of DNA or RNA into cells, vectors primarily used for replication of DNA or RNA, and vectors primarily used for expression of transcription and/or translation of DNA or RNA. The vectors also include vectors having a variety of the above-mentioned functions. The vector may be a polynucleotide capable of being transcribed and translated into a polypeptide when introduced into a suitable host cell. Typically, the vector can produce the desired expression product by culturing a suitable host cell containing the vector.

In this application, the term "cell" generally refers to an individual cell, cell line or cell culture that can contain or has contained a plasmid or vector including a nucleic acid molecule described herein, or is capable of expressing an antigen-binding protein described herein. things. The cells may include progeny of a single host cell. Due to natural, accidental or intentional mutations, the progeny cells may not necessarily be identical in morphology or genome to the original parent cells, but they may be able to express the antibodies or antigen-binding fragments thereof described in this application. The cells can be obtained by transfecting cells in vitro using the vectors described in this application. The cells may be prokaryotic cells (e.g. Escherichia coli) or eukaryotic cells (e.g. yeast cells, e.g. COS cells, Chinese hamster ovary (CHO) cells, HeLa cells, HEK293 cells, COS-1 cells, NSO cells or myeloma cells). In some cases, the cells may be mammalian cells. For example, the mammalian cells may be CHO-K1 cells.

In this application, the term "pharmaceutical composition" generally refers to a formulation that allows the biological activity of the active ingredient to be present in a form that is effective and does not contain additional ingredients that would have unacceptable toxicity to the subject to whom the composition is to be administered.

In this application, the term "treatment" generally refers to a clinical intervention intended to alter the natural course of the disease in the individual being treated, and may be to achieve prevention or treatment during the clinical course of the disease. Desirable therapeutic effects include, but are not limited to, preventing the onset or recurrence of disease, alleviating symptoms, attenuating any direct or indirect pathological consequences of disease, preventing metastasis, reducing the rate of disease progression, ameliorating or relieving disease status, and alleviating or improving prognosis. In some cases, antibodies can be used to delay disease development or slow disease progression.

As used herein, the term "administering" generally refers to administering to a subject (eg, a patient) a dose of a compound (eg, an anti-cancer therapeutic agent) or a pharmaceutical composition (eg, a pharmaceutical composition comprising an anti-cancer therapeutic agent) Methods. Apply Administration may be by any suitable means, including parenteral, intrapulmonary and intranasal, and, if required for local treatment, intralesional administration. Parenteral infusion includes, for example, intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration.

In this application, the term "tumor" generally refers to all neoplastic cell growth and proliferation (whether malignant or benign) and all precancerous and cancerous cells and tissues. In this application, the tumor may be a tumor with high expression of PD-1 or PD-L1 in cells and tissues. Tumors may include solid tumors and/or non-solid tumors.

In this application, the term "homologue" generally refers to an amino acid sequence or a nucleotide sequence that has certain homology to a wild-type amino acid sequence and a wild-type nucleotide sequence. The term "homology" may be equated with sequence "identity." Homologous sequences may include amino acid sequences that may be at least 80%, 85%, 90%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to the subject sequence. . Typically, homologs will contain active sites, etc. that are identical to the subject amino acid sequence. Homology can be considered in terms of similarity (ie, amino acid residues with similar chemical properties/functions), or homology can be expressed in terms of sequence identity. In this application, reference to a sequence having a percent identity with any one of the SEQ ID NOs of an amino acid sequence or a nucleotide sequence means that the sequence has said percent identity over the entire length of the SEQ ID NO mentioned. the sequence of.

In this application, the term "between" usually means that the C-terminus of a certain amino acid fragment is directly or indirectly connected to the N-terminus of the first amino acid fragment, and its N-terminus is directly or indirectly connected to the C-terminus of the second amino acid fragment. indirect connection. In the light chain, for example, the N-terminus of the L-FR2 is directly or indirectly connected to the C-terminus of the LCDR1, and the C-terminus of the L-FR2 is directly or indirectly connected to the N-terminus of the LCDR2. For another example, the N terminus of the L-FR3 is directly or indirectly connected to the C terminus of the LCDR2, and the C terminus of the L-FR3 is directly or indirectly connected to the N terminus of the LCDR3. In the heavy chain, for example, the N-terminus of the H-FR2 is directly or indirectly connected to the C-terminus of the HCDR1, and the C-terminus of the H-FR2 is directly or indirectly connected to the N-terminus of the HCDR2. For another example, the N terminus of the H-FR3 is directly or indirectly connected to the C terminus of the HCDR2, and the C terminus of the H-FR3 is directly or indirectly connected to the N terminus of the HCDR3. In this application, the "first amino acid fragment" and the "second amino acid fragment" can be any amino acid fragments that are the same or different.

In this application, the term "comprises" generally means including, encompassing, containing or encompassing. In some cases, it also means "for" or "composed of".

In this application, the term "about" generally refers to a variation within the range of 0.5% to 10% above or below the specified value, such as 0.5%, 1%, 1.5%, 2%, 2.5%, above or below the specified value. 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10%.

Detailed description of the invention

On the one hand, the present application provides an antigen-binding protein, which may have one or more of the following properties: (1) Capable of binding to primate-derived PD-L1 protein with a _KD value of 3.56E-09M or lower; and (2) capable of stimulating immune cells to secrete cytokines, for example, stimulating lymphocytes to secrete IL-2. The binding affinity of the primate PD-L1 antigen binding protein to PD-L1 can be determined by any method known in the art. In some cases, binding affinity can be determined by surface plasmon resonance (SPR), enzyme-linked immunoassay (ELISA), binding antigen precipitation, equilibrium dialysis, or biofilm interference (BLI). In some cases, the binding affinity and _K value of the PD-L1 antigen-binding protein to PD-L1 can be determined by biofilm interference (BLI). For example, the ForteBio Octet Molecular Interaction Analyzer can be used to analyze the binding kinetics between antigens and antibodies. For example, the antigen-binding protein of the present application may comprise a multispecific antigen-binding protein, such as a bispecific antibody.

For example, the antigen-binding protein of the present application can bind to primate-derived PD-L1 with a _KD value of about 3.56E-09M or lower. For example, the value of K _D can be about 1E-08M or less, about 9E-09M or less, about 8E-09M or less, about 7E-09M or less, about 6E-09M or less, About 5E-09M or less, about 4E-09M or less, about 3E-09M or less, about 2E-09M or less, about 1E-09M or less, about 5E-10M or less, about 1E - A value of 10 M or less binding to human-derived PD-L1, for example, as detected using the FortieBio Octet Molecular Interaction Analyzer. In another aspect, the antigen-binding protein described in the present application can block the binding of PD-1 to PD-L1.

For example, the antigen-binding protein described in the present application may comprise the antibody heavy chain variable region VH, and the antigen-binding protein in the present application may comprise HCDR1, HCDR2 and HCDR3 of the VH shown in any one of SEQ ID NOs: 7 to 11. For example, the CDRs of this application can be divided according to any dividing rules in this field.

For example, the antigen-binding protein described in the present application may comprise the antibody heavy chain variable region VH, and the antigen-binding protein in the present application may comprise HFR1, HFR2, HFR3 and HFR4 of the VH shown in any one of SEQ ID NOs: 7 to 11.

For example, the antigen-binding protein described in the present application can comprise the antibody heavy chain variable region VH, and the antigen-binding protein in the present application can comprise HCDR1, HCDR2 and HCDR3 of the VH shown in any one of SEQ ID NOs: 7 to 8.

For example, the antigen-binding protein of the present application may comprise HCDR1, HCDR2 and HCDR3 of EVHLQQSGAALVKPGASVKMSCKASGYTFTDFWVNWVKQSHGNSLEWIGEIWPNSGATNFNENFKGKATLTVDRSTSTAYLDLTRLTSEDSAIYYCTRELRRPPFTYWGQGASVTVSS (SEQ ID NO: 7).

For example, the antigen-binding protein of the present application may comprise the HCDR1, HCDR2 and HCDR3 of QVQLVQSGAEVKKPGASVKVSCKASGYTFTDFWVNWVRQAPGQGLEWIGEIWPNSGATNFNENFKGRATLTVDRSISTAYMELSRLRSDDTAVYYCTRELRRPPFTYWGQGTLVTVSS (SEQ ID NO: 8).

For example, the antigen-binding protein described in the present application may comprise an antibody heavy chain variable region VH. The antigen-binding protein of the present application may HCDR1, HCDR2 and HCDR3 of the VH shown in any one of SEQ ID NOs: 9 to 10 may be included.

For example, the antigen-binding protein of the present application may comprise HCDR1, HCDR2 and HCDR3 of EVQLVESGSALVKPGASVKMSCKASGYTFTDFWVNWVKQSHGKSLEWIGEIWPNSGTTNFNEKFRGKATLTVDKSTSTAYMELSRLTSEDSAIYYCTRELRRPPFTYWGQGTLVTVSS (SEQ ID NO: 9).

For example, the antigen-binding protein of the present application may comprise the HCDR1, HCDR2 and HCDR3 of QVQLVQSGAEVKKPGASVKVSCKASGYTFTDFWVNWVRQAPGQGLEWMGEIWPNSGTTNFAQKFQGRVTMTVDKSISTAYMELSRLRSDDTAVYYCTRELRRPPFTYWGQGTLVTVSS (SEQ ID NO: 10).

For example, the antigen-binding protein described in the present application may comprise the antibody heavy chain variable region VH, and the antigen-binding protein described in the present application may comprise HCDR1, HCDR2 and HCDR3 of the VH shown in SEQ ID NO: 11.

For example, the antigen-binding protein of the present application may comprise the HCDR1, HCDR2 and HCDR3 of QVQLVQSGAEVKKPGASVKVSCKASGYTFTDFWVNWVRQAPGQGLEWMGEIWPNYGTTNFAQKFQGRVTMTVDKSISTAYMELSRLRSDDTAVYYCTRELRRPPFTYWGQGTLVTVSS (SEQ ID NO: 11).

For example, the antigen-binding protein described in the present application may comprise the antibody heavy chain variable region VH, the VH may comprise HCDR1, HCDR2 and HCDR3, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 6. For example, the CDR of this application can be divided according to Chothia division rules.

For example, the antigen-binding protein may comprise an antibody heavy chain variable region VH, the VH may comprise HCDR1, HCDR2 and HCDR3, and the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 5. For example, the HCDR2 may include WPNX ₁ GX ₂ , where X ₁ may be S or Y and X ₂ may be A or T. For example, compared to the amino acid sequence shown in SEQ ID NO: 5, the HCDR2 may comprise at least amino acid substitutions at positions selected from the group consisting of: X ₁ and X ₂ . For example, the HCDR2 may comprise the amino acid sequence shown in any one of SEQ ID NOs: 2 to 4.

For example, the antigen-binding protein may comprise an antibody heavy chain variable region VH, the VH may comprise HCDR1, HCDR2 and HCDR3, and the HCDR2 may comprise an amino acid sequence selected from the group consisting of: SEQ ID NO: 2 to 4.

For example, the antigen-binding protein may comprise an antibody heavy chain variable region VH, the VH may comprise HCDR1, HCDR2 and HCDR3, and the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 1.

For example, the antigen-binding protein may comprise an antibody heavy chain variable region VH, and the VH may comprise HCDR1, HCDR2 and HCDR3, the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 6, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 5, and the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 1 Amino acid sequence.

For example, the antigen-binding protein may comprise an antibody heavy chain variable region VH, the VH may comprise HCDR1, HCDR2 and HCDR3, the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 6, the HCDR2 may comprise selected From the amino acid sequence shown in the following group: SEQ ID NO: 2 to 4, and the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 1.

For example, the antigen-binding protein may comprise an antibody heavy chain variable region VH, the VH may comprise HCDR1, HCDR2 and HCDR3, the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 6, the HCDR2 may comprise SEQ The amino acid sequence shown in ID NO: 2, and the HCDR1 may include the amino acid sequence shown in SEQ ID NO: 1.

For example, the antigen-binding protein may comprise an antibody heavy chain variable region VH, the VH may comprise HCDR1, HCDR2 and HCDR3, the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 6, the HCDR2 may comprise SEQ The amino acid sequence shown in ID NO: 3, and the HCDR1 may include the amino acid sequence shown in SEQ ID NO: 1.

For example, the antigen-binding protein may comprise an antibody heavy chain variable region VH, the VH may comprise HCDR1, HCDR2 and HCDR3, the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 6, the HCDR2 may comprise SEQ The amino acid sequence shown in ID NO: 4, and the HCDR1 may include the amino acid sequence shown in SEQ ID NO: 1.

For example, the antigen-binding protein may comprise an antibody heavy chain variable region VH, and the VH may comprise an amino acid sequence selected from the group consisting of SEQ ID NOs: 7 to 11.

For example, the antigen binding protein may comprise an antibody heavy chain constant region CH, and the CH is derived from an IgG constant region. For example, the antigen binding protein may comprise an antibody heavy chain constant region CH, and the CH is derived from an IgGl constant region.

For example, the antigen-binding protein may comprise an antibody heavy chain constant region CH, and the CH may comprise an amino acid sequence selected from the group consisting of SEQ ID NOs: 16 to 18.

For example, the antigen-binding protein may comprise an antibody heavy chain, and the heavy chain may comprise an amino acid sequence selected from the group consisting of SEQ ID NOs: 19 to 21.

For example, the antigen-binding protein described in the present application may comprise the antibody light chain variable region VL, and the antigen-binding protein in the present application may comprise LCDR1, LCDR2 and LCDR3 of the VL shown in SEQ ID NO: 26. For example, the CDRs of this application can be divided according to any dividing rules in this field.

For example, the antigen-binding protein described in the present application can comprise the light chain variable region VL, and the antigen-binding protein in the present application can comprise LFR1, LFR2, LFR3 and LFR4 of the VL shown in SEQ ID NO: 26.

For example, the antigen-binding protein described in the present application can comprise the light chain variable region VL, and the antigen-binding protein in the present application can comprise LCDR1, LCDR2 and LCDR3 of DIQLTQSPSFLSASVGDRVTITCKASQDVGTAVAWYQQKPGKAPKLLIYWASTRHTGVPSRFSGSGSGTEFTLTISSLQPEDFATYFCQQYSSYPWTFGGGTKVEIK (SEQ ID NO: 26).

For example, the antigen-binding protein may comprise an antibody light chain variable region VL, the VL may comprise LCDR1, LCDR2 and LCDR3, and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 25. For example, the CDR of this application can be divided according to Chothia division rules.

For example, the antigen-binding protein may comprise an antibody light chain variable region VL, the VL may comprise LCDR1, LCDR2 and LCDR3, and the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 24.

For example, the antigen-binding protein may comprise an antibody light chain variable region VL, the VL may comprise LCDR1, LCDR2 and LCDR3, and the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 23.

For example, the antigen-binding protein may comprise an antibody light chain variable region VL, the VL may comprise LCDR1, LCDR2 and LCDR3, the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 25, the LCDR2 may comprise SEQ The amino acid sequence shown in ID NO: 24, and the LCDR1 may include the amino acid sequence shown in SEQ ID NO: 23.

For example, the antigen-binding protein may comprise an antibody light chain variable region VL, and the VL may comprise the amino acid sequence shown in SEQ ID NO: 26.

For example, the antigen-binding protein may comprise an antibody light chain constant region CL, and the CL may comprise the amino acid sequence shown in SEQ ID NO: 27.

For example, the antigen-binding protein may comprise an antibody light chain, and the light chain may comprise the amino acid sequence shown in SEQ ID NO: 28.

For example, the antigen-binding protein described in the present application may comprise the antibody heavy chain variable region VH. The antigen-binding protein in the present application may comprise HCDR1, HCDR2 and HCDR3 of the VH shown in any one of SEQ ID NO: 7 to 11, and the present invention The antigen-binding protein described in the application can include the antibody light chain variable region VL. The antigen-binding protein of the application can include LCDR1, LCDR2 and LCDR3 of the VL shown in SEQ ID NO: 26.

For example, the antigen-binding protein may comprise an antibody heavy chain variable region VH, the VH may comprise HCDR1, HCDR2 and HCDR3, the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 6, the HCDR2 It may comprise an amino acid sequence selected from the following group: SEQ ID NO: 2 to 4, and the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 1; the antigen-binding protein may comprise an antibody light chain variable region VL, the VL may include LCDR1, LCDR2 and LCDR3, the LCDR3 may include the amino acid sequence shown in SEQ ID NO: 25, the LCDR2 may include the amino acid sequence shown in SEQ ID NO: 24, and the LCDR1 may Contains the amino acid sequence shown in SEQ ID NO: 23.

For example, the antigen-binding protein may comprise an antibody heavy chain variable region VH, the VH may comprise HCDR1, HCDR2 and HCDR3, the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 6, the HCDR2 may comprise SEQ The amino acid sequence shown in ID NO: 2, and the HCDR1 can include the amino acid sequence shown in SEQ ID NO: 1; the antigen-binding protein can include the antibody light chain variable region VL, and the VL can include LCDR1, LCDR2 and LCDR3, the LCDR3 may include the amino acid sequence shown in SEQ ID NO:25, the LCDR2 may include the amino acid sequence shown in SEQ ID NO:24, and the LCDR1 may include the amino acid sequence shown in SEQ ID NO:23 sequence.

For example, the antigen-binding protein may comprise an antibody heavy chain variable region VH, the VH may comprise HCDR1, HCDR2 and HCDR3, the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 6, the HCDR2 may comprise SEQ The amino acid sequence shown in ID NO: 3, and the HCDR1 can include the amino acid sequence shown in SEQ ID NO: 1; the antigen-binding protein can include the antibody light chain variable region VL, and the VL can include LCDR1, LCDR2 and LCDR3, the LCDR3 may include the amino acid sequence shown in SEQ ID NO:25, the LCDR2 may include the amino acid sequence shown in SEQ ID NO:24, and the LCDR1 may include the amino acid sequence shown in SEQ ID NO:23 sequence.

For example, the antigen-binding protein may comprise an antibody heavy chain variable region VH, the VH may comprise HCDR1, HCDR2 and HCDR3, the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 6, the HCDR2 may comprise SEQ The amino acid sequence shown in ID NO: 4, and the HCDR1 can include the amino acid sequence shown in SEQ ID NO: 1; the antigen-binding protein can include the antibody light chain variable region VL, and the VL can include LCDR1, LCDR2 and LCDR3, the LCDR3 may include the amino acid sequence shown in SEQ ID NO:25, the LCDR2 may include the amino acid sequence shown in SEQ ID NO:24, and the LCDR1 may include the amino acid sequence shown in SEQ ID NO:23 sequence.

For example, the antigen-binding protein of the present application may comprise HCDR1, HCDR2 and HCDR3, the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 6, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 2, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 6. The HCDR1 may include the amino acid sequence shown in SEQ ID NO: 1; the antigen-binding protein may include LCDR1, LCDR2 and LCDR3, and the LCDR3 may include the amino acid sequence shown in SEQ ID NO: 25 column, the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 24, and the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 23.

For example, the antigen-binding protein of the present application may include HCDR1, HCDR2 and HCDR3. The HCDR3 may include the amino acid sequence shown in SEQ ID NO: 6, the HCDR2 may include the amino acid sequence shown in SEQ ID NO: 3, and the HCDR3 may include the amino acid sequence shown in SEQ ID NO: 3. The HCDR1 may include the amino acid sequence shown in SEQ ID NO: 1; the antigen-binding protein may include LCDR1, LCDR2 and LCDR3, the LCDR3 may include the amino acid sequence shown in SEQ ID NO: 25, and the LCDR2 may include SEQ The amino acid sequence shown in ID NO: 24, and the LCDR1 may include the amino acid sequence shown in SEQ ID NO: 23.

For example, the antigen-binding protein of the present application may include HCDR1, HCDR2 and HCDR3. The HCDR3 may include the amino acid sequence shown in SEQ ID NO: 6, the HCDR2 may include the amino acid sequence shown in SEQ ID NO: 4, and the HCDR3 may include the amino acid sequence shown in SEQ ID NO: 4. The HCDR1 may include the amino acid sequence shown in SEQ ID NO: 1; the antigen-binding protein may include LCDR1, LCDR2 and LCDR3, the LCDR3 may include the amino acid sequence shown in SEQ ID NO: 25, and the LCDR2 may include SEQ The amino acid sequence shown in ID NO: 24, and the LCDR1 may include the amino acid sequence shown in SEQ ID NO: 23.

The antigen-binding protein described in the present application may include the antibody heavy chain variable region VH. The antigen-binding protein described in the present application may include the VH shown in any one of SEQ ID NO: 7 to 11, and the antigen-binding protein described in the present application may include Antibody light chain variable region VL, the antigen-binding protein of the present application can include the VL shown in SEQ ID NO: 26.

For example, the antigen-binding protein may bind PD-L1 or a functionally active fragment thereof.

For example, the PD-L1 may comprise PD-L1 derived from humans and/or monkeys.

For example, the antigen-binding protein may comprise an antibody or antigen-binding fragment thereof.

For example, the antibody may be selected from the group consisting of murine antibodies, chimeric antibodies, humanized antibodies, and fully human antibodies.

For example, the antigen-binding fragment may be selected from the group consisting of Fab, Fab', F(ab) ₂ , Fv fragment, F(ab') ₂ , scFv, di-scFv, VHH and dAb.

In another aspect, the present application provides a polypeptide, which may comprise a first targeting moiety, and the first targeting moiety may comprise an antigen-binding protein of the present application.

For example, the polypeptide may further comprise a second targeting moiety capable of binding PD-1 or a functionally active fragment thereof.

For example, the PD-1 may comprise PD-1 derived from humans and/or monkeys.

For example, the second targeting moiety may comprise an antibody or antigen-binding fragment thereof.

For example, the second targeting portion described in the present application can comprise the antibody heavy chain variable region VH, and the VH of the second targeting portion can comprise HCDR1, HCDR2 and HCDR3 of the VH shown in SEQ ID NO: 15. For example, the CDRs of this application can be divided according to any dividing rules in this field.

For example, the second targeting part described in the present application includes the antibody heavy chain variable region VH, and the VH of the second targeting part may include HFR1, HFR2, HFR3 and HFR4 of the VH shown in SEQ ID NO: 15.

For example, the antigen-binding protein of the present application may comprise HCDR1, HCDR2 and HCDR3 of EVQLVESGGGLVKPGGSLRLSCAASGFTFSNYDMSWVRQAPGKGLEWVSTISGGGSYTYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCVSPYYGMEYWGQGTLVTVSS (SEQ ID NO: 15).

For example, the second targeting portion may comprise an antibody heavy chain variable region VH, the VH of the second targeting portion may comprise HCDR1, HCDR2 and HCDR3, and the HCDR3 of the second targeting portion may comprise SEQ ID The amino acid sequence shown in NO:14. For example, the CDR of this application can be divided according to Chothia division rules.

For example, the second targeting portion may comprise an antibody heavy chain variable region VH, the VH of the second targeting portion may comprise HCDR1, HCDR2 and HCDR3, and the HCDR2 of the second targeting portion may comprise SEQ ID The amino acid sequence shown in NO:13.

For example, the second targeting portion may comprise an antibody heavy chain variable region VH, the VH of the second targeting portion may comprise HCDR1, HCDR2 and HCDR3, and the HCDR1 of the second targeting portion may comprise SEQ ID The amino acid sequence shown in NO:12.

For example, the second targeting portion may comprise an antibody heavy chain variable region VH, and the VH of the second targeting portion may comprise the amino acid sequence shown in SEQ ID NO: 15.

For example, the second targeting moiety may comprise an antibody heavy chain constant region CH, and the CH of the second targeting moiety is derived from an IgG constant region.

For example, the second targeting moiety may comprise an antibody heavy chain constant region CH, and the CH of the second targeting moiety is derived from an IgG1 constant region.

For example, the second targeting portion may comprise an antibody heavy chain constant region CH, and the CH of the second targeting portion may comprise an amino acid sequence selected from the group consisting of SEQ ID NOs: 16 to 18.

For example, the second targeting moiety may comprise an antibody heavy chain, and the heavy chain of the second targeting moiety may comprise the amino acid sequence shown in SEQ ID NO: 22.

For example, the second targeting part described in the present application may comprise the antibody light chain variable region VL, and the second targeting part in the present application may comprise LCDR1, LCDR2 and LCDR3 of the VL shown in SEQ ID NO: 26. For example, the CDRs of this application can be divided according to any dividing rules in this field.

For example, the second targeting part described in the present application may comprise the light chain variable region VL, and the second targeting part in the present application may comprise LFR1, LFR2, LFR3 and LFR4 of the VL shown in SEQ ID NO: 26.

For example, the second targeting part described in the present application may comprise the light chain variable region VL, and the second targeting part in the present application may comprise LCDR1, LCDR2 and LCDR3 of DIQLTQSPSFLSASVGDRVTITCKASQDVGTAVAWYQQKPGKAPKLLIYWASTRHTGVPSRFSGSGSGTEFTLTISSLQPEDFATYFCQQYSSYPWTFGGGTKVEIK (SEQ ID NO: 26).

For example, the second targeting moiety may comprise an antibody light chain variable region VL, the VL of the second targeting moiety may comprise LCDR1, LCDR2 and LCDR3, and the LCDR3 of the second targeting moiety may comprise SEQ ID The amino acid sequence shown in NO:25. For example, the CDR of this application can be divided according to Chothia division rules.

For example, the second targeting portion may comprise an antibody light chain variable region VL, the VL of the second targeting portion may comprise LCDR1, LCDR2 and LCDR3, and the LCDR2 of the second targeting portion may comprise SEQ ID The amino acid sequence shown in NO:24.

For example, the second targeting portion may comprise an antibody light chain variable region VL, the VL of the second targeting portion may comprise LCDR1, LCDR2 and LCDR3, and the LCDR1 of the second targeting portion may comprise SEQ ID The amino acid sequence shown in NO:23.

For example, the second targeting portion may comprise an antibody light chain variable region VL, and the VL of the second targeting portion may comprise the amino acid sequence shown in SEQ ID NO: 26.

For example, the second targeting portion may comprise an antibody light chain constant region CL, and the CL of the second targeting portion may comprise the amino acid sequence shown in SEQ ID NO: 27.

For example, the second targeting moiety may comprise an antibody light chain, and the light chain of the second targeting moiety may comprise the amino acid sequence shown in SEQ ID NO: 28.

For example, the second targeting portion may comprise an antibody heavy chain variable region VH, the VH of the second targeting portion may comprise HCDR1, HCDR2 and HCDR3, and the HCDR3 of the second targeting portion may comprise SEQ ID NO. : The amino acid sequence shown in 14, the HCDR2 of the second targeting part may comprise the amino acid sequence shown in SEQ ID NO: 13, and the HCDR1 of the second targeting part may comprise the amino acid sequence shown in SEQ ID NO: 12 Amino acid sequence; the second targeting portion may include the antibody light chain variable region VL, and the VL of the second targeting portion may include LCDR1, LCDR2 and LCDR3, the LCDR3 of the second targeting portion may comprise the amino acid sequence shown in SEQ ID NO: 25, the LCDR2 of the second targeting portion may comprise the amino acid sequence shown in SEQ ID NO: 24, and the LCDR1 of the second targeting portion may comprise the amino acid sequence shown in SEQ ID NO: 23.

For example, the polypeptide of the present application may comprise a first targeting moiety and a second targeting moiety, the first targeting moiety may comprise HCDR1, HCDR2 and HCDR3, and the HCDR3 of the first targeting moiety may comprise SEQ ID NO: The amino acid sequence shown in 6, the HCDR2 of the first targeting part can include the amino acid sequence shown in SEQ ID NO: 2, and the HCDR1 of the first targeting part can include the amino acid shown in SEQ ID NO: 1 Sequence; the first targeting portion may include LCDR1, LCDR2 and LCDR3, the LCDR3 of the first targeting portion may include the amino acid sequence shown in SEQ ID NO: 25, and the LCDR2 of the first targeting portion may include The amino acid sequence shown in SEQ ID NO: 24, and the LCDR1 of the first targeting part can include the amino acid sequence shown in SEQ ID NO: 23; the second targeting part can include HCDR1, HCDR2 and HCDR3, so The HCDR3 of the second targeting portion may comprise the amino acid sequence shown in SEQ ID NO: 14, the HCDR2 of the second targeting portion may comprise the amino acid sequence shown in SEQ ID NO: 13, and the second targeting portion Part of HCDR1 may include the amino acid sequence shown in SEQ ID NO: 12; the second targeting part may include LCDR1, LCDR2 and LCDR3, and the LCDR3 of the second targeting part may include SEQ ID NO: 25 Amino acid sequence, the LCDR2 of the second targeting portion may include the amino acid sequence shown in SEQ ID NO: 24, and the LCDR1 of the second targeting portion may include the amino acid sequence shown in SEQ ID NO: 23.

For example, the polypeptide of the present application may comprise a first targeting moiety and a second targeting moiety, the first targeting moiety may comprise HCDR1, HCDR2 and HCDR3, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 6 , the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 3, and the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 1; the first targeting part may comprise LCDR1, LCDR2 and LCDR3, so The LCDR3 may include the amino acid sequence shown in SEQ ID NO: 25, the LCDR2 may include the amino acid sequence shown in SEQ ID NO: 24, and the LCDR1 may include the amino acid sequence shown in SEQ ID NO: 23; The second targeting portion may include HCDR1, HCDR2 and HCDR3, the HCDR3 of the second targeting portion may include the amino acid sequence shown in SEQ ID NO: 14, and the HCDR2 of the second targeting portion may include SEQ ID NO: The amino acid sequence shown in SEQ ID NO: 13, and the HCDR1 of the second targeting part may include the amino acid sequence shown in SEQ ID NO: 12; the second targeting part may include LCDR1, LCDR2 and LCDR3, and the second targeting part may include LCDR1, LCDR2 and LCDR3. The LCDR3 of the targeting portion may comprise the amino acid sequence shown in SEQ ID NO: 25, the LCDR2 of the second targeting portion may comprise the amino acid sequence shown in SEQ ID NO: 24, and the LCDR1 of the second targeting portion may Contains the amino acid sequence shown in SEQ ID NO: 23.

For example, the polypeptide of the present application can include a first targeting portion and a second targeting portion. The first targeting portion can include HCDR1, HCDR2 and HCDR3. The HCDR3 can include the amino acid sequence shown in SEQ ID NO: 6 , the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 4, and the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 1; the first targeting part may comprise LCDR1, LCDR2 and LCDR3, so The LCDR3 may include the amino acid sequence shown in SEQ ID NO: 25, the LCDR2 may include the amino acid sequence shown in SEQ ID NO: 24, and the LCDR1 may include the amino acid sequence shown in SEQ ID NO: 23; The second targeting portion may include HCDR1, HCDR2 and HCDR3, the HCDR3 of the second targeting portion may include the amino acid sequence shown in SEQ ID NO: 14, and the HCDR2 of the second targeting portion may include SEQ ID NO: The amino acid sequence shown in 13, and the HCDR1 of the second targeting part can include the amino acid sequence shown in SEQ ID NO: 12; the second targeting part can include LCDR1, LCDR2 and LCDR3, and the second targeting part can include LCDR1, LCDR2 and LCDR3. The LCDR3 of the targeting portion may comprise the amino acid sequence shown in SEQ ID NO: 25, the LCDR2 of the second targeting portion may comprise the amino acid sequence shown in SEQ ID NO: 24, and the LCDR1 of the second targeting portion may Contains the amino acid sequence shown in SEQ ID NO: 23.

For example, the polypeptide of the present application can include a first targeting portion and a second targeting portion, the first targeting portion can include VH and VL, and the VH of the first targeting portion can include SEQ ID NO: 7 to The sequence shown in any one of 11, the VL of the first targeting part may include the sequence shown in SEQ ID NO: 26; the second targeting part may include VH and VL, the second targeting part may Part of the VH may include the sequence shown in SEQ ID NO: 15, and the VL of the second targeting part may include the sequence shown in SEQ ID NO: 26.

For example, the first targeting moiety is directly or indirectly linked to the second targeting moiety. For example, the polypeptides have a common light chain. For example, a polypeptide of the present application has multiple antibody light chain variable regions, one or more of which have the same sequence. For example, a polypeptide of the present application has multiple antibody light chains, one or more of which have the same antibody variable region sequence. For example, a polypeptide of the present application has multiple antibody light chains, one or more of which have the same sequence. For example, the polypeptide of the present application has multiple antibody light chains with identical light chain variable region sequences. For example, a polypeptide of the present application has multiple antibody light chains with identical sequences.

In this application, a part of each heavy chain or light chain amino acid sequence of the antigen-binding protein is homologous to the corresponding amino acid sequence in the antibody from a specific species, or belongs to a specific class. For example, the variable and constant portions of the light and heavy chains are derived from the variable and constant regions of an antibody from one animal species (eg, human). In this application, the homolog may be one that shares at least about 85% (e.g., Having at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or higher) sequence homology to a protein or polypeptide.

In this application, homology generally refers to similarity, similarity or association between two or more sequences. Alignment for the purpose of determining percent sequence homology can be accomplished in a variety of ways known in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. One skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms required to achieve maximal alignment within the full-length sequences being compared or within the sequence region of interest. The homology can also be determined by the following methods: FASTA and BLAST. A description of the FASTA algorithm can be found in "Improved Tools for Biological Sequence Comparison" by W.R. Pearson and D.J. Lipman, Proc. Natl. Acad. Sci., 85: 2444-2448, 1988; and D.J. Lipman and W.R. Pearson, "Fast and Sensitive Protein Similarity Searches," Science, 227: 1435-1441, 1989. A description of the BLAST algorithm can be found in "A basic local alignment search tool" by S. Altschul, W. Gish, W. Miller, E.W. Myers, and D. Lipman, Journal of Molecular Biology, 215: 403-410 , 1990.

In another aspect, the present application provides a nucleic acid encoding the antigen-binding protein of the present application and/or the polypeptide of the present application. For example, each of the one or more nucleic acid molecules may encode the entire antigen-binding protein or a portion thereof (e.g., HCDR1-3, LCDR1-3, VL, VH, light chain or one or more of the heavy chains).

In another aspect, the present application provides a vector, which may comprise the nucleic acid of the present application. In addition, other genes may be included in the vector, such as marker genes that allow selection of the vector in appropriate host cells and under appropriate conditions. In addition, the vector may contain expression control elements that allow correct expression of the coding region in an appropriate host. For example, the vector is an expression vector.

On the other hand, the present application provides a cell, which may comprise the antigen-binding protein of the present application, the polypeptide of the present application, and/or the nucleic acid of the present application. For example, each or each host cell may contain one or more nucleic acid molecules or vectors described herein. For example, each or each host cell may comprise multiple (eg, 2 or more) or multiple (eg, 2 or more) nucleic acid molecules or vectors described herein.

On the other hand, the present application provides a method for preparing the antigen-binding protein of the present application and/or the polypeptide of the present application, which method includes culturing under conditions that allow the expression of the antigen-binding protein and/or the polypeptide. cells according to the present application. For example, by using appropriate culture medium, appropriate temperature and culture time, etc., these methods are understood by those of ordinary skill in the art.

On the other hand, the present application provides a conjugate, which may comprise the antigen-binding protein of the present application and/ Or the polypeptide of the present application.

On the other hand, the present application provides a pharmaceutical composition, which may comprise the antigen-binding protein of the present application, the polypeptide of the present application, the nucleic acid of the present application, the vector of the present application, the cells of the present application, and/ or a conjugate of the present application, and optionally a pharmaceutically acceptable carrier. The pharmaceutically acceptable adjuvant is non-toxic to the recipient at the dosage and concentration used. The pharmaceutical composition in the present application may also contain more than one active compound, usually one that does not adversely affect each other's properties. Application active ingredients.

On the other hand, the present application provides a kit, which may include the antigen-binding protein of the present application, the polypeptide of the present application, the nucleic acid of the present application, the vector of the present application, the cells of the present application, and the conjugate of the present application. compounds, and/or pharmaceutical compositions of the present application.

On the other hand, the present application provides a method for influencing cells to produce cytokines. The method may comprise administering the antigen-binding protein of the present application, the polypeptide of the present application, the nucleic acid of the present application, the vector of the present application, and the cells of the present application. , the conjugate of the present application, the pharmaceutical composition of the present application, and/or the kit of the present application. For example, the method may be an ex vivo or in vitro method. For example, the method may be a non-therapeutic method.

For example, the cells may comprise lymphocytes. For example, T lymphocytes. For example, the cytokine may comprise an interleukin or/and a functionally active fragment thereof. For example, the cytokine may comprise interleukin 2 (IL-2) or/and functionally active fragments thereof.

On the other hand, the present application provides a method for promoting cell bridging, which method may comprise administering the polypeptide of the present application, the nucleic acid of the present application, the vector of the present application, the cell of the present application, the conjugate of the present application, the The pharmaceutical composition of the application, and/or the kit of the application. For example, the method may be an ex vivo or in vitro method. For example, the method may be a non-therapeutic method.

For example, the cell bridging can include reducing the distance between PD-L1 positive cells and PD-1 positive cells. For example, the cell bridging may include increasing the ratio of PD-L1 positive cells and PD-1 positive cells that are double positive, for example, the cell ratio may be detected by flow cytometry.

On the other hand, the present application provides an antigen-binding protein of the present application, a polypeptide of the present application, a nucleic acid of the present application, a vector of the present application, a cell of the present application, a conjugate of the present application, and a pharmaceutical composition of the present application. , and/or the use of the kit of the present application in the preparation of medicines for preventing, alleviating and/or treating tumors. For example, the tumor may comprise a PD-L1 high-expressing tumor and/or a PD-L1-positive tumor. For example, the tumor may comprise a solid tumor. For example, the tumor may comprise lung cancer. For example, "PD-L1 high-expressing tumors and/or PD-L1-positive tumors" generally refers to cancers or tumors that contain cancer cells with higher than normal PD-L1 levels.

On the other hand, the present application provides an antigen-binding protein of the present application, a polypeptide of the present application, a nucleic acid of the present application, The vector of the present application, the cells of the present application, the conjugate of the present application, the pharmaceutical composition of the present application, and/or the kit of the present application can be used to prevent, alleviate and/or treat tumors. For example, the tumor may comprise a PD-L1 high-expressing tumor and/or a PD-L1-positive tumor. For example, the tumor may comprise a solid tumor. For example, the tumor may comprise lung cancer.

On the other hand, the present application provides a method for preventing, alleviating and/or treating tumors, which includes administering the antigen-binding protein of the present application, the polypeptide of the present application, the nucleic acid of the present application, the vector of the present application, and the cells of the present application. , the conjugate of the present application, the pharmaceutical composition of the present application, and/or the kit of the present application. For example, the tumor may comprise a PD-L1 high-expressing tumor and/or a PD-L1-positive tumor. For example, the tumor may comprise a solid tumor. For example, the tumor may comprise lung cancer.

Without intending to be limited by any theory, the following examples are only to illustrate the proteins, preparation methods and uses of the present application, and are not intended to limit the scope of the invention of the present application.

Example

The sequence information for this application can look like this:

Example 1 Phage display library construction and screening

In order to construct a common light chain double antibody against PD-1 and PD-L1, based on the existing anti-PD-1 monoclonal antibody 41D2HzL4H3 (can be found in the application number PCT/CN2022/075593 submitted on February 21, 2022 ), select its light chain sequence and recombine it with the antibody heavy chain variable region (VH) sequence amplified from rat spleen cells immunized with human PD-L1 to construct a common light chain phage antibody single chain (scFv) library. Using a phage antibody single-chain library, two rounds of panning were performed against biotin-labeled human PD-L1 (hereinafter referred to as h PD-L1-Biotin, Acro, Catalog No.: PD1-H82E5), and positive enrichment was obtained. The enriched phage was infected with Escherichia coli strain SS320 (purchased from Lucigen) to prepare phage plasmid.

Example 2 Yeast display library construction and screening

Use the panned phage plasmid as a template to design primers for polymerase chain reaction (PCR) to amplify scFv or VH and VK gene fragments; the PCR-amplified scFv gene fragments are recovered and co-transfected with the yeast display plasmid into the Saccharomyces cerevisiae strain EBY100 (purchased from ATCC), the scFv is inserted into the yeast display plasmid through homologous recombination of Saccharomyces cerevisiae, thereby displaying single-chain antibodies on the surface of the yeast cell wall. The constructed library number is JYYDL168; the PCR amplified VH and VK gene fragments are recovered Then, it was co-transfected into Saccharomyces cerevisiae strain EBY100 with the yeast display plasmids pJYY132-Y and pJYY129-X to construct a yeast display library displaying Fab-form antibodies, library number JYYDL169. After electroporation, the libraries JYYDL168 and JYYDL169 were cultured overnight in 100 mL of SD-Trp medium (Clontech, Cat. No.: 630308) and SD-Trp-Leu (Clontech, Cat. No.: 630316) at 30°C and 225 rpm; 1.0× of each was taken. Resuspend 10 ⁸ bacteria in 20 mL YPGP induction medium (2% galactose, 2% peptone, 1% yeast extract, 0.54% Na ₂ HPO ₄ , 0.86% NaH ₂ PO ₄ ·H ₂ O), 20°C, Incubate at 225 rpm for 24 hours and place in a 4°C refrigerator until use.

After library induction, measure the OD ₆₀₀ of the bacterial solution. Calculate the number of cells based on 1OD as 1.0×10 ⁷ . Take 3.0×10 ⁷ cells each for JYYDL168 and JYYDL169 and perform flow staining and sorting according to the following steps: 1. Use 1mL 1× Wash once with PBSA (1×PBS+1% BSA), centrifuge at 3000 rpm for 3 minutes (the following centrifugation conditions are all under these conditions) and discard the supernatant; 2. Add 1000 μL of 1×PBSA containing 100 nM h PD-L1-Biotin to each tube. , incubate at room temperature for 30 minutes, centrifuge to remove the supernatant; 3. Add 1mL 1×PBSA and centrifuge to remove the supernatant, add 1000μL 1×PBSA containing 300nM Tab2 (anti-PD-L1 antibody prepared in-house) to each tube, and incubate at room temperature for 30 minutes. Centrifuge to remove the supernatant; 4. Add 1mL 1× PBSA, centrifuge to remove the supernatant, and add 1000 μL to each tube containing Mouse Anti Human IgG Fc-APC (Biolegend product number: 409306, 1:400 volume ratio dilution), SA-PE (eBioscience, product number : 12-4317-8, diluted 1:200) in 1×PBSA, incubate at room temperature for 20 minutes, and centrifuge to remove the supernatant; 5. Add 1mL of 1×PBSA and centrifuge to remove the supernatant; 6. Add 2mL of 1×PBSA to resuspend the cells. , perform flow cytometry sorting to collect cell populations with weak APC and strong PE signals; 7. After sorting, cells are spread on corresponding auxotrophic plates and cultured statically at 30°C for 3 days.

Example 3 Sequencing and flow cytometric staining identification of monoclonal yeast colonies

From the monoclonal plates of the JYYDL168 and JYYDL169 rounds of screening products, 92 monoclones were selected for sequencing. Finally, 26 unique antibody sequences were obtained from the JYYDL168 library and 14 unique antibody sequences were obtained from the JYYDL169 library. Perform flow cytometry staining analysis on the corresponding yeast monoclonal colonies, and take 1×10 ⁶ cells each for staining according to the scheme in Table 1. The strength of the combination of Scheme 1 with h PD-L1-Biotin is determined by the PE mean fluorescence signal intensity (MFI) It reflects that the stronger the PE fluorescence intensity, the stronger the binding force between the surface and human PD-L1; similarly, scheme 2 reflects the binding strength of the clone to Cyno PD-L1-Bio, and scheme 3 reflects the binding strength of the clone to irrelevant antigens. , proving the binding specificity of the single clone; Scheme 4 uses Tab2 as a control to perform competitive flow staining to analyze whether the epitopes of each clone and Tab2-binding antigen are the same. The stronger the APC signal, the better the clone and the control antibody bind to the antigen. The epitopes are different, whereas the binding epitopes are the same. After each single clone was stained and analyzed according to the four schemes in Table 1, clones with strong signals in Schemes 1 and 2 were selected, and clones with strong signals in Schemes 3 and 4 were removed. The results are shown in Table 2.

Table 1 Scheme for identification of monoclonal yeast colonies by flow cytometry

Table 2 Flow cytometric staining analysis results of yeast monoclonal colonies

Example 4 Expression of candidate antibodies

According to the results of Example 3, the VH sequences of Y82B4 and Y82F52 clones were selected to construct the IgG1 LALA subtype, and were paired with the light chain plasmid of 41D2HzL4H3 respectively for expression and purification. The candidate antibody numbers were Ab1910T21 and Ab1910T22. The expression results are shown in Table 3 .

Table 3 Candidate antibody expression and purification data

Example 5 Candidate Antibody Binding Assay

In order to determine the binding of Atezolimumab, Ab1910T21, and Ab1910T22 antibodies to human and monkey PD-L1, Octet RED96e (Fortébio) was used to determine the binding of candidate antibodies to human PD-L1 (Acro, Cat. No.: PD1-H5229) and monkey PD-L1 (Acro, Catalog number: PD1-C52H4) affinity. The antigen and antibody were diluted with 1×PBST (1×PBS: Sangon, B548117-0500; 0.02% Tween 20: sigma, P1379). The concentration of human PD-L1 was 50nM, the concentration of monkey PDL1 was 30nM, and the antibody was used The concentrations are all 33.3nM. Add the candidate antibody sample to a 96-well plate (Greiner bio-one, 655209) at 200 μL/well, set the software parameters, the temperature is 30°C, and the frequency for collecting standard kinetic signals is 5.0Hz; pre-wet the AHC sensor (Fortébio) with 1×PBST , item number: 18-0015) for 10 minutes, and then go on the machine for testing. Each cycle includes the following steps: 1) Immerse in buffer for 60 seconds; 2) Detect whether the antigen binds non-specifically to the sensor; 3) Regenerate with 10mM glycine solution at pH 1.7; 4) Immerse in buffer for 60 seconds; 5) Antibody solidifies on On the sensor, the time is 23s; 6) The sensor is immersed in the buffer for 180s; 7) The antigen and antibody are combined, the time is 180s; 8) The dissociation of the antigen and antibody, the time is 10 minutes; 9) The sensor is regenerated. For the 1:1 binding mode of antigen-antibody, the association rate (K _on ) and dissociation rate (K _off ) were measured to calculate the equilibrium dissociation constant (K _D ) of the antibody. The results are shown in Table 4 and Table 5 respectively. It can be seen from the table that the antibodies Ab1910T21 and Ab1910T22 of the present application have relatively strong affinity to human or monkey PD-L1.

Table 4 Affinity determination of candidate antibodies and human PD-L1

Table 5 Affinity determination of candidate antibodies and monkey PD-L1

Example 6 In vitro functional evaluation of candidate antibodies

Further evaluate the activity of candidate antibodies Ab1910T21, Ab1910T22 and the reference antibody Atezolizumab in activating mixed lymphocytes to release IL-2, as follows:

Use cell culture medium (1640+2% FBS) to revive human dendritic DC cells, and adjust the DC cell density to 1×10 ⁵ - 1×10 ⁷ cells/mL, then add mitomycin C with a final concentration of 50 μg/mL, treat in the dark at 37 degrees for 30 minutes, add 10 mL of medium to terminate, centrifuge at 400g for 10 minutes, and then wash once with 10 mL of medium. Gradient dilution of anti-PD-L1 antibody: The highest final concentration of the antibody is 2.5 μg/mL (prepared concentration is 10 μg/mL), 10-fold gradient dilution (5 concentration points + 1 0 concentration point), and then the corresponding cell culture plate (Corning , Cat. No.: 3599), add 50 μL of prepared anti-PD-L1 antibody. Collect DC cells treated with human peripheral blood lymphocytes PBMC and mitomycin C, adjust the DC cell density to 2×10 ⁵ cells/mL, and then add cells to the culture plate at 50 μL per well, that is, the number of DC cells is 1 ×10 ⁴ cells/well; adjust the PBMC cell density to 2×10 ⁶ cells/mL, and then add cells to the culture plate at 100 μL per well, that is, the number of PBMC cells is 2×10 ⁵ cells/well. Place the cell culture plate in a 37°C, 5% carbon dioxide cell culture incubator for 3 days. After 3 days, centrifuge at 300g for 5 minutes. Collect the supernatant and use the Human IL-2 ELISA kit (Novus, product number: VAL110) to detect the IL-2 content. The detection method was carried out strictly in accordance with the instructions of the kit, and the results are shown in Figure 1. As can be seen from Figure 1, the Ab1910T21 and Ab1910T22 antibodies of the present application have better activity in activating mixed lymphocytes to release IL-2 than the control antibody Atezolizumab. All antibodies will continue to be evaluated after humanization.

Example 7 Humanization of murine antibodies

Through sequence comparison of the VH of Ab1910T21 and Ab1910T22 candidate antibodies, the human Antibody Germline Gene (Data source: IMGT) with the highest homology was selected as the humanization design framework. The heavy chain is framed by IGHV1-2*06 or IGHV1-46*01, IGHJ4*01. The antibody variable regions are numbered according to Chothia rules, the antibody CDR regions are defined according to Chothia, and the amino acids in the antibody heavy chain variable region are humanized based on sequence alignment and variable region structural information. After humanization of Ab1910T21 and Ab1910T22, the VH sequences T21hzH3 and T22hzH2 were constructed into IgG1 LALA subtypes respectively, which were paired with the light chain plasmid of 41D2HzL4H3 for expression and purification. The candidate antibody numbers and expression results are shown in Table 6.

Table 6 Humanized antibody expression and purification data

Example 8 Humanized Antibody Affinity Determination

The affinity determination of Ab1910T21, Ab1910T22 antibodies before and after humanization and the control antibody Atezolizumab with human and monkey PD-L1 was the same as in Example 4. The results are shown in Table 7 and Table 8 respectively.

Table 7 Affinity determination of humanized antibodies and human PD-L1

Table 8 Affinity determination of humanized antibodies and monkey PD-L1

Example 9 In vitro functional evaluation of humanized antibodies

It can be seen from Example 8 that the humanized antibodies 2005K4T21hz3 and 2005K4T22hz2 have strong affinity with human or monkey PD-L1. The same as Example 5, we further evaluated the ability of the antibodies before and after humanization and the reference antibody Atezolizumab to activate mixed lymphocytes to release IL-2. Activity, the results are shown in Figures 2 and 3. Figure 2 shows that Ab1910T22 humanized antibody 2005K4T22hz2 still has the function of enhancing T cell activation and releasing IL-2 in vitro after humanization; Figure 3 shows that Ab1910T21 humanized antibody 2005K4T21hz3 still has the function of enhancing T cells in vitro after humanization. The function of T cell activation and release of IL-2, among which the function of 2005K4T21hz3 is better than that of the benchmark antibody Atezolizumab.

Example 10 Design and expression of co-light chain bispecific antibodies

Based on the results of Examples 8 and 9, a co-light chain double antibody was constructed using the 2005K4T21hz3 and 2005K4T22hz2 humanized antibody VH sequences and the 41D2HzL4H3 antibody sequence. IgG1 LALA D265S and knob and Hole designs were selected, as shown in Figure 4; among them, the Hole of BAb2005.05 The CDR2 in the sequence contains the PTM site of NS, which was mutated into NY, and the antibody was named BAb2005.05.2. All antibodies were expressed and purified. The antibody numbers and expression results are shown in Table 9.

Table 9 Co-light chain bispecific antibody expression

Example 11 Co-light chain bispecific antibody affinity determination

Select the co-light chain bispecific antibodies BAb2005.04, BAb2005.05, BAb2005.05.2 and the maternal PD-L1 monoclonal antibody Ab2005-H.1 (same as 2005K4T22hz2) and the maternal PD-1 monoclonal antibody Ab2005-K (same as 41D2HzL4H3 ), and the PD-L1 control antibody Atezolizumab and PD-1 control antibody Pembrolizumab were measured against human PD-L1, monkey PD-L1 or human PD-1 (Acro, Cat. No.: PD1-H5221), monkey PD-1 (Acro, (Product number: PD1-C5223), in which the concentration of human PD-1 and monkey PD-1 were both 100 nM. The remaining method steps were completely the same as in Example 4. The results are shown in Tables 10 to 13 respectively. Table 10 shows that the affinity of co-light chain bispecific antibodies BAb2005.04, BAb2005.05, and BAb2005.05.2 to human PDL1 is equivalent to that of the parent PD-L1 monoclonal antibody Ab2005-H.1, but weaker than the control antibody Atezolizumab. Table 11 shows that the affinity of BAb2005.04, BAb2005.05, and BAb2005.05.2 to monkey PDL1 is equivalent to the parent PD-L1 monoclonal antibody Ab2005-H.1 and stronger than the control antibody Atezolizumab. Table 12 shows that the affinity of BAb2005.04, BAb2005.05, and BAb2005.05.2 to human PD1 is equivalent to the parent PD-1 monoclonal antibody Ab2005-K and stronger than the control antibody Pembrolizumab. Table 13 shows that the affinity of BAb2005.04, BAb2005.05, and BAb2005.05.2 to monkey PD1 is equivalent to the parent PD-1 monoclonal antibody Ab2005-K, but weaker than the control antibody Pembrolizumab.

Table 10 Affinity determination of co-light chain bispecific antibodies and human PD-L1

Table 11 Affinity determination of co-light chain bispecific antibodies and monkey PD-L1

Table 12 Affinity determination of co-light chain bispecific antibodies and human PD-1

Table 13 Affinity determination of co-light chain bispecific antibodies and monkey PD-1

Example 12 Co-light chain bispecific antibodies can stimulate mixed lymphocytes to release IL-2

Same as Example 5, further evaluate the co-light chain bispecific antibodies BAb2005.04, BAb2005.05, BAb2005.05.2 and the maternal PD-L1 monoclonal antibody Ab2005-H.1 and the maternal PD-1 monoclonal antibody Ab2005-K, And the PD-L1 control antibody Atezolizumab and PD-1 control antibody Pembrolizumab activate the activity of mixed lymphocytes to release IL-2. The results are shown in Figure 5, BAb2005.04, BAb2005.05, BAb2005.05.2 and the parent PD-L1 monoclonal antibody Ab2005. -H.1 and the parent PD-1 monoclonal antibody Ab2005-K have the function of enhancing T cell activation and releasing IL-2 in vitro, and their functions are better than the PD-L1 control antibody Atezolizumab and the PD-1 control antibody Pembrolizumab.

Example 13 Co-light chain bispecific antibody has cell bridging function

In addition, the cell bridging function method was used to evaluate the co-light chain bispecific antibodies BAb2005.04, BAb2005.05, BAb2005.05.2 and the maternal PD-L1 monoclonal antibody Ab2005-H.1 and the maternal PD-1 monoclonal antibody Ab2005-K. To close the distance between CHOK1-PD-L1 and CHOK1-PD-1 cells, the specific steps are as follows:

First, CHOK1-PD-1 was stained with 3 μM CFSE dye (Invitrogen, Cat. No.: C34554), and CHOK1-PD-L1 cells were stained with 1 μM CellTrace ^TM Red dye (Invitrogen, Cat. No.: C34564). The specific staining method is: The cells were centrifuged at 400 g for 10 min, and the cell pellet was resuspended in suspension (PBS+2% FBS). Then filter through a 40 μm mesh, adjust the cell density to 1x 10 ⁷ cells/mL, add an equal volume of the corresponding 2-fold concentration dye, and mix immediately upon addition. After thorough mixing, shake with a vibrator for 3 seconds, stain in a water bath at 37 degrees Celsius for 5 minutes, and then use 10 times the volume of solution (PBS+10% FBS) to terminate the staining, which requires a water bath at 37 degrees Celsius for 5 minutes. Then, centrifuge at 400 g for 10 min and wash twice with the solution. Finally, the cell pellet was resuspended in experimental medium (PBS+1% BSA), and the cell density was adjusted to 8.33×10 ⁶ cells/mL, 60 μL per well, that is, 5×10 ⁵ cells/well.

Then, use culture medium to dilute the antibody in a gradient: the highest final concentration of the antibody is 22.5 μg/mL (prepared concentration is 45 μg/mL), 3-fold gradient dilution (11 concentration points + 1 0 concentration point), and simultaneously dilute the parent PD- 1. The final concentration of monoclonal antibody Ab2005-K and negative control antibody anti-HEL Human IgG1 is 90 μg/mL (prepared concentration is 180 μg/mL).

Next, for the PD-L1 side, add the prepared cells and antibodies in equal volumes (i.e., 60 μL cells + 60 μL antibodies) to the corresponding cell culture plate (Corning, Cat. No. 3799). Specifically, the wells on the PD-L1 side are CHOK1-PD-L1+ serially diluted co-light chain bispecific antibodies, and the control wells are CHOK1-PD-L1+ gradiently diluted parent monoclonal antibody combination. In addition, for the PD-1 side, add the prepared cells and antibodies to the corresponding cell culture plate as 60μL cells + 150μL antibody + 90μL medium. The specific setting is CHOK1-PD-1+Ab2005-K/anti-HEL Human IgG1. Cells and antibodies were added to the cell culture plate, mixed and incubated at 4°C for 2 hours.

Finally, add the cell and antibody mixture on the PD-L1 side and the cell and antibody mixture on the PD-1 side to the new cell culture plate at a ratio of 1:5, mix and incubate at 4 degrees Celsius for 48 hours. The samples were used to detect the proportion of double-positive cells using a flow cytometer (Beckman, model: cytoflex). The results are shown in Figure 6. The co-light chain bispecific antibodies BAb2005.04, BAb2005.05, and BAb2005.05.2 can shorten the distance between CHOK1-PD-L1 and CHOK1-PD-1 cells. Therefore, the proportion of double-positive cells changes with the double-antibody increases with increasing concentration. However, adding the maternal PD-L1 monoclonal antibody Ab2005-H.1 and the maternal PD-1 monoclonal antibody Ab2005-K at the same time does not have a cell bridging function, and adding the maternal PD-1 monoclonal antibody Ab2005.05 at the same time Anti-Ab2005-K can block the cell bridging function of the double antibody.

Example 14 Pharmacodynamic study of co-light chain bispecific antibody in human lung cancer NCI-H292 mouse tumor model

NPG mice were purchased from Beijing Weitongda Animal Breeding Co., Ltd., 5 weeks old, female, 56 in total. Frozen PBMC were purchased from Miaoshun (Shanghai) Biotechnology Co., Ltd. NCI-H292 human lung cancer cells were purchased from ATCC and cultured in RPMI-1640 medium supplemented with 10% FBS in a 37°C incubator containing _CO2 . Before the cells were continuously cultured for ten generations, approximately 4×10 ⁶ NCI-H292 cells were suspended in 50 μL PBS and an equal volume of 1×10 ⁶ PBMC cells were mixed, and then mixed with an equal volume of Matrigel, and inoculated into the The inoculation volume was 200 μL per mouse. On the day of inoculation, 56 mice were randomly divided into 7 groups according to their weight, with 8 mice in each group. The day of group administration was defined as day 0. Group G1 was administered with isotype control, group G2 was administered with Ab2005-K at a dose of 5 mg/kg; group G3 was administered with Ab2005-H.1 at a dose of 5 mg/kg; group G4 was administered with Ab2005-K combined with Ab2005-H.1 at a dose of 5mg/kg+5mg/kg; G5 group was administered BAb2005.05, dose 5mg/kg; G6 group was administered BAb2005.05.2, dose 5mg/kg; G7 group was administered BAb2005.04, dose 5mg/kg. All administration methods were intraperitoneal injection, twice a week, for a total of 6 administrations.

On the 30th day after grouping, the average tumor volume of group G1 was 688.30±50.11mm ³ and that of group G2 The average tumor volume of the G3 group was 6.00±6.00mm ³ , the average tumor volume of the G3 group was 8.55±8.55mm ³ , the average tumor volume of the G4 group was 13.65±6.61mm ³ , the average tumor volume of the G5 group was 6.16±4.20mm ³ , and the average tumor volume of the G6 group was 6.00±6.00mm 3 . The tumor volume was 5.80±5.80mm ³ , and the average tumor volume in the G7 group was 0.00±0.00mm ³ . The G2 group has a tumor inhibition rate of 101.45%, the G3 group has a tumor inhibition rate of 101.45%, the G4 group has a tumor inhibition rate of 100.87%, the G5 group has a tumor inhibition rate of 101.69%, and the G6 group has a tumor inhibition rate of 101.21%. The G7 group had a tumor inhibition rate of 101.50%. The G2 and G3 groups had extremely significant statistical differences compared with the G1 group, G2 vs G1, P <0.001; G3 vs G1, P < 0.001. It was proved that both Ab2005-K and Ab2005-H.1 single drugs can significantly inhibit tumor growth. There was a very significant statistical difference between the G4 group and the G1 group, G4 vs G1, P<0.001. Compared with the G1 group, the G5, G6 and G7 groups all had statistical differences, G5 vs G1, P<0.001; G6 vs G1, P<0.001; G7 vs G1, P<0.001. It was proved that BAb2005.05, BAb2005.05.2 and BAb2005.04 can significantly inhibit tumor growth. The results are shown in Figure 7.

The foregoing detailed description is provided by way of explanation and example, and is not intended to limit the scope of the appended claims. Various modifications to the embodiments described herein will be apparent to those of ordinary skill in the art and remain within the scope of the appended claims and their equivalents.

Claims

An antigen-binding protein comprising an antibody heavy chain variable region VH, the VH comprising HCDR1, HCDR2 and HCDR3, and the HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 6.
The antigen-binding protein as claimed in claim 1, said antigen-binding protein comprising an antibody heavy chain variable region VH, said VH comprising HCDR1, HCDR2 and HCDR3, and said HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 5 .
The antigen-binding protein of any one of claims 1-2, said antigen-binding protein comprising an antibody heavy chain variable region VH, said VH comprising HCDR1, HCDR2 and HCDR3, and said HCDR2 comprising selected from the following group Amino acid sequence shown: SEQ ID NO: 2 to 4.
The antigen-binding protein of any one of claims 1-3, said antigen-binding protein comprising an antibody heavy chain variable region VH, said VH comprising HCDR1, HCDR2 and HCDR3, and said HCDR1 comprising SEQ ID NO: The amino acid sequence shown in 1.
The antigen-binding protein according to any one of claims 1-4, wherein the antigen-binding protein comprises an antibody heavy chain variable region VH, and the VH comprises an amino acid sequence selected from the following group: SEQ ID NO: 7 to 11.
The antigen-binding protein of any one of claims 1-5, which comprises an antibody heavy chain constant region CH, and the CH is derived from an IgG constant region.
The antigen-binding protein of any one of claims 1-6, which comprises an antibody heavy chain constant region CH, and the CH is derived from an IgG1 constant region.
The antigen-binding protein according to any one of claims 1-7, said antigen-binding protein comprising an antibody heavy chain constant region CH, and said CH comprising an amino acid sequence selected from the following group: SEQ ID NO: 16 to 18.
The antigen-binding protein of any one of claims 1-8, wherein the antigen-binding protein comprises an antibody heavy chain, and the heavy chain comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 19 to 21 .
The antigen-binding protein of any one of claims 1-9, wherein the antigen-binding protein comprises an antibody light chain variable region VL, the VL comprises LCDR1, LCDR2 and LCDR3, and the LCDR3 comprises SEQ ID NO: The amino acid sequence shown in 25.
The antigen-binding protein of any one of claims 1-10, said antigen-binding protein comprising an antibody light chain variable region VL, said VL comprising LCDR1, LCDR2 and LCDR3, and said LCDR2 comprising SEQ ID NO: The amino acid sequence shown in 24.
The antigen-binding protein of any one of claims 1-11, said antigen-binding protein comprising an antibody light chain variable region VL, said VL comprising LCDR1, LCDR2 and LCDR3, and said LCDR1 comprising SEQ ID NO: twenty three The amino acid sequence shown.
The antigen-binding protein according to any one of claims 1-12, wherein the antigen-binding protein comprises an antibody light chain variable region VL, and the VL comprises the amino acid sequence shown in SEQ ID NO: 26.
The antigen-binding protein according to any one of claims 1-13, wherein the antigen-binding protein comprises an antibody light chain constant region CL, and the CL comprises the amino acid sequence shown in SEQ ID NO: 27.
The antigen-binding protein according to any one of claims 1-14, wherein the antigen-binding protein comprises an antibody light chain, and the light chain comprises the amino acid sequence shown in SEQ ID NO: 28.
The antigen-binding protein according to any one of claims 1-15, which is capable of binding PD-L1 or a functionally active fragment thereof.
The antigen-binding protein of claim 16, wherein the PD-L1 comprises PD-L1 derived from humans and/or monkeys.
The antigen-binding protein of any one of claims 1-17, which comprises an antibody or an antigen-binding fragment thereof.
The antigen-binding protein of claim 18, wherein the antibody is selected from the group consisting of murine antibodies, chimeric antibodies, humanized antibodies and fully human antibodies.
The antigen-binding protein according to any one of claims 18-19, the antigen-binding fragment is selected from the following group: Fab, Fab', F(ab) 2 , Fv fragment, F(ab') 2 , scFv, di-scFv, VHH and dAb.
A polypeptide comprising a first targeting moiety comprising the antigen-binding protein of any one of claims 1-20.
The polypeptide of claim 21, further comprising a second targeting moiety capable of binding PD-1 or a functionally active fragment thereof.
The polypeptide of claim 22, wherein the PD-1 comprises PD-1 derived from humans and/or monkeys.
The polypeptide of any one of claims 22-23, said second targeting moiety comprises an antibody or antigen-binding fragment thereof.
The polypeptide of claim 24, wherein the antibody is selected from the group consisting of murine antibodies, chimeric antibodies, humanized antibodies and fully human antibodies.
The polypeptide of any one of claims 24-25, wherein the antigen-binding fragment is selected from the group consisting of: Fab, Fab', F(ab) 2 , Fv fragment, F(ab') 2 , scFv, di- scFv, VHH and dAb.
The polypeptide of any one of claims 22-26, the second targeting portion comprises an antibody heavy chain variable region VH, the VH of the second targeting portion comprises HCDR1, HCDR2 and HCDR3, and the of the second targeting part HCDR3 contains the amino acid sequence shown in SEQ ID NO:14.
The polypeptide of any one of claims 22-27, the second targeting portion comprises an antibody heavy chain variable region VH, the VH of the second targeting portion comprises HCDR1, HCDR2 and HCDR3, and the The second targeting portion of HCDR2 contains the amino acid sequence shown in SEQ ID NO: 13.
The polypeptide of any one of claims 22-28, the second targeting portion comprises an antibody heavy chain variable region VH, the VH of the second targeting portion comprises HCDR1, HCDR2 and HCDR3, and the The second targeting portion of HCDR1 contains the amino acid sequence shown in SEQ ID NO: 12.
The polypeptide of any one of claims 22-29, the second targeting portion includes the antibody heavy chain variable region VH, and the VH of the second targeting portion includes SEQ ID NO: 15 Amino acid sequence.
The polypeptide of any one of claims 22-30, the second targeting moiety comprises an antibody heavy chain constant region CH, and the CH of the second targeting moiety is derived from an IgG constant region.
The polypeptide of any one of claims 22-31, the second targeting moiety comprises an antibody heavy chain constant region CH, and the CH of the second targeting moiety is derived from an IgGl constant region.
The polypeptide of any one of claims 22-32, the second targeting portion comprises an antibody heavy chain constant region CH, and the CH of the second targeting portion comprises an amino acid sequence selected from the group shown below :SEQ ID NO: 16 to 18.
The polypeptide of any one of claims 22-33, the second targeting portion includes an antibody heavy chain, and the heavy chain of the second targeting portion includes the amino acid sequence shown in SEQ ID NO: 22.
The polypeptide of any one of claims 22-34, the second targeting portion comprises an antibody light chain variable region VL, the VL of the second targeting portion comprises LCDR1, LCDR2 and LCDR3, and the LCDR3 of the second targeting portion includes the amino acid sequence shown in SEQ ID NO: 25.
The polypeptide of any one of claims 22-35, the second targeting portion comprises an antibody light chain variable region VL, the VL of the second targeting portion comprises LCDR1, LCDR2 and LCDR3, and the The second targeting portion of LCDR2 contains the amino acid sequence shown in SEQ ID NO: 24.
The polypeptide of any one of claims 22-36, the second targeting portion comprises an antibody light chain variable region VL, the VL of the second targeting portion comprises LCDR1, LCDR2 and LCDR3, and the The second targeting portion of LCDR1 contains the amino acid sequence shown in SEQ ID NO: 23.
The polypeptide of any one of claims 22-37, the second targeting portion includes the antibody light chain variable region VL, and the VL of the second targeting portion includes SEQ ID NO: 26 Amino acid sequence.
The polypeptide of any one of claims 22-38, the second targeting portion includes the antibody light chain constant region CL, and the CL of the second targeting portion includes the amino acid shown in SEQ ID NO: 27 sequence.
The polypeptide of any one of claims 22-39, the second targeting portion includes an antibody light chain, and the light chain of the second targeting portion includes the amino acid sequence shown in SEQ ID NO: 28.
The polypeptide of any one of claims 22-40, wherein the first targeting moiety is directly or indirectly connected to the second targeting moiety.
The polypeptide of any one of claims 21-41, having a common light chain.
A nucleic acid encoding the antigen-binding protein described in any one of claims 1-20 and/or the polypeptide described in any one of claims 21-42.
A vector comprising the nucleic acid of claim 43.
A cell comprising the antigen-binding protein of any one of claims 1-20, the polypeptide of any one of claims 21-42, and/or the nucleic acid of claim 43.
A method for preparing the antigen-binding protein according to any one of claims 1-20 and/or the polypeptide according to any one of claims 21-42, said method comprising: making the antigen-binding protein and/or Or the cell according to claim 45 is cultured under conditions for expression of the polypeptide.
A conjugate comprising the antigen-binding protein of any one of claims 1-20 and/or the polypeptide of any one of claims 21-42.
A pharmaceutical composition comprising the antigen-binding protein of any one of claims 1-20, the polypeptide of any one of claims 21-42, the nucleic acid of claim 43, The vector of claim 44, the cell of claim 45, and/or the conjugate of claim 47, and optionally a pharmaceutically acceptable carrier.
A kit comprising the antigen-binding protein of any one of claims 1-20, the polypeptide of any one of claims 21-42, the nucleic acid of claim 43, The vector of claim 44, the cell of claim 45, the conjugate of claim 47, and/or the pharmaceutical composition of claim 48.
A method of affecting cells to produce cytokines, the method comprising administering the antigen-binding protein described in any one of claims 1-20, the polypeptide described in any one of claims 21-42, or the method described in claim 43 The nucleic acid of claim 44, the cell of claim 45, the conjugate of claim 47, the pharmaceutical composition of claim 48, and/or the kit of claim 49 .
The method of claim 50, said cells comprise lymphocytes.
The method of any one of claims 50-51, wherein the cytokine comprises an interleukin or/its functionally active fragment.
The method of any one of claims 50-52, wherein the cytokine comprises interleukin 2 (IL-2) or/its function active fragment.
A method for promoting cell bridging, the method comprising administering the polypeptide of any one of claims 21-42, the nucleic acid of claim 43, the vector of claim 44, and the cell of claim 45 , the conjugate of claim 47, the pharmaceutical composition of claim 48, and/or the kit of claim 49.
The method of claim 54, said cell bridging comprising reducing the distance between PD-L1 positive cells and PD-1 positive cells.
The antigen-binding protein of any one of claims 1-20, the polypeptide of any one of claims 21-42, the nucleic acid of claim 43, the vector of claim 44, the vector of claim 45 The use of the cell described in claim 47, the pharmaceutical composition described in claim 48, and/or the kit described in claim 49 in the preparation of a medicine, the medicine being used for prevention, alleviation and/or treat tumors.
The use according to claim 56, wherein the tumor comprises a PD-L1 high-expressing tumor and/or a PD-L1-positive tumor.
The use of any one of claims 56-57, wherein the tumor comprises a solid tumor.
The use of any one of claims 56-58, wherein the tumor comprises lung cancer.