Anti-CD28 antibody and its uses

Abstract

An antibody or an antigen-binding fragment thereof. The antibody or its antigen-binding fragment may specifically bind to a CD28 protein, such as the human CD28 protein. Additionally, a multispecific binding molecule, such as a multispecific antibody that specifically binds to CD28 and one or more tumor-associated antigens, is provided. A nucleic acid molecule encoding the antibody, an expression vector for expressing the antibody, a host cell, and a method for preparing the same are further provided. Additionally, diagnostic and therapeutic methods utilizing the antibody are provided.

Description

Technology Field

[0001] Cross-reference regarding related applications

[0002] This application is based on and claims priority to Chinese Patent Application No. 202410047645.4 filed on January 12, 2024, the entirety of which is incorporated herein by reference.

[0003] The present invention provides an antibody or an antigen-binding fragment thereof capable of specifically binding to a CD28 protein, e.g., a human CD28 protein. The present invention also provides a multispecific binding molecule, e.g., a multispecific antibody, that specifically binds to CD28 and one or more tumor-associated antigens. The present invention further provides a nucleic acid molecule encoding an antibody, an expression vector for expressing the antibody, a host cell, and a method for producing the same. The present invention also provides diagnostic and therapeutic methods utilizing the antibody of the present invention. Background Technology

[0004] T cell activation involves the stimulation of highly specific T cell receptors (TCRs) by antigen-presenting cells, such as dendritic cells, which present their specific antigens on class II major histocompatibility (MHC) complexes, and can also be promoted by co-stimulatory molecules such as CD28. CD28 is a 44 kDa disulfide-coupled homomeric receptor glycosylated at five different sites. CD28 is expressed on T cells (95% of resting CD4+ cells and 50% of resting CD8+ T cells in human peripheral blood) and plasmablasts, providing co-stimulatory signals necessary for T cell activation and survival. Co-stimulation occurs when CD28 on the surface of T cells binds to CD80 (B7-1) and CD86 (B7-2) on antigen-presenting cells. CD80 expression is upregulated upon activation of antigen-presenting cells (APCs), whereas CD86 is constitutively expressed in APCs. CD28 co-stimulation of helper T cells enhances the transcription of IL2R and IL2, leading to T cell proliferation; induces the expression of Bcl XL, leading to T cell survival; and increases the production of IL-4 (which leads to Th2 differentiation), IFNγ, IL-1, TNF, IL-5, various chemokines, and their receptors.

[0005] In addition, CD28 induces the expression or upregulation of several other co-stimulatory and regulatory molecules, including ICOS, 4-1BB, and CTLA-4, as well as CD40L, which is essential for T cell and B cell interaction.

[0006] Several monoclonal antibodies against CD28, such as TGN1412 (Tegenero, US20060188493A1), are known in the art. This is a CD28 super-agonist monoclonal antibody that preferentially activates regulatory T (Treg) cells in the absence of TCR co-stimulation. However, when this antibody was used for therapeutic purposes involving T cell activation, a Phase I clinical trial resulted in rapid multi-organ failure, namely severe cytokine release syndrome (cytokine storm), in all six volunteers.

[0007] Therefore, there is still a need in the industry to develop safe anti-CD28 antibodies suitable for use as therapeutic agents, as well as multispecific antigen-binding molecules constructed based thereon that specifically bind to CD28 and other target antigens.

[0008] One aspect of the present invention relates to an anti-CD28 antibody or an antigen-binding fragment thereof.

[0009] In some embodiments, the anti-CD28 antibody of the present invention or its antigen-binding fragment specifically binds to CD28 (e.g., human CD28). In some embodiments, the anti-CD28 antibody of the present invention or its antigen-binding fragment binds to CD28 (e.g., human CD28) at a binding affinity (K) of about 50 nM, 45 nM, 40 nM, 35 nM, 30 nM, 25 nM, 20 nM, 18 nM, 15 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, or 0.6 nM or less, or a value between these. D ) binds. In some embodiments, the anti-CD28 antibody of the present invention or its antigen-binding fragment has a binding affinity (K) of about 0.5 nM or more to CD28 (e.g., human CD28). D Combines with ).

[0010] In some embodiments, the anti-CD28 antibody of the present invention or its antigen-binding fragment can activate the CD28 signaling pathway.

[0011] In some embodiments, the anti-CD28 antibody of the present invention or its antigen-binding fragment may activate T cells (e.g., human peripheral T cells such as CD4 or CD8 T cells) and, for example, activate T cells to release cytokines, for example, TNF (e.g., TNFα) or interleukin (e.g., IL2).

[0012] In some embodiments, the anti-CD28 antibody of the present invention or its antigen-binding fragment can activate human T cells, e.g., human peripheral T cells, to kill tumor cells.

[0013] Another aspect of the present invention relates to a multispecific binding molecule constructed based on the CD28 antibody of the present invention (e.g., a multispecific antibody such as a bispecific antibody), comprising a target-binding region from the anti-CD28 antibody of the present invention and optionally one or more additional target-binding regions (antigen-binding regions), for example, regions that specifically bind to one or more tumor-associated antigens (TAAs).

[0014] In one embodiment, the present invention relates to a multispecific antibody, e.g., a bispecific antibody, that specifically binds to CD28 and one or more TAAs. In some embodiments, the TAA is CLDN18.2 or PSMA, e.g., human CLDN18.2 or human PSMA.

[0015] In one embodiment, the present invention relates to a bispecific antibody that specifically binds to CD28 (e.g., human CD28) and CLDN18.2 (e.g., human CLDN18.2).

[0016] In one embodiment, the present invention relates to a bispecific antibody that specifically binds to CD28 (e.g., human CD28) and PSMA (e.g., human PSMA).

[0017] In some embodiments, the bispecific antibody of the present invention can effectively activate the CD28 signaling pathway.

[0018] In some embodiments, the bispecific antibody of the present invention can activate human peripheral T cells, for example, by activating T cells (e.g., CD4 or CD8 T cells) to release cytokines, for example, TNF (e.g., TNFα) or interleukin (e.g., IL2).

[0019] In some embodiments, the bispecific antibody of the present invention can activate human T cells, e.g., human peripheral T cells, to kill tumor cells.

[0020] In some embodiments, the bispecific antibody of the present invention may specifically bind to CD28 (e.g., human CD28) and TAA (e.g., human TAA, e.g., CLDN18.2 or PSMA).

[0021] In some embodiments, the bispecific antibody of the present invention, for example, a bispecific antibody specifically binding to CD28 and CLDN18.2 or to CD28 and PSMA, has antitumor activity, for example. In vivo It has antitumor activity.

[0022] In some embodiments, the bispecific antibody of the present invention may be used in combination with other therapeutic agents (e.g., other antibodies such as anti-PD1 antibodies) for antitumor use. Brief explanation of the drawing

[0023] Fig. 1: Results of the assay on the Jurkat-NF-κB activation activity of the anti-CD28 chimeric antibody; Fig. 2: Results of human peripheral T cell activation by anti-CD28 humanized antibody, AB: in the absence of CD3 antibody, CD: in the presence of CD3 antibody; Fig. 3: Schematic diagram of a CD28 bispecific antibody; Fig. 4: Activation of NFkB Jurkat reporter by CLDN18.2×CD28 bispecific antibody; Fig. 5: Results of CLDN18.2×CD28 bispecific antibody-mediated tumor cell killing by human peripheral T cells; Fig. 6: Results of cytokine release from peripheral T cells activated by CLDN18.2×CD28 bispecific antibody, A: in the absence of CD3 antibody, B: in the presence of CD3 antibody; Fig. 7: Results of cell surface affinity assay of PSMA×CD28 bispecific antibodies, A: PSMA affinity, B: CD28 affinity; Fig. 8: Activation results of NFkB Jurkat reporter by PSMA×CD28 bispecific antibody, A: with CD3 antibody added, B: with PSMA×CD3 bispecific antibody added; Fig. 9: Results of PSMA×CD28 bispecific antibody-mediated tumor cell killing by human peripheral T cells; Fig. 10: Results of cytokine IL2 release from peripheral T cells activated by PSMA×CD28 bispecific antibodies, A: in the absence of CD3 antibody (-signal 1), B: in the presence of CD3 antibody (+signal 1); Fig. 11: PSMA×CD28 bispecific antibody as monotherapy In vivo Results of a monotherapy efficacy study, A: Administration regimen; b: Test results; Fig. 12: PSMA×CD28 bispecific antibody in combination with anti-PD1 antibody In vivo Efficacy study results, A: Administration regimen; b: Test results. Specific details for implementing the invention

[0024] It should be understood that the present invention is not limited to the specific methods, protocols, examples, and reagents described herein, and that they may vary. It should also be understood that the terms used herein are intended only to describe specific embodiments rather than to limit the scope of the invention, which is defined solely by the appended claims.

[0025] I. Definition

[0026] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention pertains.

[0027] For the purposes of describing this specification, the following definitions are used, and where appropriate, terms used in the singular form may also be included in the plural form, and vice versa. It should be understood that the terms used herein are intended to describe specific embodiments only and are not intended to be limiting.

[0028] The term "about" used in combination with a number is intended to encompass a range of numbers having a lower limit that is 5% (e.g., 4%, 3%, 2%, or 1%) smaller than a specific number and an upper limit that is 5% (e.g., 4%, 3%, 2%, or 1%) larger than a specific number.

[0029] As used herein, the term “and / or” refers to any one of the options, any two or more of the options, or all of the options.

[0030] Where "first" and "second" are referred to herein, this is merely to distinguish two domains or two chains and does not indicate the location of the two domains in any way.

[0031] As used herein, the terms “comprise” or “include” are intended to mean that the described elements, integers, or steps are included, but do not exclude any other elements, integers, or steps. Unless otherwise indicated, as used herein, the terms “comprise” or “include” also encompass cases consisting of the described elements, integers, or steps. For example, when referring to an antibody variable region “comprised” of a specific sequence, the term is intended to also encompass an antibody variable region consisting of that specific sequence.

[0032] As used herein, the term "CD28" refers to an antigen expressed on T cells as a co-stimulatory receptor. Human CD28 comprises, for example, the amino acid sequence described in NCBI registry number NP_006130.1. In some embodiments, CD28 is human CD28.

[0033] As used herein, "antibody binding to CD28," "anti-CD28 antibody," or "antibody specifically binding to CD28" refers to an antibody capable of binding to monomeric CD28 or its extracellular domain with appropriate affinity, or to dimeric CD28 or its extracellular domain. The antibody and antigen-binding fragment of the present invention may bind to soluble CD28 and / or CD28 expressed on the cell surface. Soluble CD28 includes natural CD28 proteins as well as recombinant CD28 protein variants, such as monomeric and dimeric CD28 constructs lacking a transmembrane domain or otherwise independent of the cell membrane.

[0034] In some aspects, the anti-CD28 antibodies described herein also encompass multispecific antibodies, such as bispecific antibodies that specifically bind to both CD28 and other target antigens.

[0035] General information regarding the amino acid and nucleotide sequences of human immunoglobulin light and heavy chains is provided in Kabat, EA et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991).

[0036] The multispecific antibody of the present invention may comprise a linker. As used herein, the term “linker” refers to any molecule that enables direct linkage of different portions of the multispecific antibody. Examples of linkers for establishing covalent bonds between different portions of the multispecific antibody include, but are not limited to, peptide linkers and non-protein polymers comprising polyethylene glycol (PEG), polypropylene glycol, polyalkylene oxide, or copolymers of polyethylene glycol and polypropylene glycol. In some embodiments, the term “peptide linker” according to the present invention refers to an amino acid sequence, wherein the sequence links the amino acid sequences of different portions of the multispecific antibody to one another. Preferably, the peptide linker is of sufficient length to link two entities in such a manner that the two entities maintain their stereotypes relative to one another without interfering with the desired activity. The peptide linker may or may not predominantly comprise the following amino acid residues: Gly, Ser, Ala, or Thr.

[0037] The terms “Fc domain” or “Fc region” are used herein to define a C-terminal region of an immunoglobulin heavy chain comprising at least a portion of a constant region. The term includes the Fc region of the natural sequence and the variant Fc region. The natural immunoglobulin “Fc domain” comprises two or three constant domains, namely a CH2 domain, a CH3 domain, and an optional CH4 domain. For example, in a natural antibody, the immunoglobulin Fc domain comprises second and third constant domains (CH2 and CH3 domains) derived from two heavy chains of IgG, IgA, and IgD antibodies; or second, third, and fourth constant domains (CH2, CH3, and CH4 domains) derived from two heavy chains of IgM and IgE antibodies. Unless otherwise noted herein, amino acid residues of the Fc region or heavy chain constant region are numbered according to the EU numbering system (also known as the EU index) as described, for example, in Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD, 1991. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Two Fc regions may form a dimeric Fc by dimerization, and two different Fc may form a heterodimeric Fc by heterodimerization. In the present invention, the terms “Fc region”, “Fc portion” and “dimeric Fc (e.g., heteromeric Fc)” do not include the heavy chain variable region VH and light chain variable region VL of the immunoglobulin, as well as the heavy chain constant region CH1 and light chain constant region CL, but in some cases may include a hinge region at the N-terminus of the heavy chain constant region.In one embodiment, the human IgG heavy chain Fc region extends from Asp221, or from Cys226, or from Asp231 to the carboxyl-terminus of the heavy chain.

[0038] In one embodiment, the human IgG1 Fc region polypeptide (containing a portion of the hinge region) comprises or consists of the following amino acid sequence:

[0039] DKTHTCPPCPA PELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA KGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 94); or

[0040] DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 95).

[0041] In one embodiment, the Fc region is derived from a human Fc region. In one embodiment, the Fc region comprises all or part of a human constant region. The antibody Fc region is directly involved in complement activation, C1q binding, C3 activation, and Fc receptor binding. In one embodiment, the Fc region is a human Fc region. In one embodiment, the Fc region belongs to the human IgG4 subclass. In one embodiment, the Fc region belongs to the human IgG1 subclass. In one embodiment, the Fc region is the Fc region of human IgG1, IgG2, IgG3, or IgG4.

[0042] In the present invention, “heterodimeric Fc scaffold” refers to a scaffold comprising two different Fc regions or formed by the dimerization of two different Fc regions, which may form a multispecific antibody, e.g., a bispecific antibody, by being connected at its N-terminus or C-terminus to an antigen-binding domain (e.g., a variable region of the heavy chain and / or light chain of an antibody or antigen-binding fragment of an antibody capable of binding to a target molecule, or a soluble moiety of a ligand or receptor capable of binding to a target molecule).

[0043] The term "CH1 domain" refers to a portion of the antibody heavy chain polypeptide extending from EU position 118 to EU position 220 (EU numbering scheme). In one embodiment, the CH1 domain comprises or consists of the amino acid sequence ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC (SEQ No. 74).

[0044] In some embodiments, CH1 may include a portion of the hinge region. In some embodiments, the Fc region may include a portion of the hinge region.

[0045] In this document, specific amino acid residues in the antibody IgG constant region are described according to the EU numbering system. For example, "S364" refers to serine at EU position 364. Amino acid mutations at specific positions in the constant region are indicated as "original amino acid, amino acid position, mutated amino acid." For example, "S364R" refers to the substitution of serine (S) at EU position 364 with arginine (R). When describing combinations of mutations, the combined mutations are connected by a plus (+), "-", or "and". "S364R+D399K", "S364R-D399K", or "S364R and D399K" indicate that the Fc region contains both mutations S364R and D399K. If multiple mutation possibilities exist at a specific position, this is indicated herein by the symbol " / ". For example, the mutation "K370T / S" indicates that the residue K at position 370 can be substituted with a T or S residue.

[0046] In the case of polypeptide sequences, "conservative modification" includes substitutions, deletions, or additions to a polypeptide sequence that do not substantially alter the desired functional activity of the polypeptide sequence. For example, conservative substitutions often involve replacing an amino acid with a chemically similar amino acid. Tables of conservative substitutions providing functionally similar amino acids are widely known in the art. Eight groups of amino acids that are conservative substitutions for each other are listed below: 1) Alanine (A) and Glycine (G); 2) Aspartic acid (D) and Glutamic acid (E); 3) Asparagine (N) and Glutamine (Q); 4) Arginine (R) and Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), and Valine (V); 6) Phenylalanine (F), Tyrosine (Y), and Tryptophan (W); 7) Serine (S) and Threonine (T); and 8) cysteine ​​(C) and methionine (M). In some embodiments, the term “conservative sequence modification” is used to refer to an amino acid modification that does not significantly affect or alter the binding properties of the antibody molecule or binding protein molecule of the present invention, comprising an amino acid sequence, to the antigen of interest. For example, the conservatively modified variant maintains a binding affinity of at least 80%, 85%, 90%, 95%, 98%, 99%, e.g. 100%–110% or more to the antigen of interest compared to the parent antibody or binding protein.

[0047] As used herein, the term "vector" refers to a nucleic acid molecule capable of amplifying another nucleic acid to which it is connected. The term includes vectors that function as self-replicating nucleic acid structures, as well as vectors that are integrated into the genome of an introduced host cell. Some vectors may direct the expression of a nucleic acid operably connected to them. Such vectors are referred to herein as "expression vectors."

[0048] The term "binding molecule" refers to any molecule capable of specifically binding to a target, for example, an antibody, its antigen-binding fragment, or a fusion protein.

[0049] The term "target" refers to the binding target substance to which the binding molecule is directed. The target may be an antigen, a ligand, or a receptor. The term "antigen" refers to a molecule that triggers an immune response. Such immune response may involve antibody production or the activation of specific immune cells, or both. Those skilled in the art will understand that any macromolecule, including substantially any protein or peptide, may be used as an antigen. Additionally, the antigen may be derived from recombinant or genomic DNA. As used herein, the term "epitope" refers to a moiety of an antigen that specifically interacts with an antibody molecule. Where the binding molecule of the present invention relates to a target-binding region derived from an antibody, "target" and "antigen" are used interchangeably. In some embodiments, the antigen is a tumor-associated antigen (TAA).

[0050] The term “tumor-associated antigen (TAA)” as used herein relates to a protein that is preferably specifically expressed in a limited number of tissues and / or organs under normal conditions or at a specific stage of development, and is expressed or abnormally expressed in one or more tumor or cancer tissues. In the context of the present invention, the tumor-associated antigen is preferably associated with the cell surface of cancer cells and is preferably not expressed or is expressed only rarely in normal tissues. In some embodiments, the tumor-associated antigen is a human tumor-associated antigen. In some embodiments, the tumor-associated antigen is CLDN18.2, such as human CLDN18.2, or PSMA, such as human PSMA.

[0051] As used herein, the term “target-binding region” refers to a part of a binding molecule that binds to a specific target or antigen, e.g., a multispecific binding molecule or a bispecific binding molecule. The target-binding region may be, e.g., the antibody or immunoglobulin itself or an antibody fragment. Such target-binding regions may or may not have a tertiary structure independent of the rest of the binding molecule and may or may not bind to their targets as distinct entities. The target-binding region may also be a receptor or ligand, or a domain of a receptor capable of binding to a ligand. In the case of a multispecific antibody or a bispecific antibody, the “target-binding region” is also referred to as the “antigen-binding region.”

[0052] As used herein, the term “antigen-binding region” refers to any portion of an antibody or its antigen-binding fragment that binds to a specific target or antigen, e.g., a multispecific antibody or a bispecific antibody. The antigen-binding region may be, e.g., the antibody or immunoglobulin itself or an antibody fragment. Such antigen-binding regions may or may not have a tertiary structure independent of the remainder of the multispecific antibody or bispecific antibody, and may or may not bind to its antigen / epitope as a distinct entity. Where the binding molecule of the present invention relates to a target-binding region derived from an antibody, “target-binding region” and “antigen-binding region” are used interchangeably.

[0053] The term “multispecific binding molecule” refers to at least a bispecific multispecific binding molecule, e.g., a bispecific binding molecule, i.e., the molecule comprises at least a first target-binding region and a second target-binding region, wherein the first target-binding region binds to one target or antigen and the second target-binding region binds to another antigen or target. The multispecific binding molecule according to the present invention also encompasses a multispecific binding molecule comprising a plurality of target-binding regions / sites. In some embodiments, the multispecific binding molecule of the present invention is a multispecific antibody. In some embodiments, the bispecific binding molecule of the present invention is a bispecific antibody.

[0054] The term “single-specific” as used herein refers to polypeptide / protein molecules having one or more target-binding regions, each of which binds to the same site or structure of the same target or the same epitope of the same antigen.

[0055] As used herein, the term “multispecific” binding molecule, e.g., antibody, refers to a binding molecule having at least two target-binding regions, each of which binds to a different site / structure of the same target or to a different target. A multispecific binding molecule is a binding molecule having binding specificity for at least two different targets or sites / structures. In one embodiment, a bispecific binding molecule having binding specificity for a first target and a second target is provided herein.

[0056] As used herein, the term “multispecific” antibody refers to an antibody having at least two antigen-binding regions, each of which binds to a different epitope of the same antigen or a different epitope of a different antigen. A multispecific antibody is an antibody having binding specificity for at least two different antigens or epitopes. In one embodiment, a bispecific antibody having binding specificity for a first antigen and a second antigen is provided herein.

[0057] When "first antigen-binding region" is referred to in a multispecific antibody or a bispecific antibody, it refers to a binding region that binds to a first antigen and is not intended to limit the number of such antigen-binding regions included in the antibody. For example, a multispecific antibody or a bispecific antibody may include one or more first antigen-binding regions. For example, a bispecific antibody may include a first antigen-binding region and a second antigen-binding region, but may include one or more first antigen-binding regions and one or more second antigen-binding regions.

[0058] When "first target-binding region" is referred to in relation to a binding molecule, it refers to a binding region that binds to a first target and is not intended to limit the number of such target-binding regions included in the binding molecule. For example, a multispecific binding molecule or a bispecific binding molecule may include one or more first target-binding regions. For example, a bispecific binding molecule may include a first target-binding region and a second target-binding region, and may include one or more first target-binding regions and one or more second target-binding regions.

[0059] Where it is stated that "the target or antigen-binding domain is derived from an antibody," this means that the binding domain constituting the target / antigen-binding domain is a binding domain of an antibody that specifically binds to an antigen, or is derived therefrom. For example, a fragment of the antigen-binding domain that specifically binds to an antigen, such as Fab, is a corresponding fragment of an antibody such as Fab, or is derived therefrom; or the heavy chain variable region and / or light chain variable region of the antigen-binding domain is a heavy chain variable region and / or light chain variable region of an antibody, or is derived therefrom; or CDRs 1, 2, 3, 4, 5, or 6 of the antigen-binding domain are CDRs of an antibody.

[0060] The term "derived from" means that a fragment within the antigen-binding domain is substantially identical to the fragment of the antibody of its origin, but has mutations such as substitutions, deletions, or additions at one or more sites. In one specific embodiment, the mutation is not present in the antibody's CDR.

[0061] The terms “whole antibody” and “whole-length antibody” are used interchangeably herein to refer to antibody molecules having the structure of natural immunoglobulin molecules. In the case of a conventional 4-chain IgG antibody, the whole-length antibody comprises two heavy chains (H) and two light chains (L) connected to each other by disulfide bonds. In the case of a heavy-chain antibody having only heavy chains and lacking light chains, the whole-length antibody comprises two heavy chains (H) connected to each other by disulfide bonds. In the case of a conventional 4-chain IgG antibody, the whole-length antibody heavy chain generally consists of a heavy chain variable region (abbreviated as VH herein) and a heavy chain constant region, wherein the heavy chain constant region comprises at least three domains CH1, CH2, and CH3. The whole-length antibody light chain consists of a light chain variable region (abbreviated as VL herein) and a light chain constant region, wherein the light chain constant region consists of one domain CL. Each heavy chain variable region VH or each light chain variable region consists of three CDRs and four FRs arranged in the following order from amino-terminus to carboxyl-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The term “antibody fragment” comprises a portion of an intact antibody. In a preferred embodiment, the antibody fragment is an antigen-binding fragment.

[0062] The antibody term "antigen-binding fragment" refers to a molecule distinct from a full-length antibody, wherein the antigen-binding fragment comprises a portion of the full-length antibody but can bind to the antigen of the full-length antibody or compete with the full-length antibody (i.e., the full-length antibody from which the antigen-binding fragment is derived) for binding to the antigen. Antigen-binding fragments can be produced by recombinant DNA technology or by enzymatic or chemical cleavage of intact antibodies. Antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv, single-chain Fv, diabodies, single-domain antibodies (sdAb), and nanobodies. For example, the Fab fragment can be obtained by papain digestion of a full-length antibody. Additionally, F(ab')2, a dimer of Fab', is a divalent antibody fragment produced by pepsin digestion at the sub-disulfide bond site within the hinge region of an intact antibody. F(ab')2 can be reduced under neutral conditions by breaking disulfide bonds within the hinge region, thereby converting the F(ab')2 dimer into a Fab' monomer. The Fab' monomer is essentially a Fab fragment having a hinge region. The Fv fragment consists of the VL and VH domains of a single arm of an antibody. While the two domains VL and VH of the Fv fragment can be encoded by separate genes, the two domains can also be linked via a synthetic linker peptide using a recombination method, so that the VL and VH regions are paired to form a single protein chain, which is a single-strand Fv (scFv).

[0063] “Fab fragment” and “Fab” are used interchangeably herein to refer to an immunoglobulin fragment composed of two polypeptide chains comprising an immunoglobulin heavy chain variable domain VH, a heavy chain constant domain CH1, a light chain variable domain VL, and a light chain constant domain CL, wherein one polypeptide chain comprises one constant region selected from VH, CH1, and CL from N-terminus to C-terminus, and the other polypeptide chain comprises another constant region selected from VL, CL, and CH1 from N-terminus to C-terminus, wherein the VH and VL domains are paired to form an antigen-binding site. In the same, the Fab polypeptide chain comprising the heavy chain constant domain CH1 is also referred to as the “Fab heavy chain”; correspondingly, the Fab polypeptide chain comprising the light chain constant domain CL is also referred to as the “Fab light chain”.

[0064] The "complementary-determining region," "CDR region," or "CDR" is a region within an antibody variable domain that is highly variable in sequence, forms a structurally defined loop ("hypervariable loop"), and / or contains antigen-contact residues ("antigen-contact site"). The CDR is primarily responsible for binding to antigenic epitopes. CDRs in the heavy and light chains are generally referred to as CDR1, CDR2, and CDR3, and are numbered sequentially from the N-terminus. CDRs located in the antibody's heavy chain variable domain are referred to as CDRH1, CDRH2, and CDRH3, while CDRs located in the antibody's light chain variable domain are referred to as CDRL1, CDRL2, and CDRL3. For a given amino acid sequence of the light chain variable region or heavy chain variable region, the precise amino acid sequence boundaries of each CDR are based, for example, on the 3D structure of the antibody and the topology of the CDR loop, such as Chothia (Chothia et al. (1989) Nature 342: 877-883; Al-Lazikani et al., "Standard conformations for the canonical structures of immunoglobulins", Journal of Molecular Biology, 273: 927-948 (1997)), Kabat (Kabat et al., Sequences of Proteins of Immunological Interest, 4th Ed., US Department of Health and Human Services, National Institutes of Health (1987)), AbM (University of Bath), Contact (University College London), and the International ImMunoGeneTics database (IMGT) (www.imgt.cines.It may be determined using any one or a combination of several widely known antibody CDR assignment schemes, including North CDR definitions based on affinity propagation clustering using fr / ) and multiple crystal structures. Unless otherwise noted, the terms “CDR” or “CDR sequence” as used herein encompass a CDR sequence determined by any one or a combination of the schemes described above. A CDR may also be determined based on having the same Kabat numbering position as a reference CDR sequence (e.g., any of the exemplary CDRs of the present invention).

[0065] In some embodiments, the CDR of the heavy chain variable region of the antibody of the present invention is defined according to Kabat or Chothia, or a combination of Kabat and Chothia (hereinafter referred to as "Kabat & Chothia"; corresponding to H26-H32 under the Kabat numbering system). In some embodiments, the CDR of the light chain variable region of the antibody of the present invention is determined according to Kabat.

[0066] In one embodiment, in the anti-CD28 antibody of the present invention, CDRH1 is defined according to the Kabat & Chothia system, CDRH2 and CDRH3 are each defined according to the Kabat system, and CDRL is each defined according to the Kabat system.

[0067]

[0068] The percentage of identity between sequences is calculated as follows.

[0069] To determine the percentage of identity between two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison (e.g., a gap may be introduced in one or both of the first and second amino acid sequences or nucleic acid sequences for optimal alignment, or non-homologous sequences may be ignored for comparison). In a preferred embodiment, for comparison purposes, the length of the aligned reference sequence is at least 30%, preferably at least 40%, more preferably at least 50% or 60%, and even more preferably at least 70%, 80%, 90%, or 100% of the reference sequence length. Then, the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are compared. If one position of the first sequence is occupied by the same amino acid residue or nucleotide at the corresponding position of the second sequence, the molecules are identical at this position.

[0070] "Humanized antibodies" are antibodies that retain the antigen-specific reactivity of non-human antibodies and have lower immunogenicity when administered to humans as a therapeutic agent. This can be achieved, for example, by retaining the non-human antigen-binding site and substituting the remainder of the antibody with its human counterpart (i.e., substituting a portion of the variable region not involved in binding with a corresponding portion of the human antibody).

[0071] As used herein, the terms “anti-,” “binding,” or “specific binding” mean that the binding effect is selective for a target or antigen and can be distinguished from unwanted or non-specific interactions. The ability of a binding site to bind to a specific target or antigen may be determined by flow cytometry or enzyme-linked immunosorbent assay (ELISA), or by conventional binding assays known in the art, such as radioimmunoassay (RIA), bio-layer interferometry (BLI), MSD assay, or surface plasmon resonance (SPR).

[0072] "Affinity" or "bond affinity" refers to the intrinsic bond affinity that reflects the interaction between the members of a bond pair. The affinity of molecule X for its partner Y is generally the dissociation constant (K), which is the ratio of the dissociation rate constant to the bond rate constant. D It can be expressed as ) (each K dis and K on ). Affinity can be measured by conventional methods known in the art. One specific method for measuring affinity is the ForteBio kinetic binding assay described herein.

[0073] The term "host cell" refers to a cell into which an exogenous polynucleotide has been introduced, and includes the offspring of such cells. Host cells include "transformers" and "transformed cells," which include primary transformed cells and offspring derived therefrom.

[0074] The terms “individual” and “object” are used interchangeably and refer to mammals. Mammals include, but are not limited to, domesticated animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In particular, the object is a human.

[0075] The term "treatment" (or "to treat" or "treating") refers to delaying, stopping, halting, alleviating, stopping, reducing, or reversing the onset of symptoms, complications, or biochemical markers of a disease, or alleviating symptoms, or stopping or inhibiting the further progression of a disease, condition, or illness.

[0076] The term "prevention" (or "to prevent" or "preventing") includes suppressing the occurrence or progression of a disease or illness, or the symptoms of a specific disease or illness.

[0077] The term “therapeutic agent” as used herein encompasses any substance effective in preventing or treating tumors, e.g., cancer, including chemotherapy agents, cytokines, cytotoxic agents, additional antibodies (e.g., immune checkpoint molecular antibodies), small molecule drugs, or immunomodulators (e.g., immunosuppressants or agonists).

[0078] The term “combination therapy” refers to the administration of two or more therapeutic agents or therapeutic modalities for treating the disease described herein. Such administration includes, for example, co-administering these therapeutic agents substantially simultaneously in a single capsule containing a fixed ratio of active ingredients. Alternatively, such administration includes co-administering active ingredients in various or distinct containers (e.g., tablets, capsules, powders, and liquids). Powders and / or liquids may be reconstituted or diluted to the desired dose prior to administration. Additionally, such administration includes using each type of therapeutic agent in a sequential manner, either at approximately the same time or at different times. In either case, the therapeutic regimen will provide the beneficial effects of the drug combination in treating the disease or condition described herein.

[0079] The term “pharmaceutical combination or combination product” refers to a non-fixed combination product or a fixed combination product, including but not limited to kits and pharmaceutical compositions. The term “non-fixed combination” means that active ingredients (e.g., (i) the antibody of the present invention and (ii) additional therapeutic agents) are administered to a patient as separate entities simultaneously or sequentially (without specific time limits or at the same or different time intervals), wherein such administration provides the patient with two or more active agents that are prophylactically or therapeutically effective. The term “fixed combination” means that two or more active agents are administered to a patient simultaneously as a single entity. The doses and / or time intervals of the two or more active agents are preferably selected so that the combined use of the ingredients may result in a greater therapeutic effect for the disease or condition than that achieved by the use of any one ingredient alone. Each ingredient may be present in a separate formulation, and the formulations may be the same or different.

[0080] The term "pharmaceutical composition" refers to a composition in which the biological activity of the active ingredient contained therein exists in a form that enables the biological activity of the active ingredient and does not contain additional ingredients having unacceptable toxicity to the subject to whom the composition is administered.

[0081] The term "pharmaceutical adjuvant" refers to diluents, adjuvants (e.g., Freund adjuvants (complete and incomplete)), excipients, carriers, stabilizers, etc. administered together with the active substance.

[0082] "Subject / patient / entity sample" refers to a collection of cells or fluids obtained from a patient or subject. Sources of tissue or cell samples may be solid tissues from fresh, frozen, and / or preserved organ or tissue samples or biopsy or puncture samples; body fluids such as tears, vitreous fluid, cerebrospinal fluid, amniotic fluid, peritoneal fluid, or interstitial fluid.

[0083] II. Anti-CD28 Antibody

[0084] In some embodiments, the anti-CD28 antibody of the present invention or its antigen-binding fragment comprises three complementary-determining regions (CDRHs) from the heavy chain variable region: CDRH1, CDRH2, and CDRH3. In some embodiments, the anti-CD28 antibody of the present invention or its antigen-binding fragment comprises three complementary-determining regions (CDRLs) from the light chain variable region: CDRL1, CDRL2, and CDRL3. In some embodiments, the anti-CD28 antibody of the present invention or its antigen-binding fragment comprises three complementary-determining regions (CDRHs) from the heavy chain variable region and three complementary-determining regions (CDRLs) from the light chain variable region.

[0085] In some aspects, the anti-CD28 antibody of the present invention or its antigen-binding fragment comprises a heavy chain variable region (VH). In some aspects, the anti-CD28 antibody of the present invention or its antigen-binding fragment comprises a light chain variable region (VL). In some aspects, the anti-CD28 antibody of the present invention or its antigen-binding fragment comprises a heavy chain variable region (VH) and a light chain variable region (VL). In some embodiments, the heavy chain variable region comprises three complementary-determining regions (CDRHs) from the heavy chain variable region: CDRH1, CDRH2, and CDRH3. In some embodiments, the light chain variable region comprises three complementary-determining regions (CDRLs) from the light chain variable region: CDRL1, CDRL2, and CDRL3.

[0086] In some embodiments, the heavy chain variable region VH of the anti-CD28 antibody of the present invention is

[0087] (i) comprising or composed of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NOs 4, 5, 13, 23, 24, 69, 72, or 75; or

[0088] (ii) comprising or composed of the amino acid sequence of SEQ ID NOs 4, 5, 13, 23, 24, 69, 72 or 75; or

[0089] (iii) comprising or composed of an amino acid sequence having one or more (preferably 10 or fewer, more preferably 5, 4, 3, 2 or 1 or fewer) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence of SEQ ID NOs 4, 5, 13, 23, 24, 69, 72 or 75, wherein preferably, the amino acid changes do not occur in the CDR.

[0090] In some embodiments, the light chain variable region VL of the anti-CD28 antibody of the present invention is

[0091] (i) comprising or composed of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NOs 9, 17, 19, 28, 70, 73, or 78; or

[0092] (ii) comprising or composed of the amino acid sequence of SEQ ID NOs 9, 17, 19, 28, 70, 73 or 78; or

[0093] (iii) comprising or composed of an amino acid sequence having one or more (preferably 10 or fewer, more preferably 5, 4, 3, 2 or 1 or fewer) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence of SEQ ID NOs 9, 17, 19, 28, 70, 73 or 78, wherein preferably, the amino acid changes do not occur in the CDR.

[0094] In some embodiments, three complementary-determining regions (CDRHs), CDRH1, CDRH2, and CDRH3 from the heavy chain variable region of the anti-CD28 antibody of the present invention are

[0095] (i) three complementarity-determining regions CDRH1, CDRH2, and CDRH3 included in the VH described in SEQ ID NOs 4, 5, 13, 23, 69, 72, or 75, or

[0096] (ii) a sequence comprising, collectively across all three CDRH regions, at least one and five, four, three, two or fewer amino acid changes (preferably amino acid substitutions, more preferably conservative substitutions) compared to the sequence of (i);

[0097] Here, CDRH can be defined according to any system for determining CDR, e.g., Kabat, AbM, Chothia, Contact, or IMGT system, or a combination thereof;

[0098] Preferably, CDRH1 is defined according to the Kabat & Chothia system, and CDRH2 and CDRH3 are each defined according to the Kabat system.

[0099] In some embodiments, three complementary-determining regions (CDRLs), CDRL1, CDRL2, and CDRL3 from the light chain variable region of the anti-CD28 antibody of the present invention are

[0100] (i) three complementarity-determining regions CDRL1, CDRL2, and CDRL3 included in the VL described in SEQ ID NOs 9, 17, 19, 28, 70, 73, or 78, or

[0101] (ii) a sequence comprising, collectively across all three CDRL regions, at least one and five, four, three, two, or one or fewer amino acid changes (preferably amino acid substitutions, more preferably conservative substitutions) compared to the sequence of (i);

[0102] Here, CDRL can be defined according to any system for determining CDR, e.g., Kabat, AbM, Chothia, Contact, or IMGT system, or a combination thereof;

[0103] Preferably, CDRL1-3 are each determined according to Kabat.

[0104] In some embodiments, CDRH1 comprises or is composed of an amino acid sequence described in SEQ ID NO. 1, 10 or 20 or an amino acid sequence having one, two, or three amino acid changes (preferably amino acid substitutions, more preferably conservative substitutions) compared therewith.

[0105] In some embodiments, CDRH2 comprises or is composed of an amino acid sequence described in SEQ ID NOs. 2, 11, 21 or 76 or an amino acid sequence having one, two, or three amino acid changes (preferably amino acid substitutions, more preferably conservative substitutions) compared therewith.

[0106] In some embodiments, CDRH3 comprises or is composed of an amino acid sequence described in SEQ ID NOs. 3, 12, 22 or 77 or an amino acid sequence having one, two, or three amino acid changes (preferably amino acid substitutions, more preferably conservative substitutions) compared therewith.

[0107] In some embodiments, CDRL1 comprises or is composed of an amino acid sequence described in SEQ ID NOs. 6, 14, 18, 25, or 71, or an amino acid sequence having one, two, or three amino acid changes (preferably amino acid substitutions, more preferably conservative substitutions) compared therewith.

[0108] In some embodiments, CDRL2 comprises or is composed of an amino acid sequence described in SEQ ID NO. 7, 15, or 26 or an amino acid sequence having one, two, or three amino acid changes (preferably amino acid substitutions, more preferably conservative substitutions) compared therewith.

[0109] In some embodiments, CDRL3 comprises or is composed of an amino acid sequence described in SEQ ID NO. 8, 16, or 27 or an amino acid sequence having one, two, or three amino acid changes (preferably amino acid substitutions, more preferably conservative substitutions) compared therewith.

[0110] In some embodiments, in the anti-CD28 antibody of the present invention,

[0111] CDRH1 comprises or consists of the amino acid sequence of SEQ ID NO. 1; CDRH2 comprises or consists of the amino acid sequence of SEQ ID NO. 2; CDRH3 comprises or consists of the amino acid sequence of SEQ ID NO. 3; CDRL1 comprises or consists of the amino acid sequence of SEQ ID NO. 6; CDRL2 comprises or consists of the amino acid sequence of SEQ ID NO. 7; and / or CDRL3 comprises or consists of the amino acid sequence of SEQ ID NO. 8;

[0112] CDRH1 comprises or consists of the amino acid sequence of SEQ ID NO. 10; CDRH2 comprises or consists of the amino acid sequence of SEQ ID NO. 11; CDRH3 comprises or consists of the amino acid sequence of SEQ ID NO. 12; CDRL1 comprises or consists of the amino acid sequence of SEQ ID NO. 14, 18, or 71; CDRL2 comprises or consists of the amino acid sequence of SEQ ID NO. 15; and / or CDRL3 comprises or consists of the amino acid sequence of SEQ ID NO. 16;

[0113] CDRH1 comprises or consists of the amino acid sequence of SEQ ID NO. 20; CDRH2 comprises or consists of the amino acid sequence of SEQ ID NO. 21 or 76; CDRH3 comprises or consists of the amino acid sequence of SEQ ID NO. 22 or 77; CDRL1 comprises or consists of the amino acid sequence of SEQ ID NO. 25; CDRL2 comprises or consists of the amino acid sequence of SEQ ID NO. 26; and / or CDRL3 comprises or consists of the amino acid sequence of SEQ ID NO. 27;

[0114] CDRH1 comprises or consists of the amino acid sequence of SEQ ID NO. 20; CDRH2 comprises or consists of the amino acid sequence of SEQ ID NO. 21; CDRH3 comprises or consists of the amino acid sequence of SEQ ID NO. 22; CDRL1 comprises or consists of the amino acid sequence of SEQ ID NO. 25; CDRL2 comprises or consists of the amino acid sequence of SEQ ID NO. 26; and / or CDRL3 comprises or consists of the amino acid sequence of SEQ ID NO. 27; or

[0115] CDRH1 comprises or consists of the amino acid sequence of SEQ ID NO. 20; CDRH2 comprises or consists of the amino acid sequence of SEQ ID NO. 76; CDRH3 comprises or consists of the amino acid sequence of SEQ ID NO. 77; CDRL1 comprises or consists of the amino acid sequence of SEQ ID NO. 25; CDRL2 comprises or consists of the amino acid sequence of SEQ ID NO. 26; and / or CDRL3 comprises or consists of the amino acid sequence of SEQ ID NO. 27.

[0116] In some specific embodiments of the present invention, the anti-CD28 antibody of the present invention or its antigen-binding fragment comprises VH and VL, wherein

[0117] VH comprises or is composed of an amino acid sequence described in SEQ ID NO. 4, 5, or 72 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity therewith, and VL comprises or is composed of an amino acid sequence described in SEQ ID NO. 9 or 73 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity therewith;

[0118] VH comprises or is composed of an amino acid sequence described in SEQ ID NO. 4 or 5 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity therewith, and VL comprises or is composed of an amino acid sequence described in SEQ ID NO. 9 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity therewith;

[0119] VH comprises or is composed of the amino acid sequence described in SEQ ID NO. 72 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity therewith, and VL comprises or is composed of the amino acid sequence described in SEQ ID NO. 73 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity therewith;

[0120] VH comprises or is composed of an amino acid sequence described in SEQ ID NO. 13 or 69 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity therewith, and VL comprises or is composed of an amino acid sequence described in SEQ ID NO. 17, 19, or 70 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity therewith;

[0121] VH comprises or is composed of the amino acid sequence described in SEQ ID NO. 13 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity therewith, and VL comprises or is composed of the amino acid sequence described in SEQ ID NO. 17 or 19 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity therewith;

[0122] VH comprises or is composed of the amino acid sequence described in SEQ ID NO. 69 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity therewith, and VL comprises or is composed of the amino acid sequence described in SEQ ID NO. 70 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity therewith;

[0123] VH comprises or is composed of an amino acid sequence described in SEQ ID NO. 23, 24, or 75 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity therewith, and VL comprises or is composed of an amino acid sequence described in SEQ ID NO. 28 or 78 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity therewith;

[0124] VH comprises or is composed of the amino acid sequence described in SEQ ID NO. 23 or 24 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity therewith, and VL comprises or is composed of the amino acid sequence described in SEQ ID NO. 28 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity therewith; or

[0125] VH comprises or is composed of the amino acid sequence described in SEQ ID NO. 75 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity therewith, and VL comprises or is composed of the amino acid sequence described in SEQ ID NO. 78 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity therewith.

[0126] In some specific embodiments of the present invention, the anti-CD28 antibody of the present invention or its antigen-binding fragment comprises the following:

[0127] Three complementary-determining regions CDRH1, CDRH2, and CDRH3 contained in the VH described in SEQ ID NO. 4, 5, or 72, and three complementary-determining regions CDRL1, CDRL2, and CDRL3 contained in the VL described in SEQ ID NO. 9 or 73;

[0128] Three complementary-determining regions CDRH1, CDRH2, and CDRH3 contained in the VH described in SEQ ID NO. 13 or 69, and three complementary-determining regions CDRL1, CDRL2, and CDRL3 contained in the VL described in SEQ ID NO. 17, 19, or 70; or

[0129] Three complementary-determining regions CDRH1, CDRH2, and CDRH3 contained in the VH described in SEQ ID NO. 23, 24, or 75, and three complementary-determining regions CDRL1, CDRL2, and CDRL3 contained in the VL described in SEQ ID NO. 28 or 78;

[0130] Preferably, CDRH and CDRL here may be determined according to any system for determining CDR, e.g., Kabat, AbM, Chothia, Contact, or IMGT systems, or a combination thereof; e.g., CDRH1 is determined according to the Kabat & Chothia system, CDRH2 and CDRH3 are each determined according to Kabat; and CDRL1-3 are each determined by the Kabat system.

[0131] In some specific embodiments of the present invention, the anti-CD28 antibody of the present invention or its antigen-binding fragment comprises CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3, wherein

[0132] CDRH1 is composed of the amino acid sequence of SEQ ID NO. 1; CDRH2 is composed of the amino acid sequence of SEQ ID NO. 2; CDRH3 is composed of the amino acid sequence of SEQ ID NO. 3; CDRL1 is composed of the amino acid sequence of SEQ ID NO. 6; CDRL2 is composed of the amino acid sequence of SEQ ID NO. 7; CDRL3 is composed of the amino acid sequence of SEQ ID NO. 8;

[0133] CDRH1 is composed of the amino acid sequence of SEQ ID NO. 10; CDRH2 is composed of the amino acid sequence of SEQ ID NO. 11; CDRH3 is composed of the amino acid sequence of SEQ ID NO. 12; CDRL1 is composed of the amino acid sequence of SEQ ID NO. 14, 18, or 71; CDRL2 is composed of the amino acid sequence of SEQ ID NO. 15; CDRL3 is composed of the amino acid sequence of SEQ ID NO. 16;

[0134] CDRH1 is composed of the amino acid sequence of SEQ ID NO. 20; CDRH2 is composed of the amino acid sequence of SEQ ID NO. 21; CDRH3 is composed of the amino acid sequence of SEQ ID NO. 22; CDRL1 is composed of the amino acid sequence of SEQ ID NO. 25; CDRL2 is composed of the amino acid sequence of SEQ ID NO. 26; CDRL3 is composed of the amino acid sequence of SEQ ID NO. 27, or

[0135] CDRH1 is composed of the amino acid sequence of SEQ ID NO. 20; CDRH2 is composed of the amino acid sequence of SEQ ID NO. 76; CDRH3 is composed of the amino acid sequence of SEQ ID NO. 77; CDRL1 is composed of the amino acid sequence of SEQ ID NO. 25; CDRL2 is composed of the amino acid sequence of SEQ ID NO. 26; and CDRL3 is composed of the amino acid sequence of SEQ ID NO. 27.

[0136] In some specific embodiments of the present invention, the anti-CD28 antibody of the present invention or its antigen-binding fragment comprises VH and VL, wherein

[0137] VH comprises or is composed of the amino acid sequence described in SEQ ID NO. 4, and VL comprises or is composed of the amino acid sequence described in SEQ ID NO. 9;

[0138] VH comprises or is composed of the amino acid sequence described in SEQ ID NO. 5, and VL comprises or is composed of the amino acid sequence described in SEQ ID NO. 9;

[0139] VH comprises or is composed of the amino acid sequence described in SEQ ID NO. 13, and VL comprises or is composed of the amino acid sequence described in SEQ ID NO. 17;

[0140] VH comprises or is composed of the amino acid sequence described in SEQ ID NO. 13, and VL comprises or is composed of the amino acid sequence described in SEQ ID NO. 19;

[0141] VH comprises or is composed of the amino acid sequence described in SEQ ID NO. 23, and VL comprises or is composed of the amino acid sequence described in SEQ ID NO. 28;

[0142] VH comprises or is composed of the amino acid sequence described in SEQ ID NO. 24, and VL comprises or is composed of the amino acid sequence described in SEQ ID NO. 28;

[0143] VH comprises or is composed of the amino acid sequence described in SEQ ID NO. 72, and VL comprises or is composed of the amino acid sequence described in SEQ ID NO. 73;

[0144] VH comprises or is composed of the amino acid sequence described in SEQ ID NO. 69, and VL comprises or is composed of the amino acid sequence described in SEQ ID NO. 70; or

[0145] VH comprises or is composed of the amino acid sequence described in SEQ ID NO. 75, and VL comprises or is composed of the amino acid sequence described in SEQ ID NO. 78.

[0146] In one embodiment of the present invention, the amino acid modification described herein includes amino acid substitution, insertion, or deletion. In a preferred embodiment, the amino acid modification described herein occurs in a region outside the CDR (e.g., FR). More preferably, the amino acid modification described herein occurs in a region outside the heavy chain variable region and / or outside the light chain variable region. Preferably, the amino acid modification described herein is an amino acid substitution, preferably a conservative substitution.

[0147] In some embodiments, the anti-CD28 antibody of the present invention or its antigen-binding fragment is an antibody of the IgG1, IgG2, IgG3, or IgG4 form, for example, an antibody of the IgG1 form or its antigen-binding fragment.

[0148] In some embodiments, the anti-CD28 antibody of the present invention or its antigen-binding fragment further comprises an antibody heavy chain constant region HC. In some embodiments, the anti-CD28 antibody of the present invention or its antigen-binding fragment further comprises an antibody light chain constant region LC. In some embodiments, the anti-CD28 antibody of the present invention or its antigen-binding fragment further comprises a heavy chain constant region HC and a light chain constant region LC. In some embodiments, the heavy chain constant region of the present invention comprises an Fc region.

[0149] In some embodiments, the heavy chain constant region of the anti-CD28 antibody of the present invention is the constant region of (human) IgG1, IgG2, IgG3, or IgG4, e.g., the constant region of IgG1.

[0150] In some embodiments, the IgG1 heavy chain constant region suitable for use in the antibody molecule of the present invention is

[0151] (i) comprising or composed of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO. 29 or 79;

[0152] (ii) comprising or composed of the amino acid sequence of SEQ ID NO. 29 or 79; or

[0153] (iii) comprises or consists of an amino acid sequence having one or more (preferably 10 or fewer, more preferably 5, 4, 3, 2 or fewer) amino acid changes (preferably amino acid substitution, more preferably conservative amino acid substitution) compared to the amino acid sequence of SEQ ID NO. 29 or 79.

[0154] In some embodiments, the light chain constant region of the anti-CD28 antibody of the present invention is a (human) kappa or lambda light chain constant region, e.g., a kappa light chain constant region.

[0155] In some embodiments, the light chain invariant region is

[0156] (i) comprising or composed of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence of SEQ ID NO. 30;

[0157] (ii) comprising or composed of the amino acid sequence of SEQ ID NO. 30; or

[0158] (iii) comprises or consists of an amino acid sequence having one or more (preferably 10 or fewer, more preferably 5, 4, 3, 2 or fewer) amino acid changes (preferably amino acid substitution, more preferably conservative amino acid substitution) compared to the amino acid sequence of SEQ ID NO. 30.

[0159] In some embodiments, the anti-CD28 antibody of the present invention or its antigen-binding fragment comprises an antibody heavy chain. In some embodiments, the anti-CD28 antibody of the present invention or its antigen-binding fragment comprises an antibody light chain. In some embodiments, the anti-CD28 antibody of the present invention or its antigen-binding fragment comprises a heavy chain and a light chain. In some embodiments, the anti-CD28 antibody of the present invention or its antigen-binding fragment comprises two heavy chains and two light chains. In some embodiments, the heavy chain comprises or is composed of a heavy chain variable region and a heavy chain constant region. In some embodiments, the light chain comprises or is composed of a light chain variable region and a light chain constant region.

[0160] In some embodiments, the anti-CD28 antibody of the present invention or its antigen-binding fragment has one or more of the following characteristics:

[0161] (i) exhibiting the same or similar binding affinity and / or specificity for CD28 as the antibody of the present invention;

[0162] (ii) inhibiting the binding of the antibody of the present invention to CD28 (e.g., competitively inhibiting);

[0163] (iii) binding to an epitope that is identical to or overlaps with the antibody of the present invention;

[0164] (iv) competing with the antibody of the present invention for binding to CD28;

[0165] (v) having one or more biological characteristics of the antibody of the present invention.

[0166] In some embodiments, the anti-CD28 antibody is a monoclonal antibody.

[0167] In some embodiments, the anti-CD28 antibody is a humanized antibody. Humanization can be achieved by replacing one or more amino acid residues, specifically the framework region sequence, in the heavy and light chain variable regions of a non-human natural antibody with residues at corresponding positions in the variable region of a human normal antibody. Methods for humanizing antibodies are widely known in the art. Typically, humanization substitution is performed in a manner that preserves the favorable binding properties of the antibody. Assays for determining biological properties of humanized antibodies, such as binding affinity, are widely known in the art for determining and selecting suitable mutations or combinations of mutations of the humanized residues.

[0168] In one embodiment, the anti-CD28 antibody of the present invention is a full-length antibody. In one embodiment, the anti-CD28 antibody of the present invention also comprises an antibody fragment (e.g., an antigen-binding fragment) thereof, preferably selected from Fab, Fab', Fab'-SH, Fv, a single-chain antibody (e.g., scFv), (Fab')2, a single-domain antibody (e.g., VHH), a domain antibody (dAb), a heavy-chain antibody, or a linear antibody. In one embodiment, the anti-CD28 antibody fragment of the present invention is Fab comprising CH1 and CL as well as VH and VL as described herein.

[0169] In one embodiment, the anti-CD28 antibody of the present invention may also be a bispecific antibody or a multispecific antibody that specifically binds to CD28 and one or more additional antigens (e.g., TAA).

[0170] III. Multispecific Binding Molecules

[0171] In one aspect of the invention, the invention relates to a multispecific binding molecule capable of specifically binding to CD28 and specifically binding to one or more additional targets or antigens. In some embodiments, the additional antigen is a tumor-associated antigen (TAA) such as CLDN18.2 or PSMA. In some embodiments, the multispecific binding molecule is a multispecific antibody, e.g., a bispecific antibody.

[0172] In some embodiments, the antibody of the present invention is a bispecific antibody. The term “bispecific antibody” refers to an antibody comprising a first antigen-binding region and a second antigen-binding region, wherein the first antigen-binding region binds to one antigen or epitope and the second antigen-binding region binds to another antigen or another epitope. Thus, the bispecific antibody according to the present invention comprises specificity for two different antigens, or for two different epitopes of a single antigen. Bispecific antibody forms include IgG-like antibodies (Fan et al. (2015) Journal of Hematology & Oncology. 8: 130). The most common type of IgG-like antibody comprises two Fab regions and two Fc regions, and the heavy and light chains of each Fab may be derived from separate monoclonal antibodies. The bispecific antibody of the present invention may be prepared using bispecific antibody forms or techniques known in the art. Specific exemplary bispecific forms that can be used in the context of the present invention can be found, for example, in Labrijn, et al., Bispecific antibodies: a mechanistic review of the pipeline, Nature Reviews Drug Discovery, 2019, 18(8):1-24.

[0173] Accordingly, one aspect of the present invention relates to a bispecific antibody, which is

[0174] It comprises a first antigen-binding region and a second antigen-binding region, wherein the first antigen-binding region specifically binds to CD28 and the second antigen-binding region specifically binds to a tumor-associated antigen. In some embodiments, the tumor-associated antigen is selected from PSMA or CLDN18.2.

[0175] In some embodiments, the first antigen-binding region is derived from the anti-CD28 antibody described herein or its antigen-binding fragment, e.g., the Fab of the anti-CD28 antibody described herein. In some embodiments, the first antigen-binding region that specifically binds to CD28 comprises 1, 2, 3, 4, 5, or 6 CDRs of the anti-CD28 antibody described herein. In some embodiments, the first antigen-binding region comprises 1, 2, or 3 heavy chain variable region CDRs of the anti-CD28 antibody described herein, namely CDRH1, CDRH2, and CDRH3. In some embodiments, the first antigen-binding region comprises 1, 2, or 3 light chain variable region CDRs of the anti-CD28 antibody described herein, namely CDRL1, CDRL2, and CDRL3. In some embodiments, the first antigen-binding region comprises 3 heavy chain variable region CDRs and 3 light chain variable region CDRs of the anti-CD28 antibody described herein. In some embodiments, the first antigen-binding region comprises a heavy chain variable region of the anti-CD28 antibody described herein. In some embodiments, the first antigen-binding region comprises a light chain variable region of the anti-CD28 antibody described herein. In some embodiments, the first antigen-binding region comprises a heavy chain variable region and a light chain variable region of the anti-CD28 antibody described herein. In some embodiments, the first antigen-binding region comprises a Fab of the anti-CD28 antibody described herein.

[0176] In some embodiments, the second antigen-binding region is derived from an anti-TAA (e.g., CLDN18.2 or PSMA, e.g., human CLDN18.2 or human PSMA) antibody or its antigen-binding fragment, e.g., an anti-CLDN18.2 antibody or its antigen-binding fragment, or an anti-PSMA antibody or its antigen-binding fragment.

[0177] A first antigen-binding region suitable for use in the bispecific antibody of the present invention may comprise or be composed of the anti-CD28 full-length antibody of the present invention or an antigen-binding fragment thereof, as long as it can specifically bind to CD28, and may include, but is not limited to, a full-length antibody, half-antibody, Fab, Fab', Fab'-SH, Fv, single-chain antibody (e.g., scFv), or (Fab')2, a single-domain antibody (e.g., VHH), a domain antibody (dAb), a heavy-chain antibody, or a linear antibody that specifically binds to CD28.

[0178] A second antigen-binding region suitable for use in the bispecific antibody of the present invention may comprise or be composed of an anti-TAA antibody or an antigen-binding fragment thereof, as long as it can specifically bind to TAA, and includes, but is not limited to, full-length antibodies, half-antibodies, Fab, Fab', Fab'-SH, Fv, single-chain antibodies (e.g., scFv), and (Fab')2, single-domain antibodies (e.g., VHH), domain antibodies (dAb), heavy-chain antibodies, or linear antibodies that specifically bind to TAA.

[0179] In some embodiments, a second antigen-binding region suitable for use in the bispecific antibody of the present invention may comprise, but is not limited to, an anti-CLDN18.2 antibody or its antigen-binding fragment (e.g., the anti-CLDN18.2 antibody or its antigen-binding fragment disclosed in CN202010570517.X (WO2021254481A1), e.g., an HB37A6 antibody or its antigen-binding fragment) that can specifically bind to CLDN18.2, and may comprise, for example, but not limited to, a full-length antibody, half-antibody, Fab, Fab', Fab'-SH, Fv, single-chain antibody (e.g., scFv), and (Fab')2, a single-domain antibody (e.g., VHH), a domain antibody (dAb), a heavy-chain antibody or a linear antibody that specifically binds to CLDN18.2.

[0180] In some embodiments, a second antigen-binding region suitable for use in the bispecific antibody of the present invention may comprise or be composed of an anti-PSMA antibody or its antigen-binding fragment (e.g., an anti-PSMA antibody or its antigen-binding fragment disclosed in EP1851250B1, e.g., a 1C3 antibody or its antigen-binding fragment) as long as it can specifically bind to PSMA, and may comprise, but is not limited to, full-length antibodies, half-antibodies, Fab, Fab', Fab'-SH, Fv, single-chain antibodies (e.g., scFv), and (Fab')2, single-domain antibodies (e.g., VHH), domain antibodies (dAb), heavy-chain antibodies, or linear antibodies that specifically bind to PSMA.

[0181] In some embodiments, the bispecific antibody of the present invention is an IgG-like bispecific antibody. In some embodiments, the IgG-like bispecific antibody of the present invention refers to a bispecific antibody comprising an Fc dimer (e.g., a heteromeric Fc scaffold). Accordingly, in some embodiments, the bispecific antibody of the present invention comprises an Fc dimer, e.g., a heteromeric Fc scaffold.

[0182] In one embodiment, the bispecific antibody may comprise one or more first antigen-binding regions. In one embodiment, the bispecific antibody may comprise one or more second antigen-binding regions.

[0183] III-1 Second Antigen-Binding Zone

[0184] In some embodiments, the second antigen-binding region specifically binds to a tumor-associated antigen (TAA) or an immune checkpoint molecule.

[0185] In some embodiments, the tumor-associated antigen is CLDN18.2.

[0186] In some embodiments, the second antigen-binding region is an antigen-binding region that specifically binds to CLDN18.2. In some embodiments, the second antigen-binding region that specifically binds to CLDN18.2 is derived from an antibody that specifically binds to CLDN18.2, for example, an antibody that specifically binds to CLDN18.2 disclosed in CN202010570517.X (WO2021254481A1) (the whole of which is incorporated herein), for example, HB37A6 disclosed herein.

[0187] In some embodiments, the second antigen-binding region that specifically binds to CLDN18.2 comprises 1, 2, 3, 4, 5, or 6 CDRs of an antibody known to specifically bind to CLDN18.2 (e.g., the CLDN18.2 antibody of CN202010570517.X (WO2021254481A1), e.g., HB37A6 disclosed herein). In some embodiments, the second antigen-binding region comprises 1, 2, or 3 heavy chain variable region CDRs (i.e., CDRH1, CDRH2, and CDRH3) of an antibody known to specifically bind to CLDN18.2 (e.g., the CLDN18.2 antibody of CN202010570517.X (WO2021254481A1), e.g., HB37A6 disclosed herein). In some embodiments, the second antigen-binding region comprises one, two, or three light chain variable region CDRs (i.e., CDRL1, CDRL2, and CDRL3) of an antibody known to specifically bind to CLDN18.2 (e.g., the CLDN18.2 antibody of CN202010570517.X (WO2021254481A1), e.g., HB37A6 disclosed herein). In some embodiments, the second antigen-binding region comprises three heavy chain variable region CDRs and three light chain variable region CDRs of an antibody known to specifically bind to CLDN18.2 (e.g., the CLDN18.2 antibody of CN202010570517.X (WO2021254481A1), e.g., HB37A6 disclosed herein). In some embodiments, the second antigen-binding region comprises a heavy chain variable region of an antibody known to specifically bind to CLDN18.2 (e.g., the CLDN18.2 antibody of CN202010570517.X (WO2021254481A1), e.g., HB37A6 disclosed herein). In some embodiments, the second antigen-binding region comprises a CLDN18.2 antibody known to specifically bind to CLDN18.2 (e.g., the CLDN18 antibody of CN202010570517.X (WO2021254481A1).2. It comprises a light chain variable region of an antibody, e.g., HB37A6 disclosed herein. In some embodiments, the second antigen-binding region comprises a heavy chain variable region and a light chain variable region of an antibody known to specifically bind to CLDN18.2 (e.g., the CLDN18.2 antibody of CN202010570517.X (WO2021254481A1), e.g., HB37A6 disclosed herein). In some embodiments, the second antigen-binding region comprises a Fab of an antibody known to specifically bind to CLDN18.2 (e.g., the CLDN18.2 antibody of CN202010570517.X (WO2021254481A1), e.g., HB37A6 disclosed herein).

[0188] In some embodiments, a second antigen-binding region that specifically binds to CLDN18.2 comprises three complementary-determining regions (CDRHs) from a heavy chain variable region: CDRH1, CDRH2, and CDRH3, wherein CDRH1, CDRH2, and CDRH3 are CDRH1, CDRH2, and CDRH3 of the heavy chain variable region described in SEQ ID NO. 86; preferably, wherein CDRH may be determined according to any system for determining CDR, e.g., Kabat, AbM, Chothia, Contact, or IMGT system, or a combination thereof. In some embodiments, a second antigen-binding region that specifically binds to CLDN18.2 comprises three complementary-determining regions (CDRLs) from a light chain variable region: CDRL1, CDRL2, and CDRL3, wherein CDRL1, CDRL2, and CDRL3 are CDRL1, CDRL2, and CDRL3 of the light chain variable region described in SEQ ID NO. 90; Preferably, the CDRL here may be determined according to any system for determining the CDR, for example, the Kabat, AbM, Chothia, Contact, or IMGT system, or a combination thereof.

[0189] In some embodiments, in a second antigen-binding region that specifically binds to CLDN18.2, CDRH1 comprises or is composed of the amino acid sequence of SEQ ID NO. 87; CDRH2 comprises or is composed of the amino acid sequence of SEQ ID NO. 88; CDRH3 comprises or is composed of the amino acid sequence of SEQ ID NO. 89; CDRL1 comprises or is composed of the amino acid sequence of SEQ ID NO. 91; CDRL2 comprises or is composed of the amino acid sequence of SEQ ID NO. 92; and / or CDRL3 comprises or is composed of the amino acid sequence of SEQ ID NO. 93.

[0190] In some embodiments, a second antigen-binding region that specifically binds to CLDN18.2 comprises VH, wherein VH is

[0191] Complementary-determining regions (CDRHs) from a heavy chain variable region, comprising CDRH1, CDRH2, and CDRH3, wherein CDRH1 comprises or consists of the amino acid sequence of SEQ ID NO. 87, CDRH2 comprises or consists of the amino acid sequence of SEQ ID NO. 88, and CDRH3 comprises or consists of the amino acid sequence of SEQ ID NO. 89; or

[0192] It includes or is composed of the amino acid sequence described in SEQ ID NO. 86.

[0193] In some embodiments, a second antigen-binding region that specifically binds to CLDN18.2 comprises VL, wherein VL is

[0194] Complementary-determining regions (CDRHs) from the light chain variable region, CDRH1, CDRH2, and CDRH3, wherein CDRL1 comprises or consists of the amino acid sequence of SEQ ID NO. 91, CDRL2 comprises or consists of the amino acid sequence of SEQ ID NO. 92, and CDRL3 comprises or consists of the amino acid sequence of SEQ ID NO. 93; or

[0195] It includes or is composed of the amino acid sequence described in SEQ ID NO. 90.

[0196] In some embodiments, a second antigen-binding region that specifically binds to CLDN18.2 comprises three complementary-determining regions (CDRHs) from a heavy chain variable region: CDRH1, CDRH2, and CDRH3, and three complementary-determining regions (CDRLs) from a light chain variable region: CDRL1, CDRL2, and CDRL3, wherein CDRH1 comprises or is composed of the amino acid sequence of SEQ ID NO. 87; CDRH2 comprises or is composed of the amino acid sequence of SEQ ID NO. 88; CDRH3 comprises or is composed of the amino acid sequence of SEQ ID NO. 89; CDRL1 comprises or is composed of the amino acid sequence of SEQ ID NO. 91; CDRL2 comprises or is composed of the amino acid sequence of SEQ ID NO. 92; and CDRL3 comprises or is composed of the amino acid sequence of SEQ ID NO. 93.

[0197] In some embodiments, a second antigen-binding region that specifically binds to CLDN18.2 comprises VH and VL, wherein VH comprises or is composed of the sequence described in SEQ ID NO. 86; and / or VL comprises or is composed of the sequence described in SEQ ID NO. 90.

[0198] In some embodiments, the second antigen-binding region that specifically binds to CLDN18.2 is a Fab fragment.

[0199] In some embodiments, the second antigen-binding region is an antigen-binding region that specifically binds to PSMA. In some embodiments, the second antigen-binding region that specifically binds to PSMA is derived from an antibody that specifically binds to PSMA, for example, an antibody that specifically binds to PSMA disclosed in EP1851250B1, for example, 1C3 disclosed therein.

[0200] In some embodiments, the second antigen-binding region that specifically binds to PSMA comprises 1, 2, 3, 4, 5, or 6 CDRs of an antibody known to specifically bind to PSMA (e.g., the PSMA antibody disclosed in EP1851250B1, the whole of which is incorporated herein by reference, e.g., 1C3 disclosed herein). In some embodiments, the second antigen-binding region comprises 1, 2, or 3 heavy chain variable region CDRs (i.e., CDRH1, CDRH2, and CDRH3) of an antibody known to specifically bind to PSMA (e.g., the PSMA antibody disclosed in EP1851250B1, e.g., 1C3 disclosed herein). In some embodiments, the second antigen-binding region comprises one, two, or three light chain variable region CDRs (i.e., CDRL1, CDRL2, and CDRL3) of an antibody known to specifically bind to PSMA (e.g., the PSMA antibody disclosed in EP1851250B1, e.g., 1C3 disclosed herein). In some embodiments, the second antigen-binding region comprises three heavy chain variable region CDRs and three light chain variable region CDRs of an antibody known to specifically bind to PSMA (e.g., the PSMA antibody disclosed in EP1851250B1, e.g., 1C3 disclosed herein). In some embodiments, the second antigen-binding region comprises a heavy chain variable region of an antibody known to specifically bind to PSMA (e.g., the PSMA antibody disclosed in EP1851250B1, e.g., 1C3 disclosed herein). In some embodiments, the second antigen-binding region comprises a light chain variable region of an antibody known to specifically bind to PSMA (e.g., the PSMA antibody disclosed in EP1851250B1, e.g., 1C3 disclosed herein). In some embodiments, the second antigen-binding region comprises a heavy chain variable region and a light chain variable region of an antibody known to specifically bind to PSMA (e.g., the PSMA antibody disclosed in EP1851250B1, e.g., 1C3 disclosed herein).In some embodiments, the second antigen-binding region comprises a Fab of an antibody known to specifically bind to PSMA (e.g., the PSMA antibody disclosed in EP1851250B1, e.g., 1C3 disclosed therein).

[0201] In some embodiments, a second antigen-binding region that specifically binds to PSMA comprises three complementary-determining regions (CDRHs) from a heavy chain variable region: CDRH1, CDRH2, and CDRH3, wherein CDRH1, CDRH2, and CDRH3 are CDRH1, CDRH2, and CDRH3 of the heavy chain variable region described in SEQ ID NO. 47; preferably, CDRH may be determined according to any system for determining CDR, e.g., Kabat, AbM, Chothia, Contact, or IMGT system, or a combination thereof. In some embodiments, a second antigen-binding region that specifically binds to PSMA comprises three complementary-determining regions (CDRLs) from a light chain variable region: CDRL1, CDRL2, and CDRL3, wherein CDRL1, CDRL2, and CDRL3 are CDRL1, CDRL2, and CDRL3 of the light chain variable region described in SEQ ID NO. 49; Preferably, CDRL may be determined according to any system for determining CDR, e.g., Kabat, AbM, Chothia, Contact, or IMGT system, or a combination thereof. In some embodiments, in a second antigen-binding region that specifically binds to PSMA, CDRH1 comprises or is composed of the amino acid sequence of SEQ ID NO. 96; CDRH2 comprises or is composed of the amino acid sequence of SEQ ID NO. 97; CDRH3 comprises or is composed of the amino acid sequence of SEQ ID NO. 98; CDRL1 comprises or is composed of the amino acid sequence of SEQ ID NO. 99; CDRL2 comprises or is composed of the amino acid sequence of SEQ ID NO. 100; and / or CDRL3 comprises or is composed of the amino acid sequence of SEQ ID NO. 101.

[0202] In some embodiments, a second antigen-binding region that specifically binds to PSMA comprises VH, wherein VH is

[0203] Complementary-determining regions (CDRHs) from a heavy chain variable region, comprising CDRH1, CDRH2, and CDRH3, wherein CDRH1 comprises or consists of the amino acid sequence of SEQ ID NO. 96, CDRH2 comprises or consists of the amino acid sequence of SEQ ID NO. 97, and CDRH3 comprises or consists of the amino acid sequence of SEQ ID NO. 98; or

[0204] It includes or is composed of the amino acid sequence described in SEQ ID NO. 47.

[0205] In some embodiments, a second antigen-binding region that specifically binds to PSMA comprises VL, wherein VL is

[0206] Complementary-determining regions (CDRHs) from the light chain variable region, CDRH1, CDRH2, and CDRH3, wherein CDRL1 comprises or consists of the amino acid sequence of SEQ ID NO. 99, CDRL2 comprises or consists of the amino acid sequence of SEQ ID NO. 100, and CDRL3 comprises or consists of the amino acid sequence of SEQ ID NO. 101; or

[0207] It includes or is composed of the amino acid sequence described in SEQ ID NO. 49.

[0208] In some embodiments, the second antigen-binding region that specifically binds to PSMA comprises three complementary-determining regions (CDRHs) from the heavy chain variable region: CDRH1, CDRH2, and CDRH3, and three complementary-determining regions (CDRLs) from the light chain variable region: CDRL1, CDRL2, and CDRL3, wherein CDRH1 comprises or is composed of the amino acid sequence of SEQ ID NO. 96; CDRH2 comprises or is composed of the amino acid sequence of SEQ ID NO. 97; CDRH3 comprises or is composed of the amino acid sequence of SEQ ID NO. 98; CDRL1 comprises or is composed of the amino acid sequence of SEQ ID NO. 99; CDRL2 comprises or is composed of the amino acid sequence of SEQ ID NO. 100; and CDRL3 comprises or is composed of the amino acid sequence of SEQ ID NO. 101.

[0209] In some embodiments, a second antigen-binding region that specifically binds to PSMA comprises VH and VL, wherein VH comprises or is composed of the sequence described in SEQ ID NO. 47; and VL comprises or is composed of the sequence described in SEQ ID NO. 49.

[0210] In some embodiments, the second antigen-binding region that specifically binds to PSMA is a Fab fragment.

[0211] III-2 Fab Short Story

[0212] In some embodiments, the first antigen-binding region and / or the second antigen-binding region of the present invention is a Fab fragment. A Fab fragment suitable for use as an antigen-binding region of a multispecific binding molecule of the present invention, e.g., a bispecific antibody, is composed of two polypeptide chains comprising the VH, CH1, VL, and CL domains of the antibody, wherein VH is paired with VL and CH1 is paired with CL to form an antigen-binding region. In some embodiments, in the Fab, one chain comprises or is composed of VH and CH1 (i.e., VH-CH1) from the N-terminus to the C-terminus, and the other chain comprises or is composed of VL and CL (i.e., VL-CL) from the N-terminus to the C-terminus. In some embodiments, in the multispecific antibody of the present invention, the Fab may be connected to the N-terminus of the Fc domain of the antibody through the C-terminus of the chain comprising VH. Preferably, Fab comprises a VH-CH1 chain and a VL-CL chain and can be linked to the Fc domain of the antibody through the C-terminus of CH1 of the VH-CH1 chain. In some embodiments, the linkage is direct or through a linker. As described herein, a Fab chain comprising or consisting of VH-CH1 is also referred to as a Fab heavy chain, and a Fab chain comprising or consisting of VL-CL is also referred to as a Fab light chain.

[0213] In some embodiments, CH1 is CH1 from IgG1, IgG2, IgG3, or IgG4, preferably CH1 from IgG1. In some embodiments, CH1 is

[0214] (i) comprising or composed of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence of SEQ ID NO. 74;

[0215] (ii) comprising or composed of the amino acid sequence of SEQ ID NO. 74; or

[0216] (iii) comprises or consists of an amino acid sequence having one or more (preferably 10 or fewer, more preferably 5, 4, 3, 2 or fewer) amino acid changes (preferably amino acid substitution, more preferably conservative amino acid substitution) compared to the amino acid sequence of SEQ ID NO. 74.

[0217] In some embodiments, CL is a kappa light chain invariant region or a lambda light chain invariant region. In some embodiments, CL is a kappa light chain invariant region. In some embodiments, CL is

[0218] (i) comprising or composed of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence of SEQ ID NO. 30;

[0219] (ii) comprising or composed of the amino acid sequence of SEQ ID NO. 30; or

[0220] (iii) comprises or consists of an amino acid sequence having one or more (preferably 10 or fewer, more preferably 5, 4, 3, 2 or fewer) amino acid changes (preferably amino acid substitution, more preferably conservative amino acid substitution) compared to the amino acid sequence of SEQ ID NO. 30.

[0221] In some embodiments, the first antigen-binding region is a Fab that specifically binds to CD28, wherein the Fab fragment is derived from the anti-CD28 antibody of the present invention and comprises the heavy chain variable region VH and the light chain variable region VL of the anti-CD28 antibody of the present invention. In some embodiments, the Fab heavy chain of the Fab that specifically binds to CD28 as the first antigen-binding region comprises or is composed of VH and CH1, wherein VH is the VH of the anti-CD28 antibody of the present invention. In some embodiments, the Fab light chain of the Fab that specifically binds to CD28 as the first antigen-binding region comprises or is composed of VL and CL, wherein VL is the VL of the anti-CD28 antibody of the present invention.

[0222] In some embodiments, the Fab heavy chain that specifically binds to CD28 is

[0223] (i) comprising or composed of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO. 103, 104, or 105;

[0224] (ii) comprising or composed of the amino acid sequence of SEQ ID NO. 103, 104 or 105; or

[0225] (iii) comprises or consists of an amino acid sequence having one or more (preferably 10 or fewer, more preferably 5, 4, 3, 2 or fewer) amino acid changes (preferably amino acid substitution, more preferably conservative amino acid substitution) compared to the amino acid sequence of SEQ ID NO. 103, 104 or 105.

[0226] In some embodiments, the Fab light chain that specifically binds to CD28 is

[0227] (i) comprising or composed of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO. 36, 38, or 40;

[0228] (ii) comprising or composed of the amino acid sequence of SEQ ID NO. 36, 38 or 40; or

[0229] (iii) comprises or consists of an amino acid sequence having one or more (preferably 10 or fewer, more preferably 5, 4, 3, 2 or fewer) amino acid changes (preferably amino acid substitution, more preferably conservative amino acid substitution) compared to the amino acid sequence of SEQ ID NO. 36, 38 or 40.

[0230] In some embodiments, the Fab heavy chain that specifically binds to CD28 comprises or is composed of the amino acid sequence described in SEQ ID NO. 103, and the Fab light chain comprises or is composed of the amino acid sequence described in SEQ ID NO. 36;

[0231] In some embodiments, the Fab heavy chain specifically binding to CD28 comprises or is composed of the amino acid sequence described in SEQ ID NO. 104, and the Fab light chain comprises or is composed of the amino acid sequence described in SEQ ID NO. 38; or

[0232] In some embodiments, the Fab heavy chain that specifically binds to CD28 comprises or is composed of the amino acid sequence described in SEQ ID NO. 105, and the Fab light chain comprises or is composed of the amino acid sequence described in SEQ ID NO. 40.

[0233] In some embodiments, the second antigen-binding region is a Fab that specifically binds to TAA, wherein the Fab fragment is derived from an anti-TAA antibody and comprises the heavy chain variable region VH and the light chain variable region VL of the anti-TAA antibody (e.g., anti-CLDN18.2 antibody or anti-PSMA antibody). In some embodiments, the second antigen-binding region is a Fab that specifically binds to CLDN18.2, wherein the Fab fragment is derived from an anti-CLDN18.2 antibody and comprises the heavy chain variable region VH and the light chain variable region VL of the anti-CLDN18.2 antibody. In some embodiments, the second antigen-binding region is a Fab that specifically binds to PSMA, wherein the Fab fragment is derived from an anti-PSMA antibody and comprises the heavy chain variable region VH and the light chain variable region VL of the anti-PSMA antibody.

[0234] In some embodiments, the second antigen-binding region Fab comprises VH or VL of the second antigen-binding region described herein, or comprises VH and VL of the second antigen-binding region described herein.

[0235] In some embodiments, the Fab heavy chain that specifically binds to CLDN18.2 is

[0236] (i) comprising or composed of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence of SEQ ID NO. 102;

[0237] (ii) comprising or composed of the amino acid sequence of SEQ ID NO. 102; or

[0238] (iii) comprises or consists of an amino acid sequence having one or more (preferably 10 or fewer, more preferably 5, 4, 3, 2 or fewer) amino acid changes (preferably amino acid substitution, more preferably conservative amino acid substitution) compared to the amino acid sequence of SEQ ID NO. 102.

[0239] In some embodiments, the Fab light chain that specifically binds to CLDN18.2 is

[0240] (i) comprising or composed of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence of SEQ ID NO. 34;

[0241] (ii) comprising or composed of the amino acid sequence of SEQ ID NO. 34; or

[0242] (iii) comprises or consists of an amino acid sequence having one or more (preferably 10 or fewer, more preferably 5, 4, 3, 2 or fewer) amino acid changes (preferably amino acid substitution, more preferably conservative amino acid substitution) compared to the amino acid sequence of SEQ ID NO. 34.

[0243] In some embodiments, the Fab heavy chain that specifically binds to CLDN18.2 comprises or is composed of the amino acid sequence described in SEQ ID NO. 102, and the Fab light chain comprises or is composed of the amino acid sequence described in SEQ ID NO. 34.

[0244] In some embodiments, the Fab heavy chain that specifically binds to PSMA is

[0245] (i) comprising or composed of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence of SEQ ID NO. 106;

[0246] (ii) comprising or composed of the amino acid sequence of SEQ ID NO. 106; or

[0247] (iii) comprises or consists of an amino acid sequence having one or more (preferably 10 or fewer, more preferably 5, 4, 3, 2 or fewer) amino acid changes (preferably amino acid substitution, more preferably conservative amino acid substitution) compared to the amino acid sequence of SEQ ID NO. 106.

[0248] In some embodiments, the Fab light chain that specifically binds to PSMA is

[0249] (i) comprising or composed of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence of SEQ ID NO. 50;

[0250] (ii) comprising or composed of the amino acid sequence of SEQ ID NO. 50; or

[0251] (iii) comprises or consists of an amino acid sequence having one or more (preferably 10 or fewer, more preferably 5, 4, 3, 2 or fewer) amino acid changes (preferably amino acid substitution, more preferably conservative amino acid substitution) compared to the amino acid sequence of SEQ ID NO. 50.

[0252] In some embodiments, the Fab heavy chain that specifically binds to PSMA comprises or is composed of the amino acid sequence described in SEQ ID NO. 106, and the Fab light chain comprises or is composed of the amino acid sequence described in SEQ ID NO. 50.

[0253] III-3 Fc area

[0254] In some embodiments, one or more target-binding regions (antigen-binding regions) of the multispecific binding molecule of the present invention, such as a multispecific antibody (e.g., a bispecific antibody), further comprise an Fc region, wherein the included Fc region may be the same or different.

[0255] In some embodiments, the first Fc region and the second Fc region may be different and dimerized to form a heterodimeric Fc scaffold.

[0256] In the present invention, the Fc region refers to a C-terminal region of an immunoglobulin heavy chain comprising at least a portion of an invariant region, and may include an Fc region of the natural sequence and a variant Fc region. The Fc region of the natural sequence includes naturally occurring Fc sequences of various immunoglobulins, such as Fc regions of various Ig subclasses or allotypes thereof (Gestur Vidarsson et al., IgG subclasses and allotypes: from structure to effector functions, 20 October 2014, doi: 10.3389 / fimmu.2014.00520). In some embodiments, the Fc region of the present invention comprises CH2 and CH3 of the antibody. In some embodiments, the antibody Fc region may also have an IgG hinge region or a portion of an IgG hinge region at the N-terminus, for example, an IgG1 hinge region or a portion of an IgG1 hinge region, for example, sequences D221 to P230 according to EU numbering. The hinge region may contain mutations.

[0257] Unless otherwise indicated herein, amino acid residues in the Fc region are numbered according to the EU numbering system, also known as the EU index, as described in Kabat, EA et al., Sequences of Proteins of Immunological Interest, 5th Ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991), NIH Publication 91-3242.

[0258] In some embodiments, the Fc region is human IgG Fc, e.g., human IgG1 Fc, human IgG2 Fc, human IgG3 Fc, or human IgG4 Fc. In one embodiment, the Fc region comprises or is composed of the amino acid sequence of SEQ ID NO: 94 or 95 or an amino acid sequence having at least 90% identity with respect to it, e.g., 95%, 96%, 97%, 98%, 99%, or more identity.

[0259] As is understood by those skilled in the art, in order to facilitate the formation of a heterodimer by the multispecific antibody of the present invention, the Fc region included in the multispecific antibody of the present invention may include a mutation favorable for the heterodimerization of the first Fc region and the second Fc region. In one embodiment, a mutation is introduced into the CH3 region of the two Fc regions.

[0260] Methods for promoting heterodimerization of Fc regions are known in the art. For example, the CH3 regions of a first Fc region and the CH3 regions of a second Fc region are engineered in a complementary manner so that each CH3 region (or heavy chain containing it) can no longer homodimerize with itself and is forced heterodimerized with another complementarily engineered CH3 region (thereby heterodimerization occurs between the CH3 regions of the first and second Fc regions, and no homodimer is formed between two first CH3 regions or between two second CH3 regions). For example, corresponding Knob and Hole mutations are introduced into the first Fc region and the second Fc region based on Knob-into-Hole technology. For example, US 5,731,168; US 7,695,936; See Ridgway et al., Prot Eng 9, 617-621 (1996) and Carter, J Immunol Meth 248, 7-15 (2001).

[0261] Based on Innobody technology, corresponding mutations may also be introduced into the first Fc region and the second Fc region. For example, see PCT / CN2021 / 143141 (the entirety of which is incorporated herein by reference).

[0262] In one specific embodiment, the first CH3 region comprises an S364R / K mutation (preferably S364R) and optionally one or more additional mutations. In some embodiments, the second CH3 region comprises a K370S / T / A / V mutation (preferably K370S) and optionally one or more additional mutations. In some embodiments, the first CH3 region comprises an S364R / K mutation and the second CH3 region comprises a K370S / T / A / V mutation. In some embodiments, the first CH3 region comprises an S364R mutation and the second CH3 region comprises a K370S mutation.

[0263] In some embodiments, the first CH3 region comprises the S364R / K (preferably S364R) mutation and the D399K / R (preferably D399K) mutation. In some embodiments, the second CH3 region comprises the K370S / T / A / V (preferably K370S) mutation and the K409D / E (preferably K409D) mutation. In some embodiments, the first CH3 region comprises S364R / K+D399K / R, and the second CH3 region comprises K370S / T / A / V+Y349T / S / A / V. In some embodiments, the first CH3 region comprises S364R+D399K, and the second CH3 region comprises K370S+Y349T. In some embodiments, the first CH3 region further comprises E375N / Q (preferably E375N) and / or T350V / A (preferably T350V). In some embodiments, the second CH3 region further comprises K409D / E (preferably K409D), Q347D / E (preferably Q347D), and / or T350V / A (preferably T350V).

[0264] In some embodiments, the first CH3 region includes S364R+D399K, and the second CH3 region includes K370S+Y349T+K409D. In some embodiments, the first CH3 region additionally includes E357N. In some embodiments, the second CH3 region additionally includes Q347D. In some embodiments, the first CH3 region additionally includes E357N, and the second CH3 region additionally includes Q347D. In some embodiments, the first CH3 region and the second CH3 region each additionally include T350V, or both include T350V.

[0265] Accordingly, in some embodiments, the first CH3 region comprises S364R+D399K, and the second CH3 region comprises K370S+Y349T+K409D+Q347D. In some embodiments, the first CH3 region comprises S364R+D399K+E357N, and the second CH3 region comprises K370S+Y349T+K409D+Q347D. In some embodiments, the first CH3 region comprises S364R+D399K+E357N+T350V, and the second CH3 region comprises K370S+Y349T+K409D+Q347D+T350V.

[0266] In some embodiments, the first CH3 region comprises K409E / D (preferably K409E). In some embodiments, the second CH3 region comprises D399K / R (preferably D399K) or K370T / S / A / V (preferably K370T). In some embodiments, the first CH3 region comprises K409E / D (preferably K409E), and the second CH3 region comprises D399K / R (preferably D399K). In some embodiments, the first CH3 region further comprises T411R / K (preferably T411R). In some embodiments, the second CH3 region further comprises K370T / S / A / V (preferably K370T). In some embodiments, the CH3 region comprises K409E / D+T411R / K, and the second CH3 region comprises D399K / R+K370T / S / A / V. In some embodiments, the CH3 region comprises K409E+T411R, and the second CH3 region comprises D399K+K370T.

[0267] In some specific embodiments, the first CH3 region and the second CH3 region have the following five mutation combinations:

[0268]

[0269] In one embodiment, CH3 in one Fc region contains S364R and D399K mutations, and CH3 in the remaining Fc region contains Y349T, K370S, and K409D mutations.

[0270] In some embodiments, the Fc region additionally includes other mutations that facilitate heterodimer purification.

[0271] The Fc region of the binding molecule of the present invention (e.g., antibody molecule) can also be mutated to obtain desired characteristics. Mutations to the Fc region are known in the art.

[0272] In one embodiment, the Fc region has its characteristics of the effector function (e.g., the complement activation function of the Fc region) modified. In one embodiment, the effector function is reduced or eliminated compared to the wild-type isotype Fc region. In one embodiment, the effector function is reduced or eliminated using a method selected from using an Fc isotype having a naturally reduced or eliminated effector function and modifying the Fc region.

[0273] In one preferred embodiment, the Fc region has a reduced effector function mediated by the Fc region, such as a reduced or eliminated ADCC, ADCP, or CDC effector function, and includes, for example, a mutation to achieve said function.

[0274] As understood by those skilled in the art, depending on the intended use of the binding molecule (e.g., antibody molecule) of the present invention, the binding molecule (e.g., antibody molecule) of the present invention may also include modifications of the Fc domain that alter the binding affinity for one or more Fc receptors. In one embodiment, the Fc receptor is an Fcγ receptor, specifically a human Fcγ receptor. In some embodiments, the Fc region includes a mutation that reduces binding to the Fcγ receptor. For example, in some embodiments, the Fc region used in the present invention has a mutation that reduces binding to the Fcγ receptor, e.g., the L234A / L235A mutation or a deletion of proline at position P329 (P329delP). In another preferred embodiment, the Fc fragment may have a mutation that causes an increased serum half-life, e.g., a mutation that improves binding of the Fc fragment to FcRn.

[0275] Accordingly, in one specific embodiment, two Fc regions included in the antibody of the present invention, such as a multispecific antibody, are heterodimerized, and wherein

[0276] One Fc-region polypeptide contains mutations S364R and D399K, and another Fc-region polypeptide contains mutations Y349T, K370S, and K409D.

[0277] In some embodiments, the Fc region further comprises the L234A / L235A mutation and optionally a deletion of proline at position P329 (P329delP).

[0278] Accordingly, in one specific embodiment, two Fc regions included in the antibody of the present invention, such as a multispecific antibody, are heterodimerized, and wherein

[0279] a) one Fc region comprises or is composed of the amino acid sequence described in SEQ ID NO. 80, and the remaining Fc region polypeptide comprises or is composed of the amino acid sequence described in SEQ ID NO. 81; or

[0280] b) One Fc region comprises or is composed of the amino acid sequence described in SEQ ID NO. 82, and the remaining Fc region polypeptide comprises or is composed of the amino acid sequence described in SEQ ID NO. 83.

[0281] In one specific embodiment, two Fc regions included in the antibody of the present invention, such as a multispecific antibody, are heterodimerized, and wherein

[0282] a) one Fc region polypeptide comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID NO. 80; or

[0283] b) One Fc region polypeptide comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID NO. 82, and the remaining Fc region polypeptide comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID NO. 83.

[0284] Accordingly, in one specific embodiment, two Fc regions included in the binding molecule of the present invention, such as an antibody like a multispecific antibody, are heterodimerized, and wherein

[0285] a) One Fc region comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID NO. 80 and comprises mutants S364R and D399K, and the remaining Fc region polypeptide comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID NO. 81 and comprises mutants Y349T, K370S, and K409D;

[0286] b) One Fc region comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID NO. 80, and comprises mutants S364R and D399K and mutants L234A / L235A and P329delP, and the remaining Fc region comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID NO. 81, and comprises mutants Y349T, K370S, and K409D and mutants L234A / L235A and P329delP;

[0287] c) one Fc region comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID NO. 82 and comprises mutations S364R and D399K, and the remaining Fc region polypeptide comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID NO. 83 and comprises mutations Y349T, K370S, and K409D; or

[0288] d) One Fc region comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID NO. 82 and includes the mutations S364R, D399K, and L234A / L235A, and the remaining Fc region polypeptide comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID NO. 83 and includes the mutations Y349T, K370S, K409D, and L234A / L235A.

[0289] III-4. Exemplary Bispecific Antibodies

[0290] In some embodiments, the bispecific antibody of the present invention comprises a first antigen-binding region that specifically binds to a first antigen described herein and a second antigen-binding region that specifically binds to a second antigen described herein, wherein the first antigen-binding region comprises a first Fab and the second antigen-binding region comprises a second Fab, wherein the first Fab is connected to the N-terminus of a first Fc region at the C-terminus of its CH1 (directly or through a linker such as a hinge region), and the second Fab is connected to the N-terminus of a second Fc region at the C-terminus of its CH1 (directly or through a linker such as a hinge region). In one specific embodiment, the first Fab and the first Fc region constitute a first half-antibody that binds to a first antigen, and the second Fab and the second Fc region constitute a second half-antibody that binds to a second antigen, and the first half-antibody and the second half-antibody constitute the bispecific antibody of the present invention.

[0291] In one specific embodiment, the bispecific antibody is an IgG-like antibody having the configuration shown in FIG. 3. In one specific embodiment, the first Fc region and the second Fc region comprise an innovadi mutation, and optionally a mutation that reduces the effector function mediated by the Fc region or a mutation that reduces binding to the Fcγ receptor. In one embodiment, the bispecific antibody comprises or is composed of the following:

[0292] Heavy chain 1: comprises or is composed of a first Fab heavy chain variable region from the N-terminus to the C-terminus, a heavy chain invariant region CH1, and a first Fc region, wherein the heavy chain invariant region CH1 is connected from its C-terminus to the N-terminus of the first Fc region directly or through a linker (e.g., a hinge region);

[0293] Light chain 1: comprises or is composed of a first Fab light chain variable region-light chain invariant region from the N-terminus to the C-terminus;

[0294] Heavy chain 2: comprises or is composed of a second Fab heavy chain variable region from the N-terminus to the C-terminus, a heavy chain invariant region CH1, and a second Fc region, wherein the heavy chain invariant region CH1 is connected from its C-terminus to the N-terminus of the second Fc region either directly or through a linker (e.g., a hinge region);

[0295] Light chain 2: includes or is composed of a second Fab light chain variable region-light chain invariant region from the N-terminus to the C-terminus;

[0296] Preferably, the domains are directly connected to each other.

[0297] In some embodiments, the Fab heavy chain variable region and the heavy chain invariant region CH1 constitute the Fab heavy chain, i.e., the C-terminus of the Fab heavy chain variable region is connected to the N-terminus of the heavy chain invariant region CH1 (directly connected). In some embodiments, the Fab light chain variable region and the light chain invariant region constitute the Fab light chain, i.e., the C-terminus of the Fab light chain variable region is connected to the N-terminus of the light chain invariant region (directly connected).

[0298] In one specific embodiment, the first Fab is the Fab of the anti-CD28 antibody of the present invention, and the second Fab is the Fab of the anti-TAA antibody, or vice versa.

[0299] In one specific embodiment, the first Fab is the Fab of the anti-CD28 antibody of the present invention, and the second Fab is the Fab of the anti-CLDN18.2 antibody, or vice versa.

[0300] In one specific embodiment, the bispecific antibody of the present invention specifically binds to CD28 and CLDN18.2 and comprises or consists of the following:

[0301] Heavy chain 1: comprises or is composed of a Fab heavy chain variable region that specifically binds to CD28 from the N-terminus to the C-terminus, a heavy chain constant region CH1, and a first Fc region, wherein the heavy chain constant region CH1 is connected from its C-terminus to the N-terminus of the first Fc region directly or through a linker (e.g., a hinge region);

[0302] Light chain 1: Includes or is composed of a Fab light chain variable region-light chain invariant region that specifically binds to CD28 from the N-terminus to the C-terminus;

[0303] Heavy chain 2: comprising or composed of a Fab heavy chain variable region—heavy chain constant region CH1—that specifically binds to CLDN18.2 from the N-terminus to the C-terminus, wherein the heavy chain constant region CH1 is connected from its C-terminus to the N-terminus of the second Fc region either directly or through a linker (e.g., a hinge region); and / or

[0304] Light chain 2: Includes or is composed of a Fab light chain variable region-light chain constant region that specifically binds to CLDN18.2 from the N-terminus to the C-terminus, and

[0305] Optionally, the first Fc region comprises the mutations Y349T, K370S, and K409D, and the second Fc region comprises S364R and D399K, or vice versa; optionally, the first Fc region and the second Fc region each further comprise the mutation L234A / L235A, and optionally the P329delP mutation;

[0306] Preferably, the domains are directly connected to each other.

[0307] In some specific embodiments, a bispecific antibody that specifically binds to CD28 and CLDN18.2 comprises or consists of heavy chain 1, heavy chain 2, light chain 1 and light chain 2, wherein

[0308] Heavy chain 1 comprises or is composed of an amino acid sequence described in SEQ ID NO. 35, 37, or 39, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity therewith;

[0309] Light chain 1 comprises or is composed of an amino acid sequence described in SEQ ID NO. 36, 38, or 40, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity therewith;

[0310] Heavy chain 2 comprises or consists of the amino acid sequence described in SEQ ID NO. 33 or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity therewith; and / or

[0311] Light chain 2 comprises or consists of an amino acid sequence described in SEQ ID NO. 34 or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with respect to it.

[0312] In some specific embodiments, a bispecific antibody that specifically binds to CD28 and CLDN18.2 comprises or consists of heavy chain 1, heavy chain 2, light chain 1 and light chain 2, wherein

[0313] Heavy chain 1 comprises or is composed of the amino acid sequence described in SEQ ID NO. 35 or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity therewith; light chain 1 comprises or is composed of the amino acid sequence described in SEQ ID NO. 36 or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity therewith; Heavy chain 2 comprises or is composed of the amino acid sequence described in SEQ ID NO. 33 or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity therewith, and light chain 2 comprises or is composed of the amino acid sequence described in SEQ ID NO. 34 or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity therewith;

[0314] Heavy chain 1 comprises or is composed of the amino acid sequence described in SEQ ID NO. 37 or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity therewith; light chain 1 comprises or is composed of the amino acid sequence described in SEQ ID NO. 38 or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity therewith; heavy chain 2 comprises or is composed of the amino acid sequence described in SEQ ID NO. 33 or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity therewith; Light chain 2 comprises or is composed of the amino acid sequence described in SEQ ID NO. 34 or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity therewith; or

[0315] Heavy chain 1 comprises or is composed of the amino acid sequence described in SEQ ID NO. 39 or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity therewith; light chain 1 comprises or is composed of the amino acid sequence described in SEQ ID NO. 40 or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity therewith; heavy chain 2 comprises or is composed of the amino acid sequence described in SEQ ID NO. 33 or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity therewith; Light chain 2 comprises or consists of an amino acid sequence described in SEQ ID NO. 34 or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with respect to it.

[0316] In some specific embodiments, a bispecific antibody that specifically binds to CD28 and CLDN18.2 comprises or consists of heavy chain 1, heavy chain 2, light chain 1 and light chain 2, wherein

[0317] Heavy chain 1 comprises or is composed of the amino acid sequence described in SEQ ID NO. 35; light chain 1 comprises or is composed of the amino acid sequence described in SEQ ID NO. 36; heavy chain 2 comprises or is composed of the amino acid sequence described in SEQ ID NO. 33; light chain 2 comprises or is composed of the amino acid sequence described in SEQ ID NO. 34;

[0318] Heavy chain 1 comprises or is composed of the amino acid sequence described in SEQ ID NO. 37; light chain 1 comprises or is composed of the amino acid sequence described in SEQ ID NO. 38; heavy chain 2 comprises or is composed of the amino acid sequence described in SEQ ID NO. 33; light chain 2 comprises or is composed of the amino acid sequence described in SEQ ID NO. 34; or

[0319] Heavy chain 1 comprises or is composed of the amino acid sequence described in SEQ ID NO. 39; light chain 1 comprises or is composed of the amino acid sequence described in SEQ ID NO. 40; heavy chain 2 comprises or is composed of the amino acid sequence described in SEQ ID NO. 33; and light chain 2 comprises or is composed of the amino acid sequence described in SEQ ID NO. 34.

[0320] In one specific embodiment, the first Fab is the anti-CD28 Fab of the present invention, and the second Fab is the anti-PSMA Fab, or vice versa.

[0321] In one specific embodiment, the bispecific antibody of the present invention specifically binds to CD28 and PSMA and comprises or is composed of the following:

[0322] Heavy chain 1: comprises or is composed of a Fab heavy chain variable region that specifically binds to CD28 from the N-terminus to the C-terminus, a heavy chain constant region CH1, and a first Fc region, wherein the heavy chain constant region CH1 is connected from its C-terminus to the N-terminus of the first Fc region directly or through a linker (e.g., a hinge region);

[0323] Light chain 1: Includes or is composed of a Fab light chain variable region-light chain invariant region that specifically binds to CD28 from the N-terminus to the C-terminus;

[0324] Heavy chain 2: comprising or composed of a Fab heavy chain variable region—a heavy chain constant region CH1—specifically binding to PSMA from the N-terminus to the C-terminus, wherein the heavy chain constant region CH1 is connected from its C-terminus to the N-terminus of the second Fc region either directly or through a linker (e.g., a hinge region); and / or

[0325] Light chain 2: Includes or is composed of a Fab light chain variable region-light chain invariant region that specifically binds to PSMA from the N-terminus to the C-terminus, and

[0326] Optionally, the first Fc region comprises the mutations Y349T, K370S, and K409D, and the second Fc region comprises S364R and D399K, or vice versa; optionally, the first Fc region and the second Fc region each further comprise the mutation L234A / L235A, and optionally the P329delP mutation;

[0327] Preferably, the domains are directly connected to each other.

[0328] In some specific embodiments, a bispecific antibody that specifically binds to CD28 and PSMA comprises or is composed of heavy chain 1, heavy chain 2, light chain 1 and light chain 2, wherein

[0329] Heavy chain 1 comprises or is composed of an amino acid sequence described in SEQ ID NO. 51, 52, or 53, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity therewith;

[0330] Light chain 1 comprises or is composed of an amino acid sequence described in SEQ ID NO. 36, 38, or 40, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity therewith;

[0331] Heavy chain 2 comprises or consists of the amino acid sequence described in SEQ ID NO. 48 or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity therewith; and / or

[0332] Light chain 2 comprises or consists of an amino acid sequence described in SEQ ID NO. 50 or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity therewith.

[0333] In some specific embodiments, a bispecific antibody that specifically binds to CD28 and PSMA comprises or is composed of heavy chain 1, heavy chain 2, light chain 1 and light chain 2, wherein

[0334] Heavy chain 1 comprises or is composed of the amino acid sequence described in SEQ ID NO. 51 or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity therewith; light chain 1 comprises or is composed of the amino acid sequence described in SEQ ID NO. 36 or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity therewith; heavy chain 2 comprises or is composed of the amino acid sequence described in SEQ ID NO. 48 or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity therewith; Light chain 2 comprises or is composed of the amino acid sequence described in SEQ ID NO. 50 or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity therewith;

[0335] Heavy chain 1 comprises or is composed of the amino acid sequence described in SEQ ID NO. 52 or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity therewith; light chain 1 comprises or is composed of the amino acid sequence described in SEQ ID NO. 38 or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity therewith; heavy chain 2 comprises or is composed of the amino acid sequence described in SEQ ID NO. 48 or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity therewith; Light chain 2 comprises or is composed of the amino acid sequence described in SEQ ID NO. 50 or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity therewith; or

[0336] Heavy chain 1 comprises or is composed of the amino acid sequence described in SEQ ID NO. 53 or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity therewith; light chain 1 comprises or is composed of the amino acid sequence described in SEQ ID NO. 40 or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity therewith; heavy chain 2 comprises or is composed of the amino acid sequence described in SEQ ID NO. 48 or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity therewith; Light chain 2 comprises or consists of an amino acid sequence described in SEQ ID NO. 50 or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity therewith.

[0337] In some specific embodiments, a bispecific antibody that specifically binds to CD28 and PSMA comprises or is composed of heavy chain 1, heavy chain 2, light chain 1 and light chain 2, wherein

[0338] Heavy chain 1 comprises or is composed of the amino acid sequence described in SEQ ID NO. 51; light chain 1 comprises or is composed of the amino acid sequence described in SEQ ID NO. 36; heavy chain 2 comprises or is composed of the amino acid sequence described in SEQ ID NO. 48; and light chain 2 comprises or is composed of the amino acid sequence described in SEQ ID NO. 50;

[0339] Heavy chain 1 comprises or is composed of the amino acid sequence described in SEQ ID NO. 52; light chain 1 comprises or is composed of the amino acid sequence described in SEQ ID NO. 38; heavy chain 2 comprises or is composed of the amino acid sequence described in SEQ ID NO. 48; light chain 2 comprises or is composed of the amino acid sequence described in SEQ ID NO. 50; or

[0340] Heavy chain 1 comprises or is composed of the amino acid sequence described in SEQ ID NO. 53; light chain 1 comprises or is composed of the amino acid sequence described in SEQ ID NO. 40; heavy chain 2 comprises or is composed of the amino acid sequence described in SEQ ID NO. 48; and light chain 2 comprises or is composed of the amino acid sequence described in SEQ ID NO. 50.

[0341] The various components / domains described herein for multispecific binding molecules, such as bispecific antibodies, are as described herein. Unless otherwise explicitly stated to the contrary, it will be understood by those skilled in the art that any combination of any technical features described herein for various components of multispecific binding molecules, e.g., bispecific antibodies, falls within the scope of the present invention. Furthermore, unless otherwise explicitly stated to the contrary, it will be understood by those skilled in the art that the antibodies of the present invention (including antibodies of any form) may include any such combination.

[0342] IV. Polynucleotides, Vectors, and Host Cells

[0343] The present invention provides a nucleic acid encoding any of the antibody molecules of the present invention (e.g., an anti-CD28 antibody or an antigen-binding fragment thereof, or a multispecific antibody such as the bispecific antibody of the present invention).

[0344] In one aspect, the present invention provides a nucleic acid encoding any of the anti-CD28 antibody or its antigen-binding fragment, or a multispecific antibody such as a bispecific antibody.

[0345] To facilitate production and purification, an anti-CD28 antibody or its antigen-binding fragment or a multispecific antibody, such as a bispecific antibody, may be fused to a secreted signal peptide, and / or a tag peptide that facilitates purification, such as a hexahistidine tag or a biotin tag, at the N-terminus or C-terminus (e.g., C-terminus).

[0346] As is understood by those skilled in the art, due to codon degeneracy, each antibody or polypeptide amino acid sequence can be encoded by various nucleic acid sequences.

[0347] In some embodiments, the nucleic acid of the present invention is a nucleic acid encoding an amino acid sequence selected from any one of SEQ ID NOs: 4, 5, 9, 13, 17, 19, 23, 24, 28, 33, 35, 36-40, 48, 51-53, 69, 70, 72, 73, 75, 78 and 102-106, or an amino acid sequence selected from any one of SEQ ID NOs: 4, 5, 9, 13, 17, 19, 23, 24, 28, 33, 35, 36-40, 48, 51-53, 69, 70, 72, 73, 75, 78 and 102-106 and at least 85%, 90%, 91%, 92%, 93%, It includes a nucleic acid encoding an amino acid sequence having 94%, 95%, 96%, 97%, 98%, or 99% identity.

[0348] The nucleic acid sequence encoding the molecule of the present invention can be produced using methods well known in the art, for example by the nasal synthesis of solid DNA or by PCR amplification.

[0349] In one aspect, the present invention also provides a vector comprising the nucleic acid of the present invention. In one embodiment, the vector is an expression vector, e.g., a prokaryotic expression vector or a eukaryotic expression vector. The vector comprises, but is not limited to, a virus, a plasmid, a cosmid, a lambda phage, or a yeast artificial chromosome (YAC). In a preferred embodiment, the expression vector is pCDNA, e.g., pCDNA3.1.

[0350] In one aspect, the present invention also provides a host cell comprising a nucleic acid or a vector. In one embodiment, the host cell is a eukaryotic cell. In another embodiment, the host cell is selected from yeast cells and mammalian cells (e.g., CHO cells or 293 cells, e.g., HEK 293 or 293F cells, or 293FT cells, or Expi293 cells). In another embodiment, the host cell is a prokaryotic cell.

[0351] The suitable host cell is E. coli ( E. coli This includes prokaryotic microorganisms such as ) and eukaryotic microorganisms such as filamentous fungi or yeast, or various eukaryotic cells such as Chinese hamster ovary cells (CHO) and insect cells. Mammalian cell lines suitable for suspension culture may be used. Examples of useful mammalian host cell lines include monkey kidney CV1 cell lines (COS-7) transformed by SV40; human embryonic kidney cell lines (HEK 293 or 293F cells or 293FT cells or Expi293 cells), baby hamster kidney cells (BHK), monkey kidney cells (CV1), African green monkey kidney cells (VERO-76), human cervical cancer cells (HELA), dog kidney cells (MDCK), buffalo rat liver cells (BRL 3A), human lung cells (W138), human liver cells (Hep G2), CHO cells, NSO cells, and myeloma cell lines such as YO, NS0, P3X63, and Sp2 / 0. Mammalian host cell lines suitable for antibody production are known in the art. In a preferred embodiment, the host cell is a CHO or HEK293 cell, or a 293FT cell, or an Expi293 cell.

[0352] V. Production and purification of the molecule of the present invention

[0353] In another aspect, the present invention provides a method for producing an antibody molecule of the present invention (e.g., an anti-CD28 antibody or an antigen-binding fragment thereof, or a multispecific antibody such as a bispecific antibody), the method further comprises the steps of: culturing a host cell containing a polynucleotide encoding a polypeptide chain of the molecule under conditions suitable for the expression of the polypeptide chain; and optionally assembling the polypeptide chain under conditions suitable for the assembly of the polypeptide chain into a molecule to produce the molecule.

[0354] For recombinant production, a polynucleotide encoding a polypeptide chain of the molecule of the present invention may be inserted into one or more vectors for further cloning and / or expression in host cells. Expression vectors may be constructed using methods well known to those skilled in the art. Expression vectors include, but are not limited to, viruses, plasmids, cosmids, lambda phages, or yeast artificial chromosomes (YACs). Once an expression vector containing one or more polynucleotides of the present invention is prepared for expression, the expression vector may be transfected or introduced into suitable host cells. For this purpose, various techniques such as protoplast fusion, calcium phosphate precipitation, electroporation, retroviral transduction, viral transfection, particle impaction, liposome-based transfection, or other conventional techniques may be used.

[0355] Molecules prepared as described herein can be purified by known prior art such as high-performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography (e.g., protein A affinity chromatography), and size exclusion chromatography. The actual conditions used to purify a specific protein depend on factors such as net charge, hydrophobicity, and hydrophilicity, which will be obvious to those skilled in the art.

[0356] The purity of the molecules of the present invention can be determined by any one of various well-known analytical methods, including size exclusion chromatography, gel electrophoresis, high-performance liquid chromatography, etc. The physical / chemical properties and / or biological activity of the antibody molecules provided herein can be confirmed, screened, or characterized through various assays known in the art.

[0357] VI. Examination

[0358] The molecules provided herein (e.g., anti-CD28 antibodies or their antigen-binding fragments, or multispecific antibodies such as bispecific antibodies) may be identified, screened, or characterized for their physical / chemical properties and / or biological activity using various assays known in the art. Assays for determining the properties of anti-CD28 antibodies or their antigen-binding fragments or bispecific antibodies are illustrated in the Examples and include biolayer interferometry (BLI), T cell reporter gene assays, T cell activation assays, T cell-mediated killing assays, anti-tumor activity assays, etc.

[0359] VII. Immunoconjugate

[0360] In one aspect, the present invention provides an immunoconjugate comprising a molecule of the present invention (e.g., an anti-CD28 antibody or an antigen-binding fragment thereof, or a multispecific antibody such as a bispecific antibody) and one or more additional active components (e.g., an active component from a drug or therapeutic agent for treating a disease of the present invention, such as a small molecule that enhances the therapeutic effect of the molecule of the present invention).

[0361] In some embodiments, the immunoconjugate is an antibody-drug conjugate (ADC).

[0362] VIII. Pharmaceuticals and Uses

[0363] In some embodiments, the present invention further relates to a composition (e.g., a pharmaceutical composition or a pharmaceutical formulation) comprising a molecule of the present invention (e.g., an anti-CD28 antibody or an antigen-binding fragment thereof, a multispecific antibody such as a bispecific antibody, or an immunoconjugate).

[0364] In one embodiment, the composition further comprises pharmaceutically acceptable auxiliary materials known in the art, such as a pharmaceutically acceptable carrier including a buffer, and pharmaceutically acceptable excipients. In one embodiment, the composition, for example, a pharmaceutical composition, comprises a combination of the molecule of the present invention and one or more additional therapeutic agents.

[0365] The compositions of the present invention may further comprise suitable pharmaceutical auxiliary materials, such as pharmaceutical excipients comprising pharmaceutical carriers and buffers as known in the art. As used herein, "pharmaceutical carrier" includes any and all physiologically suitable solvents, dispersion media, isotonic agents, or absorption retardants, etc. For the use and application of pharmaceutical excipients, refer to the Handbook of Pharmaceutical Excipients, 8th Ed., RC Rowe, PJ Seskey and SC Owen, Pharmaceutical Press, London, Chicago. The compositions of the present invention may be in various forms. These forms include liquid, semi-solid, and solid administration forms, such as liquid solutions (e.g., injection solutions and infusion solutions), powders or suspensions, liposomes, and suppositories. The preferred form depends on the intended mode of administration and therapeutic use.

[0366] A pharmaceutical agent comprising the antibody molecule or immunoconjugate described herein may be prepared, for example, in the form of a lyophilized formulation or an aqueous solution, by mixing the antibody molecule of the present invention having a desired purity (e.g., an anti-CD28 antibody or its antigen-binding fragment, or a multispecific antibody such as a bispecific antibody) or an immunoconjugate with one or more optional pharmaceutical adjuvants.

[0367] In one aspect, the present invention provides a method for preventing or treating a disease in a subject, comprising the step of administering to the subject an effective amount of a molecule of the present invention (e.g., an anti-CD28 antibody or an antigen-binding fragment thereof, or a multispecific antibody such as a bispecific antibody or an immunoconjugate thereof), a pharmaceutical composition, a pharmaceutical combination, or a kit. In some embodiments, the disease is a tumor, for example, cancer.

[0368] In some embodiments, the present invention relates to a molecule of the present invention for use in treatment, for example, in the treatment of a tumor such as cancer (e.g., an anti-CD28 antibody or an antigen-binding fragment thereof, or a multispecific antibody such as a bispecific antibody or an immunoconjugate thereof of the present invention).

[0369] In some embodiments, the present invention relates to a method for treating a disease, e.g., a tumor such as cancer, using a molecule of the present invention (e.g., an anti-CD28 antibody or an antigen-binding fragment thereof, or a multispecific antibody such as a bispecific antibody, or an immunoconjugate thereof of the present invention), or the use thereof for treatment, or the use thereof in the manufacture of a therapeutic agent.

[0370] In some embodiments, the tumor is a solid tumor or a hematological tumor and a metastatic lesion. In one embodiment, examples of solid tumors include malignant tumors. The cancer may be in an early, mid, or advanced stage, or it may be metastatic cancer. In some embodiments, the tumor is tumor immune evasion.

[0371] In some embodiments, the tumor is a TAA-positive tumor or cancer. In some embodiments, the tumor is a CLDN18.2-positive tumor or cancer. In some embodiments, the tumor is a PSMA-positive tumor or cancer. In some embodiments, the tumor is associated with abnormal expression or abnormal activity of TAA. In some embodiments, the tumor is associated with abnormal expression or abnormal activity of CLDN18.2. In some embodiments, the tumor is associated with abnormal expression or abnormal activity of PSMA.

[0372] In some embodiments, a TAA-positive tumor or cancer refers to a tumor or cancer in which a subject having a tumor or cancer has TAA-expressing tumor cells. In some embodiments, the tumor cells of the subject express TAA, for example, at a moderate or high level. In some embodiments, the subject has TAA (e.g., at a nucleic acid or protein level or in elevated activity) compared to a healthy subject. In some embodiments, a biological sample of the subject (e.g., tumor cells or tumor tissue) has TAA at an elevated level (e.g., at a nucleic acid or protein level or in elevated activity) compared to a biological sample of the healthy subject (e.g., a corresponding tissue or cell of the healthy subject) or compared to TAA in an adjacent healthy tissue or cell of the subject. In some embodiments, TAA is CLDN18.2 or PSMA.

[0373] In some embodiments, the tumor is a gastrointestinal tumor, e.g., a gastrointestinal cancer, e.g., colon cancer, rectal cancer, or colorectal cancer, e.g., a CLDN18.2-positive gastrointestinal tumor, e.g., a gastrointestinal cancer, e.g., gastric cancer, breast cancer, colon cancer, rectal cancer, or colorectal cancer. In some embodiments, the tumor is a PSMA-positive tumor, e.g., a renal cell carcinoma such as breast cancer, lung cancer, rectal cancer, or colorectal cancer.

[0374] Depending on the therapeutic use thereof, the antibody of the present invention, its antigen-binding fragment, or immunoconjugate, or pharmaceutical composition may also be administered in combination with one or more other therapies, e.g., a treatment method and / or other therapeutic agent, for the use described herein, e.g., for the prevention and / or treatment of related diseases or conditions mentioned herein.

[0375] In some embodiments, when a molecule of the present invention (e.g., an anti-CD28 antibody or its antigen-binding fragment, a bispecific antibody, or an immunoconjugate thereof) is used to treat a tumor, the therapeutic agent is a variety of tumor-treating therapeutic agents, such as, for example, chemotherapy agents, angiogenesis inhibitors, cytokines, cytotoxic agents, additional antibodies, small molecule drugs, or immunomodulators (e.g., immune checkpoint inhibitors or agonists). In some embodiments, the therapeutic agent is a PD-1 antibody or its antigen-binding fragment. In some embodiments, when a molecule of the present invention (e.g., an anti-CD28 antibody or its antigen-binding fragment, a bispecific antibody, or an immunoconjugate thereof) is used to treat a tumor, the treatment method includes surgery, radiation therapy, partial irradiation, intensive irradiation, etc.

[0376] In some embodiments, the present invention further provides a pharmaceutical combination or a pharmaceutical combination product comprising the antibody of the present invention or its antigen-binding fragment or immunoconjugate, and one or more additional therapeutic agents, etc.

[0377] Another object of the present invention is to provide a component kit comprising the pharmaceutical combination of the present invention, preferably the kit in the form of a pharmaceutical dosage unit. Accordingly, the dosage unit may be provided according to a dosage regimen or intervals between drug administrations.

[0378] In one embodiment, the component kit of the present invention comprises the following in the same packaging:

[0379] - A first container comprising a pharmaceutical composition comprising an antibody disclosed herein or an antigen-binding fragment or an immunoconjugate thereof; and

[0380] - A second container comprising a pharmaceutical composition containing an additional therapeutic agent.

[0381] In some embodiments, when a molecule of the present invention (e.g., an anti-CD28 antibody or its antigen-binding fragment, a bispecific antibody, or its immunoconjugate of the present invention) is used to treat a tumor, the therapeutic agent is a therapeutic agent for various tumor treatments, such as, for example, a chemotherapy agent, angiogenesis inhibitor, cytokine, cytotoxic agent, additional antibody, small molecule drug, or immunomodulator (e.g., immune checkpoint inhibitor or agonist). In some embodiments, the therapeutic agent is a PD-1 antibody or its antigen-binding fragment.

[0382] In another aspect, the present invention also provides the use of the molecule of the present invention (e.g., an anti-CD28 antibody or its antigen-binding fragment, a bispecific antibody or its immunoconjugate), or a composition or combination thereof containing the same, in the uses described herein, for example, in the manufacture or preparation of a drug for the prevention or treatment of a related disease or illness mentioned herein.

[0383] In another aspect, the present invention also provides a molecule of the present invention (e.g., an anti-CD28 antibody or its antigen-binding fragment, a bispecific antibody or its immunoconjugate), or a composition, drug, formulation, or combination product comprising the same, for use in treatment, for example, treatment of a related disease or illness mentioned herein.

[0384] IX. Diagnosis and Detection

[0385] In certain embodiments, the molecules provided herein (e.g., anti-CD28 antibody or its antigen-binding fragment, bispecific antibody or its immunoconjugate) may be used to detect the presence of CD28 or TAA in biological samples.

[0386] As used herein, the term “detection” includes quantitative and qualitative detection, and exemplary detections may include immunohistochemistry, immunocytochemistry, flow cytometry (e.g., FACS), magnetic beads complexed with antibody molecules, ELISA, and PCR techniques (e.g., RT-PCR). In certain embodiments, the biological sample is a body fluid.

[0387] In a specific embodiment, the method comprises the steps of contacting a biological sample with the molecule described herein (e.g., a bispecific antibody or an immunoconjugate thereof) under conditions in which the molecule can bind to CD28 and / or TAA, and detecting whether a complex is formed between the molecule and CD28 or TAA, wherein the formation of the complex indicates the presence of CD28 and / or TAA. The method may be an in vitro or in vivo method. In one embodiment, the molecule of the present invention is used to select a target suitable for treatment using the anti-CD28 antibody or its antigen-binding fragment or the bispecific antibody or its immunoconjugate of the present invention, for example, wherein CD28 and / or TAA are biomarkers for selecting the target.

[0388] In specific embodiments, a labeled molecule of the present invention (e.g., an anti-CD28 antibody or its antigen-binding fragment, a bispecific antibody or its immunoconjugate) is provided. The label includes, but is not limited to, a label or moiety that is directly detected (e.g., a fluorescent label, a chromogenic label, an electron-density label, a chemiluminescent label, and a radioactive label) and a moiety such as an enzyme or ligand that is indirectly detected, for example, through an enzymatic reaction or molecular interaction.

[0389] In some embodiments provided herein, a sample is obtained prior to treatment using the molecule of the present invention, or a composition, drug, formulation, or combination product containing it. In some embodiments, a sample is obtained prior to treatment using another therapy. In some embodiments, a sample is obtained during or after treatment using another therapy.

[0390] In some embodiments, TAA is detected before treatment, for example, before an initial treatment or before a specific treatment after a treatment interval.

[0391] In some embodiments, a method for treating a disease according to the present invention is provided, comprising the steps of: detecting the presence of TAA in a subject (e.g., a sample, e.g., a subject sample) to determine a TAA value; comparing the TAA value with a control value (e.g., a value of a normal individual); and, if the TAA value is greater than the control value, treating the disease by administering a therapeutically effective amount of the bispecific antibody of the present invention or a composition, drug, or formulation containing the same to the subject, optionally in combination with one or more additional therapies.

[0392] X. Specific implementation example

[0393] 1.

[0394] (i). CDRH1, CDRH2 and CDRH3 are selected from three complementary-determining regions CDRH1, CDRH2 and CDRH3 included in the VH described in SEQ ID NO. 24, 23 or 75; CDRL1, CDRL2 and CDRL3 are three complementary-determining regions CDRL1, CDRL2 and CDRL3 included in the VL described in SEQ ID NO. 28 or 78;

[0395] (ii) CDRH1, CDRH2, and CDRH3 are selected from three complementary-determining regions CDRH1, CDRH2, and CDRH3 included in the VH described in any one of SEQ ID NOs 4, 5, and 72; and CDRL1, CDRL2, and CDRL3 are three complementary-determining regions CDRL1, CDRL2, and CDRL3 included in the VL described in SEQ ID NO. 9 or 73;

[0396] (iii). An anti-CD28 antibody or its antigen-binding fragment comprising three CDRs of a heavy chain variable region VH: CDRH1, CDRH2, and CDRH3, and three CDRs of a light chain variable region VL: CDRL1, CDRL2, and CDRL3, wherein CDRH1, CDRL2, and CDRL3 are selected from the three complementarity-determining regions CDRH1, CDRL2, and CDRL3 included in the VH described in SEQ ID NO. 13 or 69; and CDRL1, CDRL2, and CDRL3 are the three complementarity-determining regions CDRL1, CDRL2, and CDRL3 included in the VL described in SEQ ID NO. 17, 19, or 70.

[0397] 2.

[0398] (i). CDRH1 comprises or is composed of the amino acid sequence of SEQ ID NO. 20; CDRH2 comprises or is composed of the amino acid sequence of SEQ ID NO. 21; CDRH3 comprises or is composed of the amino acid sequence of SEQ ID NO. 22; CDRL1 comprises or is composed of the amino acid sequence of SEQ ID NO. 25; CDRL2 comprises or is composed of the amino acid sequence of SEQ ID NO. 26; CDRL3 comprises or is composed of the amino acid sequence of SEQ ID NO. 27;

[0399] (ii). CDRH1 comprises or is composed of the amino acid sequence of SEQ ID NO. 20; CDRH2 comprises or is composed of the amino acid sequence of SEQ ID NO. 76; CDRH3 comprises or is composed of the amino acid sequence of SEQ ID NO. 77; CDRL1 comprises or is composed of the amino acid sequence of SEQ ID NO. 25; CDRL2 comprises or is composed of the amino acid sequence of SEQ ID NO. 26; CDRL3 comprises or is composed of the amino acid sequence of SEQ ID NO. 27;

[0400] (iii). CDRH1 comprises or is composed of the amino acid sequence of SEQ ID NO. 1; CDRH2 comprises or is composed of the amino acid sequence of SEQ ID NO. 2; CDRH3 comprises or is composed of the amino acid sequence of SEQ ID NO. 3; CDRL1 comprises or is composed of the amino acid sequence of SEQ ID NO. 6 or 74; CDRL2 comprises or is composed of the amino acid sequence of SEQ ID NO. 7; CDRL3 comprises or is composed of the amino acid sequence of SEQ ID NO. 8;

[0401] (iv) an anti-CD28 antibody or its antigen-binding fragment comprising CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3, wherein CDRH1 comprises or is composed of the amino acid sequence of SEQ ID NO. 10; CDRH2 comprises or is composed of the amino acid sequence of SEQ ID NO. 11; CDRH3 comprises or is composed of the amino acid sequence of SEQ ID NO. 12; CDRL1 comprises or is composed of the amino acid sequence of SEQ ID NO. 14, 18 or 71; CDRL2 comprises or is composed of the amino acid sequence of SEQ ID NO. 15; and CDRL3 comprises or is composed of the amino acid sequence of SEQ ID NO. 16.

[0402] 3. In Embodiment 1 or 2, the heavy chain variable region VH is included, and the heavy chain variable region is

[0403] (i) comprising or composed of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO. 24, 4, 5, 13, 23, 69, 72, or 75;

[0404] (ii) An antibody or its antigen-binding fragment comprising or consisting of the amino acid sequence of SEQ ID NOs 24, 4, 5, 13, 23, 69, 72 or 75.

[0405] 4. In any one of embodiments 1 to 3, the light chain variable region VL is included, and the light chain variable region is

[0406] (i) comprising or composed of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NOs 28, 9, 17, 19, 70, 73, or 78;

[0407] (ii) An antibody or its antigen-binding fragment comprising or consisting of the amino acid sequence of SEQ ID NOs 28, 9, 17, 19, 70, 73 or 78.

[0408] 5. In Embodiment 1 or 2, the heavy chain variable region VH and the light chain variable region VL are included, wherein

[0409] (i). VH comprises or is composed of the amino acid sequence described in SEQ ID NO. 24 or 23 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity therewith, and VL comprises or is composed of the amino acid sequence described in SEQ ID NO. 28 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity therewith;

[0410] (ii) VH comprises or is composed of the amino acid sequence described in SEQ ID NO. 75 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity therewith, and VL comprises or is composed of the amino acid sequence described in SEQ ID NO. 78 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity therewith;

[0411] (iii). VH comprises or is composed of the amino acid sequence described in SEQ ID NO. 23, 24, or 75 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity therewith, and VL comprises or is composed of the amino acid sequence described in SEQ ID NO. 28 or 78 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity therewith;

[0412] (iv). VH comprises or is composed of the amino acid sequence described in SEQ ID NO. 4, 5, or 72 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity therewith, and VL comprises or is composed of the amino acid sequence described in SEQ ID NO. 9 or 73 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity therewith;

[0413] (v). VH comprises or is composed of the amino acid sequence described in SEQ ID NO. 4 or 5 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity therewith, and VL comprises or is composed of the amino acid sequence described in SEQ ID NO. 9 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity therewith;

[0414] (vi). VH comprises or is composed of the amino acid sequence described in SEQ ID NO. 72 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity therewith, and VL comprises or is composed of the amino acid sequence described in SEQ ID NO. 73 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity therewith;

[0415] (vii). VH comprises or is composed of the amino acid sequence described in SEQ ID NO. 13 or 69 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity therewith, and VL comprises or is composed of the amino acid sequence described in SEQ ID NO. 17, 19 or 70 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity therewith;

[0416] (viii). VH comprises or is composed of the amino acid sequence described in SEQ ID NO. 13 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity therewith, and VL comprises or is composed of the amino acid sequence described in SEQ ID NO. 17 or 19 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity therewith;

[0417] (ix). An antibody or its antigen-binding fragment comprising VH having an amino acid sequence described in SEQ ID NO. 69 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity therewith, and VL having an amino acid sequence described in SEQ ID NO. 70 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity therewith.

[0418] 6. In Embodiment 1 or 2, the heavy chain variable region VH and the light chain variable region VL are included, wherein

[0419] (i). VH comprises or is composed of the amino acid sequence described in SEQ ID NO. 24, and VL comprises or is composed of the amino acid sequence described in SEQ ID NO. 28;

[0420] (ii). VH comprises or is composed of the amino acid sequence described in SEQ ID NO. 23, and VL comprises or is composed of the amino acid sequence described in SEQ ID NO. 28;

[0421] (iii). VH comprises or is composed of the amino acid sequence described in SEQ ID NO. 72, and VL comprises or is composed of the amino acid sequence described in SEQ ID NO. 73;

[0422] (iv). VH comprises or is composed of the amino acid sequence described in SEQ ID NO. 4, and VL comprises or is composed of the amino acid sequence described in SEQ ID NO. 9;

[0423] (v). VH comprises or is composed of the amino acid sequence described in SEQ ID NO. 5, and VL comprises or is composed of the amino acid sequence described in SEQ ID NO. 9;

[0424] (vi). VH comprises or is composed of the amino acid sequence described in SEQ ID NO. 13, and VL comprises or is composed of the amino acid sequence described in SEQ ID NO. 17;

[0425] (vii). VH comprises or is composed of the amino acid sequence described in SEQ ID NO. 13, and VL comprises or is composed of the amino acid sequence described in SEQ ID NO. 19;

[0426] (viii). VH comprises or is composed of the amino acid sequence described in SEQ ID NO. 69, and VL comprises or is composed of the amino acid sequence described in SEQ ID NO. 70;

[0427] (ix). An antibody or its antigen-binding fragment in which VH comprises or is composed of the amino acid sequence described in SEQ ID NO. 75, and VL comprises or is composed of the amino acid sequence described in SEQ ID NO. 78.

[0428] 7.

[0429] (i). VH comprises or is composed of the amino acid sequence described in SEQ ID NO. 24, and VL comprises or is composed of the amino acid sequence described in SEQ ID NO. 28;

[0430] (ii). VH comprises or is composed of the amino acid sequence described in SEQ ID NO. 23, and VL comprises or is composed of the amino acid sequence described in SEQ ID NO. 28;

[0431] (iii). VH comprises or is composed of the amino acid sequence described in SEQ ID NO. 72, and VL comprises or is composed of the amino acid sequence described in SEQ ID NO. 73;

[0432] (iv). VH comprises or is composed of the amino acid sequence described in SEQ ID NO. 4, and VL comprises or is composed of the amino acid sequence described in SEQ ID NO. 9;

[0433] (v). VH comprises or is composed of the amino acid sequence described in SEQ ID NO. 5, and VL comprises or is composed of the amino acid sequence described in SEQ ID NO. 9;

[0434] (vi). VH comprises or is composed of the amino acid sequence described in SEQ ID NO. 13, and VL comprises or is composed of the amino acid sequence described in SEQ ID NO. 17;

[0435] (vii). VH comprises or is composed of the amino acid sequence described in SEQ ID NO. 13, and VL comprises or is composed of the amino acid sequence described in SEQ ID NO. 19;

[0436] (viii). VH comprises or is composed of the amino acid sequence described in SEQ ID NO. 69, and VL comprises or is composed of the amino acid sequence described in SEQ ID NO. 70;

[0437] (ix). An anti-CD28 antibody or an antigen-binding fragment thereof comprising a heavy chain variable region VH and a light chain variable region VL, wherein VH comprises or is composed of the amino acid sequence described in SEQ ID NO. 75 and VL comprises or is composed of the amino acid sequence described in SEQ ID NO. 78.

[0438] 8. In any one of embodiments 1 to 7, the heavy chain constant region HC is further included, for example, the heavy chain constant region HC of the antibody is the heavy chain constant region of IgG1, IgG2, IgG3, or IgG4, preferably the heavy chain constant region of human IgG1, and preferably the heavy chain constant region is

[0439] (i) comprising or composed of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence selected from SEQ ID NO. 29 or 79;

[0440] (ii) An antibody or its antigen-binding fragment comprising or consisting of an amino acid sequence selected from SEQ ID NO. 29 or 79.

[0441] 9. In Embodiment 7 or 8, the heavy chain constant region is an antibody or its antigen-binding fragment comprising a mutation that reduces binding to the Fcγ receptor, e.g., the LALA mutation and / or P329del.

[0442] 10. An antibody or an antigen-binding fragment thereof comprising a light chain invariant region in any one of embodiments 1 to 9, wherein, for example, the light chain invariant region is a lambda or kappa light chain invariant region.

[0443] 11. In Embodiment 10, the light chain invariant region is

[0444] (i) comprising or composed of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO. 30;

[0445] (ii) An antibody or antigen-binding fragment thereof comprising or consisting of the amino acid sequence of SEQ ID NO. 30.

[0446] 12. In any one of embodiments 1 to 11, the antibody is an antibody that is a humanized antibody or a chimeric antibody, or an antigen-binding fragment thereof.

[0447] 13. In any one of embodiments 1 to 12, the antibody is a monoclonal antibody or an antigen-binding fragment thereof.

[0448] 14. In any one of embodiments 1 to 13, the antigen-binding fragment is an antibody or an antigen-binding fragment thereof, wherein the antigen-binding fragment is an antibody fragment selected from the group consisting of Fab, Fab', Fab'-SH, Fv, a single-chain antibody (e.g., scFv), and (Fab')2, a single-domain antibody (e.g., VHH), a domain antibody (dAb), or a linear antibody.

[0449] 15. In any one of embodiments 1 to 11, the antibody is a bispecific antibody or a multispecific antibody comprising a first binding specificity for CD28 and additional binding specificity for one or more tumor-associated antigens, or an antigen-binding fragment thereof.

[0450] 16. In Embodiment 15, the tumor-associated antigen is an antibody selected from the group consisting of CLDN18.2 and PSMA or an antigen-binding fragment thereof.

[0451] 17. The first antigen-binding region specifically binds to CD28 and includes VH and VL, wherein

[0452] VH includes CDRH1, CDRH2, and CDRH3 as defined in Embodiment 1 or 2, and

[0453] VL includes CDRL1, CDRL2, and CDRL3 as defined in Embodiment 1 or 2;

[0454] VH and VL are VH and VL as defined in any one of embodiments 3 to 6;

[0455] A bispecific antibody comprising a first antigen-binding region and a second antigen-binding region, wherein the second antigen-binding region specifically binds to a tumor-associated antigen TAA or an immune checkpoint molecule.

[0456] 18. In Embodiment 17, the first antigen-binding region is a bispecific antibody that is the Fab of the anti-CD28 antibody defined in any one of Embodiments 1 to 14.

[0457] 19. In Embodiment 17 or 18, a bispecific antibody in which the second antigen-binding region is a Fab that specifically binds to a tumor-associated antigen or immune checkpoint molecule.

[0458] 20. A bispecific antibody in embodiment 18 or 19, wherein Fab, which is the first antigen-binding region or the second antigen-binding region, comprises CH1, and CH1 is CH1 from IgG1, IgG2, IgG3 or IgG4, preferably CH1 from IgG1.

[0459] 21. In Example 20, CH1 is

[0460] (i) comprising or composed of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence selected from SEQ ID NO. 74;

[0461] (ii) A bispecific antibody comprising or composed of the amino acid sequence of SEQ ID NO. 74.

[0462] 22. A bispecific antibody in any one of embodiments 18 to 21, wherein Fab functioning as a first antigen-binding region or a second antigen-binding region comprises a light chain constant region, and the light chain constant region is a kappa light chain constant region or a lambda light chain constant region.

[0463] 23. In Embodiment 22, the kappa light chain invariant region is

[0464] (i) comprising or composed of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO. 30;

[0465] (ii) A bispecific antibody comprising or composed of the amino acid sequence of SEQ ID NO. 30.

[0466] 24. In any one of embodiments 17 to 23, the bispecific antibody is an IgG-like bispecific antibody comprising an Fc dimer, wherein the two Fc regions constituting the Fc dimer are identical or different; optionally, the Fc region is a human IgG1, IgG2, IgG3, or IgG4 Fc region; and optionally, the Fc region is

[0467] (i) comprising or composed of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with an amino acid sequence selected from SEQ ID NO. 94 or 95;

[0468] (ii) A bispecific antibody comprising or composed of the amino acid sequence of SEQ ID NO. 94 or 95.

[0469] 25. A bispecific antibody in which, in Embodiment 24, the two Fc regions are different, and mutations are introduced into the first Fc region and the second Fc region to promote heteromerization between the first Fc region and the second Fc region based on Innovadi technology.

[0470] 26. An antibody or its antigen-binding fragment in Embodiment 25, wherein one Fc region's CH3 contains S364R and D399K mutations, and the remaining Fc region's CH3 contains Y349T, K370S and K409D mutations.

[0471] 27. In any one of embodiments 24 to 26, the first and / or second Fc region is a bispecific antibody comprising a mutation that reduces binding to an Fcγ receptor, e.g., an L234A / L235A mutation, and optionally P329delP.

[0472] 28. In Embodiment 26 or 27,

[0473] a) One Fc region comprises or is composed of the amino acid sequence described in SEQ ID NO. 80, and the remaining Fc region comprises or is composed of the amino acid sequence described in SEQ ID NO. 81;

[0474] b) One Fc region comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID NO. 80 and comprises mutations S364R and D399K, and the remaining Fc region comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID NO. 81 and comprises mutations Y349T, K370S, and K409D;

[0475] c) One Fc region comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID NO. 80, and comprises mutants S364R and D399K and mutants L234A / L235A and P329delP, and the remaining Fc region comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID NO. 81, and comprises mutants Y349T, K370S, and K409D and mutants L234A / L235A and P329delP;

[0476] d) One Fc region comprises or is composed of the amino acid sequence described in SEQ ID NO. 82, and the remaining Fc region comprises or is composed of the amino acid sequence described in SEQ ID NO. 83;

[0477] e) One Fc region comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID NO. 82 and comprises mutations S364R and D399K, and the remaining Fc region polypeptide comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID NO. 83 and comprises mutations Y349T, K370S, and K409D;

[0478] f) a bispecific antibody in which one Fc region comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID NO. 82 and comprises mutants S364R, D399K, and L234A / L235A, and the remaining Fc region polypeptide comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID NO. 83 and comprises mutants Y349T, K370S, K409D, and L234A / L235A.

[0479] 29. In any one of embodiments 17 to 28, the TAA is CLDN18.2, and preferably, the second antigen-binding region comprises three complementary-determining regions (CDRHs) from the heavy chain variable region: CDRH1, CDRH2, and CDRH3, and three complementary-determining regions (CDRLs) from the light chain variable region: CDRL1, CDRL2, and CDRL3, wherein

[0480] A bispecific antibody wherein CDRH1 comprises or is composed of the amino acid sequence of SEQ ID NO. 87; CDRH2 comprises or is composed of the amino acid sequence of SEQ ID NO. 88; CDRH3 comprises or is composed of the amino acid sequence of SEQ ID NO. 89; CDRL1 comprises or is composed of the amino acid sequence of SEQ ID NO. 91; CDRL2 comprises or is composed of the amino acid sequence of SEQ ID NO. 92; and CDRL3 comprises or is composed of the amino acid sequence of SEQ ID NO. 93.

[0481] 30. A bispecific antibody in Embodiment 29, wherein the second antigen-binding region comprises VH and VL, VH comprises or is composed of the sequence described in SEQ ID NO. 86; and VL comprises or is composed of the sequence described in SEQ ID NO. 90.

[0482] 31. In any one of embodiments 17 to 28, the TAA is PSMA; preferably, the second antigen-binding region comprises three complementary-determining regions (CDRHs) from the heavy chain variable region: CDRH1, CDRH2, and CDRH3; and three complementary-determining regions (CDRLs) from the light chain variable region: CDRL1, CDRL2, and CDRL3, wherein

[0483] A bispecific antibody wherein CDRH1 comprises or consists of the amino acid sequence of SEQ ID NO. 96; CDRH2 comprises or consists of the amino acid sequence of SEQ ID NO. 97; CDRH3 comprises or consists of the amino acid sequence of SEQ ID NO. 98; CDRL1 comprises or consists of the amino acid sequence of SEQ ID NO. 99; CDRL2 comprises or consists of the amino acid sequence of SEQ ID NO. 100; and CDRL3 comprises or consists of the amino acid sequence of SEQ ID NO. 101.

[0484] 32. In Embodiment 31, the second antigen-binding region comprises VH and VL, wherein VH comprises or is composed of the sequence described in SEQ ID NO. 47; and VL comprises or is composed of the sequence described in SEQ ID NO. 49, a bispecific antibody.

[0485] 33. In any one of embodiments 17 to 32, the bispecific antibody comprises a first antigen-binding region that specifically binds to CD28 and a second antigen-binding region that specifically binds to TAA, wherein the first antigen-binding region comprises a first Fab or is a first Fab, and the second antigen-binding region comprises a second Fab or is a second Fab, wherein the first Fab is connected (directly or through a linker such as a hinge region) from the C-terminus of its CH1 to the N-terminus of the first Fc region, and the second Fab is connected (directly or through a linker such as a hinge region) from the C-terminus of its CH1 to the N-terminus of the second Fc region.

[0486] 34. In Embodiment 33, the bispecific antibody is a bispecific antibody that is an IgG-like antibody having the configuration shown in FIG. 3.

[0487] 35. In Embodiment 33 or 34, the bispecific antibody comprises a first Fab as a first antigen-binding region that specifically binds to CD28 and a second Fab as a second antigen-binding region that specifically binds to TAA.

[0488] Heavy chain 1: comprises or is composed of a first Fab heavy chain variable region from the N-terminus to the C-terminus, a heavy chain invariant region CH1, and a first Fc region, wherein the heavy chain invariant region CH1 is connected from its C-terminus to the N-terminus of the first Fc region directly or through a linker (e.g., a hinge region);

[0489] Light chain 1: comprises or is composed of a first Fab light chain variable region-light chain invariant region from the N-terminus to the C-terminus;

[0490] Heavy chain 2: comprises or is composed of a second Fab heavy chain variable region from the N-terminus to the C-terminus, a heavy chain invariant region CH1, and a second Fc region, wherein the heavy chain invariant region CH1 is connected from its C-terminus to the N-terminus of the second Fc region either directly or through a linker (e.g., a hinge region);

[0491] Light chain 2: includes or is composed of a second Fab light chain variable region-light chain invariant region from the N-terminus to the C-terminus;

[0492] Preferably, each domain is a directly linked bispecific antibody.

[0493] 36. In Embodiment 35, TAA is CLDN18.2, where

[0494] Heavy chain 1 comprises or is composed of the amino acid sequence described in SEQ ID NO. 35 or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity therewith; light chain 1 comprises or is composed of the amino acid sequence described in SEQ ID NO. 36 or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity therewith; heavy chain 2 comprises or is composed of the amino acid sequence described in SEQ ID NO. 33 or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity therewith; Light chain 2 comprises or is composed of the amino acid sequence described in SEQ ID NO. 34 or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity therewith;

[0495] Heavy chain 1 comprises or is composed of the amino acid sequence described in SEQ ID NO. 37 or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity therewith; light chain 1 comprises or is composed of the amino acid sequence described in SEQ ID NO. 38 or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity therewith; heavy chain 2 comprises or is composed of the amino acid sequence described in SEQ ID NO. 33 or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity therewith; Light chain 2 comprises or is composed of the amino acid sequence described in SEQ ID NO. 34 or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity therewith;

[0496] Heavy chain 1 comprises or is composed of the amino acid sequence described in SEQ ID NO. 39 or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity therewith; light chain 1 comprises or is composed of the amino acid sequence described in SEQ ID NO. 40 or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity therewith; heavy chain 2 comprises or is composed of the amino acid sequence described in SEQ ID NO. 33 or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity therewith; Light chain 2 is a bispecific antibody comprising or composed of an amino acid sequence described in SEQ ID NO. 34 or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity therewith.

[0497] 37. In Embodiment 35, TAA is PSMA, where

[0498] Heavy chain 1 comprises or is composed of the amino acid sequence described in SEQ ID NO. 51 or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity therewith; light chain 1 comprises or is composed of the amino acid sequence described in SEQ ID NO. 36 or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity therewith; heavy chain 2 comprises or is composed of the amino acid sequence described in SEQ ID NO. 48 or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity therewith; Light chain 2 comprises or is composed of the amino acid sequence described in SEQ ID NO. 50 or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity therewith;

[0499] Heavy chain 1 comprises or is composed of the amino acid sequence described in SEQ ID NO. 52 or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity therewith; light chain 1 comprises or is composed of the amino acid sequence described in SEQ ID NO. 38 or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity therewith; heavy chain 2 comprises or is composed of the amino acid sequence described in SEQ ID NO. 48 or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity therewith; Light chain 2 comprises or is composed of the amino acid sequence described in SEQ ID NO. 50 or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity therewith;

[0500] Heavy chain 1 comprises or is composed of the amino acid sequence described in SEQ ID NO. 53 or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity therewith; light chain 1 comprises or is composed of the amino acid sequence described in SEQ ID NO. 40 or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity therewith; heavy chain 2 comprises or is composed of the amino acid sequence described in SEQ ID NO. 48 or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity therewith; Light chain 2 is a bispecific antibody comprising or composed of an amino acid sequence described in SEQ ID NO. 50 or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity therewith.

[0501] 38. A nucleic acid molecule comprising a nucleic acid sequence that encodes any one of the chains of an antibody or an antigen-binding fragment according to any one of Embodiments 1 to 16, or any one of the chains of a bispecific antibody according to any one of Embodiments 17 to 37.

[0502] 39. Preferably, the expression vector comprises a nucleic acid molecule according to Embodiment 38, which is pCDNA, e.g., pCDNA3.1.

[0503] 40. Preferably, the host cell is a prokaryotic or eukaryotic cell, e.g., 293 cell or CHO cell, e.g., Expi293 cell, a host cell comprising a nucleic acid molecule according to Embodiment 38 or an expression vector according to Embodiment 39.

[0504] 41. A method for manufacturing an antibody or an antigen-binding fragment thereof according to any one of embodiments 1 to 16, or a bispecific antibody according to any one of embodiments 17 to 37, comprising the steps of: culturing a host cell containing a nucleic acid molecule according to embodiment 38 or an expression vector according to embodiment 39 under conditions suitable for expressing a chain of antibodies; and optionally recovering the antibody from the host cell (or host cell medium).

[0505] 42. An immunoconjugate comprising an antibody or an antigen-binding fragment thereof according to any one of embodiments 1 to 16, or a bispecific antibody according to any one of embodiments 17 to 37.

[0506] 43. A pharmaceutical composition, drug, or formulation comprising an antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 16, a bispecific antibody according to any one of embodiments 17 to 37, or an immunoconjugate according to embodiment 42, and optionally a pharmaceutical adjuvant.

[0507] 44. A pharmaceutical combination product comprising an antibody or an antigen-binding fragment according to any one of embodiments 1 to 16, or a bispecific antibody according to any one of embodiments 17 to 37, or an immunoconjugate according to embodiment 42, and one or more additional therapeutic agents, wherein preferably, the therapeutic agent is a therapeutic agent for treating various tumors, such as a chemotherapy agent, an angiogenesis inhibitor, a cytokine, a cytotoxic agent, another antibody, a small molecule drug, or an immunomodulator (e.g., an immune checkpoint inhibitor or agonist); preferably, the additional antibody is a PD-1 antibody; an antibody according to any one of embodiments 42; or an immunoconjugate according to embodiment 42.

[0508] 45. A method comprising the step of administering to a subject an effective amount of an antibody or an antigen-binding fragment thereof according to any one of embodiments 1 to 16, or a bispecific antibody according to any one of embodiments 17 to 37, or an immunoconjugate according to embodiment 42, or a pharmaceutical composition or formulation according to embodiment 43, or a pharmaceutical combination product according to embodiment 44, in order to prevent or treat a tumor in a subject.

[0509] 46. ​​In the embodiment 45, the tumor is a solid tumor or a hematological tumor, e.g., a TAA-positive tumor or cancer; optionally, the tumor is a CLDN18.2-positive gastrointestinal tumor, e.g., gastric cancer, breast cancer, colon cancer, rectal cancer, or colorectal cancer; or the tumor is a PSMA-positive tumor, e.g., renal cell carcinoma, such as breast cancer, lung cancer, rectal cancer, or colorectal cancer.

[0510] 47. The method of Embodiment 45 or 46 further comprises the step of administering in combination with an additional therapy and / or an additional therapeutic agent, such as a treatment method (e.g., surgical therapy or radiotherapy), preferably, the therapeutic agent is any therapeutic agent for treating a tumor, such as a chemotherapy agent, an angiogenesis inhibitor, a cytokine, a cytotoxic agent, an additional antibody, a small molecule drug, or an immunomodulator (e.g., an immune checkpoint inhibitor or agonist); preferably, the additional antibody is a PD-1 antibody.

[0511] Example 1: Discovery and Affinity Testing of Murine Anti-CD28 Antibody

[0512] Immunization using CD28 antigen

[0513] Human CD28 extracellular domain protein (Acro, Cat CD8-H52Hc) was emulsified with Complete Freund's Adjuvant (sigma, Cat# F5881) and used to immunize Balb / c mice (purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.). After 2 weeks, the protein was emulsified with Incomplete Freund's Adjuvant (sigma, F5506) and used to immunize mice by subcutaneous injection once every 2 weeks for a total of 3 times (50 μg of protein per mouse).

[0514] Cell fusion and high-throughput screening

[0515] When serum titers met the requirements, mouse spleens were collected to prepare a B lymphocyte suspension, which was electrofused with SP2 / 0 myeloma cells (ATCC). The fused cells were diluted to 1-2 × E4 cells / mL in selection medium (1640 medium containing 20% ​​FBS and 1 × HAT) and plated in 96-well plates, with 100 μL of the cell suspension added to each well. On day 7 after fusion, the medium was replaced with screening medium (1640 medium containing 10% FBS and 1 × HT). On day 10 of culture (or thereafter, depending on cell growth status), the supernatant was collected for detection.

[0516] Hybridoma cells specifically expressing anti-CD28 antibodies were screened by flow cytometry (FACS). Cells to be tested (huCD28 / GS-CHO, self-constructed) were counted, diluted to 1E6 cells / mL, and added to a U-bottom 96-well plate at a rate of 100 μL / well. The mixture was centrifuged at 500 g for 5 minutes, and the cell medium was removed. The supernatant of the hybridoma culture from the 96-well plate was added to a U-type plate at a rate of 100 μL / well, and the cells were resuspended and incubated on ice for 30 minutes. The mixture was centrifuged at 500 g for 5 minutes, and the supernatant was removed. The cells were washed once with PBS. The mixture was centrifuged at 500 g for 5 minutes, and the PBS was removed. 100 μL of APC-labeled anti-mouse Fab secondary antibody (Jackson Immunoresear, Cat# 115-606-071) (diluted 1:500 with PBS) was added to each well, and 100 μL of PE-labeled anti-human Fc secondary antibody (Biolegend, Cat# 409304) was added as the positive control antibody (TGN1412 (Creative BioLabs TAB-064LC)). The mixture was incubated in the dark on ice for 30 minutes, centrifuged at 500 g for 5 minutes, and the supernatant was removed. Cells were washed once with PBS, resuspended in 50 μL of 1× PBS, and analyzed by FACS. CD28 activation tests were performed on the supernatant of positive clones. Clones with strong activation were subcloned by the restriction dilution method, and monoclonal cells were selected.

[0517] Subcloning of positive hybridoma cells

[0518] Subcloning by restriction dilution: A 96-well plate was prepared, and 200 μL of medium was added to each well. Based on the screening medium, HAT was replaced with HT (Gibco, Cat# 11067-030) to obtain the medium, while other components remained the same. Cells from positive wells selected by fusion screening were prepared into a cell suspension, and 100 μL of the cell suspension was added to each well of the first row and mixed thoroughly. Subsequently, 100 μL of the cell suspension from the first row was added to the second row and mixed thoroughly, after which 100 μL of the cell suspension was added to the next row. The above steps were repeated. The 96-well plate was left to stand for 30 minutes, and the cells were observed and counted under a microscope. A corresponding volume containing 100 cells was added to 20 mL of medium, the mixture was thoroughly mixed, and plated at a rate of 200 μL / well. After one week, the cells were observed under a microscope, and monoclonal wells were identified and marked.

[0519] When the cell confluence of each well reached 50% or more, cells were detected using the aforementioned high-throughput screening method. Target-positive wells were selected, and after proliferation culture, the cells were cryopreserved.

[0520] Preparation of chimeric antibodies

[0521] The gene sequences of the antibody light and heavy chains of the hybridoma candidate clones obtained from the above experiment were secured and constructed using human-mouse chimeric antibodies 61F3G, 5B11D8, and 4F12B5. The corresponding sequences of the antibody light and heavy chains are as follows, and the antibody heavy chain constant region is the human IgG1 heavy chain constant region (Sequence No. 79). The variable regions and CDR sequences are presented in Table 1 below.

[0522] Approximately 5E6 cells were taken from each newly cultured strain, and RNA was extracted (Macherey-Nagel, Cat# 740984.250). cDNA was obtained by performing reverse transcription using the PrimeScript II 1st Strand cDNA Synthesis Kit (Takara). Upstream primers were designed from the nucleotide sequences located in the FR1 region of the 5' end, and downstream primers were designed from the nucleotides located in the antibody constant region or the FR4 region. Gene fragments from the light and heavy chain variable regions of the antibody were amplified and ligated into a T vector (Mighty TA-cloning Kit, Takara), and single clones were selected and sequenced. The sequencing results were analyzed and aligned using MEGA7 software.

[0523] A clone having accurate and paired antibody light and heavy chain variable region sequences was obtained by alignment, and the gene fragments of the light and heavy chain variable regions were ligated into the pcDNA3.1 vector (containing the IgG1 heavy chain constant region (Sequence No. 79) or the kappa light chain constant region (Sequence No. 30)) respectively by the homologous recombinase (Exnase®II, Cat. No. C112-01) of Nanjing Vazyme Biotech Co., Ltd., wherein the IgG1 subtype was selected for the constant region, and expression plasmids of light and heavy chain antibodies were obtained.

[0524] [Table 1] Comparison Table of CDR Light and Heavy Chain Combinations of CD28 Chimeric Antibodies

[0525]

[0526] Expi293F cells (purchased from Gibco) were cultured in Expi293F medium (Gibco, REF# A14351-01). Cell density was measured the day before transfection (viability had to be greater than 95%), and the cells were recultured after adjusting the cell density to 3E6 cells / mL with fresh Expi293F medium. On the day of transfection, the cell density was adjusted to 3E6 cells / mL.

[0527] Opti-MEM medium (Gibco, REF# 31985-070), equivalent to 1 / 10 of the final transfection volume, was used as the transfection buffer, and the plasmid of the chimeric antibody to be transfected was added at a ratio of 1 mg / L, where the light-to-heavy chain plasmid ratio was 1:1. The mixture was thoroughly mixed, and then PEIMax (Polysciences Inc. Cat# 24765-1) was added at a DNA:PEI mass ratio of 1:3. The mixture was then thoroughly mixed and incubated at room temperature for 20 minutes. Subsequently, the resulting mixture was carefully poured into the Expi293F cell suspension while rotating. Cells were cultured in a shaker under culture conditions of 8% CO2, 36.5°C, and 120 rpm.

[0528] After incubating for 16–18 hours, the cell suspension was supplemented with 2% (v / v) of 200 g / L Feed (100 g / L Phytone Peptone + 100 g / L Difco Select Phytone), a glucose solution with a final concentration of 5 g / L, and valproic acid sodium salt (Merk, Cat# P4543-100G) with a final concentration of 2.2 mM, and mixed thoroughly and carefully. Cells were cultured continuously at 8% CO2, 36.5°C, and 120 rpm for 7 days prior to sample collection. The cell culture medium was thoroughly mixed with diatomite (Sartorius, Cat. No. 1000037025) (1 L of cell culture medium mixed with 40 g of diatomite), and the mixture was filtered using a 0.22 μm disposable vacuum filtration device.

[0529] Purification of target proteins by affinity chromatography: A HiTrap MabSelect PrismA (GE Healthcare, Cat# 17549853) affinity chromatography column was selected for affinity capture. Before purification, 10–20 column volumes of 0.1 M NaOH were passed through the piping and the affinity chromatography column, and subsequently, the piping and column were washed with 10–20 column volumes of distilled water. The packing column was equilibrated with 5 column volumes of 1× PBS (Gibco). After passing the filtered cell culture medium through the column, the packing column was washed with 10 column volumes of 1× PBS to remove non-specific binding proteins. The packing was washed with 5 column volumes of elution buffer (100 mM sodium citrate, pH 3.5). The eluent was collected and adjusted to pH 6.0 with 2 M Tris. After the purity was determined to be acceptable, the eluent was filtered to remove bacteria and used for subsequent functional analysis.

[0530] Affinity assay of chimeric anti-CD28 antibodies

[0531] In this study, the binding kinetics parameter (KD) of the antibody-antigen interaction of the present invention was determined by biolayer interferometry (BLI). BLI affinity testing was performed according to the existing method (Estep, P et al., High throughput solution Based measurement of antibody-antigen affinity and epitope binning. MAbs, 2013.5(2): 270-8).

[0532] Thirty minutes before the experiment, an appropriate number of AHC sensors (18-5060, Sartorius) were taken according to the number of samples and immersed in SD buffer (1× PBS, 0.1% BSA, 0.05% Tween-20). The aforementioned anti-CD28 antibody and human CD28 recombinant protein (CD8-H52Hc-1 mg, ACRO) were each diluted to 100 n.

[0533] SD buffer, antibody solution, and CD28 recombinant protein were added to 96-well black polystyrene microplates (Greiner, 655209), respectively. Detection was performed using a Fortebio Octet Red96e; the plate layout was configured based on sample locations, and sensor locations were selected accordingly. The instrument settings were as follows: The operating procedure was as follows: at a stirring speed of 1000 rpm and a temperature of 30°C, baseline equilibration for 120 seconds, sample loading for 100 seconds for antibody immobilization, baseline equilibration for 120 seconds, antigen binding for 100 seconds, and dissociation for 120 seconds. After the experiment was completed, KD values ​​were analyzed using ForteBio Octet analysis software. The results are as follows. Table 2 It is presented in.

[0534] From the kinetic parameters of the affinity experiment, it was confirmed that these chimeric CD28s have a strong affinity for human CD28 recombinant protein.

[0535] [Table 2] Affinity of anti-CD28 antibodies for CD28

[0536]

[0537] Example 2: Detection of T cell reporter activation by murine anti-CD28 antibody

[0538] To determine whether the anti-CD28 antibody can effectively recognize human CD28 and activate CD28 signaling, Jurkat NFkB (Gimman Biotech, GM-C07855) reporter T cell line was selected as the study subject to determine the activity of the CD28 antibody. The specific procedure is as follows:

[0539] 1. Anti-human IgG (Jackson ImmuneResearch, 109-005-098) antibody (1 μg / mL) was diluted with PBS and added to 96-well flat-bottom plates (Shanghai Yuhong Scientific Co., Ltd.; WHB-96-03) at a rate of 100 μL / well. The plates were incubated overnight in a 4°C refrigerator to allow the antibody to bind sufficiently to the bottom of the plates.

[0540] 2. After incubation was complete, the unbound antibody solution on the plate was discarded, PBS was added at a rate of 100 μL / well, the plate was washed once, and the PBS was discarded.

[0541] 3. Cultured Jurkat NFkB cells were centrifuged and resuspended in assay buffer (1% FBS (Gibco, SV30208.02), RPMI 1640 (Gibco, 22400-071)), and 50 μL of the cell suspension was added to each well to make up 50,000 cells per well.

[0542] 4. The chimeric CD28 antibody (2 μg / mL) to be tested was diluted with assay buffer, with or without CD3 antibody (Acro, CDE-M120A-1MG, final concentration 0.5 μg / mL), and added to the plate at a rate of 50 μL / well. The plates were incubated in a 5% CO2 incubator at 37°C for 6 hours.

[0543] 5. After the experiment was completed, the plate was removed and equilibrated at room temperature on the workbench, and 100 μL of Bio-Glo detection reagent (Promega, G7940) was added. The plate was detected using a microplate reader (Molecular Devices, spectra MAX i3x).

[0544] The results indicated that, in the absence of a first signal provided by the CD3 (OKT3, Acro, CDE-M120A-1MG) antibody, the control antibodies TGN1412 (Tegenero, US20060188493A1, abbreviated as TGN in this invention), 5B11D8, and 61F3G3 were able to activate the NFkB signal downstream of CD28, whereas 4F12B5 could not activate it ( Fig. 1 ). In the presence of CD3 antibodies, all CD28 antibodies in this study were able to activate CD28 signaling.

[0545] Example 3: Humanization of murine anti-CD28 antibody

[0546] The chimeric antibodies obtained in Examples 1-2 were humanized according to conventional methods. Accordingly, humanized antibodies hz5B11D8-a, hz5B11D8-b, hz61F3G3-a, hz61F3G3-b, hz4F12B5-a, and hz4F12B5-b were obtained. Their CDR sequences, light chain variable region and heavy chain variable region sequences, and amino acid sequences of the light and heavy chains are from the sequence list and Table 3 As presented in [the document]. A human IgG1 sequence (Sequence No. 29) was selected for the heavy chain constant region, and a CL-kappa constant region (Sequence No. 30) was selected for the light chain constant region corresponding to the kappa type of the variable region. Subsequently, the heavy chain and light chain sequences of the antibody were constructed in the expression vector pcDNA3.1 (Invitrogen, V790-20), and Expi293 cells (Invitrogen, A14527) were transfected to obtain the corresponding humanized antibody. The specific transfection procedure was the same as in Example 1.

[0547] [Table 3] Comparison Table of Light and Heavy Chain Combinations of Humanized CD28 Antibodies with CDR Mutations

[0548]

[0549] Example 4: Preparation of Humanized Anti-CD28 Antibody and Affinity Testing

[0550] Antibody preparation: Expi293F cells (purchased from Gibco) were cultured in Expi293F medium (Gibco, REF# A14351-01). Cell density was measured the day before transfection (viability had to be greater than 95%), and additional cultures were performed using fresh Expi293F medium adjusted to a cell density of 3E6 cells / mL. On the day of transfection, the cell density was adjusted to 3E6 cells / mL.

[0551] Opti-MEM medium (Gibco, REF# 31985-070), equivalent to 1 / 10 of the final transfection volume, was used as the transfection buffer, and the plasmid of the humanized monoclonal antibody to be transfected (plasmid obtained in Example 3) was added at a ratio of 1 mg / L, where the ratio of light chain to heavy chain plasmid was 1:1. The mixture was thoroughly mixed, PEIMax (Polysciences Inc. Cat# 24765-1) was added at a DNA:PEI mass ratio of 1:3, and the mixture was thoroughly mixed and incubated at room temperature for 20 minutes. Subsequently, the resulting mixture was carefully poured into the Expi293F cell suspension while rotating. The cells were cultured in a shaker under culture conditions of 8% CO2, 36.5°C, and 120 rpm.

[0552] After incubating for 16–18 hours, the cell suspension was supplemented with 2% (v / v) of 200 g / L Feed (100 g / L Phytone Peptone + 100 g / L Difco Select Phytone), a glucose solution with a final concentration of 5 g / L, and valproic acid sodium salt (Merk, Cat# P4543-100G) with a final concentration of 2.2 mM, and mixed thoroughly and carefully. Cells were cultured continuously at 8% CO2, 36.5°C, and 120 rpm for 7 days prior to sample collection. The cell culture medium was thoroughly mixed with diatomite (Sartorius, Cat. No. 1000037025) (1 L of cell culture medium mixed with 40 g of diatomite), and the mixture was filtered using a 0.22 μm disposable vacuum filtration device.

[0553] Purification of target proteins by affinity chromatography: A HiTrap MabSelect PrismA (GE Healthcare, Cat# 17549853) affinity chromatography column was selected for affinity capture. Prior to purification, 10–20 column volumes of 0.1 M NaOH were passed through the piping and the affinity chromatography column, followed by washing the piping and column with 10–20 column volumes of distilled water. The packed column was equilibrated with 5 column volumes of 1× PBS (Gibco). After passing the filtered cell culture medium through the column, the packed column was washed with 10 column volumes of 1× PBS to remove non-specific binding proteins. The packing was washed with 5 column volumes of elution buffer (100 mM sodium citrate, pH 3.5). The eluent was collected and adjusted to pH 6.0 with 2 M Tris; after the purity was determined to be acceptable, the eluent was filtered to remove bacteria and used for subsequent functional analysis.

[0554] The affinity of the humanized CD28 antibody of the present invention was determined using the biolayer interferometer (BLI) technique mentioned in Example 1. The affinity of the humanized molecule is shown in the table below ( Table 4 It is presented in ).

[0555] [Table 4] Affinity of humanized antibodies for CD28

[0556]

[0557] Example 5: Detection of T cell activation by humanized anti-CD28 antibody

[0558] To verify whether the humanized CD28 antibodies still maintain their activity, we investigated whether these antibodies could effectively activate human peripheral T cells in vitro. The specific procedure was as follows:

[0559] 1. PBMC cells (Milestone) were removed one day prior, centrifuged at 25°C and 400 G for 4 minutes, resuspended in growth medium (Gibco, 1640), and placed in a culture flask. DNase I (SIGMA, D5025-375KU) was added at a final concentration of 2 μg / mL. The cells were incubated overnight in a 37°C, 5% CO2 incubator.

[0560] 2. The CD28 humanized antibody to be tested was diluted with PBS (1 μg / well) (or added simultaneously with the CD3 antibody (Acro, CDE-M120A-1MG, 0.1 μg / well)) and added to a 96-well flat-bottom plate, and 100 μL / well of the CD28 humanized antibody to be tested was added. The plate was incubated overnight in a 4°C refrigerator to allow the antibody to bind sufficiently to the bottom of the plate.

[0561] 3. On the day of the experiment, after completing antibody incubation, the unbound antibody solution in the plate was discarded. Each well was washed once with 100 μL of PBS, and then the PBS was discarded.

[0562] 4. The PBMC cells thawed the previous day were removed, and the suspended cells were transferred to a centrifuge tube. After centrifugation at 25°C and 400 G for 4 minutes, the cell density was adjusted using assay buffer (RPMI 1640, 1% FBS). The cell concentration was adjusted to 100 μL per well (100,000 cells / well), and the cells were plated onto antibody-coated 96-well flat-bottom plates and incubated in a 37°C, 5% CO2 incubator.

[0563] 5. Four hours before the end of cell culture, the 96-well plate was removed from the incubator, and 50 μL of supernatant from each well was pipetted into a new 96-well flat-bottom plate for cytokine assay. The plate was frozen in a -40°C refrigerator. A protease transport inhibitor (Invitrogen, 00-4980-93) was added to the 96-well flat-bottom plate at a rate of 50 μL / well, and the plate was incubated for 4 hours in a 37°C, 5% CO2 incubator.

[0564] 6. After completing incubation, antibody staining for flow cytometry was prepared. First, a 96-well flat-bottom plate was centrifuged at 25°C and 400 G for 4 minutes, and the culture supernatant was discarded. The cells were resuspended in PBS, transferred to a new 96-well round-bottom plate, centrifuged at 25°C and 400 G for 4 minutes, and the PBS wash buffer was discarded.

[0565] 7. A pre-prepared surface blocking dye mixture was added at a rate of 50 microliters per well: Fc blocker (BD, 564219, 1:200 PBS) and survival / death dye (Invitrogen, L34968A 1:100 PBS). The plates were incubated in the dark at room temperature for 10 minutes. After incubation, 50 μL of the 1:100 cell surface dye mixture in FACS buffer—PD1 antibody (Biolegend, 329918), CD4 antibody (Biolegend, 329918), or CD8 antibody (Biolegend, 344724)—was added directly to each well, and the plates were incubated in the dark at 4°C for 30 minutes.

[0566] 8. After completing incubation with surface dye, the mixture was centrifuged at 25°C and 400 G for 4 minutes, and any dye not bound to the cells was discarded. The cells were resuspended in FACS wash buffer (2 mM EDTA, 0.5% BSA, PBS) and washed twice by centrifugation.

[0567] 9. Fix / Perm buffer (ebioscience 00-5523-00 Invitrogen, 00-8333-56) was added at a rate of 100 μL / well. To permeate the cell membrane, the plates were incubated in the dark at room temperature for 30 minutes.

[0568] 10. After completing incubation, the plate was centrifuged at 25°C and 400 G for 4 minutes, and the Fix / Perm buffer was discarded. The cells were resuspended in PBE (Invitrogen, 00-8333-56) wash buffer and washed twice by centrifugation.

[0569] 11. 50 μL / well of TNF-α antibody (Biolegend, 502946), an intracellular dye, was added, and the plate was incubated in the dark at room temperature for 30 minutes.

[0570] 12. After completing incubation, the mixture was centrifuged at 25°C and 400 G for 4 minutes, and any dye not bound to the cells was discarded. The cells were resuspended in PBE wash buffer and washed twice, and the wash buffer was discarded.

[0571] 13. Cells were resuspended in FACS wash buffer at 150 μL / well and analyzed on a flow cytometer (BD FACS SYMPHONY A3).

[0572] The results indicated that in the absence of a first signal provided by the CD3 antibody, the control antibody TGN1412 (abbreviated as TGN in this study) could activate CD4 T and CD8 T cells to release TNFα independently of the first signal. Figs. 2a-b The chimeric antibodies 5B11D8 and 61F3G3 of the present invention exhibited CD28 super-agonist activity, whereas 4F12B5 was a conventional CD28 agonist antibody. After humanization, hz5B11D8-a and hz61F3G3-a had reduced activity compared to the parent, but hz5B11D8-b, hz61F3G3-b, hz4F12B5-a and hz4F12B5-b had activity similar to the parent.

[0573] In the presence of a first signal provided by a CD3 antibody ( Figs. 2c-d All of the antibodies of the present invention can activate CD4 and CD8 T cells to different degrees to release TNF.

[0574] Example 6: Construction and Preparation of CLDN18.2×CD28 Bispecific Antibody

[0575] To detect the monovalent affinity and activation ability of tumor-associated antigen-dependent T cells of CD28 humanized antibodies, CLDN18.2×CD28 bispecific antibodies BsAb-1, BsAb-2, BsAb-3, and BsAb-BM-1 targeting Claudin18.2 were constructed in this study ( Table 5 ). Bispecific antibody molecules are in the "1 + 1" format ( Fig. 3a) and, where the variable region targeting CLDN18.2 was derived from HB37A6 (CN202010570517.X(WO2021254481A1), sequence information is shown in the sequence list), and the variable region sequence of the anti-CD28 antibody was derived from the variable region sequences of the humanized antibodies of the present invention hz5B11D8-a, hz61FG3-a, and hz4F12B5-b and TGN1412 (Tegenero, US20060188493A1, the sequences thereof are shown in sequence numbers 41-44 of the sequence list) ( Table 6 ). The Fc fragment of the antibody was in the Innovadi (CN116888149A bispecific antibody technology) IgG1 LALA delP format, and the schematic of the antibody is Fig. 3a It is depicted in.

[0576] Accordingly, the heavy chain of the CLDN18.2-targeting arm of the bispecific antibody is described in SEQ ID NO. 33, and the light chain is described in SEQ ID NO. 34. The heavy chain of the CD28-targeting arm of the bispecific antibody of the present invention is described in SEQ ID NOs. 35, 37, and 39, respectively, and the light chain is described in SEQ ID NOs. 36, 38, and 40, respectively. The heavy chain and light chain of the control CD28 (TGN1412) used in the bispecific antibody of the present invention are described in SEQ ID NO. 42 and SEQ ID NO. 44, respectively.

[0577] [Table 5] CLDN18.2×CD28 Bispecific Antibody Combinations

[0578]

[0579] Sequences corresponding to the heavy and light chains of HB37A6: SEQ ID NO. 33 and SEQ ID NO. 34.

[0580] Sequences corresponding to the heavy and light chains of TGN1412: SEQ ID NO. 42 and SEQ ID NO. 44.

[0581] [Table 6] Sequence information of CLDN18.2×CD28 bispecific antibody

[0582]

[0583] Preparation of bispecific antibodies

[0584] The bispecific antibody of the present invention was prepared by separately expressing and purifying two parent proteins, and then recombining them through in vitro reduction and oxidation. The production route of the bispecific antibody was as follows:

[0585] Plasmid construction: The sequence of the diabody of the present invention was synthesized using Genewiz. Light chain and heavy chain variable regions corresponding to the bispecific antibody ( Table 6 ) was cloned into a pcDNA3.1 vector (Invitrogen, V790-20) containing the corresponding heavy or light chain constant region to obtain the heavy and light chain plasmids for the CLDN18.2-targeting subarm and the heavy and light chain plasmids for the CD28-targeting subarm.

[0586] Expi293F cells (purchased from Gibco) were cultured in Expi293F medium (Gibco, REF# A14351-01). Cell density was measured the day before transfection (viability had to be greater than 95%), and the cells were recultured using fresh Expi293F medium, adjusted to a cell density of 3E6 cells / mL. On the day of transfection, the cell density was adjusted to 3E6 cells / mL.

[0587] Opti-MEM medium (Gibco, REF# 31985-070), equivalent to 1 / 10 of the final transfection volume, was used as the transfection buffer, and DNA to be transfected was added at a ratio of 1 mg / L, where the light-to-heavy chain plasmid ratio was 1:1. After thoroughly mixing the mixture, PEIMax (Polysciences Inc. Cat# 24765-1) was added at a DNA:PEI mass ratio of 1:3, and the mixture was thoroughly mixed and incubated at room temperature for 20 minutes. Subsequently, the resulting mixture was gently poured into the Expi293F cell suspension while rotating. Cells were cultured in a shaker under culture conditions of 8% CO2, 36.5°C, and 120 rpm.

[0588] After incubating for 16-18 hours, the cell suspension was supplemented with 2% (v / v) of a 200 g / L feed (100 g / L Phytone Peptone + 100 g / L Difco Select Phytone), a glucose solution with a final concentration of 5 g / L, and sodium valproate salt (Merk, Cat# P4543-100G) with a final concentration of 2.2 mM, and gently mixed. Cells were cultured continuously for 7 days at 8% CO2, 36.5°C, and 120 rpm prior to sample collection. The cell culture medium was thoroughly mixed with diatomite (Sartorius, Cat. No. 1000037025) (1 L of cell culture medium mixed with 40 g of diatomite), and the mixture was filtered using a 0.22 μm disposable vacuum filtration device.

[0589] Purification of target proteins by affinity chromatography: A HiTrap MabSelect PrismA (GE Healthcare, Cat# 17549853) affinity chromatography column was selected for affinity capture. Before purification, 10–20 column volumes of 0.1 M NaOH were passed through the piping and the affinity chromatography column, and subsequently, the piping and column were washed with 10–20 column volumes of distilled water. The packed column was equilibrated with 5 column volumes of 1× PBS (Gibco). After passing the filtered cell culture medium through the column, the packed column was washed with 10 column volumes of 1× PBS to remove non-specific binding proteins. The packing was washed with 5 column volumes of elution buffer (100 mM sodium citrate, pH 3.5), and the eluent was collected and adjusted to pH 6.0 with 2 M Tris. The eluent was filtered to remove bacteria, and after determining that the purity was acceptable, it was used for subsequent functional analysis.

[0590] In vitro reduction oxidation: Protein collections from affinity chromatography were mixed in a 1:1 molar ratio. An appropriate amount of GSH was added, and the reaction mixture was adjusted to pH 8.0 with 2 M Tris. The reaction system was incubated overnight at room temperature. The reactant was buffered with PBS and left to stand at 4°C for future use.

[0591] Purification of bispecific antibodies by ion exchange chromatography: A Mono S 5 / 50 GL ion exchange chromatography column (obtained from GE Healthcare) was selected and mounted on an AKTApure system (GE Healthcare). The AKTApure system equipped with the Mono S 5 / 50 GL ion exchange chromatography column was treated with 0.5 M NaOH for 2 hours to remove endotoxins, after which the system and column were washed with distilled water. The column was equilibrated with 5–10 column volumes of loading buffer (20 mM NaPO4, pH 6.6) until conductivity and pH stabilized. The protein obtained by affinity chromatography was diluted 10-fold with the loading buffer prior to loading. The column was re-equilibrated with 5 column volumes of loading buffer. Linear elution was performed over a total of 30 column volumes with a gradient of 0-40% elution buffer (20 mM NaPO4, 1 M NaCl, pH 6.6), and samples were collected based on UV absorbance values.

[0592] The purity of the samples collected from all fractionation tubes was analyzed by size exclusion chromatography (SEC). Based on the SEC results, samples in fractionation tubes with a purity greater than 95% were combined.

[0593] The purified bispecific antibody solution was centrifuged in a 15 mL ultrafiltration centrifuge tube at 4,500 rpm for 30 minutes. The protein was diluted with PBS and centrifuged again at 4,500 rpm for 30 minutes, and this procedure was repeated several times to exchange the buffer. The antibodies after buffer exchange were aggregated, and the antibody concentration was measured. Qualitative and quantitative analyses of the composition and content of the bispecific antibodies were further performed using a combination of capillary electrophoresis (CE-SDS) and liquid chromatography-mass spectrometry (LC-MS). The obtained bispecific antibodies ( Table 5 (listed in) was used in the study described in the subsequent examples.

[0594] Affinity assay of CLDN18.2×CD28 bispecific antibody

[0595] To characterize the affinity of the two moiety of the bispecific antibody CD28, the equilibrium dissociation constant (KD) of the binding of the bispecific antibody of the present invention to human CD28 and Claudin 18.2 proteins was determined using biolayer interferometry (BLI) in this study. For specific methods, refer to Example 1.

[0596] The test results (Table 7) confirmed that the CLDN18.2 moiety of the bispecific antibody has a high affinity for the Claudin18.2 protein. The CD28 moiety of the Diabody showed a consistent difference in affinity.

[0597] [Table 7] Detection of Affinity of Two Moities of CLDN18.2×CD28 Bispecific Antibody

[0598]

[0599] Example 7: In vitro activity assay of CLDN18.2×CD28 bispecific antibody

[0600] 7.1 Detection of NFkB Reporter Activation by CLDN18.2×CD28 Bispecific Antibody

[0601] In previous studies, the inventors verified that CD28 monoclonal antibodies can activate T cells by effectively activating the CD28 signaling pathway. In this study, we aimed to verify whether CD28 can possess excellent agonist activity against T cells even after being assembled into a bispecific antibody. First, the activation characteristics of the CLDN18.2×CD28 bispecific antibody against NFkB Jurkat reporter cells were verified in vitro. In this research system, the inventors needed to apply Claudin18.2-expressing tumor cells to provide a "hand-grip" that enables the CLDN18.2-targeting arm of the CLDN18.2×CD28 bispecific antibody to effectively bind to tumor cells. Additionally, it was necessary to verify the CD28-targeting arm in this study. For this reason, a co-culture system of the CD28-expressing T cell line Jurkat and Claudin18.2-expressing tumor cells was introduced in this study. The overall research process was as follows:

[0602] Construction of DANG-18.2 cells:

[0603] The full-length gene of human CLDN18.2 (UniProt ID: P56856-2) was constructed into the vector pWPT-GFP (Addgene, 12255) to replace its GFP sequence, and for viral packaging, the vector was transfected into HEK293T (ATCC, CRL-3216) cells along with the lentivirus packaging vectors psPAX2 (Addgene, 12260) and pMD2.G (Addgene, 12259). After 48 and 72 hours of culture, the culture supernatant was collected, and the lentivirus was concentrated using PEG8000. After transfecting pancreatic cancer DAN-G cells (DSMZ, Catalog No. ACC 249) with an enriched virus, cells expressing CLDN18.2 were sorted using a flow cytometry classifier (MoFlo XDP, Beckman Coulter) to obtain the tumor cell line DANG-18.2, which was stably transfected with CLDN18.2.

[0604] Activation of NFkB reporter by CLDN18.2×CD28 bispecific antibody

[0605] Treatment of target cells and effector cells: Cultured DANG-18.2 cell lines were removed from T75 simple culture flasks and centrifuged at 25°C and 400 G for 4 minutes. Cell density was adjusted using growth medium. Cell concentrations were adjusted based on 100 μL per well (40,000 cells / well), and the cells were thoroughly mixed and plated onto 96-well white culture plates. The 96-well white culture plates coated with target cells were removed, and the culture medium was discarded.

[0606] According to the experimental design, the CLDN18.2×CD28 bispecific antibody was serially diluted ( Table 5 ). The antibody was serially diluted fourfold from a concentration of 100 nM to the 10th concentration gradient using assay buffer (1% FBS, 1640). 50 μL of the antibody to be detected (IgG, Jackson ImmuneResearch, 109-005-098) was added to each well of a plate coated with target cells.

[0607] Cultured NFkB Jurkat cell lines (Gimman Biotech, GM-C07855) were removed from T75 simple culture flasks and centrifuged at 25°C and 400 G for 4 minutes. Cell density was adjusted using assay buffer, and cell concentration was adjusted based on 50 μL per well (50,000 cells / well). The mixture was thoroughly mixed, and OKT3 (final concentration 0.5 μg / mL) was added simultaneously. The mixture was thoroughly mixed, added to 96-well white culture plates, and incubated at 37°C in a 5% CO2 incubator for 6 hours.

[0608] A 96-well white culture plate was removed from the incubator, and 100 μL of Bio-Glo Luciferase Reagent (Promega, G7940) solution was added to each well. After incubation at room temperature for 2 minutes, detection was performed using a microplate reader (Molecular Devices, spectra MAX i3x).

[0609] The result is Fig. 4 As shown in [figure], the CD28 monoclonal antibody TGN was shown to be unable to effectively activate CD28 signaling. In the presence of CLDN18.2, which provides a "handle," the CD28 bispecific antibodies BsAb-1, BsAb-2, and BsAb-3 were all able to effectively activate downstream NFkB signaling of the CD28 signaling pathway, and the signal became stronger as the concentration increased.

[0610] 7.2. In Vitro T Cell Killing Experiment Mediated by CLDN18.2×CD28 Bispecific Antibody

[0611] To further verify the in vitro activity of the CD28 bispecific antibody, the inventors determined whether the CLDN18.2×CD28 bispecific antibody could activate human peripheral T cells to kill tumor cells. In this study, the surface of the tumor cells provided a primary signal by expressing aAPC. Additionally, CLDN18.2 was transfected onto the surface of the tumor cells as a specific recognition target for one of the antibody's arm types. The overall experimental procedure was as follows:

[0612] CLDN18.2-aAPC-CHO K1 was built as follows:

[0613] The full-length human CLDN18.2 gene (UniProt ID: P56856-2) was constructed into the vector pWPT-GFP (Addgene, 12255) to replace its GFP sequence, and aAPC-CHO K1 (Promega, J1250) cells were transfected with this vector for viral packaging in combination with the lentivirus packaging vectors psPAX2 (Addgene, 12260) and pMD2.G (Addgene, 12259). Culture supernatants were collected after 48 and 72 hours of incubation, and the lentivirus was concentrated using a Lenti-X Concentrator (Takara, Cat# 631232, Lot# 2109261A). Hamster ovarian aAPC-CHO K1 cells (Promega, J1250) were transfected with the concentrated virus, and then a flow cytometry classifier (BD FACSAria TM Cells expressing CLDN18.2 were sorted by FACS analysis using a Fusion Cell Sorter to obtain the cell line CLDN18.2-aAPC-CHO K1, which is stably transfected with CLDN18.2.

[0614] The T cell killing process mediated by CLDN18.2×CD28 was as follows:

[0615] 1. Target cell seeding

[0616] Cultured CLDN18.2-aAPC-CHO K1 were removed and digested with trypsin, and the cells were resuspended in growth medium to stop digestion. The cells were centrifuged, and cell density was adjusted using assay buffer. Cells were seeded into 96-well round-bottom plates at 100 μL per well (10,000 cells / well) and cultured for 16–20 hours, and assay buffer was added to the margin wells at a rate of 200 μL / well.

[0617] 2. Effector cell treatment

[0618] One cryopreserved PBMC vial was removed, thawed in a 37°C water bath, and centrifuged at 25°C and 400 G for 4 minutes. CD8+ T cells were isolated according to the instructions of EasySep Human CD8+ T Cell (STEMCELL, 19053). Cells were resuspended in RPMI 1640 medium and incubated overnight in a 37°C, 5% CO2 incubator. The next day, cells were removed and centrifuged at 25°C and 400 G for 4 minutes. Cell concentrations were adjusted using assay buffer. CD8+ T cells were added to a 96-well round-bottom plate at a concentration of 50 μL per well (50,000 cells / well), with an effector cell to target cell ratio of 5:1.

[0619] 3. Dilution of antibodies

[0620] The antibody to be tested was serially diluted according to the experimental design. The antibody was serially diluted fourfold from a starting concentration of 50 nM to the 10th concentration gradient. The diluent was assay buffer. 50 μL of the serially diluted antibody was pipetted into 96-well round-bottom plates coated with CD8+ T cells and CLDN18.2-aAPC-CHO K1 cells, and incubated for 48 hours in a 37°C, 5% CO2 incubator with a total volume of 200 μL in each well.

[0621] 4. To lyse the target cells, 20 μL of 10× lysis buffer was added to the wells of the cell control. The plates were incubated in a 5% CO2 incubator at 37°C for 45 minutes. Detection reagents were prepared according to the instructions of the Radio Cytotoxicity Assay Kit (Promega, G1781). Cells in a 96-well round-bottom plate were centrifuged to pellet. 50 μL of mixed buffer was added to the 96-well round-bottom culture plate, and 50 μL of the culture supernatant from the experimental well plate was transferred to each well. The plates were incubated in the dark at room temperature for 30 minutes. Stop buffer was added at a rate of 50 μL / well, and the plates were read within 1 hour using a microplate reader (Molecular Devices, spectra MAX i3x) at a wavelength of 490 nm or 492 nm.

[0622] result( Fig. 5 ) indicated that the CD28 bispecific antibody to be tested can effectively activate T cells and kill tumor cells in the presence of the first signal and cell surface claudin 18.2.

[0623] 7.3 In vitro T cell activation experiment by CLDN18.2×CD28 bispecific antibody

[0624] To further verify the activity of the CLDN18.2×CD28 antibodies in the present invention, the inventors investigated whether these antibodies could effectively activate human peripheral T cells in vitro. The specific procedure was as follows:

[0625] 1. PBMC cells were removed one day prior, centrifuged at 25°C and 400 G for 4 minutes, resuspended in growth medium, transferred to a culture flask, and DNase I (SIGMA, D5025-375KU) was added at a final concentration of 2 μg / mL. The cells were cultured overnight in a 37°C, 5% CO₂ incubator.

[0626] 2. CLDN18.2×CD28 bispecific antibody diluted with PBS (1 μg / well) or CLDN18.2×CD28 bispecific antibody diluted with CD3 antibody (Acro, CDE-M120A-1MG, 0.1 μg / well) as needed was added to a 96-well flat-bottom plate, and 100 μL of the antibody to be tested was added per well. The plate was incubated overnight in a 4°C refrigerator to allow the antibody to bind sufficiently to the bottom of the plate.

[0627] 3. On the day of the experiment, after completing antibody incubation, the unbound antibody solution in the plate was discarded, and the plate was washed once by adding 100 μL of PBS to each well, and then the PBS was discarded.

[0628] 4. The PBMC cells thawed the previous day were removed, and the suspended cells were transferred to a centrifuge tube. After centrifugation at 25°C and 400 G for 4 minutes, the cell density was adjusted using assay buffer (RPMI 1640, 1% FBS). The cell concentration was adjusted to 100 μL per well (100,000 cells / well), and the cells were plated onto antibody-coated 96-well flat-bottom plates and incubated in a 37°C, 5% CO2 incubator.

[0629] 5. Four hours before the end of cell culture, the 96-well plate was removed from the incubator, and 50 μL of supernatant from each well was pipetted into a new 96-well flat-bottom plate for cytokine detection. The plate was frozen in a -40°C refrigerator. A protease transport inhibitor (Invitrogen, 00-4980-93) was added to the 96-well flat-bottom plate at a rate of 50 μL / well, and the plate was incubated for 4 hours in a 37°C, 5% CO2 incubator.

[0630] 6. After completing incubation, antibody staining for flow cytometry was prepared. First, a 96-well flat-bottom plate was centrifuged at 25°C and 400 G for 4 minutes, and the culture supernatant was discarded. The cells were resuspended in PBS, transferred to a new 96-well round-bottom plate, centrifuged at 25°C and 400 G for 4 minutes, and the PBS wash buffer was discarded.

[0631] 7. A pre-prepared surface blocking dye mixture was added at a rate of 50 microliters per well: Fc blocker (BD, 564219, 1:200 PBS) and survival / death dye (Invitrogen, L34968A 1:100 PBS). The plates were incubated in the dark at room temperature for 10 minutes. After incubation, 50 μL of the 1:100 cell surface dye mixture in FACS buffer—PD1 antibody (Biolegend, 329918), CD4 antibody (Biolegend, 329918), and CD8 antibody (Biolegend, 344724)—was added directly to each well, and the plates were incubated in the dark at 4°C for 30 minutes.

[0632] 8. After completing incubation with surface dye, the mixture was centrifuged at 25°C and 400 G for 4 minutes, and any dye not bound to the cells was discarded. The cells were resuspended in FACS wash buffer (2 mM EDTA, 0.5% BSA, PBS) and washed twice by centrifugation.

[0633] 9. Fix / Perm buffer (ebioscience 00-5523-00 Invitrogen, 00-8333-56) was added at a rate of 100 μL / well. To permeate the cell membrane, the plates were incubated in the dark at room temperature for 30 minutes.

[0634] 10. After completing incubation, the plate was centrifuged at 25°C and 400 G for 4 minutes, and the Fix / Perm buffer was discarded. The cells were resuspended in PBE (Invitrogen, 00-8333-56) wash buffer and washed twice by centrifugation.

[0635] 11. Intracellular dye mixture: A mixed solution of IFN-γ antibody (Biolegend, 506504) and TNF-α antibody (Biolegend, 502946) was added at a rate of 50 μL / well, and the plate was incubated in the dark at room temperature for 30 minutes.

[0636] 12. After completing incubation, the mixture was centrifuged at 25°C and 400 G for 4 minutes, and any dye not bound to the cells was discarded. The cells were resuspended in PBE wash buffer and washed twice, and the wash buffer was discarded.

[0637] 13. Cells were resuspended in FACS wash buffer at 150 μL / well and analyzed on a flow cytometer (BD FACS SYMPHONY A3).

[0638] The results indicated that, in the absence of a first signal provided by a CD3 antibody, the control antibody BsAb-BM-1 (monovalent TGN) can activate CD4 T cells to release TNFα independently of the first signal ( Fig. 6 ), it was further confirmed that TGN, as a single-arm CD28 antibody, also exhibits super-agonist properties (Fig. 6a). Under the same conditions, the three CD28 bispecific antibodies of the present invention did not exhibit CD28 super-agonist activity. In the presence of a first signal provided by the CD3 antibody, all CD28 bispecific antibodies effectively activated T (CD4 and CD8) cells ( Fig. 6b ), was able to release different levels of the cytokine TNFα.

[0639] Example 8: Construction and Preparation of PSMA×CD28 Bispecific Antibody

[0640] To further evaluate the function of CD28 humanized antibodies, a PSMA×CD28 bispecific antibody targeting human PSMA was constructed in this study ( Table 8 ). Bispecific antibody molecules are in the "1 + 1" format ( Fig. 3b ) and here the variable region targeting PSMA is 1C3(EP1851250B1, sequence information is Table 9 It was derived from (as shown in), and the anti-CD28 variable region sequence was derived from the variable region sequences of the humanized antibodies hz5B11D8-a, hz61FG3-a, and hz4F12B5-b of the present invention ( Table 9 ). The Fc fragment of the antibody was in the Innovadi (bispecific antibody technology of CN116888149A) IgG1 LALA format, and the schematic diagram of the antibody Fig. 3b As shown in [figure]. Accordingly, the heavy chain of the PSMA-targeting arm 1C3 portion of the bispecific antibody is described in SEQ ID NO. 48, and the light chain is described in SEQ ID NO. 50. In the bispecific antibody of the present invention, the heavy chain of the CD28-targeting arm is described in SEQ ID NOs. 51, 52, and 53, respectively, and the light chain is described in SEQ ID NOs. 36, 38, and 40, respectively.

[0641] The control PSMA×CD28 antibody (REGN5678; US20190389951A1) used in the present invention has the heavy chain of SEQ ID NO. 55 and the light chain of SEQ ID NO. 57 for the PSMA-targeting arm, and the heavy chain of SEQ ID NO. 59 and the light chain of SEQ ID NO. 57 for the CD28-targeting arm.

[0642] [Table 8] PSMA×CD28 bispecific antibody combinations

[0643]

[0644] [Table 9] Table of PSMA×CD28 bispecific antibodies

[0645]

[0646] In all PSMA×CD3 bispecific antibodies in this study, the sequence used for PSMA is derived from patent (WO2017023761A1), and the antibody variable region sequence of the CD3-targeting arm is derived from (CN202180066476A); the sequences for the molecule's light chain, heavy chain, and their respective variable regions are Table 10 They are listed in [documents]. The heavy and light chain sequences corresponding to the PSMA-targeting arm are described in SEQ ID NO. 61 and SEQ ID NO. 63, respectively, and the heavy and light chain sequences corresponding to the CD3-targeting arm are described in SEQ ID NO. 65 and SEQ ID NO. 68, respectively.

[0647] [Table 10] Sequence information of PSMA×CD3 bispecific antibodies

[0648]

[0649] Preparation of PSMA×CD28 bispecific antibodies

[0650] The bispecific antibodies BsAb-4, BsAb-5, BsAb-6, and PSMA×CD3 molecules of the present invention were prepared by separately expressing and purifying two parent proteins, and then recombining them through in vitro reduction and oxidation. The control bispecific antibody REGN5678 was prepared by intracellular assembly.

[0651] Plasmid construction: The sequence of the diabody of the present invention was synthesized using Genewiz. Light chain and heavy chain variable regions corresponding to the bispecific antibody ( Table 9-10 By cloning ) into a pcDNA3.1 vector (Invitrogen, V790-20) containing a corresponding heavy or light chain invariant region, heavy and light chain plasmids for PSMA-targeting sub-arms, heavy and light chain plasmids for CD28-targeting sub-arms, and heavy and light chain plasmids for CD3-targeting sub-arms were obtained, respectively.

[0652] The production routes of the four bispecific antibody molecules BsAb-4, BsAb-5, BsAb-6, and PSMA×CD3 can be found in Example 7. The method for the expression and purification of the control bispecific antibody REGN5678 was based on the protocol described in Andrew D. Tustian et al., (2016) (“Development of purification processes for fully human bispecific antibodies based upon upon modification of protein A binding affinity”, mAbs, 8:4, 828-838).

[0653] Table 8-10 The PSMA×CD28 and PSMA×CD3 bispecific antibody molecules listed in [this document] were prepared and obtained, and used in the study described in the subsequent examples.

[0654] Example 9: In vitro activity assay of PSMA×CD28 bispecific antibody

[0655] 9.1 Cell Surface Affinity Assessment of PSMA×CD28 Bispecific Antibodies

[0656] To evaluate the activity of CD28 bispecific antibodies, the inventors first detected the affinity of several antibodies to the cell surface in vitro.

[0657] First, tumor cells MC38_hPSMA expressing human PSMA were constructed, and the specific construction process was as follows:

[0658] The full-length human PSMA gene (UniProt ID: Q04609-1) was constructed into the vector pWPT-GFP (Addgene, 12255) and its GFP sequence was replaced. For viral packaging, the said vector was transfected into HEK293T (ATCC, CRL-3216) cells along with the lentivirus packaging vectors psPAX2 (Addgene, 12260) and pMD2.G (Addgene, 12259). Culture supernatants were collected after 48 and 72 hours, and the lentivirus was concentrated using a Lenti-X Concentrator (Takara, Cat# 631232, Lot# 2109261A). Colon cancer MC38 cells (Nanjing Cobioer Biosciences) were transfected with the concentrated virus, and then a flow cytometry classifier (BD FACSAria) TM Cells expressing PSMA were sorted using a Fusion Cell Sorter to obtain the tumor cell line MC38_hPSMA, which is stably transfected with PSMA.

[0659] Next, human CD28-expressing tumor cells 024-hCD28 CHO GS were constructed as follows:

[0660] The full-length human CD28 gene (UniProt ID: P10747) was constructed in the vector pXC17.4 (Lonza), and the resulting plasmid was digested with PvuI (NEB, R3150L) to produce a linear fragment. The linear plasmid was delivered to CHO-GS cells (Lonza) by electroporation using an electroporator (Bio Rad, GENEPULSER XCELL, 300 V, 900 μF, resistance infinite (∞), 2 mm). After transfection, the cells were transferred to CD CHO culture medium (Gibco, 10743-029) at 37°C, 1% SP4 (Lonza, BESP1076E) was added, and the cells were cultured at 37°C, 6% CO2, and a shaker at 120 rpm. 16–30 hours after electroporation, cells were centrifuged at 1000 rpm for 5 minutes, and the supernatant was discarded. Cells were resuspended in fresh medium supplemented with SP4, MSX (SIGMA, M5379-250 mg) was added to a final concentration of 50 μM, the cell density was adjusted to 5E5 cells / mL, and culture was continued. Starting from day 7, cell density and viability were measured every 1–2 days. When cell viability exceeded 90%, a flow cytometry classifier (BD FACSAria) was used. TM Cells expressing human CD28 were sorted using a Fusion Cell Sorter to obtain the tumor cell line 024-hCD28 CHO GS, which is stably transfected with CD28.

[0661] Affinity test

[0662] Cultured 024-hCD28 CHO GS and MC38_hPSMA cell lines were removed from T75 simple culture flasks and centrifuged at 25°C and 400 G for 4 minutes. Cell density was adjusted using wash buffer (2 mM EDTA, 0.5% BSA, PBS), and cell concentration was adjusted based on 50 μL per well (100,000 cells / well). Cells were thoroughly mixed and plated into 96-well round-bottom culture plates. The antibody to be tested was serially diluted according to the experimental design. Using FACS wash buffer, the antibody was serially diluted fourfold from an initial concentration of 100 nM to the 10th concentration gradient. 50 μL of the antibody to be tested was added to each well of the cell-coated plate. The plates were incubated in a 4°C refrigerator for 30–60 minutes to allow the antibody to bind well to the cell surface. After completing incubation, the plate was centrifuged at 25°C and 400 G for 4 minutes, and any unbound antibodies in the plate were discarded. 100 μL of FACS wash buffer was added to each well to resuspend the cells. The mixture was centrifuged at 25°C and 400 G for 4 minutes, and the wash solution was discarded. Diluted PE-hIgG-heavy chain (Biolegend, 410708) was added to the well plate at a rate of 100 μL / well to bind to the heavy chain of the test antibody. The plate was incubated at room temperature under dark conditions for 20–30 minutes. After completing incubation, the plate was centrifuged at 25°C and 400 G for 4 minutes, and any unbound antibodies in the plate were discarded. 100 μL of FACS wash buffer was added to each well to resuspend the cells. The mixture was centrifuged at 25°C and 400 G for 4 minutes, and the wash solution was discarded. Finally, the cells were resuspended in 100 μL of FACS wash buffer per well and prepared for analysis on a flow cytometer (BD FACS SYMPHONY A3).

[0663] The results showed that all bispecific antibodies exhibited very strong affinity for the PSMA target ( Fig. 7 ), the total affinity of BsAb-4, BsAb-5, and BsAb-6 for PSMA on the cell surface was shown to be similar to the affinity of REGN5678 ( Fig. 7a In terms of affinity for the CD28 target, BsAb-4, BsAb-5, and BsAb-6 all showed binding ( Fig. 7b ).

[0664] 9.2 Detection of In Vitro NFkB Reporter Activation by PSMA×CD28 Bispecific Antibody

[0665] Next, the inventors further verified whether CD28 retains potent agonist activity against T cells even when assembled into a bispecific antibody. First, the activation of NFkB Jurkat reporter cells by the PSMA×CD28 bispecific antibody was verified in vitro. In this study system, tumor cells expressing human PSMA were used to provide a “handle” that enables the PSMA-targeted arm of the bispecific antibody to effectively bind to the tumor cells. Additionally, it was necessary to verify the CD28-targeted arm in this study. For this reason, the CD28-expressing T cell line Jurkat was introduced into this study. The overall study process was as follows:

[0666] Treatment of target and effector cells

[0667] The cultured MC38_hPSMA cell line was removed from a T75 simple culture flask and centrifuged at 25°C and 400 G for 4 minutes. Cell density was adjusted using growth medium, with a cell concentration of 100 μL per well (40,000 cells / well). The cells were thoroughly mixed and plated onto 96-well white culture plates (WHB). The 96-well white culture plates (WHB) coated with target cells were removed, and the culture medium was discarded.

[0668] According to the experimental design, the PSMA×CD28 bispecific antibody was serially diluted. Using assay buffer (1% FBS, 1640), the antibody was serially diluted fourfold from an initial concentration of 100 nM to the 10th concentration gradient. The antibody to be tested was added at a rate of 50 μL / well to plates coated with target cells.

[0669] The cultured NFkB Jurkat cell line was removed from a T75 simple culture flask and centrifuged at 25°C and 400 G for 4 minutes. Cell density was adjusted using assay buffer, and cell concentration was adjusted based on 50 μL per well (50,000 cells / well). The mixture was thoroughly mixed, and either the CD3 antibody (Acro, CDE-M120A-1 mg, final concentration: 0.5 μg / mL) or the PSMA×CD3 bispecific antibody (final concentration: 10 pM) was added simultaneously. The mixture was thoroughly mixed, added to a 96-well white culture plate (WHB), and incubated at 37°C in a 5% CO2 incubator for 6 hours.

[0670] A 96-well white culture plate (WHB) was removed from the incubator, and 100 μL of Bio-Glo Luciferase Reagent (Promega, G7940) solution was added to each well. After incubation at room temperature for 2 minutes, detection was performed using a microplate reader (Molecular Devices, spectra MAX i3x).

[0671] In this study, two first signals were used to evaluate the effect of CD28 bispecific antibodies on the activation of CD28 signals.

[0672] First, a CD3 antibody was selected as the first signal. Fig. 8a All CD28 bispecific antibodies described were able to activate downstream NF-κB signaling of CD28 on Jurkat cells.

[0673] Second, to evaluate T cell activation induced by CD28 bispecific antibodies, a low concentration of PSMA×CD3 bispecific antibody was selected as the first signal. Fig. 8b As shown in [figure], all tested PSMA×CD28 bispecific antibodies were able to activate the NFkB reporter, and the trend is Fig. 8a It was similar to what was observed in.

[0674] 9.3. In vitro T cell killing assay mediated by PSMA×CD28 bispecific antibodies

[0675] Next, the inventors further tested whether the PSMA×CD28 bispecific antibody could activate human peripheral T cells to kill tumor cells. In this study, aAPC was incorporated into the surface of tumor cells to provide the first signal. Additionally, human PSMA was transfected onto the surface of the tumor cells as a specific recognition target for one of the antibody's targeting arms. The overall experimental procedure was as follows:

[0676] PSMA-aAPC-CHO K1 was constructed as follows:

[0677] The full length of the human PSMA gene (UniProt ID: Q04609-1) was constructed into the vector pWPT-GFP (Addgene, 12255) to replace the GFP sequence within it, and the virus was packaged by transfecting aAPC-CHO K1 (Promega, J1250) cells with the said vector together with the lentivirus packaging vectors psPAX2 (Addgene, 12260) and pMD2.G (Addgene, 12259). After 48 and 72 hours of culture, the culture supernatant was collected, and the lentivirus was concentrated using a Lenti-X Concentrator (Takara, Cat# 631232, Lot# 2109261A). After transfecting hamster ovary aAPC-CHO K1 cells with a concentrated virus, PSMA-expressing cells were selected using a flow cytometry classifier (MoFlo XDP, Beckman Coulter) to obtain the PSMA-aAPC-CHO K1 cell line stably transfected with PSMA.

[0678] PSMA×CD28 mediated the killing of tumor cells by T cells, and the specific process was as follows:

[0679] 1. Target cell seeding

[0680] Cultured PSMA-aAPC-CHO K1 was taken and digested with trypsin, and digestion was stopped by resuspending the cells in growth medium. The cells were centrifuged, and the cell density was adjusted using assay buffer. Cells were seeded into 96-well round-bottom plates at 100 μL / well (10,000 cells / well) and cultured for 16–20 hours, and assay buffer was added to the margin wells at 200 μL / well.

[0681] 2. Effector cell treatment

[0682] One cryopreserved PBMC vial was removed, thawed in a 37°C water bath, and centrifuged at 25°C and 400 G for 4 minutes. CD8+ T cells were isolated according to the instructions of EasySep Human CD8+ T Cell (STEMCELL, 19053). Cells were resuspended in RPMI 1640 medium and incubated overnight in a 37°C, 5% CO2 incubator. The next day, cells were collected and centrifuged at 25°C and 400 G for 4 minutes. Cell concentration was adjusted with assay buffer. CD8+ T cells were added to a 96-well round-bottom plate at a concentration of 50 μL per well (50,000 cells / well), with an effector cell:target cell ratio of 5:1.

[0683] 3. Dilution of antibodies

[0684] The antibody to be tested was serially diluted according to the experimental design. The antibody was serially diluted fourfold from an initial concentration of 50 nM to the 10th concentration gradient. The diluent was assay buffer. 50 μL of the serially diluted antibody was pipetted into 96-well round-bottom plates coated with CD8+ T cells and PSMA-aAPC-CHO K1 cells and incubated in a 5% CO2 incubator at 37°C for 48 hours, with a total volume of 200 μL per well.

[0685] 4. To lyse the target cells, 20 μL of 10× lysis buffer was added to the wells of the cell control. The plates were incubated in a 5% CO2 incubator at 37°C for 45 minutes. Detection reagents were prepared according to the instructions of the Radio Cytotoxicity Assay Kit (Promega, G1781). Cells in a 96-well round-bottom plate were centrifuged to pellet. 50 μL of mixed buffer was added to the 96-well round-bottom culture plate, and 50 μL of the culture supernatant from the experimental well plate was transferred to each well. The plates were incubated in the dark at room temperature for 30 minutes. Stop buffer was added at a rate of 50 μL / well, and the plates were read within 1 hour using a microplate reader (Molecular Devices, spectra MAX i3x) at a wavelength of 490 nm or 492 nm.

[0686] result( Fig. 9 It was indicated that all three PSMA×CD28 bispecific antibodies could activate T cells and produce a killing effect on PSMA-expressing tumor cells. Regarding the degree of target cell death, BsAb-4, which has a strong affinity for CD28, exhibited the strongest CD28 activating activity and was able to mediate T cells to kill tumor cells at low concentrations. BsAb-5 had the weakest killing activity because its CD28-targeting arm had the lowest affinity. The overall killing level mediated by the BsAb-6 bispecific antibody was also attenuated due to a decrease in affinity for CD28.

[0687] Example 10: T cell activation assay against PSMA×CD28 bispecific antibody

[0688] To further evaluate the activity of the PSMAxCD28 antibodies, the inventors tested whether these antibodies could effectively activate human peripheral T cells in vitro to release the cytokine IL2. The specific process is as follows:

[0689] 1. PBMC cells were taken one day prior, centrifuged at 25°C and 400 G for 4 minutes, resuspended in growth medium, and placed in a culture flask. DNase I (SIGMA, D5025-375KU) was added to a final concentration of 2 μg / mL. The cells were incubated overnight in a 37°C, 5% CO2 incubator.

[0690] 2. The PSMA×CD28 antibody was diluted with PBS (1 μg / well) (or the CD3 antibody (Acro, CDE-M120A-1MG, 0.1 μg / well) was added together as needed), added to a 96-well flat-bottom plate, and the antibody to be tested was added at a rate of 100 μL / well. The plate was incubated overnight in a refrigerator at 4°C to allow the antibody to bind sufficiently to the bottom of the plate.

[0691] 3. On the day of the experiment, after completing antibody incubation, the unbound antibody solution in the plate was discarded. Each well was washed once with 100 μL of PBS, and then the PBS was discarded.

[0692] 4. The PBMC cells thawed the previous day were removed, and the suspended cells were transferred to a centrifuge tube. After centrifugation at 25°C and 400 G for 4 minutes, the cell density was adjusted using assay buffer (RPMI 1640, 1% FBS). The cell concentration was adjusted to 100 μL / well (100,000 cells / well), and the cells were plated onto antibody-coated 96-well flat-bottom plates and incubated in a 5% CO2 incubator at 37°C.

[0693] 5. Four hours before the end of cell culture, the 96-well plate was removed from the incubator, and 50 μL of supernatant from each well was pipetted into a new 96-well round-bottom plate for cytokine detection.

[0694] 6. According to the instructions of the IL2 Human ELISA KIT (Mabtech, 3445-1H-20), Human IL-2 antibody was diluted with PBS and added to a 96-well flat-bottom plate at a rate of 50 μL / well. The plate was covered with a sealing film and incubated overnight in a refrigerator at 4°C.

[0695] 7. Unbound purified antibodies were removed from the 96-well flat-bottom plate, and PBST buffer (0.05% Tween 20) was added at a rate of 100 μL / well and washed 6 times. Then, assay buffer (1000 mL of PBS, 500 μL of Tween 20, and 5 g of BSA) was added at a rate of 100 μL / well, and the mixture was incubated at room temperature for 1 hour.

[0696] 8. After incubation, a 96-well flat-bottom plate was taken, the liquid in the plate was discarded, 50 μL of the prepared sample supernatant and standard substance was added, and the plate was incubated at room temperature for 2 hours.

[0697] 9. A 96-well flat-bottom plate was taken, the liquid in the plate was discarded, and the plate was washed 6 times. 50 μL of diluted biotin antibody (Mabtech, 3445-1H-20) was added to each well, and the plate was incubated on a plate shaker at room temperature for 1 hour.

[0698] 10. A 96-well flat-bottom plate was taken, the liquid in the plate was discarded, and the plate was washed 6 times. 50 μL of diluted HRP was added to each well, and the plate was incubated at room temperature for 1 hour.

[0699] 11. A 96-well flat-bottom plate was taken, the liquid in the plate was discarded, and the plate was washed 6 times. 50 μL of TMB was added to each well, and after the colorimetric reaction was completed, 50 μL of stop solution was added to each well. The values ​​were read at a wavelength of 450 nm within 1 hour using a microplate reader (Molecular Devices, spectra MAX i3x).

[0700] The results were in the absence of the first signal CD3 antibody (-signal 1) ( Fig. 10a ), the control antibody TGN1412 (abbreviated as TGN in this study) demonstrated that it could activate T cells to release IL2 independently of the first signal, confirming that TGN is a CD28 super-agonist antibody. Under the same conditions, the three CD28 bispecific antibodies of the present invention, BsAb-4, BsAb-5, and BsAb-6, were unable to activate T cells, indicating that they are conventional CD28 agonist antibodies. In the presence of the first signal (+ signal 1) provided by the CD3 antibody, all CD28 antibodies were able to effectively activate T cells to release IL2 ( Fig. 10b ).

[0701] Example 11: In vivo antitumor activity assay of PSMA×CD28 bispecific antibody

[0702] To further evaluate the activity of the PSMA×CD28 bispecific antibody, the inventors tested the in vivo anti-tumor activity of the PSMA×CD28 bispecific antibody in a mouse tumor model.

[0703] Mice: 6-8 week old hCD28 humanized mice (Shanghai Model Organisms Center, Inc.) were placed in an SPF environment for 3-5 days prior to the experiment to better adapt to the current rearing environment.

[0704] In all experiments, the major and minor axes of the tumor were measured twice a week using a vernier caliper, and the tumor volume (mm³) was calculated from the measurement data. The formula was as follows:

[0705] Tumor volume (mm³) = 0.5 × major axis (mm) × minor axis (mm) × minor axis (mm)

[0706] hPSMA-MC38 cells expressing human PSMA were resuspended in 1× PBS (Gibco, 10010049) at an appropriate concentration, and tumor cells were subcutaneously implanted into the right abdomen of mice at an inoculation dose of 1E6 / mouse ( Fig. 11a Mice were grouped so that the average initial dose volume of each group was essentially the same when the tumor volume reached 80-100 mm3 on approximately 7 days after inoculation. After grouping, 10 mg / kg of PSMAxCD28 bispecific antibody was injected intraperitoneally into the mice, and the effect of the drug on tumor growth was observed.

[0707] The results indicated that in an MC38 tumor model overexpressing human PSMA, a single dose of 10 mg / kg of PSMAxCD28 could inhibit tumor cell growth to some extent ( Fig. 11b REGN5678 had a relatively weak tumor suppressive effect, and the TGI was only 42.5%. BsAb-4 and BsAb-6( Fig. 11c, each line represents a mouse ) was able to produce a stronger tumor suppression effect than REGN5678 under the same conditions.

[0708] Example 12: In vivo antitumor activity of PSMA×CD28 bispecific antibody in combination with PD1 antibody

[0709] To further evaluate the activity of the PSMA×CD28 bispecific antibody, the in vivo antitumor activity of the CD28 bispecific antibody combined with the PD1 antibody was detected in a mouse tumor model.

[0710] The anti-mouse PD1 antibody was derived from patent (WO2018184964A1), and its sequence is shown below. The manufacturing process was the same as in Example 1.

[0711] The entire procedure was performed as in Example 12. hPSMA-MC38 cells expressing human PSMA were resuspended in 1× PBS (Gibco, 10010049) at an appropriate concentration, and tumor cells were subcutaneously implanted into the right abdomen of mice at an inoculation dose of 1E6 / mouse ( Fig. 12a Mice were grouped so that the average initial dose volume of each group was essentially the same when the tumor volume reached 80-100 mm³ on approximately 7 days after inoculation. After group assignment, BsAb-6 bispecific antibody, anti-PD1 antibody, or a combination thereof was administered to mice by intraperitoneal injection, and the effect of the drug on tumor growth was observed.

[0712] The results indicated that in an MC38 tumor model overexpressing human PSMA, the combined administration of 10 mg / kg of BsAb-6 and 2 mg / kg of murine PD1 antibody produced a strong synergistic effect, significantly inhibiting tumor cell growth ( Fig. 12b ). During an observation period lasting more than 40 days after a single administration, the rate of complete tumor regression reached 100%, which was higher than the rate of complete tumor regression in the monotherapy group (60%). Fig. 12c, each line represents a mouse ).

[0713] Example 13: Mouse PK experiment with PSMA×CD28 bispecific antibody

[0714] In this study, to investigate the pharmacokinetic characteristics in mice, 10 mg / kg of REGN5678 and BsAb-6 were administered to female C57BL / 6 hCD28KI mice (Shanghai Model Organisms Center, Inc.) via tail vein injection, and a total of 12 mice were used, with 6 mice per group.

[0715] Blood was collected from the eyes of mice at 0.086, 0.5, 2, 6, 24, 48 hours, 4, 7, 14, and 21 days after administration, centrifuged at 3000 rpm for 10 minutes at 4°C, and serum was collected. The antibody content in the serum was determined by ELISA, and the half-lives of REGN5678 and BsAb-6 in mice were calculated.

[0716] The experimental results are Table 11 As presented in [the document], BsAb-6 exhibited excellent stability, a long half-life (t1 / 2), and relatively excellent blood exposure in hCD28 transgenic mice. From the half-life, it was confirmed that BsAb-6 achieved stability data similar to normal monoclonal antibodies in both detection methods.

[0717] [Table 11] In vivo PK assay of PSMA×CD28 bispecific antibody in mice

[0718]

[0719] Sequence list

[0720]

[0721]

[0722]

[0723]

[0724]

[0725]

[0726]

[0727]

[0728]

[0729]

[0730]

[0731]

Claims

Claim 1 (i) CDRH1, CDRH2, and CDRH3 are selected from three complementary-determining regions CDRH1, CDRH2, and CDRH3 included in the VH described in SEQ ID NO. 24, 23, or 75; CDRL1, CDRL2, and CDRL3 are three complementary-determining regions CDRL1, CDRL2, and CDRL3 included in the VL described in SEQ ID NO. 28 or 78; or (ii) CDRH1, CDRH2, and CDRH3 are selected from three complementary-determining regions CDRH1, CDRH2, and CDRH3 included in the VH described in any one of SEQ ID NO. 4, 5, and 72; CDRL1, CDRL2, and CDRL3 are three complementary-determining regions CDRL1, CDRL2, and CDRL3 included in the VL described in SEQ ID NO. 9 or 73; (iii) an anti-CD28 antibody or its antigen-binding fragment comprising three CDRs of a heavy chain variable region VH: CDRH1, CDRH2, and CDRH3, and three CDRs of a light chain variable region VL: CDRL1, CDRL2, and CDRL3, wherein CDRH1, CDRL2, and CDRL3 are selected from three complementary-determining regions CDRL1, CDRL2, and CDRL3 included in VH as described in SEQ ID NO. 17, 19, or 70. Claim 2 (i) CDRH1 comprises or is composed of the amino acid sequence of SEQ ID NO. 20; CDRH2 comprises or is composed of the amino acid sequence of SEQ ID NO. 21; CDRH3 comprises or is composed of the amino acid sequence of SEQ ID NO. 22; CDRL1 comprises or is composed of the amino acid sequence of SEQ ID NO. 25; CDRL2 comprises or is composed of the amino acid sequence of SEQ ID NO. 26; CDRL3 comprises or is composed of the amino acid sequence of SEQ ID NO. 27; or (ii) CDRH1 comprises or is composed of the amino acid sequence of SEQ ID NO. 20; CDRH2 comprises or is composed of the amino acid sequence of SEQ ID NO. 76; CDRH3 comprises or is composed of the amino acid sequence of SEQ ID NO. 77; CDRL1 comprises or is composed of the amino acid sequence of SEQ ID NO. 25; CDRL2 comprises or is composed of the amino acid sequence of SEQ ID NO. 26; CDRL3 comprises or is composed of the amino acid sequence of SEQ ID NO. 27; (iii) CDRH1 comprises or is composed of the amino acid sequence of SEQ ID NO. 1; CDRH2 comprises or is composed of the amino acid sequence of SEQ ID NO. 2; CDRH3 comprises or is composed of the amino acid sequence of SEQ ID NO. 3; CDRL1 comprises or is composed of the amino acid sequence of SEQ ID NO. 6 or 74; CDRL2 comprises or is composed of the amino acid sequence of SEQ ID NO. 7; CDRL3 comprises or is composed of the amino acid sequence of SEQ ID NO. 8; (iv) CDRH1 comprises or is composed of the amino acid sequence of SEQ ID NO. 10; CDRH2 comprises or is composed of the amino acid sequence of SEQ ID NO. 11; CDRH3 comprises or is composed of the amino acid sequence of SEQ ID NO. 12; CDRL1 comprises or is composed of the amino acid sequence of SEQ ID NO. 14, 18 or 71; CDRL2 comprises or is composed of the amino acid sequence of SEQ ID NO. 15;CDRL3 is an anti-CD28 antibody or its antigen-binding fragment comprising CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3, which comprises or consists of the amino acid sequence of SEQ ID NO.

16. Claim 3 The invention of claim 1 or 2, comprising a heavy chain variable region VH, wherein the heavy chain variable region comprises (i) an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO. 24, 4, 5, 13, 23, 69, 72, or 75, or (ii) an antibody or its antigen-binding fragment comprising or consisting of the amino acid sequence of SEQ ID NO. 24, 4, 5, 13, 23, 69, 72, or 75. Claim 4 In any one of claims 1 to 3, the light chain variable region VL comprises (i) an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO. 28, 9, 17, 19, 70, 73, or 78, or (ii) an antibody or its antigen-binding fragment comprising or consisting of the amino acid sequence of SEQ ID NO. 28, 9, 17, 19, 70, 73, or 78. Claim 5 In claim 1 or 2, comprising a heavy chain variable region VH and a light chain variable region VL, wherein (i) VH comprises or is composed of an amino acid sequence described in SEQ ID NO. 24 or 23 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity therewith, and VL comprises or is composed of an amino acid sequence described in SEQ ID NO. 28 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity therewith; or (ii) VH comprises an amino acid sequence described in SEQ ID NO. 75 or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, (iii) VH comprises or is composed of an amino acid sequence having 98% or 99% identity therewith, and VL comprises or is composed of the amino acid sequence described in SEQ ID NO. 78 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity therewith; (iii) VH comprises or is composed of the amino acid sequence described in SEQ ID NO. 23, 24, or 75 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity therewith, and VL comprises the amino acid sequence described in SEQ ID NO. 28 or 78 or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, Includes or consists of an amino acid sequence having 98% or 99% identity;(iv) VH comprises or is composed of the amino acid sequence described in SEQ ID NO. 4, 5, or 72 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity therewith, and VL comprises or is composed of the amino acid sequence described in SEQ ID NO. 9 or 73 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity therewith; (v) VH comprises or is composed of the amino acid sequence described in SEQ ID NO. 4 or 5 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity therewith. (vi) VH comprises or is composed of the amino acid sequence described in SEQ ID NO. 9 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity therewith; (vi) VH comprises or is composed of the amino acid sequence described in SEQ ID NO. 72 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity therewith; and VL comprises or is composed of the amino acid sequence described in SEQ ID NO. 73 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity therewith;(vii) VH comprises or is composed of the amino acid sequence described in SEQ ID NO. 13 or 69 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity therewith, and VL comprises or is composed of the amino acid sequence described in SEQ ID NO. 17, 19, or 70 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity therewith; (viii) VH comprises the amino acid sequence described in SEQ ID NO. 13 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity therewith VL comprises or is composed thereof, wherein VL comprises or is composed thereof of an amino acid sequence described in SEQ ID NO. 17 or 19 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with respect thereto; (ix) VH comprises or is composed of the amino acid sequence described in SEQ ID NO. 69 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity therewith, and VL comprises or is composed of the amino acid sequence described in SEQ ID NO. 70 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity therewith, an antibody or an antigen-binding fragment thereof.; Claim 6 In claim 1 or 2, comprising a heavy chain variable region VH and a light chain variable region VL, wherein (i) VH comprises or is composed of the amino acid sequence described in SEQ ID NO. 24, and VL comprises or is composed of the amino acid sequence described in SEQ ID NO. 28; (ii) VH comprises or is composed of the amino acid sequence described in SEQ ID NO. 23, and VL comprises or is composed of the amino acid sequence described in SEQ ID NO. 28; (iii) VH comprises or is composed of the amino acid sequence described in SEQ ID NO. 72, and VL comprises or is composed of the amino acid sequence described in SEQ ID NO. 73; (iv) VH comprises or is composed of the amino acid sequence described in SEQ ID NO. 4, and VL comprises or is composed of the amino acid sequence described in SEQ ID NO. 9; (v) VH comprises or is composed of the amino acid sequence described in SEQ ID NO. 5, and VL comprises or is composed of the amino acid sequence described in SEQ ID NO. 9; and (vi) VH comprises or is composed of the amino acid sequence described in SEQ ID NO. 13 An antibody or its antigen-binding fragment composed of: (vii) VH comprising or composed of the amino acid sequence described in SEQ ID NO. 17, and VL comprising or composed of the amino acid sequence described in SEQ ID NO. 13; (viii) VH comprising or composed of the amino acid sequence described in SEQ ID NO. 69, and VL comprising or composed of the amino acid sequence described in SEQ ID NO. 70; (ix) VH comprising or composed of the amino acid sequence described in SEQ ID NO. 75, and VL comprising or composed of the amino acid sequence described in SEQ ID NO.

78. Claim 7 (i) VH comprises or is composed of the amino acid sequence described in SEQ ID NO. 24, and VL comprises or is composed of the amino acid sequence described in SEQ ID NO. 28; (ii) VH comprises or is composed of the amino acid sequence described in SEQ ID NO. 23, and VL comprises or is composed of the amino acid sequence described in SEQ ID NO. 28; (iii) VH comprises or is composed of the amino acid sequence described in SEQ ID NO. 72, and VL comprises or is composed of the amino acid sequence described in SEQ ID NO. 73; (iv) VH comprises or is composed of the amino acid sequence described in SEQ ID NO. 4, and VL comprises or is composed of the amino acid sequence described in SEQ ID NO. 9; (v) VH comprises or is composed of the amino acid sequence described in SEQ ID NO. 5, and VL comprises or is composed of the amino acid sequence described in SEQ ID NO. 9; (vi) VH comprises or is composed of the amino acid sequence described in SEQ ID NO. 13, and VL comprises or is composed of the amino acid sequence described in SEQ ID NO. 17; (vii) An anti-CD28 antibody or an antigen-binding fragment thereof comprising a heavy chain variable region VH and a light chain variable region VL, wherein VH comprises or is composed of the amino acid sequence described in SEQ ID NO. 13 and VL comprises or is composed of the amino acid sequence described in SEQ ID NO. 19; (viii) VH comprises or is composed of the amino acid sequence described in SEQ ID NO. 69 and VL comprises or is composed of the amino acid sequence described in SEQ ID NO. 70; and (ix) VH comprises or is composed of the amino acid sequence described in SEQ ID NO. 75 and VL comprises or is composed of the amino acid sequence described in SEQ ID NO.

78. Claim 8 In any one of claims 1 to 7, the heavy chain constant region HC is further included, for example, the heavy chain constant region HC of the antibody is a heavy chain constant region of IgG1, IgG2, IgG3 or IgG4, preferably a heavy chain constant region of human IgG1, and preferably the heavy chain constant region comprises (i) an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with an amino acid sequence selected from SEQ ID NO. 29 or 79, or is composed of; (ii) an antibody or its antigen-binding fragment comprising or composed of an amino acid sequence selected from SEQ ID NO. 29 or 79. Claim 9 In claim 7 or 8, the heavy chain constant region is an antibody or its antigen-binding fragment comprising a mutation that reduces binding to the Fcγ receptor, e.g., the LALA mutation and / or P329delP. Claim 10 An antibody or an antigen-binding fragment thereof comprising, in any one of claims 1 to 9, a light chain constant region, wherein, for example, the light chain constant region is a Lambda or Kappa light chain constant region. Claim 11 In claim 10, the light chain constant region comprises (i) an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO. 30, or (ii) an antibody or its antigen-binding fragment comprising or consisting of the amino acid sequence of SEQ ID NO.

30. Claim 12 In any one of paragraphs 1 to 11, the antibody is an antibody that is a humanized antibody or a chimeric antibody, or an antigen-binding fragment thereof. Claim 13 In any one of paragraphs 1 to 12, the antibody is an antibody that is a monoclonal antibody or an antigen-binding fragment thereof. Claim 14 In any one of claims 1 to 13, the antigen-binding fragment is an antibody or its antigen-binding fragment that is an antibody fragment selected from the group consisting of Fab, Fab', Fab'-SH, Fv, a single-chain antibody (e.g., scFv), and (Fab')2, a single-domain antibody (e.g., VHH), a domain antibody (dAb), or a linear antibody. Claim 15 In any one of claims 1 to 11, the antibody is a bispecific antibody or a multispecific antibody comprising a first binding specificity for CD28 and additional binding specificity for one or more tumor-associated antigens, or an antigen-binding fragment thereof. Claim 16 In paragraph 15, the tumor-associated antigen is an antibody selected from the group consisting of CLDN18.2 and PSMA or an antigen-binding fragment thereof. Claim 17 A bispecific antibody comprising a first antigen-binding region that specifically binds to CD28 and includes VH and VL, wherein VH includes CDRH1, CDRH2, and CDRH3 as defined in claim 1 or 2, and VL includes CDRL1, CDRL2, and CDRL3 as defined in claim 1 or 2; or VH and VL are VH and VL as defined in any one of claims 3 to 7; and a second antigen-binding region that specifically binds to a tumor-associated antigen TAA or an immune checkpoint molecule. Claim 18 In claim 17, the first antigen-binding region is a bispecific antibody that is the Fab of the anti-CD28 antibody defined in any one of claims 1 to 14. Claim 19 In claim 17 or 18, the second antigen-binding region is a bispecific antibody that is a Fab that specifically binds to a tumor-associated antigen or immune checkpoint molecule. Claim 20 A bispecific antibody according to claim 18 or 19, wherein Fab, which is the first antigen-binding region or the second antigen-binding region, comprises CH1, and CH1 is CH1 from IgG1, IgG2, IgG3 or IgG4, preferably CH1 from IgG1. Claim 21 In claim 20, CH1 comprises or is composed of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with an amino acid sequence selected from SEQ ID NO. 74; and (ii) a bispecific antibody comprising or composed of the amino acid sequence of SEQ ID NO.

74. Claim 22 A bispecific antibody according to any one of claims 18 to 21, wherein Fab functioning as a first antigen-binding region or a second antigen-binding region comprises a light chain constant region, and the light chain constant region is a kappa light chain constant region or a lambda light chain constant region. Claim 23 In claim 22, the kappa light chain constant region comprises (i) an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO. 30, or is composed thereof; and (ii) a bispecific antibody comprising or composed thereof of the amino acid sequence of SEQ ID NO.

30. Claim 24 In any one of claims 17 to 23, the bispecific antibody is an IgG-like bispecific antibody comprising an Fc dimer, wherein the two Fc regions constituting the Fc dimer are identical or different; optionally, the Fc region is a human IgG1, IgG2, IgG3 or IgG4 Fc region; and optionally, the Fc region comprises or is composed of (i) an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with an amino acid sequence selected from SEQ ID NO. 94 or 95; or (ii) a bispecific antibody comprising or being composed of an amino acid sequence of SEQ ID NO. 94 or 95. Claim 25 A bispecific antibody according to claim 24, wherein the two Fc regions are different, and mutations are introduced into the first Fc region and the second Fc region to promote heterodimerization between the first Fc region and the second Fc region based on Innobody technology. Claim 26 In paragraph 25, an antibody or its antigen-binding fragment wherein one Fc region's CH3 contains S364R and D399K mutations, and the remaining Fc region's CH3 contains Y349T, K370S and K409D mutations. Claim 27 A bispecific antibody comprising, in any one of claims 24 to 26, a mutation that reduces binding to the Fcγ receptor, e.g., L234A / L235A mutation, and optionally P329delP, wherein the first and / or second Fc region is a mutation that reduces binding to the Fcγ receptor. Claim 28 In claim 26 or 27, a) one Fc region comprises or is composed of the amino acid sequence described in SEQ ID NO. 80, and the remaining Fc region comprises or is composed of the amino acid sequence described in SEQ ID NO. 81; b) one Fc region comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID NO. 80 and comprises the mutations S364R and D399K, and the remaining Fc region comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID NO. 81 and comprises the mutations Y349T, K370S, and K409D; or c) one Fc region comprises at least 85%, 90%, 95%, and 95% with the amino acid sequence described in SEQ ID NO. 80, d) comprising an amino acid sequence having 96%, 97%, 98%, or 99% identity, comprising mutants S364R and D399K and mutants L234A / L235A and P329delP, wherein the remaining Fc region comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID NO. 81 and comprising mutants Y349T, K370S, and K409D and mutants L234A / L235A and P329delP; or d) one Fc region comprises or is composed of the amino acid sequence described in SEQ ID NO. 82, and the remaining Fc region comprises or is composed of the amino acid sequence described in SEQ ID NO. 83;e) One Fc region comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID NO. 82 and comprises mutations S364R and D399K, and the remaining Fc region polypeptide comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID NO. 83 and comprises mutations Y349T, K370S, and K409D; f) A bispecific antibody in which one Fc region comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID NO. 82 and comprises mutants S364R, D399K, and L234A / L235A, and the remaining Fc region polypeptide comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID NO. 83 and comprises mutants Y349T, K370S, K409D, and L234A / L235A.; Claim 29 A bispecific antibody according to any one of claims 17 to 28, wherein the TAA is CLDN18.2, and preferably, the second antigen-binding region comprises three complementary-determining regions (CDRHs) from the heavy chain variable region: CDRH1, CDRH2, and CDRH3; and three complementary-determining regions (CDRLs) from the light chain variable region: CDRL1, CDRL2, and CDRL3, wherein CDRH1 comprises or consists of the amino acid sequence of SEQ ID NO. 87; CDRH2 comprises or consists of the amino acid sequence of SEQ ID NO. 88; CDRH3 comprises or consists of the amino acid sequence of SEQ ID NO. 89; CDRL1 comprises or consists of the amino acid sequence of SEQ ID NO. 91; CDRL2 comprises or consists of the amino acid sequence of SEQ ID NO. 92; and CDRL3 comprises or consists of the amino acid sequence of SEQ ID NO.

93. Claim 30 A bispecific antibody according to claim 29, wherein the second antigen-binding region comprises VH and VL, VH comprises or is composed of the sequence described in SEQ ID NO. 86; and VL comprises or is composed of the sequence described in SEQ ID NO.

90. Claim 31 A bispecific antibody according to any one of claims 17 to 28, wherein the TAA is PSMA; preferably, the second antigen-binding region comprises three complementary-determining regions (CDRHs) from the heavy chain variable region: CDRH1, CDRH2, and CDRH3; and three complementary-determining regions (CDRLs) from the light chain variable region: CDRL1, CDRL2, and CDRL3, wherein CDRH1 comprises or consists of the amino acid sequence of SEQ ID NO. 96; CDRH2 comprises or consists of the amino acid sequence of SEQ ID NO. 97; CDRH3 comprises or consists of the amino acid sequence of SEQ ID NO. 98; CDRL1 comprises or consists of the amino acid sequence of SEQ ID NO. 99; CDRL2 comprises or consists of the amino acid sequence of SEQ ID NO. 100; and CDRL3 comprises or consists of the amino acid sequence of SEQ ID NO.

101. Claim 32 In claim 31, the second antigen-binding region comprises VH and VL, wherein VH comprises or is composed of the sequence described in SEQ ID NO. 47; and VL comprises or is composed of the sequence described in SEQ ID NO. 49, a bispecific antibody. Claim 33 A bispecific antibody according to any one of claims 17 to 32, wherein the bispecific antibody comprises a first antigen-binding region that specifically binds to CD28 and a second antigen-binding region that specifically binds to TAA, wherein the first antigen-binding region comprises a first Fab or is a first Fab, and the second antigen-binding region comprises a second Fab or is a second Fab, wherein the first Fab is connected (directly or through a linker such as a hinge region) from the C-terminus of its CH1 to the N-terminus of the first Fc region, and the second Fab is connected (directly or through a linker such as a hinge region) from the C-terminus of its CH1 to the N-terminus of the second Fc region. Claim 34 In paragraph 33, the bispecific antibody is a bispecific antibody that is an IgG-like antibody having the composition shown in FIG.

3. Claim 35 In claim 33 or 34, the bispecific antibody comprises a first Fab as a first antigen-binding region specifically binding to CD28 and a second Fab as a second antigen-binding region specifically binding to TAA, wherein the bispecific antibody comprises: heavy chain 1: a first Fab heavy chain variable region from N-terminus to C-terminus, a heavy chain constant region CH1, or a first Fc region, wherein the heavy chain constant region CH1 is connected directly to the N-terminus of the first Fc region from its C-terminus or via a linker (e.g., a hinge region); light chain 1: a first Fab light chain variable region from N-terminus to C-terminus, or is composed thereof; heavy chain 2: a second Fab heavy chain variable region from N-terminus to C-terminus, a heavy chain constant region CH1, or a second Fc region, wherein the heavy chain constant region CH1 is connected to the second Fc region from its C-terminus. Connected directly to the N-terminus or via a linker (e.g., a hinge region); light chain 2: comprising or composed of a second Fab light chain variable region-light chain constant region from the N-terminus to the C-terminus; preferably, each domain is a directly connected bispecific antibody. Claim 36 In claim 35, TAA is CLDN18.2, wherein heavy chain 1 comprises or is composed of the amino acid sequence described in SEQ ID NO. 35 or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity therewith; light chain 1 comprises or is composed of the amino acid sequence described in SEQ ID NO. 36 or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity therewith; heavy chain 2 comprises or is composed of the amino acid sequence described in SEQ ID NO. 33 or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity therewith; Light chain 2 comprises or is composed of the amino acid sequence described in SEQ ID NO. 34 or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity therewith; heavy chain 1 comprises or is composed of the amino acid sequence described in SEQ ID NO. 37 or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity therewith; light chain 1 comprises or is composed of the amino acid sequence described in SEQ ID NO. 38 or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity therewith; Heavy chain 2 comprises or is composed of the amino acid sequence described in SEQ ID NO. 33 or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity therewith; light chain 2 comprises or is composed of the amino acid sequence described in SEQ ID NO. 34 or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity therewith; heavy chain 1 comprises or is composed of the amino acid sequence described in SEQ ID NO. 39 or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity therewith;A bispecific antibody wherein light chain 1 comprises or is composed of the amino acid sequence described in SEQ ID NO. 40 or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity therewith; heavy chain 2 comprises or is composed of the amino acid sequence described in SEQ ID NO. 33 or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity therewith; and light chain 2 comprises or is composed of the amino acid sequence described in SEQ ID NO. 34 or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity therewith. Claim 37 In claim 35, TAA is PSMA, wherein heavy chain 1 comprises or is composed of the amino acid sequence described in SEQ ID NO. 51 or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity therewith; light chain 1 comprises or is composed of the amino acid sequence described in SEQ ID NO. 36 or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity therewith; heavy chain 2 comprises or is composed of the amino acid sequence described in SEQ ID NO. 48 or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity therewith; Light chain 2 comprises or is composed of the amino acid sequence described in SEQ ID NO. 50 or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity therewith; heavy chain 1 comprises or is composed of the amino acid sequence described in SEQ ID NO. 52 or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity therewith; light chain 1 comprises or is composed of the amino acid sequence described in SEQ ID NO. 38 or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity therewith; Heavy chain 2 comprises or is composed of the amino acid sequence described in SEQ ID NO. 48 or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity therewith; light chain 2 comprises or is composed of the amino acid sequence described in SEQ ID NO. 50 or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity therewith; heavy chain 1 comprises or is composed of the amino acid sequence described in SEQ ID NO. 53 or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity therewith;A bispecific antibody wherein light chain 1 comprises or is composed of the amino acid sequence described in SEQ ID NO. 40 or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity therewith; heavy chain 2 comprises or is composed of the amino acid sequence described in SEQ ID NO. 48 or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity therewith; and light chain 2 comprises or is composed of the amino acid sequence described in SEQ ID NO. 50 or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity therewith. Claim 38 A nucleic acid molecule comprising a nucleic acid sequence that encodes any one of the chains of an antibody or its antigen-binding fragment according to any one of claims 1 to 16, or any one of the chains of a bispecific antibody according to any one of claims 17 to 37. Claim 39 Preferably, the expression vector is an expression vector comprising a nucleic acid molecule according to claim 38, such as pCDNA, e.g., pCDNA3.

1. Claim 40 Preferably, the host cell is a prokaryotic or eukaryotic cell, e.g., 293 cell or CHO cell, e.g., Expi293 cell, a host cell comprising a nucleic acid molecule according to claim 38 or an expression vector according to claim 39. Claim 41 A method for producing an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 16, or a bispecific antibody according to any one of claims 17 to 37, comprising the steps of: culturing a host cell containing a nucleic acid molecule according to claim 38 or an expression vector according to claim 39 under conditions suitable for expressing a chain of antibodies; and optionally recovering the antibody from the host cell (or host cell medium). Claim 42 An immune conjugate comprising an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 16, or a bispecific antibody according to any one of claims 17 to 37. Claim 43 A pharmaceutical composition, medicament, or formulation comprising an antibody or antigen-binding fragment thereof according to any one of claims 1 to 16, or a bispecific antibody according to any one of claims 17 to 37, or an immunoconjugate according to claim 42, and optionally a pharmaceutical adjuvant. Claim 44 Preferably, the therapeutic agent is a therapeutic agent for treating various tumors, such as a chemotherapy agent, an angiogenesis inhibitor, a cytokine, a cytotoxic agent, another antibody, a small molecule drug, or an immunomodulator (e.g., an immune checkpoint inhibitor or agonist); preferably, the additional antibody is a PD-1 antibody, an antibody according to any one of claims 1 to 16 or an antigen-binding fragment thereof, or a bispecific antibody according to any one of claims 17 to 37, or an immunoconjugate according to claim 42, and a pharmaceutical combination product comprising one or more additional therapeutic agents. Claim 45 A method comprising the step of administering to a subject an effective amount of an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 16, or a bispecific antibody according to any one of claims 17 to 37, or an immunoconjugate according to claim 42, or a pharmaceutical composition or formulation according to claim 43, or a pharmaceutical combination product according to claim 44, in order to prevent or treat a tumor in a subject. Claim 46 In paragraph 45, the tumor is a solid tumor or a hematological tumor, e.g., a TAA-positive tumor or cancer; optionally, the tumor is a CLDN18.2-positive gastrointestinal tumor, e.g., gastric cancer, breast cancer, colon cancer, rectal cancer, or colorectal cancer; or the tumor is a PSMA-positive tumor, e.g., renal cell carcinoma, such as breast cancer, lung cancer, rectal cancer, or colorectal cancer. Claim 47 The method of claim 45 or 46 further comprises the step of administering in combination with an additional therapy and / or an additional therapeutic agent, such as a therapeutic modality (e.g., surgical therapy or radiotherapy), preferably, the therapeutic agent is any therapeutic agent for treating a tumor, such as a chemotherapy agent, angiogenesis inhibitor, cytokine, cytotoxic agent, additional antibody, small molecule drug, or immunomodulator (e.g., immune checkpoint inhibitor or agonist); preferably, the additional antibody is a PD-1 antibody.

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