Anti-CD28 antibody and use thereof

Abstract

An antibody or an antigen-binding fragment thereof. The antibody or the antigen-binding fragment thereof can specifically bind to a CD28 protein, such as human CD28 protein. Also provided is a multispecific binding molecule, such as a multispecific antibody, that specifically binds to CD28 and one or more tumor-associated antigens. Further provided are a nucleic acid molecule encoding the antibody, an expression vector for expressing the antibody, a host cell and a preparation method therefor. Also provided are methods of diagnosis and treatment using the antibody.

Description

Anti-CD28 antibodies and uses thereof

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application is based on the Chinese patent application with application number 202410047645.4 and application date of January 12, 2024, and claims the priority of the Chinese patent application. The entire content of the Chinese patent application is hereby introduced into this application as a reference.

[0003] The present invention provides an antibody or antigen-binding fragment thereof that can specifically bind to a CD28 protein, such as a human CD28 protein. The present invention also provides multispecific binding molecules, such as multispecific antibodies, that specifically bind to CD28 and one or more tumor-associated antigens. The present invention further provides nucleic acid molecules encoding the antibodies, expression vectors for expressing the antibodies, host cells, and methods for preparing the same. The present invention also provides diagnostic and therapeutic methods using the antibodies of the present invention.

[0004] Background of the Invention

[0005] T cell activation involves stimulating a highly specific T cell receptor (TCR) by antigen presenting cells (such as dendritic cells), which present their specific antigens on their class II major histocompatibility (MHC) complexes and can be promoted by costimulatory molecules such as CD28. CD28 is a homodimeric receptor connected by a 44KDa disulfide bond 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, and provides the costimulatory signal required for T cell activation and survival. When CD28 on the T cell surface binds to CD80 (B7-1) and CD86 (B7-2) on antigen presenting cells, costimulation occurs. CD80 expression is upregulated in the cell when antigen presenting cells (APCs) are activated, and CD86 is constitutively expressed on APCs. CD28 co-stimulation of T helper cells enhances the transcription of IL2R and IL2 (leading to T cell proliferation), induces the expression of Bcl XL (enhancing T cell survival), and increases the production of IL-4 (leading to Th2 differentiation), IFNγ, IL-1, TNF, IL-5, various chemokines and their receptors.

[0006] 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 the CD40L molecule, which is essential for the interaction between T cells and B cells.

[0007] Several CD28 monoclonal antibodies are known in the art, such as TGN1412 (Tegenero, US20060188493A1), a CD28 superagonist monoclonal antibody that preferentially activates regulatory T (TReg) cells in the absence of TCR co-stimulation. However, when used for therapeutic purposes involving T cell activation, a Phase I clinical trial of this antibody resulted in rapid multi-organ failure, i.e., severe cytokine release syndrome (cytokine storm), in all six volunteers.

[0008] Therefore, the field still needs to develop safe anti-CD28 antibodies suitable as drugs, as well as multispecific antigen-binding molecules constructed based on them that specifically bind to CD28 and other target antigens for use in treatment. Summary of the Invention

[0009] One aspect of the present invention relates to an anti-CD28 antibody or an antigen-binding fragment thereof.

[0010] In some embodiments, the anti-CD28 antibodies or antigen-binding fragments thereof of the present invention specifically bind to CD28 (e.g., human CD28). In some embodiments, the binding affinity of the anti-CD28 antibodies or antigen-binding fragments thereof of the present invention to CD28 (e.g., human CD28) is K. D In some embodiments, the K value of the anti-CD28 antibodies or antigen-binding fragments thereof of the present invention for binding to CD28 (e.g., human CD28) is less than or equal to 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 values ​​between the values ​​recited. D Values ​​greater than or equal to approximately 0.5 nM.

[0011] In some embodiments, the anti-CD28 antibodies or antigen-binding fragments thereof of the present invention are capable of activating the CD28 signaling pathway.

[0012] In some embodiments, the anti-CD28 antibodies or antigen-binding fragments thereof of the present invention are capable of activating T cells (such as human peripheral T cells, such as CD4 or CD8 T cells), for example, activating T cells to release cytokines, such as TNF, such as TNFα, or interleukins, such as IL2.

[0013] In some embodiments, the anti-CD28 antibodies or antigen-binding fragments thereof of the present invention can activate human T cells to kill tumor cells, for example, activate human peripheral T cells to kill tumor cells.

[0014] Another aspect of the present invention relates to a multispecific binding molecule (e.g., a multispecific antibody, such as a bispecific antibody) constructed based on the CD28 antibody of the present invention, which comprises a target binding region from the anti-CD28 antibody of the present invention, and optionally one or more other target binding regions (antigen binding regions), such as a target binding region (antigen binding region) that specifically binds to one or more tumor-associated antigens (TAA).

[0015] In one embodiment, the present invention relates to a multispecific antibody, such as a bispecific antibody, that specifically binds CD28 and one or more TAAs. In some embodiments, the TAA is CLDN18.2 or PSMA, such as human CLDN18.2 or human PSMA.

[0016] In one embodiment, the invention relates to a bispecific antibody that specifically binds CD28 (eg, human CD28) and CLDN18.2 (eg, human CLDN18.2).

[0017] In one embodiment, the invention relates to a bispecific antibody that specifically binds CD28 (eg, human CD28) and PSMA (eg, human PSMA).

[0018] In some embodiments, the bispecific antibodies of the present invention are capable of effectively activating the CD28 signaling pathway.

[0019] In some embodiments, the bispecific antibodies of the invention are capable of activating human peripheral T cells, for example, activating T cells (eg, CD4 or CD8 T cells) to release cytokines, for example, TNF, such as TNFα, or interleukins, such as IL2.

[0020] In some embodiments, the bispecific antibodies of the present invention can activate human T cells to kill tumor cells, for example, activate human peripheral T cells to kill tumor cells.

[0021] In some embodiments, the bispecific antibodies of the invention are capable of specifically binding to CD28 (eg, human CD28) and a TAA (eg, a human TAA, such as CLDN18.2 or PSMA).

[0022] In some embodiments, the bispecific antibodies of the invention, such as bispecific antibodies that specifically bind to CD28 and CLDN18.2 or bispecific antibodies that specifically bind to CD28 and PSMA, have anti-tumor activity, such as in vivo anti-tumor activity.

[0023] In some embodiments, the bispecific antibodies of the present invention can be combined with other therapeutic agents (eg, other antibodies such as anti-PD1 antibodies) for anti-tumor use. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1: Results of the detection of Jurkat-NFkB activation activity by anti-CD28 chimeric antibodies;

[0025] Figure 2: Activation results of human peripheral T cells activated by anti-CD28 humanized antibody, AB: without CD3 antibody, CD: with CD3 antibody;

[0026] Figure 3: Schematic diagram of CD28 bispecific antibody;

[0027] Figure 4: Results of activating NFkB Jurkat Reporter with CLDN18.2xCD28 bispecific antibody;

[0028] Figure 5: Results of CLDN18.2xCD28 bispecific antibody-mediated human peripheral T cell killing of tumor cells;

[0029] Figure 6: Cytokine release from peripheral T cells activated by CLDN18.2xCD28 bispecific antibody, A: without CD3 antibody, B: with CD3 antibody;

[0030] Figure 7: Results of PSMAxCD28 bispecific antibody cell surface affinity test, A: PSMA affinity, B: CD28 affinity;

[0031] Figure 8: Results of NFkB Jurkat Reporter activation by PSMAxCD28 bispecific antibody, A: addition of CD3 antibody, B: addition of PSMAxCD3 bispecific antibody;

[0032] Figure 9: PSMAxCD28 bispecific antibody-mediated human peripheral T cell killing of tumor cells;

[0033] Figure 10: PSMA xCD28 bispecific antibody activates peripheral T cells to release cytokine IL2, A: without CD3 antibody (-signal 1), B: with CD3 antibody (+signal 1);

[0034] Figure 11: In vivo single-agent efficacy test results of PSMA xCD28 bispecific antibody, A: Dosage regimen; B: Test results;

[0035] Figure 12: In vivo efficacy test results of PSMA xCD28 bispecific antibody combined with PD1 antibody, A: dosing regimen; B: test results.

[0036] Detailed Description of the Invention

[0037] It should be understood that the present invention is not limited to the specific methods, protocols, examples and reagents described herein, as these may vary. It should also be understood that the terminology used herein is for the purpose of describing specific embodiments only and is not intended to limit the scope of the present invention, which will only be limited by the appended claims.

[0038] I. Definition

[0039] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0040] To interpret this specification, the following definitions will apply, and wherever appropriate, terms used in the singular may also include the plural, and vice versa. It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0041] The term "about" when used in conjunction with a numerical value is meant to encompass a range of numerical values ​​having a lower limit that is 5% (e.g., 4%, 3%, 2% or 1%) less than the specified numerical value and an upper limit that is 5% (e.g., 4%, 3%, 2% or 1%) greater than the specified numerical value.

[0042] As used herein, the term "and / or" means any one of the alternatives or two or more or all of the alternatives.

[0043] When "first" and "second" are mentioned herein, it is only to distinguish the two domains or two chains, but does not indicate the positions of the two domains in any way.

[0044] As used herein, the terms "comprising" or "including" are intended to include the stated elements, integers, or steps, but do not exclude any other elements, integers, or steps. In this document, when the terms "comprising" or "including" are used, unless otherwise indicated, the context of consisting of the stated elements, integers, or steps is also encompassed. For example, when reference is made to an antibody variable region "comprising" a specific sequence, it is intended to encompass an antibody variable region consisting of that specific sequence.

[0045] The term "CD28" as used herein refers to an antigen expressed on T cells as a co-stimulatory receptor. Human CD28 includes, for example, an amino acid sequence as shown in NCBI Accession No. NP_006130.1. In some embodiments, CD28 is human CD28.

[0046] As used herein, "antibodies that bind to CD28," "anti-CD28 antibodies," or "antibodies that specifically bind to CD28" refer to antibodies that are capable of binding with suitable affinity to monomeric CD28 or its extracellular domain, or to dimeric CD28 or its extracellular domain. The antibodies and antigen-binding fragments of the present invention can bind to soluble CD28 and / or CD28 expressed on the surface of cells. Soluble CD28 includes native CD28 protein as well as recombinant CD28 protein variants, such as monomeric and dimeric CD28 constructs that lack a transmembrane domain or are otherwise not associated with a cell membrane.

[0047] In some aspects, the anti-CD28 antibodies described herein also encompass multispecific antibodies, such as bispecific antibodies, that simultaneously specifically bind CD28 and other target antigens.

[0048] General information concerning the amino acid and nucleotide sequences of human immunoglobulin light and heavy chains is given in Kabat, EA et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991).

[0049] The multispecific antibodies of the present invention may comprise a linker. As used herein, the term "linker" refers to any molecule that enables the direct connection of the different parts of a multispecific antibody. Examples of linkers for establishing covalent linkages between different parts of a multispecific antibody include peptide linkers and non-protein polymers, including but not limited to polyethylene glycol (PEG), polypropylene glycol, polyoxyalkylenes 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 connects the amino acid sequences of the various parts of the multispecific antibody together. Preferably, the peptide linker has a length that is sufficient to connect the two entities in a manner that allows them to maintain their conformations relative to each other so as not to interfere with the desired activity. The peptide linker may or may not primarily include the following amino acid residues: Gly, Ser, Ala or Thr.

[0050] The term "Fc domain" or "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. A native 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 native antibody, an immunoglobulin Fc domain comprises the second and third constant domains (CH2 domain and CH3 domain) of two heavy chains derived from IgG, IgA, and IgD class antibodies; or the second, third, and fourth constant domains (CH2 domain, CH3 domain, and CH4 domain) of two heavy chains derived from IgM and IgE class antibodies. Unless otherwise indicated herein, the amino acid residues in the Fc region or heavy chain constant region are numbered according to the EU numbering system (also referred to as the EU index) as described in Kabat et al., Sequences of Proteins of Immunological Interes, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD, 1991. However, the C-terminal lysine (Lys447) in the Fc region may or may not exist. Two Fc regions can achieve dimerization to form a dimeric Fc, and two different Fc heterodimerizations form heterodimeric Fc. In this article, the terms "Fc region", "Fc portion" and "dimeric Fc (e.g., heterodimeric Fc)" do not include the heavy chain variable region VH and light chain variable region VL of an immunoglobulin and heavy chain constant region CH1 and light chain constant region CL, but may include the hinge region at the N-terminus of the heavy chain constant region in some cases. In one embodiment, a human IgG heavy chain Fc region extends from Asp221, or from Cys226, or from Asp231, to the carboxyl-terminus of the heavy chain.

[0051] In one embodiment, the human IgG1 Fc region polypeptide (including part of the hinge region) comprises or consists of the following amino acid sequence:

[0052] DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:94); or

[0053] 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 is of the human IgG4 subclass. In one embodiment, the Fc region is of the human IgG1 subclass. In one embodiment, the Fc region is of a human IgG1, IgG2, IgG3, or IgG4 Fc region.

[0054] Herein, a "heterodimeric Fc scaffold" refers to a scaffold comprising two different Fc regions or formed by dimerization of two different Fc regions, which can be connected at its N-terminus or C-terminus to an antigen-binding domain (e.g., an antibody heavy chain and / or light chain variable region or an antibody antigen-binding fragment that can bind to a target molecule, or a soluble portion of a ligand or receptor that can bind to a target molecule) to form a multispecific antibody such as a bispecific antibody.

[0055] The term "CH1 region" refers to the portion of an antibody heavy chain polypeptide extending from EU position 118 to EU position 220 (EU numbering system). In one embodiment, the CH1 domain comprises or consists of the amino acid sequence of ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC (SEQ ID NO: 74).

[0056] In some embodiments, CH1 may comprise a portion of the hinge region. In some embodiments, the Fc region may comprise a portion of the hinge region.

[0057] References to specific amino acid residues in the constant region of an antibody IgG are made herein according to the EU numbering system. For example, "S364" refers to the serine at EU position 364. Amino acid mutations at specific positions in the constant region are indicated by (original amino acid, amino acid position, mutated amino acid). For example, "S364R" refers to the substitution of the serine (S) at EU position 364 with an arginine (R). When referring to combinations of mutations, the combined mutations are connected by a plus sign (+), a "-" sign, or an "and" sign. "S364R+D399K," "S364R-D399K," or "S364R and D399K" indicate that the Fc region contains both the S364R and D399K mutations. When multiple mutation possibilities exist at a specific position, this is indicated herein by the symbol " / ." For example, the mutation "K370T / S" indicates that the K residue at position 370 can be replaced with either a T or S residue.

[0058] For polypeptide sequences, "conservative changes" include replacements, deletions or additions to the polypeptide sequence, but do not substantially change the desired functional activity of the polypeptide sequence. For example, conservative substitutions often result in a certain amino acid being replaced with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. The following lists 8 groups of amino acids containing mutually conservative replacements: 1) alanine (A), glycine (G); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V); 6) phenylalanine (F), tyrosine (Y), tryptophan (W); 7) serine (S), threonine (T); and 8) cysteine ​​(C), methionine (M). In some embodiments, the term "conservative sequence change" is used to refer to amino acid modifications that do not significantly affect or change the target antigen binding characteristics of the antibody molecule or binding protein molecule of the present invention containing the amino acid sequence. For example, a conservatively modified variant retains at least 80%, 85%, 90%, 95%, 98%, 99% or more, such as 100-110% or more, binding affinity for the antigen of interest relative to the parent antibody or binding protein.

[0059] The term "vector," as used herein, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors that are self-replicating nucleic acid structures as well as vectors that are incorporated into the genome of a host cell into which they have been introduced. Some vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors."

[0060] The term "binding molecule" refers to any molecule capable of specifically binding to a target, such as an antibody or antigen-binding fragment or fusion protein thereof.

[0061] The term "target" refers to the object to which a binding molecule is directed. A target can be an antigen, a ligand, or a receptor. The term "antigen" refers to a molecule that triggers an immune response. This immune response may involve the production of antibodies or the activation of specific immune cells, or both. Those skilled in the art will appreciate that any macromolecule, including essentially all proteins or peptides, can be used as an antigen. In addition, antigens can be derived from recombinant or genomic DNA. As used herein, the term "epitope" refers to the portion of an antigen that specifically interacts with an antibody molecule. When the binding molecule of the present invention relates to a target binding region derived from an antibody, "target" and "antigen" can be used interchangeably. In some embodiments, the antigen is a tumor-associated antigen (TAA).

[0062] The term "tumor-associated antigen (TAA)" as used herein preferably relates to a protein that is specifically expressed in a limited number of tissues and / or organs or in a specific developmental stage under normal conditions and is expressed or abnormally expressed in one or more tumors or cancerous tissues. In the context of the present invention, a tumor-associated antigen is preferably associated with the cell surface of cancer cells and is preferably not expressed or only rarely expressed 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.

[0063] As used herein, the term "target binding region" refers to the portion of a binding molecule, such as a multispecific binding molecule or a bispecific binding molecule, that binds a specific target or antigen. The target binding region can be, for example, an antibody or immunoglobulin itself or an antibody fragment. Such a target binding region may or may not have a tertiary structure independent of the rest of the binding molecule and may or may not bind to its target as a separate entity. The target binding region can also be a receptor or a ligand, or a domain of a receptor that is capable of binding a ligand. In the case of multispecific antibodies or bispecific antibodies, the "target binding region" is also referred to as an "antigen binding region."

[0064] As used herein, the term "antigen binding region" refers to any portion of an antibody or antigen binding fragment thereof, such as a multispecific antibody or bispecific antibody, that binds a specific target or antigen. The antigen binding region can be, for example, an antibody or immunoglobulin itself or an antibody fragment. Such an antigen binding region 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 separate entity. Where the binding molecules of the present invention relate to a target binding region derived from an antibody, "target binding region" and "antigen binding region" can be used interchangeably.

[0065] The term "multispecific binding molecule" refers to a multispecific binding molecule that is at least bispecific, 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 one target or antigen and the second target binding region binds another antigen or target. Multispecific binding molecules according to the present invention also encompass multispecific binding molecules comprising multiple target binding regions / binding sites. In some embodiments, the multispecific binding molecules of the present invention are multispecific antibodies. In some embodiments, the bispecific binding molecules of the present invention are bispecific antibodies.

[0066] As used herein, the term "monospecific" refers to a polypeptide / protein molecule 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.

[0067] As used herein, the term "multispecific" binding molecule, such as an antibody, refers to a 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 that has binding specificity for at least two different targets or sites / structures. In one embodiment, provided herein is a bispecific binding molecule that has binding specificity for a first target and a second target.

[0068] 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 to different epitopes of different antigens. A multispecific antibody is an antibody that has binding specificity for at least two different antigens or epitopes. In one embodiment, provided herein is a bispecific antibody that has binding specificity for a first antigen and a second antigen.

[0069] When referring to a "first antigen-binding region" in a multispecific antibody or bispecific antibody, it refers to the binding region that binds to the first antigen, and is not intended to limit the number of such antigen-binding regions contained in the antibody. For example, a multispecific antibody or bispecific antibody may contain one or more first antigen-binding regions. For example, a bispecific antibody contains a first antigen-binding region and a second antigen-binding region, but may contain one or more first antigen-binding regions and one or more second antigen-binding regions.

[0070] When referring to a "first target binding region" in a binding molecule, it refers to the binding region that binds to the first target, and is not intended to limit the number of target binding regions contained in the binding molecule. For example, a multispecific binding molecule or a bispecific binding molecule can contain one or more first target binding regions. For example, a bispecific binding molecule comprising a first target binding region and a second target binding region can contain one or more first target binding regions and one or more second target binding regions.

[0071] When referring to "a target or antigen binding region is derived from an antibody", it means that the binding domain constituting the target / antigen binding region is or is derived from the binding domain of the antibody that specifically binds to the antigen, for example, the specific antigen-binding fragment of the antigen binding region, such as Fab, is or is derived from the corresponding fragment, such as Fab, of the antibody, or the heavy chain variable region and / or light chain variable region of the antigen binding region is or is derived from the heavy chain variable region and / or light chain variable region of the antibody, or one, two, three, four, five or six CDRs of the antigen binding region are CDRs of the antibody.

[0072] The term "derived from" means that the fragment in the antigen binding region is substantially identical to the fragment of the antibody from which it is derived, but has a mutation, such as a substitution, deletion or addition, at one or more sites. In a specific embodiment, the mutation is not in the CDR of the antibody.

[0073] The terms "whole antibody" or "full-length antibody" are used interchangeably herein and refer to antibody molecules with the structure of natural immunoglobulin molecules. In the case of conventional four-chain IgG antibodies, the full-length antibody comprises two heavy chains (H) and two light chains (L) interconnected by disulfide bonds. In the case of heavy chain antibodies having only heavy chains and lacking light chains, the full-length antibody comprises two heavy chains (H) interconnected by disulfide bonds. For conventional four-chain IgG antibodies, the full-length antibody heavy chain is generally composed of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region, wherein the heavy chain constant region comprises at least three domains CH1, CH2 and CH3. The full-length antibody light chain is composed of a light chain variable region (abbreviated herein as VL) and a light chain constant region, wherein the light chain constant region consists of one domain CL. Each heavy chain variable region VH and each light chain variable region are composed of three CDRs and four FRs, arranged in the following order from amino terminus to carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The term "antibody fragment" includes a portion of an intact antibody. In a preferred embodiment, the antibody fragment is an antigen-binding fragment.

[0074] The term "antigen-binding fragment" of an antibody is a molecule that is different from a full-length antibody and that contains a portion of the full-length antibody, but is capable of binding to the antigen of the full-length antibody or competing with the full-length antibody (i.e., the full-length antibody from which the antigen-binding fragment is derived) for antigen binding. Antigen-binding fragments can be prepared 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, Fab fragments can be obtained by digesting a full-length antibody with papain. In addition, digesting a full-length antibody with pepsin below the disulfide bonds in the hinge region produces F(ab')2, which is a dimer of Fab' and a divalent antibody fragment. F(ab')2 can be reduced under neutral conditions by breaking the disulfide bonds in the hinge region, thereby converting the F(ab')2 dimer into a Fab' monomer. A Fab' monomer is essentially a Fab fragment with a hinge region. An Fv fragment consists of the VL and VH domains of a single arm of an antibody. The two domains of the Fv fragment, VL and VH, can be encoded by separate genes, but can also be produced by recombinant methods using a synthetic linker peptide to link the two domains as a single protein chain in which the VL region and VH region are paired to form a single-chain Fv (scFv).

[0075] "Fab fragment" or "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, from N-terminus to C-terminus, VH and a constant region selected from CH1 and CL, and the other polypeptide chain comprises, from N-terminus to C-terminus, VL and another constant region selected from CL and CH1, wherein the VH domain and the VL domain pair to form an antigen binding site. Herein, the Fab polypeptide chain comprising the heavy chain constant region CH1 is also referred to as the "Fab heavy chain"; accordingly, the Fab polypeptide chain comprising the light chain constant region CL is also referred to as the "Fab light chain."

[0076] "Complementarity determining region" or "CDR region" or "CDR" is a region in an antibody variable domain that is highly variable in sequence and forms structurally defined loops ("hypervariable loops") and / or contains antigen contact residues ("antigen contact points"). CDRs are primarily responsible for binding to antigenic epitopes. The CDRs of the heavy and light chains are typically referred to as CDR1, CDR2, and CDR3, and are numbered sequentially starting from the N-terminus. The CDRs located within the antibody heavy chain variable domain are referred to as CDRH1, CDRH2, and CDRH3, while the CDRs located within the antibody light chain variable domain are referred to as CDRL1, CDRL2, and CDRL3. In a given light chain variable region or heavy chain variable region amino acid sequence, the precise amino acid sequence boundaries of each CDR can be determined using any one or a combination of a number of well-known antibody CDR assignment schemes, including, for example, Chothia based on the three-dimensional structure of antibodies and the topology of the CDR loops (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 based on antibody sequence variability (Kabat et al., Sequences of Proteins of Immunological Interest, 4th Edition, US Department of Health and Human Services, National Institutes of Health (1987)), AbM (University of Bath), Contact (University College London), International ImMunoGeneTics The database (IMGT) (www.imgt.cines.fr / ), and the North CDR definition based on affinity propagation clustering using a large number of crystal structures. Unless otherwise indicated, in the present invention, the term "CDR" or "CDR sequence" encompasses CDR sequences determined in any of the above ways or in combination. CDRs can 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).

[0077] In some embodiments, the CDRs of the heavy chain variable regions of the antibodies of the present invention are determined 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 CDRs of the light chain variable regions of the antibodies of the present invention are determined according to Kabat.

[0078] In one embodiment, CDRH1 in the anti-CD28 antibody of the present invention is defined according to the Kabat & Chothia rules, CDRH2 and CDRH3 are defined according to the Kabat rules, and CDRLs are defined according to the Kabat rules.

[0079] Calculation of sequence identity between sequences is performed as follows.

[0080] To determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of the first and second amino acid or nucleic acid sequences for optimal alignment or non-homologous sequences can be discarded for comparison purposes). In a preferred embodiment, for comparison purposes, the length of the reference sequence being aligned is at least 30%, preferably at least 40%, more preferably at least 50%, 60%, and even more preferably at least 70%, 80%, 90%, 100% of the length of the reference sequence. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide at the corresponding position in the second sequence, then the molecules are identical at that position.

[0081] A "humanized antibody" is an antibody that retains the antigen-specific reactivity of a non-human antibody while being less immunogenic when administered to humans as a therapeutic agent. This can be achieved, for example, by retaining the non-human antigen-binding site and replacing the remaining portions of the antibody with their human counterparts (i.e., replacing portions of the variable region that are not involved in binding with corresponding portions of a human antibody).

[0082] As used herein, the terms "anti," "binding," or "specific binding" mean that the binding is selective for the 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 can be determined by flow cytometry or enzyme-linked immunosorbent assay (ELISA) or conventional binding assays known in the art, such as by radioimmunoassay (RIA) or thin-layer interferometry or MSD assays or surface plasmon resonance (SPR).

[0083] "Affinity" or "binding affinity" refers to the intrinsic binding affinity that reflects the interaction between members of a binding pair. The affinity of a molecule X for its partner Y can generally be expressed by the dissociation constant (K D ) indicates that the dissociation constant is the dissociation rate constant and the association rate constant (K dis and K on ). Affinity can be measured by common methods known in the art. One specific method for measuring affinity is the ForteBio kinetic binding assay herein.

[0084] The term "host cell" refers to a cell into which an exogenous polynucleotide has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and progeny derived therefrom.

[0085] The terms "individual" or "subject" are used interchangeably and refer to mammals. Mammals include, but are not limited to, domesticated animals (e.g., cows, 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 individual is a human.

[0086] The term "treat," ..."

[0087] The term "preventing" includes the inhibition of the onset or development of a disease or condition or symptoms of a particular disease or condition.

[0088] The term "therapeutic agent" as used herein encompasses any substance effective in preventing or treating tumors, such as cancer, including chemotherapeutic agents, cytokines, cytotoxic agents, other antibodies (such as immune checkpoint molecule antibodies), small molecule drugs, or immunomodulators (such as immunosuppressants or agonists).

[0089] The term "combination therapy" refers to the administration of two or more therapeutic agents or treatment modalities to treat diseases described herein. This administration includes co-administering these therapeutic agents in a substantially simultaneous manner, such as in a single capsule with a fixed ratio of active ingredients. Alternatively, this administration includes co-administration of each active ingredient in a variety of or separate containers (such as tablets, capsules, powders, and liquids). The powder and / or liquid can be reconstituted or diluted to the desired dose before administration. In addition, this administration also includes using each type of therapeutic agent in a sequential manner 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 disorders or conditions described herein.

[0090] The term "drug combination or combination product" refers to a non-fixed combination product or a fixed combination product, including but not limited to a kit and a pharmaceutical composition. The term "non-fixed combination" means that the active ingredients (e.g., (i) an antibody of the present invention, and (ii) other therapeutic agent) are administered to a patient simultaneously, without specific time restrictions, or at the same or different time intervals, in a separate entity, wherein such administration provides two or more active agents with a preventive or therapeutically effective level in the patient's body. The term "fixed combination" means that two or more active agents are administered to a patient simultaneously in the form of a single entity. The dosage and / or time interval of the two or more active agents are preferably selected so that the combined use of the parts can produce an effect greater than that achieved by using any one component alone when treating a disease or condition. Each component can be in the form of a separate formulation, which can be the same or different.

[0091] The term "pharmaceutical composition" refers to a composition that is in form permitting the biological activity of the active ingredient contained therein to be effective, and that contains no additional ingredients that are unacceptably toxic to a subject to which the composition would be administered.

[0092] The term "pharmaceutical excipient" refers to a diluent, adjuvant (eg, Freund's adjuvant (complete and incomplete)), excipient, carrier, stabilizer, or the like, which is administered together with the active substance.

[0093] "Subject / patient / individual sample" refers to a collection of cells or fluids obtained from a patient or subject. The source of the tissue or cell sample can be a solid tissue, such as an organ or tissue sample or a biopsy sample or a puncture sample from fresh, frozen and / or preserved; a body fluid, such as tears, vitreous humor, cerebrospinal fluid, amniotic fluid (amniotic fluid), peritoneal fluid (ascites), or interstitial fluid.

[0094] II. Anti-CD28 Antibodies

[0095] In some embodiments, the anti-CD28 antibodies or antigen-binding fragments thereof of the present invention comprise three complementarity determining regions (CDRHs) from the heavy chain variable region, CDRH1, CDRH2, and CDRH3. In some embodiments, the anti-CD28 antibodies or antigen-binding fragments thereof of the present invention comprise three complementarity determining regions (CDRLs) from the light chain variable region, CDRL1, CDRL2, and CDRL3. In some embodiments, the anti-CD28 antibodies or antigen-binding fragments thereof of the present invention comprise three complementarity determining regions (CDRHs) from the heavy chain variable region and three complementarity determining regions (CDRLs) from the light chain variable region.

[0096] In some aspects, the anti-CD28 antibodies or antigen-binding fragments thereof of the present invention comprise a heavy chain variable region (VH). In some aspects, the anti-CD28 antibodies or antigen-binding fragments thereof of the present invention comprise a light chain variable region (VL). In some aspects, the anti-CD28 antibodies or antigen-binding fragments thereof of the present invention comprise 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.

[0097] In some embodiments, the heavy chain variable region VH of the anti-CD28 antibody of the present invention is

[0098] (i) comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 4, 5, 13, 23, 24, 69, 72 or 75; or

[0099] (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 4, 5, 13, 23, 24, 69, 72 or 75; or

[0100] (iii) an amino acid sequence comprising one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence of SEQ ID NO: 4, 5, 13, 23, 24, 69, 72 or 75, consisting of said amino acid sequence. Preferably, said amino acid changes do not occur in the CDR regions.

[0101] In some embodiments, the light chain variable region VL of the anti-CD28 antibody of the present invention is

[0102] (i) comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 9, 17, 19, 28, 70, 73 or 78; or

[0103] (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 9, 17, 19, 28, 70, 73 or 78; or

[0104] (iii) an amino acid sequence comprising one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence of SEQ ID NO: 9, 17, 19, 28, 70, 73 or 78, consisting of said amino acid sequence. Preferably, said amino acid changes do not occur in the CDR regions.

[0105] In some embodiments, the three complementarity determining regions (CDRHs), CDRH1, CDRH2, and CDRH3, from the heavy chain variable region of the anti-CD28 antibodies of the invention are

[0106] (i) three complementarity determining regions CDRH1, CDRH2 and CDRH3 contained in the VH represented by SEQ ID NO: 4, 5, 13, 23, 69, 72 or 75, or

[0107] (ii) a sequence comprising at least one and no more than 5, 4, 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) in the three CDRH regions relative to the sequence of (i),

[0108] wherein the CDRH can be determined according to any scheme for determining CDRs, for example, according to the Kabat, AbM, Chothia, Contact or IMGT schemes or a combination thereof;

[0109] Preferably, the CDRH1 is determined according to the Kabat & Chothia rules, and CDRH2 and CDRH3 are determined according to Kabat.

[0110] In some embodiments, the three complementarity determining regions (CDRLs), CDRL1, CDRL2, and CDRL3, from the light chain variable region of the anti-CD28 antibody of the invention are

[0111] (i) three complementarity determining regions CDRL1, CDRL2 and CDRL3 contained in the VL of SEQ ID NO: 9, 17, 19, 28, 70, 73 or 78, or

[0112] (ii) a sequence comprising at least one and no more than 5, 4, 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) in the three CDRL regions relative to the sequence of (i);

[0113] wherein the CDRL can be determined according to any scheme for determining CDRs, for example, according to the Kabat, AbM, Chothia, Contact or IMGT schemes or a combination thereof;

[0114] Preferably, the CDRL1-3 are determined according to Kabat, respectively.

[0115] In some embodiments, the CDRH1 comprises the amino acid sequence shown in SEQ ID NO: 1, 10 or 20, or an amino acid sequence having 1, 2 or 3 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) therefrom, or consists of the amino acid sequence.

[0116] In some embodiments, the CDRH2 comprises the amino acid sequence shown in SEQ ID NO: 2, 11, 21 or 76, or an amino acid sequence having 1, 2 or 3 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) therefrom, or consists of the amino acid sequence.

[0117] In some embodiments, the CDRH3 comprises the amino acid sequence shown in SEQ ID NO: 3, 12, 22 or 77, or an amino acid sequence having 1, 2 or 3 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) therein, or consists of the amino acid sequence.

[0118] In some embodiments, the CDRL1 comprises the amino acid sequence shown in SEQ ID NO: 6, 14, 18, 25 or 71, or an amino acid sequence having 1, 2 or 3 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) therefrom, or consists of the amino acid sequence.

[0119] In some embodiments, the CDRL2 comprises the amino acid sequence shown in SEQ ID NO: 7, 15 or 26, or an amino acid sequence having 1, 2 or 3 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) therefrom, or consists of the amino acid sequence.

[0120] In some embodiments, the CDRL3 comprises the amino acid sequence shown in SEQ ID NO: 8, 16 or 27, or an amino acid sequence having 1, 2 or 3 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) therefrom, or consists of the amino acid sequence.

[0121] In some embodiments, in the anti-CD28 antibodies of the present invention,

[0122] 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;

[0123] 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;

[0124] 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;

[0125] 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

[0126] 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.

[0127] In some specific embodiments of the present invention, the anti-CD28 antibody or antigen-binding fragment thereof comprises VH and VL, wherein

[0128] the VH comprises, or consists of, the amino acid sequence of SEQ ID NO: 4 or 5 or 72, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and the VL comprises, or consists of, the amino acid sequence of SEQ ID NO: 9 or 73, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto;

[0129] the VH comprises, or consists of, the amino acid sequence of SEQ ID NO: 4 or 5, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and the VL comprises, or consists of, the amino acid sequence of SEQ ID NO: 9, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto;

[0130] the VH comprising, or consisting of, the amino acid sequence of SEQ ID NO: 72, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and the VL comprising, or consisting of, the amino acid sequence of SEQ ID NO: 73, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto;

[0131] the VH comprises, or consists of, the amino acid sequence of SEQ ID NO: 13 or 69, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and the VL comprises, or consists of, the amino acid sequence of SEQ ID NO: 17 or 19 or 70, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto;

[0132] the VH comprises, or consists of, the amino acid sequence of SEQ ID NO: 13, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and the VL comprises, or consists of, the amino acid sequence of SEQ ID NO: 17 or 19, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto;

[0133] the VH comprises, or consists of, the amino acid sequence of SEQ ID NO:69, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and the VL comprises, or consists of, the amino acid sequence of SEQ ID NO:70, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto;

[0134] the VH comprises, or consists of, the amino acid sequence of SEQ ID NO: 23 or 24 or 75, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and the VL comprises, or consists of, the amino acid sequence of SEQ ID NO: 28 or 78, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto;

[0135] the VH comprises, or consists of, the amino acid sequence of SEQ ID NO: 23 or 24, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and the VL comprises, or consists of, the amino acid sequence of SEQ ID NO: 28, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; or

[0136] The VH comprises the amino acid sequence of SEQ ID NO:75, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, or consists of said amino acid sequence, and the VL comprises the amino acid sequence of SEQ ID NO:78, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, or consists of said amino acid sequence.

[0137] In some embodiments of the present invention, the anti-CD28 antibody or antigen-binding fragment thereof comprises

[0138] (i) three complementarity determining regions CDRH1, CDRH2 and CDRH3 contained in VH as shown in SEQ ID NO: 4 or 5 or 72, and three complementarity determining regions CDRL1, CDRL2 and CDRL3 contained in VL as shown in SEQ ID NO: 9 or 73;

[0139] (ii) three complementary determining regions CDRH1, CDRH2 and CDRH3 contained in VH as shown in SEQ ID NO: 13 or 69, and three complementary determining regions CDRL1, CDRL2 and CDRL3 contained in VL as shown in SEQ ID NO: 17, 19 or 70; or

[0140] (iii) three complementary determining regions CDRH1, CDRH2 and CDRH3 contained in VH as shown in SEQ ID NO: 23, 24 or 75, and three complementary determining regions CDRL1, CDRL2 and CDRL3 contained in VL as shown in SEQ ID NO: 28 or 78;

[0141] Preferably, the CDRH and CDRL can be determined according to any scheme for determining CDRs, for example, according to the Kabat, AbM, Chothia, Contact or IMGT schemes or a combination thereof; for example, the CDRH1 is determined according to the Kabat & Chothia rules, and CDRH2 and CDRH3 are determined according to Kabat; and the CDRL1-3 are determined by the Kabat scheme respectively.

[0142] In some specific embodiments of the present invention, the anti-CD28 antibody or antigen-binding fragment thereof comprises CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3, wherein

[0143] CDRH1 consists of the amino acid sequence of SEQ ID NO: 1; CDRH2 consists of the amino acid sequence of SEQ ID NO: 2; CDRH3 consists of the amino acid sequence of SEQ ID NO: 3; CDRL1 consists of the amino acid sequence of SEQ ID NO: 6; CDRL2 consists of the amino acid sequence of SEQ ID NO: 7; and CDRL3 consists of the amino acid sequence of SEQ ID NO: 8;

[0144] CDRH1 consists of the amino acid sequence of SEQ ID NO: 10; CDRH2 consists of the amino acid sequence of SEQ ID NO: 11; CDRH3 consists of the amino acid sequence of SEQ ID NO: 12; CDRL1 consists of the amino acid sequence of SEQ ID NO: 14, 18, or 71; CDRL2 consists of the amino acid sequence of SEQ ID NO: 15; and CDRL3 consists of the amino acid sequence of SEQ ID NO: 16;

[0145] CDRH1 consists of the amino acid sequence of SEQ ID NO: 20; CDRH2 consists of the amino acid sequence of SEQ ID NO: 21; CDRH3 consists of the amino acid sequence of SEQ ID NO: 22; CDRL1 consists of the amino acid sequence of SEQ ID NO: 25; CDRL2 consists of the amino acid sequence of SEQ ID NO: 26; and CDRL3 consists of the amino acid sequence of SEQ ID NO: 27; or

[0146] CDRH1 consists of the amino acid sequence of SEQ ID NO:20; CDRH2 consists of the amino acid sequence of SEQ ID NO:76; CDRH3 consists of the amino acid sequence of SEQ ID NO:77; CDRL1 consists of the amino acid sequence of SEQ ID NO:25; CDRL2 consists of the amino acid sequence of SEQ ID NO:26; and CDRL3 consists of the amino acid sequence of SEQ ID NO:27.

[0147] In some specific embodiments of the present invention, the anti-CD28 antibody or antigen-binding fragment thereof comprises VH and VL, wherein

[0148] The VH comprises or consists of the amino acid sequence shown in SEQ ID NO: 4, and the VL comprises or consists of the amino acid sequence shown in SEQ ID NO: 9;

[0149] The VH comprises or consists of the amino acid sequence shown in SEQ ID NO: 5, and the VL comprises or consists of the amino acid sequence shown in SEQ ID NO: 9;

[0150] The VH comprises or consists of the amino acid sequence shown in SEQ ID NO: 13, and the VL comprises or consists of the amino acid sequence shown in SEQ ID NO: 17;

[0151] The VH comprises or consists of the amino acid sequence shown in SEQ ID NO: 13, and the VL comprises or consists of the amino acid sequence shown in SEQ ID NO: 19;

[0152] The VH comprises or consists of the amino acid sequence shown in SEQ ID NO: 23, and the VL comprises or consists of the amino acid sequence shown in SEQ ID NO: 28;

[0153] The VH comprises or consists of the amino acid sequence shown in SEQ ID NO: 24, and the VL comprises or consists of the amino acid sequence shown in SEQ ID NO: 28;

[0154] The VH comprises or consists of the amino acid sequence shown in SEQ ID NO: 72, and the VL comprises or consists of the amino acid sequence shown in SEQ ID NO: 73;

[0155] The VH comprises or consists of the amino acid sequence shown in SEQ ID NO: 69, and the VL comprises or consists of the amino acid sequence shown in SEQ ID NO: 70; or

[0156] The VH contains or consists of the amino acid sequence shown in SEQ ID NO: 75, and the VL contains or consists of the amino acid sequence shown in SEQ ID NO: 78.

[0157] In one embodiment of the present invention, the amino acid changes described herein include amino acid substitutions, insertions, or deletions. In preferred embodiments, the amino acid changes described herein occur in regions outside the CDRs (e.g., in the FRs). More preferably, the amino acid changes described herein occur in regions outside the heavy chain variable region and / or outside the light chain variable region. Preferably, the amino acid changes described herein are amino acid substitutions, preferably conservative substitutions.

[0158] In some embodiments, the anti-CD28 antibody or antigen-binding fragment thereof of the present invention is an antibody in the form of IgG1, an antibody in the form of IgG2, an antibody in the form of IgG3, or an antibody in the form of IgG4, or an antigen-binding fragment thereof, for example, an antibody in the form of IgG1, or an antigen-binding fragment thereof.

[0159] In some embodiments, the anti-CD28 antibodies or antigen-binding fragments thereof of the present invention further comprise an antibody heavy chain constant region HC. In some embodiments, the anti-CD28 antibodies or antigen-binding fragments thereof of the present invention further comprise an antibody light chain constant region LC. In some embodiments, the anti-CD28 antibodies or antigen-binding fragments thereof of the present invention further comprise 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.

[0160] In some embodiments, the heavy chain constant region of the anti-CD28 antibody of the present invention is a constant region of (human) IgG1, IgG2, IgG3, or IgG4, such as a constant region of IgG1.

[0161] In some embodiments, the IgG1 heavy chain constant region suitable for use in the antibody molecules of the invention is

[0162] (i) comprising or consisting of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 29 or 79;

[0163] (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 29 or 79; or

[0164] (iii) comprises an amino acid sequence having one or more (preferably no more than 10 or 10, more preferably no more than 5, 4, 3, 2, 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence of SEQ ID NO: 29 or 79, or consists of said amino acid sequence.

[0165] 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, such as a Kappa light chain constant region.

[0166] In some embodiments, the light chain constant region

[0167] (i) comprising or consisting of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 30;

[0168] (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 30; or

[0169] (iii) comprises or consists of an amino acid sequence having one or more (preferably no more than 10 or 10, more preferably no more than 5, 4, 3, 2, 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence of SEQ ID NO: 30.

[0170] In some embodiments, the anti-CD28 antibodies or antigen-binding fragments thereof of the present invention comprise an antibody heavy chain. In some embodiments, the anti-CD28 antibodies or antigen-binding fragments thereof of the present invention comprise an antibody light chain. In some embodiments, the anti-CD28 antibodies or antigen-binding fragments thereof of the present invention comprise a heavy chain and a light chain. In some embodiments, the anti-CD28 antibodies or antigen-binding fragments thereof of the present invention comprise two heavy chains and two light chains. In some embodiments, the heavy chain comprises a heavy chain variable region and a heavy chain constant region, or consists of a heavy chain variable region and a heavy chain constant region. In some embodiments, the light chain comprises a light chain variable region and a light chain constant region, or consists of a light chain variable region and a light chain constant region.

[0171] In some embodiments, the anti-CD28 antibodies or antigen-binding fragments thereof of the invention have one or more of the following properties:

[0172] (i) exhibiting the same or similar binding affinity and / or specificity for CD28 as the antibodies of the invention;

[0173] (ii) inhibiting (e.g., competitively inhibiting) the binding of an antibody of the invention to CD28;

[0174] (iii) binds to the same or overlapping epitope as an antibody of the invention;

[0175] (iv) competing with the antibodies of the present invention for binding to CD28;

[0176] (v) possess one or more biological properties of an antibody of the invention.

[0177] In some embodiments, the anti-CD28 antibody is a monoclonal antibody.

[0178] In some embodiments, the anti-CD28 antibody is humanized. Humanization can be achieved by replacing one or more amino acid residues in the heavy chain variable region and light chain variable region of a non-human natural antibody, particularly the framework region sequence, with residues at corresponding positions in the variable region of a conventional human antibody. Methods for humanizing antibodies are well known in the art. Typically, humanizing substitutions are performed in a manner that maintains the favorable binding properties of the antibody. Tests for determining the biological properties of humanized antibodies, such as binding affinity, are well known in the art to determine and select suitable humanized residue mutations or combinations of mutations.

[0179] 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 encompasses antibody fragments thereof (e.g., antigen-binding fragments), preferably selected from the following antibody fragments: Fab, Fab', Fab'-SH, Fv, single-chain antibodies (e.g., scFv), (Fab')2, single-domain antibodies (e.g., VHH), dAb (domain antibody), heavy chain antibodies, or linear antibodies. In one embodiment, the anti-CD28 antibody fragment of the present invention is Fab, comprising VH and VL as described herein, as well as CH1 and CL.

[0180] 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 other antigens (eg, TAAs).

[0181] III. Multispecific Binding Molecules

[0182] In one aspect of the present invention, the present invention relates to multispecific binding molecules that are capable of specifically binding to CD28 and specifically binding to one or more other targets or antigens. In some embodiments, the other antigen is a tumor-associated antigen (TAA), such as CLDN18.2 or PSMA. In some embodiments, the multispecific binding molecule is a multispecific antibody, such as a bispecific antibody.

[0183] 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 an antigen or epitope and the second antigen binding region binds to another antigen or another epitope. Therefore, the bispecific antibody according to the present invention comprises specificity for two different antigens, or for two different epitopes of an antigen. The bispecific antibody format comprises IgG-like antibodies (Fan et al. (2015) Journal of Hematology & Oncology.8:130). The most common IgG-like antibody type comprises two Fab regions and two Fc regions, and the heavy chain and light chain of each Fab can be derived from a separate monoclonal antibody. The bispecific antibody of the present invention can be prepared using bispecific antibody formats or technologies known in the art. Specific exemplary bispecific formats that can be used in the context of the present invention are described, for example, in Labrijn, et al. Bispecific antibodies: a mechanistic review of the pipeline. Nature Reviews Drug Discovery, 2019, 18(8): 1-24.

[0184] Therefore, one aspect of the present invention relates to a bispecific antibody comprising

[0185] A first antigen binding region and a second antigen binding region, wherein the first antigen binding region specifically binds CD28 and the second antigen binding region specifically binds a tumor-associated antigen. In some embodiments, the tumor-associated antigen is selected from PSMA or CLDN18.2.

[0186] In some embodiments, the first antigen-binding region is from an anti-CD28 antibody or antigen-binding fragment thereof described herein, for example, a Fab of an 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 an anti-CD28 antibody described herein. In some embodiments, the first antigen-binding region comprises 1, 2, or 3 heavy chain variable region CDRs, i.e., CDRH1, CDRH2, and CDRH3, of an anti-CD28 antibody described herein. In some embodiments, the first antigen-binding region comprises 1, 2, or 3 light chain variable region CDRs, i.e., CDRL1, CDRL2, and CDRL3, of an anti-CD28 antibody described herein. In some embodiments, the first antigen-binding region comprises 3 heavy chain variable region CDRs and 3 light chain variable region CDRs of an anti-CD28 antibody described herein. In some embodiments, the first antigen-binding region comprises the heavy chain variable region of an anti-CD28 antibody described herein. In some embodiments, the first antigen-binding region comprises the light chain variable region of an 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 an anti-CD28 antibody described herein. In some embodiments, the first antigen binding region comprises a Fab of an anti-CD28 antibody described herein.

[0187] In some embodiments, the second antigen-binding region is from an anti-TAA (e.g., CLDN18.2 or PSMA, e.g., human CLDN18.2 or human PSMA) antibody or antigen-binding fragment thereof, e.g., an anti-CLDN18.2 antibody or antigen-binding fragment thereof, or an anti-PSMA antibody or antigen-binding fragment thereof.

[0188] The first antigen-binding region of the bispecific antibody suitable for use in the present invention may comprise or consist 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, including but not limited to, for example, full-length antibodies, half antibodies, Fab, Fab', Fab'-SH, Fv, single-chain antibodies (e.g., scFv), (Fab')2, single-domain antibodies (e.g., VHH), dAb (domain antibody), heavy chain antibodies, or linear antibodies that specifically bind to CD28.

[0189] The second antigen-binding region of the bispecific antibody suitable for use in the present invention may comprise an anti-TAA antibody or an antigen-binding fragment thereof, or consist of the same, as long as it can specifically bind to TAA, including but not limited to, for example, full-length antibodies, half antibodies, Fab, Fab', Fab'-SH, Fv, single-chain antibodies (e.g., scFv), (Fab')2, single-domain antibodies (e.g., VHH), dAb (domain antibody), heavy chain antibodies, or linear antibodies that specifically bind to TAA.

[0190] In some embodiments, the second antigen-binding region of the bispecific antibody suitable for use in the present invention may comprise an anti-CLDN18.2 antibody or an antigen-binding fragment thereof (e.g., the anti-CLDN18.2 antibody or antigen-binding fragment thereof disclosed in CN202010570517.X (WO2021254481A1) , such as the HB37A6 antibody or antigen-binding fragment thereof), or consist thereof, as long as it can specifically bind to CLDN18.2, including but not limited to, for example, full-length antibodies, half antibodies, Fab, Fab', Fab'-SH, Fv, single-chain antibodies (e.g., scFv), (Fab')2, single-domain antibodies (e.g., VHH), dAbs (domain antibodies), heavy chain antibodies, or linear antibodies that specifically bind to CLDN18.2.

[0191] In some embodiments, the second antigen-binding region of the bispecific antibody suitable for use in the present invention may comprise or consist of an anti-PSMA antibody or antigen-binding fragment thereof (e.g., the anti-PSMA antibody or antigen-binding fragment thereof disclosed in EP1851250B1, such as the 1C3 antibody or antigen-binding fragment thereof), as long as it can specifically bind to PSMA, including but not limited to, full-length antibodies, half antibodies, Fab, Fab', Fab'-SH, Fv, single-chain antibodies (e.g., scFv), (Fab')2, single-domain antibodies (e.g., VHH), dAbs (domain antibodies), heavy chain antibodies, or linear antibodies that specifically bind to PSMA.

[0192] In some embodiments, the bispecific antibodies of the present invention are IgG-like bispecific antibodies. In some embodiments, the IgG-like bispecific antibodies of the present invention refer to bispecific antibodies comprising an Fc dimer (e.g., a heterodimeric Fc scaffold). Therefore, in some embodiments, the bispecific antibodies of the present invention comprise an Fc dimer, such as a heterodimeric Fc scaffold.

[0193] 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.

[0194] III-1 Second antigen binding region

[0195] In some embodiments, the second antigen binding region specifically binds a tumor-associated antigen (TAA) or an immune checkpoint molecule.

[0196] In some embodiments, the tumor-associated antigen is CLDN18.2.

[0197] 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, such as an antibody that specifically binds to CLDN18.2 disclosed in CN202010570517.X (WO2021254481A1) (incorporated herein in its entirety), such as HB37A6 disclosed therein.

[0198] In some embodiments, the second antigen-binding region that specifically binds to CLDN18.2 comprises 1, 2, 3, 4, 5, or 6 CDRs of a known antibody that specifically binds to CLDN18.2 (e.g., the CLDN18.2 antibodies disclosed in CN202010570517.X (WO2021254481A1), such as HB37A6 disclosed therein). 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 a known antibody that specifically binds to CLDN18.2 (e.g., the CLDN18.2 antibodies disclosed in CN202010570517.X (WO2021254481A1), such as HB37A6 disclosed therein). 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 a known antibody that specifically binds to CLDN18.2 (e.g., the CLDN18.2 antibody disclosed in CN202010570517.X (WO2021254481A1), such as HB37A6 disclosed therein). In some embodiments, the second antigen-binding region comprises three heavy chain variable region CDRs and three light chain variable region CDRs of a known antibody that specifically binds to CLDN18.2 (e.g., the CLDN18.2 antibody disclosed in CN202010570517.X (WO2021254481A1), such as HB37A6 disclosed therein). In some embodiments, the second antigen-binding region comprises a heavy chain variable region of a known antibody that specifically binds to CLDN18.2 (e.g., the CLDN18.2 antibodies disclosed in CN202010570517.X (WO2021254481A1), such as HB37A6 disclosed therein). In some embodiments, the second antigen-binding region comprises a light chain variable region of a known antibody that specifically binds to CLDN18.2 (e.g., the CLDN18.2 antibodies disclosed in CN202010570517.X (WO2021254481A1), such as HB37A6 disclosed therein). In some embodiments, the second antigen-binding region comprises a heavy chain variable region and a light chain variable region of a known antibody that specifically binds to CLDN18.2 (e.g., the CLDN18.2 antibodies disclosed in CN202010570517.X (WO2021254481A1), such as HB37A6 disclosed therein). In some embodiments, the second antigen-binding region comprises a Fab of a known antibody that specifically binds to CLDN18.2 (e.g., an antibody that specifically binds to CLDN18.2 disclosed in CN202010570517.X (WO2021254481A1), such as HB37A6 disclosed therein).

[0199] In some embodiments, the second antigen-binding region that specifically binds to CLDN18.2 comprises three complementarity-determining regions (CDRHs) from the heavy chain variable region, CDRH1, CDRH2, and CDRH3, wherein the CDRH1, CDRH2, and CDRH3 are CDRH1, CDRH2, and CDRH3 of the heavy chain variable region as shown in SEQ ID NO: 86, preferably, wherein the CDRHs can be determined according to any scheme for determining CDRs, for example, according to the Kabat, AbM, Chothia, Contact, or IMGT schemes, or a combination thereof. In some embodiments, the second antigen-binding region that specifically binds to CLDN18.2 comprises three complementarity-determining regions (CDRLs) from the light chain variable region, CDRL1, CDRL2, and CDRL3, wherein the CDRL1, CDRL2, and CDRL3 are CDRL1, CDRL2, and CDRL3 of the light chain variable region as shown in SEQ ID NO: 90. Preferably, the CDRLs can be determined according to any scheme for determining CDRs, for example, according to the Kabat, AbM, Chothia, Contact, or IMGT schemes, or a combination thereof.

[0200] In some embodiments, the CDRH1 that specifically binds to the second antigen-binding region of CLDN18.2 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 / or CDRL3 comprises or consists of the amino acid sequence of SEQ ID NO: 93.

[0201] In some embodiments, the second antigen-binding region that specifically binds to CLDN18.2 comprises a VH, wherein the VH comprises

[0202] three complementarity determining regions (CDRHs) from a heavy chain variable region, 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

[0203] The amino acid sequence shown in SEQ ID NO: 86 or consists of it.

[0204] In some embodiments, the second antigen-binding region that specifically binds to CLDN18.2 comprises a VL, wherein the VL comprises

[0205] three complementarity determining regions (CDRHs) from a 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

[0206] The amino acid sequence shown in SEQ ID NO: 90 or consists of it.

[0207] In some embodiments, the second antigen-binding region that specifically binds to CLDN18.2 comprises three complementarity determining regions (CDRHs) from the heavy chain variable region, CDRH1, CDRH2, and CDRH3, and three complementarity 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.

[0208] In some embodiments, the second antigen-binding region that specifically binds to CLDN18.2 comprises VH and VL, wherein VH comprises or consists of the sequence shown in SEQ ID NO: 86; and / or VL comprises or consists of the sequence shown in SEQ ID NO: 90.

[0209] In some embodiments, the second antigen-binding region that specifically binds to CLDN18.2 is a Fab fragment.

[0210] 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, such as an antibody that specifically binds to PSMA disclosed in EP1851250B1, such as 1C3 disclosed therein.

[0211] In some embodiments, the second antigen-binding region that specifically binds to PSMA comprises 1, 2, 3, 4, 5, or 6 CDRs of a known antibody that specifically binds to PSMA (e.g., a PSMA antibody disclosed in EP1851250B1 (incorporated herein in its entirety), e.g., 1C3 disclosed therein). 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 a known antibody that specifically binds to PSMA (e.g., a PSMA antibody disclosed in EP1851250B1, e.g., 1C3 disclosed therein). In some embodiments, the second antigen-binding region comprises 1, 2, or 3 light chain variable region CDRs, i.e., CDRL1, CDRL2, and CDRL3, of a known antibody that specifically binds to PSMA (e.g., a PSMA antibody disclosed in EP1851250B1, e.g., 1C3 disclosed therein). In some embodiments, the second antigen-binding region comprises three heavy chain variable region CDRs and three light chain variable region CDRs of a known antibody that specifically binds to PSMA (e.g., a PSMA antibody disclosed in EP1851250B1, such as 1C3 disclosed therein). In some embodiments, the second antigen-binding region comprises a heavy chain variable region of a known antibody that specifically binds to PSMA (e.g., a PSMA antibody disclosed in EP1851250B1, such as 1C3 disclosed therein). In some embodiments, the second antigen-binding region comprises a light chain variable region of a known antibody that specifically binds to PSMA (e.g., a PSMA antibody disclosed in EP1851250B1, such as 1C3 disclosed therein). In some embodiments, the second antigen-binding region comprises a heavy chain variable region and a light chain variable region of a known antibody that specifically binds to PSMA (e.g., a PSMA antibody disclosed in EP1851250B1, such as 1C3 disclosed therein). In some embodiments, the second antigen-binding region comprises a Fab of a known antibody that specifically binds to PSMA (eg, an antibody that specifically binds to PSMA disclosed in EP1851250B1, such as 1C3 disclosed therein).

[0212] 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, wherein the CDRH1, CDRH2, and CDRH3 are CDRH1, CDRH2, and CDRH3 of the heavy chain variable region as set forth in SEQ ID NO: 47. Preferably, the CDRHs can be determined according to any CDR determination scheme, such as the Kabat, AbM, Chothia, Contact, or IMGT schemes, or a combination thereof. In some embodiments, the second antigen-binding region that specifically binds to PSMA comprises three complementary determining regions (CDRLs) from the light chain variable region, CDRL1, CDRL2, and CDRL3, wherein the CDRL1, CDRL2, and CDRL3 are CDRL1, CDRL2, and CDRL3 of the light chain variable region as set forth in SEQ ID NO: 49. Preferably, the CDRLs can be determined according to any CDR determination scheme, such as the Kabat, AbM, Chothia, Contact, or IMGT schemes, or a combination thereof. In some embodiments, the CDRH1 of the second antigen-binding region that specifically binds PSMA 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 / or CDRL3 comprises or consists of the amino acid sequence of SEQ ID NO:101.

[0213] In some embodiments, the second antigen-binding region that specifically binds to PSMA comprises a VH, wherein the VH comprises

[0214] three complementarity determining regions (CDRHs) from a heavy chain variable region, 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

[0215] The amino acid sequence shown in SEQ ID NO: 47 or consists of it.

[0216] In some embodiments, the second antigen-binding region that specifically binds PSMA comprises a VL, wherein the VL comprises

[0217] three complementarity determining regions (CDRHs) from a 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

[0218] The amino acid sequence shown in SEQ ID NO: 49 or consists of it.

[0219] In some embodiments, the second antigen-binding region that specifically binds PSMA comprises three complementarity determining regions (CDRHs) from a heavy chain variable region, CDRH1, CDRH2, and CDRH3, and three complementarity determining regions (CDRLs) from a 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.

[0220] In some embodiments, the second antigen-binding region that specifically binds PSMA comprises VH and VL, wherein VH comprises or consists of the sequence set forth in SEQ ID NO:47; and VL comprises or consists of the sequence set forth in SEQ ID NO:49.

[0221] In some embodiments, the second antigen binding region that specifically binds PSMA is a Fab fragment.

[0222] III-2Fab fragment

[0223] In some embodiments, the first antigen-binding region and / or the second antigen-binding region of the present invention is a Fab fragment. Suitable for use as a multispecific binding molecule of the present invention, such as a bispecific antibody antigen-binding region, is composed of two polypeptide chains comprising the VH, CH1, VL, and CL domains of an antibody, wherein the VH is paired with the VL and the CH1 is paired with the CL to form an antigen-binding region. In some embodiments, in Fab, one chain comprises VH and CH1 (i.e., VH-CH1) or consists of VH and CH1 from N-terminus to C-terminus, and the other chain comprises VL and CL (i.e., VL-CL) or consists of VL and CL from N-terminus to C-terminus. In some embodiments, in the multispecific antibodies of the present invention, Fab can be connected to the N-terminus of the Fc domain of the antibody through the C-terminus of the chain comprising VH. Preferably, the Fab comprises a VH-CH1 chain and a VL-CL chain and can be connected to the Fc domain of the antibody through the C-terminus of the CH1 of the VH-CH1 chain. In some embodiments, the connection is a direct connection or a connection via a linker. Herein, the Fab chain comprising VH-CH1 or consisting of VH-CH1 is also referred to as a Fab heavy chain, and the Fab chain comprising or consisting of VL-CL is also referred to as a Fab light chain.

[0224] In some embodiments, the CH1 is a CH1 from IgG1, IgG2, IgG3 or IgG4, preferably a CH1 from IgG1.

[0225] (i) comprising or consisting of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 74;

[0226] (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 74; or

[0227] (iii) comprises or consists of an amino acid sequence having one or more (preferably no more than 10 or 10, more preferably no more than 5, 4, 3, 2, 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence of SEQ ID NO: 74.

[0228] In some embodiments, the CL is a kappa light chain constant region or a lambda light chain constant region. In some embodiments, the CL is a kappa light chain constant region. In some embodiments, the CL

[0229] (i) comprising or consisting of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 30;

[0230] (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 30; or

[0231] (iii) comprises or consists of an amino acid sequence having one or more (preferably no more than 10 or 10, more preferably no more than 5, 4, 3, 2, 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence of SEQ ID NO: 30.

[0232] In some embodiments, the first antigen-binding region is a Fab that specifically binds to CD28, wherein the Fab fragment is derived from an anti-CD28 antibody of the present invention and comprises the heavy chain variable region VH and the light chain variable region VL of an 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 VH and CH1, or consists of VH and CH1, wherein VH is the VH of an 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 VL and CL, or consists of VL and CL, wherein VL is the VL of an anti-CD28 antibody of the present invention.

[0233] In some embodiments, the Fab heavy chain that specifically binds to CD28 comprises

[0234] (i) comprising or consisting of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 103, 104 or 105;

[0235] (ii) comprising or consisting of the amino acid sequence of SEQ ID NO: 103, 104 or 105; or

[0236] (iii) comprises an amino acid sequence having one or more (preferably no more than 10 or 10, more preferably no more than 5, 4, 3, 2, 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence of SEQ ID NO: 103, 104 or 105, or consists of said amino acid sequence.

[0237] In some embodiments, the Fab light chain that specifically binds CD28 comprises

[0238] (i) comprising or consisting of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 36, 38 or 40;

[0239] (ii) comprising or consisting of the amino acid sequence of SEQ ID NO: 36, 38 or 40; or

[0240] (iii) comprises an amino acid sequence having one or more (preferably no more than 10 or 10, more preferably no more than 5, 4, 3, 2, 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence of SEQ ID NO: 36, 38 or 40, or consists of said amino acid sequence.

[0241] In some embodiments, the Fab heavy chain that specifically binds to CD28 comprises or consists of the amino acid sequence shown in SEQ ID NO: 103, and the Fab light chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 36;

[0242] In some embodiments, the Fab heavy chain that specifically binds to CD28 comprises or consists of the amino acid sequence shown in SEQ ID NO: 104, and the Fab light chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 38; or

[0243] In some embodiments, the Fab heavy chain that specifically binds to CD28 comprises or consists of the amino acid sequence shown in SEQ ID NO: 105, and the Fab light chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 40.

[0244] In some embodiments, the second antigen-binding region is a Fab that specifically binds to TAA, wherein the Fab fragment is from an anti-TAA antibody, which comprises the heavy chain variable region VH and the light chain variable region VL of an anti-TAA antibody (e.g., an anti-CLDN18.2 antibody or an 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 from an anti-CLDN18.2 antibody, which 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 from an anti-PSMA antibody, which comprises the heavy chain variable region VH and the light chain variable region VL of the anti-PSMA antibody.

[0245] In some embodiments, the Fab as the second antigen-binding region comprises the VH or VL of the second antigen-binding region described herein, or comprises the VH and VL of the second antigen-binding region described herein.

[0246] In some embodiments, the Fab heavy chain that specifically binds to CLDN18.2 comprises

[0247] (i) comprising or consisting of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 102;

[0248] (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 102; or

[0249] (iii) comprises or consists of an amino acid sequence having one or more (preferably no more than 10 or 10, more preferably no more than 5, 4, 3, 2, 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence of SEQ ID NO: 102.

[0250] In some embodiments, the Fab light chain that specifically binds to CLDN18.2 comprises

[0251] (i) comprising or consisting of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 34;

[0252] (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 34; or

[0253] (iii) comprises an amino acid sequence having one or more (preferably no more than 10 or 10, more preferably no more than 5, 4, 3, 2, 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence of SEQ ID NO: 34, or consists of said amino acid sequence.

[0254] In some embodiments, the Fab heavy chain that specifically binds to CLDN18.2 comprises or consists of the amino acid sequence shown in SEQ ID NO: 102, and the Fab light chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 34.

[0255] In some embodiments, the Fab heavy chain that specifically binds PSMA comprises

[0256] (i) comprising or consisting of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 106;

[0257] (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 106; or

[0258] (iii) comprises an amino acid sequence having one or more (preferably no more than 10 or 10, more preferably no more than 5, 4, 3, 2, 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence of SEQ ID NO: 106, or consists of said amino acid sequence.

[0259] In some embodiments, the Fab light chain that specifically binds PSMA comprises

[0260] (i) comprising or consisting of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 50;

[0261] (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 50; or

[0262] (iii) comprises or consists of an amino acid sequence having one or more (preferably no more than 10 or 10, more preferably no more than 5, 4, 3, 2, 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence of SEQ ID NO: 50.

[0263] In some embodiments, the Fab heavy chain that specifically binds to PSMA comprises or consists of the amino acid sequence set forth in SEQ ID NO: 106, and the Fab light chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 50.

[0264] III-3 Fc region

[0265] In some embodiments, one or more target binding regions (antigen binding regions) of a multispecific binding molecule of the invention, such as a multispecific antibody (eg, a bispecific antibody), further comprise an Fc region, wherein the Fc regions comprised may be the same or different.

[0266] In some embodiments, the first and second Fc regions are different and are capable of dimerizing to form a heterodimeric Fc scaffold.

[0267] In this article, Fc district refers to the C-terminal region of the immunoglobulin heavy chain containing the constant region of at least a portion, and can include native sequence Fc district and variant Fc district.Native sequence F district covers naturally occurring various immunoglobulin Fc sequences, such as various Ig subtypes and the Fc district (Gestur Vidarsson et al., IgG subclasses and allotypes:from structure to effector functions, 20 October 2014, doi:10.3389 / fimmu.2014.00520) of its allotype.In some embodiments, Fc district of the present invention comprises antibody CH2 and CH3.In some embodiments, antibody Fc district can also carry IgG hinge region or part IgG hinge region at N end, such as, IgG1 hinge region or part IgG1 hinge region, such as according to EU numbering, the sequence of D221 to P230.In the hinge region, can contain sudden change.

[0268] Unless otherwise indicated herein, the numbering of amino acid residues in the Fc region is 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 Edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991), NIH Publication 91-3242.

[0269] In some embodiments, the Fc region is a 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 consists of an amino acid sequence of SEQ ID NO: 94 or 95, or an amino acid sequence having at least 90% identity thereto, e.g., 95%, 96%, 97%, 98%, 99% or higher identity thereto.

[0270] As will be appreciated by those skilled in the art, to promote the formation of heterodimers of the multispecific antibodies of the present invention, the Fc regions comprised by the multispecific antibodies of the present invention may include mutations that facilitate heterodimerization of the first Fc region and the second Fc region. In one embodiment, mutations are introduced into the CH3 regions of both Fc regions.

[0271] Methods for promoting heterodimerization of the Fc region are known in the art. For example, the CH3 region of the first Fc region and the CH3 region of the second Fc region are engineered in a complementary manner so that each CH3 region (or a heavy chain comprising it) can no longer homodimerize with itself but is forced to heterodimerize with other CH3 regions of complementary engineering (so that the CH3 regions of the first and second Fc regions heterodimerize and no homodimer is formed between the two first CH3 regions or the two second CH3 regions). For example, based on the Knob-into-Hole technology, corresponding Knob mutations and Hole mutations are introduced into the first Fc region and the second Fc region. This technology is described in, for example, US 5,731,168; US 7,695,936; Ridgway et al., Prot Eng 9, 617-621 (1996) and Carter, J Immunol Meth 248, 7-15 (2001).

[0272] Corresponding mutations can also be introduced into the first and second Fc regions based on the Innobody technology. For this technology, see, for example, PCT / CN2021 / 143141 (the patent is incorporated herein in its entirety).

[0273] In a specific embodiment,

[0274] The first CH3 region comprises an S364R / K mutation (preferably S364R), and optionally one or more other mutations. In some embodiments, the second CH3 region comprises a K370S / T / A / V mutation (preferably K370S), and optionally one or more other 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.

[0275] In some embodiments, the first CH3 region comprises S364R / K (preferably S364R) and D399K / R (preferably D399K) mutations. In some embodiments, the second CH3 region comprises K370S / T / A / V (preferably K370S) mutations and K409D / E (preferably K409D) mutations. 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).

[0276] In some embodiments, the first CH3 region comprises S364R + D399K, and the second CH3 region comprises K370S + Y349T + K409D. In some embodiments, the first CH3 region further comprises E357N. In some embodiments, the second CH3 region further comprises Q347D. In some embodiments, the first CH3 region further comprises E357N, and the second CH3 region further comprises Q347D. In some embodiments, the first CH3 region and the second CH3 region further comprise T350V, or both comprise T350V.

[0277] Thus, 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.

[0278] 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.

[0279] In some specific embodiments, the first and second CH3 regions have the following five mutation combinations:

[0280] In one embodiment, the CH3 of one Fc region comprises S364R and D399K mutations, and the CH3 mutations of the other Fc region comprise Y349T, K370S, and K409D mutations.

[0281] In some embodiments, the Fc region further comprises other mutations that facilitate purification of the heterodimer.

[0282] The Fc region of the binding molecules of the invention, such as antibodies, can also be mutated to obtain desired properties. Mutations in the Fc region are known in the art.

[0283] In one embodiment, the Fc region is modified with respect to the properties of the effector function of the Fc region (e.g., the complement activation function of the Fc region). In one embodiment, the effector function has been reduced or eliminated relative to a wild-type Fc region. In one embodiment, the effector function is reduced or eliminated by a method selected from the group consisting of: using an Fc isotype that naturally has reduced or eliminated effector function, and Fc region modification.

[0284] In a preferred embodiment, the Fc region has reduced effector function mediated by the Fc region, such as reduced or abolished ADCC or ADCP or CDC effector function, eg, comprises a mutation that achieves the above function.

[0285] As will be appreciated by those skilled in the art, according to the intended use of the binding molecules of the present invention, for example, antibody molecules, the binding molecules of the present invention, for example, antibody molecules, may also include modifications in the Fc domain that alter the binding affinity to one or more Fc receptors. In one embodiment, the Fc receptor is an Fcγ receptor, particularly a human Fcγ receptor. In some embodiments, the Fc region comprises a mutation that reduces binding to Fcγ receptors. For example, in some embodiments, the Fc region for use in the present invention has a mutation that reduces binding to Fcγ receptors, such as L234A / L235A mutations or a proline deletion at position P329 (P329delP). In another preferred embodiment, the Fc fragment may have a mutation that results in increased serum half-life, such as a mutation that improves the binding of the Fc fragment to FcRn.

[0286] Thus, in a specific embodiment, an antibody of the invention, such as a multispecific antibody, comprises two Fc regions that heterodimerize, wherein

[0287] One Fc-region polypeptide comprises the mutations S364R and D399K, while the other Fc-region polypeptide comprises the mutations Y349T, K370S and K409D.

[0288] In some embodiments, the Fc region further comprises L234A / L235A mutations, respectively, and optionally a proline deletion at position P329 (P329delP).

[0289] Thus, in a specific embodiment, an antibody of the invention, such as a multispecific antibody, comprises two Fc regions that heterodimerize, wherein

[0290] a) one Fc region comprises or consists of the amino acid sequence shown in SEQ ID NO: 80, and the other Fc region polypeptide comprises or consists of the amino acid sequence shown in SEQ ID NO: 81; or

[0291] b) one Fc region comprises or consists of the amino acid sequence shown in SEQ ID NO: 82, and the other Fc region polypeptide comprises or consists of the amino acid sequence shown in SEQ ID NO: 83.

[0292] In a specific embodiment, an antibody of the invention, such as a multispecific antibody, comprises two Fc regions that heterodimerize, wherein

[0293] a) one Fc region comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence set forth in SEQ ID NO: 80, and the other Fc region polypeptide comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence set forth in SEQ ID NO: 81; or

[0294] b) one Fc region comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO:82, and the other Fc region polypeptide comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO:83.

[0295] Thus, in a specific embodiment, a binding molecule of the invention, such as an antibody, such as a multispecific antibody, comprises two Fc regions that heterodimerize, wherein

[0296] a) one Fc region comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence set forth in SEQ ID NO: 80 and comprises the mutations S364R and D399K, and the other Fc region polypeptide comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence set forth in SEQ ID NO: 81 and comprises the mutations Y349T, K370S, and K409D;

[0297] b) one Fc region comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence of SEQ ID NO:80 and comprises mutations S364R and D399K and L234A / L235A mutations and P329delP, and the other Fc region polypeptide comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence of SEQ ID NO:81 and comprises mutations Y349T, K370S and K409D and L234A / L235A mutations and P329delP;

[0298] c) one Fc region comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence set forth in SEQ ID NO: 82 and comprises the mutations S364R and D399K, and the other Fc region polypeptide comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence set forth in SEQ ID NO: 83 and comprises the mutations Y349T, K370S, and K409D; or

[0299] d) one Fc region comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the sequence of amino acids set forth in SEQ ID NO:82 and comprises mutations S364R and D399K and L234A / L235A mutations, and the other Fc region polypeptide comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the sequence of amino acids set forth in SEQ ID NO:83 and comprises mutations Y349T, K370S and K409D and L234A / L235A mutations.

[0300] III-4 Exemplary bispecific antibodies

[0301] In some embodiments, the bispecific antibodies of the present invention comprise a first antigen-binding region as described herein that specifically binds to a first antigen and a second antigen-binding region as described herein that specifically binds to a second antigen, 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 at the C-terminus of its CH1 to the N-terminus of the first Fc region (via or without a linker, such as a hinge region), and the second Fab is connected at the C-terminus of its CH1 to the N-terminus of the second Fc region (via or without a linker, such as a hinge region). In a specific embodiment, the first Fab and the first Fc region constitute a first half antibody that binds to the first antigen, the second Fab and the second Fc region constitute a second half antibody that binds to the second antigen, and the first half antibody and the second half antibody constitute the bispecific antibody of the present invention.

[0302] In a specific embodiment, the bispecific antibody is an IgG-like antibody having a configuration as shown in Figure 3. In a specific embodiment, the first Fc region and the second Fc region comprise an Innobody mutation, and optionally a mutation that reduces effector function mediated by the Fc region or a mutation that reduces binding to Fcγ receptors. In one embodiment, the bispecific antibody comprises or consists of:

[0303] Heavy chain 1: comprises or consists of the following from N-terminus to C-terminus: a first Fab heavy chain variable region-a heavy chain constant region CH1-a first Fc region, wherein the heavy chain constant region CH1 is connected at its C-terminus to the N-terminus of the first Fc region via or without a linker (e.g., a hinge region);

[0304] Light chain 1: from N-terminus to C-terminus, comprises or consists of: the light chain variable region of the first Fab - the light chain constant region;

[0305] Heavy chain 2: comprises or consists of the following from N-terminus to C-terminus: a second Fab heavy chain variable region - a heavy chain constant region CH1 - a second Fc region, wherein the heavy chain constant region CH1 is connected at its C-terminus to the N-terminus of the second Fc region via or without a linker (e.g., a hinge region);

[0306] Light chain 2: From N-terminus to C-terminus, it comprises the following: light chain variable region of the second Fab - light chain constant region;

[0307] Preferably, the domains are directly linked to each other.

[0308] In some embodiments, the Fab heavy chain variable region and the heavy chain constant region CH1 constitute the Fab heavy chain, that is, the C-terminus of the Fab heavy chain variable region is connected to the N-terminus of the CH1 of the heavy chain constant region (directly connected). In some embodiments, the Fab light chain variable region and the light chain constant region constitute the Fab light chain, that is, the C-terminus of the Fab light chain variable region is directly connected to the N-terminus of the light chain constant region).

[0309] In a specific embodiment, the first Fab is a Fab of an anti-CD28 antibody of the invention and the second Fab is a Fab of an anti-TAA antibody, or vice versa.

[0310] In a specific embodiment, the first Fab is a Fab of an anti-CD28 antibody of the invention and the second Fab is a Fab of an anti-CLDN18.2 antibody, or vice versa.

[0311] In a specific embodiment, the bispecific antibody of the invention specifically binds to CD28 and CLDN18.2, and comprises or consists of:

[0312] Heavy chain 1: comprises or consists of the following from N-terminus to C-terminus: a Fab heavy chain variable region that specifically binds to CD28 - a heavy chain constant region CH1 - a first Fc region, wherein the heavy chain constant region CH1 is connected at its C-terminus to the N-terminus of the first Fc region via or without a linker (e.g., a hinge region);

[0313] Light chain 1: From N-terminus to C-terminus, it comprises or consists of: a light chain variable region of a Fab that specifically binds to CD28 - a light chain constant region;

[0314] Heavy chain 2: comprising or consisting of, from N-terminus to C-terminus: a Fab heavy chain variable region that specifically binds to CLDN18.2 - a heavy chain constant region CH1 - a second Fc region, wherein the heavy chain constant region CH1 is connected at its C-terminus to the N-terminus of the second Fc region via or without a linker (e.g., a hinge region); and / or

[0315] Light chain 2: From N-terminus to C-terminus, it comprises the following: a light chain variable region of a Fab that specifically binds to CLDN18.2 - a light chain constant region,

[0316] Optionally, the first Fc region comprises mutations Y349T, K370S and K409D, and the second Fc region comprises S364R and D399K, or vice versa; optionally, the first and second Fc regions further comprise L234A / L235A mutations, respectively, and optionally P329delP mutation;

[0317] Preferably, the domains are directly linked to each other.

[0318] In some specific embodiments, the 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

[0319] Heavy chain 1 comprises or consists of the amino acid sequence of SEQ ID NO: 35, 37 or 39, or an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto;

[0320] Light chain 1 comprises or consists of the amino acid sequence of SEQ ID NO: 36, 38, or 40, or an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto;

[0321] Heavy chain 2 comprises or consists of the amino acid sequence of SEQ ID NO: 33, or an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto; and / or

[0322] Light chain 2 comprises the amino acid sequence shown in SEQ ID NO: 34, or an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto, or consists of the amino acid sequence.

[0323] In some specific embodiments, the 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

[0324] Heavy chain 1 comprises, or consists of, the amino acid sequence of SEQ ID NO:35, or an amino acid sequence at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto; light chain 1 comprises, or consists of, the amino acid sequence of SEQ ID NO:36, or an amino acid sequence at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto; heavy chain 2 comprises, or consists of, the amino acid sequence of SEQ ID NO:33, or an amino acid sequence at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto; and light chain 2 comprises, or consists of, the amino acid sequence of SEQ ID NO:34, or an amino acid sequence at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto;

[0325] heavy chain 1 comprises, or consists of, the amino acid sequence of SEQ ID NO:37, or an amino acid sequence at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto; light chain 1 comprises, or consists of, the amino acid sequence of SEQ ID NO:38, or an amino acid sequence at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto; heavy chain 2 comprises, or consists of, the amino acid sequence of SEQ ID NO:33, or an amino acid sequence at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto; and light chain 2 comprises, or consists of, the amino acid sequence of SEQ ID NO:34, or an amino acid sequence at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto; or

[0326] Heavy chain 1 comprises, or consists of, the amino acid sequence of SEQ ID NO:39, or an amino acid sequence at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto; light chain 1 comprises, or consists of, the amino acid sequence of SEQ ID NO:40, or an amino acid sequence at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto; heavy chain 2 comprises, or consists of, the amino acid sequence of SEQ ID NO:33, or an amino acid sequence at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto; and light chain 2 comprises, or consists of, the amino acid sequence of SEQ ID NO:34, or an amino acid sequence at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0327] In some specific embodiments, the 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

[0328] Heavy chain 1 comprises or consists of the amino acid sequence of SEQ ID NO: 35; light chain 1 comprises or consists of the amino acid sequence of SEQ ID NO: 36; heavy chain 2 comprises or consists of the amino acid sequence of SEQ ID NO: 33; and light chain 2 comprises or consists of the amino acid sequence of SEQ ID NO: 34;

[0329] Heavy chain 1 comprises or consists of the amino acid sequence of SEQ ID NO: 37; light chain 1 comprises or consists of the amino acid sequence of SEQ ID NO: 38; heavy chain 2 comprises or consists of the amino acid sequence of SEQ ID NO: 33; and light chain 2 comprises or consists of the amino acid sequence of SEQ ID NO: 34; or

[0330] Heavy chain 1 comprises or consists of the amino acid sequence shown in SEQ ID NO:39; light chain 1 comprises or consists of the amino acid sequence shown in SEQ ID NO:40; heavy chain 2 comprises or consists of the amino acid sequence shown in SEQ ID NO:33; and light chain 2 comprises or consists of the amino acid sequence shown in SEQ ID NO:34.

[0331] In a specific embodiment, the first Fab is an anti-CD28 Fab of the invention and the second Fab is an anti-PSMA Fab, or vice versa.

[0332] In a specific embodiment, the bispecific antibody of the invention specifically binds to CD28 and PSMA and comprises or consists of:

[0333] Heavy chain 1: comprises or consists of the following from N-terminus to C-terminus: a Fab heavy chain variable region that specifically binds to CD28 - a heavy chain constant region CH1 - a first Fc region, wherein the heavy chain constant region CH1 is connected at its C-terminus to the N-terminus of the first Fc region via or without a linker (e.g., a hinge region);

[0334] Light chain 1: From N-terminus to C-terminus, it comprises or consists of: a light chain variable region of a Fab that specifically binds to CD28 - a light chain constant region;

[0335] Heavy chain 2: comprising or consisting of the following from N-terminus to C-terminus: a heavy chain variable region of a Fab that specifically binds to PSMA - a heavy chain constant region CH1 - a second Fc region, wherein the heavy chain constant region CH1 is connected at its C-terminus to the N-terminus of the second Fc region via or without a linker (e.g., a hinge region); and / or

[0336] Light chain 2: From N-terminus to C-terminus, it comprises the following: a light chain variable region of a Fab that specifically binds to PSMA - a light chain constant region,

[0337] Optionally, the first Fc region comprises mutations Y349T, K370S and K409D, and the second Fc region comprises S364R and D399K, or vice versa; optionally, the first and second Fc regions further comprise L234A / L235A mutations, respectively, and optionally P329delP mutation;

[0338] Preferably, the domains are directly linked to each other.

[0339] In some specific embodiments, the bispecific antibody that specifically binds CD28 and PSMA comprises or consists of heavy chain 1, heavy chain 2, light chain 1, and light chain 2, wherein

[0340] Heavy chain 1 comprises or consists of the amino acid sequence of SEQ ID NO: 51, 52 or 53, or an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto;

[0341] Light chain 1 comprises or consists of the amino acid sequence of SEQ ID NO: 36, 38, or 40, or an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto;

[0342] Heavy chain 2 comprises or consists of the amino acid sequence of SEQ ID NO: 48, or an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto; and / or

[0343] Light chain 2 comprises the amino acid sequence shown in SEQ ID NO: 50, or an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto, or consists of the amino acid sequence.

[0344] In some specific embodiments, the bispecific antibody that specifically binds CD28 and PSMA comprises or consists of heavy chain 1, heavy chain 2, light chain 1, and light chain 2, wherein

[0345] heavy chain 1 comprises, or consists of, the amino acid sequence of SEQ ID NO:51, or an amino acid sequence at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto; light chain 1 comprises, or consists of, the amino acid sequence of SEQ ID NO:36, or an amino acid sequence at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto; heavy chain 2 comprises, or consists of, the amino acid sequence of SEQ ID NO:48, or an amino acid sequence at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto; and light chain 2 comprises, or consists of, the amino acid sequence of SEQ ID NO:50, or an amino acid sequence at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto;

[0346] heavy chain 1 comprises, or consists of, the amino acid sequence of SEQ ID NO:52, or an amino acid sequence at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto; light chain 1 comprises, or consists of, the amino acid sequence of SEQ ID NO:38, or an amino acid sequence at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto; heavy chain 2 comprises, or consists of, the amino acid sequence of SEQ ID NO:48, or an amino acid sequence at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto; and light chain 2 comprises, or consists of, the amino acid sequence of SEQ ID NO:50, or an amino acid sequence at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto; or

[0347] Heavy chain 1 comprises, or consists of, the amino acid sequence of SEQ ID NO:53, or an amino acid sequence at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto; light chain 1 comprises, or consists of, the amino acid sequence of SEQ ID NO:40, or an amino acid sequence at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto; heavy chain 2 comprises, or consists of, the amino acid sequence of SEQ ID NO:48, or an amino acid sequence at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto; and light chain 2 comprises, or consists of, the amino acid sequence of SEQ ID NO:50, or an amino acid sequence at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0348] In some specific embodiments, the bispecific antibody that specifically binds CD28 and PSMA comprises or consists of heavy chain 1, heavy chain 2, light chain 1, and light chain 2, wherein

[0349] Heavy chain 1 comprises or consists of the amino acid sequence of SEQ ID NO:51; light chain 1 comprises or consists of the amino acid sequence of SEQ ID NO:36; heavy chain 2 comprises or consists of the amino acid sequence of SEQ ID NO:48; and light chain 2 comprises or consists of the amino acid sequence of SEQ ID NO:50;

[0350] Heavy chain 1 comprises or consists of the amino acid sequence of SEQ ID NO: 52; light chain 1 comprises or consists of the amino acid sequence of SEQ ID NO: 38; heavy chain 2 comprises or consists of the amino acid sequence of SEQ ID NO: 48; and light chain 2 comprises or consists of the amino acid sequence of SEQ ID NO: 50; or

[0351] Heavy chain 1 comprises or consists of the amino acid sequence shown in SEQ ID NO:53; light chain 1 comprises or consists of the amino acid sequence shown in SEQ ID NO:40; heavy chain 2 comprises or consists of the amino acid sequence shown in SEQ ID NO:48; and light chain 2 comprises or consists of the amino acid sequence shown in SEQ ID NO:50.

[0352] The various components / domains of multispecific binding molecules, such as bispecific antibodies, described herein are as described herein. Those skilled in the art will appreciate that, unless the context clearly indicates otherwise, any combination of any of the technical features of the various components of multispecific binding molecules, such as bispecific antibodies, described herein is within the scope of the present invention. Furthermore, those skilled in the art will appreciate that, unless the context clearly indicates otherwise, the antibodies of the present invention (including any form of antibodies) may comprise any such combination.

[0353] IV. Polynucleotides, Vectors, and Host Cells

[0354] The present invention provides nucleic acids encoding any of the above antibody molecules of the present invention (eg, an anti-CD28 antibody or antigen-binding fragment thereof or a multispecific antibody such as a bispecific antibody of the present invention).

[0355] In one aspect, the invention provides nucleic acids encoding any of the above anti-CD28 antibodies or antigen-binding fragments thereof or multispecific antibodies such as bispecific antibodies.

[0356] To facilitate production and purification, anti-CD28 antibodies or antigen-binding fragments thereof or multispecific antibodies such as bispecific antibodies can be fused to a secretory signal peptide at the N-terminus or C-terminus (e.g., the C-terminus), and / or a tag peptide that facilitates purification, such as a hexahistidine tag or a biotin tag.

[0357] As will be apparent to those skilled in the art, because of codon degeneracy, each antibody or polypeptide amino acid sequence can be encoded by multiple nucleic acid sequences.

[0358] In some embodiments, a nucleic acid of the invention comprises a nucleic acid that encodes 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, or 102-106, or a nucleic acid that encodes an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to 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, or 102-106.

[0359] Nucleic acid sequences encoding molecules of the present invention can be generated using methods well known in the art, such as de novo solid phase DNA synthesis or PCR amplification.

[0360] In one aspect, the present invention also provides a vector comprising a nucleic acid of the present invention. In one embodiment, the vector is an expression vector, such as a prokaryotic expression vector or a eukaryotic expression vector. Vectors include but are not limited to viruses, plasmids, cosmids, lambda phages, or yeast artificial chromosomes (YACs). In a preferred embodiment, the expression vector is pCDNA, such as pCDNA3.1.

[0361] In one aspect, the present invention also provides a host cell comprising the nucleic acid or the vector. In one embodiment, the host cell is eukaryotic. In another embodiment, the host cell is selected from yeast cells, 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 prokaryotic.

[0362] Suitable host cells include prokaryotic microorganisms such as Escherichia coli, eukaryotic microorganisms such as filamentous fungi or yeast, or various eukaryotic cells such as Chinese hamster ovary cells (CHO), insect cells, etc. Mammalian cell lines suitable for suspension culture can be used. Examples of useful mammalian host cell lines include SV40-transformed monkey kidney CV1 line (COS-7); human embryonic kidney line (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 carcinoma cells (HELA), canine kidney cells (MDCK), Buffalo rat liver cells (BRL 3A), human lung cells (W138), human liver cells (Hep G2), CHO cells, NSO cells, myeloma cell lines such as YO, NSO, P3X63 and Sp2 / 0, etc. Mammalian host cell lines suitable for producing antibodies 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.

[0363] V. Production and Purification of the Molecules of the Invention

[0364] 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 antigen-binding fragment thereof or a multispecific antibody such as a bispecific antibody), the method comprising: culturing a host cell containing a protein encoding the polypeptide chain under conditions suitable for expression of the polypeptide chain of the molecule; optionally further comprising assembling the polypeptide chains to produce the molecule under conditions suitable for assembly of the polypeptide chains into the molecule.

[0365] For recombinant production, the polynucleotide encoding the polypeptide chain of the molecule of the present invention can be inserted into one or more vectors for further cloning and / or expression in a host cell. Methods well known to those skilled in the art can be used to construct expression vectors. Expression vectors include, but are not limited to, viruses, plasmids, cosmids, lambda phages, or yeast artificial chromosomes (YACs). Once an expression vector comprising one or more polynucleotides of the present invention has been prepared for expression, the expression vector can be transfected or introduced into a suitable host cell. A variety of techniques can be used to achieve this purpose, for example, protoplast fusion, calcium phosphate precipitation, electroporation, retroviral transduction, viral transfection, gene guns, liposome-based transfection or other conventional techniques.

[0366] The molecules prepared as described herein can be purified by known techniques such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography (e.g., Protein A affinity chromatography), size exclusion chromatography, etc. The actual conditions used to purify a particular protein will also depend on factors such as net charge, hydrophobicity, hydrophilicity, etc., and these will be apparent to those skilled in the art.

[0367] The purity of the molecules of the invention can be determined by any of a variety of well-known analytical methods, including size exclusion chromatography, gel electrophoresis, high performance liquid chromatography, etc. The physical / chemical properties and / or biological activities of the antibody molecules provided herein can be identified, screened or characterized by a variety of assays known in the art.

[0368] VI. Assay

[0369] The molecules provided herein (e.g., anti-CD28 antibodies or antigen-binding fragments thereof or multispecific antibodies such as bispecific antibodies) can be identified, screened, or characterized for their physical / chemical properties and / or biological activity by various assays known in the art. The Examples illustrate methods for determining the properties of the anti-CD28 antibodies or antigen-binding fragments thereof or bispecific antibodies of the invention, such as biofilm thin layer interferometry (BLI), T cell reporter gene assays, T cell activation assays, T cell mediated killing assays, or anti-tumor activity assays.

[0370] VII. Immunoconjugates

[0371] In one aspect, the invention provides immunoconjugates comprising a molecule of the invention (e.g., an anti-CD28 antibody or antigen-binding fragment thereof or a multispecific antibody such as a bispecific antibody) and one or more other active ingredients (e.g., an active ingredient from a drug or therapeutic agent for treating a disease of the invention, such as a small molecule that enhances the therapeutic effect of the molecule of the invention).

[0372] In some embodiments, the immunoconjugate is an antibody-drug-conjugate (ADC).

[0373] VIII. Drugs and Uses

[0374] In some embodiments, the present invention also relates to compositions (eg, pharmaceutical compositions or pharmaceutical formulations) comprising a molecule of the invention (eg, an anti-CD28 antibody or antigen-binding fragment thereof or a multispecific antibody such as a bispecific antibody, or an immunoconjugate).

[0375] In one embodiment, the composition further comprises a pharmaceutical excipient, such as a pharmaceutical carrier, a pharmaceutical excipient, including a buffer, as known in the art. In one embodiment, a composition, such as a pharmaceutical composition, comprises a molecule of the invention in combination with one or more other therapeutic agents.

[0376] The compositions of the present invention can also include suitable pharmaceutical excipients, such as pharmaceutical carriers and pharmaceutical excipients as known in the art, including buffers. As used herein, "pharmaceutical carriers" include any and all solvents, dispersion media, isotonic agents or absorption delay agents that are physiologically compatible. For the use of pharmaceutical excipients and their uses, see also "Handbook of Pharmaceutical Excipients", Eighth Edition, RC Rowe, PJ Eskey and SCOwen, Pharmaceutical Press, London, Chicago. The compositions of the present invention can be in various forms. These forms, for example, include liquid, semisolid and solid dosage forms, such as liquid solutions (for example, injectable solutions and infusible solutions), powders or suspensions, liposomes and suppositories. The preferred form depends on the intended mode of administration and therapeutic use.

[0377] A medicament comprising an antibody molecule or immunoconjugate described herein can be prepared by mixing an antibody molecule (e.g., an anti-CD28 antibody or antigen-binding fragment thereof or a multispecific antibody such as a bispecific antibody) or immunoconjugate of the invention having the desired degree of purity with one or more optional pharmaceutical excipients, for example in the form of a lyophilized formulation or an aqueous solution.

[0378] In one aspect, the present invention provides a method for preventing or treating a disease in a subject, comprising administering to the subject an effective amount of a molecule of the invention (e.g., an anti-CD28 antibody or antigen-binding fragment thereof or a multispecific antibody such as a bispecific antibody or immunoconjugate thereof), pharmaceutical composition, pharmaceutical combination, or kit. In some embodiments, the disease is, for example, a tumor such as cancer.

[0379] In some embodiments, the invention relates to molecules of the invention (e.g., anti-CD28 antibodies or antigen-binding fragments thereof or multispecific antibodies such as bispecific antibodies or immunoconjugates thereof of the invention) for use in therapy, e.g., for treating tumors such as cancer.

[0380] In some embodiments, the present invention relates to methods of treating diseases, such as tumors, such as cancer, using molecules of the invention (e.g., anti-CD28 antibodies or antigen-binding fragments thereof or multispecific antibodies such as bispecific antibodies or immunoconjugates thereof of the invention), or uses for such treatment, or uses for the preparation of medicaments for such treatment.

[0381] 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 can be in the early, middle or late stages or be a metastatic cancer. In some embodiments, the tumor is a tumor that has escaped immune toxicity.

[0382] 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 a 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.

[0383] In some embodiments, a TAA-positive tumor or cancer refers to a tumor cell expressing a TAA in a subject having all tumor cancers. In some embodiments, the tumor cells of the subject express TAAs, e.g., moderately or highly express TAAs. In some embodiments, the subject has (e.g., elevated levels, e.g., nucleic acid or protein levels or activity) a TAA (e.g., compared to a healthy subject). In some embodiments, the subject has (e.g., elevated levels, e.g., nucleic acid or protein levels or activity) a TAA in a biological sample (e.g., a tumor cell or tumor tissue) (e.g., compared to a biological sample of a healthy subject (e.g., a corresponding tissue or cell in a healthy subject), or compared to a TAA in an adjacent healthy tissue or cell of the subject). In some embodiments, the TAA is CLDN18.2 or PSMA.

[0384] In some embodiments, the tumor is a gastrointestinal tumor, such as a gastrointestinal cancer, such as colon cancer, rectal cancer, or colorectal cancer, such as a CLDN18.2-positive gastrointestinal tumor, such as a gastrointestinal cancer, such as gastric cancer, breast cancer, colon cancer, rectal cancer, or colorectal cancer. In some embodiments, the tumor is a PSMA-positive tumor, such as a renal cell cancer, such as breast cancer, lung cancer, rectal cancer, or colorectal cancer.

[0385] Depending on their therapeutic use, the antibodies or antigen-binding fragments thereof or immunoconjugates or pharmaceutical compositions of the present invention may also be administered in combination with one or more other therapies, such as treatment modalities and / or other therapeutic agents, for the purposes described herein, e.g., for preventing and / or treating the relevant diseases or conditions mentioned herein.

[0386] In some embodiments, when the molecules of the present invention (e.g., anti-CD28 antibodies or antigen-binding fragments thereof or bispecific antibodies or immunoconjugates thereof of the present invention) are used to treat tumors, the therapeutic agents are, for example, various therapeutic agents for treating tumors, such as chemotherapeutics, angiogenesis inhibitors, cytokines, cytotoxic agents, other 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 antigen-binding fragment thereof. In some embodiments, when the molecules of the present invention (e.g., anti-CD28 antibodies or antigen-binding fragments thereof or bispecific antibodies or immunoconjugates thereof of the present invention) are used to treat tumors, treatment modalities include surgery; radiotherapy, local irradiation or focused irradiation, etc.

[0387] In some embodiments, the present invention also provides a pharmaceutical combination or pharmaceutical combination product comprising an antibody or antigen-binding fragment thereof or immunoconjugate of the present invention, and one or more other therapeutic agents.

[0388] Another object of the present invention is to provide a kit comprising the pharmaceutical combination of the present invention, preferably in the form of a pharmaceutical dosage unit, whereby dosage units can be provided according to a dosing regimen or a drug administration interval.

[0389] In one embodiment, the kit of parts of the present invention comprises in the same package:

[0390] - a first container containing a pharmaceutical composition comprising an antibody, antigen-binding fragment thereof, or immunoconjugate of the present invention;

[0391] - A second container containing a pharmaceutical composition comprising an additional therapeutic agent.

[0392] In some embodiments, when the molecules of the present invention (e.g., anti-CD28 antibodies or antigen-binding fragments thereof or bispecific antibodies or immunoconjugates thereof) are used to treat tumors, the therapeutic agent is, for example, a variety of therapeutic agents for treating tumors, such as chemotherapeutic agents, angiogenesis inhibitors, cytokines, cytotoxic agents, other 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 antigen-binding fragment thereof.

[0393] In other aspects, the present invention provides the use of the molecules of the present invention (e.g., anti-CD28 antibodies or antigen-binding fragments thereof or bispecific antibodies or immunoconjugates thereof, etc.) or compositions or combination products comprising the same in the production or preparation of medicaments for the uses described herein, such as for preventing or treating the relevant diseases or conditions mentioned herein.

[0394] In other aspects, the present invention also provides molecules of the present invention (e.g., anti-CD28 antibodies or antigen-binding fragments thereof or bispecific antibodies or immunoconjugates thereof, etc.), or compositions or drugs or formulations or combination products comprising the same, which are used for therapy, for example, for treating the relevant diseases or conditions mentioned herein.

[0395] IX. Diagnosis and Testing

[0396] In certain embodiments, the molecules provided herein (eg, anti-CD28 antibodies or antigen-binding fragments thereof or bispecific antibodies or immunoconjugates thereof, etc.) can be used to detect the presence of CD28 or TAA in a biological sample.

[0397] The term "detection" as used herein includes quantitative or qualitative detection, and exemplary detection methods may involve immunohistochemistry, immunocytochemistry, flow cytometry (e.g., FACS), magnetic beads complexed with antibody molecules, ELISA assays, PCR-techniques (e.g., RT-PCR). In certain embodiments, the biological sample is a body fluid.

[0398] In certain embodiments, the method comprises contacting a biological sample with a molecule as described herein (e.g., a bispecific antibody or immunoconjugate thereof) under conditions that allow binding to CD28 and / or TAAs, and detecting whether a complex is formed between the molecule and CD28 or TAAs, wherein the formation of a complex indicates the presence of CD28 and / or TAAs. The method can be an in vitro or in vivo method. In one embodiment, the molecules of the invention are used to select subjects suitable for treatment with an anti-CD28 antibody or antigen-binding fragment thereof, a bispecific antibody or immunoconjugate thereof, etc. of the invention, for example, wherein CD28 and / or HTRA1 are biomarkers for selecting the subject.

[0399] In certain embodiments, labeled molecules of the invention (e.g., anti-CD28 antibodies or antigen-binding fragments thereof, bispecific antibodies or immunoconjugates thereof, etc.) are provided. Labels include, but are not limited to, directly detected labels or moieties (e.g., fluorescent labels, chromophore labels, electron-dense labels, chemiluminescent labels, and radioactive labels), as well as moieties that are indirectly detected, such as enzymes or ligands, e.g., by an enzymatic reaction or molecular interaction.

[0400] In some embodiments provided herein, the sample is obtained prior to treatment with a molecule of the invention, or a composition, medicament, formulation, or combination product comprising the same. In some embodiments, the sample is obtained prior to treatment with another therapy. In some embodiments, the sample is obtained during treatment with another therapy, or after treatment with another therapy.

[0401] In some embodiments, TAA is detected prior to treatment, eg, prior to initiation of treatment or prior to a treatment after a treatment interval.

[0402] In some embodiments, a method for treating a disease of the present invention is provided, comprising: testing a subject (e.g., a sample) (e.g., a subject sample) for the presence of a TAA, thereby determining a TAA value, comparing the TAA value with a control value (e.g., a value in a normal individual), and if the TAA value is greater than the control value, administering to the subject a therapeutically effective amount of a bispecific antibody of the present invention, or a composition, medicament, or formulation comprising the same, optionally in combination with one or more other therapies, thereby treating the disease.

[0403] X. Specific Implementation Plan

[0404] 1. An anti-CD28 antibody or antigen-binding fragment thereof, comprising three CDRs of the heavy chain variable region VH, CDRH1, CDRH2, and CDRH3, and three CDRs of the light chain variable region VL, CDRL1, CDRL2, and CDRL3, wherein

[0405] (i) The CDRH1, CDRH2 and CDRH3 are selected from the three complementary determining regions CDRH1, CDRH2 and CDRH3 contained in the VH as shown in SEQ ID NO: 24, 23 or 75; and the CDRL1, CDRL2 and CDRL3 are the three complementary determining regions CDRL1, CDRL2 and CDRL3 contained in the VL as shown in SEQ ID NO: 28 or 78;

[0406] (ii) the CDRH1, CDRH2 and CDRH3 are selected from the three complementary determining regions CDRH1, CDRH2 and CDRH3 contained in the VH as shown in any one of SEQ ID NO: 4 or 5 or 72; and the CDRL1, CDRL2 and CDRL3 are the three complementary determining regions CDRL1, CDRL2 and CDRL3 contained in the VL as shown in SEQ ID NO: 9 or 73; or

[0407] (iii). The CDRH1, CDRH2 and CDRH3 are selected from the three complementarity determining regions CDRH1, CDRH2 and CDRH3 contained in the VH as shown in SEQ ID NO: 13 or 69; and the CDRL1, CDRL2 and CDRL3 are the three complementarity determining regions CDRL1, CDRL2 and CDRL3 contained in the VL as shown in SEQ ID NO: 17, 19 or 70.

[0408] 2. An anti-CD28 antibody or antigen-binding fragment thereof comprising CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3, wherein

[0409] (i) 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 CDRL3 comprises or consists of the amino acid sequence of SEQ ID NO: 27;

[0410] (ii) 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 CDRL3 comprises or consists of the amino acid sequence of SEQ ID NO: 27;

[0411] (iii) 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 or 74; CDRL2 comprises or consists of the amino acid sequence of SEQ ID NO: 7; and CDRL3 comprises or consists of the amino acid sequence of SEQ ID NO: 8; or

[0412] (iv). 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 or 18 or 71; CDRL2 comprises or consists of the amino acid sequence of SEQ ID NO: 15; and CDRL3 consists of the amino acid sequence of SEQ ID NO: 16.

[0413] 3. The antibody or antigen-binding fragment thereof according to embodiment 1 or 2, comprising a heavy chain variable region VH, wherein the heavy chain variable region

[0414] (i) comprising or consisting of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 24, 4, 5, 13, 23, 69, 72 or 75; or

[0415] (ii) comprising or consisting of the amino acid sequence of SEQ ID NO: 24, 4, 5, 13, 23, 69, 72 or 75.

[0416] 4. The antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 3, comprising a light chain variable region VL, wherein the light chain variable region

[0417] (i) comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 28, 9, 17, 19, 70, 73 or 78; or

[0418] (ii) comprising or consisting of the amino acid sequence of SEQ ID NO: 28, 9, 17, 19, 70, 73 or 78.

[0419] 5. The antibody or antigen-binding fragment thereof according to embodiment 1 or 2, comprising a heavy chain variable region VH and a light chain variable region VL, wherein

[0420] (i) the VH comprises, or consists of, the amino acid sequence of SEQ ID NO: 24 or 23, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and the VL comprises, or consists of, the amino acid sequence of SEQ ID NO: 28, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto;

[0421] (ii) the VH comprises, or consists of, the amino acid sequence of SEQ ID NO: 75, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and the VL comprises, or consists of, the amino acid sequence of SEQ ID NO: 78, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto;

[0422] (iii) the VH comprises, or consists of, the amino acid sequence of SEQ ID NO: 23, 24, or 75, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and the VL comprises, or consists of, the amino acid sequence of SEQ ID NO: 28 or 78, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto;

[0423] (iv) the VH comprises, or consists of, the amino acid sequence of SEQ ID NO: 4 or 5 or 72, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and the VL comprises, or consists of, the amino acid sequence of SEQ ID NO: 9 or 73, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto;

[0424] (v) the VH comprises, or consists of, the amino acid sequence of SEQ ID NO: 4 or 5, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and the VL comprises, or consists of, the amino acid sequence of SEQ ID NO: 9, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto;

[0425] (vi) the VH comprises, or consists of, the amino acid sequence of SEQ ID NO: 72, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and the VL comprises, or consists of, the amino acid sequence of SEQ ID NO: 73, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto;

[0426] (vii) the VH comprises, or consists of, an amino acid sequence as set forth in SEQ ID NO: 13 or 69, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and the VL comprises, or consists of, an amino acid sequence as set forth in SEQ ID NO: 17 or 19 or 70, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto;

[0427] (viii) the VH comprises, or consists of, the amino acid sequence of SEQ ID NO: 13, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and the VL comprises, or consists of, the amino acid sequence of SEQ ID NO: 17 or 19, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; or

[0428] (ix) the VH comprises, or consists of, the amino acid sequence of SEQ ID NO: 69, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and the VL comprises, or consists of, the amino acid sequence of SEQ ID NO: 70, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0429] 6. The antibody or antigen-binding fragment thereof according to embodiment 1 or 2, comprising a heavy chain variable region VH and a light chain variable region VL,

[0430] (i) The VH comprises or consists of the amino acid sequence shown in SEQ ID NO: 24, and the VL comprises or consists of the amino acid sequence shown in SEQ ID NO: 28;

[0431] (ii) the VH comprises or consists of the amino acid sequence shown in SEQ ID NO: 23, and the VL comprises or consists of the amino acid sequence shown in SEQ ID NO: 28;

[0432] (iii) the VH comprises or consists of the amino acid sequence shown in SEQ ID NO: 72, and the VL comprises or consists of the amino acid sequence shown in SEQ ID NO: 73;

[0433] (iv) the VH comprises or consists of the amino acid sequence shown in SEQ ID NO: 4, and the VL comprises or consists of the amino acid sequence shown in SEQ ID NO: 9;

[0434] (v) the VH comprises the amino acid sequence shown in SEQ ID NO: 5 or consists of the amino acid sequence, and the VL comprises the amino acid sequence shown in SEQ ID NO: 9 or consists of the amino acid sequence;

[0435] (vi) the VH comprises or consists of the amino acid sequence shown in SEQ ID NO: 13, and the VL comprises or consists of the amino acid sequence shown in SEQ ID NO: 17;

[0436] (vii) the VH comprises or consists of the amino acid sequence shown in SEQ ID NO: 13, and the VL comprises or consists of the amino acid sequence shown in SEQ ID NO: 19;

[0437] (viii) the VH comprises or consists of the amino acid sequence shown in SEQ ID NO: 69, and the VL comprises or consists of the amino acid sequence shown in SEQ ID NO: 70; or

[0438] (ix) The VH comprises or consists of the amino acid sequence shown in SEQ ID NO: 75, and the VL comprises or consists of the amino acid sequence shown in SEQ ID NO: 78.

[0439] 7. An anti-CD28 antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region VH and a light chain variable region VL,

[0440] (i) The VH comprises or consists of the amino acid sequence shown in SEQ ID NO: 24, and the VL comprises or consists of the amino acid sequence shown in SEQ ID NO: 28;

[0441] (ii) the VH comprises or consists of the amino acid sequence shown in SEQ ID NO: 23, and the VL comprises or consists of the amino acid sequence shown in SEQ ID NO: 28;

[0442] (iii) the VH comprises or consists of the amino acid sequence shown in SEQ ID NO: 72, and the VL comprises or consists of the amino acid sequence shown in SEQ ID NO: 73;

[0443] (iv) the VH comprises or consists of the amino acid sequence shown in SEQ ID NO: 4, and the VL comprises or consists of the amino acid sequence shown in SEQ ID NO: 9;

[0444] (v) the VH comprises the amino acid sequence shown in SEQ ID NO: 5 or consists of the amino acid sequence, and the VL comprises the amino acid sequence shown in SEQ ID NO: 9 or consists of the amino acid sequence;

[0445] (vi) the VH comprises or consists of the amino acid sequence shown in SEQ ID NO: 13, and the VL comprises or consists of the amino acid sequence shown in SEQ ID NO: 17;

[0446] (vii) the VH comprises or consists of the amino acid sequence shown in SEQ ID NO: 13, and the VL comprises or consists of the amino acid sequence shown in SEQ ID NO: 19;

[0447] (viii) the VH comprises or consists of the amino acid sequence shown in SEQ ID NO: 69, and the VL comprises or consists of the amino acid sequence shown in SEQ ID NO: 70; or

[0448] (ix) The VH comprises or consists of the amino acid sequence shown in SEQ ID NO: 75, and the VL comprises or consists of the amino acid sequence shown in SEQ ID NO: 78.

[0449] 8. The antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 7, further comprising a heavy chain constant region HC, for example, the antibody heavy chain constant region HC is the heavy chain constant region of IgG1, IgG2, IgG3 or IgG4, preferably the heavy chain constant region of human IgG1, preferably, the heavy chain constant region

[0450] (i) comprises or consists of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from SEQ ID NO: 29 or 79; or

[0451] (ii) comprises or consists of an amino acid sequence selected from SEQ ID NO: 29 or 79.

[0452] 9. The antibody or antigen-binding fragment thereof of embodiment 7 or 8, wherein the heavy chain constant region comprises a mutation that reduces binding to Fcγ receptors, such as LALA mutation and / or P329del.

[0453] 10. The antibody or antigen-binding fragment thereof according to any one of embodiments 1-9, which comprises a light chain constant region, for example, the light chain constant region is a lambda or kappa light chain constant region.

[0454] 11. The antibody or antigen-binding fragment thereof according to embodiment 10, wherein the light chain constant region

[0455] (i) comprises or consists of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 30; or

[0456] (ii) comprising or consisting of the amino acid sequence of SEQ ID NO: 30.

[0457] 12. The antibody or antigen-binding fragment thereof according to any one of embodiments 1-11, wherein the antibody is a humanized antibody or a chimeric antibody.

[0458] 13. The antibody or antigen-binding fragment thereof according to any one of embodiments 1-12, wherein the antibody is a monoclonal antibody.

[0459] 14. The antibody or antigen-binding fragment thereof according to any one of embodiments 1-13, wherein the antigen-binding fragment is an antibody fragment selected from the group consisting of: Fab, Fab', Fab'-SH, Fv, single-chain antibody (e.g., scFv), (Fab')2, single-domain antibody (e.g., VHH), dAb (domain antibody), or linear antibody.

[0460] 15. The antibody or antigen-binding fragment thereof of any one of embodiments 1-11, wherein the antibody is a bispecific antibody or a multispecific antibody comprising a first binding specificity for CD28 and additional binding specificities for one or more tumor-associated antigens.

[0461] 16. The antibody or antigen-binding fragment thereof of embodiment 15, wherein the tumor-associated antigen is selected from CLDN18.2 or PSMA.

[0462] 17. A bispecific antibody comprising a first antigen-binding region and a second antigen-binding region,

[0463] The first antigen binding region specifically binds to CD28 and comprises VH and VL, wherein

[0464] The VH comprises CDRH1, CDRH2, CDRH3 as defined in embodiment 1 or 2 and the VL comprises CDRL1, CDRL2 and CDRL3 as defined in embodiment 1 or 2; or

[0465] The VH and VL are VH and VL as defined in any one of embodiments 3-6;

[0466] The second antigen-binding region specifically binds to a tumor-associated antigen or an immune checkpoint molecule.

[0467] 18. The bispecific antibody of embodiment 17, wherein the first antigen-binding region is the Fab of the anti-CD28 antibody defined in any one of embodiments 1-14.

[0468] 19. The bispecific antibody of embodiment 17 or 18, wherein the second antigen binding region is a Fab that specifically binds to a tumor-associated antigen or an immune checkpoint molecule.

[0469] 20. The bispecific antibody of embodiment 18 or 19, wherein the Fab serving as the first antigen binding region or the second antigen binding region comprises CH1, wherein the CH1 is CH1 from IgG1, IgG2, IgG3 or IgG4, preferably CH1 from IgG1.

[0470] 21. The bispecific antibody of embodiment 20, wherein the CH1

[0471] (i) comprises or consists of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from SEQ ID NO: 74; or

[0472] (ii) comprising or consisting of the amino acid sequence of SEQ ID NO: 74.

[0473] 22. The bispecific antibody of any one of embodiments 18-21, wherein the Fab serving as the first antigen binding region or the second antigen binding region comprises a light chain constant region, wherein the light chain constant region is a Kappa light chain constant region or a Lambda light chain constant region.

[0474] 23. The bispecific antibody of embodiment 22, wherein the Kappa light chain constant region

[0475] (i) comprises or consists of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 30; or

[0476] (ii) comprising or consisting of the amino acid sequence of SEQ ID NO: 30.

[0477] 24. The bispecific antibody of any one of embodiments 17-23, wherein the bispecific antibody is an IgG-like bispecific antibody comprising an Fc dimer, wherein the two Fc regions constituting the Fc dimer are the same or different, optionally, the Fc region is human IgG1, IgG2, IgG3 or IgG4 Fc, optionally, the Fc region

[0478] (i) comprises or consists of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from SEQ ID NO: 94 or 95; or

[0479] (ii) comprising or consisting of the amino acid sequence of SEQ ID NO: 94 or 95.

[0480] 25. The bispecific antibody of embodiment 24, wherein the two Fc regions are different, wherein based on Innobody technology, mutations are introduced into the first Fc region and the second Fc region to promote heterodimerization of the first Fc region and the second Fc region.

[0481] 26. The antibody or antigen-binding fragment thereof of embodiment 25, wherein the CH3 of one Fc region comprises S364R and D399K mutations, and the CH3 mutations of the other Fc region comprise Y349T, K370S and K409D mutations.

[0482] 27. The bispecific antibody of any one of embodiments 24-26, wherein the first and / or second Fc region comprises a mutation that reduces binding to Fcγ receptors, such as L234A / L235A mutations, and optionally P329delP.

[0483] 28. The bispecific antibody of embodiment 26 or 27, wherein

[0484] a) one Fc region comprises, or consists of, the amino acid sequence of SEQ ID NO: 80, and the other Fc region comprises, or consists of, the amino acid sequence of SEQ ID NO: 81;

[0485] b) one Fc region comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence set forth in SEQ ID NO:80 and comprises the mutations S364R and D399K, and the other Fc region comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence set forth in SEQ ID NO:81 and comprises the mutations Y349T, K370S, and K409D;

[0486] c) one Fc region comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence of SEQ ID NO:80 and comprises mutations S364R and D399K and L234A / L235A mutations and delP, and the other Fc region comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence of SEQ ID NO:81 and comprises mutations Y349T, K370S and K409D and L234A / L235A mutations and delP;

[0487] d) one Fc region comprises or consists of the amino acid sequence shown in SEQ ID NO: 82, and the other Fc region comprises or consists of the amino acid sequence shown in SEQ ID NO: 83;

[0488] e) one Fc region comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence of SEQ ID NO: 82 and comprises the mutations S364R and D399K, and the other Fc region polypeptide comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence of SEQ ID NO: 83 and comprises the mutations Y349T, K370S, and K409D; or

[0489] f) one Fc region comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the sequence of amino acids set forth in SEQ ID NO: 82 and comprises mutations S364R and D399K and L234A / L235A mutations, and the other Fc region polypeptide comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the sequence of amino acids set forth in SEQ ID NO: 83 and comprises mutations Y349T, K370S and K409D and L234A / L235A mutations.

[0490] 29. The bispecific antibody of any one of embodiments 17-28, wherein the TAA is CLDN18.2, preferably, the second antigen-binding region comprises three complementarity-determining regions (CDRHs) from the heavy chain variable region, CDRH1, CDRH2, and CDRH3, and three complementarity-determining regions (CDRLs) from the light chain variable region, CDRL1, CDRL2, and CDRL3, wherein

[0491] 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.

[0492] 30. The bispecific antibody of embodiment 29, wherein the second antigen-binding region comprises VH and VL, wherein VH comprises or consists of the sequence shown in SEQ ID NO: 86; and VL comprises or consists of the sequence shown in SEQ ID NO: 90.

[0493] 31. The bispecific antibody of any one of embodiments 17-28, wherein the TAA is PSMA, preferably, the second antigen-binding region comprises three complementarity-determining regions (CDRHs) from the heavy chain variable region, CDRH1, CDRH2, and CDRH3, and three complementarity-determining regions (CDRLs) from the light chain variable region, CDRL1, CDRL2, and CDRL3, wherein

[0494] 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.

[0495] 32. The bispecific antibody of embodiment 31, wherein the second antigen-binding region comprises VH and VL, wherein VH comprises or consists of the sequence shown in SEQ ID NO: 47; and VL comprises or consists of the sequence shown in SEQ ID NO: 49.

[0496] 33. The bispecific antibody of any one of embodiments 17-32, 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 or is a first Fab, and the second antigen binding region comprises or is a second Fab, wherein the first Fab is connected at the C-terminus of its CH1 to the N-terminus of the first Fc region (via or without a linker, such as a hinge region), and the second Fab is connected at the C-terminus of its CH1 to the N-terminus of the second Fc region (via or without a linker, such as a hinge region).

[0497] 34. The bispecific antibody of embodiment 33, wherein the bispecific antibody is an IgG-like antibody having the configuration shown in Figure 3.

[0498] 35. The bispecific antibody of embodiment 33 or 34, comprising 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, wherein the bispecific antibody comprises

[0499] Heavy chain 1: comprises or consists of the following from N-terminus to C-terminus: a first Fab heavy chain variable region-a heavy chain constant region CH1-a first Fc region, wherein the heavy chain constant region CH1 is connected at its C-terminus to the N-terminus of the first Fc region via or without a linker (e.g., a hinge region);

[0500] Light chain 1: from N-terminus to C-terminus, comprises or consists of: the light chain variable region of the first Fab - the light chain constant region;

[0501] Heavy chain 2: comprises or consists of the following from N-terminus to C-terminus: a second Fab heavy chain variable region - a heavy chain constant region CH1 - a second Fc region, wherein the heavy chain constant region CH1 is connected at its C-terminus to the N-terminus of the second Fc region via or without a linker (e.g., a hinge region);

[0502] Light chain 2: from N-terminus to C-terminus, comprises or consists of: the light chain variable region of the second Fab - the light chain constant region;

[0503] Preferably, each domain is directly linked.

[0504] 36. The bispecific antibody of embodiment 35, wherein TAA is CLDN18.2, and wherein

[0505] Heavy chain 1 comprises, or consists of, the amino acid sequence of SEQ ID NO:35, or an amino acid sequence at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto; light chain 1 comprises, or consists of, the amino acid sequence of SEQ ID NO:36, or an amino acid sequence at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto; heavy chain 2 comprises, or consists of, the amino acid sequence of SEQ ID NO:33, or an amino acid sequence at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto; and light chain 2 comprises, or consists of, the amino acid sequence of SEQ ID NO:34, or an amino acid sequence at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto;

[0506] heavy chain 1 comprises, or consists of, the amino acid sequence of SEQ ID NO:37, or an amino acid sequence at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto; light chain 1 comprises, or consists of, the amino acid sequence of SEQ ID NO:38, or an amino acid sequence at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto; heavy chain 2 comprises, or consists of, the amino acid sequence of SEQ ID NO:33, or an amino acid sequence at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto; and light chain 2 comprises, or consists of, the amino acid sequence of SEQ ID NO:34, or an amino acid sequence at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto; or

[0507] Heavy chain 1 comprises, or consists of, the amino acid sequence of SEQ ID NO:39, or an amino acid sequence at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto; light chain 1 comprises, or consists of, the amino acid sequence of SEQ ID NO:40, or an amino acid sequence at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto; heavy chain 2 comprises, or consists of, the amino acid sequence of SEQ ID NO:33, or an amino acid sequence at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto; and light chain 2 comprises, or consists of, the amino acid sequence of SEQ ID NO:34, or an amino acid sequence at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0508] 37. The bispecific antibody of embodiment 35, wherein the TAA is PSMA, and wherein

[0509] heavy chain 1 comprises, or consists of, the amino acid sequence of SEQ ID NO:51, or an amino acid sequence at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto; light chain 1 comprises, or consists of, the amino acid sequence of SEQ ID NO:36, or an amino acid sequence at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto; heavy chain 2 comprises, or consists of, the amino acid sequence of SEQ ID NO:48, or an amino acid sequence at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto; and light chain 2 comprises, or consists of, the amino acid sequence of SEQ ID NO:50, or an amino acid sequence at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto;

[0510] heavy chain 1 comprises, or consists of, the amino acid sequence of SEQ ID NO:52, or an amino acid sequence at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto; light chain 1 comprises, or consists of, the amino acid sequence of SEQ ID NO:38, or an amino acid sequence at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto; heavy chain 2 comprises, or consists of, the amino acid sequence of SEQ ID NO:48, or an amino acid sequence at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto; and light chain 2 comprises, or consists of, the amino acid sequence of SEQ ID NO:50, or an amino acid sequence at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto; or

[0511] Heavy chain 1 comprises, or consists of, the amino acid sequence of SEQ ID NO:53, or an amino acid sequence at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto; light chain 1 comprises, or consists of, the amino acid sequence of SEQ ID NO:40, or an amino acid sequence at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto; heavy chain 2 comprises, or consists of, the amino acid sequence of SEQ ID NO:48, or an amino acid sequence at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto; and light chain 2 comprises, or consists of, the amino acid sequence of SEQ ID NO:50, or an amino acid sequence at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0512] 38. A nucleic acid molecule encoding any one chain of the antibody or antigen-binding fragment thereof according to any one of embodiments 1-16, or any one chain of the bispecific antibody according to any one of embodiments 17-37, or consisting of said nucleic acid sequence.

[0513] 39. An expression vector comprising the nucleic acid molecule of embodiment 38, preferably, the expression vector is pCDNA, such as pCDNA3.1.

[0514] 40. A host cell comprising the nucleic acid molecule of embodiment 38 or the expression vector of embodiment 39, preferably, the host cell is prokaryotic or eukaryotic, such as 293 cells or CHO cells, such as Expi293 cells.

[0515] 41. A method for preparing the antibody or antigen-binding fragment thereof of any one of embodiments 1-16 or the bispecific antibody of any one of embodiments 17-37, the method comprising culturing a host cell containing the nucleic acid molecule of embodiment 38 or the expression vector of embodiment 39 under conditions suitable for expression of the antibody chains, and optionally recovering the antibody from the host cell (or host cell culture medium).

[0516] 42. An immunoconjugate comprising the antibody or antigen-binding fragment thereof of any one of embodiments 1-16 or the bispecific antibody of any one of embodiments 17-37.

[0517] 43. A pharmaceutical composition, medicament or formulation comprising the antibody or antigen-binding fragment thereof of any one of embodiments 1-16 or the bispecific antibody of any one of embodiments 17-37, or the immunoconjugate of embodiment 42 and optionally a pharmaceutically acceptable excipient.

[0518] 44. A pharmaceutical combination product comprising the antibody or antigen-binding fragment thereof of any one of embodiments 1-16 or the bispecific antibody of any one of embodiments 17-37, or the immunoconjugate of embodiment 42, and one or more other therapeutic agents, preferably, the therapeutic agents are various therapeutic agents for treating tumors, such as chemotherapeutic agents, angiogenesis inhibitors, cytokines, cytotoxic agents, other antibodies, small molecule drugs or immunomodulators (such as immune checkpoint inhibitors or agonists), preferably, the other antibodies are PD-1 antibodies.

[0519] 45. A method for preventing or treating a tumor in a subject, comprising administering to the subject an effective amount of the antibody or antigen-binding fragment thereof of any one of embodiments 1-16, or the bispecific antibody of any one of embodiments 17-37, or the immunoconjugate of embodiment 42, or the pharmaceutical composition or formulation of embodiment 43; or the pharmaceutical combination product of embodiment 44.

[0520] 46. ​​The method of embodiment 45, wherein the tumor is a solid tumor or a blood tumor, such as a TAA-positive tumor or cancer, optionally the tumor is a CLDN18.2-positive gastrointestinal tumor, such as a gastrointestinal cancer, such as gastric cancer, breast cancer, colon cancer, rectal cancer or colorectal cancer; or the tumor is a PSMA-positive tumor, such as a renal cell cancer, such as breast cancer, lung cancer, rectal cancer or colorectal cancer.

[0521] 47. The method according to any one of embodiments 45-46, wherein the method further comprises administering the drug in combination with other therapies, such as treatment modalities (e.g., surgery or radiotherapy) and / or other therapeutic agents. Preferably, the therapeutic agents are various therapeutic agents for treating tumors, such as chemotherapeutic agents, angiogenesis inhibitors, cytokines, cytotoxic agents, other antibodies, small molecule drugs or immunomodulators (e.g., immune checkpoint inhibitors or agonists). Preferably, the other antibodies are PD-1 antibodies.

[0522] Example 1: Discovery and affinity testing of mouse anti-CD28 antibodies

[0523] CD28 antigen immunization

[0524] Human CD28 extracellular segment protein (Acro, Cat CD8-H52Hc) was emulsified with Freund's complete adjuvant (sigma, Cat#F5881) and then immunized with Balb / c mice (purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd.). Two weeks later, it was emulsified with Freund's incomplete adjuvant (sigma, F5506) and then immunized three times, with subcutaneous injection once every two weeks (50 micrograms of protein per mouse).

[0525] Cell fusion and high-throughput screening

[0526] When the serum titer meets the required level, the mouse spleen is removed to prepare a B lymphocyte suspension, which is then electrofused with SP2 / 0 myeloma cells (ATCC). The fused cells are diluted to 1-2 × E₄ cells / mL in selective medium (1640 medium containing 20% ​​FBS and 1x HAT) and plated onto a 96-well plate, with 100 μL of cell suspension added to each well. Seven days after fusion, the selective medium (1640 medium containing 10% FBS and 1x HAT) is replaced. The supernatant is collected and analyzed after culturing for 10 days (or longer, depending on the cell growth status).

[0527] Hybridoma cells specifically expressing anti-CD28 antibodies were screened by flow cytometry (FACS). Count the cells to be tested (huCD28 / GS-CHO, constructed in-house) and dilute to 1E6 cells / mL. Add 100 μL / well of the culture medium to a U-bottom 96-well plate. Centrifuge at 500 g for 5 minutes, and remove the cell culture medium. Add the supernatant from the hybridoma 96-well plate to the U-bottom plate and resuspend the cells, adding 100 μL per well. Incubate on ice for 30 minutes. Centrifuge at 500 g for 5 minutes, remove the supernatant, and wash the cells once with PBS. Centrifuge at 500g for 5 minutes to remove PBS. Add 100 μl of APC-conjugated secondary antibody (Jackson Immunoresearch, Cat#115-606-071) (1:500 dilution in PBS) to each well. For the positive control antibody (TGN1412 (Creative BioLabs TAB-064LC)), add 100 μl of PE-conjugated secondary antibody (Biolegend, Cat#409304) to each well. Incubate on ice for 30 minutes in the dark. Centrifuge at 500g for 5 minutes to remove the supernatant, and wash the cells once with PBS. Resuspend the cells in 50 μl of 1× PBS and analyze on a FACS machine. Supernatants from positive clones were used for CD28 activation assays. Strongly activating clones were subcloned by limiting dilution to isolate single clones.

[0528] Positive hybridoma cell subcloning

[0529] Limiting dilution subcloning procedure: Prepare a 96-well plate and add 200 μl of culture medium to each well. This medium is the same as the screening medium except that HAT is replaced with HT (Gibco, Cat#11067-030). Prepare a cell suspension from the cells in the positive wells screened for fusion. Take 100 μl of the cell suspension from the first row and add it to each well in the first row, mixing thoroughly. Then, take 100 μl of the cell suspension from the first row and add it to the second row. After thorough mixing, take 100 μl and add it to the next row. Repeat the above steps. Let the 96-well plate stand for 30 minutes and observe and count under a microscope. Take the volume corresponding to 100 cells and add 20 ml of culture medium. Mix well and plate, 200 μl per well. After one week, observe under a microscope to determine and mark the wells with single clones.

[0530] When the cell confluence in each well reaches more than 50%, the high-throughput screening method is used for detection, and the target positive wells are picked out. After expansion culture, the cells are frozen.

[0531] Preparation of chimeric antibodies

[0532] The antibody light and heavy chain gene sequences were retrieved from the hybridoma candidate clones obtained in the above experiments, and human-mouse chimeric antibodies 61F3G, 5B11D8, and 4F12B5 were constructed from them. The corresponding sequences of the antibody light and heavy chains are shown below. The antibody heavy chain constant region is a human IgG1 heavy chain constant region (SEQ ID NO: 79). The variable region and CDR sequences are shown in Table 1.

[0533] Freshly cultured cell lines (approximately 5E6 cells per strain) were collected and RNA was extracted (Macherey-Nagel, Cat# 740984.250). cDNA was obtained by reverse transcription using the PrimeScript II 1st Strand cDNA Synthesis Kit (Takara). Upstream primers were designed based on base sequences in the 5' FR1 region, and downstream primers were designed based on bases in the antibody constant region or FR4 region to amplify the antibody light chain and heavy chain variable region gene fragments. These fragments were ligated into a T-vector (Mighty TA-cloning Kit, Takara), and single clones were selected for sequencing. The sequencing results were analyzed and compared using MEGA7 software.

[0534] After comparison, the antibody light and heavy chain variable region sequences were correct and matched clones, and their light and heavy chain variable region gene fragments were respectively passed through the homologous recombinase ( II, catalog number: C112-01) was ligated into a pcDNA3.1 vector (containing an IgG1 heavy chain constant region (SEQ ID NO: 79) or a Kappa light chain constant region (SEQ ID NO: 30)), wherein the constant region selected the IgG1 subtype to obtain expression plasmids for light and heavy chain antibodies.

[0535] Table 1: Comparison of CD28 chimeric antibody CDR light and heavy chain combinations

[0536] Expi293F cells (purchased from Gibco) were cultured with Expi293F medium (Gibco, REF#A14351-01). The cell density was measured one day before transfection (viability must be greater than 95%) and adjusted to 3E6 cells / ml with fresh Expi293F medium for continued culture. On the day of transfection, the cell density was adjusted to 3E6 cells / ml.

[0537] Take 1 / 10 of the final transfection volume of Opti-MEM medium (Gibco, REF#31985-070) as the transfection buffer, add the plasmid of the chimeric antibody to be transfected at a ratio of 1 mg / L, where the light and heavy chain plasmids are in a 1:1 ratio, mix well, add PEIMax (Polysciences Inc. Cat#24765-1) at a DNA:PEI mass ratio of 1:3, mix well, incubate at room temperature for 20 minutes, and then gently pour the mixture into the Expi293F cell suspension while shaking. The cells are cultured on a shaker under the conditions of 8% CO2, 36.5°C, and 120 rpm.

[0538] After 16-18 hours of culture, 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 to a final concentration of 5 g / L, and valproic acid sodium salt (Merk, Cat# P4543-100G) to a final concentration of 2.2 mM. The suspension was gently mixed and cultured for 7 days at 8% CO₂, 36.5°C, and 120 rpm before sampling. The cell suspension was then mixed with diatomaceous earth (Sartorius, Cat 1000037025) (40 g diatomaceous earth per 1 L of cell suspension) and filtered using a 0.22 μm disposable vacuum filter.

[0539] Affinity chromatography to purify the target protein: A HiTrap MabSelect PrismA (GE Healthcare, Cat#17549853) affinity chromatography column was used for affinity capture. Before purification, 10-20 column volumes of 0.1 M NaOH were passed through the tubing and affinity chromatography column, and then the tubing 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); the filtered cell feed was passed through the column, and the packed column was washed with 10 column volumes of 1× PBS to remove non-specific binding proteins; the packed column was rinsed with 5 column volumes of elution buffer (100 mM sodium citrate, pH 3.5), the eluate was collected, the pH was adjusted to 6.0 with 2 M Tris, and the column was sterilized by filtration. After passing the purity test, it was used for subsequent functional analysis.

[0540] Affinity testing of chimeric anti-CD28 antibodies

[0541] In this study, biofilm thin layer interferometry (BLI) was used to determine the binding kinetics (KD) of the antibody-antigen binding of the present invention. BLI affinity determination was performed according to existing methods (Estep, P et al., High throughput solution-based measurement of antibody-antigen affinity and epitope binning. MAbs, 2013. 5(2): p. 270-8).

[0542] Half an hour before the experiment, depending on the number of samples, an appropriate number of AHC sensors (18-5060, Sartorius) were soaked in SD buffer (1x PBS, 0.1% BSA, 0.05% Tween-20). The aforementioned anti-CD28 antibody and human CD28 recombinant protein (CD8-H52Hc-1 mg, ACRO) were diluted to 100 nM.

[0543] SD buffer, antibody solution, and CD28 recombinant protein were added to a 96-well black polystyrene microplate (Greiner, 655209). Detection was performed using a ForteBio Octet Red96e, with the plate arranged according to the sample position and the sensor position selected. Instrument parameters were set as follows: Run steps: 120 s baseline equilibration, 100 s addition of immobilized antibody, 120 s baseline equilibration, 100 s antigen binding, and 120 s dissociation, at a speed of 1000 rpm and a temperature of 30°C. After the experiment, KD values ​​were analyzed using ForteBio Octet analysis software. The results are shown in Table 2 below.

[0544] The affinity experimental kinetic parameters show that these chimeric CD28s have a strong affinity with human CD28 recombinant protein.

[0545] Table 2: Affinity of anti-CD28 antibodies to CD28

[0546] Example 2: Detection of T cell reporter activation by mouse anti-CD28 antibodies

[0547] To verify whether anti-CD28 antibodies can effectively recognize human CD28 and activate CD28 signaling, we selected Jurkat NFkB (Jiman Bio, GM-C07855) T cell line reporter cells as our research object to determine the activity of CD28 antibodies. The specific process is as follows:

[0548] 1. Dilute anti-human IgG (Jackson Immune Research, 109-005-098) in PBS (1 μg / mL) and add 100 μL per well to a 96-well flat-bottom plate (Shanghai Wohong WHB-96-03). Incubate overnight at 4°C to allow the antibody to fully bind to the plate bottom.

[0549] 2. After incubation, pour off the unbound antibody solution in the plate, add 100 μl of PBS to each well, wash once and discard the PBS.

[0550] 3. The cultured Jurkat NFkB cells were centrifuged and resuspended in assay buffer (1% FBS (Gibco, SV30208.02), RPMI 1640 (Gibco, 22400-071)). 50 μl was added to each well to ensure that there were 50,000 cells per well.

[0551] 4. Dilute the chimeric CD28 antibody to be tested (2 μg / mL) with assay buffer plus or minus CD3 antibody (Acro, CDE-M120A-1MG, 0.5 μg / mL final concentration) and add 50 μL to each well of the plate. Incubate in a 5% CO2, 37°C incubator for 6 h.

[0552] 5. After the experiment, the plate was removed and placed on the test bench to equilibrate to room temperature. 100 μl of Bio-Glo detection reagent (Promega, G7940) was added and the plate was detected on a microplate reader (Molecular Divices, Spectra MAX i3x).

[0553] The results (Figure 1) show that, in the absence of a primary signal provided by an antibody against CD3 (OKT3, Acro, CDE-M120A-1MG), the control antibodies TGN1412 (Tegenero, US20060188493A1, referred to herein as TGN), 5B11D8, and 61F3G3 were able to activate NFkB signaling downstream of CD28, while 4F12B5 was not. In the presence of an antibody against CD3, all of the CD28 antibodies in this study were able to activate CD28 signaling.

[0554] Example 3: Humanization of murine anti-CD28 antibody

[0555] According to conventional methods, the chimeric antibodies obtained in Example 1-2 were humanized. Thus, humanized antibodies were obtained: hz5B11D8-a, hz5B11D8-b, hz61F3G3-a, hz61F3G3-b and hz4F12B5-a and hz4F12B5-b, whose CDR sequences, light chain variable regions and heavy chain variable region sequences, and the amino acid sequences of the light and heavy chains are shown in the sequence table and Table 3. The heavy chain constant region selected the human IgG1 sequence (SEQ ID NO: 29), and the light chain constant region selected the corresponding constant region CL-Kappa (SEQ ID NO: 30) according to whether the variable region is Kappa. The heavy chain sequence and light chain sequence of the antibody were then constructed into the expression vector pcDNA3.1 (Invitrogen, V790-20), and transfected using Expi293 cells (Invitrogen, A14527) to obtain the corresponding humanized antibodies. The specific transfection process is the same as in Example 1.

[0556] Table 3: Comparison of CD28 humanized CDR mutant antibody combinations, light and heavy chain combinations

[0557] Example 4: Preparation of humanized anti-CD28 antibodies and affinity testing

[0558] Antibody preparation: Expi293F cells (purchased from Gibco) were cultured in Expi293F medium (Gibco, REF#A14351-01). The day before transfection, the cell density was measured (viability >95%) and the cell density was adjusted to 3E6 cells / mL in fresh Expi293F medium. The cell density was adjusted to 3E6 cells / mL on the day of transfection.

[0559] 1 / 10 of the final transfection volume of Opti-MEM medium (Gibco, REF#31985-070) was used as the transfection buffer, and the plasmid of the humanized monoclonal antibody to be transfected (the plasmid obtained in Example 3) was added at a ratio of 1 mg / L, wherein the light and heavy chain plasmids were in a 1:1 ratio. Mix well, add PEIMax (Polysciences Inc. Cat#24765-1) at a DNA:PEI mass ratio of 1:3, mix well, and incubate at room temperature for 20 minutes. Then, the mixture was gently poured into the Expi293F cell suspension while shaking. The cells were cultured in a shaker under the conditions of 8% CO2, 36.5°C, and 120 rpm.

[0560] After 16-18 hours of culture, 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 to a final concentration of 5 g / L, and valproic acid sodium salt (Merk, Cat# P4543-100G) to a final concentration of 2.2 mM. The suspension was gently mixed and cultured for 7 days at 8% CO₂, 36.5°C, and 120 rpm before sampling. The cell suspension was then mixed with diatomaceous earth (Sartorius, Cat 1000037025) (40 g diatomaceous earth per 1 L of cell suspension) and filtered using a 0.22 μm disposable vacuum filter.

[0561] Affinity chromatography to purify the target protein: A HiTrap MabSelect PrismA (GE Healthcare, Cat#17549853) affinity chromatography column was used for affinity capture. Before purification, 10-20 column volumes of 0.1 M NaOH were passed through the tubing and affinity chromatography column, and then the tubing 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); the filtered cell feed was passed through the column, and the packed column was washed with 10 column volumes of 1× PBS to remove non-specific binding proteins; the packed column was rinsed with 5 column volumes of elution buffer (100 mM sodium citrate, pH 3.5), the eluate was collected, the pH was adjusted to 6.0 with 2 M Tris, and the column was sterilized by filtration. After passing the purity test, it was used for subsequent functional analysis.

[0562] The affinity of the humanized CD28 antibody of the present invention was determined using the biofilm thin layer interferometry (BLI) technique mentioned in Example 1. The affinity of the humanized molecules is shown in the following table (Table 4).

[0563] Table 4: Affinity of humanized antibodies to CD28

[0564] Example 5: Detection of T cell activation by humanized anti-CD28 antibodies

[0565] To verify whether the humanized CD28 antibodies still maintain activity, we tested whether these antibodies can effectively activate human peripheral T cells in vitro. The specific process is as follows:

[0566] 1. Remove PBMC cells (Miaoshun Biotechnology) one day in advance, centrifuge at 25°C, 400g, 4min, resuspend in growth medium (Gibco, 1640), place in culture flask, add DNase I (SIGMA, D5025-375KU) at a final concentration of 2 μg / ml, and culture overnight in a 5% CO2, 37°C incubator.

[0567] 2. Dilute the humanized CD28 antibody to be tested (1 μg / well) in PBS (or add CD3 antibody (Acro, CDE-M120A-1MG, 0.1 μg / well) simultaneously to a 96-well flat-bottom plate). Add 100 μl of the humanized CD28 antibody to be tested to each well. Incubate overnight at 4°C to allow the antibody to fully bind to the bottom of the plate.

[0568] 3. On the day of the experiment, after the antibody incubation is completed, pour off the unbound antibody solution in the plate, add 100 μl of PBS to each well, wash once and discard the PBS.

[0569] 4. Remove the PBMCs that were revived the previous day and pour the suspended cells into a centrifuge tube. Centrifuge at 25°C, 400g, for 4 minutes. Adjust the cell density with assay buffer (RPMI 1640, 1% FBS) to 100 μl per well (100,000 cells / well). Plate the cells into a 96-well flat-bottom plate coated with the antibody. Incubate in a 5% CO2, 37°C incubator.

[0570] 5. 4 hours before the end of cell culture, remove the 96-well plate from the incubator and transfer 50 μl of supernatant per well to a fresh 96-well round-bottom plate for cytokine detection. Store frozen at -40°C. Add 50 μl of protease transport inhibitor (Invitrogen, 00-4980-93) to each well of the 96-well flat-bottom plate and incubate in a 5% CO2, 37°C incubator for 4 hours.

[0571] 6. After incubation, prepare for flow cytometry antibody staining. First, centrifuge the 96-well flat-bottom plate at 25°C, 400g, for 4 minutes and discard the supernatant. Resuspend the cells in PBS and transfer them to a new 96-well round-bottom plate. Centrifuge at 25°C, 400g, for 4 minutes and discard the PBS.

[0572] 7. Add 50 μl of the pre-prepared surface blocking dye mixture: Fc blocking (BD, 564219, 1:200 in PBS) and dead / active dye (Invitrogen L34968A, 1:100 in PBS) to each well. Incubate at room temperature in the dark for 10 minutes. After incubation, add 50 μl of a 1:100 cell surface dye mixture: PD1 antibody (Biolegend, 329918), CD4 antibody (Biolegend, 329918), or CD8 antibody (Biolegend, 344724) in FACS buffer directly to each well and incubate at 4°C in the dark for 30 minutes.

[0573] 8. After the surface dye incubation is complete, centrifuge at 25°C, 400g, 4 minutes to remove unbound dye. Resuspend the cells in FACS wash buffer (2mM EDTA, 0.5% BSA, PBS) and wash twice by centrifugation.

[0574] 9. Add 100 μL / well of Fix / Perm buffer (ebioscience 00-5523-00 Invitrogen, 00-8333-56) and incubate at room temperature in the dark for 30 min to penetrate the cell membrane.

[0575] 10. After incubation, centrifuge at 25°C, 400g, 4 minutes, and discard the Fix / Perm buffer. Resuspend the cells in PBE (Invitrogen, 00-8333-56) and centrifuge, discarding the solution.

[0576] 11. Add intracellular dye TNF-α antibody (Biolegend, 502946) at 50 μl / well and incubate at room temperature in the dark for 30 minutes.

[0577] 12. After incubation, centrifuge at 25°C, 400g, 4 minutes to remove any unbound dye. Resuspend the cells in PBE buffer, wash twice, and discard the buffer.

[0578] 13. Resuspend the cells in FACS wash buffer, 150 μl / well, and detect on a BD FACS SYMPHONY A3 instrument.

[0579] The results showed (Figure 2A-B) that in the absence of the first signal provided by the CD3 antibody, the control antibody TGN1412 (referred to as TGN in this study) was able to activate CD4T and CD8T cells independently of the first signal and release TNFα. The chimeric antibodies 5B11D8 and 61F3G3 in the present invention exhibited CD28 super agonist activity, while 4F12B5 was a conventional CD28 agonist antibody. After humanization, hz5B11D8-a and hz61F3G3-a had decreased activity compared to their parents, while hz5B11D8-b, hz61F3G3-b, hz4F12B5-a and hz4F12B5-b had similar activity to their parents.

[0580] In the presence of the first signal provided by CD3 antibodies ( FIG. 2C-D ), the antibodies of the present invention can activate CD4 and CD8 T cells to varying degrees to release TNF.

[0581] Example 6: Construction and preparation of CLDN18.2xCD28 bispecific antibody

[0582] To test the monovalent affinity of humanized CD28 antibodies and their ability to activate tumor-associated antigen-dependent T cells, we constructed CLDN18.2xCD28 bispecific antibodies BsAb-1, BsAb-2, BsAb-3, and BsAb-BM-1 (Table 5) that target Claudin18.2. The bispecific antibody molecule is in a "1+1" format (Figure 3A), in which the variable region targeting CLDN18.2 is derived from HB37A6 (CN202010570517.X (WO2021254481A1), sequence information is shown in the sequence listing), and the variable region sequence of anti-CD28 is derived from the variable region sequence of the humanized antibodies hz5B11D8-a, hz61FG3-a, hz4F12B5-b and TGN1412 (Tegenero, US20060188493A1, sequence is shown in the sequence listing SEQ ID NO: 41-44) in the present invention (Table 6). The Fc fragment of the antibody adopts the Innobody (bispecific antibody technology comes from CN116888149A) IgG1 LALA delP format. The schematic diagram of the antibody is shown in Figure 3A.

[0583] Therefore, the heavy chain of the CLDN18.2 end portion of the bispecific antibody is SEQ ID NO: 33, and the light chain is SEQ ID NO: 34. The heavy chains of the CD28 end portion of the bispecific antibody of the present invention are SEQ ID NOs: 35, 37, and 39, respectively, and the light chains are SEQ ID NOs: 36, 38, and 40, respectively. The heavy chain of the control CD28 (TGN1412) end portion used in the bispecific antibody of the present invention is SEQ ID NO: 42, and the light chain is SEQ ID NO: 44.

[0584] Table 5: CLDN18.2xCD28 dual antibody combinations

[0585] The corresponding sequences of the HB37A6 heavy chain and light chain are: SEQ ID NO: 33 and SEQ ID NO: 34.

[0586] The corresponding sequences of the TGN1412 heavy chain and light chain are: SEQ ID NO: 42 and SEQ ID NO: 44.

[0587] Table 6: CLDN18.2xCD28 bispecific antibody sequence information

[0588] Preparation of bispecific antibodies

[0589] The bispecific antibodies in this invention are prepared by first expressing and purifying the two parent proteins separately and then recombining them in vitro through reduction and oxidation. The bispecific antibody preparation route is as follows:

[0590] Plasmid construction: The sequences of the bispecific antibodies of the present invention were synthesized by Jinweizhi. The corresponding light chain variable regions and heavy chain variable regions (Table 6) of the bispecific antibodies were loaded into the pcDNA3.1 vector (Invitrogen, V790-20) containing the corresponding heavy chain constant region or light chain constant region, respectively, to obtain the heavy chain plasmid and light chain plasmid for the CLDN18.2 end and the heavy chain plasmid and light chain plasmid for the CD28 end.

[0591] Expi293F cells (purchased from Gibco) were cultured with Expi293F medium (Gibco, REF#A14351-01). The cell density was measured one day before transfection (viability must be greater than 95%) and adjusted to 3E6 cells / ml with fresh Expi293F medium for continued culture. On the day of transfection, the cell density was adjusted to 3E6 cells / ml.

[0592] Take 1 / 10 of the final transfection volume of Opti-MEM medium (Gibco, REF#31985-070) as the transfection buffer, add the DNA to be transfected at a ratio of 1 mg / L, where the light and heavy chain plasmids are in a 1:1 ratio, mix well, add PEIMax (Polysciences Inc. Cat#24765-1) at a DNA:PEI mass ratio of 1:3, mix well, incubate at room temperature for 20 minutes, and then gently pour the mixture into the Expi293F cell suspension while shaking. The cells are cultured in a shaker under the conditions of 8% CO2, 36.5°C, and 120 rpm.

[0593] After 16-18 hours of culture, 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 to a final concentration of 5 g / L, and valproic acid sodium salt (Merk, Cat# P4543-100G) to a final concentration of 2.2 mM. The suspension was gently mixed and cultured for 7 days at 8% CO₂, 36.5°C, and 120 rpm before sampling. The cell suspension was then mixed with diatomaceous earth (Sartorius, Cat 1000037025) (40 g diatomaceous earth per 1 L of cell suspension) and filtered using a 0.22 μm disposable vacuum filter.

[0594] Affinity chromatography to purify the target protein: A HiTrap MabSelect PrismA (GE Healthcare, Cat#17549853) affinity chromatography column was used for affinity capture. Before purification, 10-20 column volumes of 0.1 M NaOH were passed through the tubing and affinity chromatography column, and then the tubing 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); the filtered cell feed was passed through the column, and the packed column was washed with 10 column volumes of 1× PBS to remove non-specific binding proteins; the packed column was rinsed with 5 column volumes of elution buffer (100 mM sodium citrate, pH 3.5), the eluate was collected, the pH was adjusted to 6.0 with 2 M Tris, and the column was filtered and sterilized for further ion exchange chromatography.

[0595] In vitro reduction and oxidation: Mix the protein fractions from affinity chromatography at a 1:1 molar ratio, add an appropriate amount of GSH, and adjust the reaction pH to 8.0 with 2M Tris. Incubate at room temperature overnight. Exchange the reaction mixture into PBS and store at 4°C until needed.

[0596] Ion exchange chromatography purification of bispecific antibodies: A Mono S 5 / 50GL (GE Healthcare) ion exchange chromatography column was used and placed in an AKTApure system (GE healthcare). The AKTApure system equipped with a Mono S 5 / 50GL ion exchange chromatography column was detoxified with 0.5 M NaOH for 2 hours, and then 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 the conductivity and pH stabilized. The protein obtained by affinity chromatography was diluted 10-fold with loading buffer and then loaded. The column was re-equilibrated with 5 column volumes of loading buffer. A linear elution gradient of 0-40% elution buffer (20 mM NaPO4, 1 M NaCl, pH 6.6) was performed for a total of 30 column volumes, and samples were collected based on UV absorbance.

[0597] The purity of the collected samples in each fraction was tested by size exclusion chromatography (SEC), and the samples in the fractions with a purity greater than 95% were combined based on the SEC results.

[0598] The purified bispecific antibody solution was centrifuged at 4500 rpm for 30 minutes using 15 ml of ultrafiltration centrifuge tubes. The protein was diluted with PBS and centrifuged at 4500 rpm for 30 minutes. The reaction was repeated several times to replace the buffer. The antibodies after buffer replacement were merged and the antibody concentration was measured. Capillary electrophoresis (CE-SDS) and liquid chromatography-mass spectrometry (LC-MS) were further used to qualitatively and quantitatively analyze the composition and content of the bispecific antibody. The bispecific antibodies obtained in Table 5 were used for the following examples.

[0599] CLDN18.2xCD28 bispecific antibody affinity testing

[0600] To characterize the affinity of the bispecific anti-CD28 antibody at both ends, this study used thin-layer interferometry (BLI) to determine the equilibrium dissociation constant (KD) of the bispecific antibody of the present invention binding to human CD28 and Claudin18.2 protein. For specific methods, please refer to Example 1.

[0601] After testing, it was found (Table 7) that the CLDN18.2 end of the bispecific antibody had a high affinity for the Claudin18.2 protein. The affinity of the CD28 end of the bispecific antibody showed some differences.

[0602] Table 7: Affinity detection of both ends of CLDN18.2xCD28 bispecific antibodies

[0603] Example 7: In vitro activity detection of CLDN18.2xCD28 bispecific antibody

[0604] 7.1 NFkB Reporter Activation Detection with CLDN18.2xCD28 Diabody

[0605] In previous studies, we have verified that CD28 monoclonal antibodies can effectively activate the CD28 signaling pathway and thus activate T cells. Here we want to verify whether CD28 can still have good stimulatory activity on T cells after being assembled into a bispecific antibody. First, we verified the activation properties of the CLDN18.2xCD28 bispecific antibody on NFkB Jurkat Reporter cells in vitro. In this research system, we need to apply it to tumor cells expressing Claudin18.2 to provide a "handle" so that the CLDN18.2 at one end of the CLDN18.2xCD28 bispecific antibody can effectively bind to the tumor cells. In addition, the CD28 end also needs to be verified in the study. For this reason, this study introduced a co-culture system of the CD28-expressing T cell line Jurkat and Claudin18.2-expressing tumor cells. The entire research process is as follows:

[0606] Construction of DANG-18.2 cells:

[0607] The full-length gene for human CLDN18.2 (UniProt ID: P56856-2) was constructed into the pWPT-GFP vector (Addgene, 12255), replacing the GFP sequence. This construct was then co-transfected with the lentiviral packaging vectors psPAX2 (Addgene, 12260) and pMD2.G (Addgene, 12259) into HEK293T (ATCC, CRL-3216) cells for viral packaging. The culture supernatants were collected after 48 and 72 hours of culture, and the lentiviral vectors were concentrated using PEG8000. The concentrated lentiviral vectors were then used to transfect pancreatic cancer DAN-G cells (DSMZ, Cat. No. ACC 249). CLDN18.2-expressing cells were then isolated using a flow cytometer (MoFlo XDP, Beckman Coulter) to generate the DANG-18.2 tumor cell line stably transfected with CLDN18.2.

[0608] CLDN18.2xCD28 dual antibody activates NFkB reporter

[0609] Treatment of target and effector cells: Remove cultured DANG-18.2 cells from a T75 flask and centrifuge at 25°C, 400g, for 4 minutes. Adjust the cell density to 100 μl per well (40,000 cells / well) using growth medium. Mix thoroughly and plate the cells into a 96-well culture plate. Remove the 96-well culture plate coated with target cells and discard the culture medium.

[0610] According to the experimental design, serial dilutions of the CLDN18.2xCD28 dual antibody (Table 5) were performed, starting at 100 nM and followed by a 4-fold dilution series for a total of 10 dilutions. The antibodies were diluted in assay buffer (1% FBS, 1640). 50 μL of the test antibody (IgG, Jackson Immune Research, 109-005-098) was added to each well of a plate containing target cells.

[0611] NFkB Jurkat cells (Jiman Bio, GM-C07855) were removed from a T75 flask and centrifuged at 400g for 4 minutes at 25°C. Cell density was adjusted with assay buffer to 50,000 cells / well (50 μl). Mix thoroughly and add OKT3 (final concentration 0.5 μg / ml). Mix thoroughly and add to a 96-well plate. Incubate in a 5% CO2, 37°C incubator for 6 hours.

[0612] The 96-well culture white plate was taken out from the incubator, and 100 μL / well of Bio-Glo Luciferase Reagent (Promega, G7940) solution was added. After 2 minutes at room temperature, the plate was detected using a microplate reader (Molecular Divices, Spectra MAX i3x).

[0613] The results are shown in Figure 4. The CD28 monoclonal antibody TGN was unable to effectively activate CD28 signaling. In the presence of CLDN18.2 as a "hook," CD28 bispecific antibodies BsAb-1, BsAb-2, and BsAb-3 were all able to effectively activate NFkB signaling downstream of the CD28 signaling pathway, with the signal becoming stronger with increasing concentration.

[0614] 7.2 CLDN18.2xCD28 Diabody-Mediated T Cell Cytotoxicity Experiment in Vitro

[0615] To further validate the in vitro activity of the CD28 bispecific antibody, we tested whether the CLDN18.2xCD28 bispecific antibody could activate human peripheral T cells and subsequently kill tumor cells. In this study, an aAPC expressed on the tumor cell surface provided the primary signal. Furthermore, CLDN18.2 was transfected onto the tumor cell surface as a specific target for the antibody. The experimental procedure is as follows:

[0616] CLDN18.2-aAPC-CHO K1 was constructed as follows:

[0617] The full-length gene of human CLDN18.2 (UniProt ID: P56856-2) was constructed into the vector pWPT-GFP (Addgene, 12255), and the GFP sequence was replaced. It was co-transfected with the lentiviral packaging vectors psPAX2 (Addgene, 12260) and pMD2.G (Addgene, 12259) into aAPC-CHO K1 (Promega, J1250) cells for viral packaging. The culture supernatant was collected after 48 hours and 72 hours of culture, and the lentivirus was concentrated using a Lenti-X Concentrator (Takara, Cat#631232, Lot#2109261A). The concentrated virus was used to transfect hamster ovary aAPC-CHO K1 cells (Promega, J1250), and then the cells were separated using a flow cytometer (BD FACSAria TM The cells expressing CLDN18.2 were sorted using a Fusion Cell Sorter to obtain the cell line CLDN18.2-aAPC-CHO K1 stably transfected with CLDN18.2.

[0618] The CLDN18.2xCD28-mediated T cell killing process is as follows:

[0619] 1. Target Cell Plating

[0620] Remove the cultured CLDN18.2-aAPC-CHO K1 cells and digest them with trypsin. Terminate the digestion process by resuspending the cells in growth medium and centrifuging them. Adjust the cell density with assay buffer and plate the cells at 100 μl per well (10,000 cells / well) in a 96-well round-bottom plate. Culture for 16-20 hours. Add assay buffer to the edge wells at 200 μl / well.

[0621] 2. Effector Cell Treatment

[0622] Remove one frozen PBMC, thaw in a 37°C water bath, and centrifuge at 25°C, 400g, for 4 minutes. Isolate CD8+ T cells according to the EasySep Human CD8+ T Cell (STEMCELL, 19053) instructions. Resuspend in RPMI 1640 medium and incubate overnight in a 5% CO2, 37°C incubator. Remove the cells the next day and centrifuge at 25°C, 400g, for 4 minutes. Adjust the cell concentration with assay buffer and plate 50 μl per well (50,000 cells / well) of CD8+ T cells into a 96-well round-bottom plate at a 5:1 effector:target ratio.

[0623] 3. Dilute Antibodies

[0624] Serially dilute the test antibody according to the experimental design, starting at 50 nM. Dilute the antibody in assay buffer in a 4-fold series for a total of 10 dilutions. Add 50 μl of the serially diluted antibody to a 96-well round-bottom plate containing CD8+ T cells and CLDN18.2-aAPC-CHO K1 cells. Incubate in a 5% CO2, 37°C incubator for 48 hours, with a total volume of 200 μl per well.

[0625] 4. Add 20 μl of 10x Lysis Buffer to the control wells to lyse the target cells. Incubate in a 5% CO2, 37°C incubator for 45 minutes. Prepare the detection reagents according to the instructions of the Radio Cytotoxicity Assay Kit (Promega, G1781). Centrifuge the 96-well round-bottom plate to sediment the cells. Transfer 50 μl of Mix Buffer to a 96-well flat-bottom plate and add 50 μl of the supernatant from the experimental plate to each well. Incubate at room temperature in the dark for 30 minutes. Add 50 μl of Stop Buffer per well and read the assay within 1 hour at 490 nm or 492 nm on a microplate reader (Molecular Devices, Spectra MAX i3x).

[0626] The results ( FIG5 ) showed that several CD28 bispecific antibodies tested were able to effectively activate T cells and kill tumor cells in the presence of the first signal and cell surface Claudin18.2.

[0627] 7.3 In vitro T cell activation experiments with CLDN18.2xCD28 diabody

[0628] To further validate the activity of the CLDN18.2xCD28 antibodies of the present invention, we tested whether these antibodies could effectively activate human peripheral T cells in vitro. The specific process is as follows:

[0629] 1. Remove PBMC cells one day in advance, centrifuge at 25°C, 400g, 4 minutes, resuspend in growth medium, place in a culture flask, add DNase I (SIGMA, D5025-375KU) at a final concentration of 2 μg / mL, and culture overnight in a 5% CO2, 37°C incubator.

[0630] 2. Dilute the CLDN18.2xCD28 dual antibody (1 μg / well) or, if needed, the CD3 antibody (Acro, CDE-M120A-1MG, 0.1 μg / well) in PBS and add 100 μL of the antibody to be tested to each well of a 96-well flat-bottom plate. Incubate overnight at 4°C to allow the antibody to fully bind to the bottom of the plate.

[0631] 3. On the day of the experiment, after the antibody incubation is completed, pour off the unbound antibody solution in the plate, add 100 μl of PBS to each well, wash once and discard the PBS.

[0632] 4. Remove the PBMCs that were revived the previous day and pour the suspended cells into a centrifuge tube. Centrifuge at 25°C, 400g, for 4 minutes. Adjust the cell density with assay buffer (RPMI 1640, 1% FBS) to 100 μl per well (100,000 cells / well). Plate the cells into a 96-well flat-bottom plate coated with the antibody. Incubate in a 5% CO2, 37°C incubator.

[0633] 5. 4 hours before the end of cell culture, remove the 96-well plate from the incubator and transfer 50 μl of supernatant per well to a fresh 96-well round-bottom plate for cytokine assays. Freeze at -40°C. Add 50 μl of protease transport inhibitor (Invitrogen, 00-4980-93) to each well of the 96-well flat-bottom plate and incubate in a 5% CO2, 37°C incubator for 4 hours.

[0634] 6. After incubation, prepare for flow cytometry antibody staining. First, centrifuge the 96-well flat-bottom plate at 25°C, 400g, for 4 minutes and discard the supernatant. Resuspend the cells in PBS and transfer them to a new 96-well round-bottom plate. Centrifuge at 25°C, 400g, for 4 minutes and discard the PBS.

[0635] 7. Add 50 μl of the pre-prepared surface blocking dye mixture: Fc blocking (BD, 564219, 1:200 in PBS) and dead / viable dye (Invitrogen L34968A, 1:100 in PBS) to each well. Incubate at room temperature in the dark for 10 minutes. After incubation, add 50 μl of a 1:100 cell surface dye mixture: PD1 antibody (Biolegend, 329918), CD4 antibody (Biolegend, 329918), and CD8 antibody (Biolegend, 344724) in FACS buffer directly to each well. Incubate at 4°C in the dark for 30 minutes.

[0636] 8. After the surface dye incubation is complete, centrifuge at 25°C, 400g, 4 minutes to remove unbound dye. Resuspend the cells in FACS wash buffer (2mM EDTA, 0.5% BSA, PBS) and wash twice by centrifugation.

[0637] 9. Add 100 μL / well of Fix / Perm buffer (ebioscience 00-5523-00 Invitrogen, 00-8333-56) and incubate at room temperature in the dark for 30 min to penetrate the cell membrane.

[0638] 10. After incubation, centrifuge at 25°C, 400g, 4 minutes, and discard the Fix / Perm buffer. Resuspend the cells in PBE (Invitrogen, 00-8333-56) and centrifuge, discarding the solution.

[0639] 11. Add intracellular dye mixture: IFN-γ antibody (Biolegend, 506504) and TNF-α antibody (Biolegend, 502946) mixture, 50 μL / well, and incubate at room temperature in the dark for 30 minutes.

[0640] 12. After incubation, centrifuge at 25°C, 400g, 4 minutes to remove any unbound dye. Resuspend the cells in PBE buffer, wash twice, and discard the buffer.

[0641] 13. Resuspend the cells in FACS wash buffer, 150 μl / well, and detect on a BD FACS SYMPHONY A3 instrument.

[0642] The results showed (Figure 6) that in the absence of a first signal provided by the CD3 antibody, the control antibody BsAb-BM-1 (monovalent TGN) was able to activate CD4 T cells to release TNFα independently of the first signal, further confirming that TGN, as a single-arm CD28 antibody, also exhibited super-agonistic properties (Figure 6A). Under the same conditions, three CD28 bispecific antibodies of the present invention did not exhibit CD28 super-agonistic activity. In the presence of a first signal provided by the CD3 antibody, all CD28 bispecific antibodies were able to effectively activate T (CD4 and CD8) cells (Figure 6B), releasing different levels of the cytokine TNFα.

[0643] Example 8: Construction and preparation of PSMAxCD28 bispecific antibody

[0644] To further test the functionality of humanized CD28 antibodies, a PSMAxCD28 bispecific antibody targeting human PSMA was constructed in this study (Table 8). The bispecific antibody molecule adopts a "1+1" format (Figure 3B). The PSMA-targeting variable region is derived from 1C3 (EP1851250B1, sequence information is shown in Table 9), while the anti-CD28 variable region sequences are derived from the variable region sequences of the humanized antibodies hz5B11D8-a, hz61FG3-a, and hz4F12B5-b of the present invention (Table 9). The antibody Fc fragment adopts the IgG1 LALA format of Innobody (bispecific antibody technology is derived from CN116888149A). The schematic diagram of the antibody is shown in Figure 3B. Therefore, the heavy chain of the 1C3 portion of the PSMA-targeting bispecific antibody is SEQ ID NO:48, and the light chain is SEQ ID NO:50. The heavy chains of the CD28 end portions of the bispecific antibodies of the present invention are SEQ ID NOs: 51, 52 and 53, respectively, and the light chains are SEQ ID NOs: 36, 38 and 40, respectively.

[0645] The control bispecific antibody used in the present invention is PSMAxCD28: REGN5678 (US20190389951A1). The PSMA-side heavy chain is SEQ ID NO: 55, the light chain is SEQ ID NO: 57, the CD28-side heavy chain is SEQ ID NO: 59, and the light chain is SEQ ID NO: 57.

[0646] Table 8: PSMAxCD28 dual antibody combinations

[0647] Table 9: PSMAxCD28 bispecific antibody information

[0648] For all PSMAxCD3 bispecific antibodies obtained in this study, the PSMA sequence was derived from patent (WO2017023761A1), and the variable region sequence of the CD3-terminal antibody was derived from (CN202180066476A). The light chain, heavy chain, and corresponding variable region sequences of the molecule are shown in Table 10. The heavy and light chain sequences corresponding to the PSMA end are SEQ ID NO: 61 and SEQ ID NO: 63, respectively, and the heavy and light chain sequences corresponding to the CD3 end are SEQ ID NO: 65 and SEQ ID NO: 68, respectively.

[0649] Table 10: PSMAxCD3 bispecific antibody information

[0650] Preparation of PSMAxCD28 dual antibody

[0651] The four bispecific antibodies (BsAb-4, BsAb-5, BsAb-6, and PSMAxCD3) in this invention were prepared by separately expressing and purifying two parent proteins and then recombining them in vitro through reduction-oxidation. The control bispecific antibody, REGN5678, was prepared by intracellular assembly.

[0652] Plasmid construction: The sequences of the bispecific antibodies of the present invention were synthesized by GeneWeizhi. The corresponding light chain variable regions and heavy chain variable regions (Tables 9-10) in the bispecific antibodies were loaded into the pcDNA3.1 vector (Invitrogen, V790-20) containing the corresponding heavy chain constant regions and light chain constant regions, respectively, to obtain the heavy chain plasmid and light chain plasmid at the PSMA end, the heavy chain plasmid and light chain plasmid at the CD28 end, and the heavy chain plasmid and light chain plasmid at the CD3 end.

[0653] The preparation routes of the four bispecific antibodies BsAb-4, BsAb-5, BsAb-6, and PSMAxCD3 can be referred to Example 7. The expression and purification methods of the control bispecific antibody REGN5678 are referred to (Andrew D. Tustian et al. (2016) Development of purification processes for fully human bispecific antibodies based upon modification of protein A binding avidity, mAbs, 8:4, 828-838).

[0654] The PSMAxCD28 and PSMAxCD3 bispecific antibodies shown in Tables 8-10 were prepared and obtained for use in the studies described in the following examples.

[0655] Example 9: In vitro activity detection of PSMAxCD28 bispecific antibodies

[0656] 9.1 PSMAxCD28 dual antibody cell surface affinity detection

[0657] To test the activity of our CD28 bispecific antibodies, we first tested the affinity of several antibodies on the cell surface in vitro.

[0658] First, we constructed MC38_hPSMA tumor cells expressing human PSMA. The specific construction is as follows:

[0659] The full-length gene of human PSMA (UniProt ID: Q04609-1) was constructed into the vector pWPT-GFP (Addgene, 12255), and the GFP sequence was replaced. It was co-transfected with the lentiviral packaging vectors psPAX2 (Addgene, 12260) and pMD2.G (Addgene, 12259) into HEK293T (ATCC, CRL-3216) cells for viral packaging. The culture supernatants were collected after 48 hours and 72 hours of culture, and the lentivirus was concentrated using Lenti-X Concentrator (Takara, Cat#631232, Lot#2109261A). The concentrated virus was used to transfect colon cancer MC38 cells (Nanjing Kebai Biological), and then the cells were separated using a flow cytometer (BD FACSAria TM The PSMA-expressing cells were sorted using Fusion Cell Sorter to obtain the tumor cell line MC38_hPSMA stably transfected with PSMA.

[0660] Next, we constructed the human CD28-expressing tumor cell line 024-hCD28 CHO GS. The specific construction is as follows:

[0661] The full-length human CD28 gene (UniProt ID: P10747) was constructed into the pXC17.4 vector (Lonza). The plasmid was digested into linear fragments using PvuI (NEB, R3150L). The linearized plasmid was electroporated into CHO-GS (Lonza) cells using a Bio-Rad GENEPULSER XCELL, 300V, 900uF, Resistance infinity (∞), 2mm. After transfection, the cells were transferred to CD CHO medium (Gibco, 10743-029) supplemented with 1% SP4 (Lonza, BESP1076E) at 37°C and cultured in a shaker at 37°C, 6% CO2, and 120 rpm. After 16-30 hours of electro-transfection, centrifuge at 1000 rpm for 5 minutes, discard the supernatant, resuspend the cells in fresh medium supplemented with SP4, add 50 μM MSX (SIGMA M5379-250MG), adjust the cell density to 5E5 / ml, and continue culturing. Starting from Day 7, check the cell density and viability every 1-2 days. When the cell viability reaches above 90%, use flow cytometry (BD FACSAria TM The cells expressing human CD28 were sorted using Fusion Cell Sorter to obtain the tumor cell line 024-hCD28 CHO GS stably transfected with CD28.

[0662] Affinity testing

[0663] Remove cultured 024-hCD28 CHO GS and MC38_hPSMA cell lines from T75 flasks and centrifuge at 400g for 4 minutes at 25°C. Adjust cell density with FACS wash buffer (2mM EDTA, 0.5% BSA, PBS) and plate the cells to a concentration of 50 μl per well (100,000 cells / well). Mix thoroughly and plate the cells into a 96-well round-bottom plate. Prepare a serial dilution of the test antibody according to the experimental design, starting at 100 nM and then diluting the antibody in a 4-fold dilution series for a total of 10 dilutions. Add 50 μl of the test antibody to each well of the plate containing the cells. Incubate in a refrigerator at 4°C for 30-60 minutes to allow for full binding of the antibody to the cell surface. After incubation, centrifuge the plate at 25°C, 400g for 4 minutes to remove any unbound antibody. Add 100 μl of FACS wash buffer to each well and resuspend the cells. Centrifuge the plate at 25°C, 400g for 4 minutes and discard the wash buffer. Add 100 μl of PE-hIgG-heavy chain diluent (Biolegend, 410708) to each well to allow the heavy chain of the antibody to be tested to bind. Incubate at room temperature in the dark for 20-30 minutes. After incubation, centrifuge the plate at 25°C, 400g for 4 minutes to remove any unbound antibody. Add 100 μl of FACS wash buffer to each well and resuspend the cells. Centrifuge the plate at 25°C, 400g for 4 minutes and discard the wash buffer. Finally, add 100 μl of FACS wash buffer to each well to resuspend the cells and prepare for analysis on a BD FACS SYMPHONY A3 instrument.

[0664] The results showed (Figure 7) that the bispecific antibodies all exhibited strong affinity for PSMA, with the overall PSMA cell surface affinity of BsAb-4, BsAb-5, and BsAb-6 comparable to the PSMA activity of REGN5678 (Figure 7A). In terms of affinity for CD28, BsAb-4, BsAb-5, and BsAb-6 all showed binding (Figure 7B).

[0665] 9.2 PSMAxCD28 dual antibody detection of NFkB reporter activation

[0666] Next, we further verified whether CD28 can still have a good stimulatory activity on T cells after being assembled into a bispecific antibody. First, we verified the activation properties of the PSMAxCD28 bispecific antibody on NFkB Jurkat Reporter cells in vitro. In this research system, we need to apply it to tumor cells expressing human PSMA to provide a "handle" so that the PSMA on one end of the PSMAxCD28 bispecific antibody can effectively bind to the tumor cells. In addition, the CD28 end also needs to be verified in the study. For this purpose, this study introduced the CD28-expressing T cell line Jurkat. The entire research process is as follows:

[0667] Target and effector cell processing

[0668] Remove the cultured MC38_hPSMA cells from a T75 flask and centrifuge at 25°C, 400g, for 4 minutes. Adjust the cell density with growth medium to 100 μl per well (40,000 cells / well). Mix thoroughly and plate the cells into a 96-well white plate (WHB). Remove the 96-well white plate (WHB) coated with target cells and discard the culture medium.

[0669] According to the experimental design, serially dilute the PSMAxCD28 antibody, starting at 100 nM. Dilute the antibody in a 4-fold gradient, for a total of 10 dilutions. Dilute the antibody in assay buffer (1% FBS, 1640). Add 50 μL of the antibody to each well of the plate containing target cells.

[0670] NFkB Jurkat cells were removed from a T75 flask and centrifuged at 400g for 4 minutes at 25°C. Cell density was adjusted with assay buffer to 50,000 cells / well (50 μl per well). Mix thoroughly, then add CD3 antibody (Acro, CDE-M120A-1MG, final concentration 0.5 μg / ml) or PSMAxCD3 dual antibody (final concentration 10 pM). Mix thoroughly and then add to a 96-well white plate (WHB). Incubate in a 5% CO2, 37°C incubator for 6 hours.

[0671] The 96-well white culture plate (WHB) was taken out from the incubator, and 100 μL / well of Bio-Glo Luciferase Reagent (Promega, G7940) solution was added. After 2 minutes at room temperature, the cells were detected using a microplate reader (Molecular Divices, Spectra MAX i3x).

[0672] In this study, we applied two primary signals to verify the effects of CD28 bispecific antibodies on CD28 signaling activation.

[0673] First, we used anti-CD3 antibody as the first signal. As shown in Figure 8A, several CD28 dual antibodies can activate the NFkB signal downstream of CD28 in Jurkat cells.

[0674] Next, we used low-concentration PSMAxCD3 bispecific antibodies as the primary signal to examine T cell activation by CD28 bispecific antibodies. As shown in Figure 8B, several PSMAxCD28 bispecific antibodies tested activated the NFkB reporter, with a similar trend to Figure 8A.

[0675] 9.3 PSMAxCD28 Diabody-Mediated T Cell Killing Experiment in Vitro

[0676] Next, we further tested whether the PSMAxCD28 bispecific antibody could activate human peripheral T cells and kill tumor cells. In this study, an aAPC was integrated on the surface of our tumor cells to provide the first signal. In addition, human PSMA was transfected on the tumor cell surface as a specific recognition target on one end of the antibody. The entire experimental process is as follows:

[0677] PSMA-aAPC-CHO K1 was constructed as follows:

[0678] The full-length gene for human PSMA (UniProt ID: Q04609-1) was constructed into the pWPT-GFP vector (Addgene, 12255), replacing the GFP sequence. The vector was then co-transfected with the lentiviral packaging vectors psPAX2 (Addgene, 12260) and pMD2.G (Addgene, 12259) into aAPC-CHO K1 (Promega, J1250) cells for viral packaging. The culture supernatants were collected after 48 and 72 hours of culture and concentrated using a Lenti-X Concentrator (Takara, Cat# 631232, Lot# 2109261A). The concentrated virus was used to transfect hamster ovary aAPC-CHO K1 cells, and then the PSMA-expressing cells were sorted using a flow cytometer (MoFlo XDP, Beckman Coulter) to obtain the PSMA-aAPC-CHO K1 cell line stably transfected with PSMA.

[0679] PSMAxCD28 mediates T cell killing of tumor cells. The specific process is as follows:

[0680] 1. Target Cell Plating

[0681] Remove the cultured PSMA-aAPC-CHO K1 cells and digest them with trypsin. Resuspend the cells in growth medium to terminate the digestion process and centrifuge. Adjust the cell density with assay buffer and plate the cells into a 96-well round-bottom plate at 100 μl per well (10,000 cells / well) and culture for 16-20 hours. Add assay buffer to the edge wells at 200 μl / well.

[0682] 2. Effector Cell Treatment

[0683] Remove one frozen PBMC, thaw in a 37°C water bath, and centrifuge at 25°C, 400g, for 4 minutes. Isolate CD8+ T cells according to the EasySep Human CD8+ T Cell (STEMCELL, 19053) instructions. Resuspend in RPMI 1640 medium and incubate overnight in a 5% CO2, 37°C incubator. Remove the cells the next day and centrifuge at 25°C, 400g, for 4 minutes. Adjust the cell concentration with assay buffer and plate 50 μl per well (50,000 cells / well) of CD8+ T cells into a 96-well round-bottom plate at a 5:1 effector:target ratio.

[0684] 3. Dilute Antibodies

[0685] Antibody was serially diluted according to the experimental design, starting at 50 nM and followed by 10 4-fold dilutions using assay buffer. Fifty microliters of the serially diluted antibody was added to a 96-well round-bottom plate containing CD8+ T cells and PSMA-aAPC-CHO K1 cells. The cells were incubated in a 5% CO2, 37°C incubator for 48 hours, with a total volume of 200 microliters per well.

[0686] 4. Add 20 μl of 10x Lysis Buffer to the control wells to lyse the target cells. Incubate in a 5% CO2, 37°C incubator for 45 minutes. Prepare the detection reagents according to the instructions of the Radio Cytotoxicity Assay Kit (Promega, G1781). Centrifuge the 96-well round-bottom plate to sediment the cells. Transfer 50 μl of Mix Buffer to a 96-well flat-bottom plate and add 50 μl of the supernatant from the experimental plate to each well. Incubate at room temperature in the dark for 30 minutes. Add 50 μl of Stop Buffer per well and read the assay within 1 hour at 490 nm or 492 nm on a microplate reader (Molecular Devices, Spectra MAX i3x).

[0687] The results (Figure 9) show that all three PSMAxCD28 bispecific antibodies can activate T cells and kill PSMA-expressing tumor cells. In terms of the degree of target cell death, BsAb-4, which has a strong affinity for CD28, has the strongest CD28 activation activity and can mediate T cell killing of tumor cells at low concentrations. BsAb-5 has the lowest affinity for CD28 and its killing activity is relatively weak. Due to the decrease in CD28 affinity, the overall mediated killing level of BsAb-6 is also weakened.

[0688] Example 10: Detection of activated T cells using PSMAxCD28 bispecific antibodies

[0689] To further validate the activity of PSMAxCD28 antibodies, we tested whether these antibodies could effectively activate human peripheral T cells in vitro and release the cytokine IL2. The specific process is as follows:

[0690] 1. Remove PBMC cells one day in advance, centrifuge at 25°C, 400g, 4 minutes, resuspend in growth medium, place in a culture flask, add DNase I (SIGMA, D5025-375KU) at a final concentration of 2 μg / mL, and culture overnight in a 5% CO2, 37°C incubator.

[0691] 2. Dilute PSMAxCD28 antibody (1 μg / well) or, if needed, CD3 antibody (Acro, CDE-M120A-1MG, 0.1 μg / well) in PBS and add 100 μL of the antibody to be tested to each well of a 96-well flat-bottom plate. Incubate overnight at 4°C to allow the antibody to fully bind to the bottom of the plate.

[0692] 3. On the day of the experiment, after the antibody incubation is completed, pour off the unbound antibody solution in the plate, add 100 μl of PBS to each well, wash once and discard the PBS.

[0693] 4. Remove the PBMCs that were revived the previous day and pour the suspended cells into a centrifuge tube. Centrifuge at 25°C, 400g, for 4 minutes. Adjust the cell density with assay buffer (RPMI 1640, 1% FBS) to 100 μl per well (100,000 cells / well). Plate the cells into a 96-well flat-bottom plate coated with the antibody. Incubate in a 5% CO2, 37°C incubator.

[0694] 5. 4 hours before the end of cell culture, remove the 96-well plate from the incubator and transfer 50 μl of supernatant from each well to a new 96-well round-bottom plate for cytokine detection.

[0695] 6. According to the instructions of IL2 Human ELISA KIT (Mabtech, 3445-1H-20), dilute Human IL-2 antibody with PBS and add 50 μl per well to a 96-well flat-bottom plate. Incubate in a refrigerator at 4°C overnight and cover with sealing film.

[0696] 7. Pour off any unbound purified antibody from the 96-well plate and add 100 μL of PBST buffer (0.05% Tween 20) to each well. Rinse six times. Then, add 100 μL of Assay buffer (1000 mL PBS, 500 μL Tween 20, 5 g BSA) to each well and incubate at room temperature for 1 hour.

[0697] 8. After incubation, remove the 96-well plate, pour out the liquid in the plate, add the prepared sample supernatant and 50 μl of standard, and incubate at room temperature for 2 hours.

[0698] 9. Remove the 96-well plate, pour out the liquid in the plate, rinse six times, add 50 μl of biotin antibody diluent (Mabtech, 3445-1H-20) to each well, and incubate on a plate shaker at room temperature for 1 hour.

[0699] 10. Take out the 96-well plate, pour out the liquid in the plate, rinse 6 times, add 50 μl of HRP diluent to each well, and incubate at room temperature for 1 hour.

[0700] 11. Remove the 96-well plate, pour out the liquid in the plate, rinse six times, add 50 μl of TMB to each well, wait for the color development reaction to be complete, add 50 μl of stop solution to each well, and read the value at 450 nm wavelength on a microplate reader (Molecular Divices, Spectra MAX i3x) within 1 hour.

[0701] The results showed that in the absence of the first signal CD3 antibody (-signal 1) (Figure 10A), the control antibody TGN1412 (abbreviated as TGN in this study) was able to activate T cells and release IL2 independently of the first signal, confirming that TGN is a CD28 super agonist antibody. Under the same conditions, three CD28 bispecific antibodies of the present invention, BsAb-4, BsAb-5, and BsAb-6, were unable to activate T cells, behaving as conventional CD28 agonist antibodies. In the presence of the first signal provided by the CD3 antibody (+signal 1), all CD28 antibodies were able to effectively activate T cells and release IL2 (Figure 10B).

[0702] Example 11: In vivo anti-tumor activity detection of PSMAxCD28 bispecific antibody

[0703] To further verify the activity of PSMAxCD28 bispecific antibody, we tested the anti-tumor activity of PSMAxCD28 bispecific antibody in an in vivo mouse tumor model.

[0704] Mice: hCD28 humanized mice (Shanghai Model Organisms) aged 6-8 weeks were placed in an SPF laboratory environment for 3-5 days before the experiment to allow them to better adapt to the current living environment.

[0705] In all experiments, the long and short diameters of the tumors were measured twice a week using a vernier caliper, and the tumor volume (mm3) was calculated using the following formula:

[0706] Tumor volume (mm3) = 0.5 * long diameter (mm) * short diameter (mm) * short diameter (mm)

[0707] hPSMA-MC38 cells expressing human PSMA were resuspended in 1x PBS (Gibco, 10010049) at an appropriate concentration and inoculated subcutaneously on the right flank of mice at a seeding rate of 1E6 cells / mouse (Figure 11A). Approximately 7 days after inoculation, when tumors reached 80-100 mm³, mice were grouped, ensuring that the average initial dose volume within each group was essentially the same. Following grouping, 10 mg / kg of the PSMAxCD28 bispecific antibody was intraperitoneally injected into the mice to observe its effect on tumor growth.

[0708] Results showed that in the human PSMA-overexpressing MC38 tumor model, a single dose of 10 mg / kg of PSMAxCD28 was able to inhibit tumor cell growth to a certain extent (Figure 11B). REGN5678 had a relatively weak tumor suppression effect, with a TGI of only 42.5%. BsAb-4 and BsAb-6 (Figure 11C, each line represents a mouse) produced stronger tumor suppression effects than REGN5678 under the same conditions.

[0709] Example 12: In vivo anti-tumor activity detection of PSMAxCD28 dual antibody combined with PD1 antibody

[0710] To further verify the activity of PSMAxCD28 bispecific antibody, we tested the anti-tumor activity of CD28 bispecific antibody combined with PD1 antibody in an in vivo mouse tumor model.

[0711] The anti-mouse PD1 antibody is from patent (WO2018184964A1) and its sequence is as follows. The preparation process is the same as in Example 1.

[0712] The entire procedure was the same as in Example 12. hPSMA-MC38 cells expressing human PSMA were resuspended in 1x PBS (Gibco, 10010049) at an appropriate concentration and subcutaneously implanted in the right flank of mice at an inoculum of 1E6 cells / mouse (Figure 12A). Approximately 7 days after inoculation, when tumors reached 80-100 mm³, mice were grouped, with the average initial dose volume for each group being essentially the same. After grouping, the BsAb-6 bispecific antibody, anti-PD-1 antibody, or a combination thereof was administered intraperitoneally to the mice, and the effects of the drugs on tumor growth were observed.

[0713] The results showed that in the human PSMA-overexpressing MC38 tumor model, the combination of 10 mg / kg BsAb-6 and 2 mg / kg mouse PD1 antibody produced a strong synergistic effect, significantly inhibiting tumor cell growth (Figure 12B). After a single dose, the complete tumor regression rate reached 100% over an observation period lasting more than 40 days, which was higher than the complete tumor regression rate of the single-drug group (60%) (Figure 12C, each line represents one mouse).

[0714] Example 13: PK Experiment of PSMAxCD28 Bispecific Antibody in Mice

[0715] In this study, 12 female C57BL / 6hCD28KI mice (Shanghai Model Organisms) were injected with 10 mg / kg of REGN5678 and BsAb-6 via tail vein administration to investigate their pharmacokinetic properties in mice.

[0716] After dosing, mice were bled via the eyeball at 0.086 hours, 0.5 hours, 2 hours, 6 hours, 24 hours, 48 ​​hours, 4 days, 7 days, 14 days, and 21 days. The blood was centrifuged at 3000 rpm for 10 minutes at 4°C, and serum was collected. Serum antibody levels were measured by ELISA, and the in vivo half-lives of REGN5678 and BsAb-6 in mice were calculated.

[0717] The experimental results are shown in Table 11. BsAb-6 has good stability, a long half-life (t1 / 2), and relatively good blood exposure in hCD28 transgenic mice. The half-life shows that BsAb-6 achieves stability data similar to that of normal monoclonal antibodies in both detection methods.

[0718] Table 11: In vivo PK assay of PSMAxCD28 dual antibody in mice

[0719] Sequence Listing

Claims

1. An anti-CD28 antibody or an antigen-binding fragment thereof, which comprises three CDRs of the heavy-chain variable region VH, CDRH1, CDRH2 and CDRH3, and three CDRs of the light-chain variable region VL, CDRL1, CDRL2 and CDRL3, wherein (i). said CDRH1, CDRH2 and CDRH3 are selected from the three complementarity-determining regions CDRH1, CDRH2 and CDRH3 contained in the VH as shown in SEQ ID NO: 24, 23 or 75; and said CDRL1, CDRL2 and CDRL3 are the three complementarity-determining regions CDRL1, CDRL2 and CDRL3 contained in the VL as shown in SEQ ID NO: 28 or 78; (ii). said CDRH1, CDRH2 and CDRH3 are selected from the three complementarity-determining regions CDRH1, CDRH2 and CDRH3 contained in the VH as shown in any one of SEQ ID NO: 4, 5 or 72; and said CDRL1, CDRL2 and CDRL3 are the three complementarity-determining regions CDRL1, CDRL2 and CDRL3 contained in the VL as shown in SEQ ID NO: 9 or 73; or (iii). said CDRH1, CDRH2 and CDRH3 are selected from the three complementarity-determining regions CDRH1, CDRH2 and CDRH3 contained in the VH as shown in SEQ ID NO: 13 or 69; and said CDRL1, CDRL2 and CDRL3 are the three complementarity-determining regions CDRL1, CDRL2 and CDRL3 contained in the VL as shown in SEQ ID NO: 17, 19 or 70.

2. An anti-CD28 antibody or an antigen-binding fragment thereof, which comprises CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3, wherein (i). 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 CDRL3 comprises or consists of the amino acid sequence of SEQ ID NO: 27; (ii). 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 CDRL3 comprises or consists of the amino acid sequence of SEQ ID NO: 27; (iii). 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 or 74; CDRL2 comprises or consists of the amino acid sequence of SEQ ID NO:7; and CDRL3 comprises or consists of the amino acid sequence of SEQ ID NO:8; or (iv). 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 or 18 or 71; CDRL2 comprises or consists of the amino acid sequence of SEQ ID NO:15; and CDRL3 comprises or consists of the amino acid sequence of SEQ ID NO:

16.

3. The antibody or antigen-binding fragment thereof according to claim 1 or 2, which comprises a heavy chain variable region VH, wherein the heavy chain variable region (i) comprises or consists 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; or (ii) comprises or consists of the amino acid sequence of SEQ ID NO:24, 4, 5, 13, 23, 69, 72 or 75.

4. The antibody or antigen-binding fragment thereof according to any one of claims 1-3, which comprises a light chain variable region VL, wherein the light chain variable region (i) comprises or consists 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:28, 9, 17, 19, 70, 73 or 78; or (ii) comprises or consists of the amino acid sequence of SEQ ID NO:28, 9, 17, 19, 70, 73 or 78.

5. The antibody or antigen-binding fragment thereof according to claim 1 or 2, which comprises a heavy chain variable region VH and a light chain variable region VL, wherein (i). The VH contains the amino acid sequence shown 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 thereto, or consists of the amino acid sequence, and the VL contains the amino acid sequence shown 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 thereto, or consists of the amino acid sequence; (ii). The VH contains the amino acid sequence shown 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 thereto, or consists of the amino acid sequence, and the VL contains the amino acid sequence shown 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 thereto, or consists of the amino acid sequence; (iii). The VH contains the amino acid sequence shown 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 thereto, or consists of the amino acid sequence, and the VL contains the amino acid sequence shown 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 thereto, or consists of the amino acid sequence; (iv). The VH contains the amino acid sequence shown 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 thereto, or consists of the amino acid sequence, and the VL contains the amino acid sequence shown 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 thereto, or consists of the amino acid sequence; (v). The VH contains the amino acid sequence shown 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 thereto, or consists of the amino acid sequence, and the VL contains the amino acid sequence shown 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 thereto, or consists of the amino acid sequence; (vi). The VH contains the amino acid sequence shown 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 thereto or consists of said amino acid sequence, and the VL contains the amino acid sequence shown 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 thereto or consists of said amino acid sequence; (vii). The VH contains the amino acid sequence shown 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 thereto or consists of said amino acid sequence, and the VL contains the amino acid sequence shown in SEQ ID NO: 17 or 19 or 70 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto or consists of said amino acid sequence; (viii). The VH contains the amino acid sequence shown 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 thereto or consists of said amino acid sequence, and the VL contains the amino acid sequence shown 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 thereto or consists of said amino acid sequence; or (ix). The VH contains the amino acid sequence shown 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 thereto or consists of said amino acid sequence, and the VL contains the amino acid sequence shown 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 thereto or consists of said amino acid sequence.

6. The antibody or antigen-binding fragment thereof of claim 1 or 2, which comprises a heavy chain variable region VH and a light chain variable region VL, wherein (i). The VH contains the amino acid sequence shown in SEQ ID NO: 24 or consists of said amino acid sequence, and the VL contains the amino acid sequence shown in SEQ ID NO: 28 or consists of said amino acid sequence; (ii). The VH contains the amino acid sequence shown in SEQ ID NO: 23 or consists of said amino acid sequence, and the VL contains the amino acid sequence shown in SEQ ID NO: 28 or consists of said amino acid sequence; (iii). The VH contains the amino acid sequence shown in SEQ ID NO: 72 or consists of said amino acid sequence, and the VL contains the amino acid sequence shown in SEQ ID NO: 73 or consists of said amino acid sequence; (iv). The VH contains the amino acid sequence shown in SEQ ID NO: 4 or consists of said amino acid sequence, and the VL contains the amino acid sequence shown in SEQ ID NO: 9 or consists of said amino acid sequence; (v). The VH contains the amino acid sequence shown in SEQ ID NO: 5 or consists of said amino acid sequence, and the VL contains the amino acid sequence shown in SEQ ID NO: 9 or consists of said amino acid sequence; (vi). The VH contains the amino acid sequence shown in SEQ ID NO: 13 or consists of said amino acid sequence, and the VL contains the amino acid sequence shown in SEQ ID NO: 17 or consists of said amino acid sequence; (vii). The VH contains the amino acid sequence shown in SEQ ID NO: 13 or consists of said amino acid sequence, and the VL contains the amino acid sequence shown in SEQ ID NO: 19 or consists of said amino acid sequence; (viii). The VH contains the amino acid sequence shown in SEQ ID NO: 69 or consists of said amino acid sequence, and the VL contains the amino acid sequence shown in SEQ ID NO: 70 or consists of said amino acid sequence; or (ix). The VH contains the amino acid sequence shown in SEQ ID NO: 75 or consists of said amino acid sequence, and the VL contains the amino acid sequence shown in SEQ ID NO: 78 or consists of said amino acid sequence.

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

8. The antibody or antigen-binding fragment thereof according to any one of claims 1-7, which further comprises a heavy chain constant region HC. 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. Preferably, the heavy chain constant region (i) contains an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence selected from SEQ ID NO:29 or 79 or consists of said amino acid sequence; or (ii) contains the amino acid sequence selected from SEQ ID NO:29 or 79 or consists of said amino acid sequence.

9. The antibody or antigen-binding fragment thereof according to claim 7 or 8, wherein the heavy chain constant region contains a mutation that reduces binding to Fcγ receptor, such as LALA mutation and / or P329delP.

10. The antibody or antigen-binding fragment thereof according to any one of claims 1-9, which comprises a light chain constant region. For example, the light chain constant region is the Lambda or Kappa light chain constant region.

11. The antibody or antigen-binding fragment thereof according to claim 10, wherein the light chain constant region (i) contains an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO:30 or consists of said amino acid sequence; or (ii) contains the amino acid sequence of SEQ ID NO:30 or consists of said amino acid sequence.

12. The antibody or antigen-binding fragment thereof according to any one of claims 1-11, wherein the antibody is a humanized antibody or a chimeric antibody.

13. The antibody or antigen-binding fragment thereof according to any one of claims 1-12, wherein the antibody is a monoclonal antibody.

14. The antibody or antigen-binding fragment thereof according to any one of claims 1-13, wherein the antigen-binding fragment is an antibody fragment selected from: Fab, Fab’, Fab’-SH, Fv, single-chain antibody (such as scFv), (Fab’)2, single-domain antibody (such as VHH), dAb (domain antibody) or linear antibody.

15. The antibody or antigen-binding fragment thereof according to any one of claims 1-11, wherein the antibody is a bispecific antibody or a multispecific antibody, which comprises a first binding specificity for CD28 and other binding specificities for one or more tumor-associated antigens.

16. The antibody or antigen-binding fragment thereof according to claim 15, wherein the tumor-associated antigen is selected from CLDN18.2 or PSMA.

17. A bispecific antibody, which comprises a first antigen-binding region and a second antigen-binding region, wherein the first antigen-binding region specifically binds to CD28 and comprises VH and VL, wherein the VH comprises CDRH1, CDRH2, CDRH3 as defined in claim 1 or 2 and the VL comprises CDRL1, CDRL2 and CDRL3 as defined in claim 1 or 2; or the VH and VL are VH and VL as defined in any one of claims 3-7; the second antigen-binding region specifically binds to a tumor-associated antigen TAA or an immune checkpoint molecule.

18. The bispecific antibody according to claim 17, wherein the first antigen-binding region is the Fab of the anti-CD28 antibody as defined in any one of claims 1-14.

19. The bispecific antibody according to claim 17 or 18, wherein the second antigen-binding region is the Fab that specifically binds to a tumor-associated antigen or an immune checkpoint molecule.

20. The bispecific antibody according to claim 18 or 19, wherein the Fab serving as the first antigen-binding region or the second antigen-binding region comprises CH1, wherein the CH1 is CH1 from IgG1, IgG2, IgG3 or IgG4, preferably CH1 from IgG1.

21. The bispecific antibody according to claim 20, wherein the CH1 (i) comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence selected from SEQ ID NO:74 or consists of the amino acid sequence; or (ii) comprises the amino acid sequence of SEQ ID NO:74 or consists of the amino acid sequence.

22. The bispecific antibody according to any one of claims 18-21, wherein the Fab serving as the first antigen-binding region or the second antigen-binding region comprises a light chain constant region, wherein the light chain constant region is a Kappa light chain constant region or a Lambda light chain constant region.

23. The bispecific antibody according to claim 22, wherein the Kappa light chain constant region (i) comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 30 or consists of said amino acid sequence; or (ii) comprises the amino acid sequence of SEQ ID NO: 30 or consists of said amino acid sequence.

24. The bispecific antibody according to any one of claims 17 - 23, wherein the bispecific antibody is an IgG-like bispecific antibody comprising an Fc dimer, wherein the two Fc regions constituting the Fc dimer are the same or different, optionally, the Fc region is a human IgG1, IgG2, IgG3 or IgG4 Fc, optionally, the Fc region (i) comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to an amino acid sequence selected from SEQ ID NO: 94 or 95 or consists of said amino acid sequence; or (ii) comprises the amino acid sequence of SEQ ID NO: 94 or 95 or consists of said amino acid sequence.

25. The 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 based on the Innobody technology to promote heterodimerization of the first Fc region and the second Fc region.

26. The antibody or antigen-binding fragment thereof according to claim 25, wherein the CH3 of one Fc region comprises the S364R and D399K mutations, and the CH3 mutation of the other Fc region comprises the Y349T, K370S and K409D mutations.

27. The bispecific antibody according to any one of claims 24 - 26, wherein the first and / or second Fc region comprises mutations that reduce binding to Fcγ receptors, such as the L234A / L235A mutation, and optionally P329delP.

28. The bispecific antibody according to claim 26 or 27, wherein a) one Fc region comprises the amino acid sequence shown in SEQ ID NO: 80 or consists of it, and the other Fc region comprises the amino acid sequence shown in SEQ ID NO: 81 or consists of it; b) one Fc region comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence shown in SEQ ID NO: 80 and comprises the S364R and D399K mutations, while the other Fc region comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence shown in SEQ ID NO: 81 and comprises the Y349T, K370S and K409D mutations; c) One Fc region comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence shown in SEQ ID NO:80 and comprises the mutations S364R, D399K, and the L234A / L235A mutations, and P329delP, while the other Fc region comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence shown in SEQ ID NO:81 and comprises the mutations Y349T, K370S, K409D, and the L234A / L235A mutations, and P329delP; d) One Fc region comprises or consists of the amino acid sequence shown in SEQ ID NO:82, and the other Fc region comprises or consists of the amino acid sequence shown in SEQ ID NO:83; e) One Fc region comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence shown in SEQ ID NO:82 and comprises the mutations S364R and D399K, while the other Fc region polypeptide comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence shown in SEQ ID NO:83 and comprises the mutations Y349T, K370S, and K409D; or f) One Fc region comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence shown in SEQ ID NO:82 and comprises the mutations S364R, D399K, and the L234A / L235A mutations, while the other Fc region polypeptide comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence shown in SEQ ID NO:83 and comprises the mutations Y349T, K370S, K409D, and the L234A / L235A mutations.

29. The bispecific antibody of any one of claims 17 - 28, wherein the TAA is CLDN18.2, and preferably, the second antigen - binding region comprises 3 complementarity - determining regions from the heavy - chain variable region (CDRH), CDRH1, CDRH2, and CDRH3, and 3 complementarity - determining regions from the light - chain variable region (CDRL), 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, CD RL2 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.

30. The bispecific antibody of claim 29, wherein the second antigen-binding region comprises VH and VL, wherein VH comprises the sequence shown in SEQ ID NO:86 or consists of said sequence; and VL comprises the sequence shown in SEQ ID NO:90 or consists of said sequence.

31. The bispecific antibody of any one of claims 17-28, wherein the TAA is PSMA, and preferably, the second antigen-binding region comprises three complementarity-determining regions (CDRH) from the heavy-chain variable region, CDRH1, CDRH2, and CDRH3, and three complementarity-determining regions (CDRL) 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, CD RL2 comprises the amino acid sequence of SEQ ID NO:100 or consists thereof, and CDRL3 comprises the amino acid sequence of SEQ ID NO:101 or consists thereof.

32. The bispecific antibody of claim 31, wherein the second antigen-binding region comprises VH and VL, wherein VH comprises the sequence shown in SEQ ID NO:47 or consists of said sequence; and VL comprises the sequence shown in SEQ ID NO:49 or consists of said sequence.

33. The bispecific antibody of any one of claims 17-32, wherein the bispecific antibody comprises a first antigen-binding region that specifically binds CD28 and a second antigen-binding region that specifically binds a TAA, wherein the first antigen-binding region comprises or is a first Fab, and the second antigen-binding region comprises or is a second Fab, wherein the first Fab is connected to the N-terminus of the first Fc region at the C-terminus of its CH1 (via or without a linker, such as a hinge region), and the second Fab is connected to the N-terminus of the second Fc region at the C-terminus of its CH1 (via or without a linker, such as a hinge region).

34. The bispecific antibody of claim 33, wherein the bispecific antibody is an IgG-like antibody having the configuration shown in Figure 3.

35. The bispecific antibody of claim 33 or 34, which comprises a first Fab as the first antigen-binding region that specifically binds CD28 and a second Fab as the second antigen-binding region that specifically binds a TAA, wherein the bispecific antibody comprises Heavy chain 1: From the N-terminus to the C-terminus, it comprises or consists of: the heavy-chain variable region of the first Fab - the heavy-chain constant region CH1 - the first Fc region, wherein the heavy-chain constant region CH1 is connected to the N-terminus of the first Fc region at its C-terminus via or without a linker (such as a hinge region); Light chain 1: From the N-terminus to the C-terminus, it comprises or consists of: the light-chain variable region of the first Fab - the light-chain constant region; Heavy chain 2: From the N-terminus to the C-terminus, it comprises or consists of: the heavy-chain variable region of the second Fab - the heavy-chain constant region CH1 - the second Fc region, wherein the heavy-chain constant region CH1 is connected to the N-terminus of the second Fc region at its C-terminus via or without a linker (such as a hinge region); and / or Light chain 2: From the N-terminus to the C-terminus, it comprises or consists of: the light-chain variable region of the second Fab - the light-chain constant region; Preferably, each domain is directly connected.

36. The bispecific antibody according to claim 35, wherein the TAA is CLDN18.2, and wherein heavy chain 1 comprises the amino acid sequence shown in SEQ ID NO: 35, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identity thereto or consisting of said amino acid sequence; light chain 1 comprises the amino acid sequence shown in SEQ ID NO: 36, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identity thereto or consisting of said amino acid sequence; heavy chain 2 comprises the amino acid sequence shown in SEQ ID NO: 33, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identity thereto or consisting of said amino acid sequence; and light chain 2 comprises the amino acid sequence shown in SEQ ID NO: 34, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identity thereto or consisting of said amino acid sequence; heavy chain 1 comprises the amino acid sequence shown in SEQ ID NO: 37, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identity thereto or consisting of said amino acid sequence; light chain 1 comprises the amino acid sequence shown in SEQ ID NO: 38, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identity thereto or consisting of said amino acid sequence; heavy chain 2 comprises the amino acid sequence shown in SEQ ID NO: 33, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identity thereto or consisting of said amino acid sequence; and light chain 2 comprises the amino acid sequence shown in SEQ ID NO: 34, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identity thereto or consisting of said amino acid sequence; or Heavy chain 1 comprises the amino acid sequence shown in SEQ ID NO: 39, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identity thereto or consisting of said amino acid sequence; light chain 1 comprises the amino acid sequence shown in SEQ ID NO: 40, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identity thereto or consisting of said amino acid sequence; heavy chain 2 comprises the amino acid sequence shown in SEQ ID NO: 33, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identity thereto or consisting of said amino acid sequence; and light chain 2 comprises the amino acid sequence shown in SEQ ID NO: 34, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identity thereto or consisting of said amino acid sequence.

37. The bispecific antibody according to claim 35, wherein the TAA is PSMA, and wherein Heavy chain 1 comprises the amino acid sequence shown in SEQ ID NO: 51, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identity thereto or consisting of said amino acid sequence; light chain 1 comprises the amino acid sequence shown in SEQ ID NO: 36, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identity thereto or consisting of said amino acid sequence; heavy chain 2 comprises the amino acid sequence shown in SEQ ID NO: 48, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identity thereto or consisting of said amino acid sequence; and light chain 2 comprises the amino acid sequence shown in SEQ ID NO: 50, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identity thereto or consisting of said amino acid sequence; Heavy chain 1 comprises the amino acid sequence shown in SEQ ID NO:52, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identity thereto or consisting of said amino acid sequence; light chain 1 comprises the amino acid sequence shown in SEQ ID NO:38, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identity thereto or consisting of said amino acid sequence; heavy chain 2 comprises the amino acid sequence shown in SEQ ID NO:48, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identity thereto or consisting of said amino acid sequence; and light chain 2 comprises the amino acid sequence shown in SEQ ID NO:50, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identity thereto or consisting of said amino acid sequence; or Heavy chain 1 comprises the amino acid sequence shown in SEQ ID NO:53, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identity thereto or consisting of said amino acid sequence; light chain 1 comprises the amino acid sequence shown in SEQ ID NO:40, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identity thereto or consisting of said amino acid sequence; heavy chain 2 comprises the amino acid sequence shown in SEQ ID NO:48, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identity thereto or consisting of said amino acid sequence; and light chain 2 comprises the amino acid sequence shown in SEQ ID NO:50, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identity thereto or consisting of said amino acid sequence.

38. A nucleic acid molecule encoding any one of the chains of the antibody or antigen-binding fragment thereof according to any one of claims 1-16, or any one of the chains of the bispecific antibody according to any one of claims 17-37, or consisting of said nucleic acid sequence.

39. An expression vector comprising the nucleic acid molecule of claim 38, preferably, said expression vector is pCDNA, such as pCDNA3.

1.

40. A host cell comprising the nucleic acid molecule of claim 38 or the expression vector of claim 39, preferably, said host cell is prokaryotic or eukaryotic, such as 293 cells or CHO cells, such as Expi293 cells.

41. A method for preparing the antibody or antigen-binding fragment thereof according to any one of claims 1-16 or the bispecific antibody according to any one of claims 17-37, the method comprising culturing a host cell of the nucleic acid molecule according to claim 38 or the expression vector according to claim 39 under conditions suitable for the expression of the chains of the antibody, and optionally recovering the antibody from the host cell (or the host cell culture medium).

42. An immunoconjugate comprising the antibody or antigen-binding fragment thereof according to any one of claims 1-16 or the bispecific antibody according to any one of claims 17-37.

43. A pharmaceutical composition, drug or preparation comprising the antibody or antigen-binding fragment thereof according to any one of claims 1-16 or the bispecific antibody according to any one of claims 17-37, or the immunoconjugate of claim 42, and optionally a pharmaceutically acceptable excipient.

44. A pharmaceutical combination product comprising the antibody or antigen-binding fragment thereof according to any one of claims 1-16 or the bispecific antibody according to any one of claims 17-37, or the immunoconjugate of claim 42, and one or more other therapeutic agents. Preferably, the therapeutic agent is various therapeutic agents for treating tumors, such as chemotherapeutic agents, angiogenesis inhibitors, cytokines, cytotoxic agents, other antibodies, small molecule drugs or immunomodulators (such as immune checkpoint inhibitors or agonists). Preferably, the other antibody is a PD-1 antibody.

45. A method for preventing or treating a tumor in a subject, comprising administering to the subject an effective amount of the antibody or antigen-binding fragment thereof according to any one of claims 1-16 or the bispecific antibody according to any one of claims 17-37, or the immunoconjugate of claim 42, or the pharmaceutical composition or preparation of claim 43; or the pharmaceutical combination product of claim 44.

46. The method of claim 45, wherein the tumor is a solid tumor or a hematological tumor, such as a TAA-positive tumor or cancer. Optionally, the tumor is a CLDN18.2-positive gastrointestinal tumor, such as a gastrointestinal cancer, such as gastric cancer, breast cancer, colon cancer, rectal cancer or colorectal cancer; or the tumor is a PSMA-positive tumor, such as renal cell cancer, such as breast cancer, lung cancer, rectal cancer or colorectal cancer.

47. The method according to any one of claims 45-46, wherein the method further comprises administering in combination with other therapies such as treatment modalities (such as surgery or radiotherapy) and / or other therapeutic agents. Preferably, the therapeutic agent is various therapeutic agents for treating tumors, such as chemotherapeutic agents, angiogenesis inhibitors, cytokines, cytotoxic agents, other antibodies, small molecule drugs or immunomodulators (such as immune checkpoint inhibitors or agonists). Preferably, the other antibody is a PD-1 antibody.

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