Bispecific antibody aiming at phosphatidylinositol proteoglycan 3 and application thereof

Abstract

Provided are bispecific antigen-binding molecules or antigen-binding fragments thereof for GPC3, derivatives comprising the bispecific antigen-binding molecules or antigen-binding fragments thereof, and pharmaceutical compositions. In addition, the invention also provides related application of the bispecific antigen binding molecule or the antigen binding fragment of the bispecific antigen binding molecule in the aspect of cancer treatment.

Description

A bispecific antibody targeting glypican 3 and its application

[0001] This application requests priority to the Chinese patent application filed with the Patent Office of China on November 1, 2022, with application number CN202211366621.2, and invention name “A bispecific antibody against phosphatidylinositol protein glycan 3 and its application”, and the Chinese patent application filed with the Patent Office of China on October 19, 2023, with application number CN202311361967.8, and invention name “A bispecific antibody against phosphatidylinositol protein glycan 3 and its application”, the entire contents of which are incorporated by reference into this application. Technical Field

[0002] This article belongs to the field of immunology and relates to bispecific antigen-binding molecules targeting glypican 3 (GPC3) and CD3. In addition, this article also relates to polynucleotides, vectors, and host cells encoding such bispecific antigen-binding molecules. This article also relates to pharmaceutical compositions comprising bispecific antigen-binding molecules and related applications in treating and preventing cancer. Background Art

[0003] Glypican 3 is a proteoglycan that attaches to the cell surface via a glycerophosphatidylinositol (GPI) anchor. It belongs to the heparan sulfate glycoprotein (HSPG) family and is primarily involved in regulating cell proliferation, adhesion, and migration. GPC3 is highly expressed in normal embryonic tissues (including the liver and placenta), but is expressed at very low levels or not at all in normal adult tissues (FEBS J, 2013, 280(10):2471-6; Am J Surg Pathol, 2008, 32(3):433-44). Studies have found that GPC3 is specifically highly expressed in liver cancer tissues and is present in 70%-80% of hepatocellular carcinoma (HCC) patients. Compared with normal liver tissue, the average increase in GPC3 expression in liver cancer is 21.7 times. Studies have found that the 5-year survival rate of patients with GPC3-positive liver cancer is lower than that of patients with GPC3-negative liver cancer, and the prognosis of patients with low GPC3 expression is generally better than that of patients with high GPC3 expression. GPC3 controls the proliferation of liver cancer cells by affecting signaling pathways such as Wnt and YAP, thereby affecting tumor growth and metastasis. Compared with alpha-fetoprotein, GPC3 has more significant sensitivity and specificity in the detection of early liver cancer and is more practical (Eur Rev Med Pharmacol Sci, 2015, 19(19):3655-73.; Cancer Res, 1997, 57(22):5179-84.). Therefore, GPC3 has become an auxiliary diagnostic marker and therapeutic target for liver cancer. In addition, GPC3 expression is also shown in several other tumors, including lung cancer, squamous cell carcinoma, testicular non-seminoma, and liposarcoma (Am J Clin Pathol, 2008, 129(6):899-906.). As an innovative target, the development of antibody drugs targeting GPC3 has been slow. Patent documents such as WO2004022597A1, WO2004022739A1, WO2009041062A1, and WO2014097648A1 disclose information on GPC3 antibodies and treatment methods. In clinical practice, Chugai Pharmaceutical's GPC3 monoclonal antibody Codrituzumab (GC-33) is the first antibody targeting GPC3. Its main principle is to kill tumor cells through antibody-dependent cell-mediated cytotoxicity (ADCC). Currently in Phase II clinical trials, the results showed that Codrituzumab had no significant difference in overall survival and progression-free survival compared with placebo, but it can improve the prognosis of patients with GPC3-overexpressing hepatocellular carcinoma.

[0004] T cell-directed tumor cell killing approaches have demonstrated significant anti-tumor effects in many animal models and have recently made significant progress in clinical cancer treatment. However, high side effects, particularly high cytokine release, have been a major obstacle to the successful implementation of T cell-based clinical therapies. Therefore, leveraging the potential of T cells to develop novel, highly effective, and low-toxic bispecific T cell antibodies is crucial. Currently, only one bispecific binding molecule that simultaneously recognizes GPC3 and CD3ε is known to have entered clinical trials: ERY974, developed by Chugai Pharmaceutical based on GC33. Currently in Phase I clinical trials, dose escalation was halted due to the observation that a severe cytokine storm was produced at a low dose (0.81 μg / kg). Therefore, there remains an urgent need in the art to develop GPC3 / CD3 bispecific binding molecules with higher activity, lower toxicity, a wider therapeutic safety window, and the ability to produce stronger pharmacodynamics at lower doses.

[0005] SUMMARY OF THE INVENTION

[0006] This article provides a bispecific antibody against GPC3 and CD3, which can recruit T cells to tumor sites through the CD3 target and specifically kill tumor cells with high expression of GPC3.

[0007] This article relates to a bispecific antigen-binding molecule, which comprises a first antigen-binding domain that recognizes glypican 3 (GPC3); and a second antigen-binding domain that recognizes the CD3 subunit of the T-cell receptor; wherein the first antigen-binding domain comprises HCDR1, HCDR2 and HCDR3, and the HCDR1, HCDR2 and HCDR3 are derived from the heavy chain variable region shown in the sequence of SEQ ID NO:10.

[0008] It should be noted that the division of the CDRs and FRs in the antibody variable regions of the present disclosure is determined according to the Kabat definition. Other naming and numbering systems, such as Chothia, IMGT, or AHo, are also known to those skilled in the art. Therefore, humanized antibodies containing one or more CDRs derived from any naming system based on the antibody sequences of the present disclosure are clearly within the scope of the present disclosure.

[0009] In one embodiment, the first antigen binding domain comprises the HCDRs shown in SEQ ID NOs: 11-13; preferably, the sequence of HCDR 1 is shown in SEQ ID NO: 11; the sequence of HCDR2 is shown in SEQ ID NO: 12; and the sequence of HCDR3 is shown in SEQ ID NO: 13.

[0010] In one embodiment, the second antigen binding domain comprises HCDR1, HCDR2 and HCDR3, and the HCDR1, HCDR2 and HCDR3 are derived from the heavy chain variable region shown in SEQ ID NO:1; and it further comprises LCDR1, LCDR2 and LCDR3, and the LCDR1, LCDR2 and LCDR3 are derived from the light chain variable region shown in SEQ ID NO:2.

[0011] In one embodiment, the second antigen-binding domain comprises the CDRs shown in SEQ ID NOs: 4-9; preferably, the sequence of HCDR 1 is shown in SEQ ID NO: 4; the sequence of HCDR2 is shown in SEQ ID NO: 5; the sequence of HCDR3 is shown in SEQ ID NO: 6; the sequence of LCDR1 is shown in SEQ ID NO: 7; the sequence of LCDR2 is shown in SEQ ID NO: 8; and the sequence of LCDR3 is shown in SEQ ID NO: 9.

[0012] The present invention also relates to a bispecific antigen-binding molecule having one or more of the following features: (1) the first antigen-binding domain is a nanobody; preferably, it is a humanized camelid nanobody; (2) the bispecific antibody comprises heavy chain constant regions CH2 and CH3, and does not contain a light chain constant region and / or a heavy chain constant region CH1; preferably, the heavy chain constant region comprises an Fc domain or a variant Fc; more preferably, the Fc is derived from a mouse or human; (3) the bispecific antigen-binding molecule comprises an Fc domain, wherein the Fc domain is an IgG Fc domain; preferably, the Fc domain is an IgG1 domain or an IgG4 domain; (4) the bispecific antigen-binding molecule comprises an Fc domain, wherein the two polypeptide sequences constituting the Fc domain have different sequences from each other, and the amino acid residues at position 366 of the Knob chain of the two polypeptides constituting the Fc domain are mutated to tryptophan according to EU numbering, and the amino acid residues at position 366 of the other Hole chain are mutated to serine, amino acid residues at position 368 are mutated to alanine, and amino acid residues at position 407 are mutated to valine according to EU numbering; preferably, the Knob chain comprises S354C and T366W amino acid substitutions, and the Hole chain comprises Y349C, T366S, L368A, and Y407V amino acid substitutions; more preferably, the Hole chain further comprises H435R substitution; further preferably, the Fc segment further comprises L234A and L235A substitutions; further preferably, the Fc segment further comprises K447A substitution at the last position of the C-terminus;

[0013] (5) The bispecific antigen-binding molecule includes an Fc domain, wherein the sequence of the Fc domain is shown in SEQ ID NO: 14 and / or SEQ ID NO: 15.

[0014] In one embodiment, the bispecific antigen-binding molecule comprises a first antigen-binding domain and a second antigen-binding domain, wherein the first antigen-binding domain comprises the sequence shown in SEQ ID NO: 16 or 18, and / or the second antigen-binding domain comprises the sequence shown in SEQ ID NO: 17.

[0015] The present invention also relates to an isolated polynucleotide encoding the bispecific antigen-binding molecule or fragment thereof described in any one of the preceding claims; or a recombinant vector comprising the isolated polynucleotide; or a host cell comprising the isolated polynucleotide or recombinant vector.

[0016] The present invention also relates to a method of producing a bispecific antigen binding molecule as described in any of the foregoing.

[0017] The present invention also relates to a pharmaceutical composition comprising: a pharmaceutically acceptable carrier, and one or more of the group consisting of any one of the aforementioned bispecific antigen-binding molecules, polynucleotides, recombinant vectors and / or host cells.

[0018] The present invention also relates to the use of a bispecific antigen binding molecule or pharmaceutical composition in the preparation of a medicament for treating and / or preventing a disease in an individual in need thereof; preferably, the disease is cancer; more preferably, the cancer is a GPC3-positive tumor; further preferably, the cancer is selected from liver cancer, lung cancer, and skin cancer.

[0019] The present invention also relates to a method for treating and / or preventing a disease associated with GPC3 expression in a subject, comprising administering to the subject a therapeutically and / or preventively effective amount of a bispecific antigen binding molecule or pharmaceutical composition according to any of the foregoing; preferably, the disease associated with GPC3 expression is cancer; more preferably, the cancer is selected from liver cancer, lung cancer, and skin cancer.

[0020] The present invention also relates to a pharmaceutical composition for treating and / or preventing a disease associated with GPC3 expression in a subject, comprising administering to the subject a therapeutically and / or preventatively effective amount of a bispecific antigen binding molecule or pharmaceutical composition according to any of the foregoing; preferably, the disease associated with GPC3 expression is cancer; more preferably, the cancer is selected from liver cancer, lung cancer, and skin cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The accompanying drawings further illustrate the novel features disclosed in this specification. The features and advantages disclosed in this specification can be better understood with reference to these drawings, but it should be understood that these drawings are only used to illustrate specific implementations of the principles disclosed herein and are not intended to limit the scope of the appended claims.

[0022] FIG1 shows the structures of the bispecific antigen-binding molecules G100 and G156.

[0023] FIG2 shows the binding of bispecific antigen-binding molecules G100 and G156 and a control antibody to HepG2 cells naturally expressing hGPC3.

[0024] FIG3 shows the binding of bispecific antigen-binding molecules G100 and G156 and a control antibody to Jurkat cells naturally expressing hCD3.

[0025] FIG4 shows the results of a cytotoxicity experiment (TDCC) of CD3+ T cells against HepG2 cells naturally expressing hGPC3 mediated by the bispecific antigen-binding molecules G100 and G156.

[0026] FIG5 shows the results of T cell activation of HepG2 cells naturally expressing hGPC3 by the bispecific antigen-binding molecules G100 and G156 and a control antibody.

[0027] FIG6 shows the results of IL-2 secretion by the bispecific antigen-binding molecules G100 and G156 and a control antibody on HepG2 cells naturally expressing hGPC3.

[0028] FIG7 shows the results of IFNγ secretion by the bispecific antigen-binding molecules G100 and G156 and a control antibody on HepG2 cells naturally expressing hGPC3.

[0029] FIG8 shows the results of TNF-α secretion by the bispecific antigen-binding molecules G100 and G156 and a control antibody on HepG2 cells naturally expressing hGPC3.

[0030] FIG9 shows the effect of the bispecific antibody G100 of the present invention on tumor growth in a subcutaneous transplantation tumor model of human hepatoma cell line HepG2 mixed with PBMC.

[0031] Detailed Description of the Invention

[0032] the term

[0033] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0034] Before describing this invention in detail below, it should be understood that this invention is not limited to the specific methodologies, protocols 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 this invention. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which this invention belongs.

[0035] Certain embodiments disclosed herein include numerical ranges, and certain aspects of this article may be described in terms of ranges. Unless otherwise stated, it should be understood that numerical ranges or descriptions in terms of ranges are for the sole purpose of brevity and convenience and should not be considered as strict limitations on the scope of this article. Therefore, descriptions in terms of ranges should be considered to specifically disclose all possible subranges and all possible specific numerical points within the range, as these subranges and numerical points have been clearly stated herein. Regardless of the width of the numerical value, the above principles apply equally. When describing in terms of ranges, the range includes the endpoints of the range.

[0036] As used herein, the term "antigen binding molecule" refers in its broadest sense to a molecule that specifically binds to an antigenic determinant. Examples of antigen binding molecules are immunoglobulins and their derivatives, such as fragments.

[0037] The term "bispecific" means that the antigen binding molecule is able to specifically bind to two different antigenic determinants. Typically, a bispecific antigen binding molecule comprises two antigen binding sites, each of which is specific for a different antigenic determinant. In certain embodiments, a bispecific antigen binding molecule is able to simultaneously bind to two antigenic determinants, particularly two antigenic determinants expressed on two different cells.

[0038] The term "antigen" refers to a substance that is recognized and specifically bound by an antibody or antigen-binding fragment. In a broad sense, an antigen can include any immunogenic fragment or determinant of a selected target, including a single epitope, multiple epitopes, a single domain, multiple domains, or a complete extracellular domain (ECD) or protein. Peptides, proteins, glycoproteins, polysaccharides, and lipids, portions thereof, and combinations thereof, can all constitute antigens. Non-limiting exemplary antigens include tumor antigens or pathogen antigens, etc. "Antigen" can also refer to a molecule that elicits an immune response. Any form of antigen or cell or preparation containing the antigen can be used to generate antibodies specific for the antigenic determinant. The antigen can be an isolated full-length protein, a cell surface protein (e.g., immunized with cells expressing at least a portion of the antigen on their surface), or a soluble protein (e.g., immunized with only the ECD portion of the protein), or a protein construct (e.g., an Fc antigen). The antigen can be produced in genetically modified cells. Any of the aforementioned antigens can be used alone or in combination with one or more immunogenicity-enhancing adjuvants known in the art. The DNA encoding the antigen can be genomic or non-genomic (e.g., cDNA) and can encode at least a portion of the ECD sufficient to elicit an immunogenic response. Any vector can be used to transform cells in which the antigen is expressed, including but not limited to adenoviral vectors, lentiviral vectors, plasmids, and non-viral vectors such as cationic lipids.

[0039] The term "epitope" refers to a site on an antigen that specifically binds to an immunoglobulin or antibody. An epitope can be formed by adjacent amino acids or non-adjacent amino acids juxtaposed by tertiary folding of the protein. Epitopes formed by adjacent amino acids are generally retained after exposure to denaturing solvents, while epitopes formed by tertiary folding are generally lost after treatment with denaturing solvents. Epitopes generally exist in a unique spatial conformation and include at least 3-15 amino acids. Methods for determining the epitope bound by a given antibody are well known in the art and include immunoblotting and immunoprecipitation assays. Methods for determining the spatial conformation of an epitope include techniques in the art and those described herein, such as X-ray crystallography and two-dimensional nuclear magnetic resonance.

[0040] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to polymers of amino acids of any length. A polymer may be linear, cyclic, or branched, it may contain modified amino acids, particularly conservatively modified amino acids, and it may be interrupted by non-amino acids. The term also includes modified amino acid polymers, such as those that have been modified by sulfation, glycosylation, lipidation, acetylation, phosphorylation, iodination, methylation, oxidation, proteolytic processing, prenylation, racemization, selenoylation, transfer-RNA-mediated amino additions such as arginylation, ubiquitination, or any other manipulation such as conjugation to a labeling component. As used herein, the term "amino acid" refers to natural and / or non-natural or synthetic amino acids, including glycine and the D or L optical isomers, as well as amino acid analogs and peptide mimetics. A polypeptide or amino acid sequence "derived from" a specified protein refers to the source of the polypeptide. The term also includes polypeptides expressed by a specified nucleic acid sequence.

[0041] The term "amino acid modification" (or "modified amino acid") includes amino acid substitutions, insertions, and / or deletions in a polypeptide sequence. As used herein, "amino acid substitution" or "substitution" or "replacement" refers to the replacement of an amino acid at a specific position in a parent polypeptide sequence with another amino acid. For example, the substitution S32A refers to the replacement of serine at position 32 with alanine.

[0042] "Specific binding" means that the binding is selective for the antigen and can be distinguished from unwanted or non-specific interactions. The ability of an antibody to bind to a specific antigenic determinant can be determined by enzyme-linked immunosorbent assay (ELISA) or other techniques familiar to those skilled in the art, such as surface plasmon resonance (SPR) technology (analyzed on a BIAcore instrument) (Liljeblad et al., Glyco J, 17, 323-329 (2000)) and traditional binding assays (Heeley Endocr, Res, 28, 217-229 (2002)).

[0043] "Affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., a receptor) and its binding partner (e.g., a ligand). Unless otherwise indicated, "binding affinity" as used herein refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antigen binding portion and an antigen, or a receptor and its ligand). The affinity of a molecule X for its partner Y can generally be expressed as a dissociation constant (KD), which is the ratio of the dissociation and association rate constants (koff and kon, respectively). Thus, equivalent affinities can comprise different rate constants as long as the ratio of the rate constants remains the same. Affinity can be measured by well-established methods known in the art, including those described herein. A specific method for measuring affinity is surface plasmon resonance (SPR).

[0044] As used herein, the term "valent" refers to the presence of a specified number of antigen binding regions in an antigen binding molecule.

[0045] "Antigen binding site" refers to the site of an antigen binding molecule, i.e., one or more amino acid residues, that provides interaction with the antigen. For example, the antigen binding site of an antibody comprises amino acid residues from the complementarity determining region (CDR).

[0046] The term "antibody" herein is used in the broadest sense to encompass a variety of antibody structures including, but not limited to, monoclonal antibodies, polyclonal antibodies, and antibody fragments, so long as they exhibit the desired antigen-binding activity.

[0047] As used herein, "GPC3," also known as glypican 3 or glypican 3, is a proteoglycan that attaches to the cell surface via a glycerophosphatidylinositol (GPI) anchor. It belongs to the heparan sulfate glycoprotein (HSPG) family and is primarily involved in regulating cell proliferation, adhesion, and migration. The GPC3 gene is located on the human X chromosome (Xq26.1) and encodes a GPC3 protein precursor containing 580 amino acid residues (Nat Genet, 1996, 12(3):241-7). The GPC3 core protein is approximately 70 kDa and is rich in a unique sequence of 14 cysteine ​​residues (Cys). Furin protease cleaves the core protein (cleavage site is between Arg358 and Cys359) to produce a soluble N-terminal peptide of approximately 40 kDa and a membrane-bound C-terminal peptide of approximately 30 kDa containing two heparan sulfate (HS) sugar chains (Eur J Cancer, 2011, 47(3):333-8.; Proc Natl Acad Sci USA, 2011, 108(32):13112-7.).

[0048] As used herein, the terms "anti-GPC3 antibody," "anti-GPC3 Nanobody," "antibody against GPC3," and "Nanobody against GPC3" refer to antibodies that can bind to a GPC3 protein or a fragment thereof with sufficient affinity so that the antibody can be used as a diagnostic and / or therapeutic agent targeting GPC3. Murine-derived GPC3 protein is denoted as mGPC3, cynomolgus monkey-derived GPC3 protein is denoted as cynoGPC3, and human-derived GPC3 protein is denoted as huGPC3. "Anti-human GPC3 antibody," "anti-human GPC3 Nanobody," "anti-huGPC3 antibody," "anti-huGPC3 Nanobody," "antibody against huGPC3," and "Nanobody against huGPC3" specifically refer to antibodies that can bind to a human GPC3 protein or a fragment thereof with sufficient affinity so that the antibody can be used as a diagnostic and / or therapeutic agent targeting human GPC3.

[0049] As used herein, an "anti-CD3 antibody" refers to an antibody that specifically binds to a single CD3 chain (e.g., CD3(γ), CD3(δ), or CD3(ε)) or a complex formed by two or more single CD3 chains (e.g., a complex of more than one CD3(ε) chain, a complex of a CD3(γ) chain and a CD3(ε) chain, or a complex of a CD3(δ) chain and a CD3(ε) chain). In certain embodiments, the anti-CD3 antibody specifically binds to CD3(γ), CD3(δ), or CD3(ε), or any combination thereof, more preferably, to CD3(ε). "Anti-human CD3 antibodies" and "anti-hCD3 antibodies" refer to antibodies that specifically bind to human-derived CD3.

[0050] It should be noted that the division of CDRs and FRs in the antibody variable regions herein is determined according to the Kabat definition. Other naming and numbering systems, such as Chothia, IMGT, or AHo, are also known to those skilled in the art. Therefore, based on the antibody sequences herein, humanized antibodies comprising one or more CDRs derived from any naming system are clearly within the scope of this disclosure.

[0051] The term "sequence identity" or "sequence similarity" or "sequence homology" refers to the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in a reference polypeptide sequence, after aligning the sequences (and introducing gaps, if necessary) to obtain maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Sequence alignment can be performed to determine percent amino acid sequence identity using various methods in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or MEGALIGN (DNASTAR) software. One skilled in the art can determine appropriate parameters for measuring alignment, including any algorithm required to achieve maximum alignment over the full length of the compared sequences.

[0052] Conventional immunoglobulin is a tetramer, consisting of two heavy chains and two light chains, with a combined molecular weight of approximately 150kDa. In camel family (Camelidae) members, a considerable proportion of serum antibodies are homodimeric IgG, with a molecular weight of approximately 80kD (Hamers-Casterman et al., 1993, Nature, 363, 446-448). These heavy chain immunoglobulins (Ig) comprise three domains, and their variable regions are referred to as VHH (variable domain of heavy chain of heavy-chain antibody). Recombinant VHH (approximately 12 to 14kD) constitutes a complete antigen-binding domain and demonstrates a broad antigen-binding spectrum. Expand their hypervariable regions and demonstrate unique characteristics, such as three to four (interacting with conventional antibody VL) hydrophobic framework residues replaced by more hydrophilic amino acids. To stabilize the enlarged CDRs, VHHs may have additional disulfide bonds, in addition to the conventional disulfide bonds, between CDR1 and CDR3 in dromedary camels and between CDR2 and CDR3 in llamas (Harmsen and De Haard, 2007, Appl Microbiol Biotechnol., 77, 13-22; Muyldermans, 2001, J Biotechnol., 74, 277-302). The enlarged CDR3 loop can adopt a convex conformation, whereas the conventional paratope is constrained to a concave or planar structure (Muyldermans, 2001, J Biotechnol., 74, 277-302). These features allow VHHs to recognize unique epitopes that are poorly immunogenic for conventional antibodies (Lafaye, 2009, Mol Immuno., 46, 695-704; Wernery, 2001, J Vet Med B Infect Dis Vet Public Health., 48, 561-568). Although VHHs are defined as monovalent antibodies, any avidity effect is implicitly excluded and is measured as in vitro IC 50 The biological activity of the bivalent antibody can be similar to that of conventional bivalent antibody molecules (Thys et al., 2010, Antiviral Res., 87, 257-264).

[0053] In certain embodiments, the present invention relates to chimeric camelid / human antibodies, particularly chimeric antibodies in which the VH and / or VL domains are entirely camelid sequences (e.g., llama or alpaca), while the remainder of the antibody is entirely human sequences. In some preferred embodiments herein, "humanized" or "germlined" camelid antibodies and camelid / human chimeric antibodies are also included, in which the VH and / or VL domains comprise one or more amino acid substitutions in the framework regions relative to a camelid VH and / or VL domain obtained by active immunization. The "humanization" process increases the percentage of sequence identity with the human germline VH or VL domain by replacing the non-matching amino acid residues in the starting camelid VH or VL domain with the corresponding residues in the human germline VH or VL domain.

[0054] Natural, recombinant VHH or VH are encompassed herein.

[0055] A VHH according to this invention can be in the form of a monomer or in the form of a homomultimer, such as a homodimer or a homotrimer.

[0056] The antibodies herein include camel-derived antibodies, chimeric antibodies, and humanized antibodies, preferably humanized antibodies.

[0057] The term "chimeric antibody" refers to a construct in which a portion of the heavy and / or light chain is identical or homologous to corresponding sequences in antibodies from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical or homologous to corresponding sequences in antibodies from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies. In a narrower sense, a chimeric antibody comprises all or most of the selected murine heavy and light chain variable regions operably linked to human light and heavy chain constant regions. The constant region sequences, or variants or derivatives thereof, can be operably associated with the disclosed heavy and light chain variable regions using standard molecular biology techniques to provide full-length anti-GPC3 antibodies that can be used per se or incorporated into the present invention.

[0058] The term "humanized antibody" is a hybrid immunoglobulin containing the minimum sequence derived from a non-human immunoglobulin, immunoglobulin chain or its fragment. In most cases, a humanized antibody is a human immunoglobulin (receptor antibody), wherein the residue from the CDR of the receptor is replaced by the residue from the CDR of a non-human species (donor antibody) with required specificity, affinity and performance, such as mouse, rat, rabbit or primate. In some cases, the framework region residues of the human immunoglobulin are replaced by corresponding non-human residues. In some cases, a "back mutation" can be introduced into a humanized antibody, wherein the residue in one or more FRs of the variable region of the receptor human antibody is replaced by the corresponding residue from the non-human species donor antibody. Such a back mutation can contribute to the appropriate three-dimensional configuration of one or more grafted CDRs and therefore improve affinity and antibody stability. Antibodies from various donor species can be used, and these donor species include but are not limited to mouse, rat, rabbit or non-human primate. In addition, a humanized antibody can be included in a receptor antibody or in a donor antibody without finding new residues, so as to further improve antibody performance.

[0059] The term "single-domain antibody", also known as nanobody, can be defined as an amino acid sequence having the following (general) structure: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. FR1-FR4 refer to framework regions 1-4, respectively, and CDR1-CDR3 refer to complementarity determining regions 1-3, respectively. "VHH" refers to the variable antigen-binding domain of heavy chain antibodies from the Camelidae family (camel, dromedary, llama, alpaca, etc.) (see Nguyen, 2000 EMBO J., 19, 921-930; Muyldermans, 2001, J Biotechnol., 74, 277-302 and reviewed by Vanlandschoot, 2011, Antiviral Res., 92, 389-407).

[0060] The term "scFv" refers to a fusion protein comprising at least one antibody fragment comprising a light chain variable region and at least one antibody fragment comprising a heavy chain variable region, wherein the light and heavy chain variable regions are contiguous (e.g., via a synthetic linker such as a short flexible polypeptide linker) and can be expressed as a single-chain polypeptide, and wherein the scFv retains the specificity of the intact antibody from which it is derived. Unless otherwise specified, scFv can have the VL and VH variable regions in any order (e.g., relative to the N-terminus and C-terminus of the polypeptide), and scFv can include VL-peptide linker-VH or can include VH-peptide linker-VL.

[0061] Five major classes of antibodies are known in the art: IgA, IgD, IgE, IgG, and IgM. The corresponding heavy chain constant domains are called α, δ, ε, γ, and μ, respectively. IgG and IgA can be further divided into different subclasses, for example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4, and IgA can be divided into IgA1 and IgA2. The light chains of antibodies from any vertebrate species can be assigned to one of two clearly distinct types, called κ and λ, based on the amino acid sequence of their constant domains.

[0062] In the case of IgG, IgA, and IgD antibodies, the constant region comprises three domains called CH1, CH2, and CH3 (IgM and IgE have a fourth domain, CH4). In the IgG, IgA, and IgD classes, the CH1 and CH2 domains are separated by a flexible hinge region, which is a segment of variable length rich in proline and cysteine. Each class of antibodies further comprises interchain and intrachain disulfide bonds formed by paired cysteine ​​residues.

[0063] The term "Fc" is used herein to define the C-terminal region of an immunoglobulin heavy chain, i.e., the two polypeptide chains that form a dimer comprising the C-terminal constant region of an immunoglobulin heavy chain that can stabilize its own association. The term includes native sequence Fc regions and variant Fc regions. Although the boundaries of the Fc region of an IgG heavy chain can vary slightly, the human IgG heavy chain Fc region is generally defined as extending from Cys226 or Pro230 to the carboxyl terminus of the heavy chain, e.g., an IgG Fc domain comprises the IgG CH2 and IgG CH3 constant domains. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also known as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0064] " Modifications that promote the binding of the first and second subunits of the Fc domain " are operations of the peptide backbone or post-translational modifications of the Fc domain subunits that reduce or prevent the polypeptide comprising the Fc domain subunit from binding to the same polypeptide to form a homodimer. The modifications that promote binding used herein include, in particular, separate modifications to each of the two Fc domain subunits (i.e., the first and second subunits of the Fc domain) that it is desired to bind, wherein the modifications are complementary to each other, so as to promote the binding of the two Fc domain subunits. For example, the modifications that promote binding can change the structure or charge of one or both of the Fc domain subunits so as to make their binding spatially or electrostatically favorable, respectively. Thus, (hetero) dimerization occurs between a polypeptide comprising the first Fc domain subunit and a polypeptide comprising the second Fc domain subunit, which can be different in the sense that the other components fused to each subunit (e.g., Fab fragment) are different. In some embodiments, the modifications that promote binding comprise amino acid mutations in the Fc domain, specifically, amino acid substitutions. In a specific embodiment, the binding-promoting modifications comprise separate amino acid mutations, in particular, amino acid substitutions, in each of the two subunits of the Fc domain.

[0065] There are several approaches to modify the CH3 domain of the Fc domain to enhance heterodimerization, which are described in detail in, for example, WO96 / 27011, WO98 / 050431, EP1870459, WO2007 / 110205, WO2007 / 147901, WO2009 / 089004, WO2010 / 129304, WO2011 / 90754, WO2011 / 143545, WO2012 / 058768, WO2013 / 157954, and WO2013 / 096291. Typically, in all such approaches, both the CH3 domain of the first polypeptide chain of the Fc domain and the CH3 domain of the second polypeptide chain of the Fc domain are engineered in a complementary manner such that each CH3 domain (or the heavy chain comprising it) is no longer able to homodimerize with itself but is forced to heterodimerize with the other complementarily engineered CH3 domain (such that the first and second CH3 domains heterodimerize and homodimers are not formed between the two first CH3 domains or the two second CH3 domains).

[0066] In a specific embodiment, the modification that promotes the association of the first polypeptide chain and the second polypeptide chain of the Fc domain is a so-called "knob-into-hole" modification, which comprises a "knob" modification in one of the two polypeptide chains of the Fc domain and a "hole" modification in the other of the two polypeptide chains of the Fc domain.

[0067] The knob-into-hole technique is described in, for example, US 5731168; US 7695936; Ridgway et al., Prot Eng, 9, 617-621 (1996) and Carter, J Immunol Meth, 248, 7-15 (2001). Generally, the method involves introducing a protuberance ("knot") at the interface of a first polypeptide chain and introducing a corresponding cavity ("hole") in the interface of a second polypeptide chain so that the protuberance can be placed in the cavity to promote heterodimer formation and hinder homodimer formation. The protuberance is constructed by replacing the small amino acid side chains from the interface of the first polypeptide chain with larger side chains (e.g., tyrosine or tryptophan). A complementary cavity of the same or similar size as the protuberance is created in the interface of the second polypeptide chain by replacing the large amino acid side chains with smaller amino acid side chains (e.g., alanine or threonine).

[0068] Thus, in a specific embodiment, in the CH3 domain of the first polypeptide chain of the Fc domain of the bispecific antigen binding molecule herein, one amino acid residue is replaced with an amino acid residue having a larger side chain volume, thereby generating a protuberance within the CH3 domain of the first polypeptide chain, which can be positioned within the cavity within the CH3 domain of the second polypeptide chain, and in the CH3 domain of the second polypeptide chain of the Fc domain, one amino acid residue is replaced with an amino acid residue having a smaller side chain volume, thereby generating a cavity within the CH3 domain of the second polypeptide chain, into which the protuberance within the CH3 domain of the first polypeptide chain can be positioned.

[0069] Preferably, the amino acid residue with a larger side chain volume is selected from the group consisting of arginine (R), phenylalanine (F), tyrosine (Y), and tryptophan (W). Preferably, the amino acid residue with a smaller side chain volume is selected from the group consisting of alanine (A), serine (S), threonine (T), and valine (V).

[0070] Protuberances and cavities can be generated by altering the nucleic acid encoding the polypeptide, for example by site-specific mutagenesis or by peptide synthesis.

[0071] Herein, "knob-Fc" refers to an antibody containing the T366W point mutation in the Fc region, creating a knob-like structure. In contrast, "hole-Fc" refers to an antibody containing the T366S, L368A, and Y407V point mutations in the Fc region, creating a hole-like structure. Knob-Fc and hole-Fc are more likely to form heterodimers due to steric hindrance. To reduce the formation of hole-hole homodimers, the H435R mutation in the hole can reduce protein A binding during purification. To further promote heterodimer formation, point mutations S354C and Y349C can be introduced into the knob-Fc and hole-Fc, respectively, to further enhance heterodimer formation through disulfide bonds. Furthermore, to eliminate or weaken the ADCC effect induced by the antibody Fc, substitution mutations L234A and L235A can be introduced into the Fc region. For example, Knob-Fc and Hole-Fc are preferably represented herein as SEQ ID NOs: 14 and 15, respectively. In a bispecific antigen-binding molecule, Knob-Fc and Hole-Fc can serve as both the Fc region of the first polypeptide chain and the Fc region of the second polypeptide chain. In the same bispecific antigen-binding molecule, the Fc regions of the first polypeptide chain and the second polypeptide chain cannot both be Knob-Fc and Hole-Fc.

[0072] It should be noted that the CDR and FR divisions of the monoclonal antibody variable regions herein are determined according to the Kabat definition. Other naming and numbering systems, such as Chothia, IMGT, or AHo, are also known to those skilled in the art. Therefore, humanized antibodies comprising one or more CDRs derived from any naming system based on the monoclonal antibody sequences herein are clearly within the scope of this disclosure.

[0073] The terms "homologous" and "heterogeneous" used in this article are relative concepts, which can refer to the fact that different elements in a construct have the same or different origins. They can also refer to the fact that after the construction of the construct is completed, some elements that originally had the same origin, i.e., "homologous", have been transformed and changed compared to other original elements that have not been transformed, thus becoming "heterogeneous".

[0074] By "fused" is meant that the components (eg, scFv antibody and Fc domain subunit) are linked by peptide bonds, either directly or via one or more peptide linkers.

[0075] The term "linker" refers to any tool for connecting two different functional units (e.g., antigen binding fragments). The types of linkers include, but are not limited to, chemical linkers and polypeptide linkers. The sequence of a polypeptide linker ("peptide linker") is not limited. Peptide linkers are preferably non-immunogenic and flexible, such as those comprising serine and glycine sequences. Depending on the specific construct, the linker can be long or short.

[0076] The term "antibody-dependent cell-mediated cytotoxicity" (ADCC) refers to the binding of antibodies to antigenic epitopes of virus-infected cells or tumor cells, and the binding of their Fc segments to Fc receptors (FcR) on the surface of killer cells (NK cells, macrophages, etc.), mediating the direct killing of target cells by killer cells.

[0077] The term "modification" as used herein includes any manipulation of the backbone or post-translational modification of a naturally occurring or recombinant polypeptide or fragment thereof. Modification includes modification of the amino acid sequence, glycosylation pattern or side chain groups of individual amino acids, as well as combinations of these methods.

[0078] The term "pharmaceutical composition" refers to a preparation 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 preparation would be administered.

[0079] The term "pharmaceutical carrier" or "pharmaceutically acceptable carrier" refers to a diluent, adjuvant (eg, Freund's adjuvant (complete and incomplete)), excipient, or vehicle with which the therapeutic agent is administered.

[0080] The term "effective amount" refers to a dose of a pharmaceutical formulation comprising an active ingredient herein that produces the desired effect in a treated patient after administration to the patient in a single or multiple doses. The effective amount can be readily determined by the attending physician, who is skilled in the art, by considering a variety of factors such as ethnic differences; weight, age, and health status; the specific disease involved; the severity of the disease; the response of the individual patient; the specific antibody administered; the mode of administration; the bioavailability characteristics of the administered formulation; the selected dosing regimen; and the use of any concomitant therapy.

[0081] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably to refer to cells into which exogenous nucleic acids have been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include primary transformed cells and progeny derived therefrom, without regard to the number of generations. Progeny may not be identical to the parent cell in nucleic acid content, but may contain mutations. Mutant progeny having the same function or biological activity as that screened or selected in the initially transformed cells are included herein. Host cells are any type of cell system that can be used to produce the bispecific antigen binding molecules herein. Host cells include cultured cells, such as mammalian cultured cells, such as Jurkat cells, PBMC cells, HepG2 cells, Hep3B cells, and Huh-7 cells, or hybridoma cells, yeast cells, insect cells, and plant cells, as well as cells contained in transgenic animals, transgenic plants, or cultured plants or animal tissues.

[0082] The term "transfection" as used herein refers to the introduction of exogenous nucleic acid into eukaryotic cells. Transfection can be achieved by various means known in the art, including calcium phosphate-DNA coprecipitation, DEAE-dextran-mediated transfection, polybrene-mediated transfection, electroporation, microinjection, liposome fusion, lipofection, protoplast fusion, retroviral infection, and biolistics.

[0083] The term "stable transfection" or "stable transfection" refers to the introduction and integration of exogenous nucleic acid, DNA or RNA, into the genome of the transfected cell. The term "stable transfectant" refers to a cell that has stably integrated the foreign DNA into its genomic DNA.

[0084] The term "isolated polynucleotide" refers to a nucleic acid molecule, DNA or RNA that has been taken out of its natural environment. For example, for the purposes of this article, a recombinant polynucleotide encoding a polypeptide contained in a vector is considered to be isolated. Other examples of isolated polynucleotides include recombinant polynucleotides maintained in heterologous host cells or (partially or substantially) purified polynucleotides in solution. Isolated polynucleotides include polynucleotide molecules contained in cells that typically contain the polynucleotide molecules, but the polynucleotide molecules are present outside the chromosome or at a chromosomal position that is different from its natural chromosomal position. Isolated RNA molecules include in vivo or in vitro RNA transcripts herein, as well as positive and negative strand forms and double-stranded forms. Isolated polynucleotides or nucleic acids herein also include such molecules produced synthetically. In addition, polynucleotides or nucleic acids may or may not include regulatory elements, such as promoters, ribosome binding sites or transcription terminators.

[0085] Methods for producing and purifying antibodies and antigen-binding fragments are well known in the art and can be found in, for example, Chapters 5-8 and 15 of the Cold Spring Harbor Laboratory Manual of Antibody Laboratory Techniques. The antibodies or antigen-binding fragments of the present invention are engineered to incorporate one or more human FR regions into non-human CDR regions. Human FR germline sequences can be obtained from the ImMunoGeneTics (IMGT) website (http: / / imgt.cines.fr) or from The Immunoglobulin Facts Book (2001) ISBN: 012441351.

[0086] The engineered antibodies or antigen-binding fragments thereof can be prepared and purified by conventional methods. For example, cDNA sequences encoding heavy and light chains can be cloned and recombined into expression vectors. The recombinant immunoglobulin expression vector can be stably transfected into CHO cells. As a more recommended existing technology, mammalian expression systems will lead to glycosylation of antibodies, especially at the highly conserved N-terminus of the Fc region. Stable clones are obtained by expressing antibodies that specifically bind to human antigens. Positive clones are expanded in serum-free culture medium in bioreactors to produce antibodies. The culture fluid that secretes antibodies can be purified and collected by conventional techniques. The antibodies can be filtered and concentrated by conventional methods. Soluble mixtures and polymers can also be removed by conventional methods, such as molecular sieves and ion exchange.

[0087] As used herein, the term "individual" or "subject" refers to any animal, such as a mammal or marsupial. Individuals herein include, but are not limited to, humans, non-human primates (e.g., cynomolgus monkeys or rhesus monkeys or other types of macaques), mice, pigs, horses, donkeys, cattle, sheep, rats, and poultry of any kind.

[0088] As used herein, the terms "disease," "condition," or "disorder" refer to any change or disorder that damages or interferes with the normal function of a cell, tissue, or organ. For example, the term "disease" includes, but is not limited to, tumors, pathogen infection, autoimmune diseases, T-cell dysfunction, or immune tolerance defects (e.g., transplant rejection).

[0089] As used herein, the term "treatment" refers to clinical intervention aimed at altering the course of a disease in an individual or cell, and can be either preventative or interventional in the clinical pathological process. Therapeutic effects include, but are not limited to, preventing the onset or recurrence of a disease, alleviating symptoms, reducing any direct or indirect pathological consequences of a disease, preventing metastasis, slowing the progression of a disease, improving or relieving the condition, and alleviating or improving the prognosis. DETAILED DESCRIPTION

[0090] The present invention will be further described below with reference to specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of this invention. Experimental methods in the following examples, where specific conditions are not specified, were generally performed according to conventional conditions, such as those described in Molecular Cloning Manual (J. Sambrook et al., eds., Molecular Cloning Manual, 3rd edition, Science Press, 2002), or according to the conditions recommended by the manufacturer.

[0091] DNA sequencing

[0092] The DNA sequence was determined by double-strand sequencing.

[0093] Antibody preparation and screening

[0094] Methods for preparing monoclonal antibodies are known in the art. One method that can be used is the method of Kohler G. et al. (1975) "Continuous Cultures Of Fused Cells Secreting Antibody Of Predefined Specificity," Nature, 256: 495-497, or a modified form thereof.

[0095] Antigen binding domain

[0096] For example, the light chain variable region (VL) and heavy chain variable region (VH) of the CD3 antigen binding domain scFv are derived from the sequences disclosed in WO2021022304A2. Exemplary anti-CD3 CDR, VH, VL or scFv sequences are shown in Table 1:

[0097] Table 1 Sequence Listing of the CD3 Antigen Binding Domain in the Bispecific Antigen Binding Molecules

[0098] Exemplary GPC3 antigen binding domain sequences are shown in Table 2:

[0099] Table 2 Sequence Listing of the GPC3 Antigen Binding Domain in the Bispecific Antigen Binding Molecules

[0100] In some embodiments disclosed herein, the molecular structure of the bispecific antigen-binding molecule is shown in Figure 1 and is composed of two peptide chains. For example, when the Nanobody VHH targets GPC3 and the scFv antibody targets CD3, two molecular structures (Formats) of bispecific antigen-binding molecules with excellent effects were obtained after screening for the above targets. See Table 3 below for details. Format 1 and Format 2 in Table 3 correspond to Figure 1A and Figure 1B, respectively.

[0101] Table 3 Molecular structures of bispecific antigen-binding molecules

[0102] Note: Linker represents a peptide linker, which is used to connect the antigen-binding domains and the Fc region.

[0103] The peptide linker used to connect the antigen-binding domain and the Fc region can be selected from any other peptide linker that can be used to connect antibody functional domains, and is not limited to the peptide linkers limited by the following sequences. Exemplary peptide linkers can be G4S, (G4S)2, (G4S)3, (G4S)4, (G4S)1A, (G4S)2A, (G4S)3A, (G4S)4A, EPKSSDKTHTCPPCP or DKTHTCPPCP.

[0104] As shown in Table 3, the bispecific antigen binding molecule can also include an Fc region (i.e., an Fc domain). The Fc region can be Knob-Fc and Hole-Fc, and the Fc region can maintain normal half-life and good stability of the antibody. In a preferred embodiment, through the design of two chains, the purpose of greatly reducing the probability of mispairing is achieved, and the homogeneity of the sample and the yield of the target antibody can be further improved. Exemplary, the bispecific antigen binding molecule is a bispecific heterodimer, consisting of a Knob-Fc chain and a Hole-Fc chain. The structure of the Knob-Fc chain includes amino acid replacements at two sites, S354C and T366W, and the structure of the Hole-Fc chain includes amino acid replacements at four sites, Y349C, T366S, L368A, and Y407V. And, for ease of purification of the bispecific antigen binding molecule, the Hole-Fc chain can also be replaced by H435R. In addition, to reduce the ADCC activity of the antibody, the Fc segment can also be replaced by L234A and L235A. For example, as shown in Table 4, the modified Knob-Fc chain sequence is shown in SEQ ID NO: 14, and the Hole-Fc chain sequence is shown in SEQ ID NO: 15. In addition, to prevent the breakage between the variable region of the anti-human GPC3 nanobody and the C-terminus of the heavy chain, the last K at the C-terminus of the heavy chain can be mutated to A, and an amino acid substitution of K447A can be performed.

[0105] Table 4 Sequence list of different Fc

[0106] Example

[0107] Example 1: Preparation, expression and purification of bispecific antigen-binding molecules

[0108] The molecular structure of the bispecific antigen-binding molecule G100 is Format-1, and the specific sequence is shown in Table 5. The molecular structure of the bispecific antigen-binding molecule G156 is Format-2, and the specific sequence is shown in Table 6.

[0109] The DNA sequences encoding the amino acids G100 and G156 of the aforementioned bispecific antigen-binding molecules were cloned into prokaryotic or eukaryotic expression vectors, which were then introduced into prokaryotes or eukaryotes to express the polypeptide or antigen-binding fragment. After sequencing, completely correct clones were selected for transfection and expression, followed by cell culture. After 8-10 days of cell culture, the expression supernatant was collected, centrifuged at high speed to remove cell debris, and affinity purified using a Protein A column. The column was rinsed with PBS until the A280 reading dropped to baseline. The target protein was eluted with an acidic eluent at pH 3.0-3.5 and neutralized with 1M Tris-HCl, pH 8.0-9.0. After the eluted sample was appropriately concentrated, the solution was exchanged into PBS for aliquoting. The final purified humanized antibody was subjected to SDS-PAGE and HPLC purity analysis and A280 concentration determination.

[0110] Table 5 Sequence Listing of Bispecific Antigen Binding Molecule G100

[0111] Table 6 Sequence Listing of Bispecific Antigen Binding Molecule G156

[0112] Example 2: Construction of anti-CD3-GPC3 bispecific antibody and its transient transfection expression in eukaryotic cells

[0113] The target gene fragments of the aforementioned bispecific antibody and anti-CD3 positive monoclonal antibody control molecule were cloned into the pTT5 expression vector to prepare transfection-grade expression plasmids. HEK293E or Expi293 cells were inoculated with the plasmids in serum-free medium and cultured on a shaker at 37°C and 8% CO2. After 6 days of cell culture, the supernatant was collected for purification. The purified bispecific antibody was analyzed for purity by SDS-PAGE and HPLC, and its A280 concentration was determined.

[0114] Example 3: Affinity detection test of anti-CD3-GPC3 bispecific antibody

[0115] A. Affinity testing of the anti-CD3-GPC3 bispecific antibody against cells expressing human GPC3 and human CD3

[0116] FACS was used to detect the binding of the anti-CD3-GPC3 bispecific antibody to human liver cancer cells HepG2 (ATCC, Catalog No.: HB-8065) ​​that naturally express human GPC3 and Jurkat cells (ATCC, Catalog No.: TIB-152) that naturally express human CD3.

[0117] The above cells were collected and resuspended in FACS buffer (PBS + 1% BSA), and the cell concentration was adjusted to 1×10 5 The number of cells was added to a 96-well plate, and then antibodies were added in sequence according to the pre-set concentration (8 concentrations of 4-fold dilution starting from 200nM). Among them, the negative control was human IgG1AA (Negative-IgG1AA, hereinafter referred to as NC, Baiying Bio, catalog number: B109802), and the anti-CD3 positive monoclonal antibody control molecule was h160c9-IgG1AA (hereinafter referred to as 160c9AA). After incubation on a shaker at 4°C for 2h, the cells were washed twice by centrifugation with FACS buffer, and then fluorescently labeled anti-human IgG secondary antibody was added, 100μL per well. After incubation on a shaker at 4°C for 0.5h, the cells were washed twice by centrifugation with FACS buffer, and the cells were filtered into a new 96-well plate. The prepared samples were then detected on a flow cytometer, and the mean fluorescence intensity (hereinafter referred to as MFI) of each concentration was calculated by the software, and then the half-binding concentration (hereinafter referred to as EC) was calculated by GraphPad software. 50 ) and the highest mean fluorescence intensity (Top MFI), and the results are shown in Table 7 and Figures 2 and 3.

[0118] Table 7 Affinity of anti-CD3-GPC3 bispecific antibodies to hGPC3 and hCD3

[0119] The results showed that the G100 and G156 bispecific antibodies specifically bound to HepG2 cells and Jurkat cells in a concentration-dependent manner, and the binding effect was very good. At the same time, the affinity of the bispecific antibodies to human GPC3 was much higher than that to human CD3. The EC binding of G100 to human GPC3 was 50 The binding ability of G100 to human CD3 is higher than that of G156. The binding ability of G100 and G156 to hCD3 antigen is much weaker than that of CD3 positive control antibody 160C9AA.

[0120] B. In vitro binding affinity and kinetics of anti-CD3-GPC3 bispecific antibodies to human / monkey CD3

[0121] In this example, surface plasmon resonance (SPR) was used to determine the affinity and kinetic properties of bispecific antibodies binding to human CD3 E&D protein (Acro, Catalog No. CDD-H52W1) and monkey CD3 E&D protein (Acro, Catalog No. CDD-C52W4) using a Biacore 8K instrument. Human and monkey CD3 E&D proteins were immobilized on the experimental channel of a CM5 chip via amino coupling. The test antibodies G100 and G156 were then diluted to 250 nM in 1× HBS-EP+ (10 mM HEPES, pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.05% P20) running buffer. The antibodies were then serially diluted to 0.06 nM using this buffer. This yielded a series of antibody solutions with concentrations ranging from 0.06 nM to 250 nM. These solutions were then passed through the experimental and reference channels for binding, followed by dissociation, to generate binding and dissociation curves for each sample. The raw data were analyzed using Biacore Insight Evaluation Software (version 2.0.15.12933) and a 1:1 fitting model was used. The obtained bispecific antibody affinity and kinetic experimental data results are shown in Table 8.

[0122] Table 8 Binding affinity and kinetics of anti-CD3-GPC3 bispecific antibodies to human and monkey CD3 E & D proteins

[0123] The results showed that the bispecific antibodies G100 and G156 had high affinity for both human and monkey CD3E&D proteins.

[0124] C. In vitro binding affinity and kinetics of anti-CD3-GPC3 bispecific antibodies to human / monkey GPC3

[0125] In this example, surface plasmon resonance (SPR) was used to determine the affinity and kinetic properties of bispecific antibodies binding to human GPC3 protein (Acro, Catalog No. GP3-H52H4) and monkey GPC3 protein (Acro, Catalog No. GP3-C522a) using a Biacore 8K instrument. The anti-human Fc antibody protein was immobilized on the experimental channel of a CM5 chip via amino coupling. Human and monkey GPC3 were then diluted to 250 nM in 1×HBS-EP+ (10 mM HEPES, pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.05% P20) running buffer. The solutions were then serially diluted to 1.95 nM using this buffer. This yielded a concentration series of 1.95 to 250 nM. These solutions were then passed sequentially across the experimental and reference channels for binding, followed by dissociation, to generate binding and dissociation curves for each sample. The raw data were analyzed using Biacore Insight Evaluation Software (version 2.0.15.12933) and a 1:1 fitting model was used. The obtained bispecific antibody affinity and kinetic experimental data results are shown in Table 9.

[0126] Table 9 Binding affinity and kinetics of anti-CD3-GPC3 bispecific antibodies to human and monkey GPC3 proteins

[0127] The results showed that the bispecific antibodies G100 and G156 had good affinity for both human and monkey GPC3 proteins, and the affinities were comparable, and the affinities between species were also comparable.

[0128] Example 4: In vitro functional experiments of anti-CD3-GPC3 bispecific antibodies

[0129] AT cell-mediated cytotoxicity assay (TDCC)

[0130] HepG2 cells were used as target cells, and T cells were isolated from healthy human PBMCs (peripheral blood mononuclear cells) as effector cells. Cytotoxicity assay reagent (Roche, Cat. No. 04744934001) was used to detect the release of cellular lactate dehydrogenase (LDH), which was used as an indicator of cell killing.

[0131] HepG2 cells were collected by centrifugation, the supernatant was discarded, and the cells were resuspended in cell buffer (EMEM + 10% FBS) and the cell density was adjusted to 1×10 4The number of cells was transferred to 96-well plates with a volume of 100 μL. After overnight, 50 μL of pre-prepared antibodies with different concentration gradients (8 concentrations starting from 100 nM) and control sample working solution were added after the cells adhered to the wall. Then, 50 μL of pre-prepared T cells were added according to the effector cell: target cell ratio of 10:1, that is, 1×10 per well. 5 The cells were incubated in a cell culture incubator (37°C, 5% CO2) for approximately 24 hours. After incubation, the 96-well plate was removed and centrifuged at 1500 rpm for 10 minutes. The supernatant was carefully aspirated and 20 μL of cell lysis buffer was added to each well. After the cells were fully lysed, the plates were centrifuged at 2000 rpm for 5 minutes. 50 μL of cell supernatant was transferred to a new 96-well plate. 50 μL of LDH detection reagent was added according to the instructions of the LDH detection kit and the plates were incubated at room temperature for 20-30 minutes. Detection was performed on a microplate reader at a detection wavelength of 492 nm and a reference wavelength of 650 nm.

[0132] The percentage of cell lysis caused by TDCC effect was calculated using the following formula:

[0133] % cell lysis = 100% × (sample release - target cell / effector cell release) / (maximum release - target cell release)

[0134] Maximum release is the absorbance value obtained in wells containing Triton X-100-treated target cells, target / effector cell mixed release is the absorbance value obtained in wells containing both target and effector cells, target cell release is the absorbance value obtained in wells containing only target cells, and sample release is the absorbance value obtained in wells containing antibody, target cells, and effector cells. EC50 and maximum lysis were calculated using GraphPad software, and the results are shown in Table 10 and Figure 4.

[0135] BT cell activation experiment

[0136] The T cell activation experiment is mainly identified by measuring the cell activation marker CD25+CD69+%. The cells after the TDCC experiment are collected in a 96-well plate, washed twice by centrifugation with FACS buffer (PBS+1% BSA), and resuspended with 100μL FACS buffer. Then, a certain amount of anti-human BV421-CD25 (BD, Catalog No.: 562442) and anti-human PE-CD69 (BD, Catalog No.: 555531) mixture is added and incubated at 4°C for 30min. After washing twice by centrifugation with FACS buffer, the cells are filtered into a new 96-well plate, and then the prepared samples are detected on a flow cytometer. The percentage of CD25+CD69+ in CD3+T cells is calculated by software, and the half-binding concentration (hereinafter referred to as EC) is calculated by GraphPad software.50 ) and the highest percentage (CD25+CD69+%), and the results are shown in Table 10 and Figure 5.

[0137] Table 10 TDCC activity and T cell activation of anti-CD3-GPC3 bispecific antibodies

[0138] The results showed that both bispecific antibodies G100 and G156 had strong TDCC activity and T cell activation effects. The TDCC activity of bispecific antibody G100 was stronger than that of bispecific antibody G156, and its T cell activation effect was comparable to that of bispecific antibody G156.

[0139] C. Cytokine Quantification Experiment

[0140] Cytokine quantification experiments (including IL-2, TNF-α and IFNγ) were mainly performed using Elisa Kit (Biolegend, catalog numbers: 431804, 430204 and 430104). The supernatant after centrifugation was collected and diluted according to the set ratio (IL-2 detection sample was diluted 1:8, TNF-α detection sample was diluted 1:5, and IFNγ detection sample was diluted 1:25) and then detected by the Kit. The results were detected on a microplate reader with a detection wavelength of 450nm and a reference wavelength of 650nm. GraphPad software was used to calculate EC 50 The results are shown in Table 11. In addition, Figures 6-8 show the release of cytokines IL-2, IFNγ, and TNF-α when T cells isolated from fresh healthy human PBMCs were used as effector cells and GPC3-positive HepG2 cells were used as target cells to detect the TDCC effect of T cells on target cells mediated by the anti-CD3-GPC3 bispecific antibody.

[0141] Table 11 IL-2, TNF-α and IFNγ secretion of anti-CD3-GPC3 bispecific antibodies

[0142] The results showed that both bispecific antibodies G100 and G156 could induce cells to release IL-2, IFNγ and TNF-α, and the release ability of G100 was stronger than that of G156.

[0143] Example 5: In vivo efficacy experiments of anti-CD3-GPC3 bispecific antibodies

[0144] PBMCs were obtained and counted, then co-cultured with Mitomycin C-treated HepG2 cells in RPMI-1640 medium (containing 10 ng / ml IL-2 and 10% FBS) for 6 days. After 6 days, PBMCs were harvested and mixed with freshly digested HepG2 cells and inoculated subcutaneously into NCG mice (Shanghai Lidi Biotechnology Co., Ltd.) to establish a humanized HepG2 model. All experimental animals were housed in individually ventilated boxes at a constant temperature and humidity of 20.0°C to 26.0°C, 40-70% humidity, 10-20 air changes / h, and a 12h / 12h light / dark cycle.

[0145] The experiment was divided into a PBS control group, a 0.63 mg / kg G100 treatment group, and a 1.89 mg / kg G100 treatment group. Six mice were injected intraperitoneally twice a week (BIW) for 6 doses (see Table 12). The daily behavior of the animals was monitored every day after administration for a total of 18 days. During the entire experiment, the length and width of the tumor and the tumor volume (mm) were measured twice a week with a vernier caliper. 3 ) = 0.5 × (long diameter of tumor × short diameter of tumor 2 ) was calculated. Relative tumor inhibition rate (TGI) (%): TGI% = (1-T / C) × 100%. T / C% represents the relative tumor growth rate, i.e., the percentage of tumor volume or weight in the treatment group and the PBS control group at a specific time point; T and C represent the tumor volume (TV) or tumor weight (TW) in the treatment group and the PBS control group, respectively, at a specific time point. All data are expressed as mean ± SEM. Significant differences in tumor volume and weight between the treatment group and the control group were compared using the Student's t-test; p < 0.05 was considered significant.

[0146] As shown in Figure 9, in the subcutaneous transplant tumor model of human liver cancer cells HepG2 mixed with PBMC, the bispecific antibody G100 showed a good anti-tumor effect. The final average tumor volume of each group (PBS, 0.63 mg / kg G100, 1.89 mg / kg G100) was: 791.58 mm 3 、455.65mm 3 、271.79mm 3 The tumor inhibition rates (TGI) of the 0.63 mg / kg G100 group and the 1.89 mg / kg G100 group were 46.28% and 71.57%, respectively.

[0147] Furthermore, no significant weight loss was observed in NCG mice in any of the dosing groups, indicating that NCG mice tolerated the anti-CD3-GPC3 bispecific antibody well at this dose. These results demonstrate that the anti-CD3-GPC3 bispecific antibody exhibits excellent anti-tumor efficacy and a high safety profile.

[0148] Table 12 Experimental groups and dosing regimens

[0149] The embodiments herein described above are merely exemplary, and any person skilled in the art will recognize or be able to ascertain the equivalents of numerous specific compounds, materials, and operations without requiring undue experimentation. All such equivalents are within the scope of this invention and are encompassed by the claims.

Claims

1. A bispecific antigen-binding molecule comprising a first antigen-binding domain that recognizes glypican 3 (GPC3); and a second antigen-binding domain that recognizes the CD3 subunit of the T-cell receptor; wherein: The first antigen-binding domain comprises HCDR1, HCDR2 and HCDR3, and the HCDR1, HCDR2 and HCDR3 are derived from the heavy chain variable region shown in SEQ ID NO:

10.

2. The bispecific antigen-binding molecule according to claim 1, wherein The first antigen-binding domain comprises HCDRs as shown in SEQ ID NOs: 11-13; preferably, the sequence of HCDR 1 is shown in SEQ ID NO: 11; the sequence of HCDR2 is shown in SEQ ID NO: 12; and the sequence of HCDR3 is shown in SEQ ID NO:

13.

3. The bispecific antigen-binding molecule according to claim 1 or 2, wherein: The second antigen-binding domain comprises HCDR1, HCDR2 and HCDR3, wherein the HCDR1, HCDR2 and HCDR3 are derived from the heavy chain variable region shown in SEQ ID NO: 1; and further comprises LCDR1, LCDR2 and LCDR3, wherein the LCDR1, LCDR2 and LCDR3 are derived from the light chain variable region shown in SEQ ID NO:

2.

4. The bispecific antigen binding molecule according to any one of the preceding claims, wherein The second antigen-binding domain comprises the CDRs shown in SEQ ID NOs: 4-9; preferably, the sequence of HCDR 1 is shown in SEQ ID NO: 4; the sequence of HCDR2 is shown in SEQ ID NO: 5; the sequence of HCDR3 is shown in SEQ ID NO: 6; the sequence of LCDR1 is shown in SEQ ID NO: 7; the sequence of LCDR2 is shown in SEQ ID NO: 8; and the sequence of LCDR3 is shown in SEQ ID NO:

9.

5. The bispecific antigen binding molecule according to any preceding claim, further comprising one or more of the following features: (1) The first antigen-binding domain is a nanobody; preferably, it is a humanized camelid nanobody; (2) The bispecific antigen-binding molecule comprises heavy chain constant regions CH2 and CH3, and does not contain a light chain constant region and / or a heavy chain constant region CH1; preferably, the heavy chain constant region comprises an Fc domain or a variant Fc; more preferably, the Fc is derived from mouse or human; (3) The bispecific antigen-binding molecule comprises an Fc domain, wherein the Fc domain is an IgG Fc domain; preferably, the Fc domain is an IgG1 domain or an IgG4 domain; (4) The bispecific antigen-binding molecule includes an Fc domain, wherein the two polypeptide chains constituting the Fc domain have different sequences from each other, and the amino acid residues at position 366 of the Knob chain of the two polypeptides constituting the Fc domain are mutated to tryptophan according to EU numbering, and the amino acid residues at position 366 of the other Hole chain are mutated to serine, the amino acid residue at position 368 is mutated to alanine, and the amino acid residue at position 407 is mutated to valine according to EU numbering; preferably, the Knob chain includes S354C and T366W amino acid substitutions, and the Hole chain includes Y349C, T366S, L368A and Y407V amino acid substitutions; more preferably, the Hole chain also includes H435R substitution; further preferably, the Knob chain and / or Hole chain also include L234A and L235A substitutions; further preferably, the Knob chain and / or Hole chain also include K447A substitution at the last position of the C-terminus. (5) The bispecific antigen-binding molecule includes an Fc domain, wherein the Fc domain includes a Knob chain and a Hole chain, the sequence of the Knob chain is shown in SEQ ID NO: 1, and the sequence of the Hole chain is shown in SEQ ID NO:

15.

6. A bispecific antigen-binding molecule comprising a first peptide chain and a second peptide chain, wherein the sequence of the first peptide chain is as shown in SEQ ID NO: 16 or 18, and / or the sequence of the second peptide chain is as shown in SEQ ID NO:

17.

7. A polynucleotide encoding the bispecific antigen binding molecule or fragment thereof according to any preceding claim; or a recombinant vector comprising the isolated polynucleotide; or a host cell comprising the isolated polynucleotide or recombinant vector.

8. A method for producing the bispecific antigen-binding molecule according to any one of claims 1 to 6.

9. A pharmaceutical composition comprising: a pharmaceutically acceptable carrier, and one or more of the group consisting of the bispecific antigen-binding molecule of any one of claims 1 to 6, the polynucleotide of claim 7, a recombinant vector and / or a host cell.

10. Use of the bispecific antigen binding molecule of any one of claims 1 to 6 or the pharmaceutical composition of claim 9 in the preparation of a medicament for treating and / or preventing a disease in an individual in need thereof; preferably, the disease is cancer; more preferably, the cancer is a GPC3-positive tumor; further preferably, the cancer is selected from hepatocellular carcinoma, gastric cancer, esophageal cancer, ovarian cancer, or non-squamous non-small cell lung cancer.

11. A method for treating and / or preventing a GPC3-expressing disease in a subject, or a pharmaceutical composition for treating and / or preventing a GPC3-expressing disease in a subject, comprising administering to the subject an effective amount of the bispecific antigen binding molecule of any one of claims 1-6 or the pharmaceutical composition of claim 9; preferably, the disease is cancer; more preferably, the cancer is selected from hepatocellular carcinoma, gastric cancer, esophageal cancer, ovarian cancer, or non-squamous non-small cell lung cancer.

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