WO2023186112A1

WO2023186112A1 - Multispecific antibody targeting ceacam and cd3 and use thereof

Info

Publication number: WO2023186112A1
Application number: PCT/CN2023/085539
Authority: WO
Inventors: 罗羿; 陈连娣; 缪小牛; 黄威峰; 王超; 彭绍岗; 董田甜; 曾竣玮
Original assignee: 普米斯生物技术(珠海)有限公司
Priority date: 2022-04-02
Filing date: 2023-03-31
Publication date: 2023-10-05
Also published as: CN116925232A

Abstract

The present invention relates to a multispecific antibody targeting a carcinoembryonic antigen-related cell adhesion molecule (CEACAM) and CD3, a pharmaceutical composition comprising the multispecific antibody, and use thereof in the treatment of a tumor.

Description

Multispecific antibodies targeting CEACAM and CD3 and their uses

Technical field

The present invention relates to multispecific antibodies targeting carcinoembryonic antigen-related cell adhesion molecule (CEACAM) and CD3, pharmaceutical compositions comprising the multispecific antibodies, and their use for treating tumors.

Background technique

Carcinoembryonic antigen (CEACAM) family members include carcinoembryonic antigen-related adhesion molecule 5 (CEACAM5) and carcinoembryonic antigen-related adhesion molecule 6 (CEACAM6), which are anchored on the cell membrane through covalent binding to glycophosphatidylinositol (GPI). Thereby participating in the regulation of a variety of cell functions such as cell chemotactic movement, intercellular adhesion, etc. (Kinoshita, T., Biosynthesis and biology of mammalian GPI-anchored proteins. Open Biol, 2020.10(3):p.190290.). CEACAM5/CEACAM6 is highly expressed in a variety of tumor tissues, including colon cancer, pancreatic cancer, gastric cancer, non-small cell lung cancer, breast cancer, head and neck squamous cell carcinoma, endometrial cancer and bladder cancer (Blumenthal, R.D., H.J.Hansen, and D.M. Goldenberg,Inhibition of adhesion,invasion,and metastasis by antibodies targeting CEACAM6(NCA-90)and CEACAM5(Carcinoembryonic Antigen).Cancer Res,2005.65(19):p.8809-17.;Blumenthal,R.D.,et al.,Expression patterns of CEACAM5 and CEACAM6 in primary and metastatic cancers. BMC Cancer, 2007.7:p.2.).

Bispecific antibodies can target CD3 on T cells on one end and target tumor cell surface receptors such as CEA on the other end to achieve the effect of directly connecting tumor cells and T cells and killing tumor cells. Different from traditional treatments such as chemotherapy, targeted therapy or immunotherapy, this type of bispecific antibodies can directly activate all CD3-positive T cells and induce cancer cell apoptosis without the assistance of other signals (Klinger ,M.,et al.,Harnessing T cells to fight cancer with BiTE(R)antibody constructs--past developments and future directions.Immunol Rev, 2016.270(1):p.193-208.). Genentech designed a CEAxCD3 bispecific antibody Cibisatamb based on this mechanism. In a clinical study of microsatellite stable (MSS) colorectal cancer with a total of 15 patients, combined with the immune checkpoint inhibitor Atezolizumab, 6 The patient's tumor achieved partial response (PR). However, a major problem with CD3 bispecific antibodies is that they overactivate T cells and cause toxicity to normal tissues.

Therefore, there is a need in this field to develop new CEAxCD3 bispecific antibodies to reduce the possibility of T cell overactivation, improve safety, and provide a better therapeutic window.

Contents of the invention

The inventors of the present application have developed multispecific antibodies against CEACAM and CD3, which reduce Fc-mediated T cell activation by adjusting the respective affinity to CEACAM and CD3, adjust the ratio of tumor antigen binding sites to CD3 binding sites, and Relative position and other methods allow it to have a better treatment window, thus providing the following aspects.

multispecific antibodies

In one aspect, the invention provides a multispecific antibody comprising a first antigen-binding domain targeting carcinoembryonic antigen-related cell adhesion molecule (CEACAM) and a second antigen-binding domain targeting CD3, wherein, The first antigen-binding domain includes a heavy chain variable region (VH) and a light chain variable region (VL);

The VH includes CDR-H1 shown in SEQ ID NO:38, CDR-H2 shown in SEQ ID NO:39 and CDR-H3 shown in SEQ ID NO:40;

The VL includes CDR-L1 shown in SEQ ID NO:41, CDR-L2 shown in SEQ ID NO:42, and CDR-L3 shown in SEQ ID NO:43.

In certain embodiments, the first antigen binding domain includes a VH as set forth in SEQ ID NO: 1 or a variant thereof and a VL as set forth in SEQ ID NO: 2 or a variant thereof; said variant At least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least Sequences that are 98%, at least 99%, or 100% sequence identical, or have one or several amino acid substitutions, deletions, or additions (e.g., 1, 2, 3, 4, or 5 amino acids) substitution, deletion or addition); preferably, the substitution is a conservative substitution.

In certain embodiments, the second antigen binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein:

(1) The VH includes CDR-H1 shown in SEQ ID NO:44, CDR-H2 shown in SEQ ID NO:53 and CDR-H3 shown in SEQ ID NO:54; the VL includes SEQ ID NO : CDR-L1 shown in SEQ ID NO: 52, CDR-L2 shown in SEQ ID NO: 48, CDR-L3 shown in SEQ ID NO: 49;

(2) The VH includes CDR-H1 shown in SEQ ID NO:44, CDR-H2 shown in SEQ ID NO:45 and CDR-H3 shown in SEQ ID NO:46; the VL includes SEQ ID NO :CDR-L1 shown in SEQ ID NO:47, CDR-L2 shown in SEQ ID NO:48, CDR-L3 shown in SEQ ID NO:49;

(3) The VH includes CDR-H1 shown in SEQ ID NO:44, CDR-H2 shown in SEQ ID NO:50 and CDR-H3 shown in SEQ ID NO:51; the VL includes SEQ ID NO :CDR-L1 shown in 52, CDR-L2 shown in SEQ ID NO:48, CDR-L3 shown in SEQ ID NO:49; or,

(4) The VH includes CDR-H1 shown in SEQ ID NO:44, CDR-H2 shown in SEQ ID NO:50 and CDR-H3 shown in SEQ ID NO:55; the VL includes SEQ ID NO :CDR-L1 shown in SEQ ID NO:52, CDR-L2 shown in SEQ ID NO:48, CDR-L3 shown in SEQ ID NO:49.

In certain embodiments, the second antigen binding domain comprises:

(1) VH as shown in SEQ ID NO:7 or its variants and VL as shown in SEQ ID NO:6 or its variants;

(2) VH as shown in SEQ ID NO:3 or its variants and VL as shown in SEQ ID NO:4 or its variants;

(3) VH as set forth in SEQ ID NO:5 or a variant thereof and VL as set forth in SEQ ID NO:6 or a variant thereof; or,

(4) VH as shown in SEQ ID NO:8 or its variants and VL as shown in SEQ ID NO:6 or its variants;

wherein said variant has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96% , a sequence that has at least 97%, at least 98%, at least 99%, or 100% sequence identity, or has one or several amino acid substitutions, deletions, or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions); preferably, the substitutions are conservative substitutions.

M-body structure

In certain embodiments, the multispecific antibodies of the invention have an M-body structure, and the multispecific antibodies comprise:

(i) a first peptide chain comprising the VH of the first antigen-binding domain, the heavy chain CH1 domain, the VL of the second antigen-binding domain, and the heavy chain CH3 domain;

(ii) a second peptide chain comprising the VH of the first antigen-binding domain, the heavy chain CH1 domain, the VH of the second antigen-binding domain, and the heavy chain CH3 domain;

and

(iii) A third peptide chain comprising the VL and light chain constant region (CL) of the first antigen binding domain; preferably, the CL is a kappa light chain constant region.

In certain embodiments, the first antigen binding domain is a Fab and the second antigen binding domain is an Fv.

In certain embodiments, the heavy chain CH1 domain of the first peptide chain is capable of forming a dimer with the CL of the third peptide chain. In certain embodiments, the heavy chain CH1 domain of the second peptide chain is capable of forming a dimer with the CL of the third peptide chain. In certain embodiments, the heavy chain CH3 domain of the first peptide chain is identical to the heavy chain CH3 domain of the second peptide chain. The heavy chain CH3 domain forms a dimer.

In certain embodiments, the multispecific antibody comprises one of the first peptide chain, one of the second peptide chain, and two of the third peptide chain.

In certain embodiments, the heavy chain CH1 domain and the heavy chain CH3 domain of the first and second peptide chains are of the same isotype. In certain embodiments, the heavy chain CH1 domain and the heavy chain CH3 domain of the first and second peptide chains are IgG, such as IgG1, IgG2, IgG3, or IgG4.

In certain embodiments, the heavy chain CH3 domain of the first peptide chain and/or the heavy chain CH3 domain of the second peptide chain comprises a modification.

In certain embodiments, the modification promotes dimerization of both. As a result, (hetero)dimerization occurs between the first peptide chain and the second peptide chain to form a complex.

Such modifications are known to those skilled in the art and may include separate modifications to each of the two Fc domain subunits (i.e., the first and second monomers of the Fc domain) to which association is desired, wherein said The modifications are complementary to each other, thereby promoting association of the two Fc domain subunits. For example, modifications that promote association may alter the structure or charge of one or both Fc domain subunits to promote their association sterically or electrostatically, respectively. For example, modifications that promote association include amino acid mutations (eg, amino acid substitutions) in the Fc domain.

In certain embodiments, the modification is in the CH3 domain of the Fc domain.

In certain embodiments, the CH3 domains of both monomers of the Fc domain comprise amino acid substitutions.

In certain embodiments, the modifications comprise a "node" modification in one of the two heavy chain CH3 domains of the first and second peptide chains and a "hole" modification in the other of the two heavy chain CH3 domains. ” modification to form the “knob-into-hole” modification. Node-into-acupoint 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). Generally, this method involves the introduction of ridges ("knobs") at the interface of a first polypeptide and corresponding cavities ("cavities") in the interface of a second polypeptide, such that the ridges can be placed within the cavities so that Promotes heterodimer formation and hinders homodimer formation. Bumps are constructed by replacing small amino acid side chains from the first polypeptide interface with larger side chains, such as tyrosine or tryptophan. Complementary cavities of the same or similar size as the ridges are created in the interface of the second polypeptide by replacing large amino acid side chains with smaller amino acid side chains, such as alanine or threonine.

In certain exemplary embodiments, one amino acid in the CH3 domain of the first monomer is replaced with an amino acid residue with a larger side chain volume, thereby forming a ridge within the CH3 domain of the first monomer, so One amino acid in the CH3 domain of the second monomer is replaced with an amino acid residue with a smaller side chain volume, thereby forming a complementary cavity with the same or similar size as the bulge in the CH3 domain of the second monomer; Alternatively, the second monomer One amino acid in the CH3 domain is replaced with an amino acid residue with a larger side chain volume, thereby forming a bulge in the CH3 domain of the second monomer, and one amino acid in the CH3 domain of the first monomer is replaced with an amino acid residue with a larger side chain volume. Amino acid residues of small side chain volume, thereby forming a complementary cavity within the CH3 domain of the first monomer with the same or similar size as the ridge.

In certain embodiments, the amino acid residue with a larger side chain volume is selected from the group consisting of arginine (R), phenylalanine (P), tyrosine (Y), tryptophan (W) group.

In certain embodiments, the amino acid residue with smaller side chain volume is selected from the group consisting of alanine (A), serine (S), threonine (T), valine (V).

In certain exemplary embodiments, the CH3 domain comprising the "node" modification comprises the amino acid sequence shown in SEQ ID NO: 11; the CH3 domain comprising the "hole" modification comprises the amino acid sequence shown in SEQ ID NO: The amino acid sequence shown in 12.

In certain embodiments, the heavy chain CH3 domain of the first peptide chain and/or the heavy chain CH3 domain of the second peptide chain may also comprise mutations or chemical modifications to alter their effector functions (e.g., reduce or enhance Antibody-dependent cellular cytotoxicity (ADCC), reduced or enhanced antibody-dependent cellular phagocytosis (ADCP), and/or reduced or enhanced complement-dependent cytotoxicity (CDC)).

In certain embodiments, the multispecific antibody based on the M-body structure may further comprise an albumin-binding polypeptide to extend half-life. Those skilled in the art understand that albumin-binding polypeptides can extend the in vivo half-life of the active molecule to which they are linked, without adversely affecting the original biological activity of the active molecule. In certain embodiments, the albumin-binding polypeptide is an antibody or antibody fragment capable of specifically binding albumin. In certain embodiments, the albumin-binding polypeptide is a single domain antibody capable of specifically binding albumin. In certain embodiments, the albumin-binding polypeptide comprises the sequence set forth in SEQ ID NO: 37.

In certain embodiments, the first peptide chain and/or the second peptide chain further comprise an albumin-binding polypeptide at the C-terminus of the heavy chain CH3 domain thereof. In certain embodiments, the first peptide chain further comprises an albumin-binding polypeptide at the C-terminus of its heavy chain CH3 domain.

In certain embodiments, each adjacent domain in the first, second and third peptide chains in the M-body structure is optionally connected by a peptide linker. In certain embodiments, the peptide linker is a peptide linker comprising one or more glycine (G) and/or one or more serine (S) (e.g., a flexible peptide comprising (G4S)n, where n is 1, 2, 3 or 4, such as the sequence shown in SEQ ID NO:13), or contains the sequence shown in SEQ ID NO:14 or 15.

In certain embodiments, the first peptide chain, the second peptide chain and the third peptide chain in the M-body structure The variable and constant regions are directly connected without linkers.

In certain exemplary embodiments, the multispecific antibody based on the M-body structure comprises:

(1) A first peptide chain including the sequence shown in SEQ ID NO:29, a second peptide chain including the sequence shown in SEQ ID NO:31 and a third peptide chain including the sequence shown in SEQ ID NO:28;

(2) A first peptide chain including the sequence shown in SEQ ID NO: 26, a second peptide chain including the sequence shown in SEQ ID NO: 27, and a third peptide chain including the sequence shown in SEQ ID NO: 28;

(3) A first peptide chain including the sequence shown in SEQ ID NO:29, a second peptide chain including the sequence shown in SEQ ID NO:30, and a third peptide chain including the sequence shown in SEQ ID NO:28; or,

(4) A first peptide chain including the sequence shown in SEQ ID NO:29, a second peptide chain including the sequence shown in SEQ ID NO:32, and a third peptide chain including the sequence shown in SEQ ID NO:28.

Fc-based structure

In certain embodiments, the multispecific antibodies of the invention have an Fc-based structure, said multispecific antibodies comprising:

(i) a first peptide chain comprising the first antigen binding domain, the VH of the second antigen binding domain and the heavy chain constant region (CH);

(ii) a second peptide chain comprising the first antigen-binding domain and an Fc domain monomer capable of monomerizing with the Fc domain of the heavy chain constant region of the first peptide chain; The bodies form dimers;

(iii) A third peptide chain comprising the VL and light chain constant region (CL) of the second antigen binding domain; preferably, the CL is a kappa light chain constant region.

In certain embodiments, the first antigen binding domain is a scFv. It is known in the art that the stability of scFv can be enhanced by the formation of interchain disulfide bonds in the structure. Thus, in certain embodiments, the scFv contains a disulfide bond. The method of introducing disulfide bonds into scFv is well known to those skilled in the art. For example, it can be achieved by introducing cysteine (C) into VH and VL of scFv respectively. In certain embodiments, the VH and VL of the first antigen-binding domain each comprise a residue located in the FR region that is mutated to cysteine (C), so that the scFv formed by the VH and VL can comprise Disulfide bonds. In certain embodiments, one residue in the FR2 region of VH and one residue in the FR4 region of VL of the first antigen binding domain are mutated to cysteine (C).

In certain embodiments, the second antigen binding domain is a Fab.

In certain embodiments, the CH1 domain of the heavy chain constant region of the first peptide chain and the CL of the third peptide chain are capable of forming a dimer.

In certain embodiments, the multispecific antibody comprises a first peptide chain, a second peptide chain, and a third Tripeptide chain.

In certain embodiments, the heavy chain constant region of the first peptide chain and the Fc domain monomer of the second peptide chain are of the same isotype. In certain embodiments, the isotype is IgG, such as IgG1, IgG2, IgG3, or IgG4.

In certain embodiments, the heavy chain constant region Fc domain monomer of the first peptide chain and the Fc domain monomer of the second peptide chain comprise modifications.

In certain embodiments, the modification is in the CH3 domain of the Fc domain.

In certain embodiments, the modifications comprise a "knot" modification in one of the Fc domain monomers of the first peptide chain and the Fc domain monomer of the second peptide chain and a "knot" modification in the other of the two. "hole" modification to form "knob-into-hole" modification. Node-into-acupoint 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). Generally, this method involves the introduction of ridges ("knobs") at the interface of a first polypeptide and corresponding cavities ("cavities") in the interface of a second polypeptide, such that the ridges can be placed within the cavities so that Promotes heterodimer formation and hinders homodimer formation. Bumps are constructed by replacing small amino acid side chains from the first polypeptide interface with larger side chains, such as tyrosine or tryptophan. Complementary cavities of the same or similar size as the ridges are created in the interface of the second polypeptide by replacing large amino acid side chains with smaller amino acid side chains, such as alanine or threonine.

In certain exemplary embodiments, one amino acid in the CH3 domain of the first monomer is replaced with an amino acid residue with a larger side chain volume, thereby forming a ridge within the CH3 domain of the first monomer, so One amino acid in the CH3 domain of the second monomer is replaced with an amino acid residue with a smaller side chain volume, thereby forming a complementary cavity with the same or similar size as the bulge in the CH3 domain of the second monomer; Alternatively, one amino acid in the CH3 domain of the second monomer is replaced with an amino acid residue with a larger side chain volume, thereby forming a bulge in the CH3 domain of the second monomer, and the CH3 of the first monomer One amino acid in the domain is replaced with a smaller side chain volume of amino acid residues, thereby forming a complementary cavity within the CH3 domain of the first monomer having the same or similar size as the ridge.

In certain exemplary embodiments, the Fc domain monomer comprising a "node" modification comprises the amino acid sequence shown in SEQ ID NO: 56; the Fc domain monomer comprising a "hole" modification comprises as The amino acid sequence shown in SEQ ID NO:10. In certain exemplary embodiments, the heavy chain constant region of the first peptide chain comprises the amino acid sequence set forth in SEQ ID NO: 9, and/or the Fc domain monomer of the second peptide chain comprises as The amino acid sequence shown in SEQ ID NO:10.

In certain embodiments, the Fc domain monomer of the first peptide chain and/or the Fc domain monomer of the second peptide chain comprises a mutation or chemical modification to alter its effector function (e.g., reduced or enhanced antibody dependent cellular cytotoxicity (ADCC), reduced or enhanced antibody-dependent cellular phagocytosis (ADCP) and/or reduced or enhanced complement-dependent cellular cytotoxicity (CDC)). In certain embodiments, the Fc domain monomer of the first peptide chain and/or the Fc domain monomer of the second peptide chain comprises a LALA mutation (L234A, L235A).

In certain embodiments, the first antigen-binding domain is a scFv and has a structure represented by VH-L-VL or VL-L-VH, where L is a peptide linker. In certain embodiments, the L is a peptide linker comprising one or more glycine (G) and/or one or more serine (S), for example, a flexible peptide comprising (G4S)n, where n is 1, 2, 3 or 4. In certain embodiments, the VH of the scFv is compared to SEQ ID NO: 1, and one amino acid in its FR region (e.g., FR2 region) is replaced with cysteine (C). In certain embodiments, the VL of the scFv has one amino acid in its FR region (e.g., FR4 region) substituted with cysteine (C) compared to SEQ ID NO:2. In certain embodiments, the first antigen binding domain has the structure represented by VH-L-VL. In certain exemplary embodiments, the first antigen binding domain comprises the amino acid sequence set forth in SEQ ID NO: 18.

In certain embodiments, each adjacent domain in the first, second and third peptide chains in the Fc-based structure is connected by a peptide linker. In certain embodiments, the peptide linker is a peptide linker comprising one or more glycine (G) and/or one or more serine (S) (e.g., a flexible peptide comprising (G4S)n, where n is 1, 2, 3 or 4, such as the sequence shown in SEQ ID NO:13), or contains the sequence shown in SEQ ID NO:14 or 15.

In certain embodiments, the variable regions and constant regions in the first and third peptide chains in the Fc-based structure are directly connected without being connected through a linker.

In certain exemplary embodiments, the Fc-based structure-based multispecific antibody comprises:

(1) A first peptide chain including the sequence shown in SEQ ID NO: 19, a second peptide chain including the sequence shown in SEQ ID NO: 20 and a third peptide chain including the sequence shown in SEQ ID NO: 21;

(2) A first peptide chain including the sequence shown in SEQ ID NO:22, a second peptide chain including the sequence shown in SEQ ID NO:20 and a third peptide chain including the sequence shown in SEQ ID NO:23;

(3) A first peptide chain including the sequence shown in SEQ ID NO:24, a second peptide chain including the sequence shown in SEQ ID NO:20, and a third peptide chain including the sequence shown in SEQ ID NO:23; or,

(4) A first peptide chain including the sequence shown in SEQ ID NO:25, a second peptide chain including the sequence shown in SEQ ID NO:20, and a third peptide chain including the sequence shown in SEQ ID NO:23.

In certain embodiments, the multispecific antibody based on the Fc-based structure may further comprise an albumin-binding polypeptide to extend half-life. In certain embodiments, the albumin-binding polypeptide is an antibody or antibody fragment capable of specifically binding albumin. In certain embodiments, the albumin-binding polypeptide is a single domain antibody capable of specifically binding albumin. In certain embodiments, the albumin-binding polypeptide comprises the sequence set forth in SEQ ID NO: 37.

Preparation of antibodies

The multispecific antibodies of the present invention can be prepared by various methods known in the art, such as by genetic engineering recombinant technology. For example, the DNA molecules encoding them are obtained by chemical synthesis or PCR amplification. The resulting DNA molecule is inserted into an expression vector and then transfected into host cells. Then, the transfected host cells are cultured under specific conditions and express the multispecific antibody of the invention.

For example, a multispecific antibody of the invention can be produced by co-expressing multiple polynucleotides encoding individual polypeptide chains of the multispecific antibody. The polypeptide chains produced by coexpression can be associated, for example, via disulfide bonds or other means to form functional multispecific antibodies. For example, the light chain portion of the Fab fragment can be encoded by separate polynucleotides from the portion of the multispecific antibody that contains the heavy chain portion of the Fab fragment (which portion can further comprise an Fc domain monomer and optionally other antigen-binding domains) . When co-expressed, a polypeptide comprising the heavy chain portion of the Fab fragment will associate with a polypeptide comprising the light chain portion of the Fab fragment to form a Fab fragment. As another example, a portion of a multispecific antibody provided herein that includes one of the two Fc domain monomers (which portion may further include an antigen-binding domain) may be combined with a portion that includes the other of the two Fc domain monomers. The portion, which may further comprise an antigen-binding domain, is encoded by a separate polynucleotide. When co-expressed, the two Fc domain monomers associate to form the Fc domain.

In another aspect, the invention provides an isolated nucleic acid molecule encoding a multispecific antibody of the invention or The nucleotide sequence of at least one of its peptide chains.

In certain embodiments, the isolated nucleic acid molecule comprises a nucleotide sequence encoding each peptide chain of the multispecific antibody of the invention, and the nucleotide sequence encoding each peptide chain is present in the same or on different isolated nucleic acid molecules.

In another aspect, the invention provides vectors (eg, expression vectors) comprising nucleic acid molecules encoding the above-described isolated nucleic acids.

In certain embodiments, the vectors comprise nucleotide sequences encoding each peptide chain of the multispecific antibody of the invention, and the nucleotide sequences encoding each peptide chain are present in the same or different vectors superior. For example, the vector of the present invention includes: a first vector including a nucleotide sequence encoding a first peptide chain, a second vector including a nucleotide sequence encoding a second peptide chain, and a nucleoside encoding a third peptide chain. The third vector of the acid sequence.

In another aspect, the invention provides a host cell comprising a nucleic acid molecule or vector as described above. Such host cells include, but are not limited to, prokaryotic cells such as bacterial cells (e.g., E. coli cells), and eukaryotic cells such as fungal cells (e.g., yeast cells), insect cells, plant cells, and animal cells (e.g., mammalian cells, e.g., small mouse cells, human cells, etc.).

In another aspect, the invention provides a method for preparing a multispecific antibody of the invention, comprising culturing a host cell as described above under conditions that allow protein expression, and recovering the host cell culture from the cultured host cell. Multispecific antibodies.

combination

In another aspect, the invention provides a composition comprising a multispecific antibody of the invention and an immune checkpoint inhibitor.

In certain embodiments, the immune checkpoint is selected from PD-1, PD-L1, CTLA-4, TIM-3, Lag-3, TIGIT, CD73, VISTA, B7-H3, or any combination thereof.

In certain embodiments, the immune checkpoint inhibitor is a PD-1 or PD-L1 antibody or antibody fragment. In certain exemplary embodiments, the immune checkpoint inhibitor is a PD-1 antibody or antibody fragment having the VH shown in SEQ ID NO: 57 and the VL shown in SEQ ID NO: 58.

In certain embodiments, the composition comprises a multispecific antibody having an M-body structure as described herein and an immune checkpoint inhibitor, such as a PD-1 or PD-L1 antibody or antibody fragment. In certain exemplary embodiments, the immune checkpoint inhibitor is a PD-1 antibody or antibody fragment having the VH shown in SEQ ID NO: 57 and the VL shown in SEQ ID NO: 58.

In certain embodiments, the multispecific antibodies of the invention and the immune checkpoint inhibitors act as separate groups Available separately or as mixed components.

pharmaceutical composition

In another aspect, the invention provides pharmaceutical compositions comprising a multispecific antibody of the invention, an isolated nucleic acid molecule, a vector, a host cell or a composition, and a pharmaceutically acceptable carrier and/or excipient.

In certain embodiments, the pharmaceutical compositions comprise a multispecific antibody of the invention.

In certain embodiments, the pharmaceutical composition comprises a composition as described above.

The pharmaceutical compositions of the present invention are formulated in dosage forms compatible with their intended route of administration. Examples of routes of administration include parenteral administration, e.g., intravenous administration, intradermal administration, subcutaneous administration, oral administration (e.g., inhalation administration), transdermal administration (i.e., topical administration), transdermal administration (i.e., topical administration), Mucosal and rectal administration. Solutions or suspensions for parenteral, intradermal or subcutaneous administration applications may include the following components: sterile diluents such as water for injection, saline solution, fixed oils, polyethylene glycol, glycerol, propylene glycol or Other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl paraben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetate, citrate or Phosphates, and agents used to adjust tonicity, such as sodium chloride or glucose. The pH can be adjusted with acids or bases such as hydrochloric acid or sodium hydroxide. Formulations for parenteral administration may be enclosed in ampoules, disposable syringes or multi-dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the ready preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, polyoxyethylene castor oil ELTM, or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that syringability exists. It must be stable under the conditions of manufacture and storage, and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier may be a solvent or dispersion medium including, for example, water, ethanol, polyols (eg, glycerol, propylene glycol, liquid polyethylene glycol, etc.), and suitable mixtures thereof. Protection against microorganisms can be achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In many cases it will be preferable to include isotonic agents such as sugars, polyols such as mannitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

therapeutic use

In another aspect, the invention provides a method for preventing and/or treating tumors, comprising administering to a subject in need thereof a multispecific antibody, an isolated nucleic acid molecule, a vector, a host cell, a composition or drug combination things. The invention also provides the multispecific antibodies, isolated nucleic acid molecules, vectors, host cells, compositions or pharmaceutical compositions of the invention for use in preventing and/or treating tumors, or in preparation for preventing and/or treating tumors. Use in oncology drugs.

In certain embodiments, the tumor is carcinoembryonic antigen-related cell adhesion molecule (CEACAM) positive.

In certain embodiments, the tumor is CEACAM5 and/or CEACAM6 positive.

In certain embodiments, the tumor is selected from the group consisting of non-small cell lung cancer, small cell lung cancer, renal cell carcinoma, colorectal cancer, ovarian cancer, breast cancer, pancreatic cancer, gastric cancer, bladder cancer, esophageal cancer, mesothelioma, Melanoma, head and neck cancer, thyroid cancer, sarcoma, prostate cancer, glioblastoma, cervical cancer, thymus cancer, leukemia, lymphoma, myeloma, mycoses fungoids, Merkel cells Cancer and other hematological malignancies, such as classic Hodgkin lymphoma (CHL), primary mediastinal large B-cell lymphoma, T-cell/histiocytic B-cell-rich lymphoma, EBV-positive and -negative PTLD and EBV-related Diffuse large B-cell lymphoma (DLBCL), plasmablastic lymphoma, extranodal NK/T-cell lymphoma, nasopharyngeal carcinoma and HHV8-related primary effusion lymphoma, Hodgkin lymphoma, central nervous system Systemic (CNS) tumors, such as primary CNS lymphoma, spinal tumors, and brainstem gliomas.

In certain embodiments, the tumor is a solid tumor.

In certain embodiments, the tumor is selected from the group consisting of colon cancer, pancreatic cancer, gastric cancer, non-small cell lung cancer, breast cancer, head and neck squamous cell carcinoma, endometrial cancer, and bladder cancer.

In certain embodiments, the methods include administering to a subject a composition of the invention, wherein the multispecific antibody and immune checkpoint inhibitor can be administered simultaneously, separately, or sequentially.

In certain embodiments, the methods comprise co-administering (eg, simultaneously, separately, or sequentially) a multispecific antibody having an M-body structure according to the invention and an immune checkpoint inhibitor, e.g., PD, to a subject -1 or PD-L1 antibodies or antibody fragments. In certain exemplary embodiments, the immune checkpoint inhibitor is a PD-1 antibody or antibody fragment having the VH shown in SEQ ID NO: 57 and the VL shown in SEQ ID NO: 58.

The multispecific antibodies, compositions or pharmaceutical compositions containing them of the present invention can be formulated into any dosage form known in the medical field, for example, tablets, pills, suspensions, emulsions, solutions, gels, capsules, Powders, granules, elixirs, lozenges, suppositories, injections (including injections, sterile powders for injection and concentrated solutions for injection), inhalants, sprays, etc. The preferred dosage form depends on the intended mode of administration and therapeutic use. The multispecific antibodies, compositions or pharmaceutical compositions containing them of the invention should be sterile and stable under the conditions of production and storage. One preferred dosage form is an injection. Such injections may be sterile injectable solutions. For example, sterile injectable solutions may be prepared by incorporating the requisite dosage of the active ingredient in an appropriate solvent and, optionally, other desired ingredients (including, but not limited to, pH adjusters, surfactants, etc.). Active agent, adjuvant, ionic strength enhancer, isotonic agent, antiseptic agent, diluent, or any combination thereof), followed by filter sterilization. Additionally, sterile injectable solutions may be prepared as sterile lyophilized powders (for example, by vacuum drying or freeze drying) for ease of storage and use. Such sterile lyophilized powder can be dispersed in a suitable carrier before use, such as water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (such as 0.9% (w/v) NaCl), Glucose solutions (eg 5% glucose), surfactant containing solutions (eg 0.01% polysorbate 20), pH buffer solutions (eg phosphate buffer solution), Ringer's solution and any combination thereof.

The multispecific antibodies, compositions, or pharmaceutical compositions containing the same of the invention may be administered by any suitable method known in the art, including, but not limited to, oral, buccal, sublingual, ophthalmic, topical, parenteral, Rectum, intraleaf sheath, intracytoplasmic reticulum, inguinal, intravesical, topical (eg, powder, ointment, or drops), or nasal route. However, for many therapeutic uses, the preferred route/mode of administration is parenteral (eg intravenous or bolus injection, subcutaneous injection, intraperitoneal injection, intramuscular injection). The skilled artisan will understand that the route and/or mode of administration will vary depending on the intended purpose. In certain embodiments, the multispecific antibodies, compositions, or pharmaceutical compositions containing the same of the invention are administered by intravenous injection or bolus injection.

The multispecific antibodies, compositions, or pharmaceutical compositions containing the same of the invention may be formulated in dosage unit form for ease of administration. Dosage unit form refers to physically discrete units suitable as unitary dosages for the subjects to be treated; each unit contains a predetermined quantity of active ingredient calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.

The multispecific antibodies, compositions or pharmaceutical compositions containing them of the invention can be administered alone or in combination with another pharmaceutically active agent (eg, anti-tumor agent) or additional therapy (eg, anti-tumor therapy).

The subject described herein can be a mammal, such as a human.

Definition of Terms

In the present invention, unless otherwise stated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Moreover, the virology, biochemistry, and immunology laboratory procedures used in this article are routine procedures widely used in the corresponding fields. Meanwhile, in order to better understand the present invention, definitions and explanations of relevant terms are provided below.

When the terms "such as," "such as," "such as," "including," "including," or variations thereof are used herein, these terms will not be considered limiting terms and will instead be interpreted to mean "without limitation ” or “without limitation.”

Unless otherwise indicated herein or clearly contradicted by context, the terms "a" and "an" as well as "the" and similar referents shall be used in the context of describing the invention (especially in the context of the following claims). Interpreted to cover both the singular and the plural.

The term "antibody" as used herein refers to an immunoglobulin-derived molecule capable of specifically binding to a target antigen through at least one antigen-binding site located in its variable region. The target antigen. When referring to the term "antibody", it includes not only intact antibodies but also antigen-binding fragments capable of specifically binding a target antigen, unless the context clearly indicates otherwise. An "intact antibody" typically consists of two pairs of polypeptide chains, each pair having a light chain (LC) and a heavy chain (HC). Antibody light chains can be classified into kappa (kappa) and lambda (lambda) light chains. Heavy chains can be classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. Within the light and heavy chains, the variable and constant regions are connected by a "J" region of approximately 12 or more amino acids, and the heavy chain also contains a "D" region of approximately 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of 3 domains (CH1, CH2 and CH3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one domain, CL. The constant domain is not directly involved in the binding of antibodies to antigens, but exhibits a variety of effector functions, such as mediating the interaction of immunoglobulins with host tissues or factors, including various cells of the immune system (e.g., effector cells) and classical complement. Binding of the first component of the system (C1q). The VH and VL regions can also be subdivided into regions of high variability called complementarity determining regions (CDRs), interspersed with more conservative regions called framework regions (FRs). Each _VH and _VL consists of 3 CDRs and 4 FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions (VH and VL) of each heavy chain/light chain pair respectively form the antigen-binding site. The assignment of amino acids to each region or domain can follow Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk (1987) J. Mol. Biol. 196:901 Definition of -917; Chothia et al. (1989) Nature 342:878-883.

As used herein, the term "multispecific antibody" refers to an antibody that has binding specificity for at least two (eg, two, three, or four) different antigens (or epitopes). Multispecific antibodies contain multiple antigen-binding domains with binding specificities for different antigens (or epitopes), thereby being able to bind to at least two different binding sites and/or target molecules. Each antigen-binding domain comprised by a multispecific antibody can be independently selected from a full-length antibody (e.g., an IgG antibody) or an antigen-binding fragment thereof (e.g., an Fv fragment, a Fab fragment, an F(ab')2 fragment, or a scFv). In some cases, the individual antigen binding domains are linked by a peptide linker. In certain embodiments, the multispecific antibodies of the invention can be bispecific antibodies. In certain embodiments, the multispecific antibodies of the invention may be trispecific antibodies.

As used herein, the term "complementarity determining region" or "CDR" refers to the amino acid residues in the variable region of an antibody that are responsible for antigen binding. The variable regions of the heavy chain and light chain each contain three CDRs, named CDR1 and CDR2. and CDR3. The precise boundaries of these CDRs can be defined according to various numbering systems known in the art, for example according to the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991), Chothia numbering system (Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883) or IMGT numbering system (Lefranc et al. , Dev. Comparat. Immunol. 27:55-77, 2003). For a given antibody, one skilled in the art will readily identify the CDRs defined by each numbering system. Moreover, the correspondence between different numbering systems is well known to those skilled in the art (see, for example, Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003).

As used herein, the term "framework region" or "FR" residues refers to those amino acid residues in an antibody variable region other than the CDR residues as defined above.

The term "antibody" is not limited to any particular method of producing the antibody. This includes, for example, recombinant antibodies, monoclonal antibodies, and polyclonal antibodies. The antibodies may be of different isotypes, for example, IgG (eg, IgGl, IgG2, IgG3 or IgG4 subtypes), IgA1, IgA2, IgD, IgE or IgM antibodies.

As used herein, the term "Fab fragment" means an antibody fragment consisting of a light chain comprising VL and CL and a heavy chain fragment comprising VH and CH1.

As used herein, the term "Fv" means an antibody fragment consisting of the VL and VH domains of a single arm of an antibody. Fv fragments are generally considered to be the smallest antibody fragments that can form a complete antigen-binding site.

As used herein, the term "scFv" refers to a single polypeptide chain comprising VL and VH domains connected by a linker (see, e.g., Bird et al., Science 242:423 -426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); and Pluckthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, Roseburg and Moore, eds., Springer-Verlag, New York, pp. 269-315 (1994)). Such scFv molecules may have the general structure: _NH2 -VL-linker-VH-COOH or _NH2 -VH-linker-VL-COOH. Suitable prior art linkers consist of repeated GGGGS amino acid sequences or variants thereof. For example, a linker having the amino acid sequence (GGGGS) ₄ can be used, but variants thereof can also be used (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444-6448). In some cases, a disulfide bond may also exist between VH and VL of scFv.

As used herein, the term "Fc domain" or "Fc region" means a portion of the heavy chain constant region that includes CH2 and CH3. A "monomer" of an Fc domain refers to one of the two polypeptides that form a dimeric Fc domain. The Fc fragment of an antibody has many different functions but does not participate in antigen binding. "Effector functions" mediated by the Fc region include Fc receptor binding; Clq binding and complement-dependent cytotoxicity (CDC); antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (such as B cell receptors); and B cell activation, etc. In some embodiments, the Fc region includes hinge, CH2, and CH3. When the Fc region includes a hinge, the hinge mediates dimerization between the two Fc-containing polypeptides. The Fc region can be of any antibody heavy chain constant region isotype, such as IgGl, IgG2, IgG3 or IgG4.

The Fc domain may include either a native Fc region or a variant Fc region. Native Fc regions include amino acid sequences consistent with the amino acid sequences of Fc regions found in nature, for example, native sequence human Fc regions include native sequence human IgG1 Fc regions (non-A and A allotypes); native sequence human IgG2 Fc regions; native sequence human Fc regions IgG3 Fc region; and native sequence human IgG4 Fc region, and naturally occurring variants thereof. A variant Fc region includes an amino acid sequence that differs from the amino acid sequence of a native sequence Fc region due to at least one amino acid modification. In some embodiments, a variant Fc region may possess altered effector functions (e.g., Fc receptor binding, antibody glycosylation, number of cysteine residues, effector cell function, or complement function) compared to the native Fc region. . In some embodiments, variant Fc regions can possess modifications that promote dimerization.

As used herein, the term "identity" is used to refer to the match of sequences between two polypeptides or between two nucleic acids. 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 may be introduced in the first amino acid sequence or nucleic acid sequence to best match the second amino acid or nucleic acid sequence). Good comparison). The amino acid residues or nucleotides at the corresponding amino acid positions or nucleotide positions are then compared. Molecules are identical when a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence. The percent identity between two sequences is a function of the number of identical positions shared by the sequences (ie, percent identity = number of identical overlapping positions/total number of positions x 100%). In certain embodiments, both sequences are the same length.

Determination of percent identity between two sequences can also be accomplished using mathematical algorithms. One non-limiting example of a mathematical algorithm for comparison of two sequences is the algorithm of Karlin and Altschul, 1990, Proc. Improved in .Acad.Sci.U.S.A.90:5873-5877. Such algorithms were integrated into the NBLAST and XBLAST programs of Altschul et al., 1990, J. Mol. Biol. 215:403.

As used herein, the term "variant", in the context of polypeptides (including polypeptides), also refers to a polypeptide or peptide comprising an amino acid sequence that has been altered by introducing substitutions, deletions, or additions of amino acid residues. In some cases, the term "variant" also refers to a polypeptide or peptide that has been modified (ie, by covalently linking any type of molecule to the polypeptide or peptide). For example, and without limitation, polypeptides may be modified, e.g., by glycosylation, acetylation, PEGylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, Attached to cellular ligand or other proteins, etc. Derivatized polypeptides or peptides can be produced by chemical modification using techniques known to those skilled in the art, including, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, and the like. Furthermore, a variant has a similar, identical or improved function to the polypeptide or peptide from which it is derived.

As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and the antigen against which it is directed. The strength or affinity of a specific binding interaction can be expressed by the equilibrium dissociation constant (K _D ) of the interaction. In the present invention, the term " _KD " refers to the dissociation equilibrium constant of a specific antibody-antigen interaction, which is used to describe the binding affinity between an antibody and an antigen. The smaller the equilibrium dissociation constant, the tighter the antibody-antigen binding, and the higher the affinity between the antibody and the antigen.

The specific binding properties between two molecules can be determined using methods known in the art. One approach involves measuring the rate at which antigen binding site/antigen complexes form and dissociate. Both the "association rate constant" (ka or kon) and the "dissociation rate constant" (kdis or koff) can be calculated from the concentration and the actual rates of association and dissociation (see Malmqvist M, Nature, 1993, 361 :186-187). The ratio kdis/kon is equal to the dissociation constant _KD (see Davies et al., Annual Rev Biochem, 1990; 59:439-473). K _D , kon and kdis values can be measured using any valid method, for example surface plasmon resonance (SPR) can be used to measure the dissociation constant in Biacore, bioluminescence interferometry or Kinexa can also be used to measure the dissociation constant.

As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When the vector can express the protein encoded by the inserted polynucleotide, the vector is called an expression vector. The vector can be introduced into the host cell through transformation, transduction or transfection, so that the genetic material elements it carries can be expressed in the host cell. Vectors are well known to those skilled in the art, including but not limited to: plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC) ; Phages such as lambda phage or M13 phage and animal viruses, etc. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papillomaviruses, Polyoma vacuolating viruses (such as SV40). A vector can contain a variety of expression-controlling elements, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, the vector may also contain an origin of replication site.

As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, which includes, but is not limited to, prokaryotic cells such as E. coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, etc. Insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells.

As used herein, the term "conservative substitution" means one that does not adversely affect or alter the amino acid sequence comprising the List the amino acid substitutions for the expected properties of the protein/peptide. For example, conservative substitutions can be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include those in which an amino acid residue is replaced with an amino acid residue having a similar side chain, e.g., one that is physically or functionally similar to the corresponding amino acid residue (e.g., has similar size, shape, charge, chemical properties, including ability to form covalent bonds or hydrogen bonds, etc.). Families of amino acid residues with similar side chains have been defined in the art. These families include those with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine , asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), non-polar side chains (such as alanine, valine, leucine, isoleucine amino acids, proline, phenylalanine, methionine), β-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, Phenylalanine, tryptophan, histidine) amino acids. Therefore, it is preferred to replace the corresponding amino acid residue with another amino acid residue from the same side chain family. In addition, amino acid residues can be divided into categories defined by optional physical and functional properties. For example, alcohol-containing residues (S and T), aliphatic residues (I, L, V, and M), cycloalkenyl-related residues (F, H, W, and Y), hydrophobic residues (A, C, F, G, H, I, L, M, R, T, V, W and Y), negatively charged residues (D and E), polar residues (C, D, E, H, K , N, Q, R, S and T), positively charged residues (H, K and R), small residues (A, C, D, G, N, P, S, T and V), polar Small residues (A, G and S), residues involved in turn formation (A, C, D, E, G, H, K, N, Q, R, S, P and T), flexible residues (Q , T, K, S, G, P, D, E and R). Methods for identifying conservative substitutions of amino acids are well known in the art (see, e.g., Brummell et al., Biochem. 32:1180-1187 (1993); Kobayashi et al., Protein Eng. 12(10):879-884 (1999) ; and Burks et al. Proc. Natl Acad. Set USA 94:412-417 (1997), which is incorporated herein by reference).

The twenty conventional amino acids involved in this article have been prepared following conventional usage. See, e.g., Immunology-A Synthesis (2nd Edition, E.S. Golub and D.R. Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. In the present invention, the terms "polypeptide" and "protein" have the same meaning and are used interchangeably. And in the present invention, amino acids are generally represented by one-letter and three-letter abbreviations well known in the art. For example, alanine can be represented by A or Ala.

As used herein, the term "pharmaceutically acceptable carrier and/or excipient" means a carrier and/or excipient that is pharmacologically and/or physiologically compatible with the subject and the active ingredient, They are well known in the art (see, e.g., Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995) and include, but are not limited to: pH adjusters, surfactants, adjuvants, ionic strength enhancers Agents, diluents, agents to maintain osmotic pressure, agents to delay absorption, preservatives. For example, pH adjusting agents include, but are not limited to, phosphate buffer. Surfactants include, but are not limited to, cationic, anionic or nonionic surfactants such as Tween-80. Ionic strength enhancers include, but are not limited to, sodium chloride. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, etc. Agents that maintain osmotic pressure include, but are not limited to, sugar, NaCl, and the like. Agents that delay absorption include, but are not limited to, monostearate and gelatin. Diluents include, but are not limited to, water, aqueous buffers (such as buffered saline), alcohols and polyols (such as glycerol), and the like. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as thimerosal, 2-phenoxyethanol, parabens, chlorobutanol, phenol, sorbic acid, etc. Stabilizers have the meaning commonly understood by those skilled in the art, which can stabilize the desired activity of active ingredients in medicines, including but not limited to sodium glutamate, gelatin, SPGA, sugars (such as sorbitol, mannitol, starch, sucrose) , lactose, dextran, or glucose), amino acids (such as glutamic acid, glycine), proteins (such as dry whey, albumin or casein) or their degradation products (such as lactalbumin hydrolyzate), etc.

As used herein, the term "prevention" refers to a method performed to prevent or delay the occurrence of a disease or condition or symptom in a subject. As used herein, the term "treatment" refers to a method performed to obtain a beneficial or desired clinical result. For the purposes of the present invention, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, reduction of the extent of the disease, stabilization (i.e., no worsening) of the state of the disease, delaying or slowing the progression of the disease, ameliorating or alleviating the disease. status, and relief of symptoms (whether partial or complete), whether detectable or undetectable. In addition, "treatment" may also refer to prolonging survival compared to expected survival if not receiving treatment.

As used herein, the term "subject" refers to a mammal, such as a primate mammal, such as a human. In certain embodiments, the subject (eg, human) has a tumor (eg, a CEACAM and/or CD3 positive tumor).

Beneficial effects of the invention

The present invention provides new multispecific antibodies against CEACAM and CD3, which can promote the targeting and recruitment of T cells to tumor cells by binding to CD3 present on T cells and CEACAM on tumor cells, inducing MHC-independent Targeting tumor T cell toxicity without causing overactivation of T cells, it has significantly improved safety, giving it a better therapeutic window. Therefore, the multispecific antibodies of the present invention have important clinical value.

The embodiments of the present invention will be described in detail below with reference to the accompanying drawings and examples, but those skilled in the art will understand that the following drawings and examples are only used to illustrate the present invention and do not limit the scope of the present invention. According to attached The various objects and advantageous aspects of the present invention will become apparent to those skilled in the art from the following detailed description of the drawings and preferred embodiments.

Description of drawings

Figure 1 shows a schematic structural diagram of the bispecific antibody in Example 1.

Figure 2 shows the results of the binding activity assay of the bispecific antibody in Example 2 on LS174T and LoVo cells expressing CEACAM5/6.

Figure 3 shows the measurement results of the binding activity of the bispecific antibody to CD3-expressing Jurkat cells in Example 3.

Figure 4 shows the measurement results of the activity of the bispecific antibody in activating the NFAT signaling pathway in Jurkat cells in Example 4.

Figure 5 shows the measurement results of the activity of bispecific antibodies inducing primary T cells to kill tumor cells in Example 5.

Figure 6 shows the measurement results of the activity of the bispecific antibody in inducing PBMC cytokine release in Example 6.

Figure 7 shows the correlation measurement results between activation of T cells induced by bispecific antibodies and antigen expression in Example 7.

Figure 8 shows the anti-tumor effect of the bispecific antibody in Example 8 in a tumor-bearing model inoculated subcutaneously with LS174T colorectal cancer tumor cells.

Figure 9 shows the anti-tumor effect of the bispecific antibody in Example 9 combined with Anti-PD1 mAb in a tumor-bearing model in which LS174T colorectal cancer tumor cells were subcutaneously inoculated.

Figure 10 shows the results of pharmacokinetic (PK) determination of the bispecific antibody in rhesus monkeys in Example 10.

sequence information

A description of the sequences covered by this application is provided in the table below.

Table 1: Sequence information (CDRs are numbered according to kabat)

Example

The invention will now be described with reference to the following examples which are intended to illustrate but not to limit the invention. Unless otherwise specified, the molecular biology experimental methods and immunoassay methods used in the present invention basically refer to J. Sambrook et al., Molecular Cloning: Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, 1989, and The method was carried out according to the method described in F.M. Ausubel et al., Compiled Experimental Guide to Molecular Biology, 3rd Edition, John Wiley & Sons, Inc., 1995; the use of restriction enzymes was in accordance with the conditions recommended by the product manufacturer. Those skilled in the art will appreciate that the examples describe the invention by way of example and are not intended to limit the scope of the invention as claimed.

Example 1. Cloning and expression of anti-CEAxCD3 bispecific antibodies

1. In this example, 8 anti-CEA x CD3 bispecific antibodies were constructed, namely:

CEAxCD3 BiAb Fc-based 1: consists of 3 polypeptide chains. Its structural schematic is shown in Figure 1A. Peptide chain #1 has the amino acid sequence shown in SEQ ID NO 19, which contains the anti-CEA-based monoclonal antibody hM4.3 (Patent application number: hUM05-3 in WO2020244526A1) The amino acid sequence of the anti-CEA single chain antibody (SEQ ID NO 18), the anti-CD3 monoclonal antibody ADI22523 (Patent application number: WO2018208864_A1) heavy chain variable region amino acid sequence ( SEQ ID NO 3) and human IgG1 Knob mutant amino acid sequence (LALA mutation introduced to reduce Fc function, SEQ ID NO 9). The C-terminus of the anti-CEA single-chain antibody amino acid sequence (SEQ ID NO 18) was connected to the anti-CD3 monoclonal antibody ADI22523 heavy chain variable region through a flexible peptide of 11 amino acid residues Linker-1 (SEQ ID NO 13). The N-terminus of the amino acid sequence (SEQ ID NO3), and the C-terminus of the heavy chain variable region of the anti-CD3 monoclonal antibody ADI22523 was connected to the human IgG1 with Knob mutation amino acid sequence (LALA mutation was introduced to reduce Fc function, SEQ ID NO 9 ). Peptide chain #2 has the amino acid sequence shown in SEQ ID NO 20, which contains the amino acid sequence of the single-chain antibody against CEA (SEQ ID NO. NO 18), and is connected to the N of human IgG1 Fc with Hole mutation amino acid sequence (LALA mutation introduced to reduce Fc function, SEQ ID NO 10) through a flexible peptide of 11 amino acid residues Linker-1 (SEQ ID NO 13) end. Peptide chain #3 has the amino acid sequence shown in SEQ ID NO 21, which contains the amino acid sequence of the light chain variable region of the anti-CD3 monoclonal antibody ADI22523 (SEQ ID NO 4), and is connected to the human kappa light chain constant at its C-terminal Region (CL) amino acid sequence (SEQ ID NO 17).

CEAxCD3 BiAb Fc-based 2: Composed of 3 polypeptide chains, its structural diagram is shown in Figure 1A. Peptide chain #1 has the amino acid sequence shown in SEQ ID NO 22, which contains the anti-CEA-based monoclonal antibody hM4.3 (Patent application number: hUM05-3 in WO2020244526A1) constructed anti-CEA single chain antibody amino acid sequence (SEQ ID NO 18), anti-CD3 monoclonal antibody ADI26908 (Patent application number: WO2018208864_A1) heavy chain variable region amino acid sequence ( SEQ ID NO 5) and human IgG1 Knob mutation amino acid sequence (LALA mutation introduced to reduce Fc function, SEQ ID NO 9). The C-terminus of the anti-CEA single-chain antibody amino acid sequence (SEQ ID NO 18) is connected to the anti-CD3 monoclonal antibody ADI26908 heavy chain variable region through a flexible peptide of 11 amino acid residues Linker-1 (SEQ ID NO 13) The N-terminus of the amino acid sequence (SEQ ID NO 5), and the C-terminus of the heavy chain variable region of the anti-CD3 monoclonal antibody ADI26908 was connected to the human IgG1 with Knob mutation amino acid sequence (LALA mutation was introduced to reduce Fc function, SEQ ID NO 9). Peptide chain #2 has the amino acid sequence shown in SEQ ID NO 20, which contains the anti-CEA single-chain antibody amino acid sequence (SEQ ID NO 18) and is flexible through the 11 amino acid residues Linker-1 (SEQ ID NO 13) The peptide is linked to the N-terminus of human IgG1 Fc with a Hole mutation amino acid sequence (LALA mutation introduced to reduce Fc function, SEQ ID NO 10). Peptide chain #3 has the amino acid sequence shown in SEQ ID NO 23, which contains the amino acid sequence of the light chain variable region of the anti-CD3 monoclonal antibody ADI26908 (SEQ ID NO 6), and is connected to the human kappa light chain constant at its C-terminal Region (CL) amino acid sequence (SEQ ID NO 17).

CEAxCD3 BiAb Fc-based 3: consists of 3 polypeptide chains. Its structural diagram is shown in Figure 1A. Peptide chain #1 has the amino acid sequence shown in SEQ ID NO 24, which contains the anti-CEA-based monoclonal antibody hM4.3 (Patent application number: hUM05-3 in WO2020244526A1) The amino acid sequence of the anti-CEA single chain antibody (SEQ ID NO 18), the anti-CD3 monoclonal antibody ADI26913 (Patent application number: WO2018208864_A1) heavy chain variable region amino acid sequence ( SEQ ID NO 7) and the human IgG1Knob mutant amino acid sequence (LALA mutation introduced to reduce Fc function, SEQ ID NO 9). The C-terminus of the anti-CEA single-chain antibody amino acid sequence (SEQ ID NO 18) was connected to the anti-CD3 monoclonal antibody ADI26913 heavy chain variable region through a flexible peptide of 11 amino acid residues Linker-1 (SEQ ID NO 13). The N-terminus of the amino acid sequence (SEQ ID NO 7), and the C-terminus of the heavy chain variable region of the anti-CD3 monoclonal antibody ADI26913 was connected to human IgG1 with Knob mutation amino acid sequence (LALA mutation introduced to reduce Fc function, SEQ ID NO 9). Peptide chain #2 has the amino acid sequence shown in SEQ ID NO 20, which contains the anti-CEA single-chain antibody amino acid sequence (SEQ ID NO 18) and is flexible through the 11 amino acid residues Linker-1 (SEQ ID NO 13) The peptide is linked to the N-terminus of the human IgG1 Fc with the Hole mutated amino acid sequence (LALA mutation introduced to reduce Fc function, SEQ ID NO 10). Peptide chain #3 has the amino acid sequence shown in SEQ ID NO 23, which contains the amino acid sequence of the light chain variable region of the anti-CD3 monoclonal antibody ADI26908 (SEQ ID NO 6), and is connected to the human kappa light chain constant at its C-terminal Region (CL) amino acid sequence (SEQ ID NO 17).

CEAxCD3 BiAb Fc-based 4: Composed of 3 polypeptide chains, its structural schematic is shown in Figure 1A. Peptide chain #1 has the amino acid sequence shown in SEQ ID NO 25, which contains the anti-CEA-based monoclonal antibody hM4.3 (Patent application number: hUM05-3 in WO2020244526A1) Anti-CEA single chain antibody amino acid sequence (SEQ ID NO 18) constructed, anti-CD3 monoclonal antibody ADI26915 (Patent application number: WO2018208864_A1) heavy chain variable region amino acid sequence ( SEQ ID NO 8) and human IgG1 Knob mutation amino acid sequence (LALA mutation introduced to reduce Fc function, SEQ ID NO 9). The C-terminus of the anti-CEA single-chain antibody amino acid sequence (SEQ ID NO 18) is connected to the anti-CD3 monoclonal antibody ADI26915 heavy chain variable region through a flexible peptide of 11 amino acid residues Linker-1 (SEQ ID NO 13) The N-terminus of the amino acid sequence (SEQ ID NO 8), and the C-terminus of the heavy chain variable region of the anti-CD3 monoclonal antibody ADI26915 was connected to the human IgG1 with Knob mutation amino acid sequence (LALA mutation was introduced to reduce Fc function, SEQ ID NO 9). Peptide chain #2 has the amino acid sequence shown in SEQ ID NO 20, which contains the anti-CEA single-chain antibody amino acid sequence (SEQ ID NO 18) and is flexible through the 11 amino acid residues Linker-1 (SEQ ID NO 13) The peptide is linked to the N-terminus of human IgG1 Fc with a Hole mutation amino acid sequence (LALA mutation introduced to reduce Fc function, SEQ ID NO 10). Peptide chain #3 has the amino acid sequence shown in SEQ ID NO 23, which contains the amino acid sequence of the light chain variable region of the anti-CD3 monoclonal antibody ADI26908 (SEQ ID NO 6), and is connected to the human kappa light chain constant at its C-terminal Region (CL) amino acid sequence (SEQ ID NO 17).

The sequence information of the above four Fc-based bispecific antibodies is summarized in the table below.

Table 2-1: Bispecific antibodies with Fc-based structure

CEAxCD3 BiAb M-body 1: Composed of 3 polypeptide chains, its structural diagram is shown in Figure 1B. Peptide chain #1 has the amino acid sequence shown in SEQ ID NO 26, which contains the anti-CEA monoclonal antibody hM4.3 ( Patent application number: hUM05-3 in WO2020244526A1) heavy chain variable region amino acid sequence (SEQ ID NO 1), anti-CD3 monoclonal antibody ADI22523 (patent application number: WO2018208864_A1) light chain variable region amino acid sequence (SEQ ID NO 4), human IgG1 CH3 Knob mutation amino acid sequence (SEQ ID NO 11) and the albumin binding region amino acid sequence (SEQ ID NO 37) of the anti-human albumin single domain antibody Sonelokimab (patent application number: US8188223). The anti-CEA monoclonal antibody heavy chain variable region amino acid sequence (SEQ ID NO 1) is connected to the human IgG1 heavy chain CH1 amino acid sequence (SEQ ID NO 16), and then through 15 amino acid residues Linker-2 (SEQ ID NO 14 ) is connected to the anti-CD3 monoclonal antibody ADI22523 light chain variable region amino acid sequence (SEQ ID NO 4), and then connected to human IgG1 CH3 Knob through the flexible peptide of 5 amino acid residues Linker-3 (SEQ ID NO 15) Mutate the amino acid sequence (SEQ ID NO 11), and finally connect the albumin binding region amino acid sequence of the anti-human albumin single domain antibody Sonelokimab (SEQ ID NO 37) through a flexible peptide of 11 amino acid residues Linker-1 (SEQ ID NO 13) ). Peptide chain #2 has the amino acid sequence shown in SEQ ID NO 27, which contains the heavy chain variable region amino acid sequence of the anti-CEA monoclonal antibody hM4.3 (SEQ ID NO 1) connected to the human IgG1 heavy chain CH1 amino acid sequence (SEQ ID NO 16), and then connected to the anti-CD3 monoclonal antibody ADI22523 heavy chain variable region amino acid sequence (SEQ ID NO 3) through a flexible peptide of 15 amino acid residues Linker-2 (SEQ ID NO 14), and finally through 5 A flexible peptide of amino acid residue Linker-3 (SEQ ID NO 15) is linked to the human IgG1 CH3 Hole mutant amino acid sequence (SEQ ID NO 12). Peptide chain #3 has the amino acid sequence shown in SEQ ID NO 28, which contains the light chain variable region amino acid sequence (SEQ ID NO 2) Connect the human kappa light chain constant region (CL) amino acid sequence (SEQ ID NO 17).

CEAxCD3 BiAb M-body 2: consists of 3 polypeptide chains. Its structural diagram is shown in Figure 1B. Peptide chain #1 has the amino acid sequence shown in SEQ ID NO 29, which contains the anti-CEA monoclonal antibody hM4.3 ( Patent application number: hUM05-3 in WO2020244526A1) heavy chain variable region amino acid sequence (SEQ ID NO 1), anti-CD3 monoclonal antibody ADI26908 (patent application number: WO2018208864_A1) light chain variable region amino acid sequence (SEQ ID NO 6), human IgG1 CH3 Knob mutation amino acid sequence (SEQ ID NO 11) and The albumin binding region amino acid sequence (SEQ ID NO 37) of the anti-human albumin single domain antibody Sonelokimab (patent application number: US8188223). The anti-CEA monoclonal antibody heavy chain variable region amino acid sequence (SEQ ID NO 1) was connected to the human IgG1 heavy chain CH1 amino acid sequence (SEQ ID NO 16), and then the 15 amino acid residues Linker-2 (SEQ ID NO 14 ), the anti-CD3 monoclonal antibody ADI26908 light chain variable region amino acid sequence (SEQ ID NO 6) was connected to the flexible peptide, and then the human IgG1 CH3 Knob was connected to the flexible peptide of Linker-3 (SEQ ID NO 15) of 5 amino acid residues. Mutate the amino acid sequence (SEQ ID NO 11), and finally connect the albumin binding region amino acid sequence of the anti-human albumin single domain antibody Sonelokimab (SEQ ID NO 37) through a flexible peptide of 11 amino acid residues Linker-1 (SEQ ID NO 13) ). Peptide chain #2 has the amino acid sequence shown in SEQ ID NO 30, which includes the heavy chain variable region amino acid sequence of the anti-CEA monoclonal antibody hM4.3 (SEQ ID NO 1) connected to the human IgG1 heavy chain CH1 amino acid sequence (SEQ ID NO 16), and then connect the anti-CD3 monoclonal antibody ADI26908 heavy chain variable region amino acid sequence (SEQ ID NO 5) through the flexible peptide of 15 amino acid residues Linker-2 (SEQ ID NO 14), and finally through 5 A flexible peptide of amino acid residues Linker-3 (SEQ ID NO 15) is linked to the human IgG1 CH3 Hole mutant amino acid sequence (SEQ ID NO 12). Peptide chain #3 has the amino acid sequence shown in SEQ ID NO 28, which contains the light chain variable region amino acid sequence (SEQ ID NO. 2) Connect the human kappa light chain constant region (CL) amino acid sequence (SEQ ID NO 17).

CEAxCD3 BiAb M-body 3: consists of 3 polypeptide chains. Its structural diagram is shown in Figure 1B. Peptide chain #1 has the amino acid sequence shown in SEQ ID NO 29, which contains the anti-CEA monoclonal antibody hM4.3 ( Patent application number: hUM05-3 in WO2020244526A1) heavy chain variable region amino acid sequence (SEQ ID NO 1), anti-CD3 monoclonal antibody ADI26908 (patent application number: WO2018208864_A1) light chain variable region amino acid sequence (SEQ ID NO 6), human IgG1 CH3 Knob mutation amino acid sequence (SEQ ID NO 11) and the albumin binding region amino acid sequence (SEQ ID NO 37) of the anti-human albumin single domain antibody Sonelokimab (Patent application number: US8188223). The anti-CEA monoclonal antibody heavy chain variable region amino acid sequence (SEQ ID NO 1) was connected to the human IgG1 heavy chain CH1 amino acid sequence (SEQ ID NO 16), and then the 15 amino acid residues Linker-2 (SEQ ID NO 14 ), the anti-CD3 monoclonal antibody ADI26908 light chain variable region amino acid sequence (SEQ ID NO 6) was connected to the flexible peptide, and then the human IgG1 CH3 Knob was connected to the flexible peptide of Linker-3 (SEQ ID NO 15) of 5 amino acid residues. Mutate the amino acid sequence (SEQ ID NO 11), and finally connect the albumin binding region amino acid sequence of the anti-human albumin single domain antibody Sonelokimab (SEQ ID NO 37) through a flexible peptide of 11 amino acid residues Linker-1 (SEQ ID NO 13) ). Peptide chain #2 has the amino acid sequence shown in SEQ ID NO. 31, which contains the amino acid sequence of the heavy chain variable region of the anti-CEA monoclonal antibody hM4.3 (SEQ ID NO. 1) Connect the human IgG1 heavy chain CH1 amino acid sequence (SEQ ID NO 16), and then connect the anti-CD3 monoclonal antibody ADI26913 heavy chain variable region amino acids through the flexible peptide of 15 amino acid residues Linker-2 (SEQ ID NO 14) sequence (SEQ ID NO 7), and finally the human IgG1 CH3 Hole mutant amino acid sequence (SEQ ID NO 12) is connected through a flexible peptide of 5 amino acid residues Linker-3 (SEQ ID NO 15). Peptide chain #3 has the amino acid sequence shown in SEQ ID NO 28, which contains the light chain variable region amino acid sequence (SEQ ID NO. 2) Connect the human kappa light chain constant region (CL) amino acid sequence (SEQ ID NO 17).

CEAxCD3 BiAb M-body 4: consists of 3 polypeptide chains. Its structural diagram is shown in Figure 1B. Peptide chain #1 has the amino acid sequence shown in SEQ ID NO 29, which contains the anti-CEA monoclonal antibody hM4.3 ( Patent application number: hUM05-3 in WO2020244526A1) heavy chain variable region amino acid sequence (SEQ ID NO 1), anti-CD3 monoclonal antibody ADI26908 (patent application number: WO2018208864_A1) light chain variable region amino acid sequence (SEQ ID NO 6), human IgG1 CH3 Knob mutation amino acid sequence (SEQ ID NO 11) and the albumin binding region amino acid sequence (SEQ ID NO 37) of the anti-human albumin single domain antibody Sonelokimab (patent application number: US8188223). The anti-CEA monoclonal antibody heavy chain variable region amino acid sequence (SEQ ID NO 1) is connected to the human IgG1 heavy chain CH1 amino acid sequence (SEQ ID NO 16), and then through 15 amino acid residues Linker-2 (SEQ ID NO 14 ) is connected to the anti-CD3 monoclonal antibody ADI26908 light chain variable region amino acid sequence (SEQ ID NO 6), and then connected to human IgG1 CH3 Knob through the flexible peptide of 5 amino acid residues Linker-3 (SEQ ID NO 15) Mutate the amino acid sequence (SEQ ID NO 11), and finally connect the albumin binding region amino acid sequence of the anti-human albumin single domain antibody Sonelokimab (SEQ ID NO 37) through a flexible peptide of 11 amino acid residues Linker-1 (SEQ ID NO 13) ). Peptide chain #2 has the amino acid sequence shown in SEQ ID NO 32, which contains the heavy chain variable region amino acid sequence of the anti-CEA monoclonal antibody hM4.3 (SEQ ID NO 1) connected to the human IgG1 heavy chain CH1 amino acid sequence (SEQ ID NO 16), and then connected to the anti-CD3 monoclonal antibody ADI26915 heavy chain variable region amino acid sequence (SEQ ID NO 8) through a flexible peptide of 15 amino acid residues Linker-2 (SEQ ID NO 14), and finally through 5 A flexible peptide of amino acid residue Linker-3 (SEQ ID NO 15) is linked to the human IgG1 CH3 Hole mutant amino acid sequence (SEQ ID NO 12). Peptide chain #3 has the amino acid sequence shown in SEQ ID NO 28, which contains the light chain variable region amino acid sequence (SEQ ID NO 2) Connect the human kappa light chain constant region (CL) amino acid sequence (SEQ ID NO 17).

The sequence information of the above four bispecific antibodies with M-body structures is summarized in the table below.

Table 2-2: Bispecific antibodies with M-body structure

2. In this example, the positive control molecule Cibisatamb was constructed:

CEAxCD3 BiAb control molecule Cibisatamb: The control molecule Cibisatamb (patent application number: WO2011023787) consists of 4 polypeptide chains. Peptide chain #1 has the amino acid sequence shown in SEQ ID NO:33, and peptide chain #2 has the amino acid sequence shown in SEQ ID NO:34. The amino acid sequence shown is, peptide chain #3 has the amino acid sequence shown in SEQ ID NO: 35, and peptide chain #4 has the amino acid sequence shown in SEQ ID NO: 36.

3. Expression and purification

The ExpiCHO ^TM expression system kit (purchased from Thermo) was used to transfer the coding expression plasmid into Expi-CHO cells. The transfection method was in accordance with the product instructions. After 5 days of cell culture, the supernatant was collected using protein A magnetic beads (purchased from Gold Sri) sorting method to purify the target protein. Resuspend the magnetic beads in an appropriate volume of binding buffer (PBS+0.1% Tween 20, pH 7.4) (1-4 times the volume of the magnetic beads), add it to the sample to be purified, and incubate at room temperature for 1 hour, shaking gently during the period. The sample was placed on a magnetic stand (purchased from Beaver), the supernatant was discarded, and the magnetic beads were washed three times with binding buffer. Add elution buffer (0.1M sodium citrate, pH 3.2) 3-5 times the volume of the magnetic beads, shake at room temperature for 5-10 minutes, place it back on the magnetic stand, collect the elution buffer, and transfer it to the neutralization buffer added (1M Tris, pH 8.54) in a collection tube and mix well. CEACAM5/6xCD3 TCEs and control antibody hM4.3, Cibisatamb were thus obtained.

Example 2. Bispecific antibodies bind to CEACAM5/6

The expanded cultured human rectal tumor cells LS174T (purchased from ATCC, CL-188) and LoVo (purchased from Addexbio, C0009011) (self-expressing CEACAM5/6) was digested with 0.25% EDTA trypsin, washed once with culture medium and then adjusted to a cell density of 2×10 ⁶ cells/ml. Add 100 μl/well to a 96-well flow plate, centrifuge and set aside. Add 100 μl/well of the bispecific antibody after serial dilution to the above-mentioned 96-well flow cytometry plate with cells, incubate at 4°C for 60 min, and wash twice with PBS. Add 100 μl/well of Goat anti-human IgG-Fc (PE) (Abcam, ab98596) diluted 1000 times with 2% BSA solution, incubate at 4°C for 60 min, and wash twice with PBS. Finally, 100 μl/well of PBS was added to resuspend the cells, and the cells were detected on a CytoFlex (Beckman) flow cytometer and the corresponding mean fluorescence intensity (MFI) was calculated.

In the measurement experiment of the above method, the experimental results are shown in Figure 2. The bispecific antibody of the present invention is consistent with CEACAM5/6 expressed by human rectal tumor cells LS174T (Figure 2A, 2B) and LoVo (Figure 2C, 2D). combine. Cell binding of the M-body construct was comparable to the control antibody.

Example 3. Bispecific antibodies bind to CD3

Adjust the cell density of the expanded cultured Jurkat cells to 2×10 ⁶ cells/ml, add 100 μl/well to a 96-well flow plate, and centrifuge for later use. Add 100 μl/well of the bispecific antibody after serial dilution to the above-mentioned 96-well flow cytometry plate with cells, incubate at 4°C for 60 min, and then wash twice with PBS. Add 100 μl/well of Goat anti-human IgG-Fc (PE) (Abcam, ab98596) diluted 1000 times with 2% BSA solution, incubate at 4°C for 60 min, and then wash twice with PBS. Finally, 100 μl/well of PBS was added to resuspend the cells, and the cells were detected on a CytoFlex (Beckman) flow cytometer and the corresponding MFI was calculated.

In the measurement experiment using the above method, the experimental results are shown in Figure 3. As shown in Figure 3A, the bispecific antibody of the Fc-based structure of the present invention has binding activity to Jurkat cells. Fc-based molecule No. 1 showed the strongest cell binding, followed by structures 2 to 4, among which Cell binding of construct 3 was similar to that of the control antibody. As shown in Figure 3B, the binding of M-body bispecific antibody to Jurkat cells was weak and lower than that of the control antibody.

Example 4. Bispecific antibodies activate the NFAT signaling pathway in Jurkat cells

Target cells LS174T were transfected with effector cells NFAT Luciferase/Jurkat (Jurkat (ATCC, TIB-152 ^TM ) cells at 3×10 ⁴ /well and 1.2×10 ⁵ /well) expressing luc2P/NFAT-R-hygro vector. (Promega, E8481), that is, the cells are obtained) or only effector cells were inoculated into a 96-well cell culture white bottom plate, and serially diluted bispecific antibodies were added and incubated for 6 hours. Use the Bio-Glo luciferase assay system (Promega, G7940) kit to develop the color and collect the chemiluminescence signal with a microplate reader. Control antibodies included Cibisatamb, as well as the CD3 antibody OKT3 (purchased from Biolegend (317302)), CD3 mAb-3 (combined with anti- Same as the CD3 monoclonal antibody ADI26913 (patent application number: WO2018208864_A1).

The results are shown in Figure 4. As shown in Figures 4A and 4C, the CEA×CD3 bispecific antibody can activate the NFAT signaling pathway in Jurkat cells under the influence of target cells LS174T. In the absence of target cells, as shown in Figure 4B and 4D, a certain degree of non-specific activation was observed for the Fc-based bispecific antibody and the control molecule, while the M-body structure under the same high concentration conditions No non-specific activation was detected, demonstrating improved safety.

Example 5. Bispecific antibodies induce Primary T cells to kill tumor cells

The target cells LS174T were stained using the CellTrace ^TM Violet kit and seeded into a 96-well transparent bottom black-edged cell culture plate at 1×10 ⁴ cells/well. Collect the PBMC that were revived one day in advance, use a T cell isolation kit (Stemcell, 17951) to isolate the T cells in the PBMC and add 5 × 10 ⁴ cells/well into the target cell wells, and then gradually dilute the bispecific Antibodies were added to the cell wells and incubated for 48 hours. After 48 hours, use Cytation 5 to collect the DAPI fluorescence signal and calculate the corresponding killing intensity.

The results are shown in Figure 5. Both Fc-based and M-body CEAxCD3 double antibodies can induce primary T cells to kill CEACAM5/CEACAM6-positive cells LS174T in a dose-dependent manner. However, CD3 monoclonal antibody molecules and CEA monoclonal antibody molecules as negative controls did not induce killing. The positive control molecule Cibisatamb induced primary T cells to kill LS174T cells, and its activity was comparable to M-body3 or Fc-based 4.

Example 6. Bispecific antibodies induce cytokine release from PBMCs

The target cells LS174T were stained using the CellTrace ^TM Violet kit and seeded into a 96-well transparent bottom black-edged cell culture plate at 1×10 ⁴ cells/well. Collect the PBMCs that were revived one day in advance and add them to the target cell wells at a rate of 1×10 ⁵ per well. Then, add the bispecific antibody after serial dilution to the cell wells and incubate for 48 hours, then collect the supernatant. The levels of IL-2 and IFNγ in the supernatant were detected using human IL-2 ELISA kit (Invitrogen, 88-7025-77) and human IFNγ ELISA kit (Invitrogen, 88-7316-77) and the procedures recommended by the supplier were followed.

The results are shown in Figure 6. Both Fc-based and M-body CEAxCD3 dual antibodies can induce the release of IL2 (Figure 6A) or IFN-γ (Figure 6B) from PBMC in the presence of LS174T in a dose-dependent manner. The activity of the positive control molecule Cibisatamb is comparable to M-body3 or Fc-based 4.

Example 7. The activation of T cells induced by bispecific antibodies is related to the expression level of the antigen

The target cells LS174T or LS174T-low (flow sorting) were stained using the CellTrace ^TM Violet kit, and seeded into a 96-well transparent bottom black-edged cell culture plate at 1×10 ⁴ cells/well. Collect the PBMC that were revived one day in advance, use a T cell isolation kit (Stemcell, 17951) to isolate the T cells in the PBMC and add 5 × 10 ⁴ cells/well into the target cell wells, and then gradually dilute the bispecific Antibodies were added to the cell wells and incubated for 48 hours. After 48 hours, use Cytation 5 to collect the DAPI fluorescence signal and calculate the corresponding killing intensity. Additionally, the cell supernatant was collected after collecting the DAPI fluorescence signal using Cytation 5. The levels of IL-2 and IFNγ in the supernatant were detected using human IL-2 ELISA kit (Invitrogen, 88-7025-77) and human IFNγ ELISA kit (Invitrogen, 88-7316-77) and the procedures recommended by the supplier were followed.

The results are shown in Figure 7. The expression of CEACAM5/6 in target cells LS174T-low was much lower than that of LS174T (Figure 7A). Both the M-body 3 double antibody and the positive control molecule Cibisatamb can induce the killing of LS174T cells by Primary T cells, and can induce stronger cell killing in the presence of LS174T cells (Figure 7B). In addition, compared with LS174T cells, the co-incubation conditions of LS174T-low cells induced Primary T cells to release less IL-2 (Figure 7C) or IFN-γ (Figure 7D).

Example 8. Study on the tumor inhibitory activity of bispecific antibodies

In this experiment, M-NSG mice were subcutaneously inoculated with LS174T colorectal cancer tumor cells to establish a tumor-bearing model and determine the anti-tumor effect of the anti-CEAxCD3 bispecific antibody. Expand enough LS174T cells in vitro, collect the cells after trypsin digestion, wash them three times with PBS and count them. According to the number of LS174T tumor cells and 1×10E6 PBMC per mouse, inoculate them into females for 8 weeks. M-NSG mice (purchased from Shanghai Southern Model) were subcutaneously injected into the right abdomen. Observe the tumor formation of tumor cells under the skin of mice every day. Use vernier calipers to measure the maximum width axis W and maximum long axis L of the subcutaneous tumor on the right abdomen of each animal. Use an electronic balance to weigh the weight of each mouse. Calculate the subcutaneous tumor volume in the right abdomen of each mouse according to the tumor volume T=1/2×W×W×L. Mice with tumors that were too large and those that were too small were eliminated, and the mice were evenly divided into 3 groups according to the average tumor volume, with 6 mice in each group. Group the patients according to the group dosing schedule in Table 6 and inject corresponding doses of antibodies.

Table 3: Experimental protocol for tumor inhibitory activity of bispecific antibodies

The mouse tumor volume and mouse body weight were measured every 2 days. The results are shown in Figure 8. Compared with the PBS group, Yang The sex control Cibisatamb 2.5mg/kg can inhibit tumor growth to a certain extent. The bispecific antibody M-body 3 2mg/kg shows a significant inhibitory effect on tumor growth, and the anti-tumor effect is better than Cibisatamab 2.5mg/kg.

Example 9. Study on the tumor inhibitory activity of bispecific antibodies combined with immune checkpoint inhibitors

In this experiment, B-NDG B2M KO plus mice were subcutaneously inoculated with LS174T colorectal cancer tumor cells to establish a tumor-bearing model and determine the anti-tumor effect of anti-CEAxCD3 bispecific antibodies. Sufficient LS174T cells were cultured and expanded in vitro, collected after trypsin digestion, washed three times with PBS and counted. The number of LS174T tumor cells and 1.2×10E6 PBMC per mouse was inoculated into females for 8 weeks. B-NDG B2M KO plus mice (purchased from Biocytogen) were harvested subcutaneously on the right side of the abdomen. Observe the tumor formation of tumor cells under the skin of mice every day. Use vernier calipers to measure the maximum width axis W and maximum long axis L of the subcutaneous tumor on the right abdomen of each animal. Use an electronic balance to weigh the weight of each mouse. Calculate the subcutaneous tumor volume in the right abdomen of each mouse according to the tumor volume T=1/2×W×W×L. Mice with tumors that were too large and those that were too small were eliminated, and the mice were evenly divided into 4 groups according to the average tumor volume, with 6 mice in each group. Group the patients according to the group dosing schedule in Table 6 and inject corresponding doses of antibodies.

Table 4: Experimental protocol for tumor inhibitory activity of bispecific antibodies

The mouse tumor volume and mouse body weight were measured every 2 days. The results are shown in Figure 9. Compared with the PBS group, Anti-PD1mAb (in-house), which has the VH shown in SEQ ID NO: 57 and the VL shown in SEQ ID NO: 58) 3mg/kg can Inhibiting tumor growth to a certain extent, the bispecific antibody M-body 3 2mg/kg showed a significant inhibitory effect on tumor growth; while the Anti-PD1mAb+M-body 3 combination group had a better anti-tumor effect than the two single drugs, showing showed significant synergistic anti-tumor effect.

Example 10. PK of bispecific antibodies in rhesus monkeys

One healthy adult rhesus monkey (Jiangsu Dingtai Pharmaceutical Research (Group) Co., Ltd.) was selected and given a single intravenous infusion of 2.5 mg/kg M-body 3 injection. Samples were collected before administration (0h) and after administration. 2min, 1h, 3h, 8h, 24h, 48h, 72h, 120h, 168h after the end of the whole blood and serum separation, use ELISA method to detect the blood drug concentration and calculate the pharmacokinetic parameters to evaluate M-body 3 Injection pharmacokinetic characteristics; the ELISA method includes coating CEACAM5 protein (purchased from SinoBiological), using M-body 3 antibody A linear standard curve was established. Peroxidase AffiniPure Rabbit Anti-Human IgG and Fcγfragment specific (purchased from Jackson Immuno) were used as secondary antibodies for detection. Finally, TMB chromogenic solution (purchased from SOLARBIO) was used to detect the HRP signal. The results are shown in Figure 10. The half-life of M-body 3 in rhesus monkeys is approximately 116 hours.

Although the specific embodiments of the present invention have been described in detail, those skilled in the art will understand that various modifications and changes can be made to the details based on all teachings that have been published, and these changes are within the protection scope of the present invention. . The full scope of the present invention is given by the appended claims and any equivalents thereof.

Claims

A multispecific antibody comprising a first antigen-binding domain targeting carcinoembryonic antigen-related cell adhesion molecule (CEACAM) and a second antigen-binding domain targeting CD3, wherein the first antigen-binding domain comprises a heavy Chain variable region (VH) and light chain variable region (VL);

The VH includes CDR-H1 shown in SEQ ID NO:38, CDR-H2 shown in SEQ ID NO:39 and CDR-H3 shown in SEQ ID NO:40;

The VL includes CDR-L1 shown in SEQ ID NO:41, CDR-L2 shown in SEQ ID NO:42, and CDR-L3 shown in SEQ ID NO:43.
The multispecific antibody of claim 1, wherein the first antigen binding domain includes a VH as shown in SEQ ID NO: 1 or a variant thereof and a VH as shown in SEQ ID NO: 2 or a variant thereof VL; the variant has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96% , a sequence that has at least 97%, at least 98%, at least 99%, or 100% sequence identity, or has one or several amino acid substitutions, deletions, or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions); preferably, the substitutions are conservative substitutions.
The multispecific antibody of claim 1 or 2, wherein the second antigen-binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein:

(1) The VH includes CDR-H1 shown in SEQ ID NO:44, CDR-H2 shown in SEQ ID NO:53 and CDR-H3 shown in SEQ ID NO:54; the VL includes SEQ ID NO : CDR-L1 shown in SEQ ID NO: 52, CDR-L2 shown in SEQ ID NO: 48, CDR-L3 shown in SEQ ID NO: 49;

(2) The VH includes CDR-H1 shown in SEQ ID NO:44, CDR-H2 shown in SEQ ID NO:45 and CDR-H3 shown in SEQ ID NO:46; the VL includes SEQ ID NO :CDR-L1 shown in SEQ ID NO:47, CDR-L2 shown in SEQ ID NO:48, CDR-L3 shown in SEQ ID NO:49;

(3) The VH includes CDR-H1 shown in SEQ ID NO:44, CDR-H2 shown in SEQ ID NO:50 and CDR-H3 shown in SEQ ID NO:51; the VL includes SEQ ID NO : CDR-L1 shown in SEQ ID NO: 52, CDR-L2 shown in SEQ ID NO: 48, CDR-L3 shown in SEQ ID NO: 49; or,

(4) The VH includes CDR-H1 shown in SEQ ID NO:44, CDR-H2 shown in SEQ ID NO:50 and CDR-H3 shown in SEQ ID NO:55; the VL includes SEQ ID NO :CDR-L1 shown in 52, CDR-L2 shown in SEQ ID NO:48, CDR-L3 shown in SEQ ID NO:49.
The multispecific antibody of claim 3, wherein the second antigen binding domain comprises:

(1) VH as shown in SEQ ID NO:7 or its variants and VL as shown in SEQ ID NO:6 or its variants;

(2) VH as shown in SEQ ID NO:3 or its variants and VL as shown in SEQ ID NO:4 or its variants;

(3) VH as set forth in SEQ ID NO:5 or a variant thereof and VL as set forth in SEQ ID NO:6 or a variant thereof; or,

(4) VH as shown in SEQ ID NO:8 or its variants and VL as shown in SEQ ID NO:6 or its variants;

wherein said variant has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96% , a sequence that has at least 97%, at least 98%, at least 99%, or 100% sequence identity, or has one or several amino acid substitutions, deletions, or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions); preferably, the substitutions are conservative substitutions.
The multispecific antibody of any one of claims 1-4, wherein the multispecific antibody comprises:

(i) a first peptide chain comprising the VH of the first antigen-binding domain, the heavy chain CH1 domain, the VL of the second antigen-binding domain, and the heavy chain CH3 domain;

(ii) a second peptide chain comprising the VH of the first antigen-binding domain, the heavy chain CH1 domain, the VH of the second antigen-binding domain, and the heavy chain CH3 domain;

and

(iii) a third peptide chain comprising the VL and light chain constant region (CL) of the first antigen-binding domain; preferably, the CL is a kappa light chain constant region;

Preferably, the first antigen-binding domain is Fab, and the second antigen-binding domain is Fv;

Preferably, the heavy chain CH1 domain of the first peptide chain can form a dimer with the CL of the third peptide chain; preferably, the heavy chain CH1 domain of the second peptide chain can form a dimer with the CL of the third peptide chain. The CL of the tripeptide chain forms a dimer; preferably, the heavy chain CH3 domain of the first peptide chain forms a dimer with the heavy chain CH3 domain of the second peptide chain;

Preferably, the multispecific antibody comprises one of the first peptide chain, one of the second peptide chain and two of the third peptide chain.
The multispecific antibody of claim 5, wherein the heavy chain CH1 junction of the first peptide chain and the second peptide chain The structural domain and the heavy chain CH3 domain are of the same isotype;

Preferably, the heavy chain CH1 domain and the heavy chain CH3 domain of the first peptide chain and the second peptide chain are IgG, such as IgG1, IgG2, IgG3 or IgG4.
The multispecific antibody of claim 5 or 6, wherein the heavy chain CH3 domains of the first peptide chain and the second peptide chain comprise modifications to promote dimerization of the two;

Preferably, the modification is an amino acid substitution;

Preferably, the modification comprises a "node" modification in one of the two heavy chain CH3 domains of the first peptide chain and the second peptide chain and a "hole" modification in the other of the two, so that Form "knob-into-hole" modification;

Preferably, the CH3 structural domain containing the "node" modification includes the amino acid sequence shown in SEQ ID NO:11; Preferably, the CH3 structural domain including the "hole" modification includes the amino acid sequence shown in SEQ ID NO:12 amino acid sequence.
The multispecific antibody of any one of claims 5-7, wherein the first peptide chain and/or the second peptide chain further comprises an albumin-binding polypeptide at the C-terminus of its heavy chain CH3 domain;

Preferably, the albumin-binding polypeptide is an antibody or antibody fragment capable of specifically binding albumin;

Preferably, the albumin-binding polypeptide is a single domain antibody capable of specifically binding albumin;

Preferably, the albumin-binding polypeptide comprises the sequence shown in SEQ ID NO:37.
The multispecific antibody of any one of claims 5-8, wherein each adjacent domain in the first, second and third peptide chains is optionally connected by a peptide linker;

Preferably, the peptide linker is a peptide linker comprising one or more glycine (G) and/or one or more serine (S) (for example, a flexible peptide comprising (G4S)n, for example, as shown in SEQ ID NO: 13 sequence), or contains the sequence shown in SEQ ID NO: 14 or 15.
The multispecific antibody of any one of claims 5-9, wherein the multispecific antibody comprises:

(1) A first peptide chain including the sequence shown in SEQ ID NO:29, a second peptide chain including the sequence shown in SEQ ID NO:31 and a third peptide chain including the sequence shown in SEQ ID NO:28;

(2) a first peptide chain comprising the sequence shown in SEQ ID NO: 26, a second peptide chain comprising the sequence shown in SEQ ID NO: 27 and a third peptide chain comprising the sequence shown in SEQ ID NO: 28;

(3) A first peptide chain including the sequence shown in SEQ ID NO:29, a second peptide chain including the sequence shown in SEQ ID NO:30, and a third peptide chain including the sequence shown in SEQ ID NO:28; or,

(4) A first peptide chain including the sequence shown in SEQ ID NO:29, a second peptide chain including the sequence shown in SEQ ID NO:32, and a third peptide chain including the sequence shown in SEQ ID NO:28.
The multispecific antibody of any one of claims 1-4, wherein the multispecific antibody comprises:

(i) a first peptide chain comprising the first antigen binding domain, the VH of the second antigen binding domain and the heavy chain constant region (CH);

(ii) a second peptide chain comprising the first antigen-binding domain and an Fc domain monomer capable of monomerizing with the Fc domain of the heavy chain constant region of the first peptide chain; The bodies form dimers;

(iii) a third peptide chain comprising the VL of the second antigen-binding domain and a light chain constant region (CL); preferably, the CL is a kappa light chain constant region;

Preferably, the first antigen-binding domain is scFv, and the second antigen-binding domain is Fab;

Preferably, the CH1 domain of the heavy chain constant region of the first peptide chain and the CL of the third peptide chain are capable of forming a dimer;

Preferably, the multispecific antibody comprises a first peptide chain, a second peptide chain and a third peptide chain.
The multispecific antibody of claim 11, wherein the heavy chain constant region of the first peptide chain and the Fc domain monomer of the second peptide chain are of the same isotype;

Preferably, the isotype is IgG, such as IgGl, IgG2, IgG3 or IgG4.
The multispecific antibody of claim 11 or 12, wherein the heavy chain constant region Fc domain monomer of the first peptide chain and the Fc domain monomer of the second peptide chain comprise modifications to promote duplication of both. polymerization;

Preferably, the modification is an amino acid substitution;

Preferably, the modifications comprise a "node" modification in one of the Fc domain monomers of the first peptide chain and the Fc domain monomer of the second peptide chain and a "hole" modification in the other of the two. , to form a "knob-into-hole" modification;

Preferably, the Fc domain monomer comprising the "node" modification comprises the amino acid sequence shown in SEQ ID NO:56; Preferably, the Fc domain monomer comprising the "hole" modification comprises the amino acid sequence shown in SEQ ID NO :The amino acid sequence shown in 10;

Preferably, the heavy chain constant region of the first peptide chain includes the amino acid sequence shown in SEQ ID NO:9, and/or the Fc domain monomer of the second peptide chain includes the amino acid sequence shown in SEQ ID NO:10. The amino acid sequence shown.
The multispecific antibody of any one of claims 11-13, wherein the Fc domain monomer of the first peptide chain and/or the Fc domain monomer of the second peptide chain comprises a mutation or chemical modification to change Their effector functions (e.g., reduced or enhanced antibody-dependent cellular cytotoxicity (ADCC), reduced or enhanced antibody-dependent cellular phagocytosis (ADCP), and/or reduced or enhanced complement-dependent cytotoxicity (CDC)) ;

Preferably, the Fc domain monomer of the first peptide chain and/or the Fc domain monomer of the second peptide chain comprises a LALA mutation (L234A, L235A).
The multispecific antibody according to any one of claims 11 to 14, wherein the first antigen-binding domain is scFv and has a structure represented by VH-L-VL or VL-L-VH, wherein L is a peptide linker;

Preferably, the L is a peptide linker comprising one or more glycine (G) and/or one or more serine (S), such as a flexible peptide comprising (G4S)n;

Preferably, the first antigen-binding domain has a structure represented by VH-L-VL.
The multispecific antibody of any one of claims 11-15, wherein each adjacent domain in the first, second and third peptide chains is optionally connected by a peptide linker;

Preferably, the peptide linker is a peptide linker comprising one or more glycine (G) and/or one or more serine (S) (for example, a flexible peptide comprising (G4S)n, for example, as shown in SEQ ID NO: 13 sequence), or contains the sequence shown in SEQ ID NO: 14 or 15.
The multispecific antibody of any one of claims 11-16, wherein the multispecific antibody comprises:

(1) A first peptide chain including the sequence shown in SEQ ID NO: 19, a second peptide chain including the sequence shown in SEQ ID NO: 20 and a third peptide chain including the sequence shown in SEQ ID NO: 21;

(2) A first peptide chain including the sequence shown in SEQ ID NO:22, a second peptide chain including the sequence shown in SEQ ID NO:20 and a third peptide chain including the sequence shown in SEQ ID NO:23;

(3) A first peptide chain including the sequence shown in SEQ ID NO:24, a second peptide chain including the sequence shown in SEQ ID NO:20, and a third peptide chain including the sequence shown in SEQ ID NO:23; or,

(4) The first peptide chain including the sequence shown in SEQ ID NO:25, and the first peptide chain including the sequence shown in SEQ ID NO:20 dipeptide chain and a third peptide chain comprising the sequence shown in SEQ ID NO:23.
An isolated nucleic acid molecule comprising a nucleotide sequence encoding the multispecific antibody of any one of claims 1-17 or at least one peptide chain thereof.
A vector comprising the isolated nucleic acid molecule of claim 18;

Preferably, the vector comprises a nucleotide sequence encoding each peptide chain of the multispecific antibody of any one of claims 1-17, and the nucleotide sequence encoding each peptide chain is present in the same or different on the carrier;

Preferably, the vector contains a nucleotide sequence encoding each peptide chain of the multispecific antibody of any one of claims 5-10, and the nucleotide sequence encoding each peptide chain is present in the same or on different carriers;

Preferably, the vector contains a nucleotide sequence encoding each peptide chain of the multispecific antibody of any one of claims 11-17, and the nucleotide sequence encoding each peptide chain is present in the same or on a different carrier.
A host cell comprising the isolated nucleic acid molecule of claim 18 or the vector of claim 19.
A method for preparing the multispecific antibody of any one of claims 1-17, comprising culturing the host cell of claim 20 under conditions that allow protein expression, and recovering from the cultured host cell culture The multispecific antibody.
A composition comprising the multispecific antibody of any one of claims 1-17 and an immune checkpoint inhibitor;

Preferably, the immune checkpoint is selected from PD-1, PD-L1, CTLA-4, TIM-3, Lag-3, TIGIT, CD73, VISTA, B7-H3, or any combination thereof;

Preferably, the immune checkpoint inhibitor is a PD-1 or PD-L1 antibody or antibody fragment.
A pharmaceutical composition comprising the multispecific antibody of any one of claims 1-17, the isolated nucleic acid molecule of claim 18, the vector of claim 19, the host cell of claim 20, or The composition of claim 22, and pharmaceutically acceptable carriers and/or excipients.
The multispecific antibody of any one of claims 1-17, the isolated nucleic acid molecule of claim 18, the vector of claim 19, the host cell of claim 20, the nucleic acid molecule of claim 22 composition, or The use of the pharmaceutical composition according to claim 23 in the preparation of medicaments for preventing and/or treating tumors;

Preferably, the tumor is carcinoembryonic antigen-related cell adhesion molecule (CEACAM) positive;

Preferably, the tumor is CEACAM5 and/or CEACAM6 positive;

Preferably, the tumor is selected from the group consisting of non-small cell lung cancer, small cell lung cancer, renal cell carcinoma, colorectal cancer, ovarian cancer, breast cancer, pancreatic cancer, gastric cancer, bladder cancer, esophageal cancer, mesothelioma, melanoma, head Neck cancer, thyroid cancer, sarcoma, prostate cancer, glioblastoma, cervical cancer, thymus cancer, leukemia, lymphoma, myeloma, mycoses fungoids, Merkel cell carcinoma and other malignancies Hematologic disorders, such as classic Hodgkin lymphoma (CHL), primary mediastinal large B-cell lymphoma, T-cell/histiocytic B-cell-rich lymphoma, EBV-positive and -negative PTLD, and EBV-related diffuse large B-cell lymphoma cell lymphoma (DLBCL), plasmablastic lymphoma, extranodal NK/T-cell lymphoma, nasopharyngeal carcinoma and HHV8-related primary effusion lymphoma, Hodgkin lymphoma, central nervous system (CNS) Tumors, such as primary CNS lymphoma, spinal tumors, and brainstem glioma;

Preferably, the tumor is a solid tumor;

Preferably, the tumor is selected from the group consisting of colon cancer, pancreatic cancer, gastric cancer, non-small cell lung cancer, breast cancer, head and neck squamous cell carcinoma, endometrial cancer, and bladder cancer.
A method for preventing and/or treating tumors, comprising administering to a subject in need the multispecific antibody of any one of claims 1-17, the isolated nucleic acid molecule of claim 18, The vector of claim 19, the host cell of claim 20, the composition of claim 22, or the pharmaceutical composition of claim 23;

Preferably, the tumor is carcinoembryonic antigen-related cell adhesion molecule (CEACAM) positive;

Preferably, the tumor is CEACAM5 and/or CEACAM6 positive;

Preferably, the tumor is selected from the group consisting of non-small cell lung cancer, small cell lung cancer, renal cell carcinoma, colorectal cancer, ovarian cancer, breast cancer, pancreatic cancer, gastric cancer, bladder cancer, esophageal cancer, mesothelioma, melanoma, head Neck cancer, thyroid cancer, sarcoma, prostate cancer, glioblastoma, cervical cancer, thymus cancer, leukemia, lymphoma, myeloma, mycoses fungoids, Merkel cell carcinoma and other malignancies Hematologic disorders, such as classic Hodgkin lymphoma (CHL), primary mediastinal large B-cell lymphoma, T-cell/histiocytic B-cell-rich lymphoma, EBV-positive and -negative PTLD, and EBV-related diffuse large B-cell lymphoma cell lymphoma (DLBCL), plasmablastic lymphoma, extranodal NK/T-cell lymphoma, nasopharyngeal carcinoma and HHV8-related primary effusion lymphoma, Hodgkin lymphoma, central nervous system (CNS) Tumors, such as primary CNS lymphoma, spinal tumors, and brainstem glioma;

Preferably, the tumor is a solid tumor;

Preferably, the tumor is selected from colon cancer, pancreatic cancer, gastric cancer, non-small cell lung cancer, breast cancer, head and neck squamous cell carcinoma, endometrial cancer, and bladder cancer;

Preferably, the subject is a mammal, such as a human.