Anti-CEACAM5 / 6 dual-binding antibody

Abstract

Antibodies that bind to both CEACAM5 and CEACAM6, and their antigen-binding fragments, as well as their uses, nucleic acids encoding the antibodies and antigen-binding fragments, vectors containing nucleic acids, and host cells containing nucleic acids or vectors are disclosed herein. Also disclosed are pharmaceutical compositions and conjugates containing antibodies, and methods of treatment by administering antibodies.

Description

[Technical Field]

[0001] This invention relates to an antibody that binds to both CEACAM5 and CEACAM6, its antigen-binding fragment, and its use. [Background technology]

[0002] Carcinoembryonic antigens (CEAs) are glycoproteins involved in cell adhesion. CEAs were first identified in 1965 as proteins normally expressed by the fetal intestine during the first six months of pregnancy (Gold and Freedman, J Exp Med, 121, 439, 1965), and have been found in pancreatic, liver, and colon cancers. The CEA family belongs to the immunoglobulin superfamily. The CEA family, consisting of 18 genes, is subdivided into two protein subgroups: the carcinoembryonic antigen-associated cell adhesion molecule (CEACAM) subgroup and the pregnancy-specific glycoprotein subgroup (Kammerer & Zimmermann, BMC Biology 2010, 8:12). In humans, the CEACAM subgroup consists of seven members: CEACAM1, CEACAM3, CEACAM4, CEACAM5, CEACAM6, CEACAM7, and CEACAM8.

[0003] CEACAM5 (Carcinoembryonic Antigen-Associated Cell Adhesion Molecule 5), also known as CEA or CD66e, is a well-known tumor-associated antigen that has been used for many years as a biomarker in the diagnosis of cancers. CEACAM5 expression is highly correlated with certain cancers. It is upregulated in colorectal, rectal, lung, pancreatic, and gastric cancers, with changes of up to several hundred times compared to normal tissue. The distribution profile of CEACAM5 makes it a very good target for therapeutics such as antibodies and ADCs (antibody-drug conjugates). For example, Sanofi's SAR408701 (Tusamitamab / Labtansine) contains the anti-CEACAM5 antibody SAR408377 (Tusamitamab; also known as huMab2-3), which is covalently bound to the cytotoxic agent DM4, a potent microtubule-destabilizing maytansinoid, via an N-succinimidyl 4-(2-pyridyldithio)butyrate (SPDB) linker.

[0004] CEACAM6 (Carcinoembryonic Antigen-Associated Cell Adhesion Molecule 6), also known as NCA90 or CD66c, is another well-known tumor-associated antigen used as a biomarker in cancer diagnosis. CEACAM6 expression is also highly correlated with certain cancers, including colorectal cancer, lung cancer, pancreatic cancer, and gastric cancer. Due to its distribution profile, CEACAM6 is also a very good target for therapeutic agents such as antibodies and ADCs.

[0005] In cancers where CEACAM5 and CEACAM6 are co-expressed, one may have higher expression than the other. Therefore, antibodies targeting both CEACAM5 and CEACAM6 can cover more cancer cells in the heterogeneous tumor microenvironment and thus reach a larger patient population. Consequently, developing antibodies that simultaneously target CEACAM5 and CEACAM6 is important in tumor immunotherapy. [Overview of the project]

[0006] The present invention provides an antibody that binds to both CEACAM5 and CEACAM6, as well as an antigen-binding fragment thereof.

[0007] In a first aspect, the present invention relates to an antibody or antigen-binding fragment thereof that binds to both CEACAM5 and CEACAM6, wherein the antibody comprises a heavy chain variable region (VH) and a light chain variable region (VL). (1) VH contains HCDR1-3 of VH having the amino acid sequence shown in SEQ ID NO: 26, and VL contains LCDR1-3 of VL having the amino acid sequence shown in SEQ ID NO: 27. (2) VH comprises HCDR1-3 of VH having the amino acid sequence shown in SEQ ID NO: 7, and VL comprises LCDR1-3 of VL having the amino acid sequence shown in SEQ ID NO: 8. (3) VH comprises HCDR1-3 of VH having the amino acid sequence shown in SEQ ID NO: 17, and VL comprises LCDR1-3 of VL having the amino acid sequence shown in SEQ ID NO: 18, or (4) The present invention provides an antibody or antigen-binding fragment thereof, wherein VH comprises HCDR1-3 of VH having the amino acid sequence shown in SEQ ID NO: 31, and VL comprises LCDR1-3 of VL having the amino acid sequence shown in SEQ ID NO: 32.

[0008] In some embodiments, the present invention relates to an antibody or antigen-binding fragment thereof that binds to both CEACAM5 and CEACAM6, wherein the antibody comprises a heavy chain variable region (VH) and a light chain variable region (VL). (1) VH comprises HCDR1-3 containing the amino acid sequences described in SEQ ID NOs. 21, 22, and 23, respectively, and VL comprises LCDR1-3 containing the amino acid sequences described in SEQ ID NOs. 24, 5, and 25, respectively. (2) VH comprises HCDR1-3 containing the amino acid sequences described in SEQ ID NOs: 1, 2, and 3, respectively, and VL comprises LCDR1-3 containing the amino acid sequences described in SEQ ID NOs: 4, 5, and 6, respectively. (3) VH comprises HCDR1-3 containing the amino acid sequences described in SEQ ID NOs. 11, 12, and 13, respectively, and VL comprises LCDR1-3 containing the amino acid sequences described in SEQ ID NOs. 14, 15, and 16, respectively, or (4) VH comprises HCDR1 to 3 containing the amino acid sequences described in SEQ ID NOs: 1, 30, and 3, respectively, and VL comprises LCDR1 to 3 containing the amino acid sequences described in SEQ ID NOs: 4, 5, and 6, respectively, providing an antibody or antigen-binding fragment thereof.

[0009] In some embodiments, the present invention relates to an antibody or antigen-binding fragment thereof that binds to both CEACAM5 and CEACAM6, wherein the antibody comprises a heavy chain variable region (VH) and a light chain variable region (VL). (1) VH contains an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 26, and VL contains an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 27. (2) VH contains an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 7, and VL contains an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 8. (3) VH contains an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 17, and VL contains an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 18, or (4) VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 31, and VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 32, to provide an antibody or an antigen-binding fragment thereof.

[0010] In a preferred embodiment, the present invention is an antibody or an antigen-binding fragment thereof that binds to both CEACAM5 and CEACAM6, wherein the antibody comprises a heavy chain variable region (VH) and a light chain variable region (VL), (1) VH comprises the amino acid sequence shown in SEQ ID NO: 26, and VL comprises the amino acid sequence shown in SEQ ID NO: 27, (2) VH comprises the amino acid sequence shown in SEQ ID NO: 7, and VL comprises the amino acid sequence shown in SEQ ID NO: 8, (3) VH comprises the amino acid sequence shown in SEQ ID NO: 17, and VL comprises the amino acid sequence shown in SEQ ID NO: 18, or (4) VH comprises the amino acid sequence shown in SEQ ID NO: 31, and VL comprises the amino acid sequence shown in SEQ ID NO: 32, to provide an antibody or an antigen-binding fragment thereof.

[0011] In some embodiments, the antibody comprises a heavy chain (HC) and a light chain (LC), (1) HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 28, and LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 29, (2) The heavy chain (HC) contains an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 9, and the light chain (LC) contains an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 10. (3) The heavy chain (HC) contains an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 19, and the light chain (LC) contains an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 20, or (4) The heavy chain (HC) contains an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 33, and the light chain (LC) contains an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 34.

[0012] In a preferred embodiment, the antibody comprises a heavy chain (HC) and a light chain (LC). (1) The heavy chain (HC) contains the amino acid sequence shown in SEQ ID NO: 28, and the light chain (LC) contains the amino acid sequence shown in SEQ ID NO: 29. (2) The heavy chain (HC) contains the amino acid sequence shown in SEQ ID NO: 9, and the light chain (LC) contains the amino acid sequence shown in SEQ ID NO: 10. (3) The heavy chain (HC) contains the amino acid sequence shown in SEQ ID NO: 19, and the light chain (LC) contains the amino acid sequence shown in SEQ ID NO: 20, or (4) The heavy chain (HC) contains the amino acid sequence shown in SEQ ID NO: 33, and the light chain (LC) contains the amino acid sequence shown in SEQ ID NO: 34.

[0013] In some embodiments, the antibody is a mouse antibody, a chimeric antibody, a humanized antibody, or a human antibody.

[0014] In some embodiments, the antibody is an isotype selected from the group consisting of IgG, IgA, IgM, IgE, and IgD.

[0015] In some embodiments, the antibody is a subtype selected from the group consisting of IgG1, IgG2, IgG3, and IgG4.

[0016] In some embodiments, the antigen-binding fragment is selected from the group consisting of Fab, Fab', F(ab')2, Fd, Fd', Fv, scFv, ds-scFv, and dAb.

[0017] In some embodiments, the antibody is a monoclonal antibody, a bispecific antibody, or a multispecific antibody.

[0018] In some embodiments, the antibody is monovalent, bivalent, or polyvalent.

[0019] In some embodiments, the antibody or antigen-binding fragment is fluorescently labeled, radiolabeled, or has a drug moiety.

[0020] In some embodiments, an antibody or antigen-binding fragment is bound to a drug moiety, which is selected from the group consisting of microtubule inhibitors, antibiotics, DNA synthesis inhibitors, topoisomerase inhibitors, RNA polymerase II inhibitors, and RNA spliceosome inhibitors.

[0021] In preferred embodiments, the drug portion is selected from the group consisting of MMAE, MMAF, duocalmycin, DM1, DM4, SN-38, Dxd, calicheamicin, doxorubicin, and PBD benzodiazepines.

[0022] In some embodiments, the antibody or its antigen-binding fragment is bound to the drug moiety by a linker.

[0023] In some embodiments, the linker includes a severable linker or a non-severable linker.

[0024] In some embodiments, the cleavable linker is selected from the group consisting of acid-unstable linkers, hydrophilic linkers, protease-sensitive linkers, photo-unstable linkers, hydrazone linkers, dimethyl linkers, and disulfide-containing linkers.

[0025] In preferred embodiments, the linker is selected from the group consisting of MC (6-maleimidocaproyl), Val-Cit (valine-citrulline), PABC (para-aminobenzyloxycarbonyl), DMEA (dimethylethylamine), Val-Cit-PABC, MC-Val-Cit-PABC, MC-Val-Cit-PABC-DMEA, GGFG (glycine-glycine-phenylalanine-glycine), MC-GGFG-aminomethyl, AcBut (4-(4-acetylphenoxy)-butanoic acid), and AcBut-dimethylhydrazide, with MC-Val-Cit-PABC being preferred.

[0026] In a second aspect, the present invention provides a bispecific antibody comprising an antibody or antigen-binding fragment thereof according to the first aspect of the present invention, and a second antigen-binding region that specifically binds to a tumor-associated antigen, an immune cell antigen, or an immune checkpoint molecule.

[0027] In a third aspect, the present invention provides a nucleic acid comprising a nucleotide sequence encoding an antibody or antigen-binding fragment thereof according to a first aspect of the present invention, or a bispecific antibody according to a second aspect of the present invention.

[0028] In a fourth aspect, the present invention provides a vector comprising nucleic acid according to a third aspect of the present invention.

[0029] In a fifth aspect, the present invention provides a host cell comprising a nucleic acid according to a third aspect of the present invention or a vector according to a fourth aspect of the present invention.

[0030] In a sixth aspect, the present invention provides an antibody-drug conjugate (ADC) comprising an antibody or antigen-binding fragment thereof according to a first aspect of the present invention or a bispecific antibody according to a second aspect of the present invention and a drug portion conjugated thereto via a linker.

[0031] In some embodiments, the drug portion is selected from the group consisting of microtubule inhibitors, antibiotics, DNA synthesis inhibitors, topoisomerase inhibitors, RNA polymerase II inhibitors, and RNA spliceosome inhibitors.

[0032] In preferred embodiments, the drug portion is selected from the group consisting of MMAE, MMAF, duocalmycin, DM1, DM4, SN-38, Dxd, calicheamicin, doxorubicin, and PBD benzodiazepines.

[0033] In some embodiments, the linker includes a severable linker or a non-severable linker.

[0034] In some embodiments, the cleavable linker is selected from the group consisting of acid-unstable linkers, hydrophilic linkers, protease-sensitive linkers, photo-unstable linkers, hydrazone linkers, dimethyl linkers, and disulfide-containing linkers.

[0035] In preferred embodiments, the linker is selected from the group consisting of MC (6-maleimidocaproyl), Val-Cit (valine-citrulline), PABC (para-aminobenzyloxycarbonyl), DMEA (dimethylethylamine), Val-Cit-PABC, MC-Val-Cit-PABC, MC-Val-Cit-PABC-DMEA, GGFG (glycine-glycine-phenylalanine-glycine), MC-GGFG-aminomethyl, AcBut (4-(4-acetylphenoxy)-butanoic acid), and AcBut-dimethylhydrazide, with MC-Val-Cit-PABC being preferred.

[0036] In a seventh aspect, the present invention provides a pharmaceutical composition comprising (i) an antibody or antigen-binding fragment thereof according to a first aspect of the present invention, or a bispecific antibody according to a second aspect of the present invention, or a nucleic acid according to a third aspect of the present invention, or a vector according to a fourth aspect of the present invention, or a host cell according to a fifth aspect of the present invention, or an antibody-drug conjugate according to a sixth aspect of the present invention, and optionally (ii) a pharmaceutically acceptable carrier or excipient.

[0037] In some embodiments, the composition further comprises a second therapeutic agent. Preferably, the second therapeutic agent is selected from the group consisting of antibodies, chemotherapeutic agents, siRNA, antisense oligonucleotides, polypeptides, and small molecule drugs.

[0038] In an eighth aspect, the present invention provides a method for treating cancer in a subject, comprising administering to the subject an effective amount of the antibody or antigen-binding fragment thereof, a bispecific antibody, a nucleic acid, a vector, a host cell, an antibody-drug conjugate, or a pharmaceutical composition of the present invention.

[0039] In some specific embodiments, the cancer is a cancer associated with the expression of CEACAM5 and / or CEACAM6. In preferred embodiments, the cancer is selected from the group consisting of small intestine cancer, colorectal cancer, gastric cancer, lung cancer, cervical cancer, pancreatic cancer, esophageal cancer, ovarian cancer, thyroid cancer, bladder cancer, endometrial cancer, breast cancer, liver cancer, prostate cancer, and skin cancer.

[0040] In some embodiments, the method further comprises administering a second therapeutic agent to a subject. Preferably, the second therapeutic agent is selected from antibodies, chemotherapeutic agents, siRNAs, antisense oligonucleotides, polypeptides, and small molecule drugs.

[0041] In a ninth aspect, the present invention provides the use of the present invention's antibodies or their antigen-binding fragments, bispecific antibodies, nucleic acids, vectors, host cells, antibody-drug conjugates, or pharmaceutical compositions in the manufacture of a pharmacopoeia for treating cancer in a subject.

[0042] In some embodiments, the cancer is a cancer associated with the expression of CEACAM5 and / or CEACAM6. In preferred embodiments, the cancer is selected from the group consisting of small intestine cancer, colorectal cancer, gastric cancer, lung cancer, cervical cancer, pancreatic cancer, esophageal cancer, ovarian cancer, thyroid cancer, bladder cancer, endometrial cancer, breast cancer, liver cancer, prostate cancer, and skin cancer.

[0043] In some embodiments, the pharmaceutical further comprises a second therapeutic agent. Preferably, the second therapeutic agent is selected from antibodies, chemotherapeutic agents, siRNAs, antisense oligonucleotides, polypeptides, and small molecule drugs.

[0044] In some embodiments, the pharmaceutical agent is administered in combination with a second therapeutic agent. Preferably, the second therapeutic agent is selected from antibodies, chemotherapeutic agents, siRNAs, antisense oligonucleotides, polypeptides, and small molecule drugs.

[0045] In a tenth aspect, the present invention provides antibodies or antigen-binding fragments thereof, bispecific antibodies, nucleic acids, vectors, host cells, antibody-drug conjugates, or pharmaceutical compositions for use in methods of treating cancer in a subject.

[0046] In some embodiments, the cancer is a cancer associated with the expression of CEACAM5 and / or CEACAM6. In preferred embodiments, the cancer is selected from the group consisting of small intestine cancer, colorectal cancer, gastric cancer, lung cancer, cervical cancer, pancreatic cancer, esophageal cancer, ovarian cancer, thyroid cancer, bladder cancer, endometrial cancer, breast cancer, liver cancer, prostate cancer, and skin cancer.

[0047] In some embodiments, a second therapeutic agent is further administered to the subject. Preferably, the second therapeutic agent is selected from antibodies, chemotherapeutic agents, siRNA, antisense oligonucleotides, polypeptides, and small molecule drugs.

[0048] In the eleventh aspect, the present invention relates to a method for detecting the presence or level of CEACAM5 and / or CEACAM6 in a sample, (a) Contacting the sample with the antibody or antigen-binding fragment thereof described in any one of claims 1 to 10, (b) A method is provided which includes determining the presence or level of CEACAM5 and / or CEACAM6 in a sample by detecting the binding of an antibody to the sample.

[0049] In a preferred embodiment, this method is used for non-diagnostic purposes.

[0050] In a twelfth aspect, the present invention relates to a method for diagnosing cancer associated with the expression of CEACAM5 and / or CEACAM6 in a subject, (a) Obtaining biological samples from the subject, (b) Contacting a sample with the antibody of the present invention or its antigen-binding fragment, (c) detecting the binding of an antibody to a sample, This invention provides a method for identifying a subject as having cancer based on an increase in the binding of an antibody or its antigen-binding fragment to a sample compared to the binding of an antibody or its antigen-binding fragment to a control sample.

[0051] In a thirteenth aspect, the present invention relates to a method for imaging cancer associated with the expression of CEACAM5 and / or CEACAM6 in a subject, (a) Administering the antibody of the present invention or its antigen-binding fragment to a subject, wherein the antibody is conjugated to a detectable marker, (b) A method is provided which includes detecting the presence of a marker.

[0052] In some embodiments, the detectable marker is 111 In, preferably, marker detection is performed by single-photon emission computed tomography. In other embodiments, the detectable marker is 89 The marker is Zr, and preferably, marker detection is performed by positron emission tomography. [Brief explanation of the drawing]

[0053] [Figure 1]The procedure for immunization and single B cell cloning to generate anti-CEACAM5 / 6 antibodies is described. [Figure 2] This shows the binding of an anti-CEACAM5 / 6 double-binding antibody to the human CEACAM5 protein. [Figure 3] This shows the binding of an anti-CEACAM5 / 6 double-binding antibody to the cyno CEACAM5 protein. [Figure 4] This shows the binding of an anti-CEACAM5 / 6 double-binding antibody to the human CEACAM6 protein. [Figure 5] This shows that the anti-CEACAM5 / 6 double-binding antibody does not bind to the human CEACAM1 protein. [Figure 6] This shows that the anti-CEACAM5 / 6 double-binding antibody does not bind to the human CEACAM3 protein. [Figure 7] This shows that the anti-CEACAM5 / 6 double-binding antibody does not bind to the human CEACAM8 protein. [Figure 8] This shows the binding of an anti-CEACAM5 / 6 double-conjugated antibody to 293T cells overexpressing human CEACAM5. [Figure 9] This shows the binding of an anti-CEACAM5 / 6 double-binding antibody to 293T cells overexpressing human CEACAM6. [Figure 10] This shows the binding of an anti-CEACAM5 / 6 double-conjugated antibody to CHO-K1 cells overexpressing cyno CEACAM5 or cyno CEACAM6. [Figure 11] This shows the binding of anti-CEACAM5 / 6 double-conjugated antibodies to cancer cell lines LS174T, BxPC3, and A549. [Figure 12] This study demonstrates the internalization of anti-CEACAM5 / 6 double-binding antibodies in 293T cell lines overexpressing human CEACAM5 or human CEACAM6, as well as in cancer cell lines LS 174T, HPAC (pancreatic cancer), and BxPC3. [Figure 13-1]This study demonstrates the internalization of anti-CEACAM5 / 6 double-conjugated antibodies on cancer cell lines LS 174T, A549 (lung cancer), BxPC3, and HPAC in the presence of 100 nM soluble human CEACAM5 or human CEACAM6 with soluble protein interference. [Figure 13-2] This study demonstrates the internalization of anti-CEACAM5 / 6 double-conjugated antibodies on cancer cell lines LS 174T, A549 (lung cancer), BxPC3, and HPAC in the presence of 100 nM soluble human CEACAM5 or human CEACAM6 with soluble protein interference. [Figure 14] This report evaluates the antitumor activity of anti-CEACAM5 / 6 ADC against MKN45 gastric cancer in a CDX mouse model. [Figure 15] This study evaluates the antitumor activity of anti-CEACAM5 / 6 ADC against LS 174T colorectal cancer in a CDX mouse model.

[0054] array Table 1 shows the amino acid sequences of the CDR (Kabat and Chothia composite system), heavy chain variable region (VH), light chain variable region (VL), full heavy chain (HC), and light chain (LC) of the anti-CEACAM5 / 6 antibodies PR304169, PR304171, PR304294, and PR304713 of the present invention. Table 2 shows the amino acid sequences of the CDR (Kabat and Chothia composite system), heavy chain variable region (VH), light chain variable region (VL), full heavy chain (HC), and light chain (LC) of the anti-CEACAM5 reference antibody PR303561 (tusamitamab) and the anti-CEACAM6 reference antibody PR303563 (tinuririmab).

[0055] [Table 1-1]

[0056] [Table 1-2]

[0057] [Table 1-3]

[0058] [Table 2-1]

[0059] [Table 2-2]

[0060] Detailed description of the invention The aforementioned features and advantages of the present invention, as well as any additional features and advantages thereof, will be more clearly understood below by reading the detailed description of the embodiments in conjunction with the drawings.

[0061] The embodiments described herein with reference to the drawings are descriptive and illustrative, and are used to understand the invention in general terms. The embodiments should not be construed as limiting the scope of the invention. Identical or similar elements, and elements having the same or similar function, are indicated by the same reference numerals throughout this description.

[0062] Unless otherwise indicated or defined, all terms used have their ordinary meanings in the art, as will be obvious to those skilled in the art. See standard handbooks, e.g., Leuenberger, HGW, Nagel, B., and Klbl, H. eds., "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)", Helvetica Chimica Acta (1995), CH-4010, Basel, Switzerland; Sambrook et al, "Molecular Cloning: A Laboratory Manual" (2nd edition), Vols. 1-3, Cold Spring Harbor Laboratory Press (1989); F. Ausubel et al eds., "Current protocols in molecular biology", Green Publishing and Wiley InterScience, New York (1987); Roitt et al, "Immunology (6th edition)", Mosby / Elsevier, Edinburgh (2001); and Janeway et al., "Immunobiology (6th edition)", Garland Science Publishing / Churchill Livingstone, New York (2005), as well as the general background techniques mentioned above.

[0063] As used herein, the singular forms "a," "an," and "the" include multiple references unless the context clearly indicates otherwise. Therefore, for example, a reference to "antibody" includes multiple antibodies.

[0064] Unless otherwise indicated or defined, the term “comprise,” and its variations such as “comprises” and “comprising,” mean the inclusion of the described element or process or group of elements or processes, but not the exclusion of any other element or process or group of elements or processes. The term “comprising” includes “including” and “consisting,” for example, a composition “comprising” X may consist of X alone, or it may include something additional, such as X + Y.

[0065] The term "approximately" in relation to numerical values ​​is optional and can mean, for example, x ± 10% or x ± 5%.

[0066] As used herein, the term “antibody” refers to an immunoglobulin molecule that has the ability to specifically bind to a particular antigen. Antibodies often contain variable and constant regions in both the heavy and light chains. The variable regions of the heavy and light chains of an antibody contain binding domains that interact with the antigen. The constant region of an antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and components of the complement system, such as C1q, the first component in the classical pathway of complement activation. Most antibodies have a heavy chain variable region (VH) and a light chain variable region (VL), which together form the antigen-binding portion of the antibody.

[0067] The “light chain variable region” (VL) or “heavy chain variable region” (VH) consists of four “framework” regions interrupted by three “complementarity-determining regions” or “CDRs.” The framework regions help align the CDRs for specific binding of the antigen to the epitope. The CDRs contain amino acid residues of the antibody that are primarily involved in antigen binding. From the amino terminus to the carboxyl terminus, both the VL and VH domains contain the following framework (FR) and CDR regions: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. CDR1, 2, and 3 of the VL domain are also referred to herein as LCDR1, LCDR2, and LCDR3, respectively. CDR1, 2, and 3 of the VH domain are also referred herein as HCDR1, HCDR2, and HCDR3, respectively.

[0068] The assignment of amino acids to each VL and VH domain follows any conventional definition of the CDR. Conventional definitions include the Kabat definition (Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, MD, 1987 and 1991)); the Chothia definition (Chothia & Lesk, J. Mol. Biol. 196:901-917, 1987; Chothia et al., Nature, 342:878-883 (1989)); the Chothia and Kabat CDR complex (also called Chothia and Kabat CDR), where each CDR is a complex of Chothia and Kabat CDRs; the AbM definition used by Oxford Molecular's antibody modeling software; and the contact definition by Martin et al. (world wide web bioinfo.org.uk / abs). Kabat provides a widely used numbering convention (the Kabat numbering system) in which the same number is assigned to corresponding residues between different heavy chains or between different light chains.

[0069] This disclosure includes CDRs defined according to any of these numbering systems, but preferred embodiments include CDRs defined in combination with Chothia and Kabat.

[0070] [Table 3]

[0071] In Table 3, Laa~Lbb can refer to the amino acid sequence from position aa (according to the Chothia numbering system) to position bb (according to the Chothia numbering system) starting from the N-terminus of the antibody light chain, and Haa~Hbb can refer to the amino acid sequence from position aa (according to the Chothia numbering system) to position bb (according to the Chothia numbering system) starting from the N-terminus of the antibody heavy chain. For example, L24~L34 can refer to the amino acid sequence from position 24 to 34 (according to the Chothia numbering system) starting from the N-terminus of the antibody light chain, and H26~H32 can refer to the amino acid sequence from position 26 to 32 (according to the Chothia numbering system) starting from the N-terminus of the antibody heavy chain.

[0072] As used herein, the term “antibody” should be understood in its broadest sense and includes monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, antibody fragments, and multispecific antibodies (e.g., bispecific antibodies) containing at least two different antigen-binding regions. Antibodies may contain further modifications such as amino acids not naturally occurring, mutations in the Fc region, and mutations in glycosylation sites. Antibodies also include post-translationally modified antibodies, fusion proteins containing antigenic determinants of antibodies, and immunoglobulin molecules containing any other modifications to antigen recognition sites, insofar as these antibodies exhibit the desired biological activity.

[0073] As used herein, the term “antigen-binding fragment” of an antibody refers to one or more fragments of an antibody that possess the ability to specifically bind to an antigen (e.g., CEACAM5 or CEACAM6). It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody.

[0074] Examples of antibody antigen-binding fragments include: (i) Fab fragments, which are monovalent fragments consisting of VLC, VHC, CL, and CH1 domains; (ii) F(ab')2 fragments, which are bivalent fragments containing two Fab fragments linked by disulfide bonds in the hinge region; (iii) Fab' fragments, which are essentially Fab fragments having part of the hinge region (see FUNDAMENTALIMMUNOLOGY (Paul ed.3.sup.rd ed.1993)); (iv) Fd fragments consisting of VH and CH1 domains; (v) Fd' fragments having VH and CH1 domains, and one or more cysteine ​​residues at the C-terminus of the CH1 domain; (vi) Fv fragments consisting of VL and VH domains of a single arm of the antibody; and (vii) dAb fragments consisting of a VH domain (Ward et al., (1989) Nature (341:544-546); (viii) isolated complementarity-determining regions (CDRs); (ix) heavy chain variable regions containing nanobodies, a single variable domain, and two constant domains. Furthermore, the two domains of the Fv fragment, VL and VH, are encoded by separate genes, but can be joined by a synthetic linker that allows them to be produced using recombination as a single protein chain (known as single-chain Fv (scFv); see, for example, Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single-chain antibodies are also intended to be included in the term "antigen-binding fragment" of the antibody. Furthermore, the term also includes “linear antibodies” comprising a pair of tandem Fd segments (VH-CH1-VH-CH1) that form an antigen-binding domain together with a complementary light chain polypeptide, and any modified version of the aforementioned fragments that retain antigen-binding activity.

[0075] These antigen-binding fragments are obtained using prior art known to those skilled in the art, and the fragments are screened for usefulness in the same manner as intact antibodies.

[0076] As used herein, the terms "bind" or "specifically bind" refer to a non-random binding reaction between two molecules, such as between an antibody and its target antigen. The binding specificity of an antibody can be determined based on affinity and / or avidity. Affinity, represented by the equilibrium dissociation constant (KD) of the antigen from the antibody, is a measure of the strength of the binding between an epitope and the antigen-binding site on the antibody, and the smaller the KD value, the stronger the binding between the epitope and the antibody. Alternatively, affinity can also be expressed as an affinity constant (KA), which is 1 / KD.

[0077] Avidity is a measure of the strength of the binding between an antibody and a related antigen. Avidity is related to both the affinity between an epitope and its antigen-binding site on the antibody and the number of related binding sites present on the antibody. Typically, an antibody has a dissociation constant (KD) of 10 -5 ~10 -12 M or less, preferably 10 -7 ~10 -12 M or less, more preferably 10 -8 ~10 -12 M, and / or a binding affinity of at least 10 7 M -1 , preferably at least 10 8 M -1 , more preferably at least 10 9 M -1 , for example at least 10 12 M -1 . Any K -4 value greater than 10 D M is generally considered to indicate non-specific binding. Specific binding of an antibody to an antigen or epitope can be determined in any suitable manner known per se, including, for example, Scatchard analysis and / or competitive binding assays, such as radioimmunoassay (RIA), enzyme immunoassay (EIA), biolayer interferometry (BLI) assay, and sandwich competition assay, and various modifications thereof known per se in the art.

[0078] The term "epitope" refers to the site on an antigen to which an antibody binds. Epitopes can be formed from a sequence of amino acids or from discontinuous amino acids juxtaposed by the tertiary folding of one or more proteins. Epitopes formed from a sequence of amino acids (also known as linear epitopes) are typically retained upon exposure to denaturing solvents, while epitopes formed by tertiary folding (also known as conformational epitopes) are typically lost upon treatment with denaturing solvents. Epitopes typically contain at least three, more commonly five or eight to ten, amino acids in a specific spatial conformation. Epitopes define the minimal binding site of an antibody and are therefore specific targets for the antibody or its antigen-binding fragment.

[0079] As used herein, the term “sequence identity” refers to the degree to which two sequences (amino acids) have the same residues at the same positions in an alignment. For example, “the amino acid sequence is X% identical to sequence number Y” means the % identity of the amino acid sequence to sequence number Y, and further details that X% of the residues in the amino acid sequence are identical to the residues in the sequence disclosed in sequence number Y. Generally, computer programs are used for such calculations. Exemplary programs for comparing and aligning pairs of sequences include ALIGN (Myers and Miller, 1988), FASTA (Pearson and Lipman, 1988; Pearson, 1990), and gapped BLAST (Altschul et al., 1997), BLASTP, BLASTN, or GCG (Devereux et al., 1984).

[0080] Furthermore, when determining the degree of sequence identity between two amino acid sequences, those skilled in the art may take into account so-called "conservative" amino acid substitutions, which can generally be described as amino acid substitutions in which an amino acid residue is replaced by another amino acid residue with a similar chemical structure, and which have little or no effect on the function, activity or other biological properties of the polypeptide.

[0081] Such conservative substitutions are preferably those in which one amino acid from the following groups (a) to (e) is replaced by another amino acid residue from the same group: (a) small aliphatic residues, nonpolar residues, or slightly polar residues: Ala, Ser, Thr, Pro, and Gly; (b) polar negative-charged residues and their (uncharged) amides: Asp, Asn, Glu, and Gln; (c) polar positive-charged residues: His, Arg, and Lys; (d) large aliphatic nonpolar residues: and (e) aromatic residues: Phe, Tyr, and Trp.

[0082] Particularly preferred conservative substitutions are as follows: Ala to Gly or Ser; Arg to Lys; Asn to Gln or His; Asp to Glu; Cys to Ser; Gln to Asn; Glu to Asp; Gly to Ala or Pro; His to Asn or Gln; Ile to Leu or Val; Leu to Ile or Val; Lys to Arg, Gln, or Glu; Met to Leu, Tyr, or Ile; Phe to Met, Leu, or Tyr; Ser to Thr; Thr to Ser; Trp to Tyr; Tyr to Trp; and / or Phe to Val, Ile, or Leu.

[0083] As used herein, the term “monoclonal antibody” refers to an antibody obtained from a substantially homogeneous antibody population; that is, each antibody constituting the population is identical except for a small amount of naturally occurring mutations. Monoclonal antibodies are highly specific and directed toward a single antigen. The term “monoclonal antibody” as used herein is not limited to antibodies produced by hybridoma technology and should not be construed as requiring antibody production by any particular method.

[0084] In the context of this invention, the term "bispecific antibody" should be understood as an antibody having two distinct antigen-binding regions defined by different antibody sequences. This can be understood as binding to different targets, but also includes binding to different epitopes within a single target.

[0085] As used herein, the term “tumor-associated antigen” refers to an antigen that is differentially expressed in cancer cells compared to normal cells and can therefore be used to target cancer cells.

[0086] As used herein, the term “vector” refers to a nucleic acid molecule that can transport another nucleic acid to which it is linked.

[0087] As used herein, the term “host cell” refers to a cell into which an expression vector has been introduced.

[0088] The term "pharmaceutically acceptable" means that the carrier or adjuvant is compatible with the other components of the composition, is substantially harmless to its recipient, and / or is approved or appropriable for inclusion in a pharmaceutical composition for parenteral administration to humans.

[0089] As used herein, terms such as “treatment” and “to treat” mean administering a drug or performing a procedure for the purpose of obtaining an effect. The effect may be prophylactic in that it completely or partially prevents a disease or its symptoms, and / or therapeutic in that it partially or completely cures a disease and / or its symptoms. As used herein, “treatment” may include treatment of a disease or disorder (e.g., cancer) in mammals, in particular humans, and may include (a) preventing the occurrence of a disease or its symptoms in subjects who are susceptible to the disease but have not yet been diagnosed with it (e.g., including diseases associated with or that can be caused by a primary disease), (b) inhibiting the disease, i.e., stopping its development, and (c) reducing the disease, i.e., causing a regression of the disease. "Treatment" can mean any sign of success in treating, improving, or preventing cancer, including any objective or subjective parameters, such as reduction; remission; gradual reduction of symptoms or making the disease state more tolerable to the patient; slowing the rate of degeneration or decline; or preventing the final stage of degeneration from becoming more debilitating. Treatment or improvement of symptoms is based on one or more objective or subjective parameters, including the results of a physician's examination. Accordingly, the term "treat" includes the administration of antibodies, compositions, or conjugates disclosed herein to prevent, delay, alleviate, halt, or inhibit the onset of symptoms or conditions associated with a disease (e.g., cancer). The term "therapeutic effect" means the reduction, elimination, or prevention of the disease, symptoms of the disease, or side effects of the disease in a subject.

[0090] As used herein, the term “effective dose” means an amount sufficient to achieve treatment of a disease when administered to a subject for that purpose.

[0091] As used herein, the term “subject” refers to any mammalian subject to which diagnosis, treatment, or therapy is desired. “Mammal” for treatment purposes refers to any animal classified as a mammal, including humans, livestock and farm animals, as well as laboratory animals, zoo animals, sporting animals, or pet animals, such as dogs, horses, cats, cattle, sheep, goats, pigs, mice, rats, rabbits, guinea pigs, and monkeys.

[0092] The terms “cyno CEACAM5,” “cynomolgus macaques CEACAM5,” and “cynomolgus macaques CEACAM5” are used interchangeably herein and refer to cynomolgus macaques CEACAM5. This term includes any CEACAM5 variants, isoforms, and species homologs that are naturally expressed by cynomolgus macaques cells or expressed on cells transfected with the gene or cDNA encoding cynomolgus macaques CEACAM5 that is naturally expressed on cynomolgus macaques cells.

[0093] The terms “cyno CEACAM6,” “cynomolgus macaques CEACAM6,” and “cynomolgus macaques CEACAM6” are used interchangeably herein and refer to cynomolgus macaques CEACAM6. This term includes any CEACAM6 variants, isoforms, and species homologs that are naturally expressed by cynomolgus macaques cells or expressed on cells transfected with the gene or cDNA encoding cynomolgus macaques CEACAM6 that is naturally expressed on cynomolgus macaques cells.

[0094] Anti-CEACAM5 / 6 double binding antibody In a first aspect, the present invention provides an antibody or antigen-binding fragment thereof that binds to both CEACAM5 and CEACAM6, wherein the antibody comprises a heavy chain variable region (VH) and a light chain variable region (VL), and (1) VH comprises HCDR1-3 of VH having the amino acid sequence described in SEQ ID NO: 26, and VL comprises LCDR1-3 of VL having the amino acid sequence described in SEQ ID NO: 27, (2) VH comprises HCDR1-3 of VH having the amino acid sequence shown in SEQ ID NO: 7, and VL comprises LCDR1-3 of VL having the amino acid sequence shown in SEQ ID NO: 8, (3) VH comprises HCDR1-3 of VH having the amino acid sequence shown in SEQ ID NO: 17, and VL comprises LCDR1-3 of VL having the amino acid sequence shown in SEQ ID NO: 18, or (4) VH comprises HCDR1-3 of VH having the amino acid sequence described in SEQ ID NO: 31, and VL comprises LCDR1-3 of VL having the amino acid sequence described in SEQ ID NO: 32, wherein the antibody or antigen-binding fragment thereof is provided.

[0095] In some embodiments, LCDR1-3 and HCDR1-3 are defined by the EU Kabat definition / numbering system. In some embodiments, LCDR1-3 and HCDR1-3 are defined by the Chothia definition / numbering system. In some embodiments, LCDR1-3 and HCDR1-3 are defined by the AbM definition / numbering system. In some preferred embodiments, LCDR1-3 and HCDR1-3 are defined by a combined Kabat and Chothia numbering system.

[0096] In some embodiments, the antibody comprises a heavy chain variable region (VH) and a light chain variable region (VL), where (1) VH comprises HCDR1-3 containing the amino acid sequences described in SEQ ID NOs. 21, 22, and 23, respectively, and VL comprises LCDR1-3 containing the amino acid sequences described in SEQ ID NOs. 24, 5, and 25, respectively; (2) VH comprises HCDR1-3 containing the amino acid sequences described in SEQ ID NOs. 1, 2, and 3, respectively, and VL comprises LCDR1-3 containing the amino acid sequences described in SEQ ID NOs. 4, 5, and 6, respectively; (3) VH comprises HCDR1-3 containing the amino acid sequences described in SEQ ID NOs. 11, 12, and 13, respectively, and VL comprises LCDR1-3 containing the amino acid sequences described in SEQ ID NOs. 14, 15, and 16, respectively; (4) VH comprises HCDR1-3 containing the amino acid sequences described in SEQ ID NOs. 1, 30, and 3, respectively, and VL comprises LCDR1-3 containing the amino acid sequences described in SEQ ID NOs. 4, 5, and 6, respectively.

[0097] In some embodiments, the CDR is determined by a combined Kabat and Chothia system.

[0098] In some embodiments, the antibody comprises a heavy chain variable region (VH) and a light chain variable region (VL), (1) VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 26, and VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 27, (2) VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 7, and VL comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 8 (3) VH contains an amino acid sequence having 0% sequence identity with SEQ ID NO: 17, and VL contains an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 18, or (4) VH contains an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 31, and VL contains an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 32.

[0099] In some embodiments, VH includes a functional variant of the amino acid sequence described in any one of SEQ ID NOs: 26, 7, 17, and 31, formed by the insertion, deletion, and / or substitution of one or more amino acids therein, provided that an antibody containing VH containing the functional variant retains the ability to bind to both CEACAM5 and CEACAM6. In some embodiments, VL includes a functional variant of the amino acid sequence described in any one of SEQ ID NOs: 27, 8, 18, and 32, formed by the insertion, deletion, and / or substitution of one or more amino acids therein, provided that an antibody containing VL containing the functional variant retains the ability to bind to both CEACAM5 and CEACAM6.

[0100] A functional variant contains or consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity with respect to the amino acid sequence of the parent polypeptide.

[0101] In the context of functional variants, the number of inserted, deleted, and / or substituted amino acids is preferably 40% or less, more preferably 35% or less, more preferably 1-33%, more preferably 5-30%, more preferably 10-25%, and more preferably 15-20% of the total number of amino acids in the parent amino acid sequence. For example, the number of inserted, deleted, and / or substituted amino acids may be 1-20, preferably 1-10, more preferably 1-7, even more preferably 1-5, and most preferably 1-2. In a preferred embodiment, the number of inserted, deleted, and / or substituted amino acids is 1, 2, 3, 4, 5, 6, or 7.

[0102] In some embodiments, insertions, deletions, and / or substitutions can be performed in framework (FR) regions, such as FR1, FR2, FR3, and / or FR4.

[0103] In some embodiments, the substitution of one or more amino acids may be a conservative substitution of one or more amino acids. Such a conservative substitution is preferably one in which one amino acid from the following groups (a) to (e) is replaced by another amino acid residue from the same group: (a) small aliphatic residues, nonpolar residues, or slightly polar residues: Ala, Ser, Thr, Pro, and Gly; (b) polar negatively charged residues and their (uncharged) amides: Asp, Asn, Glu, and Gln; (c) polar positively charged residues: His, Arg, and Lys; (d) large aliphatic nonpolar residues: and (e) aromatic residues: Phe, Tyr, and Trp.

[0104] Particularly preferred conservative substitutions are as follows: Ala to Gly or Ser; Arg to Lys; Asn to Gln or His; Asp to Glu; Cys to Ser; Gln to Asn; Glu to Asp; Gly to Ala or Pro; His to Asn or Gln; Ile to Leu or Val; Leu to Ile or Val; Lys to Arg, Gln, or Glu; Met to Leu, Tyr, or Ile; Phe to Met, Leu, or Tyr; Ser to Thr; Thr to Ser; Trp to Tyr; Tyr to Trp; and / or Phe to Val, Ile, or Leu.

[0105] In a preferred embodiment, the present invention relates to an antibody or antigen-binding fragment thereof that binds to both CEACAM5 and CEACAM6, wherein the antibody comprises a heavy chain variable region (VH) and a light chain variable region (VL). (1) VH contains the amino acid sequence shown in SEQ ID NO: 26, and VL contains the amino acid sequence shown in SEQ ID NO: 27. (2) VH contains the amino acid sequence shown in SEQ ID NO: 7, and VL contains the amino acid sequence shown in SEQ ID NO: 8. (3) VH contains the amino acid sequence shown in SEQ ID NO: 17, and VL contains the amino acid sequence shown in SEQ ID NO: 18, or (4) VH provides an antibody or antigen-binding fragment thereof, comprising the amino acid sequence shown in SEQ ID NO: 31, and VL provides an amino acid sequence shown in SEQ ID NO: 32.

[0106] In some embodiments, the antibody comprises a heavy chain (HC) and a light chain (LC), (1) the HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 28, and the LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 29, (2) the HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 9, and the LC comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 10 (3)HC contains an amino acid sequence having sequence identity with at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 19, and LC contains an amino acid sequence having sequence identity with at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 20, or (4)HC contains an amino acid sequence having sequence identity with at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 33, and LC contains an amino acid sequence having sequence identity with at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 34.

[0107] In some embodiments, the heavy chain includes a functional variant of the amino acid sequence described in any one of SEQ ID NOs: 28, 9, 19, and 33, formed by the insertion, deletion, and / or substitution of one or more amino acids therein, provided that the antibody including the heavy chain containing the functional variant retains the ability to bind to both CEACAM5 and CEACAM6. In some embodiments, the light chain includes a functional variant of the amino acid sequence described in any one of SEQ ID NOs: 29, 10, 20, and 34, formed by the insertion, deletion, and / or substitution of one or more amino acids therein, provided that the antibody including the light chain containing the functional variant retains the ability to bind to both CEACAM5 and CEACAM6.

[0108] A functional variant contains or consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity with respect to the amino acid sequence of the parent polypeptide.

[0109] In some embodiments, the number of inserted, deleted, and / or substituted amino acids is preferably 40% or less, more preferably 35% or less, more preferably 1 to 33%, more preferably 5 to 30%, more preferably 10 to 25%, and more preferably 15 to 20% of the total number of amino acids in the parent amino acid sequence. For example, the number of inserted, deleted, and / or substituted amino acids may be 1 to 50, preferably 1 to 20, more preferably 1 to 10, and even more preferably 1 to 5. In preferred embodiments, the number of inserted, deleted, and / or substituted amino acids is 1, 2, 3, 4, 5, 6, or 7.

[0110] In some embodiments, insertions, deletions, and / or substitutions may be performed in framework (FR) regions, e.g., FR1, FR2, FR3, and / or FR4; and / or steady-state regions, e.g., CL, CH1, CH2, and / or CH3.

[0111] In some embodiments, the substitution of one or more amino acids may be a conservative substitution of one or more amino acids. Examples of conservative substitutions are as described above.

[0112] In a preferred embodiment, the antibody comprises a heavy chain (HC) and a light chain (LC), (1) HC contains the amino acid sequence shown in SEQ ID NO: 28, and LC contains the amino acid sequence shown in SEQ ID NO: 29. (2) HC contains the amino acid sequence shown in SEQ ID NO: 9, and LC contains the amino acid sequence shown in SEQ ID NO: 10. (3) HC contains the amino acid sequence shown in SEQ ID NO: 19, and LC contains the amino acid sequence shown in SEQ ID NO: 20, or (4) HC contains the amino acid sequence shown in SEQ ID NO: 33, and LC contains the amino acid sequence shown in SEQ ID NO: 34.

[0113] In some embodiments, the antibody is a mouse antibody, a chimeric antibody, a humanized antibody, or a human antibody.

[0114] Based on the amino acid sequence of the constant region of the heavy chain of an antibody, immunoglobulin molecules can be classified into five classes (isotypes): IgA, IgD, IgE, IgG, and IgM, and further classified into different subtypes such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The light chain of an antibody can be classified as either a lambda (λ) chain or a kappa (κ) chain based on the amino acid sequence of the light chain. The antibodies disclosed herein may be any of the above classes or subtypes.

[0115] In some embodiments, the antibody may be an isotype selected from the group consisting of IgG, IgA, IgM, IgE, and IgD. In some embodiments, the antibody may be a subtype selected from the group consisting of IgG1, IgG2, IgG3, and IgG4. In preferred embodiments, the antibody is an IgG1 antibody.

[0116] The antibodies disclosed herein may be intact antibodies or antigen-binding fragments thereof. Antigen-binding fragments may be any fragment of an antibody that retains the ability to bind to both CEACAM5 and CEACAM6. Examples of antigen-binding fragments include, but are not limited to, Fab fragments; F(ab')2 fragments; Fab' fragments; Fd fragments; Fd' fragments; Fv fragments; scFv fragments; dAb fragments; isolated complementarity-determining regions (CDRs); nanobodies; linear antibodies containing a pair of tandem Fd segments (VH-CH1-VH-CH1); and any modified version of any of the aforementioned fragments that retain antigen-binding activity.

[0117] In some embodiments, the antigen-binding fragment can be selected from the group consisting of Fab, Fab', F(ab')2, Fv, scFv, and ds-scFv. In preferred embodiments, the antigen-binding fragment is Fab or scFv.

[0118] In some embodiments, the antibody includes an Fc region. In some embodiments, the Fc region may be any isotype including but not limited to IgG1, IgG2, IgG3, and IgG4, and may contain one or more mutations or modifications. In one embodiment, the Fc region is or is derived from the IgG1 isotype and optionally has one or more mutations or modifications. In one embodiment, the Fc region is human IgG1 Fc.

[0119] In one embodiment, the Fc region is effector function deficient. For example, the Fc region may be an IgG1 isotype or a non-IgG1 type, such as IgG2, IgG3, or IgG4, which are mutated to have reduced or even eliminated ability to mediate effector functions such as ADCC. Such mutations are described, for example, in Dall'Acqua WF et al., J Immunol. 177(2):1129-1138 (2006) and Hezareh M, J Virol.; 75(24):12161-12168 (2001). In some embodiments, the Fc region of the antibody includes wild-type IgG1 Fc with L234A, L235A, and G237A mutations.

[0120] In some embodiments, the antibody is mutated at one or more post-translational modification sites. In one embodiment, the Fc region includes a mutation that removes the receptor site for Asn-binding glycosylation, or is manipulated to eliminate the effector function of the antibody.

[0121] Post-translational modifications (PTMs) are widely observed in proteins expressed in mammalian cells. Aside from conserved PTM sites in antibodies, such as the conserved N-glycosylation site on the IgG1 antibody CH2 domain, other PTM sites occurring within the antigen-binding site (i.e., the CDR region) of an antibody can reduce antigen-binding activity or chemical stability. For example, deamidation or isomerization can make the molecule unstable and heterogeneous. To reduce sequence tendencies, PTM motifs can be removed by mutation. VH or VL sequences were scanned for the presence of PTM motifs, such as isomerization motifs (e.g., DG). Then, "hotspot" residues (e.g., D or G in the DG motif) were mutated to either the corresponding residue in the germline sequence or other residues with similar biophysical properties.

[0122] In some embodiments, the antibody is a monoclonal antibody, a bispecific antibody, or a multispecific antibody. In some embodiments, the antibody is monovalent, bivalent, or polyvalent.

[0123] bispecific antibody In a second embodiment, the application provides a bispecific antibody or a multispecific antibody. In some embodiments, the antibody is a bispecific antibody further comprising a second antigen-binding region that binds to a second antigen. In some embodiments, the second antigen may be a tumor-associated antigen, an immune checkpoint molecule, or an immune cell antigen.

[0124] Numerous tumor-associated antigens (TIAs) associated with specific cancers have been identified in the field. In some embodiments, TIAs are antigens that can potentially stimulate a clear tumor-specific immune response. Some of these antigens are encoded by normal cells, but are not necessarily expressed by normal cells. These antigens can be characterized as antigens that are normally silent (i.e., not expressed) in normal cells, antigens that are expressed only during specific stages of differentiation, and antigens that are expressed over time, such as embryonic and fetal antigens. Other cancer antigens are encoded by oncogenes (e.g., activated ras oncogene), suppressor genes (e.g., mutant p53), and mutant cytogenes such as fusion proteins produced by internal deletions or chromosomal translocations. Other cancer antigens can be encoded by viral genes, such as those harbored on RNA and DNA oncoviruses. Many other TIAs are known and / or commercially available, and can also be produced by those skilled in the art.

[0125] Examples of tumor-associated antigens include, but are not limited to, 5T4, alpha-fetoprotein, CA-125, mesothelin, CD19, CD20, CD22, CD23, CD30, CD33, CD40, CD56, CD79, CD78, CD123, CD138, c-Met, CSPG4, IgM, type C lectin-like molecule 1 (CLL-1), EGFR, EGFRvIII, epithelial tumor antigens, ERBB2, FLT3, folate-binding proteins, GD2, GD3, HIV-1 envelope glycoprotein gp41, HIV-1 envelope glycoprotein gpl20, melanoma-associated antigens, CD200R1, MUC-1, mutant p53, mutant ras, ROR1, VEGFR2, and combinations thereof.

[0126] In some embodiments, the second antigen is a T cell antigen. In some embodiments, the T cell antigen can be selected from the group consisting of T cell receptor (TCR), CD3, CD4, CD8, CD16, CD25, CD28, CD44, CD62L, CD69, ICOS, 41-BB (CD137), and NKG2D, or any combination thereof, or 8, CD44, CD62L, CD69, ICOS, 41-BB (CD137), and NKG2D, or any combination thereof.

[0127] In some embodiments, the second antigen is an immune checkpoint molecule. In some embodiments, the immune checkpoint molecule can be selected from the group consisting of PD-1, PD-L1, CTLA-4, and the like.

[0128] In some embodiments, the bispecific antibody comprises a single polypeptide chain or multiple (e.g., two or four) polypeptide chains, each comprising a first antigen-binding region, a second antigen-binding region, and optionally an Fc region.

[0129] The Fc region can be any isotype including but not limited to IgG1, IgG2, IgG3, and IgG4, and may contain one or more mutations or modifications. In one embodiment, the Fc region is or is derived from the IgG1 isotype and optionally has one or more mutations or modifications. In one embodiment, the Fc region is human IgG1 Fc.

[0130] In one embodiment, the Fc region is an effector function deficiency. For example, the Fc region may be an IgG1 isotype or a non-IgG1 type, such as IgG2, IgG3, or IgG4, which are mutated to have reduced or even eliminated ability to mediate effector functions such as ADCC. Such mutations are described, for example, in Dall'Acqua WF et al., J Immunol. 177(2):1129-1138 (2006) and Hezareh M, J Virol.; 75(24):12161-12168 (2001).

[0131] In one embodiment, the Fc region includes a mutation that removes the receptor site for Asn-binding glycosylation, or is otherwise manipulated to alter its glycosylation properties. For example, in the IgG1 Fc region, the N297Q mutation can be used to remove the Asn-binding glycosylation site. Therefore, in certain embodiments, the Fc region includes an IgG1 wild-type sequence having the N297Q mutation.

[0132] In further embodiments, the Fc region is glycoengineered to reduce fucose and thus enhance ADCC, for example, by adding a compound to the culture medium during antibody production, as described in U.S. Patent Application No. 2009317869, or by using FUT8 knockout cells, as described in van Berkel et al. (2010) Biotechnol. Bioeng. 105:350, or by using FUT8 knockout cells, as described in Yamane-Ohnuki et al. (2004) Biotechnol. Bioeng 87:614. Alternatively, ADCC may be optimized by using methods such as those described in Umana et al. (1999) Nature Biotech 17:176. In further embodiments, the Fc region is engineered to enhance complement activation, as described in Natsume et al. (2009) Cancer Sci. 100:2411.

[0133] nucleic acid In a third aspect, the present invention provides nucleic acids comprising a nucleotide sequence encoding an anti-CEACAM5 / 6 double-conjugated antibody or an antigen-binding fragment thereof as disclosed herein, or a bispecific antibody or an antigen-binding fragment thereof as disclosed herein.

[0134] The terms "polynucleotide" or "nucleic acid" include both single-stranded and double-stranded nucleotide polymers. Nucleic acids can be ribonucleotides, deoxyribonucleotides, or modified forms of either type of nucleotide. Such modifications include base modifications such as bromouridine and inosine derivatives, ribose modifications such as 2',3'-dideoxyribose, and internucleotide ligation modifications such as phosphorothioates, phosphorodithioates, phosphoroselenoates, phosphorodiselenoates, phosphoranilothioates, phosphoraniladetes, and phosphoramidates.

[0135] For example, the present invention provides a nucleic acid molecule encoding any one of the heavy chain variable region sequences disclosed herein. The present invention also provides a nucleic acid molecule that is at least 90%, at least 95%, at least 98%, or at least 99% identical to a nucleic acid encoding any one of the heavy chain variable region sequences disclosed herein.

[0136] For example, the present invention provides a nucleic acid molecule encoding any one of the light chain variable region sequences disclosed herein. The present invention also provides a nucleic acid molecule that is at least 90%, at least 95%, at least 98%, or at least 99% identical to a nucleic acid encoding any one of the light chain variable region sequences disclosed herein.

[0137] For example, the present invention provides a nucleic acid molecule encoding (i) any one of the heavy chain variable region sequences disclosed herein and (ii) any one of the light chain variable region sequences disclosed herein. The present invention also provides a nucleic acid molecule that is at least 90%, at least 95%, at least 98%, or at least 99% identical to a nucleic acid encoding (i) any one of the heavy chain variable region sequences disclosed herein and (ii) any one of the light chain variable region sequences disclosed herein.

[0138] For example, the present invention provides a nucleic acid molecule encoding a heavy chain variable region sequence, which includes one of the heavy chain variable region sequences disclosed herein.

[0139] In some embodiments, the present invention provides nucleic acid molecules encoding heavy chain variable region sequences comprising any one of the three CDR sequences disclosed herein.

[0140] The present invention also provides nucleic acid molecules encoding heavy chain variable region sequences, each containing a CDR sequence that is at least 90%, at least 95%, at least 98%, or at least 99% identical to any one of the heavy chain variable region sequences disclosed herein.

[0141] In some embodiments, the present invention provides nucleic acid molecules encoding heavy chain variable region sequences comprising CDR1, CDR2, and CDR3 sequences that are at least 90%, at least 95%, at least 98%, or at least 99% identical to any one of the three CDR sequences disclosed herein.

[0142] For example, the present invention provides a nucleic acid molecule encoding a light chain variable region sequence, which includes one of the CDR sequences disclosed herein.

[0143] In some embodiments, the present invention provides nucleic acid molecules encoding a light chain variable region sequence comprising any one of the three CDR sequences disclosed herein.

[0144] The present invention also provides nucleic acid molecules encoding a light chain variable region sequence, which comprises a CDR sequence that is at least 90%, at least 95%, at least 98%, or at least 99% identical to any one of the light chain variable region sequences disclosed herein.

[0145] In some embodiments, the present invention provides nucleic acid molecules encoding light chain variable region sequences comprising CDR1, CDR2, and CDR3 sequences that are at least 90%, at least 95%, at least 98%, or at least 99% identical to any one of the three CDR sequences disclosed herein.

[0146] For example, the present invention provides a nucleic acid molecule encoding (i) a heavy chain variable region sequence comprising a CDR sequence from any one of the heavy chain variable region sequences disclosed herein, and (ii) a light chain variable region sequence comprising a CDR sequence from any one of the light chain variable region sequences disclosed herein. In some embodiments, the present invention provides a nucleic acid molecule encoding (i) a heavy chain variable region sequence comprising a CDR sequence from any one of the three CDR sequences disclosed herein, and (ii) a light chain variable region sequence comprising a CDR sequence from any one of the three CDR sequences disclosed herein. The present invention also provides a nucleic acid molecule encoding (i) a heavy chain variable region sequence comprising a CDR sequence that is at least 90%, at least 95%, at least 98%, or at least 99% identical to a CDR sequence from any one of the heavy chain variable region sequences disclosed herein, and (ii) a light chain variable region sequence comprising a CDR sequence that is at least 90%, at least 95%, at least 98%, or at least 99% identical to a CDR sequence from any one of the light chain variable region sequences disclosed herein. In some embodiments, the present invention provides nucleic acid molecules encoding (i) heavy chain variable region sequences comprising CDR1, CDR2, and CDR3 sequences that are at least 90%, at least 95%, at least 98%, or at least 99% identical to any one of the three CDR sequences disclosed herein, and (ii) light chain variable region sequences comprising CDR1, CDR2, and CDR3 sequences that are at least 90%, at least 95%, at least 98%, or at least 99% identical to any one of the three CDR sequences disclosed herein.

[0147] In some embodiments, the nucleic acid is ribonucleic acid (RNA) or deoxyribonucleic acid (DNA). In some embodiments, the present invention provides ribonucleic acid (RNA) comprising a nucleotide sequence encoding an anti-CEACAM5 / 6 double-conjugated antibody or its antigen-binding fragment as disclosed herein, or a bispecific antibody or its antigen-binding fragment as disclosed herein. In some embodiments, the present invention provides deoxyribonucleic acid (DNA) comprising a deoxynucleotide sequence encoding an anti-CEACAM5 / 6 double-conjugated antibody or its antigen-binding fragment as disclosed herein, or a bispecific antibody or its antigen-binding fragment as disclosed herein.

[0148] In some embodiments, deoxyribonucleic acid (DNA) can be introduced into human cells in vivo. In some embodiments, the deoxyribonucleic acid (DNA) of the present invention is contained in a vector or delivery agent. In some embodiments, the deoxyribonucleic acid (DNA) of the present invention is incorporated into the genome of a cell.

[0149] In some embodiments, ribonucleic acid (RNA) can be introduced into human cells in vivo. In some embodiments, the ribonucleic acid (RNA) of the present invention is contained in a vector or a delivery agent.

[0150] In some specific embodiments, the ribonucleic acid (RNA) comprising a nucleotide sequence encoding the anti-CEACAM5 / 6 double-binding antibody or its antigen-binding fragment, or the bispecific antibody or its antigen-binding fragment, disclosed herein is mRNA. In some embodiments, the mRNA of the present invention is contained in a vector or delivery system (e.g., a lipidosome). In some embodiments, the mRNA may be introduced in vivo into cells of the human body via a vector or delivery system (e.g., a lipidosome) to express the CEACAM5 antibody of the present invention in vivo.

[0151] vector In a fourth aspect, the present invention further provides a vector comprising a nucleic acid comprising a nucleotide sequence encoding an anti-CEACAM5 / 6 double-conjugated antibody or an antigen-binding fragment thereof as disclosed herein, or a bispecific antibody or an antigen-binding fragment thereof as disclosed herein.

[0152] In some embodiments, the vector is a recombinant expression vector capable of expressing a polypeptide containing a heavy chain or light chain variable region of an anti-CEACAM5 / 6 double-binding antibody. For example, the present invention provides a recombinant expression vector containing any of the above nucleic acid molecules.

[0153] Any vector may be suitable for this disclosure. In some embodiments, the vector is a viral vector. In some embodiments, the vector is a retroviral vector, a DNA vector, a mouse leukemia virus vector, an SFG vector, a plasmid, an RNA vector, an adenovirus vector, a baculovirus vector, an Epstein-Barr virus vector, a papovavirus vector, a vaccinia virus vector, a herpes simplex virus vector, an adenovirus-associated vector (AAV), a lentivirus vector, or any combination thereof. Suitable exemplary vectors include, for example, pGAR, pBABE-puro, pBABE-neo largeTcDNA, pBABE-hygro-hTERT, pMKO.1 GFP, MSCV-IRES-GFP, pMSCV PIG (Puro IRES GFP empty plasmid), pMSCV-loxp-dsRed-loxp-eGFP-Puro-WPRE, MSCV IRES luciferase, pMIG, MDH1-PGK-GFP_2.0, TtRMPVIR, pMSCV-IRES-mCherry FP, pRetroX GFP T2A Cre, pRXTN, pLncEXP, and pLXIN-Luc.

[0154] Recombinant expression vectors can be any suitable recombinant expression vector. Suitable vectors include plasmids and viruses, and vectors designed for proliferation and expansion, or for expression, or both. For example, vectors may be selected from the pUC series (Fermentas Life Sciences, Glenburnie, Maryland), the pBluescript series (Stratagene, La Jolla, California), the pET series (Novagen, Madison, Wisconsin), the pGEX series (Pharmacia Biotech, Uppsala, Sweden), and the pEX series (Clontech, Palo Alto, California). Bacteriophage vectors such as λGT10, λGT11, λZapII (Stratagene), λEMBL4, and λNM1149 can also be used. Examples of plant expression vectors useful in the context of this disclosure include pBI01, pBI101.2, pBI101.3, pBI121, and pBIN19 (Clontech). Examples of animal expression vectors useful in the context of this disclosure include pcDNA, pEUK-Cl, pMAM, and pMAMneo (Clontech).

[0155] Recombinant expression vectors can be prepared using standard recombinant DNA techniques, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd edition, Cold Spring Harbor Press, New York, Cold Spring Harbor, 2001; and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons, New York, 1994. Expression vector constructs, whether circular or linear, can be prepared to include a functional replication system in prokaryotic or eukaryotic host cells. The replication system may be derived from, for example, ColEl, 2μ plasmid, λ, SV40, bovine papillomavirus, etc.

[0156] The vectors of the present invention can be introduced into cells. In some embodiments, the vectors of the present invention can be introduced into cells in vitro or ex vivo. Optionally, the cells introduced with the vectors can then be administered into the body of the subject. In some embodiments, the vectors of the present invention can be introduced into cells in vivo.

[0157] For example, the vector may be an adenovirus vector comprising a nucleotide sequence encoding an anti-CEACAM5 / 6 double-conjugated antibody or its antigen-binding fragment as disclosed herein, or a bispecific antibody or its antigen-binding fragment as disclosed herein. The vector may be administered into the body of a subject and subsequently enter the target cells in vivo, thereby integrating the nucleotide sequence encoding the anti-CEACAM5 / 6 double-conjugated antibody or its antigen-binding fragment as disclosed herein, or a bispecific antibody or its antigen-binding fragment as disclosed herein, into the cell genome, and subsequently the cell expresses the anti-CEACAM5 / 6 double-conjugated antibody or its antigen-binding fragment as disclosed herein.

[0158] host cell In a fifth aspect, the present invention further provides a host cell comprising a nucleic acid or a vector disclosed herein.

[0159] Any cell may be used as a host cell for the nucleic acids or vectors of this disclosure. In some embodiments, the cell may be a prokaryotic cell, a fungal cell, a yeast cell, or a higher eukaryotic cell such as a mammalian cell. Suitable prokaryotic cells include, but are not limited to, bacteria, such as Gram-negative or Gram-positive organisms, such as Enterobacteriaceae, such as Escherichia, such as Escherichia coli; Enterobacter; Erwinia; Klebsiella; Proteus; Salmonella, such as Salmonella typhimurium; Serratia, such as Serratia marcescens and Shigella; rod-shaped bacteria such as Bacillus subtilis and B. licheniformis; Pseudomonas such as Pseudomonas aeruginosa; and Streptomyces. In some embodiments, the cells are human cells. In some embodiments, the cells are immune cells. In some embodiments, the host cells include, for example, CHO cells, e.g., CHOS cells and CHOK1 cells, or HEK293 cells, e.g., HEK293A, HEK293T, and HEK293FS.

[0160] The host cells of the present invention are prepared by introducing a vector or nucleic acid disclosed herein in vitro or ex vivo. The host cells of the present invention may be administered into a subject's body, and the host cells express an anti-CEACAM5 / 6 double-conjugate antibody or its antigen-binding fragment disclosed herein in vivo to treat a disease disclosed herein.

[0161] The present invention further provides host cells into which any of the above vectors have been introduced. The present invention further provides a method for producing the antibodies and antibody fragments of the present invention by culturing the host cells under conditions that enable the production of antibodies or antibody fragments, and recovering the antibodies and antibody fragments thus produced.

[0162] Conjugates and Antibody-Drug Conjugates The antibody or antigen-binding fragment of the present invention can bind to a chemical portion to form a conjugate.

[0163] In the context of this disclosure, “conjugate” is an antibody or antibody fragment (such as an antigen-binding fragment) covalently bound to a chemical portion. The chemical portion may be, for example, a drug, toxin, therapeutic agent, detectable label, protein, nucleic acid, lipid, nanoparticle, carbohydrate, or recombinant virus. Antibody conjugates are often referred to as “immunoconjugates.” When the conjugate includes an antibody linked to a drug (e.g., a cytotoxic agent), the conjugate is often referred to as an “antibody-drug conjugate” or “ADC.”

[0164] The terms “conjugated” or “linked” may refer to the process of combining two polypeptides into a single, continuous polypeptide molecule. In one embodiment, an antibody is conjugated to a chemical moiety. In another embodiment, an antibody conjugated to a chemical moiety is further conjugated to a lipid or other molecule, or to a protein or peptide, to increase its half-life in the body. Conjugation may be by either chemical or recombinant means. In one embodiment, conjugation is chemical, and the reaction between the antibody moiety and the chemical moiety generates a covalent bond formed between the two molecules to form a single molecule. A peptide linker (short peptide sequence) may optionally be included between the antibody and the chemical moiety.

[0165] The chemical moiety can be linked to the antibody of the present invention using any number of means known to those skilled in the art. Both covalent and non-covalent linking means may be used. The procedure for linking the chemical moiety to the antibody varies according to the chemical structure of the chemical moiety. Polypeptides typically contain various functional groups, such as carboxylic acid (COOH), free amine (-NH2), or sulfhydryl (-SH) groups, which are available for reaction with suitable functional groups on the antibody, resulting in the linking of the chemical moiety. Alternatively, the antibody may be derivatized to expose or link further reactive functional groups. Derivatization may involve linking to any of a number of known linker molecules. The linker can be any molecule used to link the antibody to the chemical moiety. The linker can form covalent bonds to both the antibody and the chemical moiety. Suitable linkers are well known to those skilled in the art and include, but are not limited to, linear or branched carbon linkers, heterocyclic carbon linkers, or peptide linkers. When the antibody and chemical moieties are polypeptides, the linker can be attached to the constituent amino acids via their side chain groups (e.g., via disulfide linkage to cysteine) or to the α-carbon amino group and carboxyl group of the terminal amino acids.

[0166] In some situations, it is desirable to release a chemical portion from the antibody when the immunoconjugate reaches its target site. Therefore, in these situations, the immunoconjugate contains a ligature that can be cleaved near the target site.

[0167] The cleavage of the linker, which releases the chemical portion from the antibody, can be facilitated by enzymatic activity or by conditions in which the immunoconjugate is exposed either inside or near the target site.

[0168] Considering the numerous methods reported for conjugating various radiodiagnostic compounds, radiotherapeutic compounds, labels (such as enzymes or fluorescent molecules), drugs, toxins, and other agents to antibodies, a person skilled in the art will be able to determine a suitable method for conjugating a given agent to an antibody or other polypeptide.

[0169] The antibodies disclosed herein can be derivatized or linked to another molecule (such as another peptide or protein). Generally, antibodies or parts thereof are derivatized so that their binding to a target antigen is not adversely affected by the derivatization or labeling. For example, an antibody can be functionally linked (by chemical coupling, gene fusion, non-covalent association or other means) to one or more other molecular entities such as another antibody (e.g., a bispecific antibody or diabody), a detection agent, a pharmaceutical, and / or a protein or peptide that can mediate the association of the antibody or antibody moiety with another molecule (such as a streptavidin core region or polyhistidine tag).

[0170] One type of derivatized antibody is produced by crosslinking two or more antibodies (of the same or different types). Suitable crosslinking agents include heterobifunctional (e.g., m-maleimidobenzoyl-N-hydroxysuccinimide ester) or homobifunctional (e.g., disuccinimidyl severate) crosslinking agents having two distinctly reactive groups separated by a suitable spacer. Such linkers are commercially available.

[0171] In some embodiments of the conjugates disclosed herein, the chemical portion is selected from the group consisting of therapeutic agents, detectable portions, and immunostimulatory molecules.

[0172] In some embodiments, the therapeutic agent may be an immunomodulator, a radiocompound, an enzyme (e.g., perforin), a chemotherapeutic agent (e.g., cisplatin), or a toxin, but is not limited thereto. In some embodiments, the therapeutic agent may be a tubulin inhibitor such as meitansine, geldanamycin, or a tubulin binder (e.g., auristatin), or a secondary groove binder such as calicheamicin.

[0173] Other suitable therapeutic agents include, for example, low molecular weight cytotoxic agents, i.e., compounds with a molecular weight of less than 700 daltons that have the ability to kill mammalian cells. Such compounds may also contain toxic metals that can have cytotoxic effects. Furthermore, it should be understood that these low molecular weight cytotoxic agents also include prodrugs, i.e., compounds that decompose or are converted under physiological conditions to release cytotoxic agents. Examples of such drugs include cisplatin, meitansine derivatives, rakelmycin, calicheamicin, docetaxel, etoposide, gemcitabine, ifosfamide, irinotecan, melphalan, mitoxantrone, solfimer sodium photophylline II, temozolomide, topotecan, trimethrexate glucuronide, auristatin E, vincristine, and doxorubicin; peptide cytotoxic agents, i.e., proteins or fragments thereof that have the ability to kill mammalian cells, for example. Examples include lysine, diphtheria toxin, Pseudomonas bacterial exotoxin A, DNase and RNase; radionuclides, i.e., unstable isotopes of elements that decay with the simultaneous emission of one or more α-particles, β-particles, or γ-rays, such as iodine-131, rhenium-186, indium-111, yttrium-90, bismuth-210, bismuth-213, actinium-225, and astatine-213, and chelating agents may be used to promote the association of these radionuclides to molecules or their polymers.

[0174] In some embodiments, the detectable portion can be selected from the group consisting of biotin, streptavidin, enzymes or their catalytically active fragments, radionuclides, nanoparticles, paramagnetic metal ions, or fluorescent, phosphorescent, or chemiluminescent molecules. Examples of detectable portions for diagnostic purposes include fluorescent labels, radiolabels, enzymes, nucleic acid probes, and contrast agents.

[0175] Antibodies can be conjugated with detectable markers, such as those detectable by ELISA, spectrophotometry, flow cytometry, microscopy, or imaging techniques (e.g., computed tomography (CT), computed axial tomography (CAT) scans, magnetic resonance imaging (MRI), nuclear magnetic resonance imaging (NMRI), magnetic resonance imaging (MTR), ultrasound, fiber optic imaging, and laparoscopy). Specific non-limiting examples of detectable markers include fluorescent substances, chemiluminescent agents, enzyme conjugates, radioisotopes, and heavy metals or compounds (e.g., superparamagnetic iron oxide nanocrystals for detection by MRI). For example, useful detectable markers include fluorescent compounds such as fluorescein, fluorescein isothiocyanate, rhodamine, 5-dimethylamine-1-naphthalenesulfonyl chloride, phycoerythrin, and lanthanidrin photomeridians. Bioluminescent markers such as luciferase, green fluorescent protein (GFP), and yellow fluorescent protein (YFP) are also useful.

[0176] Antibodies or antigen-binding fragments can also be conjugated with enzymes useful for detection, such as horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase, and glucose oxidase. When a bispecific antibody or antigen-binding fragment is conjugated with a detectable enzyme, it can be detected by adding additional reagents used by the enzyme to produce a identifiable reaction product. For example, in the presence of the drug horseradish peroxidase, the addition of hydrogen peroxide and diaminobenzidine results in a visually detectable colored reaction product. Bispecific antibodies or antigen-binding fragments can also be conjugated with biotin and detected through indirect measurement of avidin or streptavidin binding. Note that avidin itself can be conjugated with enzymes or fluorescent labels.

[0177] Antibodies can be fused to self-labeled protein tags (e.g., HaloTag). For example, the protein tag may be cloned at the end of the constant region. HaloTag is a self-labeled protein tag derived from a bacterial enzyme (haloalkane dehalogenase) designed to covalently bind to a synthetic ligand. In some examples, the synthetic ligand includes a chloroalkane linker bound to a fluorescent substance such as a near-infrared fluorescent agent (Los et al. (2008) ACS Chem Biol. 3(6):373-82).

[0178] Antibodies can be labeled with magnetic materials such as gadolinium. Antibodies can also be labeled with lanthanides (e.g., europium and dysprosium) and manganese.

[0179] Paramagnetic particles, such as superparamagnetic iron oxide, are also useful as labels. Bispecific antibodies can also be labeled with a predetermined polypeptide epitope that is recognized by a secondary reporter (e.g., leucine zipper pair sequence, secondary antibody binding site, metal-binding domain, epitope tag). In some embodiments, the label is attached by spacer arms of varying lengths to reduce potential steric hindrance.

[0180] Antibodies can also be labeled with radiolabeled amino acids. Radiolabeling can be used for both diagnostic and therapeutic purposes. For example, radiolabeling can be used to detect the expression of a target antigen by X-ray, emission spectroscopy, or other diagnostic techniques. Examples of polypeptide labeling include the following radioisotopes or radionucleotides: 3 H, 14 C, 15 N, 35 S, 90 Y, 99 Tc, 111 In, 125 I, 131 I is one example, but it is not limited to these.

[0181] In some embodiments, the immunostimulatory molecule is an immune effector molecule that stimulates an immune response. For example, the immunostimulatory molecule may be a cytokine such as IL-2 and IFN-γ, a chemokine such as IL-8, platelet factor 4, melanoma growth-stimulating protein, complement activator; a viral / bacterial protein domain, or a viral / bacterial peptide.

[0182] In some embodiments, the antibody or antigen-binding fragment of the present invention is fluorescently labeled, radiolabeled, or conjugated to a drug moiety. In some embodiments, the antibody or antigen-binding fragment of the present invention is conjugated to a drug moiety to form an antibody-drug conjugate (ADC).

[0183] Accordingly, in a sixth aspect, the present invention provides an antibody-drug conjugate (ADC) comprising an antibody or antigen-binding fragment thereof according to a first aspect of the present invention or a bispecific antibody according to a second aspect of the present invention and a drug portion conjugated thereto via a linker.

[0184] The terms “drug portion,” “drug payload,” “therapeutic molecule,” “therapeutic payload,” “therapeutic agent,” and “therapeutic portion,” as used interchangeably herein, refer to the chemical or biological portion conjugated to a double-conjugated antibody or its antigen-binding fragment that binds to CEACAM5 and CEACAM6.

[0185] Examples of drugs that may be used in ADCs, i.e., drugs that can be conjugated to antibodies, are provided below and include antibiotics, DNA synthesis inhibitors, RNA polymerase II inhibitors, and RNA spliceosome inhibitors, microtubule inhibitors, antitumor antibiotics, immunomodulators, gene therapy vectors, alkylating agents, anti-angiogenic agents, antimetabolites, boron-containing agents, chemoprotective agents, hormones, glucocorticoids, phototherapeutic agents, oligonucleotides, radioisotopes, radiosensitizers, topoisomerase inhibitors (such as topoisomerase I inhibitors), tyrosine kinase inhibitors, and combinations thereof.

[0186] In some embodiments, the drug portion is selected from the group consisting of microtubule inhibitors, antibiotics, DNA synthesis inhibitors, topoisomerase inhibitors, RNA polymerase II inhibitors, and RNA spliceosome inhibitors.

[0187] In some embodiments, the drug portion is a microtubule inhibitor. In some specific embodiments, the drug portion is auristatin. In some specific embodiments, the drug portion is maytansine. In some specific embodiments, the drug portion is tubulicin. In some specific embodiments, the drug portion is cryptophycin. In some specific embodiments, the drug portion is rhizoxin.

[0188] In some embodiments, the drug portion is an antibiotic. In some specific embodiments, the drug portion is calicheamycin. In some specific embodiments, the drug portion is doxorubicin. In some specific embodiments, the drug portion is an anthracycline.

[0189] In some embodiments, the drug moiety is a DNA synthesis inhibitor. In some specific embodiments, the drug moiety is duocalmycin. In some specific embodiments, the drug moiety is PBD (pyrrolobenzodiazepine). In some specific embodiments, the drug moiety is IGN (indolinobenzodiazepine).

[0190] In some embodiments, the drug portion is a topoisomerase inhibitor. In some specific embodiments, the drug portion is a camptothecin analog.

[0191] In some embodiments, the drug portion is an RNA polymerase II inhibitor. In some specific embodiments, the drug portion is amanitin.

[0192] In some embodiments, the drug portion is an RNA spliceosome inhibitor selected from the group consisting of spliceostatins and tylanstatins.

[0193] In some embodiments, maytansinoids (DM1, DM2, DM3, DM4, maytansin, and anthamitosin) and their analogues are preferred.

[0194] In some embodiments, auristatins (MMAE, MMAF, MMAD, and anthamitocin) and their analogues are preferred.

[0195] In some embodiments, PBD (pyrrolobenzodiazepine, or pyrrolo[2,1-c][1,4]-benzodiazepine, e.g., SG3199) is preferred. PBD is a sequence-selective DNA alkylating antibiotic with significant antitumor properties. PBD has the ability to recognize and bind to specific sequences of DNA. One such sequence is PuGPu (purine-guanine-purine). PBD can also bind to PuGPy (purine-guanine-pyrimidine) or PyGPu sequences beyond PyGPy sequences.

[0196] In some embodiments, the PBD drug portion is SG3199, which is a cytotoxic DNA subgroove interstrand crosslinked pyrrolobenzodiazepine (PBD) dimer.

[0197] In some embodiments, camptothecin analogs are preferred. Camptothecin analogs are a type of DNA topoisomerase I inhibitor. Unlike monomethyl auristatin E, camptothecin-based therapies do not clinically cause peripheral neuropathy, suggesting that SGN-CD30C may have the potential to avoid one of the most common adverse events associated with BV.

[0198] In some specific embodiments, the camptothecin drug portion is 7-ethyl-10-hydroxycamptothecin (also known as SN38).

[0199] In some specific embodiments, the camptothecin drug portion is Dxd, a potent topoisomerase I inhibitor, or an analog or derivative thereof.

[0200] In some specific embodiments, the camptothecin drug portion is exatecan (also known as DX-8951).

[0201] In some embodiments, calicheamicin, an antitumor antibiotic and cytotoxic agent that causes double-strand DNA breaks, is preferred. N-acetylcalicheamicin is a derivative of calicheamicin and is a potent enediyne antitumor antibiotic. In other embodiments, the drug moiety is doxorubicin or its analogues or derivatives.

[0202] One or more drug moieties (e.g., therapeutic and / or diagnostic agents) may be indirectly conjugated to an anti-CEACAM5 / 6 double-binding antibody (e.g., by a linker having direct covalent or non-covalent interactions). The linker may be a chemical linker, such as homobifunctional and heterobifunctional crosslinkers, which are available from many commercial sources.

[0203] In some embodiments, the linker includes a severable linker or a non-severable linker.

[0204] A linker is susceptible to cleavage such as acid-induced cleavage, photo-induced cleavage, peptidase-induced cleavage, esterase-induced cleavage, and disulfide bond cleavage, under conditions that allow the compound or antibody to maintain its activity (a cleavable linker). Alternatively, a linker may be substantially resistant to cleavage (e.g., a stable linker or an uncleavable linker).

[0205] In some embodiments, the linker is an acid-unstable linker. In some embodiments, the linker is a photo-unstable linker. In some embodiments, the linker is a protease-sensitive linker. In some embodiments, the linker is a hydrazone linker. In some embodiments, the linker is an esterase-cleavable linker. In some embodiments, the linker is a dimethyl linker. In some embodiments, the linker is a disulfide-containing linker. In some embodiments, the linker is a hydrophilic linker. In some embodiments, the linker is a procharged linker. In some embodiments, the linker is an acid-based linker.

[0206] In some embodiments, the linker includes an acid-unstable linker. In some embodiments, the linker includes a hydrophilic linker. In some embodiments, the linker includes a protease-sensitive linker. In some embodiments, the linker includes a photo-unstable linker. In some embodiments, the linker includes a hydrazone linker. In some embodiments, the linker includes a dimethyl linker. In some embodiments, the linker includes a disulfide-containing linker.

[0207] In some embodiments, the linker may contain amino acid units. In one such embodiment, the amino acid units enable cleavage of the linker by proteases, thereby facilitating the release of the drug from the antibody-drug conjugate upon exposure to intracellular proteases such as lysosomal enzymes. Exemplary amino acid units include, but are not limited to, dipeptides, tripeptides, tetrapeptides, and pentapeptides. Exemplary dipeptides include valine-citrulline (vc or val-cit) and alanine-phenylalanine (af or ala-phe); phenylalanine-lysine (fk or phe-lys); or N-methyl-valine-citrulline (Me-valcit). Exemplary tripeptides include glycine-valine-citrulline (gly-val-cit) and glycine-glycine-glycine (gly-gly-gly). In some embodiments, linkers containing vc (valine-citrulline) units are preferred. Amino acid units can be designed and optimized in terms of their selectivity for enzymatic cleavage by specific enzymes, such as tumor-associated proteases, cathepsins B, C, and D, or plasmin proteases.

[0208] In one embodiment, the linker used in this disclosure is MC (6-maleimidocaproyl), Val-Cit (valine-citrulline), PABC (para-aminobenzyloxycarbonyl), DMEA (dimethylethylamine), Val-Cit-PABC, MC-Val-Cit-PABC, MC-Val-Cit-PABC-DMEA, CL2A, Mal-PEG8-Val-Ala-PABC, GGFG (glycine-glycine-phenylalanine-glycine), MC-GGFG-aminomethyl, AcBut (4-(4-acetylphenoxy)-butanoic acid), dimethylhydrazide (3-methyl AcBut-dimethylhydrazide, SPDP (N-succinimidyl-3-(2-pyridyldithio)propionate), SPP (N-succinimidyl-4-(2-pyridyldithio)pentanoate), SPDB (N-succinimidyl-4-(2-pyridyldithio)butanoate), Sulfo-SPDB (N-succinimidyl-4-(2-pyridyldithio)-2-sulfobutanoate), SIA (N-succinimidyliodoacetate), SIAB (N-succinimidyl(4-iodoacetyl)aminobenzoate), Maleimide PEG It is derived from crosslinking reagents such as NHS, SMCC (N-succinimidyl 4-(maleimidomethyl)cyclohexanecarboxylate), sulfo-SMCC (N-sulfosuccinimidyl 4-(maleimidomethyl)cyclohexanecarboxylate), or 2,5-dioxopyrrolidine-1-yl17-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)-5,8,11,14-tetraoxo-4,7,10,13-tetraazaheptadecane-1-oate (CX1-1).

[0209] In another embodiment, the linker used in the present disclosure is derived from a crosslinking agent such as sulfo-SPDB (N-succinimidyl-4-(2-pyridyldithio)-2-sulfobutanoate), mc (6-maleimidocaproyl), Val-Cit (valine-citrulline), PABC (para-aminobenzyl alcohol), Val-Cit-PABC, mc-Val-Cit-PABC, CL2A, mal-PEG8-Val-Ala-PABC, GGFG (glycine-glycine-phenylalanine-glycine), mc-GGFG-aminomethyl, AcBut (4-(4-acetylphenoxy)-butanoic acid), dimethylhydrazide (3-methyl-3-mercaptobutanehydrazide), AcBut-dimethylhydrazide, or SMCC (N-succinimidyl-4-(maleimidomethyl)cyclohexanecarboxylate).

[0210] In preferred embodiments, the linker is selected from the group consisting of MC (6-maleimidocaproyl), Val-Cit (valine-citrulline), PABC (para-aminobenzyloxycarbonyl), DMEA (dimethylethylamine), Val-Cit-PABC, MC-Val-Cit-PABC, MC-Val-Cit-PABC-DMEA, GGFG (glycine-glycine-phenylalanine-glycine), MC-GGFG-aminomethyl, AcBut (4-(4-acetylphenoxy)-butanoic acid), and AcBut-dimethylhydrazide. In more preferred embodiments, the linker is MC-Val-Cit-PABC.

[0211] Pharmaceutical composition In a seventh aspect, the present invention provides a pharmaceutical composition comprising (i) an antibody or antigen-binding fragment thereof according to a first aspect of the present invention, or a bispecific antibody according to a second aspect of the present invention, or a nucleic acid according to a third aspect of the present invention, or a vector according to a fourth aspect of the present invention, or a host cell according to a fifth aspect of the present invention, or an ADC according to a sixth aspect of the present invention, and optionally (ii) a pharmaceutically acceptable carrier or excipient.

[0212] The present invention provides pharmaceutical compositions comprising the antibody of the present invention. In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier. As used herein, “pharmaceutically acceptable carrier” includes any and all physiologically compatible solvents, buffers, dispersion media, coatings, antimicrobial and antifungal agents, isotonic agents, and absorption retarders. Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, or epidermal administration (e.g., by injection or infusion). For example, in some embodiments, the composition for intravenous administration is typically a solution in a sterile isotonic aqueous buffer.

[0213] The antibodies or drugs of the present invention (also referred to herein as “active compounds”), as well as their derivatives, fragments, analogues, and homologs, can be incorporated into pharmaceutical compositions suitable for administration. Such compositions typically comprise the antibody or drug and a pharmaceutically acceptable carrier. As used herein, “pharmaceutically acceptable carrier” is intended to include any and all solvents, dispersion media, coatings, antimicrobial and antifungal agents, isotonic agents and absorption retarders, etc., that are suitable for pharmaceutically effective administration. Suitable carriers are described in the latest edition of Remington's Pharmaceutical Sciences, a standard reference in the art, incorporated herein by reference. Preferred examples of such carriers or diluents include, but are not limited to, water, physiological saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Liposomes and non-aqueous vehicles (e.g., non-volatile oils) may also be used. The use of such media and drugs for pharmaceutically active substances is well known in the art. Unless any conventional media or drug is incompatible with the active compound, its use in a composition is intended. Auxiliary active compounds can also be incorporated into the composition.

[0214] The pharmaceutical compositions disclosed herein are formulated to suit their intended route of administration. Examples of routes of administration include parenteral administration, such as intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (i.e., topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application may contain the following components: sterile diluents, such as water for injection, physiological saline, fixative oil, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents; antimicrobial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates, or phosphates; and agents for adjusting tonicity, such as sodium chloride or dextrose. pH can be adjusted with an acid or base such as hydrochloric acid or sodium hydroxide. Parenteral preparations may be sealed in glass or plastic ampoules, disposable syringes, or multi-dose vials.

[0215] Suitable pharmaceutical compositions for injection include sterile aqueous solutions (if water-soluble) or dispersions, and sterile powders for the immediate preparation of sterile injection solutions or dispersions. Suitable carriers for intravenous administration include physiological saline, bacteriostatic water, Cremophor EL® (BASF, Persipane, New Jersey), or phosphate-buffered saline (PBS). In all cases, the composition must be sterile and fluid enough to be easily injected. It must be stable under manufacturing and storage conditions and protected from contamination by microorganisms such as bacteria and fungi. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. Adequate fluidity can be maintained, for example, by the use of coatings such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants. Microbial action can be prevented by various antimicrobial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, and thimerosal. In some embodiments, it may be preferable to include an isotonic agent in the composition, such as a sugar, polyalcohol, such as mannitol, sorbitol, or sodium chloride. Sustained absorption of the injectable composition can be achieved by including an absorption-delaying agent in the composition, such as aluminum monostearate or gelatin.

[0216] Sterile injectable solutions can be prepared by incorporating the required amount of the active compound into a suitable solvent, along with one or a combination of the components listed above as needed, followed by sterilization by filtration. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and other necessary components from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, the preparation method includes vacuum drying and freeze-drying, from which powders of the active component and any further desired components are obtained from a pre-sterilized filtered solution.

[0217] Oral compositions generally contain an inert diluent or food carrier. They can be encapsulated in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, lozenges, or capsules. Oral compositions may also be prepared using a fluid carrier for use as a mouthwash, where the compound in the fluid carrier is applied orally, gargled, spat out, or swallowed. Pharmaceutically compatible binders and / or adjuvant materials may be included as part of the composition. Tablets, pills, capsules, lozenges, etc. may contain any of the following ingredients or compounds of similar properties: binders such as microcrystalline cellulose, tragacanth gum, or gelatin; excipients such as starch or lactose, disintegrants such as alginic acid, Primogel, or corn starch; lubricants such as magnesium stearate or Sterotes; flow promoters such as colloidal silicon dioxide; sweeteners such as sucrose or saccharin; and flavoring agents such as peppermint, methyl salicylate, or orange flavoring.

[0218] For administration by inhalation, the compound is delivered in the form of an aerosol spray from a pressurized container or dispenser containing a suitable propellant, such as a gas like carbon dioxide, or from a nebulizer.

[0219] Systemic administration may be by transmucosal or transdermal means. In the case of transmucosal administration, a suitable penetrating agent is used in the formulation for the barrier to which the agent is to be penetrated. Such penetrating agents are generally known in the art and, for example, for transmucosal administration, include surfactants, bile salts, and fusidic acid derivatives. Transmucosal administration can be achieved by the use of nasal sprays or suppositories. For transdermal administration, the active compound is formulated in the form of an ointment, paste, gel, or cream, as is generally known in the art.

[0220] The compounds can also be prepared for rectal delivery in the form of suppositories (e.g., using conventional suppository bases such as cocoa butter and other glycerides) or retained enemas.

[0221] In one embodiment, the active compound is prepared with a carrier that protects the compound from rapid elimination from the body, such as a controlled-release formulation involving an implantation and microencapsulation delivery system. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydride, polyglycolic acid, collagen, polyorthoester, and polylactic acid may be used. Methods for preparing such formulations will be apparent to those skilled in the art. The materials are also commercially available from Alza Corporation and Nova Pharmaceuticals, Inc. Liposome suspensions (containing liposomes targeted to infected cells with monoclonal antibodies against viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, such as those described in U.S. Patent No. 4,522,811.

[0222] For ease of administration and uniformity of dosage, it is particularly advantageous to formulate oral or parenteral compositions into unit dosage forms. As used herein, a unit dosage form refers to a physically distinct unit suitable as a unit dose for the subject to be treated, each unit containing a predetermined amount of the active compound calculated to produce the desired therapeutic effect in conjunction with the necessary pharmaceutical carrier. The specifications of the unit dosage forms of the present invention are defined and directly depended on the inherent characteristics of the active compound, the specific therapeutic effect to be achieved, and the limitations inherent in the art for which such active compounds are formulated for the treatment of an individual.

[0223] The pharmaceutical composition may be contained in a container, pack, or dispenser, along with instructions for administration.

[0224] The present invention provides therapeutic compositions comprising the anti-CEACAM5 / 6 double-conjugated antibody or its antigen-binding fragment. The therapeutic compositions according to the present invention are administered together with suitable carriers, excipients, and other agents incorporated into the formulation to provide improved transfer, delivery, resistance, etc. Many suitable formulations can be found in the prescription collection known to all pharmacists: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pennsylvania. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic)-containing vesicles (such as LIPOFECTIN®), DNA conjugates, anhydrous absorbent pastes, oil-in-water and water-in-oil emulsions, emulsion carbowaxes (polyethylene glycol of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowaxes. See also Powell et al. "Compendium of excipients for parenteral formulations" PDA (1998) J Pharm Sci Technol 52:238-311.

[0225] Production method Monoclonal antibodies can be prepared using hybridoma methods, such as those described in Kohler and Milstein, Nature, 256:495 (1975). In hybridoma methods, mice, hamsters, or other suitable host animals are typically immunized with an immunizer to induce lymphocytes that produce or are capable of producing antibodies that specifically bind to the immunizer. Alternatively, lymphocytes can be immunized in vitro.

[0226] Immunotherapeutic agents typically include protein antigens, their fragments, or fusion proteins. Generally, peripheral blood lymphocytes are used when human-derived cells are desired, or spleen cells or lymph node cells are used when non-human mammalian sources are desired. The lymphocytes are then fused with immortalized cell lines using a suitable fusion agent such as polyethylene glycol to form hybridoma cells (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) pp. 59-103). Immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, bovine, and human origin. Rat or mouse myeloma cell lines are typically used. Hybridoma cells can preferably be cultured in a suitable culture medium containing one or more substances that inhibit the proliferation or survival of unfused immortalized cells. For example, if parental cells lack the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), the culture medium for hybridomas typically contains hypoxanthine, aminopterin, and thymidine ("HAT medium"), and these substances inhibit the proliferation of HGPRT-deficient cells.

[0227] Preferred immortalized cell lines are those that efficiently fuse, support stable high-level expression of antibodies by selected antibody-producing cells, and are sensitive to media such as HAT medium. More preferred immortalized cell lines are mouse myeloma lines, which can be obtained, for example, from the Salk Institute Cell Distribution Center in San Diego, California, and the American Type Culture Collection in Manassas, Burnisia. Human myeloma and mouse-human heterozygous myeloma cell lines have also been described for the production of human monoclonal antibodies. (See Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York (1987) pp. 51-63).

[0228] The culture medium in which hybridoma cells are cultured can then be assayed for the presence of monoclonal antibodies against the antigen. Preferably, the binding specificity of the monoclonal antibodies produced by hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay (e.g., radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA)). Such techniques and assays are well known in the art. The binding affinity of monoclonal antibodies can be determined, for example, by Scatchard analysis as described in Munson and Pollard, Anal. Biochem., 107:220 (1980). Furthermore, in the therapeutic application of monoclonal antibodies, it is important to identify antibodies that have high specificity and high binding affinity to the target antigen.

[0229] After the desired hybridoma cells are identified, clones can be subcloned using limiting dilution procedures and propagated by standard methods. (See Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) pp. 59-103). Suitable culture media for this purpose include, for example, Dulbecco's Modified Eagle Medium and RPMI-1640 Medium. Alternatively, hybridoma cells can be grown in vivo as ascites in mammals.

[0230] Monoclonal antibodies secreted by subclones can be isolated or purified from culture medium or ascites fluid by conventional immunoglobulin purification procedures (e.g., protein A-Sepharose chromatography, hydroxyl apatite chromatography, gel electrophoresis, dialysis, or affinity chromatography).

[0231] Monoclonal antibodies can also be produced by recombinant DNA methods, such as those described in U.S. Patent No. 4,816,567. The DNA encoding the monoclonal antibodies of the present invention can be readily isolated and sequenced using conventional procedures (for example, by using oligonucleotide probes that can specifically bind to the genes encoding the heavy and light chains of the mouse antibody). Hybridoma cells of the present invention serve as a preferred source of such DNA. After isolation, the DNA can be placed in an expression vector and then transfected into host cells such as monkey COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin proteins to obtain the synthesis of monoclonal antibodies in recombinant host cells. The DNA can also be modified, for example, by substituting the coding sequences of human heavy and light chain constant domains for homologous mouse sequences (see U.S. Patent No. 4,816,567; Morrison, Nature 368,812-13 (1994)), or by covalently bonding all or part of the coding sequence of a non-immunoglobulin polypeptide to the immunoglobulin coding sequence. Such non-immunoglobulin polypeptides can be substituted for the constant domain of the antibody of the present invention, or for the variable domain of one antigen-binding site of the antibody of the present invention, to produce a chimeric bivalent antibody.

[0232] Fully human antibodies are antibody molecules in which the entire sequence of both the light and heavy chains, including the CDR, is derived from human genes. Such antibodies are referred to herein as “humanized antibodies,” “human antibodies,” or “fully human antibodies.” Human monoclonal antibodies can be prepared using trioma technology, human B-cell hybridoma technology (see Kozbor, et al., 1983 Immunol Today 4:72), and EBV hybridoma technology for producing human monoclonal antibodies (see Cole, et al., 1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp.77-96). Human monoclonal antibodies can be used and produced by using human hybridomas (see Cote, et al., 1983. Proc Natl Acad Sci USA 80:2026-2030) or by transforming human B cells with Epstein-Barr virus in vitro (see Cole, et al., 1985 In:MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp.77-96).

[0233] Furthermore, humanized antibodies can be produced in transgenic plants as an inexpensive alternative to existing mammalian systems. For example, transgenic plants can be tobacco plants, namely Nicotiania benthamiana and Nicotiana tabaccum. Antibodies are purified from plant leaves. Stable transformation of plants can be achieved using Agrobacterium tumefaciens or particle impaction. For example, a nucleic acid expression vector containing at least heavy and light chain sequences is expressed via transformation in a bacterial culture, namely A. tumefaciens strain BLA4404. Plant infiltration can be achieved by injection. Soluble leaf extracts can be prepared by grinding leaf tissue in a mortar and by centrifugation. Isolation and purification of antibodies can be readily carried out by many methods known to those skilled in the art. Other methods for antibody production in plants are described, for example, Fischer et al., Vaccine, 2003, 21:820-5 and Ko et al., Current Topics in Microbiology and Immunology, Vol.332, 2009, pp.55-78. Accordingly, the present invention further provides any cell or plant comprising a vector that encodes or produces the antibody of the present invention.

[0234] Furthermore, the target (human) antibody may be produced in fungi. For example, the fungus could be Myceliophthora thermophila (e.g., Myceliophthora thermophila strain C1; Visser et al. (2011) Industrial Biotechnology 7(3):214-223). Other examples include species of Aspergillus (e.g., Aspergillus oryzae (Huynh et al. (2020) Fungal Biology and Biotechnology 7:7), Aspergillus niger (Ward et al. (2004) Environ. Microbiol. 70:2567-76), or Aspergillus awamori (Joosten et al. (2003) Microb. Cell Fact 2:1)), and species of Trichoderma (e.g., Trichoderma reesei (Nyyssonen et al. (1993) Biotechnology 11:591-595)). In other cases, the fungi may be yeasts, such as Saccharomyces cerevisiae, Candida boidinii, Hansenula polymorpha, Pichia methanolica, Pichia pastoris, Yarrowia lipolytica, Kluyveromyces lactis, or Ogataea minuta (Joosten et al. (2003); Suzuki et al. (2017) J Biosci Bioeng. 124:156-63).

[0235] Furthermore, human antibodies can also be produced using additional techniques, including phage display libraries. (See Hoogenboom and Winter, J.Mol.Biol. 227:381 (1991); Marks et al., J.Mol.Biol., 222:581 (1991)). Similarly, human antibodies can be produced by introducing human immunoglobulin loci into transgenic animals, such as mice in which the endogenous immunoglobulin gene is partially or completely inactivated. Upon challenge, human antibody production is observed, which is remarkably similar to that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire. This approach is described, for example, in International Publication Nos. 2006 / 008548, 2007 / 096779, 2010 / 109165, 2010 / 070263, 2014 / 141189, and 2014 / 141192.

[0236] One method for producing an antibody of interest, such as a human antibody, is disclosed in U.S. Patent No. 5,916,771. This method includes introducing an expression vector containing a nucleotide sequence encoding a heavy chain into one mammalian host cell in culture, introducing an expression vector containing a nucleotide sequence encoding a light chain into another mammalian host cell, and fusing the two cells to form a hybrid cell. The hybrid cell expresses an antibody containing both a heavy chain and a light chain.

[0237] Further improvements to this procedure include methods for identifying clinically relevant epitopes of immunogens and for selecting antibodies that bind immunospecifically to these relevant epitopes with high affinity, as disclosed in PCT International Publication No. 99 / 53049.

[0238] The antibody can be expressed using a vector containing a DNA segment encoding the single-chain antibody described above.

[0239] These may include vectors, liposomes, naked DNA, adjuvant-assisted DNA, gene guns, catheters, etc. Vectors include chemical conjugates having a targeting moiety (e.g., a ligand for a cell surface receptor) and a nucleic acid-binding moiety (e.g., polylysine), as described in International Publication No. 93 / 64701; viral vectors (e.g., DNA or RNA viral vectors); fusion proteins, which are fusion proteins containing a targeting moiety (e.g., an antibody specific to a target cell) and a nucleic acid-binding moiety (e.g., protamine), as described in PCT / US95 / 02140 (International Publication No. 95 / 22618); plasmids; phages, etc. Vectors may be chromosomal, non-chromosomal, or synthetic.

[0240] Preferred vectors include viral vectors, fusion proteins, and chemical conjugates. Retroviral vectors include Moloney's mouse leukemia virus. DNA viral vectors are preferred. These vectors include pox vectors such as orthopox or avipox vectors, herpesvirus vectors such as herpes simplex virus type 1 (HSV) vectors (see Geller, AI et al., J. Neurochem, 64:487 (1995); Lim, F., et al., in DNA Cloning: Mammalian Systems, D. Glover, Ed. (Oxford Univ. Press, England, Oxford) (1995); Geller, AI et al., Proc Natl. Acad. Sci. USA 90:7603 (1993); Geller, AI, et al., Proc Natl. Acad. Sci USA 87:1149 (1990)), and adenovirus vectors (see LeGal LaSalle et al., Science, 259:988 (1993); Davidson, et al. Examples include al.Nat.Genet.3:219(1993); see Yang, et al., J.Virol.69:2004(1995), and adeno-associated virus vectors (see Kaplitt, MGet al., Nat.Genet.8:148(1994)).

[0241] Poxvirus vectors introduce genes into the cytoplasm. Avipoxvirus vectors result in only short-term expression of nucleic acids. Adenovirus vectors, adeno-associated virus vectors, and herpes simplex virus (HSV) vectors are preferred for introducing nucleic acids into nerve cells. Adenovirus vectors produce shorter expression periods (approximately 2 months) than adeno-associated virus (approximately 4 months), which is shorter than that of HSV vectors. The specific vector selected depends on the target cell and the condition being treated. Introduction can be carried out by standard techniques, such as infection, transfection, transduction, or transformation. Examples of gene introduction methods include, for example, naked DNA, CaPO4 precipitation, DEAE dextran, electroporation, protoplast fusion, lipofection, cell microinjection, and viral vectors.

[0242] Vectors can be used to target virtually any desired target cell. For example, stereotactic injection can be used to direct a vector (e.g., adenovirus, HSV) to a desired location. Furthermore, particles can be delivered by intraventricular (icv) injection using minipump infusion systems such as the SynchroMed Infusion System. Methods based on bulk flow, known as convection, have also proven effective in delivering large molecules to dilated areas of the brain and may be useful for delivering vectors to target cells. (See Bobo et al., Proc. Natl. Acad. Sci. USA 91:2076-2080 (1994); Morrison et al., Am. J. Physiol. 266:292-305 (1994)). Other methods that can be used include catheter, intravenous, parenteral, intraperitoneal, and subcutaneous injections, as well as oral or other known routes of administration.

[0243] Treatment method The antibodies provided herein can be administered to delay or inhibit the progression of cancer associated with the expression of CEACAM5 and / or CEACAM6. In these applications, a therapeutically effective dose of the composition is administered to the subject in an amount sufficient to inhibit the proliferation, replication, or metastasis of cancer cells, or to inhibit the signs or symptoms of cancer. Suitable subjects may include those diagnosed with cancers expressing CEACAM5 and / or CEACAM6, such as small intestine cancer, colorectal cancer, gastric cancer, lung cancer, cervical cancer, pancreatic cancer, esophageal cancer, ovarian cancer, thyroid cancer, bladder cancer, endometrial cancer, breast cancer, liver cancer, prostate cancer, and skin cancer.

[0244] Provided herein are methods for treating cancers associated with the expression of CEACAM5 and / or CEACAM6 in a subject by administering a therapeutically effective dose of the antibodies described herein to the subject. In some embodiments, the cancers are small intestine cancer, colorectal cancer, gastric cancer, lung cancer, cervical cancer, pancreatic cancer, esophageal cancer, ovarian cancer, thyroid cancer, bladder cancer, endometrial cancer, breast cancer, liver cancer, prostate cancer, and skin cancer.

[0245] Administration of the antibodies disclosed herein may also be accompanied by administration of other anticancer agents or therapeutic procedures (e.g., surgical resection of tumors). Any suitable anticancer agent may be administered in combination with the antibodies disclosed herein. Exemplary anticancer agents include, but are not limited to, chemotherapeutic agents such as mitotic inhibitors, alkylating agents, antimetabolites, insertive antibiotics, growth factor inhibitors, cell cycle inhibitors, enzymes, topoisomerase inhibitors, antisurvivators, biological response modifiers, antihormones (e.g., antiandrogens), and anti-angiogenic agents. Other anticancer treatments include radiotherapy and other antibodies that specifically target cancer cells.

[0246] Another common treatment for some types of cancer is surgical intervention, such as surgical resection of metastatic tumors. Another example of treatment is radiotherapy, such as the administration of radioactive material or energy to the tumor site (e.g., external beam therapy) to eradicate the tumor or help shrink it before surgical resection.

[0247] Methods for diagnosis and detection Methods are provided herein for detecting CEACAM5 and / or CEACAM6 protein in vitro or in vivo. In some cases, the expression of CEACAM5 and / or CEACAM6 is detected in a biological sample. The sample can be any sample including, but not limited to, a blood sample, tissue from a biopsy, autopsy, and pathological specimens. Biological samples also include tissue sections, such as frozen sections taken for histological purposes. Biological samples further include body fluids such as blood, serum, plasma, sputum, cerebrospinal fluid, or urine. Biological samples are typically obtained from mammals such as humans or non-human primates. In a preferred embodiment, this method is used for non-diagnostic purposes.

[0248] A method is provided herein for determining whether a subject has cancer associated with the expression of CEACAM5 and / or CEACAM6 by contacting a sample from the subject with an anti-CEACAM5 / 6 dual-binding antibody disclosed herein and detecting the binding of the antibody to the sample. An increase in the binding of the antibody to the sample compared to the binding of the antibody to a control sample identifies that the subject has cancer.

[0249] In another embodiment, a method is provided for corroborating a diagnosis of cancer associated with the expression of CEACAM5 and / or CEACAM6 in a subject by contacting a sample from the subject diagnosed with cancer associated with the expression of CEACAM5 and / or CEACAM6 with an anti-CEACAM5 / 6 dual-binding antibody disclosed herein and detecting the binding of the antibody to the sample. An increase in the binding of the antibody to the sample compared to the binding of the antibody to a control sample corroborates the diagnosis of cancer in the subject.

[0250] In some examples of the disclosed methods, the monoclonal antibody is directly labeled.

[0251] In other examples, the method further comprises contacting a sample with a second antibody that specifically binds to a monoclonal antibody, and detecting the binding of the second antibody. An increase in the binding of the second antibody to the sample, compared to the binding of the second antibody to a control sample, detects cancer associated with the expression of CEACAM5 and / or CEACAM6 in the subject, or supports a diagnosis of cancer associated with the expression of CEACAM5 and / or CEACAM6 in the subject.

[0252] In some cases, cancers include small intestine cancer, colorectal cancer, stomach cancer, lung cancer, cervical cancer, pancreatic cancer, esophageal cancer, ovarian cancer, thyroid cancer, bladder cancer, endometrial cancer, breast cancer, liver cancer, prostate cancer, and skin cancer.

[0253] In some examples, the control sample is a sample from a subject that does not have cancer. In certain embodiments, the sample is a blood sample or a tissue sample.

[0254] In some embodiments of the diagnostic and detection method, the anti-CEACAM5 / 6 double-conjugated antibody is directly labeled with a detectable label. In another embodiment, the anti-CEACAM5 / 6 double-conjugated antibody (first antibody) is unlabeled, and a second antibody or other molecule capable of binding to the first antibody is labeled. As is well known to those skilled in the art, the second antibody is selected to be capable of specifically binding to a particular species and class of the first antibody. For example, if the first antibody is human IgG, the secondary antibody may be anti-human IgG. Other molecules capable of binding to the antibody include, but are not limited to, protein A and protein G, both of which are commercially available.

[0255] Suitable labels for antibodies or secondary antibodies include a variety of enzymes, prosthetic groups, fluorescent agents, luminescent agents, magnetic agents, and radioactive materials. Non-limiting examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase. Non-limiting examples of suitable prosthetic group complexes include streptavidin / biotin and avidin / biotin. Non-limiting examples of suitable fluorescent agents include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dancylkloride, or phycoerythrin. A non-limiting exemplary luminescent agent is luminol. A non-limiting exemplary magnetic agent is gadolinium, and a non-limiting exemplary radioactive label is... 125 I, 131 I, 35 S, or 3 Includes H.

[0256] In an alternative embodiment, CEACAM5 and / or CEACAM6 can be assayed in a biological sample by a competitive immunoassay utilizing a CEACAM5 and / or CEACAM6 protein standard labeled with a detectable substance and an unlabeled anti-CEACAM5 / 6 double-conjugated antibody. In this assay, the biological sample, the labeled CEACAM5 and / or CEACAM6 protein standard, and the anti-CEACAM5 / 6 double-conjugated antibody are combined, and the amount of the labeled CEACAM5 and / or CEACAM6 protein standard bound to the unlabeled antibody is determined. The amount of CEACAM5 and / or CEACAM6 in the biological sample is inversely proportional to the amount of the labeled CEACAM5 and / or CEACAM6 protein standard bound to the anti-CEACAM5 / 6 double-conjugated antibody.

[0257] The immunoassays and methods disclosed herein can be used for a variety of purposes. In one embodiment, an anti-CEACAM5 / 6 double-conjugated antibody may be used to detect the production of CEACAM5 and / or CEACAM6 in cells during cell culture. In another embodiment, the antibody can be used to detect the amount of CEACAM5 and / or CEACAM6 in a tissue sample or a biological sample such as blood or serum. In some examples, CEACAM5 and / or CEACAM6 are cell surface CEACAM5 and / or CEACAM6. In other examples, the CEACAM5 and / or CEACAM6 proteins are soluble (e.g., in cell culture supernatant or bodily fluid samples such as blood or serum).

[0258] In one embodiment, a kit is provided for detecting CEACAM5 and / or CEACAM6 in a biological sample, such as a blood sample or a tissue sample. For example, a biopsy can be performed to obtain a tissue sample for histological examination to support a cancer diagnosis in a subject. The kit for detecting polypeptides typically includes a monoclonal anti-CEACAM5 / 6 double-conjugated antibody, such as any of the monoclonal antibodies disclosed herein. In further embodiments, the antibody is labeled (e.g., by fluorescence, radioactivity, or enzyme labeling).

[0259] In one embodiment, the kit includes teaching materials disclosing means of using an anti-CEACAM5 / 6 double-binding antibody. These teaching materials may be written in electronic form (e.g., computer diskette or compact disk) or in visual form (e.g., video file). The kit may also include further components to facilitate the specific application for which the kit is designed. Thus, for example, the kit may further include means for detecting labels (e.g., enzyme substrates for enzyme labeling, filter sets for detecting fluorescent labels, suitable secondary labels such as secondary antibodies, etc.). The kit may further include buffers and other reagents routinely used for carrying out a particular method. Such kits and suitable contents are well known to those skilled in the art.

[0260] In one embodiment, the diagnostic kit includes an immunoassay. While the details of the immunoassay may vary depending on the specific format used, a method for detecting CEACAM5 and / or CEACAM6 in a biological sample generally involves contacting the biological sample with an anti-CEACAM5 / 6 double-conjugated antibody. The antibody can specifically bind to immunocomplexes under immunologically reactive conditions to form an immune complex, and the presence of the immune complex (bound antibody) can be detected directly or indirectly.

[0261] The antibodies disclosed herein can also be used in immunoassays, for example, radioimmunoassays (RIA), ELISA, or immunohistochemical assays, though not limited to these. The antibodies can also be used in fluorescence-activated cell sorting (FACS). FACS uses multiple color channels, low-angle and obtuse-angle light scattering detection channels, and impedance channels, among other more sophisticated levels of detection, to separate or sort cells (see U.S. Patent No. 5,061,620). Any monoclonal antibody conjugating to CEACAM5 and / or CEACAM6, such as those disclosed herein, can be used in these assays. Therefore, the antibodies can be used in conventional immunoassays, including but not limited to ELISA, RIA, FACS, tissue immunohistochemistry, Western blotting, or immunoprecipitation. [Examples]

[0262] The present invention will be further illustrated by the following examples, which are not intended to limit the invention. In the following examples, experimental procedures that do not specify conditions will be carried out according to conventional procedures and conditions, or as instructed.

[0263] Example 1. Immunization strategy and screening of single B cell cloning with anti-CEACAM5 / 6 double-conjugated antibody immunization Anti-CEACAM5 antibodies were generated in Harbour H2L2 mice using recombinant human CEACAM5 (Uniprot P06731) Fc-tagged protein, human CEACAM5 A3B3 His-tagged fragment, or CHO-K1-huCEACAM5 overexpressing cell lines as immunogens (International Publication No. 2010 / 070263(A1)). The immunization schemes are listed in Table 4.

[0264] [Table 4]

[0265] For protein immunization, each mouse was administered 50 μg of protein subcutaneously and intraperitoneally, along with an adjuvant (Sigma, S6322), for the initial booster immunization and 25 μg for subsequent booster immunizations. This immunization was performed five times every two weeks. For cellular immunization, each mouse was administered 1 × 10⁶ of protein in PBS. 7 Cells were administered five times at 3-week intervals. Final immunization was performed intraperitoneally using immunogen diluted in PBS. Serum titers were tested for recombinant human CEACAM5 His-tag, recombinant cyno CEACAM5 (XP_005589491.1) His-tag, and recombinant human CEACAM1 (Uniprot P13688) His-tag proteins (in tissue) using ELISA and FACS. Mice with high serum titers for human CEACAM5 and cyno CEACAM5 but low titers for human CEACAM1 were selected for subsequent single B-cell cloning screening.

[0266] Single B-cell cloning screening Single B-cell screening based on the Beacon® optical fluid system The Beacon (registered trademark) optical fluid system uses opto-electronic positioning (OEPTM) technology to move individual cells. The Beacon opto-electronic system is an automated biological instrument and device that can enable simultaneous biological function testing, experimental analysis, positive clone selection, and other operations under cell culture conditions. The Beacon platform can perform these tasks in an automated manner in parallel on a large scale for thousands of cells.

[0267] In this application, a plasmablast discovery workflow was used. In each experiment, up to 14k individual plasmablasts can be screened for the selection of antigen-specific antibodies secreted by positive plasmablasts. These plasmablasts secreting specific antibodies were then transferred to 96-well plates containing cell lysates for subsequent single B cell sequencing to identify the heavy and light chains of the antibodies produced by single B cells (monoclonal). The overall screening strategy and progress are shown in Figure 1.

[0268] Single B cell sequencing The single B cell sequencing method was used to recover the antibody heavy and light chain sequences from single plasmablasts. Single B cell sequencing has become a powerful tool for obtaining antibody sequences. General procedures include purification of RNA from single plasmablast lysates, reverse transcription synthesis of cDNA, amplification and purification of cDNA, amplification of heavy and light chains, cloning and transfection, and Sanger sequencing. The obtained sequences were subjected to uniqueness and cluster analysis, and then pairs of heavy and light chain DNA sequences were synthesized.

[0269] Example 2. Production and purification of anti-CEACAM5 / 6 bispecific antibodies Recombinant plasmids encoding target antibodies were transiently co-transfected into HEK293-6E or 293-F cell cultures using PEI (Polyscience, 24885). Following transfection, cells were incubated with 5% CO2 at 37°C and shaken at 120 rpm. Cell culture supernatant collected on days 6-7 was used for purification. Monoclonal antibodies were purified using protein A magnetic beads (AmMag protein A magnetic beads, Genscript, L00695).

[0270] Antibody purity was determined by SEC-HPLC (Agilent 1260 Infinity II HPLC, Welch Xtimate SEC-300 column, 1×PBS pH 7.4 used as mobile phase) and SDS-PAGE (SurePAGE, Bis-Tris, 10×8, 4~12%, 12 wells, Genscript, M00653). Four recombinant antibodies, named PRO304169, PRO304171, PRO304294, and PRO304713, were successfully expressed and purified for characterization. The amino acid sequences of the four antibodies are listed in Table 1.

[0271] Meanwhile, the anti-CEACAM5 antibody tusamitamab (SAR408377) was also produced using sequence information from SAR408377, Proposed INN List 123 Vol.34, No.2, 2020, following the procedure shown above. Furthermore, the anti-CEACAM6 antibody tinuririmab (BAY-1834942) was also produced using sequence information from Proposed INN List 83 Vol.34, No.1, 2020, following the procedure shown above. These antibodies were used as controls and assigned the codes PR003561 (for tusamitamab) and PR303563 (for tinuririmab), respectively. The amino acid sequences of these antibodies are listed in Table 2.

[0272] Example 3. Characterization of the binding properties of anti-CEACAM5 / 6 double-binding monoclonal antibody. Binding to human and cynomolgus monkey CEACAM5, and human CEACAM6, 1, 3, and 8 proteins. The binding of recombinant anti-CEACAM5 / 6 double-conjugated monoclonal antibodies to human and cynomolgus monkey CEACAM5, as well as human CEACAM6 (Uniprot P40199), human CEACAM1, 3 (NP_001806.2, Sino Biological 11933-H08H), and 8 (NP_001807.2, Acro Bio CE8-H5224), was detected by ELISA. The anti-CEACAM5 / 6 double antibodies were serially diluted in staining buffer (PBS containing 2% FBS). Anti-CEACAM5 antibody PR003561 (tusamitamab), anti-CEACAM6 antibody PR303563 (tinuririmab), anti-CEACAM1 antibody CEACAM1PC (SinoBiological, Cat:10822-MM02, mouse monoclonal), anti-CEACAM3 antibody CEACAM3PC (SinoBiological, Cat:11933-MM03, mouse monoclonal), and anti-CEACAM8 antibody CEACAM8PC (SinoBiological, Cat:11729-MM04, mouse monoclonal) were used as controls. ELISA was used to test the binding of antibody-containing supernatant or purified antibody to antigen. Briefly, antigens were coated overnight at 4°C onto 96-well high-binding plates. The plates were washed three times with PBST, incubated with blocking buffer (2% bovine serum albumin) at 37°C for 1 hour, washed three times with PBST, and incubated with antibody-containing supernatant or purified antibody of the desired dilution or concentration at 37°C for 1 hour. The secondary antibody, anti-human Fc-HRP (Jackson Laboratory, 109-035-097), was used to detect the target antibody. TMB and stop solution were used to detect the HRP signal. The HRP signal was read using a SPECTRAMAX plate reader. EC50 was determined from the ELISA test results (see Table 5 and Figures 2-7).

[0273] As shown in Table 5 and Figures 2-7, the four antibodies PR304169, PR304171, PR304713, and PR304294 showed good binding activity to both human and cyno CEACAM5 proteins, but did not bind to CEACAM1, CEACAM3, and CEACAM8. A notable characteristic is that while all four antibodies showed good binding activity to human CEACAM6, the control antibody PR303561 (tusamitamab) did not cross-react to CEACAM6. This could potentially lead to a wider applicability when applied therapeutically. No such antibodies have been reported to date. This binding characteristic offers a broader applicability to cancer cells.

[0274] [Table 5]

[0275] Binding to human or cynomolgus monkey CEACAM5 / 6 stable cell lines The binding of recombinant anti-CEACAM5 / 6 double-conjugated antibodies to stable human or cynomolgus monkey CEACAM5 or CEACAM6 cells was detected by flow cytometry. Stable cell lines included 293T cell lines transfected to overexpress human CEACAM5 (293T-huCEACAM5 cells) or CEACAM6 (293T-huCEACAM6 cells) on the surface, and CHO-K1 cell lines transfected to overexpress cynomolgus monkey CEACAM5 (CHOK1-cyno CEACAM5 cells) or CEACAM6 (CHOK1-cyno CEACAM6 cells) on the surface. Anti-CEACAM5 / 6 double-conjugated antibodies were incubated with the stable cell lines. Anti-CEACAM5 / 6 double-conjugated antibodies were serially diluted in staining buffer (PBS containing 2% FBS). 50 μL of diluted antibody solution was incubated at 1–2 × 10⁻⁶. 5The cells were added to a 50 μL cell suspension containing individual cells and incubated at 4°C for 1 hour. The cells were washed twice with staining buffer, and 100 μL / well of 1:1000 dilution fluorescently labeled anti-human IgG antibody (Alexa Fluor® 488 AffiniPure goat anti-human IgG (H+L), Jackson Cat 109-545-088) was added. After incubation at 4°C for 1 hour, the cells were washed twice and subjected to flow cytometry. PR303561 and an unrelated IgG isotype control (Crownbio, hIgG1) were used as positive and negative controls, respectively. The results are shown in Figures 8-10 and Table 6 below.

[0276] The results showed that the anti-CEACAM5 / 6 double-binding antibody exhibited very good binding activity to both human and cynomolgus monkey CEACAM5 and CEACAM6 on the cell membrane.

[0277] [Table 6]

[0278] Binding to endogenous cell lines LS174T and BxPC3 expressing CEACAM5 and CEACAM6. The same procedure as above was followed, simply by replacing stable cell lines with LS 174T (ATCC CL-188), BxPC3 (ATCC CRL-1687), and A549 cell lines that simultaneously express different levels of membrane-bound endogenous CEACAM5 and CEACAM6. The binding results are shown in Figure 11 and Table 6 above.

[0279] Figure 11 and Table 6 show the good binding ability of anti-CEACAM5 / 6 double-conjugated antibodies to membrane-bound endogenous human CEACAM5 and CEACAM6 on LS 174T, BxPC3, and A549 cell lines. The double-conjugated antibodies showed much greater saturated binding capacity than the control antibody PR303561.

[0280] Example 4. Determination of binding affinity of anti-CEACAM5 / 6 double-binding antibody to soluble CEACAM5 and CEACAM6 using Octet. The binding kinetics of recombinant anti-CEACAM5 / 6 antibodies to CEACAM5 and CEACAM6 were analyzed using Octet. In Octet analysis, recombinant human CEACAM5 Fc-tagged protein (#Uniprot P06731) or CEACAM6 Fc-tagged protein (Uniprot P40199) were serially diluted in 1× dynamic buffer (Fortebio). Anti-CEACAM5 / 6 double-conjugated antibodies were diluted to 5 μg / mL. The diluted antibodies, antigens, and regeneration buffer (10 mM glycine, pH 1.75) were then added to 96-well plates (Greiner). The rate constants of association and dissociation were measured using an AHC sensor (Fortebio). The sensor surface was regenerated with regeneration buffer after each binding experiment. Traces were processed using ForteBio Data Analysis Software (version 8.0, Pall ForteBio, California, USA).

[0281] Table 7 summarizes the KD values ​​of anti-CEACAM5 / 6 double-conjugated antibodies.

[0282] [Table 7]

[0283] Example 5. Epitope binning of anti-CEACAM5 / 6 double-conjugated antibody using Octet In epitope binning analysis using Octet, antibodies were tested in pairs against other antibodies to see if they blocked each other's binding to the antigen's epitope. The first and second antibodies were diluted to the same concentration that could saturate the binding of the immobilized antigen. Recombinant CEACAM5-his protein was biotinylated and then used as an antibody, immobilized on an SA sensor. The first antibody was associated with the antigen, and then the second antibody. Data were analyzed using ForteBio Data Analysis Software (version 8.0, Pall ForteBio). The ratio of the RU value of the second antibody to the same antibody as the first antibody was calculated as the co-binding rate of this antibody to other antibodies. Generally speaking, rates greater than 50% and close to 100% indicate that the two antibodies bind to different epitope bins, while rates less than 20% indicate overlapping bins of the two evaluated antibodies. The co-binding rates are shown in Table 8.

[0284] As shown in Table 8, all anti-CEACAM5 / 6 double-conjugated antibodies belong to the same epitope bin as the control antibody PR303561, but differently.

[0285] [Table 8]

[0286] Example 6. Soluble CEACAM5 or CEACAM6 interference assay with anti-CEACAM5 / 6 double-bound antibody Next, these anti-CEACAM5 / 6 double-conjugated antibodies were tested for their binding to 293T-huCEACAM5, 293T-huCEACAM6, LS 174T, and BxPC3 cells expressing CEACAM5 and CEACAM6, in or out of the presence of 100 nM soluble CEACAM5 or CEACAM6. The experiments were carried out in a similar manner to that described in Example 3 (binding to endogenous cell lines LS 174T and BxPC3 expressing CEACAM5 and CEACAM6), except that 100 nM soluble CEACAM5 or CEACAM6 was added to the serially diluted antibody.

[0287] The results showed that when 100 nM soluble CEACAM5 or CEACAM6 was added, the binding of all these antibodies decreased, but most EC50 changes were within 5-fold, indicating minimal interference by soluble CEACAM5 or CEACAM6 with anti-CEACAM5 / 6 antibodies. This suggests that these antibodies have good resistance to soluble protein interference.

[0288] [Table 9]

[0289] Example 7. Internalization of antibodies on 293T-huCEACAM5, 293T-huCEACAM6, LS 174T, HPAC, or BxPC3 using pHAb amine-reactive dyes. The antigen-based internalization of anti-CEACAM5 / 6 double-conjugated antibodies on 293T-huCEACAM5, 293T-huCEACAM6, LS 174T, HPAC, or BxPC3 cells was determined using a pHAb amine-reactive dye (Promega, catalog number G9841).

[0290] pHAb dyes are pH-sensing dyes that exhibit very low fluorescence at pH > 7, and their fluorescence dramatically increases as the pH of the solution becomes more acidic. In this case, when antibodies labeled with pHAb dye bind extracellularly at neutral pH, no fluorescence is observed, or only very low fluorescence is observed. However, after internalization, the fluorescence becomes stronger in the lower pH environments of endosomes and lysosomes.

[0291] Antibodies were labeled with pHAb dyes, and the DAR was calculated according to the kit instructions. The labeled antibodies were incubated with 293T-huCEACAM5 and LS 174T at 4°C (used as a background control because this temperature theoretically results in very low internalization activity) or 37°C for 6 hours. Fluorescence with excitation maximal (Ex) at 532 nm and emission maximal (Em) at 560 nm was then detected. The final normalized results were obtained by subtracting the background from 4°C from the fluorescence intensity from 37°C and dividing these by the DAR of the antibody's pHAb dye. A higher value indicates higher internalization activity.

[0292] The internalization results are shown in Figure 12 and Table 10.

[0293] As shown in Figure 12, anti-CEACAM5 / 6 double-binding antibodies exhibit good internalization activity. Good internalization is useful for antibody drugs as it can help improve the effectiveness of antibodies used in ADCs, since most ADCs require internalization to release toxins, and can also help reduce the number of CEACAM5 or CEACAM6 on the cell membrane.

[0294] [Table 10]

[0295] Example 8. Internalization of anti-CEACAM5 / 6 double-binding antibodies on LS 174T, A549, BxPC3, and HPAC with soluble protein interference. Internalization of anti-CEACAM5 / 6 double-conjugated antibodies in LS 174T, A549, BxPC3, and HPAC cell lines with soluble protein interference was carried out according to the same procedure as shown in Example 7, except that 100 nM of soluble CEACAM5 or CEACAM6 was added to the system. The experimental results are shown in Figure 13.

[0296] The results showed no interference from soluble CEACAM5 or CEACAM6 proteins with the internalization activity of anti-CEACAM5 / 6 double-binding antibodies.

[0297] Example 9. In vitro cytotoxicity of anti-CEACAM5 / 6 antibody-MMAE conjugate ADCs In vitro cytotoxicity studies were performed using 293T-huCEACAM5, 293T-huCEACAM6, LS 174T, and BxPC3 cells. Anti-CEACAM5 / 6 double-conjugated antibodies were conjugated to MC-Val-Cit-PABC-MMAE to form ADCs with a DAR of approximately 4. MC-Val-Cit-PABC-MMAE was purchased commercially (MedChemExpress, HY-15575), and conjugation was performed as follows: The sample buffer containing the antibody was adjusted to the required pH by dialyzing overnight at 4°C, and then adjusted to a concentration of 5 mg / mL. The relative equivalent volume of TCEP (relative to protein) was added to the sample buffer, shaken, and incubated at room temperature for 3 hours. Next, the relative equivalent volume (relative to protein) of MC-Val-Cit-PABC-MMAE dissolved in DMSO was directly added to the reaction sample, and incubated at room temperature for 2-6 hours. Next, 10 equivalents of cysteine ​​(relative to protein) were added to the reaction sample to quench the reaction. Excess small molecules were removed by dialysis to obtain ADCs containing anti-CEACAM5 / 6 antibodies conjugated to MC-Val-Cit-PABC-MMAE. For cytotoxicity testing, cells were incubated with serially diluted ADCs for 6 days. Viability was measured using the CellTiter-Glo kit (Promega, G7572). Data were analyzed with GraphPad Prism to obtain IC50 for different ADCs.

[0298] As shown in Table 11, ADCs based on PR304169, PR304713, and PR304294 exhibited in vitro cytotoxicity equivalent to or better than that based on PR303561 against CEACAM5-overexpressing 293T cells, as well as LS 174T and BxPC3 cells. They also showed very good in vitro cytotoxicity in human CEACAM6-overexpressing 293T cells, demonstrating a broader applicability to cell lines.

[0299] [Table 11]

[0300] Example 10. In vivo efficacy of anti-CEACAM5 / 6 antibody-MMAE conjugate ADC. To evaluate the antitumor activity of anti-CEACAM5 / 6 ADCs, in vivo efficacy studies were performed in CDX mouse models using both MKN45 gastric tumor and LS 174T colon cancer tumor cell lines. Tumor volume was measured twice weekly in two dimensions using a caliper, and the volume was expressed using the formula: V = 0.5a × b 2 Using mm 3 This is expressed as follows, where a and b are the long and short diameters of the tumor, respectively. Tumor growth inhibition (TGI): TGI% = (1-T i / C i ) × 100. T i and C i These represent the average tumor volume of the treatment group and the control group on the specified day, respectively.

[0301] The administration scheme and results are shown in Figure 14 (A and B), Table 12, Figure 15 (A and B), and Table 13. As shown in the figures and tables, the PR304169, PR304713, and PR304294-based ADCs all showed antitumor activity equivalent to or better than that of the PR303561-based ADC.

[0302] [Table 12]

[0303] [Table 13]

Claims

1. An antibody or antigen-binding fragment thereof that binds to both CEACAM5 and CEACAM6, wherein the antibody comprises a heavy chain variable region (VH) and a light chain variable region (VL), (1) The VH comprises HCDR1 to HCDR3 of VH having the amino acid sequence shown in SEQ ID NO: 26, and the VL comprises LCDR1 to HCDR3 of VL having the amino acid sequence shown in SEQ ID NO:

27. (2) The VH comprises HCDR1 to HCDR3 of VH having the amino acid sequence shown in SEQ ID NO: 7, and the VL comprises LCDR1 to HCDR3 of VL having the amino acid sequence shown in SEQ ID NO:

8. (3) The VH comprises HCDR1 to HCDR3 of VH having the amino acid sequence shown in SEQ ID NO: 17, and the VL comprises LCDR1 to HCDR3 of VL having the amino acid sequence shown in SEQ ID NO: 18, or (4) An antibody or antigen-binding fragment thereof, wherein VH comprises HCDR1 to HCDR1 of VH having the amino acid sequence shown in SEQ ID NO: 31, and VL comprises LCDR1 to LCDR1 of VL having the amino acid sequence shown in SEQ ID NO:

32.

2. (1) The VH comprises HCDR1 to HCDR3, each containing the amino acid sequences described in SEQ ID NOs: 21, 22, and 23, and the VL comprises LCDR1 to HCDR3, each containing the amino acid sequences described in SEQ ID NOs: 24, 5, and 25. (2) The VH comprises HCDR1 to HCDR3, each containing the amino acid sequences described in SEQ ID NOs: 1, 2, and 3, and the VL comprises LCDR1 to HCDR3, each containing the amino acid sequences described in SEQ ID NOs: 4, 5, and 6. (3) The VH comprises HCDR1 to HCDR3 each containing the amino acid sequences described in SEQ ID NOs: 11, 12, and 13, and the VL comprises LCDR1 to HCDR3 each containing the amino acid sequences described in SEQ ID NOs: 14, 15, and 16, or (4) The antibody or antigen-binding fragment thereof according to claim 1, wherein VH comprises HCDR1 to HCDR3 each containing the amino acid sequences described in SEQ ID NOs: 1, 30, and 3, and VL comprises LCDR1 to HCDR3 each containing the amino acid sequences described in SEQ ID NOs: 4, 5, and 6.

3. (1) The VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 26, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:

27. (2) The VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 7, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:

8. (3) The VH contains an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 17, and the VL contains an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 18, or (4) The antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 31, and VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:

32.

4. The antibody comprises a heavy chain (HC) and a light chain (LC), (1) The HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 28, and the LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:

29. (2) The HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 9, and the LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:

10. (3) The HC contains an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 19, and the LC contains an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 20, or (4) The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein the HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 33, and the LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:

34.

5. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, wherein the antibody is a mouse antibody, a chimeric antibody, a humanized antibody, or a human antibody.

6. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, wherein the antibody is an isotype selected from the group consisting of IgG, IgA, IgM, IgE, and IgD.

7. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, wherein the antibody is a subtype selected from the group consisting of IgG1, IgG2, IgG3, and IgG4.

8. The antigen-binding fragments are Fab, Fab', and F(ab'). ２ An antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, selected from the group consisting of Fd, Fd', Fv, scFv, ds-scFv, and dAb.

9. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, wherein the antibody is a monoclonal antibody, a bispecific antibody, or a multispecific antibody.

10. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, wherein the antibody is monovalent, bivalent, or polyvalent.

11. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, wherein the antibody or antigen-binding fragment thereof is fluorescently labeled, radioactively labeled, or conjugated to a drug moiety.

12. The antibody or antigen-binding fragment thereof according to claim 11, wherein the antibody or antigen-binding fragment thereof is bound to a drug portion, and the drug portion is selected from the group consisting of microtubule inhibitors, antibiotics, DNA synthesis inhibitors, topoisomerase inhibitors, RNA polymerase II inhibitors, and RNA spliceosome inhibitors.

13. The antibody or antigen-binding fragment thereof according to claim 12, wherein the drug portion is selected from the group consisting of MMAE, MMAF, duocalmycin, DM1, DM4, SN-38, Dxd, calicheamicin, doxorubicin, and PBD (benzodiazepine).

14. The antibody or antigen-binding fragment according to claim 11 or 12, wherein the antibody or antigen-binding fragment is bound to the drug portion via a linker.

15. The antibody or antigen-binding fragment thereof according to claim 14, wherein the linker includes a cleavable linker or a non-cleavable linker.

16. The antibody or antigen-binding fragment thereof according to claim 15, wherein the cleavable linker is selected from the group consisting of an acid-unstable linker, a hydrophilic linker, a protease-sensitive linker, a photo-unstable linker, a hydrazone linker, a dimethyl linker, and a disulfide-containing linker.

17. The antibody or antigen-binding fragment thereof according to claim 15, wherein the linker is selected from the group consisting of MC (6-maleimidocaproyl), Val-Cit (valine-citrulline), PABC (para-aminobenzyloxycarbonyl), DMEA (dimethylethylamine), Val-Cit-PABC, MC-Val-Cit-PABC, MC-Val-Cit-PABC-DMEA, GGFG (glycine-glycine-phenylalanine-glycine), MC-GGFG-aminomethyl, AcBut (4-(4-acetylphenoxy)-butanoic acid), and AcBut-dimethylhydrazide, and preferably MC-Val-Cit-PABC.

18. A bispecific antibody comprising an antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, and a second antigen-binding region that specifically binds to a tumor-associated antigen, an immune cell antigen, or an immune checkpoint molecule.

19. A nucleic acid comprising an antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, or a nucleotide sequence encoding a bispecific antibody according to claim 18.

20. A vector comprising the nucleic acid described in claim 19.

21. A host cell comprising the nucleic acid described in claim 19 or the vector described in claim 20.

22. An antibody-drug conjugate (ADC) comprising an antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, or a bispecific antibody according to claim 18, and a drug portion conjugated thereto via a linker.

23. The antibody-drug conjugate according to claim 22, wherein the drug portion is selected from the group consisting of microtubule inhibitors, antibiotics, DNA synthesis inhibitors, topoisomerase inhibitors, RNA polymerase II inhibitors, and RNA spliceosome inhibitors.

24. The antibody-drug conjugate according to claim 23, wherein the drug portion is selected from the group consisting of MMAE, MMAF, duocalmycin, DM1, DM4, SN-38, Dxd, calicheamicin, doxorubicin, and PBD benzodiazepines.

25. The antibody-drug conjugate according to any one of claims 22 to 24, wherein the linker includes a cleavable linker or a non-cleavable linker.

26. The antibody-drug conjugate according to claim 25, wherein the cleavable linker is selected from the group consisting of an acid-unstable linker, a hydrophilic linker, a protease-sensitive linker, a photo-unstable linker, a hydrazone linker, a dimethyl linker, and a disulfide-containing linker.

27. The antibody-drug conjugate according to claim 25, wherein the linker is selected from the group consisting of MC (6-maleimidocaproyl), Val-Cit (valine-citrulline), PABC (para-aminobenzyloxycarbonyl), DMEA (dimethylethylamine), Val-Cit-PABC, MC-Val-Cit-PABC, MC-Val-Cit-PABC-DMEA, GGFG (glycine-glycine-phenylalanine-glycine), MC-GGFG-aminomethyl, AcBut (4-(4-acetylphenoxy)-butanoic acid), and AcBut-dimethylhydrazide, and preferably MC-Val-Cit-PABC.

28. A pharmaceutical composition comprising an antibody or antigen-binding fragment thereof according to any one of claims 1 to 17, or a bispecific antibody according to claim 18, or a nucleic acid according to claim 19, or a vector according to claim 20, or a host cell according to claim 21, or an antibody-drug conjugate according to any one of claims 22 to 27, and optionally a pharmaceutically acceptable carrier or excipient.

29. The pharmaceutical composition according to claim 28, further comprising a second therapeutic agent, the second therapeutic agent optionally selected from the group consisting of antibodies, chemotherapeutic agents, siRNA, antisense oligonucleotides, polypeptides, and small molecule drugs.

30. A method for treating cancer in a subject, comprising administering to the subject an effective amount of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 17, or a bispecific antibody according to claim 18, or a nucleic acid according to claim 19, or a vector according to claim 20, or a host cell according to claim 21, or an antibody-drug conjugate according to any one of claims 22 to 27, or a pharmaceutical composition according to claim 28 or 29.

31. The method according to claim 30, wherein the cancer is a cancer associated with the expression of CEACAM5 and / or CEACAM6.

32. The method according to claim 31, wherein the cancer is selected from the group consisting of small intestine cancer, colorectal cancer, stomach cancer, lung cancer, cervical cancer, pancreatic cancer, esophageal cancer, ovarian cancer, thyroid cancer, bladder cancer, endometrial cancer, breast cancer, liver cancer, prostate cancer, and skin cancer.

33. The method according to any one of claims 30 to 32, further comprising administering a second therapeutic agent to the subject.

34. The method according to claim 33, wherein the second therapeutic agent is selected from antibodies, chemotherapeutic agents, siRNA, antisense oligonucleotides, polypeptides, and small molecule drugs.

35. Use of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 17, or a bispecific antibody according to claim 18, or a nucleic acid according to claim 19, or a vector according to claim 20, or a host cell according to claim 21, or an antibody-drug conjugate according to any one of claims 22 to 27, or a pharmaceutical composition according to claim 28 or 29, in the manufacture of a pharmaceutical for treating cancer in a target.

36. The use according to claim 35, wherein the cancer is a cancer associated with the expression of CEACAM5 and / or CEACAM6, and preferably the cancer is selected from the group consisting of small intestine cancer, colorectal cancer, gastric cancer, lung cancer, cervical cancer, pancreatic cancer, esophageal cancer, ovarian cancer, thyroid cancer, bladder cancer, endometrial cancer, breast cancer, liver cancer, prostate cancer, and skin cancer.

37. The use according to claim 35 or 36, wherein the pharmaceutical further comprises a second therapeutic agent, optionally selected from antibodies, chemotherapeutic agents, siRNA, antisense oligonucleotides, polypeptides, and small molecule drugs.

38. The use according to claim 35 or 36, wherein the pharmaceutical agent is administered in combination with a second therapeutic agent, the second therapeutic agent optionally selected from antibodies, chemotherapeutic agents, siRNA, antisense oligonucleotides, polypeptides, and small molecule drugs.

39. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 17, or a bispecific antibody according to claim 18, or a nucleic acid according to claim 19, or a vector according to claim 20, or a host cell according to claim 21, or an antibody-drug conjugate according to any one of claims 22 to 27, or a pharmaceutical composition according to claim 28 or 29, for use in the treatment of cancer in a subject.

40. The cancer is a cancer associated with the expression of CEACAM5 and / or CEACAM6, and preferably the cancer is selected from the group consisting of small intestine cancer, colorectal cancer, gastric cancer, lung cancer, cervical cancer, pancreatic cancer, esophageal cancer, ovarian cancer, thyroid cancer, bladder cancer, endometrial cancer, breast cancer, liver cancer, prostate cancer, and skin cancer, and is an antibody or antigen-binding fragment thereof, bispecific antibody, nucleic acid, vector, host cell, antibody-drug conjugate, or pharmaceutical composition for use according to claim 39.

41. The further comprising administering a second therapeutic agent to the subject, wherein the second therapeutic agent is optionally selected from antibodies, chemotherapeutic agents, siRNA, antisense oligonucleotides, polypeptides, and small molecule drugs, the antibody or antigen-binding fragment thereof, bispecific antibody, nucleic acid, vector, host cell, antibody-drug conjugate, or pharmaceutical composition for use according to claim 39 or 40.

42. A method for detecting the presence or level of CEACAM5 and / or CEACAM6 in a sample, (a) Contacting the sample with the antibody or antigen-binding fragment thereof described in any one of claims 1 to 10, (b) A method comprising determining the presence or level of CEACAM5 and / or CEACAM6 in the sample by detecting the binding of the antibody to the sample.

43. A method for diagnosing cancer associated with the expression of CEACAM5 and / or CEACAM6 in a subject, (a) Obtaining a biological sample from the subject, (b) Contacting the sample with the antibody or antigen-binding fragment thereof described in any one of claims 1 to 10, (c) detecting the binding of the antibody to the sample, A method for identifying a subject as having the cancer, wherein an increase in the binding of the antibody or its antigen-binding fragment to a sample, compared to the binding of the antibody or its antigen-binding fragment to a control sample, is used.

44. A method for imaging cancer associated with the expression of CEACAM5 and / or CEACAM6 in a subject, (a) Administering to the subject an antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, wherein the antibody is conjugated to a detectable marker, and (b) A method comprising detecting the presence of the marker.

45. (a) The detectable marker is １１１ In, preferably the detection of the marker is by single-photon emission computed tomography, or (b) The detectable marker is ８９ The method according to claim 44, wherein the marker is Zr, and preferably the marker is detected by positron emission tomography.

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