Anti-CEACAM5 antibodies and conjugates and their use

Abstract

The present invention provides antibodies that bind to human CEACAM5 protein, as well as isolated nucleic acids and host cells comprising sequences encoding the antibodies. The present invention also provides immunoconjugates comprising the antibodies linked to growth inhibitory agents, and pharmaceutical compositions comprising the antibodies or immunoconjugates of the present invention. The present invention also provides uses of the antibodies, immunoconjugates, and pharmaceutical compositions of the present invention for the treatment or diagnosis of cancer.

Description

Technical Field

[0001] The present invention relates to an antibody that binds to human CEACAM5 protein, and an isolated nucleic acid and host cell comprising a sequence encoding said antibody. The present invention also relates to an immunoconjugate comprising said antibody conjugated to a growth inhibitor, and a pharmaceutical composition comprising the antibody or immunoconjugate of the present invention. The present invention also relates to the use of the antibody, immunoconjugate, and pharmaceutical composition of the present invention for the treatment or diagnosis of cancer.

Background Art

[0002] Carcinoembryonic antigen (CEA) is a glycoprotein involved in cell adhesion. CEA was first identified in 1965 (Gold and Freedman, J Exp Med, Vol. 121, p. 439, 1965) as a protein normally expressed in the fetal intestine during the first six months of pregnancy, and has been found in many cancers such as colorectal cancer or pancreatic cancer. The CEA family belongs to the immunoglobulin superfamily. The CEA family consisting of 18 genes is subdivided into two subgroups of proteins: the carcinoembryonic antigen-related cell adhesion molecule (CEACAM) subgroup and the pregnancy-specific glycoprotein subgroup (Kammerer & Zimmermann, BMC Biology, 2010, Vol. 8:12). In humans, the CEACAM subgroup consists of seven members: CEACAM1, CEACAM3, CEACAM4, CEACAM5, CEACAM6, CEACAM7, and CEACAM8. CEACAM5, originally identified as CEA, has been reported to be highly expressed on the surface of cancer cells such as colorectal tumor cells, gastric tumor cells, lung tumor cells, and pancreatic tumor cells, and its expression in normal tissues is limited to a few normal epithelial cells such as colon cells and esophageal epithelial cells. Therefore, CEACAM5 may constitute a suitable therapeutic target for tumor-specific targeting methods such as immunoconjugates.

[0003] The extracellular domains of CEACAM family members consist of repeat immunoglobulin-like (Ig-like) domains classified into three types—A, B, and N—based on sequence homology. CEACAM5 contains seven such domains: N, A1, B1, A2, B2, A3, and B3. On the one hand, the CEACAM5 A1, A2, and A3 domains show high sequence homology, while on the other hand, the B1, B2, and B3 domains show high sequence homology, with the A domains of human CEACAM5 showing 84–87% pairwise sequence similarity and the B domains showing 69–80% pairwise sequence similarity. Furthermore, other human CEACAM members that exhibit A and / or B domains in their structure, namely CEACAM1, CEACAM6, CEACAM7, and CEACAM8, show homology to human CEACAM5. In particular, the A and B domains of the human CEACAM6 protein show sequence homology to the A1 and A3 domains and the B1-B3 domains of human CEACAM5, respectively, and this sequence homology is even higher than that observed between the A and B domains of human CEACAM5. Anti-CEA antibodies for diagnostic or therapeutic purposes targeting CEA have been generated. Specificity for related antigens has always been mentioned as a concern in this field, for example, by Sharkey et al. (Cancer Research 50, 2823 (1990)). Due to the homologies mentioned above, some of the aforementioned antibodies show binding to repetitive epitopes of CEACAM5 present, for example, in different immunoglobulin domains and show cross-reactivity with other CEACAM family members such as CEACAM1, CEACAM6, CEACAM7, or CEACAM8, and thus may lack specificity for CEACAM5. However, for CEA-targeted therapy, anti-CEACAM5 antibodies are required to be specific such that they bind to human CEACAM5-expressing tumor cells but not to certain normal tissues expressing other CEACAM family members. CEACAM1, CEACAM6, and CEACAM8 have been described to be expressed by neutrophils of humans and non-human primates (Ebrahimmnejad et al., Exp Cell Res 260, 365 (2000); Zhao et al., J Immunol Methods 293, 207 (2004); Strickland et al., J Pathol 218, 380 (2009)) and it has been noted that they regulate granulopoiesis and play a role in the immune response. Thus, for therapeutic purposes, cross-reactivity of anti-CEACAM5 antibodies with CEACAM1, CEACAM6, CEACAM7, or CEACAM8 may decrease the therapeutic index of the compound as it increases toxicity in normal tissues. Thus, there is a need for antibodies specific for CEACAM5 that do not cross-react with other molecules of the CEACAM family for use, for example, as part of an antibody-drug conjugate (ADC) or for use in other ways that result in the death of target cells.

[0004] Furthermore, since CEACAM5 is described as being expressed in some normal cell tissues, it is desirable to develop anti-CEACAM5 antibodies that can bind to both human CEACAM5 and cynomolgus monkey (Macaca fascicularis) CEACAM5. This is because such antibodies can be easily tested in preclinical toxicological studies in cynomolgus monkeys to evaluate their safety profile. The combination of a) the need for species cross-reactivity and b) the need for specificity to human and macaque fascicularis CEACAM5, i.e., the absence of cross-reactivity with other macaque fascicularis and human CEACAM family members, adds further complexity to the development of novel anti-CEACAM5 antibodies, especially considering the overall sequence homology between human CEACAM proteins and macaque fascicularis CEACAM proteins.

[0005] Furthermore, CEACAM5 is described in the literature as a surface protein with poor internal translocation (reviewed in Schmidt et al., 2008, Cancer Immunol.Immunother., Vol. 57, p. 1879), which raises further challenges regarding antibody-drug conjugates for this target protein.

[0006] A known anti-CEACAM5 antibody is rabetuzumab (also known as hMN14) from Immunomedics (Sharkey et al., 1995, Cancer Research, Vol. 55, p. 5935). This antibody has been shown not to bind to the relevant antigen, but also does not cross-react with CEACAM5 derived from Macaca fascicularis. Rabetuzumab is also used as part of an antibody-drug conjugate (ADC), namely rabetuzumab-govitecan. Rabetuzumab-govitecan is an ADC consisting of the anti-CEACAM5 antibody rabetuzumab conjugated with the cytotoxic drug SN38 via a linker (called CL2A) containing a pH-sensitive carbonate and a short polyethylene glycol (PEG) chain. Rabetuzumab-govitecan is characterized by the significant instability of the linker structure used, leading to premature systemic loss of the cytotoxic payload after parenteral administration. This degradation process may limit antitumor activity and increase the risk of side effects. Another known anti-CEACAM5 ADC is Sanofi's SAR408701 (tusamitamablubutansine), which contains the anti-CEACAM5 antibody SAR408377 (tusamitamab; also known as huMab2-3) covalently linked to the cytotoxic agent DM4, a potent microtubule-destabilizing mytansinoid, via an N-succinimidyl 4-(2-pyridyldithio)butyrate (SPDB) linker. SAR408701 has been associated with toxic side effects (including keratitis and keratopathy) in several organs and tissues, including the cornea of ​​the eye. Furthermore, the efficacy of microtubule inhibitor-based ADCs may be limited in certain cancer indications, such as colorectal cancer. To date, no anti-CEACAM5 antibodies or ADCs have been approved for any therapeutic use in clinical practice. Generally, a small number of ADCs are approved for the treatment of solid tumors. For example, there is still a need for novel and improved therapeutic agents for the treatment of various solid tumor indications, such as cancer, including CRC, pancreatic cancer, gastric cancer, NSCLC, esophageal cancer, and prostate cancer. [Overview of the Initiative]

[0007] The present invention addresses this need and other needs in the art, particularly by providing a monoclonal antibody against CEACAM5 (which is reactive with both human protein and macaca fascicularis protein) and by providing an immunoconjugate (also referred to herein as an antibody-drug conjugate (ADC)) containing the above antibody, such immunoconjugates have cytotoxic effects, killing tumor cells in vitro and inhibiting tumor growth in vivo. The present invention relates to embodiments described in the claims and further descriptions below herein. In particular, in our attempt to generate a novel antibody against CEACAM5 in the form of an immunoconjugate, possessing characteristics optimal for therapeutic purposes, we diligently conducted research and development to select an antibody with a favorable profile and develop an immunoconjugate based on it. Unexpectedly, the inventors were able to select and generate optimized IgG containing several desired characteristics. These antibodies bind with high affinity to the A2-B2 domains of human CEACAM5 and do not recognize human CEACAM1, CEACAM6, CEACAM7, and CEACAM8 proteins. In relation to cells, these antibodies show high affinity to CEACAM5-expressing tumor cells and are internally transported. Furthermore, these antibodies also bind to the macaque fascicularis CEACAM5 protein, with affinities to the monkey protein and human protein being within 10-fold relative to each other. The antibodies of the present invention bind to the A2-B2 domains of macaque fascicularis CEACAM5 but do not recognize another macaque fascicularis CEACAM protein, CEACAM6.

[0008] Furthermore, the inventors have shown that when the antibodies they have produced are combined with a cytotoxic drug in an immunoconjugate, a cytotoxic effect against tumor cells can be induced in vitro. Additionally, the antibody conjugated to the cytotoxic drug (i.e., the immunoconjugate of the present invention) can significantly inhibit tumor growth in mice with CEACAM5-expressing tumors. The linker connecting the drug and antibody was designed to maximize systemic stability after parenteral administration. The release of exatecan from the immunoconjugate of the present invention within target cells results in very high potency and excellent bystander effects. The potent bystander effect may be beneficial in the treatment of patients with heterologous target expression. [Brief explanation of the drawing]

[0009] [Figure 1] This figure shows the binding of mAb1 to recombinant human (rh)CEACAM5 ECD or its domains N-A1-B1, A2-B2, A3-B3, or recombinant macaca fascicularis (mf)CEACAM5 ECD in an ELISA assay. [Figure 2] This figure shows the cell binding of mAb1 to MKN45 cell lines expressing CEACAM5, compared to the anti-CEACAM5 antibodies EC50:antibodies huMab2-3 and hmn-14 that bind to MKN45 cells. [Figure 3] This figure shows the internal migration of pHrodo-labeled antibodies into late endosomes and lysosomes in cells (total fluorescence intensity per cell, average of triple duplication). [Figure 4] This figure shows the fluorescence intensity per cell over a period of 700 to 1200 minutes. This represents the straight portion of the curve. The slope of the straight line was measured and compared between samples (see Example 1.6.5). [Figure 5] This figure shows IHC staining of FFPE cancer cell lines using the antibody rb8G4. [Figure 6] This figure shows the correlation between CEACAM5 mRNA expression and IHC staining in 104 cancer cell lines. [Figure 7]This figure shows IHC staining of normal human tissue using the antibody rb8G4. [Figure 8] This figure shows the expression of CEACAM5 mRNA in normal human tissue. [Figure 9] This figure shows IHC staining of human colorectal cancer tissue using the antibody rb8G4. [Figure 10] This figure shows IHC staining of human gastric cancer tissue using the antibody rb8G4. [Figure 11] This figure shows IHC staining of human esophageal cancer tissue using the antibody rb8G4. [Figure 12] This figure shows IHC staining of non-small cell lung cancer tissue using the antibody rb8G4. [Figure 13] Western blotting revealed the binding of mAb1 (Figure 13A) and rb8G4 (Figure 13B) to CEACAM5 in cancer cell line lysates. [Figure 14] This figure shows a typical SEC chromatogram illustrating the purity of the stock mAb, the conjugate after UF, and the final BDS. [Figure 15] This figure shows a typical RP-HPLC chromatogram illustrating the separation of light and heavy chains. The chromatogram shows a superposition of stock mAb, crude ADC, and final BDS. [Figure 16] This figure shows a typical chromatogram illustrating the free drug levels of the NAC standard and the final BDS. [Figure 17] This figure shows a typical SEC chromatogram indicating the purity of the stock mAb and the final BDS. [Figure 18] This figure shows a typical RP-HPLC chromatogram illustrating the separation of light and heavy chains. The chromatogram shows a superposition of the stock mAb and the final BDS. [Figure 19] This figure shows a typical chromatogram illustrating the free drug levels of the NAC standard and the final BDS. [Figure 20]This figure shows the stability of ADC in human, mouse, and cynomolgus monkey serum. The conjugated exatecan concentration was calculated using free exatecan (initial dose approximately 10 μM) (normalized data). [Figure 21] This figure shows the control stability of ADC3 in mouse serum and buffer. The conjugate SN38 concentration was calculated using free SN38 (initial dose 50 μg / mL ADC protein concentration) (denormalized). [Figure 22] This figure shows the payload release profiles of ADC1 and ADC2 in human liver lysosomes (pH 5.0). Conjugate drug concentrations were calculated using, for example, free exatecan (initial concentration approximately 10 μM exatecan). Normalized data. [Figure 23-1] This figure shows that free exatecan is identified as a lysosomal release product by ADC catabolism profiling. [Figure 23-2] Figure 23-1 continued. [Figure 24] This figure shows the in vitro efficacy of ADC1, ADC2, and free payloads against antigen-positive SK-CO-1 (Figure 24A) and SNU-16 (Figure 24B) cell lines compared to antigen-negative MDA-MB-231 (Figure 24C) cell lines. One representative experiment is shown, with triple overlapping mean ± SD values. The legend shown in Figure 24C, which assigns three different sets of data points to ADC1, ADC2, and the payload respectively, also applies to Figures 24A and 24B. [Figure 25] This figure shows a comparison of ADC1 and ADC2 cells with their respective isotype controls against the SK-CO-1 cell line. One representative experiment is shown, with the mean ± SD values ​​for triple overlap. [Figure 26]This figure shows the in vitro efficacy of ADC1, ADC2, ADC SAR DM4, ADC mAb1 DM4, and free payload against antigen-positive SK-CO-1 (Figure 26A) cell lines compared to antigen-negative MDA-MB-231 (Figure 26B). One representative experiment is shown, with triple overlap mean ± SD values. The legend shown in Figure 26B also applies to Figure 26A. [Figure 27] This figure (Figure 27A) shows the strong bystander effect of ADC1 and ADC2 on antigen-negative MDA-MB-231 cells when co-cultured with antigen-positive SK-CO-1 cells. No nonspecific effect of ADC1 or ADC2 on MDA-MB-231 cells alone was observed (Figure 27B). One representative experiment is shown with double overlap mean ± SD. [Figure 28] The figures show that the bystander effect of ADC1 and ADC2 on antigen-negative MDA-MB-231 cells when co-cultured with antigen-positive SK-CO-1 cells is stronger than that of ADC SAR DM4 (Figures 28A and 28B). There was no nonspecific effect of the test ADCs on MDA-MB-231 cells alone (Figure 28C). One representative experiment is shown with double overlap mean ± SD. [Figure 29] This figure shows the effectiveness of ADC1 and ADC2 in the CRC PDX model (COPF217) after a single treatment. [Figure 30] This figure shows the effectiveness of ADC1 in the NSCLC PDX model (LUPF160151) after a single treatment. [Figure 31] This figure shows the effectiveness of ADC1 in a gastric cancer PDX model (GAX066) after a single treatment. [Figure 32] This figure shows the effectiveness of ADC1 compared to ADC3 in a pancreatic xenograft model (HPAF-II). [Figure 33] This figure shows the effectiveness of ADC1 compared to ADC SAR DM4 in the CRC PDX model (COPF230). [Figure 34]This figure shows the effectiveness of ADC1 compared to ADC SAR DM4 in the CRC PDX model (REPF210). [Figure 35] This figure shows the effectiveness of ADC1 compared to ADC SAR DM4 in the gastric PDX model GAPF313 (interim analysis of an ongoing experiment). [Modes for carrying out the invention]

[0010] definition As used herein, “CEACAM5” refers to “carcinoembryonic antigen-associated cell adhesion molecule 5,” also known as “CD66e” (differentiation cluster 66e) or CEA. CEACAM5 is a glycoprotein involved in cell adhesion. CEACAM5 is particularly highly expressed on the surface of solid tumors, for example, colorectal cancer, gastric cancer, non-small cell lung cancer, pancreatic cancer, esophageal cancer, prostate cancer, and other solid tumors.The reference sequence for the full-length human CEACAM5, including the signal peptide (positions 1-34) and propeptide (positions 686-702), is available from the GenBank database under accession number AAA51967.1, with the following amino acid sequence: (SEQ ID NO: 1). In the Caucasian population, five non-synonymous SNPs have been identified at a frequency of more than 2%, four of which are located in the N domain of human CEACAM5 (positions 80, 83, 112, and 113), and the last one in the A2 domain (position 398). GenBank AAA51967.1 contains the major haplotypes (I80, V83, I112, I113, and E398).

[0011] A “domain” or “region” is generally defined based on sequence homology and may be any region of a protein that is often associated with a particular structural or functional entity. Members of the CEACAM family are known to be composed of Ig-like domains. In this document, the term domain is used to refer to either individual Ig-like domains, such as “N-domains,” or groups of consecutive domains, such as “A2-B2 domains.”

[0012] The domain structure of human CEACAM5 is as follows (based on the GenBank AAA51967.1 sequence; sequence number 1): TIFF0007848189000001.tif52142 Therefore, the A2-B2 domain of human CEACAM5 consists of amino acids 321-498 of Sequence ID No. 1.

[0013] The reference sequence for the macaca fascicularis CEACAM5 protein is available (NCBI reference sequence XP_005589491.1), and its amino acid sequence is as follows: [Outside 1] JPEG0007848189000002.jpg103169 (Sequence ID 2) (The signal peptide is in italics; the A2-B2 domain is in bold; the GPI anchor is underlined; the N-A1-B1 domain between the signal peptide and the A2-B2 domain is in regular font; the A3-B3 domain between the A2-B2 domain and the GPI anchor is in regular font).

[0014] A "coding sequence," or a sequence that "codes" the expression product of a polypeptide, protein, or enzyme, is a nucleotide sequence that, when expressed, results in the production of that polypeptide, protein, or enzyme; that is, the nucleotide sequence codes for the amino acid sequence of that polypeptide, protein, or enzyme. A protein coding sequence may include a start codon (usually ATG) and a stop codon. As used herein, references to specific proteins (e.g., antibodies) may include polypeptides having a natural amino acid sequence, as well as variants and modified forms, regardless of their origin or method of preparation. Proteins having a natural amino acid sequence are proteins that have the same amino acid sequence as those obtained in nature. Such natural sequence proteins may be isolated from nature or prepared using standard recombinant and / or synthetic methods. Natural sequence proteins include, in particular, naturally occurring cleaved or soluble forms, naturally occurring variant forms (e.g., alternative splice forms), naturally occurring allelic variants, and forms including post-translational modifications. Natural sequence proteins include proteins having post-translational modifications such as glycosylation, or phosphorylation, or other modifications of certain amino acid residues. The term "gene" means a DNA sequence that codes for or corresponds to a specific sequence of amino acids, including all or part of one or more proteins or enzymes, and may or may not include regulatory DNA sequences, such as promoter sequences, which determine the conditions under which a gene is expressed. Some non-structural genes are transcribed from DNA to RNA but may not be translated into amino acid sequences. Other genes can function as regulators of structural genes or as regulators of DNA transcription. In particular, the term "gene" may refer to a genomic sequence that codes for a protein, i.e., a sequence that includes regulatory elements, promoters, introns, and exon sequences.

[0015] In this specification, a sequence that is "at least 85% identical" to a reference sequence is a sequence that has 85% or higher sequence identity over its entire length compared to the entire length of the reference sequence, for example, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. Accordingly, the percentage of "sequence identity" can be determined by comparing two such sequences over their entire lengths using global pairwise alignment with the algorithm of Needleman and Wunsch (J.Mol.Biol. Vol. 48: p. 443 (1970)), for example, using the BLOSUM62 matrix and the program Needle (EMBOSS) with the following parameters: gap open = 10, gap extend = 0.5, end gap penalty = false, end gap open = 10, end gap extend = 0.5 (these are standard settings).

[0016] A "conservative amino acid substitution" is a substitution in which an amino acid residue is replaced by another amino acid residue having a side chain with similar chemical properties (e.g., charge, size, or hydrophobicity). Generally, conservative amino acid substitutions do not substantially alter the functional properties of a protein. Examples of groups of amino acids with side chains having similar chemical properties include: 1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; 2) aliphatic hydroxyl side chains: serine and threonine; 3) amide-containing side chains: asparagine and glutamine; 4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; 5) basic side chains: lysine, arginine, and histidine; 6) acidic side chains: aspartic acid and glutamic acid; and 7) sulfur-containing side chains: cysteine ​​and methionine. Conservative amino acid substitution groups can also be defined based on amino acid size.

[0017] An antibody (also called an immunoglobulin) may be a native or conventional antibody, for example, in which two heavy chains are linked to each other by disulfide bonds, and each heavy chain is linked to a light chain by disulfide bonds. There are two types of light chains: lambda(I) and kappa(k). There are five main heavy chain classes (or isotypes) that determine the functional activity of antibody molecules: IgM, IgD, IgG, IgA, and IgE. Each antibody chain contains different sequence domains (or regions). The light chain of a typical IgG antibody contains two regions: a variable region (VL) and a constant region (CL). The heavy chain of a typical IgG antibody has four regions: a variable region (VH) and a constant region (CH), the latter consisting of three constant domains (CH1, CH2, and CH3). The variable regions of both the light and heavy chains determine the binding to and specificity of the antigen. The constant regions of the light and heavy chains can confer important biological properties such as antibody chain attachment, secretion, transplacental mobility, complement binding, and binding to the Fc receptor (FcR). The Fv fragment is the N-terminal portion of the Fab fragment of the antibody and consists of a variable region of one light chain and one heavy chain.

[0018] The specificity of an antibody lies in the structural complementarity between the antibody-binding site and the antigenic determinant. The antibody-binding site is mainly composed of residues from the so-called hypervariable region or complementarity-determining region (CDR). Therefore, the complementarity-determining region (CDR) refers to the amino acid sequence that determines both the binding affinity and specificity of the antibody's Fv region. The light (L) chain and heavy (H) chain of an antibody each have three CDRs, referred to as CDR1-L, CDR2-L, CDR3-L, and CDR1-H, CDR2-H, and CDR3-H, respectively. Therefore, the antigen-binding site of a conventional antibody contains six CDRs, including the CDR sets derived from the heavy chain and light chain variable regions, respectively. The “framework region” (FR) refers to the amino acid sequence interposed between CDRs, i.e., the portion of the immunoglobulin light and heavy chain variable region that is relatively conserved among different immunoglobulins of the same species. The light and heavy chains of immunoglobulins each have four FRs, designated as FR1-L, FR2-L, FR3-L, FR4-L, and FR1-H, FR2-H, FR3-H, and FR4-H, respectively. As used herein, the “human framework region” is a framework region that is substantially identical to the framework region of a naturally occurring human antibody (approximately 85% or higher, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%).

[0019] In relation to the present invention, the CDR / FR of immunoglobulin light chains or heavy chains is determined based on IMGT regulations (Lefranc et al., Dev. Comp. Immunol., 2003, Vol. 27 (No. 1): pp. 55-77; www.imgt.org). As used herein, the term “antibody” includes conventional antibodies and their fragments, as well as single-domain antibodies and their fragments, such as the variable heavy chain of a single-domain antibody; and as used herein, the term “antibody” also includes chimeric, humanized, bispecific, or multispecific antibodies, as well as other types of genetically modified antibodies. The term “antibody” also includes monoclonal antibodies. The terms “monoclonal antibody” or “mAb,” as used herein, refer to an antibody molecule consisting of a single amino acid sequence directed against a specific antigen and should not be interpreted as requiring antibody production by any particular method. Monoclonal antibodies can be produced, for example, by a single clone of a B cell or hybridoma, but can also be produced by recombinant methods, such as genetic engineering or protein engineering.

[0020] The term "chimeric antibody," in its broadest sense, refers to a genetically modified antibody that contains one or more regions derived from one antibody and one or more regions derived from one or more other antibodies. In embodiments, the chimeric antibody includes VH and VL of an antibody derived from a non-human animal, accompanied by CH and CL of another antibody which is a human antibody in some embodiments. Any animal can be used as the non-human animal, such as a mouse, rat, hamster, or rabbit. Furthermore, a chimeric antibody may refer to a multispecific antibody that has specificity for at least two different antigens.

[0021] The term "humanized antibody" refers to an antibody that is entirely or partially of non-human origin and has been modified to avoid or minimize the immune response in humans by replacing certain amino acids in the framework regions, for example, the VH and VL regions. The constant regions of a humanized antibody are typically the human CH and CL regions. An antibody "fragment" (e.g., a conventional antibody) includes a portion of an intact antibody such as IgG, particularly the antigen-binding region or variable region of the intact antibody. Examples of antibody fragments include Fv, Fab, F(ab')2, Fab', dsFv, (dsFv)2, scFv, sc(Fv)2, diabodies, and bispecific and multispecific antibodies formed from antibody fragments. Furthermore, the conventional antibody fragment may be a heavy chain antibody or a single-domain antibody such as VHH. The term "Fab" refers to an antibody fragment with a molecular weight of approximately 50,000 Da and antigen-binding activity, in which approximately half of the N-terminal side of the heavy chain and the entire light chain are linked to each other by disulfide bonds. Fab is typically obtained from fragments produced by treating IgG with the protease papain. The term "F(ab')2" refers to an antibody fragment with a molecular weight of approximately 100,000 Da and antigen-binding activity, which is slightly larger than two identical Fab fragments linked by a disulfide bond in the hinge region. F(ab')2 is typically obtained from fragments resulting from the treatment of IgG with protease pepsin. The term "Fab'" refers to an antibody fragment with a molecular weight of approximately 50,000 Da and antigen-binding activity, obtained by cleaving the disulfide bond in the hinge region of F(ab')2.

[0022] A single-stranded Fv ("scFv") is a covalently linked VH::VL heterodimer, typically expressed from a gene fusion containing genes encoding VH and VL linked by a peptide coding linker. The human scFv fragment of the present invention includes a CDR that is maintained in an appropriate three-dimensional structure, for example, by using genetic recombination techniques. Divalent and multivalent antibody fragments may be spontaneously formed by the association of a monovalent scFv, or by coupling a monovalent scFv with a peptide linker, such as in divalent sc(Fv)2. "dsFv" is a disulfide-stabilized VH::VL heterodimer. "(dsFv)2" means two dsFv coupled by a peptide linker. The term "bispecific antibody" or "BsAb" refers to an antibody containing two different antigen-binding sites. Therefore, a BsAb can, for example, bind to two different antigens simultaneously. Genetic engineering has seen increasing use in recent years for designing, modifying, and producing antibodies or antibody derivatives with a desired set of binding characteristics and effector functions, as described, for example, in European Patent Application Publication No. 2 050 764. The term "multispecific antibody" refers to an antibody that contains two or more different antigen-binding sites. The term "diabody" refers to a small antibody fragment having two antigen-binding sites, such fragments containing a heavy chain variable domain (VH) (VH-VL) connected to a light chain variable domain (VL) on the same polypeptide chain. By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with a complementary domain on another chain, creating two antigen-binding sites. The term "hybridoma" refers to cells that produce desired monoclonal antibodies with antigen specificity, obtained by fusion of B cells, which are prepared by immunizing non-human mammals with an antigen, with myeloma cells derived from mice or the like.

[0023] "Purified" or "isolated" means, where a polypeptide (e.g., antibody) sequence or nucleotide sequence is referenced, that the indicated molecule exists in a substantially absent state of other biomolecules of the same type. The term "purified," as used herein, means that at least 75%, 85%, 95%, 96%, 97%, or 98% by mass of biomolecules of the same type are present. An "isolated" nucleic acid molecule encoding a particular polypeptide refers to a nucleic acid molecule that substantially contains no other nucleic acid molecules that do not encode the polypeptide of interest, although the molecule may contain some additional bases or moieties that do not adversely affect the fundamental characteristics of the composition. As used herein, the term "subject" means a mammal such as a rodent, cat, dog, primate, or human. In embodiments of the present invention, the subject (or patient) is human.

[0024] The antibody of the present invention The inventors have successfully generated, screened, and selected a specific anti-CEACAM5 antibody that, remarkably, exhibits a combination of features that make such an antibody ideally suited for use in cancer treatment, particularly as part of an immunoconjugate (antibody-drug conjugate). For example, the antibody of the present invention exhibits high affinity for both human and macaque fascicularis CEACAM5 proteins and does not significantly cross-react with human CEACAM1, CEACAM6, CEACAM7, and CEACAM8 proteins, nor with macaque fascicularis CEACAM6 protein. The inventors have determined the amino acid sequence of such a monoclonal antibody according to the present invention. The present invention provides isolated antibodies that bind to the human CEACAM5 protein and include CDR1-H consisting of the amino acid sequence DGSVSRGGYY (SEQ ID NO: 3), CDR2-H consisting of the amino acid sequence IYYSGST (SEQ ID NO: 4), CDR3-H consisting of the amino acid sequence ARGIAVAPFDY (SEQ ID NO: 5), CDR1-L consisting of the amino acid sequence QSVRSN (SEQ ID NO: 6), CDR2-L consisting of the amino acid sequence AAS (SEQ ID NO: 7), and CDR3-L consisting of the amino acid sequence QQYTNWPFT (SEQ ID NO: 8). These antibodies can also bind to the Macaca fascicularis CEACAM5 protein.

[0025] In embodiments of the present invention, the antibody having the six CDR sequences mentioned above is an amino acid sequence [Outside 2] The heavy chain variable region (VH) and amino acid sequence containing an amino acid sequence that is at least 85% identical to JPEG0007848189000003.jpg22166 (SEQ ID NO: 9) (CDR is shown in bold). [Outside 3] It contains a light chain variable region (VL) with an amino acid sequence that is at least 85% identical to JPEG0007848189000004.jpg16167 (SEQ ID NO: 10) (CDR is shown in bold).

[0026] In embodiments of the present invention, the antibody having the six CDR sequences mentioned above includes a heavy chain variable region (VH) containing the amino acid sequence of SEQ ID NO: 9 and a light chain variable region (VL) containing the amino acid sequence of SEQ ID NO: 10. In embodiments of the present invention, this antibody has an amino acid sequence ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG(Sequence ID 11) The heavy chain constant region (CH) and the amino acid sequence which is at least 85% identical to the above. amino acid sequence RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(Sequence ID 12) It further includes a light chain constant region (CL) containing an amino acid sequence that is at least 85% identical to the above.

[0027] In embodiments of the present invention, the antibody comprises a heavy chain constant region (CH) containing the amino acid sequence of SEQ ID NO: 11 and a light chain constant region (CL) containing the amino acid sequence of SEQ ID NO: 12.

[0028] In a more specific embodiment, the antibody of the present invention binds to the human CEACAM5 protein and the amino acid sequence EVQLQESGPGLVKPSQTLSLTCTVSDGSVSRGGYYLTWIRQHPGKGLEWIGYIYYSGSTYFNPSLRSRVTMSVDTSKNQFSLKLSSVTAADTAVYYCARGIAVAPFDYWGQGT LVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHT CPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (Sequence ID 13) Heavy chain (HC) containing an amino acid sequence that is at least 85% identical to and amino acid sequence EIVLTQSPATLSVSPGERATLSCRTSQSVRSNLAWYQQKPGQAPRLLIYAASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYTNWPFTFGPGTKVDIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (Sequence ID 14) The isolated antibody comprises a light chain (LC) containing an amino acid sequence that is at least 85% identical to that of the original antibody. In a further specific embodiment of the present invention, the antibody comprises a heavy chain (HC) containing the amino acid sequence of SEQ ID NO: 13 and a light chain (LC) containing the amino acid sequence of SEQ ID NO: 14. In a further specific embodiment of the present invention, the antibody comprises two identical heavy chains (HC) containing the amino acid sequence of SEQ ID NO: 13 and two identical light chains (LC) containing the amino acid sequence of SEQ ID NO: 14.

[0029] In some embodiments, one or more individual amino acids of the antibody of the present invention may be modified by substitution, in particular conservative substitution, in one or more of the sequences mentioned above, including the CDR sequence. Such modifications may be intended, for example, in connection with the humanization of the antibody, to remove a glycosylation site or a deamidation site. In some embodiments, the antibody of the present invention is an isolated antibody that binds to the human CEACAM5 protein and consists of two identical heavy chains (HC) with the amino acid sequence of SEQ ID NO: 13 and two identical light chains (LC) with the amino acid sequence of SEQ ID NO: 14, and this particular antibody is also referred to herein as "mAb1". In some embodiments, the antibodies of the present invention bind to the A2-B2 domains of human and macaque fascicularis CEACAM5. The present invention also provides antibodies that compete for binding to the A2-B2 domains of human and / or macaque fascicularis CEACAM5 proteins with antibodies containing the heavy and light chain variable regions of mAb1 (i.e., VH and VL, corresponding to SEQ ID NOs. 9 and 10, respectively).

[0030] The ability of a candidate antibody (hereinafter referred to as the "reference" antibody in relation to the candidate antibody) to compete with an antibody containing VH and VL of mAb1 for binding to the A2-B2 domains of the human and / or macaque fascicularis CEACAM5 protein can be easily assayed by competitive ELISA, for example, by conjugating an antigen (i.e., a polypeptide containing or consisting of fragments of human or macaque fascicularis CEACAM5, including the A2-B2 domains of human or macaque fascicularis CEACAM5, particularly the extracellular domain) to a solid support, and adding two solutions containing the candidate antibody and the reference antibody, respectively, to allow the antibodies to compete for binding to the antigen. The amount of reference antibody bound to the antigen can then be measured and compared to the amount of reference antibody bound to the antigen when measured against a negative control (e.g., a solution without the antibody). If the amount of bound reference antibody decreases in the presence of the candidate antibody compared to the amount of bound reference antibody in the presence of a negative control, it indicates that the candidate antibody competed with the reference antibody. Conveniently, the reference antibody can be labeled (e.g., by fluorescence) to facilitate its detection. Repeated assays may be performed using serial dilutions of the candidate antibody and / or the reference antibody.

[0031] In some embodiments, the antibodies of the present invention do not bind to or significantly cross-react with human CEACAM1, human CEACAM6, human CEACAM7, human CEACAM8, and macaque fascicularis CEACAM6 proteins. In some embodiments, the antibodies do not bind to or significantly cross-react with the extracellular domains of the aforementioned human and macaque fascicularis CEACAM proteins other than CEACAM5.

[0032] "Affinity" is theoretically determined by the equilibrium binding between the antibody and the antigen. Affinity is measured by the binding and dissociation rates using surface plasmon resonance (SPL) or by immunochemical assays (ELISA, FACS) using EC (Emission Control). 50 (or apparent K) DIt can be experimentally evaluated by various known methods, such as measuring EC. 50 This is the concentration of antibody that induces an intermediate response between the baseline and the maximum value, measured by ELISA (enzyme-linked immunosorbent assay) or FACS (fluorescence-activated cell sorting) after a specified exposure time to a prescribed concentration of antigen. EC of both antigens 50 If the affinity for antigen 1 (Ag1) is within a similar range, a monoclonal antibody that binds to antigen 2 (Ag2) is "cross-reactive" to antigen 2. In this application, a monoclonal antibody that binds to Ag1 is cross-reactive to Ag2 if its affinity for Ag2 is within 10 times or less than its affinity for Ag1 (e.g., within 5 times), in which case the affinity for both antigens is measured by the same method. A monoclonal antibody that binds to Ag1 is "not significantly cross-reactive" to Ag2 if its affinities to the two antigens Ag1 and Ag2 are very different. If the binding response is too low, the affinity for Ag2 may not be measurable. In this application, a monoclonal antibody that binds to Ag1 is not significantly cross-reactive to Ag2 if the binding response of the monoclonal antibody to Ag2 is less than 5% of the binding response of the same monoclonal antibody to Ag1, under the same experimental setup and antibody concentration. In practice, the antibody concentration used is EC 50 The concentration may be such as any other concentration, or it may be such that the concentration is necessary to reach the saturation plateau obtained with Ag1. A monoclonal antibody "specifically binds" to (or is "specific to") Ag1 if it is not significantly cross-reactive with Ag2.

[0033] In some embodiments, the antibodies according to the present invention have an affinity for Macaca fascicularis CEACAM5 that is within 10 times or less (e.g., within 5 times) the affinity for human CEACAM5. Therefore, the antibodies according to the present invention can be used in toxicological studies conducted in monkeys, as the toxicity profile observed in monkeys is considered relevant to predicting potential adverse effects in humans. In some embodiments, the antibody of the present invention has an affinity of ≤10 nM for human CEACAM5 or macaca fascicularis CEACAM5 or both. For example, the antibody of the present invention may have an affinity for human CEACAM5 of 1 to 10 nM, for example, an affinity for human CEACAM5 of about 6 nM. The affinity for human CEACAM5 or macaca fascicularis CEACAM5 can be determined, for example, by the EC50 value in an ELISA using soluble recombinant CEACAM5 as the capture antigen.

[0034] Alternatively, for example, the apparent dissociation constant (apparent KD) of the antibody of the present invention against the tumor cell line MKN45 (DSMZ, ACC409) may be determined by FACS analysis. In addition, it has been shown that the antibody according to the present invention can detect CEACAM5 expression, for example, by immunohistochemistry of frozen, formalin-fixed, and paraffin-embedded (FFPE) tissue sections. Any combination of the embodiments described above and below in this specification forms part of the present invention. In some embodiments, the antibody according to the present invention is a conventional antibody such as a conventional monoclonal antibody, or an antibody fragment, a bispecific or multispecific antibody. In some embodiments, the antibody according to the present invention comprises or consists of IgG or a fragment thereof.

[0035] In some embodiments, the antibody of the present invention may be, for example, a mouse antibody, a chimeric antibody, a humanized antibody, or a human antibody. Numerous methods for humanizing antibody sequences are known in the art; see, for example, the review by Almagro & Fransson (2008) Front Biosci. Vol. 13: pp. 1619-1633. One widely used method is CDR transplantation or antibody reshaping, which involves transplanting the CDR sequence of a donor antibody, generally a mouse antibody, onto a framework scaffold of a human antibody with different specificities. Since CDR transplantation may reduce the biological activity of the CDR-transplanted non-human antibody according to its binding specificity and affinity, a reverse mutation may be introduced at a selected location in the CDR-transplanted antibody to maintain the binding specificity and affinity of the parent antibody. Identification of possible reverse mutation locations can be carried out using information available in the literature and antibody databases. Amino acid residues that are candidates for reverse mutations are typically those located on the surface of the antibody molecule; embedded residues or those with low surface exposure will usually not be altered. An alternative humanization technique to CDR transplantation and reverse mutation is resurfacing, in which non-human non-surface-exposed residues are preserved, while surface residues are changed to human residues. Another alternative technique is known as "inducible selection" (Jespers et al. (1994) Biotechnology vol. 12, p. 899), which can be used to derive fully human antibodies from mouse antibodies that preserve the epitopes and binding characteristics of the parent antibody. In the case of chimeric antibodies, humanization typically involves modifying the framework region of the variable region sequence.

[0036] While amino acid residues that are part of the CDR are typically not altered in connection with humanization, in certain cases it may be desirable to modify individual CDR amino acid residues, for example, to remove glycosylation sites, deamidation sites, or undesirable cysteine ​​residues. N-linked glycosylation occurs by the attachment of oligosaccharide chains to asparagine residues in the tripeptide sequences Asn-X-Ser or Asn-X-Thr (where X is any amino acid other than Pro). Removal of N-glycosylation sites can be achieved, for example, by conservative substitution, by mutating either the Asn or Ser / Thr residue to a different residue. Deamidation of asparagine and glutamine residues may occur depending on factors such as pH and surface exposure. Asparagine residues are particularly susceptible to deamidation when they are mainly present in the sequence Asn-Gly, and less susceptible when they are present in other dipeptide sequences such as Asn-Ala. Therefore, when such a deamidation site, for example Asn-Gly, is present in the CDR sequence, it is often desirable to remove the site by a conservative substitution to remove one of the involved residues. Substitutions in the CDR sequence to remove one of the involved residues are also intended to be included in the present invention. In humanized antibodies or fragments thereof, the variable domains of the heavy and light chains may include human acceptor framework regions. Humanized antibodies may further include human constant heavy and light chain domains, if present.

[0037] In some embodiments, the antibody according to the present invention may be an antibody fragment (e.g., a humanized antibody fragment) selected from the group consisting of Fv, Fab, F(ab')2, Fab', dsFv, (dsFv)2, scFv, sc(Fv)2, and diabody. In some embodiments, the antibody according to the present invention may be a bispecific or multispecific antibody formed from antibody fragments, wherein at least one antibody fragment is a fragment of the antibody according to the present invention. The multispecific antibody is, for example, a multivalent protein complex as described in European Patent Application Publication No. 2050764 or U.S. Patent Application Publication No. 2005 / 0003403. The bispecific or multispecific antibodies according to the present invention may have specificity for (a) human and macaque fascicularis CEACAM5 proteins and (b) at least one other antigen. In some embodiments, the at least one other antigen is neither a member of the human CEACAM family nor a member of the macaque fascicularis CEACAM family. In other embodiments, the at least one other antigen may be an epitope in human or macaque fascicularis CEACAM5 other than the epitope targeted by mAb1. The antibodies of the present invention can be prepared by any technique known in the art. The antibodies according to the present invention may be used, for example, in an isolated (e.g., purified) form, or they may be contained in a vector such as a membrane vesicle or lipid vesicle (e.g., liposome).

[0038] Nucleic acids and host cells of the present invention Further aspects of the present invention relate to isolated nucleic acids comprising or comprising a nucleic acid sequence encoding the antibody of the present invention as defined above. Typically, the nucleic acid is a DNA or RNA molecule and may be contained in any suitable vector, such as a plasmid, cosmid, episome, artificial chromosome, phage, or viral vector. The terms “vector,” “cloning vector,” and “expression vector” refer to a vehicle capable of introducing a DNA or RNA sequence (e.g., an exogenous gene) into host cells in order to transform the host and promote the expression (e.g., transcription and translation) of the introduced sequence. Therefore, a further aspect of the present invention relates to a vector comprising the nucleic acid of the present invention as defined above. Such vectors may contain regulatory elements such as promoters, enhancers, and terminators to induce or direct the expression of the polypeptides described above upon administration to a target. Examples of promoters and enhancers used in expression vectors for animal cells include the initial promoter and enhancer of SV40 (Mizukami T. et al., 1987), the LTR promoter and enhancer of Moloney's mouse leukemia virus (Kuwana Y et al., 1987), and the promoter (Mason JO et al., 1985) and enhancer (Gillies SD et al., 1983) of immunoglobulin H chains.

[0039] Any animal cell expression vector can be used, as long as it can insert and express the gene encoding the human antibody C region. Examples of suitable vectors include pAGE107 (Miyaji H et al., 1990), pAGE103 (Mizukami T et al., 1987), pHSG274 (Brady G et al., 1984), pKCR (O'Hare K et al., 1981), and pSG1 beta d2-4- (Miyaji H et al., 1990). Other examples of plasmids include replication plasmids containing origins of replication, or embedded plasmids such as pUC, pcDNA, and pBR. Other examples of viral vectors include adenoviruses, retroviruses, herpesviruses, and AAV vectors. Such recombinant viruses can be produced by techniques known in the art, such as transfection of packaging cells or transient transfection with helper plasmids or viruses. Typical examples of viral packaging cells include PA317 cells, PsiCRIP cells, GPenv+ cells, and 293 cells. Detailed protocols for producing such replication-deficient recombinant viruses can be found, for example, in International Publication No. 95 / 14785, International Publication No. 96 / 22378, U.S. Patent No. 5,882,877, U.S. Patent No. 6,013,516, U.S. Patent No. 4,861,719, U.S. Patent No. 5,278,056, and International Publication No. 94 / 19478.

[0040] A further object of the present invention relates to host cells transfected, infected, or transformed with nucleic acids and / or vectors according to the present invention. The term "transformation" refers to the introduction of an "exogenous" gene, DNA, or RNA into a host cell so that it expresses the introduced gene or sequence and produces a desired substance, typically a protein or enzyme encoded by the introduced gene or sequence. A host cell that receives and expresses the introduced DNA or RNA is "transformed." The nucleic acids of the present invention can be used to produce antibodies of the present invention in a suitable expression system. The term "expression system" means, for example, a host cell and a suitable vector under suitable conditions for expressing a protein encoded by exogenous DNA that is delivered by a vector and introduced into the host cell.

[0041] Common expression systems include Escherichia coli (E. coli) host cells and plasmid vectors, insect host cells and baculovirus vectors, and mammalian host cells and vectors. Other examples of host cells, though not limited to these, include prokaryotic cells (bacteria, etc.) and eukaryotic cells (yeast cells, mammalian cells, insect cells, plant cells, etc.). Specific examples include Escherichia coli, Kluyveromyces or Saccharomyces yeasts, mammalian cell lines (e.g., Vero cells, CHO cells, 3T3 cells, COS cells, etc.), and primary or established mammalian cell cultures (e.g., those produced from lymphoblasts, fibroblasts, germ cells, epithelial cells, nerve cells, adipocytes, etc.). Examples include mouse SP2 / 0-Ag14 cells (ATCC CRL1581), mouse P3X63-Ag8.653 cells (ATCC CRL1580), CHO cells lacking the dihydrofolate reductase gene (hereinafter referred to as the "DHFR gene") (Urlaub G et al.; 1980), and rat YB2 / 3HL.P2.G11.16Ag.20 cells (ATCC CRL1662, hereafter referred to as "YB2 / 0 cells"). In some embodiments, YB2 / 0 cells are used. This is because, when expressed in these cells, the ADCC activity of chimeric or humanized antibodies is enhanced.

[0042] When expressing humanized antibodies, the expression vector may be either a type in which the genes encoding the antibody heavy chain and the genes encoding the antibody light chain are in separate vectors, or a type in which both genes are in the same vector (tandem type). From the standpoint of ease of construction of humanized antibody expression vectors, ease of introduction into animal cells, and the balance between the expression levels of the antibody heavy chain and antibody light chain in animal cells, humanized antibody expression vectors are of the tandem type (Shitara K et al., J Immunol Methods. January 3, 1994; Vol. 167 (Vols. 1-2): pp. 271-278). Examples of tandem type humanized antibody expression vectors include pKANTEX93 (International Publication No. 97 / 10354 pamphlet) and pEE18.

[0043] The present invention also relates to a method for generating recombinant host cells expressing the antibody according to the present invention, comprising the steps of (i) introducing the recombinant nucleic acid or vector described above into competent host cells in vitro or ex vivo, (ii) culturing the obtained recombinant host cells in vitro or ex vivo, and (iii) optionally selecting cells that express and / or secrete the antibody. Such recombinant host cells can be used to produce the antibodies of the present invention.

[0044] Method for producing antibodies of the present invention The antibodies of the present invention can be prepared, without limitation, by any chemical, biological, genetic, or enzymatic technique known in the art, either alone or in combination.

[0045] By knowing the amino acid sequence of a desired antibody, those skilled in the art can easily produce the antibody or immunoglobulin chain using standard techniques for polypeptide production. For example, the antibody or immunoglobulin chain can be synthesized using a commercially available peptide synthesizer (such as one from Applied Biosystems, Inc., Foster City, California) in accordance with the manufacturer's instructions, using a well-known solid-phase method. Alternatively, the antibodies and immunoglobulin chains of the present invention can be produced by recombinant DNA techniques well known in the art. For example, such polypeptides (e.g., antibodies) can be obtained as DNA expression products after incorporating the DNA sequence encoding the desired polypeptide into an expression vector and introducing such a vector into a suitable eukaryotic or prokaryotic cell host that will express the desired polypeptide, and which can then be isolated using well-known techniques.

[0046] For example, the present invention provides the following DNA sequence encoding the antibody mAb1: mAb1 heavy chain nucleotide sequence In the sequence, the mVk signal peptide is underlined. The start and stop codons are in italics. The VH region sequence is shown in bold. CDR is indicated by a double underline: [Outside 4] JPEG0007848189000005.jpg197170 (Sequence ID 15) mAb1 light chain nucleotide sequence In the sequence, the uPA signal peptide is underlined. The start and stop codons are in italics. The VL region sequence is shown in bold. CDR is indicated by a double underline: [Outside 5] JPEG0007848189000006.jpg101170 (Sequence ID 16)

[0047] The present invention relates to a method for producing the antibody of the present invention, further comprising the steps of (i) culturing transformed host cells according to the present invention, (ii) expressing the antibody, and (iii) recovering the expressed antibody. The antibodies of the present invention can be suitably separated from culture media by conventional immunoglobulin purification procedures such as protein A-Sepharose chromatography, hydroxyapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0048] In some embodiments, the humanized chimeric antibody of the present invention can be produced by first obtaining the nucleic acid sequences encoding the aforementioned humanized VL and VH regions, then inserting these sequences into an expression vector for animal cells containing genes encoding human antibody CH and human antibody CL to construct a human chimeric antibody expression vector, and finally introducing the expression vector into animal cells to express the coding sequences. The CH domain of the human chimeric antibody can be any region belonging to the human immunoglobulin heavy chain, for example, the IgG class is preferred, and any one of the subclasses belonging to the IgG class, such as IgG1, IgG2, IgG3, and IgG4, can be used. Furthermore, the CL of the human chimeric antibody can be any region belonging to the human immunoglobulin light chain, and either the kappa class or the lambda class can be used.

[0049] Methods for producing humanized antibodies or chimeric antibodies may include conventional recombinant DNA, and gene transfection techniques are well known in the art (see, for example, Morrison SL. et al. (1984) and U.S. Patent Nos. 5,202,238 and 5,204,244). Methods for producing humanized antibodies based on conventional recombinant DNA and gene transfection techniques are well known in the art (see, for example, Riechmann L. et al., 1988; Neuberger MS. et al., 1985). Antibodies can be humanized using various techniques known in the art, including, for example, the techniques disclosed in International Publication No. 2009 / 032661, CDR transplantation (European Patent No. 239,400; International Publication No. 91 / 09967; U.S. Patent No. 5,225,539; U.S. Patent No. 5,530,101; and U.S. Patent No. 5,585,089), veneering or surface remodeling (European Patent No. 592,106; European Patent No. 519,596; Padlan EA (1991); Studnicka GM et al. (1994); Roguska MA. et al. (1994)), and chain shuffling (U.S. Patent No. 5,565,332). Common recombinant DNA techniques for preparing such antibodies are also known (see European Patent Application No. 125023 and International Publication No. 96 / 02576).

[0050] The Fab of the present invention can be obtained by treating the antibody of the present invention (e.g., IgG) with a protease such as papain. Alternatively, the Fab can be produced by inserting the DNA sequences encoding both strands of the Fab of the antibody into a vector for prokaryotic or eukaryotic cell expression, and then introducing the vector into prokaryotic or eukaryotic cells (if necessary) to express the Fab. The F(ab')2 of the present invention can be obtained by treating the antibody of the present invention (e.g., IgG) with pepsin, which is a protease. Alternatively, F(ab')2 can be prepared by attaching the Fab' described below via a thioether bond or a disulfide bond. The Fab' of the present invention can be obtained by treating F(ab')2 of the present invention with a reducing agent such as dithiothreitol. Alternatively, Fab' can be produced by inserting the DNA sequence encoding the Fab' chain of the antibody into a prokaryotic cell expression vector or a eukaryotic cell expression vector, and then introducing the vector into prokaryotic or eukaryotic cells (if necessary) to carry out expression. The scFv of the present invention can be produced by obtaining the sequences of the CDR or VH and VL domains described above for the antibody of the present invention, then constructing DNA encoding the scFv fragment, inserting the DNA into a prokaryotic or eukaryotic cell expression vector, and then introducing the expression vector into prokaryotic or eukaryotic cells (if necessary) to express the scFv. To generate humanized scFv fragments, a well-known technique called CDR transplantation can be used, which includes selecting complementarity-determining regions (CDRs) according to the present invention and transplanting them into a human scFv fragment framework with a known three-dimensional structure (see, for example, International Publication No. 98 / 45322; International Publication No. 87 / 02671; U.S. Patent No. 5,859,205; U.S. Patent No. 5,585,089; U.S. Patent No. 4,816,567; and European Patent No. 0173494).

[0051] Modification of the antibody of the present invention The amino acid sequence modifications of the antibodies described herein are intended. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody. By modifying and altering the structure and encoding DNA sequence of the antibody of the present invention, it is possible to obtain a functional antibody or polypeptide that still possesses desirable characteristics. When altering the amino sequence of a polypeptide, the hydroxyl index of amino acids may be considered. The importance of the hydroxyl amino acid index in relation to the biological function of protein interactions is generally understood in the art. It is accepted that the relative hydroxyl properties of amino acids contribute to the secondary structure of the resulting protein, which in turn governs the interaction between the protein and other molecules, such as enzymes, substrates, receptors, DNA, antibodies, and antigens. Each amino acid is assigned a hydropathy index based on its hydrophobic and charge characteristics, which are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine ​​(+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamic acid (-3.5); glutamine (-3.5); aspartic acid (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).

[0052] Furthermore, a further embodiment of the present invention includes a function-conserving variant of the polypeptide of the present invention. For example, certain amino acids in a protein structure can be substituted with other amino acids without significantly losing activity. Since the biological functional activity of a protein is determined by its interaction ability and properties, it is possible to make specific amino acid substitutions in the protein sequence and, of course, its coding DNA sequence, and still obtain a protein with similar characteristics. Therefore, it is intended that various changes can be made to the antibody sequence of the present invention, or the corresponding DNA sequence encoding the polypeptide described above, without significantly losing their biological activity. It is known in the art that substituting a specific amino acid with another amino acid having a similar hydropathic index or score can result in a protein with similar biological activity, i.e., a protein with equivalent biological function. Using well-established techniques such as alanine scanning, it is also possible to identify all amino acids in the antibody or polypeptide of the present invention that can be substituted without significantly losing antigen binding. Such residues can be considered neutral because they do not participate in antigen binding or the maintenance of the antibody structure. One or more of these neutral positions can be substituted with alanine or another amino acid without altering the key characteristics of the antibody or polypeptide of the present invention.

[0053] A neutral position can be considered a position where any amino acid substitution can be incorporated. In fact, in the principle of alanine scanning, alanine is selected because its residue does not have any particular structural or chemical characteristics. It is generally accepted that if a particular amino acid can be substituted with alanine without changing the properties of the protein, then many, if not all, other amino acid substitutions are likely to be neutral. Conversely, if alanine is the wild-type amino acid, then if a particular substitution is neutral, then other substitutions are likely to be neutral as well. As outlined above, amino acid substitutions are generally based on the relative similarities of amino acid side-chain substituents, such as their hydrophobicity, hydrophilicity, charge, and size. Exemplary substitutions that take any of the above characteristics into account are well known to those skilled in the art and include arginine and lysine; glutamic acid and aspartic acid; serine and threonine; glutamine and asparagine; and valine, leucine, and isoleucine.

[0054] It may also be desirable to modify the antibodies of the present invention with respect to effector function, for example, to enhance antigen-dependent cell-mediated cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC), or to alter binding to Fc receptors, for example. This can be achieved by introducing one or more amino acid substitutions into the Fc region of the antibody. Alternatively, or in addition to this, a cysteine ​​residue can be introduced into the Fc region to enable interchain disulfide bond formation in this region. Homodimerated antibodies thus produced may exhibit improved internal migration ability and / or increased complement-mediated cell death and / or increased antibody-dependent cytotoxicity (ADCC) (Caron PC. et al., 1992; and Shopes B., 1992). In some embodiments, the antibodies of the present invention may also be antibodies having a modified amino acid sequence that results in reduced or eliminated binding to most Fcγ receptors, which can reduce uptake and toxicity in normal cells and tissues expressing such receptors, such as macrophages and hepatic sinusoidal cells. An example of such an antibody involves the substitution of two leucine (L) residues at positions 234 and 235 with alanine (A) (i.e., LALA), and this double substitution has been shown to reduce Fc binding to FcγR, and consequently reduce ADCC as well, thereby reducing complement binding / activation. Another example of such an antibody involves the substitution of P329G in addition to the LALA double substitution (i.e., PG-LALA; see, for example, Schlothauer et al., Novel human IgG1 and IgG4 Fc-engineered antibodies with completely abolished immune effector functions, Protein Engineering, Design and Selection, Vol. 29, No. 10, October 2016, pp. 457-466). Thus, in some embodiments, the antibody of the present invention may (i) include, for example, a substitution of the LALA or PG-LALA set, and (ii) otherwise have an amino acid sequence identical to one of the amino acid sequences of the antibody of the present invention described herein above, with reference to the corresponding SEQ ID NO.

[0055] Another type of amino acid modification of the antibody of the present invention, namely by deleting one or more carbohydrate moieties found in the antibody and / or by adding one or more glycosylation sites not present in the antibody, may be useful for altering the antibody's original glycosylation pattern. The presence of either the tripeptide sequence asparagine-X-serine or asparagine-X-threonine (wherein X is any amino acid other than proline) creates a site that can be glycosylated. Deletion or addition of a glycosylation site to the antibody can be conveniently achieved by modifying the amino acid sequence to include one or more of the tripeptide sequences described above (for N-linked glycosylation sites).

[0056] Another type of modification involves removing sequences that have been shown, either in silico or experimentally, to potentially result in heterogeneity of degradation products or antibody preparations. For example, deamidation of asparagine and glutamine residues may occur depending on factors such as pH and surface exposure. Asparagine residues are particularly susceptible to deamidation when present in the sequence Asn-Gly, and to a lesser degree, also susceptible when present in other dipeptide sequences such as Asn-Ala. When such deamidation sites, particularly Asn-Gly, are present in an antibody or polypeptide, it may be considered to remove the site by a conservative substitution, typically to remove one of the involved residues. Such substitutions in sequences to remove one or more involved residues are also intended to be included by the present invention.

[0057] Another type of covalent modification involves chemically or enzymatically coupling a glycoside to an antibody. These procedures are advantageous in that they do not require the production of antibodies in host cells that have the ability to glycosylate N-linked or O-linked glycosylation. Depending on the coupling mode used, sugars can be attached to (a) arginine and histidine, (b) free carboxyl groups, (c) free sulfhydryl groups such as those of cysteine, (d) free hydroxyl groups such as those of serine, threonine, or hydroxyproline, (e) aromatic residues such as those of phenylalanine, tyrosine, or tryptophan, or (f) amide groups of glutamine. For example, such a method is described in International Publication No. 87 / 05330. The removal of the carbohydrate portion of an antibody can be achieved chemically or enzymatically. Chemical deglycosylation requires exposure of the antibody to the compound trifluoromethanesulfonic acid or an equivalent compound. This treatment results in the cleavage of almost all sugars except linked sugars (N-acetylglucosamine or N-acetylgalactosamine), while the antibody remains intact. Chemical deglycosylation is described by Sojahr H. et al. (1987) and Edge, A. et al. (1981). Enzymatic cleavage of the carbohydrate portion of an antibody can be achieved by using various endoglycosidases and exoglycosidases, as described by Thotakura, NR. et al. (1987).

[0058] Another type of covalent modification of antibodies involves linking an antibody to a variety of non-proteinoid polymers, such as polyethylene glycol, polypropylene glycol, or polyoxyalkylene, in the manner described, for example, in U.S. Patent Nos. 4,640,835, 4,496,689, 4,301,144, 4,670,417, 4,791,192, or 4,179,337. Other amino acid sequence modifications known in the art can also be applied to the antibodies of the present invention.

[0059] The Immunoconjugate of the present invention The present invention provides immunoconjugates, also known herein as antibody-drug conjugates or more simply as conjugates. As used herein, these terms all have the same meaning and are synonymous. Preferred methods for preparing immunoconjugates are known in the art. The immunoconjugates of the present invention can be prepared, for example, by the in vitro methods described herein. The present invention provides an immunoconjugate comprising an antibody of the present invention (e.g., mAb1, or an antibody having the same six CDRs as mAb1, etc.) covalently linked to at least one growth inhibitor via a linker. The term "proliferation inhibitor" (also called "antiproliferation agent") refers to a molecule, compound, or composition that inhibits the proliferation of cells such as tumor cells in vitro and / or in vivo. In some embodiments, the growth inhibitor is a cytotoxic agent (also called a cytotoxic agent). In some embodiments, the growth inhibitor is a radioactive moiety. The term "cytotoxic agent," as used herein, refers to a substance that directly or indirectly inhibits or prevents the function of a cell and / or causes cell destruction. The term "cytotoxic agent" includes, for example, chemotherapeutic agents, enzymes, antibiotics, toxins such as small molecule toxins or enzymatically active toxins, toxoids, vinca, taxanes, mytansinoids or mytansinoid analogs, tomaimycin or pyrrolobenzodiazepine derivatives, cryptophycin derivatives, leptomycin derivatives, auristatin or drastatin analogs, prodrugs, topoisomerase I inhibitors, topoisomerase II inhibitors, DNA alkylating agents, antitubulin agents, CC-1065 and CC-1065 analogs. Topoisomerase I inhibitors are molecules or compounds that inhibit human topoisomerase I, an enzyme involved in changing the topology of DNA by catalyzing the transient cleavage and religation of a single strand of DNA. Topoisomerase I inhibitors are, for example, highly toxic to dividing cells of mammals. Examples of suitable topoisomerase I inhibitors include camptothecin (CPT), and its analogs such as topotecan, irinotecan, silatecan, cositecan, exatecan, lurtotecan, gimatecan, velotecan, and rubitecan.

[0060] In some embodiments, the immunoconjugate of the present invention comprises the cytotoxic agent exatecan as a growth inhibitor. Exatecan has the chemical name (1S,9S)-1-amino-9-ethyl-5-fluoro-1,2,3,9,12,15-hexahydro-9-hydroxy-4-methyl-10H,13H-benzo(de)pyrano(3’,4’:6,7)indolizino(1,2-b)quinoline-10,13-dione. Exatecan has the following structural formula (I):

Chemical formula

[0061] In further embodiments of the present invention, other CPT analogs and other cytotoxic agents such as those listed above may be used. Examples of some cytotoxic agents and conjugation methods are further shown in the pamphlet of International Publication No. WO 2008 / 010101, which is incorporated herein by reference. The term "radioactive moiety" means At 211 , Bi 212 , Er 169 , I 131 , I 125 , Y 90 , In 111 , P 32 , Re 186 , Re 188 , Sm 153 , Sr 89This refers to a chemical entity (molecule, compound, or composition, etc.) containing or consisting of a radioactive isotope suitable for the treatment of cancer, such as a radioactive isotope of Lu. Such radioactive isotopes generally emit primarily beta rays. In some embodiments, the radioactive isotope is an alpha-emitting isotope, for example, thorium-227, which emits alpha rays. Immunoconjugates can be prepared, for example, as described in International Publication No. 2004 / 091668.

[0062] In the immunoconjugate of the present invention, the antibody of the present invention is covalently linked to at least one growth inhibitor via a linker. “Linker” as used herein means a chemical moiety comprising a covalent bond and / or any atomic chain that covalently attaches the growth inhibitor to the antibody. Linkers are well known in the art and include, for example, disulfide groups, thioether groups, acid-unstable groups, photo-unstable groups, peptidase-unstable groups, and esterase-unstable groups. The conjugation of the antibody of the present invention with a cytotoxic agent or other growth inhibitor can be carried out using a variety of bifunctional protein coupling agents, including, but not limited to, the following: N-succinimidylpyridyl dithiobutyrate (SPDB), 4-[(5-nitro-2-pyridinyl)dithio]-2,5-dioxo-1-pyrrolidinyl ester (nitro-SPDB), 4-(pyridine-2-yldisulfanyl)-2-sulfobutyrate (sulfo-SPDB), N-succinimidyl(2-pyridyldithio)propionate (SPDP), succinimidyl(N-) Reimidomethyl)cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), difunctional derivatives of imide esters (such as dimethyladipimidate HCl), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azide compounds (such as bis(p-azidobenzoyl)-hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For example, lysine immunotoxins can be prepared as described by Vitetta et al. (1987). Carbon-labeled 1-isothiocyanatobenzylmethyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugating radioactive nucleotides to antibodies (International Publication No. 94 / 11026).

[0063] In embodiments of the present invention, the linker may be a “cleavable linker” that can facilitate the release of cytotoxic agents or other growth inhibitors inside or near cells, such as tumor cells. In some embodiments, the linker is a linker that can be cleaved in the endosomes of mammalian cells. For example, acid-unstable linkers, peptidase-sensitive linkers, esterase-unstable linkers, photo-unstable linkers, or disulfide-containing linkers can be used (see, for example, U.S. Patent No. 5,208,020). Furthermore, when referring to structural formulas representing immunoconjugates, the following nomenclature is used herein: the growth inhibitor and the linker are collectively referred to as the [(linker)-(growth inhibitor)] moiety, for example, the exatecan molecule and the linker are collectively referred to as the [(linker)-(exatecan)] moiety.

[0064] In some specific embodiments of the present invention, the linker is a linker that can be cleaved by the human enzyme glucuronidase. Thus, for example, the immunoconjugate of the present invention comprises a linker that can be cleaved by glucuronidase, as shown in formula (II): [ka] (II) It may have, In the formula, antibody is the antibody of the present invention, S is the sulfur atom of the antibody, and n is the number of [(linker)-(proliferation inhibitor)] moieties covalently linked to the antibody. The number n may be, for example, 1 to 10, in a more specific embodiment n is 7 to 8, and in an even more specific embodiment n is 7.5 to 8.0 (i.e., about 8). In some embodiments, S is the sulfur atom of cysteine ​​in the antibody. In some embodiments, the antibody is mAb1.

[0065] The number n is also called the "drug-to-antibody ratio" (or "DAR"). This number n should always be understood as the average number for any given immunoconjugate (or its preparation).

[0066] In other specific embodiments of the present invention, the linker is a linker cleavable by the human enzyme regmine. Thus, for example, the immunoconjugate of the present invention comprises a linker cleavable by regmine in the following formula (III): [ka] (III) It may have, In the formula, antibody is the antibody of the present invention, S is the sulfur atom of the antibody, and n is the number of [(linker)-(proliferation inhibitor)] moieties covalently linked to the antibody. The number n (also called DAR) may be, for example, 1 to 10, in a more specific embodiment n is 7 to 8, and in an even more specific embodiment n is 7.5 to 8.0 (i.e., about 8). In some embodiments, S is the sulfur atom of cysteine ​​in the antibody. In some embodiments, the antibody is mAb1.

[0067] In equations (II) and (III) above, the chemical structure between the sulfur atom of the antibody and the growth inhibitor is a linker. One such linker is also present in each of equations (IV) to (IX), which are further illustrated below. In any one of the embodiments described above that have a linker cleavable by glucuronidase or regmine, the growth inhibitor may be, for example, exatecan.

[0068] Accordingly, in some embodiments, the present invention is an immunoconjugate comprising an antibody according to the present invention covalently linked to exatecan via a linker, wherein the conjugate is given by the following formula (IV): [ka] (IV) It has, The present invention provides an immunoconjugate in which S is a sulfur atom of the antibody, and n is the number of [(linker)-(exatecan)] moieties covalently linked to the antibody. The number n (also called DAR) may be, for example, 1 to 10, in a more specific embodiment n is 7 to 8, and in an even more specific embodiment n is 7.5 to 8.0 (i.e., about 8). In some embodiments, the antibody is mAb1.

[0069] In other embodiments, the present invention relates to an immunoconjugate comprising an antibody according to the present invention covalently linked to exatecan via a linker, wherein the conjugate is defined by the following formula (V): [ka] (V) It has, The present invention provides an immunoconjugate in which S is a sulfur atom of the antibody and n is the number of [(linker)-(exatecan)] moieties covalently linked to the antibody. The number n (also called DAR) may be, for example, 1 to 10, in a more specific embodiment n is 7 to 8, and in an even more specific embodiment n is 7.5 to 8.0 (i.e., about 8). In some embodiments, the antibody is mAb1.

[0070] In some embodiments, the immunoconjugates of the present invention, such as the exatecan conjugate having the glucuronidase-cleavable or regmine-cleavable linker described above, the linker is covalently attached to the antibody at the sulfur atom of a cysteine ​​residue in the antibody. For example, this cysteine ​​residue in the antibody may be one of the cysteine ​​residues capable of forming an interchain disulfide bond (also referred to herein as an interchain disulfide bridge). Since an IgG1 antibody has four interchain disulfide bonds containing a total of eight cysteine ​​residues, attaching the linker to the antibody at the sulfur atom of such a cysteine ​​residue results in a DAR that may be up to 8, and in such cases the DAR is typically 7 to 8, such as 7.5 to 8.0 (i.e., about 8), provided that the antibody is IgG1 or has the same number of interchain disulfide bonds as IgG1.

[0071] Accordingly, in some embodiments, the present invention is an immunoconjugate comprising an antibody according to the present invention covalently linked to exatecan via a linker, wherein the conjugate is of the following formula (VI): [ka] (VI) It has, In the formula, S is the sulfur atom of the cysteine ​​in the antibody, and n is the number of [(linker)-(exatecan)] moieties covalently linked to the antibody, providing an immunoconjugate. The number n (also called DAR) may be, for example, 1 to 10, in a more specific embodiment n is 7 to 8, and in an even more specific embodiment n is 7.5 to 8.0 (i.e., about 8).

[0072] In other embodiments, the present invention provides an immunoconjugate comprising an antibody according to the present invention covalently linked to exatecan via a linker, wherein the conjugate is of the following formula (VII): [ka] (VII) It has, In the formula, S is the sulfur atom of the cysteine ​​in the antibody, and n is the number of [(linker)-(exatecan)] moieties covalently linked to the antibody, providing an immunoconjugate. The number n (also called DAR) may be, for example, 1 to 10, in a more specific embodiment n is 7 to 8, and in an even more specific embodiment n is 7.5 to 8.0 (i.e., about 8).

[0073] Any of the immunoconjugates described above may use any antibody of the present invention (as described above and below in this specification). In some embodiments, the immunoconjugate of the present invention comprises mAb1 as the antibody.

[0074] Accordingly, in some embodiments, the present invention is an immunoconjugate comprising mAb1 covalently linked to exatecan via a linker, wherein the conjugate is given by the following formula (VIII): [ka] (VIII) It has, The formula provides an immunoconjugate in which S is a sulfur atom of cysteine ​​in antibody mAb1 and n is the number of [(linker)-(exatecan)] moieties covalently linked to mAb1. The number n (also called DAR) may be, for example, 1 to 10, in a more specific embodiment n is 7 to 8, and in an even more specific embodiment n is 7.5 to 8.0 (i.e., about 8). In some embodiments, S is a sulfur atom of cysteine ​​in mAb1 capable of forming interchain disulfide bridges, and DAR is about 8. Examples of such immunoconjugates (i.e., "ADC1") are further described in the Examples.

[0075] In another embodiment, the present invention relates to an immunoconjugate comprising mAb1 covalently linked to exatecan via a linker, wherein the conjugate is given by the following formula (IX): [ka] (IX) It has, The formula provides an immunoconjugate in which S is the sulfur atom of cysteine ​​in antibody mAb1, and n is the number of [(linker)-(exatecan)] moieties covalently linked to mAb1. The number n (also called DAR) may be, for example, 1 to 10, in a more specific embodiment n is 7 to 8, and in an even more specific embodiment n is 7.5 to 8.0 (i.e., about 8). In some embodiments, S is a sulfur atom of cysteine ​​in mAb1 that can form interchain disulfide bridges, and the DAR is about 8. Examples of such immunoconjugates (i.e., ADC2) are further described in the Examples.

[0076] In other embodiments of the present invention, the linker may be a "non-cleavable linker" (e.g., an SMCC linker). The release of the proliferation inhibitor from the antibody may occur during lysosomal degradation of the antibody.

[0077] In other embodiments of the present invention, the immunoconjugate may be a fusion protein comprising the antibody of the present invention and a cytotoxic or growth-inhibiting polypeptide (as a growth inhibitor), such fusion proteins can be prepared by recombinant techniques or by peptide synthesis, i.e., by methods well known in the art. The DNA-coding molecule may include two regions of the conjugate (each encoding the antibody and the cytotoxic or growth-inhibiting polypeptide), which are either adjacent to each other or separated by a region encoding a linker peptide.

[0078] Furthermore, the antibodies of the present invention can be used in targeted enzyme prodrug therapies, such as antibody-targeted enzyme prodrug therapy, by conjugating the antibody to a prodrug-activating enzyme that converts a prodrug (e.g., a peptidyl chemotherapeutic agent, see International Publication No. 81 / 01145) into an active cytotoxic agent (e.g., see International Publication No. 88 / 07378 and U.S. Patent No. 4,975,278). The enzyme component of the immunoconjugate useful for ADEPT may include any enzyme capable of acting on the prodrug to convert the prodrug into a more active cytotoxic form. Enzymes useful in this situation include, but are not limited to, the following: alkaline phosphatases useful for converting phosphate-containing prodrugs into free drugs; aryl sulfatases useful for converting sulfate-containing prodrugs into free drugs; cytosine deaminases useful for converting non-toxic fluorocytosine into the anticancer drug 5-fluorouracil; and serratia proteases, thermolysin, subtilisin, carboxypeptidases, and cathepsins (such as cathepsin B and L) useful for converting peptide-containing prodrugs into free drugs. Proteases such as: D-alanyl carboxypeptidase useful for converting prodrugs containing D-amino acid substituents; carbohydrate-cleaving enzymes such as O-galactosidase and neuraminidase useful for converting glycosylated prodrugs into free drugs; P-lactamase useful for converting drugs derivatized with P-lactams into free drugs; and penicillin amidases such as penicillin V amidase or penicillin G amidase useful for converting drugs in which the amine nitrogen is derivatized with a phenoxyacetyl group or a phenylacetyl group, respectively, into free drugs. The enzymes can be covalently bound to the antibody of the present invention by techniques well known in the art, such as the use of the linker described above.

[0079] Preferred methods for preparing the immunoconjugates of the present invention are well known in the art (see, for example, Hermanson GT, Bioconjugate Techniques, 3rd edition, 2013, Academic Press). For example, a well known method involves conjugating a cytotoxic drug to an antibody via a linker that is covalently attached to a cysteine ​​residue in the interchain disulfide crosslink of the antibody.

[0080] In general, the immunoconjugates of the present invention are, for example, (i) A step of preparing a compound comprising a linker and a growth inhibitor (e.g., a cytotoxic drug), which is also referred to herein as a “drug-linker compound”. (ii) A step of contacting an aqueous solution of an antibody of any choice according to the present invention with a drug-linker compound solution, (iii) Optionally, the conjugate formed in (ii) is then separated from the unreacted antibody and / or drug-linker compound. It can be obtained by a method that includes [a specific method]. The aqueous solution of the antibody may be buffered with a buffer such as histidine, potassium phosphate, acetate, citrate, or N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (Hepes buffer). The buffer can be selected according to the properties of the antibody. The drug-linker compound may be dissolved in an organic polar solvent such as dimethyl sulfoxide (DMSO) or dimethylacetamide (DMA). For conjugation with the antibody's cysteine ​​residues, the antibody is subjected to reduction (e.g., using TCEP) before step (ii). Suitable reduction conditions for reducing only interchain disulfide bonds are known in the art. The reaction temperature for conjugation is typically 20–40°C. Reaction times can vary, typically 1–24 hours. The reaction between the antibody and the drug-linker compound can be monitored by size exclusion chromatography (SEC) using a refractometer and / or UV detector. If the conjugate yield is too low, the reaction time can be extended.

[0081] Those skilled in the art can use a number of different chromatographic methods to carry out the separation in step (iii). The conjugate can be purified, for example, by mixed support chromatography such as SEC, adsorption chromatography (ion exchange chromatography, IEC, etc.), hydrophobic interaction chromatography (HIC), affinity chromatography, hydroxyapatite chromatography, or high-performance liquid chromatography (HPLC) such as reversed-phase HPLC. Purification by dialysis, filtration, or diafiltration can also be used. After steps (ii) and / or (iii), the conjugate-containing solution may be subjected to an additional purification step (iv), for example, by chromatography, ultrafiltration, and / or diafiltration. For example, such an additional purification step by chromatography, ultrafiltration, and / or diafiltration may also be performed with the antibody-containing solution after the reduction reaction, if the reduction is performed before the conjugation.

[0082] The conjugate is recovered in aqueous solution at the end of such a process. The drug-to-antibody ratio (DAR) is a value that can vary depending on the properties of the antibody and drug-linker compound used, along with the experimental conditions used for conjugation ((drug-linker compound) / (antibody) ratio, reaction time, solvent, and, if applicable, properties of the co-solvent). Therefore, contact between the antibody and the drug-linker compound can result in a mixture containing several conjugates with different drug-to-antibody ratios. Thus, the DAR determined is an average value. Conjugation at cysteine ​​residues of interchain disulfide crosslinks using an antibody having four interchain disulfide crosslinks (e.g., mAb1 or any IgG1 antibody) is a well-known method in the art and offers the advantage that relatively homogeneous DARs of about 8 can be achieved by selecting reaction conditions that allow the conjugation to proceed to completion (or at least approach completion). An exemplary method that can be used to determine the DAR is the λ of the purified conjugate solution. D This method involves spectrophotometrically measuring the ratio of absorbances at 280 nm. 280 nm is a wavelength commonly used for measuring protein concentrations such as antibody concentrations. Wavelength λ D λ was selected to enable the distinction between drugs and antibodies, that is, as is immediately known to those skilled in the art. D λ is the wavelength at which the drug has high absorbance. D It is located at a distance of 280 nm sufficient to avoid substantial overlap between the absorbance peaks of the drug and antibody. For example, λ D The wavelength can be selected as 370 nm in the case of exatecan (or camptothecin or other camptothecin analogues), or as 252 nm in the case of mytansinoid molecules.

[0083] The method for calculating DAR can be derived, for example, from Antony S. Dimitrov (ed.), LLC, 2009, Therapeutic Antibodies and Protocols, Vol. 525, p. 445, Springer Science, λ D and 280nm(A 280 The absorbance of the conjugate in ) is measured using either a monomer peak from size exclusion chromatography (SEC) analysis (which allows for the calculation of the "DAR(SEC)" parameter) or a classical spectrophotometer (which allows for the calculation of the "DAR(UV)" parameter). The absorbance can be expressed as follows: A λD =(C D ×ε DλD )+(C A ×ε AλD ) A 280 =(C D ×ε D280 )+(C A ×ε A280 ) In the formula, ·C D and C A These represent the concentrations of the drug and antibody in the solution, respectively. ·ε DλD and ε D280 These are λ, respectively. D And the molar extinction coefficient of the drug at 280 nm. ·ε AλD and ε A280 These are λ, respectively. D And the molar extinction coefficient of the antibody at 280 nm. Solving these two equations with two unknowns yields the following equation. C D =[(ε A280 ×A λD )-(ε AλD ×A 280 )] / [(ε DλD ×ε A280 )-(ε AλD ×ε D280 )] C A = [A 280 -(C D ×ε D280 )] / ε A280 Next, the average DAR is calculated from the ratio of drug concentration to antibody concentration: DAR = C D / C A .

[0084] Exemplary methods for preparing the immunoconjugate of the present invention are described in the examples.

[0085] Drug-linker compounds Furthermore, the present invention provides compounds comprising a linker and a growth inhibitor (e.g., a cytotoxic drug), also referred to herein as "drug-linker compounds." For example, the present invention provides compounds of the following formula (X): [ka] (X) The present invention provides a compound or a physiologically acceptable salt thereof, which is also referred to herein as “drug-linker compound 1,” “compound DL1,” or “DL1.”

[0086] Furthermore, the present invention relates to the following formula (XI): [ka] (XI) The present invention provides a compound or a physiologically acceptable salt thereof, which is also referred to herein as “drug-linker compound 2,” “compound DL2,” or “DL2.”

[0087] These drug-linker compounds can be used to prepare the immunoconjugates of the present invention as described above and below herein.

[0088] The drug-linker compounds of the present invention (for example, those of formula (X) or (XI) as illustrated above) can be prepared by chemical synthesis, for example, as further described in the following examples.

[0089] Pharmaceutical composition The antibody or immunoconjugate of the present invention can be combined with a pharmaceutically acceptable carrier, diluent, and / or excipient, and a sustained-release matrix, optionally containing but not limited to biodegradable polymers, non-biodegradable polymers, lipids, or sugars, to form a pharmaceutical composition. Accordingly, another aspect of the present invention relates to a pharmaceutical composition comprising the antibody or immunoconjugate of the present invention and a pharmaceutically acceptable carrier, diluent, and / or excipient. "Pharmacologically" or "pharmaceutically acceptable" means molecular entities and compositions that, when administered to mammals, and as appropriate, particularly to humans, do not cause adverse reactions, allergic reactions, or other undesirable reactions. A pharmacovigilantly acceptable carrier, diluent, or excipient means a non-toxic solid, semi-solid, or liquid filler, diluent, encapsulating material, or any type of formulation aid. As used herein, "pharmaceutically acceptable carriers" include any physiologically compatible solvent, dispersion medium, coating, antibacterial agent, and antifungal agent. Suitable carriers, diluents, and / or excipients include, but are not limited to, water, amino acids, physiological saline, phosphate-buffered saline, phosphate buffer, acetate, citrate, succinate, one or more; amino acids and derivatives such as histidine, arginine, glycine, proline, and glycylglycine; inorganic salts such as NaCl or calcium chloride; sugars or polyhydric alcohols such as dextrose, glycerol, ethanol, sucrose, trehalose, and mannitol; surfactants such as polysorbate 80, polysorbate 20, and poloxamer 188; and combinations thereof. In many cases, it will be useful for the pharmaceutical composition to contain sugars, polyhydric alcohols, or isotonic agents such as sodium chloride. The formulation may also contain antioxidants such as tryptamine and / or stabilizers such as Tween 20.

[0090] The form of the pharmaceutical composition, the route of administration, the dosage, and the regimen inevitably depend on the condition to be treated, the severity of the disease, the patient's age, weight, and sex, etc. The pharmaceutical composition of the present invention can be formulated for topical, oral, parenteral, intranasal, intravenous, intramuscular, subcutaneous, or intraocular administration. In embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable vehicle for an injectable formulation. The pharmaceutically acceptable vehicle may be a dry, particularly lyophilized, composition to which an injectable solution can be constructed by adding an isotonic sterile saline solution (monosodium phosphate or disodium phosphate, and sodium chloride, potassium chloride, calcium chloride, or magnesium chloride, etc., or a mixture of such salts), or optionally sterile water or saline. The pharmaceutical composition can be administered using a drug-device combination drug. The dose used for administration can be adjusted as a function of various parameters, such as the mode of administration used, the associated pathology, or, alternatively, as a function of the desired duration of treatment. To prepare a pharmaceutical composition, an effective amount of the antibody or immunoconjugate of the present invention can be dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium. Suitable pharmaceutical forms for injectable use include sterile aqueous solutions or dispersions; formulations containing sesame oil, peanut oil, or aqueous propylene glycol; and sterile powders for the immediate preparation of sterile injectable solutions or dispersions. In all such cases, the form must be sterile, injectable without decomposition in a suitable device or system for delivery, stable under manufacturing and storage conditions, and protected from contamination by microorganisms such as bacteria and fungi.

[0091] Solutions of the active compound, as a free base or a pharmacokinetically acceptable salt, can be prepared in water and preferably mixed with a surfactant. Dispersions can also be prepared in glycerol, liquid polyethylene glycol, and mixtures thereof, and in oil. Under normal storage and use conditions, such formulations may contain preservatives that prevent microbial growth. The antibody or immunoconjugate of the present invention can be formulated into a pharmaceutical composition in neutral or salt form. Examples of pharmaceutically acceptable salts include acid addition salts (formed with free amino groups of proteins) formed with inorganic acids such as hydrochloric acid or phosphoric acid, or organic acids such as acetic acid, oxalic acid, tartaric acid, or mandelic acid. Salts formed with free carboxyl groups may also be derived from inorganic bases such as sodium, potassium, ammonium, calcium, or ferric hydroxide, as well as organic bases such as isopropylamine, trimethylamine, glycine, histidine, and procaine. Furthermore, the carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), suitable mixtures thereof, and vegetable oils. Appropriate fluidity can be maintained, for example, by the use of a coating such as lecithin, by maintaining the required particle size in the case of a dispersion, and by the use of a surfactant. Prevention of microbial activity can be achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, and thimerosal. In some cases, it may be desirable to include an isotonic agent such as sugar or sodium chloride. Long-term absorption of the injectable composition can be achieved by using absorption-delaying agents, such as aluminum monostearate and gelatin, in the composition.

[0092] Sterile injectable solutions can be prepared by incorporating the required amount of the active compound into a suitable solvent having, if necessary, any of the other components listed above, followed by filtration sterilization. Generally, dispersions can be prepared by incorporating various sterilizing active ingredients into a sterile vehicle containing a basic dispersion medium and any other necessary components listed above. In the case of sterile powders for preparing sterile injectable solutions, preparation methods include vacuum drying and freeze-drying techniques, which yield powders of the active ingredient as well as any additional desired components of the pre-sterilized filtered solution. The preparation of more concentrated or highly concentrated solutions for direct injection is also being considered, in which case, using DMSO as a solvent is expected to result in very rapid penetration, delivering high concentrations of the active ingredient to small tumor areas. Once formulated, the solution can be administered in a manner suitable for the drug formulation and in a therapeutically effective amount. The formulation can be easily administered in various dosage forms, such as the injectable solutions of the type described above, but drug-releasing capsules and the like can also be used.

[0093] For parenteral administration in aqueous solutions, for example, the solution may be appropriately buffered as needed, and the liquid diluent may first be isotonic with sufficient saline or glucose. Such aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous, and intraperitoneal administration. In this regard, the sterile aqueous media that can be used will be known to those skilled in the art in light of this disclosure. For example, one dose may be dissolved in 1 ml of isotonic NaCl solution and added to 1000 ml of subcutaneous injection solution, or injected into the proposed injection site (see, e.g., Remington's Pharmaceutical Sciences, 15th edition, pp. 1035-1038 and 1570-1580). The dose will inevitably vary depending on the condition of the subject being treated. In any case, the person responsible for administration will determine the appropriate dose for each individual subject.

[0094] The antibody or immunoconjugate of the present invention may be formulated in a therapeutic mixture, for example, containing about 0.01 to 100 milligrams per dose. In addition to antibodies or immunoconjugates formulated for parenteral administration, such as intravenous or intramuscular injection, other pharmaceutically acceptable forms include, for example, tablets or other solids for oral administration, sustained-release capsules, and any other forms currently in use.

[0095] In some embodiments, the use of liposomes and / or nanoparticles is intended for introducing polypeptides into host cells. The formation and use of liposomes and / or nanoparticles are known to those skilled in the art. Nanocapsules can generally encapsulate compounds in a stable and reproducible manner. To avoid side effects due to intracellular polymer overload, such ultrafine particles (approximately 0.1 μm in size) are generally designed using polymers that can be degraded in vivo. Biodegradable polyalkyl-cyanoacrylate nanoparticles or biodegradable polylactide or polylactide coglycolide nanoparticles that meet these requirements are intended for use in the present invention, and such particles can be readily fabricated by those skilled in the art. Liposomes are formed from phospholipids dispersed in an aqueous medium and can spontaneously form multilayer concentric bilayer vesicles (also called multilayer vesicles (MLVs)). The diameter of MLVs is generally 25 nm to 4 μm. Sonication of MLVs results in the formation of small monolayer vesicles (SUVs) with a diameter ranging from 200 to 500 A, containing an aqueous solution in the core. The physical characteristics of liposomes depend on pH, ionic strength, and the presence of divalent cations. In addition to the examples mentioned above, further pharmaceutical forms such as nanoparticles, microparticles and microcapsules, implants (e.g., lipid implants), or autocoagulation or autoemulsification systems are also being considered.

[0096] Treatment and Use The inventors have found that the antibody of the present invention (e.g., mAb1) can be internally transported as part of a CEACAM5-antibody conjugate after binding. Furthermore, the inventors have shown that such an antibody conjugated to a cytotoxic agent (exatecan) mediates a cytotoxic effect against tumor cells in vitro. The inventors have also shown that such immunoconjugates of the present invention, when used as a single injection at a dose of 10 mg / kg in a mouse xenograft model of patient-derived human colorectal cancer, induce significant antitumor activity in vivo. In fact, the immunoconjugates of the present invention exhibit broad activity in a large set of in vitro and in vivo models. Cytotoxic efficacy correlates well with target (CEACAM5) expression and is significantly lower in target-negative cells. Very good antitumor activity was shown in several cell line-derived xenograft (CDX) models and patient-derived xenograft (PDX) models of different cancer types. In a dose-range study of non-human primates, this immunoconjugate exhibited a typical side effect profile of topoisomerase I inhibitor chemotherapy, but was well tolerable. These preclinical data indicate a favorable therapeutic concentration range for subsequent clinical trials. Therefore, the antibody, immunoconjugate, and pharmaceutical composition of the present invention may be useful in cancer treatment.

[0097] Accordingly, the present invention provides antibodies, immunoconjugates, or pharmaceutical compositions for use as pharmaceuticals. For example, the present invention provides antibodies, immunoconjugates, or pharmaceutical compositions for use in the treatment of cancer. The present invention further provides a method for treating cancer, comprising administering the antibodies, immunoconjugates, or pharmaceutical compositions of the present invention to a subject in need thereof. Cancers treated with the antibodies, immunoconjugates, or pharmaceutical compositions of the present invention are preferably cancers expressing CEACAM5, and more preferably cancers overexpressing CEACAM5 compared to normal (i.e., non-tumor) cells of the same tissue origin. Cellular expression of CEACAM5 can be readily assayed, for example, by using the antibodies according to the present invention (or commercially available anti-CEACAM5 antibodies) as described in the following section, “Diagnostic Uses,” and, for example, by immunohistochemistry. In some embodiments, the cancers treated with the antibodies, immunoconjugates, or pharmaceutical compositions of the present invention are colorectal cancer, non-small cell lung cancer, pancreatic cancer, gastric cancer, cervical cancer, esophageal cancer (e.g., esophageal adenocarcinoma), bile duct cancer, breast cancer, prostate cancer, ovarian cancer, urothelial carcinoma, bladder cancer, or cancers of the stomach, uterus, endometrium, thyroid, or skin. In some specific embodiments, the cancers treated with the antibodies, immunoconjugates, or pharmaceutical compositions of the present invention are colorectal cancer, gastric cancer, non-small cell lung cancer, pancreatic cancer, esophageal cancer, or prostate cancer.

[0098] The antibody or immunoconjugate of the present invention can be used alone or in combination with any suitable growth inhibitor in cancer treatment. The antibodies of the present invention may be conjugated (linked) to a proliferation inhibitor, as described above. Therefore, the antibodies of the present invention may be useful for targeting cancer cells that express or overexpress CEACAM5 on their surface with the above-mentioned proliferation inhibitor. It is also well known that therapeutic monoclonal antibodies can cause depletion of cells possessing antigens specifically recognized by the antibody. This depletion can be mediated through at least three mechanisms: antibody-mediated cytotoxicity (ADCC), complement-dependent cytolysis, and direct inhibition of tumor growth via signaling mediated by the antigen targeted by the antibody. "Antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a form of cytotoxicity in which antibodies that bind to Fc receptors (FcRs) present on certain cytotoxic cells (e.g., natural killer (NK) cells, neutrophils, and macrophages) enable the specific binding of these cytotoxic effector cells to antigen-carrying target cells and the subsequent death of the target cells. To evaluate the ADCC activity of a target molecule, in vitro ADCC assays such as those described in U.S. Patent No. 5,500,362 or No. 5,821,337 can be performed. Complement-dependent cell injury, or CDC, refers to the lysis of target cells in the presence of complement. Activation of the classical complement pathway is initiated when the first components of the complement system bind to antibodies conjugated to their congener antigens. To evaluate complement activation, a CDC assay can be performed, for example, as described by Gazzano-Santoro et al. (Journal of Immunological Methods, March 1997; Vol. 202 (No. 2): pp. 163-171).

[0099] In some embodiments, the antibody of the present invention may have a modified amino acid sequence that reduces or eliminates binding to most Fcγ receptors, thereby reducing uptake and toxicity in normal cells and tissues expressing such receptors, such as macrophages and hepatic sinusoidal cells. Aspects of the present invention relate to a method for treating cancer, comprising administering a therapeutically effective amount of the antibody, immunoconjugate, or pharmaceutical composition of the present invention to a subject in need thereof. In the present invention, the terms “to treat” or “to cure” as used herein mean to alleviate, reduce, inhibit the progression of, or prevent one or more symptoms of a disorder or disease to which such terms apply. The terms “to treat cancer” as used herein mean to inhibit the growth of malignant cells of a tumor and / or the progression of metastases from the tumor. Such treatment may also result in regression of tumor growth, i.e., a reduction in the size of a measurable tumor. For example, such treatment may result in complete regression of the tumor or metastases.

[0100] In the therapeutic applications of the present invention, the terms “subject,” “patient,” “subject requiring it,” or “patient requiring it” refer to a subject (e.g., human or non-human mammal) that is affected by or likely to be affected by a tumor. For example, the patient may be a patient who has been determined to be sensitive to a CEACAM5-targeting therapeutic agent, particularly to an antibody or immunoconjugate according to the present invention, for example, the method described below herein. "Therapeutic effective dose" means an amount sufficient to treat the above-mentioned cancerous disease with a reasonable effect / risk ratio applicable to any medical treatment. However, it will be understood that the total daily dose of the antibodies, immunoconjugates, and pharmaceutical compositions of the present invention (collectively referred to as "therapeutic agents") will be determined by the attending physician within the bounds of sound medical judgment. A specific therapeutic effective dose level for any particular patient will depend on a variety of factors, including the disorder being treated and its severity; the activity of the specific therapeutic agent used; the patient's age, weight, general health, sex, and diet; the timing, route of administration, and excretion rate of the specific therapeutic agent used; the duration of treatment; drugs used in combination with or concurrently with the specific therapeutic agent used; and similar factors well known in the medical field. For example, it is well known to those skilled in the art to start with a dose of a compound lower than the level required to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.

[0101] The antibodies, immunoconjugates, or pharmaceutical compositions of the present invention can also be used to inhibit the progression of cancer metastasis. The antibodies, immunoconjugates, or pharmaceutical compositions of the present invention may also be used in combination with any other therapeutic intervention (e.g., adjuvant therapy) for treating cancer and / or reducing the growth of metastatic cancer. For example, the other therapeutic intervention for such a combination may be a standard of care (SOC) agent for the cancer being treated. The efficacy of treatment with antibodies, immunoconjugates, or pharmaceutical compositions according to the present invention can be easily assayed in vivo, for example, in a mouse model of cancer, by measuring, for example, the change in tumor volume, tumor regression percentage, partial regression, or complete regression between the treatment group and the control group.

[0102] Diagnostic use CEACAM5 has been reported to be highly expressed on the surface of cancer cells, such as colorectal, gastric, lung, and pancreatic tumor cells, while its expression in normal tissues is limited to a small number of normal epithelial cells, such as colonic and esophageal epithelial cells. Therefore, CEACAM5 constitutes a cancer marker and may be used, for example, to demonstrate the effectiveness of anti-cancer treatment or to detect disease recurrence. In some embodiments, the antibody of the present invention can be used as a component of an assay to determine a patient's sensitivity to a therapeutic agent, to monitor the effectiveness of an anti-cancer therapy, or to detect disease recurrence after treatment, in relation to therapies targeting CEACAM5-expressing tumors. In some embodiments, the same antibody of the present invention can be used as both a component of a therapeutic agent and a component of a diagnostic assay. Accordingly, a further aspect of the present invention relates to the use of the antibody according to the present invention for ex vivo detection of CEACAM5 expression in a biological sample derived from a subject. Another aspect of the present invention relates to the use of the antibody according to the present invention for in vivo detection of CEACAM5 expression in a subject. When used for the detection of CEACAM5, the antibody may be labeled with a detectable molecule, such as a fluorophore or an enzyme.

[0103] CEACAM5 detection is, for example, By detecting the expression of the surface protein CEACAM5 on tumor cells, a) Diagnosing the presence of cancer in the subject, b) Determining the sensitivity of cancer patients to CEACAM5-targeting therapeutic agents, particularly antibodies or immunoconjugates according to the present invention, or c) Monitoring the effectiveness of anti-CEACAM5 cancer therapy, or detecting cancer recurrence after anti-CEACAM5 cancer therapy, wherein the therapy is a therapy using the antibody or immunoconjugate according to the present invention. Even if that is the intention. In the embodiments, the antibodies are intended for in vitro or ex vivo diagnostic use. For example, CEACAM5 in a biological sample obtained from a subject can be detected in vitro or ex vivo using the antibodies of the present invention. The use of the present invention may also be in vivo. For example, the antibodies of the present invention may be administered to a subject to detect and / or quantify antibody-cell complexes, in which case the detection of the complexes indicates cancer.

[0104] The present invention provides an in vitro or ex vivo method for detecting the presence of cancer in a subject, (a) A step of contacting a biological sample derived from a subject with an antibody according to the present invention, particularly under conditions suitable for the antibody to form a complex with the biological sample. (b) A step of measuring the level of antibodies bound to the biological sample, (c) The presence of cancer is detected by comparing the measured level of the conjugated antibody with the control. An increase in the level of the conjugated antibody compared to the control indicates cancer. It also includes. Furthermore, the present invention relates to an in vitro or ex vivo method for determining the sensitivity of patients with cancer to a therapeutic agent targeting CEACAM5, particularly an antibody or immunoconjugate according to the present invention, (a) A step of contacting a biological sample derived from a patient with cancer with an antibody according to the present invention, particularly under conditions suitable for the antibody to form a complex with the biological sample. (b) A step of measuring the level of antibodies bound to the biological sample, (c) A step of comparing the measurement level of the antibody bound to the biological sample with the level of the antibody bound to the control. The present invention relates to a method by which an increase in the level of bound antibodies to the above biological sample, compared to a control, indicates that the patient is sensitive to a CEACAM5-targeting therapeutic agent. In the above method, the control may be a normal, non-cancerous biological sample of the same type, or a reference value determined to represent the antibody binding level in a normal biological sample of the same type. In the embodiments, the antibodies of the present invention are useful for diagnosing CEACAM5-expressing cancers such as colorectal cancer, gastric cancer, non-small cell lung cancer, pancreatic cancer, esophageal cancer, prostate cancer, or other solid tumors that express CEACAM5.

[0105] The present invention relates to an in vitro or ex vivo method for monitoring the effectiveness of anti-CEACAM5 cancer therapy, (a) A step of contacting a biological sample derived from a subject undergoing anti-CEACAM5 cancer treatment with the antibody according to the present invention, particularly under conditions suitable for the antibody to form a complex with the biological sample. (b) A step of measuring the level of antibodies bound to the biological sample, (c) A step of comparing the measurement level of the bound antibody with the level of the antibody bound to the control. The present invention relates to a method that includes a reduction in the level of bound antibodies to the biological sample compared to a control, indicating that the anti-CEACAM5 cancer treatment is effective. In the present invention, an increase in the level of bound antibodies to the biological sample indicates that the anti-CEACAM5 cancer treatment is ineffective. In embodiments of this method for monitoring effectiveness, the control is a biological sample of the same type as the biological sample submitted for analysis, but obtained from the control at an earlier point in the course of anti-CEACAM5 cancer treatment.

[0106] The present invention provides an in vitro or ex vivo method for detecting cancer recurrence after anti-CEACAM5 cancer therapy, (a) A step of contacting a biological sample derived from a subject who has completed anti-CEACAM5 cancer therapy with an antibody according to the present invention, particularly under conditions suitable for the antibody to form a complex with the biological sample. (b) A step of measuring the level of antibodies bound to the biological sample, (c) A step of comparing the measurement level of the bound antibody with the level of the antibody bound to the control. The method further relates to a method in which an increase in the level of binding antibodies to the above biological sample indicates cancer recurrence after anti-CEACAM5 cancer treatment. The above control may be a biological sample of the same type as the biological sample submitted for analysis, but previously obtained from the subject, i.e., at or after the completion of anti-CEACAM5 cancer treatment. The above anti-CEACAM5 cancer therapy is, for example, a therapy using an antibody or immunoconjugate according to the present invention. The above anti-CEACAM5 cancer therapy targets CEACAM5-expressing cancers such as colorectal cancer, gastric cancer, non-small cell lung cancer, pancreatic cancer, esophageal cancer, prostate cancer, or other solid tumors that express CEACAM5.

[0107] In some embodiments, the antibody of the present invention may be labeled with a detectable molecule or substance, such as a fluorescent molecule or fluorophore, a radioactive molecule, an enzyme, or any other label known in the art that provides a signal (directly or indirectly). As used herein, the term “labeled” with respect to antibodies according to the present invention is intended to encompass both direct labeling of antibodies by coupling (i.e., physically linking) a polypeptide with a detectable substance such as a radioactive agent or fluorophore (e.g., fluorescein isothiocyanate (FITC), phycoerythrin (PE), or indocyanine (Cy5)), and indirect labeling of polypeptides by reactivity with a detectable substance. The antibodies of the present invention can be labeled with radioactive molecules by any method known in the art. For example, the radioactive molecules are not limited to these, but include I 123 , I 124 In 111 Re 186 Re 188 , Tc 99 Examples of radioactive atoms for scintigraphy research include the following. Furthermore, the antibodies of the present invention may be labeled with spin labels for nuclear magnetic resonance (NMR) imaging (also known as MRI), such as iodine-123, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese, or iron.

[0108] "Biological samples" encompass a variety of sample types obtained from a subject that can be used in diagnostic or monitoring assays. Examples of biological samples, but not limited to, include blood and other fluid samples of biological origin, biopsy specimens or tissue cultures, or solid tissue samples such as cells and their offspring derived therefrom. Therefore, biological samples include clinical samples, cells in culture, cell supernatants, cell lysates, serum, plasma, biological fluids, and tissue samples such as tumor samples. In some embodiments, the biological sample may be a formalin-fixed and paraffin-embedded (FFPE) tissue sample.

[0109] Furthermore, the present invention is an in vivo method for detecting the presence of cancer in a subject, a) A step of administering to a patient an antibody according to the present invention labeled with a detectable molecule, b) A step of detecting the localization of the above antibody in the patient by, for example, detecting detectable molecules using imaging methods. Regarding methods including In the above method, the cancer may be a CEACAM5-expressing cancer, such as colorectal cancer, gastric cancer, non-small cell lung cancer, pancreatic cancer, esophageal cancer, prostate cancer, or other solid tumors that express CEACAM5. Furthermore, the antibody of the present invention may also be useful for cancer staging (for example, in radiographic imaging). The antibody of the present invention may be used alone or in combination with other cancer markers. The terms "detection" or "detected," as used herein, include qualitative and / or quantitative detection (i.e., measurement of levels) with or without reference to a control. In relation to the present invention, the term “diagnose,” as used herein, means determining the nature of a medical condition in order to identify a pathology affecting the subject, based on a considerable amount of collected data.

[0110] kit Finally, the present invention also provides a kit comprising at least one antibody or immunoconjugate of the present invention. A kit comprising the antibody of the present invention may be found to be used for the detection of the surface protein CEACAM5 or in therapeutic or diagnostic assays. The kit of the present invention may comprise the antibody coupled to a solid support, such as a tissue culture plate or beads (e.g., Sepharose beads). A kit comprising the antibody for in vitro detection and quantification of the surface protein CEACAM5, for example by ELISA or Western blotting, can be provided. Such antibodies useful for detection may be provided with labeling, such as fluorescent labeling or radiolabeling.

[0111] A brief explanation of arrays Amino acid sequence: Sequence ID 1: Human CEACAM5 protein sequence according to GenBank accession number AAA51967. Sequence ID 2: Macaca fascicularis CEACAM5 protein sequence (NCBI reference sequence XP_005589491.1). Sequence ID 3 mAb1 CDR1-H Sequence ID 4 mAb1 CDR2-H Sequence ID 5 mAb1 CDR3-H Sequence ID 6 mAb1 CDR1-L Sequence ID 7 mAb1 CDR2-L Sequence ID 8 mAb1 CDR3-L Sequence ID 9 mAb1 VH Sequence ID 10 mAb1 VL Sequence ID 11 mAb1 CH CL of mAb1, sequence number 12 Sequence ID 13 mAb1 HC LC of sequence number 14 mAb1 Nucleic acid sequence: Sequence ID 15: DNA sequence encoding the HC of mAb1 Sequence ID 16: DNA sequence encoding the LC of mAb1 Amino acid sequence: Sequence ID No. 17: HC of antibody hu8G4 Sequence ID No. 18: LC of antibody hu8G4 Sequence ID No. 19: HC of optimized antibody variant 1 Sequence ID No. 20 LC of optimized antibody variant 1 Sequence ID No. 21: LC of optimized antibody variant 2 Sequence ID No. 22: LC of optimized antibody variant 4 Sequence ID No. 23: LC of optimized antibody variant 5 Sequence ID No. 24: HC of optimized antibody variant 6 Sequence ID 25 huMab2-3 (allotype) HC Sequence ID 26 huMab2-3 LC Sequence ID 27 HMN-14 HC Sequence ID 28 hmn-14 LC HC of sequence number 29 rb8G4 LC of sequence number 30 rb8G4 [Examples]

[0112] Example 1: Anti-CEACAM5 antibody 1.1 Immunization of transgenic rats and isolation of hybridomas To generate monoclonal antibodies against the human CEACAM5 protein (carcinoembryonic antigen-associated cell adhesion molecule 5; CD66e), human immunoglobulin gene transgenic rats (OmniRat®) were obtained from Charles River Laboratories International Inc. (Wilmington, Massachusetts). Five animals were immunized four times with CEACAM5 cDNA (encoding amino acids 35-675 of the human CEACAM5 protein sequence, UniProt ID number P06731; the sequence of P06731 is identical to SEQ ID NO. 1 except that E398 is substituted with K398) which was cloned into Aldevron's proprietary immunization vector (pB8-CEA-hum-MC) and transiently transfected into OMT rat cells using a gene gun. Anti-CEACAM5 titers were evaluated by a cell-based ELISA (CELISA) assay using cells expressing CEACAM5 on the cell membrane (titer results are shown below). Immune animal serum was collected on day 31 of the immunization protocol after 4 rounds of genetic immunization (IS31d-4). Serum diluted in PBS + 3% FBS was tested by flow cytometry against mammalian cells transiently transfected with CEACAM5 cDNA cloned into Aldevron's proprietary expression vector (pB1-CEA-hum-MC). Goat anti-rat IgG R-phycoerythrin conjugate (Southern Biotech, #3030-09) was used as a secondary antibody at 10 μg / ml. All animals were sacrificed, and lymphocytes from the lymph nodes were pooled and cryopreserved for future use. The cells were fused with the Ag8 mouse myeloma cell line to create viable hybridomas. These hybridoma cells were then transferred to 10 96-well plates.

[0113] 1.2 CEACAM5 specificity To detect anti-CEACAM5 antibodies that did not bind to CEACAM1 (BGP), CEACAM3 (CGM1a), CEACAM4 (CGM7), CEACAM6 (NCA), and CEACAM8 (NCA-95), hybridoma supernatants were screened using cell-based ELISA (CELIZA). Goat anti-rat IgG R-phycoerythrin conjugate (Southern Biotech, #3030-09) was used as the secondary antibody at a concentration of 10 μg / ml. Clones that showed specificity for human CEACAM5 but not for its related proteins were transferred to a single 96-well plate, and the hybridoma supernatant was evaluated for specificity and cross-reactivity using an ELISA assay. In this assay, the 8G4 hybridoma clone and its subclones showed specificity for human CEACAM5 and cross-reactivity for Macaca fascicularis CEACAM5.

[0114] 1.3 Detection and cloning of antibody sequences Total RNA was prepared from each hybridoma clone according to the RNeasy96 protocol, Qiagen. Subsequently, the total RNA was transcribed into cDNA using a random hexamer and SuperScript (copyright) III. The obtained cDNA was quality-controlled using qPCR, and VH and Vk were amplified by PCR. The PCR products were purified using the AMpure XP PCR cleanup kit in combination with a KingFisher instrument. The VH and Vk genes of the 8G4 subclone were cloned into the target vectors hi00_pTT5_VH_ccdB and hh00_pTT5_Vk_ccdB, respectively, using homologous recombination (so-called "Lucigen-Cloning"). The reaction mixture was transformed into One Shot® Mach1®-T1R chemically competent Escherichia coli (E. coli). The correct recombinant clones were confirmed by Sanger sequencing.

[0115] 1.4 Humanization, biochemical characterization, and candidate selection of hits 8G4 and other clones were reformatted and expressed as single human IgG molecules. These were evaluated by SDS-PAGE, size exclusion chromatography (SEC), selectivity, affinity, cell binding, and potency. Based on the results, one humanized candidate antibody, designated hu8G4, was selected for amino acid sequence optimization to improve manufacturability and affinity.

[0116] The amino acid sequence of the humanized candidate antibody hu8G4 is as follows: Heavy chain: EVQLVESGPGLVKPSQTLSLTCTVSDGSVSRGGYYLTWIRQHPGKGLEWIGYIYYSGSTYFNPSLRSRLTMSVDTSKNQFSLKLSSVTAADTAVYYCARGIAVAPFDYWGQGTL VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTC PPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(Sequence ID 17) Light chain: ETTLTQSPATLSVSPGERATLSCRTSQSVRSNLAWYQQKPGQAPRLLIYAASTRATGIPARFSGSGSGTEFTLTIGSLQSEDFAVYFCQQYTNWPFTFGPGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC (Sequence ID 18)

[0117] 1.5 Biophysical enhancement strategies for hu8G4, particularly linked to mAb1 Evaluation of the variable region sequence of hu8G4 identified six non-germline amino acid residues in the light chain framework and two non-germline amino acid residues in the heavy chain framework. Evaluation of potentially post-translational modification susceptible amino acids and sequence motifs, such as deamidation motifs, surface-accessible methionine, and free cysteine, did not identify any susceptible amino acid residues. Several designed antibody sequences were generated in which a specific amino acid was substituted at that position with a germline-related amino acid. Different VH and VL optimized designs were then co-expressed, purified, and tested in HEK293 6E cells as Fab and complete IgG1 molecules (see, e.g., optimized variants 1-10 below). The amino acid sequences of the 10 optimized antibody variants in the complete IgG1 format were as follows: Variant 1 (VH1.00 / VL1.00) HC: EVQLQESGPGLVKPSQTLSLTCTVSDGSVSRGGYYLTWIRQHPGKGLEWIGYIYYSGSTYFNPSLRSRLTMSVDTSKNQFSLKLSSVTAADTAVYYCARGIAVAPFDYWGQGTL VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTC PPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(Sequence ID 19) LC: ETTLTQSPATLSVSPGERATLSCRTSQSVRSNLAWYQQKPGQAPRLLIYAASTRATGIPARFSGSGSGTEFTLTIGSLQSEDFAVYFCQQYTNWPFTFGPGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (Sequence ID 20) Variant 2 (VH1.00 / VL1.01) HC: (Sequence ID 19) LC: EIVLTQSPATLSVSPGERATLSCRTSQSVRSNLAWYQQKPGQAPRLLIYAASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYFCQQYTNWPFTFGPGTKVDIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (Sequence ID 21) Variant 3 (VH1.00 / VL1.02) HC: Sequence ID 19 LC: Sequence ID 14 Variant 4 (VH1.00 / VL1.03) HC: Sequence ID 19 LC: EIVMTQSPATLSVSPGERATLSCRTSQSVRSNLAWYQQKPGQAPRLLIYAASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYFCQQYTNWPFTFGPGTKVDIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (Sequence ID 22) Variant 5 (VH1.00 / VL1.04) HC: Sequence ID 19 LC: EIVMTQSPATLSVSPGERATLSCRTSQSVRSNLAWYQQKPGQAPRLLIYAASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYTNWPFTFGPGTKVDIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (Sequence ID 23) Variant 6 (VH1.02 / VL1.00) HC: EVQLQESGPG LVKPSQTLSL TCTVSDGSVS RGGYYLTWIR QHPGKGLEWI GYIYYSGSTY FNPSLRSRVT MSVDTSKNQF SLKLSSVTAA DTAVYYCARG IAVAPFDYWG QGTLVTVSSA STKGPSVFPL APSSKSTSGG TAALGCLVKD YFPEPVTVSW NSGALTSGVH TFPAVLQSSG LYSLSSVVTV PSSSLGTQTY ICNVNHKPSN TKVDKRVEPK SCDKTHTCPP CPAPELLGGP SVFLFPPKPK DTLMISRTPE VTCVVVDVSH EDPEVKFNWY VDGVEVHNAK TKPREEQYNS TYRVVSVLTV LHQDWLNGKE YKCKVSNKAL PAPIEKTISK AKGQPREPQV YTLPPSREEM TKNQVSLTCL VKGFYPSDIA VEWESNGQPE NNYKTTPPVL DSDGSFFLYS KLTVDKSRWQ QGNVFSCSVM HEALHNHYTQ KSLSLSPGK (Sequence ID 24) LC: Sequence ID 20 Variant 7 (VH1.02 / VL1.01) HC: Sequence ID 24 LC: Sequence ID 21 Variant 8 (VH1.02 / VL1.02) HC: Sequence ID 24 LC: Sequence ID 14 Variant 9 (VH1.02 / VL1.03) HC: Sequence ID 24 LC: Sequence ID 22 Variant 10 (VH1.02 / VL1.04) HC: Sequence ID 24 LC: Sequence ID 23

[0118] Assays using aggregate percentage by size exclusion chromatography showed that all optimized variants 1-10 performed similarly well in terms of quality retention, maintaining stability based on fluorescence-monitored thermal unfolding (FMTU), retaining binding to the MKN-45 cancer cell line, and maintaining selectivity for the target. Variant 8 (i.e., the variant containing VH1.02 and VL1.02) was selected for further development as an optimized variant with a sequence particularly similar to the germline. Next, further sequence optimization was performed on the selected variant 8, particularly to reduce its IgG Fc effector function. Compared to the parent clone, the resulting final sequence-optimized (so) clone, designated so8G4 (also referred to herein as mAb1), showed improved affinity and manufacturability, and reduced or absent binding to FcγRI, FcγRIIa, FcγRIIIa, FcγRIIIa / complex, C1q, FcγRIIb, and FcγRIIIb, while maintaining affinity to CEACAM5 and FcRn. The amino acid sequence of this final sequence-optimized antibody so8G4 (also referred to herein as mAb1) is as follows. Heavy chain (HC): Sequence ID No. 13 (as specified above in this specification) Light chain (LC): Sequence ID No. 14 (as specified above in this specification)

[0119] 1. In-vitro characterization of 1.6 mAb1 Several properties of antibody mAb1, including binding affinity, selectivity, and internal migration, were characterized using in vitro assays. 1.6.1 Binding affinity • Determine the binding affinity of the soluble antibody analyte to the captured target protein CEACAM5 (human or cynomolgus macaque fascicularis). The following experimental conditions were used with the Octet Red instrument. • A biosensor coated with streptavidin. • Biotinylation target protein concentration (ECD represents the extracellular domain): ○ 2.5 μg / ml of human_CEACAM5_ECD-his-biotin from R&D Systems (biotinized using routine methods) was captured at 1000 rpm for 900 seconds. ○5 μg / ml recombinant macaca fascicularis CEACAM5_ECD-His-biotin (biotinized using a standard method) obtained from Syngene was captured at 1000 rpm for 900 seconds. ·Analyte antibody concentration: 200, 100, 50, 25, 12.5, 6.25, 0nM. The binding affinity KD (equilibrium dissociation constant) value was determined using Octet Evaluation software from the measured binding kinetic rate constant (ka) and dissociation rate constant (kd). In this case, KD = kd / ka. • Using antibodies in Fab format

[0120] Results regarding the Fab generated from mAb1: The binding affinity KD for human CEACAM5 was 6.3 ± 1.98 nM. The binding affinity KD for CEACAM5 in cynomolgus monkeys was 14.1 ± 2.53 nM.

[0121] 1.6.2. Selectivity a) Species and domains The selectivity of mAb1 was determined by titrating the antibody from 4 nM to 0.25 pM and applying it to 1 μg / ml of bound recombinant human (rh) CEACAM5 ECD or its domains N-A1-B1, A2-B2, A3-B3 or bound recombinant macaca fascicularis (mf) CEACAM5 ECD (all obtained from Syngene) in an ELISA assay. The results are shown in Figure 1 and summarized below. The binding EC50 to rhCEACAM5 is 153.4 pM. The binding EC50 to the rhA2-B2 domain is 166.9 pM. The binding EC50 to mfCEACAM5 is 324.3 pM. No binding to rhN-A1-B1 or rhA3-B3 or BSA (bovine serum albumin serving as a negative control) was detected.

[0122] b) Different CEACAM proteins The selectivity of the Fab of mAb1 for human CEACAM5 and other human CEACAM family members was determined in an ELISA assay. The proteins were coated on a 96-well assay plate. huCEACAM5-His6 (R&D Systems #4128-CM), huCEACAM6-His6 (recombinant protein obtained from R&D Systems #3934-CM and Syngene), huCEACAM1-His6 (R&D Systems #2244-CM), huCEACAM3-His6 (Novoprotein C449), huCEACAM7-His6 (Novoprotein C926), huCEACAM8-His6 (Novoprotein C583), huPSG1-His6 (Novoprotein CC66). Each protein was coated on the plate at a concentration of 12 nM.

[0123] Results: mAb1 Fab bound to human CEACAM5 (EC50 of 3.04 nM), but did not bind to other human CEACAM family members, even when mAb1 Fab at a concentration of 1000 nM, which is 300 times higher than the EC50 for binding to human CEACAM5, was used in an ELISA assay. Furthermore, in the ELISA assay, the Fab of mAb1 did not bind to unrelated proteins (BSA) at all concentrations tested.

[0124] 1.6.3 Cell binding of mAb1 The ability of antibodies to bind to target proteins on cells was determined by titrating antibodies against cells expressing a target (e.g., human CEACAM5) and measuring the fluorescence MFI of the cells. Model cells for antibody binding comparison were the MKN45 cell line expressing human CEACAM5 and the CHO cell line expressing mfCEACAM5. Titration was performed using a 10-point × 4 dilution curve starting at a concentration of 2000 nM in assay buffer (PBS × 1 containing 1% BSA). Example data: mAb1 binds to human CEACAM5-expressing MKN-45 cell lines at an EC50 of 10.62 ± 1.6 nM. mAb1 binds to mfCEACAM5-expressing CHO cell lines at an EC50 of 4.8 ± 0.6 nM.

[0125] 1.6.4 Comparison of cell binding with known antibodies The cell binding of our lead antibody mAb1 to the MKN45 cell line expressing CEACAM5 was compared with that of the known ADC-related antibodies huMab2-3 (in the known ADC SAR408701) and hMN14 (also referred to herein as hmn-14) (in the known ADC rabetsuzumab govitecan or IMMU-130).

[0126] The amino acid sequences of the known antibodies mentioned above and used in the experiments described below in this specification were as follows: huMab2-3: HC (Arotype): EVQLQESGPGLVKPGGSLSLSCAASGFVFSSYDMSWVRQTPERGLEWVAYISSGGGITYAPSTVKGRFTVSRDNAKNTLYLQMNSLTSEDTAVYYCAAHYFGSSGPFAYWGQGT LVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTC PPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(Sequence ID 25) LC: DIQMTQSPASLSASVGDRVTITCRASENIFSYLAWYQQKPGKSPKLLVYNTRTLAEGVPSRFSGSGSGTDFSLTISSLQPEDFATYYCQHHYGTPFTFGSGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (Sequence ID 26) hmn-14: HC:EVQLVESGGGVVQPGRSLRLSCSASGFDFTTYWMSWVRQAPGKGLEWIGEIHPDSSTINYAPSLKDRFTISRDNAKNTLFLQMDSLRPEDTGVYFCASLYFGFPWFAYWGQG TPVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHT CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (Sequence ID 27) LC: DIQLTQSPSSLSASVGDRVTITCKASQDVGTSVAWYQQKPGKAPKLLIYWTSTRHTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYSLYRSFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (Sequence ID 28)

[0127] Rituximab was included as a control for comparison. The results of antibody binding to cells are shown in Figure 2 and summarized below. mAb1(so8G4) = 8.3nM huMab2-3=6nM hmn-14 = 11.8nM Average of several experiments (EC50): mAb1(so8G4)=10.4nM±1.6nM(n=12) huMab2-3 = 4.9nM ± 1.6nM (n=3) hmn-14=16nM(n=2) The cell binding of the anti-CEACAM5 antibody to the MKN45 cell line is similar to that of mAb1 and known antibodies.

[0128] 1.6.5 Internal distribution assay using Cell Discoverer A key characteristic of ADCs is their internal translocation into target-expressing cells and lysosomes; therefore, internal translocation is a key characteristic of the antibodies used as part of the ADC. The rate of antibody internal translocation into late endosomes and lysosomes (low-pH vesicles) can be monitored by directly labeling the antibody with a pH-sensitive dye (pHrodo) that emits strong fluorescence at pH levels below 6.0 upon excitation. This fluorescence can be imaged using Cell-Discoverer 7 (Zeiss), allowing for the calculation of the internal translocation rate. To analyze the internal migration rates of several antibodies, MKN45 cells were seeded at 25,000 cells / well in 96-well dark-colored transparent flat-bottom plates (Cellvis). The cells were cultured overnight in 100 μl / well of RMPI-1640 + 10% FBS (Thermo). After removing the cell medium, the cells were stained with 100 μl of 10 μg / ml Hoechst dye diluted in PBS x 1 for 15 minutes at room temperature (RT) in the dark. The cells were then washed twice with PBS x 1. Anti-CEACAM5 human IgG antibodies (so8G4 (i.e., mAb1), humab2-3, hmn-14) and anti-MerTK antibodies (Merck) were directly labeled with pHrodo, diluted to a concentration of 100 nM in heated RPMI1640 + 10% FBS without phenol red, and added to the corresponding wells. The plates were incubated in a Cell Discoverer at 37°C and 5% CO2 for 20 hours, and images were acquired every 20 minutes as further described below.

[0129] The internal migration of pHrodo-labeled antibodies into late endosomes and lysosomes was imaged using Cell-Discoverer7 (Zeiss) with fluorescence excitation at 567 nm and emission detection at 592 / 25 nm. Cell nuclei were labeled with Hoechst and imaged with excitation at 385 nm and emission detection at 425 / 30 nm. Fluorescence in each well was recorded every 20 minutes for 20 hours. Total fluorescence intensity (SFI) per cell was analyzed using Zen software (ZEN3.1) and linear regression analysis in Excel. The results are shown in Figures 3 and 4, and the slope of the straight portion of the curve (see Figure 4) is also summarized in the table below. TIFF0007848189000018.tif57141

[0130] Conclusion: 1. so8G4(mAb1) exhibits a higher average binding rate (28958±766) than humab2-3 (18917±1416) and hmn-14 (22268±3060). 2. Furthermore, so8G4(mAb1) exhibits higher internal migration strength compared to humab2-3 and hmn-14.

[0131] 1.7 Exemplary method for preparing mAb1 for use in drug-linker compound conjugation, for example. Anti-CEACAM5 antibody mAb1 was produced using recombinant CHO-K1Sv cell lines. Cell culture was performed in batch mode using a 200 L single-use bioreactor. Cells were grown at 37°C in a proprietary CHO feed batch growth medium supplemented with glucose. A mixture of proprietary medium components was supplied to the cultures on days 3, 5, 7, and 10 after inoculation. Coarsely acclimatized medium from bioreactor operation, 3 × 1.1 m 2 Millistak+Pod DOHC (Millipore MD0HC10FS1) and 1.1m 2Clarification was performed using a Millistak+Pod XOHC (Millipore #MX0HC01 FS1) filter, followed by final filtration with a Millipore Opticap XL3 0.5 / 0.2 μm filter (Millipore #KHGES03HH3). Following clarification, antibody mAb1 was purified using a standard antibody purification process consisting of a protein A capture step and an ion exchange chromatography step. Anti-CEACAM5 antibody mAb1 served as an intermediate for the generation of ADC molecules.

[0132] 1.8 Expression and purification of the human / rabbit chimeric variant of mAb1, and its use in immunohistochemistry (IHC) for formaldehyde-fixed and paraffin-embedded cell lines and human tumor tissues The human / rabbit chimeric variant of mAb1 was generated by routine recombinant methods. The human / rabbit chimeric variant of mAb1 (also referred to herein as "rb8G4") had the following amino acid sequence. Heavy chain EVQLQESGPGLVKPSQTLSLTCTVSDGSVSRGGYYLTWIRQHPGKGLEWIGYIYYSGSTYFNPSLRSRVTMSVDTSKNQFSLKLSSVTAADTAVYYCARGIAVAPFDYWGQGTLVTVSSQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSEDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHEDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK (SEQ ID NO: 29) Light chain EIVLTQSPATLSVSPGERATLSCRTSQSVRSNLAWYQQKPGQAPRLLIYAASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYTNWPFTFGPGTKVDIKRDPVAPSVLLFPPSKEELTTGTATIVCVANKFYPSDITVTWKVDGTTQQSGIENSKTPQSPEDNTYSLSSTLSLTSAQYNSHSVYTCEVVQGSASPIVQSFNRGDC (Sequence ID 30) rb8G4 was expressed in HEK cells (Expi293 suspension cells) by transient transfection and purified using MabSelect SuRe and citrate buffer. Subsequently, rb8G4 was used for IHC of formaldehyde-fixed and paraffin-embedded cell lines and human tumor tissue.

[0133] Materials and methods Cell lines and tissues Human cancer cell lines derived from the Merck cell bank were cultured, fixed with 4% buffered formaldehyde, and embedded in paraffin (FFPE). The paraffin-embedded cell lines were placed on a cell line microarray (CMA) (Zytomed). FFPE tissue sections for human organ tissue microarrays (TMA) were from amsbio (FDA standard tissue array, T8234701). FFPE human tumor samples were provided by BioIVT and Indivumed GmbH.

[0134] method For IHC staining with the anti-CEACAM-5 antibody rb8G4, 4 μm sections of formaldehyde-fixed paraffin-embedded (FFPE) cancer cell line microarrays (CMA) and human tumor tissue were mounted on charged slides (SuperFrost Ultra Plus, Thermo Fisher Scientific, or TOMO, Matsunami). The staining procedure was performed using the Discovery XT (Roche Diagnostics) staining platform. After deparaffinization, sections were heated in Tris-EDTA buffer pH 8 (CC1, Roche Diagnostics) for epitope retrieval. Sections were incubated with primary monoclonal antibody rb8G4 diluted to 0.5 or 0.7 μg / ml in phosphate-buffered saline (PBS) or antibody dilution buffer (DCS). Clone DA1E (rabbit monoclonal IgG, NEB) served as an isotype control antibody. Following the primary antibody, the HQ anti-rabbit IgG detection kit (Roche Diagnostics) was used. The slides were counterstained with hematoxylin, washed with tap water, dehydrated, and mounted on glass cover slips in Entellan Neu (VWR) permanent mounting medium.

[0135] CMA and TMA using human organ tissue were stained and scanned at a resolution of 0.46 μm / pixel using a NanoZoomer (Hamamatsu). Human tumor sections were stained and scanned at a resolution of 0.44 μm / pixel using an AxioScan.Z1 (Zeiss) instrument. CMA scans were analyzed using image analysis software HALO (Indica Labs, USA). To determine the amount of antigen present, the positive brown stained area was calculated as a percentage of the viable tissue area. Staining (arbitrary units) is calculated as antibody staining (AU) = positive tissue area % * average optical density of brown (OD range is 0-1). The maximum value of antibody staining is 100 = 100% of the tissue area is black (grayscale OD value is 1). CEACAM-5 mRNA data from cancer cell lines were obtained from the Cancer Cell Line Encyclopedia (CCLE; Broad Institute of MIT & Harvard).

[0136] result Verification in cancer cell lines and normal human tissue. The antibody rb8G4 showed a signal in the cytoplasm and plasma membrane of FFPE cancer cell lines (Figure 5). The specificity of the antibody rb8G4 against FFPE tissue / cells was demonstrated by comparing the staining signals of 104 cancer cell lines with the mRNA expression of these cell lines (CCLE dataset). The resulting Pearson correlation coefficient of r=0.88 supports the conclusion that the antibody rb8G4 (also known as SO8G4AB323) detects the CEACAM-5 epitope in FFPE tissue / cells (Figure 6). This cancer cell line microarray, along with individually selected positive and negative cell lines, served as a control matrix in staining experiments using human normal and tumor tissues. Staining of normal human tissue with the antibody rb8G4 (Figure 7) is consistent with CEACAM-5 mRNA expression (Source: http: / / www.proteinatlas.org / ENSG00000105388-CEACAM5 / tissue), further supporting the specificity of the antibody against CEACAM-5.

[0137] Human tumor tissue The antibody rb8G4 positively stained several human tumor indications, as shown for colorectal cancer (Figure 9), gastric cancer (Figure 10), esophageal cancer (Figure 11), and non-small cell lung cancer (Figure 12). The signal was localized in the cytoplasm and plasma membrane. 1.9 Flow cytometry and Western blotting using mAb1 and rb8G4 We compared the binding of mAb1, rb8G4, and commercially available anti-CEACAM5 antibodies to CEACAM5-positive and CEACAM5-negative cell lines. Method used: 5E5–1E6 cells in 5 mL polystyrene tubes were used for flow cytometry analysis using BD FACSCanto II (BD Biosciences). Staining with 10 μg / mL primary antibodies (mAb1, rb8G4, mouse monoclonal Agilent Dako #M7072 clone #IL7) and their respective fluorescently labeled secondary antibodies (donkey anti-human IgG Jackson-Dianova #709-116-149; donkey anti-mouse IgG Jackson ImmunoResearch #715-116-150; donkey anti-rabbit IgG Jackson-Dianova #711-116-152) was performed in 50 μL of 1% PBS / BSA for 20–30 minutes at 4°C. Between and after the staining steps, cells were washed three times with 1% PBS / BSA and resuspended in 500 μL of 1% PBS / BSA (containing 0.2 μg / mL DAPI for live cell gating) for flow cytometry analysis. FlowJo software (BD Biosciences) was used for data evaluation.

[0138] Results: mAb1 and rb8G4 showed binding corresponding to mRNA expression level data only to CEACAM5-positive cell lines (Table 1 below; MKN-45, NCI-H441). In contrast, commercially available antibodies showed weaker binding and were limited to high-CEACAM5 cell lines (Table 1 below; MKN-45). In conclusion, mAb1 and rb8G4 can specifically detect CEACAM5-positive cancer cells and can be used as detection agents. TIFF0007848189000019.tif88147

[0139] Furthermore, the binding of human mAb1 and rb8G4 to CEACAM5-positive and CEACAM5-negative cell line lysates was investigated by Western blotting. Method used: Western blotting was performed according to the standard protocol (Sambrook, J. & Russell, DW, 2001. Molecular Cloning: A Laboratory Manual, Vol. 1, CSHL Press). For SDS-PAGE and subsequent membrane wet blotting, RIPA cell lysates were loaded with 15 μg of total protein per lane, quantified using a BCA kit (Thermo Scientific, #23227). Criterion XT 4-12% gels (Bio-Rad, #3450125) were used in a Criterion electrophoresis cell (Bio-Rad, #1656001) with MOPS running buffer (Bio-Rad, #1610788). Protein transfer was confirmed by Ponceau staining. The membranes were washed before and between staining with primary antibodies (mAb1 or rb8G4) and secondary antibodies (anti-human IgG, Jackson ImmunoResearch #109-035-098, or anti-rabbit IgG, CellSignaling #7074) at concentrations of 0.5 μg / mL to 1 μg / mL. The stained membranes were visualized using ECL detection reagent with a Fusion FX imaging system (Vilber).

[0140] The results are shown in Figures 13A and 13B. Both antibodies bound in similar patterns corresponding to the expected migration speed of highly glycosylated CEACAM5. CEACAM5 detection by mAb1 (Figure 13A) and rb8G4 (Figure 13B) was specific to CEACAM5-positive cell lines, and its intensity correlated with mRNA expression levels. Secondary bands observed at lower intensities corresponded to the aforementioned potential secondary isoforms (Hatakeyama et al.: Novel protein isoforms of carcinoembryonic antigen are secreted from pancreatic, gastric and colorectal cancer cells. BMC Research Notes 2013, Vol. 6: 381).

[0141] Example 2: Synthesis of drug-linker compounds having a glucuronide-based linker: Drug-linker compound 1 (DL1) [ka] A synthetic route to compound 9 (also referred to herein as drug-linker compound 1 (DL1)). Chemical preparation protocol Step 1: Compound 1 [ka] (2S,3S,4S,5R,6R)-3,4,5-triacetoxy-6-bromotetrahydropyran-2-carboxylate methyl ester (8.30 g; 20.90 mmol; 1.00 equivalent) and 4-hydroxy-3-nitro-benzaldehyde (5.24 g; 31.35 mmol; 1.50 equivalent) were stirred in acetonitrile (83.00 ml; 10.00 V), to which silver(I) oxide (9.69 g; 41.80 mmol; 2.00 equivalent) was added. The reaction mixture was stirred at room temperature for 16 hours. The reaction mixture was filtered through Celite. The filtrate was concentrated under vacuum to obtain a solid. The solid was dissolved in ELISA and washed with a 10% aqueous solution of NaHCO3 to remove excess 4-hydroxy-3-nitro-benzaldehyde. The organic layer was concentrated under vacuum to obtain compound 1 as a sand-colored solid. Yield: 9.0g Yield percentage: 89.1% Analysis data: NMR: 1 H-NMR (400MHz, DMSO-d6): 9.98 (s, 1H), 8.46 (s, 1H), 8.25-8.21 (m, 1H), 7.64 (d, J=11.60Hz, 1H), 5.94 (d, J=10.00Hz, 1H), 5.51-5.44(m, 1H), 5.20~5.09(m, 2H), 4.80(d, J=13.20Hz, 1H), 3.64(s, 3H), 2.09(s, 9H).

[0142] Step 2: Compound 2 [ka] Compound 1 (9.00 g; 18.62 mmol; 1.00 equivalent) was stirred in propan-2-ol (33.00 ml; 3.67 V) and CHCl3 (167.00 ml; 18.56 V), to which silica gel 60-120 (3.60 g; 112.09 mmol; 6.02 equivalents) was added, followed by sodium borohydride (1.80 g; 46.55 mmol; 2.50 equivalents). The reaction mixture was stirred at room temperature for 1 hour. After completion, the reaction mixture was quenched with cooled H2O and filtered through Celite. The filtrate was extracted with dichloromethane and dried over Na2SO4. The solvent was concentrated to obtain compound 2 as an off-white powder. Yield: 8.70g Yield percentage: 92.4% Analysis data: LCMS: Column: ATLANTIS dC18 (50 x 4.6 mm) 5 μm; Mobile phase A: 0.1% HCOOH in H2O:ACN (95:5); B: ACN RT (min): 2.05; M+H: 503.2, Purity: 96.6%

[0143] Step 3: Compound 3 [ka] Compound 2 (8.70 g; 17.21 mmol; 1.00 equivalent) was stirred in ethyl acetate (100.00 ml; 11.49 V) and THF (100.00 ml; 11.49 V) to which palladium-supported carbon (10% mass / mass) (2.50 g; 2.35 mmol; 0.14 equivalents) was added. The reaction mixture was stirred at room temperature for 3 hours under a hydrogen atmosphere. After completion, the reaction mixture was filtered through Celite. The solvent was concentrated under vacuum to obtain compound 3 as an off-white solid. Yield: 8.5g Yield percentage: 100% Analysis data: LCMS: Column: ATLANTIS dC18 (50 x 4.6 mm) 5 μm; Mobile phase A: 0.1% HCOOH in H2O:ACN (95:5); B: ACN RT(min): 1.73; M+H: 456.10, Purity: 95.1%

[0144] Step 4: Compound 4 [ka] Compound 3 (10.00 g; 20.89 mmol; 1.00 equivalent) and (9H-fluoren-9-ylmethoxycarbonylamino)-acetic acid (7.60 g; 25.06 mmol; 1.20 equivalents) were stirred in DCM (250.00 ml; 25.00 V), to which 2-ethoxy-2H-quinoline-1-carboxylate ethyl ester (15.65 g; 62.66 mmol; 3.00 equivalents) was added at 0°C. The reaction mixture was stirred at room temperature for 16 hours. After completion, the solvent was removed under reduced pressure to obtain the crude product. The crude product was purified by column chromatography (56% siRNA: petroleum ether) to obtain a compound with a purity of 80%. The compound was further purified by washing with 30% siRNA and petroleum ether to obtain compound 4 as a white solid. Yield: 8.5g Yield percentage: 50.7% Analysis data: LCMS: Column: ATLANTIS dC18 (50 x 4.6 mm) 5 μm; Mobile phase A: 0.1% HCOOH in H2O:ACN (95:5); B: ACN RT (min): 3.03; M+H: 735.2, Purity: 81.9%

[0145] Step 5: Compound 5 [ka] Compound 4 (2.00 g; 2.49 mmol; 1.00 equivalent) was stirred in THF (40.00 ml; 20.00 V) at 0°C, to which bis-(4-nitrophenyl) carbonate (3.06 g; 9.97 mmol; 4.00 equivalent) and DIPEA (4.40 ml; 24.92 mmol; 10.00 equivalent) were added. The reaction mixture was stirred at room temperature for 12 hours. After the reaction was complete, the reaction mixture was concentrated under vacuum. The crude product was purified by column chromatography using silica gel (230-400) and petroleum ether / ethyl acetate as the eluent to obtain compound 5 as a pale yellow solid. Yield: 2.0g Yield percentage: 84.6% Analysis data: LCMS: Column: X-Bridge C8 (50 x 4.6) mm, 3.5 μm; Mobile phase: A: 0.1% TFA in MilliQ water; B: ACN RT (min): 3.24; M+H: 900.20, Purity: 94.9%

[0146] Step 6: Compound 6 [ka] Compound 5 (1.369 g; 1.00 equivalent) was dissolved in N,N-dimethylformamide (15.00 ml), and exatecan mesylate (679.7 mg; 1.00 equivalent), synthetic 4-methylmorpholine (0.422 ml; 3.00 equivalent), and 1-hydroxybenzotriazole (172.8 mg; 1.00 equivalent) were added. The reaction mixture was stirred overnight at room temperature. After stirring, the reaction suspension turned into a brown solution. Monitoring the reaction by LC-MS showed that the starting materials were completely converted. The reaction mixture was purified by RP flash chromatography. The product-containing fractions were combined, concentrated under vacuum, and freeze-dried overnight to obtain compound 6 as a yellow solid. Yield: 1.59g Yield percentage: 87.5% Analysis data: LCMS:Column:Chromolith HR RP-18e (50-4.6mm); Mobile phase A: 0.05% HCOOH in H2O; B: 0.04% HCOOH and 1% H2O in ACN; T: 40 °C; Flow rate: 3.3 ml / min; MS: 100~2000, amu positive; 1%->100%B: 0->2.0 min; 100%B: 2.0->2.5 min RT (min): 1.95; M+H: 1196.40, Purity: 84.4%

[0147] Step 7: Compound 7 [ka] Compound 6 (1.586 g; 1.00 equivalent) was dissolved in tetrahydrofuran (50.00 ml), and a solution of LiOH (lithium hydroxide hydrate (281.77 mg; 6.00 equivalent)) in water (67.100 ml) (0.1 M) was added dropwise at 0°C. The pH value was checked during the addition. The pH should not exceed 10. The addition of the LiOH solution was completed after 1.5 hours. Monitoring the reaction by LC-MS showed that the starting materials were completely converted. The reaction was quenched with citric acid solution to adjust the pH to 5. The reaction mixture was concentrated under reduced pressure. The crude product was purified by preparative HPLC. The product-containing fractions were combined and lyophilized to obtain compound 7 as a dark yellow solid. Yield: 728 mg Yield percentage: 54.8% Analysis data: LC-MS: Column: Chromolith HR RP-18e (50-4.6mm); Mobile phase A: 0.05% HCOOH in H2O; B: 0.04% HCOOH and 1% H2O in ACN; Temperature: 40℃; Flow rate: 3.3 ml / min; MS: 100-2000, AMU positive; 1%->100% B: 0->2.0 min; 100% B: 2.0->2.5 min RT (min): 1.68; M+H: 1056.30, Purity: 98.5%

[0148] Step 8: Compound 8 [ka] Compound 7 (728.000 mg; 1.00 equivalent) was dissolved in N,N-dimethylformamide (20.00 ml). Piperidine (136.513 μl; 2.00 equivalent) was added, and the solution was stirred at room temperature for a total of 4 hours. Monitoring the reaction by LC-MS showed that the starting materials were completely converted. The reaction mixture was concentrated under reduced pressure, and the crude product was purified by RP flash chromatography. The product-containing fractions were combined, the solvent was partially removed, and the mixture was freeze-dried overnight to obtain compound 8 as a yellow solid. Yield: 706 mg Yield percentage: 100% Analysis data: LCMS:Column:Chromolith HR RP-18e (50-4.6mm); Mobile phase A: 0.05% HCOOH in H2O; B: 0.04% HCOOH and 1% H2O in ACN; T: 40 °C; Flow rate: 3.3 ml / min; MS: 100~2000, amu positive; 1%->100%B: 0->2.0 min; 100%B: 2.0->2.5 min RT(min): 1.22; M+H: 834.30, Purity: 97.6%

[0149] Step 9: Compound 9 [ka] To a solution of compound 8 (854 mg; 1.00 equivalent) in dimethylformamide (30.00 ml), N-ethyldiisopropylamine (149.234 μl; 1.00 equivalent) and 3-(2,5-dioxo-2,5-dihydropyrrole-1-yl)-propionic acid 2,5-dioxo-pyrroridine-1-yl ester (233.61 mg; 1.00 equivalent) were added. The reaction mixture was stirred at room temperature for 3 hours. Monitoring the reaction by LC-MS showed that the starting materials were completely converted. The reaction mixture was concentrated under reduced pressure, and the crude product was obtained by RP flash chromatography. The product-containing fractions were combined, concentrated, and lyophilized to obtain the desired product with a purity of 91%. This substance was purified again by RP chromatography to obtain compound 9 as a yellow solid. Yield: 580 mg Yield percentage: 60.1% Analysis data: LCMS:Column:Chromolith HR RP-18e (50-4.6mm); Mobile phase A: 0.05% HCOOH in H2O; B: 0.04% HCOOH and 1% H2O in ACN; T: 40 °C; Flow rate: 3.3 ml / min; MS: 100~2000, amu positive; 1%->100%B: 0->2.0 min; 100%B: 2.0->2.5 min RT(min): 1.38; M+H: 985.30, Purity: 90% (the other 10% of isomers can be removed by HPLC) 1H NMR (500MHz, DMSO-d6) δ13.10~12.44(m, 1H), 9.08(s, 1H), 8.32(t, J=5.8Hz, 1H), 8.16(s, 1H), 8.02(d, J=8.8Hz, 1H), 7.76(d, J= 10.9Hz, 1H), 7.31(s, 1H), 7.15~7.09(m, 2H), 6.98(s, 2H), 5.48~5.38(m, 2H), 5.32~5.22(m, 3H), 5.11~5.01(m, 2H), 4.87(d, J=7 .6Hz, 1H), 3.92~3.88(m, 1H), 3.89~3.84(m, 2H), 3.65~3.61(m, 2H), 3.46~3.41(m, 1H), 3.42~3.37(m, 1H), 3.38~3.31(m, 1H), 3. 28~3.20(m, 1H), 3.15~3.07(m, 1H), 2.48~2.44(m, 2H), 2.38(s, 3H), 2.24~2.13(m, 2H), 1.94~1.80(m, 2H), 0.88(t, J=7.3Hz, 3H).

[0150] Example 3: Synthesis of a drug-linker compound having a legmine-cleavable linker: Drug-linker compound 2 (DL2) [ka] Step 1 4-[(2S)-3-carbamoyl-2-[(2S)-2-[(2S)-2-({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)propanamide]propanamide]propanamide]phenyl}methyl 4-nitrophenyl carbonate (400 mg; 0.52 mmol; 1.00 equivalent) [commercially available from Levena Biopharma US] was dissolved in N,N-dimethylformamide (5.00 ml). Exatecan mesylate (277.30 mg; 0.52 mmol; 1.00 equivalent), N-ethyldiisopropylamine (0.27 ml; 1.57 mmol; 3.00 equivalent), and 1-hydroxybenzotriazole (HOBT) (3.52 mg; 0.03 mmol; 0.05 equivalent) were added. The reaction mixture was stirred overnight at room temperature. LC / MS showed complete conversion.

[0151] The crude reaction mixture was purified by preparative HPLC and lyophilized to obtain 365 mg (0.343 mmol) of (9H-fluoren-9-yl)methyl((S)-1-(((S)-4-amino-1-((4-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolidino[1,2-b]quinoline-1-yl)carbamoyl)oxy)methyl)phenyl)amino)1,4-dioxobutan-2-yl)amino)-1-oxopropan-2-yl)amino)-1-oxopropan-2-yl)carbamate. LC / MS:[M+H]=1064.2 Preparative HPLC: Column: Sunfire Prep C18 OBD - 75.0g (250 bar) Solvent A: Water 0.1% TFA Solvent C: Solvent B: Acetonitrile 0.1% TFA

[0152] [ka] Step 2 (9H-fluoren-9-yl)methyl((S)-1-(((S)-1-(((S)-4-amino-1-((4-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolidino[1,2-b]quinoline-1-yl)carbamoyl)oxy)methyl)phenyl)amino)1,4-dioxobutan-2-yl)amino)-1-oxopropan-2-yl)amino)-1-oxopropan-2-yl)carbamate (365 mg; 0.34 mmol; 1.00 equivalent) was dissolved in N,N-(4.00 ml). Synthetic piperidine (0.07 ml; 0.69 mmol; 2.00 equivalents) was added, and the reaction solution was stirred at room temperature for 1 hour.

[0153] The reaction mixture was purified by preparative HPLC to obtain 300 mg (0.314 mmol) of 4-((S)-4-amino-2-((S)-2-((S)-2-aminopropanamide)propanamide)-4-oxobutanamide)benzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolidino[1,2-b]quinoline-1-yl)carbamate. LC / MS:[M+H]:841.3 Preparative HPLC for purification: RediSep Column: C18 130g SN:E0410A0D24BE1 Lot:262118923W Flow rate: 75ml / min Condition - Volume: 390.0 ml Eluent: A1 Water 0.1% TFA Eluent: B1 Acetonitrile 0.1% TFA

[0154] [ka] Step 3 4-((S)-4-amino-2-((S)-2-((S)-2-aminopropanamide)propanamide)-4-oxobutanamide)benzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolyl To a solution of dino[1,2-b]quinoline-1-yl)carbamate (571 mg; 0.60 mmol; 1.00 equivalent) in N,N-dimethylformamide (20 ml), N-ethyldiisopropylamine (203 μl; 1.20 mmol; 2.00 equivalent) and N-succinimidyl 3-maleimide propionate (162 mg; 0.60 mmol; 1.00 equivalent) were added. The reaction mixture was then stirred for 10 minutes and monitored by LC / MS. The reaction mixture was purified by preparative HPLC to obtain 378 mg (0.35 mmol) of DL2. LC / MS:[M+H]:992.4 Preparative HPLC for purification: RediSep column: C18 86g SN:E0410A8B46130 Lot:281729189W Flow rate: 60ml / min Conditions - Volume: 264.0 ml Eluent 1: A1 Water 0.1% TFA Eluent 2: B1 Acetonitrile 0.1% TFA Sequence analysis: Method information: A:H2O+0.05%HCOOH|B:MeCN+0.04%HCOOH+1%H2O Temperature: 40℃ | Flow rate: 3.3 ml / min | MS: 100~2000 amu positive Column: Chromolith HR RP-18e 50-4.6mm 0%->100%B:0->2.0 minutes|100%B:2.0->2.5 minutes

[0155] Example 4: Preparation of immunoconjugate: Glucuronide-based conjugate of mAb1 (referred to as ADC1) 4.1 Conjugation Process Antibody preparation Antibody mAb1 (as specified above in this specification) was thawed at 2-8°C starting 3 days before conjugation and stored at 2-8°C until use. mAb (>10g) was equilibrated to room temperature on the day of conjugation, prior to use. mAb (9.6mg / mL) was divided into smaller portions (10.0g, 1041.7mL) and diluted to 5.59mg / mL using conjugation buffer (200mM histidine, pH 6.5). The mAb solution was added to a 3L Chemglass-jacketed reactor and stirred at 50rpm at 25±2°C. Antibody reduction 7.0 mol equivalent (9.7 mL) of 50 mM TCEP solution (50 mM TCEP in conjugation buffer) was added to the mAb solution vial, and the reaction was allowed to proceed at 25 ± 2°C for 3 hours. Conjugation Drug-linker compound 1 (DL1) of formula (X) was weighed and dissolved in DMSO to prepare a 20 mM solution. 90% (148.6 mL) of the required DMSO was added to the reactor. Immediately after the addition of DMSO, 10.0 mol equivalent (38.2 mL) of the 20 mM drug-linker solution was added to the reactor. Then, the remaining 10% (18.2 mL) of the required DMSO was used to rinse the drug-linker vial, ensuring that the entire volume was transferred. After the final addition, the reaction was allowed to proceed at 25 ± 2 °C for 1 hour. The total volume during conjugation was 1997.0 mL. Note: The overall DMSO concentration in the reaction was 10% (volume / volume) (DMSO + drug linker solution).

[0156] Quench 35 mol equivalents (48.5 mL) of 50 mM NAC were added to the reactor, and the reaction was allowed to proceed at 25 ± 2°C for 30 minutes. filtration The filtered crude conjugate solution was transferred from the reactor and then filtered using Millipak Gamma Gold 60 (MPGL06GH2) to obtain 1993.6 mL of filtered crude conjugate (filter load: 324.7 g / m2 [protein], 66.5 L / m2 [solution]). diafiltration The filtered crude conjugate solution was buffer-exchanged using a Pellicon3 (30kDa) Biomax membrane (1 × 0.11 m², 300 LMH (550 mL / min), 16 psi TMP, actual load 88.5 g / m²) (DV = 1993.6 mL). The crude conjugate was buffer-exchanged for 16 diavolumes using diafiltration buffer (10 mM histidine, pH 5.5). After buffer exchange, the solution was concentrated to ≥25 mg / mL, transferred to a bottle, and the membrane was washed with diafiltration buffer. The total volume recovered from UF / DF was 361.5 mL.

[0157] Formulation The concentrated ADC (i.e., ADC1) was diluted to 20.0 mg / mL in 112.1 mL of diafiltration buffer (10 mM histidine, pH 5.5). The resulting solution was then diluted to 15.0 mL in 157.6 mL of 4× formulation buffer (10 mM histidine, 12% (mass / volume) trehalose dihydrate, 400 mM NaCl, pH 5.5) to achieve a final target bulk drug substance (BDS) concentration of 15.0 mg / mL. filtration The final formulation of ADC was filtered using a 0.2 μm Millipak Gamma Gold 40 (MPGL04GH2) filter to obtain 619.6 mL (filter load: 464.6 g / m² [protein], 31.0 L / m² [solution]) of ADC1 BDS. This material was filled into HDPE bottles and stored at ≤-65°C.

[0158] 4.2 Method: Characterization of formulation raw materials: ADC1 Size exclusion chromatography (SEC) SEC method parameters Wavelength 280nm Column: Tosoh TSKgel 7.8mm x 300mm, 5μm (P / N0008541) Mobile phase: 0.14M potassium phosphate monobasic 50 mM sodium phosphate monobasic 0.06M potassium phosphate dibasic 0.25M potassium chloride 5% IPA Injection volume: 20 μL Temperature 25℃ Flow rate: 0.5ml / min Execution time: 30 minutes Typical SEC chromatograms showing the purity of the stock mAb, the conjugate after UF, and the final BDS: Figure 14.

[0159] The BDS shown above reports a monomer purity of 1.7% HMWS and 96.9%. Reverse-phase HPLC (RP HPLC) method RP HPLC method parameters Wavelength 280nm Column PLRP-S 1000Å (50×2.1mm, 8μm), Agilent (P / N PL1912-1802) Mobile phase A: 0.01% TFA in water containing 0.1% formic acid Mobile phase B: 0.01% TFA in ACN containing 0.1% formic acid gradient TIFF0007848189000033.tif41141 Injection volume: 10 μL Column temperature 80℃ Flow rate 1.0mL / min Execution time: 30 minutes Sample Preparation: Dilute the sample to 2 mg / mL and add 40 μL to a microcentrifuge tube. Add 60 μL of buffer solution containing approximately 8 M guanidine HCl, approximately 130 mM Tris, approximately 1 mM EDTA, and pH 7.6. Add 2 μL of 500 mM DTT and vortex to mix. Incubate the sample at 37 ± 2°C for 30 ± 2 minutes.

[0160] A typical RP-HPLC chromatogram showing the separation of light and heavy chains: Figure 15. The chromatogram shows the stock mAb, crude ADC, and final BDS superimposed. In the above ADC1 BDS, a DAR of 7.9 was reported. Free drug law Free drug method parameters Wavelength 254nm Column Phenomenex Gemini, C18, 2 x 150mm, 3μm (P / N 00F-4439-B0) Mobile phase A: 0.1% formic acid in water Mobile phase B: 0.1% formic acid in acetonitrile gradient TIFF0007848189000034.tif31140 Injection volume: 10.00 μL Column temperature 50℃ Flow rate 0.75mL / min Sample preparation: Protein drop: 100 μL of active pharmaceutical ingredient + 250 μL of cold MeOH + 50 μL of 3M MgCl2. Centrifuge at 20,000 rpm for 10 minutes. Standard preparation: Mix 20 μL of 20 mM DL1 (drug-linker compound 1 in DMSO) + 20 μL of DMSO + 40 μL of MeOH + 20 μL of 200 mM NAC in diafiltration buffer. Incubate overnight to obtain 4 mM DL-NAC. Dilute 4 mM DL-NAC with MeOH to obtain 4 μM DL-NAC standard. Typical chromatograms showing free drug levels in NAC reference material and final BDS: Figure 16. In the ADC1 BDS shown above, residual free drug levels of less than 2.4% (by molar ratio) have been reported.

[0161] Example 5: Preparation of immunoconjugate: Peptide-based conjugate of mAb1 (called ADC2) 5.1 Conjugation Process Antibody preparation Antibody mAb1 (as specified above in this specification) was thawed at 2-8°C starting 3 days before conjugation and stored at 2-8°C until use. mAb (>9.5g) was equilibrated to room temperature on the day of conjugation, prior to use. mAb (9.6mg / mL) was divided into smaller portions (9.5g, 989.6mL) and diluted to 5.59mg / mL using conjugation buffer (200mM histidine, pH 6.5). The mAb solution was added to a 3L Chemglass-jacketed reactor and stirred at 50rpm at 25±2°C.

[0162] Antibody reduction 8.0 mol equivalent (10.5 mL) of 50 mM TCEP solution (50 mM TCEP in conjugation buffer) was added to an mAb solution vial, and the reaction was allowed to proceed at 25 ± 2 °C for 3 hours. diafiltration Using a Pellicon3 (30kDa) Biomax membrane (1 × 0.11 m², 300 LMH (550 mL / min), 16 psi TMP, actual load 86.3 g / m²), the reducing mAb solution was buffer-exchanged for 6 DV (DV = 1706.9 mL). The reducing antibody was buffer-exchanged using conjugation buffer (200 mM histidine, pH 6.5). After buffer exchange, the reducing mAb solution was recovered and returned to the reactor, and the membrane was washed with conjugation buffer. Conjugation Drug-linker compound 2 (DL2) of formula (XI) was weighed and dissolved in DMSO to prepare a 20 mM drug-linker solution. 90% (142.6 mL) of the required DMSO was added to the reactor. Immediately after the addition of DMSO, 9.5 mol equivalents (31.2 mL) of the 20 mM drug-linker solution were added to the reactor. Then, the remaining 10% (15.8 mL) of the required DMSO was used to rinse the drug-linker vial, ensuring that the entire volume was transferred. After the final addition, the reaction was allowed to proceed at 25 ± 2 °C for 2 hours. The total volume during conjugation was 1894.5 mL. Quench 35 mol equivalents (46 mL) of 50 mM NAC were added to the reactor, and the reaction was allowed to proceed at 25 ± 2°C for 45 minutes.

[0163] filtration The crude conjugate solution was transferred from the reactor and filtered using Millipak Gamma Gold 60 (MPGL06GH2) to obtain 1897.3 mL of filtered crude conjugate (filter load: 308.9 g / m2 [protein], 63.2 L / m2 [solution]). diafiltration The filtered crude conjugate solution was buffer-exchanged using a Pellicon3 (30 kDa) Biomax membrane (1 × 0.11 m², 300 LMH (550 mL / min), 16 psi TMP, actual load 84.2 g / m²) (DV = 1897.3 mL). The first 12 DVs were performed using conjugation buffer (200 mM histidine, pH 6.5), followed by an additional 8 DVs using standard diafiltration buffer (10 mM histidine, pH 5.5). After the buffer exchange was complete, the solution was concentrated to ≥25 mg / mL, transferred to a bottle, and the membrane was washed with diafiltration buffer. The total pool volume recovered from UF / DF was 335.7 mL. Formulation The concentrated ADC (i.e., ADC2) was diluted to 20.0 mg / mL with 84.7 mL of diafiltration buffer (10 mM histidine, pH 5.5). The resulting solution was then diluted with 138.6 mL of 4× formulation buffer (10 mM histidine, 12% (mass / volume) trehalose dihydrate, 400 mM NaCl, pH 5.5) to a final target BDS concentration of 15.0 mg / mL. filtration The final formulation of ADC was aseptically filtered using a Millipak Gamma Gold 60 (MPGL06GH2) filter to obtain 549.3 mL (filter load: 411.4 g / m2 [protein], 27.5 L / m2 [solution]) of ADC2 BDS. This material was filled into HDPE bottles and stored at ≤-65°C.

[0164] 5.2 Method: Characterization of the active pharmaceutical ingredient: ADC2 Size exclusion chromatography (SEC) SEC method parameters Wavelength 280nm Column: Tosoh TSKgel 7.8mm x 300mm, 5μm (P / N0008541) Mobile phase: 50 mM sodium phosphate monobasic 0.4M sodium perchlorate pH 6.3 Injection volume: 1 μL Column temperature: 25°C Flow rate 0.5mL / min Execution time: 30 minutes Typical SEC chromatograms showing the purity of stock mAb and final BDS: Figure 17.

[0165] The ADC2 BDS shown above has reported monomer purity of 4.2% HMWS and 95.8%. Reverse-phase HPLC (RP HPLC) method RP HPLC method parameters Wavelength 280nm Column PLRP-S 1000Å (50×2.1mm, 8μm), Agilent (P / N PL1912-1802) Mobile phase A: 0.01% TFA in water containing 0.1% formic acid Mobile phase B: 0.01% TFA in ACN containing 0.1% formic acid gradient TIFF0007848189000035.tif41141 Injection volume: 10 μL Column temperature 80℃ Flow rate 1.0mL / min Execution time: 30 minutes Sample Preparation: Dilute the sample to 2 mg / mL and add 40 μL to a microcentrifuge tube. Add 60 μL of buffer solution containing approximately 8 M guanidine HCl, approximately 130 mM Tris, approximately 1 mM EDTA, and pH 7.6. Add 2 μL of 500 mM DTT and vortex to mix. Incubate the sample at 37 ± 2°C for 30 ± 2 minutes. A typical RP-HPLC chromatogram showing the separation of light and heavy chains: Figure 18. The chromatogram shows a superposition of the stock mAb and the final BDS.

[0166] In the above ADC2 BDS, a DAR of 7.6 was reported. Free drug law Free drug method parameters Wavelength 254nm Column Phenomenex Gemini, C18, 2 x 150mm, 3μm (P / N 00F-4439-B0) Mobile phase A: 0.1% formic acid in water Mobile phase B: 0.1% formic acid in acetonitrile gradient TIFF0007848189000036.tif31140 Injection volume: 10.00 μL Column temperature 50℃ Flow rate 0.75mL / min Sample preparation: Protein drop: 100 μL of active pharmaceutical ingredient + 250 μL of cold MeOH + 50 μL of 3M MgCl2. Centrifuge at 20,000 rpm for 10 minutes. Standard preparation: Mix 20 μL of 20 mM DL2 (drug-linker compound 2 in DMSO) + 20 μL of DMSO + 40 μL of MeOH + 20 μL of 200 mM NAC in diafiltration buffer. Incubate overnight to obtain 4 mM DL-NAC. Dilute 4 mM DL-NAC with MeOH to obtain 4 μM DL-NAC standard. Typical chromatograms showing free drug levels in NAC reference material and final BDS: Figure 19. In the ADC2 BDS mentioned above, residual free drug levels of less than 1.9% (by molar ratio) have been reported.

[0167] Example 6: Analogue to ADC SAR408701 6.1 Antibodies For further comparative experiments, an analogue of Sanofi's anti-CEACAM5 ADC SAR408701 was prepared based on a monoclonal antibody having the following sequence. Heavy chain: Sequence ID 25 Light chain: Sequence ID 26

[0168] 6.2 Drug-Linker Compounds As the drug-linker molecule used to conjugate the antibody mentioned above, we used SPDB-DM4 (obtained from Levena Biopharma). Product name: SPDB-DM4 structure: [ka] Estimated mass: 994.35 Average mass observed value: 995.5(Ms+H + ) Mass spectral analysis: Matching and showed the correct MW. HPLC analysis: purity >95% Appearance: White powder

[0169] 6.3 Conjugation The antibody was thawed at 2-8°C up to 3 days before conjugation and stored at 2-8°C until use. The antibody (175 mg) was equilibrated to room temperature on the day of conjugation, just before use. The antibody (7.9 mg / mL) was diluted to 5 mg / mL using conjugation buffer (PBS pH 7.4) and 5 mM DMSO solution of SPDB-DM4 (Levena Biopharma) (8 mol equivalents relative to the antibody). The reaction solutions were mixed and incubated at 25°C for 4 hours.

[0170] 6.4 Preparative size exclusion chromatography, desalting, and filtration The reaction mixture was purified using preparative size exclusion chromatography. A Superdex 200pg (50 / 60) column was connected to an Akta Avant 25 system (GE Healthcare) and equilibrated with PBS pH 7.4 according to the manufacturer's instructions. The reaction mixture was then injected and flowed through the column at a flow rate of 10 ml / min with PBS pH 7.4 as the running buffer. The ADC-containing fraction was determined by 280 nm UV absorption, pooled, and concentrated. The ADC was concentrated using a 15 ml Amicon Ultra 50 kDa cutoff centrifuge (Merck Millipore) according to the manufacturer's instructions. The concentrated ADC was transferred to formulation buffer (10 mM histidine, 130 mM glycine, 5% sucrose, pH 5.5) using a HiPrep 26 / 10 desalting column (GE Healthcare) mounted on an Akta Avant 25 system (GE Healthcare) at a flow rate of 10 ml / min, according to the manufacturer's instructions. The obtained ADC was filtered using a 0.2 μm filter (Merck Millipore), aliquoted, and then flash-frozen in liquid nitrogen. The final concentration of the ADC was 5.82 mg / ml, and the substance was maintained at -80°C until further use. The ADC obtained from this process is referred herein as "ADC SAR DM4" or simply "ADC SAR". This ADC is an analogue of SAR408701.

[0171] Example 7: ADC based on mAb1 and SPDB-DM4 Another ADC was prepared based on antibody mAb1 (as described above in this specification) and drug-linker compound SPDB-DM4, i.e., the same drug-linker compound as ADC SAR DM4 described above. The ADC obtained from this process is referred to herein as "ADC mAb1 DM4" and was prepared as follows.

[0172] 7.1 Materials used: ·Antibody: mAb1, 1mg / mL in 10mM HEPES, pH5.8 • Conjugation buffer: 10mM HEPES, pH 5.8 • Drug-linker compound: SPDB-DM4, 2 mg / mL in DMF 7.2 Method: Conjugation: A drug-linker molecule in approximately 30-fold molar excess was used for conjugation (75 mL antibody + 7.1 mL SPDB-DM4 drug-linker), and the mixture was incubated at room temperature for 5 hours with gentle shaking. • Purification: Free drugs were removed from ADC by changing the buffer to 20 mM histidine, 150 mM NaCl, pH 6.0. • Formulation buffer: 20 mM histidine, 150 mM NaCl, pH 6.0 7.3 Details of purified ADC analysis: • Final yield: 40 mg ·Concentration: 2.2mg / mL DAR: 4.4

[0173] Example 8: Characterization of drug release from ADC1 and ADC2 8.1 Materials and Methods 8.1.1 Test Items TIFF0007848189000038.tif52153

[0174] 8.1.2 Materials All reagents and buffers were stored according to the manufacturer's instructions for use and used before the batch expiration date. TIFF0007848189000039.tif78156

[0175] 8.1.3 Equipment TIFF0007848189000040.tif41161

[0176] 8.1.4 Procedure 8.1.4.1 Serum sample preparation 2M HEPES solution: 52.1 g of HEPES was dissolved in 75 mL of MiliQ water and 15 mL of 25% HCl, adjusted to pH 7.55, and increased to 100 mL. This solution was mixed with serum at a 15% volume / volume ratio to obtain stabilized serum with a pH of 7.3-7.4. Human serum from Biowest (lot number S15594S4200) was thawed. 100 mL of serum was mixed with 15 mL of 2M HEPES buffer. Mouse serum from Biowest (lot number S18169S2160) was thawed. 100 mL of serum was mixed with 15 mL of 2M HEPES buffer. Cynomolgus monkey serum was thawed, and 8.5 mL of serum was mixed with 1.5 mL of 2M HEPES buffer. The pH was measured (7.37), and the serum was filtered sterile. 2 mL aliquots were frozen at -20°C. The prepared serum was thawed at room temperature. For subsequent free payload analysis by LC-MS, the desired ADC protein concentration was prepared as a triple duplicate of 180 μg / mL. After adding the ADC to the serum, the individual batches were mixed and divided into 20 μL aliquots. In addition, one 20 μL sample at 96 hours was pipetted for each ADC and used for a full workup analysis to measure the recovery rate. The sample at 0 hours was frozen directly at -80°C, and the remaining samples were incubated at 37°C and 5% CO2, stopping the reaction at 2 / 4 / 6 / 24 / 48 / 72 and 96-hour incubations, and then stored at -80°C.

[0177] 8.1.4.2 Preparation of human liver lysosome samples The pH was adjusted to either pH 5.0 or pH 4.0 using assay buffer. Lysosomal stability preparation: 80 μL of human liver lysosomes were prepared for triple duplication measurement (n=3) in the same manner as shown for ADC1: 2.76 μL ADC1 + 6 μL human liver lysosomes + 71.2 μL regmine assay buffer. Preparation of MeOH + PIC (1:200): 10 μL PIC III + 1990 μL MeOH. The reaction was initiated by transferring Eppendorf tubes to Thermomix preheated to 37°C. Subsequently, 10 μL aliquots were taken after 0, 1, 2, 4, 24, and 48 hours and mixed with 40 μL PIC III (1:200).

[0178] 8.2 Results ADC stability in human, mouse, and cynomolgus monkey serum (Figure 20). Conjugated exatecan concentration was calculated using free exatecan (initial dose approximately 10 μM) (normalized data). Similar profiles were obtained for human, cynomolgus monkey, and mouse serum. Only slight warhead release was observed, with mouse serum being the most prominent for ADC2 (5.9% did not contain the initial conjugated payload at 96 hours) and ADC1 (1.4%). ADC3 control stability in mouse serum and buffer (Figure 21). The concentration of conjugated SN38 was calculated using free SN38 (initial dose 50 μg / mL ADC protein concentration) (denormalized). Significant SN-38 release was observed in both matrices. Payload release profiles of ADC1 and ADC2 in human liver lysosomes (pH 5.0) (Figure 22). The concentration of the conjugated drug was calculated using, for example, free exatecan (initial concentration approximately 10 μM exatecan). Normalized data. Intermediate levels of payload release were observed in ADC1 and ADC2 cleavage-mediated payload release (both approximately 40% of the initial total conjugated payload).

[0179] ADC catabolic profiling confirmed free exatecan as a lysosomal release product (Figure 23). To confirm exatecan as the major release product, ADC1 catabolic profiling studies were conducted using human lysosome extracts. Comparison of TIC-MS and extracted ion chromatograms at various time points showed that the expected exatecan catabolic product was subsequently released from ADC1 during incubation (0 hours, 4 hours, 24 hours). The residence time of the detected catabolic product was 9.33 minutes, and the detected mass was m / z 436.1671 ([M+H]). + , C 24 H 23 The O4N4F spectrum and MS / MS spectrum are consistent with those of exatecan.

[0180] Example 9: ADC1 and ADC2 highly effectively and specifically kill cancer cells in vitro. The ability of ADC1 and ADC2 to kill cancer cells was evaluated using human cancer cell lines. ADC1 and ADC2 showed sub-nanomolecal in vitro potency against different CEACAM5-positive cell lines and only slight effect against CEACAM5-negative cell lines (Table 2 below). As shown in the exemplary dose-response curves (Figure 24A / B), ADC1 and ADC2 were very potent against the CEACAM5-positive cell lines SK-CO-1 and SNU-16. In contrast, the effect of ADC1 and ADC2 against antigen-negative MDA-MB-231 was limited to the highest concentration tested (Figure 24C).

[0181] Isotype control ADCs using the same linker payload as ADC1 and ADC2 showed a significantly lower effect on the SK-CO-1 cell line (Figure 25).

[0182] In conclusion, ADC1 and ADC2 specifically and effectively kill CEACAM5-expressing human cancer cell lines in vitro. TIFF0007848189000041.tif78142

[0183] Methods - Survival Rate Assay: The cytotoxic effect of ADC on cancer cell lines was measured by a cell viability assay. Cells were seeded in 90 μL volumes into a 96-well plate the day before treatment. The test compound (ADC or free payload) was formulated in cell culture medium at a 10-fold increase in starting concentration. The test compound was serially diluted (1:4), and 10 μL of each dilution was added to the cells in a triple-duplicate manner. The plates were cultured in a 37°C CO2 incubator for 6 days. For cell viability measurement, Cell Titer-Glo® reagent (Promega® Corp, Madison, Wisconsin) was added to each well, and the plates were treated according to the manufacturer's instructions. Luminescence signals were measured using a Varioskan plate reader (Thermo Fisher). Luminescence readings were converted to viability % relative to untreated cells. Nonlinear regression analysis using a four-parameter fitting formula (logarithmic (inhibitor) vs. response, variable slope) was performed using GraphPad Prism. The data are presented as relative cell viability % versus molar compound concentration, and error bars indicate the triple-overlap standard deviation (SD). The geometric mean of IC50 was calculated from multiple experiments. Furthermore, using the same method as described above, ADC1 and ADC2 were compared with ADC SAR DM4 in terms of their cytotoxic effects against antigen-positive SK-CO-1 cell lines and antigen-negative MDA-MB-231 cell lines. ADC1 and ADC2 showed 2.9-fold and 2.7-fold higher efficacy against SK-CO-1 cancer cells than ADC SAR DM4, respectively (Figure 26A). The nonspecific effect against antigen-negative MDA-MB-231 was slightly higher with ADC SAR DM4 compared with ADC1 and ADC2 (Figure 26B). ADC SAR DM4 and ADC mAb1 DM4 showed comparable efficacy against SK-CO-1, with a slight tendency for ADC mAb1 DM4 to show higher efficacy (Figure 26A).

[0184] Example 10: ADC1 and ADC2 mediate a potent bystander effect against antigen-negative cells in co-culture with antigen-positive cells. The ability of ADC1 and ADC2 to mediate bystander effects on nearby antigen-negative cells was evaluated using a bystander assay. ADC1 and ADC2 showed a potent bystander effect on CEACAM5-negative MDA-MB-231 cells in the presence of CEACAM5-positive SK-CO-1 cells (Figure 27A). At concentrations of 1E-9M tested in a single-culture viability assay, ADC1 and ADC2 treatment had the greatest effect on SK-CO-1 cells (Figure 26A), but no effect was observed on MDA-MB-231 cells (Figure 26B). Consistent with these findings, no nonspecific effect of ADC1 or ADC2 was observed in a bystander assay setting with MDA-MB-231 as the control (Figure 27B).

[0185] In conclusion, ADC1 and ADC2 mediate a potent bystander effect against antigen-negative cancer cells in co-culture with antigen-positive cells. These findings demonstrate the ability to effectively target tumors with heterologous target expression. Compared to ADC SAR, ADC1 and ADC2 mediated a significantly stronger bystander effect against antigen-negative cells in co-culture with antigen-positive cells (Figures 28A and 28B). ADC mAb1 DM4, which used the same antibody (i.e., mAb1) as ADC1 and ADC2, along with the drug-linker molecule (i.e., SPDB-DM4) used in ADC SAR DM4, also showed a more pronounced bystander effect than ADC SAR DM4 (Figures 28A and 28B). This indicates that mAb1 contributes to a higher bystander effect compared to ADC SAR using different antibodies than that observed with ADC1 and ADC2. In all ADCs tested, the degree of the bystander effect increased with increasing numbers of antigen-positive cells added to a given number of antigen-negative cells (comparison of Figure 28A and Figure 28B). This indicates that more ADCs are processed by antigen-positive cells, releasing a free payload that contributes to the bystander effect against antigen-negative cells. No nonspecific effects of the test ADC were observed specifically on MDA-MB-231 cells (Figure 28C).

[0186] Method - Bystander Assay The cytotoxic effect of ADCs on antigen-negative cancer cell lines in co-culture with antigen-positive cancer cell lines was measured by a bystander assay. In co-culture experiments using 750 or 3000 CEACAM5-positive SK-CO-1 cells per well, 1000 CEACAM5-negative MDA-MB-231 cells were seeded. As a control, 1000 MDA-MB-231 cells alone were seeded in parallel. Cells in a total volume of 90 μL were seeded into a 96-well plate the day before treatment. The test compound was formulated in cell culture medium to 10 times the final concentration of 1E-9M, and 10 μL was added to the cells in a double-duplicate manner. The plates were cultured in a 37°C CO2 incubator for 6 days.

[0187] Prior to immunofluorescence staining, the culture medium was removed and the cells were treated with 100% methanol (-20°C) for 30 minutes. After methanol removal and one PBS wash, the cells were treated with 2.5% paraformaldehyde (PFA) containing 0.2% Triton X-100 in PBS at room temperature for 15 minutes. After solution removal and one PBS wash, the cells were treated with 1% BSA / 0.1% Tween / 0.1% sodium azide in PBS at room temperature for at least 1 hour. Antigen-positive and antigen-negative cells were distinguished by immunofluorescence staining using 10 μg / mL human anti-CEACAM5 (mAb1) primary antibody and 1:2000 dilution donkey anti-human IgG fluorescent (phycoerythrin) labeled secondary antibody (Jackson ImmunoResearch #709-116-149). Cells were identified by nuclear staining using 1 μg / mL Hoechst 33342 (Life Technologies, catalog number H3570) dye. Staining was performed at room temperature for 30 minutes in a 1% BSA / 0.1% sodium azide PBS solution. Secondary antibody staining was performed in combination with Hoechst staining. Cells were washed three times with PBS between and after the staining steps.

[0188] The plates were imaged using a confocal quantitative imaging cell analyzer CQ1 (Yokogawa® Electric Corporation, Tokyo, Japan). Analysis was performed using the CQ1 software (Yokogawa) template "Nucleus and pseudo-Cell body," and the FCS export files were analyzed using FlowJo (BD). Antigen-positive and antigen-negative cells were distinguished and quantified based on the presence or absence of fluorescently labeled antibody staining around the nucleus. The bar graph shows the number of identified antigen-positive and antigen-negative cells per treatment condition.

[0189] Example 11: Efficacy of ADC1 and ADC2 in a mouse model of xenograft (PDX) derived from colorectal cancer (CRC) patients. The in vivo antitumor efficacy was evaluated using the human patient-derived CRC xenograft model COPF217 (Shanghai LideBiotech CO., LTD). COPF217 tumor fragments were subcutaneously transplanted into the right flank of 6-8 week old immunodeficient female mice (NU-Foxn1nu, Charles River). The tumor size was 165 mm. 3 Once the average volume was reached, 6 mice / group were treated intravenously once with a vehicle (physiological saline solution) or ADC1 or ADC2 (10 mg / kg dose each; day 0). The tumor length (L) and width (W) were measured with calipers, and the tumor volume was calculated using the formula L × (W^2) / 2. A single dose of 10 mg / kg of ADC1 or ADC2 resulted in a significant antitumor effect. Both substances exhibited comparable effects leading to tumor quiescence (Figure 29). Treatment with ADC1 or ADC2 did not have a significant effect on body weight (data not shown). Further experiments with other CRC PDX models: In additional experiments corresponding to the above-described experiments regarding COPF217, a single treatment with ADC1 resulted in tumor quiescence or tumor regression in 12 other CRC PDX models exhibiting high CEACAM5 expression.

[0190] Example 12: Efficacy of ADC1 in a non-small cell lung cancer (NSCLC) PDX mouse model The in vivo antitumor efficacy was evaluated using the human patient-derived NSCLC xenograft model LUPF160151 (Shanghai LideBiotech CO.,LTD). LUPF160151 tumor fragments were subcutaneously transplanted into the right flank of 6-8 week old immunodeficient female mice (NU-Foxn1nu, Charles River). The tumor size was 180 mm. 3 Once the average volume was reached, 5 mice / group were treated intravenously once with either a vehicle (physiological saline solution) or ADC1 (6 mg / kg; day 0). The tumor length (L) and width (W) were measured with calipers, and the tumor volume was calculated using the formula L × (W^2) / 2. A single dose of ADC1 at 6 mg / kg resulted in a significant antitumor effect (Figure 30) and did not affect body weight (data not shown). The model LUPF160151 showed heterologous CEACAM5 expression by CEACAM5-negative tumor cells adjacent to CEACAM5-positive tumor cells. Therefore, the good efficacy of ADC1 in this model demonstrates a potent bystander effect of ADC.

[0191] Example 13: Efficacy of ADC1 in a gastric cancer PDX mouse model The in vivo antitumor efficacy was evaluated using a human patient-derived gastric cancer xenograft model GAX066 (Shanghai ChemPartner CO., LTD). Tumor fragments were subcutaneously transplanted into the right flank of immunodeficient female mice (Nu / Nu mice, Beijing Vital River Lab Animal Technology Co. Ltd, 18-22g). The tumor size was 220 mm. 3 Once the average volume was reached, 6 mice / group were treated intravenously once with a vehicle (physiological saline solution) or ADC1 (3 or 10 mg / kg; day 0). The tumor length (L) and width (W) were measured with calipers, and the tumor volume was calculated using the formula L × (W^2) / 2. A single treatment with 3 or 10 mg / kg of ADC1 produced a significant antitumor effect (Figure 31) and did not affect body weight (data not shown). In 5 out of 6 tumors, treatment with 10 mg / kg resulted in complete tumor regression.

[0192] Example 14: Efficacy of ADC1 compared to ADC3 in a tumor model derived from pancreatic cell lines. The efficacy of ADC1 compared to ADC3 was evaluated using the human pancreatic cell line-derived xenograft model HPAF-II (ATCC, CRL-1997). 5×10 6 HPAF-II cells were subcutaneously injected into the right flank of 6-8 week old immunodeficient female mice (Hsd: thymus-deficient nude-Foxn1nu, Envigo). The tumor was 150 mm. 3 Once the average volume was reached, 10 mice / group were treated intravenously once with a vehicle (physiological saline solution) or ADC1 (1 mg / kg or 6 mg / kg; day 0) or ADC3 (1 mg / kg or 6 mg / kg; day 0). The tumor length (L) and width (W) were measured with calipers, and the tumor volume was calculated using the formula L × (W^2) / 2. A single dose of ADC1 at 6 mg / kg resulted in a significant antitumor effect. The effect was dose-dependent, with a single dose of 1 mg / kg resulting in only a significant but mild, transient antitumor effect. In contrast, single doses of ADC3 at the same doses did not show a significant antitumor effect at any of the doses (Figure 32). None of the treatments showed a significant effect on body weight (data not shown).

[0193] Example 15: Efficacy of ADC1 compared to ADC SAR DM4 in two CRC PDX mouse models. The in vivo antitumor efficacy was evaluated using human patient-derived CRC xenograft models COPF230 and REPF210 (Shanghai LideBiotech CO., LTD). Tumor fragments were subcutaneously transplanted into the right flank of 6-8 week old immunodeficient female mice (NU-Foxn1nu, Charles River). The tumor size was 170 mm. 3Once the average volume was reached, 6 mice / group were treated intravenously once with vehicle (physiological saline solution), ADC1 (6 mg / kg), or ADC SAR DM4 (6 mg / kg) (Day 0). The tumor length (L) and width (W) were measured with calipers, and the tumor volume was calculated using the formula L × (W^2) / 2. A single dose of ADC1 resulted in a significant antitumor effect in both the COPF230 (Figure 33) and REPF210 (Figure 34) PDX models. In contrast, a single dose of the same ADC SAR DM4 did not show an antitumor effect in either CRC PDX model (Figures 33 and 34). No significant effect on body weight was observed in any of the treatment groups (data not shown).

[0194] Example 16: Efficacy of ADC1 compared to ADC SAR DM4 in a gastric PDX mouse model (GAPF313) The in vivo antitumor efficacy was evaluated using the human patient-derived gastric xenograft model GAPF313 (Shanghai LideBiotech CO.,LTD). Tumor fragments were subcutaneously transplanted into the right flank of 6-8 week old immunodeficient female mice (NU-Foxn1nu, Charles River). The tumor size was 180 mm. 3 Once the average volume was reached, 6 mice / group were treated intravenously with a vehicle (physiological saline solution), ADC1 (4 mg / kg or 7 mg / kg Q2W × 3), or ADC SAR DM4 (4.7 mg / kg Q2W × 3) starting on day 0, every other week for 3 doses. The length (L) and width (W) of the tumor were measured with calipers, and the tumor volume was calculated using the formula L × (W^2) / 2. Interim data analysis from ongoing experiments demonstrates clear antitumor efficacy of ADC1 in this model, while ADC SAR DM4 is ineffective (Figure 35). All treatments were well tolerated (data not shown).

[0195] Example 17: Safety profile of ADC1 - Pilot toxicity study in cynomolgus monkeys To investigate the safety profile, ADC1 was administered to cynomolgus monkeys via IV infusion over 30 minutes at doses of 0, 3, 10, and 30 mg / kg at 3-week intervals (days 1, 22, and 43). Animals were sacrificed on day 50 for macroscopic and histopathological examination. The results showed that ADC1 exhibited a relatively favorable safety profile, lacking toxicity in certain organs affected by the toxic side effects of known ADCs.

Claims

1. An antibody comprising CDR1-H, CDR2-H, CDR3-H, CDR1-L, CDR2-L, CDR3-L, CDR1-L, CDR2-L, CDR3-L, and CDR3-L, which bind to the human CEACAM5 protein and consist of the amino acid sequence of SEQ ID NO: 3, CDR1-H, CDR2-H, CDR3-H, CDR1-L, CDR2-H, CDR3-L, CDR3-H, CDR1-L, CDR2-H, CDR3

2. The antibody according to claim 1, comprising a heavy chain variable region (VH) having an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 9, and a light chain variable region (VL) having an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO:

10.

3. The antibody according to claim 1 or 2, comprising a heavy chain variable region (VH) containing the amino acid sequence of SEQ ID NO: 9 and a light chain variable region (VL) containing the amino acid sequence of SEQ ID NO:

10.

4. The antibody according to any one of claims 1 to 3, comprising a heavy chain constant region (CH) having an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 11, and a light chain constant region (CL) having an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO:

12.

5. The antibody according to any one of claims 1 to 4, comprising a heavy chain constant region (CH) containing the amino acid sequence of SEQ ID NO: 11 and a light chain constant region (CL) containing the amino acid sequence of SEQ ID NO:

12.

6. The antibody according to any one of claims 1 to 5, comprising a heavy chain (HC) containing the amino acid sequence of SEQ ID NO: 13 and a light chain (LC) containing the amino acid sequence of SEQ ID NO:

14.

7. An antibody according to any one of claims 1 to 6, which is an antibody fragment.

8. The antibody according to claim 7, wherein the antibody fragment is selected from the group consisting of Fv, Fab, F(ab')2, Fab', dsFv, (dsFv)2, scFv, sc(Fv)2, and diabody.

9. The antibody according to any one of claims 1 to 8, which is a bispecific antibody or a multispecific antibody.

10. A nucleic acid comprising a nucleic acid sequence encoding the antibody according to any one of claims 1 to 9.

11. A host cell transformed with the nucleic acid described in Claim 10.

12. An immunoconjugate comprising an antibody according to any one of claims 1 to 9, covalently linked to at least one growth inhibitor via a linker.

13. The immunoconjugate according to claim 12, wherein the proliferation inhibitor is a cytotoxic agent or a radioactive moiety.

14. The immunoconjugate according to claim 12 or 13, wherein the growth inhibitor is selected from the group consisting of chemotherapeutic agents, enzymes, antibiotics, toxins such as small molecule toxins or enzymatically active toxins, toxoids, vinca, taxanes, meitansinoids or meitansinoid analogs, tomaimycin or pyrrolobenzodiazepine derivatives, cryptophycin derivatives, leptomycin derivatives, auristatin or drastatin analogs, prodrugs, topoisomerase I inhibitors, topoisomerase II inhibitors, DNA alkylating agents, antitubulin agents, CC-1065 and CC-1065 analogs.

15. The immunoconjugate according to any one of claims 12 to 14, wherein the proliferation inhibitor is exatecan.

16. The immunoconjugate according to any one of claims 12 to 15, wherein the linker is a severable linker.

17. The immunoconjugate according to any one of claims 12 to 16, wherein the linker is a linker that can be cleaved in mammalian endosomes.

18. The immunoconjugate according to any one of claims 12 to 17, wherein the linker is a linker that can be cleaved by a human enzyme selected from glucuronidase and regmine.

19. The following formula (II): (II) The immunoconjugate according to any one of claims 12 to 18, wherein S is a sulfur atom of the antibody and n is the number of [(linker)-(proliferation inhibitor)] moieties covalently linked to the antibody.

20. The following formula (III): (III) The immunoconjugate according to any one of claims 12 to 18, wherein S is a sulfur atom of the antibody and n is the number of [(linker)-(proliferation inhibitor)] moieties covalently linked to the antibody.

21. The following formula (IV): (IV) The immunoconjugate according to any one of claims 12 to 19, wherein S is a sulfur atom of the antibody and n is the number of [(linker)-(exatecan)] moieties covalently linked to the antibody.

22. The following formula (V): (V) The immunoconjugate according to any one of claims 12 to 18 and 20, wherein S is a sulfur atom of the antibody and n is the number of [(linker)-(exatecan)] moieties covalently linked to the antibody.

23. The immunoconjugate according to any one of claims 12 to 22, wherein S is the sulfur atom of cysteine ​​of the antibody.

24. The immunoconjugate according to claim 23, wherein the cysteine ​​of the antibody is one of the cysteines capable of forming interchain disulfide bonds.

25. The immunoconjugate according to any one of claims 19 to 24, wherein n is 7 to 8.

26. The immunoconjugate according to any one of claims 19 to 25, wherein n is 7.5 to 8.

0.

27. A pharmaceutical composition comprising the antibody according to any one of claims 1 to 9 or the immunoconjugate according to any one of claims 12 to 26.

28. The pharmaceutical composition according to claim 27 for the treatment of cancer.

29. The pharmaceutical composition according to claim 28, wherein the cancer is a CEACAM5-expressing cancer.

30. The pharmaceutical composition according to claim 28 or 29, wherein the cancer is colorectal cancer, gastric cancer, lung cancer, pancreatic cancer, esophageal cancer, or prostate cancer.

31. A method for detecting CEACAM5 expression in a biological sample derived from a subject by ex vivo using the antibody described in any one of claims 1 to 9.

32. The method according to claim 31, wherein the antibody is labeled with a detectable molecule.

33. Use of the antibody according to any one of claims 1 to 9 for ex vivo detection of CEACAM5 expression in a biological sample derived from a subject.

34. The use according to claim 33, wherein the antibody is labeled with a detectable molecule.

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