Anti-CEACAM5 antibody drug conjugate

Abstract

Provided herein is an antibody-drug conjugate of formula (I), comprising an anti-CEACAM5 antibody, its antigen-binding moiety, conjugated with a linker and exatecan. The disclosure also provides a method for treating cancer in a subject of interest, comprising administering the antibody-drug conjugate disclosed herein to the subject.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims the interests of U.S. Provisional Patent Application No. 63 / 558,431, filed on 27 February 2024, and U.S. Provisional Patent Application No. 63 / 678,883, filed on 2 August 2024, which are incorporated herein by reference in their entirety.

[0002] Reference to electronically submitted sequence listings The electronically submitted sequence listing content (name: 3338_337PC03_SequenceListing_ST26.xml; size: 114,947 bytes; creation date: February 9, 2025) is submitted with this application and is incorporated herein by reference in its entirety.

[0003] This disclosure provides antibody-drug conjugates (ADCs) for use in therapeutic applications, comprising an antibody or antigen-binding moiety that specifically binds to carcinoembryonic antigen-associated cell adhesion molecule-5 (CEACAM5). [Background technology]

[0004] CEACAM5 is a cell surface protein that is weakly expressed in normal epithelial tissues, including the colon, esophagus, head and neck, stomach, and cervix, but highly expressed in several tumor types, including colorectal cancer, gastrointestinal cancer, lung cancer, and breast cancer, with the highest prevalence and expression occurring in over 80% of colorectal cancers. In normal tissues, CEACAM5 protects tubular organs from microbial invasion. In tumor cells, CEACAM5 is functionally involved in cell differentiation, cell adhesion, tumor invasion, and metastasis.

[0005] Overexpression of CEACAM5 is often associated with poor prognosis. For example, in patients with stage I, II, and III colorectal cancer, 5-year survival has been found to be inversely correlated with CEACAM5 tissue expression, and in patients with stage III disease, elevated serum levels of CEACAM5 are associated with poor prognosis (Non-Patent Literature 1). Consistent overexpression of CEACAM5 in many cancers has made it a recognized tumor biomarker and recurrence indicator in patients with cancer, particularly those with colorectal cancer.

[0006] Several therapeutic approaches targeting CEACAM5 in cancer are under development. However, to date, no FDA-approved CEACAM5-targeted therapies for cancer have been approved. Therefore, there remains an urgent need for effective treatments for CEACAM5-related cancers, including antibodies specifically directed at CEACAM5 that do not cross-react with other molecules in the CEACAM family, as well as antibody-drug conjugates (ADCs) that specifically kill CEACAM5-expressing cancer cells. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Gazzah et al.,Ann Oncol.,33(4):416-425 [Overview of the project] [Means for solving the problem]

[0008] This disclosure relates to an antibody-drug conjugate (ADC) represented by formula (I): [ka] Or provide a pharmaceutically acceptable salt, stereoisomer, or solvate thereof, in the formula, [ka] This indicates that the double bond configuration is E or Z. V is H or (C1-C8) alkyl; X is R 3 -C is; Y is NR 5 , S, O, or CR 6 R 7 and; R 1 This is a polyalkylene glycol unit containing at least three alkylene glycol subunits; R 3 and R 5 ~R 7 Each of these is H, or an optionally substituted aliphatic or aromatic residue; L is the linker; C is the cytotoxic part; m is an integer in the range of 1 to 10; n is in the range of 1 to 20; AB is an anti-CEACAM5 antibody or its antigen-binding portion, (a) Heavy chain variable region (VH) including complementarity-determining regions (CDR) 1, CDR2, and CDR3 regions containing the amino acid sequences described in SEQ ID NOs. 14, 15, and 16, respectively, and light chain variable region (VL) including CDR1, CDR2, and CDR3 regions containing the amino acid sequences described in SEQ ID NOs. 19, 20, and 21, or (b) VH containing CDR1, CDR2, and CDR3 regions having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with the amino acid sequences described in SEQ ID NOs: 14, 15, and 16, respectively, and VL containing CDR1, CDR2, and CDR3 regions having at least 85%, 90%, 95%, 98%, or 99% sequence identity with the amino acid sequences described in SEQ ID NOs: 19, 20, and 21, respectively. This is an anti-CEACAM5 antibody containing or its antigen-binding moiety.

[0009] This disclosure provides an antibody-drug conjugate represented by formula (I), wherein VH and VL are, (a) The amino acid sequences described in SEQ ID NO: 38 and SEQ ID NO: 43, respectively; (b) The amino acid sequences described in SEQ ID NO: 49 and SEQ ID NO: 50, respectively; (c) The amino acid sequences described in Sequence ID No. 51 and Sequence ID No. 52, respectively; (d) The amino acid sequences described in SEQ ID NO: 67 and SEQ ID NO: 68, respectively; (e) The amino acid sequences described in SEQ ID NO: 69 and SEQ ID NO: 70, respectively; (f) The amino acid sequences described in SEQ ID NO: 71 and SEQ ID NO: 72, respectively; (g) The amino acid sequences described in SEQ ID NO: 73 and SEQ ID NO: 74, respectively; (h) The amino acid sequences described in SEQ ID NO: 75 and SEQ ID NO: 76, respectively; (i) The amino acid sequences described in SEQ ID NO: 77 and SEQ ID NO: 78, respectively; (j) The amino acid sequences described in SEQ ID NO: 79 and SEQ ID NO: 80, respectively; (k) The amino acid sequences described in SEQ ID NO: 81 and SEQ ID NO: 82, respectively; (l) The amino acid sequences described in SEQ ID NO: 83 and SEQ ID NO: 84, respectively; (m) The amino acid sequences described in SEQ ID NO: 85 and SEQ ID NO: 86, respectively; (n) The amino acid sequences described in SEQ ID NO: 87 and SEQ ID NO: 88, respectively; (o) The amino acid sequences described in SEQ ID NO: 89 and SEQ ID NO: 90, respectively; (p) The amino acid sequence described in SEQ ID NO: 91 and the amino acid sequence described in SEQ ID NO: 92, respectively; or (q) The amino acid sequences described in SEQ ID NO: 93 and SEQ ID NO: 94, respectively. It has at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity.

[0010] In certain embodiments, the anti-CEACAM5 antibody or antigen-binding portion thereof is set forth in Tables 10 and 11. In certain embodiments, the CDR, VH, VL, heavy chain, and / or light chain are set forth in Table 10. For example, the anti-CEACAM5 antibody comprises a heavy chain and a light chain comprising the amino acid sequences set forth in SEQ ID NO: 45 and SEQ ID NO: 46, respectively.

[0011] The present disclosure provides a method of preparing an ADC of formula (I), the method comprising contacting a compound of formula (III):

Chemical formula

[0012] The present disclosure provides a method of treating a CEACAM5-expressing cancer in a subject in need thereof, the method comprising administering a therapeutically effective amount of an ADC of formula (I):

Chemical formula

[0013] In some embodiments, the anti-CEACAM5 antibody or its antigen-binding moiety is described in Tables 10 and 11. In some embodiments, the CDR, VH, VL, heavy chain and / or light chain are described in Tables 10 and 11.

[0014] This disclosure relates to an antibody-drug conjugate represented by formula (II): [ka] Or provide a pharmaceutically acceptable salt, stereoisomer or solvate thereof, in the formula, [ka] This indicates that the configuration of the double bond is E or Z; o is an integer between 8 and 30; n is in the range of 4 to 8; AB is an anti-CEACAM5 antibody containing VH and VL or its antigen-binding portion, where VH and VL are (a) The amino acid sequences described in SEQ ID NO: 38 and SEQ ID NO: 43, respectively; (b) The amino acid sequences described in SEQ ID NO: 49 and SEQ ID NO: 50, respectively; (c) The amino acid sequences described in Sequence ID No. 51 and Sequence ID No. 52, respectively; (d) The amino acid sequences described in SEQ ID NO: 67 and SEQ ID NO: 68, respectively; (e) The amino acid sequences described in SEQ ID NO: 69 and SEQ ID NO: 70, respectively; (f) The amino acid sequences described in SEQ ID NO: 71 and SEQ ID NO: 72, respectively; (g) The amino acid sequences described in SEQ ID NO: 73 and SEQ ID NO: 74, respectively; (h) The amino acid sequences described in SEQ ID NO: 75 and SEQ ID NO: 76, respectively; (i) The amino acid sequences described in SEQ ID NO: 77 and SEQ ID NO: 78, respectively; (j) The amino acid sequences described in SEQ ID NO: 79 and SEQ ID NO: 80, respectively; (k) The amino acid sequences described in SEQ ID NO: 81 and SEQ ID NO: 82, respectively; (l) The amino acid sequences described in SEQ ID NO: 83 and SEQ ID NO: 84, respectively; (m) The amino acid sequences described in SEQ ID NO: 85 and SEQ ID NO: 86, respectively; (n) The amino acid sequences described in SEQ ID NO: 87 and SEQ ID NO: 88, respectively; (o) The amino acid sequences described in SEQ ID NO: 89 and SEQ ID NO: 90, respectively; (p) The amino acid sequence described in SEQ ID NO: 91 and the amino acid sequence described in SEQ ID NO: 92, respectively; or (q) The amino acid sequences described in SEQ ID NO: 93 and SEQ ID NO: 94, respectively. It has at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity.

[0015] In some embodiments, the anti-CEACAM5 antibody or its antigen-binding moiety is described in Tables 10 and 11. In some embodiments, the CDR, VH, VL, heavy chain and / or light chain are described in Tables 10 and 11.

[0016] This disclosure relates to an antibody-drug conjugate having the following structure: [ka] [ADC101], or a pharmaceutically acceptable salt, stereoisomer, or solvate thereof, is provided, in the formula, [ka] This indicates that the configuration of the double bond is E or Z; AB is an anti-CEACAM5 antibody containing VH and VL or its antigen-binding portion, where VH and VL are (a) The amino acid sequences described in SEQ ID NO: 38 and SEQ ID NO: 43, respectively; (b) The amino acid sequences described in SEQ ID NO: 49 and SEQ ID NO: 50, respectively; (c) The amino acid sequences described in Sequence ID No. 51 and Sequence ID No. 52, respectively; (d) The amino acid sequences described in SEQ ID NO: 67 and SEQ ID NO: 68, respectively; (e) The amino acid sequences described in SEQ ID NO: 69 and SEQ ID NO: 70, respectively; (f) The amino acid sequences described in SEQ ID NO: 71 and SEQ ID NO: 72, respectively; (g) The amino acid sequences described in SEQ ID NO: 73 and SEQ ID NO: 74, respectively; (h) The amino acid sequences described in SEQ ID NO: 75 and SEQ ID NO: 76, respectively; (i) The amino acid sequences described in SEQ ID NO: 77 and SEQ ID NO: 78, respectively; (j) The amino acid sequences described in SEQ ID NO: 79 and SEQ ID NO: 80, respectively; (k) The amino acid sequences described in SEQ ID NO: 81 and SEQ ID NO: 82, respectively; (l) The amino acid sequences described in SEQ ID NO: 83 and SEQ ID NO: 84, respectively; (m) The amino acid sequences described in SEQ ID NO: 85 and SEQ ID NO: 86, respectively; (n) The amino acid sequences described in SEQ ID NO: 87 and SEQ ID NO: 88, respectively; (o) The amino acid sequences described in SEQ ID NO: 89 and SEQ ID NO: 90, respectively; (p) The amino acid sequence described in SEQ ID NO: 91 and the amino acid sequence described in SEQ ID NO: 92, respectively; or (q) The amino acid sequences described in SEQ ID NO: 93 and SEQ ID NO: 94, respectively. It has at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity.

[0017] In some embodiments, the anti-CEACAM5 antibody or its antigen-binding moiety is described in Tables 10 and 11. In some embodiments, the CDR, VH, VL, heavy chain and / or light chain are described in Tables 10 and 11. For example, the anti-CEACAM5 antibody or its antigen-binding moiety includes a heavy chain and a light chain containing the amino acid sequences described in SEQ ID NO: 45 and SEQ ID NO: 46, respectively. [Brief explanation of the drawing]

[0018] [Figure 1] Tables 1A-1B show the competition and binning of the selected anti-CEACAM5 binding antibodies. [Figure 2] Figures 2A-2D are graphs showing cell-based binding of anti-CEACAM5 antibodies by flow cytometry (AF647). Anti-CEACAM5 antibodies were assayed to test their binding to cell lines expressing different levels of human CEACAM5: BXPC-3 (Figure 2A), Ls174T (Figure 2B), MKN-45 (Figure 2C), and HCT-116 (Figure 2D), respectively. [Figure 3A]Figures 3A-3C are graphs showing the percentage of cell death induced by an anti-CEACAM5 antibody delivering the cytotoxic agent MMAE conjugated to secondary VHH (inhibition%) (Figure 3A), the rate of internalization (red area / phase area) (Figure 3B), and the cytotoxic level of a selected anti-CEACAM5 antibody as a function of internalization (internalization AUC) within MKN45 cells (cytotoxicity AUC) (Figure 3C). [Figure 3B] Same as above. [Figure 3C] Same as above. [Figure 4] Figures 4A and 4B show the percentage of proliferation inhibition by anti-CEACAM5 antibodies delivering the cytotoxic agent MMAE conjugated to a VHH secondary antibody (Figure 4A), and the internalization rate (red area / phase area) of selected anti-CEACAM5 antibodies in Ls174T cells (Figure 4B). [Figure 5A] 5A is a general description of the mutation analysis performed to create a single mutant library and to select progeny antibodies that bind to human CEACAM5 and cynomolgus monkey CEACAM5. [Figure 5B] 5B shows a mutation scan of MBN001. CDR locations (by Kabat) were analyzed, and mutations against germline VH include T7S, S40A, A68T, and P84A. [Figure 5C]5C-5N are heatmaps for human CEACAM5 and cynomolgus monkey CEACAM5 created using MBN001 antibody mutation scan analysis for LCDR1 substitution (SEQ ID NO: 64, germline and parental) (Figures 5I and 5J); LCDR2 substitution (SEQ ID NO: 65, germline and parental) (Figures 5K and 5L); LCDR3 substitution (SEQ ID NO: 66, germline and parental) (Figures 5M and 5N); HCDR1 substitution (SEQ ID NO: 61, germline and parental) (Figures 5C and 5D); HCDR2 substitution (SEQ ID NO: 62, germline and parental) (Figures 5E and 5F); and HCDR3 substitution (SEQ ID NO: 63, germline and parental) (Figures 5G and 5H). Note that in the graphs described herein, ND indicates that the enrichment ratio could not be determined because the NGS count in the starting library was too low. [Figure 5D] Same as above. [Figure 5E] Same as above. [Figure 5F] Same as above. [Figure 5G] Same as above. [Figure 5H] Same as above. [Figure 5I] Same as above. [Figure 5J] Same as above. [Figure 5K] Same as above. [Figure 5L] Same as above. [Figure 5M] Same as above. [Figure 5N] Same as above. [Figure 6] Figures 6A-6B are isoaffinity plots showing improvements in on-rate and off-rate for the parental antibody MBN001 against human CEACAM5 (Figure 6A) and cynomolgus monkey CEACAM5 (Figure 6B) compared to MBN001-optimized offspring. [Figure 7A]Figures 7A-7C are a series of graphs showing the inhibition percentage (Figure 7A), internalization rate (Figure 7B), and cytotoxicity levels (Figure 7C) of MBN001 antibody and selected progeny (MBP004, MBP005, MBP007, MBP008, MBP009, MBP010, MBP006, and MBP011) as a function of internalization within MKN45 cells by an anti-CEACAM5 antibody delivering the cytotoxic agent MMAE conjugated to a VHH secondary antibody. [Figure 7B] Same as above. [Figure 7C] Same as above. [Figure 8] Figures 8A-8B are a series of graphs showing the absence of nonspecific binding to cell lines engineered to express human CEACAM1 (CHO-S; Figure 8A) and human CEACAM6 (HCT-116; Figure 8B) MBN001 and their progeny mAbs MBP003, MBP001, and MBP002. [Figure 9] Figures 9A-9C are a series of graphs showing the binding of anti-CEACAM5 mAbs MBN001, MBP001, MBP003, and MBP002 to CEACAM5-low expressing LS174T cells (Figure 9A), CEACAM5-moderate expressing BxPC-3 cells (Figure 9B), and CEACAM5-high expressing MKN45 cells (Figure 9C) by FACS. Figure 9D shows the EC50 values ​​(MBN001, MBP001, MBP002, and MBP003). [Figure 10] Figures 10A-10D are a series of graphs and tables showing the inhibition percentage of anti-CEACAM5 antibodies conjugated with compound A' across a panel of CEACAM5-expressing cell lines. The graphs show the growth inhibition percentage of selected antibodies conjugated with compound A' compared to isotype controls at indicated antibody concentrations in the low-CEACAM5-expressing cell line Ls174T (Figure 10A), the moderate-CEACAM5-expressing cell line BxPC-3 (Figure 10B), and the high-CEACAM5-expressing cell line MKN-45 (Figure 10C). Figure 10D is a table containing IC50 values ​​for antibody-drug-conjugates. [Figure 11]Figures 11A and 11B show the ADCC activity of anti-human CEACAM5 mAbs in Jurkat-NFAT-FcγRIIIa (Promega) cell assays across moderately expressing CEACAM5 BxPC3 cells (Figure 11A) and high-expressing CEACAM5 MKN45 cells (Figure 11B). [Figure 12-1] 12A-12D are a series of graphs showing the bystander killing effect at 72 and 120 hours after co-culture of MKN45 cells (Ag+; Figures 12A and 12C) and HCT-116 cells (Ag-; Figures 12B and 12D), followed by treatment with ADP001A, ADCP001B, ADCP001C (ADCs having one of the mAbs of MBP001, MBP002, or MBP003 conjugated to compound A'). [Figure 12-2] Same as above. [Figure 13-1] Figure 13A is a ribbon diagram of a 3D model of human CEACAM5 with its distinct structural domains. Boxes have been added to this diagram to highlight the A3 and B3 domains of human CEACAM5. Figure 13B shows the amino acid sequence of hCEACAM5 (UniProt registration: P06731) with shading that identifies the distinct structural domains shown in Figure 13A (SEQ ID NO: 25). [Figure 13-2] 13C is a diagram showing the peptide sequence coverage of the hCEACAM5-A3-B3 construct (SEQ ID NO: 24). [Figure 13-3] 13D is a graph showing the difference in deuterium uptake between MBN001-bound and unbound hCEACAM5. Regions in hCEACAM5 where HDX is significantly reduced upon MBN001 binding are enclosed in a box. 13E is a diagram showing annotations on the linear sequence of hCEACAM5 regarding the HDX effect of MBN001 binding. Bold residues indicate slower exchange, information is unavailable for italicized residues, and no difference was detected for linear residues. [Figure 14A] 14A is the final cryoEM map. [Figure 14B]14B-14C are ribbon diagrams of cryoEM-derived final structure-based models showing epitope and paratope interactions between human CEACAM5, the MBP001 Fab construct, and the Fab of Bin 2 mAb. [Figure 14C] Same as above. [Figure 15-1] 15A-15E are a series of graphs showing the in vivo efficacy of ADCN001 (MBN001 (hIgG1.3) conjugated to compound A'(DAR8)) in the MKN45 CDX model (Figure 15A), BxPC3 (Figures 15B and 15D), and Ls174T (Figures 15C and 15E) after a single intravenous injection of either 3 mg / kg (Figures 15A, 15B, and 15C) or 10 mg / kg (Figures 15D and 15E) of an ADC. [Figure 15-2] Same as above. [Figure 15-3] Same as above. [Figure 16A] 16A-16B are simple Western blot images and graphs showing the induction of pharmacodynamic markers of the DNA damage response, including pKAP, pCHK1, and gH2AX, in addition to the expression of the apoptosis marker cleavage caspase-3 in tumors from the MKN45 CDX model (n=3) at 6, 24, and 168 hours after a single intravenous injection of either 1 mg / kg or 10 mg / kg of ADCN001 (MBN001+ compound A' ADC). The expression intensities of the DNA damage response marker pKAP (1TF1b) (ser 824), pCHK1, gH2AX, and the apoptosis marker c-caspase-3 were normalized against the expression intensity of the loading control GAPDH. [Figure 16B] Same as above. [Figure 17-1] Figures 17A-17D are a series of graphs showing the in vivo efficacy of ADCV001 (MBV001 conjugated with compound A' (the wild-type hIgG1 form of MBN001)) in CDX models (MKN45 (Figures 17A and 17C) and BxPC3 (Figures 17B and 17D)) after a single intravenous injection of ADCV001 at either 3 mg / kg (Figures 17A and 17B) or 10 mg / kg (Figures 17C and 17D). [Figure 17-2] Same as above. [Figure 18-1] Graphs 18A-18D are a series of graphs showing the in vivo efficacy of ADCP001A (MBP001 + compound A' ADC), ADCP001B (MBP002 + compound A' ADC), and ADCP001C (MBP003 + compound A' ADC) ADCs in the CDX model (MKN45 (Figures 18A and 18C) and BxPC3 (Figures 18B and 18D)) after a single intravenous injection of either 3 mg / kg (Figures 18A and 18B) or 10 mg / kg (Figures 18C and 18D) of ADC. [Figure 18-2] Same as above. [Figure 19-1] Figures 19A-19C are a series of graphs showing the in vivo efficacy of ADCV001 and M9140 in the CDX model MKN45 after a single intravenous injection of either 3 mg / kg (Figure 19A) or 10 mg / kg (Figures 19B and 19C) of ADCV001 and Merck ADC1. Figure 19C is an enlarged plot of Figure 19B. [Figure 19-2] Same as above. [Figure 20] Figure 20 shows a Western blot image illustrating the sustained induction of pharmacodynamic markers of the DNA damage response, including pKAP1 and pCHK1, in tumors from the MKN45 CDX model (n=4) at time points 6, 48, 72, 240, and 336 hours after a single intravenous injection of 3 mg / kg of ADCV001 and M9140. [Modes for carrying out the invention]

[0019] This disclosure provides antibody-drug conjugates (ADCs) of formula (I), (II), or ADC101, pharmaceutical compositions comprising ADCs, and the use of ADCs in methods for treating diseases such as cancer.

[0020] definition To facilitate understanding of the detailed explanation below, we will first define certain terms. Further definitions will be provided throughout.

[0021] The term "alkyl," either by itself or as part of another term, generally refers to a substituted or unsubstituted linear or branched saturated hydrocarbon having the indicated number of carbon atoms; for example, "-(C1~C8)alkyl" or "-(C1~C 10 (Alkyl) refers to an alkyl group having 1 to 8 or 1 to 10 carbon atoms, respectively. If the number of carbon atoms is not specified, the alkyl group may have 1 to 8 carbon atoms. Representative linear (C1-C8) alkyl groups include, but are not limited to, -methyl, -ethyl, -n-propyl, -n-butyl, -n-pentyl, -n-hexyl, -n-heptyl, and -n-octyl; branched (C1-C8) alkyl groups include -isopropyl, -sec-butyl, -isobutyl, -tert-butyl, -isopentyl, and -2-methylbutyl. In some embodiments, the alkyl group may be unsubstituted. Optionally, the alkyl group may be substituted with, for example, one or more groups.

[0022] The term "polyalkene glycol unit" is derived from the formula -[(CH2) n -O] y - (wherein n is the number of methylene groups in the subunit and y is the number of subunits in the unit) refers to a repeating alkylene glycol subunit. The oxygen atom of the terminal subunit may be substituted with a hydrogen atom, a protecting group, or any other acceptable functional group.

[0023] The terms "substituted," "optionally substituted," and "optionally substituted" generally mean that one or more hydrogen atoms can be independently substituted by substituents, unless otherwise specified. Typical substituents, though not limited to, include -X, -R, and -O. - -OR, -SR, -S - , -NR2, =NR, -CX3, -CN, -OCN, -SCN, -N=C=O, -NCS, -NO, -NO2, =N2, -N3, -NRC(=O)R, -C(=O)R, -C(=O)NR2, -SO3 -, -SO3H, -S(=O)2R, -OS(=O)2OR, -S(=O)2NR, -S(=O)R, -OP(=O)(OR)2-P(=O)(OR)2, -PO4 3- Examples include -PO3H2, -C(=O)R, -C(=O)X, -C(=S)R, -CO2R, -CO2H, -C(=S)OR, -C(=O)SR, -C(=S)SR, -C(=O)NR2, -C(=S)NR2, or -C(=NR)NR2, where each X is independently a halogen: -F, -Cl, -Br, or -I; and each R is independently -H, -(C1~C 20 )alkyl (for example, -(C1~C 10 )alkyl or -(C1~C8)alkyl, etc.), -(C6~C 20 )aryl (for example, -(C6~C 10 )aryl or, for example, -C6-aryl, -(C3~C 14 ) Complex algebras (for example, -(C3~C 10 The substituent is a heterocycle (such as a C3-C8 heterocycle), a protecting group, or a prodrug moiety. Typical substituents include (=O).

[0024] As used herein, the term “aliphatic or aromatic residue” generally refers to aliphatic substituents such as alkyl residues, for example, but not limited to alkyl residues, which may optionally be substituted with further aliphatic and / or aromatic substituents. Non-limiting examples include nucleic acids, enzymes, coenzymes, nucleotides, oligonucleotides, monosaccharides, polysaccharides, polymers, fluorophores, and optionally substituted benzenes, insofar as the direct linkage of such molecules to a core structure (for example, the linkage of Y to the nitrogen atom in the case of R') is aliphatic. Aromatic residues are substituents, and non-limiting examples include, for example, if the direct linkage of a nucleotide to a core structure is via a phenyl residue, then the direct linkage to the core structure is an aromatic system, for example, optionally substituted phenyl, triazolyl, or pyridyl or part of a nucleotide. As used herein, the term “aromatic residue” also includes heteroaromatic residues.

[0025] As used herein, the term “antibody” includes the entire antibody and any antigen-binding moiety (i.e., “antigen-binding moiety”) or a single chain thereof. In one embodiment, “antibody” refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains linked together by disulfide bonds or their antigen-binding moieties. Each heavy chain comprises a heavy chain variable region (V as used herein). L The heavy chain constant region includes the light chain variable region (abbreviated as CH1, CH2, and CH3) in certain naturally occurring antibodies. In certain naturally occurring antibodies, each light chain has a light chain variable region (V in this specification). L It includes the (abbreviated as) and the light chain constant region. The light chain constant region contains one domain CL. V H and V L The domain can be further divided into highly variable domains called complementary determination domains (CDRs), which are interspersed with more conservative domains called framework domains (FRs). H and V L The antibody consists of three CDRs and four FRs, arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with the antigen. The constant region of the antibody can mediate the binding of the immunoglobulin to various cells of the immune system (e.g., effector cells) and to host tissues or factors, including the first component (Clq) of the classical complement system.

[0026] Antibodies are typically 10 -5 ~10 -11 Dissociation constants less than or equal to M (K D ) reflects high affinity, specifically binding to those homologous antigens. -4 Any K greater than M D Generally, this is considered to exhibit nonspecific binding. As used herein, an antibody that "specifically binds" to an antigen refers to an antibody that binds with high affinity to the antigen and substantially the same antigen, which is 10 -7 M or less, 10 -8 M or less, 1×10 -9 M or less, 1×10 -10M or less or 1 x 10 -11 K below M D This means that it has. In some embodiments, the antibody is 10 -8 M~10 -10 M or 10 -9 M~10 -11 M's K D It binds specifically to antigens, but does not bind to unrelated antigens with high affinity.

[0027] The “antibodies” as defined in this disclosure include, but are not limited to, natural and non-natural antibodies, monoclonal antibodies, polyclonal antibodies, chimeric antibodies, humanized antibodies, human antibodies, non-human antibodies, bivalent antibodies, bispecific antibodies, multispecific antibodies, single-chain antibodies, diabodies, and nanobodies.

[0028] As used herein, “isolated antibody” refers to an antibody that substantially does not contain other antibodies with different antigen specificities.

[0029] As used herein, the term “antigen-binding portion” of an antibody refers to one or more fragments of an antibody that possess the ability to specifically bind to an antigen (e.g., human and / or cynomolgus monkey CEACAM5). It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of antibody-binding fragments include (i) Fab fragments, V L , V H (ii) a monovalent fragment consisting of CL and CH1 domains; (ii) a bivalent fragment containing an F(ab')2 fragment and two Fab fragments linked by disulfide bridges in the hinge region; (iii) V H and Fd fragment consisting of CH1 domain; (iv) V of a single arm of antibody L and V H Fv fragment consisting of domains, (v)V H Examples include (vi) a dAb fragment consisting of domains (Ward et al., (1989) Nature 341:544-546), and (vi) an isolated complementarity-determining region (CDR) or (vii) a combination of two or more isolated CDRs that can be optionally linked by a synthetic linker. Furthermore, the two domains V of the Fv fragment L and VH These are encoded by separate genes, but they can be joined together by a synthetic linker using recombination methods, V L and V H It becomes possible to produce a single protein chain (known as a single-chain Fv (scFv); see, for example, Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883) in which regions pair up to form a monovalent molecule. Such single-chain antibodies are also intended to be encompassed within the term "antigen-binding moiety" of the antibody. The antigen-binding moiety can be produced by recombinant DNA technology or by enzymatic or chemical cleavage of intact immunoglobulins.

[0030] Antibody fragments within the scope of the present invention also include F(ab')2 fragments, which can be produced, for example, by enzymatic cleavage of IgG with pepsin. Fab fragments can be produced, for example, by reducing F(ab')2 with dithiothreitol or mercaptoethylamine. A Fab fragment is a VL-CL chain attached to a VH-CH1 chain by a disulfide crosslink. An F(ab')2 fragment is then two Fab fragments attached by two disulfide crosslinks. The Fab portion of the F(ab')2 molecule includes a portion of the Fc region between which disulfide crosslinks are located.

[0031] As used herein, “isotype” refers to the antibody class encoded by the heavy chain constant region gene of the antibody (e.g., IgG (including IgG1, IgG2, IgG3, and IgG4), IgM, IgA (including IgA1 and IgA2), IgD, and IgE antibodies).

[0032] Antibodies can originate from any of the generally known isotypes, including, but not limited to, IgA, secretory IgA, IgG, and IgM. IgG isotypes are divided into certain subclasses: IgG1, IgG2, IgG3, and IgG4 in humans, and IgG1, IgG2a, IgG2b, and IgG3 in mice. Immunoglobulins, such as IgG1, exist in several allotypes, each differing by at most a few amino acids.

[0033] As used herein, the term “allotype” refers to a naturally occurring variant within a particular group of isotypes, where the variant differs by several amino acids. The anti-CEACAM5 antibodies described herein may be of any allotype. The antibody referred to herein as “IgG1.3f” is an IgG1 antibody of allotype “f,” i.e., an IgG1 antibody having 214R, 356E, and 358M according to the EU index. The triple mutant (L234A, L235E, G237A) IgG1.3f variant contains the amino acid sequence described in SEQ ID NO: 30. Mutations in these residues will eliminate or reduce the binding of the antibody to the Fcγ receptor and / or C1q, and therefore reduce the activator efficacy of the Fc domain of the IgG1 component of the antibody.

[0034] As used herein, the term “hypervariable region” (sometimes referred to as “variable region”) refers to an amino acid residue of an antibody that is involved in antigen binding. The hypervariable region consists of amino acid residues derived from the "complementarity-determining region" or "CDR" (e.g., residues 24-34 (CDRL1), 50-56 (CDRL2), and 89-97 (CDRL3) in the light chain variable domain and residues 31-35 (CDRH1), 50-65 (CDRH2), and 95-102 (CDRH3) in the heavy chain variable domain; Kabat et al., (1991) Sequences of Proteins of Immunological Interest, 5th Ed Public Health Service, National Institutes of Health, Bethesda, Md.) and / or the "hypervariable loop" (i.e., residues 26-32 (L1), 50-52 (L2), and 91-96 (L3) in the light chain variable domain and residues 26-32 (H1), 53-55 (H2), and 96-101 (H3) in the heavy chain variable domain; Chothia and This includes Lesk, (1987) J.Mol.Biol.196:901-917).

[0035] As used herein, the term “framework” or “FR” residues refer to variable domain residues other than hypervariable region residues as defined herein as CDR residues. The numbering of the above residues does not necessarily correspond to the sequence numbering in the detailed attached sequence listing with respect to the Kabat numbering system. The amino acid residues in the antibody may also be Chothia, Enhanced Chothia, IMGT, Kabat / Chothia complex, Honegger (AHo), Contact, or any other conventional antibody numbering scheme.

[0036] The term "acceptor human framework" is V L Frameworks containing amino acid sequences of frameworks, or V derived from the Human Immunoglobulin Framework or the Human Consensus Framework. HThis refers to a framework. An acceptor human framework "derived from" the human immunoglobulin framework or human consensus framework may have the same amino acid sequence as the naturally occurring human immunoglobulin framework or human consensus framework, or it may have amino acid sequence changes compared to the wild-type naturally occurring human immunoglobulin framework or human consensus framework. In some embodiments, the number of amino acid changes is 10, 9, 8, 7, 6, 5, 4, 3, or 2, or 1. In some embodiments, V L The Acceptor Human Framework is V L The sequence is identical to the human immunoglobulin framework sequence or the human consensus framework sequence.

[0037] The terms "Fc region," "Fc domain," or "Fc" refer to the C-terminal region of the antibody's heavy chain. Therefore, the Fc region includes the antibody's constant region excluding the first constant region immunoglobulin domain (e.g., CH1 or CL).

[0038] "Effector function" refers to the interaction between the antibody Fc region and an Fc receptor or ligand, or the resulting biochemical events. Examples of effector functions include FcγR-mediated effector functions such as Clq binding, complement-dependent cell-mediated cytotoxicity (CDC), Fc receptor binding, ADCC, and antibody-dependent cell-mediated phagocytosis (ADCP), as well as downregulation of cell surface receptors (e.g., B cell receptors; BCRs). Such effector functions generally require the Fc region to be combined with a binding domain (e.g., an antibody variable domain).

[0039] The term “epitope” or “antigenic determinant” refers to a site on an antigen (e.g., human CEACAM5) to which an immunoglobulin or antibody specifically binds. Epitopes can be formed from both continuous amino acids (usually linear epitopes) or discontinuous amino acids juxtaposed by tertiary folding of a protein (usually structural epitopes). Epitopes formed from continuous amino acids are typically retained upon exposure to denaturing solvents, though not always, while epitopes formed by tertiary folding are typically lost upon treatment with denaturing solvents. Epitopes typically contain at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 amino acids in a specific spatial arrangement.

[0040] As used herein, the term "monoclonal antibody" refers to an antibody that exhibits single-binding specificity and affinity to a specific epitope, or a composition of antibodies in which all antibodies exhibit single-binding specificity and affinity to a specific epitope. Therefore, the term "human monoclonal antibody" refers to an antibody or antibody composition that exhibits single-binding specificity and has variable and arbitrary constant regions derived from human germline immunoglobulin sequences. In one embodiment, human monoclonal antibodies are produced by hybridomas, such as B cells obtained from transgenic non-human animals having a genome containing human heavy-chain and light-chain transgenes fused to immortalized cells, for example, transgenic mice. Examples of monoclonal antibodies include chimeric antibodies, human antibodies, and humanized antibodies, which may occur naturally or be produced by recombinant DNA.

[0041] In this specification, monoclonal antibodies also include camelized single-domain antibodies. See, for example, Muyldermans et al. (2001) Trends Biochem. Sci. 26:230; Reichmann et al. (1999) J. Immunol. Methods 231:25; International Publication No. 94 / 04678; International Publication No. 94 / 25591; U.S. Patent No. 6,005,079 (all of which are incorporated herein by reference). In one embodiment, two V having modifications such that a single-domain antibody is formed H Single-domain antibodies containing a domain are provided herein.

[0042] The term “recombinant antibody” refers to an antibody prepared, expressed, produced, or isolated by recombinant means, for example, (a) an antibody isolated from an animal (e.g., mouse) that is transgenic or transchromosome of an immunoglobulin gene (e.g., human immunoglobulin gene), or a hybridoma prepared therefrom; (b) an antibody isolated from a host cell transformed to express the antibody; (c) an antibody isolated from a recombinant combinatorial antibody library (including human antibody sequences) using phage display; and (d) an antibody prepared, expressed, produced, or isolated by any other means, including splicing an immunoglobulin gene sequence (e.g., human immunoglobulin gene) to another DNA sequence. Such recombinant antibodies may have variable and constant regions derived from human germline immunoglobulin sequences. However, in certain embodiments, such recombinant human antibodies may be subjected to in vitro mutagenesis, and therefore the V of recombinant antibodies H and V L The amino acid sequence of the region is human germline V H and V L This sequence is derived from and related to other sequences, but it is not naturally present in the in vivo human antibody germline repertoire.

[0043] "Human" antibodies refer to antibodies that have variable regions in which both the framework region and the CDR region are derived from human germline immunoglobulin sequences. Furthermore, if the antibody contains a constant region, the constant region is also derived from a human germline immunoglobulin sequence. Antibodies derived from human germline immunoglobulin sequences, including normal somatic hypermutations that alter the germline immunoglobulin sequence compared to the wild-type germline immunoglobulin sequence, are also included.

[0044] A "humanized antibody" refers to an antibody in which some, almost all, or all of the amino acids outside the CDR domain of a non-human antibody are replaced with amino acids derived from the corresponding human immunoglobulin. In one embodiment of a humanized antibody, some, almost all, or all of the amino acids outside the CDR domain are replaced with amino acids derived from human immunoglobulin, while some, almost all, or all of the amino acids within one or more CDR regions remain unchanged. Any addition, deletion, insertion, substitution, or modification of amino acids is acceptable as long as it does not render the antibody unable to bind to a particular antigen. A "humanized" antibody may retain similar antigen specificity to the original antibody.

[0045] The term "fully human antibody" refers to an antibody that contains only human immunoglobulin protein sequences. Fully human antibodies may contain mouse carbohydrate chains when produced in mice, in mouse cells, or in hybridomas derived from mouse cells. Similarly, "mouse antibody" refers to an antibody that contains only mouse immunoglobulin sequences.

[0046] A "chimeric antibody" refers to an antibody in which the variable region originates from one or more species, and the constant region originates from another species, such as an antibody in which the variable region originates from a mouse antibody and the constant region originates from a human antibody. See U.S. Patent No. 4,816,567 and Morrison et al., (1984) Proc. Natl. Acad. Sci. USA 81:6851-6855.

[0047] A "domain antibody" or "nanobody" is an immunologically functional immunoglobulin fragment containing only the variable region of the heavy chain or the variable region of the light chain. In some cases, two or more V H The regions are covalently linked by a peptide linker to produce a bivalent domain antibody. The two Vs of the bivalent domain antibody H The region may target the same or different antigens.

[0048] A "bivalent antibody" contains two antigen-binding sites. In some cases, the two binding sites have the same antigen specificity. However, a bivalent antibody can be bispecific.

[0049] A "bispecific" or "bifunctional antibody" is an artificial hybrid antibody having two different heavy / light chain pairs and two different binding sites. Bispecific antibodies can be produced by various methods, including hybridoma fusion or Fab' fragment linkage. See, for example, Songsivilai & Lachmann, Clin. Exp. Immunol. 79:315-321 (1990); Kostelny et al., J. Immunol. 148, 1547-1553 (1992). Examples of bifunctional antibodies include heterodimer antibody conjugates (e.g., two antibodies or antibody fragments bound together with different specificities), antibody / cell surface molecule conjugates (e.g., an antibody conjugated to a non-antibody molecule such as a receptor), and hybrid antibodies (e.g., an antibody with binding sites to two different antigens).

[0050] A "multispecific antibody" is an antibody that recognizes two or more different antigens or epitopes (e.g., a bispecific antibody, a tripspecific antibody).

[0051] As used herein, the terms "single-stranded Fv" or "scFv" refer to antibodies with the V of the antibody. H Domain and V LThis refers to antibody fragments containing domains, which are present in a single polypeptide chain. Generally, Fv polypeptides further contain polypeptide linkers. For an overview of scFv, see Pluckthun (1994) The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds. Springer-Verlag, New York, pp. 269-315.

[0052] As used herein, the term “diabody” refers to a small antibody fragment having two antigen-binding sites, the fragment being a light chain variable domain (V) in the same polypeptide chain. L ) connected to the heavy chain variable domain (V H )(V H -V L or V L -V H ) is included. By using a linker that is too short to allow pairing between two domains on the same chain, the domains are paired with complementary domains on another chain, generating two antigen-binding sites. The diabodies are described in more detail, for example, European Patent No. 404,097; International Publication No. 93 / 11161; and Holliger et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448. For an overview of engineered antibody variants, see Holliger and Hudson (2005) Nat. Biotechnol. 23:1126-1136.

[0053] The terms “immune cell engager” or “ICE” are used herein in reference to multifunctional molecules that contain two or more binding specificities capable of redirecting immune effector cells against cancer cells. Exemplary immune cell engagers include T cell engagers (e.g., bispecific T cell engagers or BiTEs), NK cell engagers (NKCEs), B cell engagers, dendritic cell engagers, and macrophage cell engagers.

[0054] The terms “bispecific T cell engager” and “BiTE” are used synonymously herein with respect to a bispecific molecule that links the targeting regions of two antibody and / or protein-binding domains, where one arm of the molecule is engineered to bind to a protein on the surface of a cytotoxic T cell (i.e., a T cell engager) (e.g., CD3), and the other arm is engineered to bind to a specific protein found primarily on tumor cells, e.g., CEACAM5. When both targets are bound, the BiTE molecule forms a crosslink between the cytotoxic T cell and the tumor cell, enabling the T cell to recognize and kill the tumor cell. The BiTE may or may not contain an immunoglobulin constant region.

[0055] The terms “bispecific NK cell engager” and “NKCE” are used synonymously herein to refer to bispecific molecules containing a CEACAM5 binding domain linked to the binding domain of an NK cell surface protein (i.e., an NK cell engager) by a short flexible linker region.

[0056] The term "binding to the same epitope" is used in reference to two or more antibodies that bind to the same segment of amino acid residues. Techniques for determining whether antibodies bind to the same epitope may be determined by epitope mapping methods described herein. Other methods include monitoring the binding of antibodies to antigen fragments (e.g., proteolytic fragments) or mutant variations of antigens, where loss of binding due to modification of amino acid residues in the antigen sequence is often considered an indicator of the epitope component, e.g., alanine scanning mutagenesis (Cunningham & Wells (1985) Science 244:1081), yeast display of mutant target sequence variants, or chimera analysis. Furthermore, computerized combinatorial methods for epitope mapping may also be used. These methods depend on the ability of the antibody of interest to isolate specific short-chain peptides from combinatorial phage display peptide libraries. H and VL Alternatively, antibodies having the same CDR1, 2, and 3 sequences are expected to bind to the same epitope.

[0057] An antibody that "competes with another antibody for binding to a target" refers to an antibody that (partially or completely) inhibits the binding of another antibody to its target. Whether two antibodies compete with each other for binding to a target, i.e., whether and to what extent one antibody inhibits the binding of the other antibody to its target, can be determined using known binding competition experiments, including surface plasmon resonance (SPR) and biolayer interferometry (BLI). In certain embodiments, an antibody competes with the target and inhibits the binding of another antibody to the target by at least 50%, 60%, 70%, 80%, 90%, or 100%. The level of inhibition or competition may vary depending on which antibody is a "blocking antibody" (i.e., an antibody that blocks another immune response with an antigen when combined with the antigen). Competitive assays can be performed as described, for example, in Ed Harlow and David Lane, Cold Spring Harbor. Protoc. 2006; doi:10.1101 / pdb.prot4277 or Chapter 11 of “Using Antibodies” by Ed Harlow and David Lane, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA 1999. Competitive antibodies bind to the same epitope, overlapping epitope, or adjacent epitope (as demonstrated, for example, by steric hindrance). Two antibodies “cross-compete” if the antibodies block both pathways from each other by at least 50%, i.e., regardless of which antibody comes into contact with the antigen first in the competitive experiment.

[0058] A competitive binding assay to determine whether two antibodies compete for or cross-compete for binding is, for example, flow cytometry to determine competition for binding to cells expressing CEACAM5. Other methods include surface plasmon resonance (SPR) (e.g., BIACORE®), solid-phase direct or indirect radioimmunoassay (RIA), solid-phase direct or indirect enzyme immunoassay (EIA), sandwich competition assay (see Stahli et al., Methods in Enzymology 9:242 (1983)); solid-phase direct biotin-avidin EIA (see Kirkland et al., J.Immunol. 137:3614 (1986)); solid-phase direct labeling assay, solid-phase direct labeling sandwich assay (see Harlow and Lane, Antibodies: Alaboratory Manual, Cold Spring Harbor Press (1988)); solid-phase direct labeling RIA using 1-125 labeling (see Morel et al., Mol.Immunol. 25(1):7 (1988)); solid-phase direct biotin-avidin EIA (see Cheung et al., Virology See 176:546 (1990); and directly labeled RIA (Moldenhauer et al., Scand. J. Immunol. 32:77 (1990)).

[0059] As used herein, the terms “specific binding,” “selective binding,” “selectively binding,” and “specifically binding” refer to an antibody that binds to an epitope on a given antigen. Typically, the antibody is determined by (i) surface plasmon resonance (SPR) using a given antigen as the analyte and the antibody as the ligand, for example, or by scatchard analysis of the binding of the antibody to antigen-positive cells, approximately 10 -7 Less than M, for example, about 10 -8 Less than M, 10 -9 Less than M or 10 -10 M or a lesser equilibrium dissociation constant (K D) is conjugated and binds to the predetermined antigen with an affinity at least 2-fold higher than its affinity for binding to non-specific antigens other than the predetermined antigen or an antigen closely related thereto (e.g., BSA, casein). Any K -4 exceeding about 10 D is generally considered to exhibit non-specific binding.

[0060] As used herein, the term "k assoc " or "k a " refers to the association rate of a particular antibody-antigen interaction. As used herein, the term "k dis " or "k d " refers to the dissociation rate of a particular antibody-antigen interaction. As used herein, the term "K D " is intended to refer to the dissociation, obtained from the ratio of k d to k a (i.e., k d / k a ) and expressed as molar concentration (M). The K D value of an antibody can be determined using methods well established in the art. A preferred method for determining the K D of an antibody is by using surface plasmon resonance, such as a biosensor system like the BIACORE® system, or by using flow cytometry and Scatchard analysis, or by using biolayer interferometry.

[0061] In the context of in vitro or in vivo assays using antibodies or immune complexes, the terms "EC50" or "IC50" refer to the antibody concentration that induces a response that is 50% of the maximum response, i.e., an intermediate response between the maximum response and baseline. In pharmacology, the potency of a compound is expressed as the maximum half-dose effective concentration (EC50), which refers to the concentration of the drug that induces an intermediate response between baseline and the maximum value. Expressing the potency of a compound by its EC50 value makes sense in a clinical context, but it is contrary to common sense in the context of purification based on biological activity, as the potency of a compound is inversely proportional to its EC50 value, and the most potent compounds have the lowest EC50. The maximum half-dose inhibitory concentration (IC50) is the most widely used and useful measure of drug efficacy. It indicates how much of the drug is needed to inhibit a biological process by half, and therefore provides a measure of the potency of antagonist drugs in pharmacological studies.

[0062] As used herein, the term “conjugated” refers to the association of two or more molecules. Conjugation may be covalent or non-covalent. Conjugation may also be genetic (i.e., recombinant fusion). Such conjugation may be achieved using a wide variety of techniques recognized in the art, such as chemical conjugation and recombinant protein production.

[0063] As used herein, the term “complex” is used in relation to an immune complex or antibody-drug complex comprising an anti-CEACAM5 antibody or its antigen-binding moiety, as described herein, linked to a cytotoxic drug or therapeutic agent as described herein.

[0064] As used herein, the term “linker” refers to a chemical portion comprising any atomic chain that can be used to covalently bond and / or, for example, to covalently bond a drug to an antibody. Linkers are 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. Conjugations of the antibodies of this disclosure with cytotoxic drugs or other growth inhibitors include, for example, but are not limited to, 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-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), and iminothiola This can be carried out using a variety of bifunctional protein coupling agents, including nucleotides (IT), difunctional derivatives of imide esters (such as dimethylHCl adipoimidoate), 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 in Vitetta et al (1987). Carbon-labeled 1-isothiocyanatobenzylmethyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for the conjugation of radioactive nucleotides to antibodies (International Publication No. 94 / 11026).

[0065] In certain embodiments, the linker is a “cleavable linker” that can facilitate the release of cytotoxic drugs or other growth inhibitors within or near cells, such as tumor cells. In some embodiments, the linker is a linker that can cleave in the endosomes of mammalian cells. For example, acid-unstable linkers, peptidase-sensitive linkers, esterase-unstable linkers, photo-unstable linkers, or disulfide-containing linkers (see, for example, U.S. Patent No. 5,208,020) may be used.

[0066] As used herein, the term “nucleic acid molecule” is used in reference to DNA molecules and RNA molecules. Nucleic acid molecules may be single-stranded or double-stranded and may be cDNA.

[0067] The term "isolated nucleic acid molecule" refers to an antibody or antibody fragment (e.g., V H , V L When used herein in relation to nucleic acids encoding CDR3, it is intended to refer to nucleic acid molecules in which the nucleotide sequence does not essentially contain other genomic nucleotide sequences, such as those encoding antibodies that bind to antigens other than CEACAM5, and other sequences may naturally be adjacent to nucleic acids in human genomic DNA.

[0068] As used herein, the term “vector” is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it is ligated. One type of vector is a “plasmid,” which refers to a circular double-stranded DNA loop to which an additional DNA segment can be ligated. Another type of vector is a viral vector, to which an additional DNA segment can be ligated into a viral genome. Certain vectors can autonomously replicate in the host cell into which they are introduced (e.g., bacterial vectors with bacterial origins of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be incorporated into the host cell's genome upon introduction into the host cell, thereby replicating with the host genome. Furthermore, certain vectors can be directed to the expression of a gene to which it is operably ligated. Such vectors are referred herein as “recombinant expression vectors” (or simply “expression vectors”). Generally, expression vectors useful in recombinant DNA technology are often in the form of plasmids. Hereinafter, “plasmid” and “vector” may be used synonymously, as plasmids are the most commonly used form of vectors. However, other forms of expression vectors that perform equivalent functions are also included, such as viral vectors (e.g., replication-deficient retroviruses, adenoviruses, and adeno-associated viruses).

[0069] Furthermore, the “conservative sequence modifications” of sequences described herein also include, for example, amino acid sequence modifications that do not invalidate the binding of antibodies encoded by nucleotide sequences or containing amino acid sequences to antigens. Such conservative sequence modifications include conservative nucleotide and amino acid substitutions, as well as the addition and deletion of nucleotides and amino acids. Modifications can be introduced into sequences by, for example, standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include those in which an amino acid residue is replaced by an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains are defined in the art. These families include basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), non-charged side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Therefore, predicted non-essential amino acid residues in anti-CEACAM5 antibodies may be substituted with other amino acid residues derived from the same side chain family. Methods for identifying conserved nucleotide and amino acid substitutions that do not exclude antigen binding are well known in the art (see, for example, Brummell et al., Biochem. 32:1180-1187 (1993); Kobayashi et al. Protein Eng. 12(10):879-884 (1999); and Burks et al. Proc. Natl. Acad. Sci. USA 94:412-417 (1997)). Alternatively, in another embodiment, mutations may be introduced randomly along all or part of the anti-CEACAM5 antibody coding sequence, for example by saturation mutagenesis, and the resulting modified anti-CEACAM5 antibody may be screened for binding activity.

[0070] For nucleic acids, the term "substantial homology" indicates that, when two nucleic acids, or their specified sequences, are optimally aligned and compared, they are identical in at least approximately 80% of their nucleotides, typically at least approximately 80%–85%, 85%–90%, 90%–95%, and at least approximately 98%–99.5%, with appropriate nucleotide insertions or deletions. Alternatively, substantial homology exists when segments hybridize to the chain complement under selective hybridization conditions. For polypeptides, the term "substantial homology" indicates that, when two polypeptides, or their specified sequences, are optimally aligned and compared, they are identical in at least approximately 80% of their amino acids, typically at least approximately 80%–85%, 85%–90%, 90%–95%, and at least approximately 98%–99.5%, with appropriate amino acid insertions or deletions.

[0071] The percentage of identity between two arrays is a function of the number of identical positions shared by the arrays, taking into account the number of gaps that need to be introduced for optimal alignment of the two arrays and the length of each gap (i.e., % homology = number of identical positions / total number of positions × 100). The comparison of arrays and the determination of the percentage of identity between two arrays can be achieved using mathematical algorithms, as described in the non-restrictive examples below.

[0072] The percentage of identity between two nucleotide sequences can be determined using the GAP program in the GCG software package (available at www.gcg.com) with the NWSgapdna.CMP matrix and gap weights of 40, 50, 60, 70, or 80 and length weights of 1, 2, 3, 4, 5, or 6. The percentage of identity between two nucleotides or two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (CABIOS, 4:11-17 (1989)) incorporated into the ALIGN program (version 2.0), using the PAM120 weighted residue table, a gap length penalty of 12, and a gap penalty of 4. Furthermore, the percentage of identity between two amino acid sequences can be determined using the Needleman and Wunsch (J.Mol.Biol.(48):444-453(1970)) algorithm, which is incorporated into the GAP program of the GCG software package (available at www.gcg.com) using either a Blossum 62 matrix or a PAM250 matrix, and gap weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6.

[0073] The nucleic acid and protein sequences described herein may be further used as "query sequences" to identify related sequences, for example, by performing searches against public databases. Such searches may be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. (1990) J.Mol.Biol.215:403-10. A BLAST nucleotide search can be performed using the NBLAST program with a score of 100 and a word length of 12 to obtain nucleotide sequences homologous to the nucleic acid molecules described herein. A BLAST protein search can be performed using the XBLAST program with a score of 50 and a word length of 3 to obtain amino acid sequences homologous to the protein molecules described herein. To obtain gapped alignments for comparative purposes, they may be used as described in Altschul et al., (1997) Nucleic Acids Res.25(17):3389-3402. When using the BLAST and gapped BLAST programs, the default parameters of each program (e.g., XBLAST and NBLAST) may be used. Please refer to www.ncbi.nlm.nih.gov.

[0074] As used herein, the term “recombinant host cell” (or simply “host cell”) is intended to refer to a cell that contains nucleic acids not naturally present in the cell and may be a cell into which a recombinant expression vector has been introduced. It should be understood that such a term is intended to refer not only to a specific target cell but also to the offspring of such a cell. Such offspring may not be identical to the parent cell in practice, as certain modifications may occur in subsequent generations due to either mutation or environmental influences, but they still fall within the scope of the term “host cell” as used herein.

[0075] As used herein, the term “inhibition” refers to any statistically significant reduction in biological activity, including partial and complete blockade of activity. For example, “inhibition” may refer to a statistically significant reduction of approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% in biological activity.

[0076] As used herein, the term “immunotherapy” means the treatment of a person who is suffering from a disease, or who is at risk of suffering from a disease or a relapse of a disease, by means of inducing, enhancing, suppressing or otherwise modifying an immune response.

[0077] As used herein, the terms “immunostimulating therapy” and “immunostimulatory therapy” refer to therapies that result in an increase (e.g., induction or enhancement) of the immune response in a subject, for example, to treat cancer.

[0078] As used herein, “immune cells” refers to a subset of blood cells known as leukocytes, including mononuclear cells such as lymphocytes, monocytes, macrophages, and granulocytes.

[0079] As used herein, “abnormal” is used in relation to the activity, level, or expression of a molecule that is outside the normal activity or expression level (e.g., overexpression) compared to a control or reference sample exhibiting a normal activity / expression profile. As used herein, the term “normal” is used in relation to the activity or expression level of a protein found in a healthy, sex- and age-matched population of subjects. The minimum size of this healthy population may be determined using standard statistical measures, for example, a physician may consider the incidence of disease in the general population and the desired level of statistical certainty in the outcome. In some embodiments, the normal range of biomarker activity, level, or expression is determined from a population of subjects (e.g., at least 5, 10, or 20 subjects), from a population of subjects (e.g., at least 40 or 80 subjects), and from a population of more than 100 subjects.

[0080] "T effect pedal" ("T eff ") cells refer to T cells with cytolytic activity (e.g., CD4+ and CD8+ T cells) as well as helper T(Th) cells that secrete inflammatory cytokines and activate and induce other immune cells, but do not include regulatory T cells (Treg cells).

[0081] As used herein, “administer” means the physical delivery of a CEACAM5 targeting agent, such as an ADC (containing an anti-CEACAM5 antibody or its antigen-binding moiety linked via a linker to a cytotoxic moiety described herein, either alone or in combination with another therapeutic agent), to a subject using any of the various methods and delivery systems known to those skilled in the art. Preferred routes of administration for the ADCs described herein include intravenous, intraperitoneal, intramuscular, subcutaneous, spinal, or other parenteral routes of administration, such as by injection or infusion. As used herein, the term “parenteral administration” means a mode of administration other than enteral and topical administration, usually by injection, and without limitation includes intravenous, intraperitoneal, intramuscular, intra-arterial, subarachnoid, intralymphatic, intrafocal, intracapsular, intraorbital, intracardiac, intradermal, transtracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injections and infusions, as well as in vivo electroporation. Alternatively, the antibodies described herein may be administered via parenteral routes, such as topical, cutaneous, or mucosal routes, such as intranasal, oral, vaginal, rectal, sublingual, or topical administration. Administration may be carried out, for example, once, multiple times, and / or over one or more consecutive periods of time.

[0082] As used herein, “cancer” refers to a broad group of diseases characterized by the uncontrolled proliferation of abnormal cells in the body. Uncontrolled cell division can result in the formation of malignant tumors or cells that can invade adjacent tissues and metastasize to distant parts of the body via the lymphatic system or bloodstream, and malignant tumors or cells include, but are not limited to, a variety of cancers, including carcinomas, melanomas, sarcomas, leukemias, lymphomas, germ cell tumors, and blastomas. Exemplary cancers for treatment include brain, bladder, breast, cervix, colon, head and neck, kidney, lung, non-small cell lung, mesothelioma, ovarian, prostate, stomach and uterine cancers, leukemia, and medulloblastoma.

[0083] As used herein, the term “small molecule drug” refers to a molecular entity that is not a polymer, often an organic or organometallic entity, which possesses drug activity and has a molecular weight of less than about 2 kilodaltons (kDa), less than about 1 kDa, less than about 900 daltons (Da), less than about 800 Da, or less than about 700 Da. While the term encompasses most pharmaceutical compounds called “drugs” other than proteins or nucleic acids, small peptides or nucleic acid analogs may also be considered small molecule drugs. Examples include chemotherapeutic anticancer agents and enzyme inhibitors. Small molecule drugs can be derived synthetically, semi-synthetically (i.e., from naturally occurring precursors), or biologically.

[0084] As used herein, the terms “to treat,” “to treat,” and “treatment” mean any type of intervention or process performed on a subject for the purpose of preventing, halting, alleviating, improving, inhibiting, delaying, or preventing the progression, onset, severity, or recurrence of disease-related symptoms, complications, conditions, or biochemical signs, or the administration of an active agent (e.g., an anti-CEACAM5 antibody as described herein or an ADC comprising its antigen-binding moiety linked to a cytotoxic moiety via a linker as described herein) to a subject. Treatment may be treatment of a subject with the disease or a subject without the disease (e.g., for preventive purposes).

[0085] As used herein, “adjunct” or “combination” administration (co-administration) includes the simultaneous administration of an ADC (containing the anti-CEACAM5 antibody or its antigen-binding moiety as described herein, linked via a linker to the cytotoxic moiety described herein) and one or more additional agents and / or compounds, and / or compounds in the same or different dosage forms, or co-administration in separate doses, simultaneously or sequentially. Accordingly, the ADC (containing the anti-CEACAM5 antibody or its antigen-binding moiety as described herein, linked via a linker to the cytotoxic moiety described herein), and a second, third, or more agents and / or compounds (e.g., small molecules) may be administered simultaneously in a single formulation, or may be formulated for separate administrations and administered simultaneously or sequentially.

[0086] As used herein, “combination” therapy means the administration of two or more therapeutic agents in a coordinated manner, including, but not limited to, simultaneous and sequential administration. Specifically, combination therapy encompasses both co-administration (e.g., administration of a co-formulation or simultaneous administration of separate therapeutic compositions) and sequential or sequential administration, insofar as the administration of one therapeutic agent is somehow conditioned upon the administration of another therapeutic agent. For example, one therapeutic agent may be administered only after a different therapeutic agent has been administered and acted for a predetermined period of time. (See, for example, Kohrt et al. (2011) Blood 117:2423). For example, an ADC (containing an anti-CEACAM5 antibody linked to a cytotoxic moiety described herein via a linker) may be administered first, followed (e.g., immediately thereafter) by a second agent (e.g., an antibody or its antigen-binding moiety, and an anticancer agent), or vice versa. In one embodiment, the ADC is administered before the administration of the second agent. In another embodiment, the ADC is administered with a second drug for, for example, a few minutes (e.g., within about 30 minutes) or for at least one hour. Such co-administration or sequential administration may result in both the ADC and the second drug being present simultaneously in the treated patient.

[0087] Administration of an effective amount of ADC (containing the anti-CEACAM5 antibody or its antigen-binding moiety as described herein, linked to the cytotoxic moiety described herein via a linker) alone, or in combination with another compound or agent (e.g., an immune checkpoint inhibitor such as an anti-PD-1 antibody), according to any of the methods provided herein, may result in at least one therapeutic effect, including, for example, a reduction in tumor growth or size, a decrease in the number of signs of cancer (e.g., metastatic lesions) appearing over time, complete remission, partial remission, or stabilization of the disease. For example, a treatment method may result in an equivalent clinical benefit rate (CBR = complete remission (CR) + partial remission (PR) + stable condition lasting ≥ 6 months) that is better than that achieved without ADC administration or with administration of either ADC or the second agent alone, for example, an improvement in clinical benefit rate of approximately 20%, 30%, 40%, 50%, 60%, 70%, or 80% or more.

[0088] As used herein, the terms “inhibit” and “block” (e.g., with respect to inhibition / blockage of CEACAM5 binding or functional activity) are used synonymously and encompass both partial and complete inhibition / blockage by anti-CEACAM5 antibodies or fragments thereof in ADCs, or other inhibition / blockage of functional activity by therapeutic agents. The degree of inhibition may be at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% (i.e., 2x (2-fold) or 2x (2×)), 3x, 5x, or 10x compared to a control antibody or reference antibody. Furthermore, the degree of inhibition may be 20%-95%, 20%-80%, 20%-50%, 40%-95%, 40%-80%, 40%-60%, 50%-90%, 50%-70%, 75%-95%, 75%-85%, 2x-20x, 2x-10x, 2x-5x, 4x-12x, or 4x-8x.

[0089] The term “effective dose” or “effective dosage” is defined as the amount sufficient to achieve, or at least partially achieve, the desired effect. The “therapeutic effective dose” or “therapeutic effective dosage” of a drug (e.g., an ADC (containing the anti-CEACAM5 antibody or its antigen-binding moiety as described herein, linked to the cytotoxic moiety described herein via a linker)) is any amount of the drug or therapeutic agent, when used alone or in combination with another therapeutic agent, that promotes disease regression as demonstrated by a reduction in the severity of disease symptoms, an increase in the frequency and duration of disease-asymptomatic periods, or the prevention of functional or physical impairment resulting from the distress of the disease. The therapeutic effective dose or dosage of a drug or therapeutic agent includes the “preventive effective dose” or “preventive effective dosage,” which is any amount of the drug or therapeutic agent, when administered alone or in combination with another therapeutic agent, that inhibits the onset or recurrence of the disease in a subject at risk of developing the disease or suffering a relapse of the disease. The ability of therapeutic agents to promote disease regression or inhibit the onset or recurrence of disease can be evaluated using various methods known to those skilled in the art, such as in human subjects during clinical trials, in animal model systems to predict efficacy in humans, or by assaying the activity of the agent in in vitro assays.

[0090] For example, in the treatment of tumors, a therapeutically effective dose or dosage of a drug or therapeutic agent (e.g., an ADC (containing an anti-CEACAM5 antibody or its antigen-binding moiety as described herein, linked via a linker to the cytotoxic moiety described herein)) inhibits tumor cell proliferation by at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least more than about 70%, at least about 80%, or at least about 90% compared to an untreated subject. In some embodiments, a therapeutically effective dose or dosage of a drug or therapeutic agent completely inhibits cell growth or tumor growth, i.e., inhibits cell growth or tumor growth by 100%. The ability of a compound or therapeutic agent (including an antibody) to inhibit tumor growth can be evaluated using the assays described herein. Alternatively, this property of a composition containing a compound or therapeutic agent can be evaluated by examining the ability of the composition to inhibit cell proliferation; such inhibition can be measured in vitro by assays known to those skilled in the art.

[0091] The term "patient" includes human and other mammalian subjects receiving either preventive or therapeutic treatment.

[0092] As used herein, the term “subject” includes any human or non-human animal. For example, the methods and compositions described herein may be used to treat a subject having cancer. The term “non-human animal” includes all vertebrates, e.g., mammals and non-mammals, e.g., non-human primates, sheep, cats, dogs, cattle, chickens, amphibians, and reptiles.

[0093] The term "sample" refers to tissue, bodily fluid, or cells (or any fraction thereof) taken from a patient or subject. Typically, the tissue or cells will be removed from the patient, although in vivo diagnosis is also intended. In the case of solid tumors, tissue samples can be taken from surgically removed tumors and prepared for testing. In the case of lymphoma and leukemia, lymphocytes, leukemia cells, or lymphoid tissue (e.g., leukemia cells from blood) can be obtained and appropriately prepared. Other samples, including urine, tears, serum, plasma, cerebrospinal fluid, feces, sputum, and cell extracts, may also be useful for certain cancers.

[0094] As used herein, the terms “detection” or “detected” refer to qualitative and / or quantitative detection (measuring levels) with or without reference to a control.

[0095] As used herein, the term “diagnose” means determining the nature of a medical condition in which the pathology affecting the subject is to be identified from some collected data.

[0096] As used herein, “includes” is synonymous with “contains,” “contains,” “has,” or “characterized,” and is comprehensive or open-ended and does not exclude additional elements or methods or processes not enumerated. As used herein, “consists of” excludes any elements, processes, or components not specified in the elements of the claims. As used herein, “essentially consists of” does not exclude materials or processes that do not substantially affect the basic and novel features of the claims. In each example herein, any of the terms “includes,” “essentially consists of,” and “consists of” may be optionally replaced with any of the other two terms to describe alternative aspects of the scope of the subject matter. The inventions described exemplary herein may preferably be carried out in the absence of any one or more elements, one or more limitations not specifically disclosed herein.

[0097] As used herein, the singular forms "a," "an," and "the" refer to multiple objects unless the context specifically indicates otherwise. The use of "or" or "and" means "and / or" unless otherwise specified. Furthermore, the use of other forms such as "include," "includes," and "includes" is not limited to these.

[0098] When the term "approximately" is used herein to refer to measurable values ​​such as quantities or temporal durations, it includes a variation of up to ±10% from the specified value. Unless otherwise indicated, all figures representing quantities of components or properties (e.g., molecular weight, reaction conditions) described herein should be understood to be modified by the term "approximately."

[0099] As used herein, “and / or” should be interpreted as a specific disclosure of each of the two designated features or components, with or without the other. Thus, the term “and / or” as used in phrases such as “A and / or B” includes “A and B,” “A or B,” “A” alone, and “B” alone. Similarly, the term “and / or” as used in phrases such as “A, B, and / or C” includes each of the following: A, B and C; A, B or C; A or C; A or B; B or C; A and C; A and B; B and C; A alone; B alone; and C alone.

[0100] When used herein, the enumeration of value ranges herein is intended only as a simplified way of referring individually to each distinct value contained within that range, unless otherwise specifically indicated herein, and each distinct value is incorporated herein as if it were individually enumerated herein. For example, if the concentration range is stated as 1% to 50%, values ​​such as 2% to 40%, 10% to 30%, or 1% to 3% are intended to be explicitly enumerated herein. These are merely examples of what is specifically intended, and all possible combinations of numerical values, including the enumerated minimum and maximum values, should be considered expressly described herein.

[0101] As used herein, the term “stereoisomer” refers to isomers of the same composition but differing in the arrangement of their atoms in space. Enantiomers and diastereomers are examples of stereoisomers. Geometric isomers are also examples of stereoisomers. The term “enantiomer” refers to one of a pair of molecular species that are mirror images of each other and cannot be superimposed. The term “diastereomer” refers to a stereoisomer that is not a mirror image of each other. The term “racemate” or “racemic mixture” refers to a composition consisting of equimolar amounts of two enantiomers, the composition being optically inactive. Geometric isomers of the C=C double bond can also exist in ADCs, and all such stable isomers are considered in the present invention. The cis- and trans- (or E- and Z-) geometric isomers of ADCs of the present invention are described and may be isolated as a mixture of isomers or as separated isomer forms.

[0102] The term "ADC101" refers to the compound represented by the formula: [ka] (AB is the anti-CEACAM5 antibody described herein or its antigen-binding portion; In the formula, o refers to the integer 24. It is also understood that this is identical to the compound represented by the formula: [ka] Since the two structures are presumed to be identical, the two figures are interchangeable. Either structure can be used to represent the same structure.

[0103] In some embodiments, the anti-CEACAM5 antibody or its antigen-binding moiety is described in Tables 10 and 11. In some embodiments, the CDR, VH, VL, heavy chain and / or light chain are described in Tables 10 and 11.

[0104] Various embodiments described herein are described in further detail in the following subsections.

[0105] I. Antibody-drug conjugates This disclosure provides an antibody-drug conjugate comprising an anti-CEACAM5 antibody or its antigen-binding moiety as described herein, which is linked or conjugated via a phosphorus (V) moiety (also denoted as "P5") and a linker to a cytotoxic moiety, i.e., camptothecin or its derivatives and analogs.

[0106] In some embodiments, the ADC of this disclosure is expressed by formula (I): [ka] It is represented by, or a pharmaceutically acceptable salt, stereoisomer, or solvate thereof, in which, [ka] This indicates that the configuration of the double bond is E or Z; V is H or (C1-C8) alkyl; X is R 3 -C is; Y is NR 5 , S, O, or CR 6 R 7 and; R 1 This is a polyalkylene glycol unit containing at least three alkylene glycol subunits; R 3 and R 5 ~R 7 Each of these is H, or an optionally substituted aliphatic or aromatic residue; L is the linker; C is the cytotoxic part; m is an integer in the range of 1 to 10; n is in the range of 1 to 20; AB is an anti-CEACAM5 antibody or its antigen-binding portion, (a) Heavy chain variable region (VH) including complementarity-determining regions (CDR) 1, CDR2, and CDR3 regions containing the amino acid sequences described in SEQ ID NOs. 14, 15, and 16, respectively, and light chain variable region (VL) including CDR1, CDR2, and CDR3 regions containing the amino acid sequences described in SEQ ID NOs. 19, 20, and 21, or (b) VH containing CDR1, CDR2, and CDR3 regions having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with the amino acid sequences described in SEQ ID NOs. 14, 15, and 16, respectively, and VL containing CDR1, CDR2, and CDR3 regions having at least 85%, 90%, 95%, 98%, or 99% sequence identity with the amino acid sequences described in SEQ ID NOs. 19, 20, and 21, respectively. This is an anti-CEACAM5 antibody containing or its antigen-binding moiety.

[0107] In some embodiments, R 3 is H or an optionally substituted aliphatic or aromatic residue. In some embodiments, R 3 is H or (C1-C8) alkyl. In some embodiments, R 3 H is H.

[0108] In some embodiments, R 1 Structure: [ka] It includes 3 to 100 subunits having [a certain characteristic].

[0109] In some embodiments, R 1 teeth, [ka] And, During the ceremony, [ka] This indicates the position of O; K F -H, -PO3H, -(C1~C 10) Alkyl, -(C1~C 10 ) Alkyl-SO3H, -(C2~C 10 ) Alkyl-CO2H, -(C2~C 10 ) Alkyl-OH, -(C2~C 10 ) Alkyl-NH2, -(C2~C 10 ) Alkyl-NH(C1~C3)alkyl and -(C2~C 10 ) Alkyl-N((C1~C3)alkyl)2, and is selected from the group consisting of; o is an integer in the range of 3 to 100.

[0110] In some embodiments, R 1 has the structure:

Chemical formula

Chemical formula

Chemical formula

[0111] In some embodiments, K F is H.

[0112] <00009?0>In some embodiments, o is an integer in the range of 8 to 30, for example, 8 to 16, 10 to 30, or 20 to 28, for example, 10, 11, 12, 13, 14, 22, 23, 24, 25 or 26.

[0113] In some embodiments, the linker L is cleavable.

[0114] In some embodiments, linker L can be cleaved by protease, glucuronidase, sulfatase, phosphatase, esterase, or disulfide reduction.

[0115] In some embodiments, linkers are cleaved under physiological conditions, particularly intracellularly, by, for example, lysosomes or endosomal proteases, to release the bound cytotoxic moiety. In some embodiments, cleavable linkers are designed to release the free cytotoxic moiety in an unmodified form. Examples of cleavable linkers include disulfide linkers, acid-unstable linkers, photostable linkers, peptidase-unstable linkers, and esterase-unstable linkers. Typically, peptidyl linkers are at least two amino acid long or at least three amino acid long.

[0116] Peptidase-unstable linkers can be used to cleave specific peptides inside or outside cells. In one embodiment, the cleavable linker is cleaved under mild conditions, i.e., intracellular conditions under which the activity of the cytotoxic moiety is unaffected.

[0117] Depending on the linker design, membrane-permeable (lipophilic) toxins released into target-positive cells can cross the cell membrane and kill other nearby cells, including adjacent cancer cells lacking antigen expression (bystander effect) (Kovtun, YVet al. (2006) Cancer Res. 66(6), 3214-3221). The ability of such cytotoxic drugs to mediate local bystander death is one selection criterion for ADCs according to this disclosure.

[0118] Examples of cleavage agents include cathepsins B and D, and plasmin, all of which are known to hydrolyze dipeptide drug derivatives to release the active drug within target cells. For example, peptidyl linkers cleavable by the thiol-dependent protease cathepsin-B, which is highly expressed in cancerous tissue, can be used (e.g., Phe-Leu or Gly-Phe-Leu-Gly (SEQ ID NO: 101) linker). In certain embodiments, peptidyl linkers cleavable by intracellular proteases are valine-citrulline (Val-Cit) linkers or phenylalanine-lysine (Phe-Lys) linkers. One advantage of using intracellular proteolytic release of therapeutic agents is that the drug is typically attenuated when conjugated, and the serum stability of the conjugate is typically high.

[0119] Various linkers may be used in the conjugates described herein. In some embodiments, the linker includes a peptidyl linker such as the dipeptide valine (Val)-citrulline (Cit)(Vc) which can be cleaved by cathepsins inside tumor cells. Further peptidyl linkers include, but are not limited to, Val-Cit, Ala-Val, Val-Ala-Val, Lys-Lys, Pro-Val-Gly-Val-Val (SEQ ID NO: 99), Ala-Asn-Val, Val-Leu-Lys, Ala-Ala-Asn, Cit-Cit, Val-Lys, Lys, Cit, Ser, or Glu. In some embodiments, linker L can be cleaved by proteases, such as cathepsins B and D.

[0120] In some embodiments, the linker L is given by the formula: * -AW 1~8 -B 0~1 -# It is represented as, In the formula, unit A is the first spacer unit; W is an amino acid; B is the second spacer unit, * indicates a binding site to -Y-, and # indicates a binding site to the cytotoxic moiety.

[0121] In some embodiments, the first spacer unit A has the structure: [Chemical formula] where [Chemical formula] is a 5- or 6-membered carbocyclic ring; * indicates the bonding point to -Y-, and ## indicates the bonding point to W. The preferred unit A is [Chemical formula] . <000s0966>

[0122] In some embodiments, W is a dipeptide (W2).

[0123] [[ID=2h]]In some embodiments, the dipeptide is selected from the group consisting of valine-citrulline (Val-Cit) and valine-alanine (Val-Ala).

[0124] In some embodiments, the second spacer unit B is a PAB group having the following structure: [Chemical formula] where the NH group is bonded to -W-, and the C(O) group is bonded to the cytotoxic moiety.

[0125] In some embodiments, the linker has the following structure: [Chemical formula] where W2 is a dipeptide * indicates the bonding point to Y, and # indicates the bonding point to the cytotoxic moiety.

[0126] In some embodiments, the linker L is *-A-W2-B1-#, with the structure: [Chemical formula]<000s0994> The formula has the following characteristics, where * indicates a binding point to Y, and # indicates a binding point to the cytotoxic region.

[0127] In some embodiments, C is a cytotoxic moiety. The terms “cytotoxic moiety” or, as may be, “payload” refer to the chemical or biochemical moiety conjugated to the anti-CEACAM5 antibody described herein via a linker.

[0128] In some embodiments, the cytotoxic moiety is an anticancer agent. Therefore, the drug is selected from the group consisting of meitansinoids, calicheamicin, tubulinin, amatoxin, drastatin and monomethyl auristatin E (MMAE) or monomethyl auristatin F (MMAF), pyrrolobenzodiazepine dimers, indolino-benzodiazepine dimers, emetine, radioisotopes, therapeutic proteins and peptides (or fragments thereof), kinase inhibitors, CDK inhibitors, histone deacetylase (HDAC) inhibitors, MEK inhibitors, KSP inhibitors, topoisomerase inhibitors, and their analogs or prodrugs. In preferred embodiments, the cytotoxic moiety is a topoisomerase I inhibitor. In some embodiments, the cytotoxic moiety is a naturally occurring topoisomerase I inhibitor, camptothecin, or its derivatives or analogs. In some embodiments, the cytotoxic moiety is a camptothecin derivative, such as exatecan. The structure of exatecan is shown below. [ka]

[0129] All stereoisomers of exatecan are assumed to be those of the ADCs disclosed herein.

[0130] In some embodiments, n in the ADC of equation (I) is 7 or 8. In some embodiments, n in the ADC is 5 or 6. In some embodiments, n in the ADC is 9 or 10. In some embodiments, n in the ADC is 7. In some embodiments, n in the ADC is 8.

[0131] In some embodiments, in formula (I), AB is an anti-CEACAM5 antibody disclosed herein or its antigen-binding moiety; V is H; Y is NH; R 3 H is; n is in the range of 4 to 8; R 1 Structure: [ka] It is a polyalkylene glycol unit having, During the ceremony, [ka] This indicates the position of O; K F H is; o is an integer in the range of 8 to 30; L has the following structure: [ka] It is a linker that has, In the formula, * indicates a binding site to Y, # indicates a binding site to the cytotoxic moiety; C is exatecan; and m is 1.

[0132] In some embodiments, o is an integer in the range of 8-30, 10-30, 15-30, 20-30, or 20-25. In some embodiments, o is 20, 21, 22, 23, 24, or 25.

[0133] In some embodiments, n is an integer in the range of 2 to 10. In some embodiments, o is an integer in the range of 20 to 28. In some embodiments, o is 22, 23, 24, 25, or 26.

[0134] In some embodiments, n is an integer in the range of 2 to 10.

[0135] In some embodiments, the anti-CEACAM5 antibody or its antigen-binding moiety is described in Tables 10 and 11. In some embodiments, the CDR, VH, VL, heavy chain and / or light chain are described in Tables 10 and 11.

[0136] In some embodiments, the ADC of this disclosure is given by formula (II): [ka] (II) is represented by, or a pharmaceutically acceptable salt, stereoisomer or solvate thereof, in which, o is an integer between 8 and 30; n is in the range of 4 to 8; AB is an anti-CEACAM5 antibody or its antigen-binding moiety disclosed herein (e.g., Tables 10 and 11); [ka] This indicates that the double bond configuration is E or Z. The linker can also exist as a mixture of E and Z isomers.

[0137] In some embodiments, the ADC of this disclosure is given by formula (II): [ka] It is represented by, or a pharmaceutically acceptable salt, stereoisomer, or solvate thereof, in which, [ka] This indicates that the configuration of the double bond is E or Z; n is in the range of 4 to 8; o is an integer between 8 and 30; AB is an anti-CEACAM5 antibody or the antigen-binding portion of an anti-CEACAM5 antibody that specifically binds to carcinoembryonic antigen-associated cell adhesion molecule-5 (CEACAM5), (a) Heavy chain variable region (VH) including complementarity-determining regions (CDR) 1, CDR2, and CDR3 regions containing the amino acid sequences described in SEQ ID NOs. 14, 15, and 16, respectively, and light chain variable region (VL) including CDR1, CDR2, and CDR3 regions containing the amino acid sequences described in SEQ ID NOs. 19, 20, and 21, or (b) VH containing CDR1, CDR2, and CDR3 regions having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with the amino acid sequences described in SEQ ID NOs. 14, 15, and 16, respectively, and VL containing CDR1, CDR2, and CDR3 regions having at least 85%, 90%, 95%, 98%, or 99% sequence identity with the amino acid sequences described in SEQ ID NOs. 19, 20, and 21, respectively. This is an anti-CEACAM5 antibody or the antigen-binding portion of an anti-CEACAM5 antibody that contains [the specified substance].

[0138] In some embodiments, the ADC of this disclosure is given by formula (ADC 101): [ka] It is represented by, or a pharmaceutically acceptable salt, stereoisomer, or solvate thereof, in which, [ka] This indicates that the configuration of the double bond is E or Z; AB is an anti-CEACAM5 antibody or the antigen-binding portion of an anti-CEACAM5 antibody that specifically binds to carcinoembryonic antigen-associated cell adhesion molecule-5 (CEACAM5), (a) Heavy chain variable region (VH) including complementarity-determining regions (CDR) 1, CDR2, and CDR3 regions containing the amino acid sequences described in SEQ ID NOs. 14, 15, and 16, respectively, and light chain variable region (VL) including CDR1, CDR2, and CDR3 regions containing the amino acid sequences described in SEQ ID NOs. 19, 20, and 21, or (b) VH containing CDR1, CDR2, and CDR3 regions having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with the amino acid sequences described in SEQ ID NOs. 14, 15, and 16, respectively, and VL containing CDR1, CDR2, and CDR3 regions having at least 85%, 90%, 95%, 98%, or 99% sequence identity with the amino acid sequences described in SEQ ID NOs. 19, 20, and 21, respectively. This is an anti-CEACAM5 antibody or the antigen-binding portion of an anti-CEACAM5 antibody that contains [the specified substance].

[0139] In certain embodiments, AB is an anti-CEACAM5 antibody or its antigen-binding portion as described in Tables 10 and 11. In certain embodiments, AB is an anti-CEACAM5 antibody or its antigen-binding portion, (a) Heavy chain variable region (VH) including complementarity-determining regions (CDR) 1, CDR2, and CDR3 regions containing the amino acid sequences described in SEQ ID NOs. 14, 15, and 16, respectively, and light chain variable region (VL) including CDR1, CDR2, and CDR3 regions containing the amino acid sequences described in SEQ ID NOs. 19, 20, and 21, or (b) VH containing CDR1, CDR2, and CDR3 regions having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with the amino acid sequences described in SEQ ID NOs: 14, 15, and 16, respectively, and VL containing CDR1, CDR2, and CDR3 regions having at least 85%, 90%, 95%, 98%, or 99% sequence identity with the amino acid sequences described in SEQ ID NOs: 19, 20, and 21, respectively. This is an anti-CEACAM5 antibody containing or its antigen-binding moiety.

[0140] In some embodiments, o is an integer of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30. In some embodiments, o is an integer between 20 and 30, for example, 23. In some embodiments, o is an integer between 20 and 25, for example, 24. In some embodiments, o is an integer of 24. In some embodiments, o is 24. In some embodiments, o is 25. In some embodiments, o is 23. In some embodiments, o is 22. In some embodiments, o is 21. In some embodiments, o is 20. In some embodiments, o is an integer between 26 and 30. In some embodiments, o is 26. In some embodiments, o is 27. In some embodiments, o is 28. In some embodiments, o is 29. In some aspects, o is 30. In some aspects, o is an integer between 8 and 19. In some aspects, o is 8. In some aspects, o is 9. In some aspects, o is 10. In some aspects, o is 11. In some aspects, o is 12. In some aspects, o is 13. In some aspects, o is 14. In some aspects, o is 15. In some aspects, o is 16. In some aspects, o is 17. In some aspects, o is 18. In some aspects, o is 19.

[0141] In some embodiments, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, n is 4. In some embodiments, n is 5. In some embodiments, n is 6. In some embodiments, n is 7. In some embodiments, n is 8. Preferably, o is 24 and n is 8.

[0142] This disclosure also relates to compounds of formula (III): [ka] Or provide a pharmaceutically acceptable salt, stereoisomer, or solvate thereof, wherein X, R 1Y, L, C, and m are defined in equation (I).

[0143] In some embodiments, the present disclosure relates to a method for preparing an ADC of formula (I), wherein the compound of formula (III) is a thiol-containing compound, AB-(SH) n The present invention provides a method for obtaining an ADC of formula (I) by reacting it with (wherein AB is an anti-CEACAM5 antibody disclosed herein or its antigen-binding moiety, and n is in the range of 1 to 10).

[0144] In some embodiments, the compound of formula (III) has the structure: [ka] A compound having (compound A), or a pharmaceutically acceptable salt, stereoisomer, or solvate thereof, where o is an integer in the range of 8 to 25, e.g., 24, and * represents a chiral center. All stereoisomers of compound A are assumed for the synthesis of ADCs disclosed herein.

[0145] In some embodiments, the present disclosure relates to a method for preparing an ADC of formula (II), wherein compound A is a thiol-containing compound, AB-(SH) n (Here, AB is an anti-CEACAM5 antibody or its antigen-binding moiety disclosed herein (for example, disclosed in Tables 10 and 11)) is reacted with the ADC of formula (II): [ka] The present invention provides a method for obtaining a pharmaceutically acceptable salt, stereoisomer, or solvate thereof (wherein o and n are as defined above). Methods for the selective bioconjugation reaction of ethynylphosphoneamide with cysteine-containing compounds are described in International Publication No. 2018041985A1 (published March 8, 2018), International Publication No. 2019170710A2 (published September 12, 2019), International Publication No. 2022223783A1 (published October 27, 2022), International Publication No. 2023083900A1 (published May 19, 2023), and International Publication No. 2023083919A1 (published May 19, 2023), each of which is incorporated herein by reference.

[0146] The number of cytotoxic moieties linked to the antigen-binding portion of CEACAM5-ADC (drug-to-antibody ratio: DAR) can vary and is limited only by the number of available binding sites on the antigen-binding portion and the number of drugs linked to a single linker.

[0147] The DAR value can vary depending on the properties of the antigen-binding moiety (e.g., any antibody or its antigen-binding moiety as described herein) and the drug used in conjunction with the experimental conditions used for conjugation (DAR, reaction time, solvent and / or co-solvent properties). Therefore, contact between the antibody and drug in an ADC can result in a mixture containing several different conjugates depending on the drug-to-antibody ratio, and may further contain free antibody and / or aggregates. Thus, the determined DAR is an average value. DAR can be analyzed by UV spectroscopy, monomer content by SEC-HPLC, and free drug content by RP-HPLC.

[0148] In some embodiments, the linker will link a single cytotoxic moiety to the antigen-binding moiety of the conjugate (e.g., any antibody described herein or its antigen-binding moiety). In some embodiments, the conjugate comprises two or more cytotoxic moieties, each moiety may be the same or different. A conjugate having 20 or more DARs is assumed, provided that the conjugate does not exhibit an unacceptable level of aggregation under the conditions of use and / or storage. In some embodiments, the conjugates described herein may have DARs in the range of about 1–10, 2–10, 1–8, 2–8, 1–6, 2–6, 1–4, or 2–4. In some specific embodiments, the conjugate may have a DAR of 2, 3, 4, or 5. In some embodiments, the DAR is 6. In some embodiments, the DAR is 7. In some embodiments, the DAR is 8. In some embodiments, the DAR is 9. In some embodiments, the DAR is 6 or 7. In some embodiments, DAR is 7, 7.5, or 8. In some embodiments, DAR is 7 to 8.

[0149] In some embodiments, the ADC of this disclosure has the following structure: [ka] A compound having, or a pharmaceutically acceptable salt, stereoisomer, or solvate thereof, wherein, [ka] represents the configuration of the double bond as E or Z; AB is an antibody that binds to CEACAM5 or its antigen-binding portion, including VH and VL, which contain the amino acid sequences described in SEQ ID NOs. 38 and 43, respectively.

[0150] In some aspects, this disclosure is ADC 101: [ka] Or provides a pharmaceutically acceptable salt thereof, wherein AB is an anti-CEACAM5 antibody or its antigen-binding moiety, (a) Heavy chain variable region (VH) including complementarity-determining regions (CDR) 1, CDR2, and CDR3 regions containing the amino acid sequences described in SEQ ID NOs. 14, 15, and 16, respectively, and light chain variable region (VL) including CDR1, CDR2, and CDR3 regions containing the amino acid sequences described in SEQ ID NOs. 19, 20, and 21, or (b) VH containing CDR1, CDR2, and CDR3 regions having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with the amino acid sequences described in SEQ ID NOs. 14, 15, and 16, respectively, and VL containing CDR1, CDR2, and CDR3 regions having at least 85%, 90%, 95%, 98%, or 99% sequence identity with the amino acid sequences described in SEQ ID NOs. 19, 20, and 21, respectively. This is an anti-CEACAM5 antibody containing or its antigen-binding moiety.

[0151] In some embodiments, the Disclosure provides ADC101 or a pharmaceutically acceptable salt thereof, where AB is an anti-CEACAM5 antibody comprising VH and VL, respectively, comprising the amino acid sequences described in SEQ ID NO: 49 and SEQ ID NO: 50, or an antigen-binding moiety thereof.

[0152] In some embodiments, the Disclosure provides ADC101 or a pharmaceutically acceptable salt thereof, where AB is an anti-CEACAM5 antibody comprising VH and VL, respectively, comprising the amino acid sequences described in SEQ ID NO: 49 and SEQ ID NO: 50, or an antigen-binding moiety thereof.

[0153] In some embodiments, the Disclosure provides ADC101 or a pharmaceutically acceptable salt thereof, where AB is an anti-CEACAM5 antibody comprising VH and VL, which comprise the amino acid sequences described in SEQ ID NO: 67 and SEQ ID NO: 68, respectively, or an antigen-binding moiety thereof.

[0154] In some embodiments, the Disclosure provides ADC101 or a pharmaceutically acceptable salt thereof, where AB is an anti-CEACAM5 antibody comprising VH and VL, which comprise the amino acid sequences described in SEQ ID NO: 69 and SEQ ID NO: 70, respectively, or an antigen-binding moiety thereof.

[0155] In some embodiments, the Disclosure provides ADC101 or a pharmaceutically acceptable salt thereof, where AB is an anti-CEACAM5 antibody comprising VH and VL, which comprise the amino acid sequences described in SEQ ID NO: 71 and SEQ ID NO: 72, respectively, or an antigen-binding moiety thereof.

[0156] In some embodiments, the Disclosure provides ADC101 or a pharmaceutically acceptable salt thereof, where AB is an anti-CEACAM5 antibody comprising VH and VL, which comprise the amino acid sequences described in SEQ ID NO: 73 and SEQ ID NO: 74, respectively, or an antigen-binding moiety thereof.

[0157] In some embodiments, the Disclosure provides ADC101 or a pharmaceutically acceptable salt thereof, where AB is an anti-CEACAM5 antibody comprising VH and VL, which comprise the amino acid sequences described in SEQ ID NO: 75 and SEQ ID NO: 76, respectively, or an antigen-binding moiety thereof.

[0158] In some embodiments, the Disclosure provides ADC101 or a pharmaceutically acceptable salt thereof, where AB is an anti-CEACAM5 antibody comprising VH and VL, which comprise the amino acid sequences described in SEQ ID NO: 77 and SEQ ID NO: 78, respectively, or an antigen-binding moiety thereof.

[0159] In some embodiments, the Disclosure provides ADC101 or a pharmaceutically acceptable salt thereof, where AB is an anti-CEACAM5 antibody comprising VH and VL, which comprise the amino acid sequences described in SEQ ID NO: 79 and SEQ ID NO: 80, respectively, or an antigen-binding moiety thereof.

[0160] In some embodiments, the Disclosure provides ADC101 or a pharmaceutically acceptable salt thereof, where AB is an anti-CEACAM5 antibody comprising VH and VL, which comprise the amino acid sequences described in SEQ ID NO: 81 and SEQ ID NO: 82, respectively, or an antigen-binding moiety thereof.

[0161] In some embodiments, the Disclosure provides ADC101 or a pharmaceutically acceptable salt thereof, where AB is an anti-CEACAM5 antibody comprising VH and VL, which comprise the amino acid sequences described in SEQ ID NO: 83 and SEQ ID NO: 84, respectively, or an antigen-binding moiety thereof.

[0162] In some embodiments, the Disclosure provides ADC101 or a pharmaceutically acceptable salt thereof, where AB is an anti-CEACAM5 antibody comprising VH and VL, which comprise the amino acid sequences described in SEQ ID NO: 85 and SEQ ID NO: 86, respectively, or an antigen-binding moiety thereof.

[0163] In some embodiments, the Disclosure provides ADC101 or a pharmaceutically acceptable salt thereof, where AB is an anti-CEACAM5 antibody comprising VH and VL, which comprise the amino acid sequences described in SEQ ID NO: 87 and SEQ ID NO: 88, respectively, or an antigen-binding moiety thereof.

[0164] In some embodiments, the Disclosure provides ADC101 or a pharmaceutically acceptable salt thereof, wherein A and B are anti-CEACAM5 antibodies comprising VH and VL, respectively, comprising the amino acid sequences described in SEQ ID NO: 89 and SEQ ID NO: 90, or the antigen-binding moiety thereof; and the cytotoxic moiety comprises compound 101.

[0165] In some embodiments, the Disclosure provides ADC101 or a pharmaceutically acceptable salt thereof, where AB is an anti-CEACAM5 antibody comprising VH and VL, which comprise the amino acid sequences described in SEQ ID NO: 91 and SEQ ID NO: 92, respectively, or an antigen-binding moiety thereof.

[0166] In some embodiments, the Disclosure provides ADC101 or a pharmaceutically acceptable salt thereof, where AB is an anti-CEACAM5 antibody comprising VH and VL, respectively, comprising the amino acid sequences described in SEQ ID NO: 93 and SEQ ID NO: 94, or an antigen-binding moiety thereof.

[0167] In some embodiments, the Disclosure provides ADC101 or a pharmaceutically acceptable salt thereof, where AB is an anti-CEACAM5 antibody comprising VH and VL, which comprise the amino acid sequences described in SEQ ID NO: 17 and SEQ ID NO: 22, respectively, or an antigen-binding moiety thereof.

[0168] In some embodiments, VH and VL include the amino acid sequence described in SEQ ID NO: 49 and SEQ ID NO: 50, respectively. In some embodiments, VH and VL include the amino acid sequence described in SEQ ID NO: 51 and SEQ ID NO: 52, respectively. In some embodiments, VH and VL include the amino acid sequence described in SEQ ID NO: 67 and SEQ ID NO: 68, respectively. In some embodiments, VH and VL include the amino acid sequence described in SEQ ID NO: 69 and SEQ ID NO: 70, respectively. In some embodiments, VH and VL include the amino acid sequence described in SEQ ID NO: 71 and SEQ ID NO: 72, respectively. In some embodiments, VH and VL include the amino acid sequence described in SEQ ID NO: 73 and SEQ ID NO: 74, respectively. In some embodiments, VH and VL include the amino acid sequence described in SEQ ID NO: 75 and SEQ ID NO: 76, respectively. In some embodiments, VH and VL include the amino acid sequence described in SEQ ID NO: 69 and SEQ ID NO: 70, respectively. In some embodiments, VH and VL include the amino acid sequence described in SEQ ID NO: 73 and SEQ ID NO: 74, respectively. In some embodiments, VH and VL include the amino acid sequence described in SEQ ID NO: 75 and SEQ ID NO: 76, respectively. In some embodiments, VH and VL include the amino acid sequence described in SEQ ID NO: 77 and SEQ ID NO: 78, respectively. In some embodiments, VH and VL include the amino acid sequence described in SEQ ID NO: 79 and SEQ ID NO: 80, respectively. In some embodiments, VH and VL include the amino acid sequence described in SEQ ID NO: 81 and SEQ ID NO: 82, respectively. In some embodiments, VH and VL include the amino acid sequence described in SEQ ID NO: 83 and SEQ ID NO: 84, respectively. In some embodiments, VH and VL include the amino acid sequence described in SEQ ID NO: 85 and SEQ ID NO: 86, respectively.In some embodiments, VH and VL include the amino acid sequence described in SEQ ID NO: 87 and SEQ ID NO: 88, respectively. In some embodiments, VH and VL include the amino acid sequence described in SEQ ID NO: 89 and SEQ ID NO: 90, respectively. In some embodiments, VH and VL include the amino acid sequence described in SEQ ID NO: 91 and SEQ ID NO: 92, respectively. In some embodiments, VH and VL include the amino acid sequence described in SEQ ID NO: 93 and SEQ ID NO: 94, respectively. In some embodiments, VH and VL include the amino acid sequence described in SEQ ID NO: 17 and SEQ ID NO: 22, respectively. In some embodiments, the heavy chain and light chain include the amino acid sequence described in SEQ ID NO: 45 and SEQ ID NO: 46, respectively.

[0169] In some embodiments, the present disclosure relates to an antibody-drug conjugate (ADC) having formula (II): [ka] The invention provides a pharmaceutically acceptable salt, stereoisomer, or solvate thereof, wherein n is in the range of 4 to 8; o is an integer in the range of 10 to 30; and AB is an anti-CEACAM5 antibody or its antigen-binding moiety, comprising VH and VL, respectively, which include the amino acid sequences described in SEQ ID NO: 67 and SEQ ID NO: 68.

[0170] In some embodiments, the present disclosure relates to an antibody-drug conjugate (ADC) having formula (II): [ka] The present invention provides a pharmaceutically acceptable salt, stereoisomer, or solvate thereof, wherein n is in the range of 4 to 8; o is an integer in the range of 10 to 30; and AB are anti-CEACAM5 antibodies or antigen-binding moieties thereof, comprising VH and VL, respectively, which include the amino acid sequences described in SEQ ID NO: 69 and SEQ ID NO: 70.

[0171] In some embodiments, the present disclosure relates to an antibody-drug conjugate (ADC) having formula (II): [ka] The present invention provides a pharmaceutically acceptable salt, stereoisomer, or solvate thereof, wherein n is in the range of 4 to 8; o is an integer in the range of 10 to 30; and AB is an anti-CEACAM5 antibody or its antigen-binding moiety, comprising VH and VL, respectively, which include the amino acid sequences described in SEQ ID NO: 71 and SEQ ID NO: 72.

[0172] In some embodiments, the present disclosure relates to an antibody-drug conjugate (ADC) having formula (II): [ka] The present invention provides a pharmaceutically acceptable salt, stereoisomer, or solvate thereof, wherein n is in the range of 4 to 8; o is an integer in the range of 10 to 30; and AB is an anti-CEACAM5 antibody or its antigen-binding moiety, comprising VH and VL, respectively, which include the amino acid sequences described in SEQ ID NO: 73 and SEQ ID NO: 74.

[0173] In some embodiments, the present disclosure relates to an antibody-drug conjugate (ADC) having formula (II): [ka] The present invention provides a pharmaceutically acceptable salt, stereoisomer, or solvate thereof, wherein n is an integer in the range of 4 to 8; o is an integer in the range of 10 to 30; and AB are anti-CEACAM5 antibodies or antigen-binding moieties thereof, comprising VH and VL, respectively, which include the amino acid sequences described in SEQ ID NO: 75 and SEQ ID NO: 76.

[0174] In some embodiments, the present disclosure relates to an antibody-drug conjugate (ADC) having formula (II): [ka] The present invention provides a pharmaceutically acceptable salt, stereoisomer, or solvate thereof, wherein n is an integer in the range of 4 to 8; o is an integer in the range of 10 to 30; and AB are anti-CEACAM5 antibodies or antigen-binding moieties thereof, comprising VH and VL, respectively, which include the amino acid sequences described in SEQ ID NO: 77 and SEQ ID NO: 78.

[0175] In some embodiments, the present disclosure relates to an antibody-drug conjugate (ADC) having formula (II): [ka] The present invention provides a pharmaceutically acceptable salt, stereoisomer, or solvate thereof, wherein n is in the range of 4 to 8; o is an integer in the range of 10 to 30; and AB is an anti-CEACAM5 antibody or its antigen-binding moiety, comprising VH and VL, respectively, which include the amino acid sequences described in SEQ ID NO: 79 and SEQ ID NO: 80.

[0176] In some embodiments, the present disclosure relates to an antibody-drug conjugate (ADC) having formula (II): [ka] The present invention provides a pharmaceutically acceptable salt, stereoisomer, or solvate thereof, wherein n is in the range of 4 to 8; o is an integer in the range of 10 to 30; and AB is an anti-CEACAM5 antibody or its antigen-binding moiety, comprising VH and VL, respectively, which include the amino acid sequences described in SEQ ID NO: 81 and SEQ ID NO: 82.

[0177] In some embodiments, the present disclosure relates to an antibody-drug conjugate (ADC) having formula (II): [ka] The present invention provides a pharmaceutically acceptable salt, stereoisomer, or solvate thereof, wherein n is in the range of 4 to 8; o is an integer in the range of 10 to 30; and AB is an anti-CEACAM5 antibody or its antigen-binding moiety, comprising VH and VL, respectively, which include the amino acid sequences described in SEQ ID NO: 83 and SEQ ID NO: 84.

[0178] In some embodiments, the present disclosure relates to an antibody-drug conjugate (ADC) having formula (II): [ka] The present invention provides a pharmaceutically acceptable salt, stereoisomer, or solvate thereof, wherein n is in the range of 4 to 8; o is an integer in the range of 10 to 30; and AB is an anti-CEACAM5 antibody or its antigen-binding moiety, comprising VH and VL, respectively, which include the amino acid sequences described in SEQ ID NO: 85 and SEQ ID NO: 86.

[0179] In some embodiments, the present disclosure relates to an antibody-drug conjugate (ADC) having formula (II): [ka] The present invention provides a pharmaceutically acceptable salt, stereoisomer, or solvate thereof, wherein n is in the range of 4 to 8; o is an integer in the range of 10 to 30; and AB is an anti-CEACAM5 antibody or its antigen-binding moiety, comprising VH and VL, respectively, which include the amino acid sequences described in SEQ ID NO: 87 and SEQ ID NO: 88.

[0180] In some embodiments, the present disclosure relates to an antibody-drug conjugate (ADC) having formula (II): [ka] The present invention provides a pharmaceutically acceptable salt, stereoisomer, or solvate thereof, wherein n is in the range of 4 to 8; o is an integer in the range of 10 to 30; and A and B are anti-CEACAM5 antibodies or antigen-binding moieties thereof, comprising VH and VL, respectively, which include the amino acid sequences described in SEQ ID NO: 89 and SEQ ID NO: 90.

[0181] In some embodiments, the present disclosure relates to an antibody-drug conjugate (ADC) having formula (II): [ka] The present invention provides a pharmaceutically acceptable salt, stereoisomer, or solvate thereof, wherein n is in the range of 4 to 8; o is an integer in the range of 10 to 30; and AB is an anti-CEACAM5 antibody or its antigen-binding moiety, comprising VH and VL, respectively, which include the amino acid sequences described in SEQ ID NO: 91 and SEQ ID NO: 92.

[0182] In some embodiments, the present disclosure relates to an antibody-drug conjugate (ADC) having formula (II): [ka] The present invention provides a pharmaceutically acceptable salt, stereoisomer, or solvate thereof, wherein n is in the range of 4 to 8; o is an integer in the range of 10 to 30; and AB is an anti-CEACAM5 antibody or its antigen-binding moiety, comprising VH and VL, respectively, which include the amino acid sequences described in SEQ ID NO: 93 and SEQ ID NO: 94.

[0183] In some embodiments, the present disclosure relates to an antibody-drug conjugate (ADC) having formula (II): [ka] The present invention provides a pharmaceutically acceptable salt, stereoisomer, or solvate thereof, wherein n is in the range of 4 to 8; o is an integer in the range of 10 to 30; and AB are anti-CEACAM5 antibodies or antigen-binding moieties thereof, comprising VH and VL, respectively, which include the amino acid sequences described in SEQ ID NO: 17 and SEQ ID NO: 22.

[0184] In some embodiments, n is 4, 5, 6, 7, 8, or 9. In some embodiments, n is 6. In some embodiments, n is 7. In some embodiments, n is 8. In some embodiments, n is 9.

[0185] In some embodiments, this disclosure relates to an antibody-drug conjugate (ADC) comprising formula (II): [ka] The present invention provides a pharmaceutically acceptable salt, stereoisomer, or solvate thereof, wherein n is in the range of 4 to 8; o is an integer in the range of 10 to 30; and AB are an anti-CEACAM5 antibody and its antigen-binding moiety, comprising a heavy chain and a light chain, respectively, containing the amino acid sequences described in SEQ ID NO: 45 and SEQ ID NO: 46.

[0186] ADCs can also be used to modify a given biological response, where the cytotoxic moiety should not be interpreted as being limited to classical chemotherapeutic agents. For example, the cytotoxic moiety may be a protein or polypeptide having the desired biological activity (e.g., lymphokine, tumor necrosis factor, IFNγ, growth factor).

[0187] Techniques for conjugating toxins or therapeutic portions into antibodies are publicly known, for example, Arnon et al., “Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy”, in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp.243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery”, in Controlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp.623-53 (Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review”, in Monoclonal Antibodies '84: Biological And Clinical Applications, Pinchera et al. (eds.), pp.475-506 (1985); “Analysis, Results, And Future Prospective Of The See "Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy," in Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.), pp.303-16 (Academic Press 1985), and Thorpe et al., "The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates," Immunol. Rev., 62:119-58 (1982).

[0188] IA. Anti-CEACAM5 antibody and antigen-binding moiety Anti-CEACAM5 antibodies useful for ADCs in this disclosure may be defined by specific structural characteristics.

[0189] As used herein, the terms “carcinoembryonic antigen-associated cell adhesion molecule-5” and “CEACAM5” are used synonymously with human CEACAM5 or cynomolgus monkey (Macaca fascicularis) CEACAM5, unless the context clearly indicates otherwise. The human CEACAM5 precursor polypeptide (including the signal peptide) contains the amino acid sequence described in SEQ ID NO: 1 (Genbank: AAH34671.1). The cynomolgus monkey CEACAM5 precursor polypeptide (having the signal peptide) contains the amino acid sequence described in SEQ ID NO: 3 (NCBI: XP_005589491.2). The amino acid sequences and nucleic acid sequences of human and cynomolgus monkey CEACAM5 are disclosed in Table 10.

[0190] The term "CEACAM5" further includes alleles, splice variants, and their processed morphological counterparts derived from other species and other naturally occurring alleles, splice variants, and their processed morphological counterparts, unless the context clearly indicates otherwise.

[0191] antibody sequence In some embodiments, isolated anti-CEACAM5 antibodies (e.g., recombinant humanized antibodies, chimeric antibodies, or human antibodies) or their antigen-binding moieties (e.g., useful for conjugation to the cytotoxic moieties described herein for generating ADCs, and useful for being antibodies in ADCs) are found in Table 10. The anti-CEACAM5 antibody or its antigen-binding moiety binds to and internalizes CEACAM5-expressing cells. Therefore, the anti-CEACAM5 antibody or its antigen-binding moiety is useful in ADCs (including antibodies linked to the cytotoxic moiety or their antigen-binding moiety) by effectively delivering the cytotoxic moiety to kill cells, such as cancer cells.

[0192] Anti-CEACAM5 antibodies useful for ADCs include all known forms of antibodies and other protein scaffolds with antibody-like properties. For example, an antibody may be a monoclonal antibody, a humanized antibody, a human antibody, a bispecific antibody, an immune complex, a chimeric antibody, or a protein scaffold with antibody-like properties such as fibronectin or ankyrin repeats. Antibodies may also be Fab, F(ab')2, scFv, afibody, avimer, nanobody, single-chain antibody, or domain antibody. Antibodies may also have any isotype or allotype, including any of the following isotypes: IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, secretory IgA (SIgA), IgD, IgE, and their allotypes. Full-length antibodies are produced using standard recombinant DNA technology and nucleic acids encoding a desired constant region sequence operably linked to a variable region sequence. H and V L It can be prepared from a sequence.

[0193] In some embodiments, an anti-CEACAM5 antibody useful for ADCs or its antigen-binding moiety includes a VH containing CDR1, CDR2, and CDR3 regions having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with the amino acid sequences described in SEQ ID NOs. 14, 15, and 16, respectively, and a VL containing CDR1, CDR2, and CDR3 regions having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with the amino acid sequences described in SEQ ID NOs. 19, 20, and 21, respectively. In some embodiments, an anti-CEACAM5 antibody or its antigen-binding moiety that specifically binds to CEACAM5 includes a VH containing CDR1, CDR2, and CDR3 regions containing the amino acid sequences described in SEQ ID NOs. 14, 15, and 16, respectively, and a VL containing CDR1, CDR2, and CDR3 regions containing the amino acid sequences described in SEQ ID NOs. 19, 20, and 21, respectively.

[0194] In some embodiments, an anti-CEACAM5 antibody useful for ADCs or its antigen-binding moiety includes a VH containing CDR1, CDR2, and CDR3 regions having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequences described in SEQ ID NOs. 14, 15, and 16, respectively, and a VL containing CDR1, CDR2, and CDR3 regions having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence described in SEQ ID NOs. 38. In some embodiments, an anti-CEACAM5 antibody useful for ADCs or its antigen-binding moiety includes a VH containing CDR1, CDR2, and CDR3 regions having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequences described in SEQ ID NOs. 14, 15, and 16, respectively, and a VL containing CDR1, CDR2, and CDR3 regions having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence described in SEQ ID NOs. 17.In some embodiments, the anti-CEACAM5 antibody useful for ADC or its antigen-binding moiety includes a VH comprising CDR1, CDR2, and CDR3 regions having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequences described in SEQ ID NOs. 14, 15, and 16, respectively, and at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% VL includes CDR1, CDR2, and CDR3 regions having % or 100% sequence identity, and VH includes an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence described in SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91, or SEQ ID NO: 93.

[0195] In some embodiments, an anti-CEACAM5 antibody useful for ADCs or its antigen-binding moiety includes a VH containing CDR1, CDR2, and CDR3 regions having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequences described in SEQ ID NOs. 14, 15, and 16, respectively, and a VL containing CDR1, CDR2, and CDR3 regions having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence described in SEQ ID NOs. 43. In some embodiments, an anti-CEACAM5 antibody useful for ADCs or its antigen-binding moiety includes a VH containing CDR1, CDR2, and CDR3 regions having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequences described in SEQ ID NOs. 14, 15, and 16, respectively, and a VL containing CDR1, CDR2, and CDR3 regions having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence described in SEQ ID NOs. 22.In some embodiments, the anti-CEACAM5 antibody useful for ADC or its antigen-binding moiety includes a VH comprising CDR1, CDR2, and CDR3 regions having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequences described in SEQ ID NOs. 14, 15, and 16, respectively, and at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% VL includes CDR1, CDR2, and CDR3 regions having % or 100% sequence identity, and VH includes an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence described in SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92, or SEQ ID NO: 94.

[0196] In some embodiments, the anti-CEACAM5 antibody useful for ADCs or its antigen-binding moiety includes VH and VL containing the amino acid sequences described in SEQ ID NO: 38 and SEQ ID NO: 43, respectively. In some embodiments, the anti-CEACAM5 antibody useful for ADCs or its antigen-binding moiety includes VH and VL containing the amino acid sequences described in SEQ ID NO: 49 and SEQ ID NO: 50, respectively. In some embodiments, the anti-CEACAM5 antibody useful for ADCs or its antigen-binding moiety includes VH and VL containing the amino acid sequences described in SEQ ID NO: 51 and SEQ ID NO: 52, respectively. In some embodiments, the anti-CEACAM5 antibody useful for ADCs or its antigen-binding moiety includes VH and VL containing the amino acid sequences described in SEQ ID NO: 67 and SEQ ID NO: 68, respectively. In some embodiments, the anti-CEACAM5 antibody useful for ADCs or its antigen-binding moiety includes VH and VL containing the amino acid sequences described in SEQ ID NO: 69 and SEQ ID NO: 70, respectively. In some embodiments, the anti-CEACAM5 antibody useful for ADCs or its antigen-binding moiety includes VH and VL containing the amino acid sequences described in SEQ ID NO: 71 and SEQ ID NO: 72, respectively. In some embodiments, the anti-CEACAM5 antibody useful for ADCs or its antigen-binding moiety includes VH and VL containing the amino acid sequences described in SEQ ID NO: 73 and SEQ ID NO: 74, respectively. In some embodiments, the anti-CEACAM5 antibody useful for ADCs or its antigen-binding moiety includes VH and VL containing the amino acid sequences described in SEQ ID NO: 75 and SEQ ID NO: 76, respectively. In some embodiments, the anti-CEACAM5 antibody useful for ADCs or its antigen-binding moiety includes VH and VL containing the amino acid sequences described in SEQ ID NO: 77 and SEQ ID NO: 78, respectively. In some embodiments, the anti-CEACAM5 antibody useful for ADCs or its antigen-binding moiety includes VH and VL containing the amino acid sequences described in SEQ ID NO: 79 and SEQ ID NO: 80, respectively.In some embodiments, the anti-CEACAM5 antibody or its antigen-binding moiety useful for ADCs includes VH and VL containing the amino acid sequences described in SEQ ID NO: 81 and SEQ ID NO: 82, respectively. In some embodiments, the anti-CEACAM5 antibody or its antigen-binding moiety useful for ADCs includes VH and VL containing the amino acid sequences described in SEQ ID NO: 83 and SEQ ID NO: 84, respectively. In some embodiments, the anti-CEACAM5 antibody or its antigen-binding moiety useful for ADCs includes VH and VL containing the amino acid sequences described in SEQ ID NO: 85 and SEQ ID NO: 86, respectively. In some embodiments, the anti-CEACAM5 antibody or its antigen-binding moiety useful for ADCs includes VH and VL containing the amino acid sequences described in SEQ ID NO: 87 and SEQ ID NO: 88, respectively. In some embodiments, the anti-CEACAM5 antibody or its antigen-binding moiety useful for ADCs includes VH and VL containing the amino acid sequences described in SEQ ID NO: 89 and SEQ ID NO: 90, respectively. In some embodiments, the anti-CEACAM5 antibody useful for ADCs or its antigen-binding moiety includes VH and VL containing the amino acid sequences described in SEQ ID NO: 91 and SEQ ID NO: 92, respectively. In some embodiments, the anti-CEACAM5 antibody useful for ADCs or its antigen-binding moiety includes VH and VL containing the amino acid sequences described in SEQ ID NO: 93 and SEQ ID NO: 94, respectively. In some embodiments, the anti-CEACAM5 antibody useful for ADCs or its antigen-binding moiety includes VH and VL containing the amino acid sequences described in SEQ ID NO: 17 and SEQ ID NO: 22, respectively. In some embodiments, the anti-CEACAM5 antibody includes a heavy chain and a light chain containing the amino acid sequences described in SEQ ID NO: 45 and SEQ ID NO: 46, respectively.

[0197] In some embodiments, an anti-CEACAM5 antibody, or its antigen-binding moiety, which is useful for conjugating to a cytotoxic moiety for the production of ADCs, has at least one amino acid mutation as described in Table 11.

[0198] In some embodiments, the anti-CEACAM5 antibody or its antigen-binding moiety useful for ADCs is 1 × 10 -6 Less than M K D It specifically binds to CEACAM5. In some embodiments, the anti-CEACAM5 antibody or its antigen-binding moiety useful for ADCs is 1 × 10⁻⁶ -7 Less than M K D It specifically binds to CEACAM5. In some embodiments, the anti-CEACAM5 antibody or its antigen-binding moiety useful for ADCs is 1 × 10⁻⁶ -8 Less than M K D It specifically binds to CEACAM5. In some embodiments, the anti-CEACAM5 antibody or its antigen-binding moiety useful for ADCs is 5 × 10 -9 Less than M K D It specifically binds to CEACAM5. In some embodiments, the anti-CEACAM5 antibody or its antigen-binding moiety useful for ADCs is 1 × 10⁻⁶ -9 Less than M K D It specifically binds to CEACAM5. In some embodiments, the anti-CEACAM5 antibody or its antigen-binding moiety useful for ADCs is 5 × 10 -10 Less than M K D It specifically binds to CEACAM5.

[0199] antibody binding An anti-CEACAM5 antibody or its antigen-binding moiety (e.g., useful for being an antigen-binding moiety in an ADC) binds to CEACAM5 in solution (e.g., human CEACAM5), CEACAM5 bound to a solid surface such as a microtiter plate, and / or CEACAM5 immobilized on a cell membrane (e.g., human CEACAM5). In some embodiments, an anti-CEACAM5 antibody or its antigen-binding moiety (e.g., useful for conjugation to a cytotoxic moiety described herein for generating an ADC) binds to human CEACAM5, cynomolgus monkey CEACAM5, or both.

[0200] In some embodiments, the anti-CEACAM5 antibody or its antigen-binding moiety (useful for being, for example, the antigen-binding moiety in an ADC) is measured, for example in Examples 3 and 4, by any detection method known in the art or described herein, with a K content of 100 nM or less, e.g., 90 nM or less, 80 nM or less, 70 nM or less, 60 nM or less, 50 nM or less, e.g., 40 nM or less, 30 nM or less, 20 nM or less, 10 nM or less, 5 nM or less, 3 nM or less, 1 nM or less, 0.9 nM or less, 0.8 nM or less, 0.7 nM or less, 0.6 nM or less, 0.5 nM or less, 0.4 nM or less, 0.3 nM or less, 0.2 nM or less, 0.1 nM or less. D It binds to human and / or cynomolgus monkey CEACAM5.

[0201] In some embodiments, the anti-CEACAM5 antibody or its antigen-binding moiety (e.g., useful for being the antigen-binding moiety in ADCs) is measured in human and / or cynomolgus monkey CEACAM5 by any detection method known in the art or described herein, with a range of 0.1 nM to 100 nM, 0.1 nM to 50 nM, 0.1 nM to 25 nM, 0.1 nM to 10 nM, 0.1 nM to 5 nM, and 0.1 nM K of ~2nM, 0.1nM~1nM, 0.1nM~0.5nM, 1nM~100nM, 1nM~50nM, 1nM~25nM, 1nM~10nM, 1nM~5nM, 1nM~2nM, 5nM~100nM, 5nM~50nM, 5nM~25nM, 5nM~10nM, 10nM~100nM, 10nM~50nM, 10nM~25nM, 25nM~100nM, 25nM~50nM, or 50nM~100nM D They are joined together.

[0202] The binding (and absence of binding) of an anti-CEACAM5 antibody or its antigen-binding moiety, which is useful as the antigen-binding moiety in ADCs, can be evaluated qualitatively or quantitatively by any method known in the art. Exemplary binding methods include immunohistochemistry, e.g., flow cytometry using CEACAM5-overexpressing cells (e.g., MKN-45 or HCT116-CEACAM5), e.g., surface plasmon resonance (SPR) using the BIACORE® system (Cytiva), or e.g., biolayer interferometry (BLI) using the Octet platform (ForteBio).

[0203] In some embodiments, the CEACAM5 antibody or its antigen-binding moiety (useful for being an antigen-binding moiety in ADCs) does not bind to or cross-react with other carcinoembryonic antigens (CEAs) such as CEACAM1, CEACAM3, CEACAM4, CEACAM5, CEACAM6, CEACAM7, CEACAM8, CEACAM16, CEACAM18, CEACAM19, CEACAM20, and / or CEACAM21 when evaluated, for example, by flow cytometry using cells overexpressing one of the aforementioned CEAs, or by SPR or BLI. For example, in some embodiments, the anti-CEACAM5 antibody or its antigen-binding moiety useful for ADCs binds to one of the aforementioned CEAs with a signal or affinity that does not significantly exceed the signal observed with a control antibody (e.g., an isotype control) or the signal observed in the absence of the anti-CEACAM5 antibody.

[0204] In some embodiments, an anti-CEACAM5 antibody or its antigen-binding moiety (including the CEACAM5 antibody or antigen-binding moiety and the cytotoxic moiety) useful as an antigen-binding moiety in an ADC binds to all or some of the amino acids of human CEACAM5 (SEQ ID NO: 1), as described in Example 16. In some embodiments, an anti-CEACAM5 antibody or its antigen-binding moiety useful for an ADC binds to all or some of the amino acids of human CEACAM5 (SEQ ID NO: 1), as described in Example 17.

[0205] In some embodiments, an anti-CEACAM5 antibody or its antigen-binding moiety useful for ADCs binds to (or is determined to bind to) CEACAM5-overexpressing cancer cell lines or tumor cells. In some embodiments, an anti-CEACAM5 antibody or its antigen-binding moiety useful for ADCs binds to CEACAM5 on these cells when evaluated, for example, by flow cytometry. For example, in some embodiments, at least 5%, at least 10%, at least 20%, at least 50%, at least 75%, or at least 90% of CEACAM5-expressing cells can be detected by binding to an anti-CEACAM5 antibody by any detection method known in the art or described herein (e.g., showing a signal greater than that seen with an isotype control antibody).

[0206] In some embodiments, an anti-CEACAM5 antibody useful for ADCs or its antigen-binding moiety (including the CEACAM5 antibody or antigen-binding moiety and the cytotoxic moiety) is measured by any detection method known in the art or described herein with an EC of 1000 ng / ml or less, 500 ng / ml or less, 200 ng / ml or less, 150 ng / ml or less, 100 ng / ml or less, 50 ng / ml or less, 25 ng / ml or less, 10 ng / ml or less, 5 ng / ml or less, 2 ng / ml or less, or 1 ng / ml or less. 50 It then binds to CEACAM5 expressed in cells (for example, human and / or cynomolgus monkey CEACAM5 expressed in MKN-45 cells).

[0207] In some embodiments, an anti-CEACAM5 antibody or its antigen-binding moiety (including the CEACAM5 antibody or antigen-binding moiety and the cytotoxic moiety) useful for ADCs is measured by any detection method known in the art or described herein, with a range of approximately 1 ng / ml to approximately 1000 ng / ml, approximately 1 ng / ml to approximately 500 ng / ml, approximately 1 ng / ml to approximately 200 ng / ml, approximately 1 ng / ml to approximately 100 ng / ml, approximately 1 ng / ml to approximately 50 ng / ml, approximately 1 ng / ml to approximately 25 ng / ml, approximately 1 ng / ml to approximately 10 ng / ml, approximately 1 ng / ml to approximately 5 ng / ml, approximately 5 ng / ml to approximately 500 ng / ml, approximately 5 ng / ml to approximately 200 ng / ml, approximately 5 ng / ml to approximately 100 ng / ml, and approximately 5 ng / ml to approximately 500 ng / ml. EC of approximately 50 ng / ml, approximately 5 ng / ml to approximately 25 ng / ml, approximately 5 ng / ml to approximately 10 ng / ml, approximately 10 ng / ml to approximately 500 ng / ml, approximately 10 ng / ml to approximately 200 ng / ml, approximately 10 ng / ml to approximately 100 ng / ml, approximately 10 ng / ml to approximately 50 ng / ml, approximately 10 ng / ml to approximately 25 ng / ml, approximately 25 ng / ml to approximately 500 ng / ml, approximately 25 ng / ml to approximately 200 ng / ml, approximately 25 ng / ml to approximately 100 ng / ml, approximately 50 ng / ml to approximately 500 ng / ml, approximately 50 ng / ml to approximately 200 ng / ml, approximately 50 ng / ml to approximately 100 ng / ml, approximately 100 ng / ml to approximately 500 ng / ml, or approximately 100 ng / ml to approximately 200 ng / ml 50 It then binds to CEACAM5, which is expressed in cells.

[0208] The binding of anti-CEACAM5 antibodies or their antigen-binding moieties (including the CEACAM5 antibody or antigen-binding moiety and cytotoxic moiety) to CEACAM5, which is useful for ADCs as described herein, may also be defined using quantitative immunofluorescence by flow cytometry, which makes it possible to quantify the number of antibody molecules bound per cell or the number of CEACAM5-expressing cells. In some embodiments, the number of CEACAM5 molecules expressed per cell or the number of CEACAM5-expressing cells in a cell line or tumor sample may be quantified by quantitative immunofluorescence using anti-CEACAM5 antibodies or fragments thereof as described herein.

[0209] The anti-CEACAM5 antibodies or their antigen-binding moieties (including the CEACAM5 antibody or antigen-binding moiety and the cytotoxic moiety) described herein, which are useful for ADCs, have high affinity for soluble or membrane-bound human and / or cynomolgus monkey CEACAM5, and when measured, for example, by surface plasmon resonance or other methods recognized in the art, for example, 10 -7 M or less, 10 -8 M or less, 10 -9 M or less, 10 -10 M or less, 10 -11 M or less, 10 -12M The following 10 -12 M~10 -7 M, 10 -11 M~10 -7 M, 10 -10 M~10 -7 M, or 10 -9 M~10 -7 M's K D They are joined together.

[0210] In some embodiments, the anti-CEACAM5 antibody or its antigen-binding moiety (including the CEACAM5 antibody or antigen-binding moiety and the cytotoxic moiety) described herein and useful for ADCs is soluble or membrane-bound human and / or cynomolgus monkey CEACAM5, when measured by, for example, surface plasmon resonance or other methods recognized in the art, 10 -7 M~10 -12 M, 10 -7 M~10 -11 M, 10 -7 M~10 -10 M, 10 -7 M~10 -9 M, 10 -7 M~10 -8 M, 10 -8 M~10 -12 M, 10 -8 M~10 -11 M, 10 -8 M~10 -11 M, 10 -8 M~10 -9 M, 10 -9 M~10 -12 M, 10 -9M~10 -11 M, 10 -9 M~10 -10 M, 10 -10 M~10 -12 M, 10 -10 M~10 -11 M, or 10 -11 M~10 -12 K D They are joined together.

[0211] Competitive antibodies and antibodies that bind to the same epitope Anti-CEACAM5 antibodies and antigen-binding moieties (e.g., useful as antigen-binding moieties in ADCs) described herein are distinguished by the characteristic epitopes (i.e., sites on CEACAM5) to which they bind (e.g., Examples 16 and 17). The epitopes to which an antibody or fragment binds may be determined using methods accepted in the art. An anti-CEACAM5 antibody or its antigen-binding moiety useful for an ADC is considered to bind to the same epitope as a reference anti-CEACAM5 antibody (e.g., MBN001) if it contacts, for example, one or more of the same residues on human CEACAM5 as a reference antibody, or all of the same residues in all of the same region of human CEACAM5 as the reference antibody.

[0212] Antibodies that share common epitope binding properties may be considered to belong to a common “epitope bin.” In some cases, a CEACAM5-binding “test antibody” may be determined to belong to a common “epitope bin” by comparison with the sequence of a given “reference” antibody (e.g., MBN001) known to belong to a particular epitope bin. In other cases, an epitope binning experiment may be performed to determine whether a test antibody belongs to the same “bin” as the antibody, based on its common binding properties with the reference antibody. For example, an antibody that reduces the binding of the antibodies disclosed herein to immobilized CEACAM5 protein or protein fragments, particularly by its sequence at nearly stoichiometric concentrations, is likely to bind to the same, overlapping, or adjacent epitopes and therefore may share desired functional properties with one or more of the antibodies disclosed herein.

[0213] In some embodiments, the antibody entering the same epitope bin is determined by assaying for an antibody that competes with a specific anti-CEACAM5 antibody described herein for binding to CEACAM5. Methods for determining antibody competition are known in the art.

[0214] In some embodiments, BIACORE analysis can be used to assess the competitive ability of an antibody. The ability of a test antibody to inhibit the binding of an anti-CEACAM5 antibody useful for ADCs to CEACAM5 demonstrates that the test antibody may compete with the antibody for binding to CEACAM5.

[0215] Inhibition or blockade of one antibody against another can be performed by conducting any suitable competitive inhibition experiment using methods accepted in the art or described herein, including but not limited to surface plasmon resonance (SPR) using the BIACORE® system (Cytiva), biolayer interferometry (BLI) using the Octet platform (ForteBio), enzyme-linked immunoassays (ELISA), and flow cytometry. In some embodiments, epitope binning of anti-CEACAM5 antibodies useful for ADCs can be performed using recombinant CEACAM5 protein or fragments, which are biotinylated and captured, for example, on a streptavidin biosensor, which are then bound with the first antibody until saturation is reached. In some embodiments, epitope binning can be performed using a cell-based competitive binding FACS assay.

[0216] Unless otherwise specified, an antibody is considered to compete with an anti-CEACAM5 antibody if, when used at approximately the same molar concentration as the selected antibody in a competitive ELISA experiment, as outlined in the three paragraphs above, it reduces the binding of the selected antibody to human CEACAM5 (SEQ ID NO: 1), cynomolgus monkey CEACAM5 (SEQ ID NO: 2), or its fragments by at least 20%.

[0217] In some embodiments, an anti-CEACAM5 antibody useful for ADCs or its antigen-binding moiety binds to a linear epitope. In some embodiments, an anti-CEACAM5 antibody useful for ADCs or its antigen-binding moiety binds to a three-dimensional epitope.

[0218] In some embodiments, anti-CEACAM5 antibodies useful for ADCs are screened for high affinity binding to human CEACAM5, and antibodies selected therefrom are investigated using, for example, yeast display assays in which sequence variants of CEACAM5 are presented on the surface of yeast cells, MS-based protein footprints such as HDX-MS and fast photochemical oxidation (FPOP) of proteins, as well as structural methods such as X-ray crystallography and molecular modeling, and nuclear magnetic resonance (NMR) spectroscopy, including NMR determination of the HD exchange rate of unstable amide hydrogens in CEACAM5, both in the free state and when bound in complex with the antibody of interest. Such methods can provide atomic resolution of the precise epitope bound by the antibody. In recent years, SP-cryo-EM has emerged as a complementary technique to crystallography and NMR for determining near-atomic structures suitable for drug discovery applications (Renaud et al. Nat Rev Drug Discov 2018;17:471-92; Scapin et al. Cell Chem Biol 2018; 25:1318-25; Ceska et al. Biochemical Society Transactions 2019:p.BST20180267).

[0219] Anti-CEACAM5 antibodies that bind to and compete for the same or similar epitopes as those disclosed herein may be prepared, for example, using an immunization protocol similar to that described herein in Example 1. In some embodiments, immunization may be performed using a construct containing the epitope to which the anti-CEACAM5 antibodies disclosed herein bind. The resulting antibodies may be screened for high affinity binding to human CEACAM5 by FACS, ELISA, or SPR, and / or for their ability to block the binding of the reference antibodies disclosed herein as determined by ELISA, or by blocking their ability to bind to cells expressing CEACAM5 on their surfaces, for example, by FACS or SPR. The test antibodies may be brought into contact with CEACAM5 protein, protein fragments, or CEACAM5-expressing cells before, simultaneously with, or after the addition of the reference antibody.

[0220] Alternatively, anti-CEACAM5 antibodies useful for ADCs, or variants of their antigen-binding moieties, can be obtained by mutagenesis of the cDNA sequences encoding the heavy and light chains of the antibody.

[0221] Antibody internalization In another embodiment, an anti-CEACAM5 antibody or its antigen-binding moiety useful for ADCs binds to human and / or cynomolgus monkey CEACAM5 and induces internalization of the anti-CEACAM5 antibody or its antigen-binding moiety, for example, according to the conditions and results described in the Examples. In some embodiments, the ADC (including an anti-CEACAM5 antibody, or its antigen-binding moiety linked to a cytotoxic moiety as described herein) binds to CEACAM5-expressing cells and is thereby internalized.

[0222] Identifying endogenous anti-CEACAM5 antibodies or their antigen-binding moieties useful for ADCs is crucial for the development of effective ADCs. When determining these ADC-useful anti-CEACAM5 antibodies or their antigen-binding moieties by any method known in the art, including but not limited to the use of the IncuCyte live cell analysis system, Amnis IMAGESTREAM® imaging flow cytometry, or laser scanning confocal microscopy, their ability to endogenize in cells may be evaluated.

[0223] Internalized anti-CEACAM5 antibodies or their antigen-binding moieties useful for ADCs can be characterized or ranked in terms of their “degree of internalization” or “level of internalization,” which may relate to the degree (e.g., percentage of cells) or level (total amount of internalized antibody) of internalization at a given antibody concentration (e.g., 100 nM) or after a given period (e.g., 2 minutes, 5 minutes, 10 minutes, or 30 minutes) compared to non-internalized antibodies, control antibodies such as control IgG (e.g., MBN001), or other control antibodies (e.g., benchmark antibodies).

[0224] In some embodiments, an anti-CEACAM5 antibody or its antigen-binding moiety useful for ADCs is internalized in at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of CEACAM5-expressing cells in a cell population. In some embodiments, an anti-CEACAM5 antibody or its antigen-binding moiety useful for ADCs is internalized in CEACAM5-expressing cells or a population of CEACAM5-expressing cells at levels at least 2-fold, at least 5-fold, at least 20-fold, at least 100-fold, at least 500-fold, or at least 2,000-fold higher than a control antibody (e.g., a non-internalized antibody, control IgG, another antibody, or benchmark antibody).

[0225] In some embodiments, the level of internalization of an anti-CEACAM5 antibody or its antigen-binding moiety into CEACAM5-expressing cells (e.g., MKN45 or HCT-116-hu / cyno CEACAM5) is determined by comparing the area under the time curve (AUC) immunofluorescence level with that of a reference antibody, as described, for example, in Example 6. In some embodiments, an anti-CEACAM5 antibody or its antigen-binding moiety useful for ADCs is internalized into CEACAM5-expressing cells or a population of CEACAM5-expressing cells at an antibody / cell concentration that yields an AUC immunofluorescence level at least 50%, at least 75%, at least 2x, at least 3x, at least 5x, at least 10x, at least 25x, at least 50x, or at least 100x higher than that of a control antibody described herein. In some embodiments, an anti-CEACAM5 antibody or its antigen-binding moiety useful for ADCs is internalized into CEACAM5-expressing cells according to the conditions and results described, for example, in Example 6 and Table 9.

[0226] In some embodiments, an anti-CEACAM5 antibody useful for ADCs, or its antigen-binding moiety, is defined as the point at which half of its maximum internalization is achieved, measured from the time the antibody is added to the cell. 1 / 2 It can be characterized by its "internalization rate," which is expressed by [the following]. In some embodiments, anti-CEACAM5 antibodies useful for ADCs, or the internalization rate of their antigen-binding moiety 1 / 2 Compared to the control antibody described herein, it may be enhanced or increased by at least 10%, 30%, 50%, 75%, 2x, 3x, 5x or more, and the T may be at least 10%, 30%, 50%, 75%, 2x, 3x, 5x or more. 1 / 2 This leads to a decrease in T over a 10-minute period. 1 / 2 Instead of having, anti-CEACAM5 antibodies or their antigen-binding moieties useful for ADCs show an increased rate of internalization, thereby T 1 / 2 This can be reduced to 5 minutes (i.e., a doubling of the internalization rate or T 1 / 2 (A decrease of 2 times). In some embodiments, T 1 / 2 It will be reduced by at least 10 minutes, 30 minutes, or 1 hour.

[0227] In some embodiments, an anti-CEACAM5 antibody useful for ADCs, or its antigen-binding moiety, may be characterized by its maximum level of internalization into CEACAM5-expressing cells or a population of CEACAM5-expressing cells, where the maximum level of internalization is represented by the level of internalization at a plateau in a graph representing internalization plotted against antibody concentration or time. In some embodiments, an anti-CEACAM5 antibody useful for ADCs, or its antigen-binding moiety, exhibits a maximum level of internalization that is at least 10%, 30%, 50%, 75%, 2x, 3x, 5x, or more compared to a control antibody described herein.

[0228] Another method for comparing the internalization effect of anti-CEACAM5 antibodies useful for ADCs, or their antigen-binding moieties, is to compare the level of internalization at a given antibody concentration (e.g., 100 nM) and / or a given time (e.g., 2 minutes, 5 minutes, 10 minutes, or 30 minutes).

[0229] In some embodiments, anti-CEACAM5 antibodies useful for ADCs, or their antigen-binding moieties, can be characterized by their level of internalization, the ability to internalize, which can be determined, for example, using area under the time curve (AUC) immunofluorescence analysis, which represents the antibody concentration at which 50% of the maximum level of internalization is obtained when measured from the time the antibody is added to the cells, as described in Example 6.

[0230] In some embodiments, the anti-CEACAM5 antibody or its antigen-binding moiety useful for ADCs exhibits an EC50 binding value of less than 50 nM, less than 40 nM, less than 30 nM, less than 25 nM, less than 20 nM, less than 15 nM, less than 10 nM, less than 8 nM, less than 6 nM, less than 4 nM, or less than 3 nM. In some embodiments, anti-CEACAM5 antibodies or their antigen-binding moieties useful for ADCs exhibit EC50 internalization values ​​of 1nM-50nM, 4nM-50nM, 10nM-50nM, 20nM-50nM, 30nM-50nM, 4nM-40nM, 4nM-30nM, 4nM-20nM, 8nM-40nM, 8nM-30nM, 8nM-20nM, 12nM-40nM, 12nm and 30nM, or 12nM-25nM.

[0231] In some embodiments, the binding level of an anti-CEACAM5 antibody or its antigen-binding moiety useful for ADCs may be defined in comparison to the binding level of a given control antibody as described herein, and may be expressed as a percentage of the resulting EC50 value compared to the control antibody. In some embodiments, the degree of binding reflected in the EC50 value may be enhanced by at least 10%, 30%, 50%, 75%, 2x, 3x, 5x, or more compared to the control antibody.

[0232] In some embodiments, an anti-CEACAM5 antibody or its antigen-binding moiety useful for ADCs includes a modified constant region that confers increased internalization compared to an anti-CEACAM5 antibody or its antigen-binding moiety without a modified constant region, or compared to a control antibody described herein. Modified constant regions for use in these embodiments are described in U.S. Patent No. 10,653,791, the entirety of which is incorporated herein by reference. For example, in some embodiments, an anti-CEACAM5 antibody or its antigen-binding moiety useful for ADCs includes an IgG2 hinge, or substitution of a non-IgG2 hinge with an IgG2 hinge. In some embodiments, an anti-CEACAM5 antibody or its antigen-binding moiety useful for ADCs includes a CH1 domain that is not a hinge and / or an IgG2 hinge, and / or the IgG2 CH1 domain is substituted with an IgG2 hinge and / or an IgG2 CH1 domain.

[0233] In some embodiments, the anti-CEACAM5 antibody useful for this ADC contains lysine at the C-terminus of the heavy chain. In some embodiments, the anti-CEACAM5 antibody useful for this ADC does not contain lysine at the C-terminus of the heavy chain. In some embodiments, the anti-CEACAM5 antibody useful for this disclosure is a composition comprising a mixture of at least two antibodies (one without C-terminal lysine and the other with C-terminal lysine).

[0234] In some embodiments, an anti-CEACAM5 antibody or its antigen-binding moiety useful for ADCs, including a modified constant region, exhibits an internalization rate (T) that increases by at least 10%, 30%, 50%, 75%, 2-fold, 3-fold, 5-fold, or more compared to the same anti-CEACAM5 antibody or its antigen-binding moiety without a modified constant region, or compared to a control antibody described herein. 1 / 2 (When measured by) has at least 10%, 30%, 50%, 75%, 2x, 3x, 5x or more T 1 / 2 This leads to a decrease.

[0235] Physical properties of antibodies Each antibody or its antigen-binding moiety has a unique isoelectric point (pI), which generally falls within the pH range of 6–9.5. The pI of IgG1 antibodies typically falls within the pH range of 7–9.5, while the pI of IgG4 antibodies typically falls within the pH range of 6–8. Furthermore, each antibody or its antigen-binding moiety has a characteristic melting temperature, with higher melting temperatures indicating greater overall stability in vivo (Krishnamurthy R and Manning MC (2002) Curr Pharm Biotechnol 3:361-71). Generally, T M1The initial unfolding temperature may be greater than 60°C, greater than 65°C, or greater than 70°C. The melting point of the antibody or fragment may be measured using differential scanning calorimetry (Chen et al (2003) Pharm Res 20:1952-60; Ghirlando et al (1999) Immunol Lett 68:47-52) or circular dichroism (Murray et al. (2002) J. Chromatogr Sci 40:343-9). In a further embodiment, antibodies and their antigen-binding moieties that do not degrade rapidly are selected. The degradation of the antibody or its antigen-binding moiety may be measured using capillary electrophoresis (CE) and MALDI-MS (Alexander AJ and Hughes DE (1995) Anal Chem 67:3626-32).

[0236] In some embodiments, anti-CEACAM5 antibodies or their antigen-binding moieties useful as ADCs have minimal agglutination, otherwise leading to undesirable immune responses and / or altered or undesirable pharmacokinetic properties. Generally, antibodies and their antigen-binding moieties having agglutination rates of 25% or less, 20% or less, 15% or less, 10% or less, or 5% or less are acceptable. Agglutination can be measured by several techniques, including size exclusion columns (SEC), high-performance liquid chromatography (HPLC), and light scattering.

[0237] IB. Bispecific or multispecific molecules In some embodiments, ADCs conjugated by formula (I) comprising a bispecific or multispecific molecule (e.g., a bispecific antibody or a multispecific antibody) are provided herein. In some embodiments, a bispecific molecule useful for an ADC comprises at least one binding region (e.g., an antibody or its antigen-binding portion) for a specific epitope on CEACAM5 (e.g., human CEACAM5) as described herein, and at least one other binding region that binds to another antigen. In some embodiments, a multispecific molecule useful for an ADC comprises an antibody or its antigen-binding portion disclosed herein, and at least two binding regions, each that binds to another antigen. The bispecific and / or multispecific molecule may be prepared as a full-length antibody or antibody-binding portion (e.g., an F(ab')2 antibody).

[0238] Methods for producing bispecific or multispecific molecules are known in the art (see, for example, PCT Publication Nos. 05117973 and 06091209). For example, the production of full-length bispecific or multispecific molecules, such as antibodies, can be based on the co-expression of two paired immunoglobulin heavy-light chains, where two or more chains have different specificities. Various techniques for directly producing and isolating bispecific or multispecific molecules from recombinant cell cultures are also known. For example, bispecific or multispecific molecules can be produced using leucine zippers. Another method for producing bispecific or multispecific molecules using single-stranded Fv(sFv) dimers has also been reported.

[0239] Examples of suitable bispecific or multispecific molecular platforms include, but are not limited to, bispecifically targeted (DT)-Ig (GSK / Domantis), two-in-one antibodies (Genentech), cross-linked Mab (Karmanos Cancer Center), Fcab, and mAb. 2(F-Star), CovX-body (CovX / Pfizer), dual variable domain (DVD)-Ig (Abbott), IgG-like bispecificity (ImClone / Eli Lilly), Ts2Ab (Medlmmune / AZ) and BsAb (Zymogenetics), HERCULES (Biogen Idee), TvAb (Roche), ScFv / Fc fusion, SCORPION (Emergent BioSolutions / Trubion, Zymogenetics / BMS), biaffinity retargeting technology (Fc-DART) (MacroGenics), Dual (ScFv)2-Fab (National Research Center for Antibody Medicine - China), F(ab)2 (Medarex / AMGEN), dual action or bis-Fab (Genentech), Dock-and-Lock (DNL) (ImmunoMedics), bivalent bispecificity (Biotecnol), SEED (EMD Serono), mAb 2 Examples include (F-star), Fab-Fv (UCB-Celltech), bispecific T cell engager (BiTE) (Micromet), tandem diabody (Tandab) (Affimed), biaffinity retargeting technology (DART) (MacroGenics), single-stranded diabody (Academic), TCR-like antibody (AIT, ReceptorLogics), COMBODY (Epigen Biotech), bitargeted nanobody (Ablynx), and Fc-modified IgGl (Xencor).

[0240] In some embodiments, a bispecific molecule useful for ADCs includes a first binding region (e.g., an antibody or its antigen-binding portion) that binds to CEACAM5 derivatized or linked to another functional molecule, e.g., another peptide or protein (e.g., another antibody or ligand against a receptor), to generate a bispecific molecule that binds to CEACAM5 and non-CEACAM5 target molecules. In some embodiments, a multispecific molecule includes a first binding region (e.g., an antibody or its antigen-binding portion) that binds to CEACAM5 derivatized or linked to two or more functional molecules, e.g., different peptides or proteins (e.g., other antibodies or ligands against receptors), to generate a multispecific molecule that binds to CEACAM5 and two or more non-CEACAM5 target molecules. The antibody or its antigen-binding portion may be derivatized or linked to two or more other functional molecules to generate a bispecific or multispecific molecule that binds to two or more different binding sites and / or target molecules. To produce bispecific or multispecific molecules, the antibodies or antigen-binding moieties disclosed herein may be functionally linked to one or more other binding molecules, such as another antibody, antibody fragment, peptide, receptor, or binding mimetic (e.g., by chemical coupling, gene fusion, non-covalent association, or otherwise), thereby yielding multispecific molecules.

[0241] Therefore, bispecific molecules, such as bispecific antibodies and bifunctional antibodies, are envisioned, comprising at least a first binding specificity to a specific epitope on CEACAM5 (e.g., human CEACAM5) and a second binding specificity to a second target. In some embodiments, multispecific molecules, such as multispecific antibodies and multifunctional antibodies, are envisioned, comprising at least a first binding specificity to a specific epitope on CEACAM5 (e.g., human CEACAM5), a second binding specificity to a second target, and a third binding specificity to a third target, where the second and third targets are not the same. In some embodiments, the second binding region and / or the third binding region specifically bind to a tumor-associated antigen.

[0242] In some embodiments, the second and / or third binding regions have agonist properties when binding to the target.

[0243] In some embodiments, the antibody is a triplicate antibody comprising first, second, and third binding regions, where the first binding region comprises the binding specificity of the anti-CEACAM5 antibody described herein (e.g., the antigen-binding region), and the second and third binding regions bind to two different targets (or different epitopes on the same target), for example, the targets described herein.

[0244] In some embodiments, the antibody is a bifunctional antibody comprising an anti-CEACAM5 antibody and a receptor molecule as described herein.

[0245] In one embodiment, the multispecific molecule includes, as binding specificity, at least one antibody, or its antigen-binding moiety including, for example, Fab, Fab', F(ab')2, Fv, or a single-chain Fv. The antibody may also be a light-chain or heavy-chain dimer, or any minimum fragment, such as Fv or a single-chain construct, as described in U.S. Patent No. 4,946,778 by Ladner et al.

[0246] In some embodiments, bispecific or multispecific immune cell engagers (ICEs) comprising a CEACAM5 binding domain linked to at least one binding domain of a cell surface protein in an immune effector cell by a short flexible linker region are provided herein. Exemplary immune effector cells include T cells, NK cells, B cells, dendritic cells, and macrophages. Compositions and methods for preparing and using immune cell engagers are disclosed in U.S. Patent Application Publication 2017 / 368169, which is incorporated herein by reference.

[0247] In some embodiments, an immune cell engager is a bispecific (BiTE) or tripspecific (TriKE) T cell engager molecule containing a CEACAM5-binding domain linked by a short flexible linker region to at least one binding domain (i.e., T cell engager domain) of a T cell surface protein in a T cell effector such as a cytotoxic T cell. CEACAM5-targeted BiTE or TriKE is CD8 + This allows CTLs to be brought into close proximity to CEACAM5-expressing tumor cells, resulting in high binding affinity. CD8 + CTLs, like all T cells, express a variable T cell receptor (TCR) associated with an immutable CD3 subunit. In some embodiments, CEACAM5-targeted BiTEs contain a CEACAM5-binding fragment linked to a CD3ε-binding domain, which binds to the CD3ε unit of the TCR complex to form synapses on the surface of tumor cells, directly activating T cells and inducing a cell death signaling pathway involving the subsequent release of granzymes and perforins. By binding to the CD3ε unit, CEACAM-based BiTEs can redirect the entire T cell repertoire in a manner that is not limited by TCR specificity and is potentially independent of TCR-peptide-major histocompatibility complex (MHC), thereby avoiding the possibility of MHC-I downregulation and immune evasion driven by immunotherapy. Advantageously, CEACAM5-targeted BiTEs provide a means for activating depleted T cells induced by long-term exposure to CEACAM5. Examples of T cell engager-binding domains to be included in BiTE or TriKE include CD3, TCRa, TCRp, TCRy, TCRC, ICOS, CD28, CD27, HVEM, LIGHT, CD40, 4-IBB, OX40, DR3, GITR, CD30, TIML, SLAM, CD2, CD226, or combinations thereof.

[0248] In some embodiments, the bispecific T cell engager molecule comprises a CEACAM5 binding domain linked to a checkpoint inhibitor binding domain (bispecific checkpoint inhibitor engager) by a short flexible linker region, such as CTLA-4, PD-1, PD-L1, PD-L2, LAG-3, TIM-3, galectin 9, CEACAM-1, BTLA, CD69, galectin-1, TIGIT, CD113, CD155, GPR56, VISTA, B7-H3, B7-H4, 2B4, CD48, GARP, PD1H, LAIR1, TIM-1, or TIM-4.

[0249] In some embodiments, the immune cell engager is a bispecific or triplicate natural killer (NK) cell engager (NKCE) molecule containing a CEACAM5 binding domain linked to at least one binding domain of an NK cell surface protein (i.e., an NK cell engager binding domain) by a short flexible linker region. In some embodiments, NKCE includes an antigen-binding domain or ligand that binds to (e.g., activates) CD16 (e.g., CD16a, CD16b, or both), NKp46, NKp30, NKp40, NKp44, NKp46, NKG2D, DNAM1, DAP10, CD16 (e.g., CD16a, CD16b, or both), CRTAM, CD27, PSGL1, CD96, CD100 (SEMA4D), NKp80, CD244 (also known as SLAMF4 or 2B4), SLAMF6, SLAMF7, KIR2DS2, KIR2DS4, KIR3DS1, KIR2DS3, KIR2DS5, KIR2DS1, CD94, NKG2C, NKG2E, CD160, or combinations thereof.

[0250] Bispecific or multispecific molecules can be prepared by conjugating constituent binding specificities, such as anti-FcR and anti-CEACAM5 binding specificities, using methods known in the art. For example, each binding specificity of a multispecific molecule can be generated separately and then conjugated with each other. When the binding specificity is a protein or peptide, various coupling agents or crosslinking agents can be used for covalent conjugation. Examples of crosslinking agents include protein A, carbodiimide, N-succinimidyl-S-acetyl-thioacetate (SATA), 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), o-phenylenedimaleimide (oPDM), N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), and sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC). In some embodiments, the conjugation agents are SATA and sulfo-SMCC, both of which are available from Pierce Chemical Co. (Rockford, IL).

[0251] When the binding specificity is that of an antibody, they can be conjugated via sulfhydryl linkages in the C-terminal hinge regions of two heavy chains. In some embodiments, the hinge region is modified to contain an odd number of sulfhydryl residues, e.g., one, before conjugation.

[0252] Alternatively, both binding specificities may be encoded in the same vector and expressed and assembled in the same host cell. This method is particularly useful when the multispecific molecule is an mAb×mAb, mAb×Fab, Fab×F(ab')2, or ligand×Fab fusion protein. A bispecific or multispecific molecule may be a single-chain molecule containing one single-chain antibody and a binding determinant, or a single-chain bispecific or multispecific molecule containing two binding determinants. A bispecific or multispecific molecule may contain at least two single-chain molecules. Methods for preparing bispecific or multispecific molecules are described, for example, in U.S. Patent Nos. 5,260,203; 5,455,030; 4,881,175; 5,132,405; 5,091,513; 5,476,786; 5,013,653; 5,258,498; and 5,482,858.

[0253] The binding of bispecific or multispecific molecules to their specific targets can be confirmed, for example, by enzyme-linked immunosorbent assays (ELISA), radioimmunoassays (RIA), FACS analysis, bioassays (e.g., growth inhibition), or simple Western blotting assays. Each of these assays generally detects the presence of a specific target protein-antibody complex by using a labeling reagent (e.g., antibody) specific to the complex of interest. For example, an FcR-antibody complex can be detected, for example, using an enzyme-linked antibody or antigen-binding moiety that recognizes and specifically binds to the antibody-FcR complex. Alternatively, the complex can be detected using any of various other immunoassays. For example, the antibody can be radiolabeled and used in a radioimmunoassay (RIA). Radioisotopes can be detected by means such as the use of an α-γ-β counter or scintillation counter, or by autoradiography.

[0254] IIC. Antibody manipulation ADCs or their components disclosed herein (e.g., anti-CEACAM5 antibodies or their antigen-binding moieties) may be modified or manipulated to improve their physical and functional properties.

[0255] Antibody manipulation of the Fc region ADCs or their components described herein (e.g., anti-CEACAM5 antibodies or their antigen-binding moieties) may typically involve modifications to their respective Fc regions to alter one or more of their physical or functional properties, such as effector function (e.g., antigen-dependent cytotoxicity), Fc receptor binding, serum half-life, and complement binding. Furthermore, ADCs or their components (e.g., anti-CEACAM5 antibodies or their antigen-binding moieties) may be chemically modified (e.g., one or more chemical moieties may bind to the antibody) or modified to alter their glycosylation, thereby again altering one or more properties of the antibody or fragment. In relation to Fc region modification, the numbering of residues within the Fc region is the numbering of the Kabat EU index.

[0256] The ADCs or components thereof disclosed herein (e.g., anti-CEACAM5 antibodies or their antigen-binding moieties) also include antibodies and fragments having Fc regions modified (or blocked) to provide modification of effector function as described, for example, in U.S. Patent No. 5,624,821; U.S. Patent Application Publication No. 2009 / 280114 and U.S. Patent Application Publication No. 2011 / 142858; and PCT Publication No. International Publication No. 2006 / 0057702. Such modifications may further include modifications to enhance or suppress various immune system responses, which may have beneficial effects in diagnosis and therapy.

[0257] Modification of effect pedal functions In some embodiments, an ADC or its components (e.g., an anti-CEACAM5 antibody or its antigen-binding moiety) includes a mutant Fc region modified from the parent Fc sequence (e.g., an unmodified Fc polypeptide that is later modified to generate a mutant) such that the antibody or its antigen-binding moiety increases or decreases its ability to mediate one or more effector functions and / or increases or decreases its binding to the Fc gamma receptor (FcγR). Thus, in exemplary embodiments, an ADC or its components (e.g., an anti-CEACAM5 antibody or its antigen-binding moiety) may include one or more amino acid changes that alter its affinity for an effector ligand such as the Fc receptor or the C1 component of complement. This technique is described in further detail in U.S. Patent Nos. 5,624,821 and 5,648,260.

[0258] The interaction between the constant region of antigen-binding proteins (such as anti-CEACAM5 antibodies or their antigen-binding moieties) and various Fc receptors (FcRs), including FcγRI(CD64), FcγRII(CD32), and FcγRIII(CD16), is thought to mediate effector functions of antigen-binding proteins, such as ADCC and CDC. Fc receptors are also important for antibody crosslinking, which may be important for antitumor immunity. In exemplary embodiments, modifications may be made in the Fc region to generate Fc variants that promote (a) increased or decreased antibody-dependent cell-mediated cytotoxicity (ADCC), (b) increased or decreased complement-mediated cytotoxicity (CDC), (c) increased or decreased affinity for C1q, (d) increased or decreased affinity for the Fc receptor relative to the parent Fc, and / or (e) increased or decreased pharmacokinetic stability.

[0259] Modification of the Fc region may include amino acid changes, such as substitution, deletion, insertion, glycosylation, deglycosylation, and / or addition of multiple Fc regions. Combining amino acid modifications may be particularly desirable. For example, a mutant Fc region may include, for example, two, three, four, five, or more substitutions within a specific Fc region location as specified herein. In some embodiments, the Fc region is modified by substituting at least one amino acid residue with a different amino acid residue so that the antibody has a modified affinity for the effector ligand but retains the antigen-binding ability of the parent antibody. For example, one or more amino acids selected from amino acid residues 234, 235, 236, 237, 297, 318, 320, and 322 may be substituted with a different amino acid residue. The effector ligand whose affinity is modified may be, for example, the Fc receptor or the C1 component of complement. This technique is described in further detail in both U.S. Patent Nos. 5,624,821 and 5,648,260 by Winter et al. In some embodiments, the C1q binding site can be removed from the Fc region, for example, by deleting or substituting the EKK sequence of human IgG1. In another example, one or more amino acids selected from amino acid residues 329, 331, and 322 can be substituted with different amino acid residues so that the antibody modifies C1q binding and / or reduces or eliminates complement-dependent cell-mediated cytotoxicity (CDC). This technique is described in more detail in U.S. Patent No. 6,194,551 by Idusogie et al. In yet another example, one or more amino acid residues within amino acid positions 231 and 239 are modified to alter the antibody's ability to bind to complement. This technique is described in more detail in U.S. Patent No. 6,180,377.

[0260] In some embodiments, for example, a form of anti-CEACAM5 antibody or its antigen-binding moiety lacking effector function is provided herein, having the mutant hIgG1f allotype (hIgG1.3f) described herein and containing, for example, the amino acid sequence described in SEQ ID NO: 30. The hIgG1.3f mutant is a triple mutant form of hIgG1f (L234A, L235E, G237A) lacking FcγR binding and effector function.

[0261] In some embodiments, ADCs or their components (e.g., anti-CEACAM5 antibodies or their antigen-binding moieties) can be engineered to have different affinities and selectivity for the Fc gamma receptor (FcγR) by mutating the heavy chain constant region, including the hinge and Fc domain. Mutations may be introduced to enhance or reduce FcγR binding. These mutations can increase or decrease FcγR-mediated crosslinking and / or signaling. With respect to therapeutic targets such as CEACAM5, FcγR-mediated crosslinking of anti-CEACAM5 antibodies has the potential to provide undesirable agonist signaling and toxicity if specific modifications are not introduced to circumvent this problem.

[0262] The binding sites for FcγR1, FcγRII, FcγRIII, and FcRn on human IgG1 have been mapped, and mutants with improved binding have been described (see Shields, R. Let al. (2001) J. Biol. Chem. 276:6591-6604). Specific mutations at positions 256, 290, 298, 333, 334, and 339 have been shown to improve binding to FcγRIII. Furthermore, the following combination mutants have been shown to improve FcγRIII binding: T256A / S298A, S298A / E333A, S298A / K224A, and S298A / E333A / K334A, which have been shown to enhance FcγRIIIa binding and ADCC activity (Shields et al., 2001). Other IgG1 mutants with potently enhanced binding to FcγRIIIa have been identified, including mutants with the S239D / I332E and S239D / I332E / A330L mutations, which showed the greatest increase in affinity for FcγRIIIa, decreased FcγRIIb binding, and potent cytotoxic activity in cynomolgus monkeys (Lazar et al., 2006). The introduction of triple mutations into antibodies such as alemtuzumab (CD52-specific), trastuzumab (HER2 / neu-specific), rituximab (CD20-specific), and cetuximab (EGFR-specific) resulted in highly enhanced ADCC activity in vitro, and the S239D / I332E mutant showed enhanced ability to deplete B cells in monkeys (Lazar et al., 2006).

[0263] In some embodiments, ADCs or their components (e.g., anti-CEACAM5 antibodies or their antigen-binding moieties) may be manipulated to reduce FcγR binding and the possibility of crosslinking and / or signal transduction, specifically to reduce binding of “low affinity” FcγRs hCD32a / FcγRIIa, hCD32b / FcγRIIb, hCD16a / FcγRIIa, and hCD16b / FcγRIIIb. Binding of the “high affinity” receptor CD64 / FcγRI is generally considered less of a concern due to the saturation of this receptor by serum IgG. Therefore, in some embodiments, the ADC or its components (e.g., an anti-CEACAM5 antibody or its antigen-binding portion) may include an IgG1.3 Fc region which essentially lacks binding to CD16, CD32a, CD32b, and CD64 and lacks ADCC, ADCP, and CDC functions (see U.S. Patent No. 10,077,306 and U.S. Patent Application Publication No. 2022 / 0106400).

[0264] In some embodiments, the Fc region may be manipulated to increase ADCC and / or FcγR binding by modifying one or more amino acids at the following positions: 234, 235, 236, 238, 239, 240, 241, 243, 244, 245, 247, 248, 249, 252, 254, 255, 256, 258, 262, 263, 264, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294, 295, 296, 298, 299, 30 The numbering of residues within the Fc region, 1, 303, 305, 307, 309, 312, 313, 315, 320, 322, 324, 325, 326, 327, 329, 330, 331, 332, 333, 334, 335, 337, 338, 340, 360, 373, 376, 378, 382, ​​388, 389, 398, 414, 416, 419, 430, 433, 434, 435, 436, 437, 438, or 439 (for example, as described in U.S. Patent No. 6,737,056), is the EU index numbering as described in Kabat. Exemplary substitutions include 236A, 239D, 239E, 268D, 267E, 268E, 268F, 324T, 332D, and 332E. Exemplary mutants include 239D / 332E, 236A / 332E, 236A / 239D / 332E, 268F / 324T, 267E / 268F, 267E / 324T, and 267E / 268F / 324T. Other modifications to enhance FcyR and complement interactions include, but are not limited to, substitutions of 298A, 333A, 334A, 326A, 247I, 339D, 339Q, 280H, 290S, 298D, 298V, 243L, 292P, 300L, 396L, 305I, and 396L. These and other modifications are outlined in Strohl, 2009, Current Opinion in Biotechnology 20:685-691.

[0265] In some embodiments, the Fc region is modified to reduce the ability of the anti-CEACAM5 antibody or its antigen-binding moiety described herein to mediate effector function, and / or to increase anti-inflammatory properties by modifying residues 243 and 264. In one embodiment, the Fc region of the ADC or its components (e.g., the anti-CEACAM5 antibody or its antigen-binding moiety) is modified by changing the residues at positions 243 and 264 to alanine. In another embodiment, the Fc region is modified to reduce the ability of the ADC or its components (e.g., the anti-CEACAM5 antibody or its antigen-binding moiety) to mediate effector function, and / or to increase anti-inflammatory properties by modifying residues 243, 264, 267 and 328.

[0266] Other Fc modifications to the Fc region include those that reduce or eliminate binding to FcγR and / or complement proteins, thereby reducing or eliminating the function of Fc-mediated effectors such as ADCC, ADCP, and CDC. Modifications to alter binding to FcyRllb include one or more substitutions, insertions, and deletions at positions 234, 235, 236, 237, 239, 266, 267, 268, 269, 325, 326, 327, 328, and 332, numbered according to the EU index. In one embodiment, the Fc variant provides selectively enhanced affinity to FcyRllb compared to one or more activating receptors. Exemplary substitutions include, but are not limited to, 234G, 235G, 236R, 237K, 267R, 269R, 325L, and 328R. Other Fc variants to enhance binding to FcyRllb include 235Y / 267E, 236D / 267E, 236R / 328R, 239D / 268D, 239D / 267E, 267E / 268D, 267E / 268E, and 267E / 328F. Other modifications to reduce FcyR and complement interactions include substitutions 297A, 234A, 235A, 237A, 318A, 228P, 236E, 268Q, 309L, 330S, 331S, 220S, 226S, 229S, 238S, 233P, and 234V, as well as removal of glycosylation at position 297 by mutation, enzymatic means, or production in organisms such as bacteria that do not glycosylate proteins. These and other modifications are outlined in Strohl, 2009, Current Opinion in Biotechnology 20:685-691.

[0267] In certain embodiments, the Fc region may be modified to remove the ADCC site. The ADCC site can be found, for example, in relation to the ADCC site in IgG1 in Molec.Immunol.29(5):633-9(1992). Furthermore, IgG1 mutants containing L235V, F243L, R292P, Y300L, and P396L mutations have been found to enhance binding to FcγRIIIa and simultaneously enhance ADCC activity in transgenic mice expressing human FcγRIIIa in models of B-cell malignancies and breast cancer (Stavenhagen et al.,2007; Nordstrom et al.,2011). Other Fc mutants that may be used include S298A / E333A / L334A, S239D / I332E, S239D / I332E / A330L, L235V / F243L / R292P / Y300L / P396L, and M428L / N434S. Specific examples of mutant Fc domains are disclosed, for example, in U.S. Patent No. 6,096,871 and PCT International Publication No. 97 / 34631.

[0268] Optionally, the Fc region may include amino acid residues that do not naturally exist in additional and / or alternative positions (e.g., U.S. Patent No. 5,624,821; U.S. Patent No. 6,277,375; U.S. Patent No. 6,737,056; U.S. Patent No. 6,194,551; U.S. Patent No. 7,317,091; U.S. Patent No. 8,101,720; PCT Patent Publication No. International Publication No. 00 / 42072; International Publication No. 01 / 58957; International Publication No. 02 / 06919; International See Pamphlet No. 04 / 016750; Pamphlet No. 04 / 029207 (International Publication); Pamphlet No. 04 / 035752 (International Publication); Pamphlet No. 04 / 074455 (International Publication); Pamphlet No. 04 / 099249 (International Publication); Pamphlet No. 04 / 063351 (International Publication); Pamphlet No. 05 / 070963 (International Publication); Pamphlet No. 05 / 040217 (International Publication), Pamphlet No. 05 / 092925 (International Publication), and Pamphlet No. 06 / 020114 (International Publication).

[0269] In one embodiment, the hinge region of Fc is modified such that the number of cysteine ​​residues in the hinge region is altered, for example, increased or decreased. For example, in one embodiment, the number of cysteine ​​residues in the hinge region of CH1 is increased to increase antibody stability, or decreased to enhance the assembly of light and heavy chains, or as described in U.S. Patent No. 5,677,425.

[0270] In some embodiments, alterations to the Fc region may be made to extend the biological half-life of the ADC in order to improve the usability of the material, reduce its use, and decrease the frequency of administration (Presta (2005) J. Allergy Clin. Immunol. 116:731 at 734-35). Various methods may be employed. For example, in certain embodiments, this may be achieved by increasing the binding affinity of the Fc region to the neonatal Fc receptor (FcRn). For example, one or more of the following residues may be mutated: 252, 254, 256, 433, 435, 436, as described in U.S. Patent No. 6,277,375. Specific exemplary substitutions include one or more of the following: T252L, T254S, and / or T256F. Alternatively, to extend the biological half-life, the antibody may be modified within the CH1 or CL region to contain a salvage receptor-binding epitope taken from two loops of the CH2 domain in the Fc region of IgG, as described in U.S. Patent Nos. 5,869,046 and 6,121,022 by Presta et al.

[0271] Other Fc variants for increased binding to FcRn and / or improved pharmacokinetic properties include substitutions at positions 259, 308, 428, and 434, such as 259I, 308F, 428L, 428M, 434S, 434H, 434F, 434Y, and 434M. Other mutants that increase Fc binding to FcRn include 250E, 250Q, 428L, 428F, 250Q / 428L (Hinton et al., 2004, J. Biol. Chem. 279(8):6213-6216, Hinton et al. 2006 Journal of Immunology 176:346-356), 256A, 272A, 286A, 305A, 307A, 307Q, 311A, 312A, 376A, 378Q, 380A, 382A, and 434A (Shields et al., Journal of Biology). Chemistry,2001,276(9):6591-6604), 252F, 252T, 252Y, 252W, 254T, 256S, 256R, 256Q, 256E, 256D, 256T, 309P, 31 1 S, 433R, 433S, 433I, 433P, 433Q, 434H, 434F, 434Y, 252Y / 254T / 256E, 433K / 434F / 436H, 308T / 309P / 311S (Dall'Acqua et al. Journal of Immunology,2002,169:5171-5180,Dall'Acqua et al.,2006,Journal of Biological Chemistry Examples include 281:23514-23524). Other modifications for regulating FcRn binding are described in Yeung et al., 2010, J Immunol, 182:7663-7671.

[0272] In another embodiment, the Fc hinge region may be mutated to reduce the biological half-life of the antibody or fragment. For example, as described in U.S. Patent No. 6,165,745, one or more amino acid mutations may be introduced into the CH2-CH3 domain interface region of the Fc hinge fragment so that the antibody or fragment has impaired staphylococcal protein A (SpA) binding compared to natural Fc hinge domain SpA binding.

[0273] In certain embodiments, hybrid IgG isotypes having specific biological characteristics may be used. For example, in certain embodiments, one or more regions and / or mutations derived from IgG2 or IgG4. In one embodiment, the ADC described herein comprises an IgG4 isotype antibody or fragment containing a serine-to-proline mutation at position 228 (S228P; EU index) in the hinge region of the heavy chain constant domain. This mutation has been reported to eliminate heterogeneity of the inter-heavy chain disulfide crosslinks in the hinge region (see Angal et al. above; position 241 is based on the Kabat numbering system). When using the IgG4 constant domain, it should typically contain the substitution S228P, which mimics the hinge sequence in IgG1 and thereby stabilizes the IgG4 molecule.

[0274] In another embodiment, IgG1 / IgG3 hybrid variants can be constructed by substituting the IgG1 position in the CH2 and / or CH3 region with amino acids derived from IgG3 at different positions for the two isotypes. Thus, hybrid variant IgG antibodies can be constructed containing one or more substitutions, e.g., 274Q, 276K, 300F, 339T, 356E, 358M, 384S, 392N, 397M, 4221, 435R, and 436F. In another embodiment described herein, IgG1 / IgG2 hybrid variants can be constructed by substituting the IgG2 position in the CH2 and / or CH3 region with amino acids derived from IgG1 at different positions for the two isotypes. Thus, hybrid variant IgG antibodies can be constructed containing one or more substitutions, e.g., one or more of the following amino acid substitutions: 233E, 234L, 235L, 236G (referring to the insertion of glycine at position 236), and 327A.

[0275] In some embodiments, the mutant Fc region may also involve sequence modifications in which an amino acid involved in disulfide bond formation is removed or replaced with another amino acid. Such removal may avoid reaction with other cysteine-containing proteins present in the host cell used to produce the antibodies described herein. Even if a cysteine ​​residue is removed, the single-stranded Fc domain may still form a dimeric Fc domain that is held together non-covalently. In other embodiments, the Fc region may be modified to be more compatible with selected host cells. For example, a PA sequence near the N-terminus of a typical native Fc region may be removed, which can be recognized by digestive enzymes in Escherichia coli (E. coli), such as proline iminopeptidase.

[0276] ADCs or their components disclosed herein (e.g., anti-CEACAM5 antibodies or their antigen-binding moieties) may contain one or more glycosylation sites. Such glycosylation sites may result in increased immunogenicity of the antibody or fragment, or a change in the antibody's PK due to altered antigen binding (Marshall et al. (1972) Annu Rev Biochem 41:673-702; Gala and Morrison (2004) J Immunol 172:5489-94; Wallick et al (1988) J Exp Med 168:1099-109; Spiro (2002) Glycobiology 12:43R-56R; Parekh et al (1985) Nature 316:452-7; Mimura et al. (2000) Mol Immunol 37:697-706). Glycosylation is known to occur in motifs containing the NXS / T sequence.

[0277] Accordingly, in some embodiments, the glycosylation properties of the ADCs or their components described herein (e.g., anti-CEACAM5 antibodies or their antigen-binding moieties) can be modified. For example, one or more glycosylation sites within the Fc domain can be modified or removed. Typically, glycosylated residues (e.g., asparagine) can confer a cytolytic response. Such residues can be deleted or substituted with non-glycosylated residues (e.g., alanine) to produce non-glycosylated antibodies. In certain embodiments, glycosylation can be modified, for example, to increase the antibody's affinity for the antigen. For example, one or more amino acid substitutions can be made that result in the exclusion of one or more variable region framework glycosylation sites, thereby eliminating glycosylation at those sites. The resulting non-glycosylation can increase the antibody's affinity for the antigen. Such techniques are described in further detail in U.S. Patent Nos. 5,714,350 and 6,350,861 by Co et al. Glycosylation of the constant region on N297 can be prevented by mutating the N297 residue to another residue, such as N297A, and / or by mutating an adjacent amino acid, such as 298, thereby reducing glycosylation on N297.

[0278] In addition, or alternatively, the ADCs or their components described herein (e.g., anti-CEACAM5 antibodies or their antigen-binding moieties) may be manipulated using modified types of glycosylation, such as low-fucosylated antibodies with a reduced amount of fucosyl residues or antibodies with increased branched GlcNac structures. Non-fucosylated antibodies possess a tri-mannosyl core structure of a complex N-glycan of Fc that lacks fucose residues. These glycosylated antibodies lacking core fucose residues derived from Fc N-glycans may exhibit stronger ADCCs than their fucosylated counterparts due to enhanced FcγRIIIa binding ability. Such carbohydrate modifications may be achieved, for example, by expressing antibodies in host cells with a modified glycosylation mechanism.

[0279] Cells with modified glycosylation mechanisms can be used as host cells to express recombinant antibodies described herein, thereby producing antibodies with modified glycosylation. For example, European Patent No. 1,176,195 by Hanai et al. describes a cell line having a functionally disrupted FUT8 gene encoding fucosyltransferase (i.e., α-1,6-fucosyltransferase), wherein the antibodies expressed in such cell lines exhibit low fucosylation. Recombinant host cells genetically modified to inactivate the FUT8 gene encoding α-1,6-fucosyltransferase are available. For example, see the POTELLIGENT™ technology system available from BioWa, Inc. (Princeton, NJ), in which CHOK1SV cells lacking a functional copy of the FUT8 gene produce a monoclonal antibody with enhanced ADCC activity, increased compared to the same monoclonal antibody produced in cells having a functional FUT8 gene. Appearances of the POTELLIGENT™ technical system are described in U.S. Patent Nos. 7,214,775 and 6,946,292, and in PCT publications International Publication Nos. 00 / 61739 and 02 / 31240.

[0280] Presta's PCT publication number International Publication No. 03 / 035835 describes the Lec13 cell line, a mutant CHO cell line that has a reduced ability to bind fucose to Asn(297)-linked carbohydrates and results in low fucosylation of antibodies expressed in its host cells (see also Shields, R. Let al. (2002) J. Biol. Chem. 277:26733-26740). Umana et al.'s PCT Publication No. 99 / 54342 describes cell lines that have been engineered to express glycoprotein-modified glycosyltransferases (e.g., β(1,4)-N-acetylglucosaminyltransferase III (GnTIII)) such that the antibodies expressed in the engineered cell lines show an increase in the branched GlcNac structure, resulting in increased ADCC activity of the antibodies (see also Umana et al. (1999) Nat. Biotech. 17:176-180).

[0281] Another modification of antibodies described herein is pegylation. In some embodiments, ADCs or their components described herein (e.g., anti-CEACAM5 antibodies or their antigen-binding moieties) are pegylated, for example, to increase the biological (e.g., serum) half-life of the antibody. To pegylate an antibody, the antibody, or a fragment thereof, is typically reacted with polyethylene glycol (PEG), e.g., a reactive ester or aldehyde derivative of PEG, under conditions that one or more PEG groups are bound to the antibody or antibody fragment. In some embodiments, pegylation is carried out via an acylation or alkylation reaction with a reactive PEG molecule (or a similar reactive water-soluble polymer). As used herein, the term “polyethylene glycol” is intended to encompass any form of PEG used to derivatize other proteins, such as mono(C1-C10) alkoxy- or aryloxy-polyethylene glycol or polyethylene glycol-maleimide. In certain embodiments, the antibody being pegylated is a non-glycosylated antibody. Methods for pegylation of proteins may be applied to antibodies or their antigen-binding moieties described herein. For example, see European Patent No. 0154316 by Nishimura et al. and European Patent No. 0401384 by Ishikawa et al.

[0282] Effector function can be measured in several ways, including, for example, by measuring ADCC effector function via the binding of FcγRIII to natural killer cells or via FcγRI to monocytes / macrophages. For example, the antigen-binding protein of the present invention can be evaluated for ADCC effector function in a natural killer cell assay. Examples of such assays can be found in Shields et al., 2001 J. Biol. Chem., Vol. 276, pp. 6591-6604; Chappel et al., 1993 J. Biol. Chem., Vol. 268, pp. 25124-25131; and Lazar et al., 2006 PNAS, 103; 4005-4010.

[0283] The affinity and binding properties of the Fc region to its ligand can be determined by a variety of in vitro assays (biochemical or immunoassays), including but not limited to equilibrium assays (e.g., enzyme-linked immunosorbent assay (ELISA) or radioimmunoassay (RIA)) or dynamics assays (e.g., BIACORE analysis), and other methods such as indirect binding assays, competitive inhibition assays, fluorescence resonance energy transfer (FRET), and chromatography (e.g., gel filtration). These and other methods may utilize labeling on one or more of the components being examined and / or employ a variety of detection methods, including but not limited to chromogenic, fluorescent, luminescent, or isotopic labeling. A detailed description of binding affinity and dynamics can be found in Paul, WE, ed., Fundamental Immunology, 4th Ed., Lippincott-Raven, Philadelphia (1999), which focuses on antibody-immunogen interactions.

[0284] With respect to the modifications described herein for increasing or decreasing one or more of the functional properties described herein (e.g., biochemical, immunochemical, cellular, physiological, or other biological activities that are publicly known in the art and determined using the methods described herein), the resulting increase in a given parameter may represent a statistically significant increase of at least 10% of the measured parameter, e.g., at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% (i.e., 2x), 3x, 5x, or 10x. Conversely, the resulting decrease in a measured parameter may represent a statistically significant decrease of at least 10% of the measured parameter, e.g., at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%, 3x, 5x, or 10x.

[0285] Any of the above modifications may be used alone or in combination with any of the above modifications or any of the modifications described in the following section to further enhance or reduce the effector function or other desirable properties (e.g., stability, expression).

[0286] Antibody manipulation of variable regions In some embodiments, an ADC or its components (e.g., an anti-CEACAM5 antibody or its antigen-binding moiety) is manipulated, for example, by modifying framework residues within the variable domain of the parent antibody to improve the properties of the antibody or its antigen-binding moiety. Typically, such framework modifications are made to reduce the immunogenicity of the anti-CEACAM5 antibody or its antigen-binding moiety. This is usually achieved by substituting non-CDR residues (i.e., framework residues) in the variable domain of the parent (e.g., rodent) antibody with similar residues derived from the immune repertoire of the species in which the antibody is used, e.g., human residues in the case of human therapeutics. Such antibodies are referred to as “humanized” antibodies. In some cases, it is desirable to increase the affinity or modify the specificity of the manipulated (e.g., humanized) antibody. One technique is to “reverse mutagenerate” one or more framework residues into the corresponding germline sequence. More specifically, an antibody that has undergone somatic mutation may contain framework residues different from those in the germline sequence from which the antibody originates. Such residues can be identified by comparing the antibody framework sequence with the germline sequence from which the antibody originates. Another approach is to revert one or more positions in the manipulated (e.g., humanized) antibody back to the original parent (e.g., rodent) residues to restore binding affinity that may have been lost during the process of substituting framework residues. (See, for example, U.S. Patents 5,693,762, 5,585,089, and 5,530,101).

[0287] In certain embodiments, the anti-CEACAM5 antibody in the ADC and its antigen-binding moiety are manipulated (e.g., humanized) to include modifications to the framework and / or CDR to improve their properties. Such manipulated modifications may be based on molecular modeling. Molecular models of the variable region of the parental (non-human) antibody sequence can be constructed to understand the structural features of the antibody and used to identify potential regions on the antibody that may interact with the antigen. Conventional CDRs are based on the alignment of the immunoglobulin sequence and the identification of the variable region. Kabat et al., (1991) Sequences of Proteins of Immunological Interest, Kabat, et al.; National Institutes of Health, Bethesda, Md.; 5th ed.; NIH Publ. No. 91-3242; Kabat (1978) Adv. Prot. Chem. 32:1-75; Kabat, et al., (1977) J. Biol. Chem. 252:6609-6616. Chothia et al. carefully examined the three-dimensional structure of loops in antibody crystal structures and proposed the hypervariable loop. Chothia, et al., (1987) J Mol. Biol. 196:901-917 or Chothia, et al., (1989) Nature 342:878-883. Variation exists between regions classified as "CDR" and "hypervariable loops." Subsequent studies (Raghunathan et al., (2012) J. Mol Recog. 25, 3, 103-113) analyzed several antibody-antigen crystal complexes and observed that the antigen-binding region in antibodies does not necessarily strictly fit into either a "CDR" residue or a "hypervariable" loop. Molecular models of variable regions in non-human antibodies can be used to guide the selection of regions that can potentially bind to antigens. In practice, the potential antigen-binding region based on the model differs from the conventional "CDR" loop or "hypervariable" loop. For molecular modeling, commercially available scientific software such as MOE (Chemical Computing Group) can be used. Human frameworks may be selected based on the best match with non-human sequences in both the framework and the CDR.For FR4 (Framework 4) in VH, the VJ region of the human germline is compared to the corresponding non-human region. For FR4 (Framework 4) in VL, the J-kappa and J-lambda regions of the human germline sequence are compared to the corresponding non-human region. Once a suitable human framework is identified, the CDR is grafted onto the selected human framework. In some cases, certain residues at the VL-VH interface may be retained as they are in the non-human (parent) sequence. Molecular models can also be used to identify residues that may alter the CDR's three-dimensional structure and, consequently, its binding to the antigen. In some cases, these residues are retained as they are in the non-human (parent) sequence. Molecular models can also be used to identify solvent-exposed amino acids that may result in undesirable effects such as glycosylation, deamidation, and oxidation. Developmental feasibility filters may be introduced early in the design phase to eliminate / minimize these potential problems.

[0288] Another type of framework modification involves mutating one or more residues within a framework region, or even within one or more CDR regions, to remove a T cell epitope and thereby reduce the potential immunogenicity of the antibody. This technique is also referred to as “deimmunization” and is described in more detail in U.S. Patent No. 7,125,689. In certain embodiments, immunogenicity may be reduced by modifying or removing one or more glycosylation sites in either the light chain or heavy chain immunoglobulin variable region, such as the framework region. In certain embodiments, it may be desirable to change certain amino acids, including those in the exposed side chain, to other amino acid residues to provide greater chemical stability of the final antibody, in order to avoid deamidation or isomerization. Deamidation of asparagine occurs in NG, DG, NG, NS, NA, NT, QG, or QS sequences, resulting in the generation of isoaspartic acid residues that introduce a kink into the polypeptide chain and reduce its stability (isoaspartic acid effect). Isomerization may occur in DG, DS, DA, or DT sequences. In certain embodiments, the antibodies provided herein do not contain deamide or asparagine isomerization sites. For example, asparagine (Asn) residues may be changed to Gln or Ala to reduce the likelihood of isoaspartic acid formation, particularly at any Asn-Gly sequence within a CDR.

[0289] Similar problems can occur with Asp-Gly sequences. Reissner and Aswad (2003) Cell. Mol. Life Sci. 60:1281. Isoaspartate formation can weaken or completely suppress the binding of an antibody to its target antigen. See Presta (2005) J. Allergy Clin. Immunol. 116:731 at 734.

[0290] In various embodiments, asparagine is converted to glutamine (Gln). It may also be desirable to modify the amino acids adjacent to the asparagine (Asn) or glutamine (Gln) residues to reduce the likelihood of deamide formation, which occurs at a higher rate when small amino acids are present adjacent to asparagine or glutamine. See Bischoff & Kolbe (1994) J. Chromatog. 662:261. Furthermore, to reduce the likelihood of methionine sulfur oxidation, any methionine residue in the CDR (typically Met exposed to the solvent) may be converted to Lys, Leu, Ala, or Phe, or other amino acids, which may reduce antigen-binding affinity and contribute to molecular heterogeneity in the final antibody preparation. Ibid. Additionally, to prevent or minimize potentially easily cleaved Asn-Pro peptide bonds, it may be desirable to convert any Asn-Pro combination found in the CDR to Gln-Pro, Ala-Pro, or Asn-Ala. Next, antibodies with such substitutions are screened to ensure that the substitutions do not reduce the antibody's affinity or specificity for CEACAM5, or other desired biological activity, to an unacceptable level. See Table 1 for exemplary stabilized CDR variants.

[0291] [Table 1]

[0292] Mai. Pharmaceutical composition Pharmaceutical compositions comprising ADCs and carriers (e.g., pharmaceutically acceptable carriers) disclosed herein are also provided herein. Such compositions are useful for a variety of therapeutic applications, such as cancer treatment.

[0293] In some embodiments, the pharmaceutical composition may further comprise other compounds, drugs, and / or agents for various therapeutic uses. Such compounds, drugs, and / or agents may include, for example, anticancer agents, chemotherapeutic agents, immunosuppressants, immunostimulants, immune checkpoint inhibitors, and / or anti-inflammatory agents. Exemplary compounds, drugs, and agents that may be formulated together with or separately from the ADCs described in the following sections.

[0294] As used herein, “pharmaceutically acceptable carrier” includes all physiologically compatible solvents, dispersions, coatings, antimicrobial and antifungal agents, as well as isotonic and absorption retardants. In some embodiments, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, or epidermal administration (e.g., by injection or infusion). Depending on the route of administration, the active compound, i.e., antibody, immunoconjugate, or bispecific molecule, may be coated with a substance to protect it from the action of acids and other natural conditions that may inactivate the compound.

[0295] The pharmaceutical compounds described herein may contain one or more pharmaceutically acceptable salts. A “pharmaceutically acceptable salt” is defined as a salt that retains the desired biological activity of the parent compound and does not impart any undesirable toxicological effects (see, for example, Berge, S M et al. (1977) J. Pharm. Sci. 66:1-19). Examples of such salts include acid addition salts and base addition salts. Examples of acid addition salts include those derived from non-toxic inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, and phosphorus, as well as non-toxic organic acids such as aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanes, hydroxyalkanoates, aromatic acids, and aliphatic and aromatic sulfonic acids. Examples of base addition salts include those derived from alkaline earth metals such as sodium, potassium, magnesium, and calcium, as well as non-toxic organic amines such as N,N'-dibenzylethylenediamine, N-methylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, and procaine.

[0296] The pharmaceutical compositions described herein may also contain pharmaceutically acceptable antioxidants. Examples of pharmaceutically acceptable antioxidants include: (1) water-soluble antioxidants, such as ascorbic acid, cysteine ​​hydrochloride, sodium bicarbonate, sodium metabisulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol and the like; and (3) metal chelating agents, such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid and the like.

[0297] Suitable aqueous and non-aqueous carriers that may be used in the pharmaceutical compositions described herein include water, ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifiers, and dispersants. Prevention of the presence of microorganisms can be ensured by the sterilization procedures described above and by including various antimicrobial and antifungal agents, such as parabens, chlorobutanol, phenolsorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like, in the composition. Furthermore, the inclusion of absorption-delaying agents such as aluminum monostearate and gelatin may result in prolonged absorption of the injectable pharmaceutical form.

[0298] Examples of pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions, and sterile powders for the immediate preparation of sterile injectable solutions or dispersions. Any medium or agent is intended for use in the pharmaceutical compositions described herein, unless it is incompatible with the active compound. The pharmaceutical compositions may or may not contain preservatives. Furthermore, auxiliary active compounds may be incorporated into the compositions.

[0299] The compositions described herein may be administered by one or more different methods via one or more routes of administration. The route and / or method of administration may vary depending on the desired outcome. Routes of administration for ADCs described herein include, for example, injection or infusion, such as intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal, or other parenteral administration routes. As used herein, “parenteral administration” means a mode of administration other than enteral and topical administration, usually by injection, and includes, but is not limited to, intravenous, intramuscular, intra-arterial, intrathecal, intra-articular, intra-orbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subepidermal, intra-articular, sub-articular, subarachnoid, intraspinal, epidural, and intrasternal injections and infusions.

[0300] Alternatively, the ADCs described herein may be administered via parenteral routes, such as topical, dermal, or mucosal routes, such as intranasal, oral, vaginal, rectal, sublingual, or topical.

[0301] IV. Use and Method The ADCs described herein have many in vitro and in vivo benefits as described herein.

[0302] cancer treatment In one embodiment, the Specified Public Service Provider (ADC) provides a method for treating cancer, comprising administering an effective dose to a subject in need such that the growth of a cancerous tumor is inhibited or reduced and / or regression and / or survival is achieved.

[0303] In some embodiments, the ADCs described herein may be administered in combination with additional cytotoxic agents or therapeutic agents, such as those described herein.

[0304] Cancers expressing CEACAM5 whose proliferation can be inhibited using the ADCs described herein include carcinomas, lymphomas, blastomas, sarcomas, and leukemias. More specific examples of such cancers include, but are not limited to, basal cell carcinoma, biliary tract cancer; bladder cancer; osteosarcoma; brain tumors and CNS cancers; breast cancer (e.g., estrogen receptor-positive breast cancer, HER2-positive breast cancer, triple-negative breast cancer); peritoneal cancer; cervical cancer; bile duct cancer; choriocarcinoma; colorectal cancer; connective tissue cancer; gastrointestinal cancer; endometrial cancer; esophageal cancer; eye cancer; head and neck cancer; gastric cancer (including gastrointestinal cancer); glioblastoma; liver cancer (e.g., hepatocellular carcinoma); carcinoma in situ; kidney or renal cancer; laryngeal cancer; leukemia; lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, lung gland cancer) Cancer, and squamous cell carcinoma of the lung; lymphoma, including Hodgkin lymphoma and non-Hodgkin lymphoma; melanoma; myeloma; neuroblastoma; oral cancer (e.g., lip, tongue, oral cavity, and pharynx); ovarian cancer; pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectal cancer; cancer of the respiratory system; salivary gland cancer; sarcoma; skin cancer; squamous cell carcinoma; teratoma; testicular cancer; thyroid cancer; uterine or endometrial cancer; urinary system Cancer; vulvar cancer; and other carcinomas and sarcomas; and B-cell lymphomas (including low-grade / follicular non-Hodgkin lymphoma (NHL); small lymphocytic (SL) NHL; intermediate-grade / follicular NHL; intermediate-grade diffuse NHL; high-grade immunoblastic NHL; high-grade lymphoblastic NHL; high-grade small unsevered cell NHL; bulky lesion NHL; mantle cell lymphoma; AIDS-associated lymphoma; and Waldenström macroglobulinemia); chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); hairy cell leukemia; chronic myeloblastic leukemia; and post-transplant lymphoproliferative disorders (PTLD), as well as nevus, edema (such as that associated with brain tumors), primary tumors, and abnormal angiogenesis associated with Meigs syndrome.

[0305] Further cancers that express CEACAM5 and can be treated with the ADCs described herein include metastatic pancreatic cancer, metastatic adenocarcinoma of the pancreas, gastric cancer, fibrous carcinoma, glioma, malignant glioma, diffuse pontine glioma, recurrent pediatric brain tumor renal cell carcinoma, clear cell metastatic renal cell carcinoma, metastatic castration-resistant prostate cancer, stage IV prostate cancer, metastatic melanoma, malignant melanoma, recurrent cutaneous melanoma, melanoma with brain metastasis, malignant melanoma of the head and neck, squamous non-small cell lung cancer, metastatic breast cancer, follicular lymphoma, advanced B-cell NHL, diffuse large B-cell lymphoma. HL including DLBCL, multiple myeloma, chronic myeloid leukemia, adult acute myeloid leukemia in remission, Inv(16)(p13.1q22), adult acute myeloid leukemia with CBFB-MYH11, t(16:16)(p13.1:q22), adult acute myeloid leukemia with CBFB-MYH11, t(8:21)(d22:q22), adult acute myeloid leukemia with RUNX1-RUNX1T1, t(9:11)(p22:q23), adult acute myeloid leukemia with MLLT3-MLL, tO15:17)(q22:q12) Adult acute promyelocytic leukemia with PML-RARA, alkylating agent-associated acute myeloid leukemia, Richter syndrome, adult glioblastoma, adult gliosarcoma, recurrent glioblastoma, recurrent pediatric rhabdomyosarcoma, recurrent Ewing's sarcoma / peripheral primitive neuroectodermal tumor, recurrent neuroblastoma, recurrent osteosarcoma, colorectal cancer, MSI-positive colorectal cancer, MSI-negative colorectal cancer, nasopharyngeal nonkeratinizing carcinoma, recurrent nasopharyngeal anaplastic carcinoma, cervical adenocarcinoma, cervical adenosquamous cell carcinoma; cervical squamous cell carcinoma, recurrent cervical cancer, anal canal squamous cell carcinoma, metastatic anal canal cancer These include recurrent anal canal cancer, recurrent head and neck cancer, head and neck squamous cell carcinoma, head and neck squamous cell carcinoma (HNSCC), ovarian cancer, colon cancer, advanced GI cancer, gastric adenocarcinoma, gastroesophageal junction adenocarcinoma, bone tumors, soft tissue sarcoma, osteosarcoma, thymic carcinoma, urothelial carcinoma, Merkel cell carcinoma, recurrent Merkel cell carcinoma, mycosis fungoides, Sézary syndrome, neuroendocrine carcinoma, nasopharyngeal carcinoma, basal cell carcinoma, squamous cell carcinoma, dermatofibrosarcoma protuberans, glioma, mesothelioma, myelodysplastic syndrome (MDS), myelofibrosis (MF), myeloproliferative neoplasms, and acute myeloid leukemia (AML).

[0306] In some embodiments, cancer includes colorectal cancer, breast cancer, lung cancer including non-small cell lung cancer (NSCLC), ovarian cancer, pancreatic cancer, bladder cancer, uterine / cervical cancer, prostate cancer, testicular cancer, esophageal cancer, gastric cancer, gastrointestinal cancer, colon cancer, kidney cancer, head and neck cancer, gastric cancer, germ cell cancer, bone cancer, liver cancer, thyroid cancer, skin cancer, central nervous system tumors, lymphoma, leukemia, myeloma, sarcoma, or myelodysplastic syndromes. In some embodiments, cancer treatable with this ADC includes colorectal cancer (CRC), non-small cell lung cancer (NSCLC), or gastric cancer (GC).

[0307] Cancer may be, for example, metastatic or primary cancer; desmoplastic or non-desmoplastic cancer; or recurrent cancer.

[0308] In some aspects, cancer is associated with fibrosis. In some aspects, cancer is associated with the infiltration of CD4+ regulatory T cells. In some aspects, cancer is associated with the infiltration of CD8+ regulatory T cells. In some aspects, cancer is associated with the infiltration of regulatory B cells. In some aspects, cancer is associated with the infiltration of bone marrow-derived suppressor cells. In some aspects, cancer is associated with the infiltration of tumor-associated macrophages. In some aspects, cancer is associated with the infiltration of innate lymphoid cells. In some aspects, cancer is associated with the infiltration of cancer-associated fibroblasts. In some aspects, cancer is associated with increased radiation-associated activity in the above cell types.

[0309] In some embodiments, the ADCs described herein are used to treat myelodysplastic syndromes (MDS). MDS is a diverse group of malignant diseases characterized by bone marrow failure resulting from hematopoietic defects and the production of dysplastic cells. TGF-β is a major driving factor in MDS (Geyh et al., Haematologica 2018;103:1462-71), and agents that inhibit TGF-β function have been proposed as therapeutic agents (Mies et al., Curr Hematol Malig Rep 2016;11:416-24). Furthermore, MDSCs are known to be dysregulated in MDS (Chen et al., JCI 2013;123:4595-611), and agents that reduce MDSC levels in the bone marrow are potential therapeutic agents.

[0310] In some embodiments, the cancer is resistant to checkpoint inhibitors. In some embodiments, the cancer is inherently refractory or resistant (e.g., resistant to PD-1 pathway inhibitors, PD-1 pathway inhibitors, or CTLA-4 pathway inhibitors). In some embodiments, a resistant or refractory state of cancer is acquired. In some embodiments, the ADCs described herein may be used in combination with checkpoint inhibitors to overcome the cancer's resistance to checkpoint inhibitors. In some embodiments, the ADCs described herein may be used in combination with or sequentially with checkpoint inhibitors that induce a mesenchymal phenotype, such as MAPK pathway inhibitors, to treat tumors having a mesenchymal and / or EMT signature.

[0311] In some embodiments, the ADCs described herein are used to enhance the viability of immune cells ex vivo, for example, in adoptive NK cell transfer. Thus, in some embodiments, the ADCs are used in combination with adoptive NK cells to treat cancer. In some embodiments, the ADCs described herein are used to treat tumors with MHC loss or MHC downregulation as monotherapy or in combination with NK activation or therapeutic enhancement.

[0312] Combination therapy The ADCs described herein may be used in combination with various treatments or agents known in the art (or in relation to multispecific antibodies or bifunctional partners) for the treatment of the diseases or conditions described herein.

[0313] In some embodiments, a method of treating cancer involves administering an effective amount of the ADC described herein in combination with another therapeutic agent, such as a second antibody, a therapeutic protein, or a small molecule drug, to a subject in need. In some embodiments, the therapeutic protein includes a checkpoint inhibitor. In some embodiments, the small molecule drug is a chemotherapeutic agent as described herein. In some embodiments, the other therapeutic agent includes an anticancer agent.

[0314] Suitable anticancer agents for use in combination therapies with ADCs as described herein include, but are not limited to, surgical, chemotherapeutic, growth inhibitor, cytotoxic, radiotherapy and agents used in radiotherapy, anti-angiogenic agents, apoptotic agents, antitubulin agents, and other agents that treat cancer, such as anti-HER-2 antibodies (e.g., HERCEPTIN®), anti-CD20 antibodies, epidermal growth factor receptor (EGFR) antagonists (e.g., tyrosine kinase inhibitors), HER1 / EGFR inhibitors (e.g., erlotinib (TARCEVA®), platelet-derived growth factor inhibitors). Harmful agents (e.g., GLEEVEC (imatinib mesylate)), COX-2 inhibitors (e.g., celecoxib), and fetokaines; antagonists (e.g., neutralizing antibodies) that bind to and / or neutralize the activity of one or more of the following targets: PD-L, PD-L1, PD-L2 (e.g., pembrolizumab; nivolumab; MK-3475; AMP-224; MPDL3280A; MEDI0680; MSB0010718C; and / or MEDI4736); CTLA4 (e.g., tremelimumab (PFIZER) and ipilimumab); LAG3 (e.g., BMS-986016); CD 103; TIM-3 and / or other TIM family members; anti-VEGF antibodies (e.g., bevacizumab); CEACAM1, CEACAM6 and / or other CEACAM family members; ErbB2, ErbB3, ErbB4, PDGFR-β, BlyS, APRIL, BCMA or VEGF receptors, TRAIL / Apo2, PARP inhibitors (e.g., AZD-2281, Lynparza OCEACAM5arib, rubracarbaparib; (Zejula) niraparib), DNA damage repair inhibitors (e.g., ATMi, ATRi, DNAPKi), and other bioactive and organic chemical agents, including those described in Section VII. Combinations thereof are also specifically envisioned for the methods described herein.

[0315] In some embodiments, ADCs are administered together with anticancer agents, such as EGFR inhibitors; HER2 inhibitors; histone deacetylase inhibitors; hormones; mitotic inhibitors; phosphatidylinositol-3-kinase (PI3K) inhibitors; Akt inhibitors; mammalian target of rapamycin (mTOR) inhibitors; proteasome inhibitors; poly(ADP-ribose) polymerase (PARP) inhibitors; Ras / MAPK pathway inhibitors; centrosome declustering agents; multikinase inhibitors; serine / threonine kinase inhibitors; tyrosine kinase inhibitors; VEGF / VEGFR inhibitors; microtubule targeting agents; topoisomerase toxins; or combinations thereof.

[0316] In some embodiments, ADCs are administered together with immune checkpoint inhibitors. Examples of immune checkpoint inhibitors include, but are not limited to, drugs (e.g., antibodies) that bind to PD-1, PD-L1, PD-L2, LAG-3, CTLA4, TIGIT, ICOS, OX40, PVR, PVRIG, VISTA, TIM3, SIRPα, ILT2, ILT3, ILT4, or ILT5.

[0317] Any anti-PD-1 antibody may be used in combination with an ADC in the method described herein. Various human monoclonal antibodies that bind specifically to PD-1 with high affinity are disclosed in U.S. Patent No. 8,008,449.

[0318] In some embodiments, the anti-PD-1 antibody is pembrolizumab, nivolumab, semiprimab, spartalizumab, camrelizumab, cintilimab, tislerizumab, tripalimab, dostallimab, retifanlimab, pimivalimab, dostallimab, serpullimab, zinbererimab, acrixolimab, MEDI-0680, AM-0001, STI-1110, AGEN2034, BCD-100, sasanlimab, BI 754091, or SSI-361.

[0319] In some embodiments, the anti-PD-1 antibody used in combination with the ADC comprises CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, CDRL3, VH and VL, and / or heavy and light chains, of any of the following: pembrolizumab, nivolumab, semiprimab, spartalizumab, camlizumab, cintilimab, tislerizumab, tripalimab, dostallimab, retifanlimab, pimivalimab, MEDI-0680, GLS-010, AM-0001, STI-1110, AGEN2034, BCD-100, sasanlimab, BI 754091, or SSI-361.

[0320] In some embodiments, anti-PD-1 antibodies used in combination with ADCs include nivolumab (OPDIVO®; formerly known as 5C4, BMS-936558, MDX-1106, or ONO-4538), pembrolizumab (KEYTRUDA®; formerly known as lambrolizumab and MK-3475; see International Publication No. 2008 / 156712A1), PDR001 (see International Publication No. 2015 / 112900), MEDI-0680 (formerly known as AMP-514; see International Publication No. 2012 / 145493), REGN-2810, see International Publication No. 2015 / 112800), JS001 (Liu and See Wu, 2017), BGB-A317 (see International Publication No. 2015 / 035606 and U.S. Patent Application Publication No. 2015 / 0079109), INCSHR1210 (SHR-1210; see International Publication No. 2015 / 085847; see Liu and Wu, 2017), TSR-042 (ANB011; see International Publication No. 2014 / 179664), GLS-010 (WBP3055; see Liu and The selection is made from the group consisting of Wu, 2017, AM-0001 (see International Publication No. 2017 / 123557), STI-1110 (see International Publication No. 2014 / 194302), AGEN2034 (see International Publication No. 2017 / 040790), and MGD013 (see International Publication No. 2017 / 106061).

[0321] In some embodiments, the anti-PD-1 antibody used in combination with ADCs is pembrolizumab (also known as Merck; KEYTRUDA®, lambrolizumab, and MK-3475; see, for example, International Publication No. 2008 / 156712). Pembrolizumab is a humanized monoclonal IgG4 (S228P) antibody against the human cell surface receptor PD-1 (programmed death-1 or programmed cell death-1). Pembrolizumab is described, for example, in U.S. Patent Nos. 8,354,509 and 8,900,587.

[0322] In some embodiments, the anti-PD-1 antibody used in combination with ADCs includes nivolumab (also known as OPDIVO®, 5C4, BMS-936558, MDX-1106, and ONO-4538). Nivolumab is a fully human IgG4(S228P) PD-1 immune checkpoint inhibitor antibody that selectively prevents interaction with PD-1 ligands (PD-L1 and PD-L2), thereby blocking the downregulation of antitumor T cell function (see, e.g., U.S. Patent No. 8,008,449; Wang et al., 2014 Cancer Immunol Res. 2(9):846-56).

[0323] In some embodiments, the anti-PD-1 antibody used in combination with ADCs is a semiprimab (Regeneron; also known as LIBTAYO or REGN-2810; see, for example, International Publication No. 2015 / 112800 and U.S. Patent No. 9,987,500).

[0324] In some embodiments, the anti-PD-1 antibody used in combination with ADCs is spartalizumab (Novartis; also known as PDR001; see, for example, International Publication No. 2015 / 112900 and U.S. Patent No. 9,683,048).

[0325] In some embodiments, the anti-PD-1 antibody used in combination with ADCs is camrelizumab (Jiangsu Hengrui Medicine; also known as SHR-1210 or INCSHR1210; see, for example, International Publication No. 2015 / 085847; Si-Yang Liu et al., J.Hematol.Oncol.10:136(2017)).

[0326] In some embodiments, the anti-PD-1 antibody used in combination with ADC is MEDI-0680 (AstraZeneca; also known as AMP-514; see, e.g., International Publication No. 2012 / 145493). In some embodiments, the anti-PD-1 antibody is pimivalimab (also known as JTX-4014; see, e.g., Papadopoulos, et al., 2022, IOTECH, Vol.16, Supplement 1, 100284). In some embodiments, the anti-PD-1 antibody is tripalimab (TAIZHOU JUNSHI PHARMA; also known as JS001; see, e.g., Si-Yang Liu et al., J.Hematol.Oncol.10:136(2017)). In some embodiments, the anti-PD-1 antibody is tislerizumab (Beigene; also known as BGB-A317; see, e.g., International Publication No. 2015 / 35606 and U.S. Patent Application Publication No. 2015 / 0079109). In some embodiments, the anti-PD-1 antibody is dostallimab (Tesaro Biopharmaceutical; also known as ANB011 or TSR-042; see, e.g., International Publication No. 2014 / 179664). In some embodiments, the anti-PD-1 antibody is GLS-010 (Wuxi / Harbin Gloria Pharmaceuticals; also known as WBP3055; see, e.g., Si-Yang Liu et al., J.Hematol.Oncol.10:136(2017)). In some embodiments, the anti-PD-1 antibody is AM-0001 (Armo BioSciences).

[0327] In some embodiments, the anti-PD-1 antibody is STI-1110 (Sorrento Therapeutics; see, e.g., International Publication No. 2014 / 194302). In some embodiments, the anti-PD-1 antibody is AGEN2034 (Agenus; see, e.g., International Publication No. 2017 / 040790). In some embodiments, the anti-PD-1 antibody is retifan rimab (also known as Macrogenics, MGA012, AEX-1188, and INCMGA-00012; see, e.g., International Publication No. 2017 / 19846). In some embodiments, the anti-PD-1 antibody is BCD-100 (Biocad; see, e.g., Kaplon et al., mAbs 10(2):183-203 (2018)). In some embodiments, the anti-PD-1 antibody is cintilimab (Innovent; also known as IBI308; see, e.g., International Publication No. 2017 / 024465, International Publication No. 2017 / 025016, International Publication No. 2017 / 132825, and International Publication No. 2017 / 133540). In some embodiments, the anti-PD-1 antibody is sasamrimab (Pfizer; also known as PF-06801591; see, e.g., U.S. Patent Application Publication No. 2016 / 0159905). In some embodiments, the anti-PD-1 antibody is BI 754091 (Boehringer Ingelheim; see, for example, Zettl M et al., Cancer. Res. (2018); 78(13 Suppl): Abstract 4558). In some embodiments, the anti-PD-1 antibody is SSI-361 (see, for example, Lyvgen Biopharma Holdings Limited, U.S. Patent Application Publication No. 2018 / 0346569).

[0328] For other anti-PD-1 monoclonal antibodies suitable for the methods of this disclosure, see, for example, U.S. Patent Nos. 6,808,710, 7,488,802, 8,168,757, 8,354,509 and 9,205,148, U.S. Patent Publication No. 2016 / 0272708, and International Publication Nos. 2012 / 145493 and 2008 / 156712. Pamphlets No. 2015 / 112900, No. 2012 / 145493, No. 2015 / 112800, No. 2014 / 206107, No. 2015 / 35606, No. 2015 / 085847, No. 2014 / 179664, No. 2017 / 020291, No. 2017 / 020858, and No. 201 Pamphlets No. 6 / 197367, No. 2017 / 024515, No. 2017 / 025051, No. 2017 / 123557, No. 2016 / 106159, No. 2014 / 194302, No. 2017 / 040790, No. 2017 / 133540, No. 2017 / 132827, and No. 2017 / 02 This information is provided in Pamphlets No. 4465, 2017 / 025016, 2017 / 106061, 2017 / 19846, 2017 / 024465, 2017 / 025016, 2017 / 132825, and 2017 / 133540 (each of these being incorporated by reference as a whole).

[0329] Examples of anti-PD-L1 antibodies useful in combination with ADCs using the methods of the present disclosure include the antibodies disclosed in U.S. Patent No. 9,580,507. In some embodiments, the anti-PD-L1 antibody is atezolizumab, durvalumab, avelumab, emvafolimab, cosivelimab, BMS-936559, STI-1014, CX-072, LY3300054, FAZ053, CS-1001, SHR-1316, CBT-502, KN035, or BGB-A333.

[0330] In some embodiments, the anti-PD-L1 antibody is BMS-936559 (also known as 12A4, MDX-1105; see, for example, U.S. Patent No. 7,943,743 and International Publication No. 2013 / 173223).

[0331] In some embodiments, the anti-PD-L1 antibody is STI-1014 (Sorrento; see, e.g., International Publication No. 2013 / 181634). STI-104 is designated as H6 in U.S. Patent No. 9,175,082. In some embodiments, the anti-PD-L1 antibody is CX-072 (Cytomx; see, e.g., International Publication No. 2016 / 149201). In some embodiments, the anti-PD-L1 antibody is LY3300054 (Eli Lilly Co.; see, e.g., International Publication No. 2017 / 034916). In some embodiments, the anti-PD-L1 antibody is FAZ053 (Novartis). In some embodiments, the anti-PD-L1 antibody is CK-301 (Checkpoint Therapeutics; see, e.g., Gorelik et al., AACR: Abstract 4606 (Apr 2016)). CK-301 is also known as kosivelimab. In some embodiments, the anti-PD-L1 antibody is CS-1001. See, for example, Zhou et al., Journal of Clinical Oncology, Meeting Abstract, 2020 ASCO Annual Meeting I, Lung Cancer-Non-Small Cell Metastatic, e21687, and Zhang et al., Cancer Research, 2020, 80(16_Supplement):3260. In some embodiments, the anti-PD-L1 antibody is SHR-1316. See, for example, Mu et al., Thorac Cancer, 2021 May; 12(9):1373-1381, and Wu et al., Anals of Oncology, Abstract, Vol.33, Supplement 2, S72, April 2022. In some embodiments, the anti-PD-L1 antibody is CBT-502 (also known as TQB2450; see, for example, Wei et al., Mol Cancer Ther (2018) 17(1_Supplement):A200).In some embodiments, the anti-PD-L1 antibody is KN035 (3D Med / Alphamab; also known as emvafolimab; see, for example, Zhang et al., Cell Discov. 7:3 (March 2017) and Shimizu et al., Invest New Drugs, 2022 Oct; 40(5):1021-1031).

[0332] In some embodiments, the anti-PD-L1 antibody is BGB-A333 (BeiGene; see, e.g., Desai et al., JCO 36(15suppl):TPS3113(2018) and Desai et al., 2023, British Journal of Cancer 128, 1418-1428). In certain embodiments, the PD-L1 antibody is atezolizumab. Atezolizumab is a fully humanized IgG1 monoclonal anti-PD-L1 antibody. Atezolizumab (Roche) is also known as TECENTRIQ®; MPDL3280A, RG7446. See, e.g., U.S. Patent No. 8,217,149 and Herbst et al. (2013) J. Clin. Oncol. 31(suppl):3000). Atezolizumab is designated YW243.55S70 in U.S. Patent No. 8,217,149. In certain embodiments, the PD-L1 antibody is durvalumab. Durvalumab is a human IgG1κ monoclonal anti-PD-L1 antibody. Durvalumab (AstraZeneca) is also known as IMFINZI® or MEDI-4736. Durvalumab is designated 2.14H9OPT in U.S. Patent No. 8,779,108. See, for example, International Publication No. 2011 / 066389. In certain embodiments, the PD-L1 antibody is avelumab. Avelumab is a human IgG1λ monoclonal anti-PD-L1 antibody. Avelumab (Pfizer) is also known as BAVENCIO® or MSB0010718C. Avelumab is designated as A09-246-2 in U.S. Patent No. 9,624,298. See, for example, International Publication No. 2013 / 079174.

[0333] In some embodiments, anti-CTLA-4 antibodies useful in combination with ADCs include tremelimumab, ipilimumab, botensilimab, BMS-986218, BMS-986288, BMS-986249, IBI310, MK-1308 (quavonlimab), AGEN-1884 (zarefrelimab), ONC-392, ADG116, or CS1002.

[0334] In some embodiments, MK-1308 is a useful anti-CTLA-4 antibody in combination with ADCs. MK-1308 is also known as quavonlimab. See, for example, Perets et al. 2021, Ann Oncol 32(3):395-403.

[0335] In some aspects, AGEN-1884 is a useful anti-CTLA-4 antibody in combination with ADCs. AGEN-1884 is also known as zarifremab. See, for example, International Publication No. 2016 / 196237.

[0336] In some aspects, tremelimumab is a useful anti-CTLA-4 antibody in combination with ADCs. Tremelimumab, marketed under the trademark name IMJUDO®, is a fully human monoclonal antibody used for the treatment of hepatocellular carcinoma and non-small cell lung cancer. Tremelimumab (AstraZeneca) is also known as tisilinumumab, CP-675,206; see International Publication No. 2000 / 037504 and Ribas, Update Cancer Ther. 2(3):133-39 (2007).

[0337] In some aspects, ipilimumab is a useful anti-CTLA-4 antibody in combination with ADCs. Ipilimumab (marketed under the trademark YERVOY® and initially approved for the treatment of metastatic melanoma) has since been approved for use in other cancers. Hoos et al. (2010) Semin. Oncol. 37:533; Hodi et al. (2010) N. Engl. J. Med. 363:711; Pardoll (2012) Nat. Immunol. 13(12):1129. In 2011, ipilimumab, a human antibody with a constant IgG1 region, was approved in the US and EU for the treatment of unresectable or metastatic melanoma based on improved overall survival in a Phase III trial in previously treated patients with advanced melanoma. Hodi et al. (2010) N. Engl. J. Med. 363:711. Tumor regression and disease stabilization were frequently observed. Ipilimumab is also known as MDX-010 and 10D1. See U.S. Patent No. 6,984,720.

[0338] In some embodiments, the anti-CTLA-4 antibody is an activatable anti-CTLA-4 antibody, for example, an activatable anti-CTLA-4 antibody in which the antibody's light chain contains a cleavable moiety and a masking moiety at the amino terminus. The masking moiety prevents binding to CTLA-4, but after cleavage of the cleavable moiety by a protease that is more dominant and / or active in tumors than in peripheral tissues, it is preferentially released in the tumor microenvironment (see, in particular, International Publication No. 2018 / 085555). Such preferential cleavage in the tumor microenvironment allows for complete CTLA-4 blockade and enhancement of the anti-tumor immune response, while minimizing CTLA-4 blockade in normal tissues, thereby reducing the risk of potential systemic toxicity of the anti-CTLA-4 antibody. In some embodiments, the activatable anti-CTLA-4 antibody is an activatable form of ipilimumab, such as an antibody containing a light chain modified to include a masking moiety and a cleavable moiety, as disclosed in International Publication No. 2018 / 085555. An example of an activatable anti-CTLA-4 antibody that participated in human clinical trials is BMS-986249 (NCT03369223: “A Study of BMS-986249 Alone and in Combination with Nivolumab in Advanced Solid Tumors”). In some embodiments, the anti-CTLA-4 antibody is BMS-986249.

[0339] In some embodiments, anti-CTLA-4 antibodies exhibit enhanced Fcγ receptor (CD16) binding. Whether an anti-CTLA-4 antibody exhibits enhanced Fcγ receptor binding is evaluated by comparison with ipilimumab's Fcγ receptor binding. Anti-CTLA-4 antibodies with enhanced Fcγ receptor (CD16) binding have been proposed as therapeutic agents for the treatment of cancer caused by Treg cell depletion. See, in particular, International Publication No. 2014 / 089113. In some embodiments, anti-CTLA-4 antibodies exhibit at least twofold enhanced Fcγ receptor (CD16) binding compared to ipilimumab's Fcγ receptor binding.

[0340] An example of an anti-CTLA-4 antibody that exhibits enhanced Fcγ receptor (i.e., FcγRIIIA or CD16) binding is a non-fucosylated anti-CTLA-4 antibody. In some embodiments, an anti-CTLA-4 antibody is a non-fucosylated anti-CTLA-4 antibody. A non-fucosylated anti-CTLA-4 antibody lacks a fucose residue in its N-linked glycan. In some embodiments, a non-fucosylated anti-CTLA-4 antibody is produced by expressing the antibody chain in mammalian cells under conditions that prevent fucosylation, including but not limited to the use of mammalian cells with genetic recombination that prevents fucosylation, or the proliferation of antibody-expressing cells in a medium containing one or more chemical compounds that inhibit fucosylation. In some embodiments, the genetic recombination that prevents fucosylation is inactivation of the FUT8 gene, e.g., knockout. In some embodiments, an anti-CTLA-4 antibody is a low-fucosylated anti-CTLA-4 antibody.

[0341] An exemplary non-fucosylated anti-CTLA-4 antibody that participated in human clinical trials is BMS-986218 (e.g., NCT03110107: “First-In-Human Study of Monoclonal Antibody BMS-986218 by Itself and in Combination with Nivolumab in Participants with Advanced Solid Tumors”). BMS-986218 is a non-fucosylated antibody developed to enhance the effect of CTLA-4 blockade by enhancing binding to the Fcγ receptor and thus promoting APC-mediated T cell priming. In some embodiments, the anti-CTLA-4 antibody is BMS-986218. See, for example, PCT / US18 / 19868.

[0342] In some embodiments, the Fc region of the anti-CTLA-4 antibody contains amino acid substitutions within the antibody constant region to enhance binding to the activated Fcγ receptor. Exemplary substitutions are G236A, S239D, A330L, and I332E (all residue numbering follows the EU numbering system). In some embodiments, the anti-CTLA-4 antibody contains a human IgG1 constant domain with S239D, A330L, and I332E mutations.

[0343] In some embodiments, the anti-CTLA-4 antibody is an activatable and non-fucosylated anti-CTLA-4 antibody.

[0344] A human monoclonal antibody that binds specifically to CTLA-4 with high affinity and is suitable for the method of this disclosure is disclosed in U.S. Patent No. 6,984,720. Other anti-CTLA-4 monoclonal antibodies are described, for example, in U.S. Patents No. 5,977,318, No. 6,051,227, No. 6,682,736, and No. 7,034,121 and in International Publication Nos. 2012 / 122444, 2007 / 113648, 2016 / 196237, and 2000 / 037504 (each of which is incorporated herein by reference as a whole).

[0345] In some embodiments, anti-LAG-3 antibodies useful in combination with CEACAM5 targeting agents by the method of this disclosure include relatrimab (BMS-986016), IMP731 (H5L7BW), MK4280 (28G-10, fabezerimab), REGN3767 (fianlimab), GSK2831781, humanized BAP050, IMP-701 (LAG525, yeramirimab), aLAG-3 (0414), aLAG-3 (0416), Sym022, TSR-033, TSR-075, XmAb841 (XmAb22841), MGD013 (teboterimab), BI754111, FS118, P These are 13B02-30, AVA-017, 25F7, AGEN1746, RO7247669, INCAGN02385, IBI-110, EMB-02, IBI-323, LBL-007, or ABL501.

[0346] In some embodiments, anti-LAG-3 antibodies useful in combination with CEACAM5 targeting agents include: relatrimab (BMS-986016), IMP731 (H5L7BW), MK4280 (28G-10, fabezerimab), REGN3767 (fianlimab), GSK2831781, humanized BAP050, IMP-701 (LAG525, yeramirimab), aLAG-3 (0414), aLAG-3 (0416), Sym022, TSR-033, TSR-075, XmAb841 (XmAb22841), MGD013 (teboterimab), BI754111, FS118, P Includes CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, CDRL3, VH and VL, and / or heavy and light chains, from any of 13B02-30, AVA-017, 25F7, AGEN1746, RO7247669, INCAGN02385, IBI-110, EMB-02, IBI-323, LBL-007, or ABL501.

[0347] In some embodiments, the anti-LAG-3 antibody useful in combination with ADCs includes relarimab (BMS-986016). In some embodiments, the anti-LAG-3 antibody includes IMP731 (H5L7BW). In some embodiments, the anti-LAG-3 antibody includes MK4280 (28G-10, fabezerimab). MK-4280 (28G-10, fabezerimab) is described in International Publication No. 2016028672 and U.S. Patent Application Publication No. 2020 / 0055938. In some embodiments, the anti-LAG-3 antibody includes REGN3767 (fianlimab). REGN3767 (fianlimab) is described, for example, in Burova E, et al., J. Immunother. Cancer (2016); 4 (Supp. 1): P195 and U.S. Patent No. 10,358,495. In some embodiments, the anti-LAG-3 antibody comprises GSK2831781. In some embodiments, the anti-LAG-3 antibody comprises humanized BAP050. Humanized BAP050 is described, for example, in International Publication No. 2017 / 019894. In some embodiments, the anti-LAG-3 antibody comprises IMP-701 (LAG525, yeramirimab), which is described, for example, in U.S. Patent No. 10,711,060 and U.S. Patent Application Publication No. 2020 / 0172617. In some embodiments, the anti-LAG-3 antibody comprises aLAG-3(0414). In some embodiments, the anti-LAG-3 antibody comprises aLAG-3(0416). In some embodiments, the anti-LAG-3 antibody comprises Sym022. In some embodiments, the anti-LAG-3 antibody comprises TSR-033. In some embodiments, the anti-LAG-3 antibody comprises TSR-075. In some embodiments, the anti-LAG-3 antibody comprises XmAb841(XmAb22841). In some embodiments, the anti-LAG-3 antibody comprises MGD013(teboterimab). In some embodiments, the anti-LAG-3 antibody comprises BI754111. In some embodiments, the anti-LAG-3 antibody comprises FS118. In some embodiments, the anti-LAG-3 antibody comprises P 13B02-30. In some embodiments, the anti-LAG-3 antibody comprises AVA-017.In some embodiments, the anti-LAG-3 antibody includes 25F7, which is described, for example, in U.S. Patent Application Publication No. 2011 / 0150892. In some embodiments, the anti-LAG-3 antibody includes AGEN1746. In some embodiments, the anti-LAG-3 antibody includes RO7247669. In some embodiments, the anti-LAG-3 antibody includes INCAGN02385. In some embodiments, the anti-LAG-3 antibody includes IBI-110. In some embodiments, the anti-LAG-3 antibody includes EMB-02. In some embodiments, the anti-LAG-3 antibody includes IBI-323. In some embodiments, the anti-LAG-3 antibody includes LBL-007. In some embodiments, the anti-LAG-3 antibody includes ABL501.

[0348] In general, any anti-LAG-3 antibody useful in combination with ADC may be used. Antibodies that bind to LAG-3 are described in International Publication No. 2015 / 042246 and U.S. Patent Application Publication No. 2014 / 0093511 and U.S. Patent Application Publication No. 2011 / 0150892, each of which is incorporated herein by reference in whole.Disclosures relating to the anti-LAG-3 antibodies described herein and other anti-LAG-3 antibodies useful in the methods of this disclosure include, for example, U.S. Patent No. 10,188,730, International Publication No. 2016 / 028672, International Publication No. 2017 / 106129, International Publication No. 2017 / 062888, International Publication No. 2009 / 044273, International Publication No. 2018 / 069500, International Publication No. 2016 / 126858, and International Publication No. 201 Pamphlet No. 4 / 179664, International Publication No. 2016 / 200782, International Publication No. 2015 / 200119, International Publication No. 2017 / 019846, International Publication No. 2017 / 198741, International Publication No. 2017 / 220555, International Publication No. 2017 / 220569, International Publication No. 2018 / 071500, International Publication No. 2017 / 015560, International Publication No. 2017 / 025498 Pamphlet No. 2017 / 087589, Pamphlet No. 2017 / 087901, Pamphlet No. 2018 / 083087, Pamphlet No. 2017 / 149143, Pamphlet No. 2017 / 219995, Specification of US Patent Application Publication No. 2017 / 0260271, Pamphlet No. 2017 / 086367, Pamphlet No. 2017 / 086419, Pamphlet No. 2018 / 034227 This information can be found in International Publication No. 2018 / 185046, International Publication No. 2018 / 185043, International Publication No. 2018 / 217940, International Publication No. 19 / 011306, International Publication No. 2018 / 208868, International Publication No. 2014 / 140180, International Publication No. 2018 / 201096, International Publication No. 2018 / 204374, and International Publication No. 2019 / 018730. The contents of each of these references are incorporated as a whole by reference.

[0349] Several experimental therapeutic protocols involve ex vivo activation and proliferation of antigen-specific T cells and adoptive transfer of these cells to recipients in order to generate antigen-specific T cells against tumors. Ex vivo activation in the presence of the anti-CEACAM5 antibodies described herein, with or without additional immunostimulatory therapy (e.g., immune checkpoint inhibitors), may be expected to increase the frequency and activity of adoptively transferred T cells.

[0350] In some embodiments, the ADCs described herein may also be administered in conjunction with standard therapeutic procedures or other procedures such as radiation, surgery, or chemotherapy. The ADCs may also be combined with vaccination protocols. Numerous experimental methods have been devised for the vaccination of tumors (see Rosenberg, S., 2000, Development of Cancer Vaccines, ASCO Educational Book Spring: 60-62; Logothetis, C., 2000, ASCO Educational Book Spring: 300-302; Khayat, D., 2000, ASCO Educational Book Spring: 414-428; Foon, K., 2000, ASCO Educational Book Spring: 730-738; also see Restifo, N. and Sznol, M., Cancer Vaccines, Ch. 61, pp. 3023-3043 in DeVita et al. (eds.), 1997, Cancer: Principles and Practice of Oncology, Fifth Edition). In one of these methods, the vaccine is prepared using autologous or allogeneic tumor cells. These cell vaccines have been shown to be most effective when tumor cells are transduced to express GM-CSF. GM-CSF has been shown to be a potent activator of antigen presentation for tumor vaccination (Dranoff et al. (1993) Proc. Natl. Acad. SCI USA 90:3539-43).

[0351] V. Kit A kit including the ADC and instructions for use described herein is also provided.

[0352] In some embodiments, the kit optionally includes a single-dose vial or a pre-filled syringe containing a single dose of ADC, along with instructions for use when treating cancer with the ADC as described herein.

[0353] The present disclosure is further illustrated by the following embodiments, which should not be construed as further limitations. All drawings and all references, Genbank arrays, granted patents and published patent applications referenced throughout this disclosure are expressly incorporated herein by reference. [Examples]

[0354] Unless otherwise specified, commercially available reagents mentioned in the following examples were used according to the manufacturer's instructions. Unless otherwise specified, this disclosure uses standard procedures for recombinant DNA technology, such as those described in the texts above and below: Sambrook et al., see above; Ausubel et al., Current Protocols in Molecular Biology (Green Publishing Associates and Wiley Interscience, NY, 1989); Innis et al., PCR Protocols: A Guide to Methods and Applications (Academic Press, Inc.: NY, 1990); Harlow et al., Antibodies: A Laboratory Manual (Cold Spring Harbor Press: Cold Spring Harbor, 1988); Gait, Oligonucleotide Synthesis (IRL Press: Oxford, 1984); Freshney, Animal Cell Culture, 1987; Coligan et al., Current Protocols in Immunology, 1991.

[0355] The following examples describe the isolation and characterization of anti-CEACAM5 monoclonal antibodies. The CDR sequence, variable region sequence, and full-length heavy and light chain sequences of the anti-CEACAM5 antibody are provided below, for example, in Table 10.

[0356] Example 1: Production of anti-CEACAM5 antibody Human anti-CEACAM5 monoclonal antibodies (mAbs) were generated by immunizing BMS's proprietary chimeric mouse strain. Hybridomas were generated. Positive human and cynomolgus monkey CEACAM5 cross-reactive conjugates (that do not recognize either human CEACAM1 or human CEACAM6 protein) were selected. CEACAM5 mAbs were further isolated from the immunized mice using single B cell cloning (SBC) techniques. An immunotherapy library was also generated from mouse B cells. The library was expressed by yeast display and selected against CEACAM5 to identify further human / cynomolgus monkey cross-reactive antibodies that demonstrated higher specificity than CEACAM1 and CEACAM6.

[0357] The VH and VL regions derived from the positive human CEACAM5 mAbs identified above were sequenced by NGS using the MiSeq sequencing system (Illumina). Approximately 380 sequence-specific clones were identified that bind to either human or cynomolgus monkey CEACAM5, comprising 173 sequence families (defined by 80% sequence homology in HCDR3). Of these CEACAM5-positive sequences, 188 antibodies (57 sequence families) were shown to bind to both human and cynomolgus monkey CEACAM5 expressed on HCT116 cells (as described below), and 75 clones (31 sequence families) demonstrated specificity beyond CEACAM1 and CEACAM6. Further characterization (described herein) led to the isolation of several antibodies, including MBN001. The amino acid sequences of the VH and VL CDRs are shown in Table 10.

[0358] Example 2: Epitope Binning A high-throughput SPR-based epitope binning sandwich assay was performed using the Carterra LSA platform to group anti-CEACAM5 mAbs into bins sharing a common binding epitope. Pairwise competition experiments were performed using a microarray-based 96×96 microfluidic system array with high-throughput Carterra SPR microfluidics under a classic sandwich format. Binning results were analyzed using Carterra Microfluidics binning software for heatmap generation and network plots. Competing antibody relationships allowed for clustering anti-CEACAM5 antibodies into bins, where each bin represents a family of anti-CEACAM5 antibodies that share the same blocking profile when tested against other anti-CEACAM5 antibodies. See Figure 1A. Based on the assay results, newly generated antibodies were binned into seven different epitope groups. MBN001 mAb was identified as the bin-1 conjugate for CEACAM5. The cross-reactivity with other CEACAM5 family members, including CEACAM1 and CEACAM6, was determined by SPR as described above, and epitope bins 4, 6, 7, and 8 were determined to cross-react with CEACAM1 and CEACAM6. See Figure 1B.

[0359] Example 3: Binding specificity of anti-CEACAM5 antibody in cell lines expressing CEACAM5 This example analyzed the cell binding specificity of the anti-human CEACAM5 mAbs (e.g., MBN001, MBN002, and MBN003) described in Example 6. Figures 2A, 2B, 2C, and 2D are a series of graphs showing the cell-based binding of antibodies to cell lines expressing various levels of human CEACAM5: moderate BXPC-3 (Figure 2A), low Ls174 T cells (Figure 2B), high MKN45 (Figure 2C), and negative HCT-116 (Figure 2D). Each of the antibodies tested (mAb MBN001, mAb MBN002, and mAb MBN003) bound to cells expressing human CEACAM5 equivalent to mAb control 3. Each mAb was further evaluated for binding specificity using a human colorectal cancer cell line (HCT-116) or a CHOS cell line overexpressing human CEACAM5, cynomolgus monkey CEACAM5, human CEACAM1, and human CEACAM6. As shown in Table 2, antibody MBN001 showed specific binding to human CEACAM5 and cynomolgus monkey CEACAM5, but not to human CEACAM1 or CEACAM6.

[0360] [Table 2]

[0361] The binding of CEACAM5 mAbs such as MBN002, MBP018, MBP003, MBP001, and MBP002 to human CEACAM7 (R&D Systems catalog number 9010-CM-050) was investigated by SPR using CEACAM7 mAb (R&D Systems catalog number MAB44782) as a positive control. No binding was observed for CEACAM5 mAbs (data not shown). The binding of CEACAM5 mAbs to human CEACAM8 (R&D Systems catalog number 9639-CM-050) was similarly investigated. Commercially available CEACAM8 mAb (R&D Systems catalog number MAB4246) showed binding to CEACAM8, but no binding was observed for CEACAM5 mAbs (data not shown).

[0362] Example 4: Binding kinetics of anti-CEACAM5 antibody To characterize the binding dynamics of selected anti-CEACAM5 antibody MBN001 to full-length human CEACAM5 and cynomolgus monkey CEACAM5, as well as the A3-B3 regions (all internally generated proteins) of human CEACAM5 and cynomolgus monkey CEACAM5, analytical binding studies were performed by surface plasmon resonance (SPR).

[0363] SPR analysis was performed at 37°C using a Biacore 8K instrument docked with a CM5 sensor tip primed with 1×HBSP+ running water and equilibrated to room temperature, followed by repriming of the instrument. The tip surface was activated by injecting an ethyl(dimethylaminopropyl)carbodiimide (EDC) / N-hydroxysuccinimide (NHS) mixture at 10 μL / min for 7 minutes. The activated tip surface was then immobilized with an anti-human Fc capture reagent (25 μg / mL in acetate pH5 buffer) injected at 10 μL / min for 7 minutes to obtain an immobilization level of approximately 9000 RU. The remaining tip surface was blocked by injecting ethanolamine at 10 μL / min for 7 minutes.

[0364] To analyze the kinetics of antibody binding, anti-CEACAM5 antibody (10 nm in HBSP+ buffer) was first captured on the chip at 10 μL / min for 20 seconds for binding to the Fc capture reagent, followed by CEACAM5 analytic binding by flowing each of a series of recombinant full-length CEACAM5 or A3-B3 CEACAM5 proteins, each diluted 3-fold from 500 nM to 0.23 nM in HBSP+ buffer, across the entire chip. The analytic binding time was 3 minutes at 30 μL / min; the antigen dissociation time was 10 minutes at 30 μL / min; and regeneration involved two injections of 3M magnesium chloride (MgCl2) at 30 μL / min for 30 seconds each.

[0365] Kyphotic data of anti-CEACAM5 mAbs: CEACAM5 binding was fitted to a 1:1 Langmuir bond using Rmax to obtain estimated kinetic and affinity values ​​for the corresponding interaction. Estimated binding kinetics and affinity values ​​for full-length CEACAM5(hu / cy) and selected antibodies against the A3-B3 region of CEACAM5(hu / cy) are shown in Tables 3-6.

[0366] [Table 3]

[0367] [Table 4]

[0368] [Table 5]

[0369] [Table 6]

[0370] Example 5: In vitro binding of anti-CEACAM5 antibody to CEACAM5-expressing cell lines The selected anti-CEACAM5 antibody MBN001 was evaluated for binding to CEACAM5-expressing cell lines MKN45, HCT116-huCEACAM5, and HCT116-cyCEACAM5, along with the HCT-116 parental control. The results of these experiments are shown in Table 7 (EC50 values) and Table 8 (Amax values).

[0371] [Table 7]

[0372] [Table 8]

[0373] The data show that MBN001 bound to both huCEACAM5-expressing and cynoCEACAM-5-expressing cells. Furthermore, MBN001 exhibited improved cell binding compared to the control antibody.

[0374] Example 6: Screening of anti-CEACAM5 mAbs that can be internalized into cells Anti-CEACAM5 mAbs were screened for their ability to internalize huCEACAM5-expressing cells. Internalization assays were performed using MKN-45 cell lines, HCT116-huCEACAM5 cell lines, parental HCT-116 control cell lines, and LS174T cell lines. Internalization assays were performed in 96-well plates using the IncuCyte S3 live cell analysis system. Plates were scanned for phase contrast and red fluorescence, and images were automatically analyzed using integrated INCUCYTE® software for phase confluence (a measure of cell area) and red fluorescence area. As the labeled antibody internalizes into the acidic environment of endosomes and lysosomes, the intensity of intracellular red fluorescence increases. The internalization signal is expressed as red fluorescence area normalized to total cell area (phase confluence).

[0375] In short, many (1 × 10) are derived from the parent cell lines MKN-45, HCT116-huCEACAM5, LS174T, and HCT-116. 4Viable cells were added to a 96-well plate and incubated at 37°C / 5% CO2 for approximately 4 hours before antibody treatment. Antibody treatment was prepared by combining the test antibody or isotype control antibody with PHRODO® Red Secondary Fab Reagent (ThermoFisher Scientific) to provide a final concentration of the test antibody (25 nM) relative to the PHRODO reagent (75 nM) after addition to the cells. After incubation at 37°C for 30 minutes, 50 μl of each antibody treatment was added to 50 μl of cells in the 96-well plate. The plate was then inserted into an INCUCYTE® system and incubated for a further 30 minutes at 37°C before reading with an IncuCyte detector. The IncuCyte settings were configured for a 24-hour duration with readings every 30 minutes, a 10x objective lens with 3 images / well, and both phase and red fluorescence channels.

[0376] Table 9 shows the quantitative evaluation of the internalization of antibodies MBN001, MBN002, and MBN003 as described herein and measured by area under the time curve (AUC) analysis. Internalization data were collected for selected antibodies in the MKN45, LS174T, and HCT 116-CEACAM5 cell lines. The data indicate that the tested antibodies were internalized in the test cell lines, and in many cases, the antibodies exhibited improved or equivalent internalization compared to the control antibodies.

[0377] Further assays were performed, and the data showed that MBN001 was effectively internalized by different CEACAM5-expressing cells (Figures 3B and 4B). To facilitate high-throughput cytotoxicity screening, the microtubule inhibitor MMAE was conjugated to VHH, a human κ-light chain targeter. The data showed that the MBN001-VHH complex was internalized by CEACAM5-expressing cells and had the ability to effectively deliver the cytotoxic agent and kill CEACAM5-expressing cells. See Figures 3A, 3C, and 4A.

[0378] [Table 9]

[0379] Table 10

[0380] Table 11

[0381] Table 12

[0382] Table 13

[0383] Table 14

[0384] Table 15

[0385] Table 16

[0386] Table 17

[0387] Table 18

[0388] Table 19

[0389] Example 7: Mutation scanning and optimization of anti-CEACAM5 mAb MBN001 As shown in Figure 5A, mutation scanning was performed to identify variants of MBN001 with improved affinity for human and / or cynomolgus monkey CEACAM5. A single-stranded variable fragment (scFv) library was created to enable single amino acid substitutions in CDRs using NNK oligos. For each CDR, multiple oligos were designed to enable coding for all 20 amino acids and stop codons at each position by incorporating NNK codons at each position (N=A, C, T, G and K=G, T). Kabat definitions were used for all CDRs except HCDR1, where AbM definitions were used. In some cases, CDR residues were omitted from the scan, or additional non-germline vernier zone amino acids were included in the scan. In particular, vernier zone residues in the VH at positions 49 and 94 were included in the library design. Positions 33-35 in HCDR1 and position 97 in LCDR3 were omitted from the scan. Furthermore, in library design, the following framework residues in VH were reintroduced into the germline: T7S, S40A, A68T, and P84A. Various locations (mutation scan of MBN001 for CDR location analysis) are shown in detail in Figure 5B.

[0390] The library was expressed in an mRNA display system (Xu et.al. (2002) Chemistry & Biology 9:933-942; Roberts and Szostak (1997) Proc. Natl. Acad. Sci 94:12297-12302) and selected through a single selection against human and cynomolgus monkey CEACAM5. Briefly, the DNA library was transcribed and translated to fuse the scFv protein to the encoding mRNA via puromycin binding. The scFv-mRNA fusions were exposed to biotinylated human CEACAM5 and biotinylated cynomolgus monkey CEACAM5 using separate selections. Target-bound scFv was captured with streptavidin beads, eluted, and amplified by PCR. The captured antibodies were sequenced by NGS. The enrichment ratio was calculated by dividing the frequency of each sequence in the post-selection population by the frequency of each selection in the starting population. This enrichment ratio was normalized relative to the enrichment ratio of the parent antibody sequence to generate a normalized enrichment ratio:

number

[0391] Using these normalized enrichment ratios (ERs), heatmaps were created to evaluate the effect of all single amino acid substitutions on binding to human and cynomolgus monkey CEACAM5. The error of this method was approximately 2x, with ER values ​​between 0.5 and 2 considered neutral, values ​​greater than 2 considered desirable, and values ​​less than 0.5 considered undesirable. This analysis is shown in Figure 5C (MBN001 HCDR1 human CEACAM5), Figure 5D (MBN001 HCDR1 cynomolgus monkey CEACAM5), Figure 5E (MBN001 HCDR2 human CEACAM5), Figure 5F (MBN001 HCDR2 cynomolgus monkey CEACAM5), Figure 5G (MBN001 HCDR3 human CEACAM5), Figure 5H (MBN001 HCDR3 cynomolgus monkey CEACAM5), Figure 5I (MBN001 LCDR1 human CEACAM5), Figure 5J (MBN001 LCDR1 cynomolgus monkey CEACAM5), Figure 5K (MBN001 LCDR2 human CEACAM5), Figure 5L (MBN001 LCDR2 cynomolgus monkey CEACAM5), Figure 5M (MBN001 As shown in Figure 5N (LCDR3 human CEACAM5) and Figure 5N (MBN001 LCDR3 cynomolgus monkey CEACAM5), and summarized in Table 11, a rich set of information on the effects of single amino acid substitutions was provided. Using deep mutation scanning data, many CDR sites (e.g., as shown for HCDR1-3 and LCDR1-3 in Figure 5B) are resistant to mutation, meaning that these substitutions can be made at the identified CDR sites / amino acid sequences and maintain the desired ability of antibody or antigen-binding moieties to bind to human CEACAM5 and cynomolgus monkey CEACAM5.

[0392] [Table 20]

[0393] [Table 21]

[0394] [Table 22]

[0395] [Table 23]

[0396] [Table 24]

[0397] [Table 25]

[0398] [Table 26]

[0399] Example 8: Generation and analysis of anti-CEACAM5 mAb progeny of MBN001 This example describes the generation of progeny of the anti-CEACAM5 antibody MBN001 and the characterization of the progeny antibodies. Based on deep mutation scanning data, a subset of CDR amino acid substitutions predicted to improve MBN001 binding to human CEACAM5 or cynomolgus monkey CEACAM5 was selected for further analysis. Antibody genes with single amino acid substitutions and combinations of substitutions were synthesized in an IgG expression vector, transiently transfected into HEK cells, and purified via protein A. Furthermore, the above-described framework germline reversion was also incorporated into the progeny sequences. The purified IgG1.3 CEACAM5 mAb clones were characterized using BIACORE® for high-throughput SPR-based monoclonal characterization.

[0400] The BIACORE® instrument was primed with 1×HBSP+ running buffer (Cytiva catalog no. BR100671). The CM5 tip (Cytiva catalog no. 29149604) was equilibrated to room temperature and the BIACORE® instrument was reprimed. The human antibody Fc capture kit (Cytiva catalog no. 29234600) was used for this analysis. The human antibody Fc capture reagent was immobilized on both flow cells in all eight flow channels of the CM5 tip via amine coupling under the following conditions. Immobilization was performed at a temperature of 25°C. The anti-human Fc capture reagent was diluted to a concentration of 25 ug / mL in acetate pH 5 buffer. The tip surface was activated by injecting a mixture of 1-ethyl-3-(-3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) at a rate of 10 microliters / min (uL / min) for 420 seconds. Next, an anti-human Fc capture reagent at a concentration of 25 ug / mL was injected onto the surface at a rate of 10 uL / min for 420 seconds. The remaining tip surface was blocked by injecting ethanolamine at a rate of 10 uL / min for 420 seconds. This process resulted in an immobilization level of approximately 9000 RU.

[0401] The SPR kinetics for full-length human and cynomolgus monkey CEACAM5 were established as follows. Antibody capture was performed by diluting the antibody to a concentration of 10 nM in HBSP+ buffer. Capture of 10 nM mAbs at 5 uL / min for 20 seconds achieved a capture level of approximately 100 RU. Analyte binding was performed by first constructing a titration series of full-length human and cynomolgus monkey CEACAM5 (500 nM to 0.23 nM) prepared by 3-fold dilution in HBSP+ buffer. The binding rate was 30 uL / min for 180 seconds. The dissociation rate was 30 uL / min for 600 seconds. Regeneration was performed using two injections of 3M magnesium chloride (MgCl2) at 30 uL / min for 30 seconds. Data fitting was performed using a 1:1 binding model with global Rmax. Rmax reflects the maximum response when all ligands are occupied.

[0402] SPR data showing improved affinity for huCEACAM5 and cynoCEACAM5 to the progeny antibodies are shown in Figures 6A and 6B for the progeny antibodies with values ​​reported in Tables 12A and 12B below. These indicate that each of the antibodies tested binds to both human CEACAM5 and cynomolgus monkey CEACAM5. The data showed that these progeny selectively bind to human CEACAM5 and do not bind to CEACAM1, CEACAM6, CEACAM7, and CEACAM8. It should also be noted that these progeny were prepared in the hIgG1 form, in contrast to the hIgG1.3f form described in this example. Both the hIgG1 and hIgG1.3f forms share the same heavy chain variable region and the same light chain variable region; i.e., only the CH2 region of the heavy chain was adapted so that their binding properties would be expected to be similar. In fact, the data show that acceptable agreement was observed between the hIgG1 version and the hIgG1.3f version of the offspring (e.g., 1- to 2-fold similarity in human CEACAM5 and cynomolgus monkey CEACAM5 binding / SPR values) (data not shown). The data in Figures 7A, 7B, and 7C confirm that MBN001 offspring were effectively internalized into human CEACAM5-expressing cells and that these offspring can be used to effectively deliver cytotoxic drugs and parts. The data in Figures 8A and 8B show the absence of nonspecific human CEACAM1 and human CEACAM6 cross-reactivity with MBN001 offspring mAbs MBP001, MBP003, and MBP002. Figures 9A-9D show that FACS EC50 remained constant for each clone across the cell line and that the maximum MFI increased tenfold from LS174T and BxPC3 to MKN45.

[0403] [Table 27]

[0404] [Table 28]

[0405] Example 9: Internalization analysis of recombinant IgG1.3 progeny antibodies Internalization experiments were conducted for the hIgG1.3 MBN001 progeny antibodies listed in Table 13A below. Antibody internalization was evaluated using HCT-116-huCEACAM5, HCT-116-cyCEACAM5, and MKN45 cell lines.

[0406] Further internalization experiments were conducted using MKN45 and Ls174T cell lines with the hIgG1 antibodies MBP001, MBP002, and MBP003. MKN45 or Ls174T cells were seeded at 10K cells / well (50 μL).

[0407] First, cell lines were analyzed for cell density and viability using a Vi-cell viability machine. Cell lines were diluted to 0.2E6 vc / mL in growth medium, and each volume (50 μL) was dispensed into a flat-bottomed 96-well plate to achieve a cell count of 10K cells / well. Ls174T medium contained MEM with 10% HI-FBS and 1% pen / strep. MKN45 medium contained RPMI (ATCC modified) with 10% HI-FBS and 1% pen / strep. HCT-116 medium contained McCoy's 5a with 10% HI-FBS, 1% pen / strep, and 6 μg / mL blastosidine. Cells were deposited into the wells of a 96-well plate (Corning catalog number 3595) at 37°C / 5% CO2 for approximately 4 hours.

[0408] Each test mAb and control mAb was labeled by first pre-mixing the respective antibody with pHrodo red (pH-sensitive conjugated Fab;Thermo catalog number Z25612) secondary reagent in a 3:1 dye:mAb molar ratio. A series of dilutions were prepared in growth medium to 2x the target concentration. The final target mAb concentration was 25 nM. To achieve the target mAb concentration (25 nM), a volume (50 uL) of the labeled mAb sample was added to seeded cells.

[0409] Coated plates in contact with the labeled mAb were incubated at 37°C for 30 minutes. Each plate was then read using an Incucyte instrument, and images from the red and phase channels [10x objective lens; 3 images / well] were collected for 24 hours, with plates read every 30 minutes. The data are shown in Tables 13A and 13B. The data indicate that the anti-CEACAM5 progeny antibody was internalized by the HCT-CEA cell line and the MKN45 cell line.

[0410] [Table 29]

[0411] [Table 30]

[0412] We analyzed the internalization of MBN001 and specific progeny (e.g., MBP004 and MBP019) into cynoCEACAM-5 expressing cells. Efficient internalization of MBN001 and its progeny was observed. Internalization of negative control mAbs was not observed.

[0413] Example 10 - Conjugation reaction of compound A' and anti-CEACAM5 mAb to obtain DAR8 ADC This example describes the conjugation of MBN001 and its progeny antibodies (e.g., MBP001, MBP002, MBP003) against compound A'. Table 14 lists the materials used in the conjugation method.

[0414] [Table 31]

[0415] [ka] First, each mAb was buffered in P5 conjugation buffer (50 mM Tris, 100 mM NaCl, 1 mM EDTA, pH 8.3 at 25°C) to adjust the concentration to 10 mg / ml / ml. Next, a Zeba Spin desalting column was used. The column was equilibrated with P5 conjugation buffer according to the manufacturer's instructions. Recovery rates were typically over 95%.

[0416] For mAb stock solutions with concentrations less than 10 mg / ml, concentration and buffer exchange (diafiltration) were performed using a protein concentrator spin column (Amicon Ultra) according to the manufacturer's instructions. This spin column was rinsed with P5 conjugation buffer before mAb application.

[0417] After buffer exchange, the mAb concentration was measured using a nanophotometer. P5 conjugation buffer was used as a blank. Finally, the mAb concentration was adjusted to 10 mg / ml using P5 conjugation buffer.

[0418] Next, the mAbs were transferred to an amber-colored plastic product. The conjugation reaction was carried out while protected from light. Conjugation with compound A' was performed at a molar ratio of 7 equivalents of TCEP and 10 equivalents of compound A' per 1 equivalent of MBP001 mAb. Typically, a 40 mM stock of compound A' in DMSO and a 10 mM diluted standard solution of TCEP were used. A 40 mM linker-payload stock solution was thawed. A fresh 10 mM diluted standard solution of TCEP was prepared by combining 20 μl of 0.5 M TECP-HCl pH 7.0 with 980 μl of P5 conjugation buffer. Both reagents were vortexed before use.

[0419] The calculated amount of diluted TCEP standard solution was added to the MBP001 mAb solution and mixed by gentle swirling. Immediately thereafter, the calculated amount of compound A' stock was added. The mixture was then incubated overnight at 23°C in a 50 ml amber tube, swirled at a speed of 300 rpm.

[0420] Conjugation efficiency was evaluated by LC-MS analysis. The conjugated sample was diluted to 1 mg / mL in 100 mM Tris (pH 7.5). 20 μl of the sample was reduced by adding 2 μl of 0.5 M dithiothreitol (DTT) or TCEP. The sample was analyzed by LC-MS using an Agilent 1290 Infinity UPLC system coupled to a 6530 Accurate-Mass Q-TOF. The analytical column (Waters Inc., BEH C4 column, 1.7 μm, 2.1 mm × 50 mm) was equilibrated at 60°C. The mobile phase consisted of 0.1% formic acid in water (phase A) and 0.1% formic acid in acetonitrile (phase B). The system was operated at a flow rate of 200 μl / min. The gradient conditions were as follows: held at 27% B for 0-2 minutes; slowly heated at 27-37% B for 2-9 minutes; linearly heated at 37-90% B for 9-9.5 minutes; held at 90% B for 9.5-12.3 minutes. The MS settings were as follows: polarity = positive, capillary voltage = 4.2kV, sample cone = 40V, source offset = 15V, source temperature = 140°C, desolvation temperature = 325°C. The data acquisition range was 900-3200 m / z. Deconvolution was performed using Agilent MassHunter Walkup. If any non-conjugated mAbs were still present, another 1.4 equivalents of TCEP and 2 equivalents of compound A' were added, followed by incubation for 2-4 hours. This process yielded DAR8 ADC.

[0421] Example 11: Cytotoxicity analysis of progeny antibodies This example evaluated the cytotoxicity of CEACAM5-targeted mAbs of MBN001 progeny mAbs conjugated to compound A', as described above. Cytotoxicity was measured by IC50 and AUC values ​​for cell proliferation inhibition across CEACAM5-expressing cell lines Ls174T and MKN45. The data (along with binding and internalization data) were used to select mAbs for in vivo efficacy testing.

[0422] Ls174T cells and MKN45 cells were collected using trypsin / EDTA (0.25%) solution (Gibco catalog no. 25200-056). The culture medium was removed and the cells were washed with 1×PBS (Ca+ and Mg free; Gibco catalog no. 14190-144). The cells were detached using trypsin / EDTA solution. The trypsin / EDTA solution was neutralized with complete medium. The cells were allowed to settle by swift transfer at 1400 rpm for 5 minutes. The supernatant was removed and the cells were suspended in complete medium corresponding to each cell line.

[0423] Next, the cells were counted, and the cell concentration was set to 1.0 × 10⁻⁶. 6 The concentration was adjusted to 10 cells / mL. Specific cells were diluted to the following concentration: Ls174T: 0.125 × 10⁶ 6 Cells per mL and MKN45: 0.1 × 10 6 Cells per mL. Different cell suspensions of a certain volume (20 μL) were added to each well of a plate (PerkinElmer catalog number 6007480). The plate was incubated for 20–24 hours. Dilutions of the test reagents (e.g., antibodies MBP001, MBP002, and MBP003 conjugated with compound A') were prepared, and 20 μL of each dilution was added to the plate and incubated at 37°C for 120 hours.

[0424] Cell viability was determined using the Cell TiterGlo (CTG) 2.0 cell viability assay (Promega catalog number G9242). The CTG reagent was removed from the refrigerator and equilibrated to room temperature. The assay plate was removed from the incubator and acclimatized to room temperature. White backing adhesive was applied to the bottom of the plate. 40 μL of CTG solution was added to each assay well and then mixed with an orbital shaker at 500 rpm for 2 minutes. The assay plate was left in the dark for 20 minutes. The plate cover was then removed and the luminescence was analyzed with an EnVision plate reader. The data are shown in Table 15. The data indicate that the antibodies tested bound to CEACAM5 on different CEACAM5-expressing cell lines for 96 hours, and as a result, the cytotoxic payload was delivered to the cells for effective cytotoxic death.

[0425] [Table 32]

[0426] Example 12: Analysis of cytotoxic activity and ADCC activity of anti-CEACAM5 ADCs This example analyzed the cytotoxic activity of anti-CEACAM5 ADCs ADCP001A, ADCP001B, and ADCP001C (i.e., progeny mAbs MBP003, MBP001, and MBP002 conjugated to compound A', respectively) across CEACAM5-expressing cell lines LS174T, BxPC3, and MKN45. Figures 10A, 10B, and 10C show the % growth inhibition (as % cell viability) for cells treated with the selected antibody conjugate at the indicated antibody concentrations in Ls174T (Figure 10A), a low-CEACAM5-expressing cell line; BxPC-3, a moderate-CEACAM5-expressing cell line (Figure 10B); and MKN45, a high-CEACAM5-expressing cell line (Figure 10C). The data demonstrate that the progeny ADCs described herein exhibited equivalent or improved cell proliferation inhibitory IC50 values ​​compared to the parent ADCN001 (MBN001 conjugated to compound A ADC; see Figure 10D).

[0427] ADCC activity was also analyzed using anti-human lead CEACAM5 mAbs in the Jurkat-NFAT-FcγRIIIa(Promega) cell assay (catalog number G9901) using BxPC3 and MKN45 cells as target cells. See Figures 11A and 11B. BxPC3 (a cell line with moderate CEACAM expression) and MKN45 (a cell line with high CEACAM5 expression) cells were incubated with different concentrations of CEACAM5 mAbs (IgG1 or inactive IgG1.3f). Jurkat-FcγRIIIa effector cells were then co-incubated with the target cells for 6 hours. NFAT activation, reflecting the induced ADCC response, was evaluated by determining luciferase activity. The data show that mAb progeny of MBP003, MBP001, and MBP002 had limited ADCC activity.

[0428] Example 13. Bystander death analysis of progeny anti-CEACAM5 mAb ADCs Depending on the linker design, membrane-permeable cytotoxic drugs conjugated in ADCs released into target-positive cells can cross the cell membrane and kill other nearby cells, including adjacent cancer cells lacking antigen expression (bystander effect). This example describes an assay to analyze the bystander killing properties of anti-CEACAM5 ADCs.

[0429] In short, 8000 CEACAM5 antigen-expressing MKN45 cells (antigen-positive cells; Ag+) and 2000 non-CEACAM5-expressing HCT-116 cells (antigen-negative cells, Ag-) were seeded in 96-well plates, and ADC bystander death was determined using Incucyte to count live Ag- (green) or Ag+ (red) cells as a function after treatment with different doses of the anti-CEACAM5 mAb+ compound A'ADC ADCP001A, ADCP001B, and ADCP001C (N=3 used for each treatment). Control cells were untreated. Dose-dependent cell inhibition percentages for Ag+ or Ag- cells were calculated at 72 and 120 hours by normalizing against the untreated control wells. See Figures 12A, 12B, 12C, and 12D. Each of the ADCs tested (containing any of MBP001, MBP002, or MBP003 conjugated with compound A') was found to have the ability to release and kill bystander cells using an exatecan payload.

[0430] Example 14. In vivo antitumor efficacy study of anti-CEACAM5 ADC This study analyzed the in vivo antitumor efficacy of anti-CEACAM5 ADCs in cell line-derived xenograft models (MKN45, BxPC3, and Ls174T). 4-6 week old immunodeficient female thymus-deficient nude mice (CRL 490, Charles River) or NSG mice (NOD.Cg-Prkdcscid Il2rgtm1Wjl / SzJ, stock 005557, Jackson Laboratory) were subcutaneously inoculated into the right flank with 2 × 10⁶ MKN45 cells, 5 × 10⁶ BxPC3 cells, or 1 × 10⁶ Ls174T cells. Animals with tumor volumes of 150 mm³ to 250 mm³ were randomized to a treatment group with an average tumor volume of 180 mm³ (N=7). Animals with tumors were treated with a single intravenous infusion of a buffer control (phosphate-buffered saline (PBS)) or with ADC. The length (L) and width (W) of the tumor are measured with calipers, and the tumor volume is measured using preparation L. * The calculation was performed using (W^2) / 2.

[0431] The in vivo efficacy of anti-CEACAM5 ADCN001 (MBN001 conjugated with compound A') was evaluated across MKN45, Ls174T, and BxPC3 models. Tumor growth results for MKN45, BxPC3, and Ls174T models treated with 3 mg / kg ADCN001 are shown in Figures 15A, 15B, and 15C, respectively, while tumor growth for BxPC3 and Ls174T models treated with 10 mg / kg ADCN001 are shown in Figures 15D and 15E. The data demonstrate that ADCN001 (MBN001 conjugated with compound A') exhibited antitumor effects in these models. Figures 17A–17D are a series of graphs showing the efficacy of ADCV001 (MBV001, the wild-type hIgG1 version of MBN001 conjugated with compound A') in CDX models (MKN45 and BxPC3) after a single intravenous infusion with either 3 mg / kg or 10 mg / kg of ADC. The results of tumor growth in the MKN45 and BxPC3 models treated with 3 mg / kg ADCV001 are shown in Figures 17A and 17B, respectively, while the results of treatment with 10 mg / kg MBV001 are shown in Figures 17C and 17D. The data demonstrate that ADCN001 (the wild-type IgG format ADC of MBN001 conjugated with compound A') was also effective in the MKN45 and BxPC3 models.

[0432] Descendants of the anti-CEACAM5 mAb MBN001 (i.e., MBP001, MBP002, and MBP003) were also conjugated with compound A' and tested for in vivo efficacy in the MKN45 and BxPC3 models. Tumor growth results after treatment with 3 mg / kg of ADC are shown in Figures 18A and 18B, respectively, and results after treatment with 10 mg / kg of ADC are shown in Figures 18C and 18D. These ADCs, ADCP001A, ADCP001B, and ADCP001C, demonstrated robust antitumor activity in the MKN45 and BxPC3 models. Plasma exposure of all ADCs was comparable among these three ADCs. Free payload plasma exposure of these ADCs was below the limit of quantification (data not shown).

[0433] In all of the aforementioned in vivo efficacy studies, no significant effect on body weight was observed (data not shown). Therefore, treatment with these anti-CEACAM5+ compounds A'ADC appeared to be well-tolerated throughout the course of treatment.

[0434] Example 15. Analysis of pharmacodynamic markers of the DDR pathway in mouse tumors treated with mAb MBN001 conjugated with compound A'. This example analyzed the DNA damage response pathway induced by treatment of cancer cells with ADCN001 (MBN001 conjugated to compound A', as produced in Example 10). MKN45 tumors in mice (n=3) were treated with various amounts (1 mpk or 10 mpk) of ADC. The expression of the DNA damage response markers pKAP(1TF1b)(ser 824), pCHK1, yH2AX, and the apoptosis marker c-caspase 3 was evaluated in tumor samples collected from the animals 6, 24, or 168 hours after a single intravenous infusion of ADCN001 using the loading control GAPDH. DNA damage markers pKAP1, pCHK1, and yH2AX were induced within 24 hours and maintained up to 168 hours post-administration, accompanied by induction of the apoptosis marker cleavage c-caspase 3 at 168 hours post-administration (Figure 16A). Data were quantified and normalized to the GAPDH control (Figure 16B).

[0435] Example 16. HDX epitope mapping of anti-CEACAM5 mAb MBN001 This example analyzed the binding epitope of human hCEACAM5 during interaction with the anti-CEACAM5 mAb MBN001. Hydrogen-deuterium exchange mass spectrometry (HDX-MS) investigated the conformation and conformational dynamics of the protein in solution by monitoring the rate and extent of deuterium exchange of amide hydrogen atoms in the backbone [Huang et al. 2014, Analytical and Bioanalytical Chemistry, 406, 6541-6558; Wei, et al., 2014, Drug Discovery Today, 19, 95-102]. The level of hydrogen-to-deuterium exchange depends on the solvent exposure of amide hydrogen atoms in the backbone, protein hydrogen bonding, time, and pH. HDX-MS provides reads for both hydrogen bonding and solvent exposure. The mass increase of the protein during HDX can be accurately measured by MS. By comparing the exchange rates between the bound and unbound states in the HDX experiment, useful insights into the conformational dynamics, binding, specificity, and stability of the protein can be provided. Protein regions that exhibit slower hydrogen exchange rates in the bound state compared to the unbound state (protection) indicate potential binding sites or structural stabilization. In relation to antigen / antibody interactions, antigen regions where hydrogen exchange slows in the presence of an antibody are identified as potential epitopes. Protein regions that exhibit faster hydrogen exchange in the bound state compared to the unbound state (deprotection) indicate structural destabilization. Hydrogen exchange rates are also susceptible to allosteric effects, which can complicate the interpretation of results.

[0436] method To design peptide constructs containing subdomains of CEACAM5, a complete atomic three-dimensional model of CEACAM5 was constructed using MOE software (Molecular Operating Environment (MOE) 2022.02 Chemical Computing Group ULC, 910-1010 Sherbrooke St.W., Montreal, QC H3A 2R7, Canada, 2023) with Cα coordinates from solution scattering data (Boehm, MK and Perkins, SJFEBS Lett 475, 11-16, (2000), PDB code 1E07). A ribbon diagram of the 3D model of human CEACAM5, along with its distinct structural domains, is shown in Figure 13A. The distinct domains in the protein sequence corresponding to the 3D model are shown in Figure 13B. The seven shaded regions in Figure 13B correspond to the different structural domains in Figure 13A.

[0437] Prior to the epitope mapping experiment, deuterated experiments were performed to generate a list of common peptides for the protein complex of the recombinant hCEACAM5-A3-B3 construct (SEQ ID NO: 24) and mAb MBN001 Fab at a 1:1 molar ratio of 15 μM. In the HDX-MS experiment, the labeling reaction was initiated by diluting each sample volume (5 μL) in 55 μL of D2O buffer (10 mM phosphate buffer, D2O, pH 7.0). The reaction was carried out at various time intervals: 20 seconds, 1 minute, 10 minutes, and 60 minutes. At the end of each labeling reaction period, the reaction was quenched by adding a quenching buffer (100 mM phosphate buffer, pH 2.5, 1:1, v / v, containing 4 M GdnCl and 0.4 M TCEP), and 50 μL of the quenched sample was injected into a Waters HDX-MS system for analysis. Deuterium uptake levels of common digested peptides were monitored in the absence / presentation of Fab. Differences in hydrogen exchange rates between bound and free antigens were identified by subtracting the mean deuteration value of each peptide in the bound antigen from the mean deuteration of the same peptide in the free antigen state. Changes were considered significant if they exceeded three times the propagated pooled standard uncertainty.

[0438] result The hCEACAM5-A3-B3 construct was selected for HDX experiments based on epitope binning data showing the binding of MBN001 to the A3-B3 region of hCEACAM5. For this report, the numbering of N-terminal residues on the antigen was started from 488 to align the numbering of the hCEACAM5-A3-B3 construct with the canonical sequence of hCEACAM5 (UniProt entry: P06731). The hCEACAM5 protein is a complex glycoprotein. Seven predicted N-linked glycosylation sites are present in the cleaved hCEACAM5-A3-B3 construct used in this analysis. The complexity and heterogeneity of hCEACAM5 presented a significant challenge in obtaining complete sequence coverage. After method optimization, 60.1% sequence coverage and 3.45 redundancy were achieved for the hCEACAM5-A3-B3 construct using the HDX-MS platform (Figure 13C).

[0439] In the presence of MBN001, a significant decrease in the hydrogen exchange rate on hCEACAM5 was observed in the peptide regions covering residues 588-606 and 668-685 compared to the unbound hCEACAM5 antigen (Figure 13D).

[0440] Further interpretation of HDX differences requires considering additional factors related to the HDX process [Bai et al., 1993 Proteins. 17(1):75-86]. Firstly, proline residues do not have an amide hydrogen and therefore do not report on the HDX process. Secondly, after proteolysis, the N-terminal residue of each peptide is converted from amide to amine. The amine undergoes rapid deuterium loss during analysis. Furthermore, the first amide residue (i.e., the second residue) of each peptide also undergoes rapid deuterium loss due to the influence of the N-terminal amine. Finally, overlapping regions can be used to narrow down the results.

[0441] Peptide regions 590–606 are covered by multiple duplicate peptides, all of which exhibit strong protection. Therefore, this suggests high reliability in the binding involvement of this region. The amino acids exhibiting significant protection are further narrowed down to the following: DVL 590 Y 591 G 592 PD 594 T 595 PI 597 I 598 S 599 PPD 602 S 603 S 604 Y 605 L 606 (Sequence ID 103). This region was identified as a primary epitope using HDX-MS.

[0442] The peptide region 668-685 was covered by a single peptide. After the exclusion of the first two N-terminal amino acids and the proline residue at position 681, the protective region remained long, covering 15 amino acids, i.e., IVK 670 SITVSASGTSPGLSA 685 (Sequence ID 104). Data was unavailable for the following regions on hCEACAM5:501-522, 546-574, 612-615, and 642-662; therefore, no conclusions could be drawn regarding these residues.

[0443] The overall HDX effect of MBN001 coupling to hCEACAM5 is shown in Figure 13E.

[0444] conclusion HDX-MS identified the following residues and peptide regions as potential epitopes on hCEACAM5(A3B3) upon binding to MBN001: L 590 , Y 591 , G 592 , D 594 , T 595 , I 597 , I 598 S 599 , D602 S 603 S 604 , Y 605 , L 606 , K 670 SITVSASGTSPGLSA 685 (Sequence ID 117).

[0445] Example 17. Cryo-EM analysis of 1 mAb MBP001 in bin 1. This example describes cryo-EM analysis performed on Bin 1 mAb MBP001. The CEACAM5 construct was approximately 20 kDa in size, and the MBP001 Fab was approximately 50 kDa in size. Particles significantly smaller than approximately 120 kDa are more difficult to extract and align using cryo-EM analysis. Therefore, an anti-CEACAM5 Bin 2 mAb (approximately 50 kDa) was also produced and mixed with the CEACAM5 construct and MBP001 Fab to produce a composite structure of approximately 120 kDa. This combined structure made the cryo-EM analysis much easier, and it was confirmed that the binder MBP001 in Bin 1 bound to a different epitope than that of Bin 2 mAb. The following is a description of the various steps and methods performed.

[0446] Design of Human CEACAM5 A3-B3 CEACAM5, like other members of the CEA-associated cell adhesion molecule (CEACAM) family within the immunoglobulin (Ig) gene superfamily, is a highly glycosylated multidomain protein whose domain boundaries are manually assigned in Uniprot using PROSITE annotation rules (ID # P06731; Uniprot: the Universal Protein Knowledgebase in 2023; The UniProt Consortium (2023) Nucleic Acids Research 51, D523-D531).

[0447] However, in order to design peptide constructs containing subdomains of CEACAM5, a complete atomic three-dimensional model of CEACAM5 was constructed using MOE software (Molecular Operating Environment (MOE) 2022.02 Chemical Computing Group ULC, 910-1010 Sherbrooke St.W., Montreal, QC H3A 2R7, Canada, 2023) using Cα coordinates from solution scattering data (Boehm, MK and Perkins, SJFEBS Lett 475, 11-16, (2000), PDB code 1E07). A ribbon diagram of the 3D model of human CEACAM5, along with its distinct structural domains, is shown in Figure 13A. The distinct domains in the protein sequence corresponding to the 3D model are shown in Figure 13B.

[0448] To design the A3-B3 construct for antibody screening, further binding of purification and cleavage tags was necessary. For purification, a histidine tag (amino sequence HHHHHH; SEQ ID NO: 95) was selected, and for protease cleavage, a tobacco leaf vein mottled virus (TVMV) tag (sequence ETVRFQG (SEQ ID NO: 102); Nallamsetty, Protein Expr. Purif. 38, 108-15, 2004) was selected. These were bound to the N-terminus of the A3-B3 construct. The inventors were interested in finding an antibody-binding epitope closer to the C-terminus (i.e., the B3 domain) to selectively bind to the non-shedding form of CEACAM5 rather than the shedding or soluble form (shedding occurs near the C-terminus), so the N-terminus was chosen for this purpose rather than the C-terminus. The final construct is shown as SEQ ID NO: 24.

[0449] Expression and purification of the hCEACAM5 A3-B3 reagent The human CEACAM5 A3-B3 domain protein (C-terminal region, 198 amino acid protein construct) shown below was constructed. [ka]

[0450] Human CEACAM5 A3-B3 domain protein was expressed by transient transfection of Expi293F® GnTI- cells (Thermo Fisher) with DNA encoding A3-B3 proteins using the ExpiFectamine® 293 transfection kit (Thermo Fisher). After 24 hours, the transfected cells were supplied with the enhancer provided in the kit and grown at 37°C, 8% CO2, and 150 RPM for a total of 4 days. The supernatant was collected by centrifugation using 0.22 μm filtration (Corning).

[0451] The clarified medium of A3-B3 was purified using a 5 ml Histrap excel column (Cytiva) and eluted with 250 mM imidazole phosphate buffer. The eluate from the Histrap column was further purified using a preparative HiLoad Superdex 200 16 / 60 (Cytiva) to isolate A3-B3 monomers from the agglutinating material. The monomer preparative size exclusion chromatography (SEC) fraction was pooled and filtered through a 0.22 μm syringe filter (Pall) as a sample for cryo-EM structure determination. The sample concentration was 25,690 M. -1 cm -1 The calculated molar extinction coefficient was determined by A280. Samples were examined by LC-MS, analytical SEC, and SDS-PAGE to assay quality.

[0452] Expression and purification of anti-CEACAM5 Fab Each generated Fab heavy chain (HC) shared the same variable region (VH) and constant region (CH1) as its parent mAb. Two amino acids, GG, were added to the C-terminus of the Fab HC. The Fab light chain (LC) remained identical to that of the parent mAb LC. DNA from both Fab HC and LC was synthesized for expression.

[0453] Fab of MBP001: Light chain: [ka] Heavy chain: [ka] (Including VH of sequence number 45)

[0454] Fab of bin 2 mAb: Light chain: [ka] Heavy chain: [ka]

[0455] The Fab of MBP001 and the Fab of Bin 2 mAb were expressed in Expi293 cells (Thermo Fisher) by transient transfection with Fab heavy chain (HC) and Fab light chain (LC) DNA using the same method as described above for CEACAM5.

[0456] Each clarified m...

Claims

1. Antibody-drug conjugates (ADCs) represented by formula (I): 【Chemistry 1】 or a pharmaceutically acceptable salt, stereoisomer, or solvate thereof (in the formula, 【Chemistry 2】 This indicates that the configuration of the double bond is E or Z; V is H or (C １ ~C ８ ) is alkyl; X is R ３ -C is; Y is NR ５ , S, O, or CR ６ R ７ And; R １ This is a polyalkene glycol unit containing at least three alkylene glycol subunits; R ３ and R ５ to R ７ are each H, or an optionally substituted aliphatic residue, or an optionally substituted aromatic residue; L is the linker; C is the cytotoxic part; m is an integer in the range of 1 to 10; n is in the range of 1 to 20; AB is an anti-CEACAM5 antibody or an antigen-binding portion of an anti-CEACAM5 antibody that specifically binds to carcinoembryonic antigen-associated cell adhesion molecule-5 (CEACAM5), (a) Heavy chain variable region (VH) including complementarity-determining region (CDR) 1, CDR2, and CDR3 regions containing the amino acid sequences described in SEQ ID NOs: 14, 15, and 16, respectively, and light chain variable region (VL) including CDR1, CDR2, and CDR3 regions containing the amino acid sequences described in SEQ ID NOs: 19, 20, and 21, or (b) VH containing CDR1, CDR2, and CDR3 regions having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with the amino acid sequences described in SEQ ID NOs: 14, 15, and 16, respectively; and VL containing CDR1, CDR2, and CDR3 regions having at least 85%, 90%, 95%, 98%, or 99% sequence identity with the amino acid sequences described in SEQ ID NOs: 19, 20, and 21, respectively. (This includes an anti-CEACAM5 antibody or its antigen-binding moiety.)

2. The ADC according to claim 1, wherein the VH includes an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with the amino acid sequence described in SEQ ID NO: 38, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91, or SEQ ID NO:

93.

3. The ADC according to claim 1, wherein the VH includes an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with the amino acid sequence described in Sequence ID No.

17.

4. The ADC according to claim 1, wherein the VH includes an amino acid sequence described in SEQ ID NO: 38, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91, or SEQ ID NO:

93.

5. The ADC according to claim 1, wherein the VH comprises the amino acid sequence described in Sequence ID No.

17.

6. The ADC according to any one of claims 1 to 5, wherein the VL includes an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with the amino acid sequence described in SEQ ID NO: 43, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92, or SEQ ID NO:

94.

7. The ADC according to any one of claims 1 to 5, wherein the VL includes an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with the amino acid sequence described in Sequence ID No.

22.

8. The ADC according to claim 1, wherein the VL includes an amino acid sequence described in SEQ ID NO: 43, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92, or SEQ ID NO:

94.

9. The ADC according to claim 1, wherein the VL includes the amino acid sequence described in Sequence ID No.

22.

10. The VH and VL are, (a) The amino acid sequence described in SEQ ID NO: 38 and the amino acid sequence described in SEQ ID NO: 43, respectively; (b) The amino acid sequences described in SEQ ID NO: 49 and SEQ ID NO: 50, respectively; (c) The amino acid sequence described in SEQ ID NO: 51 and the amino acid sequence described in SEQ ID NO: 52, respectively; (d) The amino acid sequences described in SEQ ID NO: 67 and SEQ ID NO: 68, respectively; (e) The amino acid sequences described in SEQ ID NO: 69 and SEQ ID NO: 70, respectively; (f) The amino acid sequences described in SEQ ID NO: 71 and SEQ ID NO: 72, respectively; (g) The amino acid sequences described in SEQ ID NO: 73 and SEQ ID NO: 74, respectively; (h) The amino acid sequences described in SEQ ID NO: 75 and SEQ ID NO: 76, respectively; (i) The amino acid sequences described in SEQ ID NO: 77 and SEQ ID NO: 78, respectively; (j) The amino acid sequences described in SEQ ID NO: 79 and SEQ ID NO: 80, respectively; (k) The amino acid sequence described in SEQ ID NO: 81 and the amino acid sequence described in SEQ ID NO: 82, respectively; (l) The amino acid sequences described in SEQ ID NO: 83 and SEQ ID NO: 84, respectively; (m) The amino acid sequence described in SEQ ID NO: 85 and the amino acid sequence described in SEQ ID NO: 86, respectively; (n) The amino acid sequence described in SEQ ID NO: 87 and the amino acid sequence described in SEQ ID NO: 88, respectively; (o) The amino acid sequences described in SEQ ID NO: 89 and SEQ ID NO: 90, respectively; (p) The amino acid sequence described in SEQ ID NO: 91 and the amino acid sequence described in SEQ ID NO: 92, respectively; or (q) The amino acid sequences described in SEQ ID NO: 93 and SEQ ID NO: 94, respectively. The ADC according to claim 1, having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity.

11. The ADC according to claim 1, wherein VH and VL have at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with the amino acid sequence described in SEQ ID NO: 17 and SEQ ID NO: 22, respectively.

12. The VH and VL are, (a) The amino acid sequence described in SEQ ID NO: 38 and the amino acid sequence described in SEQ ID NO: 43, respectively; (b) The amino acid sequences described in SEQ ID NO: 49 and SEQ ID NO: 50, respectively; (c) The amino acid sequence described in SEQ ID NO: 51 and the amino acid sequence described in SEQ ID NO: 52, respectively; (d) The amino acid sequences described in SEQ ID NO: 67 and SEQ ID NO: 68, respectively; (e) The amino acid sequences described in SEQ ID NO: 69 and SEQ ID NO: 70, respectively; (f) The amino acid sequences described in SEQ ID NO: 71 and SEQ ID NO: 72, respectively; (g) The amino acid sequences described in SEQ ID NO: 73 and SEQ ID NO: 74, respectively; (h) The amino acid sequences described in SEQ ID NO: 75 and SEQ ID NO: 76, respectively; (i) The amino acid sequences described in SEQ ID NO: 77 and SEQ ID NO: 78, respectively; (j) The amino acid sequences described in SEQ ID NO: 79 and SEQ ID NO: 80, respectively; (k) The amino acid sequence described in SEQ ID NO: 81 and the amino acid sequence described in SEQ ID NO: 82, respectively; (l) The amino acid sequences described in SEQ ID NO: 83 and SEQ ID NO: 84, respectively; (m) The amino acid sequence described in SEQ ID NO: 85 and the amino acid sequence described in SEQ ID NO: 86, respectively; (n) The amino acid sequence described in SEQ ID NO: 87 and the amino acid sequence described in SEQ ID NO: 88, respectively; (o) The amino acid sequences described in SEQ ID NO: 89 and SEQ ID NO: 90, respectively; (p) The amino acid sequence described in SEQ ID NO: 91 and the amino acid sequence described in SEQ ID NO: 92, respectively; or (q) The amino acid sequences described in SEQ ID NO: 93 and SEQ ID NO: 94, respectively. The ADC according to claim 1, including the following:

13. The ADC according to any one of claims 1 to 12, wherein C is exatecan and m is 1.

14. The ADC according to any one of claims 1 to 13, wherein V is H.

15. The ADC according to any one of claims 1 to 14, wherein Y is NH.

16. The polyalkylene glycol unit R １ However, the structure: 【Transformation 3】 An ADC according to any one of claims 1 to 15, comprising 3 to 100 subunits having the

17. R １ but, 【Chemistry 4】 And, During the ceremony, 【Transformation 5】 This indicates the position of O; K Ｆ is -H, -PO ３ H, -(C １ ~C １０ ) alkyl, -(C １ ~C １０ ) Alkyl-SO ３ H, -(C ２ ~C １０ ) Alkyl-CO ２ H, -(C ２ ~C １０ ) alkyl-OH, -(C ２ ~C １０ ) Alkyl-NH ２ , - (C ２ ~C １０ ) Alkyl-NH(C １ ~C ３ ) Alkyl and -(C ２ ~C １０ ) Alkyl-N ((C １ ~C ３ )alkyl) ２ Selected from the group consisting of; The ADC according to claim 16, wherein o is an integer in the range of 3 to 100.

18. The aforementioned polyethylene glycol unit R １ However, the structure: 【Transformation 6】 The ADC according to claim 16, comprising 3 to 100 subunits having the

19. R １ but, 【Transformation 7】 And, During the ceremony, 【Transformation 8】 This indicates the position of O; K Ｆ is -H, -PO ３ H, -(C １ ~C １０ ) alkyl, -(C １ ~C １０ ) Alkyl-SO ３ H, -(C ２ ~C １０ ) Alkyl-CO ２ H, -(C ２ ~C １０ ) alkyl-OH, -(C ２ ~C １０ ) Alkyl-NH ２ , - (C ２ ~C １０ ) Alkyl-NH(C １ ~C ３ ) Alkyl and -(C ２ ~C １０ ) Alkyl-N ((C １ ~C ３ )alkyl) ２ Selected from the group consisting of; The conjugate according to claim 17, wherein o is an integer in the range of 3 to 100.

20. K Ｆ The ADC according to claim 17 or 19, wherein H is present.

21. The ADC according to claim 17 or 19, wherein o is in the range of 8 to 30, for example, 8 to 16 or 20 to 28, for example, 10, 11, 12, 13, 14, 22, 23, 24, 25 or 26.

22. The linker L is, *-A-W １～８ -B ０～１ -#、 (In the formula, A is the first spacer unit; W is an amino acid; B is the second spacer unit; * indicates the connection point to -Y-; (# indicates a binding site to the cytotoxic region.) The ADC according to any one of claims 1 to 21, as represented by the ADC.

23. A is structure: 【Chemistry 9】 It has, During the ceremony, 【Chemistry 10】 It is a five- or six-membered carbon ring; * indicates the connection point to -Y; and The ADC according to claim 22, wherein ## indicates a bonding point to W. 【Request Item 24】 【Chemistry 11】 teeth, 【Chemistry 12】 The ADC according to claim 23.

25. W is a dipeptide (W ２ The ADC according to claim 22, which is the same as the ADC described in claim 22.

26. The ADC according to claim 25, wherein the dipeptide is selected from the group consisting of valine-citrulline (Val-Cit) and valine-alanine (Val-Ala).

27. The ADC according to claim 26, wherein the dipeptide is Val-Cit.

28. The second spacer unit B has the following structure: 【Chemistry 13】 A PAB group having, in the formula, The NH group is bonded to -W-, The ADC according to claim 22, wherein the C(O) group is bound to the cytotoxic moiety.

29. The aforementioned linker has the following structure: 【Chemistry 14】 It has, in the formula, W ２ The ADC according to claim 22, wherein the ADC is Val-Cit.

30. The aforementioned linker L has the following structure: 【Chemistry 15】 *-A-W ２ -B １ The ADC according to claim 22, wherein -#, where * indicates a binding point to Y, and # indicates a binding point to the cytotoxic portion.

31. V is H; Y is NH; R １ Structure: 【Chemistry 16】 It is a polyalkylene glycol unit having, During the ceremony, 【Chemistry 17】 This indicates the position of O; K Ｆ H is; o is an integer in the range of 8 to 30; R ３ H is; L has the following structure: [Chemistry 18] It is a linker that has, In the formula, * indicates a binding point to Y, and # indicates a binding point to the cytotoxic portion; C is exatecan; m is 1; The ADC according to claim 1, wherein n is in the range of 5 to 10.

32. The ADC according to claim 31, wherein o is an integer in the range of 15 to 30.

33. The ADC according to claim 31, wherein o is an integer in the range of 20 to 28.

34. The ADC according to claim 33, wherein o is 22, 23, 24, 25, or 26.

35. The ADC according to claim 31, wherein n is in the range of 6 to 8.

36. Antibody-drug conjugates (ADCs) represented by formula (II): 【Chemistry 19】 or a pharmaceutically acceptable salt, stereoisomer, or solvate thereof (in the formula, 【Chemistry 20】 This indicates that the configuration of the double bond is E or Z; n is in the range of 4 to 8; o is an integer between 10 and 30; AB is an anti-CEACAM5 antibody or an antigen-binding portion of an anti-CEACAM5 antibody that specifically binds to carcinoembryonic antigen-associated cell adhesion molecule-5 (CEACAM5), (a) Heavy chain variable region (VH) including complementarity-determining region (CDR) 1, CDR2, and CDR3 regions containing the amino acid sequences described in SEQ ID NOs: 14, 15, and 16, respectively, and light chain variable region (VL) including CDR1, CDR2, and CDR3 regions containing the amino acid sequences described in SEQ ID NOs: 19, 20, and 21, or (b) VH containing CDR1, CDR2, and CDR3 regions having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with the amino acid sequences described in SEQ ID NOs: 14, 15, and 16, respectively; and VL containing CDR1, CDR2, and CDR3 regions having at least 85%, 90%, 95%, 98%, or 99% sequence identity with the amino acid sequences described in SEQ ID NOs: 19, 20, and 21, respectively. (This includes an anti-CEACAM5 antibody or its antigen-binding moiety.)

37. Antibody-drug conjugates (ADCs) represented by formula (ADC101): 【Chemistry 21】 or a pharmaceutically acceptable salt, stereoisomer, or solvate thereof (in the formula, 【Chemistry 22】 This indicates that the configuration of the double bond is E or Z; AB is an anti-CEACAM5 antibody or an antigen-binding portion of an anti-CEACAM5 antibody that specifically binds to carcinoembryonic antigen-associated cell adhesion molecule-5 (CEACAM5), (a) Heavy chain variable region (VH) including complementarity-determining region (CDR) 1, CDR2, and CDR3 regions containing the amino acid sequences described in SEQ ID NOs: 14, 15, and 16, respectively, and light chain variable region (VL) including CDR1, CDR2, and CDR3 regions containing the amino acid sequences described in SEQ ID NOs: 19, 20, and 21, or (b) VH containing CDR1, CDR2, and CDR3 regions having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with the amino acid sequences described in SEQ ID NOs: 14, 15, and 16, respectively; and VL containing CDR1, CDR2, and CDR3 regions having at least 85%, 90%, 95%, 98%, or 99% sequence identity with the amino acid sequences described in SEQ ID NOs: 19, 20, and 21, respectively. (This includes an anti-CEACAM5 antibody or its antigen-binding moiety.)

38. Antibody-drug conjugates (ADCs) represented by formula (II): 【Chemistry 23】 or a pharmaceutically acceptable salt, stereoisomer, or solvate thereof (wherein n is in the range of 4 to 8; o is an integer in the range of 10 to 30; and A and B are anti-CEACAM5 antibodies containing VH and VL, respectively, which include the amino acid sequences described in SEQ ID NO: 38 and SEQ ID NO: 43, or the antigen-binding moiety thereof).

39. The ADC according to claim 38, wherein n is 8 and o is 24.

40. The ADC according to any one of claims 12, 36, or 37, wherein the VH and VL each include the amino acid sequence described in SEQ ID NO: 49 and the amino acid sequence described in SEQ ID NO:

50. The ADC according to any one of claims 12, 36, or 37, wherein the VH and VL each include the amino acid sequence described in SEQ ID NO: 51 and the amino acid sequence described in SEQ ID NO: 52, respectively.

41. The ADC according to any one of claims 12, 36, or 37, wherein the VH and VL each include the amino acid sequence described in SEQ ID NO: 67 and the amino acid sequence described in SEQ ID NO: 68, respectively.

42. The ADC according to any one of claims 12, 36, or 37, wherein the VH and VL each include the amino acid sequence described in Sequence ID No. 69 and the amino acid sequence described in Sequence ID No. 70, respectively.

43. The ADC according to any one of claims 12, 36, or 37, wherein the VH and VL each include the amino acid sequence described in SEQ ID NO: 71 and the amino acid sequence described in SEQ ID NO: 72, respectively.

44. The ADC according to any one of claims 12, 36, or 37, wherein the VH and VL each include the amino acid sequence described in SEQ ID NO: 73 and the amino acid sequence described in SEQ ID NO: 74, respectively.

45. The ADC according to any one of claims 12, 36, or 37, wherein the VH and VL each include the amino acid sequence described in SEQ ID NO: 75 and the amino acid sequence described in SEQ ID NO:

76.

46. The ADC according to any one of claims 12, 36, or 37, wherein the VH and VL each include the amino acid sequence described in Sequence ID No. 69 and the amino acid sequence described in Sequence ID No. 70, respectively.

47. The ADC according to any one of claims 12, 36, or 37, wherein the VH and VL each include the amino acid sequence described in SEQ ID NO: 73 and the amino acid sequence described in SEQ ID NO: 74, respectively.

48. The ADC according to any one of claims 12, 36, or 37, wherein the VH and VL each include the amino acid sequence described in SEQ ID NO: 75 and the amino acid sequence described in SEQ ID NO:

76.

49. The ADC according to any one of claims 12, 36, or 37, wherein the VH and VL each include the amino acid sequence described in SEQ ID NO: 77 and the amino acid sequence described in SEQ ID NO: 78, respectively.

50. The ADC according to any one of claims 12, 36, or 37, wherein the VH and VL each include the amino acid sequence described in SEQ ID NO: 79 and the amino acid sequence described in SEQ ID NO: 80, respectively.

51. The ADC according to any one of claims 12, 36, or 37, wherein the VH and VL each include the amino acid sequence described in SEQ ID NO: 81 and the amino acid sequence described in SEQ ID NO: 82, respectively.

52. The ADC according to any one of claims 12, 36, or 37, wherein the VH and VL each include the amino acid sequence described in SEQ ID NO: 83 and the amino acid sequence described in SEQ ID NO: 84, respectively.

53. The ADC according to any one of claims 12, 36, or 37, wherein the VH and VL each include the amino acid sequence described in SEQ ID NO: 85 and the amino acid sequence described in SEQ ID NO:

86.

54. The ADC according to any one of claims 12, 36, or 37, wherein the VH and VL each include the amino acid sequence described in SEQ ID NO: 87 and the amino acid sequence described in SEQ ID NO: 88, respectively.

55. The ADC according to any one of claims 12, 36, or 37, wherein the VH and VL each include the amino acid sequence described in SEQ ID NO: 89 and the amino acid sequence described in SEQ ID NO: 90, respectively.

56. The ADC according to any one of claims 12, 36, or 37, wherein the VH and VL each include the amino acid sequence described in SEQ ID NO: 91 and the amino acid sequence described in SEQ ID NO: 92, respectively.

57. The ADC according to any one of claims 12, 36, or 37, wherein the VH and VL each include the amino acid sequence described in SEQ ID NO: 93 and the amino acid sequence described in SEQ ID NO: 94, respectively.

58. The ADC according to any one of claims 12, 36, or 37, wherein the VH and VL each include the amino acid sequence described in SEQ ID NO: 17 and the amino acid sequence described in SEQ ID NO: 22, respectively.

59. The ADC according to any one of claims 12, 36, or 37, wherein the heavy chain and the light chain each include the amino acid sequence described in SEQ ID NO: 45 and the amino acid sequence described in SEQ ID NO:

46.

60. An ADC according to any one of claims 1 to 59, comprising an IgG1 constant region, an IgG2 constant region, an IgG3 constant region, an IgG4 constant region, or a variant thereof.

61. An ADC according to any one of claims 1 to 60, comprising an IgG1 antibody.

62. The ADC according to claim 62, comprising an IgG1.3f steady-state region.

63. The ADC according to any one of claims 60 to 62, wherein the constant region further comprises C-terminal lysine.

64. The ADC according to any one of claims 1 to 63, wherein the antibody is human, humanized, or a chimeric antibody.

65. The ADC according to any one of claims 1 to 64, wherein the antigen-binding portion comprises a Fab, Fab', (Fab')2, Fv, or scFv fragment.

66. The ADC according to any one of claims 1 to 65, wherein the antibody or its antigen-binding portion comprises a bispecific molecule containing the anti-CEACAM5 antibody or its antigen-binding portion, and a second binding region for binding to another antigen.

67. The ADC according to any one of claims 1 to 65, wherein the antibody or its antigen-binding portion comprises a multispecific molecule comprising the anti-CEACAM5 antibody or its antigen-binding portion and at least two binding regions, each of which binds to another antigen.

68. A pharmaceutical composition comprising an ADC according to any one of claims 1 to 67 and a pharmaceutically acceptable carrier.

69. The pharmaceutical composition according to claim 68, further comprising one or more further therapeutic agents.

70. A kit comprising the ADC according to any one of claims 1 to 69, and an instruction manual.

71. A method for producing an ADC according to any one of claims 1 to 70, comprising conjugating the anti-CEACAM5 antibody or its antigen-binding portion with exatecan.

72. A method for treating cancer expressing CEACAM5 in a subject requiring treatment, comprising administering to the subject a therapeutically effective amount of an ADC according to any one of claims 1 to 71 or a pharmaceutical composition according to any one of claims 68 to 71.

73. The method according to claim 72, wherein the cancer is selected from the group consisting of colorectal cancer, breast cancer, lung cancer including non-small cell lung cancer (NSCLC), ovarian cancer, pancreatic cancer, bladder cancer, uterine / cervical cancer, prostate cancer, testicular cancer, esophageal cancer, gastric cancer, gastrointestinal cancer, colon cancer, kidney cancer, head and neck cancer, gastric cancer, germ cell cancer, osteosarcoma, liver cancer, thyroid cancer, skin cancer, tumors of the central nervous system, lymphoma, leukemia, myeloma, sarcoma, and myelodysplastic syndrome.

74. The method according to claim 72 or 73, further comprising administering one or more further therapies.

75. The method according to claim 74, wherein the one or more further therapies include radiotherapy, chemotherapy, immune checkpoint inhibitor therapy, CAR-T therapy, immunosuppressive therapy, immunostimulatory therapy, cell therapy, or any combination thereof.

76. The method according to claim 74 or 75, wherein the one or more further therapies include an immune checkpoint inhibitor.

77. The method according to claim 76, wherein the immune checkpoint inhibitor comprises an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-LAG-3 antibody, an anti-CTLA-4 antibody, an anti-TIGIT antibody, an anti-TIM3 antibody, or any combination thereof.

Images