Anti-ceacam5 antibody drug conjugates

Abstract

Provided herein are antibody drug conjugates of Formula (I) comprising an anti-CEACAM5 antibody, antigen-binding portion thereof, conjugated to a linker and exatecan. The present disclosure also provides a method of treating cancer in a subject in need thereof, comprising administering to the subject an antibody drug conjugate disclosed herein.

Description

Cross-reference to related applications

[0001] This application claims the benefit of U.S. Provisional Patent Application Serial No. 63 / 558,431, filed February 27, 2024, and U.S. Provisional Patent Application Serial No. 63 / 678,883, filed August 2, 2024, which are each incorporated herein by reference in their entirety. References to sequence lists submitted electronically

[0002] The contents of the sequence list (name: 3338_337PC03_SequenceListing_ST26.xml; size: 114,947 bytes; creation date: February 9, 2025), which was submitted electronically, are submitted together with this application and are incorporated herein by reference in their entirety. Technical Field

[0003] This disclosure provides antibody-drug conjugates (ADCs) for use in therapies, the antibody-drug conjugates 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 incidence and expression in more than 80% of colorectal cancers. In normal tissues, CEACAM5 protects luminal organs from microbial invasion. In tumor cells, CEACAM5 is functionally associated with cell differentiation, cell adhesion, tumor invasion, and metastasis.

[0005] CEACAM5 overexpression is generally associated with poor prognosis. For example, in patients with stage I, II, and III colorectal cancer, 5-year survival has been inversely correlated with tissue expression of CEACAM5, and in patients with stage III disease, elevated serum levels of CEACAM5 are associated with poor prognosis (Gazzah et al., Ann Oncol. [Annals of Oncology], 33(4):416-425). Consistent overexpression of CEACAM5 in many cancers makes it a recognized tumor biomarker and indicator of recurrence in cancer patients, especially those with colorectal cancer.

[0006] Multiple therapeutic approaches targeting CEACAM5 in cancer are under development. However, to date, there are no FDA-approved CEACAM5-targeted therapies for cancer. Therefore, there remains an urgent need for effective treatments for CEACAM5-related cancers, including antibodies specifically targeting CEACAM5 that do not cross-react with other molecules in the CEACAM family, and antibody-drug conjugates (ADCs) that specifically kill CEACAM5-expressing cancer cells. Summary of the Invention

[0007] This disclosure provides an antibody-drug conjugate (ADC) having formula (I): (I) Or its pharmaceutically acceptable salts, stereoisomers or solvates, wherein: The configuration of the double bond is either E or Z. V is H or (C1-C8) alkyl; X is ; Y is NR 5 S, O or CR 6 R 7 ; R 1 It is a polyalkylene glycol unit containing at least three alkylene glycol subunits; R 3 and R 5 -R 7 Each is H, or an aliphatic or aromatic residue that is optionally substituted; L stands for connector; C represents the cytotoxic component; m is an integer ranging from 1 to 10; The range of n is from 1 to 20; and AB is an anti-CEACAM5 antibody or its antigen-binding moiety, which comprises: (a) A heavy chain variable region (VH) comprising complementarity-determining regions (CDR)1, CDR2, and CDR3 containing the amino acid sequences shown in SEQ ID NO: 14, 15, and 16, respectively; and a light chain variable region (VL) comprising CDR1, CDR2, and CDR3 containing the amino acid sequences shown in SEQ ID NO: 19, 20, and 21, or (b) VH, which comprises CDR1, CDR2, and CDR3 regions having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with the amino acid sequences shown in SEQ ID NO: 14, 15, and 16, respectively; and VL, which comprises CDR1, CDR2, and CDR3 regions having at least 85%, 90%, 95%, 98%, or 99% sequence identity with the amino acid sequences shown in SEQ ID NO: 19, 20, and 21, respectively.

[0008] This disclosure provides an antibody-drug conjugate having formula (I), wherein the VH and the VL have at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with the following amino acid sequences: (a) The amino acid sequences shown in SEQ ID NO: 38 and SEQ ID NO: 43, respectively; (b) The amino acid sequences shown in SEQ ID NO: 49 and SEQ ID NO: 50, respectively; (c) The amino acid sequences shown in SEQ ID NO: 51 and SEQ ID NO: 52, respectively; (d) The amino acid sequences shown in SEQ ID NO: 67 and SEQ ID NO: 68, respectively; (e) The amino acid sequences shown in SEQ ID NO: 69 and SEQ ID NO: 70, respectively; (f) The amino acid sequences shown in SEQ ID NO: 71 and SEQ ID NO: 72, respectively; (g) The amino acid sequences shown in SEQ ID NO: 73 and SEQ ID NO: 74, respectively; (h) The amino acid sequences shown in SEQ ID NO: 75 and SEQ ID NO: 76, respectively; (i) The amino acid sequences shown in SEQ ID NO: 77 and SEQ ID NO: 78, respectively; (j) The amino acid sequences shown in SEQ ID NO: 79 and SEQ ID NO: 80, respectively; (k) The amino acid sequences shown in SEQ ID NO: 81 and SEQ ID NO: 82, respectively; (l) The amino acid sequences shown in SEQ ID NO: 83 and SEQ ID NO: 84, respectively; (m) respectively, the amino acid sequences shown in SEQ ID NO: 85 and SEQ ID NO: 86; (n) respectively, the amino acid sequences shown in SEQ ID NO: 87 and SEQ ID NO: 88; (o) The amino acid sequences shown in SEQ ID NO: 89 and SEQ ID NO: 90, respectively; (p) Specifically, the amino acid sequences shown in SEQ ID NO: 91 and SEQ ID NO: 92, respectively; or (q) The amino acid sequences shown in SEQ ID NO: 93 and SEQ ID NO: 94, respectively.

[0009] 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 Table 10. For example, the anti-CEACAM5 antibody comprises a heavy chain and a light chain containing the amino acid sequences shown in SEQ ID NO: 45 and SEQ ID NO: 46, respectively.

[0010] This disclosure provides a method for preparing an ADC of formula (I), the method comprising: using a compound of formula (III): (III) With thiol-containing compounds AB-(SH) n The reaction is carried out to obtain the ADC.

[0011] This disclosure provides a method for treating a subject with CEACAM5-expressing cancer, the method comprising administering to the subject a therapeutically effective amount of an ADC of formula (I): (I) Or its pharmaceutically acceptable salts, stereoisomers or solvates, wherein: V is H or (C1-C8) alkyl; X is ; Y is NR 5 S, O or CR 6 R 7 ; R 1 It is a polyalkylene glycol unit containing at least three alkylene glycol subunits; R 3 and R 5 -R 7 Each is H, or an aliphatic or aromatic residue that is optionally substituted; L stands for connector; C represents the cytotoxic component; m is an integer ranging from 1 to 10; The range of n is from 1 to 20; and AB is an anti-CEACAM5 antibody or its antigen-binding moiety comprising VH and VL, wherein the VH and VL have at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with the following amino acid sequences: (a) The amino acid sequences shown in SEQ ID NO: 38 and SEQ ID NO: 43, respectively; (b) The amino acid sequences shown in SEQ ID NO: 49 and SEQ ID NO: 50, respectively; (c) The amino acid sequences shown in SEQ ID NO: 51 and SEQ ID NO: 52, respectively; (d) The amino acid sequences shown in SEQ ID NO: 67 and SEQ ID NO: 68, respectively; (e) The amino acid sequences shown in SEQ ID NO: 69 and SEQ ID NO: 70, respectively; (f) The amino acid sequences shown in SEQ ID NO: 71 and SEQ ID NO: 72, respectively; (g) The amino acid sequences shown in SEQ ID NO: 73 and SEQ ID NO: 74, respectively; (h) The amino acid sequences shown in SEQ ID NO: 75 and SEQ ID NO: 76, respectively; (i) The amino acid sequences shown in SEQ ID NO: 77 and SEQ ID NO: 78, respectively; (j) The amino acid sequences shown in SEQ ID NO: 79 and SEQ ID NO: 80, respectively; (k) The amino acid sequences shown in SEQ ID NO: 81 and SEQ ID NO: 82, respectively; (l) The amino acid sequences shown in SEQ ID NO: 83 and SEQ ID NO: 84, respectively; (m) respectively, the amino acid sequences shown in SEQ ID NO: 85 and SEQ ID NO: 86; (n) respectively, the amino acid sequences shown in SEQ ID NO: 87 and SEQ ID NO: 88; (o) The amino acid sequences shown in SEQ ID NO: 89 and SEQ ID NO: 90, respectively; (p) Specifically, the amino acid sequences shown in SEQ ID NO: 91 and SEQ ID NO: 92, respectively; or (q) The amino acid sequences shown in SEQ ID NO: 93 and SEQ ID NO: 94, respectively.

[0012] In some respects, the anti-CEACAM5 antibody or its antigen-binding moiety is described in Tables 10 and 11. In some respects, these CDRs, VHs, VLs, heavy chains, and / or light chains are described in Tables 10 and 11.

[0013] This disclosure provides an antibody-drug conjugate having formula (II): (II), or a pharmaceutically acceptable salt, stereoisomer, or solvate thereof, wherein: The configuration of the double bond is either E or Z; o is an integer from 8 to 30; The range of n is from 4 to 8; and AB is an anti-CEACAM5 antibody or its antigen-binding moiety comprising VH and VL, wherein the VH and VL have at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with the following amino acid sequences: (a) The amino acid sequences shown in SEQ ID NO: 38 and SEQ ID NO: 43, respectively; (b) The amino acid sequences shown in SEQ ID NO: 49 and SEQ ID NO: 50, respectively; (c) The amino acid sequences shown in SEQ ID NO: 51 and SEQ ID NO: 52, respectively; (d) The amino acid sequences shown in SEQ ID NO: 67 and SEQ ID NO: 68, respectively; (e) The amino acid sequences shown in SEQ ID NO: 69 and SEQ ID NO: 70, respectively; (f) The amino acid sequences shown in SEQ ID NO: 71 and SEQ ID NO: 72, respectively; (g) The amino acid sequences shown in SEQ ID NO: 73 and SEQ ID NO: 74, respectively; (h) The amino acid sequences shown in SEQ ID NO: 75 and SEQ ID NO: 76, respectively; (i) The amino acid sequences shown in SEQ ID NO: 77 and SEQ ID NO: 78, respectively; (j) The amino acid sequences shown in SEQ ID NO: 79 and SEQ ID NO: 80, respectively; (k) The amino acid sequences shown in SEQ ID NO: 81 and SEQ ID NO: 82, respectively; (l) The amino acid sequences shown in SEQ ID NO: 83 and SEQ ID NO: 84, respectively; (m) respectively, the amino acid sequences shown in SEQ ID NO: 85 and SEQ ID NO: 86; (n) respectively, the amino acid sequences shown in SEQ ID NO: 87 and SEQ ID NO: 88; (o) The amino acid sequences shown in SEQ ID NO: 89 and SEQ ID NO: 90, respectively; (p) Specifically, the amino acid sequences shown in SEQ ID NO: 91 and SEQ ID NO: 92, respectively; or (q) The amino acid sequences shown in SEQ ID NO: 93 and SEQ ID NO: 94, respectively.

[0014] In some respects, the anti-CEACAM5 antibody or its antigen-binding moiety is described in Tables 10 and 11. In some respects, these CDRs, VHs, VLs, heavy chains, and / or light chains are described in Tables 10 and 11.

[0015] This disclosure provides an antibody-drug conjugate having the following structure: [ADC 101], or a pharmaceutically acceptable salt, stereoisomer, or solvate thereof, wherein This indicates that the configuration of the double bond is E or Z; and AB is an anti-CEACAM5 antibody or its antigen-binding moiety comprising VH and VL, wherein the VH and VL have at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with the following amino acid sequences: (a) The amino acid sequences shown in SEQ ID NO: 38 and SEQ ID NO: 43, respectively; (b) The amino acid sequences shown in SEQ ID NO: 49 and SEQ ID NO: 50, respectively; (c) The amino acid sequences shown in SEQ ID NO: 51 and SEQ ID NO: 52, respectively; (d) The amino acid sequences shown in SEQ ID NO: 67 and SEQ ID NO: 68, respectively; (e) The amino acid sequences shown in SEQ ID NO: 69 and SEQ ID NO: 70, respectively; (f) The amino acid sequences shown in SEQ ID NO: 71 and SEQ ID NO: 72, respectively; (g) The amino acid sequences shown in SEQ ID NO: 73 and SEQ ID NO: 74, respectively; (h) The amino acid sequences shown in SEQ ID NO: 75 and SEQ ID NO: 76, respectively; (i) The amino acid sequences shown in SEQ ID NO: 77 and SEQ ID NO: 78, respectively; (j) The amino acid sequences shown in SEQ ID NO: 79 and SEQ ID NO: 80, respectively; (k) The amino acid sequences shown in SEQ ID NO: 81 and SEQ ID NO: 82, respectively; (l) The amino acid sequences shown in SEQ ID NO: 83 and SEQ ID NO: 84, respectively; (m) respectively, the amino acid sequences shown in SEQ ID NO: 85 and SEQ ID NO: 86; (n) respectively, the amino acid sequences shown in SEQ ID NO: 87 and SEQ ID NO: 88; (o) The amino acid sequences shown in SEQ ID NO: 89 and SEQ ID NO: 90, respectively; (p) Specifically, the amino acid sequences shown in SEQ ID NO: 91 and SEQ ID NO: 92, respectively; or (q) The amino acid sequences shown in SEQ ID NO: 93 and SEQ ID NO: 94, respectively.

[0016] In some aspects, the anti-CEACAM5 antibody or its antigen-binding moiety is described in Tables 10 and 11. In some aspects, these CDRs, VHs, VLs, heavy chains, and / or light chains are described in Tables 10 and 11. For example, the anti-CEACAM5 antibody or its antigen-binding moiety comprises a heavy chain and a light chain containing the amino acid sequences shown in SEQ ID NO: 45 and SEQ ID NO: 46, respectively. Attached Figure Description

[0017] Figure 1A-Figure 1B This is a table showing the competition and binning of selected anti-CEACAM5 binding antibodies.

[0018] Figures 2A-2DThis is a graph showing cell-based binding of anti-CEACAM5 antibodies as determined by flow cytometry (AF647). Anti-CEACAM5 antibodies were measured to test their binding with the following cell lines expressing different levels of human CEACAM5: BXPC-3 (…). Figure 2A ), Ls174T ( Figure 2B ), MKN-45 ( Figure 2C ) and HCT-116 ( Figure 2D ).

[0019] Figures 3A-3C The graph depicts the percentage of cell death (inhibition %) induced by the delivery of the cytotoxic agent MMAE, which is conjugated to the second VHH, in MKN45 cells. Figure 3A ), the internalization rate (red area / phase area) of the selected anti-CEACAM5 antibody ( Figure 3B ) and the cytotoxicity levels (cytotoxicity AUC) of these selected antibodies as a function of internalization (internalization AUC) ( Figure 3C ).

[0020] Figures 4A-4B The following graph shows the percentage of growth inhibition in Ls174T cells by delivering the cytotoxic agent MMAE conjugated to the VHH secondary antibody against CEACAM5. Figure 4A ), the internalization rate (red area / phase area) of the selected anti-CEACAM5 antibody ( Figure 4B ).

[0021] Figure 5A This is a general description of the mutation analysis performed to generate a single mutant library and to select progeny antibodies that bind to human and cynomolgus monkey CEACAM5. Figure 5B Mutation scans of MBN001 are shown. CDR positions were analyzed (according to Kabat), and mutations in germline VH include T7S, S40A, A68T, and P84A. Figures 5C-5N This is a heatmap of human CEACAM5 and cynomolgus monkey CEACAM5 generated by mutation scanning analysis using the MBN001 antibody for the following substitutions: LCDR1 substitution (SEQ ID NO: 64, pedigree and parent). Figure 5I and Figure 5J ); LCDR2 substitution (SEQ ID NO: 65, phylum and parents) Figure 5K and Figure 5L ); LCDR3 substitution (SEQ ID NO: 66, phylum and parents) ( Figure 5M and Figure 5N ); HCDR1 substitution (SEQ ID NO:61, phylum and parents) Figure 5C and Figure 5D ); HCDR2 substitution (SEQ ID NO: 62, phylum and parents) Figure 5E and Figure 5F ); and HCDR3 substitution (SEQ ID NO: 63, phylum and parents) ( Figure 5G and Figure 5H Note that in the figure described here, ND indicates that the enrichment ratio could not be determined because the NGS count in the starting library was too low.

[0022] Figures 6A-6B This is an isoaffinity plot, which shows the affinity of the parent antibody MBN001 for human CEACAM5 compared to the optimized progeny of MBN001. Figure 6A ) and cyno CEACAM5 ( Figure 6B Improvement of association and dissociation rates.

[0023] Figures 7A-7C This is a set of graphs showing the percentage of inhibition of anti-CEACAM5 antibody by delivering the cytotoxic agent MMAE conjugated to the VHH secondary antibody in MKN45 cells. Figure 7A The internalization rates of the MBN001 antibody and selected progeny (MBP004, MBP005, MBP007, MBP008, MBP009, MBP010, MBP006, and MBP011) Figure 7B ) and the cytotoxicity levels of the antibody and selected progeny as internalization changes ( Figure 7C ).

[0024] Figures 8A-8B This is a set of figures showing the absence of MBN001 and progeny mAbs MBP003, MBP001, and MBP002 in contrast to cell lines engineered to express human CEACAM1 (CHO-S). Figure 8A ) and cell lines engineered to express CEACAM6 (HCT-116; Figure 8B Nonspecific binding of ).

[0025] Figures 9A-9C This is a set of figures showing, through FACS analysis, the effects of anti-CEACAM5 mAb MBN001, MBP001, MBP003, and MBP002 on LS174T cells with low CEACAM5 expression ( Figure 9A ), CEACAM moderately expresses BxPC-3 cells ( Figure 9B ) and CEACAM5-highly expressed MKN45 cells ( Figure 9C The combination of ) . EC50 values ​​are shown in Figure 9D (MBN001, MBP001, MBP002 and MBP003).

[0026] Figures 10A-10D This is a set of figures and tables showing the percentage of inhibition of anti-CEACAM5 antibodies conjugated to compound A' in a group of CEACAM5-expressing cell lines. These figures show the percentage of growth inhibition of the selected antibodies conjugated to compound A' compared to the isotype control at the indicated antibody concentrations in the following cell lines: low CEACAM5 expression cell line Ls174T (… Figure 10A ), the moderately CEACAM5-expressing cell line BxPC-3 ( Figure 10B ) and the high CEACAM5 expression cell line MKN-45 ( Figure 10C ). Figure 10D This is a table of IC50 values ​​for antibody-drug conjugates.

[0027] Figures 11A-11B The following figure shows: In the Jurkat-NFAT-FcγRIIIa (Promega) cell assay, anti-human CEACAM5 mAb was present in CEACAM5-expressing BxPC3 cells ( Figure 11A ) and CEACAM5-highly expressed MKN45 cells ( Figure 11B ADCC activity in ).

[0028] Figures 12A-12D This is a set of images showing the effects of co-culturing MKN45 cells (Ag+); Figure 12A and Figure 12C ) and HCT-116 cells (Ag-; Figure 12B and Figure 12D ), and then treated with ADP001A, ADCP001B, ADCP001C (ADCs with MBP001, MBP002 or MBP003 conjugated to compound A') for bystander kill effect at 72 hours and 120 hours.

[0029] Figure 13A This is a strip representation of the 3D model of human CEACAM5 along with its individual structural domains. Add boxes to this figure to highlight the A3 and B3 structural domains of human CEACAM5. Figure 13B The amino acid sequence of hCEACAM5 (UniProt entry: P06731) is shown, with shaded areas indicating... Figure 13A The individual structural domain shown (SEQ ID NO: 25). Figure 13C This is a graph showing the digestion sequence coverage of the hCEACAM5-A3-B3 construct (SEQ ID NO: 24). Figure 13DThis is a graph showing the differential deuterium uptake between hCEACAM5 bound to MBN001 and unbound hCEACAM5. Regions in hCEACAM5 that exhibit a significant reduction in HDX after MBN001 binding are boxed. Figure 13E This is a diagram illustrating the HDX effect of MBN001 binding on the linear hCEACAM5 sequence. Bold residues show slower exchange, italic residues provide no information, and no differences were detected on orthographic residues.

[0030] Figure 14A This is the final CryoEM graph, and Figure 14B and Figure 14C This is a strip plot derived from cryoEM based on the final structure model, which shows epitope and complementary site interactions between the human CEACAM5 and MBP001 Fab constructs and the Fab of box 2 mAb.

[0031] Figures 15A-15E It is a set of graphs that show the effect of using 3 mg / kg ( Figure 15A , Figure 15B and Figure 15C ) or 10 mg / kg ( Figure 15D and Figure 15E Following a single intravenous injection of ADC, in the MKN45 CDX model ( Figure 15A BxPC3 Figure 15B and Figure 15D ) and Ls174T ( Figure 15C and Figure 15E The in vivo efficacy of ADCN001 (MBN001 (hIgG1.3) (DAR8) conjugated to compound A') in compound A'.

[0032] Figures 16A-16B These are simple Western blot images and the following figure illustrates the induction of pharmacodynamic markers of DNA damage response (including pKAP, pCHK1, gH2AX) in tumors (n = 3) from the MKN45 CDX model, excluding the expression of c-cysteine-3, a marker of apoptosis, at 6, 24, and 168 hours after a single intravenous injection of ADCN001 (MBN001 + compound A' ADC) at 1 mg / kg or 10 mg / kg. The expression intensities of the DNA damage response markers pKAP (1TF1b) (ser 824), pCHK1, gH2AX, and the apoptosis marker c-cysteine-3 were normalized relative to the expression intensities of the loading control GAPDH.

[0033] Figures 17A-17D It is a set of graphs that show the effect of using 3 mg / kg ( Figure 17A and Figure 17B) or 10 mg / kg ( Figure 17C and Figure 17D Following a single intravenous injection of ADCV001, in the CDX model (MKN45) Figure 17A and Figure 17C ) and BxPC3 ( Figure 17B and Figure 17D The in vivo efficacy of ADCV001 (MBV001 conjugated to compound A' in the wild-type hIgG1 form of MBN001) in compound A'.

[0034] Figures 18A-18D It is a set of graphs that show the effect of using 3 mg / kg ( Figure 18A and Figure 18B ) or 10 mg / kg ( Figure 18C and Figure 18D Following a single intravenous injection of ADC, in the CDX model (MKN45) Figure 18A and Figure 18C ) and BxPC3 ( Figure 18B and Figure 18D The in vivo efficacy of ADCP001A (MBP001 + compound A' ADC), ADCP001B (MBP002 + compound A' ADC) and ADCP001C (MBP003 + compound A' ADC) in [the study].

[0035] Figures 19A-19C It is a set of graphs that show the effect of using 3 mg / kg ( Figure 19A ) or 10 mg / kg ( Figure 19B and Figure 19C The in vivo efficacy of ADCV001 and M9140 in the CDX model MKN45 following a single intravenous injection of ADCV001 and Merck ADC1. Figure 19C yes Figure 19B Enlarged image.

[0036] Figure 20 These are Western blot images showing the sustained induction of pharmacodynamic markers of DNA damage response (including pKAP1 and pCHK1) in tumors (n = 4) from the MKN45 CDX model at 6, 48, 72, 240, and 336 hours after a single intravenous injection of 3 mg / kg ADCV001 and M9140. Detailed Implementation

[0037] This disclosure provides antibody-drug conjugates (ADCs) of formula (I) and formula (II) or ADC 101, pharmaceutical compositions comprising the ADC, and the use of the ADC in methods of treating diseases such as cancer. definition

[0038] To facilitate understanding of the following detailed explanation, some terms are first defined. Additional definitions are provided throughout the document.

[0039] The term "alkyl" itself, or as part of another term, generally refers to a straight-chain or branched saturated hydrocarbon with a specified number of carbon atoms, whether substituted or unsubstituted; for example, "-(C1-C8)alkyl" or "-(C1-C8)alkyl". 10 "alkyl" refers to an alkyl group having 1 to 8 or 1 to 10 carbon atoms. When the number of carbon atoms is not specified, the alkyl group may have 1 to 8 carbon atoms. Representative straight-chain (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-chain (C1-C8) alkyl groups include, but are not limited to, -isopentyl, -sec-butyl, -isobutyl, -tert-butyl, and -2-methylbutyl. In some aspects, the alkyl group may be unsubstituted. Optionally, the alkyl group may be substituted, for example, by one or more groups.

[0040] The term "polyalkylene glycol unit" refers to a unit having the formula -[(CH2)] n -O] y - A repeating alkylene glycol subunit, where n is the number of methylene groups in the subunit and y is the number of subunits in the unit. The oxygen atom of the terminal subunit may be substituted with a hydrogen atom, a protecting group, or any other permitted functional group.

[0041] Unless otherwise indicated, the terms "substituted," "optionally substituted," and "optionally replaceable" generally mean that one or more hydrogen atoms can be independently substituted by a substituent. Typical substituents include, but are not limited to, -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- -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, -(Cl-C20 )alkyl (e.g., -(C1-C) 10 alkyl or -(C1-C8)alkyl), -(C6-C 20 )Aryl (e.g., -(C6-C) 10 ) aryl or, for example, -C6-aryl), -(C3-C 14 Heterocyclic rings (such as, for example, -(C3-C)) 10 ( ) heterocyclic or -(C3-C8) heterocyclic, protecting group or prodrug moiety. Typical substituents also include (=O).

[0042] As used herein, the term "aliphatic or aromatic residue" generally refers to an aliphatic substituent, such as, but not limited to, an alkyl residue; however, this substituent may optionally be replaced by other aliphatic and / or aromatic substituents. As a non-limiting example, an aliphatic residue can be a nucleic acid, enzyme, coenzyme, nucleotide, oligonucleotide, monosaccharide, polysaccharide, polymer, fluorophore, optionally substituted benzene, etc., provided that the direct connection of such a molecule to the core structure (in the case of R', e.g., the connection to the nitrogen atom of Y) is aliphatic. An aromatic residue is a substituent in which the direct connection to the core structure is part of an aromatic system, such as optionally substituted phenyl, triazolyl, or pyridyl groups or nucleotides, as a non-limiting example if the direct connection of the nucleotide to the core structure is achieved, for example, via a phenyl residue. As used herein, the term "aromatic residue" also includes heteroaromatic residues.

[0043] As used herein, the term "antibody" includes the complete antibody and any antigen-binding portion (i.e., the "antigen-binding portion") or a single chain. In one aspect, "antibody" refers to a glycoprotein, or its antigen-binding portion, comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated as V in this document). H The heavy chain constant region consists of three domains: CH1, CH2, and CH3. In some naturally occurring antibodies, each light chain consists of a light chain variable region (abbreviated as V in this paper). L It consists of a light chain constant region and a structural domain CL. H and V L The region can be further subdivided into highly variable areas called complementary determinant regions (CDRs), within which more conservative regions are interspersed, called frame regions (FRs). Each V H and V LIt consists of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: 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 regions of the antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.

[0044] Antibodies typically bind specifically to their homologous antigens with high affinity, which is reflected in their dissociation constant (K). D ) is 10 -5 Up to 10 -11 M or smaller. Generally considered to be greater than approximately 10. -4 M any K D Indicates 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, meaning K D 10 -7 M or smaller, 10 -8 M or smaller, 1 x 10 -9 M or smaller, 1 x 10 -10 M or smaller, or 1 x 10 -11 M or smaller. In some respects, antibodies can reach 10 -8 M and 10 -10 Between M or 10 -9 M and 10 -11 K between M D It binds specifically to antigens, but does not bind to irrelevant antigens with high affinity.

[0045] According to this disclosure, “antibodies” include, but are not limited to, naturally occurring and non-naturally occurring antibodies, monoclonal antibodies, polyclonal antibodies, chimeric antibodies, humanized antibodies, human antibodies, non-human antibodies, bivalent antibodies, bispecific antibodies, multispecific antibodies, single-chain antibodies, biantibodies, and nanobodies.

[0046] As used in this article, "isolated antibody" refers to an antibody that does not contain other antibodies with different antigen specificities.

[0047] As used herein, the “antigen-binding portion” of a phrase antibody refers to one or more fragments of the antibody that retain the ability to specifically bind to an antigen (e.g., human and / or cynomolgus monkey CEACAM5). Fragments of full-length antibodies have been shown to perform the antigen-binding function of the antibody. Examples of binding fragments of antibodies include (i) Fab fragments, which are composed of V... L V H(ii) a monovalent fragment consisting of CL and CH1 domains; (iii) a divalent fragment consisting of two Fab fragments connected by a disulfide bridge at the hinge region; and (iv) an Fd fragment consisting of V... H (iv) The Fv fragment, consisting of the V-arm of the antibody, is composed of the CH1 domain; L and V H Domain composition; (v) dAb fragment (Ward et al., (1989) Nature 341:544-546), composed of V H The structural domains consist of (vi) separate complementary determinant regions (CDRs) or (vii) combinations of two or more separate CDRs that may optionally be connected by a synthetic joint. Furthermore, although the two structural domains V of the Fv segment... L and V H Encoded by individual genes, but which can be linked together using recombination methods via synthetic adapters, allowing them to be made into single protein chains, where V L and V H Regions pair to form monovalent molecules (called single-chain Fvs (scFvs); see, for example, Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single-chain antibodies are also intended to be encompassed within the term "antigen-binding moiety" of antibody. The antigen-binding moiety can be generated using recombinant DNA technology or through enzymatic or chemical cleavage of intact immunoglobulins.

[0048] The antibody fragments within the scope of this invention also include the F(ab')2 fragment, which can be generated by enzymatic cleavage of IgG, for example, by pepsin. The Fab fragment can be generated, for example, by reducing F(ab')2 with dithiothreitol or mercaptoethylamine. The Fab fragment is attached to the VL-CL chain of the VH-CH1 chain via a disulfide bridge. The F(ab')2 fragment consists of two Fab fragments, which are subsequently attached via two disulfide bridges. The Fab portion of the F(ab')2 molecule includes a portion of the Fc region, with the disulfide bridge located between them.

[0049] As used in this article, “isotype” refers to the class of antibodies 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).

[0050] Antibodies can be derived from any known isotype, including but not limited to IgA, secretory IgA, IgG, and IgM. IgG isotypes are divided into the following subclasses in some species: IgG1, IgG2, IgG3, and IgG4 in humans, and IgG1, IgG2a, IgG2b, and IgG3 in mice. Immunoglobulins (e.g., IgG1) exist in several allotypes, differing from each other by at most a few amino acids.

[0051] As used herein, the term "allotype" refers to a naturally occurring variant within a specific allotype group, wherein these variants differ in a few amino acids. The anti-CEACAM5 antibody described herein can belong to any allotype. The antibody referred to herein as "IgG1.3f" is an IgG1 antibody of allotype "f," i.e., having 214R, 356E, and 358M according to the EU index. The triple mutant (L234A, L235E, G237A) IgG1.3f variant contains the amino acid sequence shown 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, thereby reducing the activating efficacy of the Fc domain of the IgG1 component of the antibody.

[0052] As used herein, the term “hypervariant region” (sometimes called “variable region”) refers to the amino acid residues in an antibody responsible for antigen binding. The hypervariant region contains amino acid residues 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 Edition. Public Health Service, National Institutes of Health. (Bethesda, Maryland) and / or residues from the “high-variable ring” (i.e., residues 26-32 (L1), 50-52 (L2), and 91-96 (L3) in the light chain variable domain and 26-32 (H1), 53-55 (H2), and 96-101 (H3) in the heavy chain variable domain; Chothia and Lesk, (1987) J. Mol. Biol. [Journal of Molecular Biology] 196: 901-917).

[0053] As used herein, the term "frame" or "FR" residue refers to those variable domain residues other than the hypervariable region residues defined herein as CDR residues. The residue numbering above refers to the Kabat numbering system and does not necessarily correspond precisely to the sequence numbers in the attached sequence listing. Amino acid residues in antibodies can also be defined using other numbering systems, such as Chothia, enhanced Chothia, IMGT, Kabat / Chothia combination, Honegger (AHo), Contact, or any other conventional antibody numbering scheme.

[0054] The term "recipient human framework" refers to a V-framework that contains components derived from the human immunoglobulin framework or the human common framework. L Frame or V H The amino acid sequence of the frame. Recipient human frames “derived from” the human immunoglobulin frame or the human common frame can have the same amino acid sequence as naturally occurring human immunoglobulin frames or the human common frame, or they can have amino acid sequence variations compared to wild-type naturally occurring human immunoglobulin frames or the human common frame. In some respects, the number of amino acid variations is 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1. In some respects, V L The recipient human framework is sequence-related to V L The human immunoglobulin framework sequence or the human common framework sequence is the same.

[0055] The term "Fc region," "Fc domain," or simply "Fc" refers to the C-terminal region of the antibody's heavy chain. Therefore, the Fc region contains the antibody's constant regions, excluding the first constant region, the immunoglobulin domain (e.g., CH1 or CL).

[0056] "Effective functions" refer to the interaction between the antibody Fc region and the Fc receptor or ligand, or the resulting biochemical event. Exemplary "effective functions" include Clq binding, complement-dependent cytotoxicity (CDC), Fc receptor binding, FcγR-mediated effector functions (such as ADCC and antibody-dependent cell-mediated phagocytosis (ADCP)), and downregulation of cell surface receptors (e.g., B cell receptor; BCR). Such effector functions typically require a combination of the Fc region and a binding domain (e.g., antibody variable domain).

[0057] The term "epitope" or "antigenic determinant" refers to a site on an antigen (e.g., human CEACAM5) that specifically binds to an immunoglobulin or antibody. Epitopes can be formed from consecutive amino acids (typically linear epitopes) or from discontinuous amino acids juxtaposed through the ternary folding of a protein (typically conformational epitopes). Epitopes formed from consecutive amino acids are typically (but not always) retained upon exposure to denaturing solvents, while epitopes formed through ternary folding are typically lost upon treatment with denaturing solvents. Epitopes typically comprise 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 unique spatial conformation.

[0058] As used herein, the term "monoclonal antibody" refers to an antibody that exhibits a single binding specificity and affinity for a specific epitope, or an antibody composition in which all antibodies exhibit a single binding specificity and affinity for a specific epitope. Therefore, the term "human monoclonal antibody" refers to an antibody or antibody composition that exhibits a single binding specificity and has variable regions and optional constant regions derived from human germline immunoglobulin sequences. In one aspect, human monoclonal antibodies are produced by hybridomas comprising B cells obtained from transgenic nonhuman animals (e.g., transgenic mice) fused with immortalized cells, the transgenic nonhuman animal possessing a genome containing human heavy chain transgenes and light chain transgenes. Monoclonal antibodies include chimeric antibodies, human antibodies, and humanized antibodies, and can be naturally occurring or recombinantly generated.

[0059] The monoclonal antibodies described in this article also include camel-derived single-domain antibodies. See, for example, Muyldermans et al. (2001) Trends Biochem. Sci. 26:230; Reichmann et al. (1999) J. Immunol. Methods 231:25; WO 94 / 04678; WO 94 / 25591; U.S. Patent No. 6,005,079, which are incorporated herein by reference in their entirety. In one respect, this article provides for antibodies containing two V H Single-domain antibodies with two V-domains H The domains are modified to form single-domain antibodies.

[0060] The term "recombinant antibody" refers to antibodies prepared, expressed, produced, or isolated by recombinant means, such as (a) antibodies isolated from transgenic or transchromosomally transgenic animals (e.g., mice) or hybridomas prepared from such animals that are transgenic or transchromosomally transgenic for immunoglobulin genes (e.g., human immunoglobulin genes), (b) antibodies isolated from host cells transformed to express antibodies (e.g., from transfected tumors), (c) antibodies isolated from recombinant combined antibody libraries (e.g., containing human antibody sequences) using phage display, and (d) antibodies prepared, expressed, produced, or isolated by any other means involving splicing immunoglobulin gene sequences (e.g., human immunoglobulin genes) to other DNA sequences. Such recombinant antibodies may have variable and constant regions derived from human germline immunoglobulin sequences. However, in some respects, such recombinant human antibodies can undergo in vitro mutagenesis, thus allowing the V of the recombinant antibody to undergo further mutagenesis. H and V L The amino acid sequence of the region is as follows, although these sequences originate from human lineage V. H and V L The sequence is related to it, but it may not be naturally present in the human antibody germline library in the body.

[0061] "Human" antibodies are those possessing 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, that constant region is also derived from a human germline immunoglobulin sequence. This also includes antibodies derived from human germline immunoglobulin sequences, including normal somatic hypermutations that alter the germline immunoglobulin sequence relative to the wild-type germline immunoglobulin sequence.

[0062] "Humanized" antibodies are those in which some, most, or all of the amino acids outside the CDR domain of a non-human antibody are replaced by corresponding amino acids derived from human immunoglobulins. In one aspect of the humanized form of an antibody, some, most, or all of the amino acids outside the CDR domain have been replaced by amino acids from human immunoglobulins, while some, most, or all of the amino acids within one or more CDR domains remain unchanged. Any addition, deletion, insertion, substitution, or modification of amino acids is permitted, as long as they do not eliminate the antibody's ability to bind to a specific antigen. "Humanized" antibodies can retain antigen specificity similar to the original antibody.

[0063] The term "fully human antibody" refers to an antibody that contains only the sequence of human immunoglobulins. If produced in mice, in mouse cells, or in hybridomas derived from mouse cells, a fully human antibody may contain mouse carbohydrate chains. Similarly, a "mouse antibody" refers to an antibody that contains only the sequence of mouse immunoglobulins.

[0064] "Chimeric antibody" refers to an antibody whose variable region is derived from one or more species and whose constant region is derived from another species, such as an antibody whose variable region is derived from a mouse antibody and whose constant region is derived from a human antibody. See U.S. Patent No. 4,816,567; and Morrison et al. (1984) Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences] 81: 6851-6855.

[0065] "Domain antibodies" or "nanobodies" are immunofunctional immunoglobulin fragments 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 using peptide linkers to generate bivalent domain antibodies. The two V's of the bivalent domain antibody are... H The region can target the same or different antigens.

[0066] 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.

[0067] “Bispecific” or “bifunctional antibody” is an artificial hybrid antibody with two distinct heavy / light chain pairs and two distinct binding sites. Bispecific antibodies can be produced by a variety of methods, including hybridoma fusion or Fab fragment linkage. See, for example, Songsivilai and Lachmann, Clin. Exp. Immunol. [Clinical and Experimental Immunology] 79:315-321 (1990); Kostelny et al., J. Immunol. [Journal of Immunology] 148, 1547-1553 (1992). Bifunctional antibodies include, for example, heterodimeric antibody conjugates (e.g., two antibodies or antibody fragments linked together, each with different specificities), antibody / cell surface binding molecule conjugates (e.g., antibodies conjugated to non-antibody molecules such as receptors), and hybrid antibodies (e.g., antibodies with binding sites against two different antigens).

[0068] "Multispecific antibodies" are antibodies that recognize two or more different antigens or epitopes (e.g., bispecific antibodies, trispecific antibodies).

[0069] As used herein, the terms "single-chain Fv" or "scFv" antibody refer to a V containing an antibody. H and V LAntibody fragments containing domains, wherein these domains are present within a single polypeptide chain. Typically, Fv polypeptides further include polypeptide linkers. For a review of scFv, see Pluckthun (1994), *The Pharmacology of Monoclonal Antibodies*, Vol. 113, edited by Rosenburg and Moore, Springer-Verlag, New York, pp. 269–315.

[0070] As used herein, the term "dual antibody" refers to a small antibody fragment having two antigen-binding sites, wherein these fragments are contained within the same polypeptide chain and linked to a light chain variable domain (V). L The heavy chain variable structural domain (V) H (V) H -V L or V L -V H By using linkers that are too short to allow pairing between two domains on the same chain, these domains are forced to pair with complementary domains on another chain, resulting in two antigen-binding sites. Biantibodies are described more comprehensively in, for example, the following publications: EP 404,097; WO 93 / 11161; and Holliger et al. (1993) Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences] 90: 6444-6448. For reviews of engineered antibody variants, see Holliger and Hudson (2005) Nat. Biotechnol. [Nature Biotechnology] 23:1126-1136.

[0071] The term "immune cell connector" or "ICE" is used herein to refer to a multifunctional molecule containing two or more binding specificities that can redirect immune effector cells toward cancer cells. Exemplary immune cell connectors include T cell connectors (e.g., bispecific T cell connectors or BiTE), NK cell connectors (NKCE), B cell connectors, dendritic cell connectors, and macrophage connectors.

[0072] The terms “bispecific T-cell adaptor” and “BiTE” are used interchangeably herein to refer to a bispecific molecule that connects two target regions of antibody and / or protein binding domains, wherein one arm of the molecule is engineered to bind a protein (e.g., CD3) on the surface of a cytotoxic T cell (i.e., the T-cell adaptor), and the other arm is engineered to bind to a specific protein (e.g., CEACAM5) primarily found on tumor cells. When the two targets are connected, the BiTE molecule forms a bridge 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 include an immunoglobulin constant region.

[0073] The terms “bispecific NK cell adaptor” and “NKCE” are used interchangeably in this document to refer to a bispecific molecule containing a CEACAM5 binding domain that is linked to the binding domain of a cell surface protein of an NK cell via a short, flexible linker region (i.e., an NK cell adaptor).

[0074] The term "binding to the same epitope" is used to refer to the binding of two or more antibodies to one or more identical segments of amino acid residues. Techniques for determining whether an antibody binds to the same epitope can be determined using the epitope mapping methods described herein. Other methods involve monitoring the binding of antibodies to antigen fragments (e.g., proteolytic fragments) or to mutated variants of the antigen, where loss of binding due to modification of amino acid residues within the antigen sequence is generally considered to indicate epitope components. These other methods include alanine scanning mutagenesis (Cunningham and Wells (1985) Science [Science] 244:1081), yeast display of mutant target sequence variants, or analysis of chimeras. Additionally, computational combinatorial methods for epitope mapping can also be employed. These methods rely on the ability of the target antibody to affinity-isolate specific short peptides from a combinatorial phage-displayed peptide library. The expectation is that the same V H and V L Or antibodies with the same CDR1, 2 and 3 sequences bind to the same epitopes.

[0075] An antibody that “competes with another antibody to bind to a target” is one that inhibits (partially or completely) the binding of another antibody to a target. Whether two antibodies compete with each other to bind to a target (i.e., whether and to what extent one antibody inhibits the binding of another antibody to a target) can be determined using known binding competition experiments involving surface plasmon resonance (SPR) and biolayer interferometry (BLI). In some respects, an antibody competes with another antibody to bind to a target and inhibits the binding of the other antibody to the target by at least 50%, 60%, 70%, 80%, 90%, or 100%. The level of inhibition or competition can vary depending on which antibody is a “blocking antibody” (i.e., an antibody that blocks another immune response to an antigen when combined with that antigen). Competitive assays can be performed, for example, as described in the following literature: Ed Harlow and David Lane, ColdSpring Harb. Protoc. [Cold Spring Harbour Laboratory Protocols] 2006; doi:10.1101 / pdb.prot4277; or Ed Harlow and David Lane, Chapter 11 of “Using Antibodies”, Cold Spring Harbour Laboratory Press, Cold Spring Harbour, New York, 1999. Competitive antibodies bind to the same epitope, overlapping epitopes, or adjacent epitopes (e.g., as demonstrated by steric hindrance). Two antibodies “cross-compete” if they bidirectionally block each other by at least 50% in a competitive assay, i.e., regardless of whether one or the other antibody comes into contact with the antigen first.

[0076] Competitive binding assays used to determine whether two antibodies compete or cross-competitively bind include competitive binding to cells expressing CEACAM5, determined, for example, by flow cytometry. 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 competitive 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: A Laboratory Manual, Cold Spring Harbor Press (1988)); solid-phase direct labeling RIA using I-125 labeling (see Morel et al., Mol. Immunol. 25(1):7 (1988)); solid-phase direct biotin-avidin EIA (Cheung et al., Virology 176:546) (1990)); and direct labeling of RIA. (Moldenhauer et al., Scand. J. Immunol. [Scandinavian Journal of Immunology] 32:77 (1990)).

[0077] As used herein, the terms “specific binding,” “selective binding,” “selectively binding,” and “specific binding” refer to the binding of an antibody to an epitope on a predetermined antigen. Typically, an antibody (i) is determined, for example, by surface plasmon resonance (SPR) using a predetermined antigen as the analyte and the antibody as the ligand, or by Scatchard analysis of the binding of the antibody to antigen-positive cells, with a binding density of approximately less than 10. -7 M, such as approximately less than 10 -8 M, 10 -9 M or 10 -10 M or even lower equilibrium dissociation constant (K) D (ii) The affinity for the predetermined antigen is at least twice that for nonspecific antigens (e.g., BSA, casein) other than the predetermined antigen or closely related antigens. Generally, an affinity greater than about 10 is considered acceptable. -4 M any K D Indicates nonspecific binding.

[0078] As used in this article, the term "k" assoc "or "k a "" refers to the association rate of a specific antibody-antigen interaction, while the term "k" as used herein... dis"or "k d "K" refers to the dissociation rate of a specific antibody-antigen interaction. As used herein, the term "K" is... D "Intended to refer to the dissociation constant, which is derived from k d With k a The ratio (i.e., k) d / k a And expressed as molar concentration (M). The K of the antibody D The value can be determined using methods established in the art. The K value used to determine the antibody... D The preferred method is to use surface plasmon resonance, for example, using a biosensor system (such as the BIACORE® system or flow cytometry) and Scatchard analysis or biolayer interferometry.

[0079] In the context of in vitro or in vivo assays using antibodies or immunoconjugates, the terms "EC50" or "IC50" refer to the concentration at which an antibody induces a response equal to 50% of the maximum response (i.e., half the response between the maximum and baseline). In pharmacology, the potency of a compound is expressed as the half-maximal effective concentration (EC50), which is the concentration at which a drug induces a response between baseline and maximum. While expressing a compound's potency through its EC50 value is meaningful in a clinical context, it is counterintuitive in the context of bioactivity-guided purification because a compound's potency is inversely correlated with its EC50 value, and the most effective compounds are those with the lowest EC50. The half-maximal inhibitory concentration (IC50) is the most widely used and informative measure of drug efficacy. It indicates how much drug is needed to inhibit a biological process by half, thus providing a measure of the potency of antagonist drugs in pharmacological studies.

[0080] As used herein, the term "linkage" refers to the association of two or more molecules. Linkages can be covalent or non-covalent. Linkages can also be genetic (i.e., recombination fusion). Such linkages can be achieved using a variety of techniques recognized in the art, such as chemical conjugation and recombinant protein production.

[0081] As used herein, the term "conjugate" is used to refer to an immunoconjugate or antibody-drug conjugate comprising an anti-CEACAM5 antibody or its antigen-binding portion linked to a cytotoxic or therapeutic drug as described herein.

[0082] As used herein, the term "connector" refers to a chemical portion comprising a covalent bond and / or any atomic chain that can be used to covalently attach, for example, a drug to an antibody. Connectors are known in the art and include, for example, disulfide groups, thioether groups, acid-labile groups, light-labile groups, peptidase-labile groups, and esterase-labile groups. The conjugation of the antibodies disclosed herein with cytotoxic drugs or other growth inhibitors can be performed, for example, using a variety of bifunctional protein conjugates, including but not limited to N-succinimide pyridine dithiobutyrate (SPDB), 4-[(5-nitro-2-pyridyl)dithio]-2,5-dioxo-1-pyrrolyl butyrate (nitro-SPDB), 4-(pyridin-2-yldithio)-2-sulfonyl-butyrate (sulfon-SPDB), N-succinimide (2-pyridyl dithio)propionate (SPDP), (N -maleimidemethyl)cyclohexane-1-carboxylic acid succinimide (SMCC), iminothiacyclopentane (IT), bifunctional derivatives of imine esters (such as dimethyl diimide adipate HCl), active esters (such as disuccinimide octanoate), aldehydes (such as glutaraldehyde), diazid compounds (such as bis(p-azidobenzoyl)hexanediamine), diazido derivatives (such as bis-(p-diazobenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and difluorinated compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For example, ricin immunotoxin can be prepared as described in Vitetta et al. (1987). Carbon-labeled 1-isothiocyanobenzylmethyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugating radioactive nucleotides to antibodies (WO 94 / 11026).

[0083] In some respects, a adapter is a “cleavable adapter” that can facilitate the release of cytotoxic drugs or other growth inhibitors inside or near cells (e.g., tumor cells). In other respects, an adapter is a cleavable adapter within the endosome of a mammalian cell. For example, acid-labile adapters, peptidase-sensitive adapters, esterase-labile adapters, light-labile adapters, or disulfide-containing adapters may be used (see, for example, U.S. Patent No. 5,208,020).

[0084] As used in this article, the term "nucleic acid molecule" refers to both DNA and RNA molecules. Nucleic acid molecules can be single-stranded or double-stranded, and can be cDNA.

[0085] As this article describes, it refers to encoding antibodies or antibody fragments (e.g., V). H V LThe term “isolated nucleic acid molecule” used for nucleic acids (CDR3) is intended to refer to nucleic acid molecules in which the nucleotide sequence is substantially free of other genomic nucleotide sequences, such as those encoding antibodies that bind to antigens other than CEACAM5, which can be naturally side-joined to the nucleic acid in human genomic DNA.

[0086] 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 attached. One type of vector is a "plasmid," which is a circular double-stranded DNA loop into which an additional DNA segment can be attached. Another type of vector is a viral vector, in which an additional DNA segment can be attached to a viral genome. Some vectors are capable of autonomous replication in the host cell to which they are introduced (e.g., bacterial vectors with bacterial origins of replication and attachable mammalian vectors). Other vectors (e.g., non-attached mammalian vectors) can be integrated into the host cell's genome after being introduced into the host cell, thereby replicating along with the host genome. Furthermore, some vectors are capable of directing the expression of genes operatively attached to them. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors"). Generally, expression vectors used in recombinant DNA technologies are typically in plasmid form. In this specification, "plasmid" and "vector" are used interchangeably, as plasmids are the most commonly used form of vector. However, other forms of expression vectors that perform equivalent functions, such as viral vectors (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses), are also included.

[0087] Also provided are “conserved sequence modifications” of the sequences shown herein, such as amino acid sequence modifications that do not eliminate the binding of antibodies encoded by nucleotide sequences or antibodies containing amino acid sequences to antigens. Such conserved sequence modifications include conserved nucleotide and amino acid substitutions, as well as the addition and deletion of nucleotides and amino acids. For example, modifications can be introduced into the sequence using standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. “Conserved amino acid substitutions” include substitutions in which amino acid residues are replaced by amino acid residues having similar side chains. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), amino acids with β-branched side chains (e.g., threonine, valine, isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Therefore, the predicted non-essential amino acid residues in anti-CEACAM5 antibodies can be replaced by another amino acid residue from the same side chain family. Methods for identifying conserved substitutions of nucleotides and amino acids that do not eliminate antigen binding are well known in the art (see, for example, Brummell et al., Biochem. [Biochemistry] 32:1180-1187 (1993); Kobayashi et al., Protein Eng. [Protein Engineering] 12(10):879-884 (1999); and Burks et al., Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences] 94:412-417 (1997)). Alternatively, in another aspect, mutations can be randomly introduced (e.g., by saturation mutagenesis) along all or part of the coding sequence of the anti-CEACAM5 antibody, and the resulting modified anti-CEACAM5 antibodies can be screened for binding activity.

[0088] For nucleic acids, the term "substantial homology" indicates that, when optimally aligned and compared, two nucleic acids or their designated sequences are identical in at least about 80% of their nucleotides, typically at least about 80% to 85%, 85% to 90%, or 90% to 95% of their nucleotides, and at least about 98% to 99.5% of their nucleotides, with appropriate nucleotide insertions or deletions. Alternatively, substantial homology exists when the segment hybridizes with the complement of the strand under selective hybridization conditions. For polypeptides, the term "substantial homology" indicates that, when optimally aligned and compared, two polypeptides or their designated sequences are identical in at least about 80% of their amino acids, typically at least about 80% to 85%, 85% to 90%, 90% to 95% of their amino acids, and at least about 98% to 99.5% of their amino acids, with appropriate amino acid insertions or deletions.

[0089] The percentage of identity between two sequences is a function of the number of common positions shared by the sequences (i.e., homology% = number of common positions / total number of positions x 100), taking into account the number of gaps required to achieve optimal alignment of the two sequences and the length of each gap. The comparison of sequences and the determination of the percentage of identity between them can be accomplished using mathematical algorithms, as illustrated in the following non-limiting examples.

[0090] The GAP procedure in the GCG software package (available at www.gcg.com) can be used to determine the percentage of identity between two nucleotide sequences using the NWSgapdna.CMP matrix and vacancy weights of 40, 50, 60, 70, or 80 and length weights of 1, 2, 3, 4, 5, or 6. Alternatively, the algorithm of E. Meyers and W. Miller (CABIOS, 4:11-17 (1989)) incorporated into the ALIGN procedure (version 2.0) can be used to determine the percentage of identity between two nucleotides or two amino acid sequences using a PAM120 weighted residue table, a vacancy length penalty of 12, and a vacancy penalty of 4. Alternatively, the algorithm of Needleman and Wunsch (J. Mol. Biol. [Journal of Molecular Biology] (48):444-453 (1970)) in the GAP program, which has been incorporated into the GCG software package (available at www.gcg.com), can be used to determine the percentage of identity between two amino acid sequences using a Blossum 62 matrix or a PAM250 matrix and vacancy weights of 16, 14, 12, 10, 8, 6 or 4 and length weights of 1, 2, 3, 4, 5 or 6.

[0091] The nucleic acid and protein sequences described herein can also be used as “query sequences” for searching public databases to identify related sequences, for example. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) described by Altschul et al. (1990) J. Mol. Biol. [Journal of Molecular Biology] 215:403-10. BLAST nucleotide searches 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. BLAST protein searches 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 vacancy alignments for comparative purposes, vacancy BLAST can be used as described by Altschul et al. (1997) Nucleic Acids Res. [Nucleic Acids Research] 25(17):3389-3402. When using the BLAST and empty BLAST procedures, the default parameters for the corresponding procedures (e.g., XBLAST and NBLAST) can be used. See www.ncbi.nlm.nih.gov.

[0092] As used herein, the term "recombinant host cell" (or simply "host cell") is intended to refer to a cell containing nucleic acids not naturally present in that cell, and may be a cell into which a recombinant expression vector has been introduced. It should be understood that such terms are intended not only to refer to the specific test cell, but also to the progeny of that cell. Because subsequent generations may contain modifications due to mutations or environmental influences, such progeny may actually differ from the parent cell, but are still included within the scope of the term "host cell" as used herein.

[0093] 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” can refer to a statistically significant reduction in biological activity of approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0094] As used herein, the term "immunotherapy" refers to treatment of a subject who has a disease or is at risk of contracting or relapsing from a disease by means of methods including inducing, enhancing, suppressing or otherwise modifying the immune response.

[0095] As used herein, the term "immunostimulatory therapy" or "immunostimulatory treatment" refers to a therapy that causes an increased (e.g., induced or enhanced) immune response in a subject, thereby treating, for example, cancer.

[0096] As used in this article, “immune cells” refers to a subset of blood cells known as white blood cells, which includes mononuclear cells such as lymphocytes, monocytes, macrophages, and granulocytes.

[0097] As used herein, “abnormal” is used when a molecule’s activity or level or expression is outside the normal range (e.g., overexpression) compared to, for example, a control or reference sample exhibiting a normal activity / expression profile. The term “normal” is used herein when the activity or expression level of a protein is found in a healthy, sex- and age-matched population of subjects. The minimum size of this healthy population can be determined using standard statistical measures, for example, practitioners can consider the incidence of disease in the general population and the expected level of statistical certainty in the outcome. In some respects, the normal range for the activity, level, or expression of a biomarker is determined from a population of subjects (e.g., at least five, ten, or twenty subjects), for example from a population of at least forty or eighty subjects, and from more than 100 subjects.

[0098] "T effect" eff "" cells refer to T cells with cytolytic activity (e.g., CD4+ and CD8+ T cells) and T helper (Th) cells, which secrete inflammatory cytokines and activate and guide other immune cells, but do not include regulatory T cells (Treg cells).

[0099] As used herein, “administration” means the physical introduction of a CEACAM5 target agent, such as an ADC (comprising an anti-CEACAM5 antibody or its antigen-binding portion described herein, attached via a connector to the cytotoxic portion described herein, alone or in combination with another therapeutic agent), into a subject using any of a variety of 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 administration routes, such as by injection or infusion. As used herein, the phrase “parenteral administration” means administration methods other than enteral and topical administration (usually by injection), and includes, but is not limited to, intravenous, intraperitoneal, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, tracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, spinal, epidural, and intrasternal injections and infusions, as well as in vivo electroporation. Alternatively, the antibodies described herein can be administered via non-parenteral routes (such as topical, epidermal, or mucosal administration), for example, intranasal, oral, vaginal, rectal, sublingual, or topical administration. Administration can also be performed, for example, once, multiple times, and / or over one or more extended time periods.

[0100] As used herein, “cancer” refers to a broad group of diseases characterized by the uncontrolled growth of abnormal cells in the body. Uncontrolled cell division can lead to the formation of malignant tumors or cells that invade adjacent tissues and may metastasize to distant parts of the body via the lymphatic system or bloodstream, and includes a variety of cancers, including but not limited to, carcinomas, melanomas, sarcomas, leukemias, lymphomas, germ cell tumors, and medulloblastomas. Exemplary cancers used for treatment include brain cancer, bladder cancer, breast cancer, cervical cancer, colon cancer, head and neck cancer, kidney cancer, lung cancer, non-small cell lung cancer, mesothelioma, ovarian cancer, prostate cancer, stomach cancer, uterine cancer, leukemia, and medulloblastoma.

[0101] As used herein, the term "small molecule drug" refers to a molecular entity that is typically organic or organometallic, not a polymer, that has pharmaceutical activity, and whose molecular weight is 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. This term covers most pharmaceutical compounds that are called "drugs," excluding proteins or nucleic acids; however, small peptides or nucleic acid analogs can be considered small molecule drugs. Examples include chemotherapeutic anticancer drugs and enzyme inhibitors. Small molecule drugs can be derived synthetically, semi-synthetically (i.e., from naturally occurring precursors), or biologically.

[0102] As used herein, the terms “treat,” “treating,” and “treatment” mean any type of intervention or procedure performed on a subject, or the administration of an active agent (e.g., an ADC comprising an anti-CEACAM5 antibody or its antigen-binding portion described herein, linked to a cytotoxic portion via a connector described herein) with the aim of preventing, reversing, alleviating, improving, or suppressing disease-related symptoms, complications, signs, or biochemical indicators, or slowing or preventing their progression, development, severity, or recurrence. Treatment may be directed at a subject with the disease or a subject without the disease (e.g., for prevention).

[0103] As used herein, “adjunctive” or “combination” administration (co-administration) includes the simultaneous administration of an ADC (e.g., an anti-CEACAM5 antibody or its antigen-binding portion thereof comprising a linker to the cytotoxic portion described herein) and one or more other agents and / or compounds in the same or different dosage forms, or in combination at individual doses in parallel or sequential administration. Therefore, an ADC (comprising an anti-CEACAM5 antibody or its antigen-binding portion thereof comprising a linker to the cytotoxic portion described herein) and second, third, or more agents and / or compounds (e.g., small molecules) may be administered simultaneously as a single formulation, or formulated for single and parallel or sequential administration.

[0104] As used herein, “combination therapy” means the administration of two or more therapeutic agents in a coordinated manner, and includes, but is not limited to, parallel and sequential administration. Specifically, combination therapy encompasses co-administration (e.g., administration of a co-prepared formulation or simultaneous administration of individual therapeutic compositions) and sequential or continuous administration of both, provided that the administration of one therapeutic agent is conditional in some way on the administration of another. For example, a therapeutic agent may be administered only after different therapeutic agents have been administered and allowed to act for a prescribed period of time. (See, for example, Kohrt et al. (2011) Blood 117:2423). For example, an ADC (containing an anti-CEACAM5 antibody linked via a linker to the cytotoxic portion described herein) may be administered first, followed (e.g., immediately thereafter) by a second agent (e.g., an antibody or its antigen-binding portion and an anticancer agent), or vice versa. In one aspect, the ADC is administered before the second agent. In another aspect, the ADC is administered, for example, within minutes (e.g., within about 30 minutes) or at least one hour after the second agent. Such parallel or sequential administration can result in both the ADC and the second agent being present simultaneously in the treated patient.

[0105] According to any of the methods provided herein, administration of an effective amount of a single ADC (comprising an anti-CEACAM5 antibody or its antigen-binding portion described herein, linked via a connector to the cytotoxic portion described herein) or an ADC in combination with another compound or agent (e.g., an immune checkpoint inhibitor, such as an anti-PD-1 antibody) can produce at least one therapeutic effect, including, for example, reduction in tumor growth or size, a reduction in the number of cancer markers (e.g., metastatic lesions) that appear over time, complete remission, partial remission, or disease stabilization. For example, these treatments can produce comparable clinical benefit rates (CBR = complete remission (CR) + partial remission (PR) + disease stabilization (SD) lasting ≥ 6 months), superior to the clinical benefit rates obtained without ADC administration, or superior to the clinical benefit rates obtained with either ADC or a second agent, for example, an improvement in clinical benefit rate of approximately 20%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or more.

[0106] As used herein, the terms “inhibition” and “blocking” (e.g., inhibition / blocking of CEACAM5 binding or functional activity) are used interchangeably and encompass both partial and complete inhibition / blocking achieved by an anti-CEACAM5 antibody or fragment thereof in an ADC, or other inhibition / blocking of functional activity by a therapeutic agent. The degree of inhibition relative to a control or reference antibody can be at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% (i.e., 2-fold or 2x), 3-fold, 5-fold, or 10-fold. Additionally, the degree of inhibition can be between 20%-95%, 20%-80%, 20%-50%, 40%-95%, 40%-80%, 40%-60%, 50%-90%, 50%-70%, 75%-95%, 75%-85%, 2-fold to 20-fold, 2-fold to 10-fold, 2-fold to 5-fold, 4-fold to 12-fold, or 4-fold to 8-fold.

[0107] The term "effective dose" or "effective dosage" is defined as an amount sufficient to achieve or at least partially achieve the desired effect. A "therapeuticly effective dose" or "therapeutic effective amount" of a drug or therapeutic agent (e.g., an ADC comprising an anti-CEACAM5 antibody or its antigen-binding portion described herein, linked via a connector to the cytotoxic portion described herein) is any amount of the drug or therapeutic agent that promotes disease regression when used alone or in combination with another therapeutic agent, demonstrated by a reduction in the severity of disease symptoms, an increase in the frequency and duration of symptom-free periods, or prevention of damage or disability caused by disease distress. A therapeutically effective dose or dosage of a drug or therapeutic agent includes a "preventatively effective dose" or "preventatively effective amount," which is any amount of the drug or therapeutic agent administered alone or in combination with another therapeutic agent to a subject at risk of developing or relapsing into the disease. The ability of a therapeutic agent to promote disease regression or inhibit disease progression or relapse can be evaluated using a variety of methods known to skilled practitioners, such as in human subjects during clinical trials, in animal model systems predicting human efficacy, or by measuring the activity of the agent in in vitro assays.

[0108] For example, in the treatment of tumors, a therapeutically effective amount or dose of a drug or therapeutic agent (e.g., an ADC comprising an anti-CEACAM5 antibody or its antigen-binding portion described herein, linked via a connector to the cytotoxic portion described herein) inhibits tumor cell growth by at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least higher than 70%, at least about 80%, or at least about 90%, relative to an untreated subject. In some aspects, a therapeutically effective amount or dose of a drug or therapeutic agent completely inhibits cell or tumor growth, i.e., inhibits cell or tumor growth by 100%. The ability of a compound or therapeutic agent (including antibodies) to inhibit tumor growth can be evaluated using the assays described herein. Alternatively, this property of a composition comprising a compound or therapeutic agent can be evaluated by examining the ability of the composition to inhibit cell growth; such inhibition can be measured in vitro using assays known to a skilled practitioner.

[0109] The term "patient" includes people and other mammalian subjects who receive preventative or therapeutic treatment.

[0110] As used herein, the term "subject" includes any human or non-human animal. For example, the methods and compositions described herein can be used to treat a subject with cancer. The term "non-human animal" includes all vertebrates, such as mammals and non-mammals, such as non-human primates, sheep, cats, dogs, cattle, chickens, amphibians, and reptiles.

[0111] The term "sample" refers to tissue, body fluid, or cells (or portions thereof) taken from a patient or subject. Typically, tissue or cells are taken from the patient, but in vivo diagnostics are also considered. In the case of solid tumors, tissue samples may be taken from surgically removed tumors and prepared for testing. In the case of lymphomas and leukemias, lymphocytes, leukemia cells, or lymphoid tissue (e.g., leukemia cells from blood) may be obtained and appropriately prepared. Other samples, including, for example, urine, tears, serum, plasma, cerebrospinal fluid, stool, sputum, and cell extracts, may also be used for specific cancers.

[0112] As used herein, the term “detection” (or “detected”) refers to qualitative and / or quantitative detection (measurement level) with or without reference to a control.

[0113] As used in this article, the term “diagnosis” refers to determining the nature of a medical condition, with the intention of identifying the symptoms affecting a subject from a variety of collected data.

[0114] As used herein, “comprising” is synonymous with “including,” “containing,” “having,” or “characterized in,” and is inclusive or open-ended, and does not exclude additional, unlisted elements or method steps. As used herein, “consisting of” excludes any element, step, or component not specified in the claimed elements. As used herein, “consisting substantially of” does not exclude materials or steps that do not substantially affect the essential and novel features of the claims. In each case herein, any one of the terms “comprising,” “consisting substantially of,” and “consisting of” may optionally be replaced by any of the other two terms, thereby describing alternative aspects of the subject matter. The invention described illustratively herein can be suitably practiced in the absence of any one or more elements or limitations not expressly disclosed herein.

[0115] Unless the context clearly specifies otherwise, as used herein, the singular forms “a,” “an,” and “the” include plural indicators. Unless otherwise stated, the use of “or” or “and” means “and / or.” Furthermore, the use of the term “including” and other forms such as “include,” “includes,” and “included” is non-restrictive.

[0116] As used herein, the term “about” covers a variation of up to ±10% relative to a specified value when referring to measurable values ​​such as amount, duration, etc. Unless otherwise indicated, all figures representing quantities of, for example, components or properties (e.g., molecular weight, reaction conditions) described herein shall be understood to be modified by the term “about”.

[0117] As used herein, “and / or” is considered an explicit disclosure of each of two specified features or components being with or not with the other. Therefore, 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” covers 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.

[0118] As used herein, unless otherwise indicated herein, the ranges of values ​​listed herein are intended only as a shorthand for individually referring to each individual value falling within that range, and each individual value is incorporated into the specification as if it were listed separately herein. For example, if a concentration range is stated as 1% to 50%, it is intended that values ​​such as 2% to 40%, 10% to 30%, or 1% to 3% be explicitly listed in this specification. These are merely examples of explicit intent, and all possible combinations of values ​​between and including the listed minimum and maximum values ​​are considered to be explicitly stated in this disclosure.

[0119] As used herein, the term "stereoisomer" refers to isomers having the same composition but different spatial arrangements of their atoms. 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. The term "racemic mixture" or "racemic mixture" refers to a composition consisting of two enantiomer species in equimolar amounts, wherein the composition is not optically active. Geometric isomers of the C=C double bond can also be present in ADCs, and all such stable isomers are covered in this invention. Cis and trans (or E- and Z-) geometric isomers of the ADCs of this invention are described and can be separated as mixtures of isomers or as separate isomeric forms.

[0120] The term "ADC 101" refers to a compound having the following formula: ; AB refers to the anti-CEACAM5 antibody or its antigen-binding moiety described in this article; Where o is an integer of 24, ADC 101 should also be understood as being the same as compounds having the following formula: . Since these two structures are assumed to be identical, they are interchangeable. The same structure can be represented using either structure.

[0121] In some respects, the anti-CEACAM5 antibody or its antigen-binding moiety is described in Tables 10 and 11. In some respects, these CDRs, VHs, VLs, heavy chains, and / or light chains are described in Tables 10 and 11.

[0122] The various aspects described herein are further described in detail in the following sections. I. Antibody-drug conjugates

[0123] This disclosure provides an antibody-drug conjugate comprising the anti-CEACAM5 antibody or its antigen-binding moiety described herein, the anti-CEACAM5 antibody or its antigen-binding moiety being linked or conjugated to a cytotoxic moiety (i.e., camptothecin or its derivatives and analogs) via a phosphorus (V) moiety (also referred to as "P5") and a linker.

[0124] In some respects, the ADC disclosed herein has formula (I): (I) Or its pharmaceutically acceptable salts, stereoisomers or solvates, wherein: The configuration of the double bond is either E or Z; V is H or (C1-C8) alkyl; X is ; Y is NR 5 S, O or CR 6 R 7 ; R 1 It is a polyalkylene glycol unit containing at least three alkylene glycol subunits; R 3 and R 5 -R 7 Each is H, or an aliphatic or aromatic residue that is optionally substituted; L stands for connector; C represents the cytotoxic component; m is an integer ranging from 1 to 10; The range of n is from 1 to 20; and AB is an anti-CEACAM5 antibody or its antigen-binding moiety, which contains... (a) A heavy chain variable region (VH) comprising complementarity-determining regions (CDR)1, CDR2, and CDR3 containing the amino acid sequences shown in SEQ ID NO: 14, 15, and 16, respectively; and a light chain variable region (VL) comprising CDR1, CDR2, and CDR3 containing the amino acid sequences shown in SEQ ID NO: 19, 20, and 21; or (b) VH, which comprises CDR1, CDR2, and CDR3 regions having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with the amino acid sequences shown in SEQ ID NO: 14, 15, and 16, respectively; and VL, which comprises CDR1, CDR2, and CDR3 regions having at least 85%, 90%, 95%, 98%, or 99% sequence identity with the amino acid sequences shown in SEQ ID NO: 19, 20, and 21, respectively.

[0125] In some respects, R 3 It is H or an optionally substituted aliphatic or aromatic residue. In some respects, R 3 It is H or (C1-C8) alkyl. In some respects, R 3 It's H.

[0126] In some respects, R 1 It contains 3 to 100 subunits with the following structure:

[0127]

[0128] In some respects, R 1 yes in: Indicates the position of O; K F Selected from the following group, which consists of: -H, -PO3H, -(Cl-C 10 )alkyl, -(C 1- 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 o is an integer ranging from 3 to 100.

[0129] In some respects, R 1 It contains 3 to 50 subunits with the following structure: In some respects, R 1 yes: , in Indicates the position of O; K F Selected from the following group, which consists of: -H, -(C1-C 10 )alkyl and -(C2-C 10)alkyl-OH; and o is an integer ranging from 3 to 50.

[0130] In some respects, K F It's H.

[0131] In some respects, o is an integer ranging from 8 to 30, for example, from 8 to 16, from 10 to 30, or from 20 to 28, for example, 10, 11, 12, 13, 14, 22, 23, 24, 25, or 26.

[0132] In some respects, the L-shaped connector is cuttable.

[0133] In some respects, the linker L can be cleaved by proteases, glucuronidases, sulfatases, phosphatases, esterases, or by disulfide reduction.

[0134] In some respects, linkers are cleaved under physiological conditions, particularly intracellularly by lysosomes or endosomal proteases to release the attached cytotoxic portion. In other respects, cleavable linkers are designed to release the free cytotoxic portion in its unmodified form. Cleavable linkers include, for example, disulfide linkers, acid-labile linkers, light-labile linkers, peptidase-labile linkers, and esterase-labile linkers. Typically, peptidyl linkers are at least two or at least three amino acids long.

[0135] Peptidase unstable linkers can be used to cleave certain peptides, either intracellularly or extracellularly. On one hand, cleavable linkers are cleaved under mild conditions (i.e., conditions where the activity of the intracellular cytotoxic portion is unaffected).

[0136] Depending on the adapter design, membrane-permeable (lipophilic) toxins released within target-positive cells can cross the cell membrane and kill closely adjacent cells, including neighboring cancer cells lacking antigen expression (bystander effect) (Kovtun, YV et al. (2006) Cancer Res. 66 (6), 3214-3221). The ability of such cytotoxic drugs to mediate local bystander killing is a selection criterion based on the ADCs disclosed herein.

[0137] Cleavage agents may include, for example, cathepsins B and D, as well as plasmin, all known to hydrolyze dipeptide drug derivatives, resulting in the release of the active drug within target cells. For instance, peptide linkers that can be cleaved by cathepsin-B, a thiol-dependent protease highly expressed in cancerous tissue (e.g., Phe-Leu or Gly-Phe-Leu-Gly (SEQ ID NO:101) linkers) can be used. In certain aspects, peptide linkers cleaved by intracellular proteases are valine-citrulline (Val-Cit) linkers or phenylalanine-lysine (Phe-Lys) linkers. One advantage of using intracellular proteolytic release of the therapeutic agent is that the agent is generally attenuated upon conjugation, and the serum stability of the conjugate is generally high.

[0138] Various linkers can be used in the conjugates described herein. In some aspects, the linker comprises a peptide linker that can be cleaved by cathepsins within tumor cells, such as a dipeptide valine (Val)-citrulline (Cit) (vc). Other peptide 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 aspects, the linker L can be cleaved by proteases (e.g., cathepsins B and D).

[0139] In some respects, connector L has the following characteristics: -AW 1-8 -B 0-1 -#, Unit A is the first spacer subunit; W is an amino acid; and B is the second spacer subunit. represents the attachment point with -Y-, and # represents the attachment point with the cytotoxic portion.

[0140] In some respects, the first spacer subunit A has the following structure:

[0141] ,in It is a 5- or 6-membered carbon ring; This indicates an attachment point with -Y-, and ## indicates an attachment point with W. Preferred unit A is...

[0142] In some respects, W is a dipeptide (W2).

[0143] In some respects, the dipeptide is selected from the group consisting of valine-citrulline (Val-Cit) and valine-alanine (Val-Ala).

[0144] In some respects, the second spacer subunit B is a PAB group having the following structure:

[0145] The NH group is bonded to -W-, and the C(O) group is bonded to the cytotoxic moiety.

[0146] In some aspects, the connector has the following structure: W2 is a dipeptide. The symbol indicates the attachment point to Y, and the symbol # indicates the attachment point to the cytotoxic portion.

[0147] In some respects, connector L is -A-W2-B1-#, which has the following structure: in The symbol indicates the attachment point to Y, and the symbol # indicates the attachment point to the cytotoxic portion.

[0148] In some respects, C is the cytotoxic portion. The term "cytotoxic portion" or sometimes "payload" refers to the chemical or biochemical portion of the anti-CEACAM5 antibody described herein, which is conjugated to the antibody via a linker.

[0149] In some respects, the cytotoxic portion is an anticancer agent. Therefore, the drug can be selected from the group consisting of: maytansinoids, calicheamycin, tubulysin, amatoxin, salicylate, and aurestatins (such as monomethylaurestatin E (MMAE) or monomethylaurestatin F (MMAF)), pyrrolobenzodiazepine dimers, indoline-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 analogues or prodrugs. In a preferred embodiment, the cytotoxic portion is a topoisomerase I inhibitor. In some respects, the cytotoxic portion is a naturally occurring topoisomerase I inhibitor, camptothecin, or a derivative or analogue thereof. In some respects, the cytotoxic component is camptothecin derivatives, such as eczetidine. The structure of eczetidine is shown below. .

[0150] The ADCs disclosed in this article take into account all stereoisomers of eczetidine.

[0151] In some aspects, n in the ADC of equation (I) is 7 or 8. In some aspects, n in the ADC is 5 or 6. In some aspects, n in the ADC is 9 or 10. In some aspects, n in the ADC is 7. In some aspects, n in the ADC is 8.

[0152] In some respects, in formula (I), AB is the anti-CEACAM5 antibody disclosed herein or its antigen-binding moiety; V is H; Y is NH; R 3 H; n ranges from 4 to 8; R 1 It is a polyalkylene glycol unit with the following structure: , in: Indicates the position of O; K F It is H; and o is an integer ranging from 8 to 30; L is a connector with the following structure: in The symbol indicates the attachment point to Y, and # indicates the attachment point to the cytotoxic portion; C is eczema; and m is 1.

[0153] In some respects, o is an integer ranging from 8 to 30, from 10 to 30, from 15 to 30, from 20 to 30, or from 20 to 25. In other respects, o is 20, 21, 22, 23, 24, or 25.

[0154] In some respects, n is an integer ranging from 2 to 10. In some respects, o is an integer ranging from 20 to 28. In some respects, o is 22, 23, 24, 25, or 26.

[0155] In some respects, n is an integer ranging from 2 to 10.

[0156] In some respects, the anti-CEACAM5 antibody or its antigen-binding moiety is described in Tables 10 and 11. In some respects, these CDRs, VHs, VLs, heavy chains, and / or light chains are described in Tables 10 and 11.

[0157] In some respects, the ADC disclosed herein has formula (II): (II), Or its pharmaceutically acceptable salts, stereoisomers or solvates, wherein o is an integer from 8 to 30; The range of n is from 4 to 8; and AB refers to the anti-CEACAM5 antibody or its antigen-binding moiety disclosed herein (e.g., Tables 10 and 11); and The configuration of the double bond is either E or Z. The joint may also exist as a mixture of E and Z isomers.

[0158] In some respects, the ADC disclosed herein has formula (II): (II) Or its pharmaceutically acceptable salts, stereoisomers or solvates, wherein: The configuration of the double bond is either E or Z; The range of n is from 4 to 8; and o is an integer from 8 to 30; AB is an anti-CEACAM5 antibody or an antigen-binding moiety of an anti-CEACAM5 antibody that specifically binds to carcinoembryonic antigen-associated cell adhesion molecule 5 (CEACAM5), the anti-CEACAM5 antibody or the antigen-binding moiety of the anti-CEACAM5 antibody comprising: (a) A heavy chain variable region (VH) comprising complementarity-determining regions (CDR)1, CDR2, and CDR3 containing the amino acid sequences shown in SEQ ID NO: 14, 15, and 16, respectively; and a light chain variable region (VL) comprising CDR1, CDR2, and CDR3 containing the amino acid sequences shown in SEQ ID NO: 19, 20, and 21; or (b) VH, which comprises CDR1, CDR2, and CDR3 regions having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with the amino acid sequences shown in SEQ ID NO: 14, 15, and 16, respectively; and VL, which comprises CDR1, CDR2, and CDR3 regions having at least 85%, 90%, 95%, 98%, or 99% sequence identity with the amino acid sequences shown in SEQ ID NO: 19, 20, and 21, respectively.

[0159] In some respects, the ADC disclosed herein has the formula (ADC 101): (ADC 101) Or its pharmaceutically acceptable salts, stereoisomers or solvates, wherein: The configuration of the double bond is either E or Z; AB is an anti-CEACAM5 antibody or an antigen-binding moiety of an anti-CEACAM5 antibody that specifically binds to carcinoembryonic antigen-associated cell adhesion molecule 5 (CEACAM5), the anti-CEACAM5 antibody or the antigen-binding moiety of the anti-CEACAM5 antibody comprising: (a) A heavy chain variable region (VH) comprising complementarity-determining regions (CDR)1, CDR2, and CDR3 containing the amino acid sequences shown in SEQ ID NO: 14, 15, and 16, respectively; and a light chain variable region (VL) comprising CDR1, CDR2, and CDR3 containing the amino acid sequences shown in SEQ ID NO: 19, 20, and 21; or (b) VH, which comprises CDR1, CDR2, and CDR3 regions having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with the amino acid sequences shown in SEQ ID NO: 14, 15, and 16, respectively; and VL, which comprises CDR1, CDR2, and CDR3 regions having at least 85%, 90%, 95%, 98%, or 99% sequence identity with the amino acid sequences shown in SEQ ID NO: 19, 20, and 21, respectively.

[0160] In some embodiments, AB is an anti-CEACAM5 antibody or its antigen-binding portion described in Tables 10 and 11. In some embodiments, AB is an anti-CEACAM5 antibody or its antigen-binding portion, which comprises: (a) A heavy chain variable region (VH) comprising complementarity-determining regions (CDR)1, CDR2, and CDR3 containing the amino acid sequences shown in SEQ ID NO: 14, 15, and 16, respectively; and a light chain variable region (VL) comprising CDR1, CDR2, and CDR3 containing the amino acid sequences shown in SEQ ID NO: 19, 20, and 21, or (b) VH, which comprises CDR1, CDR2, and CDR3 regions having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with the amino acid sequences shown in SEQ ID NO: 14, 15, and 16, respectively; and VL, which comprises CDR1, CDR2, and CDR3 regions having at least 85%, 90%, 95%, 98%, or 99% sequence identity with the amino acid sequences shown in SEQ ID NO: 19, 20, and 21, respectively.

[0161] In some respects, 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 respects, o is an integer between 20 and 30, such as 23. In some respects, o is an integer between 20 and 25, such as 24. In some respects, o is an integer of 24. In some respects, o is 24. In some respects, o is 25. In some respects, o is 23. In some respects, o is 22. In some respects, o is 21. In some respects, o is 20. In some respects, o is an integer between 26 and 30. In some respects, o is 26. In some respects, o is 27. In some respects, o is 28. In some respects, o is 29. In some respects, o is 30. In some respects, o is an integer between 8 and 19. In some respects, o is 8. In some respects, o is 9. In some respects, o is 10. In some respects, o is 11. In some respects, o is 12. In some respects, o is 13. In some respects, o is 14. In some respects, o is 15. In some respects, o is 16. In some respects, o is 17. In some respects, o is 18. In some respects, o is 19.

[0162] In some respects, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some respects, n is 4. In some respects, n is 5. In some respects, n is 6. In some respects, n is 7. In some respects, n is 8. Preferably, n is 24 and n is 8.

[0163] This disclosure also provides compounds of formula (III): (III), Or its pharmaceutically acceptable salts, stereoisomers or solvates, wherein X, R 1 Y, L, C and m are defined as in equation (I).

[0164] In some respects, this disclosure provides a method for preparing an ADC of formula (I), the method comprising reacting a compound of formula (III) with a thiol-containing compound AB-(SH). n The reaction, wherein AB is the anti-CEACAM5 antibody or its antigen-binding moiety disclosed herein, and n ranges from 1 to 10, produces an ADC of formula (I):

[0165] In some respects, compounds of formula (III) have the following structures:

[0166] (Compound A) Or its pharmaceutically acceptable salt, stereoisomer, or solvation, where o is an integer ranging from 8 to 25, e.g., 24. The chiral center is indicated. All stereoisomers of compound A were considered for the synthesis of the ADC disclosed herein.

[0167] In some respects, this disclosure provides a method for preparing an ADC of formula (II), the method comprising reacting compound A with a thiol-containing compound AB-(SH). n The reaction, wherein AB is an anti-CEACAM5 antibody or its antigen-binding moiety disclosed herein (e.g., disclosed in Tables 10 and 11), is used to obtain an ADC of formula (II): (II), Or a pharmaceutically acceptable salt, stereoisomer, or solvate thereof, wherein o and n are as defined above. Methods for the selective bioconjugation of ethynylphosphonamideate with cysteine-containing compounds have been described in the following publications: WO 2018041985 A1 (published March 8, 2018), WO 2019170710A2 (published September 12, 2019), WO 2022223783 A1 (published October 27, 2022), WO 2023083900 A1 (published May 19, 2023), and WO 2023083919 A1 (published May 19, 2023), each of which is incorporated herein by reference.

[0168] The number of cytotoxic portions (drug-antibody ratio: DAR) attached to the antigen-binding portion of CEACAM5-ADC can vary and will be limited only by the number of attachment sites available on the antigen-binding portion and the number of drugs attached to a single adapter.

[0169] DAR values ​​can vary depending on the nature of the antigen-binding moiety used (e.g., any antibody or its antigen-binding moiety described herein) and the drug, as well as the experimental conditions used for conjugation (DAR, reaction time, nature of the solvent and / or co-solvent). Therefore, in an ADC, the contact between the antibody and the drug can produce a mixture containing several conjugates that differ from one another due to different drug-antibody ratios, and may further include 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.

[0170] In some aspects, the linker connects a single cytotoxic moiety to the antigen-binding moiety of the conjugate (e.g., any antibody or its antigen-binding moiety described herein). In some aspects where the conjugate contains more than one cytotoxic moiety, each moiety may be the same or different. Conjugates with a DAR of twenty or higher are considered, provided that the conjugate does not exhibit unacceptable levels of aggregation under the conditions of use and / or storage. In some aspects, the conjugates described herein may have a DAR 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 aspects, the conjugate may have a DAR of 2, 3, 4, or 5. In some aspects, the DAR is 6. In some aspects, the DAR is 7. In some aspects, the DAR is 8. In some aspects, the DAR is 9. In some aspects, the DAR is 6 or 7. In some aspects, the DAR is 7, 7.5, or 8. In some aspects, the DAR is 7–8.

[0171] In some respects, the ADC disclosed herein has the following structure: (ADC 101), Or its pharmaceutically acceptable salts, stereoisomers or solvates, wherein The configuration of the double bond is E or Z; AB is an antibody or antigen-binding moiety that binds to CEACAM5, which contains VH and VL, which contain the amino acid sequences shown in SEQ ID NO: 38 and 43, respectively.

[0172] In some respects, this disclosure provides information about the ADC 101: , Or a pharmaceutically acceptable salt thereof, wherein AB is an anti-CEACAM5 antibody or its antigen-binding moiety, the anti-CEACAM5 antibody or its antigen-binding moiety comprising (a) A heavy chain variable region (VH) comprising complementarity-determining regions (CDR)1, CDR2, and CDR3 containing the amino acid sequences shown in SEQ ID NO: 14, 15, and 16, respectively; and a light chain variable region (VL) comprising CDR1, CDR2, and CDR3 containing the amino acid sequences shown in SEQ ID NO: 19, 20, and 21; or (b) VH, which comprises CDR1, CDR2, and CDR3 regions having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with the amino acid sequences shown in SEQ ID NO: 14, 15, and 16, respectively; and VL, which comprises CDR1, CDR2, and CDR3 regions having at least 85%, 90%, 95%, 98%, or 99% sequence identity with the amino acid sequences shown in SEQ ID NO: 19, 20, and 21, respectively.

[0173] In some respects, this disclosure provides ADC 101 or a pharmaceutically acceptable salt thereof, wherein AB is an anti-CEACAM5 antibody or an antigen-binding moiety thereof comprising VH and VL, respectively, containing the amino acid sequences shown in SEQ ID NO: 49 and SEQ ID NO: 50.

[0174] In some respects, this disclosure provides ADC 101 or a pharmaceutically acceptable salt thereof, wherein AB is an anti-CEACAM5 antibody or an antigen-binding moiety thereof comprising VH and VL, respectively, containing the amino acid sequences shown in SEQ ID NO: 49 and SEQ ID NO: 50.

[0175] In some respects, this disclosure provides ADC 101 or a pharmaceutically acceptable salt thereof, wherein AB is an anti-CEACAM5 antibody or an antigen-binding moiety thereof comprising VH and VL, respectively, containing the amino acid sequences shown in SEQ ID NO: 67 and SEQ ID NO: 68.

[0176] In some respects, this disclosure provides ADC 101 or a pharmaceutically acceptable salt thereof, wherein AB is an anti-CEACAM5 antibody or an antigen-binding moiety thereof comprising VH and VL, respectively, containing the amino acid sequences shown in SEQ ID NO: 69 and SEQ ID NO: 70.

[0177] In some respects, this disclosure provides ADC 101 or a pharmaceutically acceptable salt thereof, wherein AB is an anti-CEACAM5 antibody or an antigen-binding moiety thereof comprising VH and VL, respectively, containing the amino acid sequences shown in SEQ ID NO: 71 and SEQ ID NO: 72.

[0178] In some respects, this disclosure provides ADC 101 or a pharmaceutically acceptable salt thereof, wherein AB is an anti-CEACAM5 antibody or an antigen-binding moiety thereof comprising VH and VL, respectively, containing the amino acid sequences shown in SEQ ID NO: 73 and SEQ ID NO: 74.

[0179] In some respects, this disclosure provides ADC 101 or a pharmaceutically acceptable salt thereof, wherein AB is an anti-CEACAM5 antibody or an antigen-binding moiety thereof comprising VH and VL, respectively, containing the amino acid sequences shown in SEQ ID NO: 75 and SEQ ID NO: 76.

[0180] In some respects, this disclosure provides ADC 101 or a pharmaceutically acceptable salt thereof, wherein AB is an anti-CEACAM5 antibody or an antigen-binding moiety thereof comprising VH and VL, respectively, containing the amino acid sequences shown in SEQ ID NO: 77 and SEQ ID NO: 78.

[0181] In some respects, this disclosure provides ADC 101 or a pharmaceutically acceptable salt thereof, wherein AB is an anti-CEACAM5 antibody or an antigen-binding moiety thereof comprising VH and VL, respectively, containing the amino acid sequences shown in SEQ ID NO: 79 and SEQ ID NO: 80.

[0182] In some respects, this disclosure provides ADC 101 or a pharmaceutically acceptable salt thereof, wherein AB is an anti-CEACAM5 antibody or an antigen-binding moiety thereof comprising VH and VL, respectively, containing the amino acid sequences shown in SEQ ID NO: 81 and SEQ ID NO: 82.

[0183] In some respects, this disclosure provides ADC 101 or a pharmaceutically acceptable salt thereof, wherein AB is an anti-CEACAM5 antibody or an antigen-binding moiety thereof comprising VH and VL, respectively, containing the amino acid sequences shown in SEQ ID NO: 83 and SEQ ID NO: 84.

[0184] In some respects, this disclosure provides ADC 101 or a pharmaceutically acceptable salt thereof, wherein AB is an anti-CEACAM5 antibody or an antigen-binding moiety thereof comprising VH and VL, respectively, containing the amino acid sequences shown in SEQ ID NO: 85 and SEQ ID NO: 86.

[0185] In some respects, this disclosure provides ADC 101 or a pharmaceutically acceptable salt thereof, wherein AB is an anti-CEACAM5 antibody or an antigen-binding moiety thereof comprising VH and VL, respectively, containing the amino acid sequences shown in SEQ ID NO: 87 and SEQ ID NO: 88.

[0186] In some aspects, this disclosure provides ADC 101 or a pharmaceutically acceptable salt thereof, wherein AB is an anti-CEACAM5 antibody or an antigen-binding moiety thereof comprising VH and VL containing the amino acid sequences shown in SEQ ID NO: 89 and SEQ ID NO: 90, respectively, and wherein the cytotoxic moiety comprises compound 101.

[0187] In some respects, this disclosure provides ADC 101 or a pharmaceutically acceptable salt thereof, wherein AB is an anti-CEACAM5 antibody or an antigen-binding moiety thereof comprising VH and VL, respectively, containing the amino acid sequences shown in SEQ ID NO: 91 and SEQ ID NO: 92.

[0188] In some respects, this disclosure provides ADC 101 or a pharmaceutically acceptable salt thereof, wherein AB is an anti-CEACAM5 antibody or an antigen-binding moiety thereof comprising VH and VL, respectively, containing the amino acid sequences shown in SEQ ID NO: 93 and SEQ ID NO: 94.

[0189] In some respects, this disclosure provides ADC 101 or a pharmaceutically acceptable salt thereof, wherein AB is an anti-CEACAM5 antibody or an antigen-binding moiety thereof comprising VH and VL, respectively, containing the amino acid sequences shown in SEQ ID NO: 17 and SEQ ID NO: 22.

[0190] In some aspects, VH and VL comprise the amino acid sequences shown in SEQ ID NO: 49 and SEQ ID NO: 50, respectively. In some aspects, VH and VL comprise the amino acid sequences shown in SEQ ID NO: 51 and SEQ ID NO: 52, respectively. In some aspects, VH and VL comprise the amino acid sequences shown in SEQ ID NO: 67 and SEQ ID NO: 68, respectively. In some aspects, VH and VL comprise the amino acid sequences shown in SEQ ID NO: 69 and SEQ ID NO: 70, respectively. In some aspects, VH and VL comprise the amino acid sequences shown in SEQ ID NO: 71 and SEQ ID NO: 72, respectively. In some aspects, VH and VL comprise the amino acid sequences shown in SEQ ID NO: 73 and SEQ ID NO: 74, respectively. In some aspects, VH and VL comprise the amino acid sequences shown in SEQ ID NO: 75 and SEQ ID NO: 76, respectively. In some aspects, VH and VL comprise the amino acid sequences shown in SEQ ID NO: 69 and SEQ ID NO: 70, respectively. In some aspects, VH and VL comprise the amino acid sequences shown in SEQ ID NO: 73 and SEQ ID NO: 74, respectively. In some aspects, VH and VL comprise the amino acid sequences shown in SEQ ID NO: 75 and SEQ ID NO: 76, respectively. In some aspects, VH and VL comprise the amino acid sequences shown in SEQ ID NO: 77 and SEQ ID NO: 78, respectively. In some aspects, VH and VL comprise the amino acid sequences shown in SEQ ID NO: 79 and SEQ ID NO: 80, respectively. In some aspects, VH and VL comprise the amino acid sequences shown in SEQ ID NO: 81 and SEQ ID NO: 82, respectively. In some aspects, VH and VL comprise the amino acid sequences shown in SEQ ID NO: 83 and SEQ ID NO: 84, respectively. In some aspects, VH and VL comprise the amino acid sequences shown in SEQ ID NO: 85 and SEQ ID NO: 86, respectively. In some aspects, VH and VL comprise the amino acid sequences shown in SEQ ID NO: 87 and SEQ ID NO: 88, respectively.In some aspects, VH and VL comprise the amino acid sequences shown in SEQ ID NO: 89 and SEQ ID NO: 90, respectively. In some aspects, VH and VL comprise the amino acid sequences shown in SEQ ID NO: 91 and SEQ ID NO: 92, respectively. In some aspects, VH and VL comprise the amino acid sequences shown in SEQ ID NO: 93 and SEQ ID NO: 94, respectively. In some aspects, VH and VL comprise the amino acid sequences shown in SEQ ID NO: 17 and SEQ ID NO: 22, respectively. In some aspects, the heavy chain and light chain comprise the amino acid sequences shown in SEQ ID NO: 45 and SEQ ID NO: 46, respectively.

[0191] In some respects, this disclosure provides an antibody-drug conjugate (ADC) having formula (II): (II), Or a pharmaceutically acceptable salt, stereoisomer, or solvate thereof; wherein n ranges from 4 to 8; o is an integer ranging from 10 to 30; AB is an anti-CEACAM5 antibody or its antigen-binding moiety comprising VH and VL, respectively, containing the amino acid sequences shown in SEQ ID NO: 67 and SEQ ID NO: 68.

[0192] In some respects, this disclosure provides an antibody-drug conjugate (ADC) having formula (II): (II), Or a pharmaceutically acceptable salt, stereoisomer, or solvate thereof; wherein n ranges from 4 to 8; o is an integer ranging from 10 to 30; AB is an anti-CEACAM5 antibody or its antigen-binding moiety comprising VH and VL, respectively, containing the amino acid sequences shown in SEQ ID NO: 69 and SEQ ID NO: 70.

[0193] In some respects, this disclosure provides an antibody-drug conjugate (ADC) having formula (II): (II), Or a pharmaceutically acceptable salt, stereoisomer, or solvate thereof; wherein n ranges from 4 to 8; o is an integer ranging from 10 to 30; AB is an anti-CEACAM5 antibody or its antigen-binding moiety comprising VH and VL, respectively, containing the amino acid sequences shown in SEQ ID NO: 71 and SEQ ID NO: 72.

[0194] In some respects, this disclosure provides an antibody-drug conjugate (ADC) having formula (II): Or a pharmaceutically acceptable salt, stereoisomer, or solvate thereof; wherein n ranges from 4 to 8; o is an integer ranging from 10 to 30; AB is an anti-CEACAM5 antibody or its antigen-binding moiety comprising VH and VL, respectively, containing the amino acid sequences shown in SEQ ID NO: 73 and SEQ ID NO: 74.

[0195] In some respects, this disclosure provides an antibody-drug conjugate (ADC) having formula (II): (II), Or a pharmaceutically acceptable salt, stereoisomer, or solvate thereof; wherein n is an integer ranging from 4 to 8; o is an integer ranging from 10 to 30; AB is an anti-CEACAM5 antibody or its antigen-binding moiety thereof, the anti-CEACAM5 antibody or its antigen-binding moiety comprising VH and VL respectively containing the amino acid sequences shown in SEQ ID NO: 75 and SEQ ID NO: 76.

[0196] In some respects, this disclosure provides an antibody-drug conjugate (ADC) having formula (II): (II), Or a pharmaceutically acceptable salt, stereoisomer, or solvate thereof; wherein n is an integer ranging from 4 to 8; o is an integer ranging from 10 to 30; AB is an anti-CEACAM5 antibody or its antigen-binding moiety thereof, the anti-CEACAM5 antibody or its antigen-binding moiety comprising VH and VL respectively containing the amino acid sequences shown in SEQ ID NO: 77 and SEQ ID NO: 78.

[0197] In some respects, this disclosure provides an antibody-drug conjugate (ADC) having formula (II): (II), Or a pharmaceutically acceptable salt, stereoisomer, or solvate thereof; wherein n ranges from 4 to 8; o is an integer ranging from 10 to 30; AB is an anti-CEACAM5 antibody or its antigen-binding moiety comprising VH and VL, respectively, containing the amino acid sequences shown in SEQ ID NO: 79 and SEQ ID NO: 80.

[0198] In some respects, this disclosure provides an antibody-drug conjugate (ADC) having formula (II): (II), Or a pharmaceutically acceptable salt, stereoisomer, or solvate thereof; wherein n ranges from 4 to 8; o is an integer ranging from 10 to 30; AB is an anti-CEACAM5 antibody or its antigen-binding moiety comprising VH and VL, respectively, containing the amino acid sequences shown in SEQ ID NO: 81 and SEQ ID NO: 82.

[0199] In some respects, this disclosure provides an antibody-drug conjugate (ADC) having formula (II): (II), Or a pharmaceutically acceptable salt, stereoisomer, or solvate thereof; wherein n ranges from 4 to 8; o is an integer ranging from 10 to 30; AB is an anti-CEACAM5 antibody or its antigen-binding moiety comprising VH and VL, respectively, containing the amino acid sequences shown in SEQ ID NO: 83 and SEQ ID NO: 84.

[0200] In some respects, this disclosure provides an antibody-drug conjugate (ADC) having formula (II): (II), Or a pharmaceutically acceptable salt, stereoisomer, or solvate thereof; wherein n ranges from 4 to 8; o is an integer ranging from 10 to 30; AB is an anti-CEACAM5 antibody or its antigen-binding moiety comprising VH and VL, respectively, containing the amino acid sequences shown in SEQ ID NO: 85 and SEQ ID NO: 86.

[0201] In some respects, this disclosure provides an antibody-drug conjugate (ADC) having formula (II): (II), Or a pharmaceutically acceptable salt, stereoisomer, or solvate thereof; wherein n ranges from 4 to 8; o is an integer ranging from 10 to 30; AB is an anti-CEACAM5 antibody or its antigen-binding moiety comprising VH and VL, respectively, containing the amino acid sequences shown in SEQ ID NO: 87 and SEQ ID NO: 88.

[0202] In some respects, this disclosure provides an antibody-drug conjugate (ADC) having formula (II): (II), Or a pharmaceutically acceptable salt, stereoisomer, or solvate thereof; wherein n ranges from 4 to 8; o is an integer ranging from 10 to 30; AB is an anti-CEACAM5 antibody or its antigen-binding moiety comprising VH and VL, respectively, containing the amino acid sequences shown in SEQ ID NO: 89 and SEQ ID NO: 90.

[0203] In some respects, this disclosure provides an antibody-drug conjugate (ADC) having formula (II): (II), Or a pharmaceutically acceptable salt, stereoisomer, or solvate thereof; wherein n ranges from 4 to 8; o is an integer ranging from 10 to 30; AB is an anti-CEACAM5 antibody or its antigen-binding moiety comprising VH and VL, respectively, containing the amino acid sequences shown in SEQ ID NO: 91 and SEQ ID NO: 92.

[0204] In some respects, this disclosure provides an antibody-drug conjugate (ADC) having formula (II): (II), Or a pharmaceutically acceptable salt, stereoisomer, or solvate thereof; wherein n ranges from 4 to 8; o is an integer ranging from 10 to 30; AB is an anti-CEACAM5 antibody or its antigen-binding moiety comprising VH and VL, respectively, containing the amino acid sequences shown in SEQ ID NO: 93 and SEQ ID NO: 94.

[0205] In some respects, this disclosure provides an antibody-drug conjugate (ADC) having formula (II): (II), Or a pharmaceutically acceptable salt, stereoisomer, or solvate thereof; wherein n ranges from 4 to 8; o is an integer ranging from 10 to 30; AB is an anti-CEACAM5 antibody or its antigen-binding moiety comprising VH and VL, respectively, containing the amino acid sequences shown in SEQ ID NO: 17 and SEQ ID NO: 22.

[0206] In some respects, n is 4, 5, 6, 7, 8, or 9. In some respects, n is 6. In some respects, n is 7. In some respects, n is 8. In some respects, n is 9.

[0207] In some respects, this disclosure provides an antibody-drug conjugate (ADC) comprising formula (II): (II), Or a pharmaceutically acceptable salt, stereoisomer, or solvate thereof; wherein n ranges from 4 to 8; o is an integer ranging from 10 to 30; AB is an anti-CEACAM5 antibody or its antigen-binding moiety comprising a heavy chain and a light chain respectively containing the amino acid sequences shown in SEQ ID NO: 45 and SEQ ID NO: 46.

[0208] ADCs can also be used to alter a given biological response, where the cytotoxic portion should not be considered limited to classical chemotherapeutic agents. For example, the cytotoxic portion can be a protein or peptide with the desired biological activity (e.g., lymphokines, tumor necrosis factor, IFNγ, growth factors).

[0209] Techniques for conjugating toxins or therapeutic components to antibodies are known; see, for example, Arnon et al., “Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy,” Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc., 1985); Hellstrom et al., “Antibodies For Drug Delivery,” 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,” Monoclonal Antibodies Monoclonal Antibodies '84: Biological and Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985); “Analysis, Results, and Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, 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.[Immunology Review], 62:119-58 (1982). IA. Anti-CEACAM5 antibody and antigen binding moiety

[0210] Anti-CEACAM5 antibodies that can be used in the ADCs disclosed herein can be defined by specific structural features.

[0211] Unless the context clearly indicates otherwise, as used herein, the terms “carcinoembryonic antigen-associated cell adhesion molecule 5” and “CEACAM5” are used interchangeably to refer to human CEACAM5 or cynomolgus monkey (Macaca fascicularis) CEACAM5. The human CEACAM5 precursor polypeptide (with a signal peptide) contains the amino acid sequence shown in SEQ ID NO: 1 (GenBank: AAH34671.1). The cynomolgus monkey CEACAM5 precursor polypeptide (with a signal peptide) contains the amino acid sequence shown in SEQ ID NO: 3 (NCBI: XP_005589491.2). The amino acid and nucleic acid sequences of human or cynomolgus monkey CEACAM5 are disclosed in Table 10.

[0212] Unless the context clearly indicates otherwise, the term “CEACAM5” further includes counterparts from other species and other naturally occurring alleles, splice variants, and their processed forms. antibody sequence

[0213] In some respects, isolated anti-CEACAM5 antibodies (e.g., recombinant antibodies, humanized antibodies, chimeric antibodies, or human antibodies) or their antigen-binding moieties (e.g., those that can be conjugated to the cytotoxic moieties described herein to generate an ADC and can be used as antibodies in an ADC) are listed in Table 10. Anti-CEACAM5 antibodies or their antigen-binding moieties bind to and are internalized into cells expressing CEACAM5. Therefore, anti-CEACAM5 antibodies or their antigen-binding moieties can be used in ADCs (comprising an antibody or its antigen-binding moieties linked to a cytotoxic moiety) by efficiently delivering the cytotoxic moieties to kill cells (e.g., cancer cells).

[0214] Anti-CEACAM5 antibodies that can be used in ADCs include all known forms of antibodies and other protein scaffolds with antibody-like properties. For example, antibodies can be monoclonal antibodies, humanized antibodies, human antibodies, bispecific antibodies, immunoconjugates, chimeric antibodies, or protein scaffolds with antibody-like properties (such as fibronectin or ankyrin repeat sequences). Antibodies can also be Fab, F(ab')2, scFv, avidis, avimer, nanobodies, single-chain antibodies, or domain antibodies. Antibodies can also have any isotype or allotype, including any of the following isotypes: IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, secreted IgA (SIgA), IgD, IgE, and their allotypes. Full-length antibodies can be derived from V using standard recombinant DNA technology and nucleic acids encoding desired constant region sequences to be operatively linked to variable region sequences. H and V L Sequence preparation.

[0215] In some aspects, an anti-CEACAM5 antibody or its antigen-binding portion that can be used as an ADC comprises a VH containing CDR1, CDR2, and CDR3 regions having sequence identity of 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% with the amino acid sequences shown in SEQ ID NO: 14, 15, and 16, respectively; and a VL containing CDR1, CDR2, and CDR3 regions having sequence identity of 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% with the amino acid sequences shown in SEQ ID NO: 19, 20, and 21, respectively. In some aspects, an anti-CEACAM5 antibody or its antigen-binding portion that specifically binds to CEACAM5 comprises: a VH containing CDR1, CDR2, and CDR3 regions having the amino acid sequences shown in SEQ ID NO: 14, 15, and 16, respectively; and a VL containing CDR1, CDR2, and CDR3 regions having the amino acid sequences shown in SEQ ID NO: 19, 20, and 21, respectively.

[0216] In some aspects, an anti-CEACAM5 antibody or its antigen-binding portion that can be used as an ADC comprises 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 shown in SEQ ID NO: 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 sequences shown in SEQ ID NO: 38, and wherein the VH contains an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 38. In some aspects, an anti-CEACAM5 antibody or its antigen-binding portion that can be used as an ADC comprises 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 shown in SEQ ID NO: 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 sequences shown in SEQ ID NO: 17, and wherein the VH contains an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 17.In some aspects, an anti-CEACAM5 antibody or its antigen-binding portion that can be used as an ADC comprises 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 shown in SEQ ID NO: 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 sequences shown below: SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 16. 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.

[0217] In some aspects, an anti-CEACAM5 antibody or its antigen-binding portion that can be used as an ADC comprises 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 shown in SEQ ID NO: 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 sequences shown in SEQ ID NO: 43, and wherein the VL contains an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 43. In some aspects, an anti-CEACAM5 antibody or its antigen-binding portion that can be used as an ADC comprises 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 shown in SEQ ID NO: 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 sequences shown in SEQ ID NO: 22, and wherein the VH contains an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 22.In some aspects, an anti-CEACAM5 antibody or its antigen-binding portion that can be used as an ADC comprises a VH containing CDR1, CDR2, and CDR3 regions having at least 60%, 70%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with the amino acid sequences shown in SEQ ID NO: 14, 15, and 16, respectively; and a VL containing CDR1, CDR2, and CDR3 regions having at least 60%, 70%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with the amino acid sequences shown in SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19 ... 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.

[0218] In some aspects, an anti-CEACAM5 antibody or its antigen-binding portion suitable for use in an ADC comprises VH and VL, wherein VH and VL comprise the amino acid sequences shown in SEQ ID NO: 38 and SEQ ID NO: 43, respectively. In some aspects, an anti-CEACAM5 antibody or its antigen-binding portion suitable for use in an ADC comprises VH and VL, wherein VH and VL comprise the amino acid sequences shown in SEQ ID NO: 49 and SEQ ID NO: 50, respectively. In some aspects, an anti-CEACAM5 antibody or its antigen-binding portion suitable for use in an ADC comprises VH and VL, wherein VH and VL comprise the amino acid sequences shown in SEQ ID NO: 51 and SEQ ID NO: 52, respectively. In some aspects, an anti-CEACAM5 antibody or its antigen-binding portion suitable for use in an ADC comprises VH and VL, wherein VH and VL comprise the amino acid sequences shown in SEQ ID NO: 67 and SEQ ID NO: 68, respectively. In some aspects, an anti-CEACAM5 antibody or its antigen-binding portion suitable for use in an ADC comprises VH and VL, wherein VH and VL comprise the amino acid sequences shown in SEQ ID NO: 69 and SEQ ID NO: 70, respectively. In some aspects, an anti-CEACAM5 antibody or its antigen-binding portion suitable for use in an ADC comprises VH and VL, wherein VH and VL comprise the amino acid sequences shown in SEQ ID NO: 71 and SEQ ID NO: 72, respectively. In some aspects, an anti-CEACAM5 antibody or its antigen-binding portion suitable for use in an ADC comprises VH and VL, wherein VH and VL comprise the amino acid sequences shown in SEQ ID NO: 73 and SEQ ID NO: 74, respectively. In some aspects, an anti-CEACAM5 antibody or its antigen-binding portion suitable for use in an ADC comprises VH and VL, wherein VH and VL comprise the amino acid sequences shown in SEQ ID NO: 75 and SEQ ID NO: 76, respectively. In some aspects, an anti-CEACAM5 antibody or its antigen-binding portion that can be used in an ADC comprises VH and VL, wherein VH and VL comprise the amino acid sequences shown in SEQ ID NO: 77 and SEQ ID NO: 78, respectively. In some aspects, an anti-CEACAM5 antibody or its antigen-binding portion that can be used in an ADC comprises VH and VL, wherein VH and VL comprise the amino acid sequences shown in SEQ ID NO: 79 and SEQ ID NO: 80, respectively.In some aspects, an anti-CEACAM5 antibody or its antigen-binding portion suitable for use in an ADC comprises VH and VL, wherein VH and VL comprise the amino acid sequences shown in SEQ ID NO: 81 and SEQ ID NO: 82, respectively. In some aspects, an anti-CEACAM5 antibody or its antigen-binding portion suitable for use in an ADC comprises VH and VL, wherein VH and VL comprise the amino acid sequences shown in SEQ ID NO: 83 and SEQ ID NO: 84, respectively. In some aspects, an anti-CEACAM5 antibody or its antigen-binding portion suitable for use in an ADC comprises VH and VL, wherein VH and VL comprise the amino acid sequences shown in SEQ ID NO: 85 and SEQ ID NO: 86, respectively. In some aspects, an anti-CEACAM5 antibody or its antigen-binding portion suitable for use in an ADC comprises VH and VL, wherein VH and VL comprise the amino acid sequences shown in SEQ ID NO: 87 and SEQ ID NO: 88, respectively. In some aspects, an anti-CEACAM5 antibody or its antigen-binding portion suitable for use in an ADC comprises VH and VL, wherein VH and VL comprise the amino acid sequences shown in SEQ ID NO: 89 and SEQ ID NO: 90, respectively. In some aspects, an anti-CEACAM5 antibody or its antigen-binding portion suitable for use in an ADC comprises VH and VL, wherein VH and VL comprise the amino acid sequences shown in SEQ ID NO: 91 and SEQ ID NO: 92, respectively. In some aspects, an anti-CEACAM5 antibody or its antigen-binding portion suitable for use in an ADC comprises VH and VL, wherein VH and VL comprise the amino acid sequences shown in SEQ ID NO: 93 and SEQ ID NO: 94, respectively. In some aspects, an anti-CEACAM5 antibody or its antigen-binding portion suitable for use in an ADC comprises VH and VL, wherein VH and VL comprise the amino acid sequences shown in SEQ ID NO: 17 and SEQ ID NO: 22, respectively. In some respects, the anti-CEACAM5 antibody comprises a heavy chain and a light chain, the heavy chain and the light chain comprising the amino acid sequences shown in SEQ ID NO: 45 and SEQ ID NO: 46, respectively.

[0219] In some respects, anti-CEACAM5 antibodies or their antigen-binding portions that bind to human CEACAM5 and can be conjugated to the cytotoxic portion to produce an ADC have at least one amino acid mutation as described in Table 11.

[0220] In some respects, anti-CEACAM5 antibodies or their antigen-binding portions that can be used in ADCs can be in quantities less than 1 x 10⁻⁶. -6 M of K D It specifically binds to CEACAM5. In some respects, anti-CEACAM5 antibodies or their antigen-binding moiety can be used in ADCs in quantities less than 1 x 10⁻⁶. -7 M of K D It specifically binds to CEACAM5. In some respects, anti-CEACAM5 antibodies or their antigen-binding moiety can be used in ADCs in quantities less than 1 x 10⁻⁶. -8 M of K D It specifically binds to CEACAM5. In some respects, anti-CEACAM5 antibodies or their antigen-binding moiety can be used in ADCs in quantities less than 5 x 10⁻⁶. -9 M of K D It specifically binds to CEACAM5. In some respects, anti-CEACAM5 antibodies or their antigen-binding moiety can be used in ADCs in quantities less than 1 x 10⁻⁶. -9 M of K D It specifically binds to CEACAM5. In some respects, anti-CEACAM5 antibodies or their antigen-binding moiety can be used in ADCs in quantities less than 5 x 10⁻⁶. -10 M of K D It binds specifically to CEACAM5. antibody binding

[0221] Anti-CEACAM5 antibodies or their antigen-binding portions (e.g., those that can be used as antigen-binding portions in ADCs) bind to CEACAM5 in solution (e.g., human CEACAM5), to CEACAM5 attached to a solid surface (e.g., a microtiter plate), and / or to CEACAM5 (e.g., human CEACAM5) anchored to a cell membrane. In some aspects, anti-CEACAM5 antibodies or their antigen-binding portions (e.g., those that can be conjugated to the cytotoxic portions described herein to generate ADCs) bind to human CEACAM5, cyno CEACAM5, or both.

[0222] In some respects, anti-CEACAM5 antibodies or their antigen-binding portions (e.g., can be used as antigen-binding portions in ADCs) are as follows K DCombined with human and / or cynomolgus monkey CEACAM5: 100 nM or less, such as 90 nM or less, 80 nM or less, 70 nM or less, 60 nM or less, 50 nM or less, such as 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, as measured by any detection method known in the art or described herein, for example, in Examples 3 and 4.

[0223] In some respects, anti-CEACAM5 antibodies or their antigen-binding portions (e.g., can be used as antigen-binding portions in ADCs) are as follows K D Combining with humans and / or cynomolgus monkeys CEACAM5: between 0.1 nM and 100 nM, between 0.1 nM and 50 nM, between 0.1 nM and 25 nM, between 0.1 nM and 10 nM, between 0.1 nM and 5 nM, between 0.1 nM and 2 nM, between 0.1 nM and 1 nM, between 0.1 nM and 0.5 nM, between 1 nM and 100 nM, between 1 nM and 50 nM, between 1 nM and 25 nM, between 1 nM and 10 nM, between 1 nM and 5 nM, between 1 nM and 2 nM, between 5 nM and 100 nM, between 5 nM and 50 nM, between 5 nM and 25 nM, between 5 nM and 10 nM, between 10 nM and 100 nM, between 10 nM and 100 nM. Between nM and 50 nM, between 10 nM and 25 nM, between 25 nM and 100 nM, between 25 nM and 50 nM, or between 50 nM and 100 nM, as measured by any detection method known in the art or described herein.

[0224] The binding (and non-binding) of an anti-CEACAM5 antibody or its antigen-binding portion, which can be used as the antigen-binding part in an ADC, can be qualitatively or quantitatively assessed by any method known in the art. Exemplary binding methods include immunohistochemistry, flow cytometry using, for example, cells overexpressing CEACAM5 (e.g., MKN-45 or HCT116-CEACAM5), surface plasmon resonance (SPR) using, for example, the BIACORE® system (Cytiva), or biolayer interferometry (BLI) using, for example, the Octet platform (ForteBio).

[0225] In some respects, the CEACAM5 antibody or its antigen-binding portion (which can be used as the antigen-binding portion in an ADC) 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), for example, as assessed by flow cytometry, for example, using cells overexpressing one of the aforementioned CEAs, or by SPR or BLI. For example, in some respects, the signal or affinity of the anti-CEACAM5 antibody or its antigen-binding portion used in an ADC for binding to one of the aforementioned CEAs is not significantly higher than the signal observed with a control antibody (e.g., an isotype control) or the signal observed in the absence of an anti-CEACAM5 antibody.

[0226] In some aspects, as described in Example 16, an anti-CEACAM5 antibody or its antigen-binding portion, which can be used as the antigen-binding portion in an ADC (comprising a CEACAM5 antibody or antigen-binding portion and a cytotoxic portion), can bind to all or part of the amino acids of human CEACAM5 (SEQ ID NO: 1). In some aspects, as described in Example 17, an anti-CEACAM5 antibody or its antigen-binding portion, which can be used as an ADC, can bind to all or part of the amino acids of human CEACAM5 (SEQ ID NO: 1).

[0227] In some aspects, anti-CEACAM5 antibodies or their antigen-binding portions that can be used in ADCs bind to (or are identified as binding to) cancer cell lines or tumor cells that overexpress CEACAM5. In some aspects, anti-CEACAM5 antibodies or their antigen-binding portions that can be used in ADCs bind to CEACAM5 on these cells, as assessed, for example, by flow cytometry. For example, in some aspects, at least 5%, at least 10%, at least 20%, at least 50%, at least 75%, or at least 90% of cells expressing CEACAM5 can be detected by binding to the anti-CEACAM5 antibody using any detection method known in the art or described herein (e.g., a signal exhibiting a higher signal than observed with an isotype control antibody).

[0228] In some respects, anti-CEACAM5 antibodies or their antigen-binding portions that can be used in ADCs (containing a CEACAM5 antibody or antigen-binding portion and a cytotoxic portion) can be used as follows EC 50Binding to CEACAM5 expressed on cells (e.g., human and / or cynomolgus monkey CEACAM5 expressed on, for example, MKN-45 cells): 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, as measured by any detection method known in the art or described herein.

[0229] In some respects, anti-CEACAM5 antibodies or their antigen-binding portions that can be used in ADCs (containing a CEACAM5 antibody or antigen-binding portion and a cytotoxic portion) can be used as follows EC 50 Binding to CEACAM5 expressed on cells: between approximately 1 ng / ml and approximately 1000 ng / ml, between approximately 1 ng / ml and approximately 500 ng / ml, between approximately 1 ng / ml and approximately 200 ng / ml, between approximately 1 ng / ml and approximately 100 ng / ml, between approximately 1 ng / ml and approximately 50 ng / ml, between approximately 1 ng / ml and approximately 25 ng / ml, between approximately 1 ng / ml and approximately 10 ng / ml, between approximately 1 ng / ml and approximately 5 ng / ml, between approximately 5 ng / ml and approximately 500 ng / ml, between approximately 5 ng / ml and approximately 200 ng / ml, between approximately 5 ng / ml and approximately 100 ng / ml, between approximately 5 ng / ml and approximately 50 ng / ml, between approximately 5 ng / ml and approximately 25 ng / ml, between approximately 5 ng / ml and approximately 5 ng / ml. Between approximately 10 ng / ml and approximately 10 ng / ml, between approximately 10 ng / ml and approximately 500 ng / ml, between approximately 10 ng / ml and approximately 200 ng / ml, between approximately 10 ng / ml and approximately 100 ng / ml, between approximately 10 ng / ml and approximately 50 ng / ml, between approximately 10 ng / ml and approximately 25 ng / ml, between approximately 25 ng / ml and approximately 500 ng / ml, between approximately 25 ng / ml and approximately 200 ng / ml, between approximately 25 ng / ml and approximately 100 ng / ml, between approximately 25 ng / ml and approximately 50 ng / ml, between approximately 50 ng / ml and approximately 500 ng / ml, between approximately 50 ng / ml and approximately 200 ng / ml, between approximately 50 ng / ml and approximately 100 ng / ml, between approximately 100 ng / ml and approximately 100 ng / ml. The concentration is between ng / ml and about 500 ng / ml or between about 100 ng / ml and about 200 ng / ml, as measured by any detection method known in the art or described herein.

[0230] The binding of anti-CEACAM5 antibodies or their antigen-binding portions to CEACAM5 described herein and applicable to ADCs (comprising CEACAM5 antibodies or antigen-binding portions and cytotoxic portions) can also be defined using quantitative immunofluorescence via flow cytometry. This method allows for the quantification of the number of antibody molecules bound to each cell or the number of cells expressing CEACAM5. In some aspects, the number of CEACAM5 molecules expressed per cell or the number of cells expressing CEACAM5 in a cell line or tumor sample can be quantified by quantitative immunofluorescence using the anti-CEACAM5 antibodies or fragments described herein.

[0231] The anti-CEACAM5 antibody or its antigen-binding portion described herein and applicable to ADCs (comprising a CEACAM5 antibody or antigen-binding moiety and a cytotoxic moiety) binds with high affinity to soluble or membrane-bound human and / or cynomolgus monkey CEACAM5, for example, K D 10 -7 M or smaller, 10 -8 M or smaller, 10 -9 M or smaller, 10 -10 M or smaller, 10 -11 M or smaller, 10 -12 M or smaller, 10 -12 M to 10 -7 M, 10 -11 M to 10 -7 M, 10 -10 M to 10 -7 M or 10 -9 M to 10 -7 M, as measured by, for example, surface plasmon resonance or other methods recognized in the art.

[0232] In some respects, the anti-CEACAM5 antibody or its antigen-binding portion described herein and applicable to ADCs (comprising a CEACAM5 antibody or antigen-binding portion and a cytotoxic portion) is as follows K D Binding to soluble or membrane-bound CEACAM5 in humans and / or cynomolgus monkeys: in 10 -7 M and 10 -12 Between M, in 10 -7 M and 10 -11 Between M, in 10 -7 M and 10 -10 Between M, in 10 -7 M and 10 -9 Between M, in 10 -7 M and 10 -8Between M, in 10 -8 M and 10 -12 Between M, in 10 -8 M and 10 -11 Between M, in 10 -8 M and 10 -11 Between M, in 10 -8 M and 10 -9 Between M, in 10 -9 M and 10 -12 Between M, in 10 -9 M and 10 -11 Between M, in 10 -9 M and 10 -10 Between M, in 10 -10 M and 10 -12 Between M, in 10 -10 M and 10 -11 M or in 10 -11 M and 10 -12 The distance between M, as measured by, for example, surface plasmon resonance or other methods recognized in the art. Competitive antibodies and antibodies that bind to the same epitope

[0233] The anti-CEACAM5 antibodies and antigen-binding moieties described herein (e.g., those that can be used as antigen-binding moieties in an ADC) are characterized by binding to one or more characteristic epitopes (i.e., one or more sites on CEACAM5), as in Examples 16 and 17, for example. The one or more epitopes bound by the antibody or fragment can be determined using methods recognized in the art. An anti-CEACAM5 antibody or its antigen-binding moieties that can be used in an ADC are considered to bind to the same epitopes as a reference anti-CEACAM5 antibody (e.g., MBN001) if, for example, one or more of the same residues on human CEACAM5 are contacted with a reference antibody, or all the same residues on all the same regions of human CEACAM5 are contacted with a reference antibody.

[0234] Antibodies sharing common epitope binding characteristics can be considered to fall into a common “epitope bin.” In some cases, the “test antibody” binding CEACAM5 can be determined to fall into a common “epitope bin” by comparing its sequence with that of a given “reference” antibody (e.g., MBN001) known to fall into a specific epitope bin. In other cases, epitope binning experiments can be performed to determine whether the test antibody falls into the same “bin” as the antibody based on common binding characteristics with the reference antibody. Antibodies that reduce the binding of the sequences disclosed herein to, for example, immobilized CEACAM5 protein or protein fragments (especially at approximate stoichiometric concentrations) may bind to the same, overlapping, or adjacent epitopes and therefore may share the desired functional properties with one or more antibodies as disclosed herein.

[0235] In some respects, antibodies falling into the same epitope bin are determined by measuring antibodies that compete with the specific anti-CEACAM5 antibody described herein for binding to CEACAM5. Methods for determining antibody competition are known in the art.

[0236] In some respects, BIACORE analysis can be used to assess the competitive ability of antibodies. The ability of the test antibody to inhibit the binding of an anti-CEACAM5 antibody that can be used in ADCs demonstrates that the test antibody can competitively bind to CEACAM5.

[0237] Inhibition or blockade of one antibody relative to another can be performed by any suitable competitive inhibition assay using methods recognized in the art or those described herein, including but not limited to surface plasmon resonance (SPR) using, for example, the BIACORE® system (Stopvan), biolayer interferometry (BLI) using, for example, the Octet platform (Vertex Biotech), enzyme-linked immunosorbent assay (ELISA), and flow cytometry. In some aspects, epitope binning of anti-CEACAM5 antibodies suitable for ADCs can be performed using recombinant CEACAM5 protein or fragments that are biotinylated and captured onto, for example, streptavidin biosensors, which are bound by a first antibody until saturation. In some aspects, epitope binning can be performed using cell-based competitive binding FACS assays.

[0238] Unless otherwise indicated, if an antibody reduces the binding of the selected antibody to human CEACAM5 (SEQ ID NO: 1), cynomolgus monkey CEACAM5 (SEQ ID NO: 3), or fragments thereof by at least 20% when used at approximately the same molar concentration as the selected antibody, it is considered to be competing with an anti-CEACAM5 antibody, as measured in the competitive ELISA assays outlined in the first two paragraphs.

[0239] In some respects, anti-CEACAM5 antibodies or their antigen-binding portions that can be used in ADCs bind to linear epitopes. In other respects, anti-CEACAM5 antibodies or their antigen-binding portions that can be used in ADCs bind to conformational epitopes.

[0240] In some aspects, anti-CEACAM5 antibodies are screened for potential use in ADCs based on their high affinity binding to human CEACAM5, and the selected antibodies are investigated using methods such as yeast display assays (where sequence variants of CEACAM5 are presented on the surface of yeast cells), MS-based protein footprinting methods (e.g., HDX-MS and rapid photochemical oxidation of proteins (FPOP)), and structural methods (e.g., X-ray crystal structure determination, molecular modeling, and nuclear magnetic resonance (NMR) spectroscopy, including NMR determination of the HDX exchange rate of unstable amide hydrogen in CEACAM5 when it is free and when it is bound to the target antibody in a complex). Such methods can provide atomic-level resolution of precise epitopes for antibody binding. In recent years, SP-cryo-EM has emerged as a complementary technique to crystallography and NMR for determining near-atomic level structures suitable for application in drug discovery (Renaud et al. Nat Rev Drug Discov [Nature Review: Drug Discovery] 2018; 17:471-92; Scapin et al. CellChem Biol [Cell Chemical Biology] 2018; 25:1318-25; Ceska et al. Biochemical Society Transactions [Journal of the Society for Biochemistry] 2019: p. BST20180267).

[0241] Anti-CEACAM5 antibodies that bind to and compete for the same or similar epitopes as the antibodies disclosed herein can be generated using an immunization protocol similar to that described herein (e.g., in Example 1). In some aspects, immunization can be performed using a construct containing an epitope to which the anti-CEACAM5 antibody disclosed herein binds. The resulting antibodies can be screened for binding to human CEACAM5 via FACS, ELISA, or SPR, and / or for their ability to block the binding of the reference antibody disclosed herein (e.g., determined by ELISA or by blocking the binding of the reference antibody to cells expressing CEACAM5 on their surface), for example, via FACS or SPR. The test antibody can be contacted with the CEACAM5 protein, a protein fragment, or cells expressing CEACAM5 before, simultaneously with, or after the addition of the reference antibody.

[0242] Alternatively, anti-CEACAM5 antibodies or variants of their antigen-binding moieties that can be used in ADCs can be obtained by mutagenesis of the cDNA sequences encoding the heavy and light chains of the antibody. Antibody internalization

[0243] On the other hand, depending on the conditions and results described, for example, in the examples, an anti-CEACAM5 antibody or its antigen-binding portion that can be used in an ADC can bind to CEACAM5 in humans and / or cynomolgus monkeys and induce the internalization of the anti-CEACAM5 antibody or its antigen-binding portion. In some aspects, an ADC (comprising an anti-CEACAM5 antibody or its antigen-binding portion linked to the cytotoxic portion described herein) binds to and is internalized by cells expressing CEACAM5.

[0244] Identification of internalizable anti-CEACAM5 antibodies or their antigen-binding moieties suitable for use in ADCs is important for the development of effective ADCs. The ability of anti-CEACAM5 antibodies or their antigen-binding moieties suitable for use in ADCs to be internalized into cells can be evaluated, as determined by any method well known in the art, including but not limited to the use of the IncuCyte live-cell analysis system, Amnis IMAGESTREAM® imaging flow cytometry analysis, or laser scanning confocal microscopy.

[0245] Internalizing anti-CEACAM5 antibodies or their antigen-binding portions that can be used in ADCs can be characterized or ranked in terms of their “degree of internalization” or “level of internalization”, which may involve the degree (e.g., percentage of cells) or level (total amount of internalized antibody) of internalization relative to control antibodies (e.g., MBN001) (such as non-internalizing antibodies, control IgG, or other control antibodies (e.g., baseline antibodies)) at a given antibody concentration (e.g., 100 nM) or after a given time period (e.g., 2 minutes, 5 minutes, 10 minutes, or 30 minutes).

[0246] In some aspects, the anti-CEACAM5 antibody or its antigen-binding portion that can be used in an ADC is internalized into at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of cells in a cell population that express CEACAM5. In some aspects, the level at which the anti-CEACAM5 antibody or its antigen-binding portion that can be used in an ADC is internalized into cells expressing CEACAM5 or into a cell population expressing CEACAM5 is at least 2, at least 5, at least 20, at least 100, at least 500, or at least 2,000 times that of a control antibody (e.g., a non-internalizing antibody, control IgG, other antibodies, or a baseline antibody).

[0247] In some aspects, the level of anti-CEACAM5 antibody or its antigen-binding portion internalized into CEACAM5-expressing cells (e.g., MKN45 or HCT-116-hu / cyno CEACAM5) is determined by comparing the area under the curve (AUC) immunofluorescence level relative to a reference antibody, for example, as described in Example 6. In some aspects, the antibody / cell concentration of an anti-CEACAM5 antibody or its antigen-binding portion suitable for use in an ADC, internalized into CEACAM5-expressing cells or a population of CEACAM5, results in an AUC immunofluorescence level that is at least 50%, at least 75%, at least 2-fold, at least 3-fold, at least 5-fold, at least 10-fold, at least 25-fold, at least 50-fold, or at least 100-fold higher than that of the control antibody as described herein. In some aspects, an anti-CEACAM5 antibody or its antigen-binding portion suitable for use in an ADC, for example, is internalized into CEACAM5-expressing cells according to the conditions and results shown in Example 6 and Table 9.

[0248] In some respects, anti-CEACAM5 antibodies or their antigen-binding moieties that can be used as ADCs can be characterized by their "internalization rate," which is expressed, for example, as the internalized T... 1 / 2 This is defined as the time when half of the maximum internalization is achieved, measured from the time the antibody is added to the cells. In some respects, the internalization time of anti-CEACAM5 antibodies or their antigen-binding moiety that can be used in ADCs is significantly higher than that of control antibodies as described herein. 1 / 2 It can enhance or increase by at least 10%, 30%, 50%, 75%, 2 times, 3 times, 5 times or more, resulting in T 1 / 2 Shorten by at least 10%, 30%, 50%, 75%, 2 times, 3 times, 5 times or more. For example, replace a 10-minute T... 1 / 2 Anti-CEACAM5 antibodies or their antigen-binding moieties, which can be used in ADCs, can exhibit an increased internalization rate, thereby enhancing the T... 1 / 2 Reduced to 5 minutes (i.e., internalization rate doubled or T) 1 / 2 (Shortened by half). In some respects, T 1 / 2 Shorten by at least 10 minutes, 30 minutes, or 1 hour.

[0249] In some respects, anti-CEACAM5 antibodies or their antigen-binding moiety that can be used in ADCs can be characterized by the maximum level of their internalization into CEACAM5-expressing cells or cell populations expressing CEACAM5, where the maximum internalization level is represented by the internalization level at the plateau in the following figure, which plots internalization relative to antibody concentration or time. In some respects, anti-CEACAM5 antibodies or their antigen-binding moiety that can be used in ADCs exhibit a maximum internalization level of at least 10%, 30%, 50%, 75%, 2-fold, 3-fold, 5-fold, or more compared to control antibodies as described herein.

[0250] Another way to compare the internalization efficacy of anti-CEACAM5 antibodies or their antigen-binding moieties that can be used in ADCs is to compare their internalization levels at a given antibody concentration (e.g., 100 nM) and / or at a given time (e.g., 2 minutes, 5 minutes, 10 minutes, or 30 minutes).

[0251] In some respects, anti-CEACAM5 antibodies or their antigen-binding portions that can be used in ADCs can be characterized by their internalization level, which can be determined using area under the curve (AUC) immunofluorescence analysis, representing the antibody concentration at which 50% of the maximum internalization level is achieved, as measured from the time the antibody is added to the cells, as described in Example 6.

[0252] In some respects, anti-CEACAM5 antibodies or their antigen-binding portions that can be used as ADCs exhibit EC50 binding values ​​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 respects, anti-CEACAM5 antibodies or their antigen-binding portions that can be used as ADCs exhibit EC50 internalization values ​​between 1 nM and 50 nM, between 4 nM and 50 nM, between 10 nM and 50 nM, between 20 nM and 50 nM, between 30 nM and 50 nM, between 4 nM and 40 nM, between 4 nM and 30 nM, between 4 nM and 20 nM, between 8 nM and 40 nM, between 8 nM and 30 nM, between 8 nM and 20 nM, between 12 nM and 40 nM, between 12 nM and 30 nM, or between 12 nM and 25 nM.

[0253] In some respects, the binding level of an anti-CEACAM5 antibody or its antigen-binding moiety that can be used in an ADC can be defined relative to the binding level of a given control antibody as described herein, and expressed as a percentage of the obtained EC50 value compared to the control antibody. In some respects, the degree of binding reflected in the EC50 value can be enhanced by at least 10%, 30%, 50%, 75%, 2-fold, 3-fold, 5-fold, or more compared to the control antibody.

[0254] In some aspects, an anti-CEACAM5 antibody or its antigen-binding portion suitable for use in an ADC may include a modified constant region that, relative to the same anti-CEACAM5 antibody or its antigen-binding portion without the modified constant region, or relative to a control antibody as described herein, imparts increased internalization. The modified constant regions used in these aspects are described in U.S. Patent No. 10,653,791, the contents of which are incorporated herein by reference in their entirety. For example, in some aspects, an anti-CEACAM5 antibody or its antigen-binding portion suitable for use in an ADC may include an IgG2 hinge or replace a non-IgG2 hinge with an IgG2 hinge. In some aspects, an anti-CEACAM5 antibody or its antigen-binding portion suitable for use in an ADC may include a hinge that is not an IgG2 hinge and / or an IgG2 CH1 domain that is replaced by an IgG2 hinge and / or an IgG2 CH1 domain.

[0255] In some aspects, the anti-CEACAM5 antibody that can be used in the ADC of this disclosure contains a lysine residue at the C-terminus of the heavy chain. In some aspects, the anti-CEACAM5 antibody that can be used in the ADC of this disclosure does not contain a lysine residue at the C-terminus of the heavy chain. In some aspects, the anti-CEACAM5 antibody that can be used in the ADC of this disclosure is a composition comprising a mixture of at least two antibodies, one antibody being free of a C-terminal lysine residue and the other antibody being containing a C-terminal lysine residue.

[0256] In some respects, the internalization rate (e.g., by T) of anti-CEACAM5 antibodies or their antigen-binding moieties containing modified constant regions that are suitable for ADCs, relative to the same anti-CEACAM5 antibody or its antigen-binding moieties without modified constant regions, or relative to control antibodies as described herein, is higher. 1 / 2 An increase of 10%, 30%, 50%, 75%, 2 times, 3 times, 5 times or more in the measured value leads to T 1 / 2 Shorten by at least 10%, 30%, 50%, 75%, 2 times, 3 times, 5 times or more. Antibody physical properties

[0257] Each antibody or its antigen-binding moiety will have a unique isoelectric point (pI), which typically falls within a pH range of 6 to 9.5. The pI of IgG1 antibodies typically falls within a pH range of 7–9.5, and the pI of IgG4 antibodies typically falls within a pH range of 6–8. Furthermore, each antibody or its antigen-binding moiety will have a characteristic melting temperature, with higher melting temperatures indicating greater overall in vivo stability (Krishnamurthy R and Manning MC (2002) Curr PharmBiotechnol [Contemporary Pharmaceutical Biotechnology] 3:361–71). Generally, T... M1 The initial unfolding temperature can be greater than 60°C, 65°C, or 70°C. The melting point of the antibody or fragment can be measured using differential scanning calorimetry (Chen et al. (2003) Pharm Res [Drug Research] 20:1952-60; Ghirlando et al. (1999) Immunol Lett [Immunology Communications] 68:47-52) or circular dichroism spectroscopy (Murray et al. (2002) J. Chromatogr Sci [Journal of Chromatography Science] 40:343-9). Alternatively, antibodies and their antigen-binding moieties that do not degrade rapidly are selected. Degradation of the antibody or its antigen-binding moieties can be measured using capillary electrophoresis (CE) and MALDI-MS (Alexander AJ and Hughes DE (1995) Anal Chem [Analytical Chemistry] 67:3626-32).

[0258] In some respects, anti-CEACAM5 antibodies or their antigen-binding moieties that can be used as ADCs exhibit minimal aggregation effects that would otherwise lead to the triggering of unwanted immune responses and / or altered or unfavorable pharmacokinetic properties. Typically, aggregation of the antibody and its antigen-binding moieties of 25% or less, 20% or less, 15% or less, 10% or less, or 5% or less is acceptable. Aggregation can be measured using several techniques, including size exclusion column chromatography (SEC), high-performance liquid chromatography (HPLC), and light scattering. IB. Bispecific or multispecific molecules

[0259] In some aspects, this document provides ADCs conjugated by formula (I), wherein the ADC comprises a bispecific or multispecific molecule (e.g., a bispecific antibody or a multispecific antibody). In some aspects, a bispecific molecule that can be used as an ADC comprises at least one binding region (e.g., an antibody or its antigen-binding moiety thereof) targeting a specific epitope on CEACAM5 (e.g., human CEACAM5) as described herein, and at least one other binding region binding to another antigen. In some aspects, a multispecific molecule that can be used as an ADC comprises an antibody or its antigen-binding moiety disclosed herein and at least two binding regions, each of which binds to another antigen. Bispecific and / or multispecific molecules can be prepared as full-length antibodies or antibody-binding moieties (e.g., F(ab')2 antibodies).

[0260] Methods for preparing bispecific or multispecific molecules are known in the art (see, for example, PCT Publications WO 05117973 and WO 06091209). For example, the generation of full-length bispecific or multispecific molecules (e.g., antibodies) can be based on the co-expression of two paired immunoglobulin heavy-light chains, wherein the two or more chains have different specificities. Various techniques for preparing and isolating bispecific or multispecific molecules directly from recombinant cell cultures are also known. For example, bispecific or multispecific molecules can be generated using leucine zippers. Another strategy for preparing bispecific or multispecific molecules using single-chain Fv (sFv) dimers has also been reported.

[0261] Examples of suitable bispecific or multispecific molecular platforms include, but are not limited to, dual-targeting (DT)-Ig (GSK / Domantis), two-in-one antibody (Genentech), cross-linked Mab (Karmanos Cancer Center), Fcab, and mAb. 2F-Star (F-Star), CovX (CovX / Pfizer), Dual Variable Domain (DVD)-Ig (Abbott), IgG-like Bispecifics (ImClone / EliLilly), Ts2Ab (Medlmmune / AstraZeneca), and BsAb (Zymogenetics), HERCULES (Biogen). Idee), TvAb (Roche), ScFv / Fc fusion, SCORPION (Emergent BioSolutions / Trubion, Zimogenetics / Bristol-Myers Squibb), Fc-DART (MacroGenics), Dual (ScFv)2-Fab (National Research Center for Antibody Medicine - China), F(ab)2 (Medarex / AMGEN), Dual-Action or Dual-Fab (Genentech), Dock-and-Lock (DNL) (ImmunoMedics), Bivalent Bispecific (Biotecnol), SEED (EMD Serono), mAb 2 (F-Star Company), Fab-Fv (UCB-Celltech), Bispecific T-cell connector (BiTE) (Micromet), Tandem biantibody (Tandab) (Affimed), Dual affinity retargeting technology (DART) (Macrogene), Single-chain biantibody (Academic), TCR-like antibody (AIT, Receptor Logics), COMBODY (Epigen Biotech), Dual-targeting nanobody (Ablynx), and Fc-engineered IgG1 (Xencor).

[0262] In some aspects, bispecific molecules that can be used as ADCs include a first binding region (e.g., an antibody or its antigen-binding moiety) that binds to CEACAM5, which is derived from or linked to another functional molecule (e.g., another peptide or protein (e.g., another antibody or ligand of the receptor)) to produce bispecific molecules that bind to both CEACAM5 and non-CEACAM5 target molecules. In some aspects, multispecific molecules include a first binding region (e.g., an antibody or its antigen-binding moiety) that binds to CEACAM5, which is derived from or linked to two or more functional molecules (e.g., different peptides or proteins (e.g., other antibodies or ligands of the receptor)) to produce multispecific molecules that bind to both CEACAM5 and two or more non-CEACAM5 target molecules. Antibodies or their antigen-binding moiety may be derived from or linked to more than one other functional molecule to produce bispecific or multispecific molecules that bind to more than two or more different binding sites and / or target molecules. In order to generate bispecific or multispecific molecules, the antibodies or their antigen-binding portions disclosed herein may be functionally linked (e.g., by chemical coupling, gene fusion, non-covalent association or other means) to one or more other binding molecules (such as another antibody, antibody fragment, peptide, receptor or binding mimic) to generate multispecific molecules.

[0263] Therefore, bispecific molecules (e.g., bispecific antibodies and bifunctional antibodies) are considered, which at least include a first binding specificity against a specific epitope on CEACAM5 (e.g., human CEACAM5) and a second binding specificity against a second target. In some aspects, multispecific molecules (e.g., multispecific antibodies and multifunctional antibodies) are considered, which at least include a first binding specificity against a specific epitope on CEACAM5 (e.g., human CEACAM5), a second binding specificity against a second target, and a third binding specificity against a third target, wherein the second and third targets are not identical. In some aspects, the second and / or third binding regions specifically bind to tumor-associated antigens.

[0264] In some respects, the second and / or third binding regions have excitatory properties when they bind to the target.

[0265] In some respects, the antibody is a trispecific antibody comprising a first, second, and third binding region, wherein the first binding region contains the binding specificity of the anti-CEACAM5 antibody described herein (e.g., an antigen-binding region), and the second and third binding regions bind to two different targets (or different epitopes on the same target) (e.g., the targets described herein).

[0266] In some respects, the antibody is a bifunctional antibody that comprises the anti-CEACAM5 antibody described herein and a receptor molecule.

[0267] In one aspect, a multispecific molecule contains at least one antibody or its antigen-binding moiety (including, for example, Fab, Fab', F(ab')2, Fv, or a single-chain Fv) as a binding specificity. The antibody may also be a light-chain or heavy-chain dimer, or any of its smallest fragments, such as Fv, or a single-chain construct, as described in U.S. Patent No. 4,946,778 to Ladner et al.

[0268] In some aspects, this document provides bispecific or multispecific immune cell adaptors (ICEs) comprising a CEACAM5 binding domain that is linked via a short, flexible linker region to at least one binding domain of a cell surface protein on an immune effector cell. Exemplary immune effector cells include T cells, NK cells, B cells, dendritic cells, and macrophages. Compositions and methods for preparing and using immune cell adaptors are disclosed in U.S. Patent Publication No. 2017 / 368169, the disclosure of which is incorporated herein by reference.

[0269] In some respects, immune cell adaptors are bispecific (BiTE) or trispecific (TriKE) T cell adaptor molecules that contain a CEACAM5 binding domain, which is linked via a short, flexible linker region to at least one binding domain of a T cell surface protein (i.e., a T cell adaptor domain) in T cell effectors (e.g., cytotoxic T cells). BiTEs or TriKEs targeting CEACAM5 can induce CD8+ binding. + CTLs closely approach tumor cells expressing CEACAM5, resulting in high binding affinity. CD8 + Like all T cells, CTLs express a variable T cell receptor (TCR) that associates with the invariant CD3 subunit. In some respects, BiTE targeting CEACAM5 contains a receptor linked to CD3. The CEACAM5 binding fragment of the binding domain, this CD3 CD3 binding domains of the TCR complex The unit forms synapses on the surface of tumor cells, directly activating T cells and triggering cell death signaling pathways, subsequently releasing granzymes and perforin. This is achieved through CD3 binding. The unit, CEACAM-based BiTE, is not limited by TCR specificity and can potentially redirect the entire T cell repertoire in a TCR-peptide-major histocompatibility complex (MHC)-independent manner, thereby avoiding the possibility of immunotherapy-driven MHC-I downregulation and immune escape. Advantageously, CEACAM5-targeting BiTE provides a means of activating exhausted T cells induced by prolonged exposure to CEACAM5. Exemplary T cell adaptor binding domains included in BiTE or TriKE include CD3, TRa, TCRp, TCRy, TCRC, ICOS, CD28, CD27, HVEM, LIGHT, CD40, 4-1BB, OX40, DR3, GITR, CD30, TIM1, SLAM, CD2, CD226, or combinations thereof.

[0270] In some respects, bispecific T-cell adaptor molecules include a CEACAM5 binding domain (bispecific checkpoint inhibitor adaptor) that is linked to a checkpoint inhibitor binding domain (e.g., CTLA-4, PD-1, PD-L1, PD-L2, LAG-3, TIM-3, galactagogue 9, CEACAM-1, BTLA, CD69, galactagogue-1, TIGIT, CD113, CD155, GPR56, VISTA, B7-H3, B7-H4, 2B4, CD48, GARP, PD1H, LAIR1, TIM-1, or TIM-4) via a short flexible adapter region.

[0271] In some respects, immune cell adaptors are bispecific or trispecific natural killer (NK) cell adaptor (NKCE) molecules that contain a CEACAM5 binding domain that is linked via a short, flexible linker region to at least one binding domain of an NK cell surface protein (i.e., an NK cell adaptor binding domain). In some aspects, NKCE includes an antigen-binding domain or ligand that binds to (e.g., activates) the following: 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.

[0272] Bispecific or multispecific molecules can be prepared by conjugating constitutive binding specificities (e.g., 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 individually and then conjugated to each other. When the binding specificity is a protein or peptide, covalent conjugation can be performed using a variety of coupling agents or cross-linking agents. Examples of cross-linking agents include protein A, carbodiimide, N-succinimide-S-acetyl-thioacetate (SATA), 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), o-phenylene dimaleimide (oPDM), N-succinimide-3-(2-pyridyldithio)propionate (SPDP), and 4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid sulfosuccinimide (sulfonyl-SMCC). In some respects, the binders are SATA and sulfonyl-SMCC, both of which are available from Pierce Chemical Co. (Rockford, Illinois).

[0273] When binding is specific to antibodies, they can be conjugated via thiol bonds in the C-terminal hinge regions of the two heavy chains. In some respects, prior to conjugation, the hinge regions are modified to contain an odd number (e.g., one) of thiol residues.

[0274] Alternatively, both binding specificities can 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 a mAb x mAb, mAb x Fab, Fab x F(ab')2, or ligand x Fab fusion protein. A bispecific or multispecific molecule can 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 can contain at least two single-chain molecules. Methods for preparing bispecific or multispecific molecules are described, for example, in the following patents: U.S. Patent No. 5,260,203; U.S. Patent No. 5,455,030; U.S. Patent No. 4,881,175; U.S. Patent No. 5,132,405; U.S. Patent No. 5,091,513; U.S. Patent No. 5,476,786; U.S. Patent No. 5,013,653; U.S. Patent No. 5,258,498; and U.S. Patent No. 5,482,858.

[0275] The binding of bispecific or multispecific molecules to their specific targets can be confirmed by, for example, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), FACS analysis, bioassays (e.g., growth inhibition), or simple Western blotting. Each of these assays typically detects the presence of a protein-antibody complex of particular interest using a labeled reagent (e.g., antibody) that is specific to the target complex. For example, FcR-antibody complexes can be detected using, for example, an enzyme-linked antibody or antigen-binding moiety that recognizes and specifically binds to the antibody-FcR complex. Alternatively, these complexes can be detected using any of a variety of other immunoassays. For example, antibodies can be radiolabeled and used in radioimmunoassays (RIA). Radioisotopes can be detected by means such as using an αγ-β counter or a scintillation counter or by autoradiography. IIC. Antibody Engineering

[0276] As disclosed herein, ADCs or their components (e.g., anti-CEACAM5 antibodies or their antigen-binding portions) can be modified or engineered to improve their physical and functional properties. Antibody engineering in the Fc region

[0277] The ADCs or components thereof described herein (e.g., anti-CEACAM5 antibodies or their antigen-binding moieties) may contain modifications to their respective Fc regions, typically to alter one or more physical or functional properties, such as effector function (e.g., antigen-dependent cytotoxicity), Fc receptor binding, serum half-life, and complement binding. Furthermore, the ADCs or components thereof (e.g., anti-CEACAM5 antibodies or their antigen-binding moieties) may be chemically modified (e.g., one or more chemical moieties may be attached to the antibody) or modified to alter their glycosylation, again to alter one or more properties of the antibody or fragment. In the context of Fc region modifications, the residues in the Fc region are numbered according to the Kabat EU index.

[0278] The ADCs or components thereof disclosed herein (e.g., anti-CEACAM5 antibodies or their antigen-binding portions) also include antibodies and fragments having modified (or blocked) Fc regions to provide altered effector functions, as described, for example, in the following documents: U.S. Patent No. 5,624,821; U.S. Patent Publications Nos. US 2009 / 280114 and US 2011 / 142858; and PCT Publication No. WO 2006 / 0057702. Such modifications may further include alterations that enhance or suppress a variety of immune system responses, potentially with beneficial effects in diagnosis and treatment. altered effector function

[0279] In some aspects, an ADC or a component thereof (e.g., an anti-CEACAM5 antibody or its antigen-binding portion) includes a variant Fc region that is modified (e.g., by amino acid substitution, deletion, and / or insertion) relative to a parental Fc sequence (e.g., an unmodified Fc polypeptide subsequently modified to produce a variant) to increase or decrease the ability of the antibody or its antigen-binding portion to mediate one or more effector functions and / or increase or decrease its binding to an Fc-γ receptor (FcγR) while retaining its antigen-binding capacity. Thus, in an exemplary aspect, an ADC or a component thereof (e.g., an anti-CEACAM5 antibody or its antigen-binding portion) may include one or more amino acid variations that alter the affinity for effector ligands (e.g., Fc receptors or the C1 component of complement). This method is further described in detail in U.S. Patent Nos. 5,624,821 and 5,648,260.

[0280] Interactions between constant regions of antigen-binding proteins (e.g., anti-CEACAM5 antibodies or their antigen-binding moieties) and various Fc receptors (FcRs) (including FcγRI (CD64), FcγRII (CD32), and FcγRIII (CD16)) are thought to mediate effector functions of antigen-binding proteins (e.g., ADCC and CDC). Fc receptors are also important for antibody crosslinking, which may be crucial for antitumor immunity. In an exemplary aspect, modifications can be made to the Fc region to produce 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 Fc receptors relative to the parental Fc, and / or (e) increased or decreased pharmacokinetic stability.

[0281] Alterations to the Fc region can 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 variant Fc region may contain two, three, four, five, or more substitutions, such as substitutions at specific Fc region positions identified herein. In some aspects, altering the Fc region by substituting at least one amino acid residue with a different amino acid residue results in an altered affinity of the antibody for the effector ligand while retaining 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 can be substituted with different amino acid residues. The effector ligand with altered affinity can be, for example, an Fc receptor or the C1 component of complement. This method is further described in detail in U.S. Patents 5,624,821 and 5,648,260 to Winter et al. In some aspects, the C1q binding site can be removed from the Fc region by deletion or substitution, for example, the EKK sequence of human IgG1. In another example, one or more amino acids selected from amino acid residues 329, 331, and 322 may be substituted with different amino acid residues, resulting in altered C1q binding and / or reduced or eliminated complement-dependent cytotoxicity (CDC) of the antibody. This method is further described in detail in U.S. Patent No. 6,194,551 to Idusogie et al. In another example, one or more amino acid residues within amino acid positions 231 and 239 are altered to change the antibody's ability to bind complement. This method is further described in U.S. Patent No. 6,180,377.

[0282] In some respects, this article provides an effector-free form of an anti-CEACAM5 antibody or its antigen-binding moiety having, for example, the mutant hIgG1f allotype (hIgG1.3f) described herein (e.g., containing the amino acid sequence shown in SEQ ID NO: 30). The hIgG1.3f variant is a triple mutant form of hIgG1f (L234A, L235E, G237A) lacking both FcγR binding and effector function.

[0283] In some respects, ADCs or their components (e.g., anti-CEACAM5 antibodies or their antigen-binding moieties) can be engineered to have different affinities and selectivities for the Fcγ receptor (FcγR) by mutating the constant regions of the heavy chain, including the hinge and Fc domain. Mutations can be introduced to enhance or reduce FcγR binding. These mutations can increase or decrease FcγR-mediated crosslinking and / or signaling. For therapeutic targets such as CEACAM5, FcγR-mediated crosslinking of anti-CEACAM5 antibodies has the potential to provide undesirable agonist signaling and the potential to provide toxicity without introducing certain modifications to avoid this problem.

[0284] Binding sites on human IgG1 for FcγR1, FcγRII, FcγRIII, and FcRn have been mapped, and variants with improved binding have been described (see Shields, RL et al. (2001) J. Biol. Chem. 276:6591-6604). Specific mutations at positions 256, 290, 298, 333, 334, and 339 are shown to improve binding to FcγRIII. Additionally, the following combined mutants are shown to improve FcγRIII binding: T256A / S298A, S298A / E333A, S298A / K224A, and S298A / E333A / K334A, which have been shown to exhibit enhanced FcγRIIIa binding and ADCC activity (Shields et al., 2001). Other IgG1 variants with significantly enhanced binding to FcγRIIIa have been identified, including variants with the S239D / I332E and S239D / I332E / A330L mutations, which showed a maximum increase in affinity for FcγRIIIa, a decrease in FcγRIIb binding, and strong cytotoxic activity in cynomolgus monkeys (Lazar et al., 2006). Introducing triple mutations into antibodies such as alentozumab (CD52 specific), trastuzumab (HER2 / neu specific), rituximab (CD20 specific), and cetuximab (EGFR specific) resulted in significantly enhanced in vitro ADCC activity, and the S239D / I332E variant showed enhanced ability to deplete B cells in monkeys (Lazar et al., 2006).

[0285] In some respects, ADCs or components thereof (e.g., anti-CEACAM5 antibodies or their antigen-binding portions) can be engineered to reduce the potential for FcγR binding and cross-linking and / or signaling, specifically, reducing the binding of “low-affinity” FcγRs hCD32a / FcγRIIa, hCD32b / FcγRIIb, hCD16a / FcγRIIIa, and hCD16b / FcγRIIIb. Binding to the “high-affinity” receptor CD64 / FcγRI is generally considered less of a concern because this receptor is saturated with serum IgG. Therefore, in some respects, ADCs or components thereof (e.g., anti-CEACAM5 antibodies or their antigen-binding portions) can contain an IgG1.3 Fc region that is substantially non-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 Publication No. US 2022 / 0106400).

[0286] In some respects, the Fc region can be engineered by modifying one or more amino acids at the following positions to increase ADCC and / or increase FcγR binding: 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, 3 01, 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 (as described, for example, in U.S. Patent No. 6,737,056), wherein the residues in the Fc region are numbered as in the EU index in Kabat. Exemplary substitutions include 236A, 239D, 239E, 268D, 267E, 268E, 268F, 324T, 332D, and 332E. Exemplary variants include 239D / 332E, 236A / 332E, 236A / 239D / 332E, 268F / 324T, 267E / 268F, 267E / 324T, and 267E / 268F / 324T. Other modifications for enhancing FcyR and complement interactions include, but are not limited to, substitutions for 298A, 333A, 334A, 326A, 247I, 339D, 339Q, 280H, 290S, 298D, 298V, 243L, 292P, 300L, 396L, 305I, and 396L. These and other modifications are reviewed in Strohl, 2009, Current Opinion in Biotechnology, 20:685-691.

[0287] In some respects, the Fc region is modified by modifying residues 243 and 264 to reduce the ability of the anti-CEACAM5 antibody or its antigen-binding moiety described herein to mediate effector function and / or increase anti-inflammatory properties. In one aspect, the Fc region of an ADC or its components (e.g., an anti-CEACAM5 antibody or its antigen-binding moiety) is modified by changing the residues at positions 243 and 264 to alanine. In another aspect, the Fc region is modified by modifying residues 243, 264, 267, and 328 to reduce the ability of the ADC or its components (e.g., an anti-CEACAM5 antibody or its antigen-binding moiety) to mediate effector function and / or increase anti-inflammatory properties.

[0288] Other Fc modifications to the Fc region include those that reduce or eliminate binding to FcγR and / or complement proteins, thereby reducing or eliminating Fc-mediated effector functions such as ADCC, ADCP, and CDC. Modifications that 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, where the numbering is based on the EU index. In one respect, Fc variants provide selectively enhanced affinity for FcyRllb relative 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 used 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 used to reduce FcyR and complement interactions include substitutions for 297A, 234A, 235A, 237A, 318A, 228P, 236E, 268Q, 309L, 330S, 331S, 220S, 226S, 229S, 238S, 233P, and 234V, as well as the removal of the glycosylation at position 297, either through mutation or enzymatic means or by production in organisms that do not glycosylate proteins, such as bacteria. These and other modifications are reviewed in Strohl, 2009, Current Opinion in Biotechnology, 20:685-691.

[0289] In some respects, the Fc region can be modified to remove ADCC sites. Regarding ADCC sites in IgG1, they can be found, for example, in Molec. Immunol. [Molecular Immunology] 29 (5): 633-9 (1992). Furthermore, in B-cell malignancy and breast cancer models, IgG1 mutants containing the L235V, F243L, R292P, Y300L, and P396L mutations have been found to exhibit enhanced binding to FcγRIIIa and concurrently enhanced ADCC activity in transgenic mice expressing human FcγRIIIa (Stavenhagen et al., 2007; Nordstrom et al., 2011). Other usable Fc mutants include: S298A / E333A / L334A, S239D / I332E, S239D / I332E / A330L, L235V / F243L / R292P / Y300L / P396L, and M428L / N434S. Specific examples of the variant Fc domains are disclosed, for example, in U.S. Patent No. 6,096,871 and PCT Publication No. WO97 / 34631.

[0290] Optionally, the Fc region may contain non-naturally occurring amino acid residues at other and / or alternative locations (see, for example, U.S. Patent Nos. 5,624,821; 6,277,375; 6,737,056; 6,194,551; 7,317,091; 8,101,720; PCT Patent Publication Nos. WO 00 / 42072; WO 01 / 58957; WO 02 / 06919; WO 04 / 016750; WO 04 / 029207; WO 04 / 035752; WO 04 / 074455; WO 04 / 099249; WO 04 / 063351; WO 05 / 070963; WO 05 / 040217, WO 05 / 092925 and WO 06 / 020114).

[0291] In one aspect, the hinge region of Fc is modified to alter the number of cysteine ​​residues in the hinge region, for example, by increasing or decreasing them. For example, in one aspect, the number of cysteine ​​residues in the hinge region of CH1 is increased to provide increased antibody stability, or the number is decreased to provide enhanced light and heavy chain assembly, or as described in U.S. Patent No. 5,677,425.

[0292] In some respects, the Fc region can be modified to increase the biological half-life of the ADC, thereby promoting a lower dosing frequency while improving convenience and reducing material usage (Presta (2005) J. Allergy Clin. Immunol. [Journal of Allergy and Clinical Immunology] 116:731, in 734-35). Various methods can be employed. For example, in some respects, this can be achieved by increasing the binding affinity of the Fc region to the nascent Fc receptor (FcRn). For example, one or more of the following residues can 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 prolong the biological half-life, the antibody can be modified in the CH1 or CL region to contain a rescue receptor for the binding epitope, which is derived from two loops of the CH2 domain of the Fc region of IgG, as described in U.S. Patent Nos. 5,869,046 and 6,121,022 to Presta et al.

[0293] Other Fc variants used to increase binding to FcRn and / or improve pharmacokinetic properties include substitutions at positions 259, 308, 428, and 434, including, for example, 259I, 308F, 428L, 428M, 434S, 434H, 434F, 434Y, and 434M. Other variants that enhance 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, 434A (Shields et al., Journal of Biological Chemistry, 2001, 276(9):6591-6604); 252F, 252T, 252Y, 252W, 254T, 256S, 256R, 256Q, 256E, 256D, 256T, 309P, 311S, 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) (281:23514-23524). Other modifications used to regulate FcRn binding are described in Yeung et al., 2010, J Immunol, 182:7663-7671.

[0294] On the other hand, mutations can be made in the Fc hinge region to shorten the biological half-life of the antibody or fragment. For example, one or more amino acid mutations can be introduced into the CH2-CH3 domain interface region of the Fc hinge fragment, such that SpA binding of the antibody or fragment is impaired relative to the binding of the native Fc-hinge domain to staphylococcal protein A (SpA), as described in U.S. Patent No. 6,165,745.

[0295] In some respects, heterozygous IgG isotypes with specific biological characteristics can be used. For example, in some respects, one or more regions and / or mutations from IgG2 or IgG4. In one respect, 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 corresponding to the heavy chain constant region. This mutation has been reported to eliminate the heterogeneity of the inter-heavy chain disulfide bridge in the hinge region (Angal et al., ibid.; position 241 based on the Kabat numbering system). When using the IgG4 constant domain, substitution of S228P is typically included, which mimics the hinge sequence in IgG1, thereby stabilizing the IgG4 molecule.

[0296] On the other hand, IgG1 / IgG3 heterozygous variants can be constructed by substituting the IgG1 position in the CH2 and / or CH3 regions with amino acids from different positions in two isotypes of IgG3. Thus, heterozygous variant IgG antibodies containing one or more substitutions (e.g., 274Q, 276K, 300F, 339T, 356E, 358M, 384S, 392N, 397M, 4221, 435R, and 436F) can be constructed. In other aspects described herein, IgG1 / IgG2 heterozygous variants can be constructed by substituting the IgG2 position in the CH2 and / or CH3 regions with amino acids from different positions in two isotypes of IgG1. Thus, heterozygous variant IgG antibodies containing one or more substitutions, such as one or more of the following amino acid substitutions: 233E, 234L, 235L, 236G (referring to the insertion of glycine at position 236), and 327A.

[0297] In some respects, the variant Fc region may also contain sequence alterations, where amino acids involved in disulfide bond formation are removed or replaced with other amino acids. Such removals can prevent reactions with other cysteine-containing proteins present in the host cells used to generate the antibodies described herein. Even when cysteine ​​residues are removed, the single-chain Fc domain can still form a non-covalently held dimer Fc domain. In other respects, the Fc region can be modified to make it more compatible with the selected host cells. For example, the PA sequence near the N-terminus of the typical native Fc region can be removed, as this sequence can be recognized by digestive enzymes in E. coli, such as proline iminopeptidase.

[0298] The ADCs or their components disclosed herein (e.g., anti-CEACAM5 antibodies or their antigen-binding portions) may contain one or more glycosylation sites. Such glycosylation sites may lead to increased immunogenicity of the antibody or fragment or changes in the pK of the antibody due to altered antigen binding (Marshall et al. (1972) Annu Rev Biochem [Annals of Biochemistry] 41:673-702; Gala and Morrison (2004) J Immunol [Journal of Immunology] 172:5489-94; Wallick et al. (1988) J Exp Med [Journal of Experimental Medicine] 168:1099-109; Spiro (2002) Glycobiology [Glycobiology] 12:43R-56R; Parekh et al. (1985) Nature [Nature] 316:452-7; Mimura et al. (2000) Mol Immunol [Molecular Immunology] 37:697-706). Glycosylation is known to occur at motifs containing NXS / T sequences.

[0299] Therefore, in some respects, the glycosylation properties of the ADCs described herein or their components (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 unglycosylated residues (e.g., alanine) to produce non-glycosylated antibodies. In some respects, glycosylation can be altered to, for example, increase the antibody's affinity for the antigen. For example, one or more amino acid substitutions can be made that result in the removal of one or more variable region framework glycosylation sites, thereby eliminating the glycosylation at that site. The resulting non-glycosylation can increase the antibody's affinity for the antigen. This approach is further described in detail in U.S. Patent Nos. 5,714,350 and 6,350,861 to Co et al. Glycosylation on N297 in the constant region can be prevented by mutating the N297 residue to another residue (e.g., N297A) and / or by mutating an adjacent amino acid (e.g., 298) to reduce glycosylation on N297.

[0300] Alternatively or concurrently, the ADCs described herein, or components thereof (e.g., anti-CEACAM5 antibodies or their antigen-binding moieties), can be engineered to have altered glycosylation types, such as hypofucosylated antibodies with reduced amounts of fucosylated residues or antibodies with increased bipartite GlcNac structures. Non-fucosylated antibodies possess a trimannose core structure of a complex N-glycan lacking fucosylated residues in the Fc region. Due to enhanced FcγRIIIa binding capacity, these glycosylated engineered antibodies lacking the core fucosylated residues in the Fc N-glycan can exhibit stronger ADCC than fucosylated equivalents. Such carbohydrate modifications can be achieved, for example, by expressing antibodies in host cells with altered glycosylation mechanisms.

[0301] Cells with altered glycosylation mechanisms can be used as host cells for expressing the recombinant antibodies described herein, thereby producing antibodies with altered glycosylation. For example, Hanai et al., EP 1,176,195, describe a cell line with a dysfunctional FUT8 gene (encoding fucosyltransferase, i.e., α-1,6-fucosyltransferase) such that antibodies expressed in such cell lines exhibit low fucosylation. Recombinant host cells that have been genetically modified to inactivate the FUT8 gene encoding α-1,6-fucosyltransferase are available. See, for example, the POTELLIGENT™ technology system available from BioWa, Inc. (Princeton, NJ), in which CHOK1SV cells lacking a functional copy of the FUT8 gene produce monoclonal antibodies with enhanced ADCC activity relative to the same monoclonal antibodies produced in cells with a functional FUT8 gene. Aspects of the POTELLIGENT™ technology system are described in U.S. Patent Nos. 7,214,775 and 6,946,292 and PCT Publications Nos. WO 00 / 61739 and WO 02 / 31240.

[0302] Presta's PCT publication WO 03 / 035835 describes a variant CHO cell line, Lec13, which exhibits a reduced ability to attach fucose to Asn(297)-linked carbohydrates, resulting in hypofucosylation of antibodies expressed in this host cell (see also Shields, RL et al. (2002) J. Biol. Chem. [Journal of Biochemistry] 277:26733-26740). Umana et al.'s PCT publication WO 99 / 54342 describes cell lines engineered to express glycosyltransferases that modify glycoproteins (e.g., β(1,4)-N-acetylglucosamine transferase III (GnTIII)), resulting in antibodies expressed in these engineered cell lines exhibiting an increased bipartite GlcNac structure, leading to increased ADCC activity of the antibodies (see also Umana et al. (1999) Nat. Biotech. [Nature Biotechnology] 17:176-180).

[0303] Another modification of the antibodies described herein is PEGylation. In some aspects, the ADCs or components thereof described herein (e.g., anti-CEACAM5 antibody or its antigen-binding moiety) are PEGylated to, for example, increase the antibody's biological (e.g., serum) half-life. To PEGylate an antibody, the antibody or a fragment thereof is typically reacted with polyethylene glycol (PEG) (such as a reactive ester or aldehyde derivative of PEG) under conditions where one or more PEG groups are attached to the antibody or antibody fragment. In some aspects, 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 cover any form of PEG that has been used to derivatize other proteins, such as mono(C1-C10)alkoxy- or aryloxy-polyethylene glycol or polyethylene glycol-maleimide. In some aspects, the antibody to be PEGylated is a non-glycosylated antibody. Methods for PEGylating proteins can be applied to the antibodies or their antigen-binding moieties described herein. See, for example, European Patent No. EP 0 154 316 of Nishimura et al. and European Patent No. EP 0 401 384 of Ishikawa et al.

[0304] Effector function can be measured in a variety of ways, including, for example, by binding of FcγRIII to natural killer cells or by binding of FcγRI to monocytes / macrophages to measure ADCC effector function. For example, the ADCC effector function of the antigen-binding protein of the present invention can be evaluated in a natural killer cell assay. Examples of such assays can be found in the following literature: Shields et al., 2001 J. Biol. Chem., Vol. 276, pp. 6591-6604; Chappel et al., 1993 J. Biol. Chem., Vol. 268, pp. 25124-25131; Lazar et al., 2006 PNAS, 103; 4005-4010.

[0305] The affinity and binding properties of the Fc region to its ligands can be determined by a variety of in vitro assays (based on biochemical or immunological methods), including but not limited to equilibrium methods (e.g., enzyme-linked immunosorbent assay (ELISA) or radioimmunoassay (RIA)), kinetic methods (e.g., BIACORE assay), and other methods (e.g., indirect binding assays, competitive inhibition assays, fluorescence resonance energy transfer (FRET), gel electrophoresis, and chromatography (e.g., gel filtration)). These and other methods can utilize labeling on one or more components being examined and / or employ a variety of detection methods, including but not limited to chromogenic labeling, fluorescent labeling, luminescent labeling, or isotopic labeling. A detailed description of binding affinity and kinetics can be found in Paul, WE, ed., Fundamental Immunology, 4th ed., Lippincott-Raven, Philadelphia (1999), which focuses on antibody-immunogen interactions.

[0306] Regarding the modifications described above used to increase or decrease one or more functional properties (e.g., biochemical, immunochemical, cellular, physiological, or other biological activities, as determined using methods known in the art and described herein), an increase in a given parameter may represent a statistically significant increase of at least 10% (e.g., at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%, i.e., 2-fold, 3-fold, 5-fold, or 10-fold) of the measured parameter. Conversely, a decrease in the measured parameter may represent a statistically significant decrease of at least 10% (e.g., at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%, 3-fold, 5-fold, or 10-fold) of the measured parameter.

[0307] Any of the above modifications may be used alone or in combination with any of the above modifications or those described in the next section to further enhance or reduce effector function or other desired properties (e.g., stability, expression). Antibody engineering of variable regions

[0308] In some respects, ADCs or components thereof (e.g., anti-CEACAM5 antibodies or their antigen-binding moieties) are engineered by modifying framework residues within the variable domains of the parent antibody, for example, to improve the properties of the antibody or its antigen-binding moieties. Typically, such framework modifications are performed to reduce the immunogenicity of anti-CEACAM5 antibodies or their antigen-binding moieties. This is usually achieved by replacing non-CDR residues (i.e., framework residues) in the variable domains of the parent (e.g., rodent) antibody with similar residues from an immune repertoire of the species from which the antibody is to be used (e.g., human residues in the case of human therapeutics). Such antibodies are called “humanized” antibodies. In some cases, it is desirable to increase the affinity of engineered (e.g., humanized) antibodies or to alter their specificity. One approach is to “reverse mutate” one or more framework residues to the corresponding germline sequence. More specifically, antibodies that have undergone somatic mutations may contain framework residues different from the germline sequence from which the antibody was derived. Such residues can be identified by comparing the antibody framework sequence with the germline sequence from which the antibody was derived. Another approach is to revert to the original parent (e.g., rodent) residues at one or more sites on the engineered (e.g., humanized) antibody, for example, to restore binding affinity that may have been lost during the replacement of framework residues. (See, for example, U.S. Patent Nos. 5,693,762, 5,585,089, and 5,530,101).

[0309] In some respects, anti-CEACAM5 antibodies and their antigen-binding portions in ADCs are engineered (e.g., humanized) to include modifications in the framework and / or CDR to improve their properties. Such engineering changes can be based on molecular modeling. Molecular models of the variable regions of parental (non-human) antibody sequences can be constructed to understand the structural characterization of the antibody and to identify potential regions on the antibody that can interact with the antigen. Conventional CDRs are based on the alignment of immunoglobulin sequences and the identification of variable regions. Kabat et al., (1991) Sequences of Proteins of Immunological Interest, Kabat et al.; National Institutes of Health, Bethesda, MD; 5th edition; NIH Publication No. 91-3242; Kabat (1978) Adv. Prot. Chem. 32:1-75; Kabat et al., (1977) J. Biol. Chem. 252:6609-6616. Chothia and colleagues carefully examined the conformation of rings in the crystal structures of antibodies and proposed the concept of hypervariable rings. (Chothia et al., (1987) J Mol. Biol. [Journal of Molecular Biology] 196:901-917 or Chothia et al., (1989) Nature [Nature] 342:878-883). Differences exist between regions classified as “CDR” and “hypervariable rings.” Later studies (Raghunathan et al., (2012) J. Mol Recog. [Journal of Molecular Recognition] 25, 3, 103-113) analyzed several antibody-antigen crystal complexes and observed that antigen-binding regions in antibodies do not necessarily strictly conform to “CDR” residues or “hypervariable” rings. Molecular models of the variable regions of nonhuman antibodies can be used to guide the selection of regions that can potentially bind to antigens. In fact, model-based potential antigen-binding regions differ from conventional “CDR” or “hypervariable” rings. Commercial scientific software such as MOE (Chemical Computing Group) can be used for molecular modeling. Human frames can be selected based on the best match between the frame and the CDR with the non-human sequence. For FR4 (frame 4) in VH, the human VJ region is compared with the corresponding non-human region. In the case of FR4 (frame 4) in VL, the J-κ and J-λ regions of the human sequence are compared with the corresponding non-human regions. Once a suitable human frame is identified, the CDR is transplanted into the selected human frame. In some cases, certain residues at the VL-VH interface can be preserved, such as in the non-human (parental) sequence.Molecular models can also be used to identify residues that may potentially alter the conformation of the CDR and thus change its binding to the antigen. In some cases, these residues are retained, such as in non-human (parental) sequences. Molecular models can also be used to identify solvent-exposed amino acids that can lead to undesirable effects such as glycosylation, deamidation, and oxidation. Developability filters can be introduced early in the design phase to eliminate / minimize these potential problems.

[0310] Another type of framework modification involves mutating one or more residues within the framework region or even one or more CDR regions to remove T-cell epitopes, thereby reducing the potential immunogenicity of the antibody. This approach is also known as "deimmunization" and is described in further detail in U.S. Patent No. 7,125,689. In some aspects, one or more glycosylation sites in the variable regions (such as framework regions) of light or heavy chain immunoglobulins can be modified or removed to reduce immunogenicity. In specific aspects, it is desirable to change certain amino acids containing exposed side chains to another amino acid residue to provide greater chemical stability to the final antibody, thereby avoiding deamidation or isomerization. Deamidation of asparagine can occur at NG, DG, NG, NS, NA, NT, QG, or QS sequences and results in the production of isoaspartic residues, which introduce kinks into the polypeptide chain and reduce the stability of the polypeptide chain (the isoaspartic effect). Isomerization can occur at DG, DS, DA, or DT sequences. In some aspects, the antibodies provided herein do not contain deamidation sites or asparagine isomerization sites. For example, asparagine (Asn) residues can be changed to Gln or Ala to reduce the likelihood of isoaspartic acid formation at any Asn-Gly sequence (especially within CDR).

[0311] A similar problem may occur at the Asp-Gly sequence. Reissner and Aswad (2003) Cell. Mol. Life Sci. 60:1281. Isoflavone formation can weaken or completely eliminate the binding of an antibody to its target antigen. See Presta (2005) J. Allergy Clin. Immunol. 116:731, in 734.

[0312] In all respects, asparagine is replaced with glutamine (Gln). It is also desirable to modify the amino acid adjacent to the asparagine (Asn) or glutamine (Gln) residue to reduce the likelihood of deamidation, which occurs at a higher rate when a small amino acid is present adjacent to asparagine or glutamine. See Bischoff and Kolbe (1994) J. Chromatog. [Journal of Chromatography] 662:261. Furthermore, any methionine residue (typically solvent-exposed Met) in the CDR can be replaced with Lys, Leu, Ala, or Phe or other amino acids to reduce the likelihood of thiooxidation of methionine, which may decrease antigen-binding affinity and also contribute to molecular heterogeneity in the final antibody formulation. Ibid. Additionally, to prevent or minimize potentially fragile Asn-Pro peptide bonds, it is desirable to modify any Asn-Pro combination found in the CDR to Gln-Pro, Ala-Pro, or Asn-Ala. Antibodies with such substitutions are then screened to ensure that these substitutions do not reduce the antibody's affinity or specificity for CEACAM5 or other desired biological activity to unacceptable levels. Exemplary stable CDR variants are shown in Table 1. Table 1. Exemplary stable CDR variants III. Pharmaceutical Compositions

[0313] This document also provides pharmaceutical compositions comprising an ADC as disclosed herein and a carrier (e.g., a pharmaceutically acceptable carrier). Such compositions can be used in a variety of therapeutic applications, such as cancer treatment.

[0314] In some aspects, the pharmaceutical composition may further comprise other compounds, pharmaceuticals, and / or agents for various therapeutic applications. Such compounds, pharmaceuticals, and / or agents may include, for example, anticancer agents, chemotherapeutic agents, immunosuppressants, immunostimulants, immune checkpoint inhibitors, and / or anti-inflammatory agents. Exemplary compounds, pharmaceuticals, and agents that may be formulated with or alone with this ADC are described in the next section.

[0315] As used herein, “pharmaceutically acceptable carriers” include any and all physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, and absorption delay agents. In some respects, carriers are suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, or epidermal administration (e.g., by injection or infusion). Depending on the route of administration, active compounds (i.e., antibodies, immunoconjugates, or bispecific molecules) may be coated in the material to protect the compound from acids and other natural conditions that may inactivate it.

[0316] The pharmaceutical compounds described herein may include one or more pharmaceutically acceptable salts. A “pharmaceutically acceptable salt” is a salt that retains the desired biological activity of the parent compound without conferring any undesirable toxicological effects (see, for example, Berge, SM et al. (1977) J. Pharm. Sci. [Journal of Pharmaceutical Sciences] 66:1-19). Examples of such salts include acid addition salts and base addition salts. 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, phosphorous acid, etc.) and those derived from non-toxic organic acids (such as aliphatic monocarboxylic acids and aliphatic dicarboxylic acids, phenyl-substituted alkanes, hydroxyalkanes, aromatic acids, aliphatic and aromatic sulfonic acids, etc.). Alkali addition salts include those derived from alkaline earth metals (such as sodium, potassium, magnesium, calcium, etc.) and non-toxic organic amines (such as N,N'-dibenzylethylenediamine, N-methylglucosamine, chloroprocaine, choline, diethanolamine, ethylenediamine, procaine, etc.).

[0317] The pharmaceutical compositions described herein may also include pharmaceutically acceptable antioxidants. Examples of pharmaceutically acceptable antioxidants include: (1) water-soluble antioxidants, such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, etc.; (2) oil-soluble antioxidants, such as ascorbate palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, α-tocopherol, etc.; and (3) metal chelating agents, such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, etc.

[0318] Examples of suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical compositions described herein include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, etc.) 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. The presence of microorganisms can be prevented by the sterilization procedures described above, as well as by including various antibacterial and antifungal agents (e.g., parabens, chlorobutanol, phenolic sorbic acid, etc.). It is also desirable to include isotonic agents, such as sugars and sodium chloride, in the compositions. Furthermore, prolonged absorption of injectable drug forms can be achieved by including agents that delay absorption (such as aluminum monostearate and gelatin).

[0319] Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the ad hoc preparation of sterile injectable solutions or dispersions. Consider their use in the pharmaceutical compositions described herein unless any medium or reagent is incompatible with the active compound. Pharmaceutical compositions may contain preservatives or may not contain preservatives. Complementary active compounds may be incorporated into the composition.

[0320] The compositions described herein can be administered via one or more routes of administration using a variety of methods. The route and / or manner of administration may vary depending on the desired outcome. Routes of administration for the ADCs described herein include, for example, intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal, or other parenteral administration routes, such as by injection or infusion. As used herein, the phrase “parenteral administration” means administration other than enteral and topical administration, typically by injection, and includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, tracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injections and infusions.

[0321] Alternatively, the ADCs described herein can be administered via non-parenteral routes (such as topical, epidermal, or mucosal administration), for example, intranasal, oral, vaginal, rectal, sublingual, or topical administration. IV. Uses and Methods

[0322] The ADCs described in this article have many of the in vitro and in vivo effects described herein. Cancer treatment

[0323] On one hand, this article provides a method for treating cancer, which includes administering an effective amount of ADC to a subject in need, thereby inhibiting or reducing the growth of cancerous tumors and / or achieving regression and / or prolonging survival.

[0324] In some respects, the ADCs described herein can be administered in combination with one or more other cytotoxic agents or therapeutic agents, such as those described herein.

[0325] The ADCs described in this article can be used to inhibit the growth of cancers expressing CEACAM5, including but not limited to carcinomas, lymphomas, blastomas, sarcomas, and leukemias. More specific examples of this type of cancer include, but are not limited to, basal cell carcinoma, bile duct cancer; bladder cancer; bone cancer; brain 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; digestive system cancers; endometrial cancer; esophageal cancer; eye cancer; head and neck cancer; gastric cancer (including gastrointestinal cancer); glioblastoma; liver cancer (e.g., hepatocellular carcinoma; hepatocellular tumor); intraepithelial tumor; kidney (kidney or renal) cancer; laryngeal cancer; leukemia; lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and squamous cell carcinoma); lymphoma (including Hodgkin's lymphoma and non-Hodgkin's lymphoma); melanoma; myeloma; neuroblastoma; oral cancer (e.g., lip cancer, tongue cancer, oral cancer, and pharyngeal cancer); ovarian cancer; pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectal cancer; respiratory system cancers; salivary gland cancer; and more. Adenocarcinoma; sarcoma; skin cancer; squamous cell carcinoma; teratoma; testicular cancer; thyroid cancer; uterine or endometrial cancer; urinary tract cancer; vulvar cancer; and other cancers and sarcomas; as well as 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 non-lytic cell NHL; giant mass NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenström macroglobulinemia); chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); hairy cell leukemia; chronic myeloid leukemia; and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal angiogenesis, edema (such as edema associated with brain tumors), primary tumors of origin, and Meggs syndrome associated with nevus hamartomatosis.

[0326] Other cancers expressing CEACAM5 and that can be treated with the ADCs described herein include metastatic pancreatic cancer, metastatic pancreatic adenocarcinoma, gastric cancer, fibrotic cancer, glioma, malignant glioma, diffuse endogenous pontine glioma, recurrent childhood brain tumors, 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 brain metastases, malignant head and neck melanoma, squamous cell non-small cell lung cancer, metastatic breast cancer, follicular lymphoma, advanced B-cell NHL, HL including diffuse large B-cell lymphoma (DLBCL), multiple myeloma, chronic myeloid leukemia, adult acute myeloid leukemia in remission, adult acute myeloid leukemia with Inv(16)(p13.1q22), CBFB-MYH11, and t(16:16)(p13.1:q22). Adult acute myeloid leukemia (AML) with CBFB-MYH11 and t(8:21)(d22:q22), adult AML with RUNX1-RUNX1T1 and t(9:11)(p22:q23), and adult AML with MLLT3-MLL and tO15:17)(q22:q12) PML-RARA is indicated for adult acute promyelocytic leukemia, alkylating agent-associated acute myeloid leukemia, Rector's syndrome, adult glioblastoma, adult gliosarcoma, recurrent glioblastoma, recurrent childhood 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 undifferentiated carcinoma, cervical adenocarcinoma, cervical adenosquamous carcinoma; cervical squamous cell carcinoma, recurrent cervical cancer, anal canal squamous cell carcinoma, metastatic anal canal cancer, and 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, Cezari syndrome, neuroendocrine carcinoma, nasopharyngeal carcinoma, basal cell carcinoma of the skin, squamous cell carcinoma of the skin, dermatofibrosarcoma protuberans, glioma, mesothelioma, myelodysplastic syndrome (MDS), myelofibrosis (MF), myeloproliferative neoplasms, and acute myeloid leukemia (AML).

[0327] In some respects, cancers include: 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, stomach 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 respects, cancers that can be treated with the ADCs disclosed herein include colorectal cancer (CRC), non-small cell lung cancer (NSCLC), or gastric cancer (GC).

[0328] Cancer can be, for example, metastatic or primary cancer; connective tissue proliferative or non-connective tissue proliferative cancer; or recurrent cancer.

[0329] In some respects, cancer is associated with fibrosis. In some respects, cancer is associated with the infiltration of CD4+ regulatory T cells. In some respects, cancer is associated with the infiltration of CD8+ regulatory T cells. In some respects, cancer is associated with the infiltration of regulatory B cells. In some respects, cancer is associated with the infiltration of myeloid-derived suppressor cells. In some respects, cancer is associated with the infiltration of tumor-associated macrophages. In some respects, cancer is associated with the infiltration of congenital lymphoid cells. In some respects, cancer is associated with the infiltration of cancer-associated fibroblasts. In some respects, cancer is associated with increased radiation exposure to the above cell types.

[0330] In some respects, the ADCs described herein are intended for the treatment of myelodysplastic syndromes (MDS). MDS is a diverse group of malignant disorders characterized by bone marrow failure due to defective hematopoiesis and the production of abnormally developing cells. TGF-β is a major driver of MDS (Geyh et al., Haematologica [Hematology] 2018; 103:1462-71), and agents that inhibit TGF-β function have been proposed as therapeutic agents (Mies et al., Curr Hematol Malig Rep [Current Reports on Hematologic Malignancies] 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.

[0331] In some respects, cancer is resistant to one or more checkpoint inhibitors. In some respects, cancer is inherently refractory or resistant (e.g., resistant to PD-1 pathway inhibitors, CTLA-4 pathway inhibitors, or other similar inhibitors). In some respects, a resistant or refractory state of cancer has been acquired. In some respects, the ADCs described herein can be combined with checkpoint inhibitors to overcome cancer resistance to checkpoint inhibitors. In some respects, the ADCs described herein can be used, together with checkpoint inhibitors, in combination with or sequentially with agents that induce a mesenchymal phenotype (such as MAPK pathway inhibitors), to treat tumors with mesenchymal and / or EMT characteristics.

[0332] In some respects, the ADCs described herein are used to enhance the viability of ex vivo immune cells, for example, in adoptive NK cell transfer. Therefore, in some respects, ADCs are used in combination with adoptive NK cells for cancer treatment. In some respects, the ADCs described herein are used as a monotherapy or in combination with NK activation or enhancement therapies for the treatment of tumors with MHC loss or MHC downregulation. Combination therapy

[0333] The ADCs described herein can be used in combination with a variety of treatments or agents known in the art for treating diseases or conditions as described herein (or in the case of multispecific antibodies or bifunctional couplers).

[0334] In some respects, methods of treating cancer include administering an effective amount of an ADC (antibody-adjuvant combination therapy) as described herein, in combination with another therapeutic agent, such as a secondary antibody, therapeutic protein, or small molecule drug, to a subject in need. In some respects, the therapeutic protein is a checkpoint inhibitor. In some respects, the small molecule drug is a chemotherapeutic agent as described herein. In some respects, the other therapeutic agent includes an anticancer agent.

[0335] Suitable anticancer agents for use in combination therapy with the ADCs described herein include, but are not limited to, surgical agents, chemotherapy agents, growth inhibitors, cytotoxic agents, radiotherapy and agents used for radiotherapy, anti-angiogenic agents, apoptosis agents, anti-microtubule agents, and other agents for the treatment of 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 (e.g., GL... EEVEC (imatinib mesylate), COX-2 inhibitors (e.g., celecoxib), interferons, and cytokines; antagonists (e.g., neutralizing antibodies) that bind to and / or neutralize the activity of one or more of the following targets: PD-1, PD-L1, PD-L2 (e.g., pembrolizumab; nivolumab; MK-3475; AMP-224; MPDL3280A; MEDI0680; MSB0010718C; and / or MEDI4736); CTLA4 (e.g., trimemumab (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, one or more BCMA or VEGF receptors, TRAIL / Apo2, PARP inhibitors (e.g., AZD-2281, LynparzaOCEACAM5arib, Rubraca; (Zejula) niraparib, DNA damage repair inhibitors (e.g., ATMi, ATRi, DNAPKi), and other bioactive agents and organic chemical agents, including those described in Section VII. Combinations thereof are also particularly considered for use in the methods described herein.

[0336] In some respects, anti-ADCs are administered in combination with the following: 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; multi-kinase inhibitors; serine / threonine kinase inhibitors; tyrosine kinase inhibitors; VEGF / VEGFR inhibitors; microtubule-targeting drugs; topoisomerase toxins; or combinations thereof.

[0337] In some respects, ADCs are administered in conjunction with immune checkpoint inhibitors. Exemplary immune checkpoint inhibitors include, but are not limited to, agents (e.g., antibodies) that bind to the following: PD-1, PD-L1, PD-L2, LAG-3, CTLA4, TIGIT, ICOS, OX40, PVR, PVRIG, VISTA, TIM3, SIRPα, ILT2, ILT3, ILT4, or ILT5.

[0338] In the currently described method, any anti-PD-1 antibody can be used in combination with an ADC. Various human monoclonal antibodies that specifically bind to PD-1 with high affinity have been disclosed in U.S. Patent No. 8,008,449.

[0339] In some respects, anti-PD-1 antibodies include pembrolizumab, nivolumab, cimiprizumab, spartalizumab, camrelizumab, sintilimab, tislelizumab, toripalimab, dostarlimab, retifanlimab, pimivalimab, serplulimab, zimberelimab, acrixolimab, MEDI-0680, AM-0001, STI-1110, AGEN2034, BCD-100, sasanlimab, BI 754091, or SSI-361.

[0340] In some respects, anti-PD-1 antibodies used in combination with ADCs comprise any one of the following antibodies: CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, CDRL3, VH and VL and / or heavy and light chains: pembrolizumab, nivolumab, cimiprizumab, spartazolizumab, camrelizumab, sintilimab, tislelizumab, toripalimab, dotalimab, raltivalimab, pimilimab, MEDI-0680, GLS-010, AM-0001, STI-1110, AGEN2034, BCD-100, sasanlimab, BI 754091, or SSI-361.

[0341] In some respects, the anti-PD-1 antibodies used in combination with ADCs are selected from the following group, which consists of: nivolumab (OPDIVO®; formerly known as 5C4, BMS-936558, MDX-1106, or ONO-4538), pembrolizumab (KEYTRUDA®; formerly known as lambolizumab and MK-3475; see WO 2008 / 156712A1), PDR001 (see WO 2015 / 112900), MEDI-0680 (formerly known as AMP-514; see WO 2012 / 145493), REGN-2810 (see WO 2015 / 112800), JS001 (see Liu and Wu, 2017), BGB-A317 (see WO 2015 / 112800), PDR001 (see WO 2015 / 112900), MEDI-0680 (formerly known as AMP-514; see WO 2012 / 145493), REGN-2810 (see WO 2015 / 112800), JS001 (see Liu and Wu, 2017), BGB-A317 (see WO 2015 / 112800), JS001 (see Liu and Wu, 2017), and BGB-A317 (see WO 2015 / 112900). 2015 / 035606 and US2015 / 0079109), INCSHR1210 (SHR-1210; see WO 2015 / 085847; Liu and Wu, 2017), TSR-042 (ANB011; see WO 2014 / 179664), GLS-010 (WBP3055; see Liu and Wu, 2017), AM-0001 (see WO2017 / 123557), STI-1110 (see WO 2014 / 194302), AGEN2034 (see WO 2017 / 040790), and MGD013 (see WO 2017 / 106061).

[0342] In some respects, the anti-PD-1 antibody used in combination with an ADC is pembrolizumab (Merck; also known as KEYTRUDA®, lamborghizumab, and MK-3475; see, for example, WO 2008 / 156712). Pembrolizumab is a humanized monoclonal IgG4 (S228P) antibody against the human cell surface receptor PD-1 (programmed death protein-1 or programmed cell death protein-1). Pembrolizumab is described, for example, in U.S. Patent Nos. 8,354,509 and 8,900,587.

[0343] In some respects, anti-PD-1 antibodies used in combination with ADCs include 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 blocks the interaction with PD-1 ligands (PD-L1 and PD-L2), thereby blocking the downregulation of anti-tumor T cell function (see, for example, U.S. Patent No. 8,008,449; Wang et al., 2014 Cancer Immunol Res. [Cancer Immunology Research] 2(9):846-56).

[0344] In some respects, the anti-PD-1 antibody used in combination with the ADC is cimipril (Regeneron; also known as LIBTAYO or REGN-2810; see, for example, WO 2015 / 112800 and U.S. Patent No. 9,987,500).

[0345] In some respects, the anti-PD-1 antibody used in combination with an ADC is spartazolizumab (Novartis; also known as PDR001; see, for example, WO 2015 / 112900 and U.S. Patent No. 9,683,048).

[0346] In some respects, the anti-PD-1 antibody used in combination with the ADC is camrelizumab (Jiangsu Hengrui Medicine; also known as SHR-1210 or INCSHR1210; see, for example, WO 2015 / 085847; Si-Yang Liu et al., J. Hematol. Oncol. [Oncology Symposium] 10:136 (2017)).

[0347] In some respects, the anti-PD-1 antibody used in combination with an ADC is MEDI-0680 (AstraZeneca; also known as AMP-514; see, for example, WO 2012 / 145493). In some respects, the anti-PD-1 antibody is pimilimab (also known as JTX-4014; see, for example, Papadopoulos et al., 2022, IOTECH [Industrial Technology Research], Vol. 16, Supplement 1, 100284). In some respects, the anti-PD-1 antibody is toripalimab (TAIZHOU JUNSHI PHARMA; also known as JS001; see, for example, Si-Yang Liu et al., J. Hematol. Oncol. [Oncology Symposium] 10:136 (2017)). In some respects, the anti-PD-1 antibody is tislelizumab (BeiGene; also known as BGB-A317; see, for example, WO 2015 / 35606 and US 2015 / 0079109). In some respects, the anti-PD-1 antibody is dotalimab (Tesaro Biopharmaceutical; also known as ANB011 or TSR-042; see, for example, WO 2014 / 179664). In some respects, the anti-PD-1 antibody is GLS-010 (Wuxi / Harbin Gloria Pharmaceuticals; also known as WBP3055; see, for example, Si-Yang Liu et al., J. Hematol. Oncol. [Oncology Symposium] 10:136 (2017)). In some respects, the anti-PD-1 antibody is AM-0001 (Armo BioSciences).

[0348] In some respects, the anti-PD-1 antibody is STI-1110 (Sorrento Therapeutics; see, for example, WO 2014 / 194302). In some respects, the anti-PD-1 antibody is AAGEN2034 (Agenus; see, for example, WO 2017 / 040790). In some respects, the anti-PD-1 antibody is revilimab (Macrogenes, also known as MGA012, AEX-1188, and INCMGA-00012; see, for example, WO 2017 / 19846). In some respects, the anti-PD-1 antibody is BCD-100 (Biocad; see, for example, Kaplon et al., mAbs [Monoclonal Antibodies] 10(2):183-203 (2018). In some respects, the anti-PD-1 antibody is sintilimab (Innovent; also known as IBI308; see, for example, WO 2017 / 024465, WO 2017 / 025016, WO 2017 / 132825 and WO 2017 / 133540). In some respects, the anti-PD-1 antibody is saxamer (Pfizer; also known as PF-06801591; see, for example, US 2016 / 0159905). In some respects, the anti-PD-1 antibody is BI 754091 (Boehringer Ingelheim; see, for example, Zettel M et al., Cancer). Res. [Cancer Research] (2018); 78(13 Supplement): Abstract 4558). In some respects, the anti-PD-1 antibody is SSI-361 (Lyvgen Biopharma Holdings Limited, see, for example, US 2018 / 0346569).

[0349] Other anti-PD-1 monoclonal antibodies suitable for the methods disclosed herein have been described, for example, in the following documents: U.S. Patent Nos. 6,808,710, 7,488,802, 8,168,757, 8,354,509 and 9,205,148, U.S. Publication No. 2016 / 0272708, and PCT Publications Nos. WO 2012 / 145493, WO 2008 / 156712, WO 2015 / 112900, WO 2012 / 145493, WO 2015 / 112800, WO 2014 / 206107, WO 2015 / 35606, WO 2015 / 085847, WO 2014 / 179664, WO 2017 / 020291, WO 2017 / 020858、WO 2016 / 197367、WO 2017 / 024515、WO 2017 / 025051、WO 2017 / 123557、WO 2016 / 106159、WO 2014 / 194302、WO 2017 / 040790、WO 2017 / 133540、WO 2017 / 132827、WO 2017 / 024465、WO 2017 / 025016、WO 2017 / 106061、WO 2017 / 19846、WO 2017 / 024465、WO 2017 / 025016、WO 2017 / 132825 and WO In 2017 / 133540, each of them is incorporated as a whole through reference.

[0350] Examples of anti-PD-L1 antibodies that can be used in combination with an ADC according to the methods disclosed herein include the antibodies disclosed in U.S. Patent No. 9,580,507. In some aspects, the anti-PD-L1 antibody is atezolizumab, duvarubicin, avelumab, envafolimab, cosibelimab, BMS-936559, STI-1014, CX-072, LY3300054, FAZ053, CS-1001, SHR-1316, CBT-502, KN035, or BGB-A333.

[0351] In some respects, the anti-PD-L1 antibody is BMS-936559 (also known as 12A4, MDX-1105; see, for example, U.S. Patent Nos. 7,943,743 and WO 2013 / 173223).

[0352] In some respects, the anti-PD-L1 antibody is STI-1014 (Sorrento Medical Technologies, Inc.; see, for example, WO2013 / 181634). STI-104 is designated H6 in U.S. Patent No. 9,175,082. In some respects, the anti-PD-L1 antibody is CX-072 (Cytomx, Inc.; see, for example, WO 2016 / 149201). In some respects, the anti-PD-L1 antibody is LY3300054 (Eli Lilly and Company; see, for example, WO 2017 / 034916). In some respects, the anti-PD-L1 antibody is FAZ053 (Novartis). In some respects, the anti-PD-L1 antibody is CK-301 (Checkpoint Therapeutics, Inc.; see, for example, Gorelik et al., AACR: Abstract 4606 (April 2016)). CK-301 is also known as cochilimab. In some respects, 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 respects, the anti-PD-L1 antibody is SHR-1316. See, for example, Mu et al., Thorac Cancer, May 2021; 12(9):1373-1381, and Wu et al., Anals of Oncology, Abstract, Vol. 33, Supplement 2, S72, April 2022. In some respects, the anti-PD-L1 antibody is CBT-502 (also known as TQB2450; see, for example, Wei et al., Mol Cancer Ther [Molecular Cancer Therapy] (2018) 17 (1_Supplement): A200).In some respects, the anti-PD-L1 antibody is KN035 (3D Med / Alphamab; also known as Envorimab; see, for example, Zhang et al., Cell Discov. 7:3 (March 2017) and Shimizu et al., Invest New Drugs, October 2022; 40(5):1021-1031).

[0353] In some respects, the anti-PD-L1 antibody is BGB-A333 (BeiGene; see, for example, Desai et al., JCO36 (15 Supplement): TPS3113 (2018) and Desai et al., 2023, British Journal of Cancer 128, 1418-1428). In other respects, 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, for example, US 8,217,149 and Herbst et al. (2013) J. Clin. Oncol. 31 (Supplement): 3000). Atezolizumab is designated YW243.55S70 in U.S. Patent No. 8,217,149. In some respects, the PD-L1 antibody is duvalumab. Duvalumab is a human IgG1κ monoclonal anti-PD-L1 antibody. Duvalumab (AstraZeneca) is also known as IMFINZI® or MEDI-4736. Duvalumab is designated 2.14H9OPT in U.S. Patent No. 8,779,108. See, for example, WO 2011 / 066389. In some respects, 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 A09-246-2 in U.S. Patent No. 9,624,298. See, for example, WO 2013 / 079174.

[0354] In some respects, anti-CTLA-4 antibodies that can be used in combination with ADCs include trimemumab, ipilimumab, botensilimab, BMS-986218, BMS-986288, BMS-986249, IBI310, MK-1308 (quavonlimab), AGEN-1884 (zalifrelimab), ONC-392, ADG116, or CS1002.

[0355] In some respects, the anti-CTLA-4 antibody that can be used in combination with an ADC is MK-1308. MK-1308 is also known as zivolizumab. See, for example, Perets et al. 2021, Ann Oncol [Annals of Oncology] 32(3):395-403.

[0356] In some respects, the anti-CTLA-4 antibody that can be used in combination with an ADC is AGEN-1884. AGEN-1884 is also known as zefulimab. See, for example, WO 2016 / 196237.

[0357] In some respects, the anti-CTLA-4 antibody that can be used in combination with ADCs is trimemumab. Trimemumab, sold under the brand name IMJUDO®, is a fully human monoclonal antibody used to treat hepatocellular carcinoma and non-small cell lung cancer. Trimemumab (AstraZeneca) is also known as tesimumab, CP-675,206; see WO 2000 / 037504 and Ribas, Update Cancer Ther. [Cancer Therapy Update] 2(3): 133-39 (2007)).

[0358] In some respects, the anti-CTLA-4 antibody that can be used in combination with ADCs is ipilimumab. Ipilimumab (marketed under the brand name YERVOY®) was first approved for the treatment of metastatic melanoma and has since been approved for use in other cancers. Hoos et al. (2010) Semin. Oncol. [Oncology Symposium] 37:533; Hodi et al. (2010) N. Engl. J. Med. [New England Journal of Medicine] 363:711; Pardoll (2012) Nat. Immunol. [Nature Immunology] 13(12):1129. In 2011, ipilimumab, a human antibody with a constant IgG1 region, was approved in the United States and the European Union for the treatment of unresectable or metastatic melanoma based on improvements in overall survival in previously treated patients with advanced melanoma in a phase III trial. Hodi et al. (2010) N. Engl. J. Med. [New England Journal of Medicine] 363:711. Tumor regression and disease stabilization are frequently observed. Ipilimumab is also known as MDX-010 and 10D1. See U.S. Patent No. 6,984,720.

[0359] In some respects, anti-CTLA-4 antibodies are activatable anti-CTLA-4 antibodies, such as activatable anti-CTLA-4 antibodies whose antibody light chains contain a cleavable portion and a masking portion at the amino terminus. The masking portion interferes with CTLA-4 binding, but is preferentially released into the tumor microenvironment after the cleavable portion is cleaved by proteases, which are more prevalent and / or more active in tumors than in surrounding tissues (see, WO 2018 / 085555 for details). This preferential cleavage in the tumor microenvironment enables complete CTLA-4 blockade, thereby promoting an anti-tumor immune response, while minimizing CTLA-4 blockade in normal tissues, thus reducing the risk of potential systemic toxicity of anti-CTLA-4 antibodies. In some respects, activatable anti-CTLA-4 antibodies are activatable forms of ipilimumab, such as antibodies containing a light chain modified to include both a masking portion and a cleavable portion, as disclosed, for example, in WO2018 / 085555. An example of an activatable anti-CTLA-4 antibody that has entered human clinical trials is BMS-986249 (NCT03369223: "A Study of BMS-986249 Alone and in Combination with Nivolumab in Advanced Solid Tumors"). In some respects, the anti-CTLA-4 antibody is BMS-986249.

[0360] In some respects, anti-CTLA-4 antibodies exhibit enhanced Fcγ receptor (CD16) binding. The enhancement of Fcγ receptor binding by anti-CTLA-4 antibodies is assessed by comparison with the Fcγ receptor binding of ipilimumab. Anti-CTLA-4 antibodies with enhanced Fcγ receptor (CD16) binding have been proposed as therapeutic agents for the treatment of cancer by depleting Treg cells. See, WO 2014 / 089113, for details. In some respects, anti-CTLA-4 antibodies exhibit at least a two-fold enhancement of Fcγ receptor (CD16) binding compared to the Fcγ receptor binding of ipilimumab.

[0361] An example of an anti-CTLA-4 antibody exhibiting enhanced Fcγ receptor (i.e., FcγRIIIA or CD16) binding is a non-fucosylated anti-CTLA-4 antibody. In some aspects, anti-CTLA-4 antibodies are non-fucosylated anti-CTLA-4 antibodies. Non-fucosylated anti-CTLA-4 antibodies lack fucosylate residues in their N-linked glycans. In some aspects, non-fucosylated anti-CTLA-4 antibodies are generated by expressing an antibody chain in mammalian cells under conditions that prevent fucosylation, including but not limited to using mammalian cells with genetic modifications that prevent fucosylation, or growing the antibody-expressing cells in a culture medium containing one or more compounds that inhibit fucosylation. In some aspects, the genetic modification preventing fucosylation is the inactivation (e.g., knockout) of the FUT8 gene. In some aspects, anti-CTLA-4 antibodies are hypofucosylated anti-CTLA-4 antibodies.

[0362] An exemplary non-fucosylated anti-CTLA-4 antibody that has entered 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 CTLA-4 blocking by strengthening binding to the Fcγ receptor, thereby promoting APC-mediated T cell priming. In some respects, the anti-CTLA-4 antibody is BMS-986218. See, for example, PCT / US18 / 19868.

[0363] In some aspects, the Fc region of the anti-CTLA-4 antibody contains amino acid substitutions in the antibody constant region to enhance binding to the activated Fcγ receptor. Exemplary substitutions are G236A, S239D, A330L, and I332E (all residue numbers are according to the EU numbering system). In some aspects, the anti-CTLA-4 antibody contains a human IgG1 constant domain with mutations in S239D, A330L, and I332E.

[0364] In some respects, anti-CTLA-4 antibodies are activatable non-fucosylated anti-CTLA-4 antibodies.

[0365] Human monoclonal antibodies that bind to CTLA-4 with high affinity and specificity, suitable for the methods disclosed herein, have been disclosed in U.S. Patent No. 6,984,720. Other anti-CTLA-4 monoclonal antibodies have been described, for example, in U.S. Patent Nos. 5,977,318, 6,051,227, 6,682,736, and 7,034,121, and International Publications WO 2012 / 122444, WO 2007 / 113648, WO 2016 / 196237, and WO 2000 / 037504, each of which is incorporated herein by reference in its entirety.

[0366] In some respects, the anti-LAG-3 antibodies that can be used in combination with CEACAM5-targeting agents according to the methods disclosed herein are piracetamab (BMS-986016), IMP731 (H5L7BW), MK4280 (28G-10, favezelimab), REGN3767 (fianlimab), GSK2831781, humanized BAP050, IMP-701 (LAG525, ieramilimab), aLAG-3 (0414), aLAG-3 (0416), Sym022, TSR-033, TSR-075, XmAb841 (XmAb22841), MGD013 (tebotelimab), BI754111, FS118, P 13B02-30, AVA-017, 25F7, AGEN1746, RO7247669, INCAGN02385, IBI-110, EMB-02, IBI-323, LBL-007, ABL501.

[0367] In some respects, anti-LAG-3 antibodies that can be used in combination with CEACAM5-targeting agents comprise any of the following CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, CDRL3, VH and VL and / or heavy and light chains: renalalimumab (BMS-986016), IMP731 (H5L7BW), MK4280 (28G-10, favezzilimab), REGN3767 (furanlimumab), GSK2831781, humanized BAP050, IMP-701 (LAG525). (e.g., terpolitab), aLAG-3 (0414), aLAG-3 (0416), Sym022, TSR-033, TSR-075, XmAb841 (XmAb22841), MGD013 (terpolitab), BI754111, FS118, P13B02-30, AVA-017, 25F7, AGEN1746, RO7247669, INCAGN02385, IBI-110, EMB-02, IBI-323, LBL-007 or ABL501.

[0368] In some respects, anti-LAG-3 antibodies that can be used in combination with ADCs include renalalimab (BMS-986016). In some respects, anti-LAG-3 antibodies include IMP731 (H5L7BW). In some respects, anti-LAG-3 antibodies include MK4280 (28G-10, favismab). MK-4280 (28G-10, favismab) is described in WO 2016028672 and U.S. Publication No. 2020 / 0055938. In some respects, anti-LAG-3 antibodies include REGN3767 (ferrilimab). REGN3767 (ferrilimab) is described, for example, in Burova E et al., J. Immunother. Cancer (2016);4(Supplement 1):P195 and U.S. Patent No. 10,358,495. In some aspects, anti-LAG-3 antibodies include GSK2831781. In some aspects, anti-LAG-3 antibodies include humanized BAP050. Humanized BAP050 is described, for example, in WO2017 / 019894. In some aspects, anti-LAG-3 antibodies include IMP-701 (LAG525, eralimumab). IMP-701 (LAG525; eralimumab) is described, for example, in U.S. Patent No. 10,711,060 and U.S. Publication No. 2020 / 0172617. In some aspects, anti-LAG-3 antibodies include aLAG-3 (0414). In some aspects, anti-LAG-3 antibodies include aLAG-3 (0416). In some aspects, anti-LAG-3 antibodies include Sym022. In some aspects, anti-LAG-3 antibodies include TSR-033. In some aspects, anti-LAG-3 antibodies include TSR-075. In some aspects, anti-LAG-3 antibodies include XmAb841 (XmAb22841). In some aspects, anti-LAG-3 antibodies include MGD013 (terpolimab). In some aspects, anti-LAG-3 antibodies include BI754111. In some aspects, anti-LAG-3 antibodies include FS118. In some aspects, anti-LAG-3 antibodies include P 13B02-30. In some aspects, anti-LAG-3 antibodies include AVA-017. In some aspects, anti-LAG-3 antibodies include 25F7. 25F7 is described, for example, in U.S. Publication No. 2011 / 0150892. In some aspects, anti-LAG-3 antibodies include AAGEN1746. In some aspects, anti-LAG-3 antibodies include RO7247669. In some aspects, anti-LAG-3 antibodies include INCAGN02385. In some respects, anti-LAG-3 antibodies include IBI-110. In some respects, anti-LAG-3 antibodies include EMB-02. In some respects, anti-LAG-3 antibodies include IBI-323. In some respects, anti-LAG-3 antibodies include LBL-007.In some respects, anti-LAG-3 antibodies include ABL501.

[0369] Generally, any anti-LAG-3 antibody that can be used in combination with an ADC can be used. Antibodies that bind to LAG-3 have been disclosed in International Publication No. WO / 2015 / 042246 and US Publication Nos. 2014 / 0093511 and 2011 / 0150892, which are incorporated herein by reference in their entirety. Disclosures relating to the anti-LAG-3 antibody described herein and other anti-LAG-3 antibodies that can be used in the methods disclosed herein can be found, for example: US 10,188,730, WO 2016 / 028672, WO 2017 / 106129, WO2017 / 062888, WO 2009 / 044273, WO 2018 / 069500, WO 2016 / 126858, WO 2014 / 179664, WO2016 / 200782, WO 2015 / 200119, WO 2017 / 019846, WO 2017 / 198741, WO 2017 / 220555, WO2017 / 220569, WO 2018 / 071500, WO 2017 / 015560, WO 2017 / 025498, WO 2017 / 087589, WO2017 / 087901, WO 2018 / 083087, WO 2017 / 149143, WO 2017 / 219995, US 2017 / 0260271、WO2017 / 086367、WO 2017 / 086419、WO 2018 / 034227、WO 2018 / 185046、WO 2018 / 185043、WO2018 / 217940、WO 19 / 011306、WO 2018 / 208868、WO 2014 / 140180、WO 2018 / 201096, WO2018 / 204374, and WO 2019 / 018730. The contents of each of these references are incorporated in their entirety through citation.

[0370] Several experimental treatment regimens involve the in vitro activation and expansion of antigen-specific T cells, and the adoptive transfer of these cells to recipients to generate tumor-targeting antigen-specific T cells. It is anticipated that in vitro activation in the presence of the anti-CEACAM5 antibody described herein, with or without additional immunostimulatory therapy (e.g., immune checkpoint inhibitors), could increase the frequency and activity of adoptively transferred T cells.

[0371] In some respects, the ADCs described herein can also be administered in conjunction with standard care or another treatment, such as radiation, surgery, or chemotherapy. ADCs can be combined with vaccination regimens. Numerous experimental strategies for cancer vaccination have been designed (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; see also Restifo, N. and Sznol, M., Cancer Vaccines, Chapter 61, pp. 3023-3043, DeVita et al. (eds.), 1997, Cancer: Principles and Practice). In one of these strategies, vaccines are prepared using autologous or allogeneic tumor cells. These cell vaccines have been shown to be most effective when the tumor cells are transduced to express GM-CSF. GM-CSF has been shown to be an effective activator of antigen presentation for tumor vaccination (Dranoff et al. (1993) Proc. Natl. Acad. Sci USA [Proceedings of the National Academy of Sciences] 90: 3539-43). V. Reagent Kit

[0372] A kit containing the ADC described herein and instructions for use are also provided.

[0373] In some respects, the kit contains an ADC in unit dosage form, such as in a single-dose vial or a single-dose pre-loaded syringe, optionally contained in a single vial or container, and instructions for use, for example, in the treatment of cancer with an ADC as described herein.

[0374] The following examples further illustrate this disclosure, and these examples should not be construed as further limitations. All figures and references, Genbank sequences, granted patents, and published patent applications cited throughout this disclosure are expressly incorporated herein by reference. Example

[0375] Unless otherwise instructed, use the commercially available reagents mentioned in the following examples according to the manufacturer's instructions. Unless otherwise stated, this disclosure uses standard procedures for recombinant DNA technology, such as those described above and in the following textbooks: Sambrook et al., ibid.; 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, 1988); Gait, Oligonucleotide Synthesis (IRL Press, Oxford, 1984); Freshney, Animal Cell Culture, 1987; Coligan et al., Current Protocols in Immunology, 1991.

[0376] The following examples illustrate 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 antibodies are provided below, for example in Table 10. Example 1: Production of anti-CEACAM5 antibodies

[0377] Human anti-CEACAM5 monoclonal antibodies (mAbs) were generated by immunizing a BMS-specific chimeric mouse strain. Hybridomas were generated. Positive human / cynomolgus monkey CEACAM5 cross-reactive conjugates (not recognizing human CEACAM1 or human CEACAM6 proteins) were selected. CEACAM5 mAbs were also isolated from the mice immunized as described above using a single B-cell cloning (SBC) method. Immunotherapy libraries were also generated from mouse B cells. These libraries were expressed by yeast display and selected against CEACAM5 to identify additional human / cynomolgus monkey cross-reactive antibodies exhibiting specificity beyond that of CEACAM1 and CEACAM6.

[0378] The VH and VL regions of the identified positive human CEACAM5 mAbs were sequenced by NGS using a MiSeq sequencing system (Illumina). Approximately 380 sequence-unique clones binding to human or cynomolgus monkey CEACAM5 were identified, comprising 173 sequence families (as 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 (described below), and 75 clones (31 sequence families) exhibited specificity exceeding that of CEACAM1 and CEACAM6. Further characterization (as described herein) led to the isolation of multiple antibodies, including MBN001. The amino acid sequences of the VH and VL CDRs are provided in Table 10. Example 2: Tabletop Sub-bins

[0379] High-throughput SPR-based epitope binning sandwich assays were performed using the Carterra LSA platform to group anti-CEACAM5 mAbs into bins sharing common binding epitopes. Pairwise competition experiments were conducted in a classic sandwich configuration using a 96 x 96 microfluidic system array based on microarrays via high-throughput Carterra SPR microfluidics. Binning results were analyzed using Carterra microfluidic binning software for heatmap generation and network diagram plotting. Competitive antibody relationships allowed anti-CEACAM5 antibodies to cluster into bins, where bins represent families of anti-CEACAM5 antibodies that share the same blocking spectrum when tested against other anti-CEACAM5 antibodies. See also Figure 1A Based on the assay results, the newly generated antibodies were binned into seven different epitope groups. MBN001 mAb was identified as bin 1 conjugate of CEACAM5. Cross-reactivity with other CEACAM5 family members, including CEACAM1 and CEACAM6, was determined by SPR as described above, and epitopes bins 4, 6, 7, and 8 were determined to be cross-reactive with CEACAM1 and CEACAM6. See also Figure 1B . Example 3: Binding specificity of anti-CEACAM5 antibody in CEACAM5-expressing cell lines

[0380] This example analyzes the cell binding specificity of the anti-human CEACAM5 mAb (e.g., MBN001, MBN002, and MBN003) described in Example 6. Figure 2A , Figure 2B , Figure 2C and Figure 2D This is a set of figures showing cell-based binding of antibodies against cell lines expressing different levels of human CEACAM5: intermediate BXPC-3 ( Figure 2A ), low Ls174T cells ( Figure 2B ), High MKN45 ( Figure 2C ) and negative HCT-116 ( Figure 2D Each of the tested antibodies (mAb MBN001, mAb MBN002, and mAb MBN003) bound cells expressing human CEACAM5 comparablely to mAb control 3. The binding specificity of each of these mAbs was further evaluated using a human colorectal cancer cell line (HCT-116) or CHOS cell line overexpressing human CEACAM5, cynomolgus monkey CEACAM5, human CEACAM1, and human CEACAM6. As shown in Table 2, antibody MBN001 exhibited specific binding to human CEACAM5 and cynomolgus monkey CEACAM5, but not specific binding to human CEACAM1 or CEACAM6. Table 2. Binding specificity of anti-CEACAM5 mAb to cell lines expressing human and cynomolgus monkey CEACM5 and / or CEACAM6.

[0381] Using CEACAM7 mAb (R&D Systems Catalog No. MAB44782) as a positive control, the binding of CEACAM5 mAbs (such as MBN002, MBP018, MBP003, MBP001, and MBP002) to human CEACAM7 (R&D Systems Catalog No. 9010-CM-050) was examined by SPR. No binding of CEACAM5 mAb was observed (data not shown). Similarly, the binding of CEACAM5 mAb to human CEACAM8 (R&D Systems Catalog No. 9639-CM-050) was examined. Commercial CEACAM8 mAb (R&D Systems Catalog No. MAB4246) showed binding to CEACAM8, but no binding of CEACAM5 mAb was observed (data not shown). Example 4: Binding kinetics of anti-CEACAM5 antibody

[0382] Surface plasmon resonance (SPR) analysis was used to characterize the binding kinetics of the selected anti-CEACAM5 antibody MBN001 with full-length human CEACAM5 and cynomolgus monkey CEACAM5, as well as with the A3-B3 region (all internally generated proteins) of human CEACAM5 and cynomolgus monkey CEACAM5.

[0383] SPR analysis was performed using a Biacore 8K instrument at 37°C, which was started with 1xHBSP+ running water and docked with a CM5 sensor chip equilibrated to room temperature, followed by instrument restart. The chip surface was activated by injecting a mixture of ethyl(dimethylaminopropyl)carbodiimide (EDC) / N-hydroxysuccinimide (NHS) at 10 µL / min for 7 min. The activated chip surface was then fixed with an anti-human Fc capture reagent (25 µg / mL, in pH 5 acetate buffer) injected at 10 µL / min for 7 min to produce a fixation level of approximately 9000 RU. The remaining chip surface was blocked by injecting ethanolamine at 10 µL / min for 7 min.

[0384] 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 with the Fc capture reagent. This was followed by CEACAM5 analyte binding, in which each of a series of recombinant full-length CEACAM5 or A3-B3 CEACAM5 proteins diluted 3x in HBSP+ buffer from 500 nM to 0.23 nM was flowed through the chip. Analyte association time was 3 min at 30 µL / min; antigen dissociation time was 10 min at 30 µL / min; and regeneration involved two injections of 3M magnesium chloride (MgCl2) at 30 µL / min for 30 seconds each.

[0385] Kinetic data of anti-CEACAM5 mAb:CEACAM5 binding were fitted to 1:1 Langmuir binding with Rmax to provide estimates of the kinetics and affinity values ​​of the corresponding interactions. The estimated binding kinetics and affinity of the selected antibodies to full-length CEACAM5(hu / cy) and the A3-B3 region of CEACAM5(hu / cy) are shown in Tables 3-6. Table 3. Kinetics of binding between anti-CEACAM5 mAb and full-length huCEACAM5. Table 4. Kinetics of binding between anti-CEACAM5 mAb and full-length cyCEACAM5. Table 5. Kinetics of binding between anti-CEACAM5 mAb and A3-B3 huCEACAM5. Table 6. Kinetics of binding between anti-CEACAM5 mAb antibody and A3-B3 cyCEACAM5. Example 5: In vitro binding of anti-CEACAM5 antibody in CEACAM5-expressing cell lines

[0386] The binding of the selected anti-CEACAM5 antibody MBN001 to the CEACAM5-expressing cell lines MKN45, HCT116-huCEACAM5, and HCT116-cyCEACAM5, as well as the parental control HCT-116, was evaluated. The results of these experiments are shown in Tables 7 (EC50 values) and 8 (Amax values). Table 7. EC50 values ​​of selected CEACAM5 mAb inhibitors Table 8: Amax values ​​of selected CEACAM5 mAb inhibitors The data show that MBN001 binds to both the huCEACAM5 expression line and the cynoCEACAM-5 expression line. Furthermore, MBN001 exhibits improved cell binding compared to the control antibody. Example 6: Screening for anti-CEACAM5 mAb that can be internalized into cells

[0387] Anti-CEACAM5 mAbs were screened for their ability to internalize into cells expressing huCEACAM5. Internalization assays were performed using the MKN-45 cell line, the HCT116-huCEACAM5 cell line, the parental HCT-116 control cell line, and the LS174T cell line. Internalization assays were performed in 96-well plates using the IncuCyte S3 live cell analysis system. Phase contrast and red fluorescence were scanned from the plates, and the confluence (measurement of cell area) and red fluorescent object area of ​​the images were automatically analyzed using integrated INCUCYTE® software. The intensity of red fluorescence inside the cells increased as the labeled antibody internalized into the acidic environment of the endosomes and lysosomes. The internalization signal was expressed as the red fluorescent object area normalized relative to the total cell area (confluence).

[0388] In short, a certain quantity (1 x 10) 4Live cells from the MKN-45, HCT116-huCEACAM5, LS174T, and HCT-116 parental cell lines were added to 96-well plates and incubated at 37°C / 5% CO2 for approximately four hours, followed by antibody treatment. Antibody treatment was prepared by combining the test antibody or allotype control antibody with the PHRODO™ Red Second Fab Reagent (Thermo Fisher Scientific) to provide a final concentration of test antibody (25 nM) and PHRODO reagent (75 nM) after cell addition. After incubation at 37°C for 30 minutes, 50 µl of each antibody treatment was added to 50 µl of cells in each 96-well plate. These plates were then inserted into the INCUCYTE® system and incubated further at 37°C for 30 minutes, followed by reading using an IncuCyte detection machine. Set the IncuCyte settings to a 24-hour duration, reads every 30 minutes, 10x objectives, 3 images / wells, and both a phase channel and a red fluorescence channel.

[0389] Table 9 presents a quantitative assessment of the internalization of antibodies MBN001, MBN002, and MBN003 as described herein and as measured by area under the curve (AUC) analysis. Internalization data for the selected antibodies were collected in MKN45, LS174T, and HCT 116-CEACAM5 cell lines. The data show that the tested antibodies were internalized into the test cell lines, and in many cases, the antibodies exhibited improved or comparable internalization compared to the control antibodies.

[0390] Further assays were performed, and the data showed that MBN001 was effectively internalized by cells expressing different CEACAM5 levels. Figure 3B and Figure 4B To facilitate high-throughput cytotoxicity screening, MMAE (a microtubule inhibitor) was conjugated to VHH (a target for the human κ light chain). Data showed that the MBN001-VHH complex had the ability to be internalized by CEACAM5-expressing cells and efficiently delivered the cytotoxic agent to kill CEACAM5-expressing cells. See also Figure 3A , Figure 3C and Figure 4A . Table 9. Internalization of antibodies in HCT-116-huCEACAM5 and MKN-45 cell lines. Table 10. Summary table of amino acid sequences and nucleic acid sequences Example 7: Mutation scanning and optimization of anti-CEACAM5 mAb MBN001

[0391] Mutation scanning was performed to identify variants of MBN001 with improved affinity for CEACAM5 in humans and / or cynomolgus monkeys, such as... Figure 5A As described in [the document]. Single-stranded variable fragment (scFv) libraries were generated, which allowed for single amino acid substitutions in CDRs using NNK oligonucleotides. For each CDR, multiple oligonucleotides were designed with an NNK codon (N = A, C, T, G and K = G, T) incorporated at each position to allow encoding all 20 amino acids plus a stop codon at each position. Kabat definitions were used for all CDRs except HCDR1, which used AbM definitions. In some cases, CDR residues were omitted from the scan, or additional non-germ region amino acids were included in the scan. Specifically, the vernier residues at positions 49 and 94 in VH were included in the library design. Positions 33-35 in HCDR1 and position 97 in LCDR3 were omitted from the scan. Additionally, in the library design, the following framework residues in VH were reverted to germline: T7S, S40A, A68T, P84A. Diversified positions (MBN001 mutation scans for CDR position analysis) are detailed in [the document]. Figure 5B middle.

[0392] 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 subjected to single-round selection targeting human and cynomolgus monkey CEACAM5. In short, the DNA library underwent transcription and translation by fusing the scFv protein to the encoding mRNA via puromycin linker. In individual selection, the scFv-mRNA fusion was exposed to biotinylated human CEACAM5 and biotinylated cynomolgus monkey CEACAM5. Target-bound scFvs were captured, eluted, and amplified by streptavidin beads. 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 initial population. The enrichment ratio is normalized relative to the enrichment ratio of the parent antibody sequence to produce a normalized enrichment ratio:

[0393] Using these normalized enrichment ratios (ERs), heatmaps were generated to assess the effect of each individual amino acid substitution on binding to CEACAM5 in humans and cynomolgus monkeys. The error in this method is approximately 2-fold, therefore ER values ​​of 0.5 to 2 are considered neutral, values ​​greater than 2 are considered favorable, and values ​​below 0.5 are considered unfavorable. This analysis provides a rich set of information regarding the effects of individual amino acid substitutions, as shown below: Figure 5C (MBN001 HCDR1 person CEACAM5) Figure 5D (MBN001HCDR1 Crab-eating macaque CEACAM5) Figure 5E (MBN001 HCDR2 people CEACAM5) Figure 5F (MBN001 HCDR2 Crab-eating macaque CEACAM5) Figure 5G (MBN001 HCDR3 people CEACAM5) Figure 5H (MBN001 HCDR3 Crab-eating macaque CEACAM5) Figure 5I (MBN001 LCDR1 person CEACAM5) Figure 5J (MBN001 LCDR1 Crab-eating macaque CEACAM5) Figure 5K (MBN001 LCDR2 person CEACAM5) Figure 5L (MBN001 LCDR2 Crab-eating macaque CEACAM5) Figure 5M (MBN001 LCDR3 CEACAM5) and Figure 5N(MBN001 LCDR3 cynomolgus monkey CEACAM5), and summarized in Table 11. Using deep mutation scan data, many CDR locations are shown (e.g., as...). Figure 5B (As shown in HCDR1-3 and LCDR1-3) tolerance mutations, which means that these substitutions can be made at the identified CDR positions / amino acid sequences and maintain the desired ability of the antibody or antigen-binding moiety to bind to human CEACAM5 and cynomolgus monkey CEACAM5. Example 8: Generation and analysis of MBN001's CEACAM5 mAb-resistant progeny

[0394] This example describes the generation and characterization of the progeny of the anti-CEACAM5 antibody MBN001. A subset of CDR amino acid substitutions predicted to improve the binding of MBN001 to human or cynomolgus monkey CEACAM5 based on deep mutation scanning data were selected for further analysis. Antibody genes with single amino acid substitutions and combinations thereof were synthesized in an IgG expression vector, transiently transfected into HEK cells, and purified via protein A. Furthermore, germline reversion of the aforementioned framework was incorporated into the progeny sequence. The purified IgG1.3 CEACAM5 mAb clone was characterized using BIACORE® for high-throughput SPR-based monoclonal characterization.

[0395] Start the BIACORE® instrument with 1X HBSP+ run buffer (Stenofan catalog number BR100671). Equilibrate the CM5 chip (Stenofan catalog number 29149604) to room temperature and restart the BIACORE® instrument. Perform the analysis using the Human Antibody Fc Capture Kit (Stenofan catalog number 29234600). Immobilize the human antibody Fc capture reagent to two flow cells of all eight flow channels of the CM5 chip via amine coupling using the following conditions: Immobilization was performed at 25°C. Dilute the anti-human Fc capture reagent to a concentration of 25 μg / mL in acetate pH 5 buffer. Activate the chip surface by injecting a mixture of 1-ethyl-3-(-3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) at a rate of 10 μL / min for 420 seconds. A concentration (25 μg / mL) of anti-human Fc capture reagent was then injected onto the surface at a rate of 10 μL / min for 420 seconds. The remaining chip surface was sealed by injecting ethanolamine at a rate of 10 μL / min for 420 seconds. The fixation level produced by this method was approximately 9000 RU.

[0396] SPR kinetics for full-length antibodies and cyno CEACAM5 were established as follows. Antibody capture was performed by diluting the antibody to a concentration of 10 nM in HBSP+ buffer. A capture level of approximately 100 RU was achieved by capturing 10 nM mAb at 5 μL / min for 20 seconds. Analyte binding was performed by first preparing a modified series of full-length antibodies and cyno CEACAM5, which were prepared by diluting the antibody three-fold in HBSP+ buffer (from 500 nM to 0.23 nM). The association rate was 30 μL / min for 180 seconds. The dissociation rate was 30 μL / min for 600 seconds. Regeneration was performed twice by injecting 3M magnesium chloride (MgCl2) at 30 μL / min for 30 seconds. Data were fitted using a 1:1 binding model with global Rmax. Rmax reflects the maximum response when all ligands are occupied.

[0397] SPR data demonstrating the improved affinity of huCEACAM5 and cynoCEACAM5 for progeny antibodies are shown in Figure 6A and Figure 6BIn the is affinity plots, the values ​​of the progeny antibodies are reported in Tables 12A and 12B below, which show the binding of each of the tested antibodies to both human CEACAM5 and cyno CEACAM5. The data show that these progeny antibodies selectively bind to human CEACAM5 and not to CEACAM1, CEACAM6, CEACAM7, and CEACAM8. Note that these progeny antibodies were also prepared in the hIgG1 form, not 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; that is, only the CH2 region of the heavy chain is modified, so that their binding characteristics are expected to be similar. In fact, the data show acceptable consistency observed between the hIgG1 and hIgG1.3f versions of the progeny antibodies (e.g., one to two times similarity in human CEACAM5 and cyno CEACAM5 binding / SPR values) (data not shown). Figure 7A , Figure 7B and Figure 7C The data confirm that MBN001 progeny are effectively internalized into cells expressing human CEACAM5, and that these progeny can be used to effectively deliver cytotoxic agents and fractions. Figure 8A and Figure 8B The data showed that there was no nonspecific cross-reactivity between human CEACAM1 and human CEACAM6 and the MBN001 progeny mAbs MBP001, MBP003 and MBP002. Figures 9A-9D The results show that FACS EC50 remained constant in each clone's cell line, with the maximum MFI increasing 10-fold from LS174T and BxPC3 to MKN45. Table 12A. Binding data of hIgG1.3f progeny antibodies Table 12B. Binding data of hIgG1 progeny antibodies Example 9: Internalization analysis of recombinant IgG1.3 progeny antibodies

[0398] Internalization experiments were performed on the hIgG1.3 MBN001 progeny antibody, as listed in Table 13A below. Antibody internalization was evaluated using HCT-116-huCEACAM5, HCT-116-cyCEACAM5, and MKN45 cell lines.

[0399] Additional internalization experiments were performed using MKN45 and Ls174T cell lines to detect hIgG1 antibodies MBP001, MBP002, and MBP003. MKN45 or Ls174T cells were seeded at 10K cells / well (50 μL).

[0400] Cell density and viability of the cell lines were first analyzed using the Vi-cell viability analyzer. Cell lines were diluted to 0.2E6 vc / mL in growth medium, and a specific volume (50 μL) of each cell line was aliquoted into flat-bottomed 96-well plates to achieve a cell count of 10,000 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 blast fungicide. Cells were allowed to adhere to the wells of the 96-well plates (Corning catalog number 3595) at 37°C / 5% CO2 for approximately 4 hours.

[0401] Each test mAb and control mAb was labeled as follows: First, the corresponding antibody was premixed with pHrodo Red (conjugated to a pH-sensitive Fab; Thermo Fisher Scientific catalog number Z25612) as a second reagent at a dye:mAb molar ratio of 3:1. A series of dilutions were prepared in growth medium at 2X the target concentration. The final target mAb concentration was 25 nM. A volume (50 μL) of the labeled mAb sample was added to the plated cells to achieve the target mAb concentration (25 nM).

[0402] Plates coated with the labeled mAb were incubated at 37°C for 30 minutes. Each plate was then read using an Incucyte device, which collects images from the red and phase channels [10X objectives; 3 images / well] for a duration of 24 hours, with readings every 30 minutes. Data are shown in Tables 13A and 13B. Data show that the anti-CEACAM5 progenitor antibody was internalized in both the HCT-CEA and MKN45 cell lines. Table 13A. Internalization data of hIgG1.3 progeny antibodies Table 13B. Internalization data of hIgG1 progeny antibodies

[0403] The internalization of MBN001 and certain progeny groups (e.g., MBP004 and MBP019) in cells expressing cynoCEACAM-5 was analyzed. Effective internalization of MBN001 and its progeny was observed. Internalization of the negative control mAb was not observed. Example 10 - Conjugation reaction with compound A' and anti-CEACAM5 mAb to produce DAR8 ADC

[0404] This example describes the conjugation of MBN001 and its progeny (e.g., MBP001, MBP002, MBP003) antibodies to compound A'. Table 14 lists the materials used in the conjugation method. Table 14. Reagents and Consumables (Compound A')

[0405] First, each mAb was buffer-exchanged in P5 conjugation buffer (50 mM Tris, 100 mM NaCl, 1 mM EDTA, pH 8.3, 25°C) and adjusted to a concentration of 10 mg / mL. Next, a Zeba rotary desalting column was used. These columns were equilibrated with P5 conjugation buffer according to the manufacturer's instructions. Recovery yields were typically >95%.

[0406] For mAb stock solutions with concentrations below 10 mg / ml, concentrate and exchange buffer (distillation) using a protein concentrator spin column (Amicon Ultra) according to the manufacturer's instructions. Rinse these spin columns with P5 conjugation buffer before applying the mAb.

[0407] After buffer exchange, mAb concentrations were 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.

[0408] Next, the mAb is transferred to an amber-colored plastic container. The conjugation reaction is carried out in the dark. Conjugation with compound A' is 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 solution of compound A' in DMSO and a 10 mM TCEP working solution are used. The 40 mM linker-load stock solution is thawed. A fresh 10 mM TCEP working solution is prepared by combining 20 µl of 0.5 M TECP-HCl (pH 7.0) with 980 µl of P5 conjugation buffer. Both reagents are vortexed before use.

[0409] The calculated amount of TCEP working solution was added to the solution of MBP001 mAb and mixed by gentle rotation. Immediately afterwards, the calculated amount of compound A' stock solution was added. The mixture was then incubated overnight at 23°C in an amber 50 ml tube and rotated at 300 rpm.

[0410] The 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 with a 6530 Accurate-Mass Q-TOF. The analytical column (Waters Inc., BEH C4 column, 1.7 μm, 2.1 mm x 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: 0–2 min., maintained at 27% B; 2–9 min., slowly ramped from 27% B to 37% B; 9–9.5 min., linearly ramped from 37% B to 90% B; 9.5–12.3 min., maintained at 90% B. MS settings were as follows: polarity = positive, capillary voltage = 4.2 kV, sampling cone = 40 V, source offset = 15 V, source temperature = 140°C, desolvation temperature = 325°C. Data acquisition range was 900–3200 m / z. Deconvolution was performed using Agilent MassHunter Walkup. If any unconjugated mAbs remained, an additional 1.4 equivalents of TCEP and 2 equivalents of compound A' were added, followed by incubation for 2–4 hours. This method yielded a DAR8 ADC. Example 11: Cytotoxicity analysis of progeny antibodies

[0411] As described above, this example evaluates the cytotoxicity of CEACAM5-targeting mAbs conjugated to compound A' via MBN001 progeny mAbs. Cytotoxicity was measured by IC50 and AUC values ​​of cell growth inhibition in the CEACAM5-expressing Ls174T and MKN45 cell lines. Data (along with binding and internalization data) were used to select mAbs for in vivo efficacy assays.

[0412] Ls174T and MKN45 cells were harvested using a trypsin / EDTA (0.25%) solution (Gibco catalog number 25200-056). The culture medium was removed, and the cells were washed with 1x PBS (Ca+ and Mg-free; Gibco catalog number 14190-144). Cells were isolated using the trypsin / EDTA solution. The trypsin / EDTA solution was neutralized with complete culture medium. The cells were centrifuged at 1400 rpm for five minutes. The supernatant was removed, and the cells were resuspended in complete culture medium corresponding to each cell line.

[0413] The cells were then counted, and the cell concentration was adjusted to 1.0 x 10⁻⁶. 6 Cells / mL. Dilute specific cells to the following concentration: Ls174T: 0.125 x 10⁻⁶ cells / mL. 6 Cells / mL and MKN45: 0.1 x 10 6 Cells / mL. Add a volume (20 µL) of different cell suspensions to each well of a plate (PerkinElmer catalog number 6007480). Incubate the plate for 20–24 hours. Prepare dilutions of the test reagents (e.g., antibodies MBP001, MBP002, and MBP003 conjugated to compound A') and add 20 µL of the dilutions to the plate, allowing incubation at 37°C for 120 h.

[0414] 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 allowed to equilibrate to room temperature. The assay plate was removed from the incubator and allowed to acclimatize to room temperature. A white adhesive was applied to the bottom of the plate. A volume (40 µL) of CTG solution was added to each well and mixed on a track shaker at 500 rpm for 2 minutes. The assay plate was placed in the dark for 20 minutes. The plate cap was then removed, and the luminescence was analyzed on an Envision plate reader. The data are shown in Table 15. The data show that the tested antibody bound to CEACAM5 on different CEACAM5-expressing cell lines for over 96 hours, enabling the delivery of cytotoxic payloads to cells for effective cytotoxic killing. Table 15. Cytotoxicity data of progeny antibodies Example 12: Analysis of cytotoxicity and ADCC activity of anti-CEACAM5 ADC

[0415] This study analyzed the cytotoxic activity of anti-CEACAM5 ADCs ADCP001A, ADCP001B, and ADCP001C (i.e., the progeny mAbs MBP003, MBP001, and MBP002 conjugated to compound A', respectively) in CEACAM5-expressing cell lines LS174T, BxPC3, and MKN45. Figure 10A , Figure 10B and Figure 10C The following cell lines are shown to show the percentage (in terms of cell viability%) of cell growth inhibition when treated with the selected antibody conjugate at the specified antibody concentration: Ls174T ( Figure 10A), low CEACAM5 expression cell lines; BxPC-3, medium CEACAM5 expression cell lines ( Figure 10B ); and MKN45, a cell line with high CEACAM5 expression ( Figure 10C Data show that, with parental ADCN001 (conjugated to compound A, MBN001 ADC; see [link]). Figure 10D In comparison, the progeny ADCs described in this paper exhibit comparable or even improved IC50 values ​​for cell growth inhibition.

[0416] In the Jurkat-NFAT-FcγRIIIa (Promega) cell assay (catalog number G9901), BxPC3 and MKN45 cells were used as target cells, and ADCC activity was analyzed using anti-human leader CEACAM5 mAb. See also Figure 11A and Figure 11B BxPC3 (a CEACAM-moderately expressed cell line) and MKN45 (a CEACAM5-highly expressed cell line) cells were incubated with different concentrations of CEACAM5 mAb (IgG1 or inactive IgG1.3f). Jurkat-FcγRIIIa effector cells were then co-incubated with target cells for six hours. NFAT activation, reflecting the induced ADCC response, was assessed by determining luciferase activity. Data showed that the mAb progeny of MBP003, MBP001, and MBP002 possessed limited ADCC activity. Example 13. Bystander kill analysis of progeny resistance to CEACAM5 mAb ADC

[0417] Depending on the adapter design, membrane-permeable cytotoxic drugs conjugated in ADCs released within target-positive cells can cross the cell membrane and kill closely adjacent cells, including neighboring cancer cells lacking antigen expression (bystander effect). This case was assayed to analyze the bystander killing profile of anti-CEACAM5 ADCs.

[0418] In short, 8000 MKN45 cells expressing the CEACAM5 antigen (antigen-positive cells; Ag+) and 2000 HCT-116 cells not expressing CEACAM5 (antigen-negative cells, Ag-) were plated in 96-well plates to determine ADC bystander killing after treatment with different doses of anti-CEACAM5 mAb+ compounds A' ADC ADCP001A, ADCP001B, and ADCP001C (N = 3 for each treatment). Incucyte, used to count the function of viable Ag- (green) or Ag+ (red) cells, was used. Control cells were untreated. At 72 and 120 hours, the dose-dependent percentage of Ag+ or Ag- cells was calculated by normalization relative to untreated control wells. See also Figure 12A , Figure 12B , Figure 12C and Figure 12D Each tested ADC (including MBP001, MBP002, or MBP003 conjugated with compound A') was found to have the ability to release the eczema payload and kill bystander cells with the eczema payload. Example 14. Testing the in vivo antitumor efficacy of anti-CEACAM5 ADC

[0419] This study analyzed the in vivo antitumor efficacy of anti-CEACAM5 ADCs in cell line-derived xenograft models (MKN45, BxPC3, and Ls174T). Two x 10⁶ MKN45 cells, five x 10⁶ BxPC3 cells, or one x 10⁶ Ls174T cell were subcutaneously inoculated into the right abdomen of four- to six-week-old immunodeficient female athymic nude mice (CRL 490, Charles River) or NSG mice (NOD.Cg-Prkdcscid Il2rgtm1Wjl / SzJ, catalog number 005557, Jackson Laboratory). Animals with tumor volumes between 150 mm³ and 250 mm³ were randomly assigned to treatment groups, with a mean tumor volume of 180 mm³ (N = 7). Tumor-bearing animals were treated with a buffer control (phosphate-buffered saline (PBS)) or a single intravenous injection of the ADC. Measure the tumor length (L) and width (W) using calipers, and use the formula L (W^2) / 2 calculates the tumor volume.

[0420] The in vivo potency of anti-CEACAM5 ADCN001 (MBN001 conjugated to compound A') was evaluated in MKN45, Ls174T, and BxPC3 models. Tumor growth results in MKN45, BxPC3, and Ls174T models treated with 3 mg / kg ADCN001 are provided in [the table below]. Figure 15A , Figure 15B and Figure 15C In addition, tumor growth in BxPC3 and Ls174T models treated with 10 mg / kg ADCN001 was improved. Figure 15D and Figure 15E The data show that ADCN001 (MBN001 conjugated to compound A') has antitumor activity in these models. Figures 17A-17DThis is a set of figures illustrating the efficacy of ADCV001 (MBV001 conjugated to compound A', the wild-type hIgG1 form of MBN001) in CDX models (MKN45 and BxPC3) following a single intravenous injection of 3 mg / kg or 10 mg / kg of ADC. Tumor growth results in the MKN45 and BxPC3 models treated with 3 mg / kg ADCV001 are provided in [figures to be inserted]. Figure 17A and Figure 17B In addition, tumor growth results of the MKN45 model and the BxPC3 model treated with 10 mg / kg MBV001 are provided in [the following text is missing from the original] Figure 17C and Figure 17D The data show that ADCN001 (the wild-type IgG form of MBN001 conjugated with compound A') is also effective in the MKN45 and BxPC3 models.

[0421] The progeny of anti-CEACAM5 mAb MBN001 (i.e., MBP001, MBP002, and MBP003) were also conjugated with compound A', and in vivo efficacy was tested in MKN45 and BxPC3 models. Tumor growth results after treatment with 3 mg / kg ADC are provided separately. Figure 18A and Figure 18B In addition, the tumor growth results after treatment with 10 mg / kg ADC were provided in [the study / analysis]. Figure 18C and Figure 18D These ADCs, ADCP001A, ADCP001B, and ADCP001C, demonstrated robust antitumor activity in the MKN45 and BxPC3 models. Plasma exposure to the total ADC was comparable across the three ADCs. Plasma exposure to the free payload of these ADCs was below the limit of quantitation (data not shown).

[0422] In all of the above in vivo efficacy studies, no significant effect on body weight was observed (data not shown). Therefore, treatment with these anti-CEACAM5+ compounds A'ADCs was well tolerated throughout the treatment course. Example 15. Analysis of pharmacodynamic markers of the DDR pathway in mouse tumors treated with mAb MBN001 conjugated to compound A'

[0423] This example analyzes the DNA damage response pathway induced by treatment of cancer cells with ADCN001 (such as MBN001 conjugated to compound A' generated in Example 10). MKN45 tumors in mice (n = 3) were treated with different amounts (1 mpk or 10 mpk) of ADC. The expression of DNA damage response markers pKAP(1TF1b) (ser 824), pCHK1, γH2AX, and the apoptosis marker c-cysteine ​​3 was evaluated in tumor samples harvested from animals at 6, 24, or 168 hours after a single intravenous injection of ADCN001, compared with the control GAPDH. DNA damage markers pKAP1, pCHK1, and γH2AX were induced within 24 hours of administration and persisted until 168 hours after administration, while the apoptosis marker cleaved c-cysteine ​​3 was induced at 168 hours after administration. Figure 16A The data were quantified and normalized relative to the GAPDH control. Figure 16B ). Example 16. HDX epitope plotting of CEACAM5 mAb MBN001

[0424] This example analyzes the binding epitope of human hCEACAM5 after interaction with anti-CEACAM5 mAb MBN001. Hydrogen-deuterium exchange mass spectrometry (HDX-MS) probes protein conformation and conformational dynamics in solution by monitoring the rate and extent of deuterium exchange between main-chain amide hydrogen atoms [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-deuterium exchange depends on solvent accessibility of main-chain amide hydrogen atoms, protein hydrogen bonds, time, and pH. HDX-MS provides readouts for both hydrogen bonds and solvent accessibility. The mass increase of the protein after HDX can be precisely measured by MS. Comparing the exchange rates between bound and unbound states in HDX experiments can provide valuable insights into protein conformational dynamics, binding, specificity, and stability. Protein regions exhibiting slower hydrogen exchange rates (protection) in both bound and unbound states indicate potential binding sites or structural stability. In the context of antigen / antibody interactions, antigenic regions with slowed hydrogen exchange in the presence of antibodies are identified as potential epitopes. Protein regions exhibiting faster hydrogen exchange rates (deprotection) in both bound and unbound states indicate structural destabilization. Hydrogen exchange rates are also sensitive to allosteric effects, which can complicate the interpretation of results. method

[0425] For the purpose of designing peptide constructs including the CEACAM5 subdomain, a full-atom 3D model of CEACAM5 was constructed using MOE software (Molecular Operating Environment (MOE) 2022.02 ChemComputing Group LLC, 910-1010 Sherbrooke St. W., Montreal, QC H3A 2R7, Canada, 2023) with Cα coordinates from solution scattering data (Boehm, MK and Perkins, SJ FEBS Lett [Federation of European Biochemical Societies Letters] 475, 11-16, (2000), PDB No. 1E07). The band representation of the 3D model of human CEACAM5 along with its individual domains is shown in... Figure 13A In the middle. Individual domains in the protein sequence corresponding to the 3D model are shown. Figure 13B middle. Figure 13B The seven shaded areas correspond to Figure 13A Different structural domains in it.

[0426] Prior to epitope mapping experiments, dedeuteration experiments were performed to generate a list of common peptides for the recombinant hCEACAM5-A3-B3 construct (SEQ ID NO: 24) and the protein complex of the hCEACAM5-A3-B3 construct with the Fab of mAb MBN001 (15 µM, 1:1 molar ratio). In HDX-MS experiments, a volume (5 µL) of each sample was diluted to 55 µL of D2O buffer (10 mM phosphate buffer, D2O, pH 7.0) to initiate the labeling reaction. These reactions were carried out for different time periods: 20 s, 1 min, 10 min, and 60 min. At the end of each labeling reaction period, the reaction was quenched by adding quenching buffer (100 mM phosphate buffer with 4 M GdnCl and 0.4 M TCEP, pH 2.5, 1:1, v / v), and 50 µL of the quenched sample was injected into the Waters HDX-MS system for analysis. Deuterium uptake levels of co-digested peptides were monitored in the absence and presence of Fab. Differences in hydrogen exchange rates between bound and free antigens were identified by subtracting the average deuteration of the same peptide in the bound antigen from the average deuteration of each peptide in the free antigen state. Changes were considered significant if they exceeded three times the pooling uncertainty of the propagation criteria. result

[0427] Based on epitope binning data indicating the binding of MBN001 to the A3-B3 region of hCEACAM5, the hCEACAM5-A3-B3 construct was selected for HDX experiments. For the purposes of this report, the N-terminal residues on the antigen were numbered starting at 488 to align the hCEACAM5-A3-B3 construct number 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 truncated hCEACAM5-A3-B3 construct used for this analysis. The complexity and heterogeneity of hCEACAM5 pose a significant challenge to obtaining full 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 ).

[0428] In the presence of MBN001, a significantly reduced hydrogen exchange rate on hCEACAM5 was observed in the peptide regions covering residues 588 to 606 and residues 668 to 685 compared to the unbound hCEACAM5 antigen. Figure 13D ).

[0429] Further interpretation of HDX differences requires consideration of additional factors associated with the HDX process [Bai et al., 1993 Proteins. [Proteins] 17(1):75-86]. First, proline residues do not have an amide hydrogen and are therefore not reported in the HDX process. Next, after proteolysis, the N-terminal residues of each peptide are converted from amides to amines. Amines undergo 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.

[0430] Peptide region 590-606 is covered by multiple overlapping peptides, all of which exhibit strong protection. This suggests a high probability that this region is involved in binding. The range of amino acids with significant protection is further narrowed down to: 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 (SEQ ID NO:103) HDX-MS indicates that this region is the primary epitope.

[0431] The peptide region 668-685 is covered by a single peptide. Even after excluding the first two N-terminal amino acids and the proline residue at position 681, the protected region is still quite long, covering 15 amino acids, namely: IVK 670 SITVSASGTSPGLSA 685 (SEQ ID NO: 104) No data are available for the following regions on hCEACAM5: 501-522, 546-574, 612-615, and 642-662, therefore no conclusions can be drawn regarding these residues.

[0432] The overall HDX effect of MBN001 combined on hCEACAM5 is shown in Figure 13E middle. in conclusion

[0433] HDX-MS identified the following residues and peptide regions as potential epitopes on hCEACAM5 (A3B3) when bound to MBN001: L 590 Y 591 G 592 D 594 T 595 I 597 I 598 S 599 D 602 S 603 S 604 Y 605 L 606 K 670 SITVSASGTSPGLSA 685 (SEQ ID NO: 117) Example 17. Cryo-EM analysis of box 1 mAb MBP001

[0434] This example describes the cryo-EM analysis of box 1 mAb MBP001. The CEACAM5 construct is approximately 20 kDa in size, and the MBP001 Fab is approximately 50 kDa. Using cryo-EM analysis, particles significantly smaller than approximately 120 kDa were more difficult to pick and match. Therefore, a CEACAM5-resistant box 2 mAb (approximately 50 kDa) was also generated and mixed with the CEACAM5 construct and the MBP001 Fab to produce a complex structure of approximately 120 kDa. This composite structure made cryo-EM analysis easier and confirmed that the box 1 conjugate MBP001 binds to different epitopes with the box 2 mAb. The following is a description of the different steps and methods performed. Design of CEACAM5 A3-B3

[0435] Like other members of the CEA-associated cell adhesion molecule (CEACAM) family within the immunoglobulin (Ig) gene superfamily, CEACAM5 is a highly glycosylated multidomain protein whose domain boundaries have been artificially assigned using the PROSITE annotation rules in Uniprot (ID P06731; Uniprot: Universal Protein Knowledge Base, 2023; The UniProt Consortium (2023) Nucleic Acids Research 51, D523-D531).

[0436] However, for the purpose of designing peptide constructs including the CEACAM5 subdomain, a full-atom 3D model of CEACAM5 was constructed using Cα coordinates from solution scattering data using MOE software (Molecular Operation Environment (MOE) 2022.02 Chemical Computing Group LLC, 910-1010 Sherbrooke West Street, Montreal H3A 2R7, Quebec, Canada, 2023.) (Boehm, MK and Perkins, SJ FEBS Lett [Circular of the Federation of European Biochemical Societies] 475, 11-16, (2000), PDB No. 1E07). The band representation of the 3D model of human CEACAM5 along with its individual domains is shown in... Figure 13A In the middle. Individual domains in the protein sequence corresponding to the 3D model are shown. Figure 13B middle.

[0437] Designing the A3-B3 construct for antibody screening further required the attachment of purification and cleavage tags. For purification, a histidine tag (amino sequence HHHHHH; SEQ ID NO: 95) was chosen, and for protease cleavage, a tobacco vein mottle virus (TVMV) tag (sequence ETVRFQG (SEQ ID NO: 102); Naallamsetty, ProteinExpr. Purif. [Protein Expression and Purification] 38, 108-15, 2004) was chosen. These were attached to the N-terminus of the A3-B3 construct. The N-terminus was chosen over the C-terminus for this purpose because we were interested in finding antibody-binding epitopes closer to the C-terminus (i.e., the B3 domain) to selectively bind to the non-detached rather than the detached or soluble form of CEACAM5 (detachment occurs near the C-terminus). The final construct is shown in SEQ ID No: 24. Expression and purification of hCEACAM5 A3-B3 reagent

[0438] The human CEACAM5 A3-B3 domain protein (C-terminal region, 198 amino acid protein construct) shown below was constructed. HHHHHHETVRFQGPKPSISSNNSKPVEDKDAVAFTCEPEAQNTTYLWWVNGQSLPVSPRLQLSNGNRTLTLFNVTRNDARAYVCGIQNSVSANRSDPVTLDVLYGPDTPIISPPDSSYLSGANLNLSCHSASNPSPQYSWRINGIPQQHTQVLFIAKITPNNNGTYACFVSNLATGRNNSIVKSITVSASGTSPGLSA (SEQ ID NO: 96)

[0439] The human CEACAM5 A3-B3 domain protein was expressed in Expi293F™ GnTI- cells (Thermo Fisher Scientific) by transiently transfecting ExpiFectamine™ 293 transfection kit (Thermo Fisher Scientific) with DNA encoding the A3-B3 protein. After 24 hours, the transfected cells were fed with the enhancers provided in the kit and grown for a total of 4 days at 37°C, 8% CO2, and 150 RPM. The supernatant was harvested by centrifugation and filtration using a 0.22 μm filter (Corning).

[0440] Clarified medium containing A3-B3 was purified from a 5 ml Histrap Excel column (Stenofan) and eluted with 250 mM imidazole-phosphate buffer. The eluent from the Histrap column was further purified using a preparative HiLoad Superdex 200 16 / 60 (Stenofan) to separate the A3-B3 monomer from the aggregate. The monomer preparative size exclusion chromatography (SEC) fractions were combined and filtered through a 0.22 μm syringe filter (Pall) as the sample for CryoEM structure determination. The calculated 25,690 M N₂ was used via A280. - ¹ cm - The molar extinction coefficient¹ determined the sample concentration. For quality determination, the samples were examined by LC-MS, analytical SEC, and SDS-PAGE. Expression and purification of anti-CEACAM5 Fab

[0441] Each Fab heavy chain (HC) produced shares the same variable region (VH) and constant region (CH1) as its parent mAb. Two amino acids, GG, are added to the C-terminus of the Fab HC. The Fab light chain (LC) remains identical to the parent mAb LC. The DNA from which Fab HC and LC were synthesized is used for expression. Fab of MBP001: Light chain: EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQID NO 46) Heavy chain: QVQLVESGGGVVQPGRSLRLSCAASGIYFSSHGMHWVRQAPGKGLEWVTFISYDGSYKSYVDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCATGLTGTGAFDIWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCGG (SEQ ID NO: 100) (comprising the VH of SEQ ID NO: 45) Fab of Box 2 mAb: Light chain: DIQLTQSPSFLSASVGDRVTITCRASQGISTYLAWYQQKPGKAPKFLIYAEKTLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQLASYPFTFGPGTKVDIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ IDNO: 97) Heavy chain: QVQLQESGPGLVKPSGTLSLTCAVSGGSISSSDWWSWVRQPPGKGLEWIGEIYHQGSTNYNPSLKSRVTISVDKSKNQFSLKLSSVTAADTAVYYCARASSSGYYGHDVWGQGT TVTVSSASTKGPSVFPLAPSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCGG (SEQ ID NO: 98)

[0442] Using t...

Claims

1. An antibody-drug conjugate (ADC) having formula (I): (I) Or its pharmaceutically acceptable salts, stereoisomers or solvates, wherein: The configuration of the double bond is either E or Z; V is H or (C1-C8) alkyl; X is ; Y is NR 5 S, O or CR 6 R 7 ; R 1 It is a polyalkylene glycol unit containing at least 3 alkylene glycol subunits; R 3 and R 5 -R 7 Each is H, or an optional substituted aliphatic residue, or an optional substituted aromatic residue; L stands for connector; C represents the cytotoxic component; m is an integer ranging from 1 to 10; The range of n is from 1 to 20; and AB is an anti-CEACAM5 antibody or an antigen-binding moiety of an anti-CEACAM5 antibody that specifically binds to carcinoembryonic antigen-associated cell adhesion molecule 5 (CEACAM5), the anti-CEACAM5 antibody or the antigen-binding moiety of the anti-CEACAM5 antibody comprising: (a) A heavy chain variable region (VH) comprising complementarity-determining regions (CDR)1, CDR2, and CDR3 containing the amino acid sequences shown in SEQ ID NO: 14, 15, and 16, respectively; and a light chain variable region (VL) comprising CDR1, CDR2, and CDR3 containing the amino acid sequences shown in SEQ ID NO: 19, 20, and 21; or (b) VH, which comprises CDR1, CDR2, and CDR3 regions having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with the amino acid sequences shown in SEQ ID NO: 14, 15, and 16, respectively; and VL, which comprises CDR1, CDR2, and CDR3 regions having at least 85%, 90%, 95%, 98%, or 99% sequence identity with the amino acid sequences shown in SEQ ID NO: 19, 20, and 21, respectively.

2. The ADC of claim 1, wherein the VH comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with the amino acid sequence shown 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 of claim 1, wherein the VH comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:

17.

4. The ADC of claim 1, wherein the VH comprises the amino acid sequence shown 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 of claim 1, wherein the VH comprises the amino acid sequence shown in SEQ ID NO:

17.

6. The ADC of any one of claims 1-5, wherein the VL comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with the amino acid sequence shown 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 of any one of claims 1-5, wherein the VL comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:

22.

8. The ADC of claim 1, wherein the VL comprises the amino acid sequence shown 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 of claim 1, wherein the VL comprises the amino acid sequence shown in SEQ ID NO:

22.

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

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

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

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

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

15. The ADC as claimed in any of the preceding claims, wherein Y is NH.

16. The ADC as claimed in any of the preceding claims, wherein the polyalkylene glycol unit R 1 It contains 3 to 100 subunits with the following structure: 。 17. The ADC of claim 16, wherein R 1 yes in: Indicate the position of O; K F Selected from the following group, which consists of: -H, -PO3H, -(Cl-C 10 )alkyl, -(C 1- 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 o is an integer ranging from 3 to 100.

18. The ADC of claim 16, wherein the polyethylene glycol unit R 1 It contains 3 to 100 subunits with the following structure: 。 19. The conjugate of claim 17, wherein R 1 yes: in Indicate the position of O; K F Selected from the following group, which consists of the following: -H, -PO3H, -(Cl-C 10 )alkyl, -(C 1- 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 o is an integer ranging from 3 to 100.

20. The ADC of claim 17 or 19, wherein K F It is H.

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

22. The ADC as claimed in any one of claims 1 to 21, wherein the connector L has the following formula: -A-W 1-8 -B 0-1 -#, in A is the first spacer subunit; W is an amino acid; B is the second spacer subunit; Indicates the attachment point to the -Y-; and # indicates the attachment point to the cytotoxic portion.

23. The ADC of claim 22, wherein A has the following structure: ， in It is a 5- or 6-membered carbon ring; Indicates the attachment point to the -Y-; and ## indicates the attachment point to W.

24. The ADC of claim 23, wherein yes .

25. The ADC of claim 22, wherein W is a dipeptide (W2).

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

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

28. The ADC of claim 22, wherein the second spacer subunit B is a PAB group having the following structure: ,in The NH group is bonded to -W-, and The C(O) group is bonded to the cytotoxic moiety.

29. The ADC of claim 22, wherein the connector has the following structure: , where W2 is Val-Cit.

30. The ADC of claim 22, wherein the connector L has the following structure -A-W2-B1-#: in The symbol indicates the attachment point to the Y symbol, and the symbol # indicates the attachment point to the cytotoxic portion.

31. The ADC of claim 1, wherein: V is H; Y is NH; R 1 It is a polyalkylene glycol unit with the following structure: in: Indicate the position of O; K F It is H; o is an integer ranging from 8 to 30; R 3 It is H; L is a connector with the following structure: in The # indicates the attachment point to the Y, and the # indicates the attachment point to the cytotoxic portion; C is icitrazone; m is 1; and The range of n is from 5 to 10.

32. The ADC of claim 31, wherein o is an integer ranging from 15 to 30.

33. The ADC of claim 31, wherein o is an integer ranging from 20 to 28.

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

35. The ADC of claim 31, wherein n ranges from 6 to 8.

36. An antibody-drug conjugate (ADC) having formula (II): (II) Or its pharmaceutically acceptable salts, stereoisomers or solvates, wherein: The configuration of the double bond is either E or Z; The range of n is from 4 to 8; and o is an integer between 10 and 30; AB is an anti-CEACAM5 antibody or an antigen-binding moiety of an anti-CEACAM5 antibody that specifically binds to carcinoembryonic antigen-associated cell adhesion molecule 5 (CEACAM5), the anti-CEACAM5 antibody or the antigen-binding moiety of the anti-CEACAM5 antibody comprising: (a) A heavy chain variable region (VH) comprising complementarity-determining regions (CDR)1, CDR2, and CDR3 containing the amino acid sequences shown in SEQ ID NO: 14, 15, and 16, respectively; and a light chain variable region (VL) comprising CDR1, CDR2, and CDR3 containing the amino acid sequences shown in SEQ ID NO: 19, 20, and 21; or (b) VH, which comprises CDR1, CDR2, and CDR3 regions having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with the amino acid sequences shown in SEQ ID NO: 14, 15, and 16, respectively; and VL, which comprises CDR1, CDR2, and CDR3 regions having at least 85%, 90%, 95%, 98%, or 99% sequence identity with the amino acid sequences shown in SEQ ID NO: 19, 20, and 21, respectively.

37. An antibody-drug conjugate (ADC) having the formula (ADC 101): (ADC 101) Or its pharmaceutically acceptable salts, stereoisomers or solvates, wherein: The configuration of the double bond is either E or Z; AB is an anti-CEACAM5 antibody or an antigen-binding moiety of an anti-CEACAM5 antibody that specifically binds to carcinoembryonic antigen-associated cell adhesion molecule 5 (CEACAM5), the anti-CEACAM5 antibody or the antigen-binding moiety of the anti-CEACAM5 antibody comprising: (a) A heavy chain variable region (VH) comprising complementarity-determining regions (CDR)1, CDR2, and CDR3 containing the amino acid sequences shown in SEQ ID NO: 14, 15, and 16, respectively; and a light chain variable region (VL) comprising CDR1, CDR2, and CDR3 containing the amino acid sequences shown in SEQ ID NO: 19, 20, and 21; or (b) VH, which comprises CDR1, CDR2, and CDR3 regions having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with the amino acid sequences shown in SEQ ID NO: 14, 15, and 16, respectively; and VL, which comprises CDR1, CDR2, and CDR3 regions having at least 85%, 90%, 95%, 98%, or 99% sequence identity with the amino acid sequences shown in SEQ ID NO: 19, 20, and 21, respectively.

38. An antibody-drug conjugate (ADC) having formula (II): (II), Or a pharmaceutically acceptable salt, stereoisomer, or solvate thereof, wherein n ranges from 4 to 8; o is an integer ranging from 10 to 30; AB is an anti-CEACAM5 antibody or its antigen-binding moiety comprising VH and VL, respectively, containing the amino acid sequences shown in SEQ ID NO: 38 and SEQ ID NO:

43.

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

40. The ADC of any one of claims 12, 36 or 37, wherein the VH and the VL respectively comprise the amino acid sequence shown in SEQ ID NO:49 and the amino acid sequence shown in SEQ ID NO:

50. The ADC of any one of claims 12, 36 or 37, wherein the VH and the VL respectively comprise the amino acid sequence shown in SEQ ID NO: 51 and the amino acid sequence shown in SEQ ID NO:

52.

41. The ADC of any one of claims 12, 36 or 37, wherein the VH and the VL respectively comprise the amino acid sequence shown in SEQ ID NO: 67 and the amino acid sequence shown in SEQ ID NO:

68.

42. The ADC of any one of claims 12, 36 or 37, wherein the VH and the VL respectively comprise the amino acid sequence shown in SEQ ID NO:69 and the amino acid sequence shown in SEQ ID NO:

70.

43. The ADC of any one of claims 12, 36 or 37, wherein the VH and the VL respectively comprise the amino acid sequence shown in SEQ ID NO:71 and the amino acid sequence shown in SEQ ID NO:

72.

44. The ADC of any one of claims 12, 36 or 37, wherein the VH and the VL respectively comprise the amino acid sequence shown in SEQ ID NO:73 and the amino acid sequence shown in SEQ ID NO:

74.

45. The ADC of any one of claims 12, 36 or 37, wherein the VH and the VL respectively comprise the amino acid sequence shown in SEQ ID NO:75 and the amino acid sequence shown in SEQ ID NO:

76.

46. ​​The ADC of any one of claims 12, 36 or 37, wherein the VH and the VL respectively comprise the amino acid sequence shown in SEQ ID NO:69 and the amino acid sequence shown in SEQ ID NO:

70.

47. The ADC of any one of claims 12, 36 or 37, wherein the VH and the VL respectively comprise the amino acid sequence shown in SEQ ID NO:73 and the amino acid sequence shown in SEQ ID NO:

74.

48. The ADC of any one of claims 12, 36 or 37, wherein the VH and the VL respectively comprise the amino acid sequence shown in SEQ ID NO:75 and the amino acid sequence shown in SEQ ID NO:

76.

49. The ADC of any one of claims 12, 36 or 37, wherein the VH and the VL respectively comprise the amino acid sequence shown in SEQ ID NO:77 and the amino acid sequence shown in SEQ ID NO:

78.

50. The ADC of any one of claims 12, 36 or 37, wherein the VH and the VL respectively comprise the amino acid sequence shown in SEQ ID NO:79 and the amino acid sequence shown in SEQ ID NO:

80.

51. The ADC of any one of claims 12, 36 or 37, wherein the VH and the VL respectively comprise the amino acid sequence shown in SEQ ID NO:81 and the amino acid sequence shown in SEQ ID NO:

82.

52. The ADC of any one of claims 12, 36 or 37, wherein the VH and the VL respectively comprise the amino acid sequence shown in SEQ ID NO:83 and the amino acid sequence shown in SEQ ID NO:

84.

53. The ADC of any one of claims 12, 36 or 37, wherein the VH and the VL respectively comprise the amino acid sequence shown in SEQ ID NO:85 and the amino acid sequence shown in SEQ ID NO:

86.

54. The ADC of any one of claims 12, 36 or 37, wherein the VH and the VL respectively comprise the amino acid sequence shown in SEQ ID NO:87 and the amino acid sequence shown in SEQ ID NO:

88.

55. The ADC of any one of claims 12, 36 or 37, wherein the VH and the VL respectively comprise the amino acid sequence shown in SEQ ID NO:89 and the amino acid sequence shown in SEQ ID NO:

90.

56. The ADC of any one of claims 12, 36 or 37, wherein the VH and the VL respectively comprise the amino acid sequence shown in SEQ ID NO:91 and the amino acid sequence shown in SEQ ID NO:

92.

57. The ADC of any one of claims 12, 36 or 37, wherein the VH and the VL respectively comprise the amino acid sequence shown in SEQ ID NO:93 and the amino acid sequence shown in SEQ ID NO:

94.

58. The ADC of any one of claims 12, 36 or 37, wherein the VH and the VL respectively comprise the amino acid sequence shown in SEQ ID NO:17 and the amino acid sequence shown in SEQ ID NO:

22.

59. The ADC of any one of claims 12, 36 or 37, wherein the heavy chain and the light chain respectively comprise the amino acid sequence shown in SEQ ID NO: 45 and the amino acid sequence shown in SEQ ID NO:

46.

60. The ADC as claimed in any of the preceding claims, wherein the ADC comprises an IgG1 constant region, an IgG2 constant region, an IgG3 constant region, an IgG4 constant region, or a variant thereof.

61. The ADC as claimed in any of the preceding claims, wherein the ADC comprises an IgG1 antibody.

62. The ADC of claim 61, wherein the ADC comprises an IgG1.3f constant region.

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

64. The ADC as claimed in any of the preceding claims, wherein the antibody is a human antibody, a humanized antibody, or a chimeric antibody.

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

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

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

68. A pharmaceutical composition comprising an ADC as described in any one of the preceding claims and a pharmaceutically acceptable carrier.

69. The pharmaceutical composition of claim 68, further comprising one or more additional therapeutic agents.

70. A kit comprising an ADC as described in any of the preceding claims and instructions for use.

71. A method for generating an ADC as described in any of the preceding claims, the method comprising conjugating the anti-CEACAM5 antibody or its antigen-binding portion to eczema.

72. A method of treating a subject with cancer expressing CEACAM5, the method comprising administering to the subject a therapeutically effective amount of an ADC as described in any of the preceding claims or a pharmaceutical composition as described in any of the preceding claims.

73. The method of 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, stomach cancer, gastrointestinal cancer, colon cancer, kidney cancer, head and neck cancer, stomach cancer, germ cell cancer, bone cancer, liver cancer, thyroid cancer, skin cancer, central nervous system tumors, lymphoma, leukemia, myeloma, sarcoma, and myelodysplastic syndrome.

74. The method of claim 72 or claim 73, further comprising administering one or more additional therapies.

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

76. The method of claim 74 or claim 75, wherein the one or more additional therapies comprise immune checkpoint inhibitors.

77. The method of 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.

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