Anti-CEA antibody drug conjugates and methods of use

Abstract

The present disclosure provides antibody drug conjugates comprising antibodies and antigen binding fragments thereof that bind to human CEA and linker-payloads, pharmaceutical compositions comprising the anti-CEA antibody drug conjugates, and the use of the anti-CEA antibody drug conjugates for the treatment of CEA-related diseases or disorders.

Description

Cross - Reference to Related Applications

[0001] This application claims the priority benefits of PCT Application No. PCT / CN2022 / 134067, titled "Anti - CEA Antibody Drug Conjugates and Methods of Use", filed on November 24, 2022, and PCT Application No. PCT / CN2023 / 107003, titled "Anti - CEA Antibody Drug Conjugates and Methods of Use", filed on July 12, 2023. These PCT applications are hereby incorporated by reference in their entireties. Reference to Electronic Sequence Listing

[0002] This application contains a sequence listing that has been electronically submitted in.XML format and is hereby incorporated by reference in its entirety. The.XML copy created on November 20, 2023, is named "01368 - 0008 - 00PCT.xml" and is 156,334 bytes in size. The sequence listing contained in this.XML file is part of the specification and is hereby incorporated by reference in its entirety herein. Technical Field

[0003] Disclosed herein are anti - CEA antibody - drug conjugates (ADCs) comprising an antibody or an antigen - binding fragment thereof that binds to human CEA and is covalently linked to a growth inhibitor, and their therapeutic uses. Background Art

[0004] Antibody - drug conjugates (ADCs) are chimeric molecules that combine antibody specificity (to recognize antigens such as tumor - associated antigens (TAAs) and bind to them with high affinity) with the potent enzymatic activity of a drug (such as a toxin) to induce target cell death. Current ADCs have some therapeutic limitations, and thus there is a need to develop new prototypes with optimized properties.

[0005] Carcinoembryonic antigen (CEA, also known as CEACAM5 or CD66e) is a glycoprotein with a molecular weight of approximately 70 - 100 kDa, depending on the amount of glycosylation present. CEA is a TAA and was originally described as an oncofetal protein in colorectal cancer. CEA is present at low levels in adult tissues of epithelial origin such as the colon, stomach, tongue, cervix, and prostate. CEA is restricted to the apical surface in non-tumor cells but is present throughout the cell membrane in cancer cells. CEA overexpression has been observed in many types of cancer, including colorectal cancer, pancreatic cancer, lung cancer, gastric cancer, hepatocellular carcinoma, breast cancer, and thyroid cancer. For example, CEA is found in the columnar epithelial cells and goblet cells of the colon. In tumors arising from these tissue types, CEA expression increases from the apical membrane to the cell surface and enters the bloodstream once removed from the cell surface. CEA is continuously released from tumor cells and reaches detectable concentrations in peripheral blood, so CEA quantification is often used for cancer diagnosis. Thus, in the prognosis and management of cancer, CEA can be used as a diagnostic tumor marker to determine elevated CEA levels in the blood of cancer patients.

[0006] CEA is also considered a useful tumor-associated antigen for targeted therapies. Retroviral constructs displaying anti-CEA scFv have been generated to deliver the inducible nitric oxide synthase (iNOS) gene to CEA-expressing cancer cells. Anti-CEA antibodies conjugated to radioisotopes have been used to demonstrate radiation specificity for CEA-expressing tumors. The radioisotope approach has been extended to anti-CEA antibody-drug conjugates (ADCs), such as by conjugating anti-CEA antibodies to monomethyl auristatin E (MMAE).

[0007] However, one of the problems encountered with anti-CEA antibodies is cross-reactivity. CEA is highly homologous to other CEACAM family members. For example, human CEA shows 84% homology to CEACAM6, 77% homology to CEACAM8, and 73% identity to CEACAM1. Thus, there is a need for anti-CEA antibodies that are specific for CEA and do not cross-react significantly with human CEACAM1, human CEACAM6, human CEACAM7, or human CEACAM8. In addition, there remains a need for anti-CEA ADCs with high anti-tumor activity as well as potent and non-cross-reactive anti-CEA targeting specificity. SUMMARY OF THE INVENTION

[0008] This disclosure encompasses at least the following embodiments.

[0009] This disclosure relates to antibody-drug conjugates (ADCs) that comprise an antibody or an antigen-binding fragment thereof (Ab) capable of specifically binding to human CEA and a cytotoxic agent (D).

[0010] In certain embodiments, the antibody or antigen-binding fragment thereof (Ab) comprises: (i) Three heavy-chain CDRs: HCDR1 comprising the amino acid sequence shown in SEQ ID NO:24, HCDR2 comprising the amino acid sequence shown in SEQ ID NO:25, HCDR3 comprising the amino acid sequence shown in SEQ ID NO:26, and three light-chain CDRs: LCDR1 comprising the amino acid sequence shown in SEQ ID NO:27, LCDR2 comprising the amino acid sequence shown in SEQ ID NO:28, LCDR3 comprising the amino acid sequence shown in SEQ ID NO:23; or (ii) Three heavy-chain CDRs: HCDR1 comprising the amino acid sequence shown in SEQ ID NO:7, HCDR2 comprising the amino acid sequence shown in SEQ ID NO:8, HCDR3 comprising the amino acid sequence shown in SEQ ID NO:9, and Three light-chain CDRs: LCDR1 comprising the amino acid sequence shown in SEQ ID NO:10, LCDR2 comprising the amino acid sequence shown in SEQ ID NO:11, LCDR3 comprising the amino acid sequence shown in SEQ ID NO:6; or (iii) Three heavy-chain CDRs: HCDR1 comprising the amino acid sequence shown in SEQ ID NO:41, HCDR2 comprising the amino acid sequence shown in SEQ ID NO:42, HCDR3 comprising the amino acid sequence shown in SEQ ID NO:43, and three light-chain CDRs: LCDR1 comprising the amino acid sequence shown in SEQ ID NO:44, LCDR2 comprising the amino acid sequence shown in SEQ ID NO:45, LCDR3 comprising the amino acid sequence shown in SEQ ID NO:40.

[0011] In certain embodiments, the present disclosure relates to an antibody-drug conjugate comprising the formula: Ab-(C-L-(D) m ) n or a pharmaceutically acceptable salt, solvate, or hydrate thereof; wherein Ab is the antibody or an antigen-binding fragment thereof; C is a conjugation moiety; L is a linker; D is a cytotoxic agent; m is an integer from 1 to 8; and n is 1 to 10.

[0012] In certain embodiments, m is 1. It is understood that when m is 1, the antibody-drug conjugate comprises (e.g., has) the formula: Ab-(C-L-D) n .

[0013] In certain embodiments, C is a formula selected from (C-I), (C-Ia), (C-Ib), (C-II), (C-III), (C-IIIa), or (C-IV): and * marks the bond connecting C to Ab. In certain embodiments, C is a formula selected from:

[0014] In certain embodiments, C is (C-Ic): and * marks the bond connecting C to Ab.

[0015] In certain embodiments, C comprises (e.g., has) the formula (C-I), (C-Ia), (C-II), (C-III), (C-IIIa), or (C-IV): wherein * marks the bond connecting the conjugation moiety to Ab.

[0016] In certain embodiments, L comprises (e.g., has) the formula (L-I), (L-II), or (L-III): wherein Su is a hydrophilic residue; and * marks the bond connecting the linker to the conjugation moiety.

[0017] In certain embodiments, Su is

[0018] In certain embodiments, Su is

[0019] In certain embodiments, Su is

[0020] In certain embodiments, Su is

[0021] In certain embodiments, L is where the * marks the bond connecting L to C.

[0022] In certain embodiments, the cytotoxic agent (D) is a topoisomerase inhibitor.

[0023] In certain embodiments, D is: where the values of the variables (e.g., Y, R 3 , R 4 ) are as described herein.

[0024] In certain embodiments, D has the following structural formula: where the values of the variables (e.g., R 7 , R 8 ) are as described herein.

[0025] In certain embodiments, the cytotoxic agent (D) is

[0026] In certain embodiments, the cytotoxic agent (D) is

[0027] In certain embodiments, D is

[0028] In certain embodiments, C-L-(D) m is: where the * marks the bond connecting C to Ab.

[0029] In certain embodiments, C-L-(D) m is: where the * marks the bond connecting C to Ab.

[0030] In certain embodiments, C-L-(D) m is: Wherein the bond connecting C and Ab is marked with an asterisk.

[0031] In certain embodiments, C-L-(D) m is: Wherein the bond connecting C and Ab is marked with an asterisk.

[0032] In certain embodiments, the antibody-drug conjugate is: Or a tautomer, pharmaceutically acceptable salt, solvate or hydrate thereof, wherein Ab is an anti-CEA antibody or an antigen-binding fragment thereof as described herein, and n is as described herein, for example, between 1 and 10, preferably about 7, 8 or 9.

[0033] In certain embodiments, the antibody-drug conjugate has the following formula: Or a tautomer, pharmaceutically acceptable salt, solvate or hydrate thereof, wherein Ab and n are as described herein.

[0034] In certain embodiments, the antibody-drug conjugate has the following formula: Or a tautomer, pharmaceutically acceptable salt, solvate or hydrate thereof, wherein Ab and n are as described herein.

[0035] In certain embodiments, the antibody-drug conjugate has the following formula: Or a tautomer, pharmaceutically acceptable salt, solvate or hydrate thereof, wherein Ab and n are as described herein.

[0036] In certain embodiments, n is from 3 to 10, such as from 4 to 10, from 5 to 10, from 6 to 10 or from 7 to 9. In certain embodiments, n is about 8.

[0037] In certain embodiments, the present disclosure relates to an antibody-drug conjugate comprising an anti-CEA antibody or an antigen-binding fragment, wherein the antibody or antigen-binding fragment comprises: (i) Three heavy-chain CDRs: HCDR1 comprising the amino acid sequence shown in SEQ ID NO:24, HCDR2 comprising the amino acid sequence shown in SEQ ID NO:25, An HCDR3 comprising the amino acid sequence shown in SEQ ID NO:26, and Three light chain CDRs: An LCDR1 comprising the amino acid sequence shown in SEQ ID NO:27, An LCDR2 comprising the amino acid sequence shown in SEQ ID NO:28, An LCDR3 comprising the amino acid sequence shown in SEQ ID NO:23; or (ii) Three heavy chain CDRs: An HCDR1 comprising the amino acid sequence shown in SEQ ID NO:7, An HCDR2 comprising the amino acid sequence shown in SEQ ID NO:8, An HCDR3 comprising the amino acid sequence shown in SEQ ID NO:9, and Three light chain CDRs: An LCDR1 comprising the amino acid sequence shown in SEQ ID NO:10, An LCDR2 comprising the amino acid sequence shown in SEQ ID NO:11, An LCDR3 comprising the amino acid sequence shown in SEQ ID NO:6; or (iii) Three heavy chain CDRs: An HCDR1 comprising the amino acid sequence shown in SEQ ID NO:41, An HCDR2 comprising the amino acid sequence shown in SEQ ID NO:42, An HCDR3 comprising the amino acid sequence shown in SEQ ID NO:43, and Three light chain CDRs: An LCDR1 comprising the amino acid sequence shown in SEQ ID NO:44, An LCDR2 comprising the amino acid sequence shown in SEQ ID NO:45, An LCDR3 comprising the amino acid sequence shown in SEQ ID NO:40; or (iv) A heavy chain variable region comprising SEQ ID NO:31 and a light chain variable region comprising SEQ ID NO:32; (v) A heavy chain variable region comprising SEQ ID NO:48 and a light chain variable region comprising SEQ ID NO:49; or (vi) A heavy chain variable region comprising SEQ ID NO:14 and a light chain variable region comprising SEQ ID NO:15; or (vii) a heavy chain variable region comprising a sequence having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 31 and a light chain variable region comprising a sequence having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 32; or (viii) a heavy chain variable region comprising a sequence having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 48 and a light chain variable region comprising a sequence having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 49; or (ix) a heavy chain variable region comprising a sequence having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 14 and a light chain variable region comprising a sequence having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 15.

[0038] In certain embodiments, the antibody or antigen-binding fragment is a monoclonal antibody, a human engineered antibody, a single-chain antibody (scFv), a Fab fragment, a Fab' fragment or an F(ab')2 fragment.

[0039] In some embodiments, the antibody or antigen-binding fragment comprises an scFv that comprises a VH having the amino acid sequence of SEQ ID NO: 14 and a VL having the amino acid sequence of SEQ ID NO: 15.

[0040] In some embodiments, the antibody or antigen-binding fragment comprises an scFv that comprises a VH having the amino acid sequence of SEQ ID NO: 31 and a VL having the amino acid sequence of SEQ ID NO: 32.

[0041] In some embodiments, the antibody or antigen-binding fragment comprises an scFv that comprises a VH having the amino acid sequence of SEQ ID NO: 48 and a VL having the amino acid sequence of SEQ ID NO: 49.

[0042] In certain embodiments, the antibody or antigen-binding fragment comprises an scFv having the amino acid sequence of SEQ ID NO: 14, SEQ ID NO: 31 or SEQ ID NO: 48.

[0043] In certain embodiments, the antibody or its antigen-binding fragment comprises a heavy chain constant region of the IgG1, IgG2, IgG3 or IgG4 subclass and / or a light chain constant region of the κ or λ type.

[0044] In certain embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region of the IgG1 subclass and a light chain constant region of the κ type.

[0045] In certain embodiments, the present disclosure relates to an antibody-drug conjugate of any of the following formulas: or a tautomer, pharmaceutically acceptable salt, solvate or hydrate thereof; wherein n is from 4 to 10, such as 4, 5, 6, 7, 8, 9 or 10; Ab is an antibody that binds CEA or an antigen-binding fragment thereof; and the antibody or antigen-binding fragment comprises: (i) three heavy chain CDRs: HCDR1 comprising the amino acid sequence shown in SEQ ID NO:24, HCDR2 comprising the amino acid sequence shown in SEQ ID NO:25, HCDR3 comprising the amino acid sequence shown in SEQ ID NO:26, and three light chain CDRs: LCDR1 comprising the amino acid sequence shown in SEQ ID NO:27, LCDR2 comprising the amino acid sequence shown in SEQ ID NO:28, LCDR3 comprising the amino acid sequence shown in SEQ ID NO:23; or (ii) three heavy chain CDRs: HCDR1 comprising the amino acid sequence shown in SEQ ID NO:7, HCDR2 comprising the amino acid sequence shown in SEQ ID NO:8, HCDR3 comprising the amino acid sequence shown in SEQ ID NO:9, and three light chain CDRs: LCDR1 comprising the amino acid sequence shown in SEQ ID NO:10, LCDR2 comprising the amino acid sequence shown in SEQ ID NO:11, LCDR3 comprising the amino acid sequence shown in SEQ ID NO:6; or (iii) three heavy chain CDRs: HCDR1 comprising the amino acid sequence shown in SEQ ID NO:41, HCDR2 comprising the amino acid sequence shown in SEQ ID NO:42, HCDR3 comprising the amino acid sequence shown in SEQ ID NO:43, and Three light chain CDRs: LCDR1 comprising the amino acid sequence shown in SEQ ID NO:44, LCDR2 comprising the amino acid sequence shown in SEQ ID NO:45, LCDR3 comprising the amino acid sequence shown in SEQ ID NO:40; or (iv) a heavy chain variable region comprising SEQ ID NO:31 and a light chain variable region comprising SEQ ID NO:32; (v) a heavy chain variable region comprising SEQ ID NO:48 and a light chain variable region comprising SEQ ID NO:49; or (vi) a heavy chain variable region comprising SEQ ID NO:14 and a light chain variable region comprising SEQ ID NO:15; or (vii) a heavy chain variable region comprising a sequence having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:31 and a light chain variable region comprising a sequence having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:32; or (viii) a heavy chain variable region comprising a sequence having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:48 and a light chain variable region comprising a sequence having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:49; or (ix) a heavy chain variable region comprising a sequence having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:14 and a light chain variable region comprising a sequence having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:15.

[0046] In certain embodiments, n is about 8.

[0047] In certain embodiments, the present disclosure relates to a pharmaceutical composition comprising an antibody-drug conjugate as described above and herein and a pharmaceutically acceptable carrier.

[0048] In certain embodiments, the present disclosure relates to a method of treating a subject (e.g., a patient) having a CEA-related disease or disorder (e.g., a cell that expresses or accumulates CEA), the method comprising administering to a subject (e.g., a patient) in need thereof an effective amount of an antibody-drug conjugate described herein or a pharmaceutical composition comprising the antibody-drug conjugate. In some embodiments, the cell that expresses or accumulates CEA is a cancer cell.

[0049] In certain embodiments, the present disclosure relates to an anti-CEA antibody conjugated to a compound having the following formula: C-L-D or a pharmaceutically acceptable salt, solvate, or hydrate thereof; wherein C is a conjugation moiety; L is a linker; and D is a cytotoxic agent.

[0050] In certain embodiments, C comprises the following formula (C-I'), (C-II'), (C-III'), or (C-IV'):

[0051] In certain embodiments, L comprises the following formula (L-I), (L-II), or (L-III): wherein Su is a hydrophilic residue; and * denotes the bond by which the linker is attached to the conjugation moiety.

[0052] In certain embodiments, Su is

[0053] In certain embodiments, Su is

[0054] In certain embodiments, the cytotoxic agent (D) is

[0055] In certain embodiments, the cytotoxic agent (D) is

[0056] In certain embodiments, the compound is or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[0057] In certain embodiments, the present disclosure relates to a method of producing an anti-CEA antibody-drug conjugate as described above, the method comprising: (i) Culturing a host cell transformed with an isolated nucleic acid comprising a sequence encoding an anti-CEA antibody or an antigen-binding fragment thereof, wherein the antibody or fragment thereof comprises a) a heavy chain comprising the amino acid sequence of SEQ ID NO:99 and a light chain comprising the amino acid sequence of SEQ ID NO:100, or sequences having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to a heavy chain comprising the amino acid sequence of SEQ ID NO:99 and a light chain comprising the amino acid sequence of SEQ ID NO:100, wherein certain CDRs of the heavy and light chains shown in bold / underlined in Table 20 are retained; or b) three heavy chain CDRs: HCDR1 comprising the amino acid sequence as shown in SEQ ID NO:24, HCDR2 comprising the amino acid sequence as shown in SEQ ID NO:25, HCDR3 comprising the amino acid sequence as shown in SEQ ID NO:26, and three light chain CDRs: LCDR1 comprising the amino acid sequence as shown in SEQ ID NO:27, LCDR2 comprising the amino acid sequence as shown in SEQ ID NO:28, LCDR3 comprising the amino acid sequence as shown in SEQ ID NO:23; or c) three heavy chain CDRs: HCDR1 comprising the amino acid sequence as shown in SEQ ID NO:7, HCDR2 comprising the amino acid sequence as shown in SEQ ID NO:8, HCDR3 comprising the amino acid sequence as shown in SEQ ID NO:9, and three light chain CDRs: LCDR1 comprising the amino acid sequence as shown in SEQ ID NO:10, LCDR2 comprising the amino acid sequence as shown in SEQ ID NO:11, LCDR3 comprising the amino acid sequence as shown in SEQ ID NO:6; or d) three heavy chain CDRs: HCDR1 comprising the amino acid sequence as shown in SEQ ID NO:41, HCDR2 comprising the amino acid sequence as shown in SEQ ID NO:42, An HCDR3 comprising the amino acid sequence shown in SEQ ID NO:43, and Three light chain CDRs: An LCDR1 comprising the amino acid sequence shown in SEQ ID NO:44, An LCDR2 comprising the amino acid sequence shown in SEQ ID NO:45, An LCDR3 comprising the amino acid sequence shown in SEQ ID NO:40; or e) A heavy chain variable region comprising SEQ ID NO:31 and a light chain variable region comprising SEQ ID NO:32; f) A heavy chain variable region comprising SEQ ID NO:48 and a light chain variable region comprising SEQ ID NO:49; or g) A heavy chain variable region comprising SEQ ID NO:14 and a light chain variable region comprising SEQ ID NO:15; or h) A heavy chain variable region comprising a sequence having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:31 and a light chain variable region comprising a sequence having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:32; or i) A heavy chain variable region comprising a sequence having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:48 and a light chain variable region comprising a sequence having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:49; or j) A heavy chain variable region comprising a sequence having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:14 and a light chain variable region comprising a sequence having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:15; and (ii) Expressing the antibody or its antigen-binding fragment; (iii) Recovering the expressed antibody or its antigen-binding fragment; and (iv) Optionally conjugating or linking at least one compound to the antibody or its fragment using a linker such that an antibody-drug conjugate is formed.

[0058] In certain embodiments, provided is the use of any one of the antibody-drug conjugates described herein (e.g., in the form of a pharmaceutical composition) for the treatment described herein (e.g., treating a subject having cells that express and / or accumulate CEA).

[0059] In certain embodiments, provided are the antibody-drug conjugates described herein (e.g., in the form of a pharmaceutical composition) for use as described herein (e.g., for treating a subject having cells that express and / or accumulate CEA).

[0060] In certain embodiments, provided is the use of any one of the antibody-drug conjugates described herein (e.g., in the form of a pharmaceutical composition) in the manufacture of a medicament for treatment as described herein (e.g., treating a subject having cells that express and / or accumulate CEA).

[0061] In certain embodiments, provided is a kit that comprises any one or more of the antibody-drug conjugates disclosed herein (e.g., in the form of a composition) and instructions for use thereof. In some embodiments, the kit further comprises instructions for a detection assay, wherein the antibody-drug conjugate forms a complex with CEA, and the complex is detected by an assay comprising an enzyme-linked immunosorbent assay (ELISA), a radioimmunoassay (RIA), and / or a Western blot.

[0062] In certain embodiments, the present disclosure relates to a kit that comprises an anti-CEA antibody-drug conjugate and instructions for use thereof, the antibody-drug conjugate comprising an antibody or an antigen-binding fragment thereof, the antibody or the antigen-binding fragment thereof comprising: (i) three heavy-chain CDRs: HCDR1 comprising the amino acid sequence as set forth in SEQ ID NO:24, HCDR2 comprising the amino acid sequence as set forth in SEQ ID NO:25, HCDR3 comprising the amino acid sequence as set forth in SEQ ID NO:26, and three light-chain CDRs: LCDR1 comprising the amino acid sequence as set forth in SEQ ID NO:27, LCDR2 comprising the amino acid sequence as set forth in SEQ ID NO:28, LCDR3 comprising the amino acid sequence as set forth in SEQ ID NO:23; or (ii) three heavy-chain CDRs: HCDR1 comprising the amino acid sequence as set forth in SEQ ID NO:7, HCDR2 comprising the amino acid sequence as set forth in SEQ ID NO:8, HCDR3 comprising the amino acid sequence as set forth in SEQ ID NO:9, and Three light chain CDRs: LCDR1 comprising the amino acid sequence shown in SEQ ID NO: 10, LCDR2 comprising the amino acid sequence shown in SEQ ID NO: 11, LCDR3 comprising the amino acid sequence shown in SEQ ID NO: 6; or (iii) Three heavy chain CDRs: HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 41, HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 42, HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 43, and Three light chain CDRs: LCDR1 comprising the amino acid sequence shown in SEQ ID NO: 44, LCDR2 comprising the amino acid sequence shown in SEQ ID NO: 45, LCDR3 comprising the amino acid sequence shown in SEQ ID NO: 40; or (iv) A heavy chain variable region comprising SEQ ID NO: 31 and a light chain variable region comprising SEQ ID NO: 32; (v) A heavy chain variable region comprising SEQ ID NO: 48 and a light chain variable region comprising SEQ ID NO: 49; or (vi) A heavy chain variable region comprising SEQ ID NO: 14 and a light chain variable region comprising SEQ ID NO: 15; or (vii) A heavy chain variable region comprising a sequence having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 31 and a light chain variable region comprising a sequence having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 32; or (viii) A heavy chain variable region comprising a sequence having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 48 and a light chain variable region comprising a sequence having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 49; or (ix) A heavy chain variable region comprising a sequence having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:14 and a light chain variable region comprising a sequence having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:15.

[0063] In certain embodiments, the present disclosure relates to a kit comprising an anti-CEA antibody-drug conjugate and instructions for use thereof, wherein the antibody-drug conjugate comprises an antibody, the antibody comprising: a. A VH sequence comprising the sequence shown in SEQ ID NO:31, or a VH sequence comprising a sequence having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:31, and a VL sequence comprising the sequence shown in SEQ ID NO:32, or a VL sequence comprising a sequence having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:32; b. A VH sequence comprising the sequence shown in SEQ ID NO:48, or a VH sequence comprising a sequence having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:48, and a VL sequence comprising the sequence shown in SEQ ID NO:49, or a VL sequence comprising a sequence having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:49; or c. A VH sequence comprising the sequence shown in SEQ ID NO:14, or a VH sequence comprising a sequence having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:14, and a VL sequence comprising the sequence shown in SEQ ID NO:15, or a VL sequence comprising a sequence having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:15.

[0064] The present disclosure is not intended to and should not be construed as representing the full extent and scope of the present disclosure. In addition, references herein to "the present disclosure" or aspects thereof should be understood to refer to certain embodiments of the present disclosure and should not be construed as limiting all embodiments to a particular description. The present disclosure is set forth in various levels of detail in the present disclosure, the detailed description, and the drawings, and the inclusion or exclusion of elements, components, etc. in the present disclosure is not intended to limit the scope of the present disclosure. Features from any of the disclosed embodiments can be used in combination with each other without limitation. Additionally, other features and advantages of the present disclosure will become apparent to those of ordinary skill in the art by considering the following detailed description and the drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0065] Figure 1 A schematic diagram showing shedding CEA (sCEA), chimeric CEA (CHIM), CEACAM6, and CEA variants (CEA-v) is shown. In CEA, domains N, A1, B1, A2, B2, A3, B3, and the GPI linker (GPI) are labeled; in CEACAM6, domains N', A', and B' are labeled.

[0066] Figure 2A -B depicts the phylogenetic tree of the VH ( Figure 2A ) and VL ( Figure 2B ) regions of the anti-CEA domain B3 antibody. The VH and VL sequences of the candidate anti-CEA antibodies were aligned using the Megalign TM software of DNASTAR. Sequence homology is shown in the phylogenetic tree.

[0067] Figure 3A An affinity assay of the purified murine anti-CEA antibody BGA7592 for the chimeric construct (CHIM) by surface plasmon resonance (SPR) is shown. Different lines represent the binding ability of BGA7592 to CHIM at different concentrations, where the top line shows the binding ability of BGA7592 to CHIM at the highest concentration, and serial dilutions of CHIM form the other lines. The ascending curve of each line shows the association rate, and the descending curve shows the dissociation rate.

[0068] Figure 3B The binding profile of BGA7592 obtained by antigen ELISA is depicted.

[0069] Figure 4A -B shows the effect of soluble CEA (sCEA) on the binding of CEA antibodies to patient-derived MKN45 gastric adenocarcinoma cells. Figure 4A The binding profiles of anti-domain B3 antibodies in the presence or absence of soluble CEA (sCEA) are shown; Figure 4Bis shown in the form of a histogram Figure 4A antibody binding profiles.

[0070] Figure 5A -B shows the randomized sites of an antibody library that generates affinity matured antibody light chain CDR (LCDR) regions ( Figure 5A )(SEQ ID NO:82, 83, 84 respectively) and heavy chain CDR (HCDR) regions ( Figure 5B )(SEQ ID NO:80, 81 and 3 respectively) for the humanized BGA7592 antibody.

[0071] Figure 6 shows the amino acid changes in the BGA7592 light chain CDR region after four rounds of selection.

[0072] Figure 7 shows the binding of the affinity matured humanized BGA7592 variant to LOVO cells obtained by flow cytometry.

[0073] Figure 8 shows the binding of an anti-CEA antibody to MKN45 cells as measured by flow cytometry.

[0074] Figure 9A and 9B are bar graphs showing the off-target binding of antibody BGA5384 to various CEACAM family members obtained by antigen ELISA ( Figure 9A ) and flow cytometry ( Figure 9B ).

[0075] Figure 10 shows the effect of soluble CEA on the binding of BGA5384 to MKN45 cells expressing CEA in the presence of various concentrations of soluble CEA.

[0076] Figure 11 is a graph showing the antibody-dependent cell cytotoxicity (ADCC) of antibody BGA6710 in vitro.

[0077] Figure 12 shows the effect of the BGA6710 antibody on tumor volume in a murine cancer model.

[0078] Figure 13 shows the killing curves of CEA antibodies conjugated to the maytansinoid compound DM4 against cells with different CEA expression levels.

[0079] Figure 14 shows the killing curves of CEA antibodies conjugated to auristatin MMAE against cells with different CEA expression levels.

[0080] Figure 15Shows the killing curves of CEA antibody conjugated with topoisomerase inhibitor DXD against cells with different CEA expression levels.

[0081] Figure 16 Shows the killing curves of various free cytotoxic agents against MKN45 cells (CEA high) with a CEA-specific antibody binding capacity (SABC) of ~200K.

[0082] Figure 17 Shows the cytotoxic effects of eight ADCs against MKN45 cells (CEA high).

[0083] Figure 18 Shows the cytotoxic effects of eight ADCs against patient-derived cells (lung adenocarcinoma) (CEA medium) from H2122.

[0084] Figure 19 Shows the cytotoxic effects of eight ADCs against patient-derived cells (colorectal adenocarcinoma) (CEA low) from LS174T.

[0085] Figure 20 Shows the cytotoxic effects of eight ADCs against patient-derived cells (breast adenocarcinoma) (CEA negative) from MB-231.

[0086] Figure 21 Shows the anti-tumor efficacy of different concentrations of BGA-7650 and BGA-9962 relative to the control in a cell line-derived xenograft (CDX) model using MKN-45 cells (CEA high).

[0087] Figure 22 Shows the anti-tumor efficacy of different concentrations of BGA-7650 and BGA-9962 relative to the control in a CDX model using SW-1463 cells (CEA medium) (rectal adenocarcinoma).

[0088] Figure 23 Shows the anti-tumor efficacy of different concentrations of BGA-7650 and BGA-9962 relative to the control in a CDX model using H2122 cells (CEA low) (lung adenocarcinoma).

[0089] Figure 24 Shows the anti-tumor efficacy of different concentrations of BGA-9962 and BGA-7650 relative to the control in a patient-derived xenograft (PDX) model of gastric cancer (GC).

[0090] Figure 25 Is a graph of the concentrations of various antibodies, ADCs, and free cytotoxic agents over time in Balb / c nude mice (non-tumor-bearing).

[0091] Figure 26Figure showing the in vivo DAR of two ADCs over time in mice (single dose, iv 3mpk Balb / c nude mice, n = 3 per group). List of Abbreviations Definitions

[0092] Unless specifically defined hereinbelow or elsewhere in this document, all technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art.

[0093] As used herein (including in the appended claims), singular forms of words such as "a", "an", and "the" include their respective plural forms unless the context clearly dictates otherwise.

[0094] Unless specifically stated or apparent from the context, as used herein, the term "about" refers to a value or a component within an acceptable error range of a particular value or component as determined by one of ordinary skill in the art, which will depend in part on how the value or component is measured or determined, i.e., the limitations of the measuring system. For example, "about" can mean within one or more standard deviations in the practice of the art. "About" can mean a range of up to 10% (i.e., ±10%). Thus, "about" can be understood to be within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, 0.01%, or 0.001% greater or less than the stated value. For example, about 5 mg can include any amount between 4.5 mg and 5.5 mg. Additionally, especially for biological systems or processes, these terms can mean up to one order of magnitude or up to 5-fold of a value. When a particular value or component is provided in this disclosure, unless otherwise stated, the meaning of "about" should be assumed to be within the acceptable error range of that particular value or component.

[0095] The term "or" is used to mean "and / or" and can be used interchangeably with the term "and / or" unless the context clearly dictates otherwise.

[0096] The term "carcinoembryonic antigen" or "CEA" refers to a glycoprotein of approximately 70 - 100 kDa, also known as CEACAM5 or CD66e. The amino acid sequence of human CEA, SEQ ID NO:52, can also be found under accession number P06731 or NM_004363.2.

[0097] As used herein, the term "administer" when applied to an animal, human, subject, cell, tissue, organ, or biological fluid means the contacting of an exogenous drug, therapeutic agent, diagnostic agent, or composition with the animal, human, subject, cell, tissue, organ, or biological fluid. The treatment of cells encompasses the contacting of a reagent with the cells and the contacting of a reagent with a fluid, wherein the fluid contacts the cells.

[0098] As used herein, the terms "subject" or "patient" include any living organism, preferably an animal, more preferably a mammal (e.g., rat, mouse, dog, cat, rabbit, primate), and most preferably a human (e.g., a patient suffering from or at risk of suffering from a disorder described herein).

[0099] In one aspect, "treating" any disease or disorder means ameliorating the disease or disorder (i.e., slowing or arresting or reducing the development of the disease or at least one of its clinical symptoms). In another aspect, "treating" means alleviating or ameliorating at least one physical parameter, including those parameters that may not be discernible to the patient. In yet another aspect, "treating" means modulating the disease or disorder physically (e.g., stabilization of discernible symptoms), physiologically (e.g., stabilization of physical parameters), or both.

[0100] As used herein, the term "affinity" refers to the strength of the interaction between an antibody and an antigen. Within an antigen, the variable regions of the antibody interact with the antigen at multiple sites through non-covalent forces. Generally, the more interactions, the stronger the affinity.

[0101] As used herein, the term "antibody" refers to a polypeptide of the immunoglobulin family that can bind a corresponding antigen non-covalently, reversibly, and in a specific manner. For example, a naturally occurring IgG antibody is a tetramer comprising at least two heavy (H) chains and two light (L) chains that are interconnected by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region consists of three domains, CH1, CH2, and CH3. Each light chain consists of a light chain variable region (abbreviated herein as VL or Vκ) and a light chain constant region. The light chain constant region consists of one domain, CL. The VH and VL regions can be further subdivided into hypervariable regions, called complementarity determining regions (CDRs), which are interspersed with more conserved regions, called framework regions (FRs). Each VH and VL 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, and FR4. The variable regions of the heavy and light chains contain binding domains that interact with the antigen. The constant region of the antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (Clq).

[0102] The positions of the CDRs and framework regions can be determined using a variety of definitions well-known in the art, such as Kabat, Chothia, AbM, and IMGT (see, e.g., Johnson et al., Nucleic Acids Res., 29:205-206 (2001); Chothia and Lesk, J. Mol. Biol., 196:901-917 (1987); Chothia et al., Nature, 342:877-883 (1989); Chothia et al., J. Mol. Biol., 227:799-817 (1992); Al-Lazikani et al., J. Mol. Biol., 273:927-748 (1997); Lefranc, M.-P., The Immunologist, 7, 132-136 (1999); Lefranc, M.-P. et al., Dev. Comp. Immunol., 27, 55-77 (2003)).

[0103] The term "antibody" includes, but is not limited to, monoclonal antibodies, human antibodies, humanized antibodies, chimeric antibodies, and anti-idiotypic (anti-Id) antibodies. The antibody can be of any isotype / class (e.g., IgG, IgE, IgM, IgD, IgA, and IgY) or subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2).

[0104] In some embodiments, the anti-CEA antibody comprises at least one antigen-binding site. In some embodiments, the anti-CEA antibody comprises an antigen-binding fragment from a CEA antibody described herein. In some embodiments, the anti-CEA antibody is isolated or recombinant.

[0105] The term "monoclonal antibody" or "mAb" or "Mab" as used herein means a population of antibodies that are substantially homogeneous, i.e., the antibody molecules in the population are identical in amino acid sequence except for possible naturally occurring mutations that may be present in minor amounts. In contrast, conventional (polyclonal) antibody preparations typically include a variety of different antibodies that have different amino acid sequences in their variable domains, particularly their CDRs, and which typically are specific for different epitopes. The modifier "monoclonal" indicates the character of the antibody obtained from a substantially homogeneous population of antibodies and should not be construed as requiring that the antibody be made by any particular method. Monoclonal antibodies can be obtained by methods known to those of skill in the art. See, e.g., Kohler et al., Nature 1975 256:495-497; U.S. Patent No. 4,376,110; Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY 1992; Harlow et al., ANTIBODIES: a LABORATORY MANUAL, Cold Spring Harbor Laboratory 1988; and Colligan et al., CURRENT PROTOCOLS IN IMMUNOLOGY 1993. The antibodies disclosed herein can be of any immunoglobulin class (including IgG, IgM, IgD, IgE, IgA), and any subclass thereof (e.g., IgG1, IgG2, IgG3, IgG4). Hybridomas that produce monoclonal antibodies can be cultured in vitro or in vivo. High titers of monoclonal antibodies can be obtained in in vivo production, wherein cells from a single hybridoma are injected intraperitoneally into a mouse, e.g., a naïve Balb / c mouse, to produce ascites fluid containing a high concentration of the desired antibody. Monoclonal antibodies of isotype IgM or IgG can be purified from such ascites fluid, or from culture supernatant, using column chromatography methods well known to those of skill in the art.

[0106] Unless otherwise indicated, "antigen-binding fragment" means an antigen-binding fragment of an antibody, i.e., an antibody fragment that retains the ability to specifically bind an antigen to which the full-length antibody binds, e.g., a fragment that retains one or more CDR regions. Examples of antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules, such as single-chain Fv (ScFv); nanobodies and antibodies formed from antibody fragments; and bicyclic peptides (Hurov, K. et al., 2021. Journal for ImmunoTherapy of Cancer, 9(11)).

[0107] As used herein, an antibody or antigen-binding antibody fragment "specifically binds" or "selectively binds" to an antigen (e.g., a protein) to mean that, compared to other proteins, the antibody exhibits preferential binding to the target, but such specificity does not require absolute binding specificity. A "specific" or "selective" binding reaction determines the presence of an antigen in a heterogeneous population of proteins and other biological products (e.g., in blood, serum, plasma, or tissue samples). Thus, under certain specified immunoassay conditions, an antibody or its antigen-binding fragment specifically binds to a particular antigen at least twice the background level and does not specifically bind to other antigens present in the sample in significant amounts. In one aspect, under specified immunoassay conditions, an antibody or its antigen-binding fragment specifically binds to a particular antigen at a value of at least ten-fold compared to the background binding level and does not specifically bind to other antigens present in the sample in significant amounts.

[0108] The term "human antibody" as used herein means an antibody that contains only human immunoglobulin protein sequences. If produced in a mouse, mouse cell, or hybridoma derived from a mouse cell, a human antibody may contain murine carbohydrate chains. Similarly, a "mouse antibody" or "rat antibody" means an antibody that contains only mouse or rat immunoglobulin protein sequences, respectively.

[0109] The term "humanized" or "humanized antibody" means an antibody form that contains sequences from non-human (e.g., murine) antibodies as well as human antibodies. Such antibodies contain a minimal sequence derived from a non-human immunoglobulin. Generally speaking, a humanized antibody will contain substantially all of at least one and usually two variable domains, wherein all or substantially all of the hypervariable loops correspond to those of the non-human immunoglobulin, and all or substantially all of the FRs are those of human immunoglobulin sequences. A humanized antibody will also optionally contain at least a portion of the immunoglobulin constant region (Fc), typically at least a portion of a human immunoglobulin. When it is necessary to distinguish a humanized antibody from the parental rodent antibody, the prefix "hum", "hu", "Hu", or "h" is added to the antibody clone name. The humanized form of a rodent antibody will generally contain the same CDR sequences as the parental rodent antibody, but may include certain amino acid substitutions to increase affinity, increase the stability of the humanized antibody, remove post-translational modifications, or for other reasons.

[0110] The term "equilibrium dissociation constant" or "K D " or "M" refers to the dissociation rate constant (kd, time -1 ) divided by the association rate constant (ka, time -1 , M -l ). The equilibrium dissociation constant can be measured using any known method in the art. The antibodies of the present disclosure will generally have a value of less than about 10 -7 or 10 -8M (e.g., less than about 10 -9 M or 10 -10 M, and in some aspects less than about 10 -11 M, 10 -12 M or 10 -13 M) for the equilibrium dissociation constant.

[0111] As used herein, the terms "cancer" or "tumor" are used in their broadest sense as understood in the art and refer to a physiological disorder in a mammal that is generally characterized by unregulated cell growth. In the context of the present disclosure, cancer or tumor is not limited to a particular type or location.

[0112] In the context of the present disclosure, when referring to an amino acid sequence, the term "conservative substitution" means that the original amino acid is replaced by a new amino acid that substantially does not change the chemical, physical, and / or functional properties of the antibody or fragment, such as its binding affinity for CEA. Common conservative substitutions of amino acids are well known in the art.

[0113] The terms "improve", "increase", "inhibit", and "decrease" refer to values relative to a baseline or other reference measurement. In some embodiments, an appropriate reference measurement may include a measurement in a particular system (e.g., in a single individual) in the absence of an agent or treatment (e.g., before and / or after) or in the presence of an appropriate equivalent reference agent under otherwise equivalent conditions. In some embodiments, an appropriate reference measurement may include a measurement in an equivalent system known or expected to respond in an equivalent manner in the presence of the relevant agent or treatment.

[0114] As used herein, the term "knob-into-hole" technology refers to amino acids that direct the pairing of two polypeptides together in vitro or in vivo by introducing a spatial protrusion (knob) into one polypeptide and a pocket or cavity (hole) into the other polypeptide at the interface where the two polypeptides interact. For example, it has been used at the Fc:Fc binding interface of an antibody, the C L :C H I interface or the V H / V LAn interface is introduced for a chaperone (see, for example, US2011 / 0287009, US2007 / 0178552, WO 96 / 027011, WO 98 / 050431, and Zhu et al., 1997, Protein Science 6:781-788). In some embodiments, the chaperone ensures correct pairing of two different heavy chains during antibody production. For example, an antibody having chaperone amino acids in its Fc region may also contain a single variable domain linked to each Fc region, or may further contain different heavy chain variable domains paired with similar or different light chain variable domains. Chaperone technology can also be used in the VH or VL regions to ensure correct pairing. Examples of algorithms suitable for determining percent sequence identity and sequence similarity are the BLAST algorithms, which are described in Altschul et al., Nuc. Acids Res. 25:3389-3402, 1977; and Altschul et al., J. Mol. Biol. 215:403-410, 1990. Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information. This algorithm involves first identifying high-scoring sequence pairs (HSPs) by identifying short word lengths W in the query sequence that, when aligned with the same word length in a database sequence, match or satisfy some positive-valued threshold score T. T is referred to as the neighborhood word score threshold. These initial neighborhood word hits serve as seeds for a search to find longer HSPs that contain them. The word hits are extended in both directions along each sequence until the cumulative alignment score can no longer increase. For nucleotide sequences, parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for a mismatched residue; always <0) are used to calculate the cumulative score. For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction is stopped when the cumulative alignment score drops by an amount X from its maximum achieved value; due to the accumulation of one or more negative-scoring residue alignments, the cumulative score tends to zero or lower; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) defaults to a word length (W) of 11, an expectation value (E) of 10, M = 5, N = -4, and compares both strands. For amino acid sequences, the BLAST program defaults to a word length of 3, an expectation value (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff, (1989) Proc. Natl. Acad. Sci. USA 89:10915) for an alignment (B) of 50, M = 5, N = -4, and compares both strands.

[0115] The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90:5873-5787, 1993). One measure of similarity provided by the BLAST algorithm is the minimum sum probability (P(N)), which provides an indication of the probability that a match occurs by chance between two nucleotide or amino acid sequences. For example, if the minimum sum probability in a comparison of a test nucleic acid with a reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001, then the nucleic acid is considered to be similar to the reference sequence.

[0116] The percent identity between two amino acid sequences can also be determined using the following algorithm: E. Meyers and W. Miller, Comput. Appl. Biosci. 4:11-17, (1988), which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. In addition, the percent identity between two amino acid sequences can be determined using the algorithm of Needleman and Wunsch, J. Mol. Biol. 48:444-453 (1970), which has been incorporated into the GAP program in the GCG software package, using a BLOSUM62 matrix or a PAM250 matrix, a gap weight of 16, 14, 12, 10, 8, 6, or 4, and a length weight of 1, 2, 3, 4, 5, or 6.

[0117] The term "nucleic acid" may be used interchangeably herein with the term "polynucleotide" and refers to deoxyribonucleotides or ribonucleotides and polymers thereof in single-stranded or double-stranded form. The term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, or non-naturally occurring, have similar binding properties as the reference nucleic acid, and are metabolized in a manner similar to the reference nucleotides. Examples of such analogs include, but are not limited to, phosphorothioates, phosphoramidates, methylphosphonates, chiral methylphosphonates, 2'-O-methyl ribonucleotides, and peptide-nucleic acids (PNA).

[0118] The term "operably linked" in the context of nucleic acids refers to the functional relationship between two or more polynucleotide (e.g., DNA) segments. Typically, it refers to the functional relationship between a transcriptional regulatory sequence and a transcriptional sequence. For example, a promoter or enhancer sequence is operably linked to a coding sequence if it stimulates or regulates the transcription of the coding sequence in a suitable host cell or other expression system. Generally, a promoter transcriptional regulatory sequence that is operably linked to a transcriptional sequence is physically contiguous with the transcriptional sequence, i.e., they are cis-acting. However, some transcriptional regulatory sequences (such as enhancers) do not need to be physically contiguous or adjacent to the coding sequence whose transcription they enhance.

[0119] In some aspects, the present disclosure provides compositions, such as pharmaceutically acceptable compositions, that comprise an anti-CEA antibody as described herein formulated with at least one pharmaceutically acceptable excipient. As used herein, the term "pharmaceutically acceptable excipient" includes any and all solvents, dispersion media, isotonic agents, absorption delaying agents, and the like that are physiologically compatible. The excipient can be suitable for intravenous, intramuscular, subcutaneous, parenteral, rectal, spinal, or epidermal administration (e.g., by injection or infusion).

[0120] The term "therapeutically effective amount" or "effective amount" as used herein refers to the amount of an agent that is sufficient to effect such treatment of a disease, disorder, or at least one clinical symptom of a disease or disorder when administered to a subject to treat the disease, disorder, or symptom. A "therapeutically effective amount" can vary with the agent, the disease, disorder, and / or symptom of the disease or disorder, the severity of the disease, disorder, and / or symptom of the disease or disorder, the age of the subject to be treated, and / or the weight of the subject to be treated. The appropriate amount in any given case will be apparent to those skilled in the art or can be determined by routine experimentation. In the case of combination therapy, a "therapeutically effective amount" refers to the total amount of the combination components.

[0121] The term "combination therapy" refers to the administration of two or more therapeutic agents to treat a therapeutic condition or disorder. Such administration encompasses co-administering these therapeutic agents in a substantially simultaneous manner. Such administration also encompasses co-administering in multiple containers or formulations (e.g., capsules, powders, and liquids) or in separate containers or formulations for each active ingredient. The powder and / or liquid can be reconstituted or diluted to the desired dose prior to administration. Additionally, "combination therapy" encompasses using each type of therapeutic agent in a sequential manner at approximately the same time or at different times. In either case, the treatment regimen will provide the beneficial effects of the drug combination in treating the conditions or disorders described herein.

[0122] As used herein, the phrase "in combination with" means that the anti-CEA ADC is administered to a subject either concomitantly with, immediately before, or immediately after the administration of an additional therapeutic agent. In certain embodiments, the anti-CEA ADC is administered as a co-formulation with the additional therapeutic agent.

[0123] The term "toxin" or "payload" or "cytotoxic agent" is used herein to refer to a molecule that inhibits or reduces the expression of a molecule in a cell, inhibits or reduces the function of a cell, induces apoptosis and / or causes cell death. The term includes radioisotopes, chemotherapeutic agents, and toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant, or animal origin, including fragments and / or variants thereof. Examples of cytotoxic agents include, but are not limited to, auristatins (e.g., auristatin E, auristatin F, MMAE, and MMAF), auromycins, maytansinoids, pyrrolobenzodiazepines (PBDs), ricin, ricin A-chain, combrestatin, duocarmycins, dolastatins, doxorubicin, daunomycin, paclitaxel, cisplatin, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, mitoxantrone, actinomycin, diphtheria toxin, Pseudomonas exotoxin (PE) A, PE40, abrin, abrin A-chain, modeccin A-chain, alpha-sarcin, gelonin, mitogellin, restrictocin, phenomycin, enomycin, curicin, crotin, and calicheamicin, and radioisotopes such as At211, I131, I125, Y90, Re186, Re188, Sm153, Bi212 or 213, P32, and Lu177.

[0124] "Alkyl" groups are saturated straight-chain or branched acyclic hydrocarbons having from 1 to 10 carbon atoms, typically from 1 to 8 carbon atoms or in some embodiments from 1 to 6, 1 to 4, or 2 to 6 carbon atoms. Representative alkyl groups include -methyl, -ethyl, -n-propyl, -n-butyl, -n-pentyl, and -n-hexyl; while saturated branched alkyls include -isopropyl, -sec-butyl, -isobutyl, -tert-butyl, -isopentyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, etc. Alkyl groups can be substituted or unsubstituted. In certain embodiments, when the alkyl groups described herein are said to be "substituted", they can be substituted by any one or more of the substituents found in the exemplary compounds and embodiments disclosed herein and the following: halogen (chlorine, iodine, bromine, or fluorine); hydroxy; alkoxy; alkoxyalkyl; amino; alkylamino; carboxy; nitro; cyano; thiol; thioether; imine; imide; amidine; guanidine; enamine; aminocarbonyl; acylamino; phosphonic acid group; phosphine; thiocarbonyl; sulfonyl; sulfone; sulfonamide; ketone; aldehyde; ester; urea; carbamate; oxime; hydroxylamine; alkoxyamine; aralkyloxyamine; N-oxide; hydrazine; acylhydrazine; hydrazone; azide; isocyanate; isothiocyanate; cyanate; thiocyanate; B(OH)2; or O(alkyl)aminocarbonyl.

[0125] "Alkenyl" groups are straight-chain or branched acyclic hydrocarbons having from 2 to 10 carbon atoms, typically from 2 to 8 carbon atoms and including at least one carbon-carbon double bond. Representative straight-chain and branched (C2-C8) alkenyls include -vinyl, -allyl, -1-butenyl, -2-butenyl, -isobutenyl, -1-pentenyl, -2-pentenyl, -3-methyl-1-butenyl, -2-methyl-2-butenyl, -2,3-dimethyl-2-butenyl, -1-hexenyl, 2-hexenyl, -3-hexenyl, -1-heptenyl, -2-heptenyl, -3-heptenyl, -1-octenyl, -2-octenyl, 3-octenyl, etc. The double bond of the alkenyl group can be non-conjugated or conjugated with another unsaturated group. Alkenyl groups can be unsubstituted or substituted.

[0126] The "cycloalkyl" group is a saturated or partially saturated cyclic alkyl group of 3 to 10 carbon atoms having a single cyclic ring or multiple fused or bridged rings which may optionally be substituted by 1 to 3 alkyl groups. In some embodiments, the cycloalkyl group has 3 to 8 ring members, and in other embodiments the number of ring carbon atoms ranges from 3 to 5, 3 to 6, or 3 to 7. By way of example, such cycloalkyl groups include monocyclic structures (such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, 1-methylcyclopropyl, 2-methylcyclopentyl, 2-methylcyclooctyl, etc.) or polycyclic or bridged ring structures (such as adamantyl, etc.). Examples of unsaturated cycloalkyl groups include cyclohexenyl, cyclopentenyl, cyclohexadienyl, butadienyl, pentadienyl, hexadienyl, etc. The cycloalkyl group may be substituted or unsubstituted. By way of example, such substituted cycloalkyl groups include cyclohexanone, etc.

[0127] The "aryl" group is an aromatic carbocyclic group of 6 to 14 carbon atoms having a single ring (e.g., phenyl) or multiple fused rings (e.g., naphthyl or anthracenyl). In some embodiments, the aryl group contains 6 - 14 carbon atoms, and in other embodiments contains 6 to 12 or even 6 to 10 carbon atoms in the ring portion of the group. Specific aryls include phenyl, biphenyl, naphthyl, etc. The aryl group may be substituted or unsubstituted. The phrase "aryl group" also includes groups containing fused rings, such as fused aromatic - aliphatic ring systems (e.g., indanyl, tetrahydronaphthyl, etc.).

[0128] A "heteroaryl" group is an aryl ring system having one to four heteroatoms as ring atoms in a heteroaromatic ring system, with the remaining atoms being carbon atoms. In some embodiments, the heteroaryl group contains 5 to 6 ring atoms, and in other embodiments contains 6 to 9 or even 6 to 10 atoms in the ring portion of the group. Suitable heteroatoms include oxygen, sulfur, and nitrogen. In certain embodiments, the heteroaromatic ring system is monocyclic or bicyclic. Non-limiting examples include, but are not limited to, groups such as the following: pyrrolyl, pyrazolyl, imidazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, thiazolyl, pyrrolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, thienyl, benzothienyl, furyl, benzofuryl (e.g., isobenzofuran-1,3-diimine), indolyl, azaindolyl (e.g., pyrrolo-pyridyl or 1H-pyrrolo[2,3-b]pyridyl), indazolyl, benzimidazolyl (e.g., 1H-benzo[d]imidazolyl), imidazopyridyl (e.g., azabenzimidazolyl, 3H-imidazo[4,5-b]pyridyl or 1H-imidazo[4,5-b]pyridyl), pyrazolopyridyl, triazolopyridyl, benzotriazolyl, benzoxazolyl, benzothiazolyl, benzothiadiazolyl, isoxazolopyridyl, thianaphthyl, purinyl, xanthinyl, adeninyl, guaninyl, quinolinyl, isoquinolinyl, tetrahydroquinolinyl, quinoxalinyl, and quinazolinyl groups.

[0129] "Heterocyclic group" is a non-aromatic cycloalkyl group in which one to four ring carbon atoms are independently replaced by heteroatoms independently selected from the group consisting of O, S, and N. In some embodiments, the heterocyclic group includes 3 to 10 ring members, while other such groups have 3 to 5, 3 to 6, or 3 to 8 ring members. The heterocyclic group can also be bonded to other groups at any ring atom (i.e., at any carbon or heteroatom of the heterocycle). The heterocyclic group can be substituted or unsubstituted. The heterocyclic group encompasses unsaturated, partially saturated, and saturated ring systems, such as imidazolyl, imidazolinyl, and imidazolidinyl groups. The phrase heterocyclic group includes fused ring species, including those containing fused aromatic and non-aromatic groups, such as benzotriazolyl, 2,3-dihydrobenzo[l,4]dioxinyl, and benzo[l,3]dioxolyl. The phrase also includes bridged polycyclic systems containing heteroatoms, such as, but not limited to, quinuclidinyl.Representative examples of heterocyclic groups include, but are not limited to, aziridinyl, azetidinyl, pyrrolidinyl, imidazolidinyl, pyrazolidinyl, thiazolidinyl, tetrahydrothienyl, tetrahydrofuryl, dioxolanyl, furyl, thienyl, pyrrolyl, pyrrolinyl, imidazolyl, imidazolinyl, pyrazolyl, pyrazolinyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, thiazolyl, thiazolinyl, isothiazolyl, thiadiazolyl, oxadiazolyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, tetrahydropyranyl (e.g., tetrahydro-2H-pyranyl), tetrahydrothiopyranyl, oxathiane, dioxyl, dithialkyl, pyranyl, pyridyl, pyrimidinyl, pyridazinyl, pyrazinyl, triazinyl, dihydropyridyl, dihydrodithiinyl, dihydrodithionyl, homopiperazinyl, quinuclidinyl, indolyl, dihydroindolyl, isoindolyl, azaindolyl (pyrrolopyridyl), indazolyl, indazinyl, benzotriazolyl, benzimidazolyl, benzofuryl, benzothienyl, benzothiazolyl, benzoxadiazolyl, benzoxazinyl, benzodithiinyl, benzoxathiinyl, benzothiazinyl, benzoxazolyl, benzothiazolyl, benzothiadiazolyl, benzo[l,3]dioxolanyl, pyrazolopyridyl, imidazopyridyl (azabenzimidazolyl; e.g., 1H-imidazo[4,5-b]pyridyl or 1H-imidazo[4,5-b]pyridin-2(3H)-one), triazolopyridyl, isoxazolopyridyl, purinyl, xanthinyl, adeninyl, guaninyl, quinolyl, isoquinolyl, quinazolinyl, quinoxalinyl, quinazolinyl, cinnolinyl, phthalazinyl, naphthyridinyl, pteridinyl, thianaphthyl, dihydrobenzothiazinyl, dihydrobenzofuryl, dihydroindolyl, dihydrobenzodioxinyl, tetrahydroindolyl, tetrahydroindazolyl, tetrahydrobenzimidazolyl, tetrahydrobenzotriazolyl, tetrahydropyrrolopyridyl, tetrahydropyrazolopyridyl, tetrahydroimidazopyridyl, tetrahydrotriazolopyridyl, and tetrahydroquinolyl groups. Representative substituted heterocyclic groups can be mono-substituted or substituted more than once (such as, but not limited to, pyridyl or morpholinyl groups, which are 2-, 3-, 4-, 5-, or 6-substituted) or disubstituted by various substituents (such as those listed below).

[0130] The "cycloalkylalkyl" group is a group of the formula: -alkyl-cycloalkyl, where alkyl and cycloalkyl are defined above. Substituted cycloalkylalkyl groups can be substituted at the alkyl, cycloalkyl, or both the alkyl and cycloalkyl moieties of the group. Representative cycloalkylalkyl groups include, but are not limited to, cyclopentylmethyl, cyclopentylethyl, cyclohexylmethyl, cyclohexylethyl, and cyclohexylpropyl. Representative substituted cycloalkylalkyl groups can be mono-substituted or substituted more than once.

[0131] "Arylalkyl" group is a group of the formula: -alkyl-aryl, where alkyl and aryl are as defined above. Substituted arylalkyl groups can be substituted at the alkyl, aryl, or both the alkyl and aryl moieties of the group. Representative arylalkyl groups include, but are not limited to, benzyl and phenethyl groups, and fused (cycloalkylaryl)alkyl groups such as 4-ethyl-indanyl.

[0132] "Heterocyclylalkyl" group is a group of the formula: -alkyl-heterocyclyl, where alkyl and heterocyclyl are as defined above. Substituted heterocyclylalkyl groups can be substituted at the alkyl, heterocyclyl, or both the alkyl and heterocyclyl moieties of the group. Representative heterocyclylalkyl groups include, but are not limited to, 4-ethyl-morpholinyl, 4-propylmorpholinyl, furan-2-ylmethyl, furan-3-ylmethyl, pyridin-3-ylmethyl, (tetrahydro-2H-pyran-4-yl)methyl, (tetrahydro-2H-pyran-4-yl)ethyl, tetrahydrofuran-2-ylmethyl, tetrahydrofuran-2-yl ethyl, and indol-2-ylpropyl.

[0133] "Halogen" is chlorine, iodine, bromine, or fluorine.

[0134] "Alkoxy" group is -O-(alkyl), where alkyl is defined above.

[0135] "Alkoxyalkyl" group is -(alkyl)-O-(alkyl), where each alkyl is independently as defined above.

[0136] "Amine" group is a group of the formula: -NH2.

[0137] "Hydroxylamine" group is a group of the formula: N(R # )OH or NHOH, where R # is a substituted or unsubstituted alkyl, cycloalkyl, cycloalkylalkyl, aryl, arylalkyl, heterocyclyl, or heterocyclylalkyl group as defined herein.

[0138] "Alkoxyamine" group is a group of the formula: -N(R # )O-alkyl or -NHO-alkyl, where R # is as defined above.

[0139] "Aryloxyamine" group is a group of the formula: N(R # )O-aryl or NHO aryl, where R # is as defined above.

[0140] "Alkylamine" group is a group of the formula: NH alkyl or N(alkyl)2, where each alkyl is independently as defined above.

[0141] "Aminocarbonyl" group is a group of the formula: -C(=O)N(R # )2, -C(=O)NH(R# ) or C(=O)NH2, where each R # is as defined above.

[0142] The "amido" group is a group of the formula: NHC(=O)(R # ) or N(alkyl)C(=O)(R # ), where each alkyl and R # is independently as defined above.

[0143] The "O(alkyl)aminocarbonyl" group is a group of the formula: -O(alkyl)C(=O)N(R # )2, -O(alkyl)C(=O)NH(R # ) or -O(alkyl)C(=O)NH2, where each R # is independently as defined above.

[0144] The "N-oxide" group is a group of the formula: -N + -O - .

[0145] The "carboxyl" group is a group of the formula: C(=O)OH.

[0146] The "ketone" group is a group of the formula: C(=O)(R # ), where each R # is as defined above.

[0147] The "aldehyde" group is a group of the formula: -CH(=O).

[0148] The "ester" group is a group of the formula: C(=O)O(R # ) or OC(=O)(R # ), where R # is as defined above.

[0149] The "urea" group is a group of the formula: -N(alkyl)C(=O)N(R # )2, -N(alkyl)C(=O)NH(R # ), -N(alkyl)C(=O)NH2, -NHC(=O)N(R # )2, -NHC(=O)NH(R # ) or NHC(=O)NH2 # , where each alkyl and R # is independently as defined above.

[0150] The "imine" group is a group of the formula: -N=C(R # )2 or -C(R # )=N(R # ), where each R# Independently defined as above.

[0151] The "imide" group is a group of the formula: -C(=O)N(R#)C(=O)(R # ) or N((C=O)(R # ))2, where each R # is independently defined as above.

[0152] The "carbamate" group is a group of the formula: -OC(=O)N(R # )2, -OC(=O)NH(R # ) or -N(R # )C(=O)O(R # ) or -NHC(=O)O(R # ), where each R # is independently defined as above.

[0153] The "amidine" group is a group of the formula: -C(=N(R # ))N(R # )2, -C(=N(R # ))NH(R # ), -C(=N(R # ))NH2, -C(=NH)N(R # )2, -C(=NH)NH(R # ), -C(=NH)NH2, -N=C(R # )N(R # )2, -N=C(R # )NH(R # ), -N=C(R # )NH2, -N(R # )C(R # )=N(R # ), -NHC(R # )=N(R # ), -N(R # )C(R # )=NH or -NHC(R # )=NH, where each R # is independently defined as above.

[0154] The "guanidine" group is a group of the formula: -N(R # )C(=N(R # ))N(R # )2, -NHC(=N(R # ))N(R # )2, -N(R # )C(=NH)N(R# )2、-N(R # )C(=N(R # ))NH(R # )、-N(R # )C(=N(R # ))NH2、-NHC(=NH)N(R # )2, -NHC(=N(R # ))NH(R # )、-NHC(=N(R # ))NH2、-NHC(=NH)NH(R # ), -NHC(=NH)NH2, -N=C(N(R # )2)2, -N=C(NH(R # ))2 or -N=C(NH2)2, wherein each R # Independently as defined above.

[0155] An "enamine" group is a group of the formula: -N(R # )C(R # )=C(R # )2. -NHC(R # )=C(R # )2、-C(N(R # )2)=C(R # )2、-C(NH(R # ))=C(R # )2, -C(NH2)=C(R # )2、-C(R # )=C(R # )(N(R # )2) C(R # )=C(R # )(NH(R # )) or -C(R # )=C(R # )(NH2), where each R # Independently as defined above.

[0156] An "oxime" group is a group of the formula: -C(=NO(R # ))(R # )、-C(=NOH)(R # )、-CH(=NO(R # )) or -CH(=NOH), wherein each R # Independently as defined above.

[0157] A "hydrazide" group is a group of the formula: -C(=O)N(R #)N(R # )2, -C(=O)NHN(R # )2, -C(=O)N(R # )NH(R # ), -C(=O)N(R # )NH2, -C(=O)NHNH(R # )2 or -C(=O)NHNH2, where each R # is independently defined as above.

[0158] The "hydrazine" group is a group of the formula: -N(R # )N(R # )2, -NHN(R # )2, -N(R # )NH(R # ), -N(R # )NH2, -NHNH(R # )2 or -NHNH2, where each R # is independently defined as above.

[0159] The "hydrazone" group is a group of the formula: -C(=N-N(R # )(R # )2)(R # ), -C(=NNH(R # ))(R # )2, -C(=N-NH2)(R # )2, -N(R # )(N=C(R # ))2) or -NH(N=C(R # )2), where each R

[0160] The "azide" group is a group of the formula: -N3.

[0161] The "isocyanate" group is a group of the formula: N=C=O.

[0162] The "isothiocyanate" group is a group of the formula: N=C=S.

[0163] The "cyanate" group is a group of the formula: OCN.

[0164] The "thiocyanate" group is a group of the formula: SCN.

[0165] The "thioether" group is a group of the formula: -S(R # ), where R # is defined as above.

[0166] The "thiocarbonyl" group is a group of the formula: -C(=S)(R# ), wherein R # is as defined above.

[0167] The "sulfinyl" group is a group of the formula: -S(=O)(R # ), wherein R # is as defined above.

[0168] The "sulfone" group is a group of the formula: -S(=O)2(R # ), wherein R # is as defined above.

[0169] The "sulfonylamino" group is a group of the formula: -NHSO2(R # ) or -N(alkyl)SO2(R # ), wherein each alkyl and R # is as defined above.

[0170] The "sulfonamide" group is a group of the formula: -S(=O)2N(R # )2 or -S(=O)2NH(R # ) or -S(=O)2NH2, wherein each R # is independently as defined above.

[0171] The "phosphonate" group is a group of the formula: -P(=O)(O(R # ))2, -P(=O)(OH)2, -OP(=O)(O(R # ))(R # ) or -OP(=O)(OH)(R # ), wherein each R # is independently as defined above.

[0172] The "phosphine" group is a group of the formula: -P(R # )2, wherein each R # is independently as defined above.

[0173] When the groups (other than alkyl groups) described herein are said to be "substituted", they may be substituted by any suitable one or more substituents. Illustrative examples of substituents are those found in the exemplary compounds and examples disclosed herein, and halogen (chlorine, iodine, bromine, or fluorine); alkyl; hydroxy; alkoxy; alkoxyalkyl; amino; alkylamino; carboxy; nitro; cyano; thiol; thioether; imine; imide; amidine; guanidine; enamine; aminocarbonyl; acylamino; phosphonate; phosphine; thiocarbonyl; sulfinyl; sulfone; sulfonamide; ketone; aldehyde; ester; urea; carbamate; oxime; hydroxylamine; alkoxyamine; aralkyloxyamine; N-oxide; hydrazine; acylhydrazine; hydrazone; azide; isocyanate; isothiocyanate; cyanate; thiocyanate; oxygen (═O); B(OH)2, O(alkyl)aminocarbonyl; cycloalkyl, which may be monocyclic or fused or non-fused polycyclic (e.g., cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl); or heterocyclic group, which may be monocyclic or fused or non-fused polycyclic (e.g., pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl or thiazinyl); monocyclic or fused or non-fused polycyclic aryl or heteroaryl (e.g., phenyl, naphthyl, pyrrolyl, indolyl, furyl, thienyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, triazolyl, tetrazolyl, pyrazolyl, pyridyl, quinolinyl, isoquinolinyl, acridinyl, pyrazinyl, pyridazinyl, pyrimidinyl, benzimidazolyl, benzothienyl or benzofuryl); aryloxy; aralkyloxy; heterocycloxy; and heterocycloalkoxy.

[0174] As used herein, the term "pharmaceutically acceptable salt" refers to salts prepared from pharmaceutically acceptable non-toxic acids or bases (including inorganic and organic acids and bases).

[0175] As used herein and unless otherwise specified, the term "solvate" means a compound or its salt further comprising a stoichiometric or non-stoichiometric amount of a solvent bound by non-covalent intermolecular forces. In one embodiment, the solvate is a hydrate.

[0176] As used herein and unless otherwise specified, the term "hydrate" means a compound or its salt further comprising a stoichiometric or non-stoichiometric amount of water bound by non-covalent intermolecular forces.

[0177] All pharmaceutically acceptable salts, solvates and / or hydrates of the compounds depicted herein are within the scope of this disclosure.

[0178] As used herein and unless otherwise indicated, the term "prodrug" means a compound derivative that can be hydrolyzed, oxidized, or otherwise reacted under biological conditions (in vitro or in vivo) to provide an active compound, particularly a compound. Examples of prodrugs include, but are not limited to, derivatives and metabolites of a compound that include a biolyzable moiety, such as a biolyzable amide, a biolyzable ester, a biolyzable carbamate, a biolyzable carbonate, a biolyzable acylurea, and a biolyzable phosphate analog. In certain embodiments, a prodrug of a compound having a carboxyl functional group is a lower alkyl ester of a carboxylic acid. The carboxylic acid ester is conveniently formed by esterifying any carboxylic acid moiety present on the molecule. Prodrugs can generally be prepared using well-known methods, such as those described in Burger's Medicinal Chemistry and Drug Discovery, 6th Edition (edited by Donald J. Abraham, 2001, Wiley) and Design and Application of Prodrugs (edited by H. Bundgaard, 1985, Harwood Academic Publishers Gmfh).

[0179] As used herein and unless otherwise specified, the term "stereoisomer" or "stereoisomerically pure" means a stereoisomer of a compound that is substantially free of other stereoisomers of that compound. For example, a stereoisomerically pure compound having one chiral center will be substantially free of the opposite enantiomer of that compound. A stereoisomerically pure compound having two chiral centers will be substantially free of other diastereomers of that compound. Typical stereoisomerically pure compounds contain greater than about 80% by weight of one stereoisomer of the compound and less than about 20% by weight of other stereoisomers of the compound, greater than about 90% by weight of one stereoisomer of the compound and less than about 10% by weight of other stereoisomers of the compound, greater than about 95% by weight of one stereoisomer of the compound and less than about 5% by weight of other stereoisomers of the compound, or greater than about 97% by weight of one stereoisomer of the compound and less than about 3% by weight of other stereoisomers of the compound. These compounds can have chiral centers and can exist in the form of racemates, individual enantiomers or diastereomers, and mixtures thereof. All such isomeric forms (including mixtures thereof) are included in the embodiments disclosed herein. The uses of the stereoisomerically pure forms of such compounds and the uses of mixtures of these forms are covered by the embodiments disclosed herein. For example, mixtures containing equal or unequal amounts of enantiomers of a particular compound can be used in the methods and compositions disclosed herein. Standard techniques (such as chiral columns or chiral resolving agents) can be used to asymmetrically synthesize or resolve these isomers. See, for example, Jacques, J. et al., Enantiomers, Racemates and Resolutions (Wiley Interscience, New York, 1981); Wilen, S.H. et al., Tetrahedron 33:2725 (1977); Eliel, E.L., Stereochemistry of Carbon Compounds (McGraw Hill, NY, 1962); and Wilen, S.H., Tables of Resolving Agents and Optical Resolutions, page 268 (edited by E.L. Eliel, Univ. of Notre Dame Press, Notre Dame, IN, 1972).

[0180] It should also be noted that the compounds can include E and Z isomers or mixtures thereof, as well as cis and trans isomers or mixtures thereof. In certain embodiments, the compounds are separated as cis or trans isomers. In other embodiments, the compounds are mixtures of cis and trans isomers.

[0181] "Tautomers" refer to isomeric forms of a compound that are in equilibrium with each other. The concentrations of these isomeric forms will depend on the environment in which the compound is present and can vary, for example, depending on whether the compound is a solid or in an organic or aqueous solution. For example, in an aqueous solution, pyrazole can exhibit the following isomeric forms, which are referred to as tautomers of each other:

[0182] As will be readily understood by those skilled in the art, a wide variety of functional groups and other structures can exhibit tautomerism and all tautomers of a compound are within the scope of this disclosure.

[0183] It should also be noted that the compounds can contain non-natural proportions of atomic isotopes at one or more atoms. For example, the compounds can be radiolabeled with radioactive isotopes (such as tritium ( 3 H), iodine-125 ( 125 I), sulfur-35 ( 35 S) or carbon-14 ( 14 C)), or can be isotopically enriched, such as enriched with deuterium ( 2 H), carbon-13 ( 13 C) or nitrogen-15 ( 15 N). As used herein, "isotopologues" are isotopically enriched compounds. The term "isotopically enriched" refers to atoms having an isotopic composition different from the natural isotopic composition of the atom. "Isotopically enriched" can also refer to a compound that contains at least one atom having an isotopic composition different from the natural isotopic composition of the atom. The term "isotopic composition" refers to the amount of each isotope of a given atom. Radiolabeled and isotopically enriched compounds can be used as therapeutic agents (e.g., for the treatment of cancer and inflammation), research reagents (e.g., binding assay reagents) and diagnostic agents (e.g., in vivo imaging agents). All isotopic variations of the compounds described herein (whether radioactive or not) should be covered by the scope of the embodiments provided herein. In some embodiments, isotopologues of the compounds are provided, for example, these isotopologues are compounds enriched with deuterium, carbon-13 or nitrogen-15.

[0184] It should be noted that if there is a difference between the depicted structure and the name of the structure, the depicted structure will be given greater weight.

[0185] As used herein, "alkynyl" refers to a monovalent hydrocarbon radical moiety containing at least two carbon atoms and one or more carbon-carbon triple bonds. The alkynyl is optionally substituted and can be straight-chain, branched-chain or cyclic. Alkynyl includes but is not limited to those groups having: 2-20 carbon atoms, i.e., C 2-20 alkynyl; having 2-12 carbon atoms, i.e., C 2-12 alkynyl; having 2-8 carbon atoms, i.e., C 2-8 alkynyl; having 2-6 carbon atoms, i.e., C 2-6 alkynyl; and having 2-4 carbon atoms, i.e., C 2-4 alkynyl. Examples of alkynyl moieties include but are not limited to ethynyl, propynyl and butynyl.

[0186] As used herein, "haloalkyl" refers to an alkyl as defined above, wherein the alkyl includes at least one substituent selected from the group consisting of halogen (e.g., fluorine (F), chlorine (Cl), bromine (Br) or iodine (I)). Examples of haloalkyl include but are not limited to -CF3, -CH2CF3, –CCl2F and –CCl3.

[0187] As used herein, "haloalkoxy" refers to an alkoxy as defined above, wherein the alkoxy includes at least one substituent selected from the group consisting of halogen (e.g., F, Cl, Br or I).

[0188] As used herein, "arylalkyl" refers to the monovalent moiety of a group that is an alkyl compound, wherein the alkyl compound is substituted with an aromatic substituent, i.e., the aromatic compound includes a single bond to the alkyl group, and wherein the group is located on the alkyl group. The arylalkyl group is bonded to the indicated chemical structure via the alkyl group. The arylalkyl can be represented by the following structures, for example, B-CH2-, B-CH2-CH2-, B-CH2-CH2-CH2-, B-CH2-CH2-CH2-CH2-, B-CH(CH3)-CH2-CH2-, B-CH2-CH(CH3)-CH2-, where B is an aromatic moiety such as phenyl. The arylalkyl is optionally substituted, i.e., the aryl group and / or the alkyl group can be substituted as disclosed herein. Examples of arylalkyl include but are not limited to benzyl.

[0189] As used herein, "alkylaryl" refers to the monovalent moiety of a group that is an aryl compound, where the aryl compound is substituted with an alkyl substituent, i.e., the aryl compound includes a single bond to an alkyl group and where the group is located on the aryl group. The alkylaryl is bonded to the indicated chemical structure via the aryl group. The alkylaryl can be represented by structures such as -B-CH3, -B-CH2-CH3, -B-CH2-CH2-CH3, -B-CH2-CH2-CH2-CH2-CH3, -B-CH(CH3)-CH2-CH3, -B-CH2-CH(CH3)-CH3, where B is an aromatic moiety such as phenyl. The alkylaryl is optionally substituted, i.e., the aryl group and / or the alkyl group can be substituted as disclosed herein. Examples of alkylaryl include but are not limited to toluoyl.

[0190] As used herein, "aryloxy" refers to the monovalent moiety of a group that is an aromatic compound, where the ring atoms are carbon atoms and where the ring is substituted with an oxygen group, i.e., the aromatic compound includes a single bond to an oxygen atom and where the group is located on the oxygen atom, e.g., for phenoxy is C6H5-O-. The aryloxy substituent is bonded to the compound it substitutes via this oxygen atom. The aryloxy is optionally substituted. Aryloxy includes but is not limited to those groups having: 6 to 20 ring carbon atoms, i.e., C 6-20 aryloxy; 6 to 15 ring carbon atoms, i.e., C 6-15 aryloxy; and 6 to 10 ring carbon atoms, i.e., C 6-10 aryloxy. Examples of aryloxy moieties include but are not limited to phenoxy, naphthyloxy, and anthryloxy.

[0191] As used herein, the term "residue" refers to the chemical moiety that remains after an internal chemical reaction of a compound. For example, the term "amino acid residue" or "N-alkyl amino acid residue" refers to the product of an amide coupling or peptide coupling of an amino acid or N-alkyl amino acid with a suitable coupling partner; where, for example, a water molecule is expelled after the amide or peptide coupling of the amino acid or N-alkyl amino acid, resulting in a product having an amino acid residue or N-alkyl amino acid residue incorporated therein.

[0192] As used herein, "sugar" or "sugar group" or "sugar residue" refers to a carbohydrate moiety that may contain 3-carbon (trisaccharide) units, 4-carbon (tetrasaccharide) units, 5-carbon (pentasaccharide) units, 6-carbon (hexasaccharide) units, 7-carbon (heptasaccharide) units, or combinations thereof, and can be a monosaccharide, disaccharide, trisaccharide, tetrasaccharide, pentasaccharide, oligosaccharide, or any other polysaccharide. In some cases, "sugar" or "sugar group" or "sugar residue" includes furanose (e.g., ribofuranose, fructofuranose) or pyranose (e.g., glucopyranose, galactopyranose) or combinations thereof. In some cases, "sugar" or "sugar group" or "sugar residue" includes aldose or ketose or combinations thereof. Non-limiting examples of monosaccharides include ribose, deoxyribose, xylose, arabinose, glucose, mannose, galactose, and fructose. Non-limiting examples of disaccharides include sucrose, maltose, lactose, lactulose, and trehalose. Other "sugar" or "sugar group" or "sugar residue" includes polysaccharides and / or oligosaccharides, including but not limited to amylose, amylopectin, glycogen, inulin, and cellulose. In some cases, "sugar" or "sugar group" or "sugar residue" is an amino sugar. In some cases, "sugar" or "sugar group" or "sugar residue" is a glucosamine residue (1-amino-1-deoxy-D-glucitol) that is linked via its amino group to the rest of the molecule to form an amide bond (i.e., glucosamide) with the rest of the molecule.

[0193] As used herein, "inorganic acid residue" refers to orthophosphoric acid and pyrophosphoric acid, phosphoric acid and sulfuric acid residues.

[0194] As used herein, "organic acid residue" refers to residues of alkane carboxylic acids, amino acids, or oligopeptides. In one embodiment, the alkane carboxylic acid is formic acid, acetic acid, propionic acid, butyric acid, valeric acid, caproic acid, enanthic acid, caprylic acid, pelargonic acid, capric acid, undecanoic acid, lauric acid, tridecanoic acid, myristic acid, pentadecanoic acid, palmitic acid, margaric acid, stearic acid, nonadecanoic acid, or eicosanoic acid. In one embodiment, the alkane carboxylic acid is formic acid, acetic acid, propionic acid, or butyric acid.

[0195] Certain groups, moieties, substituents, and atoms are depicted by a wavy line intersecting one or more bonds to indicate the atom through which these groups, moieties, substituents, and atoms are bonded. For example, a phenyl group substituted with a propyl group is depicted as: Having the following structure:

[0196] As used herein, unless otherwise specified, a description showing a substituent bonded to a cyclic group (e.g., aromatic, heteroaromatic, fused ring, and saturated or unsaturated cycloalkyl or heterocycloalkyl) via a bond between ring atoms is intended to indicate that the cyclic group can be substituted by the substituent at any ring position in the cyclic group or on any ring in a fused ring group according to the techniques described herein or techniques known in the art to which this disclosure pertains.

[0197] Unless otherwise specified, a description showing a substituent bonded to an acyclic group via a bond between two atoms is intended to indicate that the substituent can be bonded to either atom of the bond through which the substituent bond passes according to the techniques described herein or techniques known in the art to which this disclosure pertains. Thus, for example, covers Detailed Description

[0198] This disclosure provides anti-CEA antibody-drug conjugates (ADCs). This disclosure also provides anti-CEA ADCs that comprise an antibody having desired pharmacokinetic characteristics and other desired properties and can thus be used to reduce the likelihood of cells that express or accumulate CEA or to treat a subject having such cells (such as a cancer characterized by the expression or accumulation of CEA). This disclosure further provides a pharmaceutical composition comprising the anti-CEA ADC and methods of preparing and using the anti-CEA ADC or the pharmaceutical composition comprising the anti-CEA ADC. In some embodiments, the anti-CEA ADC can be used to treat CEA-related diseases and disorders. Anti-CEA antibody

[0199] This disclosure provides anti-CEA ADCs that comprise an anti-CEA antibody or an antigen-binding fragment thereof that specifically binds to CEA. The antibodies or antigen-binding fragments of this disclosure include, but are not limited to, the antibodies or antigen-binding fragments produced as described below.

[0200] This disclosure provides an antibody or antigen-binding fragment that specifically binds to CEA, wherein the antibody or antigen-binding fragment comprises a VH domain having the amino acid sequence of SEQ ID NO: 14, 31, or 48 (Table 1). This disclosure also provides an antibody or antigen-binding fragment that specifically binds to CEA, wherein the antibody or antigen-binding fragment comprises a heavy-chain CDR (HCDR) having the amino acid sequence of any one of the HCDRs listed in Table 1. In one aspect, this disclosure provides an antibody or antigen-binding fragment that specifically binds to CEA, wherein the antibody comprises one, two, three, or more HCDRs (alternatively, consisting of these HCDRs) having the amino acid sequence of any one of the HCDRs listed in Table 1.

[0201] The present disclosure provides an antibody or antigen-binding fragment that specifically binds to CEA, wherein the antibody or antigen-binding fragment comprises a VH domain as described in Table 1 or any one of the groups of HCDRs in Table 1, and a VL domain having the amino acid sequence of SEQ ID NO: 15, 32, or 49 (Table 1). The present disclosure also provides an antibody or antigen-binding fragment that specifically binds to CEA, wherein the antibody or antigen-binding fragment comprises an LCDR having the amino acid sequence of any one of the light chain CDRs (LCDRs) listed in Table 1. Specifically, the present disclosure provides an antibody or antigen-binding fragment that specifically binds to CEA, the antibody or antigen-binding fragment comprising one, two, three, or more LCDRs (alternatively, consisting of these LCDRs) having the amino acid sequence of any one of the LCDRs listed in Table 1.

[0202] The present disclosure provides an antibody or antigen-binding fragment that specifically binds to CEA, wherein the antibody or antigen-binding fragment comprises (i) a heavy chain variable region comprising SEQ ID NO: 31 and a light chain variable region comprising SEQ ID NO: 32; or (ii) a heavy chain variable region comprising SEQ ID NO: 48 and a light chain variable region comprising SEQ ID NO: 49; or (iii) a heavy chain variable region comprising SEQ ID NO: 14 and a light chain variable region comprising SEQ ID NO: 15; or (iv) three heavy chain CDRs: HCDR1 comprising the amino acid sequence as shown in SEQ ID NO: 24, HCDR2 comprising the amino acid sequence as shown in SEQ ID NO: 25, HCDR3 comprising the amino acid sequence as shown in SEQ ID NO: 26, and three light chain CDRs: LCDR1 comprising the amino acid sequence as shown in SEQ ID NO: 27, LCDR2 comprising the amino acid sequence as shown in SEQ ID NO: 28, LCDR3 comprising the amino acid sequence as shown in SEQ ID NO: 23; or (v) three heavy chain CDRs: HCDR1 comprising the amino acid sequence as shown in SEQ ID NO: 7, HCDR2 comprising the amino acid sequence as shown in SEQ ID NO: 8, HCDR3 comprising the amino acid sequence as shown in SEQ ID NO: 9, and Three light chain CDRs: LCDR1 comprising the amino acid sequence shown in SEQ ID NO:10, LCDR2 comprising the amino acid sequence shown in SEQ ID NO:11, LCDR3 comprising the amino acid sequence shown in SEQ ID NO:6; or (vi) Three heavy chain CDRs: HCDR1 comprising the amino acid sequence shown in SEQ ID NO:41, HCDR2 comprising the amino acid sequence shown in SEQ ID NO:42, HCDR3 comprising the amino acid sequence shown in SEQ ID NO:43, and Three light chain CDRs: LCDR1 comprising the amino acid sequence shown in SEQ ID NO:44, LCDR2 comprising the amino acid sequence shown in SEQ ID NO:45, LCDR3 comprising the amino acid sequence shown in SEQ ID NO:40.

[0203] This disclosure provides an ADC comprising an antibody or antigen-binding fragment that specifically binds to CEA, wherein the antibody or antigen-binding fragment comprises a VH domain having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the sequence shown in SEQ ID NO:14, 31 or 48 (Table 1).

[0204] This disclosure provides an ADC comprising an antibody or antigen-binding fragment that specifically binds to CEA, wherein the antibody or antigen-binding fragment comprises a VH domain having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the VH sequence of (i), (ii) or (iii); and a VL domain having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the VL sequence of (i), (ii) or (iii): (i) A heavy chain variable region comprising SEQ ID NO:31 and a light chain variable region comprising SEQ ID NO:32; (ii) A heavy chain variable region comprising SEQ ID NO:48 and a light chain variable region comprising SEQ ID NO:49; and (iii) A heavy chain variable region comprising SEQ ID NO:14 and a light chain variable region comprising SEQ ID NO:15.

[0205] In some aspects, no more than 1, 2, 3, 4, or 5 amino acids have been altered (e.g., via insertion, deletion, or substitution) in these CDR regions when compared to the CDR regions depicted in the sequences described in Table 1.

[0206] Other antibodies of the disclosure include those in which the amino acids or nucleic acids encoding the amino acids have been altered but have at least 60%, 70%, 80%, 90%, 95%, or 99% percent identity to the variable region sequences described in Table 1. In some aspects, no more than 1, 2, 3, 4, or 5 amino acids have been altered (e.g., via insertion, deletion, or substitution) in these variable regions when compared to the variable regions depicted in the sequences described in Table 1, while retaining substantially the same therapeutic activity.

[0207] The disclosure also provides nucleic acid sequences encoding VH, VL, full-length heavy chains, and full-length light chains of antibodies that specifically bind to CEA. Such nucleic acid sequences can be optimized for expression in mammalian cells. Table 1. Amino Acid and Nucleic Acid Sequences

[0208] The disclosure provides ADCs comprising an antibody and its antigen-binding fragments and any payloads described herein, wherein the antibody and its antigen-binding fragments bind to an epitope of human CEA.

[0209] The disclosure also provides ADCs comprising an antibody and its antigen-binding fragments, wherein the antibody and its antigen-binding fragments bind to the same epitope as an anti-CEA antibody having one or more of the sequences disclosed in Table 1. Thus, additional antibodies and their antigen-binding fragments can be identified based on their ability to cross-compete (e.g., competitively inhibit the binding of other antibodies in a statistically significant manner) with other antibodies in a binding assay. Testing the ability of a test antibody to inhibit the binding of the antibodies of Table 1 and their antigen-binding fragments to CEA demonstrates that the test antibody can compete with the antibody of Table 1 or its antigen-binding fragment for binding to CEA. Without being bound by any one theory, such an antibody can bind to the same or a related (e.g., structurally similar or spatially proximal) epitope on CEA as the antibody or its antigen-binding fragment with which it competes. In certain aspects, an antibody that binds to the same epitope on CEA as the antibody or its antigen-binding fragment of Table 1 is a human or humanized monoclonal antibody. Such a human or humanized monoclonal antibody can be prepared and isolated as described herein. Antibody Linker

[0210] It should also be understood that the domains and / or regions of the polypeptide chains of the antibodies disclosed herein can be separated by linkers of various lengths. In some embodiments, the antigen-binding domain is separated from CL, CH1, hinge, CH2, CH3, or the entire Fc region from each other by a linker region. For example, VL1-CL-(linker)-VH2-CH1. Such linker regions can contain randomly sorted amino acids, or a restricted set of amino acids. Such linker regions can be flexible or rigid (see US2009 / 0155275).

[0211] In some embodiments, a linker can be used to conjugate a toxin or payload to a compound between the disclosed antibodies. In some embodiments, the linker is cleavable under intracellular conditions such that cleavage of the linker releases the toxin / payload from the antibody in the intracellular environment. In still other embodiments, the linker unit is non-cleavable and the toxin is released, for example, by antibody degradation. The linker can be, but is not limited to, a cleavable linker, a non-cleavable linker, a hydrophilic linker, a pre-charged linker, or a dicarboxylic acid-based linker. Dimerization of specific amino acids

[0212] In one embodiment, the antibodies disclosed herein contain at least one dimerization-specific amino acid alteration. The dimerization-specific amino acid alteration produces a "mortar and pestle" interaction and increases the assembly of the correct antibody. The dimerization-specific amino acids can be within the CH1 domain or the CL domain or a combination thereof. Examples of dimerization-specific amino acids for pairing the CH1 domain with other CH1 domains (CH1-CH1) and the CL domain with other CL domains (CL-CL) can be found at least in the disclosures of WO2014082179, the WO 2015181805 family, and WO 2017059551. The dimerization-specific amino acids can also be within the Fc domain and can be combined with the dimerization-specific amino acids within the CH1 or CL domain. In one embodiment, the present disclosure provides an antibody comprising at least one pair of dimerization-specific amino acids. Alteration of the framework of the Fc region

[0213] In various aspects, the Fc region is altered by substituting at least one amino acid residue with a different amino acid residue to alter the effector function of the antibody. For example, one or more amino acids can be substituted with different amino acid residues such that the antibody has an altered affinity for effector ligands but retains the antigen-binding ability of the parental antibody. The effector ligands with altered affinity can be, for example, Fc receptors or the C1 component of complement. This method is described, for example, in U.S. Patent Nos. 5,624,821 and 5,648,260 to Winter et al.

[0214] In another aspect, one or more amino acid residues can be replaced with one or more different amino acid residues such that the antibody has altered C1q binding and / or reduced or eliminated complement-dependent cytotoxicity (CDC). This method is described, for example, in U.S. Patent No. 6,194,551 to Idusogie et al.

[0215] In another aspect, one or more amino acid residues are altered to change the ability of the antibody to fix complement. This method is described, for example, in Published WO 94 / 29351 to Bodmer et al. In certain aspects, one or more amino acids of the antibody or antigen-binding fragment thereof are replaced with one or more allotypic amino acid residues of the IgG1 subclass and κ isotype. Allotypic amino acid residues also include, but are not limited to, the heavy chain constant regions of the IgG1, IgG2, and IgG3 subclasses and the light chain constant region of the κ isotype, as described by Jefferis et al., MAbs. [Monoclonal Antibodies] 1:332-338 (2009).

[0216] In another aspect, the Fc region is modified by modifying one or more amino acids to increase the ability of the antibody to mediate antibody-dependent cytotoxicity (ADCC) and / or increase the affinity of the antibody for Fcγ receptors. This method is described, for example, in Published WO 00 / 42072 to Presta. In addition, the binding sites for FcγRI, FcγRII, FcγRIII, and FcRn on human IgG1 have been mapped, and variants with improved binding have been described (see Shields et al., J. Biol. Chem. [Journal of Biological Chemistry] 276:6591-6604, 2001).

[0217] In another aspect, the glycosylation of the antibody is modified. For example, aglycosylated antibodies can be prepared (i.e., the antibody lacks or has reduced glycosylation). For example, glycosylation can be altered to increase the affinity of the antibody for the "antigen". Such carbohydrate modifications can be achieved, for example, by altering one or more glycosylation sites within the antibody sequence. For example, one or more amino acid substitutions can be made that result in the elimination of one or more variable region framework glycosylation sites, thereby eliminating glycosylation at that site. Such aglycosylation can increase the affinity of the antibody for the antigen. This method is described, for example, in U.S. Patent Nos. 5,714,350 and 6,350,861 to Co et al.

[0218] Additionally or alternatively, antibodies with altered glycosylation patterns can be prepared, such as hypofucosylated antibodies with a reduced amount of fucosyl residues or antibodies with an increased bisecting GlcNAc structure. Such altered glycosylation patterns have been shown to increase the ADCC ability of antibodies. This carbohydrate modification can be achieved, for example, by expressing the antibody in a host cell with an altered glycosylation pathway. Cells with an altered glycosylation pathway have been described in the art and can be used as host cells in which to express a recombinant antibody to produce an antibody with altered glycosylation. For example, EP 1,176,195 by Hang et al. describes a cell line with a functionally disrupted FUT8 gene that encodes a fucosyltransferase, such that antibodies expressed in this cell line exhibit hypofucosylation. Published WO 03 / 035835 by Presta describes a variant CHO cell line, Lecl3 cells, which have a reduced ability to attach fucose to Asn(297)-linked carbohydrates, also resulting in hypofucosylation of antibodies expressed in this host cell (see also Shields et al., (2002) J. Biol. Chem. 277:26733-26740). WO 99 / 54342 by Umana et al. describes a cell line engineered to express a glycosyltransferase that modifies glycoproteins (e.g., β(1,4)-N-acetylglucosaminyltransferase III (GnTIII)), such that antibodies expressed in the engineered cell line exhibit an increased bisecting GlcNAc structure, which results in increased ADCC activity of the antibody (see also Umana et al., Nat. Biotech. 17:176-180, 1999).

[0219] On the other hand, if it is desired to reduce ADCC, many previous reports have shown that the human antibody subclass IgG4 has only moderate ADCC and little or no CDC effector function (Moore G.L. et al., 2010 MAbs, 2:181-189). However, native IgG4 has been found to be less stable under stress conditions such as in acidic buffers or at elevated temperatures (Angal, S. 1993 Mol Immunol, 30:105-108; Dall'Acqua, W. et al., 1998 Biochemistry, 37:9266-9273; Aalberse et al., 2002 Immunol, 105:9-19). Reduction of ADCC can be achieved by operably linking the antibody to an IgG4 Fc engineered with a combination of alterations that reduce FcγR binding or C1q binding activity, thereby reducing or eliminating ADCC and CDC effector functions. Considering the physicochemical properties of antibodies as biopharmaceuticals, one of the less desirable intrinsic properties of IgG4 is that its two heavy chains dynamically separate in solution to form half-antibodies, which results in the generation of bispecific antibodies in vivo via a process called "Fab-arm exchange" (Van der Neut Kolfschoten M. et al., 2007 Science, 317:1554-1557). Mutation of serine at position 228 (EU numbering system) to proline has been shown to inhibit IgG4 heavy chain separation (Angal, S. 1993 Mol Immunol, 30:105-108; Aalberse et al., 2002 Immunol, 105:9-19). It has been reported that some amino acid residues in the hinge region and the γ Fc region have an impact on the interaction of antibodies with Fcγ receptors (Chappel S.M. et al., 1991 Proc. Natl. Acad. Sci. USA, 88:9036-9040; Mukherjee, J. et al., 1995 FASEB J, 9:115-119; Armour, K.L. et al., 1999 Eur J Immunol, 29:2613-2624; Clynes, R.A. et al., 2000 Nature Medicine, 6:443-446; Arnold J.N., 2007 Annu Rev Immunol, 25:21-50).In addition, some rare IgG4 isotypes in the population can also give rise to different physicochemical properties (Brusco, A. et al., 1998 Eur J Immunogenet, 25:349-55; Aalberse et al., 2002 Immunol, 105:9-19). To generate antibodies with low ADCC and CDC but good stability, the hinge region and Fc region of human IgG4 can be modified and many changes can be introduced. These modified IgG4 Fc molecules can be found in US Patent No. 8,735,553 to Li et al., SEQ ID NO:83-88. Antibody production

[0220] The antibodies and antigen-binding fragments thereof of the disclosed ADCs can be produced by any means known in the art, including but not limited to recombinant expression of antibody tetramers, chemical synthesis, and enzymatic digestion, while full-length monoclonal antibodies can be obtained by, for example, hybridoma or recombinant production. Recombinant expression can be from any suitable host cell known in the art, such as mammalian host cells, bacterial host cells, yeast host cells, insect host cells, etc.

[0221] The present disclosure further provides polynucleotides encoding the antibodies described herein, such as polynucleotides encoding heavy or light chain variable regions or segments containing complementary determining regions as described herein. In some aspects, the polynucleotide encoding the heavy chain variable region has at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% nucleic acid sequence identity to the polynucleotide represented by SEQ ID NO:16, SEQ ID NO:33, or SEQ ID NO:50. In some aspects, the polynucleotide encoding the light chain variable region has at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% nucleic acid sequence identity to the polynucleotide selected from SEQ ID NO:17, 34, or 51.

[0222] The polynucleotides of the present disclosure can encode the variable region sequences of anti-CEA antibodies. They can also encode the variable and constant regions of the antibodies. Some polynucleotide sequences encode polypeptides containing the variable regions of the heavy and light chains of exemplary anti-CEA antibodies.

[0223] The present disclosure also provides expression vectors and host cells for producing anti-CEA antibodies. The choice of expression vector depends on the intended host cell of the expression vector. Typically, the expression vector contains a promoter and other regulatory sequences (e.g., enhancers) operably linked to a polynucleotide encoding an anti-CEA antibody chain or antigen-binding fragment. In some aspects, an inducible promoter is used to prevent expression of the inserted sequence except under the control of inducing conditions. Inducible promoters include, for example, arabinose, lacZ, metallothionein promoters or heat shock promoters. Cultures of transformed organisms can be expanded under non-inducing conditions without biasing the population towards coding sequences for which the host cell better tolerates their expression products. In addition to the promoter, other regulatory elements can be included for efficient expression of the anti-CEA antibody or its antigen-binding fragment. These elements can include the ATG start codon and adjacent ribosome-binding sites or other sequences. Additionally, the efficiency of expression can be enhanced by including an enhancer suitable for the cell system used (see, for example, Scharf et al., Results Probl. Cell Differ. [Results and Problems in Cell Differentiation] 20:125, 1994; and Bittner et al., Meth. Enzymol. [Methods in Enzymology], 153:516, 1987). For example, the SV40 enhancer or CMV enhancer can be used to increase expression in mammalian host cells.

[0224] Host cells for carrying and expressing anti-CEA antibody vectors can be prokaryotic or eukaryotic. Escherichia coli is a prokaryotic host that can be used for cloning and expressing the polynucleotides of the present disclosure. Other suitable microbial hosts include bacilli (such as Bacillus subtilis) and other Enterobacteriaceae (such as Salmonella, Serratia, and various Pseudomonas species). In these prokaryotic hosts, expression vectors can also be prepared that typically contain expression control sequences (e.g., origin of replication) compatible with the host cell. Additionally, any number of various well-known promoters can be present, such as the lactose promoter system, the tryptophan (trp) promoter system, the β-lactamase promoter system, or the promoter system from bacteriophage λ. The promoter typically optionally controls expression with an operator sequence and has a ribosome-binding site sequence, etc. to initiate and complete transcription and translation. Other microorganisms such as yeast can also be used to express anti-CEA antibodies. A combination of insect cells and baculovirus vectors can also be used.

[0225] In other aspects, mammalian host cells are used to express and produce the anti-CEA antibodies of the present disclosure. Examples include hybridoma cell lines that express endogenous immunoglobulin genes or mammalian cell lines carrying exogenous expression vectors. These include any normally dying or normal or abnormally immortalized animal or human cells. For example, several suitable host cell lines capable of secreting intact immunoglobulins have been developed, including CHO cell lines, various COS cell lines, HEK 293 cells, myeloma cell lines, transformed B cells, and hybridomas. The use of mammalian tissue cell cultures to express polypeptides is generally discussed, for example, in Winnacker, From Genes to Clones, VCH Publishers, New York, N.Y., 1987. Expression vectors for mammalian host cells can include expression control sequences such as origins of replication, promoters, and enhancers (see, e.g., Queen et al., Immunol. Rev. 89:49-68, 1986) and necessary processing information sites such as ribosome binding sites, RNA splicing sites, polyadenylation sites, and transcription terminator sequences. These expression vectors typically contain promoters derived from mammalian genes or mammalian viruses. Suitable promoters can be constitutive, cell type-specific, stage-specific, and / or regulatable or inducible. Useful promoters include, but are not limited to, the metallothionein promoter, the constitutive adenovirus major late promoter, the dexamethasone-inducible MMTV promoter, the SV40 promoter, the MRP polIII promoter, the constitutive MPSV promoter, the tetracycline-inducible CMV promoter (such as the human immediate early CMV promoter), the constitutive CMV promoter, and promoter-enhancer combinations known in the art. Antibody-drug conjugate

[0226] The antibodies disclosed herein can be combined with cytotoxic agents (the "D" or "P" herein) to form antibody-drug conjugates. Cytotoxic agents can be any molecule that inhibits or reduces the expression of a molecule in a cell, inhibits or reduces cell function, induces apoptosis, and / or causes cell death. Examples of cytotoxic agents include those described herein. In an embodiment, the cytotoxic agent is a topoisomerase inhibitor.

[0227] In an embodiment, the antibody-drug conjugate has the formula A: Ab-(C-L-(D) m ) n (A), or a pharmaceutically acceptable salt, solvate, or hydrate thereof, wherein: Ab is the antibody or an antigen-binding fragment thereof; C is a conjugation moiety; L is a linker; D is a cytotoxic agent; m is an integer from 1 to 8; and n is from 1 to 10. In a specific embodiment, m is 1.

[0228] In an embodiment, the antibody-drug conjugate has the formula A-1: Ab-(C-L-D) n (A-1), or a pharmaceutically acceptable salt, solvate or hydrate thereof, wherein: Ab is the antibody or an antigen-binding fragment thereof; C is a conjugation moiety; L is a linker; D is a cytotoxic agent; m is an integer from 1 to 8; and n is from 1 to 10.

[0229] In an embodiment, n is from 3 to 10, such as from 4 to 10, from 5 to 10, from 6 to 10 or from 7 to 9. In certain embodiments, n is about 8.

[0230] International Publication No. WO 2023 / 125530 (the entire content of which is incorporated herein by reference) discloses antibody-drug conjugates in which the linker payload moiety is suitable for the context of the present disclosure, as well as linker payloads suitable for the context of the present disclosure. In some embodiments, the linker payload is the linker payload disclosed in WO 2023 / 125530.

[0231] In an embodiment, the antibody-drug conjugate has the formula (I): or a pharmaceutically acceptable salt, tautomer, solvate, stereoisomer, enantiomer, isotopomer or prodrug thereof, wherein BA is Ab, and the variable is described with respect to the antibody-drug conjugate of the present disclosure (e.g., the antibody-drug conjugate of formula A or A-1); L is a covalent linker; PA is a payload residue (e.g., cytotoxic agent (D)), and the variable is described with respect to the antibody-drug conjugate of the present disclosure (e.g., the antibody-drug conjugate of formula A or A-1); and the subscript x is from 1 to 30 (e.g., n), and the variable is described with respect to the antibody-drug conjugate of the present disclosure (e.g., the antibody-drug conjugate of formula A or A-1). In some cases, x is from 1 to 4. In some cases, x is about 1. In some cases, x is about 2. In some cases, x is about 3. In some cases, x is about 4.

[0232] In another embodiment, the antibody-drug conjugate has the formula (Ia): or a pharmaceutically acceptable salt, tautomer, solvate, stereoisomer, enantiomer, isotopomer or prodrug thereof, wherein RG 1 is a reactive group residue; RG 2 is an optional reactive group residue; SP 1 and SP 2 are each independently an optional spacer residue; HG is a hydrophilic residue; PAB is an optional self-immolative unit; subscript p is 0 or 1; and subscript x is from 1 to 30. The values of the remaining variables (e.g., AA 2 , AA 3 ) and alternative values of the variables (e.g., x, p, PAB, HG, RG 1 , RG 2 , SP 1 , SP 2 , BA) are as described elsewhere herein.

[0233] In some embodiments, x is from 1 to 15. In some embodiments, x is from 2 to 10. In some embodiments, x is from 3 to 9. In one embodiment, x is about 3. In one embodiment, x is about 4. In one embodiment, x is about 5. In one embodiment, x is about 6. In one embodiment, x is about 7. In one embodiment, x is about 8. In one embodiment, x is about 9.

[0234] In some embodiments of the compound of formula (Ia), AA 2 comprises the formula (W): and AA 3 is a dipeptide residue of –valine-alanine–, –valine-citrulline– or ; wherein R 6 is -CH3 or –(CH2)3-NHC(=O)NH2.

[0235] In some embodiments, is

[0236] In some embodiments, AA 3 is wherein R 6 is -CH3 or –(CH2)3-NHC(=O)NH2. In another embodiment, R 6 is -CH3.

[0237] In some embodiments, PAB represents -NH-CH2-O-, Formula (Y1): or Formula (Y2): wherein represents the bond through which PAB is bonded to the adjacent group in the formula.

[0238] In some embodiments, PAB is -NH-CH2-O-.

[0239] In some embodiments, RG 1 is -(succinimido-3-yl-N)— In some embodiments, RG 1 is

[0240] In some embodiments, RG 1 is wherein EWG is an electron-withdrawing group such as -CN, -NO2, halogen, -CF3, -C(=O)OR 1 or -C(=O)R 1 , and R 1 is a substituted or unsubstituted alkyl, a substituted or unsubstituted cycloalkyl, a substituted or unsubstituted aryl, a substituted or unsubstituted heterocycloalkyl or a substituted or unsubstituted heteroaryl.

[0241] In some embodiments, RG 1 is

[0242] In some embodiments, RG 1 is wherein EWG is an electron-withdrawing group such as -CN, -NO2, halogen, -CF3, -C(=O)OR 1 or -C(=O)R 1 , and R 1 is a substituted or unsubstituted alkyl, a substituted or unsubstituted cycloalkyl, a substituted or unsubstituted aryl, a substituted or unsubstituted heterocycloalkyl or a substituted or unsubstituted heteroaryl.

[0243] In some embodiments, RG 1 is an open heterocycle, such as formed by The product produced by the conjugation of the maleimide ring with the antibody. In this regard, it should be understood that the conjugation of the antibody with the maleimide ring can occur at either of the two carbons in the carbon-carbon double bond of the maleimide. Similarly, in the case of ring opening, the conjugation can occur at either of the two carbon atoms in the double bond. In some embodiments, RG 1 is In some embodiments, RG 1 is In some embodiments, RG 1 is In some embodiments, RG 1 is In some embodiments, RG 1 is In some embodiments, RG 2 is a bond, -C(=O)-NH-, or -NHC(=O)-. In some embodiments, RG 2 is -C(=O)-NH-.

[0244] In some embodiments, SP 1 is -(CH2) n1 -C(═O)-, -(CH2CH2O) n2 -CH2CH2-C(═O)-, -CH[-(CH2) n3 -COOH]-C(═O)-, -CH2-C(═O)-NH-(CH2) n4 -C(═O)-, -CH2-C(═O)-NH-(CH2) n3 -C(═O)-NH-(CH2) n4 -C(═O)- or —C(═O)—(CH2) n5 -C(═O)—, where each of n1, n2, n3, n4, and n5 independently represents an integer from 1 to 8. In some embodiments, SP 1 is *-CH2C(O)N(H)CH2CH2C(O)-, where the asterisk marks the bond connected to RG 1 In some embodiments, SP 1 is *-(CH2)5C(O)-, where the asterisk marks the bond connected to RG 1 In some embodiments, SP 1 is *-C(H)(CH2NH2)-(CH2)2OC(O)N(H)(CH2)2C(O)-, where the asterisk marks the bond connected to RG 1 In some embodiments, SP

[0245] In some embodiments, SP 2 is -(CH2) n6-; and n6 represents an integer from 1 to 8. In some embodiments, n6 is 2.

[0246] In some embodiments, HG is where each n7 is independently 1 - 15; each n8 is independently 0 or 1; each n9 is independently 1 or 2; each n10 is independently an integer from 4 to 16, such as 4, 8, or 12; each n11 is independently an integer from 0 to 5; n12 is an integer from 0 to 3; d is 0 - 3; R 2 is H or Me; R 3 is -OH, -NH2, -NHCH2-CH2-(PEG) x -OH or -NHCH2-CH2-(PEG) x -OMe; R 4 is OH or NH2; and each of X, Y, and Z is independently -CH2-, -NH-, -S-, or -O-.

[0247] In some embodiments, HG is where each n7 is independently 1 - 15; each n8 is independently 0 or 1; each n9 is independently 1 or 2; each n10 is independently an integer from 4 to 16, such as 4, 8, or 12; d is 0 - 3; R 2 is H or Me; R 3 is -OH, -NH2, -NHCH2-CH2-(PEG) x -OH or -NHCH2-CH2-(PEG) x -OMe; R 4 is OH or NH2.

[0248] In some embodiments, HG is where each n8 is independently 0 or 1; and R 1 is H or Me.

[0249] In some embodiments, HG is

[0250] In some embodiments, HG is Each n11 is independently an integer from 0 to 5; n12 is an integer from 0 to 3; and each of X, Y, and Z is independently -CH2-, -NH-, -S-, or -O-.

[0251] In some embodiments, HG is -NHSO2NH2, -SO3H, -SO2NH2, -PO3H2, and RG 2 is a bond.

[0252] In some embodiments, each PA independently represents a chromophore functional group.

[0253] In some embodiments, each chromophore functional group is independently a functional group selected from the group consisting of: a class or subclass of xanthophore, erythrophore, iridophore, leucophore, melanophore, and cyanophore; a class or subclass of fluorophore molecules, which are fluorescent compounds that re-emit light upon illumination; a class or subclass of visual phototransduction molecules; a class or subclass of photophore molecules; a class or subclass of luminescent molecules; and a class or subclass of luciferin compounds.

[0254] In some embodiments, each PA is independently selected from the group consisting of: monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), monomethyl auristatin D (MMAD), maytansine (DM1 / DM4), paclitaxel, docetaxel, epothilone B, epothilone A, CYT997, auristatin tyramine phosphate, auristatin aminoquinoline, halofuginone, calicheamicin θ, 7-ethyl-10-hydroxy-camptothecin (SN-38), pyrrolobenzodiazepine (PBD), pancratistatin, cyclophosphate, Cribrostatin-6, kitastatin, Turbostatin 1-4, halofuginone, eribulin, hemiasterlin, PNU, and silastatin.

[0255] In some embodiments, each PA independently represents formula (D1): wherein R 4 、R 5a and R 5b Each of and is independently hydrogen, a sugar residue, a substituted or unsubstituted inorganic or organic acid residue, a substituted or unsubstituted C 1-8alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted non-aromatic heterocyclic group, substituted or unsubstituted cycloalkylalkyl or substituted or unsubstituted heterocyclicalkyl; R 5a and R 5b together with the atoms to which they are attached form a substituted or unsubstituted cycloalkyl, substituted or unsubstituted non-aromatic heterocyclic group.

[0256] In some embodiments, R 4 is hydrogen, and wherein each of R 5a and R 5b is independently H, CH3 or CF3; or R 5a and R 5b together with the atoms to which they are attached form a substituted or unsubstituted cycloalkyl, substituted or unsubstituted non-aromatic heterocyclic group.

[0257] In some embodiments, R 4 is hydrogen, and wherein each of R 5a and R 5b is independently H, CH3 or CF3; or R 5a and R 5b together with the atoms to which they are attached form a substituted or unsubstituted cycloalkyl, substituted or unsubstituted non-aromatic heterocyclic group.

[0258] In some embodiments, each PA independently represents

[0259] In some embodiments, each PA independently represents formula (D2): wherein ring B is a substituted or unsubstituted cycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heterocyclic group or substituted or unsubstituted heteroaryl.

[0260] In some embodiments, each PA independently represents

[0261] In some embodiments, each PA independently represents formula (D3): wherein S 2 is an enzyme-hydrolyzable hydrophilic group.

[0262] In some embodiments, S 2 is hydrogen or represents one of the following formulas:

[0263] In some embodiments, each PA independently represents formula (E1): wherein each of R 7 and R 8 is independently hydrogen, halogen, or alkyl.

[0264] In some embodiments, R 7 and R 8 are hydrogen.

[0265] In some embodiments, R 7 and R 8 are methyl.

[0266] In some embodiments, R 7 is methyl and R 8 is F.

[0267] In some embodiments, the carbon to which R 7 and R 8 are attached has the S configuration.

[0268] In some embodiments, the carbon to which R 7 and R 8 are attached has the R configuration.

[0269] In some embodiments, each PA independently represents the following formula:

[0270] In some embodiments, each PA is independently Dxd, or independently represents the following formula:

[0271] In some embodiments, each PA independently represents the following formula:

[0272] In some embodiments, AA 2 is glycine or an amino acid residue of .

[0273] In some embodiments of the compound of formula (Ia) or a pharmaceutically acceptable salt, tautomer, solvate, stereoisomer, enantiomer, isotopomer, or prodrug thereof, AA 2 comprises formula (W): and AA 3 is – glycine - glycine - phenylalanine - glycine - or a tetrapeptide residue of.

[0274] In another embodiment, the antibody - drug conjugate has the formula (Ib): or a pharmaceutically acceptable salt, tautomer, solvate, stereoisomer, enantiomer, isotopomer or prodrug thereof, wherein AA 2 comprises the formula (W): and AA 1 is – valine - alanine -, – valine - citrulline - or a dipeptide residue of; wherein R 6 is - CH3 or –(CH2)3 - NHC(=O)NH2. The values of the remaining variables (e.g., x, p, BA, HG, RG 1 、RG 2 、SP 1 、SP 2 、PAB, PA) and the alternative values of the variables (e.g., AA 1 、AA 2 ) are as described elsewhere herein, for example, with respect to the compounds of formula Ia.

[0275] In some embodiments, AA 2 comprises the formula (W): and AA 1 is – glycine - glycine - phenylalanine - glycine - or a tetrapeptide residue of.

[0276] In another embodiment, the antibody - drug conjugate has the formula (Ic): or a pharmaceutically acceptable salt, tautomer, solvate, stereoisomer, enantiomer, isotopomer or prodrug thereof, wherein AA 3 is – valine - alanine -, – valine - citrulline - or a dipeptide residue of; wherein R 6is -CH3 or –(CH2)3-NHC(=O)NH2. The values of the remaining variables (e.g., BA, RG 1 , SP 1 , PAB, p, PA, x) and the alternative values of the variables (AA 3 , R 6 ) are as described elsewhere herein, for example, with respect to the compounds of formula Ia.

[0277] In some embodiments (e.g., embodiments of the compounds of formula (Ic)), AA 3 is – glycine - glycine - phenylalanine - glycine - or a tetrapeptide residue of.

[0278] In some embodiments, the antibody - drug conjugate is selected from one of the following compounds, or a pharmaceutically acceptable salt, tautomer, solvate, stereoisomer, enantiomer, isotopomer or prodrug thereof:

[0279] The PCT application No. PCT / CN2022 / 123665 (the entire content of which is incorporated herein by reference) discloses antibody - drug conjugates in which the linker - payload portion is suitable for the context of the present disclosure, as well as linker - payloads suitable for the context of the present disclosure. In some embodiments, the linker - payload is the linker - payload disclosed in PCT / CN2022 / 123665.

[0280] In some embodiments (e.g., embodiments of the compounds of formula I), PA is a residue of: wherein Y is -A-B-C'-D'-H; A is a bond, CR 1 R 2 or N-R 1 ; B is a bond, -C(=O)- or -C(=O)O-; C' is a bond or a divalent group, wherein the divalent group is an unsubstituted or substituted C 1-8 alkyl, unsubstituted or substituted cycloalkyl, unsubstituted or substituted heterocyclic group, unsubstituted or substituted aryl or unsubstituted or substituted heteroaryl; D' is a bond, NH or O; R 1 and R 2 each independently is hydrogen, halogen, substituted or unsubstituted alkyl or substituted or unsubstituted alkoxy; or R 1 and R 2 together with the atom to which they are attached form an unsubstituted or substituted cycloalkyl, unsubstituted or substituted heterocyclic group, unsubstituted or substituted aryl or unsubstituted or substituted heteroaryl; and R 3 and R 4 each independently is hydrogen, halogen, substituted or unsubstituted alkyl or substituted or unsubstituted alkoxy; or R 3 and R 4 together with the atom to which they are attached form an unsubstituted or substituted cycloalkyl, unsubstituted or substituted heterocyclic group, unsubstituted or substituted aryl or unsubstituted or substituted heteroaryl. In some embodiments, when R 3 is methyl and R 4 is F, Y is not -NH-C(=O)-C-D-H.

[0281] In an embodiment, among the residues of the payload depicted above, Y is -A-B-C'-D'- and is the result of removing -H from -A-B-C'-D'-H. It should be understood that although the payload residue can be produced by removing a hydrogen atom from the payload depicted herein, it can also be produced by removing a hydroxyl group (such as the hydroxyl group formed when D' is O in the payload depicted above (or the corresponding hydroxyl group in any other payload structure depicted herein)).

[0282] In some embodiments, PA is a residue of the following: wherein A is CR 1 R 2 , NH or N-R 1 ; B is a bond, -C(=O)- or -C(=O)O-; R 1 and R 2 each independently is H or C 1-4 alkyl; R 3 and R 4 each independently is hydrogen, halogen, substituted or unsubstituted alkyl or substituted or unsubstituted alkoxy; or R 3 and R 4 together with the atoms to which they are attached form an unsubstituted or substituted cycloalkyl, unsubstituted or substituted heterocyclic group, unsubstituted or substituted aryl or unsubstituted or substituted heteroaryl; R 5 and R 6 each independently is hydrogen, halogen, substituted or unsubstituted alkyl or substituted or unsubstituted alkoxy; and n is 1, 2, 3, 4 or 5.

[0283] In some embodiments, A is -CH2-, and B is a bond.

[0284] In some embodiments, R 5 and R 6 are hydrogen, and n is 1, 2 or 3.

[0285] In some embodiments, R 3 is methyl, and R 4 is F.

[0286] In some embodiments, PA is a residue of the following:

[0287] In some embodiments, R 3 and R 4 together with the atoms to which they are attached form an unsubstituted or substituted m-dioxolane ring.

[0288] In some embodiments, PA is a residue of the following:

[0289] In some embodiments, A is -N(CH3)-, and B is a bond.

[0290] In some embodiments, R 5 and R 6 are hydrogen, and n is 2.

[0291] In some embodiments, PA is a residue of the following:

[0292] In some embodiments, A is -NH-, and B is -C(=O)O-. In further embodiments, R 5 and R 6 are hydrogen, and n is 2.

[0293] In some embodiments, PA is a residue of:

[0294] In some embodiments, A is -NH-, and B is -C(=O)-. In further embodiments, R 5 and R 6 are hydrogen, and n is 2.

[0295] In some embodiments, PA is a residue of:

[0296] In some embodiments, R 3 is Cl, R 4 is F, and B is -C(=O)-.

[0297] In some embodiments, PA is a residue

[0298] In some embodiments, R 3 is methyl, R 4 is Cl, and B is -C(=O)-.

[0299] In some embodiments, PA is a residue

[0300] In some embodiments, R 3 and R 4 together with the atoms to which they are attached form an unsubstituted or substituted heterocyclic group.

[0301] In some embodiments, R 3 and R 4 together with the atoms to which they are attached form an unsubstituted or substituted m-dioxolene ring, and B is -C(=O)-.

[0302] In some embodiments, PA is a residue of:

[0303] In some embodiments, PA is a residue of: wherein the values and alternative values of the variables (e.g., R 3 , R 4 ) are as described elsewhere herein.

[0304] In some embodiments, R 3 is methyl; and R 4 is Cl.

[0305] In some embodiments, PA is a residue of the following:

[0306] In some embodiments, R 3 is Cl; and R 4 is F.

[0307] In some embodiments, PA is a residue of the following:

[0308] In some embodiments, R 3 is F; and R 4 is F.

[0309] In some embodiments, PA is a residue of the following:

[0310] In some embodiments, R 3 is H; and R 4 is F.

[0311] In some embodiments, PA is a residue of the following:

[0312] In some embodiments, R 3 is H; and R 4 is OH.

[0313] In some embodiments, PA is a residue of the following:

[0314] In some embodiments, R 3 is methyl; and R 4 is methyl.

[0315] In some embodiments, PA is a residue of the following:

[0316] In some embodiments, R 3 is methoxy; and R 4 is F.

[0317] In some embodiments, PA is a residue of the following:

[0318] In some embodiments, R 3 is H; and R 4 is methoxy.

[0319] In some embodiments, PA is a residue of the following:

[0320] In some embodiments, R 3 is H; and R 4 is Cl.

[0321] In some embodiments, PA is a residue of:

[0322] In some embodiments, R 3 and R 4 together with the atoms to which they are attached form an unsubstituted or substituted heterocyclic group.

[0323] In some embodiments, R 3 and R 4 together with the atoms to which they are attached form an unsubstituted or substituted dioxolane ring.

[0324] In some embodiments, PA is a residue of:

[0325] In some embodiments, PA is a residue of:

[0326] In some embodiments, PA is a residue of:

[0327] In some embodiments, PA is a residue of:

[0328] In some embodiments, PA is a residue of:

[0329] In some embodiments, PA is a residue of:

[0330] In some embodiments, PA is a residue of: wherein R 7' and R 8' each independently is hydrogen or a substituted or unsubstituted alkyl group; or R 7' and R 8' together with the nitrogen atom to which they are attached form an unsubstituted or substituted heterocyclic group or an unsubstituted or substituted heteroaryl group.

[0331] In some embodiments, PA is a residue of:

[0332] In some embodiments, the antibody-drug conjugate has formula (V): or a pharmaceutically acceptable salt, tautomer, solvate, stereoisomer, enantiomer, isotopomer or prodrug thereof, wherein the values and alternative values of the variables (e.g., A, B, C', D', L, R 3 , R 4 and x) are as described elsewhere herein.

[0333] In some embodiments, the antibody-drug conjugate has a structure of formula (VIIIa), (VIIIb) or (VIIIc): or a pharmaceutically acceptable salt, tautomer, solvate, stereoisomer, enantiomer, isotopomer or prodrug thereof, wherein the values and alternative values of the variables (e.g., L, R 7 , R 8 and x) are as described elsewhere herein. In some embodiments, the antibody-drug conjugate has a structure of any one of the following formulas: or a pharmaceutically acceptable salt, tautomer, solvate, stereoisomer, enantiomer, isotopomer or prodrug thereof, wherein the values and alternative values of the variables (e.g., L and x) are as described elsewhere herein.

[0334] In some embodiments, L is The keys marked with an asterisk are connected to BA.

[0335] In some embodiments, L is The keys marked with an asterisk are connected to BA.

[0336] In some embodiments, L is: where the values and alternative values of the variables (e.g., RG 1 , SP 1 , AA 2 , AA 3 , PAB, p, SP 2 , RG 2 and HG) are as described herein. In some embodiments, L is: where the values and alternative values of the variables (e.g., RG 1 , SP 1 , AA 1 , AA 2 , PAB, p, SP 2 , RG 2 and HG) are as described elsewhere herein.

[0337] In some embodiments, L is: where the values and alternative values of the variables (e.g., RG 1 , SP 1 , AA 3 , PAB and p) are as described elsewhere herein.

[0338] In some embodiments, -AA 2 (SP 2 -RG 2 -HG)-AA 3 -(PAB) p - is where the * mark is the key connected to SP 1 connection.

[0339] In some embodiments, the antibody-drug conjugate is selected from the following, or a pharmaceutically acceptable salt, tautomer, solvate, stereoisomer, enantiomer, isotopomer or prodrug thereof, where Ab is any of the anti-CEA antibodies disclosed herein:

[0340] This text contemplates all possible combinations of the linker and the payload. In this regard, it should be understood that L as used herein in the context of Formulas I - VIII encompasses the C - L of the compound of Formula A or A - 1. Additionally, it should be understood that C as used in the context of Formula A or A - 1 corresponds to RG 1 -SP 1 .

[0341] In an embodiment, D is: wherein Y is -A - B - C - D - *, where * marks the bond by which D is attached to L; A is a bond, CR 1 R 2 or N - R 1 ; B is a bond, -C(=O)- or -C(=O)O-; C is a bond or a divalent group, where the divalent group is an unsubstituted or substituted C 1-8 alkyl, unsubstituted or substituted cycloalkyl, unsubstituted or substituted heterocycloalkyl, unsubstituted or substituted aryl, or unsubstituted or substituted heteroaryl; D is a bond, NH or O; R 1 and each of R 2 is independently hydrogen, halogen, substituted or unsubstituted alkyl, or substituted or unsubstituted alkoxy; or R 1 and R 2 together with the atoms to which they are attached form an unsubstituted or substituted cycloalkyl, unsubstituted or substituted heterocycloalkyl, unsubstituted or substituted aryl, or unsubstituted or substituted heteroaryl; R 3 and each of R 4 is independently hydrogen, halogen, substituted or unsubstituted alkyl, or substituted or unsubstituted alkoxy; or R 3 and R 4 together with the atoms to which they are attached form an unsubstituted or substituted cycloalkyl, unsubstituted or substituted heterocycloalkyl, unsubstituted or substituted aryl, or unsubstituted or substituted heteroaryl.

[0342] In an embodiment, D is wherein each of R 7 and R 8 is independently hydrogen, halogen, or alkyl.

[0343] In an embodiment, the cytotoxic agent has the following formula:

[0344] In certain embodiments, the cytotoxic agent (D) is In certain embodiments, D is

[0345] Each antibody-drug conjugate may include one or more cytotoxic agent molecules, such as one, two, three, four, five, six, seven, or eight molecules. The number of cytotoxic agent molecules conjugated to a single antibody or antibody fragment can be described as the drug-to-antibody ratio (DAR). In the formula Ab-(C-L-(D) m ) n the DAR is the product of m and n.

[0346] The cytotoxic agent can be conjugated directly to the anti-CEA antibody or indirectly to the anti-CEA antibody via a linker (L). In an embodiment, the linker is cleavable, such as by enzymatic cleavage, to release the cytotoxic agent. In an embodiment, such as when the cytotoxic agent is hydrophobic, the linker is hydrophilic. In an embodiment, the linker has the following formula, where the bond at which * marks L can be conjugated to the conjugation moiety (C):

[0347] In the foregoing formula, Su can be a sugar-like moiety. This moiety can be derived from natural or non-natural sugars. This moiety can be hydrophilic. In an embodiment, such as when the cytotoxic agent is hydrophobic, including the hydrophilic Su moiety can reduce the likelihood of antibody-drug conjugate aggregation and thereby reduce the clearance rate in vivo.

[0348] In an embodiment, Su is a hydrophilic residue.

[0349] In an embodiment, Su is where n8 is 0 or 1; R 6 is -OR 2 -N(H)R 2 -C(O)OR 2 -C(O)N(H)R 2 -CH2-OR 2 -CH2-N(H)R 2 -CH2-C(O)OR 2 or -CH2-C(O)N(H)R 2 ; and R 2is hydrogen or methyl. In certain embodiments, n8 is 1. In certain embodiments, n8 is 0. In certain embodiments, R 2 is hydrogen. In certain embodiments, R 2 is methyl. In certain embodiments, R 6 is -OR 2 、-N(H)R 2 、-C(O)OR 2 or -C(O)N(H)R 2 。In certain embodiments, R 6 is -CH2-OR 2 、-CH2-N(H)R 2 、-CH2-C(O)OR 2 or -CH2-C(O)N(H)R 2 。In certain embodiments, R 6 is -CH2-C(O)N(H)R 2 ,such as -CH2-C(O)NH2.

[0350] In an embodiment, Su is where n8 is 0 or 1; R 5 is -OH, -NH2, -C(O)OH, -C(O)NH2, -CH2-OH or -CH2-NH2. In certain embodiments, Su is In certain embodiments, Su is In certain embodiments, n8 is 0. In certain embodiments, n8 is 1. In certain embodiments, R 5 is -CH2OH. In certain embodiments, R 5 is -C(O)OH.

[0351] In an embodiment, Su is In certain embodiments, Su is

[0352] In an embodiment, Su is In certain embodiments, Su is

[0353] In an embodiment, Su is:

[0354] In an embodiment, L is

[0355] The antibody-drug conjugates disclosed herein may include a conjugate moiety (C). The conjugate moiety may be indirectly conjugated to a cytotoxic agent via a linker. The conjugate moiety may help avoid or reduce the deconjugation of the cytotoxic agent in vivo, which may help maintain a stable drug-to-antibody ratio (DAR). The conjugate moiety may have the following formula before conjugation to an antibody, for example:

[0356] When the conjugate moiety is directly conjugated to an anti-CEA antibody, the conjugate moiety may have the following formula, where the * marks the bond by which the conjugate moiety is linked to the antibody:

[0357] The conjugate moiety, linker, sugar moiety, and cytotoxic agent may be included in the antibody-drug conjugates disclosed herein in any combination. Non-limiting examples of conjugate moiety (before conjugation)-linker-cytotoxic agent combinations include the following:

[0358] Each antibody-drug conjugate may include more than one conjugate moiety-linker-cytotoxic agent compound (C-L-D), such as one, two, three, four, five, six, seven, eight, nine, or ten C-L-Ds. In embodiments, each antibody-drug conjugate includes from 1 to 10, such as from 3 to 10, from 4 to 10, from 5 to 10, from 6 to 10, from 7 to 9, or about 8.

[0359] In certain embodiments, -C-L-(D) m is: where the * marks the bond by which C is linked to the Ab. In certain embodiments, -C-L-(D) m is: where the * marks the bond by which C is linked to the Ab. In certain embodiments, -C-L-(D) m is: In certain embodiments, C-L-(D) m is:

[0360] In embodiments, the antibody-drug conjugate is

[0361] In certain embodiments, the antibody-drug conjugate has the following formula: or a tautomer, pharmaceutically acceptable salt, solvate or hydrate thereof, wherein Ab and n are as described herein. In certain embodiments, the antibody-drug conjugate has the following formula: or a tautomer, pharmaceutically acceptable salt, solvate or hydrate thereof, wherein Ab and n are as described herein. In certain embodiments, the antibody-drug conjugate has the following formula: or a tautomer, pharmaceutically acceptable salt, solvate or hydrate thereof, wherein Ab and n are as described herein. Methods of preparing antibody-drug conjugates

[0362] The antibody-drug conjugates disclosed herein can be produced by any method known in the art. In one embodiment, a host cell transformed with an isolated nucleic acid comprising a sequence encoding an anti-CEA antibody or an antigen-binding fragment thereof is cultured under suitable culture conditions. The antibody or an antigen-binding fragment thereof is thereby expressed and can be recovered from the cell culture.

[0363] A cytotoxic agent is conjugated to the antibody or an antigen-binding fragment thereof using a linker disclosed herein to produce an antibody-drug conjugate. In an embodiment, a conjugation moiety is also conjugated to the linker, such as between the antibody and the linker. Methods of treatment

[0364] The antibody-drug conjugates disclosed herein can be used in a variety of applications, including but not limited to methods for treating CEA-related disorders or diseases. In one aspect, CEA-related disorders or diseases are characterized by cells that overexpress or accumulate CEA. In some embodiments, the cells are cancerous.

[0365] In certain aspects, the method comprises administering to a subject in need thereof (e.g., a patient) an effective amount of an anti-CEA antibody-drug conjugate. Subjects can include but are not limited to subjects having cancer that expresses CEA, cancer that accumulates CEA, CEA-reactive cancer, and subjects having gastric or rectal cancer and metastases thereof. In an embodiment, the cancer is lung cancer (e.g., non-small cell lung cancer), gastrointestinal cancer (e.g., gastric cancer) or colorectal cancer (e.g., rectal cancer).

[0366] The antibody-drug conjugates disclosed herein can be administered by any suitable means, including parenterally, intraluminally, and intranasally, and (if desired for local treatment) intralesionally. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Administration can be by any suitable route, for example, by injection, such as intravenous or subcutaneous injection, depending in part on whether the administration is short-term or long-term. A variety of dosing regimens are contemplated herein, including but not limited to single administration or multiple administrations at different time points, bolus administration, and pulsed infusion.

[0367] The antibodies or antigen-binding fragments or antibody-drug conjugates disclosed herein can be formulated, dosed, and administered in a manner consistent with good medical practice. Factors to be considered in this regard include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site to which the agent is to be delivered, the method of administration, the dosing regimen, and other factors known to the medical practitioner. The antibodies need not but optionally can be formulated with one or more agents currently used to prevent or treat the disorder being studied. The effective amount of such other agents depends on the amount of antibody present in the formulation, the type of disorder or treatment, and the other factors discussed above. These are generally used at the same dose and route of administration as described herein, or at about 1%-99% of the doses described herein, or at any dose and by any route determined to be appropriate empirically / clinically. Combination Therapy

[0368] In one aspect, the anti-CEA antibody-drug conjugate can be used in combination with other therapeutic agents. Other therapeutic agents that can be used with the anti-CEA antibody-drug conjugate disclosed herein include but are not limited to chemotherapeutic agents (e.g., paclitaxel or paclitaxel agents (e.g., ) Docetaxel; Carboplatin; Topotecan; Cisplatin; Irinotecan, Doxorubicin, Lenalidomide, 5-Azacytidine, Ifosfamide, Oxaliplatin, Pemetrexed Disodium, Cyclophosphamide, Etoposide, Decitabine, Fludarabine, Vincristine, Bendamustine, Chlorambucil, Busulfan, Gemcitabine, Melphalan, Pentostatin, Mitoxantrone, Pemetrexed Disodium), tyrosine kinase inhibitors (such as EGFR inhibitors) (such as Erlotinib), multi-kinase inhibitors (such as MGCD265, RGB-286638), CD-20 targeting agents (such as Rituximab, Ofatumumab, RO5072759, LFB-R603), CD52 targeting agents (such as Alemtuzumab), Prednisolone, Darbepoetin Alfa, Lenalidomide, Bcl-2 inhibitors (such as Olimersen Sodium), Aurora kinase inhibitors (such as MLN8237, TAK-901), Proteasome inhibitors (such as Bortezomib), CD-19 targeting agents (such as MEDI-551, MOR208), MEK inhibitors (such as ABT-348), JAK-2 inhibitors (such as INCB018424), mTOR inhibitors (such as Temsirolimus, Everolimus), BCR / ABL inhibitors (such as Imatinib), ET-A receptor antagonists (such as ZD4054), TRAIL receptor 2 (TR-2) agonists (such as CS-1008), EGEN-001 or Polo-like kinase 1 inhibitors (such as BI 672).

[0369] In another aspect, the anti-CEA antibody-drug conjugate can be used in combination with an anti-PD1 antibody. The anti-PD1 antibody can include, but is not limited to, tislelizumab, pembrolizumab, and nivolumab. Tislelizumab is disclosed in US 8,735,553. Pembrolizumab (previously known as MK-3475), as disclosed by Merck in US 8,354,509 and US 8,900,587, is a humanized IgG4-K immunoglobulin that targets the PD1 receptor and inhibits the binding of the PD1 receptor ligands PD-L1 and PD-L2. Pembrolizumab has been approved for the indications of metastatic melanoma and metastatic non-small cell lung cancer (NSCLC), and clinical studies are ongoing for the treatment of head and neck squamous cell carcinoma (HNSCC) and refractory Hodgkin lymphoma (cHL). Nivolumab (as disclosed by Bristol-Meyers Squibb) is a fully human IgG4-K monoclonal antibody. Nivolumab (clone 5C4) is disclosed in US Patent No. US 8,008,449 and WO 2006 / 121168. Nivolumab is approved for the treatment of melanoma, lung cancer, kidney cancer, and Hodgkin lymphoma. Drug Compositions and Preparations

[0370] Also provided are compositions (including pharmaceutical preparations) that comprise: an anti-CEA antibody-drug conjugate comprising an anti-CEA antibody or an antigen-binding fragment thereof, or a polynucleotide comprising a sequence encoding an anti-CEA antibody or antigen-binding fragment, and a toxic drug conjugate. These compositions may also comprise a suitable carrier, such as a pharmaceutically acceptable excipient well known in the art, including buffers.

[0371] A pharmaceutical preparation of an anti-CEA antibody-drug conjugate as described herein is prepared by mixing such an antibody or antigen-binding fragment and antibody-drug conjugate of desired purity with one or more optional pharmaceutically acceptable carriers in the form of a lyophilized preparation or an aqueous solution (Remington's Pharmaceutical Sciences, 16th Edition, Osol, A. Ed. (1980)). Pharmaceutically acceptable carriers are generally non-toxic to the recipient at the dosages and concentrations employed and include, but are not limited to: buffers, such as phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (such as octadecyl dimethyl benzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butanol, or benzyl alcohol; alkyl esters of p-hydroxybenzoic acid, such as methyl p-hydroxybenzoate or propyl p-hydroxybenzoate; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); polypeptides of low molecular weight (less than about 10 residues); proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions, such as sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants, such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers herein further include interstitial drug dispersants, such as soluble neutral active hyaluronidase glycoprotein (sHASEGP), such as human soluble PH-20 hyaluronidase glycoprotein, such as rHuPH20( Baxter International, Inc.). Certain exemplary sHASEGP and methods of use, including rHuPH20, are described in U.S. Patent Nos. 7,871,607 and 2006 / 0104968. In one aspect, sHASEGP is combined with one or more additional glycosaminoglycanases, such as chondroitinase.

[0372] Exemplary lyophilized antibody formulations are described in US Pat. No. 6,267,958. Aqueous antibody formulations include those described in US Pat. No. 6,171,586 and WO 2006 / 044908, the latter formulations comprising a histidine-acetate buffer.

[0373] Sustained release preparations may be prepared. Suitable examples of sustained release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody or antibody drug conjugate, which matrices are in the form of shaped articles, eg films, or microcapsules.

[0374] Preparations for in vivo administration are generally sterile. Sterility can be readily achieved, for example, by filtration through sterile filtration membranes. Examples Example 1: Generation of anti-CEA monoclonal antibodies CEA recombinant protein for immunoassays and binding assays

[0375] Several recombinant proteins were designed and expressed for antibody screening (see Table 2). Antibodies against CEA cross-react with human and macaque (Macaca mulatta) CEA in the peri-membrane region containing domain B3 (amino acids 596-674 of SEQ ID NO:52, see Beauchemin et al., "Isolation and characterization of full-length functional cDNA clones for human carcinoembryonic antigen." Mol. Cell Biol., 1987, 7(9):3321-3330). These antibodies lack off-target binding to other human CEACAM members.

[0376] The cDNA coding regions of full-length human CEA (SEQ ID NO: 52), macaque CEA (SEQ ID NO: 53), and full-length human CEACAM6 (SEQ ID NO: 54) were ordered based on the GenBank sequence. For human CEA (accession number: NM_004363.2), the gene can be obtained from Sinobio, catalog number HG11077-UT. For macaque CEA (accession number: NM_001047125), the gene can be obtained from GenScript TM ), catalog number OMB23865D. For human CEACAM6 (accession number: NM_002483.4), the gene can be obtained from Xinbainuo, catalog number HG10823-UT. The schematic diagram of CEA fusion protein is shown in Figure 1As shown. It has been reported that the spliced variants of human CEA are co-expressed with the full-length CEA on tumors (Peng et al., PloS one [Public Library of Science: General], 7, e36412-e36412 (2012)), and thus the variant (CEA-v) was prepared. To generate this construct, the coding region of the extracellular domain (ECD) consisting of amino acids (AA) 1-687 of huCEA (SEQ ID NO:55), the region of amino acids (AA) 1-690 of simian CEA (SEQ ID NO:56), and the region of amino acids (AA) 1-320 of CEACAM6 (SEQ ID NO:57) were PCR amplified. The regions of CEA amino acids (AA) 1-78 (SEQ ID NO:58) and amino acids 398-687 of CEA (SEQ ID NO:59) were PCR amplified and then conjugated by overlap PCR to prepare the CEA variant (CEA-v) (SEQ ID NO:60). Alternatively, the region of CEACAM6 amino acids (AA) 1-273 (SEQ ID NO:61) and the perimembrane region of domain B3 containing CEA amino acids (AA) 596-687 (SEQ ID NO:62) were PCR amplified and then conjugated by overlap PCR to prepare the chimeric construct (CHIM) (SEQ ID NO:63). Then all constructs were cloned into a pcDNA3.1-based expression vector (Invitrogen, Carlsbad, CA, USA), and their C-termini were fused with a 6xHis tag respectively, thereby generating five recombinant fusion protein expression plasmids CEA, simian CEA, CEACAM6, CEA-v, and CHIM. To generate the recombinant fusion proteins, the CEA, simian CEA, CEACAM6, CEA-v, and CHIM plasmids were transiently transfected into a HEK293-based mammalian cell expression system (generated in-house) and cultured in a CO2 incubator equipped with a rotary shaker for 5-7 days. The supernatant containing the recombinant protein was collected and centrifuged to clarify. The recombinant proteins were purified using Ni-NTA agarose (catalog number R90115, Invitrogen). All recombinant proteins were dialyzed against phosphate-buffered saline (PBS) and stored in small aliquots in an -80 °C freezer. Stable expression in cell lines

[0377] To establish a stable cell line expressing full-length human CEA (accession number: NM_004363.2), the CEA-expressing cDNA was cloned into the retroviral vector pFB-Neo (catalog number 217561, Agilent, USA). Amphotropic retroviral vectors were generated according to a previous protocol (Zhang et al., Blood. 2005 106(5):1544-51). The viral vector containing human CEA was transduced into L929 (American Type Culture Collection (ATCC), Manassas, VA, USA) and CT26 cells (American Type Culture Collection, Manassas, VA, USA) to generate human CEA-expressing cell lines. High-expressing cell lines were selected by culturing in complete RPMI 1640 medium containing 10% FBS and G418, and then verified by FACS binding assays. Immunization, Hybridoma Fusion and Cloning

[0378] Eight- to twelve-week-old Balb / c mice (HFK BIOSCIENCE CO., LTD, Beijing, China) were immunized intraperitoneally (i.p.) with 500 μL of 1×10 7 L929 / huCEA cells with or without a water-soluble adjuvant (catalog number KX0210041, KangBiQuan, Beijing, China). The procedure was repeated two weeks later to boost antibody production. Two weeks after the third immunization, the soluble CEA (sCEA) binding of mouse sera was evaluated by ELISA and FACS. Splenocytes were isolated using standard techniques (Colligan JE et al., CURRENT PROTOCOLS IN IMMUNOLOGY, 1993) and fused with the murine myeloma cell line SP2 / 0 cells (American Type Culture Collection, Manassas, VA, USA). CEA Binding Activity of Antibodies was Evaluated by ELISA and FACS

[0379] To screen for antibodies that bind to human CEA but not to CEACAM6 or sCEA, antibodies that bind to CHIM but not to sCEA, CEACAM6, and CEA-v, and antibodies that bind to CHIM, sCEA, and CEA-v but not to CEACAM6 were screened and re-screened. The supernatants of hybridoma clones were initially screened by ELISA (slightly modified) as described in (Methods in Molecular Biology (2007) 378:33-52). Briefly, sCEA, CHIM, CEACAM6, or CEA-v were coated at a low concentration of 3 μg / ml in 96-well plates. Color development was performed using an HRP-conjugated anti-mouse IgG antibody (Catalog No. 7076S, Cell Signaling Technology, USA) and a substrate (Catalog No. 00-4201-56, eBioscience, USA), and the absorbance signal at 450 nm was measured using a microplate reader (SpectraMax Paradigm TM , Molecular Devices, USA). ELISA-positive clones were further verified by FACS using L929 / huCEA and / or MKN45 cells (ATCC). MKN45 cells were derived from human gastric cancer. Cells expressing CEA (10 5 cells / well) were incubated with the ELISA-positive hybridoma supernatant and then bound to an AlexaFluro-647-labeled goat anti-mouse IgG antibody (Catalog No. A0473, Beyotime Biotechnology, China). Cell fluorescence was quantified using a flow cytometer (Guava easyCyte TM 8HT, Merck-Millipore, USA).

[0380] Functional assays were performed on the conditioned media from hybridomas that showed positive signals in the FACS screening and bound to CHIM rather than CEACAM6 and sCEA to evaluate the effect of the presence of sCEA on the binding of CEA antibodies to CEA-expressing cells (see examples below). Antibodies with the desired binding specificity and functional activity were further subcloned and characterized. Hybridoma Subcloning and Adaptation to Serum-Free or Low-Serum Media

[0381] After preliminary screening mainly by ELISA, FACS and functional assays, positive hybridoma clones are subcloned by limiting dilution. The top antibody subclones verified by functional assays are adapted to grow in CDM4MAb medium (Catalog No. SH30801.02, Hyclone, USA) containing 3% FBS. Expression and purification of monoclonal antibodies

[0382] Hybridoma cells are cultured in CDM4MAb medium (Catalog No. SH30801.02, Hyclone) and incubated in a CO2 incubator at 37 °C for 5 to 7 days. The conditioned medium is collected by centrifugation and filtered through a 0.22 μm membrane before purification. The supernatant containing murine antibodies is applied according to the protocol in the manufacturer's instructions and bound to a Protein A column (Catalog No. 17127901, GE Life Sciences). This procedure usually yields antibodies with a purity higher than 90%. The Protein A affinity-purified antibodies are dialyzed against PBS or further purified using a HiLoad 16 / 60 Superdex TM 200 column (Catalog No. 17531801, GE Life Sciences) to remove aggregates. The protein concentration is determined by measuring the absorbance at 280 nm. The final antibody preparation is stored in aliquots at -80 °C in a refrigerator. Table 2: Amino acid and nucleic acid sequences Example 2. Cloning and sequence analysis of CEA antibodies

[0383] According to the manufacturer's protocol, total RNA was harvested from murine hybridoma cells using the Ultrapure RNA Kit (Catalog No. 74104, QIAGEN, Germany) to prepare total RNA. The first-strand cDNA was synthesized using a cDNA synthesis kit from Invitrogen (Catalog No. 18080-051), and PCR amplification of the VH and VL genes of murine monoclonal antibodies was performed using a PCR kit (Catalog No. CW0686, CWBio, Beijing, China). Oligonucleotide primers for antibody cDNA clones of the heavy-chain variable region (VH) and κ light-chain variable region (VL) were synthesized based on previously reported sequences (Brocks et al., Mol Med. 2001 7(7):461-9). The PCR products were then subcloned into the pEASY-Blunt cloning vector (Catalog No. CB101-02, TransGen, China) and sequenced. The amino acid sequences of the VH and VL regions were determined from the DNA sequencing results.

[0384] Monoclonal antibodies were analyzed by comparing sequence homology and grouped based on sequence similarity (Figure 2). Complementary determining regions (CDRs) were defined by sequence annotation according to the IMGT system (Lefranc et al., 1999 Nucleic Acids Research 27:209-212). The amino acid sequence of the representative clone BGA7592 is listed in Table 3. Table 3: Amino acid sequence Example 3. Determination of the binding profile of purified murine anti-CEA antibody

[0385] By using BIAcore TM T-200 (GE Healthcare) surface plasmon resonance (SPR) assay to characterize the specific binding to CEA (as shown by ELISA and FACS) and the binding kinetics of the CEA antibody without sCEA interference ( Figure 3A )). Briefly, anti-mouse IgG antibody was immobilized on an activated CM5 biosensor chip (Catalog No. BR100530, GE Healthcare). The purified murine antibody was passed over the chip surface and captured by the anti-mouse IgG antibody. Then, serial dilutions (6.0 nM to 2150 nM) of purified CHIM, CEA-v, CEA, or simian CEA recombinant proteins were passed over the chip surface, and changes in surface plasmon resonance signals were analyzed using a one-to-one Langmuir binding model (BIA evaluation software, GE Healthcare) to calculate the association rate (kon ) and dissociation rate (k off ). The equilibrium dissociation constant (K D ) was calculated as the ratio k off / k on . The binding affinity profile of BGA7592 is shown in Table 4 below. Table 4: Comparison of BGA7592 Binding Affinity by SPR antigen <![CDATA[K D (M)]]> sCEA ND sCEA-v 1.50E-07 CHIM 1.20E-07 monkey CEA ND (undetectable due to weak binding)

[0386] The binding profile of BGA7592 was examined via antigen ELISA. Binding of purified BGA7592 to huCEA and cynomolgus CEA was observed, indicating that BGA7592 is a weak binder of soluble huCEA and cynomolgus CEA, or that soluble CEA has a different conformation when immobilized ( Figure 3B ). For this experiment, sCEA, CHIM, cynomolgus CEA (“cynoCEA”), CEA-v or bovine serum albumin (BSA) were coated overnight at 4 °C in a 96-well plate at a high concentration of 10 μg / ml. BGA7592 or the control antibody ab4451 (Catalog No. ab4451, abcam, USA) was incubated at a concentration of 2 μg / ml for 1 hour. Color development was carried out using an HRP-conjugated anti-mouse IgG antibody (Catalog No. 7076S, Cell Signaling Technology, USA) and a substrate (Catalog No. 00-4201-56, Invitrogen, USA), and the absorbance signal at a wavelength of 450 nm was measured using a microplate reader (SpectraMax Paradigm, Molecular Devices, USA). Example 4. Effect of Recombinant Soluble CEA on the Binding of BGA7592 to CEA-Expressing Cells

[0387] The effect of the presence of soluble CEA on the specific binding of various CEA antibodies to CEA-expressing cells was evaluated by flow cytometry. Briefly, in the presence of an additional 20 μg / ml of recombinant soluble CEA protein, cells expressing human CEA (10 5 cells / well) were incubated with 2 μg / ml of purified murine monoclonal CEA antibody, followed by binding to an Alexa Fluor-647-labeled goat anti-mouse IgG antibody (Catalog No. A0473, Beyotime Biotechnology, China). Cellular fluorescence was quantified using a flow cytometer (Guava easyCyte TM 8HT, Merck-Millipore, USA). As Figure 4A and 4B shown, the binding of BGA7592 to CEA-expressing cells was not affected by the presence of soluble CEA. Example 5. Humanization of a murine anti - human CEA antibody mAb Humanization and Engineering

[0388] For the humanization of BGA7592, sequence comparison was performed in the human immunoglobulin gene databases of IMGT and NCBI to search for sequences in the human germline IgG genes that had a high degree of homology with the cDNA sequences of the variable regions of BGA7592. Human IGVH and IGVL genes that were present at high frequencies in the human antibody repertoire (Glanville et al., 2009 PNAS [Proceedings of the National Academy of Sciences of the United States of America] 106:20216 - 20221) and were highly homologous to BGA7592 were selected as templates for humanization. Prior to humanization, the heavy - chain and light - chain variable domains of BGA7592 were fused to the wild - type human IgG1 constant region (SEQ ID NO:87) named human IgG1wt and the human κ constant (CL) region (SEQ ID NO:88) (Table 5), respectively. Table 5: Amino Acid Sequences

[0389] Humanization was carried out by CDR grafting (Methods in Molecular Biology [Molecular Biology Methods], Volume 248: antibody Engineering, Methods and Protocols [Antibody Engineering, Methods and Protocols], Humana Press) and the BGA7592 antibody was engineered in the form of human IgG1. In the first round of humanization, mutations of amino acid residues from murine to human in the framework regions were guided by the modeled 3D structures, and murine framework residues that were structurally important for maintaining the canonical structure of the CDRs were retained in the first version of the humanized antibody, BGA7592 - 1 (the amino acid sequences of the heavy chain and light chain are shown in SEQ ID NO:89 and 90) (Table 6). Table 6: Amino Acid Sequences

[0390] Specifically, the CDRs of BGA7592-1 VL were transplanted into the framework of the human germline variable gene IGVK1-27 that retained two murine framework residues (N66 and V68) (the amino acid sequence of the light chain variable domain is shown in SEQ ID NO:92). The CDRs of BGA7592-1 VH were transplanted into the framework of the human germline variable gene IGVH1-46 that retained five murine framework (L39, I53, Y55, N66, S68) residues (the amino acid sequence of the heavy chain variable domain is shown in SEQ ID NO:91) (Table 7). Table 7: Amino Acid Sequences

[0391] BGA7592-1 was constructed into a full-length human antibody form using expression vectors developed in-house by the company. These expression vectors contain the constant region of wild-type human IgG1 and have easily adaptable subcloning sites. Expression and preparation of the BGA7592-1 antibody were achieved by co-transfecting the above two constructs into 293G cells and purifying using a Protein A column (Catalog No. 17543802, General Life Sciences). The purified antibody was concentrated to 0.5 - 5 mg / mL in PBS and stored in aliquots in an -80 °C freezer.

[0392] Using BGA7592-1, additional numbers of single or multiple amino acid changes were made to convert human residues in the VH and VL framework regions to the corresponding murine germline residues, which included V68A, R72A, and V79A in VH and V43S in VL. This resulted in BGA7592-2 (V68A, R72A in VH), BGA7592-3 (V79A in VH), BGA7592-4 (V68A, R72A, V79A in VH), BGA7592-5 (V43S in VL), BGA7592-6 (V68A, R72A in VH and V43S in VL), BGA7592-7 (V79A in VH and V43S in VL), and BGA7592-8 (V68A, R72A, V79A in VH and V43S in VL). All antibodies containing the modifications had similar binding activity to BGA7592-1, and none of the changes eliminated binding.

[0393] To remove the post-translational modification (PTM) sites, further engineering was carried out by introducing mutations in the CDR and framework regions based on the BGA75921 sequence. These mutations included the N52T, N54Q, N59S, N102G, N104Q, and S61A amino acid changes in the VH region. This yielded BGA7592-1A (N52T (VH)), BGA7592-1B (N54Q (VH)), BGA7592-1C (N59S (VH)), BGA7592-1D (N102G (VH)), BGA7592-1E (N104Q (VH)), and BGA7592-1F (N54Q, N59S, S61A (VH)), and all antibodies had binding specificities similar to BGA7592-1, and none of the changes eliminated binding. While maintaining specificity, amino acid composition and expression levels were also considered. All humanized mutations were carried out using primers containing mutations at specific positions and a site-directed mutagenesis kit (Catalog No. FM111-02, TransGen Biotech Co., Ltd., Beijing, China). The required mutations were verified by sequence analysis. Compared with BGA7592-1, BGA7592-1F had significantly reduced binding affinity, no glycosylation sites, but had a high expression level (Table 8). Table 8: Amino acid sequence Example 6. Generation of an affinity maturation library

[0394] A phagemid was constructed using the phagemid vector pCANTAB 5E (GE Healthcare) by standard molecular biology techniques. The phagemid was designed to display the BGA7592-1F Fab fragment on the surface of the M13 phage as a fusion to the N-terminus of the gene-3 minor coat protein fragment. There was an amber stop codon before the gene-3 sequence to allow direct expression of the Fab fragment from the phagemid clone. The phagemid was used as a template to construct a phage display library containing 10 8 unique members.

[0395] Two libraries (H-AM, L-AM) were constructed, randomizing the CDR positions in the heavy and light chains, respectively. All three CDRs were randomized in each library, but each CDR had at most one mutation in each clone, except for HCDR3, which could have two simultaneous mutations. Each position was randomized with the NNK codon (IUPAC code) encoding any amino acid or the amber stop codon. The combined heavy and light chain libraries were designed to have 5.0×10 6The potential diversity of the unique full-length clones, without stop codons or cysteine codons, and with expected distributions of approximately 0.02%, 1.1%, 17%, and 82% of the clones having 0, 1, 2, and 3 mutations, respectively. Due to primer design in the HCDR3 region, a small fraction of the heavy-chain clones are expected to have 4 mutations. As a first step, DNA fragments were amplified using pCANTAB 5E (as a template) and primers containing randomized CDR3 positions (see Figure 5A and 5B ). The PCR products were then gel-purified and assembled with primers containing randomized CDR2 positions. The process was repeated with primers targeting randomized CDR1 positions. The resulting PCR products of the heavy or light chains were then assembled with their corresponding CH or CL fragments by overlap PCR. The fragments were further assembled by overlap PCR with the light or heavy chains without mutations. The resulting fragments were then gel-purified and ligated to pCANTAB 5E after NcoI / NotI digestion. The purified ligation products were transformed into TG1 bacteria by electroporation. Sequencing of 48 clones from each library confirmed the randomization at each position (data not shown), although not all amino acid mutations were observed at each position due to limited sampling depth. Approximately 52% and 55% of the light-chain and heavy-chain libraries had full-length random clones sufficient to cover all the potential diversity designed, and even with moderate incorporation biases in oligonucleotide synthesis and library construction, 10 8 independent clones were generated. Example 7. Generation of Affinity-Matured Humanized BGA7592 Variants Library Selection and Screening

[0396] Generation of humanized BGA7592 Fab with affinity maturation by phage display using a standard protocol (Silacci et al., (2005) Proteomics, 5, 2340 - 50; Zhao et al., (2014) PLoS One, 9, e111339). For the first and second rounds of selection, competitive selection against immobilized CHIM was performed in immunotubes (catalog number 470319, ThermoFisher). Briefly, the immunotubes were coated overnight at 4 °C with 1 ml of CHIM (5 μg / ml in PBS). In the presence of various concentrations of BGA7592 - 1FIgG (round 1, 1 μg / ml; round 2, 5 μg / ml), all affinity maturation libraries were incubated with the coated immunotubes for 1 hour. For the third and fourth rounds of selection, cell panning was performed using L929 / huCEA cells (round 3) or LOVO cells (ATCC CCL - 229) (round 4), with HEK293 cells as the depletion cells. After four rounds of selection, individual clones were picked and supernatants containing phage were prepared using a standard protocol. ELISA - positive clones were sequenced and the mutation sites were analyzed. Analysis of mutation frequency in CDRs

[0397] After four rounds of selection, the mutation frequencies in each CDR were relatively high, ranging from 17% in HCDR3 to 95% in LCDR2. Regarding the heavy chain, approximately half of the clones identified in the H - AM library were identical to the parental clone. The other clones contained a back - mutation at Q54N in HCDR2.

[0398] When analyzing the light chain, the mutations were more diverse. Two sites were mutated in almost all clones in LCDR1. Light chain residues 29 and 31 were mutated from Ile to Gln and from Gly to Gln in 47.09% and 35.29% of the clones, respectively. Position 29 not only had a high frequency of Gln mutations but also had a subset of clones mutated to tyrosine. Position 31 not only had a high frequency of Gln mutations but also had approximately a 12.5% chance of mutating to Leu. Due to library design limitations, no mutations at positions 29 and 31 were found to be combined with each other. However, mutations in each of these two sites were usually combined with mutations in other CDRs. Regarding LCDR2, only A51 was mutated in at least 64.71% of the clones, but without any obvious pattern, which included large hydrophobic and polar residues such as Tyr, Phe, Thr, and Asn. Regarding LCDR3, two sites were mutated in at least 50% of the clones. Light chain residues 90 and 92 were mutated from His to Leu and from Tyr to Leu in 11.76% and 47.06% of the clones, respectively.Figure 6 The sequence differences in the light chain CDR regions after four rounds of selection are shown. Expression of the selected humanized BGA7592 variant

[0399] Combinations of mutations were made. The light chain variable regions from the selected phage clones were subcloned into a human κ light chain expression mammalian expression vector. The light chain expression vector was co-transfected with a mammalian expression vector expressing the BGA7592-1F (also described herein as BGA5366) heavy chain into 293G cells at a ratio of 1:1. The CEA antibody form was purified from the culture supernatant by protein A affinity chromatography (catalog number 17543802, General Life Sciences). The purified antibody was concentrated to 0.5 - 5 mg / mL in PBS and stored in aliquots at -80 °C in a refrigerator. Characterization of the affinity matured humanized BGA7592 variant

[0400] By using BIAcore TM SPR assays (Table 9) of the T-200 (General Life Sciences) and flow cytometry ( Figure 7 ) were performed to compare the affinities of BGA7592-1F (BGA5366) and other affinity matured clones. For this experiment, anti-human IgG (Fc) antibody was immobilized on an activated CM5 biosensor chip (catalog number BR100839, General Life Sciences). The anti-CEA antibody flowed over the chip surface and was captured by the anti-human Fab antibody. Then serial dilutions (1.37 nM to 333 nM) of CHIM were flowed over the chip surface, and the changes in surface plasmon resonance signals were analyzed by using a one-to-one Langmuir binding model (BIA evaluation software, General Life Sciences) to calculate the association rate (k on ) and dissociation rate (k off ). For flow cytometry, cells expressing CEA (10 5 cells / well) were incubated with various concentrations of the purified affinity matured antibodies, followed by binding to an Alexa Fluro-647 labeled anti-hu IgG Fc antibody (catalog number 409320, BioLegend, USA). Cell fluorescence was quantified using a flow cytometer (Guava easyCyte TM 8HT, Merck-Millipore, USA). The equilibrium dissociation constant (K D ) was calculated as the ratio k off / k on。It shows that BGA7592-1F-ph-L (BGA3676) (SEQ ID NO:95) and BGA7592-1F-ph-M (BGA2433) (SEQ ID NO:96) have increased affinity for the huCEA surface protein (Table 10). Table 9: Comparison of binding affinities by SPR Table 10: Amino acid sequences Example 8. Further engineering of affinity-matured humanized BGA7592 variants

[0401] Further engineering was carried out by introducing mutations in the CDRs based on the BGA7592-1F-ph-M (BGA2433) template, which included W33Y, Q54N, and S59N in VH and T51Y in VL. This generated BGA8179 (W33Y (VH)), BGA2107 (Q54N (VH)), BGA0089 (S59N (VH)), BGA1789 (T51Y (VL)), all of which had improved binding activity compared to BGA7592-1F-ph-F (BGA9521). The antibody with the greatest improvement ultimately yielded the BGA6710 antibody with (W33Y (VH), T51Y (VL)) alterations (Table 11), and the sequences are shown in Table 12. Table 11: Comparison of binding affinities to CHIM by SPR sample ID Ka (1 / Ms) Kd (1 / s) KD (M) Rmax (RU) BGA7592-1F-ph-M (BGA2433) 1.97E+04 2.48E-04 1.26E-08 97.2 BGA8179 1.99E+04 2.82E-04 1.42E-08 129.1 BGA2107 1.98E+04 3.80E-04 1.92E-08 71.1 BGA0089 1.84E+04 3.94E-04 2.15E-08 59.4 BGA1789 3.21E+04 4.63E-04 1.44E-08 92.9 BGA6710 3.15E+04 4.75E-04 1.51E-08 112.4 Table 12: Amino acid and nucleic acid sequences of BGA6710 Example 9. Optimization of BGA6710

[0402] To further improve the biochemical / biophysical properties, BGA6710 was optimized by introducing substitutions in the CDR and framework regions (Table 13). Large hydrophobic residues were selected and changed to polar residues, except for K13 and Q53, which were selected based on the observed differences between human VH germlines. Considerations included amino acid composition, thermal stability (Tm), surface hydrophobicity, and isoelectric point (pI), while maintaining functional activity. As described in Example 6, variants were expressed in Fab form by cloning into the vector pCANTAB-5E. The CEA binding of the supernatants containing Fab was then screened by ELISA and SPR analysis. Variants with no significant decrease in affinity were selected and residues tolerant to substitution were identified. It was demonstrated that L92E in the light chain and K13E, Q54E, Y57D / E, and Y57K in the heavy chain had minimal effects on affinity.

[0403] Accordingly, variants of BGA6710 in IgG form with single identified mutations or combinations were expressed and purified as described in Example 8. SPR studies and FACS analysis were performed and summarized in Table 14. It was confirmed that the specificity and epitope were not altered due to the introduced amino acid substitutions (data not shown). In summary, the results showed that these single or combined mutations (K13E, Q54E, Y57D, and Y57K in the heavy chain, L92E in the light chain) had minimal effects on affinity, except for L92E, which slightly decreased the binding affinity to CEA. In summary, the Y57K substitution optimized the expression, CEA binding, and affinity of the BGA6710 antibody, resulting in BGA5384 (Table 1). Table 13: Summary of residues for substitution residue AA substitution H:K13 E H:Y32 H, N, Q, D, E, K H:Y33 H, N, Q, D, E, K H:Q53 A, D, G, N, S, T, Y, R, H H:Y57 H, N, Q, D, E, K H:Y100 H, N, Q, D, E, K H:Y105 H, N, Q, D, E, K L:V15 T, P, L L:Y30 H, N, Q, D, E, K L:Y32 H, N, Q, D, E, K L:Y49 H, N, Q, D, E, K L:P80 S, T, A L:L92 H, N, Q, D, E, K Table 14: Summary of affinity measurements of BGA6710 variants by SPR Example 10. Binding profile of anti-CEA antibody BGA5384

[0404] BGA5384 and the previously disclosed CEA antibody (designated antibody 2F1 in U.S. Patent Application Publication No. 2012 / 0251529) were produced in human IgG1 form and their binding kinetics were characterized by SPR using a BIAcore TM T-200 (General Life Sciences)

[0405] To obtain these data, anti-human IgG (Fc) antibody was immobilized on an activated CM5 biosensor chip (Catalog No. BR100839, General Life Sciences). The BGA5384 antibody was flowed over the chip surface and captured by the anti-human Fab antibody. Then, serial dilutions (1.37 nM to 2150 nM) of soluble huCEA or cynoCEA (Catalog No.: CE5-C52H5, Acrobiosystem Co., Ltd., Beijing) were flowed over the chip surface, and the changes in surface plasmon resonance signals were analyzed using a one-to-one Langmuir binding model (BIA evaluation software, General Life Sciences) to calculate the association rate (k on ) and dissociation rate (k off ). The equilibrium dissociation constant (K D ) was calculated as the ratio k off / k on . The BGA5384 and 2F1 control antibodies exhibited different binding affinities. BGA5384 has a very high affinity for human CEA and also has a considerable affinity for cynoCEA, as shown in Table 15. Table 15: Comparison of binding affinities of anti-CEA antibodies by SPR

[0406] For flow cytometry, MKN45 cells expressing CEA (10 5 cells / well) were incubated with various concentrations of purified affinity-matured antibodies, followed by binding to Alexa Fluor-647-labeled anti-hu IgG Fc antibody (Catalog No. 409320, Bethyl Laboratories, Inc., USA). Cell fluorescence was quantified using a flow cytometer (Guava easyCyte TM 8HT, Merck-Millipore, USA). As Figure 8 shown, BGA5384 exhibited specific binding to native CEA on live cells in a dose-responsive manner (as measured by mean fluorescence intensity MFI), with an EC 50 of 2.92 μg / ml. Example 11. Evaluation of off-target specificity

[0407] The off-target specificity of BGA5384 was evaluated via ELISA and flow cytometry. For flow cytometry, CEACAM3 (SEQ ID NO:65), CEACAM7 (SEQ ID NO:66), or CEACAM8 (SEQ ID NO:67) was transiently transfected into HEK293 cells (10 5cells / well), and then incubated with 2 μg / ml purified BGA5384, followed by binding with Alexa Fluor-647-labeled anti-huIgG Fc antibody (Catalog No. 409320, Biolegend, USA). Cell fluorescence was quantified using a flow cytometer (Guava easyCyte TM 8HT, Merck-Millipore, USA). For antigen ELISA, CEACAM1 (SEQ ID NO:64) (Catalog No. 10822-H08H, SinoBiological, China), CHIM (SEQ ID NO:63), CEA (SEQ ID NO:55) or CEACAM6 (SEQ ID NO:57) was coated overnight at 4 °C in 96-well plates at a concentration of 10 μg / ml. Color development was performed using an HRP-conjugated anti-human Fc (Fc-specific) IgG antibody (Catalog No. A0170, Sigma, USA) and a substrate (Catalog No. 00-4201-56, Invitrogen, USA), and the absorbance signal at a wavelength of 450 nm was measured using a microplate reader (SpectraMax Paradigm, Molecular Devices, USA). As Figure 9A-9B shown, no cross-reactivity with other CEACAM family members was observed; BGA5384 was specific only for CEA( Figure 9A -CEACAM3 in B). Example 12. Effect of soluble huCEA on the binding of BGA5384 to CEA-expressing cells

[0408] To determine whether soluble CEA (sCEA) has any effect on the specific binding of BGA5384, different concentrations (0, 0.5, 1 or 2 μg / ml) of recombinant soluble CEA were pre-mixed and incubated with (0.01 - 100 μg / ml) BGA5384 for 5 minutes. Then the mixture was incubated with 2 × 10 5 CEA-expressing cells such as MKN45 cells for 30 minutes at 4 °C. The cells were stained with a secondary antibody anti-huFc-APC (Catalog No. 409320, Biolegend, USA) and analyzed by flow cytometry. In the presence of 2 μg / ml recombinant sCEA, the binding of BGA5384 to CEA-expressing cells was not affected. This result is shown for MKN45 cells( Figure 10 ), and indicates that BGA5384 is specific for the membrane-bound form of CEA. Example 13. Potent ADCC effect of BGA6710 on CEA + tumor cells

[0409] To determine whether wild-type IgG1-form BGA6710 can induce antibody-dependent cell cytotoxicity (ADCC), NK92MI cells expressing CD16 (V158) (NK92MI / CD16V) were used as effector cells and co-cultured with mouse colon cancer cells expressing CEA (CT26 - ATCC CRL-2638). In the presence of BGA6710 at the indicated concentrations (0.00005 - 5 μg / ml), co-culture was performed at an E:T ratio of 1:1 for 5 hours, and cytotoxicity was determined by lactate dehydrogenase (LDH) release. The amount of LDH in the supernatant was measured using the CytoTox TM 96 Non-Radioactive Cytotoxicity Assay Kit (Promega, Madison, WI) and the amount of specific lysis was calculated according to the manufacturer's instructions. As Figure 11 shown, BGA6710 can induce ADCC in vitro, with an EC 50 of approximately 6.7 ng / ml. Example 14. In Vivo Antitumor Efficacy of BGA6710

[0410] To determine the in vivo efficacy of BGA6710 against CEA + tumor cells, NK92MI / CD16V cells (5 x 10 6 cells) were mixed with CT26 / CEA cells (10 6 cells) and injected subcutaneously into NCG mice. Starting from the day of tumor injection, BGA6710 (0.12, 0.62, or 3.1 mg / kg) or vehicle control (7 mice per group) was administered twice a week. Compared with the vehicle, the 3.1 mg / kg dose of BGA6710 showed a small amount of tumor inhibition, although the difference from the vehicle control was not statistically significant (P > 0.05)( Figure 12 ). Example 15. Synthesis of Cytotoxic Agent (Payload) Table 16: List of Payloads UPLC Analysis Method:

[0411] Method A: Mobile phase A: Aqueous solution of 0.1% FA, B: MeCN; Gradient: 10% B for 0.2 min, 10% - 95% B for 5.8 min, 95% B for 0.5 min; Flow rate: 0.6 mL / min; Column: ACQUITY BEH C18 1.7 μm.

[0412] Method B: Mobile phase A: Aqueous solution of 0.1% FA, B: MeCN; Gradient: 10% B is maintained for 0.5 min, 10% - 90% B is maintained for 2.5 min, 90% B is maintained for 0.2 min; Flow rate: 0.6 mL / min; Column: ACQUITY BEH C18 1.7μm.

[0413] Method C: Mobile phase A: Aqueous solution of 0.1% FA, B: MeCN; Gradient: 10% B is maintained for 0.2 min, 10% - 90% B is maintained for 1.3 min, 90% B is maintained for 0.3 min; Flow rate: 0.6 mL / min; Column: ACQUITY BEH C18 1.7μm.

[0414] P1 and P2 (Table 16) are commercially available and were purchased from MedChemExpress Co., Ltd. in Shanghai. Synthesis procedures for payloads P3 and P4

[0415] Payload P3

[0416] Step 1: N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[d']pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-3-hydroxy-2,2-dimethylpropanamide (P3). To a mixture of P3a (5.0 mg, 0.042 mmol) and HATU (16 mg, 0.042 mmol) in DMF (1 mL), add DIEA (21 μL, 16 mg, 0.13 mmol) and exatecan mesylate (23 mg, 0.043 mmol, purchased from MedChemExpress Co., Ltd.). Stir the resulting brown mixture at room temperature for 2 hours. After completion of the reaction, purify the mixture by preparative HPLC (TFA) (Method: Column: XBridge Prep C18 OBD 5um 19*150mm; Mobile phase: A - water (0.1% TFA): B - acetonitrile; Flow rate: 20 mL / min), lyophilize the fractions to obtain P3 (15 mg, 65.5% yield), as a white powder. MS (ESI) m / z: 536.4 [M+H] + . 11H NMR (400 MHz, DMSO-d6) δ 8.00 (d, J = 8.4 Hz, 1H), 7.79 (d, J = 11.2 Hz, 1H), 7.31 (s, 1H), 6.52 (s, 1H), 5.59–5.54 (m, 1H), 5.42 (s, 2H), 5.18 (q, J = 19.2 Hz, 2H), 4.87 (t, J = 5.2 Hz, 1H), 3.45 (dd, J = 10.2, 4.8 Hz, 1H), 3.41–3.28 (m, 1H), 3.15 (t, J = 5.6 Hz, 2H), 2.40 (s, 3H), 2.24–2.07 (m, 2H), 1.92–1.80 (m, 2H), 1.11 (d, J = 7.6 Hz, 6H), 0.87 (t, J = 7.2 Hz, 3H). Payload P4

[0417] Step 1: Diethyl 2-fluoro-2-methylmalonate (P4b). A solution of compound P4a (10.00 g, 57.40 mmol) in THF (200 mL) was cooled to 0 °C. 60% NaH (3.21 g, 80.37 mmol) in oil was added portionwise to the mixture, and the mixture was stirred at 0 °C for 30 minutes. Then N-fluoro-N-(phenylsulfonyl)benzenesulfonamide (NSFI, 19.91 g, 63.20 mmol) was added portionwise to the mixture at 0 °C, and then the mixture was warmed to room temperature and stirred for 16 hours. After completion of the reaction, the suspension was filtered and the filtrate was concentrated. PE (100 mL) was added to the residue, the precipitate was filtered off, and the filtrate was concentrated to give compound P4b (12.50 g, crude) as a pale yellow oil. 1 1H NMR (400 MHz, CDCl3) δ 4.30 (q, J = 7.2 Hz, 4H), 1.79 (d, J = 22.0 Hz, 3H), 1.31 (t, J = 7.2 Hz, 6H). 19 19F NMR (376 MHz, CDCl3) δ -157.50.

[0418] Step 2: 3-Ethoxy-2-fluoro-2-methyl-3-oxopropanoic acid (P4c). A solution of KOH (321 mg) in H2O (50 μL) and EtOH (2 mL) was added dropwise to a solution of compound P4b (1.00 g, 5.20 mmol) in EtOH (5 mL) at 0 °C. The mixture was stirred at room temperature for 2 h. The mixture was diluted with 20 (mL), washed with DCM (20 mL * 3). The aqueous solution was adjusted to pH = 3 with 1N HCl, then extracted with EtOAc (50 mL * 3). The organic layers were dried, combined and dried over anhydrous Na2SO4, filtered and concentrated to give compound P4c (470 mg, 55.0% yield) as a colorless oil. 1 H NMR (400 MHz, CDCl3) δ 8.31 (br s, 1H), 4.32 (q, J = 7.2 Hz, 2H), 1.83 (d, J = 22.0 Hz, 3H), 1.33 (t, J = 7.2 Hz, 3H). 19 F NMR (376 MHz, CDCl3) δ -157.59.

[0419] Step 3: 2-Fluoro-3-hydroxy-2-methylpropanoic acid (P4d). 2M LiBH4 (1.22 mL, 2.44 mmol) was added to a solution of compound P4c (200 mg, 1.22 mmol) in isopropanol (4 mL) at 0 °C. The mixture was stirred at room temperature for 2 h. The mixture was quenched dropwise with 2N HCl (1.22 mL) at 0 °C. The mixture was diluted with H2O (10 mL), extracted with EtOAc (50 mL * 3). The organic layers were combined and dried over anhydrous Na2SO4, filtered and concentrated to give compound P4d (92 mg, 61.7% yield) as a colorless oil. 1 H NMR (400 MHz, CDCl3) δ 4.01–3.81 (m, 2H), 1.58 (d, J = 21.2 Hz, 3H). 19 F NMR (376 MHz, CDCl3) δ -163.98.

[0420] Step 4: N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[d']pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-2-fluoro-3-hydroxy-2-methylpropanamide (P4). To a solution of compound P4d (23 mg, 0.19 mmol) in DMF (2 mL) was added irinotecan mesylate (50 mg, 0.094 mmol), HATU (54 mg, 141 mmol), and DIEA (36 mg, 0.28 mmol). The mixture was stirred at room temperature for 1 h. The mixture was purified by preparative HPLC (FA) (method: column: XBridge Prep C18 OBD 5um 19*150 mm; mobile phase: A - water (0.1% formic acid): B - acetonitrile; flow rate: 20 mL / min), and the fractions were lyophilized to give two isomers:

[0421] Isomer 1: P4: white solid, (11 mg, 21.9% yield). UPLC-MS, RT = 3.52 min. 1 1H NMR (400 MHz, DMSO-d6) δ 9.06 (dd, J = 9.0, 2.8 Hz, 1H), 8.00 (d, J = 10.9 Hz, 1H), 7.54 (s, 1H), 6.75 (s, 1H), 5.82 (d, J = 8.0 Hz, 1H), 5.65 (s, 2H), 5.43 (dt, J = 77.8, 12.4 Hz, 3H), 4.17–3.91 (m, 1H), 3.83 (ddd, J = 18.0, 12.4, 5.6 Hz, 1H), 3.40–3.27 (m, 1H), 2.62 (s, 3H), 2.50–2.34 (m, 2H), 2.22–1.98 (m, 2H), 1.81 (d, J = 21.4 Hz, 3H), 1.11 (t, J = 7.2 Hz, 3H); MS(ESI) m / z: 540.3 [M + H] + .

[0422] Isomer 2: P4-1: white solid, (8.4 mg, 16.6% yield). UPLC-MS, RT = 3.86 min. 11H NMR (400 MHz, DMSO-d6) δ 8.72 (dd, J = 8.4, 2.4 Hz, 1H), 7.78 (d, J = 11.2 Hz, 1H), 7.31 (s, 1H), 6.52 (s, 1H), 5.58 (d, J = 8.0 Hz, 1H), 5.42 (s, 2H), 5.32 - 5.05 (m, 3H), 3.83 (dd, J = 26.8, 12.0 Hz, 1H), 3.61 (dd, J = 21.6, 12.0 Hz, 1H), 3.22–3.07 (m, 2H), 2.46–2.30 (m, 3H), 2.28–2.05 (m, 2H), 2.02–1.74 (m, 2H), 1.45 (d, J = 21.4 Hz, 3H), 0.87 (t, J = 7.2 Hz, 3H); MS (ESI) m / z: 540.3 [M+H] + 。 Example 16. Synthesis of Linker-Payload Table 17: Linker-Payload List

[0423] LD2-1 and LD2-2 (Table 17) are commercially available and were purchased from MedChemExpress Co., Ltd. in Shanghai. Synthesis Procedures of Linker-Cytotoxic Agents LD2-3 to LD2-8 Linker-Cytotoxic Agent LD2-3 Step 1: (5S,8S)-1-(9H-Fluoren-9-yl)-5-isopropyl-8,14,14-trimethyl-3,6,9-trioxo-2,12-dioxa-4,7,10-triazapentadecane-15-carboxylic acid benzyl ester (LD2-3c). LD2-3a (300 mg, 0.62 mmol, synthesized according to the reported procedure: ACS Med.Chem.Lett. [ACS Medicinal Chemistry Letters] 2019, 10, 1386 - 1392 and US9808537B2), LD2-3b (260 mg, 1.25 mmol) and A white suspension mixture of molecular sieve in anhydrous THF (10 mL) was stirred at room temperature for 10 minutes. Sc(OTf)3 (368 mg, 0.75 mmol) was added, and the resulting yellow suspension was stirred at room temperature for 4 hours. The yellow suspension mixture was filtered through a pad of diatomaceous earth and washed with EtOAc (30 mL). The combined organic layers were washed with saturated NaHCO3 (30 mL) and brine (30 mL), dried over Na2SO4, filtered, and the filtrate was concentrated in vacuo to give a residue. It was purified by silica gel column (MeOH / DCM = 0% - 5%), and the fractions were concentrated in vacuo to give LD2-3c (274 mg, 69.8% yield) as a white solid. MS (ESI) m / z: 652.6 [M+Na] + 。

[0424] Step 2: Benzyl 3-(((S)-2-((S)-2-amino-3-methylbutanamido)propanamido)methoxy)-2,2-dimethylpropanoate (LD2-3d). Et2NH (477 mg, 5.53 mmol) was added to a solution of LD2-3c (274 mg, 0.44 mmol) in DMF (5 mL). The mixture was stirred at room temperature for 20 minutes. The reaction mixture was concentrated in vacuo and co-evaporated with toluene twice to give LD2-3d (275 mg, crude) as a brown oil. MS (ESI) m / z: 430.4 [M+Na] + 。

[0425] Step 3: Benzyl (5S,8S,11S)-5-(3-((((2R,3S,4R,5S)-5-(2-amino-2-oxoethyl)-3,4-dihydroxytetrahydrofuran-2-yl)methyl)amino)-3-oxopropyl)-1-(9H-fluoren-9-yl)-8-isopropyl-11,17,17-trimethyl-3,6,9,12-tetraoxo-2,15-dioxa-4,7,10,13-tetraazaoctadec-18-oate (LD2-3f). HATU (198 mg, 0.52 mmol) and DIPEA (168 mg, 1.30 mmol) were added to a solution of LD2-3d (275 mg, crude) and LD2-3e (282 mg, 0.52 mmol, purchased from WuXi AppTec) in DMF (5 mL). The mixture was stirred at room temperature for 10 minutes. The mixture was purified by reverse phase (C18, 60 g, 30% - 70%), and the fractions were lyophilized to give LD2-3f (370 mg, 91.5% yield) as a brown solid. MS (ESI) m / z: 953.8 [M+Na] + 。

[0426] Step 4: (5S,8S,11S,17R)-5-(3-((((2R,3S,4R,5S)-5-(2-amino-2-oxoethyl)-3,4-dihydroxytetrahydrofuran-2-yl)methyl)amino)-3-oxopropyl)-1-(9H-fluoren-9-yl)-17-fluoro-8-isopropyl-11,17-dimethyl-3,6,9,12-tetraoxo-2,15-dioxa-4,7,10,13-tetraazaoctadec-18-oic acid (LD2-3g). Pd / C (10%, 600 mg) was added to a solution of compound LD2-3f (3.01 g, 3.21 mmol) in a co-solvent DMF-MeOH (40 mL, 1:1, v:v). The mixture was stirred under H2 atmosphere (15 psi) for 7 h. The mixture was filtered through a Celite pad, concentrated, and the compound LD2-3g (2.50 g, crude) was obtained as a white solid. MS (ESI) m / z: 863.7 [M+Na] + 。

[0427] Step 5: ((6S,9S,12S)-1-((2R,3S,4R,5S)-5-(2-amino-2-oxoethyl)-3,4-dihydroxytetrahydrofuran-2-yl)-19-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[d']pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-9-isopropyl-12,18,18-trimethyl-3,7,10,13,19-pentaoxo-16-oxa-2,8,11,14-tetraazanonadec-6-yl)carbamic acid (9H-fluoren-9-yl) methyl ester (LD2-3h). Compound LD2-3g (692 mg, 1.30 mmol), HATU (675 mg, 1.78 mmol) and DIEA (459 mg, 3.55 mmol) were added to a solution of compound irinotecan mesylate (1000 mg, 1.18 mmol, purchased from MedChemExpress Co., Ltd.) in DMF (20 mL). The mixture was stirred at room temperature for 30 min. The mixture was concentrated and purified by silica gel column chromatography (eluent: DCM / MeOH = 0% to 20%) to give the title compound LD2-3h (1.32 g, 88.6% yield) as an off-white solid. MS (ESI) m / z: 1282.1 [M+Na] + 。

[0428] Step 6: (S)-2-Amino-N5-(((2R,3S,4R,5S)-5-(2-amino-2-oxoethyl)-3,4-dihydroxytetrahydrofuran-2-yl)methyl)-N1-((S)-1-(((S)-1-(((3-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[d']pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-2,2-dimethyl-3-oxopropoxy)methyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)pentanediamide (LD2-3i). To a solution of compound LD2-3h (1000 mg, 0.79 mmol) in DMF (20 mL) was added Et2NH (580 mg, 7.93 mmol). The mixture was stirred at room temperature for 30 minutes. The mixture was concentrated under high vacuum to give compound LD2-3i (825 mg, crude), as an off-white solid, which was used directly without further purification. MS(ESI) m / z: 1036.9 [M+H] + .

[0429] Step 7: (S)-N5-(((2R,3S,4R,5S)-5-(2-amino-2-oxoethyl)-3,4-dihydroxytetrahydrofuran-2-yl)methyl)-2-(3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetamido)propanamido)-N1-((S)-1-(((S)-1-(((3-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[d']pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-2,2-dimethyl-3-oxopropoxy)methyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)glutaramide (LD2-3). To a solution of LD2-3j (15 mg, 0.067 mmol) in anhydrous DMF (1.0 mL) was added HATU (26 mg, 0.067 mmol) and DIEA (0.017 mL, 0.097 mmol), and the mixture was stirred at room temperature for 15 minutes. Then LD2-3i (50 mg, 0.048 mmol) was added to the above mixture, and the mixture was stirred at room temperature for 10 minutes. The resulting solution was purified by preparative HPLC (method: column: XBridge Prep C18 OBD 5um 19*150mm; mobile phase: A - water (0.1% TFA): B - acetonitrile; flow rate: 20 mL / min), and the fractions were lyophilized to give LD2-3 (37 mg, 50.9% yield) as a yellow solid. MS (ESI) m / z: 1266.7 [M+Na] + . Linker-cytotoxic agent LD2-4

[0430] Step 1: (S)-11-benzyl-1-(9H-fluoren-9-yl)-20,20-dimethyl-3,6,9,12,15-pentaoxo-2,18-dioxa-4,7,10,13,16-pentaazapentacosan-21-yl benzoate (LD2-4c). To a solution of LD2-4a (250 mg, 0.40 mmol) and LD2-3b (83 mg, 0.40 mmol) in THF (5 mL) was added Molecular sieve. The mixture was stirred at room temperature for 10 minutes, then Sc(OTf)3 (195 mg, 0.40 mmol) was added and the reaction was allowed to proceed further at room temperature for 16 hours. The suspension mixture was filtered through a Celite pad, and the filter cake was washed with THF (10 mL). Then the filtrate was quenched by adding saturated NaHCO3 (10 mL) and extracted with EtOAc (30 mL * 2). After separation, the combined organic layers were washed with brine (50 mL), dried over Na2SO4, filtered, and the filtrate was concentrated under vacuum to give a residue. The residue was further purified by silica gel column chromatography (A - DCM; B - MeOH, MeOH / DCM = 0% - 5%) to give LD2 - 4c (90 mg, 29.2% yield), as a white solid. MS (ESI) m / z: 800.5 [M + Na] + .

[0431] Step 2: (S)-11-Benzyl-1-(9H-fluoren-9-yl)-20,20-dimethyl-3,6,9,12,15-pentaoxo-2,18-dioxa-4,7,10,13,16-pentaazanonacos-21-oic acid (LD2-4d). To a solution of LD2-4c (80 mg, 0.10 mmol) in MeOH (3 mL) was added wet Pd / C (20 mg). The black suspension was purged with a H2 balloon three times and then reacted under a H2 balloon at room temperature for 2 hours. After completion of the reaction, the black suspension was filtered through a Celite pad and the filter cake was washed with MeOH. The combined organic layers were concentrated under vacuum to give LD2-4d (61 mg, 84.8% yield). MS (ESI) m / z: 710.4 [M + Na] + .

[0432] Step 3: ((S)-7-Benzyl-17-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[d']pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-16,16-dimethyl-2,5,8,11,17-pentaoxo-14-oxa-3,6,9,12-tetraazapentadecyl)carbamic acid (9H-fluoren-9-yl)methyl ester (LD2-4f). DIEA (43 μL, 34 mg, 0.26 mmol) was added to a mixture of LD2-4d (60 mg, 0.087 mmol) and HATU (33 mg, 0.087 mmol) in DMF (2 mL). The mixture was allowed to react at room temperature for 10 minutes. Irinotecan mesylate (46 mg, 0.087 mmol) was added and the mixture was allowed to react for an additional 1 hour at the same temperature. After completion of the reaction, the mixture was filtered and the filtrate was purified by preparative HPLC (Method: Column: XBridge Prep C18 OBD 5um 19*150mm; Mobile phase: A - water (0.1% formic acid): B - acetonitrile; Flow rate: 20 mL / min), to give LD2-4f (85 mg, 88.2% yield). MS (ESI) m / z: 1105.5 [M+H] + .

[0433] Step 4: 3-(((S)-13-Amino-7-benzyl-3,6,9,12-tetraoxo-2,5,8,11-tetraazatridecyl)oxy)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[d']pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-2,2-dimethylpropanamide (LD2-4g). Et2NH (64 μL, 46 mg, 0.62 mmol) was added to a solution of LD2-4f (85 mg, 0.062 mmol) in DMF (2 mL). The mixture was stirred at room temperature for 0.5 hour. After completion of the reaction, the mixture was concentrated in vacuo to give LD2-4g (86 mg, crude) as a yellow solid. MS (ESI) m / z: 883.5 [M+H] + .

[0434] Step 6: ((6S,15S)-15-Benzyl-25-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[d']pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-24,24-dimethyl-3,7,10,13,16,19,25-heptaoxo-1-((2S,3R,4R,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)-22-oxa-2,8,11,14,17,20-hexaazapentacos-6-yl)carbamic acid (9H-fluoren-9-yl)methyl ester (LD2-4i). To a solution of LD2-4g (86 mg, crude) and LD2-4h (43 mg, 0.079 mmol, purchased from Wuxi WuXi AppTec Co., Ltd.) in DMF (1.5 mL) was added DIEA (26 μL, 21 mg, 0.16 mmol). The mixture was stirred at room temperature for 1.5 h. After completion of the reaction, the mixture was purified by preparative HPLC (FA) (method: column: XBridge Prep C18 OBD 5um 19*150 mm; mobile phase: A - water (0.1% formic acid): B - acetonitrile; flow rate: 20 mL / min), and the fractions were lyophilized to give LD2-4i (70 mg, 62.6% yield) as a white powder. MS (ESI) m / z: 1410.7 [M+H] + .

[0435] Step 7: (S)-2-Amino-N1-((S)-7-benzyl-17-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[d']pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-16,16-dimethyl-2,5,8,11,17-pentaoxo-14-oxa-3,6,9,12-tetraazadecyl)-N5-(((2S,3R,4R,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)methyl)pentanediamide (LD2-4j). To a solution of LD2-4i (70 mg, 0.050 mmol) in DMF (1 mL) was added Et2NH (51 μL, 36 mg, 0.50 mmol). The mixture was stirred at room temperature for 0.5 h. After completion of the reaction, the mixture was concentrated in vacuo to give LD2-4j (71 mg, crude) as a yellow solid. MS (ESI) m / z: 1188.2 [M+H] + .

[0436] Step 8: (S)-N 1 -((S)-7-Benzyl-17-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[d']pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-16,16-dimethyl-2,5,8,11,17-pentaoxo-14-oxa-3,6,9,12-tetraazapentadecyl)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-N 5 -(((2S,3R,4R,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)methyl)glutaramide (LD2-4). To a solution of LD2-4k (19 mg) in DMF (3 mL) was added HATU (34 mg, 0.088 mmol) and DIEA (10 μL, 7.6 mg, 0.059 mmol). The resulting yellow solution was stirred at room temperature for 5 minutes, then LD2-4j (71 mg, crude) was added. The mixture was stirred at room temperature for 60 minutes. After completion of the reaction, the mixture was purified by preparative HPLC (FA) (method: column: XBridge Prep C18 OBD 5um 19*150mm; mobile phase: A - water (0.1% formic acid): B - acetonitrile; flow rate: 20 mL / min), and the fractions were lyophilized to give LD2-4 (32 mg, 26.3% yield) as a white powder. MS (ESI) m / z: 1381.1 [M+H] + 。 Linker-Cytotoxin LD2-5

[0437] LD2-5 (30 mg, 50.7% yield) was synthesized according to the synthetic procedure of LD2-4. MS (ESI) m / z: 1408.1 [M+Na] + 。 Linker-Cytotoxin LD2-6

[0438] Step 1: N-((((9H-Fluoren-9-yl)methoxy)carbonyl)-L-valyl)-O-((2R,3R,4S,5S,6S)-3,4,5-triacetoxy-6-(methoxycarbonyl)tetrahydro-2H-pyran-2-yl)-L-serine (LD2-6b). To a mixture of LD2-6a (4.10 g, 4.92 mmol, purchased from MedChemExpress Co., Ltd.) in MeOH (50 mL), THF (100 mL) and DCM (20 mL) was added wet Pd / C (400 mg, 10% purity). The black suspension was purged with an H2 balloon three times and then stirred at room temperature for 1 h. The black suspension was filtered through a Celite pad and washed with MeOH (200 mL). The organic layers were combined and concentrated in vacuo to afford LD2-6b (3.65 g, 99.8% yield) as an off-white solid. MS (ESI) m / z: 743.6 [M+H] + .

[0439] Step 2: (2R,3R,4S,5S,6S)-2-((S)-2-((S)-2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanamido)-3-((2-(benzyloxy)-2-oxoethyl)amino)-3-oxopropoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (LD2-6d). To a solution of LD2-6b (3.65 g, 4.92 mmol) and LD2-6c (1.66 g, 4.92 mmol) in DMF (50 mL) was added HATU (1.87 g, 4.92 mmol) and DIEA (1.59 g, 12.29 mmol). The mixture was stirred at room temperature for 30 min. The mixture was purified by FCC (MeOH / DCM = 0% - 10%) and the fractions were concentrated in vacuo to afford LD2-6d (3.80 g, 86.9% yield) as an off-white foamy solid. MS (ESI) m / z: 890.7 [M+H] + .

[0440] Step 3: N-((((9H-Fluoren-9-yl)methoxy)carbonyl)-L-valyl)-O-((2R,3R,4S,5S,6S)-3,4,5-triacetoxy-6-(methoxycarbonyl)tetrahydro-2H-pyran-2-yl)-L-seryl-glycine (LD2-6e). To a mixture of LD2-6d (3.80 g, 4.27 mmol) in MeOH (150 mL) and DCM (50 mL) was added wet Pd / C (400 mg, 10% purity). The black suspension was purged with a H2 balloon three times and then stirred at room temperature for 40 minutes. The black suspension was filtered through a Celite pad and washed with MeOH (150 mL). The organic layers were combined and concentrated in vacuo to give LD2-6e (3.30 g, 96.6% yield) as an off-white solid. MS (ESI) m / z: 800.7 [M+H] + .

[0441] Step 4: (2R,3R,4S,5S,6S)-2-((S)-2-((S)-2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanamido)-3-((acetoxymethyl)amino)-3-oxopropoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (LD2-6f). To a solution of LD2-6e (3.30 g, 4.13 mmol) in DMF (30 mL) was added Pb(OAc)4 (2.74 g, 6.19 mmol), Cu(OAc)2 (74.9 mg, 0.41 mmol) and HOAc (247.8 mg, 4.13 mmol). The resulting dark mixture was purged with a N2 balloon three times and then stirred at 65 °C for 40 minutes, and the mixture turned dark blue. The mixture was diluted with EtOAc (300 mL), washed with brine (100 mL * 3), dried over Na2SO4, filtered and concentrated in vacuo to give a residue. It was purified by FCC (MeOH / DCM = 0 - 10%) and the fractions were concentrated in vacuo to give LD2-6f (2.81 g, 83.4% yield) as a pale yellow solid. MS (ESI) m / z: 836.6 [M+Na] + .

[0442] Step 5: (2R,3R,4S,5S,6S)-2-((S)-2-((S)-2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanamido)-3-(((3-(Benzyloxy)-2,2-dimethyl-3-oxopropoxy)methyl)amino)-3-oxopropoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (LD2-6h). A white suspension mixture of LD2-6f (300 mg, 0.37 mmol), LD2-3b (154 mg, 0.74 mmol) and molecular sieve (200 mg) in anhydrous THF (10 mL) was stirred at room temperature for 10 minutes. Sc(OTf)3 (218 mg, 0.44 mmol) was added, and the resulting yellow suspension was stirred at room temperature for 4 hours. The yellow suspension mixture was filtered through a Celite pad and washed with EtOAc. The combined organic layers were washed with saturated NaHCO3 (30 mL) and brine (30 mL), dried over Na2SO4, filtered, and the filtrate was concentrated in vacuo to give a residue. It was purified by silica gel column (MeOH / DCM = 0% - 5%), and the fractions were concentrated in vacuo to give LD2-6h (275 mg, 77.5% yield) as a white foam solid. MS(ESI) m / z: 984.8 [M+Na] + .

[0443] Step 6: (5S,8S)-1-(9H-Fluoren-9-yl)-5-isopropyl-14,14-dimethyl-3,6,9-trioxo-8-((((2R,3R,4S,5S,6S)-3,4,5-triacetoxy-6-(methoxycarbonyl)tetrahydro-2H-pyran-2-yl)oxy)methyl)-2,12-dioxo-4,7,10-triazapentadecanoic acid (LD2-6j). To a solution of LD2-6h (275 mg, 0.29 mmol) in MeOH (10 mL) was added wet Pd / C (55 mg, 10% purity). The black suspension was purged with H2 balloon three times and then stirred at room temperature for 2 hours. The mixture was filtered through a syringe filter and washed with MeOH (15 mL), and concentrated in vacuo to give LD2-6j (230 mg, crude) as a white foam solid. MS(ESI) m / z: 894.6 [M+Na] + .

[0444] Step 7: (2R,3R,4S,5S,6S)-2-((S)-2-((S)-2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanamido)-3-(((3-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[d']pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-2,2-dimethyl-3-oxopropoxy)methyl)amino)-3-oxopropoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (LD2-6k). To a mixture of LD2-6j (230 mg, crude), irinotecan mesylate (140 mg, 0.26 mmol) and HATU (100 mg, 0.26 mmol) in DMF (5 mL) was added DIEA (102 mg, 0.79 mmol). The resulting brown mixture was stirred at room temperature for 1 h. The mixture was diluted with EtOAc (20 mL), washed with brine (20 mL * 3), dried over Na2SO4, filtered, and concentrated in vacuo to give a residue. It was purified by FCC (MeOH / DCM = 0% - 3%), concentrated in vacuo to give LD2-6k (325 mg, 95.6% yield) as an off-white foamy solid. MS (ESI) m / z: 1289.9 [M+H] + .

[0445] Step 8: (2S,3S,4S,5R,6R)-6-((S)-2-((S)-2-Amino-3-methylbutanamido)-3-(((3-(((1S,9S)-9-Ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[d']pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-2,2-dimethyl-3-oxopropoxy)methyl)amino)-3-oxopropoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (LD2-6l). To a solution of LD2-6k (325 mg, 0.25 mmol) in DMF (5 mL) was added Et2NH (523 mg, 5.06 mmol). The mixture was stirred at room temperature for 20 minutes. LCMS showed the reaction was complete, then it was concentrated under vacuum to give the crude product. It was dissolved in MeOH (6 mL), K2CO3 (174.7 mg, 1.26 mmol) was added and the mixture was stirred at room temperature for 10 minutes, then H2O (2 mL) was added to the mixture and it was stirred at room temperature for 30 minutes. The mixture was acidified to pH = 3 with saturated KHSO4 at 0 °C, filtered and purified by preparative HPLC (0.1% FA), and the fractions were lyophilized to give LD2-6l (140 mg, 59.7% yield) as a pale yellow solid. MS (ESI) m / z: 927.4 [M+H] + . 1 1H NMR (400 MHz, d6-DMSO) δ 9.56 (s, 1H), 8.39 (s, 1H), 8.07 (d, J = 8.4 Hz, 1H), 7.77 (d, J = 11.2 Hz, 1H), 7.31 (s, 1H), 6.52 (s, 1H), 5.54 (dd, J = 13.2, 7.2 Hz, 1H), 5.43 (s, 2H), 5.18 (dd, J = 41.6, 18.8 Hz, 2H), 5.09–5.02 (m, 1H), 4.96 (s, 1H), 4.62 (dd, J = 10.0, 6.8 Hz, 1H), 4.56–4.44 (m, 2H), 4.19 (d, J = 7.6 Hz, 1H), 3.82 (dd, J = 10.8, 6.8 Hz, 1H), 3.61 (dd, J = 11.6, 6.4 Hz, 2H), 3.17 - 3.05 (m, 4H), 2.94 (t, J = 8.0 Hz, 1H), 2.39 (s, 3H), 2.11 (dt, J = 21.3, 7.6 Hz, 2H), 2.03–1.93 (m, 2H), 1.92–1.78 (m, 3H), 1.12 (d, J = 8.0 Hz, 6H), 0.87 (dd, J = 13.0, 6.6 Hz, 9H).

[0446] Step 9: Methyl 4-(5-(methylthio)-1,2,4-thiadiazol-3-yl)benzoate (LD2-6o). To a solution of compound LD2-6m (100 mg, 0.47 mmol) in toluene (4 mL) and H2O (1 mL) was added compound LD2-6n (110 mg, 0.57 mmol), K2CO3 (168 mg, 0.95 mmol) and Pd(dppf)Cl2 . DCM (35 mg, 0.047 mmol). The mixture was stirred at 110 °C under N2 atmosphere for 3 h. The mixture was filtered through a pad of Celite, diluted with EtOAc (100 mL), and washed with brine (50 mL×4). The organic layer was dried over anhydrous Na2SO4, filtered, and concentrated. The crude product was purified by flash column chromatography (eluting with PE / EtOAc = 0% - 40%). Compound LD2-6o (56 mg, 44.4% yield) was obtained as an off-white solid. MS (ESI) m / z: 267.1 [M+H] + 。

[0447] Step 10: 4-(5-(Methylthio)-1,2,4-thiadiazol-3-yl)benzoic acid (LD2-6p). To a solution of compound LD2-6o (54 mg, 0.20 mmol) in MeOH (3 mL) and H2O (1 mL) was added LiOH (17 mg, 0.41 mmol). The mixture was stirred at room temperature for 2 h. The mixture was adjusted to pH 7 and purified by preparative HPLC (FA conditions) to give compound LD2-6p (36 mg, 70.3% yield) as a white solid. MS (ESI) m / z: 253.1 [M+H] + 。

[0448] Step 11: 4-(5-(Methylsulfonyl)-1,2,4-thiadiazol-3-yl)benzoic acid (LD2-6q). To a solution of compound LD2-6p (35 mg, 0.14 mmol) in DCM (3 mL) and THF (3 mL) was added m-CPBA (96 mg, 0.55 mmol). The mixture was stirred at room temperature for 16 h. The mixture was concentrated and purified by preparative HPLC (method: column: XBridgePrep C18 OBD 5um 19*150mm; mobile phase: A - water (0.1% TFA): B - acetonitrile; flow rate: 20 mL / min). Compound LD2-6q (12 mg, 99% purity) was obtained as a white solid. MS (ESI) m / z: 284.8 [M+H] + 。

[0449] Step 12: (2S,3S,4S,5R,6R)-6-((S)-3-(((3-(((1S,9S)-9-Ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[d']pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-2,2-dimethyl-3-oxopropoxy)methyl)amino)-2-((S)-3-methyl-2-(4-(5-(methylsulfonyl)-1,2,4-thiadiazol-3-yl)benzamido)butanamido)-3-oxopropoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (LD2-6). HATU (9. mg, 0.024 mmol) and DIEA (5.6 mg, 0.043 mmol) were added to a solution of compound LD2-6q (7.4 mg, 0.026 mmol) in DMF (2 mL). The mixture was stirred at room temperature for 30 minutes. Compound LD2-6l (20 mg, 0.022 mmol) was added to the mixture and stirred at room temperature for 15 minutes. The reaction was purified by preparative HPLC (method: column: XBridge Prep C18 OBD 5um 19*150 mm; mobile phase: A - water (0.1% TFA): B - acetonitrile; flow rate: 20 mL / min), to give compound LD2-6 (7.6 mg, 29.5% yield) as a white solid. MS (ESI) m / z: 1193.5 [M+H] + . Linker-Cytotoxin LD2-7

[0450] LD2-7 (32 mg, 50.9% yield) was synthesized according to the procedure of Step 7 of LD2-3. MS (ESI) m / z: 1303.0 [M+H] + . Linker-Cytotoxin LD2-8

[0451] Step 1: Methyl (R)-3-(((benzyloxy)carbonyl)amino)-4-((tert-butoxycarbonyl)amino)butanoate (LD2-8b). LD2-8a (2.00 g, 5.68 mmol) and K2CO3 (863 mg, 6.24 mmol) were added to DMF (10 mL), and then CH3I (1.61 g, 11.35 mmol) was added dropwise at 0 °C. The resulting mixture was stirred at 0 °C for 20 minutes and allowed to warm to 25 °C and further stirred at 25 °C for 60 minutes. The reaction process was monitored by TLC (PE / EA) and LCMS. After complete reaction, the reaction mixture was diluted with EA (80 mL) and washed with brine (30 mL * 3) and H2O (30 mL * 2). The organic layer was dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to give the methyl ester of LD2-8b (2.08 g, quantitative) as a pale yellow solid. MS (ESI) m / z: 267.2 [M - Boc + H] + .

[0452] Step 2: (4-Hydroxybutane-1,2-diyl)(R)-bis(benzyloxycarbonyl) tert-butylamine (LD2-8c). LD2-8b (1.00 g, 2.73 mmol) was dissolved in MeOH (15 mL), and then LiBH4 (2 M stock solution in THF, 6.80 mL) was added at 0 °C. The resulting mixture was stirred at 25 °C for 2 hours. The reaction process was monitored by LCMS and TLC. After complete reaction, saturated aqueous NH4Cl solution (10 mL) was added to quench the reaction. The reaction mixture was diluted with H2O (80 mL) and extracted with EA (50 mL * 3). The combined organic layers were washed with brine (40 mL * 2) and water (40 mL * 2), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure, and further purified by flash column chromatography (PE / EA) to give LD2-8c (760 mg, 82.3% yield) as a white solid. MS (ESI) m / z: 239.2 [M - Boc + H]+.

[0453] Step 3: (4-(((4-Nitrophenoxy)carbonyl)oxy)butane-1,2-diyl)(R)-bis(tert-butyl N-carbobenzoxy-L-aspartate) (LD2-8e). LD2-8c (300 mg, 0.89 mmol) and LD2-8d (405 mg, 1.33 mmol) were dissolved in DMF (5 mL), and then DIEA (229 mg, 1.77 mmol) was added. The resulting mixture was stirred at 25 °C for 1.5 h. After complete reaction, the reaction mixture was diluted with EA (100 mL) and washed with brine (35 mL * 2) and water (35 mL * 2). The organic layer was dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to give LD2-8e as a white solid (371 mg, 83.1% yield). MS (ESI) m / z: 404.4 [M - Boc + H] + .

[0454] Step 4: (4-(((3-(Dimethylamino)-3-oxopropyl)carbamoyl)oxy)butane-1,3-diyl)(S)-bis(9H-fluoren-9-ylmethyl N-carbamate) (LD2-8g). LD2-8e (420 mg, 0.83 mmol) and LD2-8f (149 mg, 1.67 mmol) were dissolved in DMF (5 mL), and then aqueous NaHCO3 solution (1 M, 5 mL) was added. The resulting mixture was stirred at 25 °C for 2.5 h. After complete reaction, the reaction mixture was concentrated and purified by flash column chromatography (DCM / MeOH) to give LD2-8g as a pale yellow solid (365 mg, 96.5% yield). MS (ESI) m / z: 354.4 [M - Boc + H] + .

[0455] Step 5: (R)-7-(2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)-2,2-dimethyl-4,11-dioxo-3,10-dioxa-5,12-diazapentadecanoic acid (LD2-8h). LD2-8g (360 mg, 0.79 mmol) was dissolved in MeOH (18 mL), and then Pd / C (wet base, 108 mg) was added. The resulting mixture was stirred at room temperature under H2 (15 psi) for 2 h. After complete reaction, the reaction mixture was filtered and concentrated under reduced pressure to give LD2-8h as a clear syrup (252 mg, 99.4% yield). The crude product was used directly in the next step without purification. MS (ESI) m / z: 320.3 [M + H] + .

[0456] Step 6: (R)-7-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-2,2-dimethyl-4,11-dioxo-3,10-dioxa-5,12-diazapentadec-15-oic acid (LD2-8j). LD2-8h (250 mg, 0.78 mmol) and LD2-8i (243 mg, 1.57 mmol) were dissolved in a mixed solvent of ACN (8 mL) and aqueous NaHCO3 solution (1 M, 16 mL). The resulting mixture was stirred at 0 °C for 1 h and further stirred at 25 °C until the reaction was complete. Then the reaction mixture was acidified with aqueous KHSO4 solution (20 mL) and extracted with EA (35 mL * 3). The combined organic layers were dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to give a yellow oil as the crude product, which was purified by flash column chromatography to give LD2-8j (280 mg, 89.6% yield) as a white solid. MS (ESI) m / z: 422.3 [M+Na] + . 1 1H NMR (400 MHz, d6-DMSO) δ 12.48 (s, 1H), 7.03 - 7.01 (m, 2H), 6.99 (s, 2H), 4.08 - 4.03 (m, 3H), 3.86 - 3.83 (m, 2H), 3.14 - 3.11 (m, 2H), 2.35 (t, J = 7.2 Hz, 2H), 2.16 - 2.09 (m, 1H), 1.9LD2-8.84 (m, 1H), 1.32 (s, 9H).

[0457] Step 7: ((8S,11S,14S)-14-(3-((((2R,3S,4R,5S)-5-(2-amino-2-oxoethyl)-3,4-dihydroxytetrahydrofuran-2-yl)methyl)amino)-3-oxopropyl)-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H'12'-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-11-isopropyl-2,2,8-trimethyl-1,7,10,13,16-pentaoxo-4-oxa-6,9,12,15-tetraazaoctadec-18-yl)carbamic acid (R)-4-((tert-butoxycarbonyl)amino)-3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)butyl ester (LD2-8l). LD2-8l (32 mg, 50.9% yield) was synthesized according to the procedure of Step 7 of Example LD2-3. MS (ESI) m / z: 1418.1 [M+H] + 。

[0458] Step 8: ((8S,11S,14S)-14-(3-((((2R,3S,4R,5S)-5-(2-amino-2-oxoethyl)-3,4-dihydroxytetrahydrofuran-2-yl)methyl)amino)-3-oxopropyl)-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H'12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-11-isopropyl-2,2,8-trimethyl-1,7,10,13,16-pentaoxo-4-oxa-6,9,12,15-tetraazaoctadec-18-yl)carbamic acid (R)-4-amino-3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)butyl ester (LD2-8). LD2-8 l (22 mg, 0.016 mmol) was dissolved in a mixed solvent of DCM (2 mL), and then ZnBr2 (151 mg, 0.67 mmol) was added. The resulting suspension was stirred at 40 °C for 12 h. After complete reaction, the reaction mixture was filtered and concentrated. The residue was diluted with a mixed solvent of CH3CN / 0.1% FA aqueous solution and purified by preparative HPLC (method: column: XBridge Prep C18 OBD 5um 19*250mm; mobile phase: A - water (0.1% TFA): B - acetonitrile; flow rate: 20 mL / min), to obtain compound LD2-8 (13 mg, 61.7% yield), as a white solid. MS(ESI) m / z: 1318.1 [M+H] + 。 Example 17. Preparation and evaluation method of antibody-drug conjugate (ADC)

[0459] Refer to the preparation of ADC BGA7650 (Table 18). An organic solvent (e.g., DMSO, DMF, DMA, PG, acetonitrile, 0 - 25% v / v) and linker-payload (2 - 25 equivalents, 10 mM stock solution in an organic solvent) were gradually added to a reaction buffer (PBS buffer, pH 6.0 - 9.0) with anti-CEA antibody tusamitamab (1 - 20 mg / mL) at 0 - 37 °C within 0.5 - 48 h. The solution buffer was exchanged (rotary desalting column, ultrafiltration, and dialysis) into a storage buffer (e.g., pH 5.5 - 6.5 histidine acetate buffer, with optional additives such as sucrose, trehalose, Tween 20, 60, 80).

[0460] Drug-to-antibody ratio (DAR) determination: LCMS method. LC-MS analysis was performed under the following measurement conditions: LC-MS System: Vanquish Flex UHPLC and Orbitrap Exploris 240 Mass Spectrometer Column: MAbPac TM RP, 2.1 * 50 mm, 4 μm, Thermo Scientific TM Column Temperature: 80 °C Mobile Phase A: Aqueous solution of 0.1% formic acid (FA); Mobile Phase B: Acetonitrile solution containing 0.1% formic acid (FA); Gradient Program: 25% B - 25% B (0 min - 2 min), 25% B - 50% B (2 min - 18 min), 50% B - 90% B (18 min - 18.1 min), 90% B - 90% B (18.1 min - 20 min), 90% B - 25% B (20 min - 20.1 min), 25% B - 25% B (20.1 min - 25 min) Injection Volume: 2 μg; MS Parameters: Obtain full and denatured MS data at a setting of R = 15k in HMR mode, and use ReSpect TM BioPharma Finder TM in the 4.0 software for deconvolution using the ReSpect TM algorithm and sliding window integration.

[0461] Preparation of DAR 8 Antibody - Drug Conjugates. The antibody in conjugation buffer (concentration 0.5 - 25 mg / mL, PBS buffer pH 6.0 - 8.5) was incubated for 10 minutes at a reduction temperature (0 - 40 °C), and 8 - 15 equivalents of TECP solution (5 mM stock solution in PBS buffer) was added to the reaction mixture, and the reduction reaction was maintained at the reduction temperature for 1 - 8 hours. After cooling the reduction mixture to 0 - 25 °C, organic solvents (e.g., DMSO, DMF, DMA, PG, acetonitrile, 0 - 25% v / v) and linker - payload stock solution (10 - 25 equivalents, 10 mM stock solution in organic solvent) were added step - by - step. The conjugation solution was placed at 0 - 25 °C for 1 - 3 hours, and the reaction was quenched with N - acetylcysteine (1 mM stock solution). The solution was buffer - exchanged (spin - desalting column, ultrafiltration, and dialysis) into a storage buffer (e.g., pH 5.5 - 6.5 histidine acetate buffer, with optional additives such as sucrose, trehalose, Tween 20, 60, 80).

[0462] Conjugated maleimide hydrolysis. After the conjugation step, the ADC buffer was exchanged into a ring-opening buffer (pH 7.0 - 9.0, PBS, borate or Tris buffer), and the solution was incubated at 22 or 37 °C for 5 - 48 hours. The ring-opening process was monitored via reduced LCMS. Once the hydrolysis of the conjugated maleimide was complete, the resulting ADC buffer was exchanged via dialysis into an alkaline Tris pH 8.0 - 8.5 buffer or an acidic histidine-acetate pH 5.0 - 6.5 buffer.

[0463] ADC Characterization. ADC examples were prepared by following the above procedure for the DAR 8 profile. All ADCs were characterized via the following analytical methods. The drug-to-antibody ratio (DAR) of the ADCs was determined by the LCMS method or the hydrophobic interaction column (HIC) method. The SEC purity of the constructed ADCs was >95%. DAR Determination

[0464] LCMS Method. LC-MS analysis was performed under the following measurement conditions: LC-MS System: Vanquish Flex UHPLC and Orbitrap Exploris 240 mass spectrometer Column: MAbPac TM RP, 2.1*50mm, 4μm, Thermo Scientific TM Column Temperature: 80 °C Mobile Phase A: 0.1% formic acid (FA) aqueous solution Mobile Phase B: Acetonitrile solution containing 0.1% formic acid (FA) Gradient Program: 25% B - 25% B (0 min - 2 min), 25% B - 50% B (2 min - 18 min), 50% B - 90% B (18 min - 18.1 min), 90% B - 90% B (18.1 min - 20 min), 90% B - 25% B (20 min - 20.1 min), 25% B - 25% B (20.1 min - 25 min) Injection Volume: 1 μg MS Parameters: Full and denatured MS data were obtained at a setting of R = 15k in HMR mode and deconvolution was performed using the ReSpect TM BioPharma Finder TM algorithm and sliding window integration in Thermo Scientific TM software version 4.0.

[0465] HIC Method. HPLC analysis was performed under the following measurement conditions: HPLC system: Waters ACQUITY ARC HPLC system Detector: Measurement wavelength: 280 nm Column: Tosoh Bioscience 4.6 μm ID × 3.5 cm, 2.5 μm butyl - porous resin column Column temperature: 25 °C Mobile phase A: 1.5 M ammonium sulfate, 50 mM phosphate buffer, pH 7.0 Mobile phase B: 50 mM phosphate buffer, 25% (V / V) isopropanol, pH 7.0 Gradient program: 0%B - 0%B (0 min - 2 min), 0%B - 100%B (2 min - 15 min), 100%B - 100%B (15 min - 16 min), 100%B - 0%B (16 min - 17 min), 0%B - 0%B (17 min - 20 min) Injection volume: 20 μg ADC purity: SEC method

[0466] HPLC analysis was performed under the following measurement conditions: HPLC system: Waters H - Class UPLC system Detector: Measurement wavelength: 280 nm Column: ACQUITY UPLC BEH200 SEC 1.7um 4.6x150mm, Waters Column temperature: Room temperature Mobile phase A: 200 mM phosphate buffer, 250 mM potassium chloride, 15% isopropanol, PH 7.0 Gradient program: Isocratic elution at a flow rate of 0.3 mL / min for 10 min Injection volume: 20 μg ADC hydrophobicity evaluation: HIC

[0467] ADCs with greater hydrophobic characteristics elute at a later retention time in HIC chromatography.

[0468] HPLC analysis was performed under the following measurement conditions: Method 1 HPLC system: Waters ACQUITY ARC HPLC system Detector: Measurement wavelength: 280 nm Column: Tosoh Bioscience 4.6 μm ID × 3.5 cm, 2.5 μm butyl - porous resin column Column temperature: 25 °C Mobile phase A: 1.5 M ammonium sulfate, 50 mM phosphate buffer, pH 7.0 Mobile phase B: 50 mM phosphate buffer, 25% (V / V) isopropanol, pH 7.0 Gradient program: 0% B - 0% B (0 min - 2 min), 0% B - 100% B (2 min - 15 min), 100% B - 100% B (15 min - 16 min), 100% B - 0% B (16 min - 17 min), 0% B - 0% B (17 min - 20 min) Injection volume: 20 μg Method 2

[0469] HPLC system: Waters ACQUITY ARC HPLC system Detector: Measurement wavelength: 280 nm Column: MABPac HIC-10, 5 μm, 4.6×10 mm (Thermo) Column temperature: 25 °C Mobile phase A: 1.5 M ammonium sulfate, 50 mM sodium phosphate, pH 7.0 Mobile phase B: 50 mM sodium phosphate, pH 7.0 Gradient program: 20% B - 20% B (0 min - 1 min), 0% B - 0% B (1 min - 35 min), 20% B - 20% B (35 min - 40 min) Flow rate: 0.5 mL / min Sample preparation: Dilute the sample to 0.5 mg / mL with the initial mobile phase. Table 18: The constructed conjugate Tisotumab sequence >LC DIQMTQSPASLSASVGDRVTITCRASENIFSYLAWYQQKPGKSPKLLVYNTRTLAEGVPSRFSGSGSGTDFSLTISSLQPEDFATYYCQHHYGTPFTFGSGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ IDNO:105) >HC EVQLQESGPGLVKPGGSLSLSCAASGFVFSSYDMSWVRQTPERGLEWVAYISSGGGITYAPSTVKGRFTVSRDNAKNTLYLQMNSLTSEDTAVYYCAAHYFGSSGPFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:106) Cell line information MKN45 (JCRB, JCRB0254)

[0470] MKN45 is a cell line that exhibits a round morphology. It is loosely attached to the stroma isolated from the gastric tissue of a 62-year-old female with gastric cancer in 1998. MKN45 was purchased from JCRB. The basal medium for MKN45 is RPMI-1640 medium (Gibco 22400089). To prepare the complete growth medium, the following components were added to the basal medium: fetal bovine serum to a final concentration of 10% (Gibco, 10099-141C). The cell line was grown at 37 °C in a humidified 5% CO2 atmosphere and tested regularly for the presence of mycoplasma using the MycoAlert TM PLUS Mycoplasma Detection Kit (Lonza, LT07-710). SNU-16 (ATCC, CRL-5974)

[0471] SNU-16 is a cell line that exhibits an epithelial morphology and was isolated from the ascites of a 33-year-old Asian female gastric cancer patient before chemotherapy in 1987, and SNU16 was purchased from ATCC. The basal medium for SNU16 is RPMI-1640 medium (Gibco 22400089). To prepare the complete growth medium, the following components were added to the basal medium: fetal bovine serum to a final concentration of 10% (Gibco, 10099-141C). The cell line was grown at 37 °C in a humidified 5% CO2 atmosphere and tested regularly for the presence of mycoplasma using the MycoAlert TM PLUS Mycoplasma Detection Kit (Lonza, LT07-710). NCI-H2122 (ATCC, CRL-5985)

[0472] NCI-H2122 cells are lymphoblasts isolated from the metastatic tumor of the pleural effusion of a 46-year-old female smoker in 1989, and NCI-H2122 was purchased from ATCC. The basal medium for NCI-H2122 is RPMI-1640 medium (Gibco 22400089). To prepare the complete growth medium, the following components were added to the basal medium: fetal bovine serum to a final concentration of 10% (Gibco, 10099-141C). The cell line was grown at 37 °C in a humidified 5% CO2 atmosphere and tested regularly for the presence of mycoplasma using the MycoAlert TM PLUS Mycoplasma Detection Kit (Lonza, LT07-710). LS174T (ATCC, CL-188)

[0473] LS174T is a cell line that exhibits an epithelial morphology. It was isolated from the colon of a 58-year-old white female adenocarcinoma patient with colorectal cancer, and LS174T was purchased from ATCC. The basal medium for LS174T is Eagle's Minimum Essential Medium (30 - 2003) formulated by ATCC. To prepare the complete growth medium, the following components were added to the basal medium: fetal bovine serum to a final concentration of 10% (Gibco, 10099 - 141C). The cell line was grown at 37 °C in a humidified 5% CO2 atmosphere and tested regularly for the presence of mycoplasma using MycoAlert TM PLUS Mycoplasma Detection Kit (Lonza, LT07 - 710). MDA - MB - 231 (ATCC, HTB - 26)

[0474] MDA - MB - 231 is an epitheloid cell isolated from the breast of a 40-year-old white female with adenocarcinoma, and MDA - MB - 231 was purchased from ATCC. The basal medium for MDA - MB - 231 is RPMI - 1640 medium (Gibco 22400089). To prepare the complete growth medium, the following components were added to the basal medium: fetal bovine serum to a final concentration of 10% (Gibco, 10099 - 141C). The cell line was grown at 37 °C in a humidified 5% CO2 atmosphere and tested regularly for the presence of mycoplasma using MycoAlert TM PLUS Mycoplasma Detection Kit (Lonza, LT07 - 710). HCT116 (ATCC, CCL - 247)

[0475] The HCT116 cell line was isolated from the colon of an adult male with colorectal cancer, has a mutation in codon 13 of the ras proto-oncogene, and was purchased from ATCC. The basal medium for MDA - MB - 231 is RPMI - 1640 medium (Gibco 22400089). To prepare the complete growth medium, the following components were added to the basal medium: fetal bovine serum to a final concentration of 10% (Gibco, 10099 - 141C). The cell line was grown at 37 °C in a humidified 5% CO2 atmosphere and tested regularly for the presence of mycoplasma using MycoAlert TM PLUS Mycoplasma Detection Kit (Lonza, LT07 - 710). Additional cell lines

[0476] The SW1463 cells are derived from human colorectal adenocarcinoma and express moderate levels of CEA.

[0477] NCI-N87 expresses low levels of CEA and is derived from gastric cancer.

[0478] HT29 cells have low or even negative CEA expression and are derived from human colorectal adenocarcinoma. Table 19: Cell lines and their CEA expression levels Example 18. In vitro cell killing of CEA antibodies conjugated with various payloads

[0479] According to Example 17, conjugate the BGA5384 antibody (Table 20) with in-house generated linkers and various payloads. Conjugate BGA5384 with maytansine DM4 ( Figure 13 ), auristatin MMAE ( Figure 14 ), and topoisomerase DXD (BGA2588) ( Figure 15 ). Table 20: Amino acid sequence of BGA5384

[0480] To determine the cell killing of each ADC, cell lines with different CEA expression levels (Example 17; Table 20) were seeded in 96-well plates and incubated overnight at 37 °C. Serial dilutions of the ADC were added, and then the cells were cultured for 6 days and cell viability was measured. As Figure 13-15 shown, all CEA antibody-drug conjugates showed good cell killing in high to medium CEA-expressing cells at low concentrations of CEA ADC. In CEA cells with low to low-negative to negative expression, high concentrations of CEA ADC were required to show cell killing. These data indicate that the BGA5384 antibody can be conjugated with various payloads and achieve cell killing in a range of CEA-expressing cells. Example 19. Payload sensitivity: MKN45

[0481] The effect of the payload on MKN45 cells is shown in Figure 16 . On day 0, cells were harvested with 0.25% trypsin-EDTA, seeded at 5,000 cells / well in 96-well plates (655090, Greiner), and incubated overnight at 37 °C, 5% CO2. On day 1, the payload compound (5-fold dilutions) was added to the plates. The cell and compound mixture was incubated at 37 °C, 5% CO2 for 6 days. On day 6, 100 μL of detection reagent (G7573, ) was used to collect the live cell signal; by Tecan Read the signal. Analyze the data using GraphPad Prism 9.0.0. All killing concentrations were repeated twice or three times (Table 21 and Figure 16 ). "SABC" refers to specific antibody binding capacity. Table 21: Effects of Payloads on MKN45 Cells Example 20. ADC Direct Cell Killing Assay

[0482] Figure 17-20 The cell activities of ADCs with 8 different constructs (see Table 18) in patient-derived cell lines of MKN45 (gastric cancer), H2122 (lung adenocarcinoma), LS174T (colorectal adenocarcinoma), and MB-231 (breast cancer) were shown respectively.

[0483] On day 0, cells were harvested with 0.25% trypsin-EDTA and plated at 5,000 cells / well (MKN45 or Ls174T) or 2,000 cells / well (NCI-H2122 or MDA-MB-231) in 96-well plates (655090, Greiner) and incubated overnight at 37 °C, 5% CO2. On day 1, ADCs (5-fold dilutions) were added to the plates. The cell and ADC mixtures were incubated at 37 °C, 5% CO2 for 6 days. On day 6, 100 μL of detection reagent (G7573, ) was used to collect the signal of viable cells; the signal was read by Tecan Read the signal. Analyze the data using GraphPad Prism 9.0.0. All killing concentrations were repeated twice or three times. The results for each of MKN45 (Table 22 and Figure 17 ), NCI-H2122 (Table 23 and Figure 18 ), Ls174T (Table 24 and Figure 19 ), and MDA-MB0231 (Table 25 and Figure 20 ) cells were presented. Table 22: Cell Activities of ADCs on MKN45 Cell Lines Table 23: Cell Activities of ADCs on NCI-H2122 Cell Lines Table 24: Cell Activities of ADCs on Ls147T Cell Lines Table 25: Cell Activities of ADCs on MDA-MB-231 Cell Lines Example 21. Efficacy of ADC BGA7650 and BGA9962 in cell line-derived xenograft models.

[0484] Cell line-derived xenograft (CDX) models using MKN-45 (CEA high expression) (gastric adenocarcinoma), SW-1463 (CEA moderate) (rectal adenocarcinoma), and NCI-H2122 (CEA low) (lung adenocarcinoma) were generated as described below.

[0485] Cells were cultured in RPMI-1640 medium supplemented with 10% (v / v) fetal bovine serum, 100 U / ml penicillin, and 100 μg / mL streptomycin. On the day of implantation, cells were collected and resuspended in cold (4 °C) serum-free RPMI-1640 medium. The cell density was adjusted to 2 (MKN-45), 1.5 (SW1463), or 4 (NCI-H2122) × 10 7 cells / mL, and the cells were placed on ice before inoculation.

[0486] Six- to eight-week-old female mice were purchased and housed in ventilated cages, given food and water ad libitum, and acclimated for approximately 1 week before inoculation. MKN-45, SW-1463, and NCI-H2122 tumors were induced by subcutaneous injection of 2.0 (MKN-45), 3.0 (SW1463), or 8.0 (NCI-H2122) × 10 6 cells into NCG, NCG, and Balb / c nude mice on the right flank.

[0487] The experiment was conducted approximately 2 - 3 weeks after injection of cancer cells. For tumor volume measurement, all tumors were measured with calipers, and the tumor volume was calculated using the formula V = 0.5(a × b 2 ) where "a" is the tumor length and "b" is the tumor width and / or height. When the tumor volume reached approximately 200 mm 3 , on day 0, the mice were randomly divided into 5 groups with 8, 9, and 9 animals in the vehicle, BGA7650, and BGA9962 groups, respectively. After ensuring that the average tumor volumes at the start of all cohorts were approximately equal, on treatment day 1, the animals were administered vehicle, BGA7650 (1.3 mg / kg or 4 mg / kg; or "mpk"), and BGA9962 (2 mg / kg or 6 mg / kg) intravenously. The animal body weights and tumor volumes were measured twice a week. Data were expressed as mean tumor volume ± standard error of the mean (SEM). Tumor growth inhibition (TGI) was calculated using the following formula: % TGI = [1 – (treatment Tt – treatment T0) / (vehicle Tt – vehicle T0)] × 100% Treatment Tt = mean tumor volume of the group administered on day t Treatment T0 = mean tumor volume of the group administered on day 0 Vehicle Tt = mean tumor volume of the vehicle group on day t Vehicle T0 = mean tumor volume of the vehicle group on day 0 Results

[0488] In cell line-derived xenograft (CDX) models using MKN-45( Figure 21 ), SW-1463( Figure 22 ), and NCI-H2122( Figure 23 ), compared with the vehicle group, the BGA9962 and BGA7650 treatment groups slowed tumor growth. In these three CDX models, both BGA9962 and BGA7650 showed dose-dependent efficacy( Figure 21 , 22 and 23). BGA7650 at 4 mg / kg rather than 1.3 mg / kg induced significant anti-tumor efficacy. BGA9962 at 2 mg / kg showed superior anti-tumor efficacy compared with BGA7650 at 1.3 mg / kg and was comparable to the efficacy of BGA7650 at 4 mg / kg. Additionally, BGA9962 at 6 mg / kg significantly reduced tumor growth and showed higher anti-tumor effects than BGA7650 at two doses (1.3 and 4 mg / kg). All animals tolerated the treatment well without significant weight loss or abnormal clinical observations. Example 22. Efficacy of BGA7650 and BGA9962 in a human patient-derived gastric cancer xenograft model

[0489] In a human patient-derived gastric cancer (“GC”) xenograft model initiated as described in Example 21( Figure 24 ), the anti-tumor effects of BGA7650 and BGA9962 were evaluated. Single-dose treatment with 4 mg / kg BGA7650, 2 mg / kg BGA9962, or 6 mg / kg BGA9962 induced tumor growth inhibition (TGI) rates of 22% (P = 0.8444), 106% (P < 0.0001), and 107% (P < 0.0001), respectively, on day 24 of treatment. BGA9962 at two doses (2 mg / kg and 6 mg / kg) showed significantly higher anti-tumor activity than BGA7650 at 4 mg / kg( Figure 24 and Table 26). All animals tolerated the treatment well without significant weight loss or abnormal clinical observations. Table 26: TGI measurement results of BGA7650 and BGA9962 on day 24 of treatment ADC TGI (%) at T24 BGA7650 4mg / kg 22 BGA9962 2mg / kg 106 BGA9962 6mg / kg 107 Example 23. Single-dose PK at 3 mg / kg (i.v.) in non-tumor-bearing mice

[0490] A single dose of BGA9962 or BGA7650 at 3 mg / kg (i.v.) was administered to Balb / c nude (non-tumor-bearing) mice as described in Example 21, and pharmacokinetics were evaluated. Compared to BGA7650, BGA9962 showed strong pharmacokinetic (PK) characteristics in Balb / c nude (non-tumor-bearing) mice (n = 3) Figure 25 ). BGA9962 exhibited superior PK compared to the comparator BGA7650. Compared to BGA7650 (triangles connected by solid lines), BGA9962 (triangles connected by dashed lines) produced lower serum-free payloads in plasma.

[0491] For the comparator BGA7650 (hollow circles connected by solid lines), a separation was observed between TAb (total antibody) and ADC, but for BGA9962 (hollow squares connected by solid lines), no separation was observed. These results confirmed that BGA9962 had a stable DAR over time compared to BGA7650. In vitro, BGA9962 was stable in mouse and human plasma, with no change in DAR after 336 hours of incubation (data not shown). Example 24. In vivo DAR quantification and comparative stability

[0492] The in vivo drug-to-antibody ratio (DAR) of BGA7650 and BGA9962 was analyzed by intact LC-MS. Serum samples (from Balb / c nude mice treated with a single i.v. dose of 3 mg / k of the ADC, n = 3 per group) were processed into payload-conjugated peptides of the heavy chain (HC) and light chain (LC) from the ADC. DAR mass spectra were analyzed to calculate the average DAR per chain, and the average DAR of the intact ADC was calculated using the following formula: DAR = (DAR(LC) + DAR(HC)) x 2.

[0493] Since BGA7650 has payload conjugation through lysine at non-specific positions, it was difficult to perform intact LC-MS DAR analysis. Instead, the in vivo DAR of BGA7650 was indirectly measured by using immunocapture followed by analysis of the ratio of total conjugated payload to total antibody concentration using anti-Fc (for total antibody detection) and LC-MS (for conjugated payload detection). Therefore, Figure 26 it was shown that BGA9962 maintained a stable DAR of 8 in vivo with minimal deconjugation, while BGA7650 deconjugated steadily over time.

Claims

1. An antibody-drug conjugate, the antibody-drug conjugate comprising: An antibody or an antigen-binding fragment thereof, the antibody or the antigen-binding fragment thereof binding to human CEA and comprising: (i) Three heavy-chain CDRs: HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 24, HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 25, HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 26, and Three light-chain CDRs: LCDR1 comprising the amino acid sequence shown in SEQ ID NO: 27, LCDR2 comprising the amino acid sequence shown in SEQ ID NO: 28, LCDR3 comprising the amino acid sequence shown in SEQ ID NO: 23; or (ii) Three heavy-chain CDRs: HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 7, HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 8, HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 9, and Three light-chain CDRs: LCDR1 comprising the amino acid sequence shown in SEQ ID NO: 10, LCDR2 comprising the amino acid sequence shown in SEQ ID NO: 11, LCDR3 comprising the amino acid sequence shown in SEQ ID NO: 6; or (iii) Three heavy-chain CDRs: HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 41, HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 42, HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 43, and Three light-chain CDRs: LCDR1 comprising the amino acid sequence shown in SEQ ID NO: 44, LCDR2 comprising the amino acid sequence shown in SEQ ID NO: 45, LCDR3 comprising the amino acid sequence shown in SEQ ID NO: 40; and Cytotoxic agent (D).

2. The antibody-drug conjugate according to claim 1, or a pharmaceutically acceptable salt, solvate or hydrate thereof, the antibody-drug conjugate having the formula Ab-(C-L-(D) m ) n , wherein Ab is the antibody or an antigen-binding fragment thereof; C is a conjugation moiety; L is a linker; D is the cytotoxic agent; m is an integer from 1 to 8; and n is from 1 to 10.

3. The antibody-drug conjugate according to claim 2, wherein m is 1.

4. The antibody-drug conjugate according to claim 2 or claim 3, wherein n is from 3 to 10.

5. The antibody-drug conjugate according to claim 4, wherein n is about 8.

6. The antibody-drug conjugate according to any one of claims 2 to 4, wherein C is a formula selected from (C-I), (C-Ia), (C-Ib), (C-II), (C-III), (C-IIIa) or (C-IV): And * marks the bond by which C is linked to Ab.

7. The antibody-drug conjugate according to claim 6, wherein C is 8. The antibody-drug conjugate according to any one of claims 2 to 7, wherein L is a formula selected from (L-I), (L-II) or (L-III): wherein Su is a hydrophilic residue; and * marks the bond by which L is linked to C.

9. The antibody-drug conjugate according to claim 8, wherein Su is 10. The antibody-drug conjugate according to claim 9, wherein Su is 11. The antibody-drug conjugate according to any one of claims 2 to 7, wherein L is Wherein the bond connecting the label L and C is indicated by an asterisk (*).

12. The antibody-drug conjugate according to any one of claims 1 to 11, wherein the cytotoxic agent is a topoisomerase inhibitor.

13. The antibody-drug conjugate according to any one of claims 2 - 12, wherein D is: Wherein Y is -A-B-C'-D'-*, where * indicates the bond connecting D and L; A is a bond, CR 1 R 2 or N-R 1 ; B is a bond, -C(=O)- or -C(=O)O-; C' is a bond or a divalent group, where the divalent group is an unsubstituted or substituted C 1-8 alkyl, unsubstituted or substituted cycloalkyl, unsubstituted or substituted heterocyclic group, unsubstituted or substituted aryl, or unsubstituted or substituted heteroaryl; D' is a bond, NH or O; R 1 and R 2 each independently is hydrogen, halogen, substituted or unsubstituted alkyl, or substituted or unsubstituted alkoxy; or R 1 and R 2 together with the atom to which they are attached form an unsubstituted or substituted cycloalkyl, unsubstituted or substituted heterocyclic group, unsubstituted or substituted aryl, or unsubstituted or substituted heteroaryl; R 3 and R 4 each independently is hydrogen, halogen, substituted or unsubstituted alkyl, or substituted or unsubstituted alkoxy; or R 3 and R 4 together with the atom to which they are attached form an unsubstituted or substituted cycloalkyl, unsubstituted or substituted heterocyclic group, unsubstituted or substituted aryl, or unsubstituted or substituted heteroaryl.

14. The antibody-drug conjugate according to any one of claims 1 - 13, wherein D is: Wherein R 7 and R 8 are each independently hydrogen, halogen, or alkyl.

15. The antibody-drug conjugate according to any one of claims 2 - 12, wherein D is selected from:

16. The antibody-drug conjugate according to claim 15, wherein D is 17. The antibody-drug conjugate according to any one of claims 2 to 5, wherein C-L-(D) m is: wherein the * indicates the bond connecting C to Ab.

18. The antibody-drug conjugate according to claim 17, wherein C-L-(D) m is:

19. The antibody-drug conjugate according to any one of claims 2-5, or a tautomer, pharmaceutically acceptable salt, solvate or hydrate thereof, wherein the antibody-drug conjugate has one of the following formulas:

20. The antibody-drug conjugate according to any one of claims 1 to 19, wherein the antibody or antigen-binding fragment comprises: (i) a heavy chain variable region comprising SEQ ID NO:31 and a light chain variable region comprising SEQ ID NO:32; (ii) a heavy chain variable region comprising SEQ ID NO:48 and a light chain variable region comprising SEQ ID NO:49; and (iii) a heavy chain variable region comprising SEQ ID NO:14 and a light chain variable region comprising SEQ ID NO:

15.

21. The antibody-drug conjugate according to any one of claims 1 to 20, wherein the antibody or antigen-binding fragment is a monoclonal antibody, a human engineered antibody, a single-chain antibody (scFv), a Fab fragment, a Fab' fragment or an F(ab')2 fragment.

22. The antibody-drug conjugate according to any one of claims 1 to 21, wherein the antibody or antigen-binding fragment comprises an scFv, and the scFv comprises a VH having the amino acid sequence of SEQ ID NO:14 and a VL having the amino acid sequence of SEQ ID NO:

15.

23. The antibody-drug conjugate according to any one of claims 1 to 21, wherein the antibody or antigen-binding fragment comprises an scFv having the amino acid sequence of SEQ ID NO:

14.

24. The antibody-drug conjugate according to any one of claims 1 to 23, wherein the antibody or its antigen-binding fragment comprises a heavy chain constant region of the IgG1, IgG2, IgG3 or IgG4 subclass and / or a light chain constant region of the κ or λ type.

25. The antibody-drug conjugate according to claim 24, wherein the antibody or its antigen-binding fragment comprises a heavy-chain constant region of the IgG1 subclass and a light-chain constant region of the κ type.

26. An antibody-drug conjugate of the following formula: or a tautomer, pharmaceutically acceptable salt, solvate or hydrate thereof, wherein: n is from 4 to 10; and Ab is an antibody or an antigen-binding fragment thereof that binds CEA and comprises: (i) three heavy-chain CDRs: HCDR1 comprising the amino acid sequence shown in SEQ ID NO:7, HCDR2 comprising the amino acid sequence shown in SEQ ID NO:8, HCDR3 comprising the amino acid sequence shown in SEQ ID NO:9, and three light-chain CDRs: LCDR1 comprising the amino acid sequence shown in SEQ ID NO:10, LCDR2 comprising the amino acid sequence shown in SEQ ID NO:11, LCDR3 comprising the amino acid sequence shown in SEQ ID NO:6; or (ii) three heavy-chain CDRs: HCDR1 comprising the amino acid sequence shown in SEQ ID NO:24, HCDR2 comprising the amino acid sequence shown in SEQ ID NO:25, HCDR3 comprising the amino acid sequence shown in SEQ ID NO:26, and three light-chain CDRs: LCDR1 comprising the amino acid sequence shown in SEQ ID NO:27, LCDR2 comprising the amino acid sequence shown in SEQ ID NO:28, LCDR3 comprising the amino acid sequence shown in SEQ ID NO:23; or (iii) three heavy-chain CDRs: HCDR1 comprising the amino acid sequence shown in SEQ ID NO:41, HCDR2 comprising the amino acid sequence shown in SEQ ID NO:42, HCDR3 comprising the amino acid sequence shown in SEQ ID NO:43, and three light-chain CDRs: LCDR1 comprising the amino acid sequence shown in SEQ ID NO:44, LCDR2 comprising the amino acid sequence shown in SEQ ID NO:45, LCDR3 comprising the amino acid sequence shown in SEQ ID NO:

40.

27. An antibody-drug conjugate, or a tautomer, pharmaceutically acceptable salt, solvate or hydrate thereof, wherein the antibody-drug conjugate has the following formula: n is from 4 to 10; and Ab is an antibody or an antigen-binding fragment thereof that binds to CEA and comprises: (i) three heavy-chain CDRs: HCDR1 comprising the amino acid sequence shown in SEQ ID NO:7, HCDR2 comprising the amino acid sequence shown in SEQ ID NO:8, HCDR3 comprising the amino acid sequence shown in SEQ ID NO:9, and three light-chain CDRs: LCDR1 comprising the amino acid sequence shown in SEQ ID NO:10, LCDR2 comprising the amino acid sequence shown in SEQ ID NO:11, LCDR3 comprising the amino acid sequence shown in SEQ ID NO:6; or (ii) three heavy-chain CDRs: HCDR1 comprising the amino acid sequence shown in SEQ ID NO:24, HCDR2 comprising the amino acid sequence shown in SEQ ID NO:25, HCDR3 comprising the amino acid sequence shown in SEQ ID NO:26, and three light-chain CDRs: LCDR1 comprising the amino acid sequence shown in SEQ ID NO:27, LCDR2 comprising the amino acid sequence shown in SEQ ID NO:28, LCDR3 comprising the amino acid sequence shown in SEQ ID NO:23; or (iii) three heavy-chain CDRs: HCDR1 comprising the amino acid sequence shown in SEQ ID NO:41, HCDR2 comprising the amino acid sequence shown in SEQ ID NO:42, HCDR3 comprising the amino acid sequence shown in SEQ ID NO:43, and three light-chain CDRs: LCDR1 comprising the amino acid sequence shown in SEQ ID NO:44, LCDR2 comprising the amino acid sequence shown in SEQ ID NO:45, LCDR3 comprising the amino acid sequence shown in SEQ ID NO:

40.

28. A pharmaceutical composition comprising the antibody-drug conjugate according to any one of claims 1 to 27 and a pharmaceutically acceptable carrier.

29. A method for treating cancer expressing CEA, the method comprising administering to a subject in need thereof an effective amount of the antibody-drug conjugate according to any one of claims 1 to 27 or the pharmaceutical composition according to claim 28.

30. The method according to claim 29, wherein the cancer expressing CEA is lung cancer, gastrointestinal cancer or colorectal cancer.

31. The method according to claim 30, wherein the lung cancer is non-small cell lung cancer.

32. The method according to claim 30, wherein the gastrointestinal cancer is gastric cancer.

33. The method according to claim 30, wherein the colorectal cancer is rectal cancer.

34. A method for producing the antibody-drug conjugate according to any one of claims 1 to 27, the method comprising: (i) Culturing a host cell transformed with an isolated nucleic acid comprising a sequence encoding the antibody or an antigen-binding fragment thereof, wherein the antibody or an antigen-binding fragment thereof comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:99 and a light chain comprising the amino acid sequence of SEQ ID NO:100; (ii) Expressing the antibody or an antigen-binding fragment thereof; (iii) Recovering the expressed antibody or an antigen-binding fragment thereof; and (iv) Conjugating the cytotoxic agent to the antibody or fragment using a linker such that an antibody-drug conjugate is formed.

35. An anti-CEA antibody-drug conjugate comprising any one of the following: BGA2588, BGA9962, BGA8357, BGA0084, BGA7413, BGA2490 and BGA0179 as shown in Table 18.

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