Anti-CEA antibody-drug conjugates and methods of use thereof

Abstract

The present disclosure provides antibody-drug conjugates comprising an antibody or antigen-binding fragment thereof that binds to human CEA and a linker payload, pharmaceutical compositions comprising the anti-CEA antibody-drug conjugates, and uses of the anti-CEA antibody-drug conjugates for the treatment of CEA-related diseases or disorders. The present disclosure relates to antibody-drug conjugates (ADCs) comprising an antibody or antigen-binding fragment (Ab) capable of specifically binding to human CEA and a cytotoxic agent (D).

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to PCT Application No. PCT / CN2022 / 134067, filed November 24, 2022, entitled "Anti-CEA Antibody Drug Conjugates and Methods of Use," and PCT / CN2023 / 107003, filed July 12, 2023, entitled "Anti-CEA Antibody Drug Conjugates and Methods of Use," which are incorporated herein by reference in their entireties.

[0002] Electronic Sequence Listing Reference This application contains a Sequence Listing that has been submitted electronically in .XML format and is incorporated herein 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 a part of the present specification and is incorporated herein by reference in its entirety.

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

[0004] Antibody-drug conjugates (ADCs) are chimeric molecules that combine the specificity of an antibody, which recognizes and binds with high affinity to an antigen, such as a tumor-associated antigen (TAA), with the potent enzymatic activity of a drug, e.g., a toxin, to induce target cell death. Current ADCs have some therapeutic limitations, necessitating the development of 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 first 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 of non-tumor cells, but is distributed throughout the cell membrane in cancer cells. Overexpression of CEA has been observed in many types of cancer, including colorectal, pancreatic, lung, gastric, hepatocellular, breast, and thyroid cancers. For example, CEA is found in columnar epithelium and goblet cells of the colon. In tumors derived from these tissue types, CEA expression increases from the apical membrane to the cell surface, where it enters the bloodstream upon removal from the cell surface. Because CEA is constitutively released from tumor cells and reaches detectable concentrations in peripheral blood, its quantification is frequently used in cancer diagnosis. Therefore, CEA is useful as a diagnostic tumor marker for determining elevated CEA levels in the blood of cancer patients in cancer prognosis and management.

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

[0007] However, one of the problems encountered with anti-CEA antibodies is cross-reactivity. CEA is highly homologous to other members of the CEACAM family. For example, human CEA shows 84% ​​homology with CEACAM6, 77% homology with CEACAM8, and 73% identity with CEACAM1. Therefore, there is a need for anti-CEA antibodies that are specific for CEA and do not significantly cross-react with human CEACAM1, CEACAM6, CEACAM7, or CEACAM8. Furthermore, there remains a need for anti-CEA ADCs that possess highly potent antitumor activity combined with potent, non-cross-reactive anti-CEA targeting specificity. Summary of the Invention [Means for solving the problem]

[0008] The present disclosure encompasses at least the following embodiments.

[0009] The present disclosure relates to an antibody drug conjugate (ADC) comprising an antibody or 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 (Ab) thereof comprises: (i) The three heavy chain CDRs: HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 24 HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 25; HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 26, and The three light chain CDRs: LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 27; LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 28; an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 23; or (ii) The three heavy chain CDRs: HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 7 HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 8; HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 9, and The three light chain CDRs: LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 10; LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 11; an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 6; or (iii) The three heavy chain CDRs: HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 41 HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 42; HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 43, and The three light chain CDRs: LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 44; LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 45; LCDR3 comprising the amino acid sequence set forth in SEQ ID NO:40.

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

[0012] In certain embodiments, m is 1. When m is 1, the antibody drug conjugate has the formula Ab-(CLD) n It will be understood that the term includes (e.g., has).

[0013] In certain embodiments, C is a formula selected from (CI), (C-Ia), (C-Ib), (C-II), (C-III), (C-IIIa), or (C-IV): [ka] [ka] and The * indicates the bond where C connects to Ab. In certain embodiments, C is a formula selected from: [ka]

[0014] In certain embodiments, C is (C-Ic): [ka] where * indicates the bond where C connects to Ab.

[0015] In certain embodiments, C is selected from the group consisting of: [ka] where * indicates the bond where the conjugator connects to the Ab.

[0016] In certain embodiments, L is represented by the following formula (LI), (L-II), or (L-III): [ka] wherein Su is a hydrophilic residue, and * indicates the bond where the linker connects to the conjugator.

[0017] In certain embodiments, Su is [ka] is.

[0018] In certain embodiments, Su is [ka] is.

[0019] In certain embodiments, Su is [ka] is.

[0020] In certain embodiments, Su is [ka] is.

[0021] In certain embodiments, L is [ka] where * indicates the bond where L connects to C.

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

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

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

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

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

[0027] In certain embodiments, D is [ka] is.

[0028] In certain embodiments, CL-(D) m is the following: [ka] [ka] where * indicates the bond connecting C to Ab.

[0029] In certain embodiments, CL-(D) m teeth: [ka] and where * indicates the bond where C connects to Ab.

[0030] In certain embodiments, CL-(D) m is the following: [ka] and where * indicates the bond where C connects to Ab.

[0031] In certain embodiments, CL-(D) m is the following: [ka] and where * indicates the bond where C connects to Ab.

[0032] In certain embodiments, the antibody drug conjugate comprises one of the following: [ka] [ka] or a tautomer, pharmaceutically acceptable salt, solvate, or hydrate thereof, where Ab is an anti-CEA antibody or antigen-binding fragment thereof described herein, and n is as described herein, e.g., 1 to 10, preferably about 7, 8, or 9.

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

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

[0037] In certain embodiments, the present disclosure provides an antibody drug conjugate comprising an anti-CEA antibody or antigen-binding fragment, wherein the antibody or antigen-binding fragment is one of the following: (i) the three heavy chain CDRs: HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 24; HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 25; HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 26, and The three light chain CDRs: LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 27; LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 28; an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 23; or (ii) three heavy chain CDRs: HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 7; HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 8; HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 9, and The three light chain CDRs: LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 10; LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 11; an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 6; or (iii) three heavy chain CDRs: HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 41; HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 42; HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 43, and The three light chain CDRs: LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 44; LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 45; an LCDR3 comprising the amino acid sequence set forth 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) An antibody drug conjugate comprising 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 a F(ab')2 fragment.

[0039] In certain embodiments, the antibody or antigen-binding fragment comprises an scFv comprising 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 certain embodiments, the antibody or antigen-binding fragment comprises an scFv comprising 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 certain embodiments, the antibody or antigen-binding fragment comprises an scFv comprising 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 antigen-binding fragment thereof comprises a heavy chain constant region of the IgG1, IgG2, IgG3, or IgG4 subclass, and / or a light chain constant region of the kappa or lambda 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 kappa type.

[0045] In certain embodiments, the present disclosure provides a compound of the formula: [ka] [ka] or a tautomer, pharmaceutically acceptable salt, solvate, or hydrate thereof, In the formula, n is 4 to 10, for example, 4, 5, 6, 7, 8, 9, or 10; Ab is an antibody or antigen-binding fragment thereof that binds to CEA, and the antibody or antigen-binding fragment is: (i) The three heavy chain CDRs: HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 24 HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 25; HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 26, and The three light chain CDRs: LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 27; LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 28; an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 23; or (ii) The three heavy chain CDRs: HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 7 HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 8; HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 9, and The three light chain CDRs: LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 10; LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 11; an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 6; or (iii) The three heavy chain CDRs: HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 41 HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 42; HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 43, and The three light chain CDRs: LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 44; LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 45; an LCDR3 comprising the amino acid sequence set forth 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) An antibody drug conjugate comprising 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-associated disease or disorder, e.g., CEA-expressing or -accumulating cells, 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 same. In some embodiments, the CEA-expressing or -accumulating cells are cancer cells.

[0049] In certain embodiments, the present disclosure provides a compound of the formula: CLD or a pharmaceutically acceptable salt, solvate, or hydrate thereof, wherein: C is the conjugator, L is a linker, D relates to anti-CEA antibodies, which are cytotoxic agents.

[0050] In certain embodiments, C is selected from the group consisting of the following formulas (C-I'), (C-II'), (C-III'), and (C-IV'): [ka] Includes.

[0051] In certain embodiments, L is represented by the following formula (LI), (L-II), or (L-III): [ka] wherein Su is a hydrophilic residue, and * indicates the bond where the linker connects to the conjugator.

[0052] In certain embodiments, Su is [ka] is.

[0053] In certain embodiments, Su is [ka] is.

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

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

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

[0057] In certain embodiments, the present disclosure provides a method of producing the above-described anti-CEA antibody drug conjugate, comprising: (i) culturing a host cell transformed with an isolated nucleic acid comprising a sequence encoding an anti-CEA antibody or antigen-binding fragment thereof, wherein the antibody or fragment thereof is 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 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) The three heavy chain CDRs: HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 24 HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 25; HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 26, and The three light chain CDRs: LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 27; LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 28; an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 23; or c) the three heavy chain CDRs: HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 7 HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 8; HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 9, and The three light chain CDRs: LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 10; LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 11; an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 6; or d) The three heavy chain CDRs: HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 41 HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 42; HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 43, and The three light chain CDRs: LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 44; LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 45; an LCDR3 comprising the amino acid sequence set forth 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) culturing a human IgG1-positive antibody comprising 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 antigen-binding fragment thereof; (iii) recovering the expressed antibody or antigen-binding fragment thereof; and (iv) conjugating or linking at least one compound to the antibody or fragment thereof, optionally with a linker, to form an antibody drug conjugate.

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

[0059] In certain embodiments, an antibody drug conjugate described herein is provided (e.g., in the form of a pharmaceutical composition) for use as described herein (e.g., for treating a subject having CEA-expressing and / or accumulating cells).

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

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

[0062] In certain embodiments, the present disclosure provides a kit comprising an anti-CEA antibody drug conjugate and instructions for its use, wherein the antibody drug conjugate is an antibody or antigen-binding fragment thereof, comprising: (i) The three heavy chain CDRs: HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 24 HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 25; HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 26, and The three light chain CDRs: LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 27; LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 28; an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 23; or (ii) The three heavy chain CDRs: HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 7 HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 8; HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 9, and The three light chain CDRs: LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 10; LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 11; an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 6; or (iii) The three heavy chain CDRs: HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 41 HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 42; HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 43, and The three light chain CDRs: LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 44; LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 45; an LCDR3 comprising the amino acid sequence set forth 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 kit comprising an antibody or antigen-binding fragment thereof, the antibody or antigen-binding fragment comprising 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 provides a kit comprising an anti-CEA antibody drug conjugate and instructions for using the same, wherein the antibody drug conjugate is selected from the group consisting of: a. a VH sequence comprising the sequence set forth in SEQ ID NO: 31, or 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 set forth in SEQ ID NO: 32, or 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 set forth in SEQ ID NO: 48, or 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 set forth 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 kit comprising an antibody comprising a VH sequence comprising the sequence set forth in SEQ ID NO: 14, or 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 set forth 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 Summary of the Invention is not intended to represent the entirety and scope of the present disclosure, and should not be construed as such. Furthermore, references made 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 the particular description. The present disclosure is described in various levels of detail in the Summary of the Invention, as well as the Detailed Description and accompanying drawings, and no limitation as to the scope of the disclosure is intended by either the inclusion or non-inclusion of elements, components, etc. in the Summary of the Invention. Features from any of the disclosed embodiments can be used in combination with each other without limitation. Furthermore, other features and advantages of the present disclosure will become apparent to those skilled in the art upon review of the following detailed description and accompanying drawings. [Brief explanation of the drawings]

[0065] [Figure 1] Schematic diagrams of sCEA, chimeric CEA (CHIM), CEACAM6, and CEA variant (CEA-v) are shown. In CEA, domains N, A1, B1, A2, B2, A3, B3, and the GPI linker (GPI) are labeled, and in CEACAM6, domains N', A', and B' are labeled. [Figure 2A] Figure 1 shows a phylogenetic tree of the anti-CEA domain B3 antibody VH region. The VH sequences of candidate anti-CEA antibodies were aligned using DNASTAR's Megalign™ software. Sequence homology is displayed in the phylogenetic tree. [Figure 2B] Figure 1 illustrates a phylogenetic tree of the anti-CEA domain B3 antibody VL region. The VL sequences of candidate anti-CEA antibodies were aligned using DNASTAR's Megalign™ software. Sequence homology is displayed in the phylogenetic tree. [Figure 3A]Figure 1 shows the affinity determination of purified murine anti-CEA antibody BGA7592 on a chimeric construct (CHIM) by surface plasmon resonance (SPR). The various lines represent the binding capacity of BGA7592 to CHIM at different concentrations, with the top line representing the binding capacity of BGA7592 to the highest concentration of CHIM, and the remaining lines representing serial dilutions of CHIM. The ascending curve for each line represents the association rate, and the descending curve represents the dissociation rate. [Figure 3B] 1 illustrates the binding profile of BGA7592 by antigen ELISA. [Figure 4A] 1 shows the effect of soluble CEA (sCEA) on the binding of CEA antibodies to patient-derived MKN45 gastric adenocarcinoma cells. 2 shows the binding profile of anti-domain B3 antibodies in the presence or absence of soluble CEA (sCEA). [Figure 4B] Figure 4A shows the effect of soluble CEA (sCEA) on the binding of CEA antibodies to patient-derived MKN45 gastric adenocarcinoma cells. The antibody binding profiles in Figure 4A are shown as histograms. [Figure 5] A to B indicate randomization sites for generating an antibody library for affinity maturation of the light chain CDR (LCDR) region (A) (SEQ ID NOs: 82, 83, and 84, respectively) and heavy chain CDR (HCDR) region (B) (SEQ ID NOs: 80, 81, and 3, respectively) of the humanized BGA7592 antibody. [Figure 6] The amino acid changes in the BGA7592 light chain CDR regions after four rounds of selection are shown. [Figure 7] Binding of affinity matured humanized BGA7592 variants to LOVO cells by flow cytometry. [Figure 8] Binding of anti-CEA antibodies to MKN45 cells as measured by flow cytometry is shown. [Figure 9] A and B are bar graphs showing off-target binding of antibody BGA5384 to various CEACAM family members by antigen ELISA (A) and flow cytometry (B). [Figure 10]1 shows the effect of soluble CEA on BGA5384 binding to CEA-expressing MKN45 cells in the presence of various concentrations of soluble CEA. [Figure 11] 1 is a graph showing antibody-dependent cellular cytotoxicity (ADCC) of antibody BGA6710 in vitro. [Figure 12] Figure 1 shows the effect of the BGA6710 antibody on tumor volume in a mouse cancer model. [Figure 13] 1 shows killing curves for cells with various CEA expression levels by a CEA antibody conjugated to the maytansine compound DM4. [Figure 14] 1 shows killing curves for cells with various CEA expression levels by a CEA antibody conjugated to auristatin MMAE. [Figure 15] 1 shows killing curves for cells with various CEA expression levels by a CEA antibody conjugated to the topoisomerase inhibitor DXD. [Figure 16] 1 shows the killing curves of MKN45 cells with a CEA-specific antibody binding capacity (SABC) of approximately 200,000 (CEA high) by various free cytotoxic agents. [Figure 17] Figure 1 shows the cell-killing effects of eight ADCs on MKN45 cells (CEA high). [Figure 18] The cell-killing effects of eight ADCs on H2122 patient-derived cells (lung adenocarcinoma) (moderate CEA level) are shown. [Figure 19] Figure 1 shows the cell-killing effects of eight ADCs on LS174T patient-derived cells (colorectal adenocarcinoma) (CEA low). [Figure 20] The cell-killing effects of eight ADCs on MB-231 patient-derived cells (breast adenocarcinoma) (CEA-negative) are shown. [Figure 21] 1 shows the antitumor effects 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). [Figure 22]1 shows the antitumor effects of different concentrations of BGA-7650 and BGA-9962 relative to the control in a CDX model using SW-1463 cells (intermediate CEA grade) (rectal adenocarcinoma). [Figure 23] 1 shows the antitumor effects of different concentrations of BGA-7650 and BGA-9962 relative to the control in a CDX model using H2122 cells (CEA low) (lung adenocarcinoma). [Figure 24] Figure 1 shows the antitumor effects of different concentrations of BGA-9962 and BGA-7650 versus control in a patient-derived xenograft (PDX) model of gastric cancer (GC). [Figure 25] Graph of concentration versus time for various antibodies, ADCs, and free cytotoxic agents in Balb / c nude mice (non-tumor bearing). [Figure 26] FIG. 1 is a graph showing the in vivo DAR over time in mice for two ADCs (single dose, iv 3mpk Balb / c nude mice, n=3 per group). DETAILED DESCRIPTION OF THE INVENTION

[0066] [Table 1-1] [Table 1-2]

[0067] definition Unless otherwise defined below or elsewhere in this document, all technical and scientific terms used herein have the meanings commonly understood by those of ordinary skill in the art.

[0068] As used in this specification, including the appended claims, singular words such as "a," "an," and "the" include their corresponding plural forms unless the context clearly dictates otherwise.

[0069] Unless otherwise specified or clear from the context, as used herein, the term "about" refers to a value or composition that falls within an acceptable error range of a particular value or composition as determined by one of ordinary skill in the art, which depends in part on how the value or composition is measured or determined, i.e., the limitations of the measurement system. For example, "about" can mean within one standard deviation or more than one standard deviation, according to practice in the art. "About" can also 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% more or less than the stated value. For example, about 5 mg can include any amount between 4.5 mg and 5.5 mg. Furthermore, particularly with respect to biological systems or processes, these terms can mean values ​​up to an order of magnitude or up to 5 times greater. When a specific value or composition is provided in this disclosure, unless otherwise specified, the meaning of "about" should be assumed to be within the tolerance range of that specific value or composition.

[0070] The term "or" is used to mean, and is used interchangeably with, the term "and / or," unless context clearly dictates otherwise.

[0071] The term "carcinoembryonic antigen" or "CEA" refers to an approximately 70-100 kDa glycoprotein, also known as CEACAM5 or CD66e. The amino acid sequence of human CEA (SEQ ID NO: 52) can also be found under accession numbers P06731 or NM_004363.2.

[0072] As used herein, the terms "administration" and "administering," when applied to an animal, human, subject, cell, tissue, organ, or biological fluid, refer to the contact of an exogenous pharmaceutical, therapeutic, diagnostic, or composition with an animal, human, subject, cell, tissue, organ, or biological fluid. Treatment of a cell encompasses contact of a reagent with the cell, as well as contact of a reagent with a fluid when the fluid is in contact with the cell.

[0073] The term "subject" or "patient" as used herein includes any 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 having or at risk of having a disorder described herein).

[0074] In one aspect, "treating" any disease or disorder refers to ameliorating the disease or disorder (i.e., delaying, preventing, or reducing the onset of the disease or at least one of its clinical symptoms). In another aspect, "treat," "treating," or "treatment" refers to alleviating or improving at least one physical parameter, including those that may not be discernible by the patient. In yet another aspect, "treat," "treating," or "treatment" refers to modulating the disease or disorder either physically (e.g., stabilizing a discernible symptom), physiologically (e.g., stabilizing a physical parameter), or both.

[0075] The term "affinity" as used herein refers to the strength of the interaction between an antibody and an antigen. Within the antigen, the variable region of the antibody interacts with the antigen at multiple sites through non-covalent forces. Generally, the more interactions, the stronger the affinity.

[0076] The term "antibody," as used herein, refers to a polypeptide of the immunoglobulin family that can bind to a corresponding antigen in a reversible and specific manner other than by covalent bonds. For example, naturally occurring IgG antibodies are tetramers containing at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain is composed 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 is composed 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 regions of hypervariability called complementarity-determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an 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 (C1q) of the classical complement system.

[0077] The locations of CDRs and framework regions can be determined using various definitions well known in the art, such as Kabat, Chothia, AbM, and IMGT (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).

[0078] The term "antibody" includes, but is not limited to, monoclonal antibodies, human antibodies, humanized antibodies, chimeric antibodies, and anti-idiotypic (anti-Id) antibodies. Antibodies 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).

[0079] 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.

[0080] As used herein, the term "monoclonal antibody" or "mAb" or "Mab" refers to a population of substantially homogeneous antibodies, i.e., the antibody molecules within 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 large number of different antibodies with different amino acid sequences within their variable domains, particularly their CDRs, which are often specific for different epitopes. The modifier "monoclonal" indicates the character of the antibody as being obtained from a population of substantially homogeneous antibodies and is not to be construed as requiring production of the antibody by any particular method. Monoclonal antibodies can be obtained by methods known to those skilled in the art. See, for example, 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, such as IgG, IgM, IgD, IgE, IgA, etc., and any subclass thereof, e.g., IgG1, IgG2, IgG3, IgG4. Hybridomas producing monoclonal antibodies can be cultivated in vitro or in vivo. High-titer monoclonal antibodies can be obtained by in vivo production, where cells from individual hybridomas are injected intraperitoneally into mice, such as pristine-primed Balb / c mice, to produce ascites containing high concentrations of the desired antibody. Monoclonal antibodies of the IgM or IgG isotype can be purified from such ascites or from the culture supernatant using column chromatography techniques well known to those skilled in the art.

[0081] Unless otherwise specified, "antigen-binding fragment" refers to an antigen-binding fragment of an antibody, i.e., an antibody fragment that retains the ability to specifically bind to the antigen bound by the full-length antibody, 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, bispecific antibodies, 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)).

[0082] As used herein, an antibody or antigen-binding antibody fragment "specifically binds" or "selectively binds" to an antigen (e.g., a protein) means that the antibody exhibits preferential binding to its target relative to other proteins, although this 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 biologics, for example, in a blood, serum, plasma, or tissue sample. Thus, under certain designated immunoassay conditions, an antibody or antigen-binding fragment thereof specifically binds to a particular antigen at least two-fold stronger than background levels and does not specifically bind in significant amounts to other antigens present in the sample. In one embodiment, under designated immunoassay conditions, an antibody or antigen-binding fragment thereof specifically binds to a particular antigen at least ten-fold stronger than background levels of binding and does not specifically bind in significant amounts to other antigens present in the sample.

[0083] The term "human antibody" herein refers to an antibody that contains only human immunoglobulin protein sequences. A human antibody may contain mouse glycosylation if produced in a mouse, a mouse cell, or a mouse cell-derived hybridoma. Similarly, a "mouse antibody" or a "rat antibody" refers to an antibody that contains only mouse immunoglobulin protein sequences or only rat immunoglobulin protein sequences, respectively.

[0084] The terms "humanized" or "humanized antibody" refer to forms of antibodies containing sequences derived from non-human (e.g., murine) and human antibodies. Such antibodies contain minimal sequence derived from non-human immunoglobulin. Generally, a humanized antibody comprises substantially all of at least one, and typically two, variable domains, with all or substantially all hypervariable loops corresponding to those of a non-human immunoglobulin and all or substantially all FR regions being those of a human immunoglobulin sequence. A humanized antibody also optionally comprises at least a portion of an immunoglobulin constant region (Fc), typically at least a portion of a human immunoglobulin constant region (Fc). When necessary to distinguish a humanized antibody from a rodent parent antibody, the prefix "hum," "hu," "Hu," or "h" is added to the name of the antibody clone. Humanized forms of rodent antibodies generally contain the same CDR sequences of the rodent parent antibody but can include certain amino acid substitutions to enhance affinity, increase the stability of the humanized antibody, remove post-translational modifications, or for other reasons.

[0085] "Equilibrium dissociation constant" or "K D The term "M" refers to the dissociation rate constant (kd, time -1 ) is the association rate constant (ka, time -1 , M -l ) The equilibrium dissociation constant can be measured using any method known in the art. Antibodies of the present disclosure generally have an equilibrium dissociation constant of about 10 -7 Less than or equal to 10 -8 Less than m, e.g., about 10 -9 Less than M or 10 -10 M or less, and in some embodiments, about 10 -11 Under M, 10 -12 Less than M or 10 -13 It is less than M.

[0086] As used herein, the terms "cancer" or "tumor" have the broadest meaning understood in the art and refer to a physiological condition in mammals that is typically characterized by unregulated cell growth. In the context of this disclosure, cancer or tumor is not limited to any particular type or location.

[0087] In the context of the present disclosure, when referring to an amino acid sequence, the term "conservative substitution" refers to the substitution of an original amino acid with a new amino acid that does not substantially alter the chemical, physical, and / or functional properties of the antibody or fragment, e.g., its binding affinity to CEA. Common conservative changes of amino acids are well known in the art.

[0088] The terms "improve," "increase," "inhibit," and "reduce" refer to values ​​relative to a baseline or other reference measurement. In some embodiments, a suitable reference measurement may include a measurement in a particular system (e.g., a single individual), other conditions being equal, in the absence of (e.g., before and / or after) the agent or treatment, or in the presence of an appropriate equivalent reference agent. In some embodiments, a suitable 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.

[0089] As used herein, the term "knob-into-hole" technology refers to amino acids that together direct the pairing of two polypeptides, either in vitro or in vivo, by introducing a spatial protuberance (knob) in one polypeptide and a socket or cavity (hole) in the other polypeptide (at the interface where they interact). For example, knob-into-hole technology can be used to engineer the Fc:Fc binding interface of an antibody, C L :C H I interface, or V H / V LIn some embodiments, knob-into-hole amino acids have been introduced into the antibody's interface (see, e.g., US2011 / 0287009, US2007 / 0178552, WO96 / 027011, WO98 / 050431, and Zhu et al., 1997, Protein Science 6:781-788). In some embodiments, knob-into-hole amino acids ensure proper pairing of two different heavy chains together during antibody production. For example, antibodies with knob-into-hole amino acids in their Fc region may further comprise a single variable domain linked to each Fc region, or may further comprise a different heavy chain variable domain paired with a similar or different light chain variable domain. Knob-into-hole technology can also be used with VH or VL regions to ensure proper pairing. An example of a suitable algorithm for determining percent sequence identity and sequence similarity is the BLAST algorithm 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 analyses is publicly available through the National Center for Biotechnology Information. This algorithm involves first identifying high-scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that match or meet a positive threshold score T when aligned with words of the same length in a database sequence. T is referred to as the neighborhood word score threshold. These initial neighborhood word hits serve as starting points for searches to find longer HSPs containing them. Word hits are extended outward along each end of each sequence as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for mismatching residues; always < 0).For amino acid sequences, a scoring matrix is ​​used to calculate the cumulative score. Extension of the word hits in each direction is halted when the cumulative alignment score falls by an amount X from the maximum achieved value, when the cumulative score becomes zero or less due to the accumulation of one or more negative-scoring residue alignments, or when either end of the 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) uses a word length (W) of 11, an expectation (E) of 10, M=5, N=-4, and a comparison of both strands as defaults. For amino acid sequences, the BLAST program uses a word length of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915), an alignment (B) of 50, M=5, N=-4, and a comparison of both strands as defaults.

[0090] The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, for example, Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90:5873-5787, 1993). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability that a match between two sequences of nucleotides or amino acids will occur by chance. For example, a nucleic acid is considered to be similar to a reference sequence if the smallest sum probability when comparing the test nucleic acid with the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.

[0091] The percent identity between two amino acid sequences can also be determined using the algorithm of 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 remainder table, a gap length penalty of 12, and a gap penalty of 4. Additionally, 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 either a BLOSUM62 matrix or a PAM250 matrix, and gap weights of 16, 14, 12, 10, 8, 6, or 4 and length weights of 1, 2, 3, 4, 5, or 6.

[0092] The term "nucleic acid" is used interchangeably herein with the term "polynucleotide" and refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single-stranded or double-stranded form. The term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which may be 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 nucleotide. Examples of such analogs include, but are not limited to, phosphorothioates, phosphoramidates, methyl phosphonates, chiral methyl phosphonates, 2-O-methyl ribonucleotides, and peptide nucleic acids (PNAs).

[0093] The term "operably linked" in the context of nucleic acids refers to a functional relationship between two or more polynucleotide (e.g., DNA) segments. Typically, this 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 an appropriate host cell or other expression system. Generally, promoter transcriptional regulatory sequences operably linked to a transcriptional sequence are physically contiguous to 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 located in close proximity to the coding sequence whose transcription they enhance.

[0094] In some embodiments, the present disclosure provides compositions, e.g., pharmaceutically acceptable compositions, comprising an anti-CEA antibody described herein formulated with at least one pharmaceutically acceptable excipient. As used herein, the term "pharmaceutically acceptable excipient" includes all physiologically compatible solvents, dispersion media, isotonic and absorption delaying agents, and the like. The excipient may be suitable for intravenous, intramuscular, subcutaneous, parenteral, rectal, spinal, or epidermal administration (e.g., by injection or infusion).

[0095] As used herein, the term "therapeutically effective amount" or "effective amount" refers to the amount of an agent that, when administered to a subject for treating a disease or at least one of the clinical symptoms of a disease or disorder, is sufficient to effect such treatment for the disease, disorder, or condition. A "therapeutically effective amount" may vary depending on the agent, the disease, disorder, and / or symptoms of the disease or disorder, the severity of the disease, disorder, and / or symptoms of the disease or disorder, the age of the subject being treated, and / or the weight of the subject being treated. The appropriate amount in any given case will be apparent to one of ordinary skill in the art or can be determined by routine experimentation. In the case of a combination therapy, a "therapeutically effective amount" refers to the total amount of the combined components.

[0096] The term "combination therapy" refers to the administration of two or more therapeutic agents to treat a therapeutic condition or disorder. Such administration includes co-administration of these therapeutic agents in a substantially simultaneous manner. Such administration also includes co-administration in multiple or separate containers or formulations (e.g., capsules, powders, and liquids) for each active ingredient. The powders and / or liquids can be reconstituted or diluted to the desired dose before administration. Furthermore, "combination therapy" encompasses the use of each type of therapeutic agent in a sequential manner, either at about the same time or at different times. In either case, the treatment regimen provides the beneficial effect of the drug combination in treating the conditions or disorders described herein.

[0097] As used herein, the phrase "in combination with" means that an anti-CEA ADC is administered to a subject simultaneously with, immediately before, or immediately after the administration of an additional therapeutic agent. In certain embodiments, an anti-CEA ADC is administered as a combination with an additional therapeutic agent.

[0098] 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 of a cell, and / or causes cell death. This term includes toxins (including fragments and / or variants thereof), such as radioisotopes, chemotherapeutic agents, and small molecule or enzymatically active toxins of bacterial, fungal, plant, or animal origin. Examples of cytotoxic agents include auristatins (e.g., auristatin E, auristatin F, MMAE, and MMAF), auromycin, maytansinoids, pyrrolobenzodiazepines (PBDs), ricin, ricin A chain, comblastatins, duocarmycins, dolastatins, doxorubicin, daunorubicin, taxol, cisplatin, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, dihydroxyanthracin dione, actinomycin, and the like. Examples of suitable exotoxins include, but are not limited to, thiatoxin, diphtheria toxin, Pseudomonas exotoxin (PE) A, PE40, abrin, abrin A chain, modeccin A chain, alpha-sarcin, gelonin, mitogenin, letostrictocin, phenomycin, enomycin, curicin, crotin, and calicheamicin, as well as radioactive isotopes such as At211, I131, I125, Y90, Re186, Re188, Sm153, Bi212 or 213, P32, and Lu177.

[0099] An "alkyl" group is a saturated straight or branched chain acyclic hydrocarbon 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; saturated branched chain alkyls include -isopropyl, -sec-butyl, -isobutyl, -tert-butyl, -isopentyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, and the like. Alkyl groups can be substituted or unsubstituted. In certain embodiments, when alkyl groups described herein are said to be "substituted," they can be substituted with any substituent or substituents (as found in the exemplary compounds and embodiments disclosed herein), as well as halogen (chloro, iodo, bromo, or fluoro), hydroxyl, alkoxy, alkoxyalkyl, amino, alkylamino, carboxy, nitro, cyano, thiol, thioether, imine, imide, amidine, guanidine, enamine, aminocarbonyl, acylamino, phosphonate, phosphine, thiocarbonyl, sulfonyl, sulfone, sulfonamide, ketone, aldehyde, ester, urea, urethane, oxime, hydroxylamine, alkoxyamine, aralkoxyamine, N-oxide, hydrazine, hydrazide, hydrazone, azide, isocyanate, isothiocyanate, cyanate, thiocyanate, B(OH), or O(alkyl)aminocarbonyl.

[0100] An "alkenyl" group is a straight-chain or branched-chain acyclic hydrocarbon having 2 to 10 carbon atoms, typically 2 to 8 carbon atoms, and containing at least one carbon-carbon double bond. Representative straight-chain and branched (C2C8) alkenyls include -vinyl, -allyl, -1-butenyl, -2-butenyl, -isobutylenyl, -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, and the like. The double bond of an alkenyl group can be unconjugated or conjugated to another unsaturated group. An alkenyl group can be unsubstituted or substituted.

[0101] A "cycloalkyl" group is a saturated or partially saturated cyclic alkyl group of 3 to 10 carbon atoms, having a single ring or multiple fused or bridged rings, which may be optionally substituted with 1 to 3 alkyl groups. In some embodiments, the cycloalkyl group has 3 to 8 ring members; in other embodiments, the number of ring carbon atoms ranges from 3 to 5, 3 to 6, or 3 to 7. Such cycloalkyl groups include, for example, single ring structures such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, 1-methylcyclopropyl, 2-methylcyclopentyl, 2-methylcyclooctyl, etc., or multiple ring or bridged ring structures such as adamantyl, etc. Examples of unsaturated cycloalkyl groups include cyclohexenyl, cyclopentenyl, cyclohexadienyl, butadienyl, pentadienyl, and hexadienyl, among others. The cycloalkyl group can be substituted or unsubstituted. Such substituted cycloalkyl groups include, for example, cyclohexanone, etc.

[0102] An "aryl" group is an aromatic carbocyclic group of 6 to 14 carbon atoms, having a single ring (e.g., phenyl) or multiple condensed rings (e.g., naphthyl or anthryl). In some embodiments, aryl groups have 6 to 14 carbons, and in other embodiments, 6 to 12 or even 6 to 10 carbon atoms in the ring portion of the group. Specific aryl groups include phenyl, biphenyl, naphthyl, and the like. Aryl groups can be substituted or unsubstituted. The phrase "aryl group" also includes groups containing condensed rings, for example, fused aromatic aliphatic ring systems (e.g., indanyl, tetrahydronaphthyl, etc.).

[0103] A "heteroaryl" group is an aryl ring system having 1 to 4 heteroatoms as ring atoms in the heteroaromatic ring system, with the remainder of the atoms being carbon atoms. In some embodiments, heteroaryl groups contain 5 to 6 ring atoms, and in other embodiments, 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, heteroaryl ring systems are monocyclic or bicyclic. Non-limiting examples include pyrrolyl, pyrazolyl, imidazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, thiazolyl, pyrrolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, thiophenyl, benzothiophenyl, furanyl, benzofuranyl (e.g., isobenzofuran-1,3-diimine), indolyl, azaindolyl (e.g., pyrrolopyridyl or 1H-pyrrolo[2,3-b]pyridyl), indazolyl, benzimidazolyl (e.g., 1H-benzo[d]imidazolyl), imidazole, and the like. These include, but are not limited to, groups such as azabenzoimidazolyl (e.g., azabenzimidazolyl, 3H-imidazo[4,5-b]pyridyl or 1H-imidazo[4,5-b]pyridyl), pyrazolopyridyl, triazolopyridyl, benzotriazolyl, benzoxazolyl, benzothiazolyl, benzothiadiazolyl, isoxazolopyridyl, thianaphthalenyl, purinyl, xanthinyl, adeninyl, guaninyl, quinolinyl, isoquinolinyl, tetrahydroquinolinyl, quinoxalinyl, and quinazolinyl groups.

[0104] A "heterocyclyl" is a non-aromatic cycloalkyl in which 1 to 4 of the ring carbon atoms are independently replaced with a heteroatom independently selected from the group consisting of O, S, and N. In some embodiments, heterocyclyl groups contain 3 to 10 ring members, while other such groups have 3 to 5, 3 to 6, or 3 to 8 ring members. A heterocyclyl can also be attached to other groups at any ring atom (i.e., any carbon atom or heteroatom of the heterocyclic ring). Heterocyclyl groups can be substituted or unsubstituted. Heterocyclyl groups encompass unsaturated, partially saturated, and saturated ring systems, such as imidazolyl, imidazolinyl, and imidazolidinyl groups. The term heterocyclyl includes fused ring species, such as those containing fused aromatic and non-aromatic groups, such as benzotriazolyl, 2,3-dihydrobenzo[1,4]dioxinyl, and benzo[1,3]dioxolyl. The phrase also includes bridged polycyclic ring systems containing heteroatoms, such as, for example, but not limited to, quinuclidyl.Representative examples of heterocyclyl groups include aziridinyl, azetidinyl, pyrrolidyl, imidazolidinyl, pyrazolidinyl, thiazolidinyl, tetrahydrothiophenyl, tetrahydrofuranyl, dioxolyl, furanyl, thiophenyl, pyrrolyl, pyrrolinyl, imidazolyl, imidazolinyl, pyrazolyl, pyrazolinyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, thiazolyl, thiazolinyl, isothiazolyl, thiadiazolyl, oxadiazolyl, piperidyl, piperazinyl, morpholinyl, thiomorpholinyl, tetrahydropi pyranyl (e.g., tetrahydro-2H-pyranyl), tetrahydrothiopyranyl, oxathiane, dioxyl, dithianyl, pyranyl, pyridyl, pyrimidinyl, pyridazinyl, pyrazinyl, triazinyl, dihydropyridyl, dihydrodithionyl, dihydrodithionyl, homopiperazinyl, quinuclidyl, indolyl, indolinyl, isoindolyl, azaindolyl (pyrrolopyridyl), indazolyl, indolizinyl, benzotriazolyl, benzimidazolyl, benzofuranyl, benzothiophenyl, benzthiazolyl, benzoxazolyl Azolyl, benzoxazinyl, benzodithiinyl, benzoxathiinyl, benzothiazinyl, benzoxazolyl, benzothiazolyl, benzothiadiazolyl, benzo[1,3]dioxolyl, pyrazolopyridyl, imidazopyridyl (azabenzimidazolyl, e.g., 1H-imidazo[4,5-b]pyridyl, or 1H-imidazo[4,5-b]pyridin-2(3H)-onyl), triazolopyridyl, isoxazolopyridyl, purinyl, xanthinyl, adeninyl, guaninyl, quinolinyl, isoquinolinyl, quinolizinyl, quinoxalyl Examples of aryl groups include, but are not limited to, phenyl, quinazolinyl, cinnolinyl, phthalazinyl, naphthyridinyl, pteridinyl, thianaphthalenyl, dihydrobenzothiazinyl, dihydrobenzofuranyl, dihydroindolyl, dihydrobenzodioxinyl, tetrahydroindolyl, tetrahydroindazolyl, tetrahydrobenzimidazolyl, tetrahydrobenzotriazolyl, tetrahydropyrrolopyridyl, tetrahydropyrazolopyridyl, tetrahydroimidazopyridyl, tetrahydrotriazolopyridyl, and tetrahydroquinolinyl groups.Representative substituted heterocyclyl groups may be mono- or more than twice substituted, for example, but not limited to, pyridyl or morpholinyl groups, which may be di-, tri-, tetra-, pentad-, or hexad-substituted, or di-substituted, with various substituents such as those listed below.

[0105] A "cycloalkylalkyl" group is a radical of the formula: -alkyl-cycloalkyl, where alkyl and cycloalkyl are defined above. Substituted cycloalkylalkyl groups can be substituted on the alkyl, cycloalkyl, or both the alkyl and cycloalkyl portions 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.

[0106] An "aralkyl" group is a radical of the formula: -alkyl-aryl, where alkyl and aryl are defined above. Substituted aralkyl groups can be substituted on the alkyl, aryl, or both the alkyl and aryl portions of the group. Representative aralkyl groups include, but are not limited to, benzyl and phenethyl groups, and fused (cycloalkylaryl)alkyl groups such as 4-ethyl-indanyl.

[0107] A "heterocyclylalkyl" group is a radical of the formula: -alkyl-heterocyclyl, where alkyl and heterocyclyl are defined above. Substituted heterocyclylalkyl groups can be substituted on the alkyl, heterocyclyl, or both the alkyl and heterocyclyl portions of the group. Representative heterocyclylalkyl groups include, but are not limited to, 4-ethylmorpholinyl, 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-ylethyl, and indol-2-ylpropyl.

[0108] "Halogen" is chloro, iodo, bromo, or fluoro.

[0109] An "alkoxy" or "alkoxyl" group is an --O(alkyl), where alkyl is as defined above.

[0110] An "alkoxyalkyl" group is -(alkyl)O(alkyl), where each alkyl is independently as defined above.

[0111] An "amine" group is a radical of the formula: --NH.sub.2.

[0112] A “hydroxylamine” group has the formula: N(R # )OH or NHOH radical, where R # is a substituted or unsubstituted alkyl, cycloalkyl, cycloalkylalkyl, aryl, aralkyl, heterocyclyl, or heterocyclylalkyl group as defined herein.

[0113] An “alkoxyamine” group is a group of the formula: —N(R # )O-alkyl or -NHO-alkyl radicals, where R # is as defined above.

[0114] An “aralkoxyamine” group has the formula: N(R # ) O-aryl or NHOaryl radicals, where R # is as defined above.

[0115] An "alkylamine" group is a radical of the formula: NH alkyl or N(alkyl) 2 , where each alkyl is independently as defined above.

[0116] An "aminocarbonyl" group is a group of the formula: -C(=O)N(R # )2, -C(=O)NH(R # ), or a radical of C(=O)NH2, where each R # is as defined above.

[0117] An "acylamino" group is a group of the formula: NHC(=O)(R # ) or N(alkyl)C(=O)(R # ) radical, where each alkyl and R # are independently as defined above.

[0118] An "O(alkyl)aminocarbonyl" group is a group of the formula: -O(alkyl)C(=O)N(R # )2, -O(alkyl)C(=O)NH(R # ), or a radical of —O(alkyl)C(═O)NH2, where each R # are independently as defined above.

[0119] An "N-oxide" group is a group of the formula: -N + -O - is a radical of

[0120] A "carboxy" group is a radical of the formula: C(=O)OH.

[0121] A "ketone" group is a group of the formula: C(=O)(R # ) radical, where R # is as defined above.

[0122] An "aldehyde" group is a radical of the formula: --CH(.dbd.O).

[0123] An "ester" group is an ester of the formula: C(=O)O(R # ) or OC(=O)(R # ) radical, where R # is as defined above.

[0124] A "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 # are independently as defined above.

[0125] An "imine" group is a group of the formula: -N=C(R # )2 or -C(R # )=N(R # ) radical, where each R # are independently as defined above.

[0126] An "imido" group is a group of the formula: -C(=O)N(R#)C(=O)(R # ) or N((C=O)(R # ))2 radical, wherein each R # are independently as defined above.

[0127] A "urethane" group has the formula: -OC(=O)N(R # )2, -OC(=O)NH(R # ), -N(R # )C(=O)O(R # ), or -NHC(=O)O(R # ) radical, where each R # are independently as defined above.

[0128] An "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 radical, and each R # are independently as defined above.

[0129] A "guanidine" group has 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 radical, where each R# are independently as defined above.

[0130] 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 # are independently as defined above.

[0131] An "oxime" group is a group of the formula: -C(=NO(R # ))(R # ), -C(=NOH)(R # ), -CH(=NO(R # )), or —CH(═NOH), where each R # are independently as defined above.

[0132] A "hydrazide" group has 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 # are independently as defined above.

[0133] A “hydrazine” group has the formula: —N(R # )N(R # )2, -NHN(R # )2, -N(R # )NH(R # ) 、 -N(R # )NH2, -NHNH(R # )2, or -NHNH2 radicals, where each R # are independently as defined above.

[0134] A "hydrazone" group has the formula: -C(=NN(R # )2)(R # )2, -C(=NNH(R # ))(R # )2, -C(=N-NH2)(R # )2, -N(R # )(N=C(R # )2), or -NH(N=C(R # )2) is a radical of the formula # are independently as defined above.

[0135] An "azido" group is a radical of the formula: -N3.

[0136] An "isocyanate" group is a radical of the formula: N=C=O.

[0137] An "isothiocyanate" group is a radical of the formula: N=C=S.

[0138] A "cyanate" group is a radical of the formula: OCN.

[0139] A “thiocyanate” group is a radical of the formula: SCN.

[0140] A “thioether” group has the formula: —S(R # ) radical, where R # is as defined above.

[0141] A "thiocarbonyl" group is a group of the formula: -C(=S)(R# ) radical, where R # is as defined above.

[0142] A "sulfinyl" group is a group of the formula: -S(=O)(R # ) radical, where R # is defined above.

[0143] A "sulfone" group is a group of the formula: -S(=O)(R # ) radical, where R # is as defined above.

[0144] A "sulfonylamino" group is a group of the formula: -NHSO(R # ) or -N(alkyl)SO2(R # ) radical, where each alkyl and R # is defined above.

[0145] A "sulfonamide" group is a group of the formula: -S(=O)N(R # )2, or -S(=O)2NH(R # ), or a radical of —S(═O)2NH2, where each R # are independently as defined above.

[0146] A "phosphonate" group has the formula: -P(=O)(O(R # ))2, -P(=O)(OH)2, -OP(=O)(O(R # ))(R # ), or -OP(=O)(OH)(R # ) radical, where each R # are independently as defined above.

[0147] A "phosphine" group has the formula: -P(R # )2 radical, wherein each R # are independently as defined above.

[0148] When groups described herein are said to be "substituted," with the exception of alkyl groups, they can be substituted with any suitable substituent or substituents. Illustrative examples of substituents include those found in the exemplary compounds and embodiments disclosed herein, as well as halogen (chloro, iodo, bromo, or fluoro), alkyl, hydroxyl, 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, urethane, oxime, hydroxylamine, alkoxyamine, aralkoxyamine, N-oxide, hydrazine, hydrazide, hydrazone, azide, isocyanate, isothiocyanate, cyanate, thiocyanate, oxygen (═O), B(OH), O(alkyl)aminocarbonyl, cycloalkyl (which may be simply and heterocyclyl (which may be a single ring or a fused or non-fused multiple rings) (e.g., cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl), or heterocyclyl (which may be a single ring or a fused or non-fused multiple rings) (e.g., pyrrolidyl, piperidyl, piperazinyl, morpholinyl, or thiazinyl), single ring or a fused or non-fused multiple rings aryl or heteroaryl (e.g., phenyl, naphthyl, pyrrolyl, indolyl, furanyl, thiophenyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, triazolyl, tetrazolyl, pyrazolyl, pyridinyl, quinolinyl, isoquinolinyl, acridinyl, pyrazinyl, pyridazinyl, pyrimidinyl, benzimidazolyl, benzothiophenyl, or benzofuranyl), aryloxy, aralkyloxy, heterocyclyloxy, and heterocyclylalkoxy.

[0149] As used herein, the term "pharmaceutically acceptable salt(s)" refers to salts prepared from pharmaceutically acceptable non-toxic acids or bases, including inorganic acids and bases and organic acids and bases.

[0150] As used herein, unless otherwise indicated, the term "solvate" means a compound or salt thereof that further includes a stoichiometric or non-stoichiometric amount of solvent bound by non-covalent intermolecular forces. In one embodiment, the solvate is a hydrate.

[0151] As used herein, unless otherwise indicated, the term "hydrate" refers to a compound or salt thereof that further includes a stoichiometric or non-stoichiometric amount of water bound by non-covalent intermolecular forces.

[0152] All pharmaceutically acceptable salts, solvates, and / or hydrates of the compounds depicted herein are within the scope of this disclosure.

[0153] As used herein, unless otherwise indicated, the term "prodrug" refers to a derivative of a compound that can be hydrolyzed, oxidized, or otherwise reacted under biological conditions (in vitro or in vivo) to yield an active compound, particularly a compound. Examples of prodrugs include, but are not limited to, derivatives and metabolites of a compound that contain a biohydrolyzable moiety, such as biohydrolyzable amides, biohydrolyzable esters, biohydrolyzable carbamates, biohydrolyzable carbonates, biohydrolyzable ureides, and biohydrolyzable phosphate analogs. In certain embodiments, prodrugs of compounds with a carboxyl functional group are lower alkyl esters of the carboxylic acid. Conveniently, the carboxylic acid esters are formed by esterifying any of the carboxylic acid moieties present on the molecule. Prodrugs are typically prepared by well-known methods, for example, as described in Burger's Medicinal Chemistry and Drug Discovery 6 th(Donald J. Abraham ed., 2001, Wiley) and Design and Application of Prodrugs (H. Bundgaard ed., 1985, Harwood Academic Publishers Gmfh).

[0154] As used herein, unless otherwise indicated, the terms "stereoisomer" or "stereoisomerically pure" refer to one 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. A typical stereoisomerically pure compound will contain greater than about 80% by weight of one stereoisomer of the compound and less than about 20% by weight of other stereoisomers of that compound, greater than about 90% by weight of one stereoisomer of the compound and less than about 10% by weight of other stereoisomers of that compound, greater than about 95% by weight of one stereoisomer of the compound and less than about 5% by weight of other stereoisomers of that 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 that compound. Compounds may contain chiral centers and may exist as racemates, individual enantiomers or diastereomers, and mixtures thereof. All such isomers, including mixtures thereof, are included in the embodiments disclosed herein. The use of stereomerically pure forms of such compounds, as well as mixtures of these forms, is encompassed by the embodiments disclosed herein. For example, mixtures containing equal or unequal amounts of the enantiomers of a particular compound can be used in the methods and compositions disclosed herein. These isomers can be asymmetrically synthesized or resolved using standard techniques, such as chiral columns or chiral resolving agents.See, for example, Jacques, J., et al., Enantiomers, Racemates and Resolutions (Wiley Interscience, New York, 1981); Wilen, SH, et al., Tetrahedron 33:2725 (1977); Eliel, E.L., Stereochemistry of Carbon Compounds (McGrawHill, NY, 1962); and Wilen, SH, Tables of Resolving Agents and Optical Resolutions p. 268 (E.L. Eliel, Ed., University of Notre Dame Press, Notre Dame, IN, 1972).

[0155] 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 isolated as either cis or trans isomers. In other embodiments, the compounds are mixtures of cis and trans isomers.

[0156] "Tautomer" refers to isomeric forms of a compound that are in equilibrium with each other. The concentration of isomeric forms may vary depending on the environment in which the compound is found, for example, whether the compound is solid or in an organic or aqueous solution. For example, in aqueous solution, pyrazole may exhibit the following isomeric forms, which are referred to as tautomers of each other: [ka]

[0157] As will be readily understood by one of ordinary skill in the art, a wide variety of functional groups and other structures may exhibit tautomerism, and all tautomers of the compounds are within the scope of the present disclosure.

[0158] It is also noted that the compounds may contain unnatural proportions of atomic isotopes at one or more of the atoms. For example, the compounds may contain radioactive isotopes, such as tritium ( 3 H), iodine-125( 125 I), sulfur 35( 35 S), or carbon-14 ( 14 C), or may be radiolabeled with, for example, deuterium ( 2 H), carbon-13( 13 C), or nitrogen-15( 15 The compound may be isotopically enriched with 1,2,3,4,5,6,6-trimethyl-1,2,4,6,7,8,8,9,9,9,10,11,12,13,14,15,16,17,18,19,19,19,20,21,22,23,24,25,26,27,28,29,29,29,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,59,60,61,62,63,64,59,59,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,79,79,70,71,71,72,74,75,76,77,78,79,79,79,79,70,71,71,71,72,73,74,75,76,77,78,79 ...0,70,71,71,71,71,72,73,74,75,76,77,78,

[0159] It should be noted that if there is a discrepancy between a depicted structure and the name for that structure, the depicted structure should prevail.

[0160] 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. Alkynyl is optionally substituted and can be straight-chained, branched, or cyclic. Alkynyl includes radicals having 2 to 20 carbon atoms, i.e., C 2-20 Alkynyl radicals, radicals having 2 to 12 carbon atoms, i.e., C 2-12Alkynyl radicals, radicals having 2 to 8 carbon atoms, i.e., C 2-8 Alkynyl radicals, radicals having 2 to 6 carbon atoms, i.e., C 2-6 Alkynyl radicals and radicals having 2 to 4 carbon atoms, i.e., C 2-4 Examples of alkynyl radicals include, but are not limited to, ethynyl, propynyl, and butynyl.

[0161] As used herein, "haloalkyl" refers to an alkyl, as defined above, containing at least one substituent selected from a halogen, e.g., fluorine (F), chlorine (Cl), bromine (Br), or iodine (I). Examples of haloalkyl include, but are not limited to, -CF, -CHCF, -CClF, and -CCl.

[0162] As used herein, "haloalkoxy" refers to an alkoxy, as defined above, that contains at least one substituent selected from a halogen, for example, F, Cl, Br, or I.

[0163] As used herein, "arylalkyl" refers to a monovalent moiety that is a radical of an alkyl compound, where the alkyl compound is substituted with an aromatic substituent. That is, the aromatic compound contains a single bond to the alkyl group, and the radical is localized on the alkyl group. The arylalkyl group is attached to the depicted chemical structure via the alkyl group. Arylalkyl can be represented by structures such as B-CH2-, B-CH2-CH2-, B-CH2-CH2-, B-CH2-CH2-CH2-, B-CH(CH3)-CH2-CH2-, B-CH2-CH(CH3)-CH2-, where B is an aromatic moiety, e.g., phenyl. Arylalkyl is optionally substituted. That is, the aryl group and / or the alkyl group can be substituted as disclosed herein. Examples of arylalkyl include, but are not limited to, benzyl.

[0164] As used herein, "alkylaryl" refers to a monovalent moiety that is a radical of an aryl compound, where the aryl compound is substituted with an alkyl substituent. That is, the aryl compound contains a single bond to the alkyl group, and the radical is localized on the aryl group. The alkylaryl group is attached to the depicted chemical structure via the aryl group. The alkylaryl can be represented by structures such as -B-CH, -B-CH-CH, -B-CH-CH-CH, -B-CH-CH-CH, -B-CH(CH)-CH-CH, -B-CH-CH(CH)-CH, where B is an aromatic moiety, e.g., phenyl. The alkylaryl is optionally substituted; that is, the aryl group and / or the alkyl group can be substituted as disclosed herein. Examples of alkylaryl include, but are not limited to, toluyl.

[0165] As used herein, "aryloxy" refers to a monovalent moiety that is a radical of an aromatic compound, where the ring atoms are carbon atoms and the ring is substituted with an oxygen radical, i.e., the aromatic compound contains a single bond to the oxygen atom and the radical is localized at the oxygen atom (e.g., in the case of phenoxy, C6H5-O-). The aryloxy substituent is attached to the compound it substitutes through this oxygen atom. The aryloxy is optionally substituted. Aryloxy includes radicals having 6 to 20 ring carbon atoms, i.e., C 6-20 Aryloxy radicals, radicals having 6 to 15 ring carbon atoms, i.e., C 6-15 Aryloxy radicals and radicals having 6 to 10 ring carbon atoms, i.e., C 6-10 Examples of aryloxy moieties include, but are not limited to, phenoxy, naphthoxy, and anthroxy.

[0166] As used herein, the term "residue" refers to the chemical moiety in a compound that remains after a chemical reaction. For example, the term "amino acid residue" or "N-alkylamino acid residue" refers to the product of amide or peptide coupling of an amino acid or an N-alkylamino acid with a suitable coupling partner, e.g., a water molecule is expelled after amide or peptide coupling of the amino acid or N-alkylamino acid, resulting in the incorporation of the amino acid residue or N-alkylamino acid residue into the product.

[0167] As used herein, a "sugar" or "sugar group" or "sugar residue" refers to a carbohydrate moiety that may contain a 3-carbon (triose) unit, a 4-carbon (tetrose) unit, a 5-carbon (pentose) unit, a 6-carbon (hexose) unit, a 7-carbon (heptose) unit, or a combination thereof, and may be a monosaccharide, disaccharide, trisaccharide, tetrasaccharide, pentasaccharide, oligosaccharide, or any other polysaccharide. In some cases, a "sugar" or "sugar group" or "sugar residue" includes a furanose (e.g., ribofuranose, fructofuranose), or a pyranose (e.g., glucopyranose, galactopyranose), or a combination thereof. In some cases, a "sugar" or "sugar group" or "sugar residue" includes an aldose or ketose, or a combination 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 "sugars" or "sugar groups" or "sugar residues" include polysaccharides and / or oligosaccharides, including, but not limited to, amylose, amylopectin, glycogen, inulin, and cellulose. In some cases, the "sugar" or "sugar group" or "sugar residue" is an amino sugar. In some cases, the "sugar" or "sugar group" or "sugar residue" is a glucamine residue (1-amino-1-deoxy-D-glucitol), which is linked to the rest of the molecule via its amino group to form an amide bond with the rest of the molecule (i.e., a glucamide).

[0168] As used herein, "inorganic acid residues" refers to orthophosphoric and pyrophosphoric acid, phosphoric acid, and sulfuric acid residues.

[0169] As used herein, "organic acid residue" refers to the residue of an alkane carboxylic acid, an amino acid, or an oligopeptide. In one embodiment, the alkane carboxylic acid is methanoic acid, ethanoic acid, propanoic acid, butanoic acid, pentanoic acid, hexanoic acid, heptanoic acid, octanoic acid, nonanoic acid, decanoic acid, undecanoic acid, dodecanoic acid, tridecanoic acid, tetradecanoic acid, pentadecanoic acid, hexadecanoic acid, heptadecanoic acid, octadecanoic acid, nonadecanoic acid, or icosanoic acid. In one embodiment, the alkane carboxylic acid is methanoic acid, ethanoic acid, propanoic acid, or butanoic acid.

[0170] Certain groups, moieties, substituents, and atoms are represented by a wavy line crossing a bond or bonds to indicate the atom to which the group, moiety, substituent, or atom is bonded. For example: [ka] The propyl-substituted phenyl group, depicted as: [ka] is.

[0171] As used herein, a diagram showing a substituent attached to a cyclic group (e.g., aromatic, heteroaromatic, fused ring, and saturated or unsaturated cycloalkyl or heterocycloalkyl) via a bond between ring atoms is meant to indicate that the cyclic group can be substituted with that substituent at any ring position of a cyclic group, or on any ring of a fused ring group, in accordance with techniques described herein or known in the art to which this disclosure pertains, unless otherwise specified.

[0172] Diagrams showing substituents attached to an acyclic group via a bond between two atoms are meant to indicate that the substituent may be attached to either atom of the bond through which the substituent bond passes, unless otherwise specified, in accordance with techniques described herein or known in the art to which this disclosure pertains. Thus, for example, [ka] teeth, [ka] Includes.

[0173] Detailed Description The present disclosure provides anti-CEA antibody-drug conjugates (ADCs). The present disclosure also provides anti-CEA ADCs comprising antibodies with desirable pharmacokinetic properties and other desirable attributes, and thus can be used to reduce the likelihood of a subject having CEA-expressing or accumulating cells, such as cancers characterized by CEA expression or accumulation, or to treat such subjects. The present disclosure further provides pharmaceutical compositions comprising the anti-CEA ADCs, and methods of making and using pharmaceutical compositions comprising the same. In some embodiments, the anti-CEA ADCs are useful for treating CEA-associated diseases and disorders.

[0174] Anti-CEA antibody The present disclosure provides anti-CEA ADCs comprising an anti-CEA antibody or antigen-binding fragment thereof that specifically binds to CEA. The antibodies or antigen-binding fragments of the present disclosure include, but are not limited to, the following antibodies or antigen-binding fragments thereof generated as described below.

[0175] The present disclosure provides antibodies or antigen-binding fragments that specifically bind 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). The present disclosure also provides antibodies or antigen-binding fragments that specifically bind 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, the present disclosure provides antibodies or antigen-binding fragments that specifically bind to CEA, wherein the antibody comprises one, two, three, or more HCDRs (or alternatively consists of one, two, three, or more HCDRs) having the amino acid sequence of any of the HCDRs listed in Table 1.

[0176] The present disclosure provides antibodies or antigen-binding fragments that specifically bind to CEA, wherein the antibodies or antigen-binding fragments comprise a VH domain set forth in Table 1, or any of the HCDR sets 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 antibodies or antigen-binding fragments that specifically bind to CEA, wherein the antibodies or antigen-binding fragments comprise a light chain CDR (LCDR) having the amino acid sequence of any one of the LCDRs listed in Table 1. In particular, the present disclosure provides antibodies or antigen-binding fragments that specifically bind to CEA, wherein the antibodies or antigen-binding fragments comprise one, two, three, or more LCDRs (or alternatively, consist of one, two, three, or more LCDRs) having the amino acid sequence of any of the LCDRs listed in Table 1.

[0177] The present disclosure provides an antibody or antigen-binding fragment that specifically binds to CEA, wherein the antibody or antigen-binding fragment is (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 set forth in SEQ ID NO: 24; HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 25; HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 26, and The three light chain CDRs: LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 27; LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 28; an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 23; or (v) three heavy chain CDRs: HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 7; HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 8; HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 9, and The three light chain CDRs: LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 10; LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 11; an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 6; or (vi) three heavy chain CDRs: HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 41; HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 42; HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 43, and The three light chain CDRs: LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 44; LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 45; LCDR3 comprising the amino acid sequence set forth in SEQ ID NO:40.

[0178] The present disclosure provides ADCs 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 set forth in SEQ ID NO: 14, 31, or 48 (Table 1).

[0179] The present disclosure provides ADCs 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.

[0180] In some embodiments, no more than 1, 2, 3, 4, or 5 amino acids are altered (e.g., via insertion, deletion, or substitution) in the CDR regions when compared to the CDR regions set forth in the sequences in Table 1.

[0181] Other antibodies of the present disclosure include those in which the amino acids or nucleic acids encoding the amino acids have been altered, but which have at least 60%, 70%, 80%, 90%, 95%, or 99% percent identity to the variable region sequences set forth in Table 1. In some embodiments, no more than 1, 2, 3, 4, or 5 amino acids have been altered (e.g., via insertion, deletion, or substitution) in the variable regions when compared to the variable regions set forth in the sequences set forth in Table 1, yet retain substantially the same therapeutic activity.

[0182] The present disclosure also provides nucleic acid sequences encoding the VH, VL, full-length heavy chain, and full-length light chain of an antibody that specifically binds to CEA, which nucleic acid sequences can be optimized for expression in mammalian cells. [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5] [Table 2-6]

[0183] The present disclosure provides ADCs comprising antibodies and antigen-binding fragments thereof that bind to an epitope of human CEA and any of the payloads described herein.

[0184] The present disclosure also provides ADCs comprising antibodies and antigen-binding fragments thereof that bind to the same epitope as an anti-CEA antibody having one or more of the sequences disclosed in Table 1. Accordingly, additional antibodies and antigen-binding fragments thereof can be identified based on their ability to cross-compete with other antibodies in binding assays (e.g., competitively inhibit binding in a statistically significant manner). The ability of a test antibody to inhibit the binding of an antibody and antigen-binding fragment thereof in Table 1 to CEA demonstrates that the test antibody can compete with the antibody or antigen-binding fragment thereof in Table 1 for binding to CEA. Without being bound by any one theory, such antibodies may bind to the same or related (e.g., structurally similar or spatially proximal) epitope on CEA as the competing antibody or antigen-binding fragment thereof. In certain embodiments, an antibody that binds to the same epitope on CEA as an antibody or antigen-binding fragment thereof in Table 1 is a human or humanized monoclonal antibody. Such human or humanized monoclonal antibodies can be prepared and isolated as described herein.

[0185] antibody linker It is also understood that the domains and / or regions of the polypeptide chains of the antibodies disclosed herein can be separated by linker regions of various lengths. In some embodiments, antigen-binding domains are separated from each other, from the CL, CH1, hinge, CH2, CH3, or the entire Fc region by linker regions. For example, VL1-CL-(linker)-VH2-CH1. For example, such linker regions can contain a random assortment of amino acids or a limited set of amino acids. Such linker regions can be flexible or rigid (see US 2009 / 0155275).

[0186] In some embodiments, a linker can be used to conjugate a compound between a toxin or payload and the disclosed antibody. In some embodiments, the linker is cleavable under intracellular conditions, and cleavage of the linker releases the toxin / payload from the antibody in the intracellular environment. In still other embodiments, the linker unit is not 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 precharged linker, or a dicarboxylic acid-based linker.

[0187] Dimerization of specific amino acids In one embodiment, the antibodies disclosed herein comprise at least one dimerization-specific amino acid change. Dimerization-specific amino acid changes result in "knobs-in-holes" interactions, increasing the recruitment of correct antibodies. The dimerization-specific amino acid can be in the CH1 domain or the CL domain, or a combination thereof. Examples of dimerization-specific amino acids used to pair a CH1 domain with another CH1 domain (CH1-CH1) and a CL domain with another CL domain (CL-CL) can be found in at least WO2014082179, WO2015181805 family, and WO2017059551. The dimerization-specific amino acid can also be in the Fc domain or combined with a dimerization-specific amino acid in the CH1 or CL domain. In one embodiment, the present disclosure provides an antibody comprising at least one dimerization-specific amino acid pair.

[0188] Fc region framework modifications In some embodiments, the Fc region is modified 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 a different amino acid residue, resulting in an antibody with altered affinity for an effector ligand while retaining the antigen-binding ability of the parent antibody. The affinity-altered effector ligand can be, for example, an Fc receptor or the C1 component of complement. This approach is described, for example, in U.S. Patent Nos. 5,624,821 and 5,648,260, both by Winter et al.

[0189] In another embodiment, one or more amino acid residues can be substituted 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 approach is described, for example, in U.S. Patent No. 6,194,551 by Idusogie et al.

[0190] In another embodiment, one or more amino acid residues are altered to modify the antibody's ability to fix complement. This approach is described, for example, in publication WO 94 / 29351 by Bodmer et al. In a specific embodiment, one or more amino acids of an antibody or antigen-binding fragment thereof of the present disclosure are replaced with one or more allotypic amino acid residues for the IgG1 subclass and kappa isotype. Allotypic amino acid residues 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 kappa isotype, as described by Jefferis et al., MAbs.1:332-338 (2009).

[0191] In another embodiment, the Fc region is modified by modifying one or more amino acids to enhance the ability of the antibody to mediate antibody-dependent cellular cytotoxicity (ADCC) and / or to increase the affinity of the antibody for Fcγ receptors. This approach is described, for example, in publication WO 00 / 42072 by Presta. Furthermore, the binding sites on human IgG1 for FcγRI, FcγRII, FcγRIII, and FcRn have been mapped, and variants with improved binding have been described (see Shields et al., J. Biol. Chem. 276:6591-6604, 2001).

[0192] In another embodiment, the glycosylation of the antibody is modified. For example, an aglycosylated antibody (i.e., an antibody lacking or reduced glycosylation) can be generated. Altering glycosylation can, for example, increase the affinity of the antibody for an "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 to remove one or more variable region framework glycosylation sites, thereby eliminating glycosylation at those sites. Such aglycosylation can increase the affinity of the antibody for an antigen. Such an approach is described, for example, in U.S. Patent Nos. 5,714,350 and 6,350,861 by Co et al.

[0193] Additionally or alternatively, antibodies can be generated with altered types of glycosylation (e.g., hypofucosylated antibodies with reduced amounts of fucosyl residues or antibodies with increased bisecting GlcNAc structures). Such altered glycosylation patterns have been shown to enhance the ADCC ability of antibodies. Such sugar modifications can be achieved, for example, by expressing the antibody in a host cell with an altered glycosylation pathway. Cells with altered glycosylation pathways have been described in the art and can be used as host cells for expressing recombinant antibodies, thereby producing antibodies with altered glycosylation. For example, EP 1,176,195 by Hang et al. describes a cell line in which the FUT8 gene, encoding fucosyltransferase, has been functionally disrupted so that the expressed antibodies exhibit hypofucosylation. Publication WO 03 / 035835 by Presta describes a mutant CHO cell line, Lecl3 cells, that has a reduced ability to attach fucose to Asn(297)-linked carbohydrates, which also results in hypofucosylation of antibodies expressed in the host cells (see also Shields et al., (2002) J. Biol. Chem. 277:26733-26740). WO 99 / 54342 by Umana et al. describes cell lines engineered to express a glycoprotein-modifying glycosyltransferase (e.g., beta(1,4)-N-acetylglucosaminyltransferase III (GnTIII)) such that antibodies expressed in the engineered cell line exhibit an increase in bisecting GlcNac structures, which results in increased ADCC activity of the antibody (see also Umana et al., Nat. Biotech. 17:176-180, 1999).

[0194] In another aspect, if reduced ADCC is desired, human antibody subclass IgG4 has been shown in many previous reports to have only moderate ADCC and little CDC effector function (Moore GL, 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). Reduced ADCC can be achieved by operably binding an antibody to an IgG4 Fc engineered with a combination of modifications that reduce FcγR binding or C1q binding activity, thereby reducing or eliminating ADCC and CDC effector function. Considering the physicochemical properties of antibodies as biological drugs, one of the more undesirable intrinsic properties of IgG4 is the dynamic separation of its two heavy chains in solution to form antibody halves, which generates bispecific antibodies in vivo through a process called "Fab arm exchange" (Van der Neut Kolfschoten M., et al., 2007 Science, 317:1554-157). Mutation of serine to proline at position 228 (EU numbering system) appeared to inhibit IgG4 heavy chain separation (Angal, S. 1993 Mol Immunol, 30:105-108; Aalberse et al., 2002 Immunol, 105:9-19).Some amino acid residues in the hinge and gamma Fc region have been reported to affect antibody interaction with Fcγ receptors (Chappel SM, et al., 1991 Proc. Natl. Acad. Sci. USA, 88:9036-9040; Mukherjee, J. et al., 1995 FASEB J, 9:115-119; Armour, KL et al., 1999 Eur J Immunol, 29:2613-2624; Clynes, RA et al., 2000 Nature Medicine, 6:443-446; Arnold JN, 2007 Annu Rev Immunol, 25:21-50). Furthermore, some IgG4 isoforms that occur rarely in the human population may also have 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, it is possible to modify the hinge and Fc regions of human IgG4 and introduce several alterations. These modified IgG4 Fc molecules can be found in SEQ ID NOs: 83-88 of U.S. Patent No. 8,735,553 by Li et al.

[0195] antibody production The antibodies and antigen-binding fragments thereof for use in the disclosed ADCs can be produced by any means known in the art, including, but not limited to, recombinant expression, chemical synthesis, and enzymatic digestion of antibody tetramers, while full-length monoclonal antibodies can be obtained, for example, by hybridoma or recombinant production. Recombinant expression can be from any suitable host cell known in the art, such as a mammalian host cell, a bacterial host cell, a yeast host cell, an insect host cell, etc.

[0196] The present disclosure further provides polynucleotides encoding the antibodies described herein, e.g., polynucleotides encoding heavy or light chain variable regions or segments comprising the complementarity determining regions described herein. In some embodiments, 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 a polynucleotide set forth in SEQ ID NO: 16, SEQ ID NO: 33, or SEQ ID NO: 50. In some embodiments, 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 a polynucleotide selected from SEQ ID NO: 17, SEQ ID NO: 34, or SEQ ID NO: 51.

[0197] The polynucleotides of the present disclosure can encode the variable region sequences of anti-CEA antibodies. The polynucleotides of the present disclosure can also encode both the variable and constant regions of the antibodies. Some of the polynucleotide sequences encode polypeptides containing both the heavy and light chain variable regions of the exemplified anti-CEA antibodies.

[0198] 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 in which the vector will be expressed. Typically, expression vectors contain a promoter and other regulatory sequences (e.g., enhancers) operably linked to the polynucleotide encoding the anti-CEA antibody chain or antigen-binding fragment. In some embodiments, 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 grown under non-inducing conditions without biasing the population toward coding sequences whose expression products are better tolerated by the host cell. In addition to a promoter, other regulatory elements can be included for efficient expression of the anti-CEA antibody or antigen-binding fragment thereof. These elements may include an ATG initiation codon and adjacent ribosome binding site or other sequences. Furthermore, the efficiency of expression can be increased by incorporating enhancers appropriate for the cell system being used (see, e.g., Scharf et al., Results Probl. Cell Differ. 20:125, 1994, and Bittner et al., Meth. Enzymol., 153:516, 1987). For example, the SV40 enhancer or CMV enhancer can be used to increase expression in mammalian host cells.

[0199] Host cells for harboring and expressing anti-CEA antibody vectors can be prokaryotic or eukaryotic. E. coli is one prokaryotic host useful 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. Expression vectors typically contain expression control sequences compatible with the host cell (e.g., an origin of replication). Additionally, various well-known promoters may be present, such as the lactose promoter system, tryptophan (trp) promoter system, beta-lactamase promoter system, or promoter systems derived from phage lambda. The promoter typically optionally controls expression via an operator sequence and contains, for example, ribosome binding site sequences for initiating and completing transcription and translation. Other microorganisms, such as yeast, can also be used to express anti-CEA antibodies. Insect cells in combination with baculovirus vectors can also be used.

[0200] In other embodiments, mammalian host cells are used to express and produce the anti-CEA antibodies of the present disclosure. Examples include hybridoma cell lines expressing endogenous immunoglobulin genes or mammalian cell lines harboring exogenous expression vectors. These include any normal mortal, or normal or abnormal immortal, 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, HEK293 cells, myeloma cell lines, transformed B cells, and hybridomas. The use of mammalian tissue cell culture to express polypeptides is generally described, for example, in Winnacker, From Genes to Clones, VCH Publishers, NY, NY, 1987. Expression vectors for mammalian host cells can include expression control sequences such as an origin of replication, a promoter, and an enhancer (see, e.g., Queen et al., Immunol. Rev. 89:49-68, 1986), as well as necessary processing information sites such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcription terminator sequences. These expression vectors usually contain promoters derived from mammalian genes or mammalian viruses. Suitable promoters can be constitutive, cell type-specific, stage-specific, and / or tunable or regulatable. 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 poly III 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.

[0201] Antibody-drug conjugates The antibodies disclosed herein may be combined with a cytotoxic agent (herein "D" or "P") to form an antibody-drug conjugate. A cytotoxic agent can be any molecule that inhibits or reduces the expression of a molecule in a cell, inhibits or reduces the function of a cell, induces apoptosis of a cell, and / or causes cell death. Examples of cytotoxic agents include those described herein. In embodiments, the cytotoxic agent is a topoisomerase inhibitor.

[0202] In embodiments, the antibody drug conjugate has the formula A: Ab-(CL-(D) m ) n (A), or a pharmaceutically acceptable salt, solvate, or hydrate thereof, wherein: Ab is an antibody or antigen-binding fragment thereof; C is a conjugator, L is a linker, D is a cytotoxic agent; m is an integer from 1 to 8, and n is 1 to 10.

[0203] In a specific embodiment, m is 1.

[0204] In embodiments, the antibody drug conjugate has the formula A-1: Ab-(CLD) n (A-1), or a pharmaceutically acceptable salt, solvate, or hydrate thereof, wherein: Ab is an antibody or antigen-binding fragment thereof; C is a conjugator, L is a linker, D is a cytotoxic agent; m is an integer from 1 to 8, and n is 1 to 10.

[0205] In embodiments, n is 3 to 10, e.g., 4 to 10, 5 to 10, 6 to 10, or 7 to 9. In certain embodiments, n is about 8.

[0206] International Publication No. WO2023 / 125530, the entire contents of which are incorporated herein by reference, discloses antibody-drug conjugates, linker-payload moieties thereof that are suitable for use in the context of the present disclosure, and linker payloads that are suitable for use in the context of the present disclosure. In some embodiments, the linker payload is a linker payload disclosed in WO2023 / 125530.

[0207] In embodiments, the antibody drug conjugate has formula (I): [ka] or a pharmaceutically acceptable salt, tautomer, solvate, stereoisomer, enantiomer, isotopic substitution, or prodrug thereof; wherein BA is Ab, the variables of which are as described for the antibody drug conjugates of the present disclosure (e.g., the antibody drug conjugates of Formula A or A-1), L is a covalent linker, PA is a payload moiety (e.g., a cytotoxic agent (D)), the variables of which are as described for the antibody drug conjugates of the present disclosure (e.g., the antibody drug conjugates of Formula A or A-1), and the subscript x is 1 to 30 (e.g., the variable n is as described for the antibody drug conjugates of the present disclosure (e.g., the antibody drug conjugates of Formula A or A-1). In some cases, x is 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.

[0208] In a further embodiment, the antibody drug conjugate has the formula (Ia): [ka] or a pharmaceutically acceptable salt thereof, a tautomer, solvate, stereoisomer, enantiomer, isotopically substituted derivative, or prodrug thereof, In the formula, RG 1 is a reactive group residue, RG 2 is an optional reactive group residue, SP 1 and SP 2 is, independently in each instance, the residue of an optional spacer group, HG is a hydrophilic residue, PAB is an optional self-immolative unit, the subscript p is 0 or 1, and the subscript x is 1 to 30. The values ​​of the remaining variables (e.g., AA 2 , A.A. 3 ) and alternative values ​​of variables (e.g., x, p, PAB, HG, RG 1 , R.G. 2 , SP 1 , SP 2 , BA) are as defined elsewhere herein.

[0209] In some embodiments, x is 1 to 15. In some embodiments, x is 2 to 10. In some embodiments, x is 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.

[0210] In some embodiments of the compound of Formula (Ia), AA 2 is the formula (W): [ka] Including AA 3 is -valine-alanine-, -valine-citrulline-, or [ka] where R 6 is -CH3, or -(CH2)3-NHC(=O)NH2.

[0211] In some embodiments, [ka] teeth, [ka] is.

[0212] In some embodiments, AA 3 teeth, [ka] where R 6 is —CH, or —(CH)—NHC(═O)NH. In a further embodiment, R 6 is -CH3.

[0213] In some embodiments, PAB is —NH—CH—O—, a group of formula (Y1): [ka] or formula (Y2): [ka] wherein: [ka] indicates the bond that connects PAB to the adjacent group of the formula.

[0214] In some embodiments, PAB is —NH—CH 2 —O—.

[0215] In some embodiments, RG 1 teeth, [ka] -(succinimide-3-yl-N)-, [ka] In some embodiments, RG 1 teeth, [ka] is.

[0216] In some embodiments, RG 1 teeth, [ka] where EWG is an electron withdrawing group, e.g., -CN, -NO, halogen, -CF, -C(=O)OR 1 , or -C(=O)R 1 and R 1 is substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heterocycloalkyl, or substituted or unsubstituted heteroaryl.

[0217] In some embodiments, RG 1 teeth, [ka] is.

[0218] In some embodiments, RG 1 teeth, [ka] where EWG is an electron withdrawing group, e.g., -CN, -NO, halogen, -CF, -C(=O)OR 1 , or -C(=O)R 1 and R 1 is substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heterocycloalkyl, or substituted or unsubstituted heteroaryl.

[0219] In some embodiments, RG 1 teeth, [ka] The ring-open heterocycle is a product obtained from conjugation of the maleimide ring of RG with an antibody. In this regard, it will be understood that conjugation of the antibody to the maleimide ring can occur at either of the two carbons in the carbon-carbon double bond of the maleimide. Similarly, in the context of a ring-open heterocycle, conjugation can occur at either of the two carbon atoms of the double bond. In some embodiments, RG 1 teeth, [ka] In some embodiments, RG 1 teeth, [ka] In some embodiments, RG 1 teeth, [ka] In some embodiments, RG 1 teeth, [ka] In some embodiments, RG 1 teeth, [ka] In some embodiments, RG 2 is a bond, —C(═O)—NH—, or —NHC(═O)—. In some embodiments, RG 2 is -C(=O)-NH-.

[0220] 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)-, wherein n1, n2, n3, n4, and n5 each independently represent an integer from 1 to 8. In some embodiments, SP 1 is *-CH2C(O)N(H)CH2CH2C(O)-, and the asterisk indicates RG 1 In some embodiments, SP 1 is *-(CH2)5C(O)-, and the asterisk indicates RG 1 In some embodiments, SP 1 is *-C(H)(CH2NH2)-(CH2)2OC(O)N(H)(CH2)2C(O)-, and the asterisk indicates RG 1 indicates the bond that connects to

[0221] In some embodiments, SP 2 is -(CH2) n6 - and n6 represents an integer from 1 to 8. In some embodiments, n6 is 2.

[0222] In some embodiments, HG is [ka] [ka] wherein each n7 is independently 1 to 15, each n8 is independently 0 or 1, each n9 is independently 1 or 2, each n10 is independently an integer of 4 to 16, for example, 4, 8, or 12, each n11 is independently an integer of 0 to 5, n12 is an integer of 0 to 3, d is 0 to 3, and R 2 is H or Me, and R 3 -OH, -NH2, -NHCH2-CH2-(PEG) x-OH, or -NHCH2-CH2-(PEG) x -OMe and R 4 is OH or NH2, and each of X, Y, and Z is independently -CH2-, -NH-, -S-, or -O-.

[0223] In some embodiments, HG is [ka] [ka] wherein each n7 is independently 1 to 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, e.g., 4, 8, or 12, d is 0 to 3, and R 2 is H or Me, and R 3 -OH, -NH2, -NHCH2-CH2-(PEG) x -OH, or -NHCH2-CH2-(PEG) x -OMe and R 4 is OH or NH2.

[0224] In some embodiments, HG is [ka] wherein each n8 is independently 0 or 1; R 1 is H or Me.

[0225] In some embodiments, HG is [ka] is.

[0226] In some embodiments, HG is [ka] wherein 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-.

[0227] In some embodiments, HG is —NHSO2NH2, —SO3H, —SO2NH2, —PO3H2, and RG 2 is a bond.

[0228] In some embodiments, each PA independently represents a chromophore functional group.

[0229] In some embodiments, each chromophore functional group is independently a functional group selected from the class or subclass of xanthophores, erythrophores, iridophores, leucophores, melanophores, and cyanophores, the class or subclass of fluorophore molecules, which are fluorescent compounds that receive light and re-emit light, the class or subclass of visual light transduction molecules, the class or subclass of photophore molecules, the class or subclass of luminescent molecules, and the class or subclass of luciferin compounds.

[0230] 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), mertansine (maytansinoid DM1 / DM4), paclitaxel, docetaxel, epothilone B, epothilone A, CYT997, auristatin tyramine phosphate, auristatin aminoquinoline, halocombstatin, calicheamicin theta, 7-ethyl-10-hydroxy-camptothecin (SN-38), pyrrolobenzodiazepine (PBD), pancratistatin, cyclophosphate, cribrostatin-6, kitastatin, turbostatin 1-4, halocombstatin, eribulin, hemiasterin, PNU, and cilstatin.

[0231] In some embodiments, each PA independently has the formula (D1): [ka] wherein R 4 , R 5a , and R 5b each independently represents hydrogen, a sugar residue, a substituted or unsubstituted inorganic or organic acid residue, a substituted or unsubstituted C 1-8 alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted non-aromatic heterocyclyl, substituted or unsubstituted cycloalkylalkyl, or substituted or unsubstituted heterocyclylalkyl; R 5a and R 5b together with the atom to which they are attached form a substituted or unsubstituted cycloalkyl, a substituted or unsubstituted non-aromatic heterocyclyl.

[0232] In some embodiments, R 4 is hydrogen, [ka] where R 5a and R 5b each is independently H, CH, or CF, or R 5a and R 5b together with the atom to which they are attached form a substituted or unsubstituted cycloalkyl, a substituted or unsubstituted non-aromatic heterocyclyl.

[0233] In some embodiments, R 4 is hydrogen, [ka] where R 5a and R 5b each is independently H, CH, or CF, or R 5a and R 5b together with the atom to which they are attached form a substituted or unsubstituted cycloalkyl, a substituted or unsubstituted non-aromatic heterocyclyl.

[0234] In some embodiments, each PA independently comprises: [ka] Represents.

[0235] In some embodiments, each PA independently has the formula (D2): [ka] wherein ring B is a substituted or unsubstituted cycloalkyl, a substituted or unsubstituted aryl, a substituted or unsubstituted heterocyclyl, or a substituted or unsubstituted heteroaryl.

[0236] In some embodiments, each PA independently comprises: [ka] Represents.

[0237] In some embodiments, each PA independently has the formula (D3): [ka] wherein S 2 is an enzymatically hydrolyzable hydrophilic group.

[0238] In some embodiments, S 2 The group is hydrogen or has the formula: [ka] represents one of the following:

[0239] In some embodiments, each PA independently has the formula (E1): [ka] wherein R 7 and R 8 Each of is independently hydrogen, halogen, or alkyl.

[0240] In some embodiments, R 7 and R 8 is hydrogen.

[0241] In some embodiments, R 7 and R 8 is methyl.

[0242] In some embodiments, R 7 is methyl and R 8 is F.

[0243] In some embodiments, R 7 and R 8 The carbon to which is attached is in the S configuration.

[0244] In some embodiments, R 7 and R 8 The carbon to which is attached is in the R configuration.

[0245] In some embodiments, each PA independently has the following formula: [ka] Represents.

[0246] In some embodiments, each PA is independently Dxd or independently has the formula: [ka] Represents.

[0247] In some embodiments, each PA independently has the following formula: [ka] Represents.

[0248] In some embodiments, AA 2 is glycine or [ka] are the amino acid residues of

[0249] In some embodiments of a compound of Formula (Ia) or a pharmaceutically acceptable salt thereof, a tautomer, a solvate, a stereoisomer, an enantiomer, an isotopic substitution, or a prodrug thereof, AA 2 is the formula (W): [ka] Including AA 3 is -glycine-glycine-phenylalanine-glycine- or [ka] is a tetrapeptide residue of

[0250] In a further embodiment, the antibody drug conjugate has the formula (Ib): [ka] or a pharmaceutically acceptable salt thereof, a tautomer, solvate, stereoisomer, enantiomer, isotopic substitution, or prodrug thereof, wherein: 2 is the formula (W): [ka] Including, AA 1 is -valine-alanine-, -valine-citrulline-, or [ka] where R 6 is -CH3, or -(CH2)3-NHC(=O)NH2. The values ​​of the remaining variables (e.g., x, p, BA, HG, RG 1 , R.G. 2 , SP 1 , SP 2 , PAB, PA) and alternative values ​​of the variables (e.g., AA 1 , A.A. 2) are as described elsewhere herein, for example, with respect to compounds of formula Ia.

[0251] In some embodiments, AA 2 is the formula (W): [ka] Including AA 1 is -glycine-glycine-phenylalanine-glycine- or [ka] is a tetrapeptide residue of

[0252] In a further embodiment, the antibody drug conjugate has the formula (Ic): [ka] or a pharmaceutically acceptable salt thereof, a tautomer, solvate, stereoisomer, enantiomer, isotopically substituted derivative, or prodrug thereof, During the ceremony, A.A. 3 is -valine-alanine-, -valine-citrulline-, or [ka] where R 6 is -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 alternative values ​​of variables (AA 3 , R 6 ) are as described elsewhere herein, for example, with respect to compounds of formula Ia.

[0253] In some embodiments (e.g., compounds of Formula (Ic)), AA 3 is -glycine-glycine-phenylalanine-glycine- or [ka] is a tetrapeptide residue of

[0254] In some embodiments, the antibody drug conjugate is selected from one of the following compounds, or a pharmaceutically acceptable salt tautomer, solvate, stereoisomer, enantiomer, isotopic substitution, or prodrug thereof: [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4] [Table 3-5] [Table 3-6] [Table 3-7] [Table 3-8] [Table 3-9] [Table 3-10] [Table 3-11] [Table 3-12] [Table 3-13] [Table 3-14] [Table 3-15] [Table 3-16]

[0255] PCT Application No. PCT / CN2022 / 123665, the entire contents of which are incorporated herein by reference, discloses antibody-drug conjugates, linker-payload moieties thereof that are suitable for use in the context of the present disclosure, and linker payloads that are suitable for use in the context of the present disclosure. In some embodiments, the linker payload is a linker payload disclosed in PCT / CN2022 / 123665.

[0256] In some embodiments (e.g., of a compound of Formula I), PA is: [ka] is a residue of Y is -AB-C'-D'-H; A is bond, CR 1 R 2 , or NR 1 and 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 heterocyclyl, unsubstituted or substituted aryl, or unsubstituted or substituted heteroaryl; D' is a bond, NH, or O; R 1 , and R 2 each is independently hydrogen, halogen, substituted or unsubstituted alkyl, or substituted or unsubstituted alkoxyl, or R 1 , and R 2together with the atom to which they are attached form an unsubstituted or substituted cycloalkyl, an unsubstituted or substituted heterocyclyl, an unsubstituted or substituted aryl, or an unsubstituted or substituted heteroaryl; and R 3 , and R 4 each is independently hydrogen, halogen, substituted or unsubstituted alkyl, or substituted or unsubstituted alkoxyl, or R 3 , and R 4 taken together with the atom to which they are attached form an unsubstituted or substituted cycloalkyl, an unsubstituted or substituted heterocyclyl, an unsubstituted or substituted aryl, or an unsubstituted or substituted heteroaryl. In some embodiments, R 3 is methyl and R 4 is F, then Y is not -NH-C(=O)-CDH.

[0257] In embodiments, in the residue of the payload, Y is -AB-C'-D'-, resulting from removal of -H from -AB-C'-D'-H. It will be understood that the payload residue may result from removal of a hydrogen atom from a payload depicted herein, but may also result from removal of a hydroxy group, such as the hydroxy group formed when D' is O in the payload depicted herein (or the corresponding hydroxy group in any of the other payload structures depicted herein).

[0258] In some embodiments, the PA is: [ka] is a residue of A is CR 1 R 2 , NH, or NR 1 and B is a bond, —C(═O)—, or —C(═O)O—; R 1 , and R 2 each independently represents H, or C 1-4 is alkyl, R 3 , and R 4 each is independently hydrogen, halogen, substituted or unsubstituted alkyl, or substituted or unsubstituted alkoxyl, or R 3 , and R 4 together with the atom to which they are attached form an unsubstituted or substituted cycloalkyl, an unsubstituted or substituted heterocyclyl, an unsubstituted or substituted aryl, or an unsubstituted or substituted heteroaryl; R 5 , and R 6 each is independently hydrogen, halogen, substituted or unsubstituted alkyl, or substituted or unsubstituted alkoxyl; n is 1, 2, 3, 4, or 5.

[0259] In some embodiments, A is -CH2- and B is a bond.

[0260] In some embodiments, R 5 and R 6 is hydrogen and n is 1, 2, or 3.

[0261] In some embodiments, R 3 is methyl and R 4 is F.

[0262] In some embodiments, the PA is [ka] is a residue of

[0263] In some embodiments, R 3 and R 4 taken together with the atoms to which they are attached form an unsubstituted or substituted dioxole ring.

[0264] In some embodiments, the PA is [ka] is a residue of

[0265] In some embodiments, A is —N(CH 3 )— and B is a bond.

[0266] In some embodiments, R 5 and R 6 is hydrogen and n is 2.

[0267] In some embodiments, the PA is [ka] is a residue of

[0268] In some embodiments, A is -NH- and B is -C(=O)O-. In further embodiments, R 5 and R 6 is hydrogen and n is 2.

[0269] In some embodiments, the PA is [ka] is a residue of

[0270] In some embodiments, A is -NH- and B is -C(=O)-. In further embodiments, R 5 and R 6 is hydrogen and n is 2.

[0271] In some embodiments, the PA is [ka] is a residue of

[0272] In some embodiments, R 3 is Cl and R 4 is F and B is -C(=O)-.

[0273] In some embodiments, the PA is [ka] is a residue of

[0274] In some embodiments, R 3 is methyl and R 4 is Cl and B is -C(=O)-.

[0275] In some embodiments, the PA is [ka] is a residue of

[0276] In some embodiments, R 3 , and R 4 taken together with the atoms to which they are attached form an unsubstituted or substituted heterocyclyl.

[0277] In some embodiments, R 3 , and R 4 together with the atoms to which they are attached form an unsubstituted or substituted dioxole ring, and B is -C(=O)-.

[0278] In some embodiments, the PA is [ka] is a residue of

[0279] In some embodiments, the PA is [ka] where the variables (e.g., R 3 , R 4 ) values ​​and alternative values ​​are as described elsewhere herein.

[0280] In some embodiments, R 3 is methyl and R 4 is Cl.

[0281] In some embodiments, the PA is [ka] is a residue of

[0282] In some embodiments, R 3 is Cl and R 4 is F.

[0283] In some embodiments, the PA is [ka] is a residue of

[0284] In some embodiments, R 3 is F and R 4 is F.

[0285] In some embodiments, the PA is [ka] is a residue of

[0286] In some embodiments, R 3 is H and R 4 is F.

[0287] In some embodiments, the PA is [ka] is a residue of

[0288] In some embodiments, R 3 is H and R 4 is OH.

[0289] In some embodiments, the PA is [ka] is a residue of

[0290] In some embodiments, R 3 is methyl and R 4 is methyl.

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

[0292] In some embodiments, R 3 is methoxyl and R 4 is F.

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

[0294] In some embodiments, R 3 is H and R 4 is methoxyl.

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

[0296] In some embodiments, R 3 is H and R 4 is Cl.

[0297] In some embodiments, the PA is [ka] is a residue of

[0298] In some embodiments, R 3 and R 4taken together with the atoms to which they are attached form an unsubstituted or substituted heterocyclyl.

[0299] In some embodiments, R 3 and R 4 taken together with the atoms to which they are attached form an unsubstituted or substituted dioxole ring.

[0300] In some embodiments, the PA is [ka] is a residue of

[0301] In some embodiments, the PA is [ka] is a residue of

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

[0303] In some embodiments, the PA is [ka] is a residue of

[0304] In some embodiments, the PA is [Table 4-1] [Table 4-2] [Table 4-3] [Table 4-4] [Table 4-5] is a residue of

[0305] In some embodiments, the PA is [Table 5-1] [Table 5-2] [Table 5-3] [Table 5-4] [Table 5-5] [Table 5-6] [Table 5-7] [Table 5-8] [Table 5-9] [Table 5-10] [Table 5-11] [Table 5-12] is a residue of

[0306] In some embodiments, the PA is [ka] where R 7’ and R 8’each independently is hydrogen, or substituted or unsubstituted alkyl, or R 7’ and R 8’ taken together with the nitrogen atom to which they are attached form an unsubstituted or substituted heterocyclyl, or an unsubstituted or substituted heteroaryl.

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

[0308] In some embodiments, the antibody drug conjugate has formula (V): [ka] or a pharmaceutically acceptable salt thereof; tautomers, solvates, stereoisomers, enantiomers, isotopic substitutions, or prodrugs thereof, wherein the values ​​and alternative value variables (e.g., A, B, C', D', L, R 3 , R 4 , and x) are as described elsewhere herein.

[0309] In some embodiments, the antibody drug conjugate has the structure of Formula (VIIIa), (VIIIb), or (VIIIc): [ka] or a pharmaceutically acceptable salt thereof; a tautomer, solvate, stereoisomer, enantiomer, isotopic substitution, or prodrug thereof, wherein the values ​​and alternative value variables (e.g., L, R 7 , R 8 , and x) are as described elsewhere herein.

[0310] In some embodiments, the antibody drug conjugate has the structure of any one of the following formulas: [ka] or a pharmaceutically acceptable salt thereof, tautomer, solvate, stereoisomer, enantiomer, isotopic substitution, or prodrug thereof, wherein the values ​​and alternative values ​​of the variables (e.g., L and x) are as defined elsewhere herein.

[0311] In some embodiments, L is [ka] where the bond indicated by an asterisk is attached to BA.

[0312] In some embodiments, L is [ka] where the bond indicated by an asterisk is attached to BA.

[0313] In some embodiments, L is: [ka] where the values ​​of the variables and substitution values ​​(e.g., RG 1 , SP 1 , A.A. 2 , A.A. 3 ,PAB,p,SP 2 RG 2 , and HG) are as described herein.

[0314] In some embodiments, L is: [ka] where the values ​​of the variables and substitution values ​​(e.g., RG 1 , SP 1 , A.A. 1 , A.A. 2 ,PAB,p,SP 2 RG 2 , and HG) are as described elsewhere herein.

[0315] In some embodiments, L is: [ka] where the values ​​of the variables and substitution values ​​(e.g., RG 1 , SP 1 , A.A. 3 , PAB, and p) are as described elsewhere herein.

[0316] In some embodiments, -AA 2 (SP 2 -RG 2 -HG)-AA 3 -(PAB) p -teeth, [ka] where * is SP 1 indicates the bond that connects to

[0317] In some embodiments, the antibody drug conjugate is selected from the following, or a pharmaceutically acceptable salt tautomer, solvate, stereoisomer, enantiomer, isotopic substitution, or prodrug thereof, wherein Ab is any of the anti-CEA antibodies disclosed herein: [Table 6-1] [Table 6-2] [Table 6-3] [Table 6-4] [Table 6-5] [Table 6-6]

[0318] All possible combinations of linkers and payloads are contemplated herein. In this regard, it will be understood that L, as used herein in the context of Formulas I-VIII, encompasses C, of ​​the compounds of Formula A or A-1. Additionally, C, as used in the context of Formula A or A-1, is a linker that is a substituted or unsubstituted aryl group, such as RG 1 -SP 1 It will be understood that this corresponds to

[0319] In an embodiment, D is [ka] wherein: Y is -ABCD-*, where * indicates the bond where D connects to L; A is bond, CR 1 R 2 , or NR 1 and 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 heterocyclyl, unsubstituted or substituted aryl, or unsubstituted or substituted heteroaryl; D is a bond, NH, or O; R 1 , and R 2 each is independently hydrogen, halogen, substituted or unsubstituted alkyl, or substituted or unsubstituted alkoxyl, or R 1 , and R 2 together with the atom to which they are attached form an unsubstituted or substituted cycloalkyl, an unsubstituted or substituted heterocyclyl, an unsubstituted or substituted aryl, or an unsubstituted or substituted heteroaryl; R 3 , and R 4 each is independently hydrogen, halogen, substituted or unsubstituted alkyl, or substituted or unsubstituted alkoxyl, or R 3 , and R 4together with the atoms to which they are attached form an unsubstituted or substituted cycloalkyl, an unsubstituted or substituted heterocyclyl, an unsubstituted or substituted aryl, or an unsubstituted or substituted heteroaryl.

[0320] In an embodiment, D is [ka] where R 7 and R 8 are each independently hydrogen, halogen, or alkyl.

[0321] In embodiments, the cytotoxic agent has the formula: [ka]

[0322] In certain embodiments, the cytotoxic agent (D) is [ka] In certain embodiments, D is [ka] is.

[0323] Each antibody-drug conjugate may contain one or more molecules of a cytotoxic agent, for example, 1, 2, 3, 4, 5, 6, 7, or 8 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). m ) n where DAR is the product of m and n.

[0324] The cytotoxic agent may be directly attached to the anti-CEA antibody or indirectly attached to the anti-CEA antibody via a linker (L). In embodiments, the linker is cleavable, such as by an enzyme, to release the cytotoxic agent. In embodiments, the linker is hydrophilic, such as when the cytotoxic agent is hydrophobic. In embodiments, the linker has the following formula, where * indicates a bond and L may be attached to a conjugator (C): [ka]

[0325] In the above formula, Su may be a sugar-like moiety. The moiety may be derived from a natural or unnatural sugar. The moiety may be hydrophilic. In embodiments, for example, if the cytotoxic agent is hydrophobic, the inclusion of a hydrophilic Su moiety may reduce the likelihood of antibody-drug conjugate aggregation, thereby reducing the rate of clearance in vivo.

[0326] In an embodiment, Su is a hydrophilic residue.

[0327] In an embodiment, Su is [ka] wherein n8 is 0 or 1, and R 6 -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 2 is 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 2is methyl. In certain embodiments, R 6 -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 , for example, —CH2—C(O)NH2.

[0328] In an embodiment, Su is [ka] wherein n8 is 0 or 1, and R 5 is -OH, -NH, -C(O)OH, -C(O)NH, -CH-OH, or -CH-NH. In certain embodiments, Su is [ka] In certain embodiments, Su is [ka] In certain embodiments, n8 is 0. In certain embodiments, n8 is 1. In certain embodiments, R 5 is —CHOH. In certain embodiments, R 5 is —C(O)OH.

[0329] In an embodiment, Su is [ka] In certain embodiments, Su is [ka] is.

[0330] In an embodiment, Su is [ka] In certain embodiments, Su is [ka] is.

[0331] In an embodiment, Su is [ka] is.

[0332] In an embodiment, L is [ka] is.

[0333] The antibody-drug conjugate disclosed herein may include a conjugator (C). The conjugator may be indirectly linked to a cytotoxic agent via a linker. The conjugator may help to prevent or reduce deconjugation of the cytotoxic agent in vivo, which may help to maintain a stable drug-to-antibody ratio (DAR). For example, the conjugator may have the following formula before conjugation with the antibody: [ka]

[0334] When the conjugator is directly attached to the anti-CEA antibody, the conjugator may have the following formula, where * indicates the bond where the conjugator connects to the antibody: [ka]

[0335] Any combination of conjugator, linker, sugar moiety, and cytotoxic agent can be included in the antibody drug conjugates disclosed herein. Non-limiting examples of conjugator (preconjugation)-linker-cytotoxic agent combinations include the following: [ka] [ka]

[0336] Each antibody drug conjugate may comprise multiple conjugator-linker-cytotoxic agent (CLD) compounds, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 CLDs. In embodiments, each antibody drug conjugate comprises 1 to 10, e.g., 3 to 10, 4 to 10, 5 to 10, 6 to 10, 7 to 9, or about 8 CLDs.

[0337] In certain embodiments, -CL-(D) m is the following: [ka] [ka] where * indicates the bond where C connects to Ab. In certain embodiments, -CL-(D) m is the following: [ka] where * indicates the bond where C connects to Ab. In certain embodiments, -CL-(D) m is the following: [ka] In certain embodiments, CL-(D) m is the following: [ka] is.

[0338] In certain embodiments, the antibody drug conjugate is: [ka] [ka]

[0339] In certain embodiments, the antibody drug conjugate has the following formula: [ka] or a tautomer, pharmaceutically acceptable salt, solvate, or hydrate thereof, where Ab and n are as described herein. In certain embodiments, the antibody drug conjugate has the following formula: [ka] or a tautomer, pharmaceutically acceptable salt, solvate, or hydrate thereof, where Ab and n are as described herein. In certain embodiments, the antibody drug conjugate has the following formula: [ka] or a tautomer, pharmaceutically acceptable salt, solvate, or hydrate thereof, wherein Ab and n are as described herein.

[0340] Methods for producing antibody-drug conjugates The antibody-drug conjugate disclosed herein can be produced by any method known in the art.In one example, the host cell transformed with the isolated nucleic acid comprising the sequence encoding anti-CEA antibody or its antigen-binding fragment is cultured under suitable culture conditions.The antibody or its antigen-binding fragment can be expressed and recovered from the cell culture.

[0341] A cytotoxic agent is conjugated to an antibody or antigen-binding fragment thereof using a linker disclosed herein to produce an antibody-drug conjugate. In embodiments, the conjugator is also attached to the linker, e.g., between the antibody and the linker.

[0342] Treatment method The antibody-drug conjugates of the present disclosure are useful in a variety of applications, including, but not limited to, methods for treating CEA-related disorders or diseases. In one aspect, the CEA-related disorders or diseases are characterized by cells that overexpress or accumulate CEA. In some embodiments, the cells are cancerous.

[0343] In certain aspects, the method comprises administering an effective amount of an anti-CEA antibody-drug conjugate to a subject (e.g., a patient) in need thereof. The subject may include, but is not limited to, a subject with a CEA-expressing cancer, a CEA-accumulating cancer, a CEA-responsive cancer, or a gastric or rectal cancer and metastasis thereof. In embodiments, 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).

[0344] The antibody-drug conjugates disclosed herein can be administered by any suitable means, including parenteral, intrapulmonary, and intranasal, including localized treatment and intralesional administration, if desired. Parenteral administration includes intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Dosing can be by any suitable route, for example, by infusion, such as intravenous or subcutaneous injection, depending in part on whether administration is short-term or long-term. Various dosing schedules are contemplated herein, including, but not limited to, single administration or multiple administrations over various time periods, bolus administration, and pulse infusion.

[0345] The antibodies or antigen-binding fragments, or antibody-drug conjugates of the present disclosure can be formulated, dosed, and administered in a manner consistent with good medical practice. Factors to consider 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 of drug delivery, the method of administration, the schedule of administration, and other factors known to medical professionals. The antibodies are optionally, but not necessarily, formulated with one or more agents currently used to prevent or treat the disorder. The effective amount of such other agents will vary depending on the amount of antibody present in the formulation, the type of disorder or treatment, and the other factors discussed above. These will generally be used in the same dosages and via the same routes of administration as those described herein, or between about 1 and 99% of the dosages described herein, or at any dosage and via any route empirically / clinically determined to be appropriate.

[0346] Combination therapy In one embodiment, 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 conjugates of the present disclosure include chemotherapeutic agents (e.g., paclitaxel or paclitaxel formulations, (e.g., Abraxane®), 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 (e.g., EGFR inhibitors (e.g., erlotinib), multikinase inhibitors (e.g., MGCD265, RGB-286638), CD20-targeted agents (e.g., rituximab), , ofatumumab, RO5072759, LFB-R603), CD52-targeted agents (e.g., alemtuzumab), prednisolone, darbepoetin alfa, lenalidomide, Bcl-2 inhibitors (e.g., oblimersen sodium), Aurora kinase inhibitors (e.g., MLN8237, TAK-901), proteasome inhibitors (e.g., bortezomib), CD19-targeted agents (e.g., MEDI-551, MOR20 8), MEK inhibitors (e.g., ABT-348), JAK-2 inhibitors (e.g., INCB018424), mTOR inhibitors (e.g., temsirolimus, everolimus), BCR / ABL inhibitors (e.g., imatinib), ET-A receptor antagonists (e.g., ZD4054), TRAIL receptor 2 (TR-2) agonists (e.g., CS-1008), EGEN-001, or polo-like kinase 1 inhibitors (e.g., BI 672).

[0347] In another embodiment, the anti-CEA antibody-drug conjugate can be used in combination with an anti-PD1 antibody. Anti-PD1 antibodies include, but are not limited to, tislelizumab, pembrolizumab, and nivolumab. Tislelizumab is disclosed in U.S. Pat. No. 8,735,553. Pembrolizumab (formerly known as MK-3475), disclosed by Merck in U.S. Pat. Nos. 8,354,509 and 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 treatment of metastatic melanoma and metastatic non-small cell lung cancer (NSCLC), and is currently undergoing clinical investigation for the treatment of head and neck squamous cell carcinoma (HNSCC) and refractory Hodgkin's lymphoma (cHL). Nivolumab (disclosed by Bristol-Meyers Squibb) is a fully human IgG4-K monoclonal antibody. Nivolumab (clone 5C4) is disclosed in U.S. Patent No. US8,008,449 and WO2006 / 121168. Nivolumab is approved for the treatment of melanoma, lung cancer, kidney cancer, and Hodgkin's lymphoma.

[0348] Pharmaceutical Compositions and Formulations Also provided are compositions containing pharmaceutical formulations comprising an anti-CEA antibody-drug conjugate comprising an anti-CEA antibody or antigen-binding fragment thereof, or a polynucleotide comprising a sequence encoding the anti-CEA antibody or antigen-binding fragment thereof, and a toxic drug conjugate. These compositions may further comprise suitable carriers, e.g., pharmaceutically acceptable excipients such as buffers, which are well known in the art.

[0349] Pharmaceutical formulations of the anti-CEA antibody-drug conjugates described herein are prepared in the form of lyophilized formulations or aqueous solutions by mixing such antibodies or antigen-binding fragments and antibody-drug conjugates having the desired degree of purity with one or more optional pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)). Pharmaceutically acceptable carriers are generally non-toxic to recipients at the dosages and concentrations used, and include buffers such as phosphate, citric acid, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzylammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl or benzyl alcohol, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins; Examples of suitable pharmaceutically acceptable carriers include, but are not limited to, 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 dextrins, 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 non-ionic surfactants such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers herein further include interstitial drug dispersion agents, such as soluble neutral active hyaluronidase glycoproteins (sHASEGPs), e.g., human soluble PH-20 hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX®, Baxter International, Inc.). Certain exemplary sHASEGPs, including rHuPH20, and methods of use are described in U.S. Patent Nos. US 7,871,607 and 2006 / 0104968.In one embodiment, the sHASEGP is combined with one or more additional glycosaminoglycanases, such as chondroitinases.

[0350] Exemplary lyophilized antibody formulations are described in U.S. Patent No. 6,267,958. Aqueous antibody formulations include those described in U.S. Patent No. 6,171,586 and WO2006 / 044908, the latter formulations including a histidine-acetate buffer.

[0351] Sustained-release preparations can 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, e.g., films, or microcapsules.

[0352] Formulations to be used for in vivo administration are generally sterile. Sterility may be readily accomplished, for example, by filtration through sterile filtration membranes. [Example]

[0353] Example 1: Generation of anti-CEA monoclonal antibodies CEA recombinant protein for immunization and binding assays For antibody screening, several recombinant proteins were designed and expressed (see Table 2). Antibodies against CEA cross-react with human and Macaca mulatta CEA in the membrane peripheral 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 with other human CEACAM members.

[0354] The cDNA coding regions of full-length human CEA (SEQ ID NO: 52), Macaca CEA (SEQ ID NO: 53), and full-length human CEACAM6 (SEQ ID NO: 54) were aligned based on GenBank sequences. For human CEA (accession number: NM_004363.2), the gene is available from Sinobio (catalog number HG11077-UT). For Macaca CEA (accession number: NM_001047125), the gene is available from GenScript™ (catalog number OMb23865D). For human CEACAM6 (accession number: NM_002483.4), the gene is available from Sinobio (catalog number HG10823-UT). A schematic diagram of the CEA fusion proteins is shown in Figure 1. It has been reported that splice variants of human CEA are co-expressed with full-length CEA in tumors (Peng et al., PloS one, 7, e36412-e36412 (2012)). Therefore, a variant (CEA-v) was prepared accordingly. To generate this construct, the coding region for the extracellular domain (ECD) consisting of amino acids (AA) 1-687 of huCEA (SEQ ID NO: 55), the region consisting of amino acids (AA) 1-690 of monkey CEA (SEQ ID NO: 56), and the region consisting of amino acids (AA) 1-320 of CEACAM6 (SEQ ID NO: 57) were amplified by PCR. The regions consisting of amino acids (AA) 1-78 of CEA (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 generate 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 CEA periplasmic region containing domain B3, amino acids (AA) 596-687 (SEQ ID NO: 62), were PCR-amplified and then conjugated by overlap PCR to generate a chimeric construct (CHIM) (SEQ ID NO: 63). All constructs were then individually cloned into a pcDNA3.1-based expression vector (Invitrogen, Carlsbad, CA, USA) with the C-terminus fused to a 6xHis tag, resulting in five recombinant fusion protein expression plasmids for CEA, monkey CEA, CEACAM6, CEA-v, and CHIM.For recombinant fusion protein production, CEA, monkey CEA, CEACAM6, CEA-v, and CHIM plasmids were transiently transfected into a HEK293-based mammalian cell expression system (generated in-house) and cultured for 5–7 days in a CO2 incubator equipped with a rotating shaker. The supernatant containing the recombinant proteins was collected and removed by centrifugation. The recombinant proteins were purified using Ni-NTA agarose (catalog no. R90115, Invitrogen). All recombinant proteins were dialyzed against phosphate-buffered saline (PBS) and stored in small aliquots at -80°C in a freezer.

[0355] Stable expression in cell lines To establish stable cell lines expressing full-length human CEA (accession number: NM_004363.2), a cDNA expressing CEA was cloned into the retroviral vector pFB-Neo (catalog number 217561, Agilent, USA). Dual-tropic retroviral vectors were generated according to a previous protocol (Zhang et al., Blood. 2005 106(5):1544-51). To generate human CEA-expressing cell lines, the viral vector containing human CEA was transduced into L929 (ATCC, Manassas, VA, USA) and CT26 cells (ATCC, Manassas, VA, USA). High-expressing cell lines were selected by culturing in complete RPMI 1640 medium containing 10% FBS with G418 and then verified by FACS binding assay.

[0356] Immunization, hybridoma fusion and cloning Eight to 12-week-old Balb / c mice (HFK BIOSCIENCE CO., LTD, Beijing, China) were injected with 500 μl of 1 × 10 Fecal IgG1 vaccine with or without water-soluble adjuvant (Cat. No. KX0210041, KangBiQuan, Beijing, China). 7Mice were immunized intraperitoneally (ip) with 100 L929 / huCEA cells. This procedure was repeated two weeks later to stimulate antibody production. Two weeks after the third immunization, mouse sera were assessed for soluble CEA (sCEA) binding by ELISA and FACS. Splenocytes were isolated and fused to the mouse myeloma cell line SP2 / 0 cells (ATCC, Manassas, VA, USA) using standard techniques (Colligan JE, et al., CURRENT PROTOCOLS IN IMMUNOLOGY, 1993).

[0357] Evaluation of antibody CEA binding activity by ELISA and FACS To screen for antibodies that bind to human CEA but not to CEACAM6 or sCEA, we screened and counterscreened for antibodies that bind to CHIM but not to sCEA, CEACAM6, and CEA-v, as well as antibodies that bind to CHIM, sCEA, and CEA-v but not to CEACAM6. Supernatants from hybridoma clones were initially screened by ELISA as described in Methods in Molecular Biology (2007) 378:33-52, with some modifications. Briefly, sCEA, CHIM, CEACAM6, or CEA-v was individually coated onto 96-well plates at a low concentration of 3 μg / ml. HRP-conjugated anti-mouse IgG antibody (Cat. No. 7076S, Cell Signaling Technology, USA) and substrate (Cat. No. 00-4201-56, eBioscience, USA) were used for development, and the absorbance signal at a wavelength of 450 nm was measured using a plate reader (SpectraMax Paradigm™, Molecular Devices, USA). ELISA-positive clones were further verified by FACS using L929 / huCEA and / or MKN45 cells (ATCC). MKN45 cells are derived from human gastric carcinoma. CEA-expressing cells (10 5The cells (0.01% / well) were incubated with ELISA-positive hybridoma supernatants and then bound with Alexa Fluro-647-labeled goat anti-mouse IgG antibody (catalog no. A0473, Beyotime Biotechnology, China). Cell fluorescence was quantified using a flow cytometer (Guava easyCyte™ 8HT, Merck-Millipore, USA).

[0358] Conditioned medium from hybridomas that showed positive signals in FACS screening and did not bind to CEACAM6 or sCEA but bound to CHIM was subjected to functional assays to evaluate the presence of sCEA in 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.

[0359] Subcloning of hybridomas and adaptation to serum-free or low-serum medium After screening primarily by ELISA, FACS, and functional assays, positive hybridoma clones were subcloned by limiting dilution. Top antibody subclones validated through functional assays were adapted for growth in CDM4MAb medium (Cat. No. SH30801.02, Hyclone, USA) containing 3% FBS.

[0360] Monoclonal antibody expression and purification Hybridoma cells were cultured in CDM4MAb medium (catalog no. SH30801.02, Hyclone) and incubated at 37°C in a CO2 incubator for 5–7 days. Prior to purification, conditioned medium was collected by centrifugation and filtration through a 0.22 μm membrane. The supernatant containing the mouse antibody was applied and bound to a Protein A column (catalog no. 17127901, GE Life Sciences) according to the manufacturer's protocol. This procedure typically yielded antibodies with purity exceeding 90%. Protein A affinity-purified antibodies were dialyzed against PBS or further purified using a HiLoad 16 / 60 Superdex™ 200 column (catalog no. 17531801, GE Life Sciences) to remove aggregates. Protein concentration was determined by measuring absorbance at 280 nm. The final antibody preparation was stored in aliquots at -80°C in a freezer. [Table 7-1] [Table 7-2] [Table 7-3] [Table 7-4] [Table 7-5] [Table 7-6] [Table 7-7] [Table 7-8] [Table 7-9] [Table 7-10] [Table 7-11] [Table 7-12] [Table 7-13] [Table 7-14] [Table 7-15]

[0361] Example 2. Cloning and sequence analysis of CEA antibodies Total RNA was prepared from mouse hybridoma cells using the Ultrapure RNA Kit (Cat. No. 74104, QIAGEN, Germany) according to the manufacturer's protocol. First-strand cDNA was synthesized using an Invitrogen cDNA synthesis kit (Cat. No. 18080-051), and PCR amplification of the VH and VL genes of mouse monoclonal antibodies was performed using a PCR kit (Cat. No. CW0686, CWBio, Beijing, China). Oligoprimers used for antibody cDNA cloning of the heavy chain variable region (VH) and kappa 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 (Cat. No. CB101-02, TransGen, China) and sequenced. The amino acid sequences of the VH and VL regions were determined from the results of DNA sequencing.

[0362] The monoclonal antibodies were analyzed by comparing sequence homology and grouped based on sequence similarity (Figure 2). The complementarity-determining regions (CDRs) were defined based on the IMGT (Lefranc et al., 1999 Nucleic Acids Research 27:209-212) system of sequence annotation. The amino acid sequence of a representative clone, BGA7592, is shown in Table 3. [Table 8]

[0363] Example 3. Determination of the binding profile of purified mouse anti-CEA antibodies CEA antibodies with specific binding to CEA, as demonstrated by ELISA and FACS, and CEA antibodies without sCEA interference, were characterized for their binding kinetics by surface plasmon resonance (SPR) assay using a BIAcore™ T-200 (GE Life Sciences) (Figure 3A). Briefly, anti-mouse IgG antibody was immobilized on an activated CM5 biosensor chip (catalog no. BR100530, GE Life Sciences). Purified mouse antibody was flowed over the chip surface and captured by the anti-mouse IgG antibody. Next, serial dilutions (6.0 nM to 2150 nM) of purified CHIM, CEA-v, CEA, or monkey CEA recombinant protein were flowed over the chip surface, and the changes in surface plasmon resonance signal were analyzed to determine the association rate (k ) using a one-to-one Langmuir binding model (BIA Evaluation Software, GE Life Sciences). on ) and dissociation rate (k off The equilibrium dissociation constant (K D ) as the ratio k off / k on The binding affinity profile of BGA7592 is shown in Table 4 below. [Table 9]

[0364] The binding profile of BGA7592 was checked by antigen ELISA. The binding of purified BGA7592 to huCEA and monkey CEA was observed, indicating that BGA7592 is a weak binder to soluble huCEA and monkey CEA, or that soluble CEA has a different conformation when immobilized (Figure 3B). In this experiment, sCEA, CHIM, monkey CEA ("cynoCEA"), CEA-v, or bovine serum albumin (BSA) was coated onto a 96-well plate at a high concentration of 10 μg / ml overnight at 4°C. BGA7592 or the control antibody ab4451 (catalog number ab4451, Abcam, USA) was incubated at a concentration of 2 μg / ml for 1 hour. HRP-conjugated anti-mouse IgG antibody (Cat. No. 7076S, Cell Signaling Technology, USA) and substrate (Cat. No. 00-4201-56, eBioscience, USA) were used for development, and the absorbance signal at a wavelength of 450 nm was measured using a plate reader (SpectraMax Paradigm, Molecular Devices, USA).

[0365] Example 4. Effect of recombinant soluble CEA on the binding of BGA7592 to CEA-expressing cells The presence of soluble CEA in the specific binding of various CEA antibodies to CEA-expressing cells was assessed by flow cytometry. Briefly, human CEA-expressing cells (10 5 Cells (1000 cells / well) were incubated with 2 μg / ml of purified CEA mouse monoclonal antibody in the presence of 20 μg / ml of recombinant soluble CEA protein, followed by binding with Alexa Fluor-647-labeled goat anti-mouse IgG antibody (catalog no. A0473, Beyotime Biotechnology, China). Cell fluorescence was quantified using a flow cytometer (Guava easyCyte™ 8HT, Merck-Millipore, USA). As shown in Figures 4A and 4B, the binding of BGA7592 to CEA-expressing cells was not affected by the presence of soluble CEA.

[0366] Example 5. Humanization of mouse anti-human CEA antibody mAb humanization and engineering For the humanization of BGA7592, human germline IgG genes were searched for sequences sharing high homology with the cDNA sequence of the BGA7592 variable region by sequence comparison in the IMGT and NCBI human immunoglobulin gene databases. Human IGVH and IGVL genes, which are frequently present in the human antibody repertoire (Glanville et al., 2009 PNAS 106:20216-20221) and share high homology with BGA7592, were selected as templates for humanization. Prior to humanization, the BGA7592 heavy and light chain variable domains were fused to the wild-type human IgG1 constant region, designated human IgG1wt (SEQ ID NO: 87), and the human kappa constant (CL) region (SEQ ID NO: 88), respectively (Table 5). [Table 10]

[0367] Humanization was performed by CDR grafting (Methods in Molecular Biology, Vol. 248: Antibody Engineering, Methods and Protocols, Humana Press), and the BGA7592 antibody was engineered in a human IgG1 format. In the first round of humanization, mutations from mouse to human amino acid residues in the framework regions were guided by simulated 3D structures, and structurally important mouse framework residues to maintain the canonical structure of the CDRs were retained in the first version of the humanized antibody BGA7592, BGA7592-1 (heavy and light chain amino acid sequences are shown in SEQ ID NOs: 89 and 90) (Table 6). [Table 11]

[0368] Specifically, the CDRs of BGA7592-1 VL were grafted onto the framework of the human germline variable gene IGVK1-27 (amino acid sequence of the light chain variable domain shown in SEQ ID NO: 92) while retaining two mouse framework residues (N66 and V68). The CDRs of BGA7592-1 VH were grafted onto the framework of the human germline variable gene IGVH1-46 (amino acid sequence of the heavy chain variable domain shown in SEQ ID NO: 91) while retaining five mouse framework residues (L39, I53, Y55, N66, S68) (Table 7). [Table 12]

[0369] BGA7592-1 was constructed as a full-length human antibody using an in-house developed expression vector containing the constant region of wild-type human IgG1 with easily adaptable subcloning sites. Expression and preparation of BGA7592-1 antibody were achieved by co-transfection of the two constructs into 293G cells and purification using a Protein A column (catalog no. 17543802, GE Life Sciences). The purified antibody was concentrated to 0.5-5 mg / mL in PBS, aliquoted, and stored in a -80°C freezer.

[0370] Additional single or multiple amino acid changes were made using BGA7592-1 to convert human residues in the framework regions of VH and VL to the corresponding mouse germline residues, including V68A, R72A, and V79A in VH and V43S in VL, respectively. This resulted in the following antibodies: 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, V43S in VL), and BGA7592-8 (V68A, R72A, V79A in VH, V43S in VL). All antibodies containing modifications had similar binding activity to BGA7592-1, and none of the changes disrupted binding.

[0371] Further engineering was performed on the BGA75921 sequence by introducing mutations into the CDR and framework regions to remove post-translational modification (PTM) sites. This included the N52T, N54Q, N59S, N102G, N104Q, and S61A amino acid changes in the VH region. This resulted in 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)). All antibodies had similar binding specificity for BGA7592-1, and none of the changes disrupted binding. While maintaining specificity, amino acid composition and expression level were also taken into consideration. All humanization mutations were generated using primers containing mutations at specific positions and a site-directed mutagenesis kit (catalog number FM111-02, TransGen, Beijing, China). Desired mutations were confirmed by sequence analysis. Compared to BGA7592-1, BGA7592-1F lacked a glycosylation site and had significantly reduced binding affinity, but had a higher expression level (Table 8). [Table 13]

[0372] Example 6. Generation of affinity maturation libraries The phagemid vector pCANTAB 5E (GE Healthcare) was used by standard molecular biology techniques to construct a phagemid designed to display the BGA7592-1F Fab fragment on the surface of M13 bacteriophage as a fusion to the N-terminus of a fragment of the minor coat protein of gene 3. An amber stop codon preceded the gene 3 sequence to allow for expression of the Fab fragment directly from the phagemid clone. The phagemid was used as a template to generate 10 8 A phage display library containing unique members was constructed.

[0373] Two libraries (H-AM and L-AM) were constructed by randomizing the CDR positions of the heavy and light chains, respectively. All three CDRs were randomized in each library, with each CDR having a maximum of one mutation per clone, except for HCDR3, which could have two simultaneous mutations. Each position was randomized with the NNK codon (IUPAC code), which encodes any amino acid or an amber stop codon. The combined heavy and light chain library design included 5.0 × 10 CDRs containing no stop or cysteine ​​codons. 6There was a potential diversity of 100 unique full-length clones, with the expected distribution of clones with 0, 1, 2, and 3 mutations being approximately 0.02%, 1.1%, 17%, and 82%, respectively. A minority of heavy chain clones were predicted to have four mutations due to the primer design in the HCDR3 region. As a first step, DNA fragments were amplified using pCANTAB 5E as a template and primers containing randomized CDR3 positions (see Figures 5A and 5B). Next, the PCR products were gel-purified and assembled using primers containing randomized CDR2 positions. This procedure was repeated using primers directed at randomized CDR1 positions. The resulting heavy or light chain PCR products were then assembled with the corresponding CH or CL fragments by overlap PCR. The fragments were further assembled with the unmutated light or heavy chain by overlap PCR. The resulting fragments were then gel-purified and ligated with pCANTAB 5E after NcoI / NotI digestion. The purified ligations were transformed into TG1 bacteria by electroporation. Sequencing of 48 clones from each library confirmed randomization at each position (data not shown). However, due to limited sampling depth, not all amino acid mutations were observed at every position. Approximately 52% and 55% of the light and heavy chain libraries contained full-length randomized clones, respectively, representing 10% of the total generated clones, even with moderate incorporation bias in oligonucleotide synthesis and library construction. 8 independent clones were sufficient to cover all potential diversity of the design.

[0374] Example 7. Generation of affinity matured humanized BGA7592 variants Library selection and screening Affinity-matured humanized BGA7592 Fab was generated by phage display using standard protocols (Silacci et al., (2005) Proteomics, 5, 2340-50; Zhao et al., (2014) PLoS One, 9, e111339). In the first and second rounds of selection, competitive selection was performed against immobilized CHIM in immunotubes (catalog no. 470319, ThermoFisher). Briefly, immunotubes were coated with 1 ml of CHIM (5 μg / ml in PBS) overnight at 4°C. All affinity-matured libraries were incubated with the coated immunotubes in the presence of various concentrations of BGA7592-1F IgG (round 1, 1 μg / ml; round 2, 5 μg / ml) for 1 hour. In the third and fourth rounds of selection, L929 / huCEA cells (round 3) or LOVO cells (ATCC CCL-229) (round 4) were used for cell panning, with HEK293 cells used as depletion cells. After four rounds of selection, individual clones were selected, and phage-containing supernatants were prepared using standard protocols. ELISA-positive clones were sequenced and analyzed for mutation sites.

[0375] Analysis of CDR mutation frequency The mutation frequency in each CDR after four rounds of selection was relatively high, ranging from 17% in HCDR3 to 95% in LCDR2. For the heavy chain, approximately half of the clones identified in the H-AM library were identical to the parental clone. The other clones contained one backmutation at Q54N in HCDR2.

[0376] Analysis of the light chain revealed even more diverse mutations. Two sites were mutated in almost all clones of LCDR1, respectively. Light chain residues 29 and 31 were mutated from Ile to Gln and from Gly to Gln in 47.09% and 35.29% of clones, respectively. Position 29 not only had a high frequency of Gln mutations, but also had a subset of clones with mutations to tyrosine. Position 31 not only had a high frequency of Gln mutations, but also had a 12.5% ​​chance of being mutated to Leu. Due to library design constraints, mutations at positions 29 and 31 were not observed in combination with each other. However, mutations at each of these two sites were often combined with mutations in other CDRs. For LCDR2, only A51 was mutated in at least 64.71% of clones, but without any clear pattern, and large hydrophobic polar residues such as Tyr, Phe, Thr, and Asn were included. For 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 Tyr to Leu in 11.76% and 47.06% of the clones, respectively. Figure 6 shows the sequence variance of the light chain CDR regions after four rounds of selection.

[0377] Expression of selected humanized BGA7592 variants Combinations of mutations were performed. The light chain variable regions from selected phage clones were subcloned into a mammalian expression vector expressing a human kappa light chain. The light chain expression vector was co-transfected into 293G cells at a 1:1 ratio with a mammalian expression vector expressing the BGA7592-1F (also referred to herein as BGA5366) heavy chain. The CEA antibody version was purified from the culture supernatant by Protein A affinity chromatography (catalog no. 17543802, GE Life Sciences). The purified antibody was concentrated to 0.5–5 mg / mL in PBS and stored in aliquots at -80°C in a freezer.

[0378] Characterization of affinity-matured humanized BGA7592 variants Affinity comparison of BGA7592-1F (BGA5366) and other affinity-matured clones was performed by SPR assay (Table 9) using a BIAcore™ T-200 (GE Life Sciences) and flow cytometry (Figure 7). In this experiment, an anti-human IgG (Fc) antibody was immobilized on an activated CM5 biosensor chip (catalog no. BR100839, GE Life Sciences). An anti-CEA antibody was flowed over the chip and captured by an anti-human Fab antibody. Next, serial dilutions of CHIM (1.37 nM to 333 nM) were flowed over the chip, and the change in surface plasmon resonance signal was analyzed to determine the association rate (k) using a one-to-one Langmuir binding model (BIA Evaluation Software, GE Life Sciences). on ) and dissociation rate (k off ) was calculated. In flow cytometry, CEA-expressing cells (10 5 Cells (per well) were incubated with various concentrations of purified affinity-matured antibodies, which were then bound with Alexa Fluro-647-labeled anti-hu IgG Fc antibody (Cat. No. 409320, BioLegend, USA). Cell fluorescence was quantified using a flow cytometer (Guava easyCyte™ 8HT, Merck-Millipore, USA). The equilibrium dissociation constant (K D ) as the ratio k off / k on BGA7592-1F-ph-L (BGA3676) (SEQ ID NO: 95) and BGA7592-1F-ph-M (BGA2433) (SEQ ID NO: 96) were shown to have improved affinity for the huCEA surface protein (Table 10). [Table 14] [Table 15]

[0379] Example 8. Further engineering of affinity matured humanized BGA7592 variants Further engineering was performed by introducing mutations into the CDRs based on the BGA7592-1F-ph-M (BGA2433) template. These included W33Y, Q54N, and S59N in the VH and T51Y in the VL. This resulted in BGA8179 (W33Y (VH)), BGA2107 (Q54N (VH)), BGA0089 (S59N (VH)), and BGA1789 (T51Y (VL)), all of which showed improved binding activity to BGA7592-1F-ph-F (BGA9521). The most improved antibody was finally the BGA6710 antibody (Table 11) with the (W33Y (VH), T51Y (VL)) changes, the sequence of which is shown in Table 12. [Table 16] [Table 17]

[0380] Example 9: Optimization of BGA6710 To further improve the biochemical / biophysical properties, BGA6710 was optimized by introducing substitutions into the CDR and framework regions (Table 13). Large hydrophobic residues were selected and changed to polar residues, with the exception of K13 and Q53, which were selected based on differences observed between human VH germlines. Amino acid composition, thermal stability (Tm), surface hydrophobicity, and isoelectronic point (pI) were considered while maintaining functional activity. As described in Example 6, variants were expressed in Fab format by cloning into the vector pCANTAB-5E. Fab-containing supernatants were then screened for CEA binding by ELISA and SPR analysis. Variants without significant affinity loss were selected, and tolerant residues for substitution were identified. L92E in the light chain and K13E, Q54E, Y57D / E, and Y57K in the heavy chain were shown to have minimal impact on affinity.

[0381] Therefore, BGA6710 variants in IgG format with the identified single mutations or combinations were expressed and purified as described in Example 8. SPR studies and FACS analyses were performed and summarized in Table 14. It was confirmed that the introduced amino acid substitutions did not alter the specificity or epitope (data not shown). Collectively, these results indicated that these single or combined mutations (K13E, Q54E, Y57D, and Y57K in the heavy chain, and L92E in the light chain) had minimal impact on affinity, except for L92E, which slightly reduced binding affinity to CEA. In summary, the Y57K change optimized the expression, CEA binding, and affinity of the BGA6710 antibody, resulting in BGA5384 (Table 1). [Table 18] [Table 19]

[0382] Example 10. Binding profile of anti-CEA antibody BGA5384 BGA5384 and a previously disclosed CEA antibody designated antibody 2F1 in U.S. Patent Application Publication No. 2012 / 0251529 were produced in a human IgG1 format and characterized for binding kinetics by SPR assay using a BIAcore™ T-200 (GE Life Sciences).

[0383] To obtain these data, anti-human IgG (Fc) antibodies were immobilized on an activated CM5 biosensor chip (catalog number BR100839, GE Life Sciences). BGA5384 antibodies were flowed over the chip surface and captured by anti-human Fab antibodies. Next, serial dilutions (1.37 nM to 2150 nM) of soluble huCEA or cynoCEA (catalog number CE5-C52H5, Acrobiosystem) were flowed over the chip surface. The changes in surface plasmon resonance signals were analyzed to calculate the association rate (k) using a one-to-one Langmuir binding model (BIA Evaluation Software, GE Life Sciences). on ) and dissociation rate (k off The equilibrium dissociation constant (K D ) as the ratio k off / k on The binding affinity of BGA5384 and the 2F1 control antibody was calculated as follows: BGA5384 and the 2F1 control antibody exhibited different binding affinities. As shown in Table 15, BGA5384 has very high affinity for human CEA and comparable affinity for cynomolgus monkey CEA. [Table 20]

[0384] Flow cytometry revealed that CEA-expressing MKN45 cells (10 5 Cells (cells / well) were incubated with various concentrations of purified affinity-matured antibodies, which were then bound with Alexa Fluor-647-labeled anti-hu IgG Fc antibody (catalog no. 409320, BioLegend, USA). Cell fluorescence was quantified using a flow cytometer (Guava easyCyte™ 8HT, Merck-Millipore, USA). As shown in Figure 8, BGA5384 had an EC (measured by mean fluorescence intensity, MFI) of 2.92 μg / ml. 50 It was shown that it specifically binds to native CEA on living cells in a dose-responsive manner.

[0385] Example 11. Assessment of off-target specificity The off-target specificity of BGA5384 was assessed by ELISA and flow cytometry. In flow cytometry, CEACAM3 (SEQ ID NO: 65), CEACAM7 (SEQ ID NO: 66), or CEACAM8 (SEQ ID NO: 67) was transfected into HEK293 cells (10 5 Cells (1000 x 1000 cells / well) were transiently transfected and then incubated with 2 μg / ml of 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™ 8HT, Merck-Millipore, USA). For antigen ELISA, CEACAM1 (SEQ ID NO: 64) (catalog no. 10822-H08H, Sino Biological, China), CHIM (SEQ ID NO: 63), CEA (SEQ ID NO: 55), or CEACAM6 (SEQ ID NO: 57) was coated onto 96-well plates at a concentration of 10 μg / ml overnight at 4°C. HRP-conjugated anti-human Fc (Fc-specific) IgG antibody (catalog no. A0170, Sigma, USA) and substrate (catalog no. 00-4201-56, eBioscience, USA) were used for development, and the absorbance signal at 450 nm was measured using a plate reader (SpectraMax Paradigm, Molecular Devices, USA). As shown in Figures 9A-9B, no cross-reactivity with other CEACAM family members was observed, and BGA5384 exhibited specificity only for CEA (CEACAM3 in Figures 9A-9B).

[0386] Example 12. Effect of soluble huCEA on the binding of BGA5384 to CEA-expressing cells To determine whether soluble CEA (sCEA) had any effect on the specific binding of BGA5384, various concentrations of recombinant soluble CEA (0, 0.5, 1, or 2 μg / ml) were premixed with BGA5384 (0.01–100 μg / ml) and incubated for 5 min. The mixture was then inoculated onto 2 × 10 MKN45 cells. 5The cells were incubated with 1000 CEA-expressing cells at 4°C for 30 minutes. The cells were stained with secondary antibody anti-huFc-APC (Cat. No. 409320, BioLegend, USA) and analyzed by flow cytometry. In the presence of 2 μg / ml of recombinant sCEA, BGA5384 binding to CEA-expressing cells was not affected. This result is shown for MKN45 cells (Figure 10), demonstrating the specificity of BGA5384 for membrane-bound CEA.

[0387] Example 13. BGA6710 is CEA + Induces potent ADCC against tumor cells To determine whether BGA6710 in wild-type IgG1 format can induce antibody-dependent cellular cytotoxicity (ADCC), CD16(V158)-expressing NK92MI cells (NK92MI / CD16V) were used as effector cells and cocultured with CEA-expressing murine colon carcinoma cells (CT26-ATCC CRL-2638). Coculture was performed for 5 hours at an E:T ratio of 1:1 in the presence of BGA6710 at the indicated concentrations (0.00005–5 μg / ml). Cytotoxicity was determined by lactate dehydrogenase (LDH) release. The amount of LDH in the supernatant was measured using the CytoTox™ 96 Non-Radioactive Cytotoxicity Assay kit (Promega, Madison, WI), and specific lysis was calculated according to the manufacturer's instructions. As shown in Figure 11, BGA6710 induced an EC of approximately 6.7 ng / ml. 50 were able to induce ADCC in vitro.

[0388] Example 14. In vivo antitumor effect of BGA6710 CEA + To measure the in vivo effect of BGA6710 on tumor cells, NK92MI / CD16V cells (5 × 10 6 ) to CT26 / CEA cells (10 6) and subcutaneously injected into NCG mice. BGA6710 (0.12, 0.62, or 3.1 mg / kg) or vehicle control was given twice a week starting from the day of tumor injection (7 mice per group). Compared to vehicle, BGA6710 at a dose of 3.1 mg / kg showed a low amount of tumor inhibition, although the difference from the vehicle control was not statistically significant (P>0.05) (Figure 12).

[0389] Example 15. Synthesis of cytotoxic agents (payloads) [Table 21]

[0390] UPLC analysis method: Method A: Mobile phase A: 0.1% FA in water, B: MeCN, Gradient: 10% B hold for 0.2 min, 10% to 95% B, 5.8 min, 95% B hold for 0.5 min, Flow rate: 0.6 mL / min, Column: ACQUITY UPLC® BEH C18 1.7 μm

[0391] Method B: Mobile phase A: 0.1% FA in water, B: MeCN, Gradient: 10% B hold for 0.5 min, 10% to 90% B for 2.5 min, 90% B hold for 0.2 min, Flow rate: 0.6 mL / min, Column: ACQUITY UPLC® BEH C18 1.7 μm

[0392] Method C: Mobile phase A: 0.1% FA in water, B: MeCN, Gradient: 10% B hold for 0.2 min, 10% to 90% B for 1.3 min, 90% B hold for 0.3 min, Flow rate: 0.6 mL / min, Column: ACQUITY UPLC® BEH C18 1.7 μm

[0393] P1 and P2 (Table 16) are commercially available and were purchased from MedChemExpress CO. LTD (Shanghai).

[0394] Synthesis procedure for payloads P3 and P4 Payload P3 [ka]

[0395] 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-benz'[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) was added DIEA (21 μL, 16 mg, 0.13 mmol) and exatecan mesylate (23 mg, 0.043 mmol, purchased from MedChemExpress CO. LTD). The resulting brown mixture was stirred at room temperature for 2 h. Upon completion of the reaction, the mixture was purified by preparative HPLC (TFA) (Method: Column: XBridge Prep C18 OBD 5um 19*150mm, Mobile phase: A-water (0.1% TFA): B-acetonitrile, Flow rate: 20mL / min), and the fractions were lyophilized to give P3 (15mg, 65.5% yield) as a white powder. MS (ESI) m / z: 536.4 [M+H] + . 1 H 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).

[0396] Payload P4 [ka]

[0397] Step 1: Diethyl 2-fluoro-2-methylmalonate (P4b). A solution of compound P4a (10.00 g, 57.40 mmol) in THF (200 mL) was dissolved in 0.5 mL of HCl. o The mixture was cooled to 0°C. 60% NaH in oil (3.21 g, 80.37 mmol) was added in small portions to the mixture. o The mixture was stirred at RT for 30 min. Then, N-fluoro-N-(phenylsulfonyl)benzenesulfonamide (NSFI, 19.91 g, 63.20 mmol) was added to the mixture at RT. o The mixture was added in portions, then warmed to room temperature and stirred for 16 hours. After the reaction was complete, the suspension was filtered, and the filtrate was concentrated. PE (100 mL) was added to the residue, the precipitate was filtered, and the filtrate was concentrated to give compound P4b (12.50 g, crude) as a pale yellow oil. 1 H 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 F NMR (376 MHz, CDCl3) δ -157.50.

[0398] Step 2: 3-Ethoxy-2-fluoro-2-methyl-3-oxopropanoic acid (P4c). To a solution of compound P4b (1.00 g, 5.20 mmol) in EtOH (5 mL) was added H 2 A solution of KOH (321 mg) in 50 μL of HCl and 2 mL of EtOH was added dropwise at 0° C. The mixture was stirred at room temperature for 2 hours. The mixture was diluted with 20 mL and washed with DCM (20 mL*3). The aqueous solution was adjusted to pH=3 with 1N HCl and then extracted with EtOAc (50 mL*3). The organic layers were combined, dried over anhydrous Na2SO4, filtered, and concentrated to give compound P4c (470 mg, 55.0% yield) as a colorless oil. 1H 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.

[0399] Step 3: 2-Fluoro-3-hydroxy-2-methylpropanoic acid (P4d). To a solution of compound P4c (200 mg, 1.22 mmol) in isopropanol (4 mL) was added 2 M LiBH (1.22 mL, 2.44 mmol). o C. The mixture was stirred at room temperature for 2 hours. The mixture was quenched by dropwise addition of 2N HCl (1.22 mL) at 0 °C, diluted with H2O (10 mL), and extracted with EtOAc (50 mL * 3). The organic layers were combined, 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.

[0400] 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-benz'[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 exatecan 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 hour. 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: 20mL / min), and the fractions were lyophilized to give two isomers:

[0401] Isomer 1: P4: White solid, (11 mg, 21.9% yield). UPLC-MS, RT=3.52 min. 1 H 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, MS (ESI) m / z: 540.3 [M+H] + .

[0402] Isomer 2: P4-1: White solid, (8.4 mg, 16.6% yield). UPLC-MS, RT=3.86 min.1 H 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, MS (ESI) m / z: 540.3 [M+H] + .

[0403] Example 16. Linker-payload synthesis [Table 22-1] [Table 22-2]

[0404] LD2-1 and LD2-2 (Table 17) are commercially available and were purchased from MedChemExpress CO. LTD (Shanghai).

[0405] Synthetic Procedures for Linker-Cytotoxic Agents LD2-3 to LD2-8 Linker-cytotoxic agent LD2-3 [ka] Step 1: Benzyl (5S,8S)-1-(9H-fluoren-9-yl)-5-isopropyl-8,14,14-trimethyl-3,6,9-trioxo-2,12-dioxa-4,7,10-triazapentadecan-15-oate (LD2-3c). A white suspension mixture of LD2-3a (300 mg, 0.62 mmol, synthesized according to reported procedures: ACS Med. Chem. Lett. 2019, 10, 1386-1392 and US9808537B2), LD2-3b (260 mg, 1.25 mmol), and 4 Å molecular sieves in anhydrous THF (10 mL) was stirred at room temperature for 10 min. Sc(OTf)3 (368 mg, 0.75 mmol) was added, and the resulting yellow suspension was stirred at room temperature for 4 h. The yellow suspension mixture was filtered through a Celite pad and washed with EtOAc (30 mL). The combined organic layers were washed with saturated NaHCO (30 mL) and brine (30 mL), dried over NaSO, filtered, and the filtrate was concentrated in vacuo to give a residue. It was purified by silica gel column (MeOH / DCM = 0% to 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] + .

[0406] Step 2: Benzyl 3-(((S)-2-((S)-2-amino-3-methylbutanamido)propanamido)methoxy)-2,2-dimethylpropanoate (LD2-3d). To a solution of LD2-3c (274 mg, 0.44 mmol) in DMF (5 mL) was added EtNH (477 mg, 5.53 mmol). The mixture was stirred at room temperature for 20 min. The reaction mixture was concentrated in vacuo and coevaporated twice with toluene to give LD2-3d (275 mg, crude) as a brown oil. MS (ESI) m / z: 430.4 [M+Na] + .

[0407] 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-tetraazaoctadecane-18-oate (LD2-3f). 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) was added HATU (198 mg, 0.52 mmol) and DIPEA (168 mg, 1.30 mmol). The mixture was stirred at room temperature for 10 min. 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] + .

[0408] 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-tetraazaoctadecan-18-oic acid (LD2-3g). To a solution of compound LD2-3f (3.01 g, 3.21 mmol) in the co-solvent DMF-MeOH (40 mL, 1:1, v:v), Pd / C (10%, 600 mg) was added. The mixture was stirred under H atmosphere (15 psi) for 7 hours. The mixture was filtered through a pad of Celite and concentrated to give compound LD2-3g (2.50 g, crude) as a white solid. MS (ESI) m / z: 863.7 [M+Na] + .

[0409] Step 5: (9H-Fluoren-9-yl)methyl((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-2,3,9,10,1 3,15-Hexahydro-1H,12H-benz'[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-tetraazanonadecane-6-yl)carbamate (LD2-3h). To a solution of compound exatecan mesylate (1000 mg, 1.18 mmol, purchased from MedChemExpress CO. LTD) in DMF (20 mL) was added compound LD2-3g (692 mg, 1.30 mmol), HATU (675 mg, 1.78 mmol), and DIEA (459 mg, 3.55 mmol). The mixture was stirred at room temperature for 30 minutes. 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, yield 88.6%) as an off-white solid. MS (ESI) m / z: 1282.1 [M+Na] + .

[0410] 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-benz'[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 EtNH (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] + .

[0411] 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)propanamide)-N1-((S)-1-(((S)-1-(((3-(((1S,9S)-9-ethyl-5-fluoro-9-hydro 4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benz'[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 (LD2-3). To a solution of LD2-3j (15 mg, 0.067 mmol) in dry DMF (1.0 mL), HATU (26 mg, 0.067 mmol) and DIEA (0.017 mL, 0.097 mmol) were added and stirred at room temperature for 15 min. Next, LD2-3i (50 mg, 0.048 mmol) was added to the above mixture and stirred at room temperature for 10 minutes. The resulting solution was purified by preparative HPLC (Method: Column XBridge Prep C18 OBD5um 19*150 mm, Mobile phase: A-water (0.1% TFA): B-acetonitrile, Flow rate: 20 mL / min, Fractions were lyophilized to obtain LD2-3 (37 mg, Yield 50.9%) as a yellow solid. MS (ESI) m / z: 1266.7 [M+Na] + .

[0412] Linker-cytotoxic agent LD2-4 [ka] Step 1: Benzyl (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-pentaazahenicosan-21-oate (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 4 Å molecular sieves. The mixture was stirred at room temperature for 10 min, then Sc(OTf) (195 mg, 0.40 mmol) was added and the mixture was further reacted at room temperature for an additional 16 h. The suspension mixture was filtered through a Celite pad, and the cake was washed with THF (10 mL). The filtrate was then quenched by adding saturated NaHCO (10 mL) and extracted with EtOAc (30 mL * 2). After separation, the combined organic layer was washed with brine (50 mL), dried over NaSO, filtered, and the filtrate was concentrated in vacuo to give a residue, which 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] + .

[0413] 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-pentaazahenicosan-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 h. After the reaction was complete, the black suspension was filtered through a pad of Celite, the cake was washed with MeOH, and 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] + .

[0414] Step 3: (9H-Fluoren-9-yl)methyl ((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-benz'[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-tetraazaheptadecyl)carbamate (LD2-4f). To a mixture of LD2-4d (60 mg, 0.087 mmol) and HATU (33 mg, 0.087 mmol) in DMF (2 mL) was added DIEA (43 μL, 34 mg, 0.26 mmol). The mixture was allowed to react at room temperature for 10 minutes. Exatecan mesylate (46 mg, 0.087 mmol) was added and the reaction was continued at the same temperature for another hour. After the reaction was complete, the mixture was filtered, and the filtrate was purified using preparative HPLC (Method: Column: XBridge Prep C18 OBD 5 um 19*150 mm, 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] + .

[0415] 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-benz'[d']pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-2,2-dimethylpropanamide (LD2-4g). To a solution of LD2-4f (85 mg, 0.062 mmol) in DMF (2 mL) was added EtNH (64 μL, 46 mg, 0.62 mmol). The mixture was stirred at room temperature for 0.5 h. Upon 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] + .

[0416] Step 6: (9H-Fluoren-9-yl)methyl ((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-benz'[d']pyrano[3',4':6,7]indolizino[1,2-b]quinoline-1-yl)methyl (2S,3R,4R,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)-22-oxa-2,8,11,14,17,20-hexaazapentacosan-6-yl)carbamate (LD2-4i). To a solution of LD2-4g (86 mg, crude) and LD2-4h (43 mg, 0.079 mmol, purchased from WuXi AppTec) 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. Upon completion of the reaction. The mixture was purified by prep-HPLC (FA) (Method: Column: XBridge Prep C18 OBD 5um 19*150mm, Mobile phase: A-water (0.1% formic acid): B-acetonitrile, Flow rate: 20mL / min, Fractions were lyophilized to give LD2-4i (70mg, Yield 62.6%) as a white powder. MS (ESI) m / z: 1410.7 [M+H] + .

[0417] 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-benz'[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-tetraazaheptadecyl)-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 EtNH (51 μL, 36 mg, 0.50 mmol). The mixture was stirred at room temperature for 0.5 h. Upon 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] + .

[0418] 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-benz'[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-tetraazaheptadecyl)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide)-N 5-(((2S,3R,4R,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)methyl)pentanediamide (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, and then LD2-4j (71 mg, crude) was added. The mixture was stirred at room temperature for 60 minutes. Upon completion of the reaction. The mixture was purified by prep-HPLC (FA) (Method: Column: XBridge Prep C18 OBD 5um 19*150mm, Mobile phase: A-water (0.1% formic acid): B-acetonitrile, Flow rate: 20mL / min), and the fractions were lyophilized to give LD2-4 (32mg, Yield 26.3%) as a white powder. MS (ESI) m / z: 1381.1 [M+H] + .

[0419] Linker-cytotoxic agent LD2-5 [ka] LD2-5 (30 mg, 50.7% yield) was synthesized according to the synthetic procedure for LD2-4. MS (ESI) m / z: 1408.1 [M+Na] + .

[0420] Linker-cytotoxic agent LD2-6 [ka] 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 a H 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 give LD2-6b (3.65 g, 99.8% yield) as an off-white solid. MS (ESI) m / z: 743.6 [M+H] + .

[0421] 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% to 10%), and the fractions were concentrated in vacuo to give LD2-6d (3.80 g, 86.9% yield) as an off-white foamy solid. MS (ESI) m / z: 890.7 [M+H] + .

[0422] 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-serylglycine (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 H balloon three times and then stirred at room temperature for 40 min. The black suspension was filtered through a pad of Celite 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] + .

[0423] Step 4: (2R,3R,4S,5S,6S)-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) (2.74 g, 6.19 mmol), Cu(OAc) (74.9 mg, 0.41 mmol), and HOAc (247.8 mg, 4.13 mmol). The resulting dark mixture was purged with a N balloon three times and then stirred at 65 °C for 40 min, turning the mixture deep 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] + .

[0424] 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 4 Å molecular sieves (200 mg) in anhydrous THF (10 mL) was stirred at room temperature for 10 min. Sc(OTf) (218 mg, 0.44 mmol) was added, and the resulting yellow suspension was stirred at room temperature for 4 h. The yellow suspension mixture was filtered through a Celite pad and washed with EtOAc. The combined organic layers were washed with saturated NaHCO (30 mL) and brine (30 mL), dried over NaSO, filtered, and the filtrate was concentrated in vacuo to give a residue. It was purified by silica gel column (MeOH / DCM = 0% to 5%), and the fractions were concentrated in vacuo to give LD2-6h (275 mg, 77.5% yield) as a white foamy solid. MS (ESI) m / z: 984.8 [M+Na] + .

[0425] 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-dioxa-4,7,10-triazapentadecan-15-oic 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 a H balloon three times and then stirred at room temperature for 2 h. The mixture was filtered through a syringe head, washed with MeOH (15 mL), and concentrated in vacuo to give LD2-6j (230 mg, crude) as a white foamy solid. MS (ESI) m / z: 894.6 [M+Na]+ .

[0426] Step 7: (2R,3R,4S,5S,6S)-2-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanamide)-3-(((3-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexyl)amino) Tetrahydro-1H,12H-benz'[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), exatecan 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, which was purified by FCC (MeOH / DCM=0%~3%) and 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] + .

[0427] Step 8: (2S,3S,4S,5R,6R)-6-((S)-2-((S)-2-amino-3-methylbutanamide)-3-(((3-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benz'[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 EtNH (523 mg, 5.06 mmol). The mixture was stirred at room temperature for 20 minutes. LCMS showed the reaction was complete and then concentrated in vacuo to give the crude product. This was dissolved in MeOH (6 mL), KCO (174.7 mg, 1.26 mmol) was added, and the mixture was stirred at room temperature for 10 minutes. Then HO (2 mL) was added to the mixture, and the mixture was stirred at room temperature for 30 minutes. The mixture was acidified with saturated KHSO at 0 °C to pH = 3. Filtration and purification by preparative HPLC (0.1% FA) and lyophilization of the fractions gave LD2-6l (140 mg, 59.7% yield) as a pale yellow solid. MS (ESI) m / z: 927.4 [M+H] + . 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, 6.8Hz, 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).

[0428] 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 HO (1 mL) was added compound LD2-6n (110 mg, 0.57 mmol), KCO (168 mg, 0.95 mmol), and Pd(dppf)Cl . DCM (35 mg, 0.047 mmol) was added. The mixture was stirred at 110 oThe mixture was stirred at RT 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 (eluted with PE / EtOAc = 0% to 40%). Compound LD2-6o (56 mg, yield 44.4%) was obtained as an off-white solid. MS (ESI) m / z: 267.1 [M+H] + .

[0429] 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 HO (1 mL) was added LiOH (17 mg, 0.41 mmol). The mixture was stirred at room temperature for 2 hours. 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] + .

[0430] 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 hours. The mixture was concentrated and purified by preparative HPLC (Method: Column: XBridge Prep C18 OBD 5 um 19*150 mm, 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] + .

[0431] 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-benz'[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). To a solution of compound LD2-6q (7.4 mg, 0.026 mmol) in DMF (2 mL), HATU (9 mg, 0.024 mmol) and DIEA (5.6 mg, 0.043 mmol) were added. 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 5 um 19*150 mm, Mobile phase: A-water (0.1% TFA): B-acetonitrile, Flow rate: 20 mL / min) to obtain compound LD2-6 (7.6 mg, 29.5% yield) as a white solid. MS (ESI) m / z: 1193.5 [M+H] + .

[0432] Linker-cytotoxic agent LD2-7 [ka] LD2-7 (32 mg, 50.9% yield) was synthesized according to the procedure in step 7 of LD2-3. MS (ESI) m / z: 1303.0 [M+H] + .

[0433] Linker-cytotoxic agent LD2-8 [ka] 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), followed by dropwise addition of CHI (1.61 g, 11.35 mmol) at 0 °C. The resulting mixture was stirred at 0 °C for 20 min, warmed to 25 °C, and further stirred at 25 °C for 60 min. The reaction process was monitored by TLC (PE / EA) and LCMS. After the reaction was completed, the reaction mixture was diluted with EA (80 mL) and washed with brine (30 mL*3) and HO (30 mL*2). The organic layer was dried over anhydrous NaSO, 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] + .

[0434] Step 2: Benzyl tert-butyl(4-hydroxybutane-1,2-diyl)(R)-dicarbamate (LD2-8c). LD2-8b (1.00 g, 2.73 mmol) was dissolved in MeOH (15 mL), followed by the addition of LiBH (2 M stock solution in THF, 6.80 mL) at 0 °C. The resulting mixture was stirred at 25 °C for 2 h. The reaction process was monitored by LCMS and TLC. After completion of the reaction, the reaction was quenched by adding saturated aqueous NH Cl (10 mL). The reaction mixture was diluted with H O (80 mL) and extracted with EA (50 mL * 3). The combined organic layer was washed with brine (40 mL * 2) and water (40 mL * 2), dried over anhydrous Na SO , filtered, 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]+.

[0435] Step 3: Benzyl tert-butyl (4-(((4-nitrophenoxy)carbonyl)oxy)butane-1,2-diyl) (R)-dicarbamate (LD2-8e). LD2-8c (300 mg, 0.89 mmol) and LD2-8d (405 mg, 1.33 mmol) were dissolved in DMF (5 mL), followed by the addition of DIEA (229 mg, 1.77 mmol). The resulting mixture was stirred at 25 °C for 1.5 h. After completion of the 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] + .

[0436] Step 4: (9H-Fluoren-9-yl)methyl tert-butyl (4-(((3-(dimethylamino)-3-oxopropyl)carbamoyl)oxy)butane-1,3-diyl)(S)-dicarbamate (LD2-8g). LD2-8e (420 mg, 0.83 mmol) and LD2-8f (149 mg, 1.67 mmol) were dissolved in DMF (5 mL), followed by the addition of aqueous NaHCO3 (1 M, 5 mL). The resulting mixture was stirred at 25 °C for 2.5 h. After the reaction was complete, 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] + .

[0437] 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-diazapentadecan-15-oic acid (LD2-8h). LD2-8g (360 mg, 0.79 mmol) was dissolved in MeOH (18 mL), followed by the addition of Pd / C (wet base, 108 mg). The resulting mixture was stirred under H2 (15 psi) at room temperature for 2 h. After the reaction was complete, 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] + .

[0438] 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-diazapentadecan-15-oic acid (LD2-8). LD2-8h (250 mg, 0.78 mmol) and LD2-8i (243 mg, 1.57 mmol) were dissolved in a mixture of ACN (8 mL) and aqueous NaHCO (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. The reaction mixture was acidified with aqueous KHSO (20 mL) and extracted with EA (35 mL). The combined organic layers were dried over anhydrous NaSO, filtered, and concentrated under reduced pressure to give a yellow oil as 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] + . 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).

[0439] Step 7: (R)-4-((tert-butoxycarbonyl)amino)-3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)butyl((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 1-9-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-tetraazaoctadecan-18-yl)carbamate (LD2-8l). LD2-8l (32 mg, 50.9% yield) was synthesized according to the procedure in Step 7 of Example LD2-3. MS (ESI) m / z: 1418.1 [M+H] + .

[0440] Step 8: (R)-4-amino-3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)butyl((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-tetraazaoctadecan-18-yl)carbamate (LD2-8). LD2-8 (22 mg, 0.016 mmol) was dissolved in a mixture of DCM (2 mL), followed by the addition of ZnBr (151 mg, 0.67 mmol). The resulting suspension was stirred at 40 °C for 12 h. After completion of the 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: 20mL / min) to obtain compound LD2-8 (13mg, Yield 61.7%) as a white solid. MS (ESI) m / z: 1318.1 [M+H] + .

[0441] Example 17. Preparation and evaluation of antibody drug conjugates (ADCs) Preparation of reference ADC BGA7650 (Table 18). Organic solvent (e.g., DMSO, DMF, DMA, PG, acetonitrile, 0-25% v / v) and linker-payload (2-25 equivalents, 10 mM stock in organic solvent) are mixed with the anti-CEA antibody tusamitamab (1-20 mg / mL) in a 0-37 mL solution. oThe solution was gradually added to a reaction buffer (PBS buffer, pH 6.0-9.0) for 0.5-48 hours at 4°C. The solution was subjected to buffer exchange (spin desalting column, ultrafiltration, and dialysis) and exchanged into a storage buffer (e.g., pH 5.5-6.5 histidine acetate buffer, with optional additives such as sucrose, trehalose, Tween 20, 60, or 80).

[0442] Determination of Drug-to-Antibody Ratio (DAR): LC-MS 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, 1,500Å, Thermo Scientific(TM) Column temperature: 80 o C Mobile phase A: 0.1% formic acid (FA) in water, Mobile phase B: Acetonitrile solution containing 0.1% formic acid (FA), Gradient program: 25% B to 25% B (0 min to 2 min), 25% B to 50% B (2 min to 18 min), 50% B to 90% B (18 min to 18.1 min), 90% B to 90% B (18.1 min to 20 min), 90% B to 25% B (20 min to 20.1 min), 25% B to 25% B (20.1 min to 25 min) Injection sample amount: 2 μg, MS parameters: Intact and denatured MS data were acquired in HMR mode with R = 15k and deconvoluted using the ReSpect™ algorithm and sliding window integration in Thermo Scientific™ BioPharma Finder™ 4.0 software.

[0443] Preparation of DAR8 antibody-drug conjugate. Antibody in conjugation buffer (concentration 0.5-25 mg / mL, PBS buffer (pH 6.0-8.5)) was incubated at reduced temperature (0-40°C) for 10 min with 8-15 equivalents of TECP solution (5 mM stock in PBS buffer). TECP solution (5 mM stock in PBS buffer) was added to the reaction mixture, and the reduction reaction was allowed to proceed at reduced temperature for 1-8 h. After the reduction mixture was cooled to 0-25°C, organic solvents (e.g., DMSO, DMF, DMA, PG, acetonitrile, 0-25% v / v) and linker-payload stock (10-25 equivalents, 10 mM stock in organic solvent) were added stepwise. The conjugation solution was allowed to stand at 0-25°C for 1-3 h, and the reaction was quenched with N-acetylcysteine ​​(1 mM stock). The solution was subjected to buffer exchange (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, etc.).

[0444] Hydrolysis of maleimides after conjugation. After the conjugation step, the ADC was buffer-exchanged into a ring-opening buffer (pH 7.0-9.0, PBS, borate, or Tris buffer), and the solution was left at 22 or 37 °C for 5-48 hours. The ring-opening process was monitored by reduced LCMS. Upon completion of hydrolysis of the conjugated maleimide, the resulting ADC was buffer-exchanged via dialysis into a basic Tris pH 8.0-8.5 buffer or an acidic histidine acetate pH 5.0-6.5 buffer.

[0445] ADC Characterization. Example ADCs were prepared by following the procedure described above with a DAR profile. All ADCs were characterized by the following analytical methods. The drug-to-antibody ratio (DAR) of the ADCs was determined by LCMS or hydrophobic interaction column (HIC) methods. The SEC purity of the constructed ADCs was all >95% pure.

[0446] DAR decision LC-MS Method. LC-MS analysis was carried out 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, 1,500Å, Thermo Scientific(TM) Column temperature: 80℃ Mobile phase A: 0.1% formic acid (FA) in water Mobile phase B: Acetonitrile solution containing 0.1% formic acid (FA) Gradient program: 25% B to 25% B (0 min to 2 min), 25% B to 50% B (2 min to 18 min), 50% B to 90% B (18 min to 18.1 min), 90% B to 90% B (18.1 min to 20 min), 90% B to 25% B (20 min to 20.1 min), 25% B to 25% B (20.1 min to 25 min) Injected sample amount: 1 μg MS parameters: Intact and denatured MS data were acquired in HMR mode with R = 15k and deconvoluted using the ReSpect™ algorithm and sliding window integration in Thermo Scientific™ BioPharma Finder™ 4.0 software.

[0447] 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 x 3.5 cm, 2.5 μm butyl non-porous resin column Column temperature: 25℃ 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 to 0%B (0 min to 2 min), 0%B to 100%B (2 min to 15 min), 100%B to 100%B (15 min to 16 min), 100%B to 0%B (16 min to 17 min), 0%B to 0%B (17 min to 20 min) Injected sample amount: 20 μg

[0448] ADC purity: SEC method HPLC analysis was carried out under the following measurement conditions: HPLC system: Waters H-Class UPLC system Detector: Measurement wavelength: 280 nm Column: ACQUITY UPLC BEH200 SEC 1.7um 4.6×150mm, Waters Column temperature: room temperature Mobile phase A: 200 mM phosphate buffer, 250 mM potassium chloride, 15% isopropyl alcohol, pH 7.0 Gradient program: 10 min isocratic elution at a flow rate of 0.3 mL / min Injected sample amount: 20 μg

[0449] ADC Hydrophobicity Assessment: HIC ADCs with strong hydrophobicity have a slower retention time in HIC chromatography.

[0450] HPLC analysis was carried out 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 x 3.5 cm, 2.5 μm butyl non-porous resin column Column temperature: 25℃ 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 to 0%B (0 min to 2 min), 0%B to 100%B (2 min to 15 min), 100%B to 100%B (15 min to 16 min), 100%B to 0%B (16 min to 17 min), 0%B to 0%B (17 min to 20 min) Injected sample amount: 20 μg

[0451] Method 2 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℃ 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 to 20%B (0 to 1 min), 0%B to 0%B (1 to 35 min), 20%B to 20%B (35 to 40 min) Flow rate: 0.5mL / min Sample preparation: Samples were diluted to 0.5 mg / mL in the initial mobile phase. [Table 23-1] [Table 23-2] Tsusamitamab sequence >LC DIQMTQSPASLSASVGDRVTITCRASENIFSYLAWYQQKPGKSPKLLVYNTRTLAEGVPSRFSGSGSGTDFSLTISSLQPEDFATYYCQHHYGTPFTFGSGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 105) >HC EVQLQESGPGLVKPGGSLSLSCAASGFVFSSYDMSWVRQTPERGLEWVAYISSGGGITYAPSTVKGRFTVSRDNAKNTLYLQMNSLTSEDTAVYYCAAHYFGSSGPFAYWGQ GTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 106)

[0452] Cell line information MKN45 (JCRB, JCRB0254) MKN45 is a cell line exhibiting rounded morphology. MKN45 is loosely stroma-bound and was isolated in 1998 from the stomach tissue of a 62-year-old woman with gastric cancer. MKN45 was purchased from JCRB. The basal medium for MKN45 is RPMI-1640 medium, Gibco 22400089. To prepare 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 periodically tested for the presence of mycoplasma using the MycoAlert™ PLUS Mycoplasma Detection Kit (Lonza, LT07-710).

[0453] SNU-16 (ATCC, CRL-5974) SNU-16 is a cell line exhibiting epithelial morphology isolated from ascites fluid from a 33-year-old Asian female gastric cancer patient before chemotherapy in 1987. SNU16 was purchased from ATCC. The basal medium for SNU16 is RPMI-1640 medium, Gibco 22400089. To make complete growth medium, the following components were added to the basal medium to a final concentration of 10%: fetal bovine serum (Gibco, 10099-141C). The cell line was grown at 37°C in a humidified 5% CO atmosphere and periodically tested for the presence of mycoplasma using the MycoAlert™ PLUS Mycoplasma Detection Kit (Lonza, LT07-710).

[0454] NCI-H2122 (ATCC, CRL-5985) NCI-H2122 cells are lymphoblasts isolated from a pleural effusion metastasis from a 46-year-old female smoker in 1989. NCI-H2122 cells were purchased from ATCC. The basal medium for NCI-H2122 is RPMI-1640 medium, Gibco 22400089. To make complete growth medium, the following components were added to the basal medium to a final concentration of 10%: fetal bovine serum (Gibco, 10099-141C). The cell line was grown at 37°C in a humidified 5% CO2 atmosphere and periodically tested for the presence of mycoplasma using the MycoAlert™ PLUS Mycoplasma Detection Kit (Lonza, LT07-710).

[0455] LS174T (ATCC, CL-188) LS174T is a cell line with epithelial morphology isolated from the colon of a 58-year-old Caucasian female patient with adenocarcinoma of the colorectum. LS174T was purchased from ATCC. The basal medium for LS174T is ATCC formulation Eagle's Minimum Essential Medium, 30-2003. To make complete growth medium, the following components were added to the basal medium to a final concentration of 10%: fetal bovine serum (Gibco, 10099-141C). The cell line was grown at 37°C in a humidified 5% CO atmosphere and periodically tested for the presence of mycoplasma using the MycoAlert™ PLUS Mycoplasma Detection Kit (Lonza, LT07-710).

[0456] MDA-MB-231 (ATCC, HTB-26) MDA-MB-231 are epithelial-like cells isolated from the mammary gland of a 40-year-old Caucasian woman with adenocarcinoma. MDA-MB-231 were purchased from ATCC. The basal medium for MDA-MB-231 is RPMI-1640 medium, Gibco 22400089. To make complete growth medium, the following components were added to the basal medium to a final concentration of 10%: fetal bovine serum (Gibco, 10099-141C). The cell line was grown at 37°C in a humidified 5% CO2 atmosphere and periodically tested for the presence of mycoplasma using the MycoAlert™ PLUS Mycoplasma Detection Kit (Lonza, LT07-710).

[0457] HCT116 (ATCC, CCL-247) The HCT116 cell line was isolated from the colon of an adult male with colon cancer and carries a mutation at codon 13 of the ras proto-oncogene. It was purchased from ATCC. The basal medium for MDA-MB-231 is RPMI-1640 medium, Gibco 22400089. To make complete growth medium, the following components were added to the basal medium to a final concentration of 10%: fetal bovine serum (Gibco, 10099-141C). The cell line was grown at 37°C in a humidified 5% CO2 atmosphere and periodically tested for the presence of mycoplasma using the MycoAlert™ PLUS Mycoplasma Detection Kit (Lonza, LT07-710).

[0458] Additional cell lines SW1463 cells are derived from a human colorectal adenocarcinoma and express moderate levels of CEA.

[0459] NCI-N87 expressed low levels of CEA and was derived from a gastric carcinoma.

[0460] HT29 cells were low to negative for CEA expression and were derived from a human colorectal adenocarcinoma. [Table 24]

[0461] Example 18. In vitro cell killing by CEA antibodies conjugated with various payloads The BGA5384 antibody (Table 20) was conjugated using in-house generated linkers and various payloads according to Example 17. BGA5384 was conjugated with the maytansinoid DM4 (Figure 13), the auristatin MMAE (Figure 14), and the topoisomerase DXD (BGA2588) (Figure 15). [Table 25]

[0462] To determine the amount of cell killing induced by each ADC, cells from lines with different CEA expression levels (Example 17, Table 20) were seeded into 96-well plates and incubated overnight at 37°C. Serially diluted ADCs were added, and the cells were then cultured for 6 days and subjected to a cell viability assay. As shown in Figures 13-15, all CEA antibody-drug conjugates demonstrated good cell killing in high-to-moderate CEA-expressing cells at low concentrations of CEA ADC. In low-to-low-negative-to-negative CEA-expressing cells, high concentrations of CEA ADC were required to demonstrate cell killing. These data demonstrate that the BGA5384 antibody can be conjugated to various payloads and can achieve cell killing in various CEA-expressing cells.

[0463] Example 19. Payload Sensitivity: MKN45 The effect of payloads on MKN45 cells is shown in Figure 16. On day 0, cells were harvested with 0.25% trypsin-EDTA and plated at 5,000 cells / well in a 96-well plate (655090, Greiner) and incubated overnight at 37°C and 5% CO2. On day 1, payload compounds were added to the plate (5-fold dilution). The cell and compound mixture was incubated at 37°C and 5% CO2 for 6 days. Signaling of viable cells was collected on day 6 with 100 μL of detection reagent (G7573, Promega®), and the signal was read by Tecan Spar®. Data were analyzed by GraphPad Prism 9.0.0. All killing concentrations were used in duplicate or triplicate (Table 21 and Figure 16). "SABC" refers to specific antibody binding capacity. [Table 26]

[0464] Example 20. ADC Direct Cell Killing Assay Figures 17-20 show the cellular activity of eight different constructed ADCs (see Table 18) in MKN45 (gastric cancer), H2122 (lung adenocarcinoma), LS174T (colorectal adenocarcinoma), and MB-231 (breast adenocarcinoma) patient-derived cell lines, respectively.

[0465] On day 0, cells were harvested using 0.25% trypsin-EDTA and plated into 96-well plates (655090, Greiner) at 5,000 cells / well (MKN45 or Ls174T) or 2,000 cells / well (NCI-H2122 or MDA-MB-231) and incubated overnight at 37°C and 5% CO2. On day 1, ADCs were added to the plates (5-fold dilution). The cell and ADC mixture was incubated at 37°C and 5% CO2 for 6 days. Signaling of viable cells was collected on day 6 with 100 μL of detection reagent (G7573, Promega®), and the signal was read using a Tecan Spar®. Data were analyzed using GraphPad Prism 9.0.0. All killing concentrations were used in duplicate or triplicate. Results are presented 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. [Table 27] [Table 28] [Table 29] [Table 30]

[0466] Example 21. Efficacy of ADCs BGA7650 and BGA9962 in cell line-derived xenograft models. Cell line-derived xenograft (CDX) models using MKN-45 (CEA high) (gastric adenocarcinoma), SW-1463 (CEA intermediate) (rectal adenocarcinoma), and NCI-H2122 (CEA low) (lung adenocarcinoma) were generated as follows.

[0467] 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 transplantation, cells were harvested and resuspended in cold (4°C) serum-free RPMI-1640 medium. Cell densities were adjusted to 2 (MKN-45), 1.5 (SW1463), or 4 (NCI-H2122) × 10 cells / ml. 7 The solution was adjusted to cells / mL and the cells were placed on ice before inoculation.

[0468] Six- to eight-week-old female mice were purchased, housed in ventilated cages, and given food and water ad libitum. They were allowed to acclimate for approximately 1 week before inoculation. MKN-45, SW-1463, and NCI-H2122 tumors were injected at 2.0 (MKN-45), 3.0 (SW1463), or 8.0 (NCI-H2122) × 10 tumor cells, respectively. 6 The cells were induced by subcutaneous injection into the right flank of NCG, NCG, and Balb / c nude mice.

[0469] The experiment was carried out approximately 2 to 3 weeks after the injection of cancer cells. For tumor volume measurement, all tumors were measured with a caliper and tumor volume was calculated using the formula V = 0.5(a × b 2 ) where "a" is the length of the tumor and "b" is the width and / or height of the tumor. 3 Upon reaching 100 mg / kg, mice were randomized into five groups, with 8, 9, and 9 animals in the vehicle, BGA7650, and BGA9962 groups, respectively, on day 0. After ensuring that the mean tumor volumes of all cohorts were approximately equal at the start, animals were intravenously administered vehicle, BGA7650 (1.3 mg / kg or 4 mg / kg, or "mpk"), and BGA9962 (2 mg / kg or 6 mg / kg) on ​​day 1 of treatment. Animal weights and tumor volumes were measured twice weekly. Data are presented as mean tumor volume ± standard error of the mean (SEM). Tumor growth inhibition (TGI) was calculated using the following formula: %TGI=[1-(Treated Tt-Treated T0) / (Vehicle Tt-Vehicle T0)]×100% Tt = mean tumor volume of the treatment group on day t Mean tumor volume of treated dose group on day T0 = 0 Vehicle Tt = mean tumor volume of the vehicle group on day t Vehicle TO = Mean tumor volume of the vehicle group on day 0

[0470] result In cell line-derived xenograft (CDX) models using MKN-45 (Figure 21), SW-1463 (Figure 22), and NCI-H2122 (Figure 23), BGA9962 and BGA7650 treatment groups slowed tumor growth compared to the vehicle group. In these three CDX models, both BGA9962 and BGA7650 demonstrated dose-dependent efficacy (Figures 21, 22, and 23). Significant antitumor effects were induced by 4 mg / kg, but not 1.3 mg / kg, of BGA7650. 2 mg / kg of BGA9962 demonstrated superior antitumor efficacy compared to 1.3 mg / kg of BGA7650 and comparable efficacy to 4 mg / kg of BGA7650. Furthermore, 6 mg / kg of BGA9962 significantly reduced tumor growth, demonstrating greater antitumor efficacy than both doses (1.3 and 4 mg / kg) of BGA7650. All animals tolerated the treatment well, without significant weight loss or abnormal clinical observations.

[0471] Example 22. Efficacy of BGA7650 and BGA9962 in a human patient-derived gastric cancer xenograft model. The antitumor effects of BGA7650 and BGA9962 were evaluated in a human patient-derived gastric cancer ("GC") xenograft model initiated as described in Example 21 (Figure 24). 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 both doses (2 mg / kg and 6 mg / kg) demonstrated significantly greater antitumor activity than 4 mg / kg BGA7650 (Figure 24 and Table 26). All animals tolerated the treatment well, without significant weight loss or abnormal clinical observations. [Table 31]

[0472] Example 2: Single-dose PK in non-tumor-bearing mice at 3.3 mg / kg (iv) As described in Example 21, a single dose of 3 mg / kg (iv) of BGA9962 or BGA7650 was administered to Balb / c nude (non-tumor-bearing) mice to evaluate pharmacokinetics. BGA9962 demonstrated a potent pharmacokinetic (PK) profile compared to BGA7650 in Balb / c nude (non-tumor-bearing) mice (n=3) (Figure 25). BGA9962 demonstrated superior PK relative to the comparator BGA7650. BGA9962 (triangles connected by dashed lines) exhibited a lower serum-free payload in plasma compared to BGA7650 (triangles connected by solid lines).

[0473] Separation between TAb (total antibody) and ADC was observed for comparator BGA7650 (solid line connecting open circles), but not for BGA9962 (solid line connecting open squares). These results confirm that the DAR of BGA9962 is stable over time compared to BGA7650. In vitro, BGA9962 is stable in mouse and human plasma, with no change in DAR after 336 hours of incubation (data not shown).

[0474] Example 24. Quantification of in vivo DAR and comparison of stability The in vivo drug-to-antibody ratios (DAR) of BGA7650 and BGA9962 were analyzed by intact LC-MS. Serum samples (n=3 per group, derived from Balb / c nude mice treated with a single iv dose of 3 mg / k ADC) were processed from the heavy chain (HC) and light chain (LC) of the ADC to the payload-conjugated peptide. The DAR mass spectra were analyzed to calculate the average DAR for each chain, and the average DAR for the intact ADC was calculated using the following formula: DAR=(DAR(LC)+DAR(HC))×2.

[0475] Because BGA7650 is payload-conjugated via lysines, not just at a specific position, it was difficult to process for intact LC-MS DAR analysis. Instead, the in vivo DAR of BGA7650 was measured indirectly by analyzing the ratio of total conjugated payload to total antibody concentration using immunocapture followed by anti-Fc (to detect total antibody) and LC-MS (to detect conjugated payload). Thus, Figure 26 shows that BGA9962 maintained a stable DAR of 8 in vivo with minimal deconjugation, while BGA7650 gradually deconjugated over time.

Claims

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

2. Formula Ab-(CL-(D) ｍ ) ｎ 2. The antibody-drug conjugate of claim 1, or a pharmaceutically acceptable salt, solvate, or hydrate thereof, wherein: Ab is the antibody or antigen-binding fragment thereof; C is a conjugator; L is a linker, D is the cytotoxic agent; m is an integer from 1 to 8; The antibody-drug conjugate, or a pharmaceutically acceptable salt, solvate, or hydrate thereof, wherein n is 1 to 10.

3. 3. The antibody drug conjugate of claim 2, wherein m is 1.

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

5. 5. The antibody drug conjugate of claim 4, wherein n is about 8.

6. The antibody-drug conjugate of any one of claims 2 to 4, wherein C is (C-I), (C-Ia), (C-Ib), (C-II), (C-III), (C-IIIa), or (C-IV): 【Chemistry 1-1】 [Chemistry 1-2] and * indicates the bond where C connects to Ab in the antibody drug conjugate.

7. C is, 【Chemistry 2】 7. The antibody-drug conjugate of claim 6, wherein:

8. The antibody-drug conjugate according to any one of claims 2 to 7, L is (LI), (L-II), or (L-III): 【Transformation 3】 wherein Su is a hydrophilic residue, and * indicates the bond connecting L to C in the antibody drug conjugate.

9. Su, 【Chemistry 4】 9. The antibody-drug conjugate of claim 8, wherein:

10. Su, 【Transformation 5】 10. The antibody-drug conjugate of claim 9, wherein:

11. L, 【Transformation 6】 8. The antibody drug conjugate of claim 2, wherein * indicates the bond connecting L to C.

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

13. The antibody drug conjugate of any one of claims 2 to 12, wherein D is 【Transformation 7】 wherein: Y is -ABC'-D'-*, where * represents the bond connecting D to L; A is a bond, CR １ R ２ , or N-R １ and 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 heterocyclyl, unsubstituted or substituted aryl, or unsubstituted or substituted heteroaryl; D' is a bond, NH, or O; R １ and R ２ Each of R is independently hydrogen, halogen, substituted or unsubstituted alkyl, or substituted or unsubstituted alkoxyl, or １ , and R ２ together with the atom to which they are attached form an unsubstituted or substituted cycloalkyl, an unsubstituted or substituted heterocyclyl, an unsubstituted or substituted aryl, or an unsubstituted or substituted heteroaryl; R ３ and R ４ Each of R is independently hydrogen, halogen, substituted or unsubstituted alkyl, or substituted or unsubstituted alkoxyl, or ３ and R ４ together with the atom to which they are attached form an unsubstituted or substituted cycloalkyl, an unsubstituted or substituted heterocyclyl, an unsubstituted or substituted aryl, or an unsubstituted or substituted heteroaryl.

14. D is, 【Transformation 8】 and In the formula, R ７ and R ８ The antibody drug conjugate of any one of claims 1 to 13, wherein each is independently hydrogen, halogen, or alkyl.

15. D is, 【Chemistry 9】 The antibody-drug conjugate of any one of claims 2 to 12, wherein the antibody-drug conjugate is selected from the group consisting of:

16. D is, 【Chemistry 10】 16. The antibody-drug conjugate of claim 15, wherein:

17. C-L-(D) ｍ but the following: 【Chemistry 11-1】 【Chemistry 11-2】 6. The antibody drug conjugate of any one of claims 2 to 5, wherein * indicates the bond connecting C to Ab.

18. C-L-(D) ｍ but the following: 【Chemistry 12】 18. The antibody-drug conjugate of claim 17, wherein:

19. 6. The antibody drug conjugate of any one of claims 2 to 5, or a tautomer, pharmaceutically acceptable salt, solvate, or hydrate thereof, wherein the antibody drug conjugate has the following formula: 【Chemistry 13-1】 【Chemistry 13-2】 or a tautomer, pharmaceutically acceptable salt, solvate, or hydrate thereof, having one of:

20. 20. The antibody drug conjugate of any one of claims 1 to 19, wherein the antibody or antigen-binding fragment is: (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) the antibody-drug conjugate, comprising a heavy chain variable region comprising SEQ ID NO: 14 and a light chain variable region comprising SEQ ID NO:

15.

21. 21. The antibody drug conjugate of 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 a F(ab')2 fragment.

22. 22. The antibody drug conjugate of any one of claims 1 to 21, wherein the antibody or antigen-binding fragment comprises an scFv comprising 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. 22. The antibody drug conjugate of 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. 24. The antibody drug conjugate of any one of claims 1 to 23, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain constant region of the IgG1, IgG2, IgG3, or IgG4 subclass, and / or a light chain constant region of the kappa or lambda type.

25. 25. The antibody drug conjugate of claim 24, wherein 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 kappa type.

26. The following formula: 【Chemistry 14】 or a tautomer, pharmaceutically acceptable salt, solvate, or hydrate thereof, wherein: n is 4 to 10, and Ab is an antibody or antigen-binding fragment thereof that binds to CEA, (i) the three heavy chain CDRs: HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 7; HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 8; HCDR3 comprising the amino acid sequence set forth in SEQ ID NO:9, and The three light chain CDRs: LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 10; LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 11; an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO:6; or (ii) three heavy chain CDRs: HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 24; HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 25; HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 26, and The three light chain CDRs: LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 27; LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 28; an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 23; or (iii) three heavy chain CDRs: HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 41; HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 42; HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 43, and The three light chain CDRs: LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 44; LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 45; an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 40, or a tautomer, pharmaceutically acceptable salt, solvate, or hydrate thereof.

27. 1. An antibody drug conjugate or a tautomer, pharmaceutically acceptable salt, solvate, or hydrate thereof, comprising: The antibody drug conjugate has the following formula: 【Chemistry 15-1】 【Chemistry 15-2】 and n is 4 to 10, and Ab is an antibody or antigen-binding fragment thereof that binds to CEA, (i) the three heavy chain CDRs: HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 7; HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 8; HCDR3 comprising the amino acid sequence set forth in SEQ ID NO:9, and The three light chain CDRs: LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 10; LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 11; an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO:6; or (ii) three heavy chain CDRs: HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 24; HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 25; HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 26, and The three light chain CDRs: LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 27; LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 28; an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 23; or (iii) three heavy chain CDRs: HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 41; HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 42; HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 43, and The three light chain CDRs: LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 44; LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 45; an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 40, or a tautomer, pharmaceutically acceptable salt, solvate, or hydrate thereof.

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

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

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

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

32. 31. The method of claim 30, wherein the gastrointestinal cancer is gastric cancer.

33. 31. The method of claim 30, wherein the colorectal cancer is rectal cancer.

34. A method for producing an antibody drug conjugate according to any one of claims 1 to 27, comprising: (i) culturing a host cell transformed with an isolated nucleic acid comprising a sequence encoding the antibody or antigen-binding fragment thereof, wherein the antibody or 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 antigen-binding fragment thereof; (iii) recovering the expressed antibody or antigen-binding fragment thereof; and (iv) conjugating the cytotoxic agent to the antibody or fragment thereof using a linker to form the antibody drug conjugate.

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

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