CEACAM5 antibody-drug conjugates and methods of use thereof

Abstract

The present invention relates to antibody-drug conjugates comprising an antibody that binds to CEACAM5 conjugated to a drug, such as a topoisomerase I inhibitor. Also provided herein are methods of treating cancer comprising administering such ADCs.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 384,214, filed November 17, 2022, European Patent Application No. 22306780.2, filed December 2, 2022, and U.S. Provisional Patent Application No. 63 / 596,943, filed November 7, 2023, the entire disclosures of which are incorporated herein by reference.

[0002] Electronic Sequence Listing Reference The contents of the electronic sequence listing (761682009941seqlist.xml, size: 12,724 bytes, and creation date: November 15, 2023) are incorporated herein by reference in their entirety.

[0003] The present application relates to antibody conjugates (ADCs) comprising an antibody that binds to CEACAM5 conjugated to a topoisomerase I inhibitor. [Background technology]

[0004] Carcinoembryonic antigen (CEA) is a glycoprotein involved in cell adhesion. CEA was first identified in 1965 as a protein normally expressed by fetal intestine during the first six months of pregnancy (Gold and Freedman, J Exp Med, 121, 439, 1965) and has been found in pancreatic, liver, and colon cancers. The CEA family belongs to the immunoglobulin superfamily. The CEA family, consisting of 18 genes, is subdivided into two subgroups of proteins: the carcinoembryonic antigen-related cell adhesion molecule (CEACAM) subgroup and the pregnancy-specific glycoprotein subgroup (Kammerer & Zimmermann, BMC Biology 2010, 8:12).

[0005] In humans, the CEACAM subfamily consists of seven members: CEACAM1, CEACAM3, CEACAM4, CEACAM5, CEACAM6, CEACAM7, and CEACAM8. Numerous studies have shown that CEACAM5 is highly expressed on the surface of colon, stomach, lung, breast, prostate, ovary, cervix, and bladder tumor cells, and weakly expressed in a few normal epithelial tissues, such as colonic columnar epithelial cells and goblet cells, gastric mucosal cervical cells, and esophageal and cervical squamous epithelial cells (Hammarstrom et al., 2002, in "Tumor markers, Physiology, Pathobiology, Technology and Clinical Applications," Eds. Diamandis EP et al., AACC Press, Washington, pp. 375). Therefore, CEACAM5 may constitute a suitable therapeutic target for tumor-specific targeting approaches, such as immunoconjugates. The present invention provides monoclonal antibodies directed against CEACAM5 and demonstrates that they can be conjugated to cytotoxic agents to induce cytotoxic activity that can kill tumor cells in vitro and induce tumor regression in vivo.

[0006] The extracellular domains of CEACAM family members are composed of repeated immunoglobulin-like (Ig-like) domains classified according to sequence homology into three types: A, B, and N. CEACAM5 contains seven such domains: N, A1, B1, A2, B2, A3, and B3.

[0007] On the one hand, the CEACAM5 A1, A2, and A3 domains, and on the other hand, the B1, B2, and B3 domains, show high sequence homology, with the A domain of human CEACAM5 exhibiting 84-87% pairwise sequence similarity and the B domain 69-80%. Furthermore, other human CEACAM members that exhibit A and / or B domains in their structures, i.e., CEACAM1, CEACAM6, CEACAM7, and CEACAM8, show homology with human CEACAM5. In particular, the A and B domains of the human CEACAM6 protein show sequence homology with the A1 and A3 domains, and with any of the B1-B3 domains of human CEACAM5, respectively, which is even higher than that observed between the A and B domains of human CEACAM5.

[0008] The design of antibody-drug conjugates (ADCs) involves consideration of various factors, including the presence of a conjugation handle on the drug for attachment to the linker, and linker technology for attaching the drug to the antibody in a conditionally stable manner, by attaching a cytotoxic agent to the antibody, typically via a linker. Thus, one strategy for cancer therapy targeting CEACAM5 is by generating an ADC comprising an antibody that binds to CEACAM5 conjugated to a cytotoxic drug. Summary of the Invention

[0009] In some embodiments, L-(QD) p Provided herein is an antibody-drug conjugate that binds to CEACAM5, having the formula: wherein L is a ligand unit comprising an antibody or antigen-binding fragment thereof that binds to CEACAM5; the subscript p is an integer ranging from 1 to 16; Q is a linker unit having a formula selected from the group consisting of: -ZA-RL-, -ZA-RL-Y-, -ZAS * -RL-, -ZAB(S * )-RL-, -ZAS* -RL-Y- and -ZAB(S * )-RL-Y- Z is the stretcher unit, A is a coupling or connector unit, B is a parallel connector unit, S * is the resolving agent, RL is a glycoside unit, Y is a spacer unit, D is a Drug unit having the formula: [ka] The wavy line indicates the site of covalent attachment to Q.

[0010] In some embodiments, L-(QD) p Provided herein is an antibody-drug conjugate that binds to CEACAM5, having the formula: In the formula, L is CDR1-H, CDR2-H, and CDR3-H of a variable heavy chain domain (VH) comprising the amino acid sequence shown in SEQ ID NO: 7; a ligand unit comprising an antibody or an antigen-binding fragment thereof that binds to CEACAM5, the antibody comprising CDR1-L, CDR2-L, and CDR3-L of a variable light chain domain (VL) that comprises the amino acid sequence shown in SEQ ID NO: 8; the subscript p is an integer ranging from 1 to 16; Q is a linker unit, D is a Drug unit, and the Drug unit is a topoisomerase I inhibitor.

[0011] In some embodiments, the antibody or antigen-binding fragment thereof comprises: CDR1-H, CDR2-H, and CDR3-H of a variable heavy chain domain (VH) comprising the amino acid sequence shown in SEQ ID NO: 7; and CDR1-L, CDR2-L, and CDR3-L of a variable light chain domain (VL) comprising the amino acid sequence shown in SEQ ID NO:8.

[0012] In some embodiments of the aforementioned antibody-drug conjugates or salts thereof, the antibody or antigen-binding fragment thereof comprises a variable heavy chain domain (VH) having at least 80%, 85%, 90%, 95%, or 99% amino acid sequence identity to the amino acid sequence of SEQ ID NO:7, and a variable light chain domain (VL) having at least 80%, 85%, 90%, 95%, or 99% amino acid sequence identity to the amino acid sequence of SEQ ID NO:8.

[0013] In some embodiments, provided herein is an antibody-drug conjugate or a salt thereof, wherein the antibody or antigen-binding fragment thereof is CDR1-H comprising the amino acid sequence shown in SEQ ID NO: 1; CDR2-H comprising the amino acid sequence shown in SEQ ID NO: 2; CDR3-H comprising the amino acid sequence shown in SEQ ID NO: 3; CDR1-L comprising the amino acid sequence shown in SEQ ID NO: 4; CDR2-L including the amino acid sequence NTR; and CDR3-L comprising the amino acid sequence shown in SEQ ID NO:6.

[0014] In some embodiments, provided herein is an antibody-drug conjugate or a salt thereof, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain having at least 80%, 85%, 90%, 95%, or 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 9 and a light chain having at least 80%, 85%, 90%, 95%, or 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 10.

[0015] In some embodiments, provided herein are antibody-drug conjugates or salts thereof, wherein the antibody or antigen-binding fragment thereof is chimeric or humanized.

[0016] In some embodiments, provided herein is an antibody-drug conjugate or a salt thereof, wherein the antibody or antigen-binding fragment is selected from the group consisting of Fv, Fab, F(ab')2, Fab', dsFv, (dsFv)2, scFv, sc(Fv)2, and a diabody.

[0017] In some embodiments, Q is a linker unit having the formula -ZA-RL-. In some embodiments, R is a glucuronide unit.

[0018] In some embodiments, R is a glucuronide unit having the formula: [ka] During the ceremony, Su is a sugar moiety; -O'- is an oxygen glycosidic bond, R 1S , R 2S and R 3S are independently hydrogen, halogen, —CN, —NO, or other electron-withdrawing or electron-donating group; the wavy line indicates a bond to Z either directly or indirectly via A or B or A and B; # indicates a bond to either D or Y, either directly or indirectly through an intervening functional group or other moiety.

[0019] In some embodiments, R is a glucuronide unit having the formula: [ka] During the ceremony, Su is a sugar moiety; O' represents the oxygen atom of a glycosidic bond that can be cleaved by a glycosidase; The wavy line with a single asterisk (*) indicates the site of covalent binding to D, The wavy line marked with a double asterisk (**) indicates the site of covalent attachment to the remainder of Q.

[0020] In some embodiments, Su is the hexose form of a monosaccharide.

[0021] In some embodiments, the glucuronide unit has the formula: [ka]

[0022] where the wavy line marked with a single asterisk (*) indicates the site of covalent attachment to D, and the wavy line marked with a double asterisk (**) indicates the site of covalent attachment to the remainder of Q.

[0023] In some embodiments, Z comprises a succinimide-alkanoyl moiety optionally having a hydrolyzed form of a succinimide ring as the succinamide moiety.

[0024] In some embodiments, Z optionally has a succinimide ring in hydrolyzed form as the succinamide moiety. [ka] and

[0025] wherein the wavy line marked with a double asterisk (**) indicates the site of covalent attachment of Q to the remainder of the formula;

[0026] The wavy line with a triple asterisk (***) indicates the point of covalent attachment of L to the sulfur atom;

[0027] R 17 is -C1-C 10 Alkylene, C1-C 10 Heteroalkylene, -C3-C8 carbocyclo, -O-(C1-C8 alkylene), -arylene, -C1-C 10 Alkylene-arylene-, -arylene-C1-C 10 Alkylene-, -C1-C 10Alkylene-(C3-C8 carbocyclo)-, -(C3-C8 carbocyclo)-C1-C 10 Alkylene, -C3-C8 heterocyclo, -C1-C 10 Alkylene-(C3-C8 heterocyclo)-, -(C3-C8 heterocyclo)-C1-C 10 Alkylene-, -C1-C 10 Alkylene-C(=O)-, C1-C 10 Heteroalkylene-C(=O)-, -C3-C8 carbocyclo-C(=O)-, -O-(C1-C8 alkylene)-C(=O)-, -arylene-C(=O)-, -C1-C 10 Alkylene-arylene-C(=O)-, -arylene-C1-C 10 Alkylene-C(=O)-, -C1-C 10 Alkylene-(C3-C8 carbocyclo)-C(=O)-, -(C3-C8 carbocyclo)-C1-C 10 Alkylene-C(=O)-, -C3-C8 heterocyclo-C(=O)-, -C1-C 10 Alkylene-(C3-C8 heterocyclo)-C(=O)-, -(C3-C8 heterocyclo)-C1-C 10 Alkylene-C(=O)-, -C1-C 10 Alkylene-NH-, -C1-C 10 Heteroalkylene-NH-, -C3-C8 carbocyclo-NH-, -O-(C1-C8 alkylene)-NH-, -arylene-NH-, -C1-C 10 Alkylene-arylene-NH-, -arylene-C1-C 10 Alkylene-NH-, -C1-C 10 Alkylene-(C3-C8 carbocyclo)-NH-, -(C3-C8 carbocyclo)-C1-C 10 Alkylene-NH-, -C3-C8 heterocyclo-NH-, -C1-C 10 Alkylene-(C3-C8 heterocyclo)-NH-, -(C3-C8 heterocyclo)-C1-C 10 Alkylene-NH-, -C1-C 10 Alkylene-S-, C1-C 10Heteroalkylene-S-, -C3-C8 carbocyclo-S-, -O-(C1-C8 alkylene)-S-, -arylene-S-, -C1-C 10 Alkylene-arylene-S-, -arylene-C1-C 10 Alkylene-S-, -C1-C 10 Alkylene-(C3-C8 carbocyclo)-S-, -(C3-C8 carbocyclo)-C1-C 10 Alkylene-S-, -C3-C8 heterocyclo-S-, -C1-C 10 Alkylene-(C3-C8 heterocyclo)-S- or -(C3-C8 heterocyclo)-C1-C 10 It is alkylene-S-.

[0028] In some embodiments, R 17 is -(CH2) 2-5 -C(=O)-.

[0029] In some embodiments, Z optionally has a succinimide ring in hydrolyzed form as the succinamide moiety. [ka] where the wavy line marked with a double asterisk (**) indicates the site of covalent attachment of Q to the remainder, and the wavy line marked with a triple asterisk (***) indicates the point of covalent attachment of L to the sulfur atom.

[0030] In some embodiments, Z is [ka] where the wavy line marked with a double asterisk (**) indicates the site of covalent attachment of Q to the remainder, and the wavy line marked with a triple asterisk (***) indicates the point of covalent attachment of L to the sulfur atom.

[0031] In some embodiments, Z is [ka] where the wavy line marked with a double asterisk (**) indicates the site of covalent attachment of Q to the remainder, and the wavy line marked with a triple asterisk (***) indicates the point of covalent attachment of L to the sulfur atom.

[0032] In some embodiments, Z is [ka] where the wavy line marked with a double asterisk (**) indicates the site of covalent attachment of Q to the remainder, and the wavy line marked with a triple asterisk (***) indicates the point of covalent attachment of L to the sulfur atom.

[0033] In some embodiments, A is a connector unit.

[0034] In some embodiments, A has the formula: [ka]

[0035] where the wavy line with double asterisk (**) indicates the site of covalent attachment to RL;

[0036] A wavy line with a single asterisk (*) indicates the point of covalent attachment to Z;

[0037] R 111are independently hydrogen, p-hydroxybenzyl, methyl, isopropyl, isobutyl, sec-butyl, -CH2OH, -CH(OH)CH3, -CH2CH2SCH3, -CH2CONH2, -CH2COOH, -CH2CH2CONH2, -CH2CH2COOH, -(CH2)3NHC(=NH)NH2, -(CH2)3NH2, -(CH2)3NHCOCH3, -(CH2)3NHCHO, -(CH2)4NHC(=NH)NH2, -(CH2)4NH2, -(CH2)4NHCOCH3, -(CH2)4NHCHO, -(CH2)3NHCONH2, -(CH2)4NHCONH2, -CH2CH2CH(OH)CH2NH2, 2-pyridylmethyl-, 3-pyridylmethyl-, 4-pyridylmethyl-, [ka] is selected from the group consisting of

[0038] Each R 100 is independently selected from the group consisting of hydrogen and —C1-C3 alkyl;

[0039] c is an independently selected integer from 1 to 10.

[0040] In some embodiments, A has the formula: [ka]

[0041] where the wavy line marked with a double asterisk (**) indicates the site of covalent attachment to RL and the wavy line marked with a single asterisk (*) indicates the point of covalent attachment to Z.

[0042] In some embodiments, -ZA- has the formula: [ka]

[0043] In the formula, the wavy line marked with a double asterisk (**) indicates the site of covalent attachment to RL, and the wavy line marked with a single asterisk (*) indicates the point of covalent attachment to L.

[0044] In some embodiments, -ZA-RL- has the formula: [ka]

[0045] where Su is the hexose form of a monosaccharide, O' represents the oxygen atom of the glycosidic bond that can be cleaved by a glycosidase, the wavy line with a double asterisk (**) indicates the site of covalent attachment to D, and the wavy line with a single asterisk (*) indicates the point of covalent attachment to L.

[0046] In some embodiments, -ZA-RL- has the formula: [ka]

[0047] where the wavy line marked with a double asterisk (**) indicates the site of covalent attachment to D, and the wavy line marked with a single asterisk (*) indicates the point of covalent attachment to L.

[0048] In some embodiments, -RL-D- has the formula: [ka]

[0049] In the formula, the wavy line with a single asterisk (*) indicates the point of covalent attachment to A.

[0050] In some embodiments, -A-RL-D has the formula: [ka]

[0051] In the formula, the wavy line with a single asterisk (*) indicates the point of covalent attachment to Z.

[0052] In some embodiments, S* is a PEG group.

[0053] In some embodiments, -QD- has the formula: [ka]

[0054] In the formula, the wavy line with a single asterisk (*) indicates the point of covalent attachment to L.

[0055] In some embodiments, -QD- has the formula: [ka]

[0056] In the formula, the wavy line with a single asterisk (*) indicates the point of covalent attachment to L.

[0057] In some embodiments, the Drug Unit is a Topoisomerase I inhibitor.

[0058] In some embodiments that may be combined with any of the embodiments described above or below, provided herein is an antibody-drug conjugate, or a salt thereof, comprising a drug unit to antibody (DAR) ratio of 1:10.

[0059] In some embodiments, the DAR is about 4 or about 8.

[0060] In some embodiments, p is an integer from about 1 to about 10.

[0061] In some embodiments, p is an integer of about 4 or about 8.

[0062] In some embodiments, the linker unit is attached to the antibody or antigen-binding fragment at a cysteine ​​amino acid residue. In some embodiments, the cysteine ​​is a naturally occurring cysteine. In some embodiments, the cysteine ​​is located in the hinge region of the antibody or antigen-binding fragment thereof. In some embodiments, the antibody or antigen-binding fragment thereof is cysteine ​​engineered.

[0063] In some aspects, provided herein is a pharmaceutical composition comprising an antibody-drug conjugate described herein or a salt thereof, and a pharmaceutically acceptable carrier.

[0064] In some aspects, provided herein are methods of treating cancer in an individual, comprising administering to the individual an antibody-drug conjugate or a salt thereof, or a pharmaceutical composition described herein.

[0065] In some aspects, provided herein is an antibody-drug conjugate or a salt thereof, or a pharmaceutical composition described herein for use in treating cancer.

[0066] In some embodiments, the cancer is a solid tumor.

[0067] In some embodiments, the cancer is selected from the group consisting of colorectal cancer, neuroendocrine cancer, gastric cancer, lung cancer, uterine cancer, cervical cancer, pancreatic cancer, esophageal cancer, ovarian cancer, thyroid cancer, bladder cancer, endometrium cancer, bladder cancer, endometrial cancer, breast cancer, liver cancer, prostate cancer, stomach cancer, bile duct cancer, and skin cancer.

[0068] In some embodiments, the cancer is selected from the group consisting of colorectal cancer, stomach cancer, gastric cancer, gastroesophageal junction cancer, lung cancer, uterine cancer, cervical cancer, pancreatic cancer, esophageal cancer, ovarian cancer, thyroid cancer, bladder cancer, endometrium cancer, bladder cancer, neuroendocrine cancer, endometrial cancer, breast cancer, liver cancer, prostate cancer, and bile duct cancer, and skin cancer. In some embodiments, the lung cancer comprises non-small cell lung cancer (NSCLC), non-squamous NSCLC (nsq-NSCLC), squamous NSCLC (sq-NSCLC), or small cell lung cancer (SCLC), or any combination thereof. In some embodiments, the pancreatic cancer comprises pancreatic ductal adenocarcinoma (PDAC).

[0069] In some embodiments, the cancer is selected from the group consisting of colon cancer, lung cancer, gastric cancer, and pancreatic cancer.

[0070] In some embodiments, the cancer is selected from the group consisting of colorectal cancer, lung cancer, gastric cancer, gastroesophageal junction cancer, neuroendocrine cancer, and pancreatic cancer.

[0071] In some embodiments, the cancer is colorectal cancer, NSCLC, SCLC, gastric cancer, gastroesophageal junction cancer, and pancreatic ductal adenocarcinoma.

[0072] In some embodiments, the cancer is primary, metastatic, or carcinomatous.

[0073] In some embodiments, the tumor expresses high levels of CEACAM5. In some embodiments, at least 50% of the tumor cells in a sample of the tumor score with an intensity greater than 2+ as measured by immunohistochemistry.

[0074] In some embodiments, the tumor expresses moderate levels of CEACAM5. In some embodiments, at least 1% and less than 50% of the tumor cells in a tumor sample are scored with an intensity of ≧2+ as measured by immunohistochemistry, or at least 50% of the tumor cells in a tumor sample are scored with an intensity of 1+ as measured by immunohistochemistry.

[0075] In some embodiments, the tumor expresses any level of CEACAM5. In some embodiments, reactivity to CEACAM5 is observed, but the CEACAM5 expression level is not considered to be moderate or high.

[0076] In some embodiments, the antibody-drug conjugate or salt thereof does not induce a significant level of toxicity in an individual.

[0077] In some embodiments, the antibody-drug conjugate or salt thereof causes a reduction in tumor volume following administration.

[0078] In some aspects, provided herein are kits comprising an antibody-drug conjugate or a salt thereof, or a pharmaceutical composition described herein. [Brief explanation of the drawings]

[0079] [Figure 1A] 1 shows the in vitro binding ability of ADC1 with a DAR of 8 to huFcRn expressed on the cell surface of HEK293 cells under pH 7.2 and pH 6.6, respectively. [Figure 1B] 1 shows the in vitro binding ability of ADC1 with a DAR of 8 to huFcRn expressed on the cell surface of HEK293 cells under pH 7.2 and pH 6.6, respectively. [Figure 2] 1 shows the evaluation of the antitumor activity (e.g., tumor volume reduction) of ADC1 against CRC patient-derived xenograft tumor CR-IGR-0002P in SCID female mice. The curves represent the median + or -MAD for each day in each group. The black arrows indicate the days of treatment (single administration). [Figure 3] 1 shows the evaluation of the antitumor activity (e.g., tumor volume reduction) of ADC1 against CRC patient-derived xenograft tumor CR-IGR-0007P in SCID female mice. The curves represent the median + or -MAD for each day in each group. The black arrows indicate the days of treatment (single administration). [Figure 4] 1 shows the evaluation of the antitumor activity (e.g., tumor volume reduction) of ADC1 against CRC patient-derived xenograft tumor CR-IGR-0048M in SCID female mice. The curves represent the median + or -MAD for each day in each group. The black arrows indicate the days of treatment (single administration). [Figure 5] 1 shows the evaluation of the antitumor activity (e.g., tumor volume reduction) of ADC1 against CRC patient-derived xenograft tumor CR-IC-0016M in SCID female mice. The curves represent the median + or -MAD for each day in each group. The black arrows indicate the days of treatment (single administration). [Figure 6] 1 shows the evaluation of the antitumor activity (e.g., tumor volume reduction) of ADC1 against the lung patient-derived xenograft tumor LUN-NIC-0014 in SCID female mice. The curves represent the median + or -MAD for each day in each group. The black arrows indicate the days of treatment (single administration). [Figure 7] 1 shows the evaluation of the antitumor activity (e.g., tumor volume reduction) of ADC1 against the lung patient-derived xenograft tumor LUN-NIC-0084 in SCID female mice. The curves represent the median + or -MAD for each day in each group. The black arrows indicate the days of treatment (single administration). [Figure 8] 1 shows the evaluation of the antitumor activity (e.g., tumor volume reduction) of ADC1 against the lung patient-derived xenograft tumor LUN-NIC-0004 in SCID female mice. The curves represent the median + or -MAD for each day in each group. The black arrows indicate the days of treatment (single administration). [Figure 9] 1 shows the evaluation of the antitumor activity (e.g., tumor volume reduction) of ADC1 against the lung patient-derived xenograft tumor LUN-NIC-0008 in SCID female mice. The curves represent the median + or -MAD for each day in each group. The black arrows indicate the days of treatment (single administration). [Figure 10] 1 shows the evaluation of the antitumor activity (e.g., tumor volume reduction) of ADC1 against the gastric patient-derived xenograft tumor STO-IND-0006 in SCID female mice. The curves represent the median + or -MAD for each day in each group. The black arrows indicate the days of treatment (single administration). [Figure 11] 1 shows the evaluation of the antitumor activity (e.g., tumor volume reduction) of ADC1 against the gastric patient-derived xenograft tumor SA-STO-0014 in SCID female mice. The curves represent the median + or -MAD for each day in each group. The black arrows indicate the days of treatment (single administration). [Figure 12] 1 shows the evaluation of the antitumor activity (e.g., tumor volume reduction) of ADC1 against the gastric patient-derived xenograft tumor STO-IND-0007 in SCID female mice. The curves represent the median + or -MAD for each day in each group. The black arrows indicate the days of treatment (single administration). [Figure 13] Figure 1 shows the evaluation of the antitumor activity of ADC1 in a panel of 16 colon patient-derived xenograft models under single-mouse study—best relative tumor regression. The best relative tumor regression or best response to ADC1 is depicted below the waterfall plot. PDX models are selected by increasing their sensitivity to ADC1. [Figure 14] Figure 1 shows the in vivo efficacy evaluation of ADC1 in a panel of 19 gastric patient-derived xenograft models under a single-mouse study format. The best relative tumor shrinkage or best response to ADC1 is depicted below the waterfall plot. PDX models are selected by increasing their sensitivity to ADC1. [Figure 15] Figure 1 shows the in vivo efficacy evaluation of ADC1 in a panel of 31 lung patient-derived xenograft models under a single-mouse study format. The best relative tumor shrinkage or best response to ADC1 is depicted below the waterfall plot. PDX models are selected by increasing their sensitivity to ADC1. [Figure 16]Figure 1 shows the in vivo efficacy evaluation of ADC1 against the pancreatic patient-derived xenograft tumor IM-PAN-011 (metastatic PDAC) subcutaneously implanted in female SCID mice. Tumor volume evolution by treatment group. Curves represent the median + or - MAD for each day in each group. Black arrows indicate days of treatment (single administration). [Figure 17] Figure 1 shows the in vivo efficacy evaluation of ADC1 against the pancreatic patient-derived xenograft tumor SA-PAN-0077 (PDAC) subcutaneously implanted in female SCID mice. Tumor volume evolution by treatment group. Curves represent the median + or - MAD for each day in each group. Black arrows indicate days of treatment (single administration). [Figure 18] Figure 1 shows the in vivo efficacy evaluation of ADC1 against pancreatic patient-derived xenograft tumor IM-PAN-0006 (PDAC) subcutaneously implanted in female SCID mice. Tumor volume evolution by treatment group. Curves represent the median + or - MAD for each day in each group. Black arrows indicate days of treatment (single administration). [Figure 19] Figure 1 shows the in vivo efficacy evaluation of ADC1 against pancreatic patient-derived xenograft tumor IM-PAN-003 (PDAC) subcutaneously implanted in female SCID mice. Tumor volume evolution by treatment group. Curves represent the median + or - MAD for each day in each group. Black arrows indicate days of treatment (single administration). [Figure 20] 1 shows the total CD PK and DAR profile in plasma after a single intravenous administration of ADC1 with a DAR of 8 at 3 mg / kg to SCID mice. DETAILED DESCRIPTION OF THE INVENTION

[0080] In some embodiments, provided herein are antibody-drug conjugates (ADCs) comprising an antibody or antigen-binding fragment thereof that binds to CEACAM5 conjugated to a topoisomerase I inhibitor. In some embodiments, the ADC comprises an antibody or antigen-binding fragment thereof that binds to CEACAM5 conjugated to camptothecin. In some embodiments, the ADC advantageously binds to CEACAM5 at nanomolar concentrations and kills distinct CEACAM5-positive CRC cells at subnanomolar concentrations with low toxicity to CEACAM5-negative cells. Without wishing to be bound by theory, the cytotoxicity of the ADCs provided herein may be mediated by their internalization, processing, and cytotoxic release in CEACAM5-expressing tumor cells. Additionally or alternatively, cytotoxicity may be mediated by a bystander effect, which allows the cytotoxic agent to diffuse to neighboring CEACAM5-negative tumor cells, resulting in cell death. In some embodiments, the bystander effect allows the payload to diffuse from antigen-positive tumor cells to neighboring antigen-negative tumor cells, resulting in cell death. In some embodiments, the ADCs are well-tolerated with significant anti-tumor activity, particularly in colorectal cancer (CRC) cell models. In some embodiments, the ADCs are well-tolerated with significant anti-tumor activity, particularly in colorectal cancer (CRC), gastric cancer (GC), gastroesophageal junction cancer (GEJ), lung cancer, and pancreatic cancer tumor models. In some embodiments, the topoisomerase I payload has been optimized for potency, reduced P-gp efflux, and enhanced bystander activity.

[0081] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0082] The specifications for chemical cloning are based on Sambrook et al.,Molecular Cloning:A Laboratory Manual 4th edition(2012)Cold Spring Harbor Laboratory Press,Cold Spring Harbor NY、Current Protocols In Molecular Biology(FMAusubel,et al.eds.,(2003))、The Series METHODS IN ENZYMOLOGY(Academic Press,Inc.)、PCR 2:A Practical Approach(MJMacPherson,BDHames and GRTaylor). eds.(1995))、Greenfield,ed.(2013)Antibodies,A Laboratory Manual,2nd edition,Cold Spring Harbor Laboratory Press、Oligonucleotide Synthesis(MJGait,ed.,1984)、Methods in Molecular Biology,Humana Press、Cell Biology:A Laboratory Notebook(JECellis,ed.,1998)Academic Press、Animal Cell Culture(RIFreshney),ed.,1987)、Introduction to Cell and Tissue Culture(JPMather and PERoberts,1998)Plenum Press、Cell and Tissue Culture Laboratory Procedures(A.Doyle,JBGriffiths,and DGNewell,eds.,1993-8)J.Wiley and Sons、Handbook of Experimental Immunology(DMWeir and CCBlackwell,eds.)、Gene Transfer Vectors for Mammalian Cells(JMMiller and MPCalos,eds.)., 1987), PCR: The Polymerase chain Reaction, (Mullis et al., eds., 1994), Current Protocols in Immunology (JEColigan et al., eds., 1991), Short Protocols in Molecular Biology (Wiley and Sons, 1999), Immunobiology (CA Janeway and P. Travers, 1997), Antibodies (P. Finch, 1997), Antibodies: A Practical Approach (D. Catty., ed., IRL Press, 1988-1989), Monoclonal Antibodies: A Practical Approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000), Using Antibodies: A Laboratory Manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999), The Antibodies (M. Zanetti and These methods are commonly understood by those skilled in the art and are widely practiced using conventional methodologies, such as those widely used methodologies described in J.D. Capra, eds., Harwood Academic Publishers, 1995), Cancer: Principles and Practice of Oncology (VT DeVita et al., eds., J.B. Lippincott Company, 1993), and updates thereof. Each of the foregoing references in this paragraph is incorporated herein by reference in its entirety.

[0083] I. Definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. For example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press, The Dictionary of Cell and Molecular Biology, 5th ed., 2013, Academic Press, and the Oxford Dictionary of Biochemistry And Molecular Biology, 2nd ed., 2006, Oxford University Press provide those skilled in the art with a general dictionary of many of the terms used in this disclosure.

[0084] Unless otherwise required by context or otherwise expressly indicated, singular terms shall include pluralities and plural terms shall include the singular.

[0085] It will be understood that aspects and embodiments of the invention described herein include "comprising," "consisting of," and / or "consisting essentially of" aspects and embodiments.

[0086] As used herein, the singular forms "a," "an," and "the" should be understood to refer to "one or more" of any list or listed members, unless otherwise indicated.

[0087] As used herein, the term "and / or" should be interpreted as a specific disclosure of each of two particular features or components, with or without the other. Thus, when used in a phrase such as "A and / or B" herein, the term "and / or" is intended to include "A and B," "A or B," "A" (alone), and "B" (alone). Similarly, when used in a phrase such as "A, B, and / or C," the term "and / or" is intended to encompass each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0088] The term "about" refers to a value or composition that is within an acceptable error range for a particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined, i.e., the limitations of the measurement system. As will be understood by one of ordinary skill in the art, references herein to "about" a value or parameter include (and describe) embodiments that are directed to the value or parameter itself. For example, a description that references "about X" includes a description of "X."

[0089] When trade names are used herein, unless the context dictates otherwise, reference to the trade name also refers to the product formulation, the generic drug, and the active pharmaceutical ingredient of the trade name product.

[0090] The terms CEACAM5 carcinoembryonic antigen-related cell adhesion molecule 5 and CD66e are used interchangeably herein and, unless otherwise specified, include any naturally occurring variants (e.g., splice variants, allelic variants), isoforms, and vertebrate species homologs of human CEACAM5. The terms encompass "full-length," unprocessed CEACAM5, as well as any form of CEACAM5 resulting from processing within cells. The amino acid sequence of an exemplary human CEACAM5 is provided in the GenBank database under accession number AAA51967.1. The amino acid sequence of one specific example of a mature human CEACAM5 protein is set forth in SEQ ID NO: 11.

[0091] An "antibody" may be a natural or conventional antibody consisting of two heavy chains linked to each other by disulfide bonds, and each heavy chain linked to a light chain by a disulfide bond. There are two types of light chains: lambda (I) and kappa (K). There are five major heavy chain classes (or isotypes) that determine the functional activity of antibody molecules: IgM, IgD, IgG, IgA, and IgE. Each chain contains distinct sequence domains. A light chain contains two domains or regions: a variable domain (VL) and a constant domain (CL). A heavy chain contains four domains: a variable domain (VH) and three constant domains (CH1, CH2, and CH3, collectively referred to as CH). The variable regions of both the light chain (VL) and the heavy chain (VH) determine binding recognition and specificity to the antigen. The constant region domains of the light chain (CL) and heavy chain (CH) confer important biological properties, such as antibody chain assembly, secretion, transplacental mobility, complement fixation, and Fc receptor (FcR) binding. The Fv fragment is the N-terminal portion of an immunoglobulin Fab fragment and consists of the variable portions of one light chain and one heavy chain. Antibody specificity resides in the structural complementarity between the antibody-combining site and an antigenic determinant. The antibody-combining site is primarily composed of residues from hypervariable or complementarity-determining regions (CDRs). In some cases, residues from non-hypervariable or framework regions (FRs) influence the overall domain structure and thus the binding site. Thus, complementarity-determining regions or CDRs refer to amino acid sequences that together define the binding affinity and specificity of the native Fv region of a native immunoglobulin binding site. The light and heavy chains of an immunoglobulin each have three CDRs: CDR1-L, CDR2-L, CDR3-L, and CDR1-H, CDR2-H, and CDR3-H. Thus, a conventional antibody antigen-binding site contains six CDRs, including a set of CDRs from each of a heavy chain V region and a light chain V region.

[0092] "Framework region" (FR) refers to the amino acid sequences located between the CDRs, i.e., those portions of the immunoglobulin light and heavy chain variable regions that are relatively conserved among different immunoglobulins in a single species. The light and heavy chains of an immunoglobulin each have four FRs, designated FR1-L, FR2-L, FR3-L, FR4-L, and FR1-H, FR2-H, FR3-H, FR4-H, respectively.

[0093] As used herein, a "human framework region" is a framework region that is substantially identical (about 85% or more, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) to the framework region of a naturally occurring human antibody. In the context of the present invention, the definitions of CDR / FR in an immunoglobulin light chain or heavy chain are determined in accordance with the IMGT definitions (Lefranc et al. Dev. Comp. Immunol., 2003, 27(1):55-77, www.imgt.org).

[0094] As used herein, the term "antibody" refers to conventional antibodies and fragments thereof, as well as single domain antibodies and fragments thereof, in particular the variable heavy chains of single domain antibodies, as well as chimeric, humanized, bispecific or multispecific antibodies.

[0095] As used herein, the terms "monoclonal antibody" or "mAb" refer to an antibody molecule of a single amino acid sequence directed against a particular antigen and should not be construed as requiring production of the antibody by any particular method. A monoclonal antibody may be produced by a single clone of B cells or hybridoma, but may also be recombinant, i.e., produced by protein engineering.

[0096] The term "chimeric antibody" in its broadest sense refers to an engineered antibody that contains one or more regions from one antibody and one or more regions from one or more other antibodies. In embodiments, a chimeric antibody comprises the VH and VL domains of an antibody derived from a non-human animal, associated with the CH and CL domains of another antibody, in embodiments, a human antibody. The non-human animal can be any animal, such as a mouse, rat, hamster, or rabbit. Chimeric antibodies can also represent multispecific antibodies, having specificity for at least two different antigens.

[0097] The term "humanized antibody" refers to an antibody that is wholly or partially of non-human origin and that has been modified, for example, by replacing certain amino acids in the framework regions of the VH and VL domains, to avoid or minimize an immune response in humans. The constant domains of a humanized antibody are most often human CH and CL domains.

[0098] A "fragment" of a (traditional) antibody comprises a portion of an intact antibody, in particular the antigen-binding or variable region of the intact antibody. Examples of antibody fragments include Fv, Fab, F(ab')2, Fab', dsFv, (dsFv)2, scFv, sc(Fv)2, diabodies, diabodies, diabodies, and multispecific antibodies formed from antibody fragments. A fragment of a traditional antibody may also be a heavy chain antibody or a single domain antibody such as a VHH.

[0099] The term "Fab" refers to an antibody fragment having a molecular weight of about 50,000 and antigen-binding activity, in which approximately the N-terminal half of the heavy chain and the entire light chain are linked together via disulfide bonds, typically obtained by treating IgG with the protease papain.

[0100] The term "F(ab')2" refers to an antibody fragment with a molecular weight of approximately 100,000 and antigen-binding activity, which is slightly larger than two identical Fab fragments linked via disulfide bonds in the hinge region. It is usually obtained between fragments by treating IgG with the protease, pepsin.

[0101] The term "Fab" refers to an antibody fragment having a molecular weight of approximately 50,000 and antigen-binding activity, which can be obtained by cleaving the disulfide bond in the hinge region of F(ab')2.

[0102] Single-chain Fv ("scFv") polypeptides are typically covalent VH::VL heterodimers expressed from gene fusions containing genes encoding VH and VL linked by a peptide-encoding linker. Human scFv fragments of the present invention comprise CDRs held in the appropriate conformation, for example, by using genetic recombination techniques. Bivalent and multivalent antibody fragments can form spontaneously by association of monovalent scFvs or can be generated by coupling monovalent scFvs with a peptide linker, such as a bivalent sc(Fv)2. A "dsFv" is a VH::VL heterodimer stabilized by a disulfide bond. "(dsFv)2" refers to two dsFvs coupled by a peptide linker.

[0103] The term "bispecific antibody" or "BsAb" refers to an antibody that combines the antigen-binding sites of two antibodies in a single molecule. Thus, BsAbs can simultaneously bind to two different antigens. Genetic engineering has been used with increasing frequency to design, modify, and generate antibodies or antibody derivatives with desired sets of binding properties and effector functions, as described, for example, in EP 2050764 A1.

[0104] The term "multispecific antibody" refers to an antibody that combines the antigen-binding sites of more than one antibody in a single molecule.

[0105] The term "diabody" refers to a small antibody fragment with two antigen-binding sites, which comprises a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) in the same polypeptide chain (VH-VL). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites.

[0106] The term "hybridoma" refers to cells obtained by subjecting B cells, prepared by immunizing a non-human mammal with an antigen, to cell fusion with myeloma cells, such as those derived from a mouse, that produce the desired monoclonal antibody with antigen specificity.

[0107] "Purified" and "isolated," when referring to a polypeptide (i.e., an antibody of the invention) or nucleotide sequence, mean that the indicated molecule is present in the substantial absence of other biological macromolecules of the same type. As used herein, the term "purified" means that at least 75%, 85%, 95%, 96%, 97%, or 98%, by weight, of the same type of biological macromolecules are present. An "isolated" nucleic acid molecule encoding a particular polypeptide refers to a nucleic acid molecule that is substantially free of other nucleic acid molecules that do not encode the polypeptide of interest, although the molecule may contain some additional bases or moieties that do not adversely affect the essential characteristics of the composition.

[0108] The terms "polypeptide" and "protein" are used interchangeably to refer to polymers of amino acid residues and are not limited to a minimum length. Such polymers of amino acid residues can contain natural or non-natural amino acid residues, including, but not limited to, dimers, trimers, peptides, oligopeptides, and multimers of amino acid residues. Both full-length proteins and fragments thereof are encompassed by the definition. The term also includes post-expression modifications of the polypeptide, such as glycosylation, sialylation, acetylation, phosphorylation, and the like. The term "polypeptide" also refers to proteins containing modifications to the native sequence, such as deletions, additions, and substitutions (generally conservative in nature), so long as the protein maintains the desired activity. The terms "polypeptide" and "protein" encompass CEACAM5 antigen-binding proteins, including antibodies, antibody fragments, or sequences having deletions, additions, and / or substitutions from one or more amino acids of the antigen-binding protein.

[0109] A "native sequence" or "naturally occurring" polypeptide includes a polypeptide having the same amino acid sequence as a polypeptide found in nature. Thus, a native sequence polypeptide can have the amino acid sequence of a naturally occurring polypeptide from any mammal. Such native sequence polypeptides can be isolated from nature or produced by recombinant or synthetic means. The term "native sequence" polypeptide specifically encompasses naturally occurring truncated or secreted forms of a polypeptide (e.g., extracellular domain sequences), naturally occurring variant forms (e.g., alternatively spliced ​​forms), and naturally occurring allelic variants of a polypeptide.

[0110] A polypeptide "variant" means a biologically active polypeptide (e.g., an antigen binding protein or antibody) having at least about 70%, 80%, or 90% amino acid sequence identity with a native or reference sequence polypeptide, after aligning the sequences and introducing gaps as necessary to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Such variants include, for example, polypeptides in which one or more amino acid residues are added or deleted from the N- or C-terminus of the polypeptide. In some embodiments, a variant has at least about 80% amino acid sequence identity. In some embodiments, a variant has at least about 90% amino acid sequence identity. In some embodiments, a variant has at least about 95% amino acid sequence identity with a native sequence polypeptide.

[0111] As used herein, "percent (%) amino acid sequence identity" and "homology" with respect to peptide, polypeptide, or antigen-binding protein (e.g., antibody) sequences are defined as the percentage of amino acid residues in a candidate sequence that are identical with amino acid residues in a particular peptide or polypeptide sequence, after aligning the sequences and introducing gaps as necessary to achieve the maximum percent sequence identity, without considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in a variety of ways that are within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or MEGALIGN™ (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. For example, the % sequence identity of a given amino acid sequence A to a given amino acid sequence B, with a given amino acid sequence B, or to a given amino acid sequence B (which can be rephrased as a given amino acid sequence A having or comprising a particular % sequence identity to a given amino acid sequence B, with a given amino acid sequence B, or to a given amino acid sequence B) is calculated as follows: 100 times the fraction X / Y where X is the number of amino acid residues scored as identical matches by the program's alignment of sequences A and B, and Y is the total number of amino acid residues in B. Unless otherwise specified, all % amino acid sequence identity values ​​used herein are calculated according to this formula using the ALIGN-2 computer program. It will be understood that if the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % sequence identity of A with B will not equal the % sequence identity of B with A.

[0112] The term "leader sequence" refers to a sequence of amino acid residues located at the N-terminus of a polypeptide that facilitates secretion of the polypeptide from mammalian cells. Leader sequences may be cleaved upon export of the polypeptide from mammalian cells to form the mature protein. Leader sequences may be natural or synthetic, and they may be heterologous or homologous to the protein to which they bind.

[0113] The term "immunoglobulin" refers to a class of structurally related glycoproteins consisting of two pairs of polypeptide chains, one pair of light (L) low-molecular-weight chains, and one pair of heavy (H) chains, all four interconnected by disulfide bonds. The structure of immunoglobulins is well characterized. See, e.g., Fundamental Immunology (Paul, W., ed., 7th ed. Raven Press, NY (2013)). Briefly, each heavy chain typically comprises a heavy chain variable region (referred to herein as V H or VH) and a heavy chain constant region (C H The heavy chain constant region is typically composed of three domains: C, ... H 1. C H 2, and C H 3. Heavy chains are generally interconnected via disulfide bonds in the so-called "hinge region." Each light chain typically contains a light chain variable region (herein referred to as V L or VL) and a light chain constant region (C L The light chain constant region typically consists of one domain, C LThe CL may be of the κ (kappa) or λ (lambda) isotype. The terms "constant domain" and "constant region" are used interchangeably herein. Immunoglobulins may be derived from any of the commonly known isotypes, including, but not limited to, IgA, secretory IgA, IgG, and IgM. IgG subclasses are also well known to those skilled in the art and include, but are not limited to, human IgG1, IgG2, IgG3, and IgG4. "Isotype" refers to the antibody class or subclass (e.g., IgM or IgG1) encoded by the heavy chain constant region genes. [Table 1]

[0114] Unless otherwise specified, the terms "CDR" and "complementarity determining region" of a given antibody or region thereof, e.g., a variable region, and the individual CDRs of an antibody or region thereof (e.g., "CDR-H1, CDR-H2"), should be understood to encompass complementarity determining regions defined by any of the known schemes described hereinabove. In some cases, a scheme for identifying a particular CDR or CDRs is specified, such as CDRs defined by the IMGT, Kabat, AbM, Chothia, or Contact methods. In other instances, the specific amino acid sequences of the CDRs are provided.

[0115] Thus, in some embodiments, the antigen binding protein comprises CDRs and / or HVRs defined by the IMGT system. In other embodiments, the antigen binding protein comprises CDRs or HVRs defined by the Kabat system. In still other embodiments, the antigen binding protein comprises CDRs or HVRs defined by the AbM system. In further embodiments, the antigen binding protein comprises CDRs or HVRs defined by the Chothia system. In still other embodiments, the antigen binding protein comprises CDRs or HVRs defined by the IMGT system.

[0116] The term "variable region" or "variable domain" refers to the domain of the heavy or light chain of an antigen-binding protein (e.g., an antibody) that is involved in binding of the antigen-binding protein (e.g., an antibody) to an antigen. The variable regions or domains of the heavy and light chains (VH and VL, respectively) of an antigen-binding protein such as an antibody can be further subdivided into regions of hypervariability (or hypervariability that may be hypervariable in the sequence and / or configuration of structurally defined loops) such as complementarity-determining regions (CDRs) interspersed with regions that are more conserved, called framework regions (FRs).

[0117] As used herein, the term "heavy chain constant region" refers to a region comprising at least three heavy chain constant domains: H 1. C H 2, and C H The heavy chain constant region refers to a region containing 3. Non-limiting exemplary heavy chain constant regions include γ, δ, and α. Non-limiting exemplary heavy chain constant regions also include ε and μ. Each heavy constant region corresponds to an antibody isotype. For example, an antibody containing a γ constant region is an IgG antibody, an antibody containing a δ constant region is an IgD antibody, and an antibody containing an α constant region is an IgA antibody. Furthermore, an antibody containing a μ constant region is an IgM antibody, and an antibody containing an ε constant region is an IgE antibody. A particular isotype can be further subdivided into subclasses. For example, IgG antibodies include, but are not limited to, IgG1 (containing a gamma 1 constant region), IgG2 (containing a gamma 2 constant region), IgG3 (containing a gamma 3 constant region), and IgG4 (containing a gamma 4 constant region) antibodies; IgA antibodies include, but are not limited to, IgA1 (containing an alpha 1 constant region) and IgA2 (containing an alpha 2 constant region) antibodies; and IgM antibodies include, but are not limited to, IgM1 and IgM2.

[0118] As used herein, the term "heavy chain" (HC) refers to a polypeptide comprising at least a heavy chain variable region, with or without a leader sequence. In some embodiments, a heavy chain also comprises at least a portion of a heavy chain constant region. As used herein, the term "full-length heavy chain" refers to a polypeptide comprising a heavy chain variable region and a heavy chain constant region, with or without a leader sequence.

[0119] As used herein, the term "light chain constant region" refers to a light chain constant domain, C L Non-limiting exemplary light chain constant regions include lambda and kappa.

[0120] As used herein, the term "light chain" (LC) refers to a polypeptide comprising at least a light chain variable region, with or without a leader sequence. In some embodiments, a light chain also comprises at least a portion of a light chain constant region. As used herein, the term "full-length light chain" refers to a polypeptide comprising a light chain variable region and a light chain constant region, with or without a leader sequence.

[0121] The "EU numbering system" or "EU index" is generally used when referring to residues in immunoglobulin heavy chain constant regions (e.g., the EU index as reported in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991). "EU index as in Kabat" refers to the residue numbering of the human IgG1 EU antibody. Unless otherwise specified herein, references to residue numbers in the constant domain of an antibody refer to residue numbering according to the EU numbering system.

[0122] The term "derivative" refers to a molecule (e.g., an antigen binding protein such as an antibody or fragment thereof) that contains a chemical modification other than an amino acid (or nucleic acid) insertion, deletion, or substitution. In certain embodiments, a derivative includes a covalent modification, including, but not limited to, chemical conjugation with a polymer, lipid, or other organic or inorganic moiety. In certain embodiments, a derivative of a particular antigen binding protein may have a longer circulating half-life than the antigen binding protein that is not chemically modified. In certain embodiments, a derivative may have improved targeting ability to a desired cell, tissue, and / or organ. In some embodiments, a derivative of an antigen binding protein is covalently modified to include one or more polymers, including, but not limited to, monomethoxy-polyethylene glycol, dextran, cellulose, or other carbohydrate-based polymers, poly-(N-vinylpyrrolidone)-polyethylene glycol, propylene glycol homopolymer, polypropylene oxide / ethylene oxide copolymer, polyoxyethylated polyols (e.g., glycerol) and polyvinyl alcohol, and mixtures of such polymers. See, for example, U.S. Patent Nos. 4,640,835, 4,496,689, 4,301,144, 4,670,417, 4,791,192, and 4,179,337.

[0123] As used herein, the term "epitope" refers to a site on an antigen (e.g., CEACAM5) that targets an antigen-binding protein (e.g., an antibody or fragment thereof) to which that antigen binds. Epitopes often consist of chemically active surface groupings of molecules, such as amino acids, polypeptides, sugar side chains, phosphoryl or sulfonyl groups, and have specific three-dimensional structural characteristics as well as specific charge characteristics. Epitopes can be formed from both adjacent amino acids of an antigen juxtaposed by tertiary folding or non-adjacent amino acids. Epitopes formed from adjacent residues are typically retained upon exposure to denaturing solvents, while epitopes formed by tertiary folding are typically lost upon treatment with denaturing solvents. In certain embodiments, an epitope can comprise, but is not limited to, at least 3, at least 4, at least 5, at least 6, or at least 7 amino acids in a unique spatial configuration. In some embodiments, an epitope refers to 3-5, 4-6, or 8-10 amino acids in a unique spatial conformation. In further embodiments, the epitope is less than 20 amino acids, less than 15 amino acids, or less than 12 amino acids, less than 10 amino acids, or less than 8 amino acids in length. An epitope can include amino acid residues directly involved in binding (also referred to as the immunodominant components of the epitope) and other amino acid residues not directly involved in binding, including amino acid residues that are effectively blocked or covered by the antigen-binding molecule (i.e., amino acids within the footprint of the antigen-binding molecule). Methods for determining the spatial conformation of an epitope include, for example, X-ray crystallography, two-dimensional nuclear magnetic resonance, and HDX-MS (see, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, GE Morris, Ed. (1996)). Once the desired epitope of an antigen is determined, an antigen-binding protein (e.g., an antibody or fragment thereof) to the epitope can be generated using established techniques. The resulting antigen binding proteins can then be screened in a competition assay to identify antigen binding proteins that bind to the same or overlapping epitopes.A method for binning antibodies based on cross-competition studies is described in WO03 / 48731.

[0124] A "non-linear epitope" or "conformational epitope" comprises non-contiguous polypeptides, amino acids, and / or carbohydrates within an antigenic protein to which an epitope-specific antibody binds.

[0125] A "linear epitope" comprises a contiguous stretch of polypeptides, amino acids, and / or carbohydrates within an antigenic protein to which an epitope-specific antigen-binding protein (e.g., an antibody or fragment thereof) binds.

[0126] A "paratope" or "antigen-binding site" is the site on an antigen-binding protein (e.g., an antibody or fragment thereof) that binds to an epitope, and typically includes amino acids that are adjacent to the epitope when the antibody is bound (see, e.g., Sela-Culang et al., 2013, Front Immunol. 4:302).

[0127] "Affinity" refers to the strength of the sum total of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). The affinity of a molecule X for its partner Y is generally determined by the dissociation constant (K d Affinity can be measured by common methods known in the art, including those described herein.

[0128] As used herein, the terms "specifically bind," "binding," or simply "bind," or other related terms in the context of binding of an antigen-binding protein to its target antigen, mean that the antigen-binding protein exhibits essentially background binding to non-target molecules. However, an antigen-binding protein (e.g., CEACAM5) that specifically binds to a target antigen may cross-react with CEACAM5 proteins from different species. Typically, CEACAM5 antigen-binding proteins exhibit a dissociation constant (K) as measured via surface plasma resonance (SPR) technology (e.g., BIACore, GE-Healthcare Uppsala, Sweden) using an antibody as the ligand and an antigen as the analyte. D ) is 10 -7 M or less, for example, about 10- 8 M or less, e.g., about 10 -9 M or less, about 10 -10 M or less or 10 -11 It specifically binds to human CEACAM5 when its molecular weight is less than M.

[0129] The term "KD" (M), as used herein, refers to the dissociation equilibrium constant of a particular antigen-binding protein-antigen interaction (e.g., an antibody-antigen interaction). Affinity and KD, as used herein, refer to the dissociation equilibrium constant of a particular antigen-binding protein-antigen interaction (e.g., an antibody-antigen interaction). D is an inverse correlation, such that higher affinity is intended to refer to a lower KD and lower affinity is intended to refer to a higher KD.

[0130] An "antibody-drug conjugate" or simply "ADC" refers to an antibody conjugated to a cytotoxic agent, such as a topoisomerase I inhibitor. The antibody-drug conjugate typically binds to a target antigen (e.g., CEACAM5) on the cell surface, followed by internalization of the antibody-drug conjugate into the cell and release of the drug.

[0131] "Cytotoxic effect" refers to the depletion, elimination, and / or killing of target cells.

[0132] A "cytotoxic agent" refers to an agent that has a cytotoxic effect on cells.

[0133] "Cytostatic activity" refers to the inhibition of cell proliferation.

[0134] A "cytostatic agent" refers to an agent that has a cytostatic effect on cells, thereby inhibiting the growth and / or proliferation of specific subsets of cells. The cytostatic agent may be conjugated to an antibody or administered in combination with an antibody.

[0135] "Fc receptor" or "FcR" describes a receptor that binds to the Fc region of an antibody. In some embodiments, the FcγR is a native human FcR. In some embodiments, the FcR binds IgG antibodies (gamma receptors) and includes receptors of the FcγRI, FcγRII, and FcγRIII subclasses, including allelic variants and alternatively spliced ​​forms of these receptors. FcγRII receptors include FcγRIIA (an "activating receptor") and FcγRIIB (an "inhibiting receptor"), which have similar amino acid sequences that differ primarily in their cytoplasmic domains. Activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) within its cytoplasmic domain. Inhibiting receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) within its cytoplasmic domain (see, e.g., Daeron, Annu. Rev. Immunol. 15:203-234 (1997)). FcRs are reviewed, for example, in Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991), Capel et al., Immunomethods 4:25-34 (1994), and de Haas et al., J. Lab. Clin. Med. 126:330-41 (1995). Other FcRs, including those identified in the future, are encompassed by the term "FcR" herein. The terms "Fc receptor" or "FcR" also include FcRn, a neonatal receptor involved in the transfer of maternal IgG to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)) and the regulation of immunoglobulin homeostasis. Methods for measuring binding to FcRn are known (see, for example, Ghetie and Ward, Immunol. Today 18(12):592-598(1997); Ghetie et al., Nature Biotechnology, 15(7):637-640(1997); Hinton et al., J. Biol. Chem. 279(8):6213-6216(2004); WO2004 / 92219 (Hinton et al.)).

[0136] "Effector function" refers to biological activities attributable to the Fc region of an antibody, which vary depending on the antibody isotype. Examples of antibody effector functions include Clq binding and complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), down-regulation of cell surface receptors (e.g., B cell receptors), and B cell activation. Such functions can be triggered, for example, by binding of an Fc effector domain to an Fc receptor on a phagocytic or lytic immune cell, or by binding of an Fc effector domain to a component of the complement system. Typically, the effects mediated by Fc-binding cells or complement components result in the inhibition and / or depletion of CD33 target cells. The Fc region of an antibody can recruit Fc receptor (FcR)-expressing cells and juxtapose them to antibody-coated target cells. Cells expressing surface FcRs for IgG, including FcγRIII (CD16), FcγRII (CD32), and FcγRIII (CD64), can act as effector cells for the destruction of IgG-coated cells. Such effector cells include monocytes, macrophages, natural killer (NK) cells, neutrophils, and eosinophils. Engagement of FcγRs by IgG activates antibody-dependent cellular cytotoxicity (ADCC) or antibody-dependent cellular phagocytosis (ADCP). ADCC involves the activation of CD16 through the secretion of pore-forming proteins and proteases. + Phagocytosis is mediated by effector cells, whereas phagocytosis is mediated by CD32 + and CD64 +mediated by effector cells (see, e.g., Fundamental Immunology, 4th ed., Paul ed., Lippincott-Raven, NY, 1997, Chapters 3, 17 and 30; Uchida et al., 2004, J. Exp. Med. 199:1659-69; Akewanlop et al., 2001, Cancer Res. 61:4061-65; Watanabe et al., 1999, Breast Cancer Res. Treat. 53:199-207).

[0137] A "human effector cell" is a leukocyte that expresses one or more FcRs and performs effector function. In certain embodiments, the cells express at least FcγRIII and perform ADCC effector function. Examples of human leukocytes that mediate ADCC include peripheral blood mononuclear cells (PBMCs), natural killer (NK) cells, monocytes, cytotoxic T cells, and neutrophils. Effector cells may be isolated from a native source, such as blood.

[0138] "Antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a mechanism of cytotoxicity in which the Fc region of an antibody bound to an antigen on the cell surface of a target cell interacts with Fc receptors (FcRs) present on certain cytotoxic effector cells (e.g., NK cells, neutrophils, and macrophages). This interaction enables these cytotoxic effector cells to subsequently kill the target cell with cytotoxic agents. NK cells, the primary cells for mediating ADCC, express only FcγRIII, whereas monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991). To assess ADCC activity of a molecule of interest, an in vitro ADCC assay, such as that described in U.S. Patent No. 5,500,362 or U.S. Patent No. 5,821,337, or U.S. Patent No. 6,737,056 (Presta), can be performed. Effector cells useful for such assays include PBMCs and NK cells. ADCC activity of a molecule of interest can also be assessed in vivo in animal models, such as those disclosed in Clynes et al. Proc. Natl. Acad. Sci. (USA) 95:652-656 (1998). Additional polypeptide variants with altered Fc region amino acid sequences (polypeptides with variant Fc regions) and increased or decreased ADCC activity are described, for example, in U.S. Patent Nos. 7,923,538 and 7,994,290.

[0139] "Complement-dependent cytotoxicity" or "CDC" refers to the lysis of target cells in the presence of complement. Activation of the classical complement pathway is initiated by the binding of the first component of the complement system (C1q) to the Fc region of antibodies (of the appropriate subclass), which are bound to their cognate antigens on target cells. This binding activates a series of enzymatic reactions leading to the formation of pores in the target cell membrane and subsequent cell death. Complement activation can also result in the deposition of complement components on the target cell surface, which promotes ADCC through binding of complement receptors (e.g., CR3) on leukocytes. To assess complement activation, a CDC assay can be performed, for example, as described in Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996). Polypeptide variants (such as antibodies having variant Fc regions) with altered Fc region amino acid sequences and increased or decreased C1q binding ability are described, for example, in U.S. Patent Nos. 6,194,551B1, 7,923,538, 7,994,290, and WO 1999 / 51642. See also, for example, Idusogie et al., J. Immunol. 164:4178-4184 (2000).

[0140] The term "antibody-dependent cellular phagocytosis" or simply "ADCP" refers to the process by which antibody-coated cells are internalized, either in whole or in part, by phagocytic immune cells (e.g., macrophages, neutrophils, and dendritic cells) that bind to the Fc region of Ig.

[0141] The terms "nucleic acid molecule," "nucleic acid," and "polynucleotide" are used interchangeably herein to refer to polymers of nucleotides of any length. Such polymers of nucleotides can contain natural and / or non-natural nucleotides and include, but are not limited to, DNA, RNA, and PNA. A "nucleic acid sequence" refers to the linear sequence of nucleotides that comprise a nucleic acid molecule or polynucleotide.

[0142] The term "vector" refers to any molecule or entity (e.g., nucleic acid, plasmid, bacteriophage, or virus) used to transfer a nucleic acid molecule into a host cell. A vector typically comprises a nucleic acid molecule engineered to contain a cloned polynucleotide or polynucleotides encoding a polypeptide or polypeptides of interest that can be propagated in a host cell. Examples of vectors include, but are not limited to, plasmids, viral vectors, and expression vectors, e.g., recombinant expression vectors. A vector can comprise one or more of an origin of replication, one or more regulatory sequences (e.g., promoters and / or enhancers) that control expression of a polypeptide of interest, and / or one or more selectable marker genes. The term includes vectors that are self-replicating nucleic acid molecules as well as vectors that integrate into the genome of a host cell into which they are introduced.

[0143] The term "expression vector" refers to a vector that is suitable for transformation of a host cell and can be used to express a polypeptide of interest in the host cell.

[0144] The terms "host cell" and "host cell line" are used interchangeably herein and refer to a cell or population of cells that may be or have been a recipient of a vector or isolated polynucleotide. Host cells can be prokaryotic or eukaryotic. Exemplary eukaryotic cells include mammalian cells, such as primate or non-primate cells, fungal cells, such as yeast, plant cells, and insect cells. Non-limiting exemplary mammalian cells include, but are not limited to, NSO cells, PER.C6® cells (Klucell), and 293 and CHO cells, and their derivatives, such as 293-6E and DG44 cells, respectively. Such terms refer not only to the original cell but also to the progeny of such cells. Certain modifications may occur in subsequent generations, for example, due to mutations or environmental influences. Such progeny are encompassed within the terms, so long as the cells possess the same function or biological activity as the original cell.

[0145] The term "control sequence" refers to a polynucleotide sequence that can influence the expression and processing of coding sequences to which it is ligated. The nature of such control sequences may depend on the host organism. In particular embodiments, control sequences for prokaryotes may include a promoter, a ribosomal binding site, and a transcription termination sequence. Control sequences for eukaryotes may include, for example, a promoter containing one or more recognition sites for transcription factors, a transcription enhancer sequence, and a transcription termination sequence. "Control sequences" may include leader sequences and / or fusion partner sequences.

[0146] As used herein, "operably linked" means that the components to which the term is applied are in a relationship that allows them to carry out their inherent functions under suitable conditions. For example, a control sequence within a vector is "operably linked" to a protein-coding sequence such that expression of the protein-coding sequence is achieved under conditions compatible with transcriptional activity of the control sequence. When two coding sequences are operably linked, the phrase means that the two DNA fragments or coding sequences are joined such that the amino acid sequences encoded by the two fragments remain in frame.

[0147] The term "transfection" refers to the uptake of foreign or exogenous DNA by a cell; a cell has been "transfected" when exogenous DNA has been introduced into the cell membrane. Several transfection techniques are well known in the art and are disclosed herein. See, e.g., Graham et al., 1973, Virology 52:456; Sambrook et al., 2001, Molecular Cloning: A Laboratory Manual, supra; Davis et al., 1986, Basic Methods in Molecular Biology, Elsevier; Chu et al., 1981, Gene 13:197. Such techniques can be used to introduce one or more exogenous DNA moieties into a suitable host cell.

[0148] The term "transformation" refers to a change in the genetic characteristics of a cell; a cell has been transformed when it has been modified to contain new DNA or RNA. For example, a cell is transformed when it is genetically modified from its native state by the introduction of new genetic material via transfection, transduction, or other techniques. After transfection or transduction, the transforming DNA can recombine with the transforming DNA of the cell by physically integrating into the cell's chromosome, or it can be maintained transiently as an episomal element without replication, or it can replicate independently as a plasmid. A cell is considered "stably transformed" when the transforming DNA is replicated with cell division.

[0149] As used herein, the term "isolated" refers to a molecule that is separated from at least some of the components in which it is typically found or produced in nature. For example, a polypeptide is referred to as "isolated" when it is separated from at least some of the components of the cell in which it was produced. If the polypeptide is secreted by the cell after expression, physically separating the supernatant containing the polypeptide from the cell in which it was produced is considered to "isolate" the polypeptide. Similarly, a polynucleotide is referred to as "isolated" when it is not part of a larger polynucleotide in which it is typically found in nature (e.g., in the case of a DNA polynucleotide, genomic DNA or mitochondrial DNA, etc.) or when it is separated from at least some of the components of the cell in which it was produced, for example, in the case of an RNA polynucleotide. Thus, a DNA polynucleotide contained in a vector within a host cell may be referred to as "isolated."

[0150] The terms "individual," "subject," or patient are used interchangeably herein to refer to animals, e.g., mammals. In some embodiments, methods are provided for treating mammals, including, but not limited to, humans, rodents, monkeys, cats, dogs, horses, cows, pigs, sheep, goats, mammalian laboratory animals, mammalian farm animals, mammalian sport animals, and mammalian pets. In some instances, the "individual" or "subject" is a human. In some examples, the term "individual" or "subject" refers to an individual or subject (e.g., a human) in need of treatment for a disease or disorder.

[0151] As used herein, "disease" or "disorder" refers to a condition that requires treatment, such as cancer.

[0152] As used herein, "cancer" and "tumor" are interchangeable terms that refer to any abnormal cell or tissue growth or proliferation in an animal. A solid tumor is an abnormal growth or mass of tissue that usually does not contain cysts or liquid areas. More specific, non-limiting examples of such cancers include neuroendocrine cancer, colon cancer, stomach cancer, lung cancer, uterine cancer, cervical cancer, pancreatic cancer, esophageal cancer, ovarian cancer, thyroid cancer, bladder cancer, endometrium cancer, breast cancer, liver cancer, prostate cancer, stomach cancer, and bile duct cancer and skin cancer.

[0153] The terms "metastatic cancer" and "metastatic disease" refer to cancer that has spread from its site of origin to another part of the body, for example, to a regional lymph node or a distant site.

[0154] As used herein, "treatment" is an approach to obtain beneficial or desired clinical results. As used herein, "treatment" encompasses any administration or application of a therapeutic agent to a disease in a mammal, including a human. Beneficial or desired clinical results include, but are not limited to, any one or more of the following: alleviation of one or more symptoms, reduction in the extent of the disease, prevention or delay of disease spread (e.g., metastasis, e.g., to the lungs or lymph nodes), prevention or delay of disease recurrence, delay or slowing of disease progression, amelioration of the disease state, inhibition of the disease or disease progression, inhibition or slowing of the disease or its progression, prevention of its onset, and remission (partial or complete). Also encompassed by "treatment" is the reduction of the pathological consequences of a proliferative disease.

[0155] In the context of cancer, the term "treating" includes any or all of inhibiting the growth of cancer cells, inhibiting the replication of cancer cells, reducing the number of cancer cells, reducing the rate of cancer cell invasion into peripheral organs, reducing the rate or extent of tumor metastasis, reducing the overall tumor burden, and alleviating one or more symptoms associated with cancer.

[0156] "Reference," as used herein, refers to any sample, standard, or level used for comparison purposes. A reference can be obtained from a healthy and / or disease-free sample. In some examples, a reference can be obtained from an untreated sample. In some examples, a reference is obtained from a non-disease sample of an untreated sample of a subject individual. In some examples, a reference is obtained from one or more healthy individuals who are not subjects or patients.

[0157] As used herein, to "inhibit" a function or activity is to reduce the function or activity when compared to the same conditions excluding the condition or parameter of interest, or when compared to another condition. For example, an antibody that inhibits tumor growth reduces the rate of tumor growth compared to the rate of tumor growth in the absence of the antibody.

[0158] An "effective amount" or "therapeutically effective amount" or "therapeutically effective dosage" of a drug or therapeutic agent is that amount of any drug or agent that, when used alone or in combination with another therapeutic agent, provides a therapeutic effect, such as protecting a subject from developing a disease or promoting regression of a disease as evidenced by a decrease in the severity of disease symptoms, an increase in the frequency and duration of disease-free periods, or prevention of functional or disability impairment due to the affliction of the disease.

[0159] "Administering" or "administration" refers to the physical introduction of a therapeutic agent into a subject using any of a variety of methods and delivery systems known to those of skill in the art. Exemplary routes of administration include intravenous, intramuscular, subcutaneous, intraperitoneal, spinal, or other parenteral routes of administration, e.g., injection or infusion (e.g., intravenous infusion). Administration can also be, for example, one time, multiple times, and / or over one or more extended periods of time.

[0160] The term "chemotherapeutic agent" refers to any chemical compound that is effective in inhibiting tumor growth. Non-limiting examples of chemotherapeutic agents include alkylating agents (e.g., nitrogen mustards, ethylenimine compounds, and alkylsulfonates), antimetabolites (e.g., folic acid, purine, or pyrimidine antagonists), antimitotic agents (e.g., antitubulin agents such as derivatives of vinca alkaloids, auristatins, and podophyllotoxins), cytotoxic antibiotics, compounds that damage or interfere with DNA expression or replication (e.g., DNA minor groove binders), and growth factor receptor antagonists, as well as cytotoxic or cytostatic agents.

[0161] The terms "pharmaceutical formulation" and "pharmaceutical composition" refer to a preparation that is in a form that allows the biological activity of the active ingredient to be effective and that does not contain additional components that are unacceptably toxic to the subject to which the formulation is administered. Such formulations may be sterile.

[0162] A "pharmaceutically acceptable carrier" refers to a non-toxic solid, semi-solid, or liquid filler, diluent, encapsulating material, formulation aid, or carrier conventional in the art for use with therapeutic agents that together comprise a "pharmaceutical composition" for administration to a subject. A pharmaceutically acceptable carrier is non-toxic to recipients at the dosages and concentrations employed and is compatible with other ingredients of the formulation. A pharmaceutically acceptable carrier is appropriate for the formulation with which it is used.

[0163] As used herein, the phrase "pharmaceutically acceptable salt" refers to a pharmaceutically acceptable organic or inorganic salt of a compound of the present invention. Exemplary salts include, but are not limited to, sulfate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, acid phosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisate, fumarate, gluconate, glucuronate, saccharate, formate, benzoate, glutamate, methanesulfonate (mesylate), ethanesulfonate, benzenesulfonate, p-toluenesulfonate, pamoate (i.e., 4,4'-methylene-bis-(2-hydroxy-3-naphthoate)) salts, alkali metal (e.g., sodium and potassium) salts, alkaline earth metal (e.g., magnesium) salts, and ammonium salts. A pharmaceutically acceptable salt may involve the inclusion of another molecule, such as an acetate ion, a succinate ion, or other counterion. The counterion may be any organic or inorganic moiety that stabilizes the charge of the parent compound. Furthermore, a pharmaceutically acceptable salt may have two or more charged atoms in its structure. When multiple charged atoms are part of a pharmaceutically acceptable salt, it may have multiple counterions. Thus, a pharmaceutically acceptable salt may have one or more charged atoms and / or one or more counterions.

[0164] As used herein, the term "compound" refers to and includes the compound itself, whether named or represented by structure, and its salt forms, regardless of whether such salt forms are explicitly stated, unless the context clearly indicates that such salt forms are excluded. The term "compound" also includes solvate forms of the compound, in which a solvent is non-covalently associated with the compound or reversibly covalently associated with the compound, such as when a carbonyl group of the compound is hydrated to form a geminal diol. Solvate forms include the compound itself and its salt forms, including hemisolvates, monosolvates, and solvates, including hydrates, and when the compound can be associated with two or more solvent molecules, the two or more solvent molecules may be the same or different.

[0165] In some cases, the compounds of the present invention include an explicit reference to one or more of the above forms, e.g., salts and solvates, and this does not refer to any solid form of the compound. However, this reference is for emphasis only and should not be interpreted as excluding any of the above-identified forms. Furthermore, if an explicit reference to salt and / or solvate forms of a compound or ligand drug conjugate composition is not made, the omission should not be interpreted as excluding salt and / or solvate forms of the compound or conjugate, unless the context makes clear that such salt and / or solvate forms are excluded.

[0166] Pharmaceutically acceptable salts are salt forms of the compounds described herein that are suitable for administration to a subject and, in some embodiments, include countercations or counteranions as described by P.H. Stahl and C.G. Wermuth, editors, Handbook of Pharmaceutical Salts: Properties, Selection and Use, Weinheim / Zurich: Wiley-VCH / VHCA, 2002.

[0167] The Linker unit is a bifunctional moiety that connects a camptothecin to a Ligand unit in an ADC. The Linker unit of the present invention has several components (e.g., in some embodiments, a Stretcher unit having a base unit, a Connector unit that may or may not be present, a parallel Connector unit that may or may not be present, a releasable linker, and a Spacer unit that may or may not be present).

[0168] As used herein, "PEG," "PEG unit," or "polyethylene glycol" refers to an organic moiety consisting of repeating ethylene-oxy subunits, which may be polydisperse, monodisperse, or discrete (i.e., having a discrete number of ethylene-oxy subunits). Polydisperse PEGs are heterogeneous mixtures of sizes and molecular weights, while monodisperse PEGs are typically purified from heterogeneous mixtures, thus providing a single chain length and molecular weight. Preferred PEG units are discrete PEGs, compounds that are synthesized stepwise without a polymerization process. Discrete PEGs provide single molecules with defined and specified chain lengths.

[0169] The PEG units provided herein comprise one or more polyethylene glycol chains, each consisting of one or more ethyleneoxy subunits covalently bonded to one another. The polyethylene glycol chains can be linked together, for example, in a linear, branched, or star-shaped configuration. Typically, at least one of the polyethylene glycol chains prior to incorporation into an ADC is derivatized at one end with an alkyl moiety substituted with an electrophilic group for covalent attachment to the carbamate nitrogen of the methylene carbamate unit (i.e., representing an example of R). Typically, the terminal ethyleneoxy subunit in each polyethylene glycol chain that is not involved in covalent attachment to the remainder of the linker unit is modified with a PEG capping unit, typically an optionally substituted alkyl such as -CH3, -CH2CH3, or -CH2CH2CO2H. A preferred PEG unit has a single polyethylene glycol chain comprising 4 to 24 -CH2CHO- subunits covalently linked in series and terminating at one end with a PEG capping unit.

[0170] Unless otherwise indicated, the term "alkyl," by itself or as part of another term, refers to a substituted or unsubstituted, straight or branched, saturated or unsaturated hydrocarbon having the indicated number of carbon atoms (e.g., "-C1-C8 alkyl" or "-C1-C 10("Alkyl" refers to alkyl groups having 1 to 8 or 1 to 10 carbon atoms, respectively. If the number of carbon atoms is not specified, the alkyl group has 1 to 8 carbon atoms. Representative straight chain "-C1-C8 alkyl" groups include, but are not limited to, -methyl, -ethyl, -n-propyl, -n-butyl, -n-pentyl, -n-hexyl, -n-heptyl, and -n-octyl; branched -C3-C8 alkyl groups include, but are not limited to, -isopropyl, -sec-butyl, -isobutyl, -tert-butyl, -isopentyl, and -2-methylbutyl; unsaturated -C2-C8 alkyl groups include, but are not limited to, -isopropyl, -sec-butyl, -isobutyl, -tert-butyl, -isopentyl, and -2-methylbutyl. Examples of alkyl groups include, but are not limited to, -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-hexyl, 2-hexyl, -3-hexyl, -acetylenyl, -propynyl, -1 butynyl, -2 butynyl, -1 pentynyl, -2 pentynyl, and -3 methyl 1 butynyl. Alkyl groups can be unsubstituted. Alkyl groups can be substituted with one or more groups. In other embodiments, the alkyl group will be saturated.

[0171] Unless otherwise indicated, "alkylene" by itself or as part of another term refers to a substituted or unsubstituted saturated, branched or straight-chain, or cyclic hydrocarbon radical of the stated number of carbon atoms, typically 1 to 10 carbon atoms, having two monovalent radical centers derived by the removal of two hydrogen atoms from the same or two different carbon atoms of a parent alkane. Typical alkylene radicals include, but are not limited to, methylene (-CH-), 1,2-ethylene (-CHCH-), 1,3-propylene (-CHCHCH-), 1,4-butylene (-CHCHCHCH-), and the like. In preferred embodiments, the alkylene is a branched or straight-chain hydrocarbon (i.e., it is not a cyclic hydrocarbon).

[0172] Unless otherwise indicated, "aryl," by itself or as part of another term, means a substituted or unsubstituted monovalent carbocyclic aromatic hydrocarbon radical of the stated number of carbon atoms, typically 6 to 20 carbon atoms, derived by the removal of one hydrogen atom from a single carbon atom of a parent aromatic ring system. Some aryl groups are represented in the exemplary structures as "Ar." Typical aryl groups include, but are not limited to, radicals derived from benzene, substituted benzene, naphthalene, anthracene, biphenyl, and the like. An exemplary aryl group is the phenyl group.

[0173] Unless otherwise indicated, an "arylene," by itself or as part of another term, is an aryl group, as defined above, having two covalent bonds (i.e., it is divalent) and which may be ortho-, meta-, or para-oriented, with phenyl as an exemplary group, as shown in the following structure: [ka]

[0174] Unless otherwise indicated, "C3-C8 heterocycle," by itself or as part of another term, refers to a monovalent substituted or unsubstituted aromatic or non-aromatic monocyclic or bicyclic ring system having 3 to 8 carbon atoms (also referred to as ring members) and 1 to 4 heteroatom ring members, independently selected from N, O, P, or S, derived by removing one hydrogen atom from a ring atom of the parent ring system. One or more N, C, or S atoms in a heterocycle can be oxidized. A ring containing a heteroatom can be aromatic or non-aromatic. A heterocycle in which all ring atoms participate in aromaticity is called a heteroaryl; otherwise, it is called a heterocycle.

[0175] Unless otherwise specified, a heterocycle is attached to its pendant group at any heteroatom or carbon atom that results in a stable structure. Such heteroaryls can be attached through an aromatic carbon of the aromatic ring system, referred to as a C-linked heteroaryl, or through a non-double-bonded N atom (i.e., non-=N-) of the aromatic ring system, referred to as an N-linked heteroaryl. Thus, nitrogen-containing heterocycles can be C-linked or N-linked, and include pyrrole moieties (such as pyrrol-1-yl (N-linked) and pyrrol-3-yl (C-linked)) and imidazole moieties (such as imidazol-1-yl and imidazol-3-yl (both N-linked), and imidazol-2-yl, imidazol-4-yl, and imidazol-5-yl moieties (all of which are C-linked)).

[0176] Unless otherwise indicated, "C3-C8 heteroaryl" refers to an aromatic C3-C8 heterocycle, where the subscript indicates the total number of carbons in the cyclic ring system of the heterocycle or the total number of aromatic carbons in the aromatic ring system of the heteroaryl, and does not imply any indication of the size of the ring system or the presence or absence of ring fusion. Representative examples of C3-C8 heterocycles include, but are not limited to, pyrrolidinyl, azetidinyl, piperidinyl, morpholinyl, tetrahydrofuranyl, tetrahydropyranyl, benzofuranyl, benzothiophene, indolyl, benzopyrazolyl, pyrrolyl, thiophenyl (thiophene), furanyl, thiazolyl, imidazolyl, pyrazolyl, pyrimidinyl, pyridinyl, pyrazinyl, pyridazinyl, isothiazolyl, and isoxazolyl.

[0177] When explicitly stated, the size of a heterocycle or heteroaryl ring system is indicated by the total number of atoms in the ring. For example, a designation as a 5- or 6-membered heteroaryl indicates the total number or aromatic atoms (i.e., 5 or 6) in the heteroaromatic ring system of the heteroaryl, but does not refer to the number of aromatic heteroatoms or aromatic carbons in the ring system. Fused heteroaryls are explicitly stated or implied by the context and are typically indicated by the number of aromatic atoms in each aromatic ring that are fused together to form the fused heteroaromatic ring system. For example, a 5- or 6-membered heteroaryl is an aromatic 5-membered ring fused to an aromatic 6-membered ring in which one or both rings have aromatic heteroatoms or heteroatoms are shared between the two rings.

[0178] A heterocycle fused to an aryl or heteroaryl is an example of an optionally substituted heterocycle, where the heterocycle is substituted by ring fusion with an aryl or heteroaryl, such that the heterocycle remains non-aromatic and is part of a larger structure by bonding to the non-aromatic portion of the fused ring system. Similarly, an aryl or heteroaryl fused to a heterocycle or carbocycle that is part of a larger structure by bonding to the aromatic portion of the fused ring system is an example of an optionally substituted aryl or heterocycle, where the aryl or heterocycle is substituted by ring fusion with a heterocycle or carbocycle.

[0179] Unless otherwise indicated, "C3-C8 heterocyclo," by itself or as part of another term, refers to a C3-C8 heterocycle, as defined above, in which one of the heterocycle's hydrogen atoms is replaced with a bond (i.e., it is divalent). Unless otherwise indicated, "C3-C8 heteroarylene," by itself or as part of another term, refers to a C3-C8 heteroaryl group, as defined above, in which one of the heteroaryl group's hydrogen atoms is replaced with a bond (i.e., it is divalent).

[0180] Unless otherwise indicated, a "C3-C8 carbocycle," by itself or as part of another term, is a 3-, 4-, 5-, 6-, 7-, or 8-membered monovalent, substituted or unsubstituted, saturated or unsaturated, non-aromatic monocyclic or bicyclic carbocyclic ring derived by the removal of one hydrogen atom from a ring atom of a parent ring system. Representative -C3-C8 carbocycles include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentadienyl, cyclohexyl, cyclohexenyl, 1,3-cyclohexadienyl, 1,4-cyclohexadienyl, cycloheptyl, 1,3-cycloheptadienyl, 1,3,5-cycloheptatrienyl, cyclooctyl, and cyclooctadienyl.

[0181] Unless otherwise indicated, "C3-C8 carbocyclo," by itself or as part of another term, refers to a C3-C8 carbocyclic group as defined above in which another one of the carbocyclic group's hydrogen atoms has been replaced with a bond (i.e., it is divalent).

[0182] Unless otherwise indicated, the term "heteroalkyl," by itself or in combination with another term, means, unless otherwise stated, a stable straight- or branched-chain hydrocarbon, or combinations thereof, which is fully saturated or contains 1 to 3 degrees of unsaturation, and consists of the stated number of carbon atoms, with 1 to 10, preferably 1 to 3, heteroatoms selected from the group consisting of O, N, Si, and S, wherein the nitrogen and sulfur atoms can be optionally oxidized, and the nitrogen heteroatom can be optionally quaternized. The O, N, and S heteroatoms can be placed at any interior position of the heteroalkyl group or at the position at which the alkyl group is attached to the remainder of the molecule. The Si heteroatom can be placed at any position of the heteroalkyl group, including the position at which the alkyl group is attached to the remainder of the molecule.

[0183] Examples include -CH2-CH2-O-CH3, -CH2-CH2-NH-CH3, -CH2-CH2-N(CH3)-CH3, -CH2-S-CH2-CH3, -CH2-CH2-S(O)-CH3, -NH-CH2-CH2-NH-C(O)-CH2-CH3, -CH2-CH2-S(O)-CH3, -CH=CH-O-CH3, -Si(CH3)3, -CH2-CH=NO-CH3, and -CH=CH-N(CH3)-CH3. Up to two heteroatoms may be consecutive, such as, for example, -CH2-NH-OCH3 and -CH2-O-Si(CH3)3. Typically, a C1-C4 heteroalkyl or heteroalkylene has 1 to 4 carbon atoms and 1 or 2 heteroatoms, and a C1-C3 heteroalkyl or heteroalkylene has 1 to 3 carbon atoms and 1 or 2 heteroatoms. In some embodiments, the heteroalkyl or heteroalkylene is saturated.

[0184] Unless otherwise stated, the term "heteroalkylene," by itself or in combination with other terms, means a divalent group derived from heteroalkyl (as described above), as exemplified by -CH-CH-S-CH-CH- and -CH-S-CH-CH-NH-CH-. For heteroalkylene groups, heteroatoms can also occupy either or both of the chain termini. Furthermore, for alkylene and heteroalkylene linking groups, no orientation of the linking group is implied.

[0185] Unless otherwise indicated, "aminoalkyl," by itself or in combination with another term, means heteroalkyl, where the alkyl portion, as defined herein, is substituted with an amino, alkylamino, dialkylamino, or cycloalkylamino group. Exemplary non-limiting aminoalkyls are -CHNH, -CHCHNH, -CHCHNHCH, and -CHCHN(CH), further including branched species such as -CH(CH)NH and -C(CH)CHNH in either the (R)- or (S)-configuration. Alternatively, aminoalkyl is an alkyl moiety, group, or substituent, as defined herein, where the sp carbon other than the radical carbon is substituted.3 Carbon has at least one sp 3 Provided that carbon remains, the sp 3 Nitrogen is alkyl sp 3 It is replaced by an amino or alkylamino moiety replacing the carbon. When an aminoalkyl moiety is referred to as a substituent on a larger structure or another moiety, the aminoalkyl is covalently attached to the structure or moiety through a carbon radical on the alkyl portion of the aminoalkyl.

[0186] Unless otherwise indicated, "alkylamino" and "cycloalkylamino," alone or in combination with another term, mean an alkyl or cycloalkyl radical as described herein, wherein the radical carbon of the alkyl or cycloalkyl radical is heterocyclic or heterocyclic, and the radical carbon ..., and the radical carbon is heterocyclic, and the radical carbon is heterocyclic, and the radical carbon is hetero 3 Any remaining carbons are replaced with nitrogen radicals. When an alkylamino is substituted at the nitrogen with another alkyl moiety, the resulting substituted radical is sometimes referred to as a dialkylamino moiety, group, or substituent, where the alkyl moieties substituting the nitrogen are independently selected.

[0187] Exemplary and non-limiting amino, alkylamino, and dialkylamino substituents include those having the structure -N(R')2, where R' is independently hydrogen or C 1-6 In cycloalkylamines, R', together with the nitrogen to which they are attached, define a heterocyclic ring. When both R's are hydrogen or alkyl, the moieties are sometimes described as primary and tertiary amino groups, respectively. When one R' is hydrogen and the other is alkyl, the moiety is sometimes described as a secondary amino group. Primary and secondary alkylamino moieties are more reactive as nucleophiles toward carbonyl-containing electrophilic centers, while tertiary amines are more basic.

[0188] "Substituted alkyl" and "substituted aryl" refer to alkyl and aryl, respectively, in which one or more hydrogen atoms, typically one, are each independently replaced with a substituent. Exemplary substituents include -X, -R', -OH, -OR', -SR', -N(R'), -N(R'), =NR', -CX, -CN, -NO, -NR'C(=O)R', -C(=O)R', -C(=O)N(R'), -S(=O)R', -S(=O)NR', ​​-S(=O)R', -OP(=O)(OR'), -P(=O)(OR'), -PO=, POH, - Examples include, but are not limited to, -C(=O)R', -C(=S)R', -COR', -CO-, -C(=S)OR', -C(=O)SR', -C(=S)SR', -C(=O)N(R'), -C(=S)N(R'), and -C(=NR)N(R'), where each X is independently selected from the group consisting of -F, -Cl, -Br, and -I; and each R' is independently selected from -H, -C-C 20 Alkyl, -C6-C 20 Aryl, -C3-C 14 It is selected from the group consisting of a heterocycle, a protecting group, and a prodrug moiety.

[0189] More typically, the substituents are selected from the group consisting of -X, -R', -OH, -OR', -SR', -N(R'), -N(R'), =NR', -NR'C(=O)R', -C(=O)R', -C(=O)N(R'), -S(=O)R', -S(=O)NR', ​​-S(=O)R', -C(=O)R', -C(=S)R', -C(=O)N(R'), -C(=S)N(R'), and -C(=NR)N(R'), wherein each X is independently and each X is selected from the group consisting of -F and -Cl, or selected from the group consisting of -X, -R', -OH, -OR', -N(R')2, -N(R')3, -NR'C(=O)R', -C(=O)N(R')2, -S(=O)2R', -S(=O)2NR', -S(=O)R', -C(=O)R', -C(=O)N(R')2, -C(=NR)N(R')2, protecting groups, and prodrug moieties; 20 Alkyl, -C6-C 20 Aryl, -C3-C14 It is selected from the group consisting of a heterocycle, a protecting group, and a prodrug moiety.

[0190] In some embodiments, the alkyl substituents are selected from the group consisting of -N(R')2, -N(R')3, and -C(=NR)N(R')2, where R ’ is hydrogen and -C1-C 20 In another embodiment, the alkyl is substituted with a series of ethyleneoxy moieties to define a PEG unit. The alkylene, carbocycle, carbocyclo, arylene, heteroalkyl, heteroalkylene, heterocycle, heterocyclo, heteroaryl, and heteroarylene groups described above may also be similarly substituted.

[0191] As used herein, "protecting group" means a moiety that prevents or reduces the ability of the atom or functional group to which it is attached to participate in unwanted reactions. Typical protecting groups for atoms or functional groups are described in Greene (1999), "PROTECTIVE GROUPS IN ORGANIC SYNTHESIS, 3RD ED.", Wiley Interscience. Protecting groups for heteroatoms such as oxygen, sulfur, and nitrogen are used in some instances to minimize or prevent their undesired reactions with electrophilic compounds. In other instances, protecting groups are used to reduce or eliminate the nucleophilicity and / or basicity of the unprotected heteroatom. A non-limiting example of a protected oxygen is -OR. PR is given by R PRis a protecting group for hydroxyl, which is typically protected as an ester (e.g., acetate, propionate, or benzoate). Other protecting groups for hydroxyl avoid interfering with the nucleophilicity of organometallic or other highly basic reagents, and hydroxyl is typically protected as an ether, including alkyl or heterocycloalkyl ethers (e.g., methyl or tetrahydropyranyl ethers), alkoxymethyl ethers (e.g., methoxymethyl or ethoxymethyl ethers), optionally substituted aryl ethers, and silyl ethers (e.g., trimethylsilyl (TMS), triethylsilyl (TES), tert-butyldiphenylsilyl (TBDPS), tert-butyldimethylsilyl (TBS / TBDMS), triisopropylsilyl (TIPS), and [2-(trimethylsilyl)ethoxy]-methylsilyl (SEM)). Nitrogen protecting groups include -NHR PR or -N(R PR )2-, and R PR at least one of R is a nitrogen atom protecting group, or R PR Both of these contain protecting groups.

[0192] A protecting group is suitable if it can prevent or avoid undesirable side effects or premature loss of the protecting group under the reaction conditions required to carry out desired chemical transformations elsewhere in the molecule, and, if necessary, during purification of the newly formed molecule, and can be removed under conditions that do not adversely affect the structural or stereochemical integrity of the newly formed molecule. By way of example and not limitation, suitable protecting groups can include those previously described for protecting functional groups. Suitable protecting groups can be those used in peptide coupling reactions.

[0193] "Aromatic alcohol" refers to an aromatic ring system substituted with a hydroxyl functional group -OH, either by itself or as part of a larger structure. Thus, aromatic alcohol refers to any aryl, heteroaryl, arylene, or heteroarylene moiety described herein that has a hydroxyl functional group bonded to an aromatic carbon of the aromatic ring system. The aromatic alcohol may be part of a larger moiety, or embedded in the larger moiety by ring fusion, as in the case where the aromatic ring system is a substituent on the moiety, and may optionally be substituted with one or more other hydroxyl substituents as described herein. Phenolic alcohols are aromatic alcohols that have a phenol group as the aromatic ring.

[0194] The term "aliphatic alcohol" by itself or as part of a larger structure refers to a moiety having a non-aromatic carbon bonded to a hydroxyl functional group -OH. The hydroxyl-bearing carbon may be unsubstituted (i.e., methyl alcohol) or may have one, two, or three optionally substituted branched or unbranched alkyl substituents to define a primary alcohol or a secondary or tertiary aliphatic alcohol having a linear or cyclic structure. When part of a larger structure, the alcohol may be a substituent of this structure by bonding through the hydroxyl-bearing carbon, through a carbon of an alkyl or other moiety described herein to the hydroxyl-bearing carbon, or through a substituent of the alkyl or other moiety. Aliphatic alcohol contemplates non-aromatic cyclic structures (i.e., carbocyclic and heterocyclic, optionally substituted) in which the hydroxyl functional group is bonded to a non-aromatic carbon of the cyclic ring system.

[0195] As used herein, "arylalkyl" or "heteroarylalkyl" refers to a substituent, moiety, or group in which an aryl moiety is linked to an alkyl moiety, i.e., arylalkyl-, where the alkyl and aryl groups are as described above, e.g., CH-CH- or CH-CH(CH)CH-. An arylalkyl or heteroarylalkyl is defined as any group in which the sp 3It is associated with a larger structure or moiety through carbon.

[0196] As used herein, "electron-withdrawing group" means a functional group or electronegative atom that draws electron density away from the atom to which it is inductively and / or via resonance, whichever is more dominant (i.e., the functional group or atom may be inductively electron-withdrawing but overall electron-donating via resonance), tending to stabilize the anion or electron-rich moiety. The electron-withdrawing effect is typically transferred inductively, albeit in a damped form, to other atoms bonded to the bonded atom made electron-deficient by the electron-withdrawing group (EWG), thus affecting the electrophilicity of more remote reactive centers. Exemplary electron-withdrawing groups include -C(=O), -CN, -NO2, -CX3, -X, -C(=O)OR', -C(=O)N(R')2, -C(=O)R', -C(=O)X, -S(=O )2R', -S(=O)2OR', -S(=O)2NHR', -S(=O)2N(R')2, -P(=O)(OR')2, -P(=O)(CH3)NHR', -NO, -N(R')3 + X is -F, -Br, -Cl, or -I; and R' in some embodiments, at each occurrence, is independently selected from hydrogen and C 1-6 It is selected from the group consisting of alkyl, as well as the specific O-linked moieties described herein, such as acyloxy.

[0197] Exemplary EWGs can also include aryl groups (e.g., phenyl), depending on the substitution and the particular heteroaryl group (e.g., pyridine). Thus, the term "electron-withdrawing group" also includes aryls or heteroaryls that are further substituted with electron-withdrawing groups. Typically, electron-withdrawing groups on aryls or heteroaryls are -C(=O), -CN, -NO2, -CX3, and -X, where independently selected X is a halogen, typically -F or -Cl. Depending on their substituents, alkyl moieties may also be electron-withdrawing groups.

[0198] "Electron-donating group," as the term is used herein, unless otherwise stated or implied by context, refers to a functional group or electropositive atom that increases the electron density of the atom to which it is inductively and / or via resonance, tending to stabilize a cation or electron-deficient system, where either is more dominant (i.e., the functional group or atom may be inductively electron-withdrawing but overall electron-donating via resonance). The electron-donating effect is typically transmitted via resonance to other atoms bonded to the bonded atom made electron-rich by the electron-donating group (EDG), thus increasing the electron density of more remote reactive centers. Typically, the electron-donating group is selected from the group consisting of -OH, -OR', -NH2, -NHR', and N(R')2, where each R' is independently selected from the group consisting of C1-C 12 alkyl, typically C1-C6 alkyl. Depending on its substituents, C6-C 24 Aryl, C5-C 24 Heteroaryl or unsaturated C1-C 12 Alkyl moieties can also be electron-donating groups, and in some aspects, such moieties are encompassed by the term electron-donating group.

[0199] "Leaving group ability" refers to the ability of an alcohol-, thiol-, amine-, or amide-containing compound corresponding to camptothecin in an ADC to be released as a free drug from the conjugate following activation of a self-immolative event within the conjugate. The release can be variable without the benefit of the methylene carbamate unit to which the camptothecin is attached (i.e., when the camptothecin is directly attached to the self-immolative moiety and has no intervening methylene carbamate unit). Good leaving groups are typically weak bases; the more acidic the functional group released from such a conjugate, the weaker the conjugate base. Thus, the leaving group ability of an alcohol-, thiol-, amine-, or amide-containing free drug derived from camptothecin will be related to the pKa of the functional group of the drug that would be released from the conjugate if a methylene carbamate unit (i.e., camptothecin is directly attached to the self-immolative moiety) were not used. Thus, a lower pKa of the functional group increases its leaving group ability. Although other factors may contribute to the release of free drug from conjugates without the benefit of a methylene carbamate unit, drugs with functional groups with lower pKa values ​​are typically better leaving groups than drugs conjugated via functional groups with higher pKa values. Another consideration is that functional groups with too low a pKa value may result in an unacceptable activity profile due to premature loss of camptothecin due to spontaneous hydrolysis. For conjugates using a methylene carbamate unit, a common functional group (i.e., carbamic acid) with a pKa value that allows efficient release of free drug without unacceptable loss of camptothecin is generated upon autolysis.

[0200] As used herein, a "succinimide moiety" refers to an organic moiety consisting of a succinimide ring system, which is typically present in one type of Stretcher unit (Z), further consisting of an alkylene-containing moiety attached to the imide nitrogen of the ring system. The succinimide moiety typically results from the Michael addition of a sulfhydryl group of a Ligand unit to the maleimide ring system of a Stretcher unit precursor (Z'). Thus, a succinimide moiety consists of a thio-substituted succinimide ring system, the imide nitrogen of which, when present in an ADC, is substituted with the remainder of the Linker unit of the ADC and, optionally, with a substituent that was present on the maleimide ring system of Z'.

[0201] As used herein, "acid-amide moiety" refers to a succinic acid having an amide substituent resulting from the thio-substituted succinimide ring system of the succinimide moiety that has undergone rupture of one of its carbonyl-nitrogen bonds by hydrolysis. Hydrolysis resulting in the succinic acid-amide moiety provides a linker unit that is less likely to suffer premature loss of the Ligand unit to which it is attached through removal of the antibody-thio substituent. Hydrolysis of the succinimide ring system of the thio-substituted succinimide moiety is expected to provide regiochemical isomers of the acid-amide moiety due to differences in the reactivity of the two carbonyl carbons of the succinimide ring system, at least in part due to any substituents present on the maleimide ring system of the Stretcher unit precursor and the thio substituent introduced by the targeting ligand.

[0202] As used herein, the term "prodrug" refers to a less biologically active or inactive compound that is converted into a more biologically active compound in the body via a chemical or biological process (i.e., chemical reaction or enzymatic biotransformation). Typically, a biologically active compound is rendered biologically inactive (i.e., converted to a prodrug) by chemically modifying the compound with a prodrug moiety. In some embodiments, the prodrug is a Type II prodrug, which is bioactivated extracellularly, e.g., in digestive fluids, or in the body's circulatory system, e.g., blood. Exemplary prodrugs are esters and β-D-glucopyranosides.

[0203] In many cases, the conjugates, linkers, and component assemblies described herein refer to reactive groups. A "reactive group" or RG is a group containing a reactive site (RS) that can form a bond with either a component of a linker unit (i.e., A, W, Y) or camptothecin D. RS is a reactive site within the reactive group (RG). Reactive groups include sulfhydryl groups that form disulfide or thioether bonds, aldehyde, ketone, or hydrazine groups that form hydrazone bonds, carboxylic acids or amino groups that form peptide bonds, carboxylic acids or hydroxy groups that form ester bonds, sulfonic acids that form sulfonamide bonds, alcohols that form carbamate bonds, and amines that form sulfonamide or carbamate bonds.

[0204] The following table illustrates exemplary functional groups that can be formed after reaction of reactive groups, reactive sites, and reactive sites. The table is not limiting. One of skill in the art will understand that the R' and R" moieties noted in the table are any organic moiety (e.g., alkyl, aryl, heteroaryl, or substituted alkyl, aryl, or heteroaryl) that is compatible with the bond formation provided upon converting RG to one of the exemplary functional groups. It will also be understood that, when applied to embodiments of the present invention, R' can represent one or more components of a self-stabilizing linker or any secondary linker, as the case may be, and R" can represent one or more components of any secondary linker, camptothecin, stabilizing unit, or detection unit, as the case may be.

[0205] A "sterile" formulation is sterile or essentially free of living microorganisms and their spores.

[0206] Various aspects of the disclosure are described in further detail in the following sections.

[0207] II.CEACAM5 The domain organization of human CEACAM5 is as follows (based on GenBank AAA51967.1 sequence, SEQ ID NO: 11): [Table 2]

[0208] Therefore, the A3-B3 domain of human CEACAM5 consists of the amino acids at positions 499 to 685 of SEQ ID NO:11.

[0209] The domain organization of Macaca fascicularis CEACAM5 is as follows (based on the cloned extracellular domain sequence, SEQ ID NO: 12): [Table 3]

[0210] III. Anti-CEACAM5 ADC In some embodiments, an ADC having the formula: L-(QD) p or a salt thereof, as provided herein: wherein L is a ligand unit comprising an antibody or antigen-binding fragment thereof that binds to CEACAM5; the subscript p is an integer from 1 to 16, and Q is a linker unit; D is a Drug unit, and the Drug unit is a topoisomerase I inhibitor. In some embodiments, the ADCs provided herein have certain advantages over other ADCs, including increased anti-tumor activity and reduced toxicity.

[0211] A.CEACAM5 antibody According to embodiments, antibodies according to the invention are specific for surface human and Macaca fascicularis CEACAM5 proteins. In embodiments, antibodies of the invention do not bind to or significantly cross-react with human CEACAM1, human CEACAM6, human CEACAM7, human CEACAM8, Macaca fascicularis CEACAM1, Macaca fascicularis CEACAM6, and Macaca fascicularis CEACAM8 proteins.

[0212] In particular, the antibodies do not bind to or significantly cross-react with the extracellular domains of the aforementioned human and Macaca fascicularis CEACAM proteins.

[0213] In particular, the antibody binds to the A3-B3 domain of CEACAM5.

[0214] One embodiment of the present invention has an affinity for human CEACAM5 or Macaca fascicularis CEACAM5, or both, which is ≦10 nM, for example, ≦5 nM, ≦3 nM, ≦1 nM or ≦0.1 nM, for example, an affinity of 0.01 nM to 5 nM, or / and an affinity of 0.1 nM to 5 nM, or an affinity of 0.1 nM to 1 nM.

[0215] Affinity for human CEACAM5 or Macaca fascicularis CEACAM5 may be determined as an EC50 value in an ELISA using soluble recombinant CEACAM5 as a capture antigen. The antibodies of the present invention may also have an apparent dissociation constant (apparent KD) of ≦25 nM, e.g., ≦20 nM, ≦10 nM, ≦5 nM, ≦3 nM, or ≦1 nM, as determined by FACS analysis on the tumor cell line MKN45 (DSMZ, ACC409) or patient-derived xenograft tumor cells (CR-IGR-034P) available from Oncodesign Biotechnology, tumor collection CReMEC. The apparent KD may be in the range of 0.01 to 20 nM, or in the range of 0.1 to 20 nM, 0.1 to 10 nM, or 0.1 to 5 nM. Furthermore, it has been shown that antibodies according to the invention are capable of detecting CEACAM5 expression by immunohistochemistry in frozen and formalin-fixed and paraffin-embedded (FFPE) tissue sections.

[0216] In some embodiments, the anti-CEACAM5 antibody comprises an Fc region. In some embodiments, the anti-CEACAM5 antibody is an Fc-competent antibody. In some embodiments, the Fc-competent antibody may induce or improve ADCC and / or ADCP activity.

[0217] In some embodiments, the anti-CEACAM5 antibody comprises one or more of a CDR1-H comprising the amino acid sequence set forth in SEQ ID NO: 1, a CDR2-H comprising the amino acid sequence set forth in SEQ ID NO: 2, a CDR3-H comprising the amino acid sequence set forth in SEQ ID NO: 3, a CDR1-L comprising the amino acid sequence set forth in SEQ ID NO: 4, a CDR2-L comprising the amino acid sequence NTR, and a CDR3-L comprising the amino acid sequence set forth in SEQ ID NO: 6.

[0218] In some embodiments, the anti-CEACAM5 antibody comprises one or more of a CDR1-H comprising the amino acid sequence set forth in SEQ ID NO: 1, a CDR2-H comprising the amino acid sequence set forth in SEQ ID NO: 2, a CDR3-H comprising the amino acid sequence set forth in SEQ ID NO: 3, a CDR1-L comprising the amino acid sequence set forth in SEQ ID NO: 4, a CDR2-L comprising the amino acid sequence NTR, and a CDR3-L comprising the amino acid sequence set forth in SEQ ID NO: 6.

[0219] In some embodiments, the antibody or antigen-binding fragment thereof comprises a VH having at least 80%, 85%, 90%, 95%, or 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 7. In some embodiments, the antibody or antigen-binding fragment thereof comprises a VL having at least 80%, 85%, 90%, 95%, or 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 8. In some embodiments, the antibody or antigen-binding fragment thereof comprises a VH having at least 80%, 85%, 90%, 95%, or 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 7, and a VL having at least 80%, 85%, 90%, 95%, or 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 8.

[0220] In some embodiments, the antigen binding protein comprises a CDR1-H, CDR2-H, and CDR3-H of a variable heavy chain domain (VH) comprising the amino acid sequence set forth in SEQ ID NO: 7. In some embodiments, the antibody or antigen-binding fragment thereof comprises a CDR1-L, CDR2-L, and CDR3-L of a variable light chain domain (VL) comprising the amino acid sequence set forth in SEQ ID NO: 8. In some embodiments, the antigen binding protein comprises a CDR1-H, CDR2-H, and CDR3-H of a variable heavy chain domain (VH) comprising the amino acid sequence set forth in SEQ ID NO: 7, and a CDR1-L, CDR2-L, and CDR3-L of a variable light chain domain (VL) comprising the amino acid sequence set forth in SEQ ID NO: 8.

[0221] In some embodiments, the anti-CEACAM5 antibody comprises one or more of a CDR1-H comprising the amino acid sequence set forth in SEQ ID NO: 1, a CDR2-H comprising the amino acid sequence set forth in SEQ ID NO: 2, a CDR3-H comprising the amino acid sequence set forth in SEQ ID NO: 3, and a VH comprising at least 80%, 85%, 90%, 95%, or 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 7. In some embodiments, the antibody or antigen-binding fragment thereof comprises a CDR1-L comprising the amino acid sequence set forth in SEQ ID NO: 4, a CDR2-L comprising the amino acid sequence NTR, a CDR3-L comprising the amino acid sequence set forth in SEQ ID NO: 6, and a VL comprising at least 80%, 85%, 90%, 95%, or 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 8.

[0222] In other embodiments, the antibody or antigen-binding fragment thereof comprises a VL comprising CDR1-L, CDR2-L, and CDR3-L, wherein the CDRs of the VL have up to 1, 2, 3, 4, or 5 overall amino acid changes relative to the corresponding CDR reference sequences, where the CDR1-L reference sequence has the amino acid sequence of SEQ ID NO: 4, the CDR2-L has the amino acid sequence set forth in SEQ ID NO: 5, and the CDR3-L has the amino acid sequence set forth in SEQ ID NO: 6. In such embodiments, the amino acid changes are typically insertions, deletions, and / or substitutions. In some of these embodiments, the set number of amino acid changes is 1-3; in other embodiments, the set number of amino acid changes is 1 or 2. In some of the foregoing embodiments, the changes are conservative amino acid substitutions.

[0223] In some embodiments, the antibody or antigen-binding fragment thereof comprises a VH comprising a CDR1-H, a CDR2-H, and a CDR3-H, wherein the CDRs of the VH have up to 1, 2, 3, 4, or 5 overall amino acid changes relative to the corresponding CDR reference sequences, where the CDR1-H reference sequence has the amino acid sequence of SEQ ID NO: 1, the CDR2-H has the amino acid sequence set forth in SEQ ID NO: 2, and the CDR3-H has the amino acid sequence set forth in SEQ ID NO: 3. In such embodiments, the amino acid changes are typically insertions, deletions, and / or substitutions. In some of these embodiments, the set of amino acid changes number from 1 to 3; in other embodiments, the set of amino acid changes number is 1 or 2. In some of the foregoing embodiments, the changes are conservative amino acid substitutions.

[0224] In some embodiments, the antibody or antigen-binding fragment thereof comprises a VH comprising CDR1-H, CDR2-H, and CDR3-H, wherein the CDRs of the VH have up to 1, 2, 3, 4, or 5 amino acid changes overall relative to the corresponding CDR reference sequences, where the CDR1-H reference sequence has the amino acid sequence of SEQ ID NO: 1, the CDR2-H has the amino acid sequence set forth in SEQ ID NO: 2, and the CDR3-H has the amino acid sequence set forth in SEQ ID NO: 3, and a VL comprising CDR1-L, CDR2-L, and CDR3-L, wherein the CDRs of the VL have up to 1, 2, 3, 4, or 5 amino acid changes overall relative to the corresponding CDR reference sequences, where the CDR1-L reference sequence has the amino acid sequence of SEQ ID NO: 4, the CDR2-L has the amino acid sequence set forth in SEQ ID NO: 5, and the CDR3-L has the amino acid sequence set forth in SEQ ID NO: 6. In such embodiments, the amino acid changes are typically insertions, deletions, and / or substitutions. In some of these embodiments, the set number of amino acid changes is 1 to 3, and in other embodiments, the set number of amino acid changes is 1 or 2. In some of the foregoing embodiments, the changes are conservative amino acid substitutions.

[0225] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain having at least 80%, 85%, 90%, 95%, or 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 9. In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain having at least 80%, 85%, 90%, 95%, or 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 10. In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain having at least 80%, 85%, 90%, 95%, or 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 9 and a light chain having at least 80%, 85%, 90%, 95%, or 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 10.

[0226] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 9. In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain comprising the amino acid sequence set forth in SEQ ID NO: 10. In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 9 and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 10.

[0227] In some embodiments, the antibody or antigen-binding fragment thereof comprises a VH domain, wherein the VH domain sequence has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to an amino acid sequence selected from any one of SEQ ID NOs: 7, provided that the antibody or antigen-binding fragment thereof retains the ability to bind to CEACAM5. In certain embodiments, such antibodies or antigen-binding fragments thereof contain substitutions (e.g., conservative substitutions), insertions, and / or deletions relative to the reference sequence (i.e., one of SEQ ID NOs: 7), provided that such antibodies or antigen-binding fragments thereof retain the ability to bind to CEACAM5. In certain embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids have been substituted, inserted, and / or deleted in any one of SEQ ID NOs: 7. In some embodiments, 1 to 5 or 1 to 3 amino acids have been substituted, inserted, and / or deleted in the VH sequence. In some of these embodiments, such substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., within the FRs). In further such embodiments, the VH comprises one, two, or three CDRs selected from (a) a CDR1-H comprising the amino acid sequence of SEQ ID NO: 1, (b) a CDR2-H comprising the amino acid sequence of SEQ ID NO: 2, and (c) a CDR3-H comprising the amino acid sequence of SEQ ID NO: 3.

[0228] In some embodiments, the antibody or antigen-binding fragment thereof comprises a VL domain, wherein the VL domain sequence has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to an amino acid sequence selected from any one of SEQ ID NOs: 8, provided that the antibody or antigen-binding fragment thereof retains the ability to bind to CEACAM5. In certain embodiments, such antibodies or antigen-binding fragments thereof contain substitutions (e.g., conservative substitutions), insertions, and / or deletions relative to the reference sequence (i.e., one of SEQ ID NOs: 8), provided that such antibodies or antigen-binding fragments thereof retain the ability to bind to CEACAM5. In certain embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids have been substituted, inserted, and / or deleted in any one of SEQ ID NOs: 8. In some embodiments, 1 to 5 or 1 to 3 amino acids have been substituted, inserted, and / or deleted in the VL sequence. In some of these embodiments, such substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., within the FRs). In further such embodiments, the VL comprises one, two, or three CDRs selected from: (a) a CDR1-L comprising the amino acid sequence of SEQ ID NO: 4; (b) a CDR2-L comprising the amino acid sequence of SEQ ID NO: 5; and (c) a CDR3-L comprising the amino acid sequence of SEQ ID NO: 6.

[0229] In a further embodiment, the antibody or antigen-binding fragment thereof comprises (a) a VH domain, wherein the VH domain sequence has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to an amino acid sequence selected from any one of SEQ ID NOs: 7, and (b) a VL domain, wherein the VL domain sequence has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to an amino acid sequence selected from any one of SEQ ID NOs: 8, provided that the antibody or antigen-binding fragment thereof retains the ability to bind to CEACAM5. In certain embodiments, such antibodies or antigen-binding fragments thereof contain substitutions (e.g., conservative substitutions), insertions, and / or deletions relative to the reference sequences (i.e., SEQ ID NO: 7 for the VH domain and SEQ ID NO: 8 for the VL domain), provided that such antibodies or antigen-binding fragments thereof retain the ability to bind to CEACAM5. In certain embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids are substituted, inserted, and / or deleted in the VH and / or VL sequences. In some embodiments, 1 to 5 or 1 to 3 amino acids are substituted, inserted, and / or deleted entirely in the VH and / or VL sequences. In other embodiments, 1 to 5 or 1 to 3 amino acids are substituted, inserted, and / or deleted entirely in the VH and VL sequences. In certain embodiments, such substitutions, insertions, or deletions occur in regions outside the HVRs (i.e., in the FRs). In further such embodiments, the VL comprises one, two, or three CDRs selected from (a) a CDR1-L comprising the amino acid sequence of SEQ ID NO: 4, (b) a CDR2-L comprising the amino acid sequence of SEQ ID NO: 5, and (c) a CDR3-L comprising the amino acid sequence of SEQ ID NO: 6, and the VH comprises one, two, or three CDRs selected from (a) a CDR1-H comprising the amino acid sequence of SEQ ID NO: 1, (b) a CDR2-H comprising the amino acid sequence of SEQ ID NO: 2, and (c) a CDR3-H comprising the amino acid sequence of SEQ ID NO: 3.

[0230] The antigen-binding protein in any of the foregoing embodiments may be an antibody in any form. Thus, the antigen-binding protein according to any of the above embodiments may be, for example, a monoclonal antibody, a multispecific antibody, a human, humanized, or chimeric antibody, and an antigen-binding fragment of any of the above, such as a single-chain antibody, a Fab fragment, a F(ab') fragment, or a fragment produced by a Fab expression library. The antibody may be of any immunoglobulin isotype (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass.

[0231] In certain embodiments, antibodies or antigen-binding fragments thereof having HVR and / or variable domain sequences described herein are antigen-binding fragments (e.g., human antigen-binding fragments), including, but not limited to, Fab, Fab' and F(ab'), Fd, single-chain Fv (scFv), single-chain antibodies, disulfide-linked Fv (sdFv), and fragments comprising either a VL or VH domain. Antigen-binding fragments comprising single-chain antibodies can comprise the variable region alone or in combination with all or a portion of the following: hinge region, CH1 domain, CH2 domain, CH3 domain, and CL domain. Antigen-binding fragments also comprising any combination of the variable region and the hinge region, CH1 domain, CH2 domain, CH3 domain, and CL domain are included in the present disclosure.

[0232] Antibodies or antigen-binding fragments thereof may be monospecific, bispecific, trispecific, or of higher order multispecificity. Multispecific antibodies may be specific for different epitopes of CEACAM5, or may be specific for both CEACAM5 and a heterologous protein. See, e.g., PCT Publication Nos. WO93 / 17715, WO92 / 08802, WO91 / 00360, WO92 / 05793, Tutt, et al., 1991, J. Immunol. 147:6069, U.S. Patent Nos. 4,474,893, 4,714,681, 4,925,648, 5,573,920, 5,601,819, and Kostelny et al., 1992, J. Immunol. 148:15471553.

[0233] In any of the embodiments described herein, one or more amino acids (e.g., 1, 2, 3, or 4) at the amino or carboxy termini of the light and / or heavy chains, e.g., the C-terminal lysine of the heavy chain, may be deleted or derivatized in some or all of the molecules in the composition. One specific example of such a modification is an antibody or antigen-binding fragment thereof in which the carboxy-terminal lysine of the heavy chain is deleted (e.g., as part of a post-translational modification). Furthermore, it should be understood that any of the sequences described herein include post-translational modifications to that particular sequence during expression of the antibody or antigen-binding fragment thereof in cell culture (e.g., CHO cell culture).

[0234] 1. Humanized antigen-binding proteins In certain embodiments, the antibody or antigen-binding fragment thereof is a humanized antibody that binds to CEACAM5. Typically, non-human antibodies are humanized to reduce immunogenicity in humans while retaining the specificity and affinity of the parent non-human antibody. Humanized antibodies are genetically engineered antibodies in which HVRs (e.g., CDRs) or portions thereof from a non-human "donor" antibody are grafted onto human "acceptor" antibody sequences (see, e.g., Queen, US Pat. Nos. 5,530,101 and 5,585,089; Winter, US Pat. No. 5,225,539; Carter, US Pat. No. 6,407,213; Adair, US Pat. No. 5,859,205; and Foote, US Pat. No. 6,881,557).

[0235] The acceptor antibody sequence can be, for example, a mature human antibody sequence, a composite of such sequences, a consensus sequence of human antibody sequences, or a germline region sequence. The human acceptor sequence can be selected for a high degree of sequence identity with the donor sequence in the variable region framework to match canonical configurations among the acceptor and donor HVRs or CDRs, among other criteria. Thus, a humanized antibody is an antibody with HVRs or CDRs, if present, entirely or substantially, from the donor antibody and variable region framework sequences and constant regions, and, if present, entirely or substantially, from human antibody sequences. Similarly, a humanized heavy chain typically derives entirely or substantially from a donor antibody heavy chain and heavy chain variable region framework sequences and heavy chain constant region, with all three HVRs or CDRs, if present, derived substantially from human heavy chain variable region framework and constant region sequences. Similarly, a humanized light chain typically derives entirely or substantially from a donor antibody light chain and light chain variable region framework sequences and light chain constant region, with all three CDRs, if present, derived substantially from human light chain variable region framework and constant region sequences. An HVR or CDR in a humanized antibody is substantially derived from a corresponding HVR or CDR in a non-human antibody when at least 80%, 85%, 90%, 95%, or 100% of the corresponding residues (as defined by Kabat) are identical between the respective HVRs or CDRs. An antibody chain variable region framework sequence or antibody chain constant region is substantially derived from a human variable region framework sequence or human constant region, respectively, when at least 80%, 85%, 90%, 95%, or 100% of the corresponding residues as defined by Kabat are identical.

[0236] Humanized antibodies often incorporate all six HVRs (e.g., CDRs, preferably as defined by Kabat) from a murine antibody, although they can also be generated with less than all HVRs or CDRs (e.g., at least three, four, or five) from a murine antibody (e.g., Pascalis et al., J. Immunol. 169:3076, 2002; Vajdos et al., Journal of Molecular Biology, 320:415-428, 2002; Iwahashi et al., Mol. Immunol. 36:1079-1091, 1999; and Tamura et al., Journal of Immunology, 164:1432-1441, 2000).

[0237] Certain amino acids from the human variable region framework residues can be selected for substitution based on their possible effect on HVR (e.g., CDR) conformation and / or binding to antigen. Examination of such possible effects can be performed by modeling, by examining the properties of amino acids at particular positions, or by empirical observation of the effects of substituting or mutagenesing specific amino acids.

[0238] For example, if an amino acid differs between a murine variable region framework residue and a selected human variable region framework residue, the human framework amino acid can be substituted with the equivalent framework amino acid from the murine antibody if the amino acid is reasonably predicted to be: (1) binds directly to the antigen noncovalently; (2) adjacent to the HVR or CDR region; (3) otherwise interacts with an HVR or CDR region (e.g., is within about 6 Å of such region); (4) mediating the interaction between heavy and light chains; or (5) is the result of somatic mutations in the mouse chain; (6) Glycosylation site.

[0239] Framework residues from classes (1) to (3) are sometimes alternatively referred to as canonical residues and Vernier residues. Canonical residues refer to framework residues that define the canonical class of donor CDR loops, which determine the structure of the CDR loops (Chothia and Lesk, J. Mol. Biol. 196, 901-917 (1987); Thornton & Martin, J. Mol. Biol., 263, 800-815, 1996). Vernier residues refer to a layer of framework residues that support the antigen-binding loop structure and play a role in fine-tuning the antibody's fit to the antigen (Foote & Winter, 1992, J. Mol. Biol. 224, 487-499).

[0240] Humanized antibodies and methods for making them are reviewed, for example, in Almagro and Fransson, (2008) Front. Biosci. 13:1619-1633, and are also described in, for example, Riechmann et al., (1988) Nature 332:323-329, Queen et al., (1989) Proc. Natl. Acad. Sci. USA 86:10029-10033, U.S. Patent Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409, Kashmiri et al., (2005) Methods 36:25-34 (describing specificity-determining region (SDR) grafting), Padlan, (1991) Mol. Immunol. 28:489-498 (describing "resurfacing"), Dall'Acqua et al., (2005) Methods 36:43-60 (describing "FR shuffling"), and Osbourn et al., (2005) Methods 36:61-68 and Klimka et al., (2000) Br. J. Cancer, 83:252-260 (describing a "guided selection" approach to FR shuffling).

[0241] Human framework regions that can be used for humanization include framework regions selected using the "best-fit" method (see, e.g., Sims et al. (1993) J. Immunol. 151:2296), framework regions derived from consensus sequences of human antibodies of particular subgroups of light or heavy chain variable regions (see, e.g., Carter et al. (1992) Proc. Natl. Acad. Sci. USA, 89:4285, and Presta et al. (1993) J. Immunol, 151:2623), human mature (somatically mutated) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, (2008) Front. Biosci. 13:1619-1633), and framework regions derived from screening FR libraries (see, e.g., Baca et al. al., (1997) J. Biol. Chem. 272:10678-10684 and Rosok et al., (1996) J. Biol. Chem. 271:22611-22618).

[0242] 2. Exemplary Antibody Constant Regions In some embodiments, the heavy and light chain variable regions of the antibodies described herein may be linked to at least a portion of a human constant region. In some embodiments, the human heavy chain constant region is of an isotype selected from IgA, IgG, and IgD. In some embodiments, the human light chain constant region is of an isotype selected from Kappa and Lambda. In some embodiments, the antibodies described herein comprise a human IgG constant region. In some embodiments, the antibodies described herein comprise a human IgG4 heavy chain constant region. In some of these embodiments, the antibodies described herein comprise a S241P mutation in the human IgG4 constant region. In some embodiments, the antibodies described herein comprise a human IgG4 constant region and a human Kappa light chain.

[0243] Throughout this specification and claims, unless otherwise specified or known to those of skill in the art, the numbering of residues in immunoglobulin heavy chains is that of the EU index as set forth in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991), expressly incorporated herein by reference. "EU index as in Kabat" refers to the residue numbering of the human IgG1 EU antibody.

[0244] Human constant regions exhibit allotypic and isoallotypic variation between different individuals; i.e., the constant region can differ between different individuals at one or more polymorphic positions. Isoallotypes differ from allotypes in that sera that recognize the isoallotype bind to non-polymorphic regions of one or more other isotypes. Reference to a human constant region includes a constant region with any naturally occurring allotype, or any substitution of a residue occupying a polymorphic position within a naturally occurring allotype. Also, there can be up to 1, 2, 5, or 10 mutations relative to a naturally occurring human constant region, such as those listed above, to decrease Fcγ receptor binding or increase binding to FcRn.

[0245] In some embodiments, one or more amino acids at the amino or carboxy termini of the light and / or heavy chains, such as the C-terminal lysine of the heavy chain, can be deleted or derivatized in a proportion or all of the molecules.

[0246] The choice of constant region depends in part on whether antibody-dependent cell-mediated cytotoxicity, antibody-dependent cellular phagocytosis, and / or complement-dependent cytotoxicity is desired. For example, human isotypes IgG1 and IgG3 have strong complement-dependent cytotoxicity, human isotype IgG2 has weak complement-dependent cytotoxicity, and human IgG4 has no complement-dependent cytotoxicity. Human IgG1 and IgG3 also induce stronger cell-mediated effector functions than human IgG2 and IgG4. The light chain constant region can be lambda or kappa.

[0247] Additionally, as described in more detail below, substitutions can be made in the constant region to reduce or increase effector functions such as complement-mediated cytotoxicity or ADCC (see, e.g., Winter et al., U.S. Patent No. 5,624,821; Tso et al., U.S. Patent No. 5,834,597; and Lazar et al., Proc. Natl. Acad. Sci. USA 103:4005, 2006), or to increase half-life in humans (see, e.g., Hinton et al., J. Biol. Chem. 279:6213, 2004).

[0248] 3. Variants The antigen-binding proteins provided herein also include amino acid sequence variants of the antigen-binding proteins provided herein. As an example, variants can be prepared that have improved antibody binding affinity and / or other biological properties. Amino acid sequence variants of antigen-binding proteins can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antigen-binding protein or by peptide synthesis. Such modifications include, for example, deletions from, and / or insertions into, and / or substitutions of, residues within the amino acid sequence of the antigen-binding protein. Any combination of deletions, insertions, and substitutions can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics, e.g., antigen binding.

[0249] In some embodiments, the antigen binding protein is a variant in that it has one or more amino acid substitutions, deletions, and / or insertions relative to the antigen binding proteins described herein. In certain such embodiments, the variant has one or more amino acid substitutions. In further such embodiments, the substitutions are conservative amino acid substitutions.

[0250] Amino acid substitutions can include, but are not limited to, the substitution of one amino acid in a polypeptide for another. Conservative amino acid substitutions can include non-naturally occurring amino acid residues, which are typically incorporated by chemical peptide synthesis rather than by synthesis in biological systems. Naturally occurring residues can be grouped into classes based on common side chain properties: (1) Hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile, (2) Neutral hydrophilicity: Cys, Ser, Thr, Asn, Gln, (3) Acidic: Asp, Glu, (4) Basic: His, Lys, Arg, (5) Residues that affect chain orientation: Gly, Pro, (6) Aromatic: Trp, Tyr, Phe. [Table 4]

[0251] Non-conservative substitutions involve exchanging a member of one of these classes for another class.

[0252] In modifying the amino acid sequence of an antigen binding protein (eg, an anti-CEACAM5 antibody), in some embodiments, the hydropathic index of amino acids can be considered. Each amino acid has been assigned a hydropathic index based on its hydrophobicity and charge characteristics as follows: isoleucine (+4.5), valine (+4.2), leucine (+3.8), phenylalanine (+2.8), cysteine / cystine (+2.5), methionine (+1.9), alanine (+1.8), glycine (-0.4), threonine (-0.7), serine (-0.8), tryptophan (-0.9), tyrosine (-1.3), proline (-1.6), histidine (-3.2), glutamic acid (-3.5), glutamine (-3.5), aspartic acid (-3.5), asparagine (-3.5), lysine (-3.9), and arginine (-4.5).

[0253] The importance of the hydropathic amino acid index in conferring interactive biological function on a protein is understood in the art. Kyte et al., 1982, J. Mol. Biol., 157:105-131. It is known that certain amino acids can be replaced with other amino acids having a similar hydropathic index or score and still retain similar biological activity. When making changes based on hydropathic index, certain embodiments include substitutions of amino acids whose hydropathic index is within ±2, certain embodiments include those within ±1, and certain embodiments include those within ±0.5.

[0254] It is also understood in the art that substitutions of like amino acids can be made effectively on the basis of hydrophilicity, particularly when the biologically functional proteins or peptides (e.g., antibodies) so generated are intended for use in immunological embodiments, as is the case here. In certain embodiments, the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with its immunogenicity and antigenicity, i.e., with a biological property of the protein.

[0255] The following hydrophilicity values ​​have been assigned to these amino acid residues: arginine (+3.0); lysine (+3.0±1); aspartate (+3.0±1); glutamate (+3.0±1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5±1); alanine (-0.5); histidine (-0.5); cysteine ​​(-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5), and tryptophan (-3.4). When making changes based on similar hydrophilicity values, certain embodiments include substitutions of amino acids whose hydrophilicity values ​​are within ±2, certain embodiments include those within ±1, and certain embodiments include those within ±0.5. Epitopes can also be identified from primary amino acid sequences on the basis of hydrophilicity. These regions are also called "epitope core regions."

[0256] For example, modifications (e.g., substitutions) can be made in HVRs to improve antibody affinity. Such changes can be made in HVR "hot spots," i.e., residues encoded by codons that undergo frequent mutation during the somatic maturation process (see, e.g., Chowdhury, Methods Mol. Biol. 207:179-196 (2008)), and / or antigen-contacting residues, and the resulting variant VH or VL are tested for binding affinity. Affinity maturation by constructing and reselecting secondary libraries is described, for example, in Hoogenboom et al. in Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, (2001)). In some embodiments of affinity maturation, diversity is introduced into the variable genes selected for maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis). A secondary library is then created. The library is then screened to identify any antibody variants with the desired affinity. Another method for introducing diversity involves an HVR-directed approach, in which several HVR residues (e.g., 4-6 residues at a time) are randomized. HVR residues involved in antigen binding can be specifically identified, for example, using alanine scanning mutagenesis or modeling. CDR-H3 and CDR-L3 are often specifically targeted.

[0257] In certain embodiments, substitutions, insertions, or deletions may be made within one or more HVRs, so long as such changes do not substantially reduce the antibody's ability to bind to antigen. For example, conservative modifications (e.g., conservative substitutions provided herein) that do not substantially reduce binding affinity may be made in HVRs. Such changes may, for example, be outside of antigen-contact residues within the HVRs. In certain embodiments of the variant VH and VL sequences provided above, each HVR is either unaltered or contains no more than one, two, or three amino acid substitutions.

[0258] A useful method for identifying antibody residues or regions that can be targeted for mutagenesis is called "alanine scanning mutagenesis," described in Cunningham and Wells (1989) Science, 244:1081-1085. In this method, a target residue or group of target residues (e.g., charged residues such as arg, asp, his, lys, and glu) is identified and replaced with neutral or negatively charged amino acids (e.g., alanine or polyalanine) to determine whether the interaction between the antibody and antigen is affected. Further substitutions can be introduced at amino acid positions that demonstrate functional sensitivity to the initial substitution. Alternatively, or in addition, a crystal structure of the antigen-antibody complex can be used to identify contact points between the antibody and antigen. Such contact residues and neighboring residues can be targeted or eliminated as candidates for substitution. Variants can be screened to determine whether they contain desired properties.

[0259] Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing 100 or more residues, as well as intrasequence insertions of single or multiple amino acid residues. An example of a terminal insertion is an antibody with an N-terminal methionyl residue. Other insertional variants of the antibody molecule include the fusion to the N- or C-terminus of the antibody to an enzyme (e.g., ADEPT) or a polypeptide which increases the serum half-life of the antibody.

[0260] Antibodies with reduced effector function include antibodies with substitutions of one or more of Fc region residues 238, 265, 269, 270, 297, 327, and 329 (U.S. Patent No. 6,737,056). Such Fc variants include Fc variants with substitutions at two or more of amino acid positions 265, 269, 270, 297, and 327, including the so-called "DANA" Fc variant with substitutions of residues 265 and 297 to alanine (U.S. Patent No. 7,332,581).

[0261] In certain embodiments, antibody variants are prepared with improved or diminished binding to FcR. (See, e.g., U.S. Pat. No. 6,737,056, WO 2004 / 056312, and Shields et al., J. Biol. Chem. 9(2):6591-6604 (2001)). In some embodiments, the antibody variant comprises an Fc region with one or more amino acid substitutions that improve ADCC, e.g., substitutions at positions 298, 333, and / or 334 of the Fc region (EU numbering of residues). For example, systemic substitution of solvent-exposed amino acids in the Fc region of human IgG1 has produced IgG variants with altered Fc^R binding affinity (Shields et al., 2001, J. Biol. Chem. 276:6591-604). When compared to the parent IgG1, a subset of these variants with substitutions at Thr256 / Ser298, Ser298 / Glu333, Ser298 / Lys334, or Ser298 / Glu333 / Lys334 to Ala show increased both binding affinity to Fc^R and ADCC activity (Shields et al., 2001, J. Biol. Chem. 276:6591-604; Okazaki et al., 2004, J. Mol. Biol. 336:1239-49).

[0262] In some embodiments, changes are made in the Fc region to alter (i.e., either improve or decrease) Clq binding and / or complement-dependent cytotoxicity (CDC), e.g., as described in U.S. Patent No. 6,194,551, WO 99 / 51642, and Idusogie et al. J. Immunol. 164:4178-4184 (2000). For example, the complement fixation activity of an antibody (both Clq binding and CDC activity) can be improved by substitutions at Lys326 and Glu333 (Idusogie et al., 2001, J. Immunol. 166:2571-2575). The same substitutions on the human IgG2 backbone can convert antibody isotypes that bind poorly to C1q and have severely deficient complement activation activity into antibody isotypes that can bind C1q and mediate CDC (Idusogie et al., 2001, J. Immunol. 166:2571-75). Several other methods have also been applied to improve the complement fixation activity of antibodies. For example, grafting the 18-amino acid carboxyl-terminal tailpiece of IgM to the carboxyl terminus of IgG significantly enhances their CDC activity. This is also observed with IgG4, which normally has no detectable CDC activity (Smith et al., 1995, J. Immunol. 154:2226-36). Furthermore, Ser444, located near the carboxy terminus of the IgG1 heavy chain, is replaced by Cys, which induces tail-to-tail dimerization of IgG1 and a 200-fold increase in CDC activity over monomeric IgG1 (Shopes et al., 1992, J. Immunol. 148:2918-22). In addition, bispecific diabody constructs with specificity for C1q also confer CDC activity (Kontermann et al., 1997, Nat. Biotech. 15:629-31).

[0263] Complement activity can be reduced by mutating at least one of amino acid residues 318, 320, and 322 of the heavy chain to a residue with a different side chain, such as Ala. Other alkyl-substituted non-ionic residues, such as Gly, Ile, Leu, or Val, or aromatic non-polar residues, such as Phe, Tyr, Trp, and Pro, in place of any one of these residues, also reduce or eliminate C1q binding. To reduce or eliminate C1q binding activity, Ser, Thr, Cys, and Met can be used at residues 320 and 322 instead of 318. Replacement of residue 318 (Glu) with a polar residue can modify, but not eliminate, C1q binding activity. Replacement of residue 297 (Asn) with Ala eliminates lytic activity but only slightly reduces affinity for C1q (approximately three-fold weaker). This change destroys the glycosylation site and the presence of carbohydrates required for complement activation. Other substitutions at this site also destroy glycosylation sites. The following mutations, and any combination thereof, also reduce C1q binding: D270A, K322A, P329A, and P311S (see WO06 / 036291).

[0264] The half-life of the antibodies provided herein can be increased or decreased to modify their therapeutic activity. FcRn is a receptor structurally similar to MHC class I antigens that noncovalently associates with β2-microglobulin. FcRn regulates the catabolism of IgG and its transcytosis across tissues (Ghetie and Ward, 2000, Annu. Rev. Immunol. 18:739-766; Ghetie and Ward, 2002, Immunol. Res. 25:97-113). IgG-FcRn interaction occurs at pH 6.0 (the pH of intracellular vesicles) but not at pH 7.4 (the pH of blood). This interaction allows IgG to return to the circulation (Ghetie and Ward, 2000, Ann. Rev. Immunol. 18:739-766; Ghetie and Ward, 2002, Immunol. Res. 25:97-113). The region on human IgG1 involved in FcRn binding has been mapped (Shields et al., 2001, J. Biol. Chem. 276:6591-604). Alanine substitutions at Pro238, Thr256, Thr307, Gln311, Asp312, Glu380, Glu382, or Asn434 of human IgG1 enhance FcRn binding (Shields et al., 2001, J. Biol. Chem. 276:6591-604). IgG1 molecules with these substitutions have a longer serum half-life. Therefore, these modified IgG1 molecules may be able to perform their effector functions, and therefore exert their therapeutic effects, for a longer period of time compared to unmodified IgG1. Other exemplary substitutions for increasing binding to FcRn include Gln at position 250 and / or Leu at position 428.Other studies have shown that binding of the Fc region to FcRn can be improved by introducing one or more substitutions at one or more of the following Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, ​​413, 424, or 434, e.g., substitution of Fc region residue 434 (see, e.g., U.S. Pat. Nos. 7,371,826 and 7,361,740).

[0265] In certain embodiments, the antibodies provided herein contain one or more modifications that increase or decrease the extent to which the antibody is glycosylated. Addition or deletion of glycosylation sites to an antibody can be conveniently accomplished by altering the amino acid sequence such that one or more glycosylation sites are created or removed.

[0266] If the antibody contains an Fc region, the carbohydrate attached thereto can be modified. Natural antibodies produced by mammalian cells typically contain branched, biantennary oligosaccharides that are generally attached by an N-linkage to Asn297 in the CH2 domain of the Fc region. See, for example, Wright et al. TIBTECH 15:26-32 (1997). The oligosaccharides can contain various carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose attached to GlcNAc in the "stalk" of the biantennary oligosaccharide structure.

[0267] Engineering this glycoform on IgG can significantly improve IgG-mediated ADCC. Addition of this glycoform or removal of fucose (Shields et al., 2002, J. Biol. Chem. 277:26733-40; Shinkawa et al., 2003, J. Biol. Chem. 278:6591-604; Niwa et al., 2004, Cancer Res. 64:2127-33) and bisecting N-acetylglucosamine modifications from this glycoform (Umana et al., 1999, Nat. Biotechnol. 17:176-180; Davies et al., 2001, 288-94) are two examples of IgG Fc engineering that improve binding between IgG Fc and Fc^R, thereby enhancing Ig-mediated ADCC activity. Antibodies containing such substitutions or engineering are included in some of the embodiments provided herein.

[0268] In certain embodiments, antibodies are provided having carbohydrate structures lacking fucose attached (directly or indirectly) to the Fc region. For example, the amount of fucose in such antibodies can be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose in the glycan at Asn297 relative to the sum of all glycans (e.g., complex, hybrid, and high-mannose structures) attached to Asn297, as measured by MALDI-TOF mass spectrometry, for example, as described in WO 2008 / 077546. Asn297 refers to the asparagine residue located at approximately position 297 of the Fc region (EU numbering of Fc region residues). However, Asn297 may also be located approximately ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300, due to minor sequence variations in antibodies. Such fucosylation variants may have improved ADCC function. See, for example, U.S. Patent Publication No. 2003 / 0157108 (Presta, L.) and U.S. Patent Application Publication No. 2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd.). Examples of publications related to "defucosylated" or "fucose-deficient" antibody variants include: US2003 / 0157108, WO2000 / 61739, WO2001 / 29246, US2003 / 0115614, US2002 / 0164328, US2004 / 0093621, US2004 / 0 132140, US2004 / 0110704, US2004 / 0110282, US2004 / 0109865, WO2003 / 085119, WO200 3 / 084570, WO2005 / 035586, WO2005 / 035778, WO2005 / 053742, WO2002 / 031140, Okazaki et al.J.Mol.Biol.336:1239-1249(2004), Yamane-Ohnuki et al.Biotech.Bioeng.87:614(2004).Examples of cell lines capable of producing defucosylated antibodies include Lec13 CHO cells, which lack protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); U.S. Patent Application No. US2003 / 0157108A1; Presta, L.; and WO2004 / 056312A1; Adams et al., especially in Example 11), and knockout cell lines, such as α-1,6-fucosyltransferase gene, FUT8, knockout CHO cells (see, e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87:614 (2004); Kanda, Y. et al., Biotechnol. Bioeng., 94(4):680-688 (2006); and WO2003 / 085107).

[0269] Further provided are other antibodies containing bisected oligosaccharides, for example, where a biantennary oligosaccharide attached to the Fc region of the antibody is bisected by GlcNAc. Such antibodies may have reduced fucosylation and / or improved ADCC function. Examples of such antibodies are described, for example, in WO 2003 / 011878 (Jean-Mairet et al.), U.S. Patent No. 6,602,684 (Umana et al.), and US 2005 / 0123546 (Umana et al.). Antibodies with at least one galactose residue in the oligosaccharide attached to the Fc region are also provided. Such antibody variants may have improved CDC function. Such antibody variants are described, for example, in WO 1997 / 30087 (Patel et al.), WO 1998 / 58964 (Raju, S.), and WO 1999 / 22764 (Raju, S.).

[0270] In some embodiments, the antibody variants provided herein comprise a substitution of a native amino acid at position 234, 235, 237, 239, 267, 298, 299, 326, 330, or 332 in the human IgG1 isotype, preferably an S239C mutation (constant region substitutions according to the EU index). The presence of the additional cysteine ​​residue allows for the formation of an interchain disulfide bond. The formation of such an interchain disulfide bond can cause steric hindrance, thereby reducing the affinity of the Fc region-FcγR binding interaction. The cysteine ​​residue introduced within or near the Fc region of the IgG constant region can also serve as a site for conjugation to a therapeutic agent (e.g., coupling of a cytotoxic drug using a thiol-specific reagent, such as a maleimide derivative of the drug). The presence of the therapeutic agent can cause steric hindrance, thereby further reducing the affinity of the Fc region-FcγR binding interaction. Other substitutions at any of positions 234, 235, 236, and / or 237 reduce affinity for Fcγ receptors, particularly the FcγRI receptor (see, e.g., US Pat. No. 6,624,821).

[0271] In other cysteine ​​engineered antibody variants, one or more reactive thiol groups are placed at accessible sites on the antibody, as further described herein, and can be used to conjugate the antibody to other moieties, such as drug moieties or linker-drug moieties, to create immunoconjugates. In certain embodiments, any one or more of the following residues may be substituted with cysteine: V205 (Kabat numbering) of the light chain, A118 (EU numbering) of the heavy chain, and 5400 (EU numbering) of the heavy chain Fc region. The generation of cysteine ​​engineered antibodies is described, for example, in U.S. Patent No. 7,521,541.

[0272] Certain antibodies or antigen-binding fragments thereof that are provided include the following modifications to the constant region:

[0273] Antigen-binding proteins provided herein include those that compete with one of the exemplary antibodies or antigen-binding fragments thereof described above for specific binding to CEACAM5. In some of these embodiments, the test and reference antibodies or antigen-binding fragments thereof cross-compete with each other. Such antibodies or antigen-binding fragments thereof may bind to the same epitope as one of the antigen-binding proteins described herein, or to an overlapping epitope. Antibodies or antigen-binding fragments thereof, including fragments that compete with the exemplary antibodies, are expected to exhibit similar functional properties (e.g., one or more of the activities described above). Exemplary antibodies or antigen-binding fragments thereof include those described above, including those having 1) heavy and / or light chains, 2) VH and / or VL, and / or 3) comprising one or more of the CDRs provided herein.

[0274] Thus, in some embodiments, the antibodies or antigen-binding fragments thereof provided include those that compete with antibodies having all six of the CDRs listed for the same antibody provided herein.

[0275] In any of the embodiments described in this section, the antibody or antigen-binding fragment thereof can have any combination or all of the activities listed herein. In further such embodiments, the test and reference antibodies or antigen-binding fragments thereof cross-compete with each other.

[0276] In some embodiments, competition or cross-competition is determined by surface plasmon resonance analysis (e.g., BIACORE®) (see, e.g., Abdiche, et al., 2009, Anal. Biochem. 386:172-180; Abdiche, et al., 2012, J. Immunol Methods 382:101-116; and Abdiche, et al., 2014 PLoS One 9:e92451).

[0277] In another embodiment, the antigen-binding proteins provided include those that bind to the same epitope as any of the antibodies or antigen-binding fragments thereof described herein. A variety of techniques are available to identify antibodies or antigen-binding fragments thereof that bind to the same epitope as one or more of the antibodies or antigen-binding fragments thereof described herein. Such methods include, for example, competitive assays as described herein, screening of peptide fragments, MS-based protein footprinting, alanine or glutamine scanning approaches, and through X-ray analysis of crystals of the antigen:antigen-binding protein complex that provide atomic resolution of the epitope.

[0278] One approach to determining the epitope or epitope region bound by a specific antibody (an "epitope region" is a region containing or overlapping with the epitope) involves assessing the binding of the antibody or its antigen-binding fragment to a peptide comprising a fragment of CEACAM5, e.g., a native or denatured fragment. A series of overlapping peptides encompassing the sequence of CEACAM5 (e.g., human CEACAM5) can be prepared and screened for binding, for example, by direct ELISA, competitive ELISA (in which peptides are assessed for their ability to prevent antibody binding to CEACAM5 bound to wells of a microtiter plate), or on a chip. Such peptide screening methods may fail to detect some discontinuous functional epitopes, i.e., functional epitopes involving amino acid residues that are not contiguous along the primary sequence of the CEACAM5 polypeptide chain.

[0279] In other embodiments, regions containing residues that contact or are buried by an antibody can be identified by mutating specific residues in CEACAM5 and determining whether the antibody or its antigen-binding fragment can bind to the mutant or variant CEACAM5 protein. By making several individual mutations, residues that play a direct role in binding or are sufficiently close to the antibody that the mutations can affect binding between the antigen-binding protein and the antigen can be identified. Knowledge of these amino acids can reveal domains or regions of the antigen that contain residues that contact or are covered by the antibody or its antigen-binding fragment. Such domains can contain the antibody's binding epitope. A common approach for such scanning techniques involves replacing amino acids in the wild-type polypeptide with arginine and / or glutamic acid residues (typically individually). These two amino acids are typically used in such scanning techniques because they are charged and bulky, and therefore have the potential to inhibit binding between the antibody and CEACAM5 in the region of CEACAM5 where the mutations are introduced. Arginines present in the wild-type antigen are replaced with glutamic acid. A variety of such individual mutants are obtained and the collected binding results are analyzed to determine which residues affect binding (see, e.g., Naneviz, T., et al., 1995, J. Biol. Chem., 270:37, 21619-21625 and Zupnick, A., et al., 2006, J. Biol. Chem., 281:29, 20464-20473).

[0280] An alternative approach to identifying epitopes is by MS-based protein footprinting, such as hydrogen / deuterium exchange mass spectrometry (HDX-MS) and fast photochemical oxidation of proteins (FPOP). Methods for performing HDX-MS are described, for example, in Wei et al. (2014) Drug Discovery Today 19:95. Methods for performing FPOP are described, for example, in Hambley and Gross (2005) J. American Soc. Mass Spectrometry 16:2057.

[0281] The epitope bound by an antibody or antigen-binding fragment thereof can also be determined by structural methods such as X-ray crystallography, molecular modeling, and nuclear magnetic resonance (NMR) spectroscopy, including NMR determination of the rate of HD exchange of labile amide hydrogens when the antigen is free and when complexed with the antibody or antigen-binding fragment thereof (see, e.g., Zinn-Justin et al. (1992) Biochemistry 31, 11335-11347, and Zinn-Justin et al. (1993) Biochemistry 32, 6884-6891).

[0282] X-ray crystallographic analysis can be achieved using any method known in the art. Examples of crystallization methods are described, for example, by Giege et al. (1994) Acta Crystallogr. D50:339-350 and McPherson (1990) Eur. J. Biochem. 189:1-23. Such crystallization approaches include microbatch (e.g., Chayen (1997) Structure 5:1269-1274), hanging drop vapor diffusion (e.g., McPherson (1976) J. Biol. Chem. 251:6300-6303), seeding, and dialysis. Once formed, the antibody:antigen crystals themselves can be studied using well-known X-ray diffraction techniques and refined using computer software such as X-PLOR (Yale University, 1992, distributed by Molecular Simulations, Inc.; see, e.g., Blundell & Johnson (1985) Meth. Enzymol. 114&115, HW Wyckoff et al., eds., Academic Press, U.S. Patent Application Publication No. 2004 / 0014194) and BUSTER (Bricogne (1993) Acta Cryst. D49:37-60; Bricogne (1997) Meth. Enzymol. 276A:361-423, Carter & Sweet, eds., Roversi et al. (2000) Acta Cryst. D56:1313-1323).

[0283] In some embodiments, the antigen binding protein has an affinity (e.g., EC 50 In some embodiments, the antibody or antigen-binding fragment thereof binds to CEACAM5 with an affinity of 5 to 10 nM, 1 to 5 nM, 500 pM to 1 nM, 100 to 250 pM, 50 to 100 pM, 10 to 50 pM, or 1 to 10 pM.

[0284] 4. Derivatives In some embodiments, the antigen binding protein is a derivative of an antigen binding protein, such as those described herein, which is a derivatized antigen binding protein that can include any molecule or substance that confers a desired property on the antigen binding protein (e.g., antibody or fragment), such as increased half-life for a particular use. Derivatized antigen binding proteins can include, for example, a detectable (or labeled) moiety (e.g., a radioactive, colorimetric, antigenic, or enzymatic molecule, or a detectable bead (such as a magnetic or electrolytic (e.g., gold) bead), a molecule that binds to another molecule (e.g., biotin or streptavidin), a therapeutic or diagnostic moiety (e.g., a radioactive, cytotoxic, or pharmaceutically active moiety), or a molecule that increases the suitability of the antigen binding protein for a particular use (e.g., administration to a subject, such as a human subject, or other in vivo or in vitro use). Examples of molecules that can be used to derivatize antigen binding proteins include albumin (e.g., human serum albumin) and polyethylene glycol (PEG). Albumin-conjugated and PEGylated derivatives of antigen binding proteins can be prepared using techniques well known in the art.

[0285] Other derivatives include covalent or aggregative conjugates of antigen-binding proteins with other proteins or polypeptides, such as by expression of recombinant fusion proteins comprising a heterologous polypeptide fused to the N- or C-terminus of the antigen-binding protein. For example, the conjugated peptide can be a heterologous signal (or leader) polypeptide, such as the yeast α-factor leader, or a peptide such as an epitope tag. Antigen-binding protein-containing fusion proteins can include peptides added to facilitate purification or identification of the antigen-binding protein (e.g., poly-His, or FLAG peptide).

[0286] 5. Oligomers Oligomers comprising one or more antigen-binding proteins are also provided. The oligomers may be in the form of covalently or non-covalently linked dimers, trimers, or higher oligomers. In embodiments, oligomers comprising two or more antigen-binding proteins are provided, with one example being a homodimer. Other oligomers include heterodimers, homotrimers, heterotrimers, homotetramers, heterotetramers, etc.

[0287] One embodiment is directed to oligomers comprising multiple CEACAM5 antigen-binding polypeptides linked via covalent or non-covalent interactions between peptide moieties fused to the CEACAM5 antigen-binding protein. Such peptides can be peptide linkers (spacers) or peptides with oligomerization-promoting properties. As described in more detail below, leucine zippers and certain polypeptides derived from antibodies are among the peptides that can promote oligomerization of antigen-binding proteins that bind to them.

[0288] In certain embodiments, the oligomer comprises 2 to 4 CEACAM5 antigen binding proteins. The CEACAM5 antigen binding protein portion of the oligomer can be in any of the forms described above, for example, variants or fragments.

[0289] In one embodiment, oligomers are prepared using immunoglobulin-derived polypeptides. The preparation of fusion proteins comprising certain heterologous polypeptides fused to various portions of antibody-derived polypeptides (including Fc domains) is described, for example, in Ashkenazi et al., 1991, Proc. Natl. Acad. Sci. USA 88:10535; Byrn et al., 1990, Nature 344:677; and Hollenbaugh et al., 1992, "Construction of Immunoglobulin Fusion Proteins," in Current Protocols in Immunology, Suppl. 4, pages 10.19.1-10.19.11.

[0290] In another embodiment, the antigen-binding protein is a dimer made by fusing the CEACAM5 antigen-binding protein to the Fc region of an antibody. The dimer can be made, for example, by inserting a gene fusion encoding the fusion protein into a suitable expression vector, expressing the gene fusion in a host cell transformed with the recombinant expression vector, and allowing the expressed fusion protein to assemble like an antibody molecule, whereby interchain disulfide bonds are formed between the Fc portions to obtain a dimer.

[0291] Alternatively, the oligomer is a fusion protein comprising multiple CEACAM5 antigen-binding proteins, with or without a peptide linker (spacer peptide). Among suitable peptide linkers are those described in U.S. Patent Nos. 4,751,180 and 4,935,233.

[0292] Another method for preparing oligomeric CEACAM5 antigen-binding protein oligomers involves the use of leucine zippers. Leucine zipper domains are peptides that promote oligomerization of the proteins in which they are found (Landschulz et al., 1988, Science 240:1759). Known leucine zippers include naturally occurring peptides and their derivatives that dimerize or trimerize. Examples of leucine zipper domains suitable for producing soluble oligomeric proteins are described in PCT Application No. WO 94 / 10308, and a leucine zipper derived from pulmonary surfactant protein D (SPD) is described in Hoppe et al., 1994, FEBS Letters 344:191. The use of modified leucine zippers that allow stable trimerization of fused heterologous proteins is described in Fanslow et al., 1994, Semin. Immunol. 6:267-278. In one approach, a recombinant fusion protein comprising a CEACAM5 antigen binding protein fragment or derivative fused to a leucine zipper peptide is expressed in a suitable host cell, and the resulting soluble oligomeric CEACAM5 antigen binding protein fragment or derivative is recovered from the culture supernatant.

[0293] 6. Multispecific antigen-binding proteins In a further aspect, the antibody or antigen-binding fragment thereof may be a multispecific antibody or antigen-binding fragment thereof, e.g., a bispecific antibody. In certain embodiments, the multispecific antibody or antigen-binding fragment thereof is a multispecific antibody having binding specificities for at least two different targets. In some of these embodiments, one of the binding specificities is for CEACAM5 and the other is for a different antigen. In other embodiments, the bispecific antibody binds to two different epitopes of CEACAM5. In some embodiments, the bispecific antibody can be used to bind to an antigen on a target cell and localize a cytotoxic agent to a cell expressing CEACAM5. Bispecific antibodies can be prepared as full-length antibodies or antibody fragments.

[0294] A variety of techniques for producing multispecific antibodies are available, including, for example, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs with different specificities (see Milstein and Cuello, Nature 305:537 (1983), WO 93 / 08829, and Traunecker et al., EMBO J. 10:3655 (1991)), and "knobs-in-holes" engineering (see, e.g., U.S. Pat. No. 5,731,168). Multispecific antibodies can be made by engineering electrostatic steering effects to create antibody Fc-heterodimeric molecules (WO 2009 / 089004 A1), cross-linking two or more antibodies or fragments (see, e.g., U.S. Pat. No. 4,676,980, and Brennan et al., Science, 229:81 (1985)), using leucine zippers to generate bispecific antibodies (see, e.g., Kostelny et al., J. Immunol., 148(5):1547-1553 (1992)), and using "diabody" technology to create bispecific antibody fragments (see, e.g., Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)), and using single-chain Fv (sFv) dimers (see, e.g., Gruber et al., J. Immunol., 90:6444-6448 (1993)). al. J. Immunol., 152:5368 (1994)), and by preparing trispecific antibodies as described, for example, in Tutt et al. J. Immunol. 147:60 (1991).

[0295] Exemplary bispecific antibody molecules provided herein include (i) two antibodies, one specific for CEACAM5 and another specific for a second target, which are conjugated together; (ii) a single antibody having one chain specific for CEACAM5 and a second chain specific for a second molecule; and (iii) a single-chain antibody specific for CEACAM5 and a second molecule. In certain embodiments, the second target / second molecule is a target other than CEACAM5. However, in other embodiments, the second target is a different region or epitope on CEACAM5, such that the bispecific antibody binds to two different epitopes on CEACAM5.

[0296] 7. Other exemplary formats An antigen-binding protein (e.g., an antibody or antigen-binding fragment thereof) can be a single polypeptide or can comprise 2, 3, 4, 5, 6, 7, 8, 9, or 10 (the same or different) polypeptides. In some embodiments where the antibody or antigen-binding fragment thereof is a single polypeptide, the antibody or antigen-binding fragment can comprise a single antigen-binding domain or two antigen-binding domains. In some embodiments where the antibody or antigen-binding fragment is a single polypeptide and comprises two antigen-binding domains, the first and second antigen-binding domains can be identical to or different from one another (and can specifically bind to the same or different antigens or epitopes).

[0297] Different portions of the antigen-binding proteins described herein, e.g., the variable domains of the antibodies described herein, can be arranged in various configurations to yield additional antigen-binding proteins. For example, in some embodiments in which the antibody or antigen-binding fragment is a single polypeptide, the first antigen-binding domain and the second antigen-binding domain (if present) can each independently be selected from the group consisting of a VH domain, a VHH domain, a VNAR domain, and an scFv. In some embodiments in which the antibody or antigen-binding fragment is a single polypeptide, the antibody or antigen-binding fragment can be a BiTE®, (scFv)2, nanobody, nanobody-HSA, DART, TandAb, scDiabody, scDiabody-CH3, scFv-CH-CL-scFv, HSAbody, scDiabody-HAS, tandem-scFv, adnectin, DARPin, fibronectin, and DEP conjugate. Additional examples of antigen-binding domains that can be used when the antibody or antigen-binding fragment is a single polypeptide are known in the art.

[0298] V H The H domain is a single monomeric variable antibody domain that can be found in camelids. NARThe domain is a single monomeric variable antibody domain that can be found in cartilaginous fish. Non-limiting embodiments of VHH domains and VNAR domains are described, for example, in Cromie et al., Curr. Top. Med. Chem. 15:2543-2557, 2016; De Genst et al., Dev. Comp. Immunol. 30:187-198, 2006; De Meyer et al., Trends Biotechnol. 32:263-270, 2014; Kijanka et al., Nanomedicine 10:161-174, 2015; Kovaleva et al., Expert. Opin. Biol. Ther. 14:1527-1539, 2014; Krah et al., Immunopharmacol. Immunotoxicol. 38:21-28, 2016; Mujic-Delic et al., Trends Pharmacol.Sci.35:247-255,2014, Muyldermans,J.Biotechnol.74:277-302,2001, Muyldermans et al.,Trends Biochem.Sci.26:230-235,2001, Muyldermans,Ann.Rev.Biochem.82:775-797,2013, Rahbarizadeh et al.,Immunol.Invest.40:299-338,2011, Van Audenhove et al.,EBioMedicine 8:40-48,2016, Van Bockstaele et al.,Curr.Opin.Investig.Drugs 10:1212-1224,2009, Vincke et al.,Methods Mol. Biol. 911:15-26, 2012, and Wesolowski et al., Med. Microbiol. Immunol. 198:157-174, 2009.

[0299] In some embodiments where the antibody or antigen-binding fragment is a single polypeptide and comprises two antigen-binding domains, the first and second antigen-binding domains can both be VHH domains, or at least one antigen-binding domain can be a VHH domain. In some embodiments where the antibody or antigen-binding fragment is a single polypeptide and comprises two antigen-binding domains, the first and second antigen-binding domains can both be VNAR domains, or at least one antigen-binding domain can be a VNAR domain. In some embodiments where the antibody or antigen-binding domain is a single polypeptide, the first antigen-binding domain is an scFv domain. In some embodiments where the antibody or antigen-binding fragment is a single polypeptide and comprises two antigen-binding domains, the first and second antigen-binding domains can both be scFv domains, or at least one antigen-binding domain can be an scFv domain.

[0300] In some embodiments, an antibody or antigen-binding fragment can comprise two or more polypeptides (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 polypeptides). In some embodiments, an antibody or antigen-binding fragment comprises two or more polypeptides, 2, 3, 4, 5, or 6 of the two or more polypeptides can be identical.

[0301] B. Drugs and Linkers Some embodiments of the invention are described below, which are not intended to limit the invention in any way, followed by a more detailed description of the components that make up the conjugates. Those skilled in the art will understand that each identified conjugate and any selected embodiment thereof is meant to include the full range of each component and linker.

[0302] In some embodiments, an ADC having the formula: L-(QD) p or a salt thereof, provided herein, wherein: L is a ligand unit comprising an antibody or antigen-binding fragment thereof that binds to CEACAM5, the subscript p is an integer from 1 to 16; Q is a linker unit having a formula selected from the group consisting of: -ZA-, -ZA-RL-, -ZA-RL-Y-, ZAS * -W-, -ZAS * -RL-, -ZAB(S * )-RL-, -ZAS * -W-RL-, -ZAS * -RL-Y- and -ZAB(S * )-RL-Y- Z is the stretcher unit, A is a coupling or connector unit, B is a parallel connector unit, S * is the resolving agent, W is a peptide unit; RL is a releasable unit, Y is a spacer unit, D is a Drug unit selected from the group consisting of: [ka] R B is a member selected from the group consisting of H, C-C alkyl, C-C haloalkyl, C-C cycloalkyl, (C-C cycloalkyl)-C-C alkyl-, phenyl, and phenyl-C-C alkyl-; R C is a member selected from the group consisting of C1-C6 alkyl and C3-C6 cycloalkyl; Each R F and R F’are independently -H, C1-C8 alkyl, C1-C8 hydroxyalkyl, C1-C8 aminoalkyl, (C1-C4 alkylamino)-C1-C8 alkyl-, N,N-(C1-C4 hydroxyalkyl)(C1-C4 alkyl)amino-C1-C8 alkyl-, N,N-di(C1-C4 alkyl)amino-C1-C8 alkyl-, N-(C1-C4 hydroxyalkyl)-C1-C8 aminoalkyl, C1-C8 alkyl-C(O)-, C1-C8 hydroxyalkyl-C(O)-, C1-C8 aminoalkyl-C(O)-, C3-C 10 Cycloalkyl, (C3-C 10 Cycloalkyl)-C1-C4 alkyl-, C3-C 10 Heterocycloalkyl, (C3-C 10 a member selected from the group consisting of heterocycloalkyl)-C1-C4 alkyl-, phenyl, phenyl-C1-C4 alkyl-, diphenyl-C1-C4 alkyl-, heteroaryl and heteroaryl-C1-C4 alkyl-; or R F and R F’ each combined with the nitrogen atom to which it is attached forms a 5-, 6-, or 7-membered ring having 0 to 3 substituents selected from the group consisting of halogen, C1-C4 alkyl, —OH, —OC1-C4 alkyl, —NH2, —NHC1-C4 alkyl, and —N(C1-C4 alkyl)2; R B , R C , R F and R F’ wherein the cycloalkyl, heterocycloalkyl, phenyl, and heteroaryl portions of the formula (I) are substituted with 0 to 3 substituents selected from the group consisting of halogen, C1-C4 alkyl, —OH, —OC1-C4 alkyl, —NH2, —NHC1-C4 alkyl, and —N(C1-C4 alkyl)2, and the point of attachment of D to Q is such that Q is -ZA-RL-, -ZA-RL-Y-, -ZAS * -RL-, -ZAB(S * )-RL-, -ZAS * -RL-Y- or -ZAB(S *)-RL-Y- (wherein R is any one of the releasable linkers disclosed herein), via a heteroatom of any one of the hydroxyl functional groups or primary or secondary amine functional groups present on CPT1, CPT2, CPT3, CPT4, CPT5, CPT6, or CPT7; or The attachment point of D to Q is -ZA-, -ZAS * -W- or -ZAB(S * )-W- or Q is -ZAS * -RL-, -ZAB(S * )-RL-, -ZAS * -W-RL- or -ZAB(S * )-W-R- (wherein R is a releasable unit other than a glucuronide unit), via the oxygen atom of a hydroxyl group substituent in the lactone ring of CPT1, CPT2, CPT3, CPT4, CPT5, CPT6 or CPT7; However, R F and R F’ is -H when the point of attachment is the nitrogen atom of the amino group of CPT6; However, -ZA-RL-, -ZA-RL-Y-, -Z AS * -RL-, -ZAB(S * )-RL-, -ZAS * -RL-Y- and -ZAB(S * When -ZA- in )-RL-Y- is other than succinimido-caproyl-β-alanyl and D is CPT1 attached via its amino group, it optionally has a succinimide ring in hydrolyzed form.

[0303] In one group of embodiments, D has the formula CPT5.

[0304] In one group of embodiments, D has the formula CPT2.

[0305] In one group of embodiments, D has the formula CPT3.

[0306] In one group of embodiments, D has the formula CPT4.

[0307] In one group of embodiments, D has the formula CPT1.

[0308] In one group of embodiments, D has the formula CPT6.

[0309] In one group of embodiments, D has the formula CPT7.

[0310] In one group of embodiments, Q has a formula selected from the group consisting of: -ZA-RL- and -ZA-RL-Y-,

[0311] wherein R is a releasable linker that is a glucuronide unit, and the groups Z, A, and Y have the meanings provided above and in any one of the embodiments specifically recited herein.

[0312] In one group of embodiments, Q has a formula selected from the group consisting of: -ZAS * -RL- and -ZAS * -RL-Y-,

[0313] wherein R L is a releasable linker that is a glucuronide unit, and the groups Z, A, S * and Y has the meaning provided above and in any one of the embodiments specifically recited herein.

[0314] In one group of embodiments, Q has a formula selected from the group consisting of: -ZAB(S * )-RL- and -ZAB(S * )-RL-Y-,

[0315] wherein R L is a releasable linker that is a glucuronide unit, and the groups Z, A, S *, B and Y have the meanings provided above and in any one of the embodiments specifically recited herein.

[0316] In another group of embodiments, Q has a formula selected from the group consisting of: -ZA- or -ZA-RL-,

[0317] wherein R L is a releasable linker other than a glucuronide unit, and the groups Z and A have the meanings provided above and in any one of the embodiments specifically recited herein.

[0318] In another group of embodiments, Q has a formula selected from the group consisting of: -ZAS * -RL- and -ZAB(S * )-RL-,

[0319] wherein R L is a releasable linker other than a glucuronide unit, and the groups Z, A, S * and B have the meanings provided above and in any one of the embodiments specifically recited herein.

[0320] In another group of embodiments, Q has a formula selected from the group consisting of: -ZAS * -W- and -ZAB(S * )-W-,

[0321] In the formula, the groups Z, A, S * , B, and W have the meanings provided above and in any one of the embodiments specifically recited herein.

[0322] In another group of embodiments, Q has a formula selected from the group consisting of: -ZAS * -W-RL- and -ZAB(S * )-W-RL-,

[0323] wherein R L is a releasable linker other than a glucuronide unit, and the groups Z, A, S * , B and W have the meanings provided above and in any one of the embodiments specifically recited herein.

[0324] In one group of embodiments, an ADC wherein Q has a formula of -ZA-RL-, -ZA-RL-Y-, -ZAS*-RL-, -ZAS*-RL-Y-, -ZAB(S*)-RL- or -ZAB(S*)-RL-Y-, and comprises a Drug Unit having the Formula CPT1 is represented by the formula: [ka] [ka]

[0325] wherein R is any one of the releasable linkers disclosed herein, preferably R is a glucuronide unit, and the groups L, Z, A, S are each * , B, and Y have the meanings provided above and in any one of the embodiments specifically recited herein, with the proviso that -ZA- in formulae CPT1iN, CPT1iiN, CPT1iiiN, CPT1ivN, CPT1vN, and CPT1viN is other than succinimide-caproyl-β-alanyl and optionally has a succinimide ring in hydrolyzed form.

[0326] In another embodiment, an ADC wherein Q has the formula -ZA-, -ZA-RL-, -ZAS*-W-, -ZAB(S*)-W-, -ZAS*-RL-, -ZAB(S*)-RL-, -ZAS*-W-RL-, and -ZAB(S*)-W-RL-, and comprises a Drug unit having the formula CPT1 is represented by the formula: [ka]

[0327] wherein R is a releasable linker other than a glucuronide unit, and the groups L, Z, A, S * , B and W have the meanings provided above and in any one of the embodiments specifically recited herein.

[0328] In another group of embodiments, an ADC wherein Q has a formula of -Z A-R-, -Z A-R-Y-, -Z A-S*-R-, -Z A-S*-R-Y-, -Z A-S*-R-Y-, -Z A-B(S*)-R- or -Z A-B(S*)-R-Y, and comprises a Drug Unit having the Formula CPT2 is represented by the formula: [ka]

[0329] wherein R is any one of the releasable linkers disclosed herein, preferably R is a glucuronide unit, and the groups L, Z, A, S are each * , B and Y have the meanings provided above and in any one of the embodiments specifically recited herein.

[0330] In another embodiment, an ADC wherein Q has the formula -ZA-, -ZA-RL-, -ZAS*-W-, -ZAB(S*)-W-, -ZAS*-RL-, -ZAB(S*)-RL-, -ZAS*-W-RL-, and -ZAB(S*)-W-RL-, and comprises a Drug unit having the formula CPT2 is represented by the formula: [ka]

[0331] wherein R is a releasable linker other than a glucuronide unit, and the groups L, Z, A, S * , B and W have the meanings provided above and in any one of the embodiments specifically recited herein.

[0332] In one group of embodiments, R of formula CPT2iOa, CPT2iiOa, CPT2iiiOa, CPT2ivOa, CPT2vOa, CPT2viOa, CPT2iOb, CPT2iiOb, CPT2iiiOb, CPT2ivOb, CPT2vOb, CPT2viOb, CPT2viiOb, or CPT2viiiOb B is a moiety selected from the group consisting of -H, C1-C8 alkyl, and C1-C8 haloalkyl.

[0333] In one group of embodiments, R of formula CPT2iOa, CPT2iiOa, CPT2iiiOa, CPT2ivOa, CPT2vOa, CPT2viOa, CPT2iOb, CPT2iiOb, CPT2iiiOb, CPT2ivOb, CPT2vOb, CPT2viOb, CPT2viiOb, or CPT2viiiOb B is selected from the group consisting of C-C cycloalkyl, (C-C cycloalkyl)-C-C alkyl-, phenyl, and phenyl-C-C alkyl-; R B wherein the cycloalkyl and phenyl portions are substituted with 0 to 3 substituents selected from the group consisting of halogen, C1-C4 alkyl, —OH, —OC1-C4 alkyl, —NH2, —NHC1-C4 alkyl, and —N(C1-C4 alkyl)2.

[0334] In another group of embodiments, an ADC wherein Q has a formula of -Z A-R-, -Z A-R-Y-, -Z A-S*-R-, -Z A-S*-R-Y-, -Z A-S*-R-Y-, -Z A-B(S*)-R- or -Z A-B(S*)-R-Y, and comprises a Drug Unit having the Formula CPT3 is represented by the formula: [ka] [ka]

[0335] wherein R is any one of the releasable linkers disclosed herein, preferably R is a glucuronide unit, and the groups L, Z, A, S are each* , B and Y have the meanings provided above and in any one of the embodiments specifically recited herein.

[0336] In another embodiment, an ADC wherein Q has the formula -ZA-, -ZA-RL-, -ZAS*-W-, -ZAB(S*)-W-, -ZAS*-RL-, -ZAB(S*)-RL-, -ZAS*-W-RL-, and -ZAB(S*)-W-RL-, and comprises a Drug unit having the formula CPT3 is represented by the formula: [ka]

[0337] wherein R is a releasable linker other than a glucuronide unit, and the groups L, Z, A, S * , B and W have the meanings provided above and in any one of the embodiments specifically recited herein.

[0338] In one group of embodiments, R of formula CPT3iOa, CPT3iiOa, CPT3iiiOa, CPT3ivOa, CPT3vOa, CPT3viOa, CPT3iO'a, CPT3iiO'a, CPT3iiiO'a, CPT3ivO'a, CPT3vO'a, CPT3viO'a, CPT3iOb, CPT3iiOb, CPT3iiiOb, CPT3ivOb, CPT3vOb, CPT3viOb, CPT3viiOb or CPT3viiiOb C is C1-C6 alkyl.

[0339] In one group of embodiments, R of formula CPT3iOa, CPT3iiOa, CPT3iiiOa, CPT3ivOa, CPT3vOa, CPT3viOa, CPT3iO'a, CPT3iiO'a, CPT3iiiO'a, CPT3ivO'a, CPT3vO'a, CPT3viO'a, CPT3iOb, CPT3iiOb, CPT3iiiOb, CPT3ivOb, CPT3vOb, CPT3viOb, CPT3viiOb or CPT3viiiOb Cis a C3-C6 cycloalkyl.

[0340] In another group of embodiments, an ADC wherein Q has a formula of -Z A-R-, -Z A-R-Y-, -Z A-R-, -Z A-R-Y-, -Z A-R-Y-, -Z A-R- or -Z A-R-Y-, and comprises a Drug Unit having the Formula CPT4 is represented by the formula: [ka]

[0341] wherein R is any one of the releasable linkers disclosed herein, preferably R is a glucuronide unit, and the groups L, Z, A, S are each * , B and Y have the meanings provided above and in any one of the embodiments specifically recited herein.

[0342] In another embodiment, an ADC wherein Q has the formula -ZA-, -ZA-RL-, -ZAS*-W-, -ZAB(S*)-W-, -ZAS*-RL-, -ZAB(S*)-RL-, -ZAS*-W-RL-, and -ZAB(S*)-W-RL-, and comprises a Drug unit having the formula CPT4 is represented by the formula: [ka] [ka]

[0343] wherein R is a releasable linker other than a glucuronide unit, and the groups L, Z, A, S * , B and W have the meanings provided above and in any one of the embodiments specifically recited herein.

[0344] In another group of embodiments, an ADC wherein Q has a formula of -Z A-R-, -Z A-R-Y-, -Z A-R-, -Z A-R-Y-, -Z A-R-, -Z A-R-Y-, -Z A-R-, or -Z A-R-Y-, and comprises a Drug unit having the Formula CPT5 is represented by the formula: [ka] [ka]

[0345] wherein R is any one of the releasable linkers disclosed herein, preferably R is a glucuronide unit, and the groups L, Z, A, S are each * , B and Y have the meanings provided above and in any one of the embodiments specifically recited herein.

[0346] In another embodiment, an ADC wherein Q has the formula -ZA-, -ZA-RL-, -ZAS*-W-, -ZAB(S*)-W-, -ZAS*-RL-, -ZAB(S*)-RL-, -ZAS*-W-RL-, and -ZAB(S*)-W-RL-, and comprises a Drug unit having the formula CPT5 is represented by the formula: [ka]

[0347] wherein R is a releasable linker other than a glucuronide unit, and the groups L, Z, A, S * , B and W have the meanings provided above and in any one of the embodiments specifically recited herein.

[0348] In another group of embodiments, an ADC wherein Q has a formula of -Z A-R-, -Z A-R-Y-, -Z A S*-R-, -Z A S*-R-Y-, -Z A S*-R-Y-, -Z A B(S*)-R- or -Z A B(S*)-R-Y, and comprises a Drug Unit having the Formula CPT6 is represented by the formula: [ka] [ka]

[0349] wherein R is any one of the releasable linkers disclosed herein, preferably R is a glucuronide unit, and the groups L, Z, A, S are each * , B, and Y have the meanings provided above and in any of the embodiments specifically recited herein.

[0350] In other embodiments, an ADC wherein Q has the formula -ZA-, -ZA-RL-, -ZAS*-W-, -ZAB(S*)-W-, -ZAS*-RL-, -ZAB(S*)-RL-, -ZAS*-W-RL-, and -ZAB(S*)-W-RL-, and comprises a Drug unit having the formula CPT6 is represented by the formula: [ka] [ka]

[0351] wherein R is a releasable linker other than a glucuronide unit, and the groups L, Z, A, S * , B and W have the meanings provided above and in any one of the embodiments specifically recited herein.

[0352] In one group of embodiments, R of formula CPT6iN, CPT6iiN, CPT6iiiN, CPT6ivN, CPT6vN or CPT6viN F is -H.

[0353] In one group of embodiments, both R of formula CPT6iOa, CPT6iiOa, CPT6iiiOa, CPT6ivOa, CPT6vOa, CPT6viOa, CPT6iOb, CPT6iiOb, CPT6iiiOb, CPT6ivOb, CPT6vOb, CPT6viOb, CPT6viiOb or CPT6viiiOb F and R F’ is -H.

[0354] In one group of embodiments, R of formula CPT6iN, CPT6iiN, CPT6iiiN, CPT6ivN, CPT6vN or CPT6viN F is a moiety selected from the group consisting of C-C alkyl, C-C hydroxyalkyl, C-C aminoalkyl, (C-C alkylamino)-C-C alkyl-, N,N-(C-C hydroxyalkyl)(C-C alkyl)amino-C-C alkyl-, N,N-di(C-C alkyl)amino-C-C alkyl-, N-(C-C hydroxyalkyl)-C-C aminoalkyl-, C-C alkyl-C(O)-, C-C hydroxyalkyl-C(O)-, and C-C aminoalkylC(O)-.

[0355] In one group of embodiments, R of formula CPT6iN, CPT6iiN, CPT6iiiN, CPT6ivN, CPT6vN or CPT6viN F is C3-C 10 Cycloalkyl, (C3-C 10 Cycloalkyl)-C1-C4 alkyl-, C3-C 10 Heterocycloalkyl, (C3-C 10 a moiety selected from the group consisting of heterocycloalkyl)-C1-C4 alkyl-, phenyl, phenyl-C1-C4 alkyl-, diphenylC1-C4 alkyl-, heteroaryl, and heteroaryl-C1-C4 alkyl-;F The cycloalkyl, heterocycloalkyl, phenyl, and heteroaryl portions of are substituted with 0 to 3 substituents independently selected from the group consisting of halogen, C1-C4 alkyl, —OH, —O1-C4 alkyl, —NH2, —NHC1-C4 alkyl, and —N(C1-C4 alkyl)2.

[0356] In one group of embodiments, R of formula CPT6iN, CPT6iiN, CPT6iiiN, CPT6ivN, CPT6vN or CPT6viN F are independently -H, C3-C 10 Cycloalkyl, (C3-C 10 Cycloalkyl)-C1-C4 alkyl-, C3-C 10 Heterocycloalkyl, (C3-C 10 a moiety selected from the group consisting of heterocycloalkyl)-C1-C4 alkyl-, phenyl, phenyl-C1-C4 alkyl-, diphenylC1-C4 alkyl-, heteroaryl, and heteroaryl-C1-C4 alkyl-; F The cycloalkyl, heterocycloalkyl, phenyl, and heteroaryl portions of are substituted with 0 to 3 substituents independently selected from the group consisting of halogen, C1-C4 alkyl, —OH, —O1-C4 alkyl, —NH2, —NHC1-C4 alkyl, and —N(C1-C4 alkyl)2.

[0357] In one group of embodiments, R of formula CPT6iOa, CPT6iiOa, CPT6iiiOa, CPT6ivOa, CPT6vOa, CPT6viOa, CPT6iOb, CPT6iiOb, CPT6iiiOb, CPT6ivOb, CPT6vOb, CPT6viOb, CPT6viiOb or CPT6viiiOb F and R F’ are combined with the nitrogen atom to which they are both attached to form a 5-, 6-, or 7-membered ring having 0 to 3 substituents independently selected from the group consisting of halogen, C1-C4 alkyl, —OH, —OC1-C4 alkyl, —NH2, —NHC1-C4 alkyl, and —N(C1-C4 alkyl)2.

[0358] In one group of embodiments, R of formula CPT6iOa, CPT6iiOa, CPT6iiiOa, CPT6ivOa, CPT6vOa, CPT6viOa, CPT6iOb, CPT6iiOb, CPT6iiiOb, CPT6ivOb, CPT6vOb, CPT6viOb, CPT6viiOb or CPT6viiiOb F and R F’ at least one of which is independently a moiety selected from the group consisting of C1-C8 alkyl, C1-C8 hydroxyalkyl, C1-C8 aminoalkyl, (C1-C4 alkylamino)-C1-C8 alkyl, N,N-(C1-C4 hydroxyalkyl)(C1-C4 alkyl)amino-C1-C8 alkyl-, N,N-di(C1-C4 alkyl)amino-C1-C8 alkyl-, N-(C1-C4 hydroxyalkyl)-C1-C8 aminoalkyl-, C1-C8 alkylC(O)-, C1-C8 hydroxyalkyl-C(O)-, and C1-C8 aminoalkyl-C(O)-. and the other is a moiety selected from the group consisting of —H, C1-C8 alkyl, C1-C8 hydroxyalkyl, C1-C8 aminoalkyl, (C1-C4 alkylamino)-C1-C8 alkyl-, N,N-(C1-C4 hydroxyalkyl)(C1-C4 alkyl)amino-C1-C8 alkyl-, N,N-di(C1-C4 alkyl)amino-C1-C8 alkyl-, N-(C1-C4 hydroxyalkyl)-C1-C8 aminoalkyl-, C1-C8 alkyl-C(O)—, C1-C8 hydroxyalkyl-C(O)—, and C1-C8 aminoalkylC(O)—.

[0359] In one group of embodiments, each R of formula CPT6iO, CPT6iiO, CPT6iiiO, CPT6ivO, CPT6vO or CPT6viO F and R F’are independently a moiety selected from the group consisting of C-C alkyl, C-C hydroxyalkyl, C-C aminoalkyl, (C-C alkylamino)-C-C alkyl-, N,N-(C-C hydroxyalkyl)(C-C alkyl)amino-C-C alkyl-, N,N-di(C-C alkyl)amino-C-C alkyl-, N-(C-C hydroxyalkyl)-C-C aminoalkyl, C-C alkyl-C(O)-, C-C hydroxyalkyl-C(O)-, and C-C aminoalkylC(O)-.

[0360] In one group of embodiments, R of formula CPT6iO, CPT6iiO, CPT6iiiO, CPT6ivO, CPT6vO or CPT6viO F and R F’ At least one of the 10 Cycloalkyl, C3-C 10 Cycloalkyl-C1-C4 alkyl, -C3-C 10 Heterocycloalkyl, (C3-C 10 a moiety selected from the group consisting of heterocycloalkyl)-C1-C4 alkyl-, phenyl, phenylC1-C4 alkyl, diphenylC1-C4 alkyl, heteroaryl, and heteroaryl-C1-C4 alkyl-; F and R F’the cycloalkyl, heterocycloalkyl, phenyl, and heteroaryl portions of are substituted with 0 to 3 substituents independently selected from the group consisting of halogen, C-C alkyl, —OH, —O-C alkyl, —NH, —NHC alkyl, and —N(C-C alkyl) and the other is a moiety selected from the group consisting of —H, C-C alkyl, C-C hydroxyalkyl, C-C amino alkyl, (C-C alkylamino)-C-C alkyl-, N,N-(C-C hydroxyalkyl)(C-C alkyl)aminoC-C alkyl-, N,N-di(C-C alkyl)aminoC-C alkyl-, N-(C-C hydroxyalkyl)-C-C amino alkyl-, C-C alkyl-C(O)—, C-C hydroxyalkyl-C(O)—, and C-C amino alkylC(O)—).

[0361] In one group of embodiments, R of formula CPT6iO, CPT6iiO, CPT6iiiO, CPT6ivO, CPT6vO or CPT6viO F and R F’ At least one of them is C3-C 10 Cycloalkyl, C3-C 10 Cycloalkyl-C1-C4 alkyl, -C3-C 10 Heterocycloalkyl, (C3-C 10 heterocycloalkyl)-C1-C4 alkyl-, phenyl, phenylC1-C4 alkyl, diphenylC1-C4 alkyl, heteroaryl, and heteroaryl-C1-C4 alkyl-; the other is independently selected from the group consisting of -H, C3-C 10 Cycloalkyl, (C3-C 10 Cycloalkyl)-C1-C4 alkyl-, C3-C 10 Heterocycloalkyl, (C3-C 10 a moiety selected from the group consisting of heterocycloalkyl)-C1-C4 alkyl-, phenyl, phenyl-C1-C4 alkyl-, diphenylC1-C4 alkyl-, heteroaryl, and heteroaryl-C1-C4 alkyl; R F and R F’The cycloalkyl, heterocycloalkyl, phenyl, and heteroaryl portions of are substituted with 0 to 3 substituents independently selected from the group consisting of halogen, C1-C4 alkyl, —OH, —O1-C4 alkyl, —NH2, —NHC1-C4 alkyl, and —N(C1-C4 alkyl)2.

[0362] In one group of embodiments, each R of formula CPT6iO, CPT6iiO, CPT6iiiO, CPT6ivO, CPT6vO or CPT6viO F and R F’ are independently -H, C3-C 10 Cycloalkyl, (C3-C 10 Cycloalkyl)-C1-C4 alkyl-, C3-C 10 Heterocycloalkyl, (C3-C 10 a moiety selected from the group consisting of heterocycloalkyl)-C1-C4 alkyl-, phenyl, phenyl-C1-C4 alkyl-, diphenylC1-C4 alkyl-, heteroaryl, and heteroaryl-C1-C4 alkyl-; F and R F’ The cycloalkyl, heterocycloalkyl, phenyl, and heteroaryl portions of are substituted with 0 to 3 substituents independently selected from the group consisting of halogen, C1-C4 alkyl, —OH, —O1-C4 alkyl, —NH2, —NHC1-C4 alkyl, and —N(C1-C4 alkyl)2.

[0363] In another group of embodiments, an ADC wherein Q has a formula of -Z A-R-, -Z A-R-Y-, -Z A S*-R-, -Z A S*-R-Y-, -Z A S*-R-Y-, -Z A B(S*)-R-, or -Z A B(S*)-R-Y-, and comprises a Drug unit having the Formula CPT7 is represented by the formula: [ka] [ka]

[0364] wherein R is any one of the releasable linkers disclosed herein, preferably R is a glucuronide unit, and the groups L, Z, A, S are each * , B and Y have the meanings provided above and in any one of the embodiments specifically recited herein.

[0365] In another embodiment, an ADC wherein Q has the formula -ZA-, -ZA-RL-, -ZAS*-W-, -ZAB(S*)-W-, -ZAS*-RL-, -ZAB(S*)-RL-, -ZAS*-W-RL-, and -ZAB(S*)-W-RL-, and comprises a Drug unit having the formula CPT5 is represented by the formula: [ka] [ka]

[0366] wherein R is a releasable linker other than a glucuronide unit, and the groups L, Z, A, S * , B and W have the meanings provided above and in any one of the embodiments specifically recited herein.

[0367] 1. Camptothecin-linker compound In some embodiments, when preparing ADCs, it may be desirable to synthesize the complete drug-linker combination prior to conjugation to a targeting agent (e.g., an antibody). In such embodiments, the camptothecin-linker compounds described herein are intermediate compounds. In these embodiments, the Stretcher unit in the camptothecin-linker compound is not yet covalently attached to a Ligand unit (i.e., is a Stretcher unit precursor, Z') and therefore has a functional group for conjugation to a targeting ligand. In one embodiment, the camptothecin-linker compound consists of a camptothecin compound (represented herein by formulas CPT1, CPT2, CPT3, CPT4, CPT5, CPT6, and CPT7) and a linker unit (Q) comprising a glucuronide unit as a releasable linker (RL) through which the Ligand unit is connected to the camptothecin.

[0368] In another embodiment, a camptothecin-linker compound comprises a camptothecin compound of formula CPT1, CPT2, CPT3, CPT4, CPT5, CPT6, or CPT7, and a Linker unit (Q) comprising a releasable linker (RL) other than a glucuronide unit to which a Ligand unit is conjugated. Thus, in any embodiment, the Linker unit comprises, in addition to RL, a Stretcher unit precursor (Z') that is a precursor to the Ligand unit and thus comprises a functional group for conjugation to a targeting agent that can connect RL (directly or indirectly) to the Ligand unit. In some of these embodiments, a resolving agent (S * ) as a side chain appendage. In any one of these embodiments, connector unit (A) is present if it is desired to add more distance between the Stretcher unit and RL.

[0369] In one group of embodiments, camptothecin-linker compounds comprise a camptothecin compound having the formula CPT1, CPT2, CPT3, CPT4, CPT5, CPT6, or CPT7, and a linker unit (Q), wherein Q comprises a releasable linker (RL), which is a glucuronide unit that is either directly attached to a Stretcher unit precursor (Z') or indirectly attached to Z' via a bond to an intervening component of the linker unit (i.e., A, S*, and / or B(S*)) of the camptothecin-linker compound, and Z' comprises a functional group capable of forming a covalent bond to a targeting agent.

[0370] In another group of embodiments, camptothecin-linker compounds comprise a camptothecin having the formula CPT1, CPT2, CPT3, CPT4, CPT5, CPT6, or CPT7, and a linker unit (Q), wherein Q comprises a releasable linker (R) other than a glucuronide unit (R) that is either directly attached to a Stretcher unit precursor (Z') or indirectly attached to Z' via a bond to an intervening component of the linker unit (i.e., A, S*, and / or B(S*)) of the camptothecin-linker compound, and Z' comprises a functional group capable of forming a covalent bond to a targeting agent.

[0371] In the context of ADCs and / or camptothecin-linker compounds, the assembly is best described in terms of its constituent parts. Although some procedures are described herein, the assembly and general conditions for preparing the conjugates and compounds will be well understood by those skilled in the art.

[0372] 2. Components Ligand Unit In some embodiments of the present invention, a Ligand unit is present. The Ligand unit (L-) is a targeting agent that specifically binds to a targeting moiety. In one group of embodiments, the Ligand unit comprises an antibody or antigen-binding fragment thereof that binds to CEACAM5. In some embodiments, the Ligand unit comprises any of the antibodies or antigen-binding fragments thereof described herein. The Ligand unit targets a specific target cell population with which the Ligand unit interacts due to the presence of its targeting component or molecule (e.g., an antibody), presents camptothecin (e.g., CPT6), and allows for the subsequent release of free drug within the target cell (i.e., intracellularly) or in the vicinity of the target cell (i.e., extracellularly). The Ligand unit L includes, but is not limited to, proteins, polypeptides, and peptides. Suitable Ligand units include, for example, antibodies, e.g., full-length antibodies and antigen-binding fragments thereof, interferons, lymphokines, hormones, growth factors and colony-stimulating factors, vitamins, nutrient transport molecules (including, but not limited to, transferrin), or any other cell-binding molecule or substance. In some embodiments, the Ligand unit (L) is derived from an antibody or non-antibody protein targeting agent.

[0373] In one group of embodiments, the Ligand unit (e.g., an antibody or antigen-binding fragment thereof that binds to CEACAM5) is attached to Q (a Linker unit) that comprises a glucuronide-releasable linker. As noted above, additional space may be provided between the camptothecin drug compound and the Ligand unit (e.g., a Stretcher unit and optionally a Connector unit, A), or attributes may be provided to the composition to increase solubility (e.g., a resolving agent, S). *Further binding components can be present in the conjugates described herein to achieve this purpose. In some of these embodiments, the Ligand unit (e.g., an antibody or antigen-binding fragment thereof that binds to CEACAM5) is attached to Z of the Linker unit via a heteroatom of the Ligand unit. Heteroatoms that can be present on the Ligand unit for such attachment include sulfur (in one embodiment, from a sulfhydryl group of the targeting ligand), oxygen (in one embodiment, from a carboxyl or hydroxyl group of the targeting ligand), and optionally substituted nitrogen (in one embodiment, from a primary or secondary amine functional group of the targeting ligand, or from an optionally substituted amide nitrogen). These heteroatoms can be present on the targeting ligand in the ligand's natural state, e.g., in a naturally occurring antibody, or can be introduced into the targeting ligand via chemical modification or biological engineering.

[0374] In one embodiment, a targeting agent that is a precursor to a Ligand unit has a sulfhydryl functional group (such as from a cysteine ​​amino acid) such that the Ligand unit is attached to a Linker unit via the sulfhydryl atom of the sulfhydryl functional group.

[0375] In yet another aspect, the targeting agent, which is a precursor of the Ligand unit, has one or more lysine residues that can be chemically modified to introduce one or more sulfhydryl groups. In these embodiments, the Ligand unit is covalently bound to the Linker unit via the sulfhydryl functional group's sulfur atom. Reagents that can be used to modify lysines in this way include, but are not limited to, N-succinimidyl S-acetylthioacetate (SATA) and 2-iminothiolane hydrochloride (Traut's Reagent).

[0376] In another embodiment, a targeting agent that is a precursor to a Ligand unit has one or more carbohydrate groups that can be modified to provide one or more sulfhydryl functional groups. The chemically modified Ligand unit in an ADC is attached to a Linker unit component (e.g., a Stretcher unit) via the sulfhydryl functional group's sulfur atom.

[0377] In yet another embodiment, a targeting agent that is a precursor of a Ligand unit has one or more carbohydrate groups that can be oxidized to provide an aldehyde (—CHO) functional group (see, e.g., Laguzza, et al., 1989, J. Med. Chem. 32(3):548-55). In these embodiments, the corresponding aldehyde interacts with a reactive site on the Stretcher unit precursor to form a bond between the Stretcher unit and the Ligand unit. Reactive sites on the Stretcher unit precursor that can interact with a reactive carbonyl-containing functional group on the targeting Ligand unit include, but are not limited to, hydrazine and hydroxylamine. Other protocols for modifying proteins for the attachment of a Linker unit (Q) or related species are described in Coligan et al., Current Protocols in Protein Science, vol. 2, John Wiley & Sons (2002), incorporated herein by reference.

[0378] In some aspects, a targeting agent that is a precursor of a Ligand unit (t) can form a bond by interacting with a reactive functional group on a Stretcher unit precursor (Z') to form a covalent bond between the Stretcher unit (Z) that structurally corresponds to the targeting agent and the Ligand unit. The functional group of Z' that has the ability to interact with a targeting agent will depend on the nature of the targeting agent that corresponds to the structure of the Ligand unit. In some embodiments, the reactive group is a maleimide that is present on the Stretcher unit prior to its attachment to form the Ligand unit (i.e., the maleimide portion of the Stretcher unit precursor). Covalent attachment of the Ligand unit to the Stretcher unit is achieved through a sulfhydryl functional group on the targeting agent that is a precursor of the Ligand unit, which interacts with the maleimide functional group of Z' to form a thio-substituted succinimide. The sulfhydryl functional group can be present on the targeting agent in its native state, e.g., at a natural residue, or can be introduced into the targeting agent by chemical modification or bioengineering.

[0379] In yet another embodiment, the Ligand unit is derived from an antibody that binds to CEACAM5, and the sulfhydryl group is formed by reduction of the antibody's interchain disulfide. Thus, in some embodiments, the Linker unit is conjugated to a cysteine ​​residue from the reduced interchain disulfide.

[0380] In yet another embodiment, the Ligand unit is derived from an antibody and a sulfhydryl functionality is chemically introduced into the antibody, for example, by the introduction of a cysteine ​​residue. Thus, in some embodiments, a Linker unit (with or without an attached camptothecin) is conjugated to the Ligand unit via the introduced cysteine ​​residue of the Ligand unit.

[0381] It has been observed that the site of drug conjugation for bioconjugates can affect many parameters, including ease of conjugation, drug linker stability, impact on the biophysical properties of the resulting bioconjugate, and in vitro cytotoxicity. Regarding drug linker stability, the site of conjugation of the drug linker moiety to the ligand unit can affect the ability of the conjugated drug linker moiety to undergo elimination reactions, which in some cases result in premature release of the free drug. Sites for conjugation on targeting agents include, for example, reduced interchain disulfides as well as selected cysteine ​​residues at engineered sites. In some embodiments, the conjugation methods for forming ADCs described herein use thiol residues at engineered sites (e.g., position 239 according to the EU index as set forth in Kabat) that are less susceptible to elimination reactions compared to conjugation methods that use thiol residues from reduced disulfide bonds. In other embodiments, the conjugation methods for forming ADCs described herein use thiol residues resulting from interchain disulfide bond reduction. In some embodiments, the Ligand unit is derived from an antibody that binds to CEACAM5.

[0382] Camptothecin Compounds Camptothecin compounds utilized in the various embodiments described herein are represented by the formula: [ka]

[0383] R B is a moiety selected from the group consisting of H, C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 cycloalkyl, (C3-C8 cycloalkyl)-C1-C4 alkyl-, phenyl, and phenyl-C1-C4 alkyl-;

[0384] R Cis a moiety selected from the group consisting of C1-C6 alkyl and C3-C6 cycloalkyl;

[0385] Each R F and R F’ are independently -H, C1-C8 alkyl, C1-C8 hydroxyalkyl, C1-C8 aminoalkyl, (C1-C4 alkylamino)-C1-C8 alkyl-, N,N-(C1-C4 hydroxyalkyl)(C1-C4 alkyl)amino-C1-C8 alkyl-, N,N-di(C1-C4 alkyl)amino-C1-C8 alkyl-, N-(C1-C4 hydroxyalkyl)-C1-C8 aminoalkyl-, C1-C8 alkylC(O)-, C1-C8 hydroxyalkyl-C(O)-, C1-C8 aminoalkyl-C(O)-, C3-C 10 Cycloalkyl, (C3-C 10 Cycloalkyl)-C1-C4 alkyl-, C3-C 10 Heterocycloalkyl, (C3-C 10 a moiety selected from the group consisting of heterocycloalkyl)-C1-C4 alkyl-, phenyl, phenyl-C1-C4 alkyl-, diphenyl-C1-C4 alkyl-, heteroaryl, and heteroaryl-C1-C4 alkyl-; or

[0386] R F and R F’ are combined with the nitrogen atom to which they are both attached to form a 5-, 6-, or 7-membered ring having 0-3 substituents independently selected from the group consisting of halogen, C1-C4 alkyl, —OH, —OC1-C4 alkyl, —NH2, —NHC1-C4 alkyl, and —N(C1-C4 alkyl)2;

[0387] R B , R C , R F and R F’wherein the cycloalkyl, heterocycloalkyl, phenyl, and heteroaryl portions are substituted with 0 to 3 substituents independently selected from the group consisting of halogen, C1-C4 alkyl, —OH, —O1-C4 alkyl, —NH2, —NHC1-C4 alkyl, and —N(C1-C4 alkyl)2.

[0388] Still other camptothecin compounds useful in the context of the ADCs and camptothecin linker compounds described herein are camptothecin compounds 14a-14z of Table I and compounds 18a-18r of Table J, as well as camptothecin compounds having five- or six-ring fused framework analogs to their structures provided as formulae CPT1, CPT2, CPT3, CPT4, CPT5, CPT6, CPT7, 14a-14z, and 18a-18r, which, in some embodiments, have additional groups, including, but not limited to, hydroxyl, thiol, amine, or amide functional groups, where an oxygen, sulfur, or optionally substituted nitrogen atom can be incorporated into the linker and released from the ADC as the free drug. In some embodiments, the functional group provides the only site on the camptothecin compound available for attachment to a linker unit (Q). The resulting drug linker portion of an ADC is a moiety that can release the active free drug at a site targeted by its ligand unit to exert a cytotoxic, cytostatic, or immunosuppressive effect.

[0389] "Free drug" refers to the drug present once released from the drug linker moiety. In some embodiments, the free drug comprises a fragment of a releasable linker or spacer unit (Y) group. The free drug comprising a fragment of a releasable linker or spacer unit (Y) is released from the remainder of the drug linker moiety via cleavage of the releasable linker or via cleavage of a bond in the spacer unit (Y) group and is biologically active upon release. In some embodiments, the free drug differs from the conjugated drug in that the functional group of the free drug for binding to the self-immolative assembly unit is no longer associated with a component of the ADC (other than the previously shared heteroatom). For example, the free hydroxyl functional group of an alcohol-containing drug can be released from the DO * H, however, in the conjugated form, the oxygen heteroatom, denoted by O*, is incorporated into the methylene carbamate unit of the self-immolative unit. Upon activation of the self-immolative moiety and release of the free drug, the covalent bond to the O* is replaced with a hydrogen atom so that the oxygen heteroatom, denoted by O*, is present as -OH on the free drug.

[0390] Linker Unit (Q) As noted above, some embodiments provide that the linker unit Q has a formula selected from the group consisting of: -ZA-RL-, -ZA-RL-Y-, -ZAS * -RL-, -ZAB(S * )-RL-, -ZAS * -RL-Y- and -ZAB(S * )-RL-Y-,

[0391] where Z is a stretcher unit, A is a coupling or connector unit, B is a branch unit, and S * is a resolving agent, R L is a releasable linker that is a glucuronide unit, Y is a spacer unit,

[0392] The point of attachment of D to Q is through the hydroxyl and any one of the heteroatoms of CPT1, CPT2, CPT3, CPT4, CPT5, CPT6 or CPT7, or the primary and secondary amines present in any one of compounds 14a-14z of Table I and compounds 18a-18r of Table J.

[0393] In other embodiments, the linker unit Q has a formula selected from the group consisting of: -ZA-, -ZA-RL-, -ZAS * -W-, -ZAB(S * )-W-, -ZAS * -RL-, -ZAB(S * )-RL-, -ZAS * -W-RL- and -ZAB(S * )-W-RL-,

[0394] where Z is a stretcher unit, A is a coupling or connector unit, B is a parallel connector unit, and S * is a resolving agent, R is a releasable linker other than a glucuronide unit, W is an amino acid unit,

[0395] The point of attachment to Q is via a hydroxyl group substituent on the lactone ring of any one of CPT1, CPT2, CPT3, CPT4, CPT5, CPT6, or CPT7, or compounds 14a-14z of Table I and compounds 18a-18r of Table J.

[0396] In one group of embodiments, Q is -ZAS * -RL- and -ZAS * -RL-Y-

[0397] In another group of embodiments, Q is -ZAB(S * )-RL- and -ZAB(S * )-RL-Y-.

[0398] In yet another group of embodiments, Q has a formula selected from the group consisting of -ZA-RL- and -ZA-RL-Y-.

[0399] Stretcher unit (Z) or (Z') The Stretcher unit (Z) is a component of an ADC or camptothecin-linker compound or other intermediate that serves to connect the Ligand unit to the remainder of the conjugate. In that respect, the Stretcher unit has a functional group that can form a bond with a functional group of a targeting ligand (e.g., an antibody) prior to attachment to the Ligand unit (i.e., the Stretcher unit precursor, Z').

[0400] In some embodiments, the Stretcher unit precursor (Z') has an electrophilic group that can interact with a reactive nucleophilic group present on a Ligand unit (e.g., an antibody) to provide a covalent bond between the Ligand unit and the Stretcher unit of a Linker unit. Nucleophilic groups on antibodies that have this capability include, but are not limited to, sulfhydryl, hydroxyl, and amino functional groups. The heteroatom of the nucleophilic group on the antibody is reactive to the electrophilic group on the Stretcher unit precursor, providing a covalent bond between the Ligand unit and the Stretcher unit of a Linker unit or Drug Linker moiety. Useful electrophilic groups for this purpose include, but are not limited to, maleimide, haloacetamide groups, and NHS esters. The electrophilic group provides a convenient site for antibody attachment to form an ADC or Ligand unit linker intermediate.

[0401] In other embodiments, the Stretcher unit precursor has a reactive site bearing a nucleophilic group that is reactive to an electrophilic group present on a Ligand unit (e.g., an antibody). Useful electrophilic groups on an antibody for this purpose include, but are not limited to, aldehyde and ketone carbonyl groups. The heteroatom of the nucleophilic group of the Stretcher unit precursor can react with an electrophilic group on an antibody to form a covalent bond to the antibody. Useful nucleophilic groups on a Stretcher unit precursor for this purpose include, but are not limited to, hydrazide, hydroxylamine, amino, hydrazine, thiosemicarbazone, hydrazine carboxylate, and arylhydrazide. The electrophilic group on an antibody provides a convenient site for antibody attachment to form an ADC or Ligand unit linker intermediate.

[0402] In some embodiments, the sulfur atom of the Ligand unit is linked to the succinimide ring system of the Stretcher unit formed by reaction of a thiol functional group of the Targeting Ligand with the maleimide moiety of the corresponding Stretcher unit precursor, hi other embodiments, the thiol functional group of the Ligand unit reacts with an alpha haloacetamide moiety by nucleophilic displacement of the halogen substituent to provide the sulfur-linked Stretcher unit.

[0403] Exemplary stretcher units for such embodiments include those having the following configuration: [ka]

[0404] R 17 The wavy line adjacent to indicates attachment to a parallel connector unit (B) or connector unit (A) when B is absent, or a splitting agent (S * ), and if B is absent, the other wavy line indicates a covalent bond to the sulfur atom of the Ligand unit, R 17 But -C1-C 10 Alkylene, C1-C 10Heteroalkylene, -C3-C8 carbocyclo, -O-(C1-C8 alkylene), -arylene, -C1-C 10 Alkylene-arylene-, -arylene-C1-C 10 Alkylene-, -C1-C 10 Alkylene-(C3-C8 carbocyclo)-, -(C3-C8 carbocyclo)-C1-C 10 Alkylene, -C3-C8 heterocyclo, -C1-C 10 Alkylene-(C3-C8 heterocyclo)-, -(C3-C8 heterocyclo)-C1-C 10 Alkylene-, -C1-C 10 Alkylene-C(=O)-, C1-C 10 Heteroalkylene-C(=O)-, -C3-C8 carbocyclo-C(=O)-, -O-(C1-C8 alkylene)-C(=O)-, -arylene-C(=O)-, -C1-C 10 Alkylene-arylene-C(=O)-, -arylene-C1-C 10 Alkylene-C(=O)-, -C1-C 10 Alkylene-(C3-C8 carbocyclo)-C(=O)-, -(C3-C8 carbocyclo)-C1-C 10 Alkylene-C(=O)-, -C3-C8 heterocyclo-C(=O)-, -C1-C 10 Alkylene-(C3-C8 heterocyclo)-C(=O)-, -(C3-C8 heterocyclo)-C1-C 10 Alkylene-C(=O)-, -C1-C 10 Alkylene-NH-, -C1-C 10 Heteroalkylene-NH-, -C3-C8 carbocyclo-NH-, -O-(C1-C8 alkylene)-NH-, -arylene-NH-, -C1-C 10 Alkylene-arylene-NH-, -arylene-C1-C 10 Alkylene-NH-, -C1-C 10 Alkylene-(C3-C8 carbocyclo)-NH-, -(C3-C8 carbocyclo)-C1-C 10 Alkylene-NH-, -C3-C8 heterocyclo-NH-, -C1-C 10Alkylene-(C3-C8 heterocyclo)-NH-, -(C3-C8 heterocyclo)-C1-C 10 Alkylene-NH-, -C1-C 10 Alkylene-S-, C1-C 10 Heteroalkylene-S-, -C3-C8 carbocyclo-S-, -O-(C1-C8 alkylene)-S-, -arylene-S-, -C1-C 10 Alkylene-arylene-S-, -arylene-C1-C 10 Alkylene-S-, -C1-C 10 Alkylene-(C3-C8 carbocyclo)-S-, -(C3-C8 carbocyclo)-C1-C 10 Alkylene-S-, -C3-C8 heterocyclo-S-, -C1-C 10 Alkylene-(C3-C8 heterocyclo)-S- or -(C3-C8 heterocyclo)-C1-C 10 It is alkylene-S-.

[0405] In some embodiments, R 17 The group optionally contains an aminoalkyl moiety, e.g., —(CH) x NH2, -(CH2) x NHR a , and -(CH2) x NR a 2, the subscript x is an integer from 1 to 4, and each R a independently, C 1-6 Alkyl and C 1-6 haloalkyl, or two R a The groups, in combination with the nitrogen to which they are attached, form an azetidinyl, pyrrolidinyl, or piperidinyl group.

[0406] Exemplary stretcher units are of the formula Za or Za-BU, where R 17 is -C1-C 10 Alkylene-C(=O)-, -C1-C 10Heteroalkylene-C(=O)-, -C3-C8 carbocyclo-C(=O)-, -O-(C1-C8 alkylene)-C(=O)-, -arylene-C(=O)-, -C1-C 10 Alkylene-arylene-C(=O)-, -arylene-C1-C 10 Alkylene-C(=O)-, -C1-C 10 Alkylene-(C3-C8 carbocyclo)-C(=O)-, -(C3-C8 carbocyclo)-C1-C 10 Alkylene-C(=O)-, -C3-C8 heterocyclo-C(=O)-, -C1-C 10 Alkylene-(C3-C8 heterocyclo)-C(=O)-, or -(C3-C8 heterocyclo)-C1-C 10 It is alkylene -C(=O)-.

[0407] Thus, some preferred embodiments are represented by formulas Za and Za-BU. [ka]

[0408] The wavy line adjacent to the carbonyl carbon atom represents B, A, or S in the above formula, depending on the presence or absence of A and / or B. * The other wavy line indicates the covalent bond of the succinimide ring carbon atom to the sulfur atom of the Ligand unit. During synthesis, the basic amino function of the Basic Unit (BU) can be protected by a protecting group.

[0409] More preferred embodiments of the stretcher units of the formula Za and Za-BU are as follows: [ka]

[0410] The wavy line adjacent to the carbonyl carbon atom represents B, A, or S in the above formula, depending on the presence or absence of A and / or B. * The other wavy line indicates the covalent bond of the succinimide ring carbon atom to the sulfur atom of the Ligand unit.

[0411] It will be understood that the Ligand unit-substituted succinimides may exist in hydrolyzed forms, which are exemplified below for the hydrolysis of Za or Za-BU, and structures representing the positional isomers from that hydrolysis have the formulae Zb and Zc, or Zb-BU and Zc-BU.

[0412] Thus, in another preferred embodiment, the Stretcher unit (Z) consists of a succinic acid-amide moiety represented by: [ka]

[0413] R 17 and the wavy line adjacent to the carbonyl carbon atom bonded to R and the wavy line adjacent to the carbon atom of the acid-amide moiety are as defined for Za or Za-BU, depending on the presence or absence of A and / or B, and R 17 is -C1-C5 alkylene-, and in Zb-BU and Zc-BU, the alkylene is replaced by a basic unit (BU), and BU is -(CH2) x NH2, -(CH2) x NHR a , or -(CH2) x N(R a )2, where the subscript x is an integer from 1 to 4, and each R a independently, C 1-6 Alkyl and C 1-6 haloalkyl, or together with the nitrogen to which they are attached, R a both define an azetidinyl, pyrrolidinyl or piperidinyl group.

[0414] In more preferred embodiments, -ZA- comprises a moiety derived from a maleimido-alkanoic acid moiety or an mDPR moiety. See, e.g., WO 2013 / 173337. In one group of embodiments, ZA- is derived from a maleimido-propionyl moiety.

[0415] Thus, in some of these more preferred embodiments, the stretcher unit (Z) consists of a succinic acid-amide moiety represented by the structure of formula Zb', Zc', (R / S)-Zb'-BU, (S)-Zb'-BU, (R / S)-Zc'-BU or (S)-Zc'-BU, as follows: [ka]

[0416] The wavy lines are as defined for Za or Za-BU.

[0417] In particularly preferred embodiments, the Stretcher unit (Z) consists of a succinimide moiety represented by the following structure: [ka]

[0418] or consisting of a succinic acid-amide moiety represented by the following structure: [ka]

[0419] Za, Zb or Zc -R 17 - is -CH2- or -CH2CH2-, or -R of Za-BU, Zb-BU or Zc-BU 17 Exemplary Stretcher units attached to a connector unit (A) comprised of Za'-BU, Zb'-BU, or Zc'-BU, where (BU)- is -CH(CHNH)-, have the following structure: [ka]

[0420] The wavy lines are as defined for Za or Za-BU.

[0421] The other Stretcher units bound to the Ligand unit (L) and the Connector unit (A) have the above structures, wherein A in any one of the above -Za-A-, -Za(BU)-A-, -Za'-A-, -Za'(BU)-A-, -Zb-A-, -Zb(BU)-A-, -Zb'-A-, -Zb'(BU)-, -Zc'-A- and Zc'(BU)-A- structures is replaced by a parallel Connector unit having the following structure: [ka]

[0422] where the subscript n ranges from 8 to 24, and R PEG is a PEG unit capping group, preferably -CH or -CHCHCOH, the asterisk (*) indicates a covalent bond to a Stretcher unit corresponding in structure to formula Za, Za', Zb', or Zc', and the wavy line indicates a covalent bond to a Releasable Linker (RL).

[0423] An exemplary Stretcher unit prior to conjugation to a Ligand unit (ie, a Stretcher unit precursor) is comprised of a maleimide moiety and is represented by a structure including the structure of formula Z'a: [ka]

[0424] The wavy line adjacent to the carbonyl carbon atom represents B, A, or S depending on the presence or absence of A and / or B in the above formula. * indicates the bond to R 17 is -(CH2) 1-5 - and optionally substituted aminoalkyl, e.g., -(CH) x NH2, -(CH2) x NHR a , and -(CH2) x N(R a )2, where the subscript x is an integer from 1 to 4, and each R aindependently, C 1-6 Alkyl and C 1-6 haloalkyl, or two R a The groups, in combination with the nitrogen to which they are attached, form an azetidinyl, pyrrolidinyl, or piperidinyl group.

[0425] Another exemplary Stretcher unit prior to conjugation to a Ligand unit (ie, a Stretcher unit precursor) is comprised of a maleimide moiety and is represented by a structure comprising the structure of formula Z'a-BU: [ka]

[0426] The wavy line adjacent to the carbonyl carbon atom represents B, A, or S depending on the presence or absence of A and / or B in the above formula. * indicates the bond to R 17 is -(CH2) 1-5 - and optionally substituted aminoalkyl, e.g., -(CH) x NH2, -(CH2) x NHR a , and -(CH2) x N(R a )2, where the subscript x is an integer from 1 to 4, and preferably R 17 is -CH2- or -CH2CH2-, the subscript x is 1 or 2, and each R a independently, C 1-6 Alkyl and C 1-6 haloalkyl, or two R a The groups, in combination with the nitrogen to which they are attached, form an azetidinyl, pyrrolidinyl, or piperidinyl group.

[0427] In some preferred embodiments of Formula Z'a, the Stretcher unit precursor is represented by one of the following structures: [ka]

[0428] wherein the wavy line adjacent to the carbonyl is as defined for Z'a or Z'a-BU.

[0429] In a more preferred embodiment, the Stretcher unit precursor (Z') is comprised of a maleimide moiety and is represented by the following structure: [ka]

[0430] wherein the wavy line adjacent to the carbonyl is as defined for Za', and the amino group is optionally protonated or protected by an amino protecting group.

[0431] It will be understood that in a Stretcher unit having a BU moiety, the amino functionality of that moiety is typically protected during synthesis by an amino protecting group, such as an acid labile protecting group (eg, BOC).

[0432] Exemplary Stretcher unit precursors are covalently linked to a connector unit comprised of the structure Z'a or Z'a-BU, and -R 17 -or-R 17 (BU)- is -CH-, -CHCH-, or -CH(CHNH)-, and has the following structure: [ka]

[0433] wherein the wavy line adjacent to the carbonyl is as defined for Z'a or Z'a-BU.

[0434] Another Stretcher unit precursor having a connector unit (A) bonded thereto has the above structure, and A in either one of the above Z'-A- and Z'(BU)-A- structures is a parallel connector unit and a resolving agent (-B(S)) having the following structure: *)-) is replaced. [ka]

[0435] where the subscript n ranges from 8 to 24, and R PEG is a PEG unit capping group, preferably -CH or -CHCHCOH, where the asterisk (*) indicates a covalent bond to a Stretcher unit precursor corresponding to the structure of formula Za or Za', and the wavy line indicates a covalent bond to R. In the examples shown herein, the PEG groups shown are meant to be illustrative of various resolving agents containing PEG groups of different lengths, and other resolving agents that can be directly attached or modified for attachment to parallel connector units.

[0436] In another embodiment, the Stretcher unit is attached to the Ligand unit via a disulfide bond between a sulfur atom of the Ligand unit and a sulfur atom of the Stretcher unit. A representative Stretcher unit of this embodiment is shown in brackets in formula Zb: [ka]

[0437] The wavy lines indicate attachment to the parallel connector unit (B) or connector unit (A) when B is not present, or to the splitting agent (S * ) and if A and B are not present, R 17 But -C1-C 10 Alkylene, C1-C 10 Heteroalkylene, -C3-C8 carbocyclo, -O-(C1-C8 alkylene), -arylene, -C1-C 10 Alkylene-arylene-, -arylene-C1-C 10 Alkylene-, -C1-C 10 Alkylene-(C3-C8 carbocyclo)-, -(C3-C8 carbocyclo)-C1-C 10 Alkylene, -C3-C8 heterocyclo, -C1-C10 Alkylene-(C3-C8 heterocyclo)-, -(C3-C8 heterocyclo)-C1-C 10 Alkylene-, -C1-C 10 Alkylene-C(=O)-, C1-C 10 Heteroalkylene-C(=O)-, -C3-C8 carbocyclo-C(=O)-, -O-(C1-C8 alkylene)-C(=O)-, -arylene-C(=O)-, -C1-C 10 Alkylene-arylene-C(=O)-, -arylene-C1-C 10 Alkylene-C(=O)-, -C1-C 10 Alkylene-(C3-C8 carbocyclo)-C(=O)-, -(C3-C8 carbocyclo)-C1-C 10 Alkylene-C(=O)-, -C3-C8 heterocyclo-C(=O)-, -C1-C 10 Alkylene-(C3-C8 heterocyclo)-C(=O)-, -(C3-C8 heterocyclo)-C1-C 10 Alkylene-C(=O)-, -C1-C 10 Alkylene-NH-, -C1-C 10 Heteroalkylene-NH-, -C3-C8 carbocyclo-NH-, -O-(C1-C8 alkylene)-NH-, -arylene-NH-, -C1-C 10 Alkylene-arylene-NH-, -arylene-C1-C 10 Alkylene-NH-, -C1-C 10 Alkylene-(C3-C8 carbocyclo)-NH-, -(C3-C8 carbocyclo)-C1-C 10 Alkylene-NH-, -C3-C8 heterocyclo-NH-, -C1-C 10 Alkylene-(C3-C8 heterocyclo)-NH-, -(C3-C8 heterocyclo)-C1-C 10 Alkylene-NH-, -C1-C 10 Alkylene-S-, C1-C 10 Heteroalkylene-S-, -C3-C8 carbocyclo-S-, -O-(C1-C8 alkylene)-S-, -arylene-S-, -C1-C 10 Alkylene-arylene-S-, -arylene-C1-C 10 Alkylene-S-, -C1-C10 Alkylene-(C3-C8 carbocyclo)-S-, -(C3-C8 carbocyclo)-C1-C 10 Alkylene-S-, -C3-C8 heterocyclo-S-, -C1-C 10 Alkylene-(C3-C8 heterocyclo)-S- or -(C3-C8 heterocyclo)-C1-C 10 It is alkylene-S-.

[0438] In yet another embodiment, the reactive group of the Stretcher unit precursor comprises a reactive site capable of forming a bond with a primary or secondary amino group of a Ligand unit (e.g., an antibody). Examples of these reactive sites include, but are not limited to, activated esters such as succinimide esters, 4-nitrophenyl esters, pentafluorophenyl esters, tetrafluorophenyl esters, anhydrides, acid chlorides, sulfonyl chlorides, isocyanates, and isothiocyanates. Representative Stretcher units of this embodiment are shown within square brackets in formulas Zci, Zcii, and Zciii: [ka]

[0439] The wavy lines indicate attachment to the parallel connector unit (B) or connector unit (A) when B is not present, or to the splitting agent (S * ) and if A and B are not present, R 17 But -C1-C 10 Alkylene, C1-C 10 Heteroalkylene, -C3-C8 carbocyclo, -O-(C1-C8 alkylene), -arylene, -C1-C 10 Alkylene-arylene-, -arylene-C1-C 10 Alkylene-, -C1-C 10 Alkylene-(C3-C8 carbocyclo)-, -(C3-C8 carbocyclo)-C1-C 10 Alkylene, -C3-C8 heterocyclo, -C1-C 10Alkylene-(C3-C8 heterocyclo)-, -(C3-C8 heterocyclo)-C1-C 10 Alkylene-, -C1-C 10 Alkylene-C(=O)-, C1-C 10 Heteroalkylene-C(=O)-, -C3-C8 carbocyclo-C(=O)-, -O-(C1-C8 alkylene)-C(=O)-, -arylene-C(=O)-, -C1-C 10 Alkylene-arylene-C(=O)-, -arylene-C1-C 10 Alkylene-C(=O)-, -C1-C 10 Alkylene-(C3-C8 carbocyclo)-C(=O)-, -(C3-C8 carbocyclo)-C1-C 10 Alkylene-C(=O)-, -C3-C8 heterocyclo-C(=O)-, -C1-C 10 Alkylene-(C3-C8 heterocyclo)-C(=O)-, -(C3-C8 heterocyclo)-C1-C 10 Alkylene-C(=O)-, -C1-C 10 Alkylene-NH-, -C1-C 10 Heteroalkylene-NH-, -C3-C8 carbocyclo-NH-, -O-(C1-C8 alkylene)-NH-, -arylene-NH-, -C1-C 10 Alkylene-arylene-NH-, -arylene-C1-C 10 Alkylene-NH-, -C1-C 10 Alkylene-(C3-C8 carbocyclo)-NH-, -(C3-C8 carbocyclo)-C1-C 10 Alkylene-NH-, -C3-C8 heterocyclo-NH-, -C1-C 10 Alkylene-(C3-C8 heterocyclo)-NH-, -(C3-C8 heterocyclo)-C1-C 10 Alkylene-NH-, -C1-C 10 Alkylene-S-, C1-C 10 Heteroalkylene-S-, -C3-C8 carbocyclo-S-, -O-(C1-C8 alkylene)-S-, -arylene-S-, -C1-C 10 Alkylene-arylene-S-, -arylene-C1-C 10 Alkylene-S-, -C1-C 10Alkylene-(C3-C8 carbocyclo)-S-, -(C3-C8 carbocyclo)-C1-C 10 Alkylene-S-, -C3-C8 heterocyclo-S-, -C1-C 10 Alkylene-(C3-C8 heterocyclo)-S- or -(C3-C8 heterocyclo)-C1-C 10 It is alkylene-S-.

[0440] In yet other embodiments, the reactive group of the Stretcher unit precursor comprises a reactive nucleophile that can react with an electrophile present on or introduced into the Ligand unit. For example, a carbohydrate moiety on a targeting Ligand can be mildly oxidized using a reagent such as sodium periodate, and the resulting electrophilic functional group (-CHO) of the oxidized carbohydrate can be condensed with a Stretcher unit precursor containing a reactive nucleophile such as a hydrazide, oxime, primary or secondary amine, hydrazine, thiosemicarbazone, hydrazine carboxylate, or arylhydrazide, as described in Kaneko, T. et al. (1991) Bioconjugate Chem. 2:133-41. Representative Stretcher units of this embodiment are shown in brackets in the formulas Zdi, Zdii, and Zdiii: [ka]

[0441] The wavy lines indicate attachment to the parallel connector unit (B) or connector unit (A), or the splitting agent (S * ) and if A and B are not present, R 17 But -C1-C 10 Alkylene, C1-C 10 Heteroalkylene, -C3-C8 carbocyclo, -O-(C1-C8 alkylene), -arylene, -C1-C 10 Alkylene-arylene-, -arylene-C1-C 10 Alkylene-, -C1-C 10 Alkylene-(C3-C8 carbocyclo)-, -(C3-C8 carbocyclo)-C1-C10 Alkylene, -C3-C8 heterocyclo, -C1-C 10 Alkylene-(C3-C8 heterocyclo)-, -(C3-C8 heterocyclo)-C1-C 10 Alkylene-, -C1-C 10 Alkylene-C(=O)-, C1-C 10 Heteroalkylene-C(=O)-, -C3-C8 carbocyclo-C(=O)-, -O-(C1-C8 alkylene)-C(=O)-, -arylene-C(=O)-, -C1-C 10 Alkylene-arylene-C(=O)-, -arylene-C1-C 10 Alkylene-C(=O)-, -C1-C 10 Alkylene-(C3-C8 carbocyclo)-C(=O)-, -(C3-C8 carbocyclo)-C1-C 10 Alkylene-C(=O)-, -C3-C8 heterocyclo-C(=O)-, -C1-C 10 Alkylene-(C3-C8 heterocyclo)-C(=O)-, -(C3-C8 heterocyclo)-C1-C 10 Alkylene-C(=O)-, -C1-C 10 Alkylene-NH-, -C1-C 10 Heteroalkylene-NH-, -C3-C8 carbocyclo-NH-, -O-(C1-C8 alkylene)-NH-, -arylene-NH-, -C1-C 10 Alkylene-arylene-NH-, -arylene-C1-C 10 Alkylene-NH-, -C1-C 10 Alkylene-(C3-C8 carbocyclo)-NH-, -(C3-C8 carbocyclo)-C1-C 10 Alkylene-NH-, -C3-C8 heterocyclo-NH-, -C1-C 10 Alkylene-(C3-C8 heterocyclo)-NH-, -(C3-C8 heterocyclo)-C1-C 10 Alkylene-NH-, -C1-C 10 Alkylene-S-, C1-C 10 Heteroalkylene-S-, -C3-C8 carbocyclo-S-, -O-(C1-C8 alkylene)-S-, -arylene-S-, -C1-C 10Alkylene-arylene-S-, -arylene-C1-C 10 Alkylene-S-, -C1-C 10 Alkylene-(C3-C8 carbocyclo)-S-, -(C3-C8 carbocyclo)-C1-C 10 Alkylene-S-, -C3-C8 heterocyclo-S-, -C1-C 10 Alkylene-(C3-C8 heterocyclo)-S- or -(C3-C8 heterocyclo)-C1-C 10 It is alkylene-S-.

[0442] In some embodiments of the invention, the stretcher unit has a mass of about 1000 daltons or less, about 500 daltons or less, about 200 daltons or less, about 30, 50, or 100 daltons to about 1000 daltons, about 30, 50, or 100 daltons to about 500 daltons, or about 30, 50, or 100 daltons to about 200 daltons.

[0443] Connector unit (A) In some embodiments, a connector unit (A) is included in an ADC or camptothecin-linker compound when it is desirable to add additional distance between the Stretcher unit (Z) or its precursor (Z') and the releasable linker. In some embodiments, the extra distance aids in activation within the RL. Thus, the connector unit (A), when present, extends the framework of the linker unit. In that respect, the connector unit (A) is covalently attached to a Stretcher unit (or its precursor) at one end and to an optional parallel connector unit or splitter (S) at the other end. * ) is covalently bonded to

[0444] Those skilled in the art will appreciate that the connector unit can be any group useful for providing attachment to the remaining linker units (Q) of the releasable linker. The connector unit can consist of, for example, one or more (e.g., 1-10, preferably 1, 2, 3, or 4) natural or unnatural amino acids, amino alcohols, amino aldehydes, diamino residues. In some embodiments, the connector unit is a single natural or unnatural amino acid, amino alcohol, amino aldehyde, or diamino residue. An exemplary amino acid that can function as a connector unit is β-alanine.

[0445] In some of these embodiments, the connector unit has the formula shown below: [ka]

[0446] The wavy line indicates the attachment of the connector unit in the ADC or camptothecin linker compound, and R 111 are independently hydrogen, p-hydroxybenzyl, methyl, isopropyl, isobutyl, sec-butyl, -CH2OH, -CH(OH)CH3, -CH2CH2SCH3, -CH2CONH2, -CH2COOH, -CH2CH2CONH2, -CH2CH2COOH, -(CH2)3NHC(=NH)NH2, -(CH2)3NH2, -(CH2)3NHCOCH3, -(CH2)3NHCHO, -(CH2)4NHC(=NH)NH2, -(CH2)4NH2, -(CH2)4NHCOCH3, -(CH2)4NHCHO, -(CH2)3NHCONH2, -(CH2)4NHCONH2, -CH2CH2CH(OH)CH2NH2, 2-pyridylmethyl-, 3-pyridylmethyl-, 4-pyridylmethyl-, [ka] is selected from the group consisting of

[0447] Each R 100are independently selected from hydrogen or -C1-C3 alkyl, preferably hydrogen or CH3, and the subscript c is an independently selected integer from 1 to 10, preferably 1 to 3.

[0448] Splitting agent (S * ) or -B(S * Representative connector units having a carbonyl group for attachment to a carboxyl group are as follows: [ka]

[0449] In each example, R 13 are independently -C1-C6 alkylene-, -C3-C8 carbocyclo-, -arylene-, -C1-C 10 Heteroalkylene, -C3-C8 heterocyclo, -C1-C 10 Alkylene-arylene-, -arylene-C1-C 10 Alkylene-, -C1-C 10 Alkylene-(C3-C8 carbocyclo)-, -(C3-C8 carbocyclo)-C1-C 10 Alkylene-, -C1-C 10 Alkylene-(C3-C8 heterocyclo)- and -(C3-C8 heterocyclo)-C1-C 10 alkylene-, and the subscript c is an integer ranging from 1 to 4. In some embodiments, R 13 is -C1-C6 alkylene and c is 1.

[0450] Splitting agent (S * ) or -B(S * Another representative connector unit having a carbonyl group for attachment to a carboxyl group is: [ka]

[0451] R 13 is -C1-C6 alkylene-, -C3-C8 carbocyclo-, -arylene-, -C1-C10 Heteroalkylene, -C3-C8 heterocyclo, -C1-C 10 Alkylene-arylene-, -arylene-C1-C 10 Alkylene-, -C1-C 10 Alkylene-(C3-C8 carbocyclo)-, -(C3-C8 carbocyclo)-C1-C 10 Alkylene-, -C1-C 10 Alkylene-(C3-C8 heterocyclo)- and -(C3-C8 heterocyclo)-C1-C 10 In some embodiments, R 13 is -C1-C6 alkylene.

[0452] Splitting agent (S * ) or -B(S * Representative connector units having an NH moiety that binds to a carboxyl group are as follows: [ka]

[0453] In each example, R 13 are independently -C1-C6 alkylene-, -C3-C8 carbocyclo-, -arylene-, -C1-C 10 Heteroalkylene, -C3-C8 heterocyclo, -C1-C 10 Alkylene-arylene-, -arylene-C1-C 10 Alkylene-, -C1-C 10 Alkylene-(C3-C8 carbocyclo)-, -(C3-C8 carbocyclo)-C1-C 10 Alkylene-, -C1-C 10 Alkylene-(C3-C8 heterocyclo)- and -(C3-C8 heterocyclo)-C1-C 10 alkylene-, and the subscript c is 1 to 14. In some embodiments, R 13 is -C1-C6 alkylene and the subscript c is 1.

[0454] Splitting agent (S * ) or -B(S* Another representative connector unit having an NH moiety that binds to the aryl group is: [ka]

[0455] In the formula, R 13 is -C1-C6 alkylene-, -C3-C8 carbocyclo-, -arylene-, -C1-C 10 Heteroalkylene, -C3-C8 heterocyclo, -C1-C 10 Alkylene-arylene-, -arylene-C1-C 10 Alkylene-, -C1-C 10 Alkylene-(C3-C8 carbocyclo)-, -(C3-C8 carbocyclo)-C1-C 10 Alkylene-, -C1-C 10 Alkylene-(C3-C8 heterocyclo)-, -(C3-C8 heterocyclo)-C1-C 10 alkylene-, -C(=O)C1-C6 alkylene- or -C1-C6 alkylene-C(=O)-C1-C6 alkylene.

[0456] Selected embodiments of the connector unit include those having the following structure: [ka]

[0457] The wavy line adjacent to the nitrogen indicates the covalent bond of the stretcher unit (Z) (or its precursor Z'), and the wavy line adjacent to the carbonyl indicates the covalent bond of the partitioning agent (S * ) or -B(S * )-, and m is an integer ranging from 1 to 6, preferably from 2 to 6, and more preferably from 2 to 4.

[0458] In some embodiments, unless otherwise specified, "connector" and "connector" are used interchangeably.

[0459] Releasable Linker (RL) The glucuronide unit is a type of releasable linker that provides a mechanism for separating the camptothecin from the ligand unit and other components of the linker unit through activation of a self-immolative cascade within the linker unit. In such embodiments, the self-immolative cascade is activated by the operation of a glycosidase on the carbohydrate moiety of the glucuronide unit. Several sugars or sugar moieties are useful in the embodiments described herein. Specific carbohydrate moieties (e.g., sugar moieties) include galactose, glucose, mannose, xylose, arabinose, mannose-6-phosphate, fucose, rhamnose, gulose, allose, 6-deoxyglucose, lactose, maltose, cellobiose, gentiobiose, maltotriose, GlcNAc, GalNAc, and maltohexaose.

[0460] A glycoside unit typically comprises a sugar moiety (Su) linked to a self-immolative spacer via an oxygen glycosidic bond. Cleavage of the oxygen glycosidic bond initiates a self-immolative reaction sequence that results in the release of the free drug. In some embodiments, the self-immolative sequence is activated from cleavage of a glucuronide unit, an exemplary glycoside unit, by β-glucuronidase. The glucuronide unit comprises an activation unit and a self-immolative spacer unit. The glucuronide unit comprises a sugar moiety (Su) linked to a self-immolative spacer unit via an oxygen glycosidic bond.

[0461] In some embodiments, the glucuronide unit comprises a sugar moiety (Su) linked via an oxygen glycosidic bond (—O′—) to the following self-immolative unit (SP): [ka]

[0462] The wavy lines indicate the connection to a Drug unit of any one of formulas CPT1, CPT2, CPT3, CPT4, CPT5, CPT6 and CPT7, or to a Spacer unit attached to a Drug unit (camptothecin compound), and either directly or via a Connector unit (A) or a Parallel Connector unit (B), a Resolving Agent (S * ), or indirectly via a combination of a connector unit and a parallel connector unit, to a Stretcher unit (Z) or its precursor (Z').

[0463] The oxygen glycosidic bond (-O'-) is typically a β-glucuronidase cleavage site (ie, Su is derived from the glucuronide), such as the glycosidic bond cleavable by human lysosomal β-glucuronidase.

[0464] In some embodiments, the glucuronide unit can be represented by the formula Ga or Gb: [ka]

[0465] where Su is a sugar moiety, -O'- represents an oxygen glycosidic bond, and R 1S , R 2S and R 3S are independently hydrogen, halogen, -CN, -NO, or other electron-withdrawing or electron-donating group; the wavy line indicates attachment to a Stretcher unit (Z) (or its precursor (Z'), directly or indirectly, via a connector unit or parallel connector units or a connector unit and parallel connector units); and # indicates attachment to a camptothecin or spacer (directly or indirectly through an intervening functional group or other moiety).

[0466] In a preferred embodiment, R 1S , R 2S and R 3S is independently selected from hydrogen, halogen, —CN, or —NO. In another preferred embodiment, R 1S, R 2S and R 3S are each hydrogen. In another preferred embodiment, R 2S is an electron-withdrawing group, preferably NO2, and R 1S and R 3S are each hydrogen.

[0467] In some such embodiments, the activatable self-immolative group capable of glycosidase cleavage to initiate a self-immolative reaction sequence is represented by the following formula Gc: [ka]

[0468] In the formula, R 4S is CHOH or —COH, the wavy line indicates a covalent bond to a Stretcher unit (Z) (or its precursor Z′) either directly or indirectly through a connector unit or a parallel connector unit or a connector unit and a parallel connector unit, and the hash mark (#) indicates a covalent bond to a methylene carbamate unit.

[0469] In some embodiments, the activatable self-immolative moiety consists of a glucuronide unit, which is represented by the following formula Gd: [ka]

[0470] The wavy line indicates a covalent bond to the Stretcher unit (Z) (or its precursor Z') directly or indirectly via a connector unit or a parallel connector unit or a connector unit and a parallel connector unit, and the hash mark (#) indicates a covalent bond of the benzylic carbon of a spacer or functional group attached to camptothecin.

[0471] Another type of releasable linker that provides a mechanism for separating camptothecin from the Ligand unit and other components of the Linker unit via activation of a self-immolative cascade within the Linker unit is a linker in which the phenylene component is a J m wherein the subscript m, indicating the number of substituents, is an integer ranging from 0 to 4, and each J is independently -C1-C8 alkyl, -O-(C1-C8 alkyl), -halogen, -nitro, or -cyano.

[0472] In some embodiments, R is a self-immolative group capable of releasing -D without the need for a separate hydrolysis step or subsequent self-immolative event. In some embodiments, -R is a PAB moiety linked to the carbonyl of -W- through the amino nitrogen atom of the PAB group and directly linked to -D through a carbonate group. In related embodiments, -R consists of a PAB moiety linked to the carbonyl of -A-, -S*-, or -B- through the amino nitrogen atom of the PAB group and directly linked to -D through a carbonate group. Without being bound to any particular theory or mechanism, a possible mechanism of drug release from a R consists of a PAB moiety in which R is directly bonded to -D through a carbonate group is shown in Toki et al. (2002) J Org. Chem. 67:1866-1872.

[0473] In some embodiments, the R L unit containing a PAB moiety is represented by the following formula: [ka]

[0474] wherein the subscript m is an integer ranging from 0 to 4, and each J is independently -C1-C8 alkyl, -O-(C1-C8 alkyl), -halogen, -nitro, or -cyano.

[0475] Other examples of self-immolative groups include, but are not limited to, aromatic compounds electronically similar to the PAB moiety, such as 2-aminoimidazole-5-methanol derivatives (Hay et al. (1999) Bioorg. Med. Chem. Lett. 9:2237) and ortho- or para-aminobenzyl acetals. Other RLs undergo cyclization upon amide bond hydrolysis, such as substituted and unsubstituted 4-aminobutyric acid amides (Rodrigues et al., Chemistry Biology, 1995, 2, 223), appropriately substituted bicyclo[2.2.1] and bicyclo[2.2.2] ring systems (Storm et al., J. Amer. Chem. Soc., 1972, 94, 5815), and 2-aminophenylpropionic acid amides (Amsberry et al., J. Org. Chem., 1990, 55, 5867).

[0476] In one embodiment, RL is a branched bis(hydroxymethyl)styrene (BHMS) unit.

[0477] In some embodiments, R has the formula: [ka]

[0478] where the wavy line marked with ** indicates the site of attachment to D, and the wavy line marked with * indicates the point of attachment of Q to an additional linker component.

[0479] In some embodiments, the RL comprises a heterocyclic "self-immolative moiety" of Formula I, II, or III attached to a drug and incorporates an amide group that, upon hydrolysis by an intracellular protease, initiates a reaction that ultimately cleaves the self-immolative moiety from the drug, such that the drug is released from the conjugate in an active form. The linker moiety further comprises a peptide sequence adjacent to the self-immolative moiety that is a substrate for an intracellular enzyme, e.g., an intracellular protease such as a cathepsin (e.g., cathepsin B), which cleaves the peptide at the amide bond shared with the self-immolative moiety. In embodiments disclosed herein, the PAB-containing RL is directly attached to the third hydroxyl of the lactone ring present in each of CPT1-CPT7, each of compounds 14-14z of Table I, or each of compounds 18a-18r of Table J.

[0480] In some embodiments, the heterocyclic self-immolative group (RL) is selected from Formulas I, II, and III: [ka]

[0481] The wavy line indicates the site of covalent attachment to the cell-specific ligand and drug moiety, and U represents O, S, or NR. 6 and Q is the CR 4 or N and V 1 , V 2 and V 3 With respect to formulas II and III, Q, V 1 and V 2 Independently, CR, provided that at least one of 4 or N, and T is O reserved from CPT1, CPT2, CPT3, CPT4, CPT5, CPT6 or CPT7;

[0482] R 1 , R 2 , R 3 and R 4 are independently H, F, Cl, Br, I, OH, -N(R 5 )2, -N(R 5 )3 +, C1-C8 alkyl halides, carboxylates, sulfates, sulfamates, sulfonates, -SO2R 5 , -S(=O)R 5 , -SR 5 , -SO2N(R 5 )2, -C(=O)R 5 , -CO2R 5 , -C(=O)N(R 5 )2, -CN, -N3, -NO2, C1-C8 alkoxy, C1-C8 halosubstituted alkyl, polyethyleneoxy, phosphonate, phosphate, C1-C8 alkyl, C1-C8 substituted alkyl, C2-C8 alkenyl, C2-C8 substituted alkenyl, C2-C8 alkynyl, C2-C8 substituted alkynyl, C6-C 20 Aryl, C6-C 20 Substituted Aryl, C1-C 20 Heterocycles and C1-C 20 substituted heterocycles, or when taken together, R 2 and R 3 forms a carbonyl (=O) or a spirocarbocycle of 3 to 7 carbon atoms,

[0483] R 5 and R 6 are independently H, C1-C8 alkyl, C1-C8 substituted alkyl, C2-C8 alkenyl, C2-C8 substituted alkenyl, C2-C8 alkynyl, C2-C8 substituted alkynyl, C6-C 20 Aryl, C6-C 20 Substituted Aryl, C1-C 20 Heterocycles and C1-C 20 substituted heterocycles;

[0484] C1-C8 substituted alkyl, C2-C8 substituted alkenyl, C2-C8 substituted alkynyl, C6-C 20 Substituted aryl and C2-C 20 Substituted heterocycles include F, Cl, Br, I, OH, -N(R 5 )2, -N(R 5 )3 +, C1-C8 alkyl halide, carboxylate, sulfate, sulfamate, sulfonate, C1-C8 alkyl sulfonate, C1-C8 alkyl amino, 4-dialkylaminopyridinium, C1-C8 alkyl hydroxyl, C1-C8 alkyl thiol, -SO2R 5 , -S(=O)R 5 , -SR 5 , -SO2N(R 5 )2, -C(=O)R 5 , -CO2R 5 , -C(=O)N(R 5 )2, -CN, -N3, -NO2, C1-C8 alkoxy, C1-C8 trifluoroalkyl, C1-C8 alkyl, C3-C 12 Carbocycle, C6-C 20 Aryl, C2-C 20 It is independently substituted with one or more substituents selected from the group consisting of heterocycle, polyethyleneoxy, phosphonate, and phosphate.

[0485] The conjugates are stable extracellularly or in the absence of enzymes capable of cleaving the amide bond of the self-immolative moiety, however, upon entry into a cell or exposure to a suitable enzyme, the amide bond is cleaved, initiating a spontaneous self-immolative reaction that results in cleavage of the bond covalently linking the self-immolative moiety to the drug, thereby releasing the drug in an undifferentiated or pharmacologically active form.

[0486] The self-immolative moieties in the conjugates of the invention incorporate one or more heteroatoms, thus providing improved solubility, improved cleavage rates, and / or reduced tendency of the conjugate to aggregate. These improvements of the heterocyclic self-immolative linker constructs of the invention relative to non-heterocyclic, PAB-type linkers in some cases result in surprising and unexpected biological properties, such as increased efficacy, reduced toxicity, and / or improved one or more desirable pharmacokinetic and / or pharmacodynamic properties.

[0487] It is understood that T in Formulas I-III is O because it is derived from the tertiary hydroxyl (—OH) on the lactone ring moiety of any one of CPT1, CPT2, CPT3, CPT4, CPT5, CPT6, CPT7, compounds 14a-14z of Table I, and compounds 18a-18r of Table J.

[0488] Without being limited to theory or any particular mechanism, the presence of an electron-withdrawing group on the heterocycle of a Formula I, II, or III linker sometimes moderates the rate of cleavage.

[0489] In one embodiment, the self-immolative moiety is a group of formula I, where Q is N and U is O or S. Such groups have non-linear structural features that improve the solubility of the conjugate. In this context, R may be H, methyl, nitro, or CF. In one embodiment, Q is N and U is O, thereby forming an oxazole ring, and R is H. In another embodiment, Q is N and U is S, thereby forming a thiazole ring at R, optionally substituted with a Me or CF group.

[0490] In another exemplary embodiment, the self-immolative moiety is 1 and V 2 is a group of formula II, wherein Q, V are independently N or CH. 1 and V 2 are each N. In another embodiment, Q and V 1 is N and V 2 In another embodiment, Q and V are 2 is N and V 1 In another embodiment, Q and V are 1 are both CH and V 2 is N. In another embodiment, Q is N and V 1 and V 2 are both CH.

[0491] In another embodiment, the self-immolative moiety is Q, V 1 , V 2 and V 3is a group of formula III, wherein each occurrence of Q is independently N or CH. In another embodiment, Q is N and V 1 , V 2 and V 3 are each N. In another embodiment, QV 1 and V 2 are CH and V 3 Each of Q, V is N. 2 and V 3 are CH and V 1 is N. In another embodiment, Q, V 1 and V 3 are CH and V 2 is N. In another embodiment, Q and V 2 are both N and V 1 and V 3 In another embodiment, Q and V are both CH. 2 are both CH and V 1 and V 3 are both N. In another embodiment, Q and V 3 are both N and V 1 and V 2 are both CH.

[0492] Without being bound by theory, Scheme 1a shows the mechanism of free drug release from a camptothecin drug unit attached via the nitrogen atom of an amine substituent to a releasable linker that is a glucuronide unit.

[0493] Scheme 1a: [ka] Fractionation Agent (S*): The ADCs described herein may contain a resolving agent (S * ) Partitioning agent moieties are useful, for example, to mask the hydrophobicity of certain camptothecin drug unit or binding unit components.

[0494] Representative resolving agents include polyethylene glycol (PEG) units, cyclodextrin units, polyamides, hydrophilic peptides, polysaccharides, and dendrimers.

[0495] When a polyethylene glycol (PEG) unit, a cyclodextrin unit, a polyamide, a hydrophilic peptide, a polysaccharide, or a dendrimer is included in Q, the groups can be present as "in-line" components or as side chain or branched components. In those embodiments where branched versions are present, the linker unit typically comprises, for example, a lysine residue (or parallel connector unit, B) that provides for simple functional conjugation of the PEG unit to the remainder of the linking unit.

[0496] Polyethylene glycol unit (PEG) To prepare the compounds of the present invention, polydisperse PEG, monodisperse PEG, and discrete PEG can be used. Polydisperse PEG is a heterogeneous mixture of sizes and molecular weights, while monodisperse PEG is typically purified from a heterogeneous mixture, thus providing a single chain length and molecular weight. Preferred PEG units are discrete PEG compounds that are synthesized stepwise without a polymerization process. Discrete PEG provides a single molecule with a defined and specified chain length.

[0497] The PEG units provided herein comprise one or more polyethylene glycol chains. In some embodiments, the polyethylene glycol chains are linked together, e.g., in a linear, branched, or star-shaped configuration. Typically, at least one of the PEG chains is derivatized at one end for covalent attachment to a suitable site on a linker unit component (e.g., B), or is derivatized at one end for covalent attachment to a suitable site on a linker unit component (e.g., ZAS*-RL-, ZAS *-R, R-Y-) can be used as an in-line (e.g., bifunctional) linking group to covalently link two of the R, R, R-Y-. Exemplary linkages within the Linker unit are by a non-conditionally cleavable bond or via a conditionally cleavable bond. Exemplary linkages are via an amide bond, an ether bond, an ester bond, a hydrazone bond, an oxime bond, a disulfide bond, a peptide bond, or a triazole bond. In some embodiments, linkages within the Linker unit are via a non-conditionally cleavable bond. In some embodiments, linkages within the Linker unit are not via an ester bond, a hydrazone bond, an oxime bond, or a disulfide bond. In some embodiments, linkages within the Linker unit are not via a hydrazone bond.

[0498] A conditionally cleavable bond refers to a bond that is not substantially susceptible to cleavage while circulating in plasma, but is susceptible to cleavage in an intracellular or intratumoral environment. A non-conditionally cleavable bond is a bond that is not substantially susceptible to cleavage in any biological environment. Chemical hydrolysis of hydrazones, reduction of disulfides, and enzymatic cleavage of peptide or glycosidic bonds are examples of conditionally cleavable bonds.

[0499] In some embodiments, the PEG unit is directly attached to the parallel connector unit B. The other end (or termini) of the PEG unit is free and unconstrained and may take the form of a methoxy, carboxylic acid, alcohol, or other suitable functional group. The methoxy, carboxylic acid, alcohol, or other suitable functional group serves as a cap for the terminal PEG subunit of the PEG unit. Unconstrained means that the PEG unit is not attached to a camptothecin, antibody, or another binding entity at its unconstrained site. Those skilled in the art will understand that a PEG unit, in addition to repeating polyethylene glycol subunits, may also contain non-PEG material (e.g., to facilitate the attachment of multiple PEG chains to one another). Non-PEG material refers to atoms within the PEG unit that are not part of the repeating -CHCHO- subunit. In some embodiments provided herein, the PEG unit comprises two monomeric PEG chains attached to one another via a non-PEG element. In other embodiments provided herein, the PEG unit comprises two linear PEG chains attached to a central core or parallel connector unit (i.e., the PEG unit itself is branched).

[0500] Several PEG conjugation methods are available to those skilled in the art [e.g., Goodson, et al. (1990) Bio / Technology 8:343 (PEGylation of interleukin-2 at its glycosylation site after site-directed mutagenesis), EP 0401384 (coupling PEG to G-CSF), Malik, et al. al., (1992) Exp. Hematol. 20:1028-1035 (PEGylation of GM-CSF using tresyl chloride), PCT Publication No. WO 90 / 12874 (PEGylation of erythropoietin containing recombinantly introduced cysteine ​​residues using cysteine-specific mPEG derivatives), U.S. Pat. No. 5,757,078 (PEGylation of EPO peptides), U.S. Pat. No. 5,672,662 (Poly(ethylene glycol) monosubstituted with propionic or butanoic acid and its functional derivatives for biotechnological applications), U.S. Pat. No. 6,077,939 (PEGylation of the N-terminal alpha carbon of peptides), Veronese et al., (1985) Appl. Biochem. Biotechnol 11:141-142 (PEGylation of the N-terminal α-carbon of peptides with PEG-nitrophenyl carbonate ("PEG-NPC") or PEG-trichlorophenyl carbonate), and Veronese (2001) Biomaterials 22:405-417 (review article on PEGylation of peptides and proteins)].

[0501] For example, PEG can be covalently attached to amino acid residues via reactive groups. A reactive group is a group (e.g., a free amino or carboxyl group) to which an activated PEG molecule can be attached. For example, N-terminal amino acid residues and lysine (K) residues have free amino groups, and C-terminal amino acid residues have free carboxyl groups. Thiol groups (e.g., as found on cysteine ​​residues) are also useful as reactive groups for attaching PEG. In addition, enzyme-assisted methods for specifically introducing activated groups (e.g., hydrazide, aldehyde, and aromatic amino groups) into the C-terminus of polypeptides have been described (see Schwarz, et al. (1990) Methods Enzymol. 184:160; Rose, et al. (1991) Bioconjugate Chem. 2:154; and Gaertner, et al. (1994) J. Biol. Chem. 269:7224).

[0502] In some embodiments, PEG molecules can be attached to amino groups using methoxylated PEGs ("mPEG") with different reactive moieties. Non-limiting examples of such reactive moieties include succinimidyl succinate (SS), succinimidyl carbonate (SC), mPEG-imidate, para-nitrophenyl carbonate (NPC), succinimidyl propionate (SPA), and cyanuric chloride. Non-limiting examples of such mPEGs include mPEG-succinimidyl succinate (mPEG-SS), mPEG2-succinimidyl succinate (mPEG2-SS), mPEG-succinimidyl carbonate (mPEG-SC), mPEG2-succinimidyl carbonate (mPEG2-SC), mPEG-imidate, mPEG-para-nitrophenyl carbonate (mPEG-NPC), mPEG-imidate, mPEG2-para-nitrophenyl mPEG-NPC, mPEG-succinimidyl propionate (mPEG-SPA), mPEG2-succinimidyl propionate (mPEG2-SPA), mPEG-N-hydroxy-succinimide (mPEG-NHS), mPEG2-N-hydroxy-succinimide (mPEG2-NHS), mPEG-cyanuric chloride, mPEG2-cyanic acid chloride, mPEG2-lysinol-NPC, and mPEG2-Lys-NHS.

[0503] Generally, at least one of the PEG chains that make up the PEG unit is functionalized so that it can be covalently attached to other linker unit components.

[0504] Functionalization may include, for example, amine, thiol, NHS ester, maleimide, alkyne, azide, carbonyl, or some other functional group. In some embodiments, the PEG unit further comprises a non-PEG material (i.e., a material not composed of -CHCHO-) that provides for coupling to other linker unit components or that facilitates the coupling of two or more PEG chains.

[0505] The presence of a PEG unit (or other resolving agent) within the linker unit can have two potential effects on the pharmacokinetics of the resulting ADC. The desired effect is reduced clearance (and consequently increased exposure) resulting from reduced nonspecific interactions induced by exposed hydrophobic elements of the ADC or by camptothecin itself. The second effect is undesirable, and is a reduced volume and rate of distribution that sometimes results from an increased molecular weight of the ADC.

[0506] Increasing the number of PEG subunits increases the hydrodynamic radius of the conjugate, typically resulting in a decreased diffusivity. In turn, decreased diffusivity typically reduces the ADC's ability to penetrate tumors (Schmidt and Wittrup, Mol Cancer Ther 2009;8:2861-2871). Due to these two competing pharmacokinetic effects, it is desirable to use a PEG that is large enough to reduce ADC clearance and thus increase plasma exposure, but not so large that it significantly reduces its diffusivity and interferes with the ADC's ability to reach its intended target cell population. For methods for selecting the optimal PEG size for a particular drug linker, see the Examples (e.g., Examples 1, 18, and 21) of US2016 / 0310612, which is incorporated herein by reference.

[0507] In one group of embodiments, the PEG units comprise one or more linear PEG chains each having at least 2 subunits, at least 3 subunits, at least 4 subunits, at least 5 subunits, at least 6 subunits, at least 7 subunits, at least 8 subunits, at least 9 subunits, at least 10 subunits, at least 11 subunits, at least 12 subunits, at least 13 subunits, at least 14 subunits, at least 15 subunits, at least 16 subunits, at least 17 subunits, at least 18 subunits, at least 19 subunits, at least 20 subunits, at least 21 subunits, at least 22 subunits, at least 23 subunits, or at least 24 subunits. In preferred embodiments, the PEG units comprise a total of at least 4 subunits, at least 6 subunits, at least 8 subunits, at least 10 subunits, or at least 12 subunits. In some such embodiments, the PEG units comprise no more than about 72 total subunits, preferably no more than about 36 total subunits.

[0508] In another group of embodiments, the PEG units comprise a total of 4 to 72, 4 to 60, 4 to 48, 4 to 36, or 4 to 24 subunits, 5 to 72, 5 to 60, 5 to 48, 5 to 36, or 5 to 24 subunits, 6 to 72, 6 to 60, 6 to 48, 6 to 36, or 6 to 24 subunits, 7 to 72, 7 to 60, 7 to 48, 7 to 36, or 7 to 24 subunits, 8 to 72, 8 to 60, 8 to 48, 8 to 36, or 8 to 24 subunits, 9 to 72, 9 to 60, 9 to 48, 9 to 36, or 9 to 24 subunits, 10 to 72, 10 to 60, 10 to 48, 10 to 36, or 10 to 24 subunits, 11 to 72, 11 to 60, 11 to 48, 11 to 36, or 11 to 24 subunits, 12 to 72, 12 to 60, 12 to 48, 12 to 36, or 12 to 24 subunits, 13 to 72, 13 to 60, 13 to 48, 13 to 36, or 13 to 24 subunits, 14 to 72, 14 to 60, 14 to 48, 14 to 36, or 14 to 24 subunits, 15 to 72, 15 to 60, 15 to 48, 15 to 36, or 15 to 24 subunits 10-48, 9-36 or 9-24 subunits, 10-72, 10-60, 10-48, 10-36 or 10-24 subunits, 11-72, 11-60, 11-48, 11-36 or 11-24 subunits, 12-72, 12-60, 12-48, 12-36 or 12-24 subunits, 13-72, 13-60, 13-48, 13-36 or 13-24 subunits, 14-72, 14-60, 14-48, 14-36 or or 14 to 24 subunits, 15 to 72, 15 to 60, 15 to 48, 15 to 36, or 15 to 24 subunits, 16 to 72, 16 to 60, 16 to 48, 16 to 36, or 16 to 24 subunits, 17 to 72, 17 to 60, 17 to 48, 17 to 36, or 17 to 24 subunits, 18 to 72, 18 to 60, 18 to 48, 18 to 36, or 18 to 24 subunits, 19 to 72, 19 to 60, 19 to 48, 19 to 36, or 19 to 24 subunits 20 to 72, 20 to 60, 20 to 48, 20 to 36 or 20 to 24 subunits, 21 to 72, 21 to 60, 21 to 48, 21 to 36 or 21 to 24 subunits, 22 to 72, 22 to 60, 22 to 48, 22 to 36 or 22 to 24 subunits, 23 to 72, 23 to 60, 23 to 48, 23 to 36 or 23 to 24 subunits, or 24 to 72, 24 to 60, 24 to 48, 24 to 36 or 24 subunits.

[0509] Exemplary linear PEG units that may be used in any of the embodiments provided herein are as follows: [ka]

[0510] The wavy lines indicate attachment sites to parallel connector units (B), and each n is independently selected from 4 to 72, 6 to 72, 8 to 72, 10 to 72, 12 to 72, 6 to 24, or 8 to 24. In some embodiments, the subscript b is about 4, about 8, about 12, or about 24.

[0511] As described herein, PEG units are selected to improve the clearance of the resulting ADC but not significantly affect the ability of the conjugate to penetrate tumors. In embodiments, PEG units selected for use preferably have between 4 subunits and about 24 subunits, more preferably between about 4 subunits and about 12 subunits.

[0512] In preferred embodiments of the present disclosure, the PEG unit is about 300 daltons to about 5 kilodaltons, about 300 daltons to about 4 kilodaltons, about 300 daltons to about 3 kilodaltons, about 300 daltons to about 2 kilodaltons, or about 300 daltons to about 1 kilodalton. In some such embodiments, the PEG unit has at least 6 subunits, or at least 8, 10, or 12 subunits. In some such embodiments, the PEG unit has at least 6 subunits, or at least 8, 10, or 12 subunits, but not more than 72 subunits, preferably not more than 36 subunits.

[0513] It will be understood that when referring to PEG subunits, and depending on the context, the number of subunits can refer to an average number, for example, when referring to a population of ADCs or camptothecin-linker compounds that utilize polydisperse PEG.

[0514] Parallel connector unit (B): In some embodiments, the ADC and camptothecin linker compound comprise parallel connector units, providing points of attachment to the resolving agent (linker units -B( As a general embodiment, PEG units can be attached to parallel connector units such as lysines as shown below, with the wavy line and asterisk indicating the covalent bond within the linker unit of the ADC or camptothecin linker compound: [ka]

[0515] Spacer unit (Y): In some embodiments, the ADCs provided herein have a spacer (Y) between the releasable linker (RL) and the camptothecin. The spacer unit can be a functional group to facilitate attachment of the RL to the camptothecin, or it can provide an additional structural component (e.g., a methylene carbamate unit) to further facilitate release of the camptothecin unit from the remainder of the conjugate.

[0516] In these embodiments, to further facilitate release of the free drug exemplary camptothecin unit as a spacer unit is represented by the following formula: [ka]

[0517] In the formula, EWG represents an electron-withdrawing group, and R 1 is —H or C1-C4 alkyl, and the subscript n is 1 or 2. In some embodiments, EWG is —CN, —NO2, —CX3, —X, C(═O)OR′, —C(═O)N(R′)2, —C(═O)R′, —C(═O)X, —S(═O)2R′, —S(═O)2OR′, —S(═O)2NHR′, —S(═O)2N(R′)2, —P(═O)(OR′)2, —P(═O)(CH3)NHR′, —NO, —N(R′)3 + wherein X is -F, -Br, -Cl, or -I; and R ’are independently selected from the group consisting of hydrogen and C1-C6 alkyl; the wavy line adjacent to the nitrogen atom in each of formulas (a), (a'), (a''), (b), and (b') is the point of covalent attachment to R L; and the wavy line adjacent to the carbonyl carbon atom in formulas (b) and (b') is the point of covalent attachment to the hydroxyl or primary or secondary amine heteroatom of a camptothecin compound of formula CPT1, CPT2, CPT3, CPT4, CPT5, CPT6, or CPT7, or any one of compounds 14a-14z of Table I, or any one of compounds 18a-18r of Table J;

[0518] Formulas (a), (a'), and (a") represent exemplary methylene carbamate units where T* is a heteroatom from a hydroxyl or primary or secondary amine functionality of a camptothecin compound of formula CPT1, CPT2, CPT3, CPT4, CPT5, CPT6, or CPT7, or any one of compounds 14a-14z of Table I, or any one of compounds 18a-18r of Table J, and the wavy line adjacent to T* is the point of covalent attachment to the remainder of the camptothecin drug unit corresponding to the structure of the camptothecin compound.

[0519] In yet other embodiments, the spacer unit is a methylene carbamate unit represented by the formula: [ka]

[0520] Formula (a1) and formula (a1′) where each R is independently —H or C1-C4 alkyl are O * from a hydroxyl substituent on the lactone ring of a camptothecin compound of formula CPT1, CPT2, CPT3, CPT4, CPT5, CPT6 or CPT7, or any one of compounds 14a-14z of Table I or any one of compounds 18a-18r of Table J, or from another hydroxyl substituent on a camptothecin compound of formula CPT5 or CPT7, or R F or R F’R of CPT6 when at least one of is C1-C8 hydroxyalkylN,N—(C1-C4 hydroxyalkyl)(C1-C4 alkyl)-amino-C1-C8 alkyl- or N—C1-C4 hydroxyalkyl-C1-C8 aminoalkyl-, C1-C8 alkylC(O)— F or R F’ represents a methylene carbamate unit, which is an oxygen atom from a hydroxyl substituent of

[0521] The wavy lines in formula (a1), formula (a1') and formula (b1) retain their previous meaning from formulas (a), (a') and (b), respectively. In formula (a1'), -CH2CH2N + The (R)2 moiety represents an exemplary basic unit in protonated form.

[0522] Without being bound by theory, Scheme 1b illustrates the mechanism of free drug release from camptothecin attached to a methylene carbamate unit in an ADC bearing a self-immolative moiety. * is a heteroatom from a hydroxyl or primary or secondary amine of the camptothecin compound that is incorporated into the methylene carbamate unit.

[0523] Scheme 1b: [ka] Subscript "p" - Drug-to-Antibody Ratio (DAR) In one group of embodiments of the invention, the subscript p represents the number of drug linker moieties on the Ligand unit (e.g., antibody) of an individual ADC, and is preferably an integer ranging from 1 to 16, 1 to 12, 1 to 10, or 1 to 8. Individual ADCs are sometimes referred to as ADC compounds. In that group of embodiments, there are 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 drug linker moieties conjugated to the Ligand unit (e.g., antibody) of an individual ADC. In another group of embodiments of the invention, the ADC describes a population of individual ADC compounds that are substantially identical except for the number of camptothecin drug linker moieties attached to each Ligand unit (i.e., ADC composition), such that the subscript p represents the average number of camptothecin drug linker moieties attached to the Ligand unit of the ADC composition. In this group of embodiments, the subscript p, representing the DAR, is a number ranging from 1 to about 16, 1 to about 12, 1 to about 10, or 1 to about 8, 2 to about 16, 2 to about 12, 2 to about 10, or 2 to about 8. In some embodiments, the value of the subscript p refers to the average drug loading and drug loading of the predominant ADC in the composition. In some embodiments, the value of the subscript p refers to the predominant drug loading of the ADC in the composition. In some embodiments, at least about 60%, e.g., at least about any of 70%, 80%, 90%, 95%, 99%, 99.9%, or 100% of the ADCs in the composition have the value of the subscript p (i.e., DAR) as their drug loading. For example, in some embodiments, an ADC having a DAR of 8 can refer to a composition in which the predominant ADC has a DAR of 8 (e.g., at least about any of 60%, 70%, 80%, 90%, 95%, 99%, 99.9%, or 100% of the ADCs have a DAR of 8), and there may be a minor amount (e.g., no more than about any of 40%, 30%, 20%, 10%, 5%, 2%, 1%, or 0.1%) of ADCs with other DARs (e.g., a DAR of 8, 7, 6, 5, or 4).

[0524] In some embodiments, conjugation is via interchain disulfides, and 1 to about 8 camptothecin linker compound molecules are conjugated to a targeting agent serving as a Ligand unit. In some embodiments, conjugation is via an introduced cysteine ​​residue as well as an interchain disulfide, and there are 1 to 10, or 1 to 12, or 1 to 14, or 1 to 16 camptothecin linker compound moieties conjugated to a Ligand unit (e.g., an antibody). In some embodiments, conjugation is via an introduced cysteine ​​residue, and there are 4 or 8 camptothecin linker compound molecules conjugated to a Ligand unit (e.g., an antibody). [Table 5-1] [Table 5-2] [Table 6-1] [Table 6-2]

[0525] In some embodiments, an antibody-drug conjugate having the formula: L-(QD) p or a salt thereof, wherein: L, CDR1-H comprising the amino acid sequence shown in SEQ ID NO: 1; CDR2-H comprising the amino acid sequence shown in SEQ ID NO: 2; CDR3-H comprising the amino acid sequence shown in SEQ ID NO: 3; CDR1-L comprising the amino acid sequence shown in SEQ ID NO: 4; CDR2-L including the amino acid sequence NTR; a CDR3-L comprising the amino acid sequence shown in SEQ ID NO: 6; and a ligand unit comprising an antibody or an antigen-binding fragment thereof that binds to CEACAM5, the subscript p is an integer ranging from 1 to 16; Q is a linker unit, D is a Drug unit having the formula: [ka] R B is a member selected from the group consisting of H, C-C alkyl, C-C haloalkyl, C-C cycloalkyl, (C-C cycloalkyl)-C-C alkyl-, phenyl, and phenyl-C-C alkyl-; R C is a member selected from the group consisting of C1-C6 alkyl and C3-C6 cycloalkyl; Each R F and R F’ are independently -H, C1-C8 alkyl, C1-C8 hydroxyalkyl, C1-C8 aminoalkyl, (C1-C4 alkylamino)-C1-C8 alkyl-, N,N-(C1-C4 hydroxyalkyl)(C1-C4 alkyl)amino-C1-C8 alkyl-, N,N-di(C1-C4 alkyl)amino-C1-C8 alkyl-, N-(C1-C4 hydroxyalkyl)-C1-C8 aminoalkyl, C1-C8 alkyl-C(O)-, C1-C8 hydroxyalkyl-C(O)-, C1-C8 aminoalkyl-C(O)-, C3-C 10 Cycloalkyl, (C3-C 10 Cycloalkyl)-C1-C4 alkyl-, C3-C 10 Heterocycloalkyl, (C3-C 10 a member selected from the group consisting of heterocycloalkyl)-C1-C4 alkyl-, phenyl, phenyl-C1-C4 alkyl-, diphenyl-C1-C4 alkyl-, heteroaryl and heteroaryl-C1-C4 alkyl-; or R F and R F’each combined with the nitrogen atom to which it is attached forms a 5-, 6-, or 7-membered ring having 0 to 3 substituents selected from the group consisting of halogen, C1-C4 alkyl, —OH, —OC1-C4 alkyl, —NH2, —NHC1-C4 alkyl, and —N(C1-C4 alkyl)2; R B , R C , R F and R F’ wherein the cycloalkyl, heterocycloalkyl, phenyl, and heteroaryl portions of the formula (I) are substituted with 0 to 3 substituents selected from the group consisting of halogen, C1-C4 alkyl, —OH, —OC1-C4 alkyl, —NH2, —NHC1-C4 alkyl, and —N(C1-C4 alkyl)2, and the point of attachment of D to Q is such that Q is -ZA-RL-, -ZA-RL-Y-, -ZAS * -RL-, -ZAB(S * )-RL-, -ZAS * -RL-Y- or -ZAB(S * )-RL-Y- (wherein R is any one of the releasable linkers disclosed herein), via a heteroatom of any one of the hydroxyl functional groups or primary or secondary amine functional groups present on CPT1, CPT2, CPT3, CPT5, CPT6, or CPT7; or The attachment point of D to Q is -ZA-, -ZAS * -W- or -ZAB(S * )-W- or Q is -ZAS * -RL-, -ZAB(S * )-RL-, -ZAS * -W-RL- or -ZAB(S * )-W-R- (wherein R is a releasable unit other than a glucuronide unit), via the oxygen atom of a hydroxyl group substituent in the lactone ring of CPT1, CPT2, CPT3, CPT5, CPT6 or CPT7; However, R F and R F’ is -H when the point of attachment is the nitrogen atom of the amino group of CPT6; However, -ZA-RL-, -ZA-RL-Y-, -Z AS * -RL-, -ZAB(S * )-RL-, -ZAS * -RL-Y- and -ZAB(S * When -ZA- in )-RL-Y- is other than succinimido-caproyl-β-alanyl and D is CPT1 attached through its amino group, optionally with the succinimide ring in hydrolyzed form, the wavy line indicates the site of covalent attachment to Q.

[0526] In some embodiments, an antibody-drug conjugate having the formula: L-(QD) p or a salt thereof, provided herein, wherein: L, CDR1-H, CDR2-H, and CDR3-H of a variable heavy chain domain (VH) comprising the amino acid sequence shown in SEQ ID NO: 7; a ligand unit comprising an antibody or an antigen-binding fragment thereof that binds to CEACAM5, the antibody comprising CDR1-L, CDR2-L, and CDR3-L of a variable light chain domain (VL) comprising the amino acid sequence shown in SEQ ID NO: 8; the subscript p is an integer ranging from 1 to 16; Q is a linker unit, D is a Drug unit having the formula: [ka] R B is a member selected from the group consisting of H, C-C alkyl, C-C haloalkyl, C-C cycloalkyl, (C-C cycloalkyl)-C-C alkyl-, phenyl, and phenyl-C-C alkyl-; R C is a member selected from the group consisting of C1-C6 alkyl and C3-C6 cycloalkyl; Each R F and R F’are independently -H, C1-C8 alkyl, C1-C8 hydroxyalkyl, C1-C8 aminoalkyl, (C1-C4 alkylamino)-C1-C8 alkyl-, N,N-(C1-C4 hydroxyalkyl)(C1-C4 alkyl)amino-C1-C8 alkyl-, N,N-di(C1-C4 alkyl)amino-C1-C8 alkyl-, N-(C1-C4 hydroxyalkyl)-C1-C8 aminoalkyl, C1-C8 alkyl-C(O)-, C1-C8 hydroxyalkyl-C(O)-, C1-C8 aminoalkyl-C(O)-, C3-C 10 Cycloalkyl, (C3-C 10 Cycloalkyl)-C1-C4 alkyl-, C3-C 10 Heterocycloalkyl, (C3-C 10 a member selected from the group consisting of heterocycloalkyl)-C1-C4 alkyl-, phenyl, phenyl-C1-C4 alkyl-, diphenyl-C1-C4 alkyl-, heteroaryl and heteroaryl-C1-C4 alkyl-; or R F and R F’ each combined with the nitrogen atom to which it is attached forms a 5-, 6-, or 7-membered ring having 0 to 3 substituents selected from the group consisting of halogen, C1-C4 alkyl, —OH, —OC1-C4 alkyl, —NH2, —NHC1-C4 alkyl, and —N(C1-C4 alkyl)2; R B , R C , R F and R F’ wherein the cycloalkyl, heterocycloalkyl, phenyl, and heteroaryl portions are substituted with 0 to 3 substituents selected from the group consisting of halogen, C1-C4 alkyl, —OH, —O1-C4 alkyl, —NH2, —NHC1-C4 alkyl, and —N(C1-C4 alkyl)2; The attachment point of D to Q is -ZA-RL-, -ZA-RL-Y-, -ZAS * -RL-, -ZAB(S * )-RL-, -ZAS * -RL-Y- or -ZAB(S *)-RL-Y- (wherein R is any one of the releasable linkers disclosed herein), via a heteroatom of any one of the hydroxyl functional groups or primary or secondary amine functional groups present on CPT1, CPT2, CPT3, CPT5, CPT6, or CPT7; or The attachment point of D to Q is -ZA-, -ZAS * -W- or -ZAB(S * )-W- or Q is -ZAS * -RL-, -ZAB(S * )-RL-, -ZAS * -W-RL- or -ZAB(S * )-W-R- (wherein R is a releasable unit other than a glucuronide unit), via the oxygen atom of a hydroxyl group substituent in the lactone ring of CPT1, CPT2, CPT3, CPT5, CPT6 or CPT7; However, R F and R F’ is -H when the point of attachment is the nitrogen atom of the amino group of CPT6; However, -ZA-RL-, -ZA-RL-Y-, -Z AS * -RL-, -ZAB(S * )-RL-, -ZAS * -RL-Y- and -ZAB(S * When -ZA- in )-RL-Y- is other than succinimido-caproyl-β-alanyl and D is CPT1 attached through its amino group, optionally with the succinimide ring in hydrolyzed form, the wavy line indicates the site of covalent attachment to Q.

[0527] In some embodiments, an antibody-drug conjugate that binds to CEACAM5 has the formula: L-(QD) p or a salt thereof, provided herein, wherein: L is a ligand unit comprising an antibody or antigen-binding fragment thereof that binds to CEACAM5, the subscript p is an integer ranging from 1 to 16; Q is a linker unit having a formula selected from the group consisting of: -ZA-RL-, -ZA-RL-Y-, -ZAS * -RL-, -ZAB(S * )-RL-, -ZAS * -RL-Y- and -ZAB(S * )-RL-Y- Z is the stretcher unit, A is a coupling or connector unit, B is a parallel connector unit, S * is the resolving agent, RL is a glycoside unit, Y is a spacer unit, D is a Drug unit having the formula: [ka] The wavy line indicates the site of covalent attachment to Q, and the antibody or antigen-binding fragment thereof is CDR1-H comprising the amino acid sequence shown in SEQ ID NO: 1, and CDR2-H comprising the amino acid sequence shown in SEQ ID NO: 2; CDR3-H comprising the amino acid sequence shown in SEQ ID NO: 3; CDR1-L comprising the amino acid sequence shown in SEQ ID NO: 4; CDR2-L including the amino acid sequence NTR; and CDR3-L comprising the amino acid sequence shown in SEQ ID NO:6.

[0528] In some embodiments, the ADCs described herein have the formula: L-(QD)8 In the formula, L is CDR1-H comprising the amino acid sequence shown in SEQ ID NO: 1; CDR2-H comprising the amino acid sequence shown in SEQ ID NO: 2; CDR3-H comprising the amino acid sequence shown in SEQ ID NO: 3; CDR1-L comprising the amino acid sequence shown in SEQ ID NO: 4; CDR2-L including the amino acid sequence NTR; and a CDR3-L comprising the amino acid sequence set forth in SEQ ID NO: 6, and an antibody or antigen-binding fragment thereof that binds to CEACAM5, QD, [ka] is.

[0529] In some embodiments, provided herein is an antibody-drug conjugate having the following formula, or a salt thereof: L-(QD)8 In the formula, L is CDR1-H, CDR2-H, and CDR3-H of a variable heavy chain domain (VH) comprising the amino acid sequence shown in SEQ ID NO: 7; and a variable light chain domain (VL) comprising CDR1-L, CDR2-L, and CDR3-L, each of which comprises the amino acid sequence set forth in SEQ ID NO: 8, and an antibody or antigen-binding fragment thereof that binds to CEACAM5, QD, [ka] is.

[0530] In some embodiments, an antibody-drug conjugate that binds to CEACAM5 has the formula: L-(QD) p or a salt thereof, provided herein, wherein: L, CDR1-H comprising the amino acid sequence shown in SEQ ID NO: 1; CDR2-H comprising the amino acid sequence shown in SEQ ID NO: 2; CDR3-H comprising the amino acid sequence shown in SEQ ID NO: 3; CDR1-L comprising the amino acid sequence shown in SEQ ID NO: 4; CDR2-L including the amino acid sequence NTR; a CDR3-L comprising the amino acid sequence shown in SEQ ID NO: 6; and a ligand unit comprising an antibody or an antigen-binding fragment thereof that binds to CEACAM5, the subscript p is an integer ranging from 1 to 16; Q is a linker unit, D is a Drug unit, and the Drug unit is a topoisomerase I inhibitor.

[0531] In some embodiments, provided herein is an antibody-drug conjugate having the following formula, or a salt thereof: L-(QD)8 In the formula, L is a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 9; a light chain comprising the amino acid sequence shown in SEQ ID NO: 10; and an antibody or antigen-binding fragment thereof that binds to CEACAM5, Q is a linker unit, D is a Drug unit, and the Drug unit is a topoisomerase I inhibitor.

[0532] In some embodiments, provided herein is an antibody-drug conjugate having the following formula, or a salt thereof: L-(QD)8 In the formula, L is a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 9; a light chain comprising the amino acid sequence shown in SEQ ID NO: 10; and an antibody or antigen-binding fragment thereof that binds to CEACAM5, QD, [ka] is.

[0533] In some embodiments, provided herein is an antibody-drug conjugate having the following formula, or a salt thereof: L-(QD)8 In the formula, L is a heavy chain having the amino acid sequence set forth in SEQ ID NO: 9; a light chain having the amino acid sequence set forth in SEQ ID NO: 10; and an antibody or antigen-binding fragment thereof that binds to CEACAM5, QD, [ka] is.

[0534] C. Methods for preparing ADCs The ADCs described herein are prepared either by sequential construction of antibody, linker, and drug units, or in a convergent manner by assembling parts following a completed assembly step. The Curtius rearrangement or chloramine synthesis can be used to provide methylene carbamate linkers (spacers) that are useful in some embodiments of the conjugates described herein.

[0535] Scheme 2: Preparation of exemplary camptothecin drug linker compounds of formula Z'-A-RL-YD, Z'-AS*-RL-YD, or Z'-AB(S*)-RL-YD, where Y is prepared using a Curtius rearrangement reaction: [ka]

[0536] Scheme 2 shows a synthetic strategy involving the Curtius rearrangement of an acyl azide derivative of the free drug, where CPT is O *The oxygen atom represented by the formula (I) is incorporated into the resulting methylene carbamate unit, Z' is a Stretcher unit precursor, R is a releasable linker, X is -A-, -AS*-, or -AB(S*), A is a connector unit, S* is a partitioning agent, and B is a camptothecin drug unit corresponding to the structure of a camptothecin compound bearing a hydroxyl functional group, which is a parallel connector unit. This strategy can be applied to camptothecin drugs containing multiple alcohols or other heteroatoms as a means to achieve regioselectivity, since there are many complementary methods of alkylation to form acyl azides, such as haloester alkylation, haloacid alkylation, or metal carbene insertion with ethyl or methyl diazoacetate (see Doyle, M. et al., Modern Catalytic Methods for Organic Synthesis with Diazo Compounds; Wiley: New York, 1998). The acyl azide is then heated with at least a stoichiometric amount of an alcohol-containing linker unit intermediate of the formula Z'-X-R-OH.

[0537] Scheme 3: Alternative preparation of exemplary camptothecin drug linker compounds of formula Z'-A-RL-YD, Z'-AS*-RL-YD or Z'-AB(S*)-RL-YD, where spacer unit Y is a methylene carbamate unit of formula (a) or formula (a') via N-chloromethylamine synthesis: [ka]

[0538] In the formula, R 1 is hydrogen or C1-C4 alkyl, R is -H or -CH2CH2SO2Me, and the other variables have their meanings from Scheme 2.

[0539] The N-chloromethylamine synthesis is an alternative to the Curtius rearrangement in that it allows for the introduction of unmodified alcohols or other heteroatom containing camptothecin compounds that are not compatible with the conditions required to form the acyl azide in Scheme 2 and may proceed via condensation with a reactive N-chloromethylamine. The methodology is also more suitable for introducing certain types of methylene carbamate units, as shown, for example, by Scheme 4.

[0540] Scheme 4 illustrates the synthesis of exemplary camptothecin-linker compounds of formula Z'-A-RL-YD, Z'-AS*-RL-YD, or Z'-AB(S*)-RL-YD, where the spacer unit (Y) is a methylene carbamate unit of formula (a"). Reaction of p-nitro-phenyl carbonate with a cyclic aminol provides the carbamate, which is then converted to a chlorocycloalkylamine for alkylation with nucleophiles from thiol, hydroxyl, amine, or amide functional groups of the free camptothecin drug. Alternatively, the carbamate can be attached to the drug-linker compound shown. To assemble the camptothecin-linker intermediate, the alkylated product can be treated with acid in the presence of a drug moiety. After deprotection of the alkylated product, the resulting free amine is condensed with 3-maleimidopropionic acid N-hydroxysuccinimide ester, which introduces a Stretcher unit precursor covalently attached to the Connector unit, thus providing a camptothecin-linker compound. The resulting camptothecin-linker compound is then condensed with a thiol-containing targeting agent to provide an ADC having a self-immolative moiety and a Spacer unit comprising a methylene carbamate unit of formula a.

[0541] Scheme 4: Preparation of exemplary camptothecin drug linker compounds of formula Z'-A-RL-YD, where spacer unit Y is a methylene carbamate unit of formula (a") [ka]

[0542] In the case of ADCs having a camptothecin-linker compound and a methylene carbamate unit, where T* is a nitrogen atom from a primary or secondary amine substituent, direct alkylation of the camptothecin compound with chloromethylamine according to the generalized procedures provided by Scheme 3 or Scheme 4 may be undesirable due to excessive or undesired over-alkylation of the nitrogen heteroatom from the amine functionality of the free drug. In those instances, the method embodied by Scheme 5 can be used.

[0543] Scheme 5: [ka] In Scheme 5, an intermediate carbamate is prepared that already has a basic unit (i.e., a dimethylaminoethyl moiety) as the R substituent of the methylene carbamate unit of formula (a1'). The nitrogen of the carbamate is condensed with formaldehyde, and the resulting intermediate is quenched with the amine functionality of an aliphatic amine-containing camptothecin drug. N* represents the nitrogen atom from that functionality. The condensation forms a methylene carbamate of formula (a1') covalently bonded to the drug unit, where R 1 is hydrogen and R is dimethylaminoethyl. The phenylnitro group is then reduced to an amine to provide a handle for the sequential introduction of the connector unit (A) and the stretcher unit precursor (Z').

[0544] IV. Expression and Production of Antigen Binding Proteins A. Nucleic Acid Molecules Encoding Antigen-Binding Proteins Nucleic acid molecules encoding the antigen-binding proteins described herein, or portions thereof, are also provided. Such nucleic acids include, for example, 1) those encoding the antigen-binding protein (e.g., an antibody or fragment thereof), or derivatives or variants thereof; 2) polynucleotides encoding one or more HVRs or CDRs located within the heavy and / or light chain, VH domain and / or VL domain, or variable domain (e.g., one, two, or all three VH HVRs or CDRs, or one, two, or all three VL HVRs or CDRs); 3) polynucleotides sufficient for use as hybridization probes, PCR primers, or sequencing primers to identify, analyze, mutate, or amplify such encoding polynucleotides; 4) antisense nucleic acids for inhibiting expression of such encoding polynucleotides; and 5) complementary sequences of the foregoing. Nucleic acids can be of any length. They can be, for example, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 750, or 1,000 or more nucleotides in length, and / or can include one or more additional sequences, e.g., regulatory sequences, and / or can be part of a larger nucleic acid, e.g., a vector. Nucleic acids can be single-stranded or double-stranded.

[0545] Nucleic acid molecules can be present in whole cells, cell lysates, or in a partially purified or substantially pure form. A nucleic acid is "isolated" or "substantially purified" when it has been purified away from other cellular components or other contaminants, such as other cellular nucleic acids (e.g., other chromosomal DNA, e.g., chromosomal DNA linked to the isolated DNA in nature) or proteins, by standard techniques, including alkali / SDS treatment, CsCl banding, column chromatography, restriction enzymes, agarose gel electrophoresis, and others well known in the art. See F. Ausubel, et al., ed. (1987) Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York. The nucleic acids described herein can be, for example, DNA or RNA, and may or may not contain intronic sequences. In certain embodiments, the nucleic acid is a cDNA molecule.

[0546] Thus, provided are nucleic acid molecules comprising polynucleotides encoding one or more chains of an anti-CEACAM5 antibody. In some embodiments, the nucleic acid molecule comprises a polynucleotide encoding a heavy chain or a light chain of the anti-CEACAM5 antibody. In some embodiments, the nucleic acid molecule comprises both a polynucleotide sequence encoding a heavy chain and a polynucleotide sequence encoding a light chain of the anti-CEACAM5 antibody. In some embodiments, a first nucleic acid molecule comprises a first polynucleotide sequence encoding a heavy chain and a second nucleic acid molecule comprises a second polynucleotide sequence encoding a light chain.

[0547] In one embodiment, the nucleic acid molecule comprises a polynucleotide encoding the VH of one of the antibodies provided herein. In another embodiment, the nucleic acid comprises a polynucleotide encoding the VL of one of the antibodies provided herein. In yet another embodiment, the nucleic acid encodes both the VH and VL of one of the antibodies provided herein. In some embodiments, the nucleic acid encodes an antibody VH comprising the amino acid sequence set forth in SEQ ID NO:7 and a VL comprising the amino acid sequence set forth in SEQ ID NO:8.

[0548] In certain embodiments, the nucleic acids encode variants of one or more of the above amino acid sequences (e.g., heavy and / or light chain amino acid sequences, or VH and / or VL amino acid sequences disclosed herein), wherein the variants have up to 25 amino acid modifications, such as up to 20 amino acid modifications, for example, up to 15, 14, 13, 12 or 11 amino acid modifications, for example, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid modifications, e.g., deletions or insertions, preferably substitutions, e.g., conservative substitutions.

[0549] Once the nucleic acids encoding the VH and VL segments are obtained, these nucleic acids can be further manipulated by standard recombinant DNA techniques, for example, to convert the variable region genes into full-length antibody chain genes, Fab fragment genes, or scFv genes, in which the VL- or VH-encoding nucleic acid is operably linked to another nucleic acid encoding another polypeptide, such as an antibody constant region or a flexible linker.

[0550] ...

Claims

1. The following antibody-drug conjugate that binds to CEACAM5 has the following formula. L-(Q-D) p or its salt, in the formula, L is a ligand unit containing an antibody that binds to CEACAM5 or an antigen-binding fragment thereof. The subscript p is an integer in the range of 1 to 16. -Q-D is, 【Chemistry 1】 And in the formula, The wavy line indicates the covalent bond site to L. Z is 【Chemistry 2】 And in the formula, The wavy line with a double asterisk (**) indicates the covalent bond site to A. The wavy line with a triple asterisk (***) indicates the covalent bond point to the sulfur atom of L, and R 17 However, - (CH 2 ) X NH 2 -C which was optionally replaced by 1 -C 10 It is alkylene-C(=O)-, and in the formula x is an integer from 1 to 4. A is a connector unit having a combination or the following formula 【Transformation 3】 And in the formula, A wavy line with a single asterisk (*) indicates a covalent bond point to Z. The wavy line with a double asterisk (**) indicates the covalent bond site to the remaining part of Q-D. R 100 and R 111 are each independently hydrogen or -C 1 -C 3 alkyl, An antibody-drug conjugate or salt thereof, where c is an integer from 1 to 5.

2. An antibody or its antigen-binding fragment that binds to CEACAM5, CDR1-H containing the amino acid sequence shown in Sequence ID No. 1 and CDR2-H containing the amino acid sequence shown in Sequence ID No. 2, CDR3-H containing the amino acid sequence shown in Sequence ID No. 3, CDR1-L containing the amino acid sequence shown in Sequence ID No. 4, CDR2-L containing the amino acid sequence NTR, CDR3-L containing the amino acid sequence shown in Sequence ID No. 6, The antibody-drug conjugate or salt thereof according to claim 1, comprising:

3. The antibody or its antigen-binding fragment The variable heavy chain domains (VH) CDR1-H, CDR2-H, and CDR3-H, which contain the amino acid sequence shown in Sequence ID No. 7, CDR1-L, CDR2-L, and CDR3-L are variable light chain domains (VLs) containing the amino acid sequence shown in Sequence ID No. 8, The antibody-drug conjugate or salt thereof according to claim 1, comprising:

4. The antibody-drug conjugate or salt thereof according to claim 1, wherein the antibody or antigen-binding fragment comprises a heavy chain having the amino acid sequence shown in SEQ ID NO: 9 and a light chain having the amino acid sequence shown in SEQ ID NO:

10.

5. Z optionally has a succinimide ring in a hydrolyzed form as the succinamide portion. 【Chemistry 4】 The antibody-drug conjugate or salt thereof according to claim 1, wherein a wavy line with a double asterisk (**) indicates a covalent bonding site to the rest of Q, and a wavy line with a triple asterisk (***) indicates a covalent bonding site to the sulfur atom of L.

6. A has the following equation, 【Transformation 5】 During the ceremony, The wavy line with a double asterisk (**) indicates the covalent bond site to the rest of Q-D. A wavy line with a single asterisk (*) indicates a covalent bond point to Z. The antibody-drug conjugate or salt thereof according to claim 1.

7. -Q-D- has the following equation, 【Transformation 6】 During the ceremony, A wavy line with a single asterisk (*) indicates a covalent bond point to L. The antibody-drug conjugate or salt thereof according to claim 6.

8. The antibody-drug conjugate or salt thereof according to claim 1, comprising a drug-to-antibody (DAR) ratio of 1:10, wherein the DAR is optionally about 4 or about 8.

9. Claim 1, wherein Q is bound to the antibody or antigen-binding fragment via a cysteine ​​amino acid residue. The antibody-drug conjugate or salt thereof as described above.

10. The antibody-drug conjugate having the following formula L-(Q-D) 8 or its salt, in the formula, L, A heavy chain having the amino acid sequence shown in Sequence ID No. 9, A light chain having the amino acid sequence shown in Sequence ID No. 10, It comprises an antibody that binds to CEACAM5, or an antigen-binding fragment thereof, Q-D is, 【Transformation 7】 An antibody-drug conjugate or a salt thereof.

11. A pharmaceutical composition comprising an antibody-drug conjugate or a salt thereof according to any one of claims 1 to 10, and a pharmaceutically acceptable carrier.

12. An antibody-drug conjugate or salt thereof according to any one of claims 1 to 10, for use in the treatment of cancer in individuals requiring cancer treatment.

13. The aforementioned cancers include colorectal cancer, stomach cancer, gastric cancer, gastroesophageal junction cancer, lung cancer, uterine cancer, cervical cancer, pancreatic cancer, esophageal cancer, ovarian cancer, thyroid cancer, bladder cancer, endometrial cancer, neuroendocrine cancer, and endometrial cancer. An antibody-drug conjugate or salt thereof for use according to claim 12, selected from the group consisting of cancer, breast cancer, liver cancer, prostate cancer, and bile duct cancer, and skin cancer, optionally, non-small cell lung cancer (NSLCC), non-squamous NSCLC (nsq-NSLCC), squamous NSCLC (sq-NSLCC), or small cell lung cancer (SCLC), pancreatic ductal adenocarcinoma (PDAC), colorectal cancer (CRC), gastric cancer (GC), gastroesophageal junction cancer (GEJ), or any combination thereof.

14. The antibody-drug conjugate or salt thereof for use according to claim 12, wherein the individual has received prior treatment before being treated with the antibody-drug conjugate or salt thereof.

15. A kit comprising an antibody-drug conjugate or a salt thereof according to any one of claims 1 to 10.

16. Antibody-drug conjugate having the following formula or a salt thereof L-(Q-D) 8 or a method for preparing the salt thereof, in the formula, L comprises an antibody that binds to CEACAM5 or an antigen-binding fragment thereof, Q-D is, 【Transformation 8】 or a salt thereof, where a wavy line marked with a single asterisk (*) indicates a covalent bond to L. The process involves conjugating the drug linker compound to an antibody or antigen-binding fragment that binds to CEACAM5 by reacting the maleimide group of the drug linker compound with the sulfhydryl group of an antibody or antigen-binding fragment that binds to CEACAM5. The drug linker compound has the following structure: 【Chemistry 9】 A method having a salt thereof.

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