Anti-CEA antibody-exatecan analogue conjugate and its medical use

Abstract

The present invention relates to an anti-CEA antibody-exatecan analogue conjugate and its pharmaceutical use. Specifically, the anti-CEA antibody-exatecan analogue conjugate is represented by the general formula (Pc-LYD), where Pc is an anti-CEA antibody or an antigen-binding fragment thereof, L is a linker unit, and Y is -O-(CR a R b ) m -CR 1 R 2 -C(O)-, -O-CR 1 R 2 -(CR a R b ) m -, -O-CR 1 R 2 -, -NH-(CR a R b ) m -CR 1 R 2 -C(O)- and -S-(CR a R b ) m -CR 1 R 2 -C(O)-, n is 1 to 10, and n is a decimal number or an integer. TIFF2023506805000077.tif4755

Description

[Technical Field]

[0001] This application claims priority from a Chinese patent application filed on December 16, 2019 (application number CN 201911294912.3).

[0002] The present disclosure relates to an anti-CEA antibody-exatecan analog conjugate, a method for preparing the same, a pharmaceutical composition containing the same, and its use in preparing drugs for treating CEA-mediated diseases or conditions, particularly in preparing anti-cancer drugs. [Background technology]

[0003] Nothing herein necessarily constitutes prior art, but rather merely provides background information relevant to the present disclosure.

[0004] Carcinoembryonic antigen (CEA, also known as CEACAM-5 and CD66e) was one of the first tumor-associated antigens discovered. It is a glycoprotein with a molecular weight of approximately 180 kDa. CEA is a member of the immunoglobulin superfamily and contains seven domains linked to the cell membrane via a glycosylphosphatidylinositol (GPI) anchor (Thompson JA, J Clin Lab Anal. 5:344-366, 1991). CEA was first discovered and reported by Gold P and Freedman SO in tissue extracts of colon cancer (Gold and Freedman 1965; Gold and Freedman, 1965). Subsequently, CEA was detected in the sera of colon cancer patients and patients with other tumors using a highly sensitive radioimmunoassay, but its content in the sera of healthy individuals and patients with other diseases was reported to be very low (Thomson, Krupey et al., 1969). CEA is highly expressed in cancer cells, where it promotes cell-cell adhesion and cell migration (Marshall J., Semin Oncol., 30(Suppl. 8):30-6, 2003). CEA is commonly expressed in epithelial tissues, including cells of the gastrointestinal, respiratory, and genitourinary tracts, as well as cells of the colon, cervix, sweat glands, and prostate (Nap et al., Tumour Biol., 9(2-3):145-53, 1988; Nap et al., Cancer Res., 52(8):2329-23339, 1992).

[0005] Antibody drug conjugates (ADCs) link monoclonal antibodies or antibody fragments to biologically active cytotoxins via stable chemical linker compounds, taking advantage of the specificity of antibodies in binding to surface antigens on normal and tumor cells and the high performance of cytotoxic substances, while avoiding the relatively low therapeutic efficacy of the former and the excessive toxicity and side effects of the latter. This means that, compared with traditional chemotherapy drugs, antibody drug conjugates can bind more precisely to tumor cells and have less impact on normal cells.

[0006] Patents for antibodies and ADC drugs targeting CEA have already been reported, such as WO2015069430. However, there is currently still a need to develop more effective and safe anti-CEA antibody-drug conjugates for better use in the treatment of CEA-associated tumors. Summary of the Invention

[0007] The present disclosure relates to an anti-CEA antibody-drug conjugate comprising an anti-CEA antibody or an antigen-binding fragment thereof, and pharmaceutical uses thereof, wherein the anti-CEA antibody or antigen-binding fragment thereof is optionally linked to a toxic drug via a linker, and comprises an antibody heavy chain variable region and a light chain variable region, wherein: i) the HCDR1 and HCDR3 of the heavy chain variable region are the same as the HCDR1 and HCDR3 of the heavy chain variable region shown in SEQ ID NO:7, the HCDR2 of the heavy chain variable region is the same as the HCDR2 of the heavy chain variable region shown in SEQ ID NO:7 or has one amino acid difference therewith, and the LCDR1, LCDR2 and LCDR3 of the light chain variable region are the same as the LCDR1, LCDR2 and LCDR3 of the light chain variable region shown in SEQ ID NO:8, ii) the HCDR1 and HCDR3 of the heavy chain variable region are the same as the HCDR1 and HCDR3 of the heavy chain variable region shown in the sequence SEQ ID NO:9, the HCDR2 of the heavy chain variable region is the same as the HCDR2 of the heavy chain variable region shown in the sequence SEQ ID NO:9 or has one amino acid difference therewith, and the LCDR1, LCDR2 and LCDR3 of the light chain variable region are the same as the LCDR1, LCDR2 and LCDR3 of the light chain variable region shown in the sequence SEQ ID NO:10; iii) the HCDR1, HCDR2 and HCDR3 of the heavy chain variable region are the same as the HCDR1, HCDR2 and HCDR3 of the heavy chain variable region shown in the sequence SEQ ID NO:11, and the LCDR1, LCDR2 and LCDR3 of the light chain variable region are the same as the LCDR1, LCDR2 and LCDR3 of the light chain variable region shown in the sequence SEQ ID NO:12, or iv) the HCDR1 and HCDR3 of the heavy chain variable region are the same as the HCDR1 and HCDR3 of the heavy chain variable region shown in SEQ ID NO:13, the HCDR2 of the heavy chain variable region is the same as the HCDR2 of the heavy chain variable region shown in SEQ ID NO:13 or has one amino acid difference therebetween, the LCDR1 and LCDR3 of the light chain variable region are the same as the LCDR1 and LCDR3 of the light chain variable region shown in SEQ ID NO:14, and the LCDR2 of the light chain variable region is the same as the LCDR2 of the light chain variable region shown in SEQ ID NO:14 or has one amino acid difference therebetween.

[0008] In some embodiments of the present disclosure, in the antibody-drug conjugate, the anti-CEA antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, v) the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 set forth in SEQ ID NO:15, SEQ ID NO:16, and SEQ ID NO:17, respectively, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 set forth in SEQ ID NO:18, SEQ ID NO:19, and SEQ ID NO:20, respectively; or the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 set forth in SEQ ID NO:15, SEQ ID NO:38, and SEQ ID NO:17, respectively, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 set forth in SEQ ID NO:18, SEQ ID NO:19, and SEQ ID NO:20, respectively; vi) the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 set forth in SEQ ID NO:21, SEQ ID NO:22, and SEQ ID NO:23, respectively, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 set forth in SEQ ID NO:24, SEQ ID NO:25, and SEQ ID NO:26, respectively; or the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 set forth in SEQ ID NO:21, SEQ ID NO:47, and SEQ ID NO:23, respectively, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 set forth in SEQ ID NO:24, SEQ ID NO:25, and SEQ ID NO:26, respectively; vii) the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 set forth in SEQ ID NO:27, SEQ ID NO:28, and SEQ ID NO:29, respectively, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 set forth in SEQ ID NO:30, SEQ ID NO:31, and SEQ ID NO:32, respectively; or viii) the heavy chain variable region comprises HCDR1 and HCDR3 set forth in SEQ ID NO:33 and SEQ ID NO:34, respectively, and HCDR2 set forth in SEQ ID NO:16 or SEQ ID NO:38, and the light chain variable region comprises LCDR1 and LCDR3 set forth in SEQ ID NO:35 and SEQ ID NO:37, respectively, and LCDR2 set forth in SEQ ID NO:36 or SEQ ID NO:64.

[0009] In some embodiments, in the antibody-drug conjugate, the anti-CEA antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, In some embodiments, in the antibody-drug conjugate, the anti-CEA antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 set forth in SEQ ID NO:33, SEQ ID NO:16, and SEQ ID NO:34, respectively, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 set forth in SEQ ID NO:35, SEQ ID NO:36, and SEQ ID NO:37, respectively; or the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 set forth in SEQ ID NO:33, SEQ ID NO:38, and SEQ ID NO:34, respectively, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 set forth in SEQ ID NO:35, SEQ ID NO:36, and SEQ ID NO:37, respectively; or the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 set forth in SEQ ID NO: 33, SEQ ID NO: 16 and SEQ ID NO: 34, respectively; and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 set forth in SEQ ID NO: 35, SEQ ID NO: 64 and SEQ ID NO: 37, respectively; Or, Preferably, the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO:33, SEQ ID NO:38 and SEQ ID NO:34, respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:35, SEQ ID NO:64 and SEQ ID NO:37, respectively.

[0010] In some embodiments of the present disclosure, in the antibody-drug conjugate, the anti-CEA antibody is a murine antibody, a chimeric antibody, or a humanized antibody.

[0011] In some embodiments of the present disclosure, in the antibody-drug conjugate, the anti-CEA antibody or antigen-binding fragment thereof of any one of the above comprises a heavy chain variable region and a light chain variable region, (a) the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO:7 or is at least 90% identical to the amino acid sequence set forth in SEQ ID NO:7, and / or the amino acid sequence of the light chain variable region is set forth in SEQ ID NO:8 or is at least 90% identical to the amino acid sequence set forth in SEQ ID NO:8; or (b) the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO:9 or is at least 90% identical to the amino acid sequence set forth in SEQ ID NO:9, and / or the amino acid sequence of the light chain variable region is set forth in SEQ ID NO:10 or is at least 90% identical to the amino acid sequence set forth in SEQ ID NO:10; or (c) the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO:11 or is at least 90% identical to the amino acid sequence set forth in SEQ ID NO:11, and / or the amino acid sequence of the light chain variable region is set forth in SEQ ID NO:12 or is at least 90% identical to the amino acid sequence set forth in SEQ ID NO:12; or (d) the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO:13 or is at least 90% identical to the amino acid sequence set forth in SEQ ID NO:13, and / or the amino acid sequence of the light chain variable region is set forth in SEQ ID NO:14 or is at least 90% identical to the amino acid sequence set forth in SEQ ID NO:14.

[0012] In some embodiments of the present disclosure, in the antibody-drug conjugate, the anti-CEA antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, (e) the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO:39, 40, 41, or 42, or has at least 90% identity to any one of the amino acid sequences set forth in SEQ ID NO:39, 40, 41, or 42, and / or the amino acid sequence of the light chain variable region is set forth in SEQ ID NO:43, 44, 45, or 46, or has at least 90% identity to any one of the amino acid sequences set forth in SEQ ID NO:43, 44, 45, or 46; Preferably, the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO:42 and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO:44; or (f) the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO:48, 49, 50, 51, or 52, or has at least 90% identity to any one of the amino acid sequences set forth in SEQ ID NO:48, 49, 50, 51, or 52, and / or the amino acid sequence of the light chain variable region is set forth in SEQ ID NO:53, 54, or 55, or has at least 90% identity to any one of the amino acid sequences set forth in SEQ ID NO:53, 54, or 55; Preferably, the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO:52 and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO:53; or (g) the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO:56, 57, or 58, or has at least 90% identity to any one of the amino acid sequences set forth in SEQ ID NO:56, 57, or 58, and / or the amino acid sequence of the light chain variable region is set forth in SEQ ID NO:59, 60, 61, 62, or 63, or has at least 90% identity to any one of the amino acid sequences set forth in SEQ ID NO:59, 60, 61, 62, or 63; Preferably, the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO:58 and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO:62; or (h) the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO:65, 66, 67, or 68, or has at least 90% identity to any one of the amino acid sequences set forth in SEQ ID NO:65, 66, 67, or 68, and / or the amino acid sequence of the light chain variable region is set forth in SEQ ID NO:69, 70, 71, 72, 73, 74, 75, or 76, or has at least 90% identity to any one of the amino acid sequences set forth in SEQ ID NO:69, 70, 71, 72, 73, 74, 75, or 76; Preferably, the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO:68, and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO:76.

[0013] In some embodiments of the present disclosure, in the antibody-drug conjugate, the anti-CEA antibody of any one of the above is a humanized antibody, the humanized antibody comprises a framework region derived from a human antibody or a framework region variant thereof, and the framework region variant has at most 10 amino acid backmutations in the light chain framework region and / or the heavy chain framework region of the human antibody, respectively; Preferably, the framework region variant is selected from any one of the following (i) to (l): (i) the framework regions of the light chain variable region comprising LCDR1, LCDR2 and LCDR3, the sequences of which are set forth in SEQ ID NO:18, SEQ ID NO:19 and SEQ ID NO:20, respectively, contain one or more amino acid backmutations selected from 46P, 47W, 49Y, 70S and 71Y; and / or the framework regions of the heavy chain variable region comprising HCDR1, the sequence of which is set forth in SEQ ID NO:15, HCDR2, the sequence of which is set forth in SEQ ID NO:16 or SEQ ID NO:38, and HCDR3, the sequence of which is set forth in SEQ ID NO:17, contain one or more amino acid backmutations selected from 38K or 46K; (j) the framework regions of the light chain variable region, including LCDR1, LCDR2, and LCDR3, whose sequences are set forth in SEQ ID NO:24, SEQ ID NO:25, and SEQ ID NO:26, respectively, contain one or more amino acid backmutations selected from 2V, 42G, 44V, and 71Y; and / or the framework regions of the heavy chain variable region, including HCDR1, whose sequence is set forth in SEQ ID NO:21, HCDR2, whose sequence is set forth in SEQ ID NO:22 or SEQ ID NO:47, and HCDR3, whose sequence is set forth in SEQ ID NO:23, contain one or more amino acid backmutations selected from 48I, 66K, 67A, 69L, 71V, 73K, 82F, 82A R (k) the framework regions of the light chain variable region, including LCDR1, LCDR2, and LCDR3, whose sequences are set forth in SEQ ID NO:30, SEQ ID NO:31, and SEQ ID NO:32, respectively, contain one or more amino acid backmutations selected from 3V, 43P, and 58V; and / or the framework regions of the heavy chain variable region, including HCDR1, HCDR2, and HCDR3, whose sequences are set forth in SEQ ID NO:27, SEQ ID NO:28, and SEQ ID NO:29, respectively, contain one or more amino acid backmutations selected from 38K, 66K, and 71V; and (l) the framework regions of the light chain variable region, including LCDR1 whose sequence is set forth in SEQ ID NO: 35, LCDR2 whose sequence is set forth in SEQ ID NO: 36 or SEQ ID NO: 64, and LCDR3 whose sequence is set forth in SEQ ID NO: 37, contain one or more amino acid backmutations selected from 4V, 36Y, 43P, 47V, 49E, 70D, and 87I; and / or the framework regions of the heavy chain variable region, including HCDR1 whose sequence is set forth in SEQ ID NO: 33, HCDR2 whose sequence is set forth in SEQ ID NO: 16 or SEQ ID NO: 38, and HCDR3 whose sequence is set forth in SEQ ID NO: 34, contain one or more amino acid backmutations selected from 2I, 38K, and 46K; (m) the framework regions of the light chain variable region, including LCDR1, LCDR2, and LCDR3, whose sequences are set forth in SEQ ID NO: 24, SEQ ID NO: 25, and SEQ ID NO: 26, respectively, contain one or more amino acid backmutations selected from 2V, 42G, 44V, and 71Y; and / or the framework regions of the heavy chain variable region, including HCDR1, whose sequence is set forth in SEQ ID NO: 21, HCDR2, whose sequence is set forth in SEQ ID NO: 22 or SEQ ID NO: 47, and HCDR3, whose sequence is set forth in SEQ ID NO: 23, contain one or more amino acid backmutations selected from 66K, 67A, 69L, 71V, 73K, 82F, 82A R Therein, the sites of reversion are numbered according to the Kabat numbering convention.

[0014] In some embodiments of the present disclosure, in the antibody-drug conjugate, the anti-CEA antibody of any one of the above is a humanized antibody, the humanized antibody comprises a framework region derived from a human antibody or a framework region variant thereof, and the framework region variant has at most 10 amino acid backmutations in the light chain framework region and / or the heavy chain framework region of the human antibody, respectively; Preferably, the framework region variant is selected from any one of the following (i) to (l): (i) the framework regions of the light chain variable region, including LCDR1, LCDR2, and LCDR3, whose sequences are set forth in SEQ ID NO:18, SEQ ID NO:19, and SEQ ID NO:20, respectively, contain one or more amino acid backmutations selected from 46P, 47W, 49Y, 70S, and 71Y; and / or the framework regions of the heavy chain variable region, including HCDR1 whose sequence is set forth in SEQ ID NO: 15, HCDR2 whose sequence is set forth in SEQ ID NO: 16 or SEQ ID NO: 38, and HCDR3 whose sequence is set forth in SEQ ID NO: 17, contain one or more amino acid backmutations selected from 38K or 46K; (j) the framework regions of the light chain variable region, including LCDR1 set forth in SEQ ID NO:24, LCDR2 set forth in SEQ ID NO:25, and LCDR3 set forth in SEQ ID NO:26, respectively, contain one or more amino acid backmutations selected from 2V, 42G, 44V, and 71Y; and / or The framework region of the heavy chain variable region, which includes HCDR1 whose sequence is set forth in SEQ ID NO: 21, HCDR2 whose sequence is set forth in SEQ ID NO: 22 or SEQ ID NO: 47, and HCDR3 whose sequence is set forth in SEQ ID NO: 23, contains 48I, 66K, 67A, 69L, 71V, 73K, 82F, 82A R (k) the framework regions of the light chain variable region, including LCDR1, LCDR2, and LCDR3, whose sequences are set forth in SEQ ID NO:30, SEQ ID NO:31, and SEQ ID NO:32, respectively, contain one or more amino acid backmutations selected from 3V, 43P, and 58V; and / or The framework regions of the heavy chain variable region, including HCDR1, HCDR2, and HCDR3, whose sequences are set forth in SEQ ID NO: 27, SEQ ID NO: 28, and SEQ ID NO: 29, respectively, contain one or more amino acid backmutations selected from 38K, 66K, and 71V; and (l) the framework regions of the light chain variable region, including LCDR1 whose sequence is set forth in SEQ ID NO:35, LCDR2 whose sequence is set forth in SEQ ID NO:36 or SEQ ID NO:64, and LCDR3 whose sequence is set forth in SEQ ID NO:37, contain one or more amino acid backmutations selected from 4V, 36Y, 43P, 47V, 49E, 70D, and 87I; and / or the framework regions of the heavy chain variable region, including HCDR1 whose sequence is set forth in SEQ ID NO: 33, HCDR2 whose sequence is set forth in SEQ ID NO: 16 or SEQ ID NO: 38, and HCDR3 whose sequence is set forth in SEQ ID NO: 34, contain one or more amino acid backmutations selected from 2I, 38K, and 46K; Therein, the sites of reversion are numbered according to the Kabat numbering convention.

[0015] In some embodiments of the present disclosure, in the antibody-drug conjugate, the anti-CEA antibody or antigen-binding fragment thereof described in any one of the above comprises an antibody heavy chain constant region and a light chain constant region, preferably the heavy chain constant region is selected from human IgG1, IgG2, IgG3, and IgG4 constant regions, and the light chain constant region is selected from human antibody κ and λ chain constant regions, more preferably the antibody comprises a heavy chain constant region having a sequence set forth in SEQ ID NO: 77 and a light chain constant region having a sequence set forth in SEQ ID NO: 78 or SEQ ID NO: 79; Most preferably, the anti-CEA antibody used in the present disclosure is (m) a heavy chain whose sequence is set forth in SEQ ID NO:80 or has at least 85% identity thereto, and / or a light chain whose sequence is set forth in SEQ ID NO:81 or has at least 85% identity thereto; (n) a heavy chain whose sequence is set forth in SEQ ID NO:82 or has at least 85% identity thereto, and / or a light chain whose sequence is set forth in SEQ ID NO:83 or has at least 85% identity thereto; (o) a heavy chain whose sequence is set forth in SEQ ID NO:84 or has at least 85% identity thereto, and / or a light chain whose sequence is set forth in SEQ ID NO:85 or has at least 85% identity thereto; or (p) a heavy chain whose sequence is set forth in SEQ ID NO:86 or has at least 85% identity thereto, and / or a light chain whose sequence is set forth in SEQ ID NO:87 or has at least 85% identity thereto.

[0016] In some embodiments of the present disclosure, the antigen-binding fragment of any one of the preceding claims is selected from a Fab, a Fab', a F(ab')2, a single-chain antibody (scFv), a dimerized V region (diabody), and a disulfide-stabilized V region (dsFv).

[0017] In some embodiments of the present disclosure, the antibody-drug conjugate described in any one of the preceding claims is an antibody-drug conjugate represented by the general formula (Pc-LYD): [ka] Among them, Y is -O-(CR a R b ) m -CR 1 R 2 -C(O)-, -O-CR 1 R 2 -(CR a R b ) m -, -O-CR 1 R 2 -, -NH-(CR a R b ) m -CR 1 R 2 -C(O)- and -S-(CR a R b ) m -CR 1 R 2 -C(O)-, R a and R b are the same or different and are each independently selected from a hydrogen atom, a deuterium atom, a halogen, an alkyl group, a haloalkyl group, a deuterated alkyl group, an alkoxy group, a hydroxy group, an amino group, a cyano group, a nitro group, a hydroxyalkyl group, a cycloalkyl group, and a heterocyclyl group, or R a and R b together with the carbon atoms to which they are attached form a cycloalkyl group and a heterocyclyl group, R 1is selected from halogen, haloalkyl, deuterated alkyl, cycloalkyl, cycloalkylalkyl, alkoxyalkyl, heterocyclyl, aryl and heteroaryl groups; R 2 is selected from a hydrogen atom, a halogen, a haloalkyl group, a deuterated alkyl group, a cycloalkyl group, a cycloalkylalkyl group, an alkoxyalkyl group, a heterocyclyl group, an aryl group, and a heteroaryl group, or R 1 and R 2 together with the carbon atoms to which they are attached form a cycloalkyl or heterocyclyl group, Or R a and R 2 together with the carbon atoms to which they are attached form a cycloalkyl or heterocyclyl group, m is an integer from 0 to 4, n is 1 to 10, and n is a decimal or an integer; L is a linker unit, Pc is an anti-CEA antibody or antigen-binding fragment thereof as described above.

[0018] In some embodiments of the present disclosure, in the antibody-drug conjugate described in any one of the above aspects, n is an integer or decimal number of 0 to 10, and may be an average value of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, preferably 1 to 8, more preferably 2 to 8, and most preferably 4 to 6, and n is an average value of a decimal number or an integer.

[0019] In some embodiments of the present disclosure, in the antibody-drug conjugate described in any one of the above, n is an average value of 3 to 5 decimals or integers.

[0020] In some embodiments of the present disclosure, in the antibody-drug conjugate described in any one of the above, n is an average value of 6 to 7 decimal or integers.

[0021] In some embodiments of the present disclosure, the antibody-drug conjugate described in any one of the above aspects is represented by the general formula (Pc-LYD), wherein: Y is -O-(CR aR b ) m -CR 1 R 2 -C(O)-, R a and R b are the same or different and are each independently selected from a hydrogen atom, a deuterium atom, a halogen atom, and an alkyl group; R 1 is a haloalkyl group or C 3-6 is a cycloalkyl group, R 2 represents a hydrogen atom, a haloalkyl group, and C 3-6 cycloalkyl groups, Or R 1 and R 2 are C together with the carbon atoms connected to them. 3-6 forming a cycloalkyl group, m is 0 or 1.

[0022] In some embodiments of the present disclosure, in the antibody-drug conjugate of any one of the preceding claims, Y is [ka] wherein the O-end of Y is linked to the linker unit L.

[0023] In some embodiments of the present disclosure, in the antibody-drug conjugate of any one of the preceding claims, Y is [ka] wherein the O-end of Y is linked to the linker unit L.

[0024] In some embodiments of the present disclosure, the antibody-drug conjugate of any one of the preceding claims is selected from the following: [ka] Among them, L is a linker unit, Pc is an anti-CEA antibody or an antigen-binding fragment thereof; n is 1 to 10, and n is a decimal number or an integer.

[0025] In some embodiments of the present disclosure, the antibody-drug conjugate of any one of the preceding claims is selected from the following: [ka] Among them, L is a linker unit, Pc is an anti-CEA antibody or an antigen-binding fragment thereof; n is 1 to 10, and n is a decimal number or an integer.

[0026] In some embodiments of the present disclosure, in the antibody-drug conjugate of any one of the preceding claims, the linker unit -L- is -L 1 -L 2 -L 3 -L 4 - and L 1 -(succinimide-3-yl-N)-WC(O)-, -CH2-C(O)-NR 3 -WC(O)- or -C(O)-WC(O)-, wherein W is C 1-8 Alkyl group, C 1-8 alkyl-cycloalkyl groups or straight-chain heteroalkyl groups of 1 to 8 atoms, said heteroalkyl groups containing 1 to 3 heteroatoms selected from N, O or S, among which said C 1-8 the alkyl group, cycloalkyl group, and linear heteroalkyl group are each independently optionally further substituted with one or more substituents selected from halogen, hydroxyl group, cyano group, amino group, alkyl group, chloroalkyl group, deuterated alkyl group, alkoxy group, and cycloalkyl group; L 2 Ha-NR 4 (CH2CH2O)p 1 CH2CH2C(O)-, -NR 4 (CH2CH2O)p 1 CH2C(O)-, -S(CH2)p 1 C(O)- or a chemical bond, among which p 1is an integer between 1 and 20, L 3 is a peptide residue consisting of 2 to 7 amino acids, wherein the amino acids are selected from amino acid residues formed by the amino acids phenylalanine, glycine, valine, lysine, citrulline, serine, glutamic acid, and aspartic acid, and are optionally further substituted with one or more substituents selected from halogen, hydroxy group, cyano group, amino group, alkyl group, chloroalkyl group, deuterated alkyl group, alkoxy group, and cycloalkyl group; L 4 is -NR 5 (CR 6 R 7 ) t -, -C(O)NR 5 , -C(O)NR 5 (CH2) t - and a chemical bond, wherein t is an integer from 1 to 6; R 3 , R 4 and R 5 are the same or different and are each independently selected from a hydrogen atom, an alkyl group, a haloalkyl group, a deuterated alkyl group, and a hydroxyalkyl group; R 6 and R 7 are the same or different and are each independently selected from a hydrogen atom, a halogen, an alkyl group, a haloalkyl group, a deuterated alkyl group, and a hydroxyalkyl group.

[0027] In some embodiments, L 1 -(succinimide-3-yl-N)-WC(O)-, -CH2-C(O)-NR 3 -WC(O)- or -C(O)-WC(O)-, wherein W is C 1-8 Alkyl group, C 1-8 Alkyl group-C 3-6 A cycloalkyl group or a straight chain heteroalkyl group of 1 to 8 chain atoms, said heteroalkyl group containing 1 to 3 heteroatoms selected from N, O or S, among which said C 1-8 Alkyl group, C 1-8 Alkyl group-C3-6 The cycloalkyl groups or straight chain heteroalkyl groups of 1 to 8 chain atoms are each independently optionally further substituted with one or more substituents selected from halogen, hydroxy, cyano, amino, alkyl, chloroalkyl, deuterated alkyl, alkoxy, and cycloalkyl groups.

[0028] In some embodiments, L 2 Ha-NR 4 (CH2CH2O)p 1 CH2CH2C(O)-, -NR 4 (CH2CH2O)p 1 CH2C(O)-, -S(CH2)p 1 C(O)- or a chemical bond, among which p 1 is an integer between 1 and 20.

[0029] In some embodiments, L 3 is a peptide residue consisting of 2 to 7 amino acids, wherein the amino acids are selected from amino acid residues formed by the amino acids phenylalanine, glycine, valine, lysine, citrulline, serine, glutamic acid, and aspartic acid, and are optionally further substituted with one or more substituents selected from halogen, hydroxyl group, cyano group, amino group, alkyl group, chloroalkyl group, deuterated alkyl group, alkoxy group, and cycloalkyl group.

[0030] In some embodiments, L 4 Ha-NR 5 (CR 6 R 7 ) t -, -C(O)NR 5 -, -C(O)NR 5 (CH2) t - and a chemical bond, wherein t is an integer of 1 to 6.

[0031] In some embodiments, R 3 , R 4 and R 5are the same or different and are each independently selected from a hydrogen atom, an alkyl group, a haloalkyl group, a deuterated alkyl group, and a hydroxyalkyl group.

[0032] In some embodiments, R 6 and R 7 are the same or different and are each independently selected from a hydrogen atom, a halogen, an alkyl group, a haloalkyl group, a deuterated alkyl group, and a hydroxyalkyl group.

[0033] In some embodiments of the present disclosure, in the antibody-drug conjugate of any one of the preceding claims, the linker unit -L- is -L 1 -L 2 -L 3 -L 4 - and L 1 teeth [ka] and s 1 is an integer between 2 and 8, L 2 is a chemical bond, L 3 is a tetrapeptide residue, preferably L 3 is the tetrapeptide residue of glycine-glycine-phenylalanine-glycine (GGFG, SEQ ID No: 92), L 4 Ha-NR 5 (CR 6 R 7 )t- and R 5 , R 6 or R 7 are the same or different and each independently represents a hydrogen atom or an alkyl group; t is 1 or 2; Among them, the above L 1 The end is connected to Pc, and L 4 The ends are connected to a Y.

[0034] In some embodiments of the present disclosure, the antibody-drug conjugate described in any one of the preceding claims is represented by the general formula (Pc-LYD) or the general formula Pc-LD, wherein -L- is [ka] is.

[0035] In some embodiments of the present disclosure, the antibody-drug conjugate described in any one of the preceding claims is represented by the general formula (Pc-LYD), or the general formula Pc-LD, in which -LY- is optionally [ka] Selected from.

[0036] In some embodiments of the present disclosure, the antibody-drug conjugate of any one of the preceding claims is represented by the general formula (Pc-L a -YD), [ka] Among them, W, L 2 , L 3 , R 5 , R 6 , R 7 is as defined for the linker unit L, Pc, n, R 1 , R 2 , m is as defined in the general formula (Pc-LYD).

[0037] In some embodiments of the present disclosure, the antibody-drug conjugate of any one of the preceding claims is represented by the general formula (Pc-L b -YD), [ka] Among them, s 1 is an integer between 2 and 8, Pc, R 1 , R2 , R 5 ~R 7 , m and n are represented by the general formula (Pc-L a -YD).

[0038] In some embodiments of the present disclosure, the antibody-drug conjugate of any one of the preceding claims is selected from the following: [ka] In this, Pc and n are as defined in the general formula (Pc-LYD).

[0039] In some embodiments of the present disclosure, the antibody-drug conjugate is selected from: [ka] wherein n is as defined in the general formula (Pc-LYD), The antibodies are as follows: Hu63-13 comprises a heavy chain whose sequence is set forth in SEQ ID NO:80 and a light chain whose sequence is set forth in SEQ ID NO:81; Hu47-14 comprises a heavy chain whose sequence is set forth in SEQ ID NO:82 and a light chain whose sequence is set forth in SEQ ID NO:83; Hu67-14 comprises a heavy chain whose sequence is set forth in SEQ ID NO:84 and a light chain whose sequence is set forth in SEQ ID NO:85; Hu103-32 comprises a heavy chain whose sequence is shown in SEQ ID NO:86 and a light chain whose sequence is shown in SEQ ID NO:87.

[0040] Alternatively, n may be a non-zero integer or decimal number from 0 to 10, preferably an integer or decimal number from 1 to 10, more preferably an integer or decimal number from 2 to 8, and most preferably an integer or decimal number from 3 to 8, and optionally n is an integer or decimal number from 3 to 5, and optionally n is an integer or decimal number from 6 to 7.

[0041] The present disclosure provides: [ka] After reducing Pc, the compound of the general formula (L a -YD, and the resulting product is subjected to a coupling reaction with the general formula (Pc-L a -YD), wherein Pc is an anti-CEA antibody or an antigen-binding fragment thereof; W, L 2 , L 3 , R 1 , R 2 , R 5 ~R 7 , m and n are represented by the general formula (Pc-L a -YD), a Further provided is a method for preparing an antibody-drug conjugate of formula (I-YD).

[0042] The present disclosure provides: [ka] The method includes the steps of reducing Pc, and then coupling with a compound of general formula (L'-D) to obtain a compound, wherein: Pc is an anti-CEA antibody or antigen-binding fragment thereof as described above, Further provided is a method for preparing an antibody-drug conjugate represented by the general formula (Pc-L'-D), wherein n is as defined in the general formula (Pc-LYD).

[0043] In another aspect, the present disclosure provides a drug composition comprising the antibody-drug conjugate of any one of the preceding claims and one or more pharmaceutically acceptable excipients, diluents, or carriers.

[0044] In another aspect, the present disclosure provides use of the antibody-drug conjugate according to any one of the preceding claims or a pharmaceutical composition comprising the same as a drug.

[0045] In another aspect, the present disclosure provides a use of the antibody-drug conjugate or a pharmaceutical composition comprising the same described in any one of the preceding claims in preparing a medicament for treating a CEA-mediated disease or condition, wherein the CEA-mediated disease or condition is a CEA-high-expressing cancer or a CEA-low-expressing cancer.

[0046] In another aspect, the present disclosure provides a use of the antibody-drug conjugate according to any one of the preceding claims or a drug composition comprising the same in preparing a drug for treating or preventing tumors and cancers, and the tumors and cancers are preferably head and neck squamous cell carcinoma, head and neck cancer, brain cancer, glioma, glioblastoma multiforme, neuroblastoma, central nervous system cancer, neuroendocrine tumor, pharyngeal cancer, nasopharyngeal cancer, esophageal cancer, thyroid cancer, malignant pleural mesothelioma, lung cancer, breast cancer, liver cancer, hepatic and gallbladder cancer, pancreatic cancer, gastric cancer, gastrointestinal cancer, intestinal cancer, colon cancer, colorectal cancer, renal cancer, clear cell renal cell carcinoma, ovarian cancer, endometrial cancer, cervical cancer, bladder cancer, prostate cancer, testicular cancer, skin cancer, melanoma, leukemia, lymphoma, bone cancer, chondrosarcoma, myeloma, multiple myeloma, myelodysplastic syndromes, myelopathic rheumatoid arthritis ... and more preferably, the lymphoma is selected from Hodgkin's lymphoma, non-Hodgkin's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, primary mediastinal large B-cell lymphoma, mantle cell lymphoma, small lymphocytic lymphoma, T-cell / histiocyte-rich large B-cell lymphoma, and lymphoplasmacytic lymphoma; the lung cancer is selected from non-small cell lung cancer and small cell lung cancer; and the leukemia is selected from chronic myeloid leukemia, acute myeloid leukemia, lymphocytic leukemia, lymphoblastic leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, and myeloid cell leukemia.

[0047] In another aspect, the present disclosure further relates to a method for treating and / or preventing a tumor, the method comprising the step of administering to a patient in need thereof a therapeutically effective amount of the antibody-drug conjugate described in any one of the preceding claims or a pharmaceutical composition comprising the same, wherein the tumor is preferably a cancer associated with high CEA expression.

[0048] In another aspect, the present disclosure further relates to a method for treating or preventing cancer, the method comprising the step of administering to a patient in need thereof a therapeutically effective amount of the antibody-drug conjugate according to any one of the preceding claims or a pharmaceutical composition comprising the same, wherein the tumor and cancer are preferably head and neck squamous cell carcinoma, head and neck cancer, brain cancer, glioma, glioblastoma multiforme, neuroblastoma, central nervous system cancer, neuroendocrine tumor, pharyngeal cancer, nasopharyngeal cancer, esophageal cancer, thyroid cancer, malignant pleural mesothelioma, lung cancer, breast cancer, liver cancer, hepatic and gallbladder cancer, pancreatic cancer, gastric cancer, gastrointestinal cancer, intestinal cancer, colon cancer, colorectal cancer, renal cancer, clear cell renal cell carcinoma, ovarian cancer, endometrial cancer, cervical cancer, bladder cancer, prostate cancer, testicular cancer, skin cancer, melanoma, leukemia, lymphoma, bone cancer, chondrosarcoma, myeloma, The preferred cancers include multiple myeloma, myelodysplastic syndrome, Kruckenberg's tumor, myeloproliferative neoplasm, squamous cell carcinoma, Ewing's sarcoma, systemic light-chain amyloidosis, and Merkel cell carcinoma. More preferably, the lymphoma is selected from Hodgkin's lymphoma, non-Hodgkin's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, primary mediastinal large B-cell lymphoma, mantle cell lymphoma, small lymphocytic lymphoma, T-cell / histiocyte-rich large B-cell lymphoma, and lymphoplasmacytic lymphoma. The preferred lung cancers are non-small cell lung cancer and small cell lung cancer. The preferred leukemias are chronic myeloid leukemia, acute myeloid leukemia, lymphocytic leukemia, lymphoblastic leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, and myeloid cell leukemia.

[0049] The active compound (e.g., a ligand-drug conjugate described herein, or a pharmaceutically acceptable salt thereof) can be in a form suitable for administration by any suitable route, preferably in a unit dose form or in a form that can be self-administered by a subject as a single dose. Unit dose forms according to the present disclosure may be lozenges, capsules, cachets, bottled liquids, powders, granules, tablets, suppositories, reconstituted powders, or liquid formulations.

[0050] The dosage of the active compound or composition used in the therapeutic methods of the present disclosure will generally vary depending on the severity of the disease, the weight of the subject, and the potency of the active compound, although as a general guide, a suitable unit dose may be 0.1 to 1000 mg.

[0051] The pharmaceutical composition of the present invention may contain one or more additives in addition to the active compound, and the additives are selected from components such as fillers, diluents, binders, wetting agents, disintegrants, excipients, etc. The composition may contain 0.1 to 99% by weight of the active compound, depending on the method of administration.

[0052] The CEA antibodies and antibody-drug conjugates provided by the present disclosure not only have good affinity for cell surface antigens, efficient endocytosis by cells, and high tumor inhibition efficiency, but also have broader drug application possibilities and are suitable for clinical drug application. [Brief explanation of the drawings]

[0053] [Figure 1] 1 shows the results of FACS detection of the binding of a humanized antibody to human CEA at the cellular level.

[0054] [Figure 2] Bystander effect cytotoxicity of ADC molecules. As shown by the data results, all ADC molecules have a very high bystander effect cytotoxicity. When MKN45 and HCT116 are co-cultured, the ADC molecules can inhibit the proliferation of both types of cells. However, when HCT116 is cultured alone, the ADC molecules bound to 2-A are almost non-toxic to the cells.

[0055] [Figure 3]The effect of ADC molecules on tumor volume in the LS174T tumor xenograft model. The data results show that, compared with the control group (PBS), all ADC molecules have the effect of inhibiting tumor volume growth. The tumor inhibition effect of the 3 mpk group is higher than that of the 1 mpk group. In the 3 mpk group, the best tumor inhibition effect is Hu63-13-2-A, followed by Hu47-14-2-A, and then Hu67-14-2-A, and the worst is Lmab-CL2A-SN38.

[0056] [Figure 4] The effect of ADC molecules on tumor weight in the LS174T tumor xenograft model was shown. Compared to the control group (PBS), all ADC molecules inhibited tumor weight gain at both low and high doses. Among the 3 MPK groups, Hu63-13-2-A had the best tumor-inhibiting effect, followed by Hu47-14-2-A and Hu67-14-2-A, and Lmab-CL2A-SN38 had the worst effect.

[0057] [Figure 5] The effect of ADC molecules on tumor volume in the MKN45 tumor xenograft model. The data results show that, compared with the control group (PBS), all ADC molecules have the effect of inhibiting tumor volume growth, with the tumor inhibition effect of the 3 mpk group being higher than that of the 1 mpk group. In the 3 mpk group, the best tumor inhibition effect was Hu67-14-2-A, followed by Hu103-32-2-A, then Hu63-13-2-A, and the worst was Lmab-CL2A-SN38.

[0058] [Figure 6] The effect of ADC molecules on tumor weight in the MKN45 tumor xenograft model was shown. Compared to the control group (PBS), all ADC molecules inhibited tumor weight gain at both low and high doses. Among the 3 MPK groups, Hu67-14-2-A had the best tumor-inhibiting effect, followed by Hu103-32-2-A, then Hu63-13-2-A, and the worst was Lmab-CL2A-SN38. DETAILED DESCRIPTION OF THE INVENTION

[0059] 1. Terminology Unless otherwise defined, all technical and scientific terms used herein are accorded the meaning commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, this application describes the preferred methods and materials. In describing and claiming this disclosure, the following terms will be used in accordance with the following definitions.

[0060] When trade names are used in this disclosure, it is intended to include formulations of products bearing that trade name, generic drugs and active drug portions of products bearing that trade name.

[0061] Unless otherwise specified, terms used in the specification and claims have the following meanings.

[0062] The term "drug" refers to a chemical substance that can alter or reveal the physiological functions and pathological conditions of the body and can be used to prevent, diagnose, and treat diseases. Drugs include cytotoxic drugs. Although there is no strict boundary between drugs and poisons, poisons refer to chemical substances that have toxic effects on the body and damage human health in relatively small doses, and any drug can also cause toxic reactions if the dose is too large.

[0063] Cytotoxic drugs are substances that inhibit or prevent the function of cells and / or cause the death or destruction of cells. In principle, cytotoxic drugs can kill tumor cells even at sufficiently high concentrations, but due to a lack of specificity, they can also cause apoptosis of normal cells while killing tumor cells, resulting in serious side effects. Cytotoxic drugs include toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, radioisotopes (e.g., At 211 , I 131 , I 125 , Y 90 ,Re 186 ,Re 188 , Sm153 , Bi 212 , P 32 and radioactive isotopes of Lu), toxic drugs, chemotherapeutic agents, antibiotics and nucleolytic enzymes.

[0064] The term "linker unit," "linker," or "linking fragment" refers to a chemical fragment or bond that is linked at one end to a ligand and at the other end to a drug, and may be linked to another linker and then to a drug.

[0065] The linker may comprise one or more linker elements. Exemplary linker elements include 6-maleimidocaproyl ("MC"), maleimidopropionyl ("MP"), valine-citrulline ("val-cit" or "vc"), alanine-phenylalanine ("ala-phe"), p-aminobenzyloxycarbonyl ("PAB"), N-succinimidyl 4-(2-pyridylthio)pentanoate ("SPP"), N-succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate ("SMCC", also referred to herein as "MCC"), and N-succinimidyl (4-iodo-acetyl)aminobenzoate ("SIAB"). The linker may include an extender, a spacer, and an amino acid unit and can be synthesized by methods known in the art, such as the method described in US2005-0238649A1. The linker may be a "cleavable linker" that facilitates drug release in cells. For example, an acid-labile linker (e.g., hydrazone), a protease-sensitive (e.g., peptidase-sensitive) linker, a photolabile linker, a dimethyl linker, or a disulfide-containing linker (Chari et al., Cancer Research 52: 127-131 (1992), U.S. Patent No. 5,208,020) can be used.

[0066] Abbreviation

[0067] The linker element is The structure is as follows: MC=6-maleimidocaproyl; [ka] Val-Cit or "vc" = valine-citrulline (an exemplary dipeptide in a protease-cleavable linker); Citrulline = 2-amino-5-ureidopentanoic acid, PAB = p-aminobenzyloxycarbonyl (an example of a "self-immolative" linker element); Me-Val-Cit = N-methyl-valine-citrulline (in which the linker peptide bond is modified so as not to be cleaved by cathepsin B); MC(PEG)6-OH = maleimidocaproyl-polyethylene glycol (can be attached to antibody cysteines); SPP = N-succinimidyl 4-(2-pyridylthio)pentanoate, SPDP = N-succinimidyl 3-(2-pyridyldithio)propionate, SMCC = succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate; Including but not limited to IT = iminothiolane.

[0068] The term "antibody-drug conjugate" refers to an antibody linked to a biologically active drug via a stable linking unit. As used herein, "antibody-drug conjugate" or antibody-drug conjugate (ADC) refers to a monoclonal antibody or antibody fragment linked to a biologically active toxic drug via a stable linking unit. The antibody can be bound to the drug directly or via a linker. The average number of drug modules per antibody (mean drug loading or drug carrying capacity, which may be indicated by the n value) can range, for example, from about 0 to about 20 drug modules per antibody; in some embodiments, each antibody is bound to 1 to about 10 drug modules; and in some embodiments, each antibody is bound to 1 to about 8 drug modules.

[0069] The term "average drug loading" or "drug loading" refers to the average amount of cytotoxic drugs loaded on each ligand in a molecule of Formula (I), and may be expressed as the ratio of the amount of drug to the amount of antibody. The drug loading ranges from 0 to 12, preferably 1 to 10, cytotoxic drugs per ligand (Pc). In embodiments of the present disclosure, the drug loading is represented by n, which may be referred to as the drug-antibody ratio (DAR) value, and is, for example, the average of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. The average amount of drug on each ADC molecule after the coupling reaction can be characterized by conventional methods, such as UV / visible spectroscopy, mass spectrometry, ELISA, and HPLC.

[0070] The three-letter and one-letter codes for amino acids used in this disclosure are as described in J. Biol. Chem, 243, p. 3558 (1968).

[0071] The term "antibody" refers to an immunoglobulin, which has a tetrapeptide chain structure consisting of two heavy chains and two light chains linked by interchain disulfide bonds. Based on the amino acid composition and sequence order of the heavy chain constant region of the immunoglobulin, immunoglobulins can be divided into five classes or immunoglobulin isotypes: IgM, IgD, IgG, IgA, and IgE, with the corresponding heavy chains being μ, δ, γ, α, and ε chains, respectively. Ig of the same class can be further divided into different subclasses based on differences in the amino acid composition of the hinge region and the number and location of heavy chain disulfide bonds; for example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4. Light chains are divided into kappa or lambda chains based on differences in the constant region. Each of the five Ig classes can have either kappa or lambda chains.

[0072] Approximately 110 amino acids near the N-terminus of full-length antibody heavy and light chains are highly variable and form the variable region (Fv region), while the remaining amino acid sequences near the C-terminus are relatively stable and form the constant region. The variable region contains three hypervariable regions (HVRs) and four framework regions (FRs) whose sequences are relatively conserved. The three hypervariable regions determine the specificity of the antibody and are also called complementarity-determining regions (CDRs). Each light chain variable region (LCVR) and heavy chain variable region (HCVR) consists of three CDR regions and four FR regions, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The three CDR regions of the light chain are referred to as LCDR1, LCDR2, and LCDR3, and the three CDR regions of the heavy chain are referred to as HCDR1, HCDR2, and HCDR3.

[0073] The terms "fully humanized antibody," "fully human antibody," or "fully human antibody" are also known as "fully humanized monoclonal antibodies," in which both the variable and constant regions of the antibody are human-derived, eliminating immunogenicity, toxicity, and side effects. The development of monoclonal antibodies has gone through four stages: mouse monoclonal antibodies, chimeric monoclonal antibodies, humanized monoclonal antibodies, and fully humanized monoclonal antibodies. The main technologies involved in the preparation of fully humanized antibodies include human hybridoma technology, EBV-mediated B lymphocyte transformation technology, phage display technology, transgenic mouse antibody preparation technology, and single B cell antibody preparation technology.

[0074] The term "antigen-binding fragment" refers to one or more fragments of an antibody that retain the ability to bind to an antigen. It has been shown that fragments of a full-length antibody can perform the antigen-binding function of an antibody. The binding fragments included in the term "antigen-binding fragment" are selected from antigen-binding fragments of Fab, Fab', F(ab')2, single-chain antibodies (scFv), dimerized V regions (diabodies), disulfide-stabilized V regions (dsFv), and peptides containing CDRs, and include, for example, (i) a Fab fragment, which is a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; (ii) a F(ab')2 fragment, which is a bivalent fragment comprising two Fab fragments linked by a disulfide bridge in the hinge region; (iii) an Fd fragment consisting of the VH and CH1 domains; (iv) an Fv fragment consisting of the VH and VL domains of a single antibody arm; (v) a single domain or dAb fragment consisting of the VH domain (Ward et al., (1989) Nature 341: 544-546); and (vi) an isolated complementarity-determining region (CDR); or (vii) a combination of two or more isolated CDRs, which can be optionally linked by a synthetic linker. Although the two domains of an Fv fragment, VL and VH, are encoded by separate genes, they can be linked by a synthetic linker using recombinant techniques to generate a single protein chain in which the VL and VH regions pair to form a monovalent molecule (called a single-chain Fv (scFv); see, for example, Bird et al. (1988) Science 242: 423-426 and Huston et al. (1988) Proc. Natl. Acad. Sci USA 85: 5879-5883). Such single-chain antibodies are also intended to be encompassed by the term "antigen-binding fragment" of an antibody. Such antibody fragments can be obtained by conventional techniques known to those skilled in the art, and the fragments are screened for functionality in the same way as intact antibodies. Antigen-binding portions can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact immunoglobulins. The antibodies may be of different isotypes, such as, for example, IgG (eg, IgG1, IgG2, IgG3 or IgG4 subtypes), IgA1, IgA2, IgD, IgE or IgM antibodies.

[0075] Typically, Fab is an antibody fragment with a molecular weight of approximately 50,000 that has antigen-binding activity and is obtained by treating an IgG antibody molecule with the protease papain (e.g., the 224th amino acid residue, which cleaves the H chain). In this fragment, the N-terminal portion of the H chain and the L chain are linked together by disulfide bonds.

[0076] F(ab')2 is an antibody fragment typically obtained by digesting the lower portion of the disulfide bond in the hinge region of IgG with the enzyme pepsin. It has a molecular weight of approximately 100,000, retains antigen-binding activity, and is comprised of two Fab regions linked at the hinge position.

[0077] Generally, Fab' is an antibody fragment having a molecular weight of about 50,000 and having antigen-binding activity, which is obtained by cleaving the disulfide bond in the hinge region of the above-mentioned F(ab')2.

[0078] Furthermore, the Fab' can be produced by inserting DNA encoding the Fab' fragment into a prokaryotic or eukaryotic expression vector and introducing the vector into a prokaryote or eukaryote to express the Fab'.

[0079] The terms "single-chain antibody," "single-chain Fv," or "scFv" refer to molecules comprising an antibody heavy chain variable domain (or VH) and an antibody light chain variable domain (or VL) linked by a linker. Such scFv molecules can have the general structure NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art linkers consist of repeated GGGGS amino acid sequences or variants thereof, for example, 1-4 repeat variants (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90: 6444-6448). Other linkers for use in the present disclosure are described in Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur. J. Immunol. 31:94-106, Hu et al. (1996), Cancer Res. 56:3055-3061, Kipriyanov et al. (1999), J. Mol. Biol. 293:41-56, and Roovers et al. (2001), Cancer Immunol.

[0080] The term "CDR" refers to one of the six major hypervariable regions in an antibody variable domain that mediates antigen binding. Typically, each heavy chain variable region has three CDRs (HCDR1, HCDR2, and HCDR3), and each light chain variable region has three CDRs (LCDR1, LCDR2, and LCDR3). The amino acid sequence boundaries of a CDR can be determined using any one of a variety of known approaches. One of the most commonly used definitions of the six CDRs is provided by Kabat EA et al. (1991) Sequences of proteins of immunological interest. NIH Publication 91-3242. As used herein, the Kabat definition of CDR applies only to CDR1, CDR2, and CDR3 in the light chain variable domain and CDR2 and CDR3 in the heavy chain variable domain. Further numbering conventions include the "Chothia" numbering convention, the "ABM" numbering convention, the "contact" numbering convention (see Martin, ACR. Protein Sequence and Structure Analysis of Antibody Variable Domains[J]. 2001), and the ImMunoGenTics (IMGT) numbering convention (Lefranc MP, Dev. Comp. Immunol., 27, 55-77(2003)).

[0081] The term "antibody framework" refers to a part of a variable domain VL or VH that serves as a support for the antigen binding loops (CDRs) of said variable domain. It is essentially a variable domain without the CDRs.

[0082] The term "epitope" or "antigenic determinant" refers to the site on an antigen that is bound by an immunoglobulin or antibody. An epitope usually includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 consecutive or non-consecutive amino acids in a unique spatial conformation. See, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, GE Morris, Ed. (1996).

[0083] The terms "specific binding," "selective binding," "selectively binds," and "specifically binds" refer to the binding of an antibody to a predetermined epitope on an antigen. Typically, an antibody binds to an epitope of about 10 -7 Smaller than M, e.g., about 10 -8 M, 10 -9 M or 10 -10 binds with an affinity (KD) less than or equal to M.

[0084] The term "KD" refers to the dissociation equilibrium constant at which an antibody and an antigen interact. Typically, antibodies (or antigen-binding fragments) of the present disclosure have a dissociation equilibrium constant of about 10 -7 Smaller than M, e.g., about 10 -8 M or 10 -9 It binds to CEA (or its epitope) with a dissociation equilibrium constant (KD) smaller than M, and in the present disclosure, for example, the KD value of the affinity between an antibody and a cell surface antigen is measured by the FACS method.

[0085] The term "competition," when used in reference to competition between antigen-binding proteins for the same epitope (e.g., neutralizing antigen-binding proteins or neutralizing antibodies), refers to competition between antigen-binding proteins as measured by the assay methods described below, in which the antigen-binding protein to be detected (e.g., an antibody or an immunologically functional fragment thereof) prevents or inhibits (e.g., reduces) specific binding between a reference antigen-binding protein (e.g., a ligand or reference antibody) and a common antigen (e.g., a CEA antigen or a fragment thereof). A variety of competitive binding assays can be used to determine whether one antigen-binding protein competes with another, including, for example, solid-phase direct or indirect radioimmunoassays (RIAs), solid-phase direct or indirect enzyme immunoassays (EIAs), sandwich competition assays (see, e.g., Stahli et al., 1983, Methods in Enzymology 9:242-253), solid-phase direct biotin-avidin EIAs (see, e.g., Kirkland et al., 1986, J. Immunol. 137:3614-3619), solid-phase direct labeling assays, solid-phase direct labeling sandwich assays (see, e.g., Harlow and Lane, 1988, Antibodies, A Laboratory Manual, Cold Spring Harbor Press), solid-phase direct labeling RIAs using an I-125 marker (see, e.g., Morel et al., 1988, Molec. Immunol. 25:3614-3619), and the like. These include solid-phase direct biotin-avidin EIA (see, e.g., Cheung et al., 1990, Virology 176: 546-552), solid-phase direct biotin-avidin EIA (see, e.g., Cheung et al., 1990, Virology 176: 546-552), and direct labeling RIA (Moldenhauer et al., 1990, Scand. J. Immunol. 32: 77-82). Typically, these assays involve the use of purified antigen bound to a solid surface or cells bearing either an unlabeled detection antigen-binding protein or a labeled reference antigen-binding protein. Competitive inhibition is measured by measuring the amount of label bound to the solid surface or cells in the presence of the antigen-binding protein to be measured. Typically, the antigen-binding protein to be measured is present in excess.Antigen-binding proteins identified by competitive assays (competing antigen-binding proteins) include antigen-binding proteins that bind to the same epitope as the reference antigen-binding protein and antigen-binding proteins that bind to an adjacent epitope sufficiently close to the epitope bound by the reference antigen-binding protein, such that the two epitopes spatially interfere with each other during binding. Further details of methods for measuring competitive binding are provided in the Examples of this application. Typically, when a competing antigen-binding protein is present in excess, specific binding between the reference antigen-binding protein and a common antigen is inhibited (e.g., reduced) by at least 40-45%, 45-50%, 50-55%, 55-60%, 60-65%, 65-70%, 70-75%, or 75% or more. In some cases, binding is inhibited by at least 80-85%, 85-90%, 90-95%, 95-97%, or 97% or more.

[0086] The term "nucleic acid molecule" refers to a DNA molecule or an RNA molecule. A nucleic acid molecule can be single-stranded or double-stranded, but preferably is double-stranded DNA. A nucleic acid is "operatively linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is operatively linked to a coding sequence if the promoter or enhancer affects the transcription of the coding sequence.

[0087] "Identity" of an amino acid sequence refers to the percentage of amino acid residues in a first sequence that are identical to those in a second sequence, with gaps introduced as necessary during alignment of the amino acid sequences to maximize the percentage of sequence identity, and with any conservative substitutions not considered part of the sequence identity. To measure the percentage of amino acid sequence identity, alignment can be achieved by several methods within the skill of the art, such as publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2, or Megalign (DNASTAR) software. Those skilled in the art can determine the parameters to be applied for measuring alignment, including any algorithms necessary to achieve maximum alignment over the entire length of the sequences being compared.

[0088] The term "expression vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. In one embodiment, the vector is a "plasmid," which refers to a circular double-stranded DNA loop into which other DNA segments can be ligated. In another embodiment, the vector is a viral vector, into which other DNA segments can be ligated into the viral genome. The vectors disclosed herein can either autonomously replicate in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors), or can integrate into the genome of a host cell after introduction and thereby replicate along with the host genome (e.g., non-episomal mammalian vectors).

[0089] Methods for producing and purifying antibodies and antigen-binding fragments well known in the art are described, for example, in Chapters 5-8 and 15 of Reisenko's Manual of Antibody Laboratory Techniques. Antigen-binding fragments can also be prepared by conventional methods. The antibodies or antigen-binding fragments described in the invention have one or more human-derived FR regions added to non-human CDR regions by genetic engineering techniques. Human FR germline sequences can be obtained from the ImMunoGeneTics (IMGT) website http: / / imgt.cines.fr by aligning the IMGT human antibody variable region germline gene database with MOE software, or from the immunoglobulin journal Lefranc, G., The Immunoglobulin Facts Book, Academic Press, 2001, ISBN 012441351.

[0090] The term "host cell" refers to a cell into which an expression vector has been introduced. Host cells may include bacterial, microbial, plant, or animal cells. Bacteria amenable to transformation include members of the Enterobacteriaceae family, such as strains of Escherichia coli and Salmonella, Bacillaceae, such as Bacillus subtilis, Pneumococcus, Streptococcus, and Haemophilus influenzae. Suitable microorganisms include Saccharomyces cerevisiae and Pichia pastoris. Suitable animal host cell lines include CHO (Chinese hamster ovary cell line) and NS0 cells.

[0091] The engineered antibodies or antigen-binding fragments of the present disclosure can be prepared and purified by conventional methods. For example, cDNA sequences encoding the heavy and light chains can be cloned and recombined into expression vectors. The recombinant immunoglobulin expression vector can be stably transfected into host cells. In a more preferred conventional technique, a mammalian expression system results in antibody glycosylation, particularly at the N-terminal end of the Fc region. Positive clones are cultured in a bioreactor to produce the antibody. The culture medium secreting the antibody can be purified by conventional techniques, for example, through a Sepharose A or G FF column. Nonspecifically bound components are washed away. The bound antibody is then eluted using a pH gradient, and the antibody fragments are detected and collected by SDS-PAGE. The antibody can be filtered and concentrated by conventional methods. Soluble mixtures and multimers may be removed by conventional methods, such as molecular sieving or ion exchange. The resulting product should be immediately frozen, e.g., at -70°C, or lyophilized.

[0092] The term "peptide" refers to a compound fragment between an amino acid and a protein, consisting of two or more amino acid molecules linked together by peptide bonds, and is a structural and functional fragment of a protein.

[0093] The term "sugar" refers to a biopolymer made up of the three elements C, H, and O, and can be divided into monosaccharides, disaccharides, and polysaccharides.

[0094] The term "alkyl group" refers to a saturated aliphatic hydrocarbon group that is a straight or branched chain group containing from 1 to 20 carbon atoms, preferably an alkyl group containing from 1 to 12 carbon atoms, more preferably an alkyl group containing from 1 to 10 carbon atoms, and most preferably an alkyl group containing from 1 to 6 carbon atoms (including 1, 2, 3, 4, 5, or 6 carbon atoms). Non-limiting examples thereof include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, n-heptyl, 2-methylhexyl, 3-methylhexyl, 4-methylhexyl, 5-methylhexyl, and the like. Examples of alkyl groups include 2,3-dimethylpentyl, 2,4-dimethylpentyl, 2,2-dimethylpentyl, 3,3-dimethylpentyl, 2-ethylpentyl, 3-ethylpentyl, n-octyl, 2,3-dimethylhexyl, 2,4-dimethylhexyl, 2,5-dimethylhexyl, 2,2-dimethylhexyl, 3,3-dimethylhexyl, 4,4-dimethylhexyl, 2-ethylhexyl, 3-ethylhexyl, 4-ethylhexyl, 2-methyl-2-ethylpentyl, 2-methyl-3-ethylpentyl, n-nonyl, 2-methyl-2-ethylhexyl, 2-methyl-3-ethylhexyl, 2,2-diethylpentyl, n-decyl, 3,3-diethylhexyl, 2,2-diethylhexyl, and various branched chain isomers thereof.More preferred are lower alkyl groups containing 1 to 6 carbon atoms, non-limiting examples of which include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, and the like. The alkyl group may be substituted or unsubstituted, and when substituted, the substituent may be substituted at any available linkage site, and the substituent is preferably one or more groups independently selected from alkyl groups, alkenyl groups, alkynyl groups, alkoxy groups, alkylthio groups, alkylamino groups, halogens, mercapto groups, hydroxy groups, nitro groups, cyano groups, cycloalkyl groups, heterocycloalkyl groups, aryl groups, heteroaryl groups, cycloalkoxy groups, heterocycloalkoxy groups, cycloalkylthio groups, heterocycloalkylthio groups, and oxo groups.

[0095] The term "heteroalkyl group" is an alkyl group containing one or more heteroatoms selected from N, O, or S, as defined above.

[0096] The term "alkylene group" refers to a saturated, straight-chain or branched-chain aliphatic hydrocarbon group having two residues derived by removing two hydrogen atoms from the same carbon atom or two different carbon atoms of a parent alkane, and is a straight-chain or branched-chain alkylene group containing 1 to 20 carbon atoms, preferably 1 to 12 carbon atoms, and more preferably 1 to 6 carbon atoms (including 1, 2, 3, 4, 5, or 6 carbon atoms). Non-limiting examples of alkylene groups include, but are not limited to, methylene (-CH-), 1,1-ethylene (-CH(CH)-), 1,2-ethylene (-CHCH)-, 1,1-propylene (-CH(CHCH)-), 1,2-propylene (-CHCH(CH)-), 1,3-propylene (-CHCHCHCH-), 1,4-butylene (-CHCHCHCHCH-), and 1,5-butylene (-CHCHCHCHCHCH-). An alkylene group may be substituted or unsubstituted, and when substituted, the substituent may be substituted at any available linkage site, and said substituent is preferably independently and optionally substituted with one or more substituents selected from alkyl groups, alkenyl groups, alkynyl groups, alkoxy groups, alkylthio groups, alkylamino groups, halogens, mercapto groups, hydroxy groups, nitro groups, cyano groups, cycloalkyl groups, heterocyclyl groups, aryl groups, heteroaryl groups, cycloalkoxy groups, heterocycloalkoxy groups, cycloalkylthio groups, heterocycloalkylthio groups, and oxo groups.

[0097] The term "alkoxy group" refers to -O-(alkyl group) and -O-(unsubstituted cycloalkyl group), where alkyl group or cycloalkyl group is defined above. Non-limiting examples of alkoxy groups include methoxy, ethoxy, propoxy, butoxy, cyclopropoxy, cyclobutoxy, cyclopentyloxy, and cyclohexyloxy groups. An alkoxy group may be optionally substituted or unsubstituted; if substituted, the substituents are preferably one or more groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, and heterocycloalkylthio groups.

[0098] The term "haloalkyl group" refers to an alkyl group, as defined above, in which a hydrogen has been replaced with one or more halogens.

[0099] The term "deuterated alkyl group" refers to an alkyl group, as defined above, in which a hydrogen has been replaced with one or more deuterium atoms.

[0100] The term "hydroxyalkyl group" refers to an alkyl group, as defined above, in which a hydrogen has been replaced with one or more hydroxy groups.

[0101] The term "hydroxy group" refers to an --OH group.

[0102] The term "halogen" refers to fluorine, chlorine, bromine or iodine.

[0103] The term "amino group" refers to -NH2.

[0104] The term "nitro group" refers to -NO2.

[0105] The term "cyano" refers to -CN.

[0106] The present disclosure further includes various deuterated forms of the compound of formula (I). Each available hydrogen atom connected to a carbon atom may be independently replaced with a deuterium atom. Those skilled in the art can synthesize deuterated forms of the compound of formula (I) by referring to relevant literature. When preparing the deuterated form of the compound of formula (I), commercially available deuterated starting materials may be used, or the compound may be synthesized by conventional techniques using deuterated reagents, including, but not limited to, deuterated borane, tritiated borane in tetrahydrofuran, lithium aluminum deuterated hydride, deuterated iodoethane, and deuterated iodomethane.

[0107] "Optionally" or "optionally" means that the subsequently described event or circumstance may, but need not, occur, and the phrase includes instances where the event or circumstance occurs and instances where it does not. For example, "a heterocyclyl group optionally substituted with an alkyl group" means that an alkyl group may, but need not, be present, and the phrase includes instances where the heterocyclyl group is substituted with an alkyl group and instances where the heterocyclyl group is not substituted with an alkyl group.

[0108] "Substituted" refers to one or more hydrogen atoms of a group, preferably up to five, more preferably one, two, or three hydrogen atoms, being independently replaced with a substituent. Substituents are located only at chemically feasible sites, and those skilled in the art can determine (by experiment or theory) possible or impossible substitutions without undue effort. For example, an amino group or hydroxy group having a free hydrogen may be unstable if bonded to a carbon atom having an unsaturated (e.g., olefinic) bond.

[0109] The term "pharmaceutical composition" is intended to include a mixture of one or more compounds described herein or physiologically / pharmaceutically acceptable salts or prodrugs thereof with other chemical components, and other components, such as physiologically / pharmaceutically acceptable carriers and excipients, intended to facilitate administration to the body and contribute to the absorption of the active ingredients to further exert their biological activity.

[0110] The term "pharmaceutically acceptable salt" or "medicinal salt" refers to a salt of an antibody-drug conjugate of the present disclosure, or a salt of an active compound described in the present disclosure, which salt is safe and effective when used in a subject and possesses the requisite biological activity; the ligand-drug conjugates of the present disclosure contain at least one amino group and can therefore form salts with acids; non-limiting examples of pharmaceutically acceptable salts include, but are not limited to, hydrochloride, hydrobromide, hydroiodide, sulfate, hydrogensulfate, citrate, acetate, succinate, ascorbate, oxalate, nitrate, sorbate, hydrogen phosphate, dihydrogen phosphate, salicylate, hydrogen citrate, tartrate, maleate, fumarate, formate, benzoate, methanesulfonate, ethanesulfonate, benzenesulfonate, and p-toluenesulfonate.

[0111] In one embodiment of the present disclosure, the cytotoxic drug is attached to a sulfhydryl group of the antibody by a linking unit.

[0112] Ligand-cytotoxic drug conjugate loading can be controlled by methods including, but not limited to: (1) Controlling the molar ratio of the linking reagent and the monoclonal; (2) Control of reaction time and temperature; (3) Selection of different reaction reagents.

[0113] The preparation of conventional pharmaceutical compositions can be referred to the Chinese Pharmacopoeia.

[0114] The term "pharmaceutically acceptable carrier" is used in conjunction with the drugs of the present disclosure and refers to a system that modifies the drug's entry into a subject and its distribution in the body, controls the drug's release rate, and delivers the drug to a target organ. Drug carrier release and targeting systems can reduce drug degradation and loss, lower side effects, and improve bioavailability. For example, polymeric surfactants, which are used as carriers, can self-assemble to form aggregates of various forms due to their unique amphiphilic structure, including micelles, microemulsions, gels, liquid crystals, and vesicles. These aggregates have the ability to encapsulate drug molecules and have good membrane permeability, making them good drug carriers.

[0115] The term "excipient" refers to an additive other than an active compound in a pharmaceutical composition, and may also be called an additive. For example, adhesives, fillers, disintegrants, and lubricants in lozenges, base materials in semi-solid preparations such as ointments and creams, and preservatives, antioxidants, flavoring agents, fragrances, cosolvents, emulsifiers, solubilizers, osmotic pressure adjusters, and coloring agents in liquid preparations can all be called excipients.

[0116] The term "diluent," also known as a filler, is primarily used to increase the weight and volume of lozenges. The addition of a diluent not only ensures a consistent volume, but also reduces the dosage deviation of the active ingredient and improves the compressibility of the drug. When the drug in a lozenge contains an oily component, an absorbent must be added to absorb the oily substance so that the lozenge can be prepared in a "dry" state. Examples include starch, lactose, inorganic calcium salts, microcrystalline cellulose, etc.

[0117] The pharmaceutical compositions may be in the form of a sterile injectable aqueous solution. Acceptable solvents and vehicles used include water, Ringer's solution, and isotonic sodium chloride solution. Sterile injectable preparations may also be sterile injectable oil-in-water microemulsions in which the active ingredient is dissolved in the oil phase. For example, the active ingredient may be dissolved in a mixture of soybean oil and lecithin. The oil solution is then added to a mixture of water and glycerin and processed to form a microemulsion. The injectable solution or microemulsion may be infused into the subject's bloodstream via local bolus injection. Alternatively, and most preferably, the solution and microemulsion are administered in a manner that maintains a constant, cyclical concentration of the compound of the present invention. To maintain such a constant concentration, a continuous intravenous delivery device may be used. An example of such a device is the Deltec CADD-PLUS™ 5400 intravenous pump.

[0118] The pharmaceutical compositions may be in the form of sterile injectable aqueous or oleaginous suspensions for intramuscular and subcutaneous administration. Such suspensions can be prepared using suitable dispersing or wetting agents and suspending agents as described above according to known techniques. Sterile injectable preparations may also be sterile injectable solutions or suspensions prepared in non-toxic parenterally acceptable diluents or solvents, such as solutions prepared in 1,3-butanediol. Sterile fixed oils can be easily used as solvents or suspending media. For this purpose, any mixed fixed oil, including synthetic monoglycerides or diglycerides, can be used. Fatty acids, such as oleic acid, can also be used to prepare injectables.

[0119] The present disclosure relates to antibody-drug conjugates (ADCs) comprising a cleavable linking arm of a specific structure, an active agent of a specific structure, and an antibody. Such ADCs are conjugates in which a toxic substance is linked to an antibody via a spacer. The antibody-drug conjugates (ADCs) exert their antitumor effects by degrading in vivo to release the active agent.

[0120] 2. Synthesis method To achieve the synthesis goal, the following synthesis technology scheme is adopted: A method for preparing a compound represented by the general formula (Pc-L a -Y-D), comprising

Chemical formula

[0121] The above specification submits details of one or more embodiments of the present disclosure. The present disclosure can be implemented or tested by any method and material similar or equivalent to the description of the present application. However, preferred methods and materials are described below. Other features, objects, and advantages of the present disclosure will become apparent from the specification and the claims. In the specification and the claims, unless otherwise specified in the context, the singular form includes plural referents. Unless otherwise defined, all technical and scientific terms used in this application have the ordinary meaning understood by those skilled in the art to which the present disclosure pertains. All patents and publications cited in the specification are incorporated by reference. The following examples are submitted to more comprehensively illustrate the preferred embodiments of the present disclosure. These examples should in no way be construed as limiting the scope of the present disclosure, and the scope of the present disclosure is limited by the claims.

[0122] Example 1: Preparation of CEA Recombinant Protein and Stably Transfected Cells I. Recombinant CEA Antigen and Sequence in Which CEA Protein is Expressed on the Cell Surface The Fc- and His-tagged human CEA amino acid sequences were encoded and cloned into mammalian cell expression vectors, respectively, and then expressed in 293E cells. After purification, the recombinant proteins were obtained and used in the experiments in the following examples. At the same time, the untagged human CEA gene, human CEACAM1 gene, and monkey CEA gene were transfected into CHO cells to form CHO cell lines expressing CEA protein on the cell surface, which were then used for antibody screening and identification. The amino acid sequences of the relevant proteins are as follows:

[0123] TIFF0007827624000017.tif75156

[0124] TIFF0007827624000018.tif96156

[0125] TIFF0007827624000019.tif75156

[0126] TIFF0007827624000020.tif80156

[0127] TIFF0007827624000021.tif80156

[0128] TIFF0007827624000022.tif64156

[0129] 2. Purification of related proteins 1. Purification of His-tagged proteins The cell expression supernatant sample was centrifuged at high speed to remove impurities. The nickel column was equilibrated with PBS buffer (pH 7.4) and washed with 2 to 5 column volumes. The supernatant sample was then applied to a Ni Sepharose Excel column at a constant flow rate. 280The column was washed with PBS buffer until the readings were reduced to the baseline, and then washed with PBS + 10 mM imidazole to remove nonspecifically bound heteroproteins. The effluent was collected. Finally, the target protein was eluted with PBS containing 300 mM imidazole, and the elution peak was collected. After concentrating the collected eluate, the sample buffer was converted to PBS using a desalting column for subsequent use in experiments.

[0130] 2. Purification of Fc-containing proteins, chimeric antibodies, and hybridoma antibodies The cell expression supernatant sample was centrifuged at high speed to remove impurities, and the Fc-containing recombinant protein and chimeric antibody expression supernatant were purified on a Protein A column, while the hybridoma expression supernatant was purified on a Protein G column. The supernatant was applied to the column at a constant flow rate. 280 The column was washed with PBS until the readings were reduced to baseline. The target protein was eluted with 100 mM acetic acid, pH 3.0, and neutralized with 1 M Tris-HCl, pH 8.0. The eluted sample was concentrated and converted to PBS before being aliquoted for use.

[0131] Example 2: Preparation of mouse anti-human CEA monoclonal antibody 1. Immunity and fusion Mice were immunized with hCEA-His protein and cyno-CEA-His protein, or hCEA-CHO cells and cynoCEA-CHO cells. The dose of protein immunization was 50 μg for the first immunization and 25 μg for subsequent immunizations. The cell immunization dose was 10 μg per immunization. 7 The mice were immunized with spleen cells once every two weeks. After three immunizations, blood samples were taken to measure serum antibody titers. Mice with high and stable serum antibody titers were selected for splenocyte fusion. The spleen lymphocytes were then fused with myeloma cells Sp2 / 0 (ATCC (Registered Small Quantity) CRL-8287) in a PEG-mediated fusion step. TM ) were fused to obtain hybridoma cells. The fused hybridoma cells were collected in a volume of 0.5 to 1 × 10 6The cells were resuspended in MC semi-solid complete medium (RPMI-1640 medium containing 20% ​​FBS, 1x HAT, 1x OPI, and 2% methyl cellulose) at a density of 1 / mL, dispensed into 35 mm cell culture dishes, and incubated at 37°C and 5% CO2 for 7–9 days. On day 7–9 after fusion, single cells were selected based on cell clone size and cloned into 96-well cell culture plates containing 200 μl / well of HT complete medium (RPMI-1640 medium containing 20% ​​FBS, 1x HT, and 1x OPI) and cultured at 37°C and 5% CO2 for 3 days for detection.

[0132] 2. Selection of hybridoma cells Primary antibody screening was performed using an enzyme-linked immunosorbent assay (ELISA) based on cell surface antigens. Cells were plated onto ELISA plates (Corning, Cat# 3599) and cultured overnight in a 37°C incubator. When the cells were completely attached to the wells and nearly filled the wells, the supernatant was removed, washed once with PBS, and cell fixative (Beyotime, Cat# P0098) was added. The plate was then left at room temperature for 45 minutes. The fixative was then removed, and the plate was washed three times with a plate washer. 5% nonfat dry milk was added and blocked at 37°C for at least 3 hours. The blocking solution was then removed, and the plate was washed three times with a plate washer. The blocked cell plate could be stored at -20°C or used immediately. Before use, gradient-diluted hybridoma cell culture supernatant was added to the plate, which was then incubated at 37°C for 1 hour and washed three times with a plate washer. 100 μl of 1:10,000 Goat anti-mouse IgG H&L (HRP) secondary antibody (Abcam, Cat# ab205719) was added, incubated at 37°C for 1 hour, washed three times with a plate washer, and 100 μl of TMB (KPL, Cat# 5120-0077) was added and incubated at 37°C for 10 minutes. The reaction was terminated by the addition of 100 μl of 1 M sulfuric acid, and the absorbance was read at 450 nm using a microplate reader. If the test antibody bound to cell surface CEA without being competitively removed by soluble CEA (sCEA), the antibody and sCEA were incubated for 30 minutes before being added to the cell plate.

[0133] Positive clones screened were amplified and cryopreserved as seeds, and then subcloned two to three times until single-cell clones were obtained. Antibodies were further prepared and purified from the selected hybridoma clones using serum-free cell culture. The resulting hybridoma antibodies were examined for binding to CEA protein on the cell surface using a flow cytometer (see Test Example 1 of the present disclosure for the method), and hybridoma cell lines with good binding activity were selected. The binding activity detection results for the monoclonal hybridoma cell lines mAb47, mAb63, mAb67, and mAb103 are shown in Table 1.

[0134] [Table 1]

[0135] 3. Hybridoma antibody sequence measurement The monoclonal hybridoma cell lines mAb47, mAb63, mAb67, and mAb103 were selected, and the monoclonal antibody sequences were cloned. The process was as follows: Hybridoma cells in the logarithmic growth phase were harvested, and RNA was extracted using Trizol (Invitrogen, Cat# 15596-018) and reverse transcribed into cDNA. The cDNA was used as a template for PCR amplification and then sent to a sequencing company for sequencing. The antibody amino acid sequences corresponding to the obtained DNA sequences are shown in Table 2 below.

[0136] [Table 2]

[0137] [Table 3]

[0138] 4. Preparation of human IgG1 chimeric antibodies The candidate molecules mAb47, mAb63, mAb67, and mAb103 obtained by screening the above hybridomas were amplified and sequenced to obtain the variable region-encoding gene sequences. Primers at both ends were designed using the sequences obtained by sequencing, and each antibody VH / VK gene fragment was assembled by PCR using the sequencing gene as a template. Homologous recombination was then performed with the expression vector pHr (a gene (CH1-Fc / CL) fragment with a signal peptide and hIgG1 / hkappa / hlambda constant regions) to construct the recombinant chimeric antibody full-length expression plasmid VH-CH1-Fc-pHr / VL-CL-pHr, and the chimeric antibodies Ch47, Ch63, Ch67, and Ch103 were further obtained.

[0139] Example 3: Humanization of a mouse anti-human CEA monoclonal antibody By aligning the IMGT database of human antibody heavy and light chain variable region germline genes with the MOE software, highly homologous heavy and light chain variable region germline genes were selected as templates, and the CDRs of the mouse antibody were grafted onto the corresponding humanized templates to form the variable region sequence FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. In the example below, the CDR amino acid residues of the antibody are annotated according to the Kabat numbering system.

[0140] 1. Humanization of mouse antibody mAb47 Highly homologous heavy and light chain variable region germline genes are selected as templates. For example, IGKV6-21*01 and IGKJ2*01 are selected as humanized light chain templates for mouse antibody mAb47, and IGHV7-4-1*02 and IGHJ6*01 are selected as humanized heavy chain templates. The CDRs of mouse antibody mAb47 are then transplanted into the corresponding humanized templates to perform humanization. The humanized back mutation design of mouse antibody mAb47 is shown in Table 4 below.

[0141] [Table 4]

[0142] Furthermore, the heavy chain variable region h47VH3 has a D61S mutation (i.e., the antibody HCDR2 sequence WINTYSGVPTYADDFKG( From SEQ ID NO:16 WINTYSGVPTYASDFKG( Amino acid mutations were made in the antibody HCDR2 to mutate the heavy chain variable region h47VH4 to SEQ ID NO: 38), resulting in the heavy chain variable region h47VH4, yet the antibody still has good activity.

[0143] The specific sequences of the murine antibody mAb47 after humanization are shown in Table 5. [Table 5]

[0144] 2. Humanization of mouse antibody mAb63 Highly homologous heavy and light chain variable region germline genes are selected as templates. For example, IGKV1-39*01 and IGKJ4*01 are selected as humanized light chain templates for mouse antibody mAb63, and IGHV1-46*01 and IGHJ1*01 are selected as humanized heavy chain templates. The CDRs of mouse antibody mAb63 are then transplanted into the corresponding humanized templates to perform humanization. The humanized back mutation design of mouse antibody mAb63 is shown in Table 6 below.

[0145] [Table 6]

[0146] Furthermore, the heavy chain variable region h63VH1 has an N54S mutation (i.e., the antibody HCDR2 sequence DIFPKNGNTDYNRKFKD( From SEQ ID NO:22 DIFPKSGNTDYNRKFKD( Amino acid mutations were made in the antibody HCDR2 to mutate the heavy chain variable region h63VH5 to SEQ ID NO: 47), resulting in the heavy chain variable region h63VH5, yet the antibody still has good activity.

[0147] The specific sequences of the murine antibody mAb63 after humanization and mutation are shown in Table 7. [Table 7]

[0148] 3. Humanization of mouse antibody mAb67 Highly homologous heavy and light chain variable region germline genes are selected as templates. For example, IGKV4-1*01 and IGKJ4*01, IGKV3-15*01 and IGKJ4*01, or IGKV1-39*01 and IGKJ4*01 are selected as humanized light chain templates for mouse antibody mAb67, and IGHV1-3*01 and IGHJ1*01, or IGHV5-51*01 and IGHJ1*01 are selected as humanized heavy chain templates. The CDRs of mouse antibody mAb67 are then transplanted into the corresponding humanized templates to perform humanization. The humanized back mutation design for mouse antibody mAb67 is shown in Table 8 below.

[0149] [Table 8]

[0150] The specific sequences of the murine antibody mAb67 after humanization are shown in Table 9. [Table 9]

[0151] 4. Humanization of mouse antibody mAb103 Highly homologous heavy and light chain variable region germline genes were selected as templates. For example, IGLV4-69*01 and IGLJ2*01 were selected as humanized light chain templates for mouse antibody mAb103, and IGHV7-4-1*02 and IGHJ1*01 were selected as humanized heavy chain templates. The CDRs of mouse antibody mAb103 were then transplanted into the corresponding humanized templates to perform humanization. The humanized back mutation design for mouse antibody mAb103 is shown in Table 10 below.

[0152] [Table 10]

[0153] Furthermore, the heavy chain h103VH1 has a D61S mutation (i.e., the antibody HCDR2 sequence WINTYSGVPTYADDFKG( From SEQ ID NO:16 WINTYSGVPTYASDFKG( Although an amino acid mutation was introduced into the antibody HCDR2 so that the antibody LCDR2 sequence changed from LKKDGSHSTGD (SEQ ID NO:36) to LKKDGSHSTGE (SEQ ID NO:64)) and a D56E mutation was introduced into the light chain h103VL3 (i.e., an amino acid mutation was introduced into the antibody LCDR2 so that the antibody LCDR2 sequence changed from LKKDGSHSTGD (SEQ ID NO:36) to LKKDGSHSTGE (SEQ ID NO:64)), the antibody still had good activity.

[0154] The specific sequence of the murine antibody mAb103 after humanization is shown in Table 11. [Table 11-1] [Table 11-2]

[0155] 5. Preparation of humanized antibodies Expression vectors for antibody light and heavy chains were constructed, and the humanized antibody light and heavy chains were cross-paired and combined. 293E cells were transfected with the supernatant, which was then cultured and purified to obtain full-length humanized antibodies. The humanized antibody heavy chain constant region may be selected from the constant regions of IgG1, IgG2, IgG3, IgG4, and their variants. For example, the humanized heavy chain variable region is linked to the humanized antibody heavy chain constant region (SEQ ID NO: 77). The humanized antibody light chain constant region may be selected from the constant regions of human kappa or lambda chains or their variants. For example, the human light chain constant region is linked to the humanized light chain variable region (SEQ ID NO: 79).

[0156] Exemplary antibody constant region sequences are as follows: TIFF0007827624000035.tif48156

[0157] TIFF0007827624000036.tif27156

[0158] TIFF0007827624000037.tif27156

[0159] For example, a humanized antibody heavy chain variable region listed in Table 5 derived from mAb47 was linked to the amino terminus of a human heavy chain IgG1 constant region having the sequence set forth in SEQ ID NO:77 to form a full-length antibody heavy chain, and a humanized antibody light chain variable region listed in Table 5 was linked to the amino terminus of a human light chain kappa constant region having the sequence set forth in SEQ ID NO:78 to form a full-length antibody light chain, resulting in a series of mAb47 humanized antibodies as shown in Table 12 below.

[0160] [Table 12]

[0161] For example, a humanized antibody heavy chain variable region listed in Table 7 derived from mAb63 was linked to the amino terminus of a human heavy chain IgG1 constant region whose sequence is set forth in SEQ ID NO:77 to form a full-length antibody heavy chain, and a humanized antibody light chain variable region listed in Table 7 was linked to the amino terminus of a human light chain kappa constant region whose sequence is set forth in SEQ ID NO:78 to form a full-length antibody light chain, resulting in a series of mAb63 humanized antibodies as shown in Table 13 below.

[0162] [Table 13]

[0163] For example, a humanized antibody heavy chain variable region described in Table 9 derived from mAb67 was linked to the amino terminus of a human heavy chain IgG1 constant region having the sequence set forth in SEQ ID NO:77 to form a full-length antibody heavy chain, and a humanized antibody light chain variable region described in Table 9 was linked to the amino terminus of a human light chain kappa constant region having the sequence set forth in SEQ ID NO:78 to form a full-length antibody light chain, resulting in a series of mAb67 humanized antibodies as shown in Table 14 below.

[0164] [Table 14]

[0165] For example, a humanized antibody heavy chain variable region listed in Table 11 derived from mAb103 was linked to the amino terminus of a human heavy chain IgG1 constant region whose sequence is set forth in SEQ ID NO:77 to form a full-length antibody heavy chain, and a humanized antibody light chain variable region listed in Table 11 was linked to the amino terminus of a human light chain λ constant region whose sequence is set forth in SEQ ID NO:79 to form a full-length antibody light chain, resulting in a series of mAb103 humanized antibodies as shown in Table 15 below.

[0166] [Table 15]

[0167] Exemplary humanized antibody light / heavy chain full length sequences are shown in Table 16 below. [Table 16-1] [Table 16-2]

[0168] Currently known two ADC molecules targeting CEA are SAR-408701 and labetozumab govitecan (also known as Lmab-CL2A-SN38), in which the antibody light / heavy chain sequences are as follows:

[0169] TIFF0007827624000044.tif59156

[0170] TIFF0007827624000045.tif37156

[0171] TIFF0007827624000046.tif59156

[0172] TIFF0007827624000047.tif37156

[0173] Each of the above antibodies was cloned, expressed, and purified using conventional gene cloning and recombinant expression methods.

[0174] Example 4: Preparation of Compounds

[0175] Experimental methods for which specific conditions are not specified in the examples of this disclosure generally follow conventional conditions or conditions recommended by the manufacturers of raw materials or products. Reagents for which a specific source is not specified are conventional, commercially available reagents.

[0176] The structure of the compound is determined by nuclear magnetic resonance (NMR) or mass spectrometry (MS). NMR measurements were performed using a Bruker AVANCE-400 nuclear magnetic resonance spectrometer. The solvents used were deuterated dimethyl sulfoxide (DMSO-d), deuterated chloroform (CDCl), and deuterated methanol (CD3OD). The internal standard was tetramethylsilane (TMS). The chemical shifts were 10 -6 Expressed in ppm.

[0177] For MS measurements, a FINNIGAN LCQAd(ESI) mass spectrometer (manufacturer: Thermo, scale: Finnigan LCQ advantage MAX) is used.

[0178] UPLC measurements are performed using a Waters Acquity UPLC SQD liquid chromatograph mass spectrometer.

[0179] For HPLC measurements, an Agilent 1200DAD high performance liquid chromatograph (Sunfire C18 150×4.6 mm chromatography column) and a Waters 2695-2996 high performance liquid chromatograph (Gimini C18 150×4.6 mm chromatography column) are used.

[0180] UV-HPLC measurements are performed using a Thermo nanodrop2000 ultraviolet spectrophotometer.

[0181] Growth inhibition rate and IC 50 The values ​​are measured using a PHERA starFS microplate reader (BMG, Germany).

[0182] Yantai Huanghai HSGF254 or Qingdao GF254 silica gel plates are used as silica gel plates for thin layer chromatography (TLC). The silica gel plate specifications for thin layer chromatography (TLC) are 0.15 mm to 0.2 mm, while the silica gel plate specifications for separating and purifying products by thin layer chromatography are 0.4 mm to 0.5 mm.

[0183] Column chromatography generally uses Yantai Huanghai 200-300 mesh silica gel as the carrier.

[0184] Known starting materials according to the present disclosure may be synthesized by adopting or following methods known in the art, or may be purchased commercially from companies such as ABCR GmbH & Co. KG, Acros Organics, Aldrich Chemical Company, Shaoyuan Chemical Technology (Accela ChemBio Inc.), and Darui Chemical.

[0185] Unless otherwise specified in the examples, all reactions are carried out in an argon or nitrogen atmosphere.

[0186] An argon or nitrogen atmosphere refers to an argon or nitrogen balloon with a volume of approximately 1 L connected to the reaction flask.

[0187] A hydrogen atmosphere refers to a hydrogen balloon with a volume of approximately 1 L attached to the reaction flask.

[0188] For the pressurized hydrogenation reaction, a Parr 3916EKX hydrogenation apparatus and a Seiran QL-500 hydrogen generator or an HC2-SS hydrogenation apparatus are used.

[0189] The hydrogenation reaction is generally carried out after three cycles of evacuation and hydrogen injection.

[0190] For microwave reactions, a CEM Discover-S 908860 microwave reactor is used.

[0191] Unless otherwise specified in the examples, the solutions used in the reactions refer to aqueous solutions.

[0192] Unless otherwise specified in the examples, the reaction temperature is room temperature.

[0193] The optimum reaction temperature is room temperature, with a temperature range of 20°C to 30°C.

[0194] In this example, a PBS buffer solution with a pH of 6.5 was prepared as follows: 8.5 g of KH2PO4, 8.56 g of K2HPO4·3H2O, 5.85 g of NaCl, and 1.5 g of EDTA were placed in a flask, the volume was adjusted to 2 L, and the contents were dissolved in an ultrasonic wave and then shaken to obtain the solution.

[0195] The eluent system for column chromatography and the developing solvent system for thin-layer chromatography used for purifying the compound include A: dichloromethane and isopropyl alcohol system, B: dichloromethane and methanol system, and C: petroleum ether and ethyl acetate system, and the volume ratio of the solvents may be adjusted depending on the polarity of the compound, or may be adjusted by adding a small amount of triethylamine and an acidic or basic reagent, etc.

[0196] Some of the compounds of the present disclosure have been characterized by Q-TOF LC / MS using an Agilent 6530 accurate mass quadrupole-time of flight mass spectrometer and an Agilent 1290-Infinity ultra-high performance liquid chromatograph (Agilent Poroshell 300SB-C8 5 μm, 2.1 × 75 mm chromatography column).

[0197] The YD drug moiety of the antibody-drug conjugate of the present disclosure is referenced in PCT / CN2019 / 107873, and the synthesis and testing of related compounds are cited in this patent, the synthesis of non-limiting examples of which are cited below.

[0198] 1. Synthesis of the toxic drug disclosed herein (S)-2-Cyclopropyl-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-2-hydroxyacetamide 1-A (R)-2-Cyclopropyl-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-2-hydroxyacetamide 1-B [ka] [ka]

[0199] To 1b (4 mg, 7.53 μmol), 2 mL of ethanol and 0.4 mL of N,N-dimethylformamide were added, the mixture was purged with argon three times, and the mixture was cooled to 0-5°C in an ice-water bath. 0.3 mL of N-methylmorpholine was added dropwise and the reaction mixture was stirred until clear. 2-Cyclopropyl-2-hydroxyacetic acid 1a (2.3 mg, 19.8 μmol, prepared according to the method disclosed in patent application WO2013106717), 1-hydroxybenzotriazole (3 mg, 22.4 μmol), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (4.3 mg, 22.4 μmol) were added sequentially to the reaction mixture, and the mixture was stirred at 0-5°C for 1 hour. The ice-water bath was removed, and the mixture was heated to 30°C and stirred for 2 hours. The reaction mixture was concentrated under reduced pressure, and the resulting crude compound 1 was purified by high-performance liquid chromatography (separation conditions: chromatography column: XBridge Prep C18 OBD 5 μm 19*250 mm, mobile phase: A-water (10 mmol NHOAc), B-acetonitrile, gradient elution, flow rate: 18 mL / min). The corresponding components were collected and concentrated under reduced pressure to give the title products (1-A: 1.5 mg, 1-B: 1.5 mg). MS m / z (ESI): 534.0 [M+1].

[0200] Single-configuration compound 1-B (relatively short retention time) UPLC analysis revealed a retention time of 1.06 min and a purity of 88% (chromatographic column: ACQUITY UPLC BEHC18 1.7 μm 2.1*50 mm, mobile phase: A - water (5 mmol NHOAc), B - acetonitrile). 1 H NMR (400 MHz, DMSO-d6): δ 8.37 (d, 1H), 7.76 (d, 1H), 7.30 (s, 1H), 6.51 (s, 1H), 5.58-5.56 (m, 1H), 5.48 (d, 1H), 5.41 (s, 2H), 5.32-5.29 (m, 2H), 3.60 (t, 1H), 3.19-3.13 (m, 1H), 2.38 (s, 3H), 2.20-2.14 (m, 1H), 1.98 (q, 2H), 1.87-1.83 (m, 1H), 1.50-1.40 (m, 1H), 1.34-1.28 (m, 1H), 0.86 (t, 3H), 0.50-0.39 (m, 4H).

[0201] Single-configuration compound 1-A (relatively long retention time) UPLC analysis revealed a retention time of 1.10 min and a purity of 86% (chromatographic column: ACQUITY UPLC BEHC18 1.7 μm 2.1*50 mm, mobile phase: A - water (5 mmol NHOAc), B - acetonitrile). 1 H NMR (400 MHz, DMSO-d6): δ 8.35 (d, 1H), 7.78 (d, 1H), 7.31 (s, 1H), 6.52 (s, 1H), 5.58-5.53 (m, 1H), 5.42 (s, 2H), 5.37 (d, 1H), 5.32 (t, 1H), 3.62 (t, 1H), 3.20-3.15 (m, 2H), 2.40 (s, 3H), 2.25-2.16 (m, 1H), 1.98 (q, 2H), 1.87-1.82 (m, 1H), 1.50-1.40 (m, 1H), 1.21-1.14 (m, 1H), 0.87 (t, 3H), 0.47-0.35 (m, 4H).

[0202] 2. Synthesis of the Linker Toxic Drug of the Present Disclosure N-((2R,10S)-10-Benzyl-2-cyclopropyl-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1,6,9,12,15-pentaoxo-3-oxa-5,8,11,14-tetraazahexadecyl-16-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide 2-A N-((2S,10S)-10-Benzyl-2-cyclopropyl-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1,6,9,12,15-pentaoxo-3-oxa-5,8,11,14-tetraazahexadecyl-16-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide 2-B [ka] [ka]

[0203] Step 1 2-Cyclopropyl-2-hydroxybenzyl acetate 2a 1a (1.3 g, 11.2 mmol, prepared by the method disclosed in patent application WO2013 / 106717) was dissolved in 50 mL of acetonitrile, and potassium carbonate (6.18 g, 44.8 mmol), benzyl bromide (1.33 mL, 11.2 mmol), and tetrabutylammonium iodide (413 mg, 1.1 mmol) were added sequentially. The reaction mixture was stirred at room temperature for 48 hours, filtered through diatomaceous earth, and the filter cake was washed with ethyl acetate (10 mL). The combined filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography using developing solvent system C to give the title product 2a (2 g, 86.9% yield).

[0204] Step 2 10-Cyclopropyl-1-(9H-fluorenyl-9-yl)-3,6-dioxo-2,9-dioxa-4,7-diazaundecan-11-oate benzyl ester 2b 2a (120.9 mg, 0.586 mmol) and 2g (180 mg, 0.489 mmol, prepared as described in patent application CN105829346A) were placed in a reaction flask, 4 mL of tetrahydrofuran was added, and the mixture was purged with argon three times. The mixture was cooled to 0-5°C in an ice-water bath, potassium tert-butoxide (109 mg, 0.98 mmol) was added, the ice bath was removed, the mixture was warmed to room temperature, and stirred for 40 min. 10 mL of ice-water was added, and the mixture was extracted with ethyl acetate (20 mL × 2) and chloroform (10 mL × 5). The combined organic phases were concentrated. The resulting residue was dissolved in 4 mL of dioxane, 2 mL of water was added, sodium bicarbonate (49.2 mg, 0.586 mmol) and 9-fluorenylmethyl chloroformate (126 mg, 0.49 mmol) were added, and the mixture was stirred at room temperature for 2 h. 20 mL of water was added, and the mixture was extracted with ethyl acetate (10 mL × 3). The organic phase was washed with saturated sodium chloride solution (20 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography using developing solvent system C to give the title product 2b (48 mg, yield 19%). MS m / z (ESI): 515.0 [M+1].

[0205] Step 3 10-Cyclopropyl-1-(9H-fluorenyl-9-yl)-3,6-dioxo-2,9-dioxa-4,7-diazaundecan-11-oic acid 2c 2b (20 mg, 0.038 mmol) was dissolved in 4.5 mL of a mixed solvent of tetrahydrofuran and ethyl acetate (V:V = 2:1), and palladium on carbon (12 mg, 10% dry) was added. The mixture was purged with hydrogen gas three times and stirred at room temperature for 1 hour. The reaction mixture was filtered through diatomaceous earth, the filter cake was washed with ethyl acetate, and the filtrate was concentrated to give crude product 2c (13 mg). This product was used in the next reaction without further purification. MS m / z (ESI): 424.9 [M+1].

[0206] Step 4 (9H-Fluorenyl-9-yl)methyl(2-(((1-cyclopropyl-2-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-2-oxoethoxy)methyl)amino)-2-oxoethyl)carbamate 2d 1b (10 mg, 18.8 μmol) was placed in a reaction flask, 1 mL of N,N-dimethylformamide was added, and the mixture was purged with argon three times. The mixture was cooled to 0-5°C in an ice-water bath, and one drop of triethylamine was added. The crude product 5c (13 mg, 30.6 μmol) and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (16.9 mg, 61.2 μmol) were added, and the mixture was stirred in an ice bath for 40 minutes. 10 mL of water was added, and the mixture was extracted with ethyl acetate (10 mL × 3). The organic phases were combined. The organic phase was washed with saturated sodium chloride solution (10 mL × 2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by thin-layer chromatography using developing solvent system B to give the title product 2d (19 mg, 73.6% yield). MS m / z (ESI): 842.1[M+1].

[0207] Step 5 2-((2-Aminoacetamino)methoxy)-2-cyclopropyl-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)acetamide 2e 2d (19 mg, 22.6 μmol) was dissolved in 2 mL of dichloromethane, 1 mL of diethylamine was added, and the mixture was stirred at room temperature for 2 hours. The reaction mixture was concentrated under reduced pressure, and 1 mL of methylbenzene was added and concentrated under reduced pressure. This process was repeated twice. 3 mL of n-hexane was added to the residue to form a slurry. After standing, the supernatant was poured off, leaving a solid. The solid residue was concentrated under reduced pressure and dried on an oil pump to obtain the crude title product 2e (17 mg). This product was used directly in the next reaction without further purification. MS m / z (ESI): 638.0[M+18].

[0208] Step 6 N-((2R,10S)-10-Benzyl-2-cyclopropyl-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1,6,9,12,15-pentaoxo-3-oxa-5,8,11,14-tetraazahexadecyl-16-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide 2-A N-((2S,10S)-10-Benzyl-2-cyclopropyl-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1,6,9,12,15-pentaoxo-3-oxa-5,8,11,14-tetraazahexadecyl-16-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide 2-B The crude product 2e (13.9 mg, 22.4 μmol) was dissolved in 0.6 mL of N,N-dimethylformamide, purged with argon three times, and cooled to 0-5°C in an ice-water bath. A 0.3 mL N,N-dimethylformamide solution of 2f (21.2 mg, 44.8 μmol, obtained by the method disclosed in patent application "EP2907824") was added, and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (18.5 mg, 67.3 μmol) was added and reacted with stirring in an ice bath for 10 minutes. The ice bath was removed, the mixture was warmed to room temperature, and stirred for 1 hour, resulting in the reaction of compound 2. The reaction mixture was purified by high-performance liquid chromatography (separation conditions: chromatography column: XBridge Prep C18 OBD 5 μm 19*250 mm, mobile phase: A-water (10 mmol of NH4OAc), B-acetonitrile, gradient elution, flow rate: 18 mL / min), and the corresponding components were collected and concentrated under reduced pressure to give the title products (2-A: 2.4 mg, 2-B: 1.7 mg). MS m / z (ESI): 1074.4 [M+1].

[0209] Single-configuration compound 2-A (relatively short retention time): UPLC analysis revealed a retention time of 1.14 min and a purity of 85% (chromatographic column: ACQUITY UPLC BEHC18 1.7 μm 2.1*50 mm, mobile phase: A - water (5 mmol NHOAc), B - acetonitrile). 1H NMR (400 MHz, DMSO-d6): δ 8.60 (t, 1H), 8.51-8.49 (d, 1H), 8.32-8.24 (m, 1H), 8.13-8.02 (m, 2H), 8.02-7.96 (m, 1H), 7.82-7.75 (m, 1H), 7.31 (s, 1H), 7.26-7.15 (m, 4H), 6.99 (s, 1H), 6.55-6.48 (m, 1H), 5.65-5.54 (m, 1H), 5.41 (s, 2H), 5.35-5.15 (m, 3H), 4.74-4.62 (m, 1H), 4.54-4.40 (m, 2H), 3.76-3.64 (m, 4H), 3.62-3.48 (m, 2H), 3.20-3.07 (m, 2H), 3.04-2.94 (m, 1H), 2.80-2.62 (m,12H), 2.45-2.30 (m, 3H), 2.25-2.15 (m, 2H), 2.15-2.04 (m, 2H), 1.93-1.78 (m, 2H), 1.52-1.39 (m, 3H), 1.34-1.12 (m, 5H), 0.87 (t, 3H), 0.64-0.38 (m, 4H).

[0210] Single-configuration compound 2-B (relatively long retention time): UPLC analysis revealed a retention time of 1.16 min and a purity of 89% (chromatographic column: ACQUITY UPLC BEHC18 1.7 μm 2.1*50 mm, mobile phase: A - water (5 mmol NHOAc), B - acetonitrile). 1H NMR (400 MHz, DMSO-d6): δ 8.68-8.60 (m, 1H), 8.58-8.50 (m, 1H), 8.32-8.24 (m, 1H), 8.13-8.02 (m, 2H), 8.02-7.94 (m, 1H), 7.82-7.75 (m, 1H), 7.31 (s, 1H), 7.26-7.13 (m, 3H), 6.99 (s, 1H), 6.55-6.48 (m, 1H), 5.60-5.50 (m, 1H), 5.41 (s, 2H), 5.35-5.15 (m, 2H), 4.78-4.68 (m, 1H), 4.60-4.40 (m, 2H), 3.76-3.58 (m, 4H), 3.58-3.48 (m, 1H), 3.20-3.10 (m, 2H), 3.08-2.97 (m, 2H), 2.80-2.72 (m, 2H), 2.45-2.30 (m, 3H), 2.25-2.13 (m, 2H), 2.13-2.04 (m, 2H), 2.03-1.94 (m, 2H), 1.91-1.78 (m, 2H), 1.52-1.39 (m, 3H), 1.34-1.12 (m, 4H), 0.91-0.79 (m, 3H), 0.53-0.34 (m, 4H).

[0211] Example 5: Preparation of antibody drug conjugates (ADCs) Analysis of drug loading in ADC stock solution Experimental objectives and principles ADC stock solution is a type of antibody crosslinker drug, and its mechanism of treatment is to deliver toxin molecules to cells through the targeting of antibodies, which then kills the cells. The amount of drug loaded plays a crucial role in the efficacy of the drug. The drug loading amount of ADC stock solution was measured using an ultraviolet method.

[0212] Experimental Method The cuvettes containing the sodium succinate buffer were placed in the reference absorption cell and the sample measurement absorption cell, respectively, and after removing the solvent blank, the cuvette containing the sample solution was placed in the sample measurement absorption cell, and the absorbance at 280 nm and 370 nm was measured. Calculation of results: The loading amount of ADC stock solution was measured by ultraviolet spectrophotometry (instrument used: Thermo nanodrop 2000 ultraviolet spectrophotometer). The principle is that the total absorbance value of ADC stock solution at a certain wavelength is equal to the sum of the absorbance values ​​of the cytotoxic drug and the monoclonal antibody at the same wavelength, i.e. (1) A 280 nm =ε mab-280 bC mab +ε Drug-280 bC Drug ε Drug-280 : The drug has an average molar extinction coefficient of 5100 at 280 nm; C Drug : drug concentration; ε mab-280 : The average molar extinction coefficient at 280 nm of the monoclonal stock solution is 214600; C mab :Concentration of monoclonal antibody stock solution; b: The optical path length is 1 cm.

[0213] Similarly, the equation for the total absorbance value of the sample at 370 nm can be obtained. (2) A 370 nm =ε mab-370 bC mab +ε Drug-370 bC Drug ε Drug-370 : The drug has an average molar extinction coefficient of 19000 at 370 nm; C Drug : drug concentration; ε mab-370 : The extinction coefficient of the monoclonal antibody stock solution at 370 nm is 0; C mab :Concentration of monoclonal antibody stock solution; b: The optical path length is 1 cm.

[0214] The two equations (1) and (2) can be used to calculate the amount of drug loaded, in combination with the data on the extinction coefficients and concentrations of the monoclonal antibody and drug at the two detection wavelengths. Drug loading = CDrug / C mab .

[0215] 1. Preparation of antibody-drug conjugate ADCs according to the present disclosure The toxic compound 1-B is a DNA topoisomerase I inhibitor that can form a complex with topoisomerase I and DNA to cause single-strand breaks in DNA, blocking DNA replication and effectively inhibiting cell proliferation in cells.

[0216] The ADC preparation process is as follows: A humanized antibody (selected from Hu63-13, Hu47-14, Hu67-14, or Hu103-32) was placed in a 0.05 M PBS buffer solution at pH 6.5 (antibody concentration: 10 mg / mL), and a 10 mM aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (Innochem, CAS: 51805-45-9, Cat# B45573) equivalent to 5.3 times the molar amount of the antibody was added. The mixture was then placed in a 37°C shaking incubator and allowed to react for 3 hours. The reaction mixture was then placed in an ice bath to cool to 25°C.

[0217] The toxic compound 2-A, equivalent to 15 times the molar amount of the antibody, was dissolved in dimethyl sulfoxide and added to the reaction mixture. The mixture was placed on a shaker at room temperature for 3 hours, after which the reaction was stopped. The reaction mixture was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer solution, pH 6.5, containing 0.01 M EDTA) to obtain the desired antibody-drug conjugate with a drug-antibody molar ratio (DAR, n value) of 6 to 8.

[0218] Those skilled in the art can obtain conjugates with different DAR values ​​by adjusting the reaction conditions and reagents. For example, by adjusting the molar ratio of antibody to TCEP and toxic compound, and adding a 10 mM aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) equivalent to 2.5 times the molar amount of antibody and a 10-fold molar amount of toxic compound 2-A, an ADC molecule with a drug-antibody molar ratio (DAR, n value) of 3 to 5 can be obtained.

[0219] Specifically, the ADCs prepared are as follows: [Table 17]

[0220] [ka]

[0221] As required by the experimental examples, the present disclosure further prepares ADCs with other DAR values, as detailed in the experimental examples.

[0222] 2. Preparation of control ADC Lmab-CL2A-SN38 (ADC-4) To evaluate the differences between the constructed ADC in this disclosure and the control ADC molecule Lmab-CL2A-SN38, the toxin CL2A-SN38 was synthesized with reference to the structure in WHO Drug Information Vol. 30, No. 1, 2016 and the method in Mol Pharm. 2015 Jun 1; 12(6):1836-47, and CL2A-SN38 was conjugated to Lmab to form the ADC molecule Lmab-CL2A-SN38. The reaction conditions were adjusted with reference to the above-mentioned ADC preparation method to obtain ADC molecules with different DAR values. The prepared ADC molecules were stored at -20°C for use.

[0223] Test Example Test Example 1: FACS binding experiment To detect the binding status of the antibody to the cell surface CEA protein, the antibody binding activity was detected by FACS using cells expressing CEA on the cell surface. The cells were collected and centrifuged at 400 g for 5 minutes at 4°C. Pre-chilled PBS containing 10% FBS was added, and the cells were centrifuged at 400 g for 5 minutes at 4°C. The above procedure was repeated twice, and the cells were collected in a 100 ml tube. 5Cells were allocated to a 96-well plate at 100 μl per well, and 100 μl of the gradient diluted antibody solution was added to each well. The cells were incubated at 4°C for 60 minutes, centrifuged, and the supernatant was removed. 250 μl of pre-chilled PBS containing 10% FBS was added to each well to resuspend the cells. The cells were centrifuged at 400 g for 5 minutes at 4°C, and the supernatant was removed. This procedure was repeated twice. 50 μl of the secondary antibody Alexa Fluor@488 sheep anti-human IgG (H+L) (Lifetechologies, Cat# A11013) diluted 1:200 was added, and the cells were incubated at 4°C for 45 minutes in the dark, centrifuged, and the supernatant was removed. 250 μl of pre-chilled PBS containing 10% FBS was added to each well to resuspend the cells. The cells were centrifuged at 400 g for 5 minutes at 4°C, and the procedure was repeated twice. 100 μl of pre-chilled PBS was added to each well to resuspend the cells. The cells were then detected by a flow cytometer (BD, Fluorescence signal values ​​were obtained using FACSverse. A higher signal indicates a higher binding activity between the antibody and the cell surface protein. The PRISM analysis software was used to generate a binding graph based on the detection results. The EC2 binding activity between the antibody and the cell surface proteins human CEA (MKN45 human gastric cancer cells, Nanjing Kebai Biotechnology Co., Ltd., Cat# CBP60488), cynoCEA-CHO, and CEACAM1-CHO was calculated by fitting. 50 The binding activities of the humanized antibodies are shown in Tables 17 and 18 below.

[0224] [Table 18]

[0225] The results of detecting the binding activity of other humanized antibodies to cell surface CEA protein (MKN45, cynoCEA-CHO) are shown in Table 18 below. [Table 19]

[0226] As is clear from the experimental results, the humanized antibodies screened in this disclosure retain binding activity similar to that of the mouse antibody and are both capable of binding to cell surface human CEA protein, while the humanized antibodies screened in this disclosure are capable of binding to cell surface monkey CEA protein and have superior binding activity to monkey CEA protein than the positive control antibody.

[0227] Test Example 2: Competition experiment with soluble CEA (sCEA) To detect whether the antibody preferentially binds to CEA on the cell membrane surface even in the presence of sCEA, gradient-diluted antibody and sCEA (5 μg / mL) of a certain solubility were pre-incubated for 30 minutes. MKN45 cells were then harvested and allocated to a 96-well plate. The gradient-diluted antibody and the antibody / sCEA pre-incubation mixture were added to each well. The mixture was incubated at 4°C for 60 minutes, centrifuged, and the supernatant was removed. The cells were washed with pre-chilled PBS containing 10% FBS. This procedure was repeated twice. 50 μl of 1:200 diluted secondary antibody, Alexa Fluor@488 sheep anti-human IgG (H+L) (Lifetechologies, Cat# A11013), was added. The mixture was incubated at 4°C for 45 minutes in the dark, centrifuged, and the supernatant was removed. The cells were washed with 250 μl of pre-chilled PBS containing 10% FBS. This procedure was repeated twice. Each well was then filled with 100 μl of pre-chilled PBS. The cells were resuspended in 100 μl of sCEA and analyzed using a flow cytometer (FACS) (BD, FACSverse) to obtain fluorescent signal values. If the signal ratio at each antibody concentration was less than 2, whether in the absence or presence of sCEA, the antibody binding curve did not change significantly in the presence of sCEA, indicating that the antibody still preferentially binds to CEA on the cell membrane surface. The experimental results are shown in Tables 19 and 20. Table 19 shows the fluorescent signal ratios for Hu63-13, Hu47-14, Hu67-14, Hu103-32, and the positive control Lmab at different antibody concentrations, without and with sCEA. Table 20 shows the maximum fluorescent signal ratios for other experimental antibodies, without and with sCEA.

[0228] [Table 20]

[0229] [Table 21]

[0230] As evident from the experimental results, the humanized antibodies Hu63-13, Hu47-14, Hu67-14, and Hu103-32 screened in this disclosure had fluorescent signal ratios less than 2 at various antibody concentrations, both without and with sCEA added. For example, the maximum ratio for Hu63-13 was 1.59, while the maximum ratio for the positive control Lmab reached 5.18, demonstrating that the antibodies screened in this disclosure are superior to the control antibodies. The humanized antibodies screened in this disclosure also had maximum fluorescent signal ratios less than 2, both without and with sCEA added, and less than the positive control antibody, indicating that the humanized antibodies screened in this disclosure still preferentially bind to CEA on the cell membrane surface in the presence of sCEA and are superior to the positive control antibody.

[0231] Test Example 3: Measurement of affinity between antibody and soluble CEA using Biacore The affinity of the humanized antibody to be measured with human and monkey soluble CEA was measured using a Biacore (GE, T200) instrument. Following the instructions in the Human Anti-Capture Reagent Kit (GE, Cat# BR-1008-39) manual, a human anti-capture antibody was covalently coupled to the Biacore instrument's biosensor chip CM5 (GE, Cat# BR-1005-30) to perform affinity capture of a fixed amount of the antibody to be measured. A series of gradient concentrations of soluble CEA antigen were then passed over the chip surface, and the reaction signal was detected in real time by the Biacore to obtain binding and dissociation curves. After each dissociation cycle, the biochip was regenerated by washing with the regeneration solution provided in the Human Anti-Capture Reagent Kit. The data obtained in the experiment were fitted with a (1:1) Langmuir model using BIAevaluation version 4.1 software to obtain affinity values. Since the present disclosure requires that the binding activity of the screened antibody to CEA on the cell membrane surface be higher than that to soluble CEA, the lower the affinity of the antibody to soluble CEA, the better. The test results for the affinity of the humanized antibody to soluble CEA are shown in Table 21 below.

[0232] [Table 22]

[0233] As is clear from the test results, the humanized antibodies Hu63-13, Hu47-14, and Hu67-14 all have relatively low affinity for soluble CEA protein, significantly lower than the control antibodies Sanofi and Lmab. This suggests that Hu63-13, Hu47-14, and Hu67-14 are less likely to be neutralized by soluble CEA in the blood in the body, and that many of these antibodies may bind to cells expressing CEA on the cell membrane surface.

[0234] Test Example 4: Endocytosis activity of anti-CEA antibody in CEA-high expressing cells MKN45 Since the anti-CEA antibody conjugate of the present disclosure can be endocytosed by cells and then release a toxin to kill the cells, the endocytic activity of the CEA antibody in cells expressing CEA can promote the activity of the ADC. To evaluate the endocytosis activity of humanized antibodies in MKN45 cells, MKN45 cells were plated in a 96-well plate (Coring, Cat# 3795) and cultured overnight. The following day, humanized CEA antibodies Hu63-13, Hu47-14, Hu67-14, and Hu103-32 were pre-incubated with iFL Green Human IgG Labeling Reagent (Invitrogen, Cat# Z25611) for 15 minutes. After the iFL reagent bound to the Fc of the humanized antibodies, the antibody-iFL complexes were added to the cell culture plate. After 6 and 24 hours, respectively, the cell culture medium was removed, washed twice with PBS, digested, and the cells were harvested. The fluorescent signal intensity in the cells was detected by FACS. After the antibody was endocytosed by the cells, iFL bound to the antibody Fc was transported into the cells. iFL was internalized within the cells, and the fluorescent signal could only be detected in an acidic environment. Therefore, the stronger the detected signal, the higher the endocytosis activity of the antibody. As shown in Figure 1, the endocytosis activity of each humanized antibody, all humanized antibodies could be endocytosed by MKN45, and the longer the time, the more endocytosed the antibody.

[0235] Test Example 5: Cytotoxicity of ADC against cancer cells with different CEA expression levels After conjugating the humanized CEA antibody to the toxin 2-A, the cytotoxicity of each ADC against cancer cell lines with different CEA expression levels was evaluated. Highly CEA-expressing MKN45 cells, moderately CEA-expressing LS174T cells, and negatively CEA-expressing HCT116 cells were plated in a 96-well plate. The next day, gradient-diluted ADC samples were added to the cells and cultured at 37°C for 5 days. 50 μl of Cell Titer-Glo reagent (Promega, Cat# G9242) was added to each well, and the cells were incubated for 10 minutes in the dark. The luminescence signal was then detected using a cell imaging detector (BioTek, Cytation 5). The results were used to generate an inhibition graph using PRISM analysis software, and the IC value of the ADC's inhibitory activity on cell proliferation was calculated by fitting. 50 The values ​​obtained are the toxicity of each ADC to the cells as follows:

[0236] [Table 23]

[0237] ADC molecules conjugated to toxin 2-A exhibit cytotoxicity dependent on CEA expression levels; the higher the CEA expression level, the more toxic the ADC is to cells. Furthermore, both high and low DAR values ​​have relatively high cytotoxicity against CEA-expressing cells and relatively low cytotoxicity against CEA-non-expressing cells. On the other hand, the control ADC molecule Lmab-CL2A-SN38 has comparable cytotoxicity against all three cell lines, showing no selectivity in cytotoxicity. IC 50 The magnitude of the ratio can indirectly reflect the safety of the ADC molecule; a larger ratio indicates a lower cytotoxicity of the ADC molecule against non-expressing CEA and a better potential safety profile in the body.

[0238] Test Example 6: Bystander effect cytotoxicity of ADC After the ADC is endocytosed into the cell, the toxin is released from the ADC and causes toxicity to the cell, and after cell death and lysis, the toxin is released from the cell and can further enter nearby cells and cause toxicity to nearby cells as well.

[0239] To evaluate the bystander cytotoxicity of the ADC, the CEA-high-expressing cell line MKN45 and the CEA-negative cell line HCT116 were co-cultured in a 6-well cell culture plate. After 24 hours, the ADC samples were added at a final concentration of 4 nM and cultured in a 37°C cell incubator for 5 days. The cells were then digested with pancreatin, harvested, and incubated with CEA Monoclonal Antibody FITC (ThermoFisher, Cat# MA1-80578) at a final concentration of 10 μg / mL for 1 hour on ice in the dark. The cells were washed twice with PBS and counted using a flow cytometer. Cells with a fluorescent signal were CEA-expressing MKN45 cells, while cells without a fluorescent signal were CEA-nonexpressing HCT116 cells.

[0240] The bystander cytotoxicity results of the ADC samples are shown in Figure 2. Both ADC molecules exhibited significant bystander cytotoxicity. When MKN45 and HCT116 were co-cultured, the ADC molecules inhibited the proliferation of both types of cells. However, when HCT116 was cultured alone, the ADC molecules conjugated to 2-A were almost non-toxic to the cells. On the other hand, the control ADC molecule, Lmab-CL2A-SN38, was highly toxic to both the co-cultured cells and the cells cultured alone.

[0241] The DAR values ​​of the ADC molecules used in the experiments are as follows: Hu63-13-2-ADAR 6.29; Hu47-14-2-A DAR 6.6; Hu67-14-2-A DAR 6.41; Lmab-CL2A-SN38 DAR 7.0.

[0242] Test Example 7: In vivo tumor-inhibiting activity of ADC molecules The in vivo efficacy of ADC molecules was evaluated using LS174T and MKN45 tumor xenograft models. SPF Balb / c nude mice (Changzhou Kavnese Laboratory Animal Co., Ltd., Certificate No. 201833814, SCXK(Su)2016-0010) were housed under the following conditions: a 12 / 12-hour light / dark cycle, a temperature of 23±1°C, and humidity of 40-50%. Standard sterilized mouse chow was administered, with food and water available ad libitum. Before the start of the experiment, the mice were acclimated to the laboratory environment for 10 days, and then LS174T cells (5×10 5 cells / animal) or MKN45 cells (4 × 10 6 cells / animal) were subcutaneously inoculated, and tumors were grown to 150 mm 3 When the mice grew to a certain size, they were randomly divided into groups with 8 mice per group. After grouping, the ADC samples were injected intraperitoneally at a dose of 1 mg / kg or 3 mg / kg, and the blank control group was injected intraperitoneally with PBS, which was administered only once. The tumor size in the mouse body was observed and measured, and the data was recorded. The formula for calculating tumor volume (V) was V = 1 / 2 × L. 長 ×L 短 2 , relative tumor volume (RTV) = V T / V0, tumor inhibition rate (%) = (C RTV -T RTV ) / C RTV (%). Of these, V0, V T are the tumor volumes at the start and end of the experiment, respectively, and C RTV , T RTV are the relative tumor volumes of the blank control group and the experimental group at the end of the experiment, respectively.

[0243] In vivo efficacy of ADCs showed that, compared with the control PBS, all ADC molecules inhibited tumor volume and weight growth at both low and high doses, with a dose-dependent effect, and the tumor-inhibiting effect of the 3 mpk group was superior to that of the 1 mpk group. In the LS174T tumor xenograft model, the results are shown in Figure 3 (changes in tumor volume) and Figure 4 (tumor weight on the final day). In the 3 mpk group, the best tumor-inhibiting effect was Hu63-13-2-A, followed by Hu47-14-2-A, and then Hu67-14-2-A, and the worst was Lmab-CL2A-SN38. In the MKN45 tumor xenograft model, the results are shown in Figure 5 (changes in tumor volume) and Figure 6 (tumor weight on the final day). Among the 3 mpk groups, Hu67-14-2-A had the best tumor inhibitory effect, followed by Hu103-32-2-A, then Hu63-13-2-A, and the worst was Lmab-CL2A-SN38.

[0244] As shown in Tables 23 and 24 below, the in vivo tumor inhibition rates of the ADC molecules were significantly dose-dependent. In the LS174T tumor xenograft model, the highest tumor inhibition rate in the low-dose group (1 mpk) was Hu63-13-2-A (55.95%), followed by Hu67-14-2-A (43.71%) and the worst was Hu47-14-2-A (23.04%). In the high-dose group (3 mpk), the highest tumor inhibition rate was Hu63-13-2-A (77.13%), followed by Hu47-14-2-A (66.87%), followed by Hu67-14-2-A (47.66%) and the worst was Lmab-CL2A-SN38 (33.44%). In the MKN45 tumor xenograft model, the tumor inhibition rate in the low-dose group (1 mpk) was highest for Hu67-14-2-A (15.88%), followed by Hu103-32-2-A (11.39%) and lowest for Hu63-13-2-A (9.89%). In the high-dose group (3 mpk), the tumor inhibition rate was highest for Hu67-14-2-A (79.51%), followed by Hu103-32-2-A (74.66%), followed by Hu63-13-2-A (60.26%) and lowest for Lmab-CL2A-SN38 (13.51%).

[0245] [Table 24]

[0246] [Table 25]

[0247] Test Example 8: In vivo pharmacokinetics test of ADC molecules A pharmacokinetic study was conducted using SD rats. SD rats (Xiper-Bikai Laboratory Animal Co., Ltd.) were randomly divided into groups of three and administered a 3 mg / kg intravenous injection. From the treatment group, 0.3 mL of whole blood was collected before administration and 5 minutes, 8 hours, 1 day, 2 days, 4 days, 7 days, 10 days, 14 days, 21 days, and 28 days after administration. No anticoagulant was added. After collection, the blood was left at 4°C for 30 minutes and centrifuged at 1000 g for 15 minutes. The upper layer of serum was collected, placed in EP tubes, and stored at -80°C. ELISA was used to detect serum concentrations, and Winnolin software was used to calculate the pharmacokinetic parameters of the test drugs. The detection results are shown below.

[0248] [Table 26]

[0249] All ADC molecules have relatively good pharmacokinetic properties, and the half-life of the antibodies is slightly longer than that of the ADC molecules: Hu63-13-2-A has a half-life of 7.1 days in vivo, and the antibody half-life is 8 days; Hu47-14-2-A has a half-life of 6.6 days in vivo, and the antibody half-life is 7.9 days; Hu67-14-2-A has a half-life of 7.1 days in vivo, and the antibody half-life is 7.5 days; and Hu103-32-2-A has a half-life of 5.63 days in vivo, and the antibody half-life is 6.58 days.

[0250] Test Example 9: In vitro plasma stability test of ADC molecules To evaluate the in vitro plasma stability of the ADC molecules, the ADC molecules were added to human and monkey plasma at a concentration of 100 μg / mL and left at 37°C for 21 days. Samples were taken once a week and the free toxin content in the plasma was analyzed and detected by LC / MS / MS (Shimadzu LC-30AD ultra-high performance liquid chromatography system, Applied Biosystems API4000 triple quadrupole tandem mass spectrometer). The detection results are shown below.

[0251] [Table 27]

[0252] A detection result of 0 indicates that the free toxin content in plasma was below the detection limit and was undetectable. All ADC molecules had good stability in human and monkey plasma. After 21 days of incubation at 37°C, the free toxin content of Hu63-13-2-A in human and monkey plasma was 0.32% and 0.4%, respectively. The free toxin content of Hu47-14-2-A in human and monkey plasma was 0.34% and 0.29%, respectively. The free toxin content of Hu67-14-2-A in human and monkey plasma was 0.4% and 0.24%, respectively. The free toxin content of Hu103-32-2-A in human and monkey plasma was 1.13% and 0.96%, respectively.

[0253] Test Example 10: In vitro growth inhibition test of tumor cells by compounds 1. Purpose of the test This experiment aimed to detect the in vitro growth inhibitory activity of the drug compounds disclosed herein on U87MG cells (Cell Bank of the Chinese Academy of Sciences, Catalog # TCHu138) and SK-BR-3 tumor cells (human breast cancer cells, ATCC, Product No. HTB-30). After treating the cells with different concentrations of the compounds in vitro and culturing them for 6 days, cell proliferation was detected using CTG (CellTiter-Glo® Luminescent Cell Viability Assay, Promega, Product No. G7573) reagent, and IC 50 The in vitro activity of the compound is evaluated based on the value.

[0254] II. Experimental Method In the present invention, in order to exemplify the method for testing the in vitro growth inhibitory activity of the compound of the present invention on tumor cells, the in vitro growth inhibitory test method on U87MG cells is taken as an example. This method can also be applied to, but is not limited to, the in vitro growth inhibitory activity test on other tumor cells.

[0255] 1. Cell culture: U87MG and SK-BR-3 cells were cultured in EMEM medium (GE, product number SH30024.01) containing 10% FBS and McCoy's 5A medium (Gibco, product number 16600-108) containing 10% FBS, respectively.

[0256] 2. Cell preparation: Logarithmic growth phase U87MG and SK-BR-3 cells were harvested and washed once with PBS (phosphate buffer solution, Shanghai Yuanpei Biotechnology Co., Ltd.). Then, 2–3 mL of trypsin (0.25% Trypsin-EDTA (1x), Gibico, Life Technologies) was added and digested for 2–3 minutes. After the cells were completely digested, 10–15 mL of cell culture medium was added to elute the digested cells. The cells were centrifuged at 1,000 rpm for 5 minutes, the supernatant was discarded, and 10–20 mL of cell culture medium was added to resuspend the cells to prepare a single-cell suspension.

[0257] 3. Seeding cells onto plates: Single cell suspensions of U87MG and SK-BR-3 were mixed uniformly and cultured in cell culture medium until the viable cell density reached 2.75 × 10 3 cells / mL and 8.25 x 10 3 The density-adjusted cell suspension was adjusted to 180 cells / mL, mixed evenly, and added to a 96-well cell culture plate at 180 μL per well. Only 200 μL of medium was added to the outer periphery of the 96-well plate. The culture plate was then incubated in an incubator (37°C, 5% CO2) for 24 hours.

[0258] 4. Preparation of compounds: Compounds were dissolved in DMSO (dimethyl sulfoxide, Shanghai Taitan Technology Co., Ltd.) to prepare a stock solution with an initial concentration of 10 mM.

[0259] The initial concentration of the small molecule compound was 500 nM, and the drug preparation method was as follows.

[0260] 30 μl of each sample to be measured was added to the first column of a 96-well U-bottom drug preparation plate, resulting in a sample concentration of 100 μM. 20 μl of DMSO was added to each well in columns 2 to 11. 10 μl of sample from column 1 was added to 20 μl of DMSO in column 2 and mixed uniformly, and 10 μl was added to column 3, and so on up to column 10. 5 μl to 95 μl of the drug from the drug preparation plate was added to each well in EMEM medium and mixed uniformly for use.

[0261] The initial concentration of ADC was 10 nM or 500 nM, and the drug preparation method was as follows:

[0262] 100 μL of each sample to be measured was added to the first column of a 96-well plate, with a sample concentration of 100 nM or 5 μM. 100 μL of PBS was added to each well in columns 2 to 11. 50 μL of the sample in column 1 was added to 100 μL of PBS in column 2 and mixed evenly. 50 μL of the sample was added to column 3, and so on up to column 10, resulting in a 3-fold dilution.

[0263] 5. Sample addition procedure: 20 μl of samples to be measured at different concentrations were added to the culture plate so that there were two duplicate wells per sample. The culture plate was incubated in an incubator (37°C, 5% CO2) for 6 days.

[0264] 6. Color development procedure: A 96-well cell culture plate was taken out, and 90 μl of CTG solution was added to each well, followed by incubation at room temperature for 10 minutes.

[0265] 7. Plate reading procedure: The 96-well cell culture plate was taken out and placed on a microplate reader (BMG labtech, PHERAstar FS), and the chemiluminescence was measured by the microplate reader.

[0266] 3. Data analysis Data was processed and analyzed using Microsoft Excel and Graphpad Prism 5. The experimental results are shown in the table below.

[0267] [Table 28]

[0268] Conclusion: The small molecule fragments in this disclosure have significant growth inhibitory activity against SK-BR-3 and U87 cells, and the chiral center has a certain effect on the inhibitory activity of the compounds. Furthermore, the present invention includes the following aspects. [Aspect 1] An antibody-drug conjugate comprising an anti-CEA antibody or antigen-binding fragment thereof, wherein the anti-CEA antibody or antigen-binding fragment thereof is optionally linked to a toxic drug by a linker, and the antibody comprises a heavy chain variable region and a light chain variable region, wherein: i) the HCDR1 and HCDR3 of the heavy chain variable region are the same as the HCDR1 and HCDR3 of the heavy chain variable region shown in SEQ ID NO:7, the HCDR2 of the heavy chain variable region is the same as the HCDR2 of the heavy chain variable region shown in SEQ ID NO:7 or has one amino acid difference therewith, and the LCDR1, LCDR2 and LCDR3 of the light chain variable region are the same as the LCDR1, LCDR2 and LCDR3 of the light chain variable region shown in SEQ ID NO:8, ii) the HCDR1 and HCDR3 of the heavy chain variable region are the same as the HCDR1 and HCDR3 of the heavy chain variable region shown in the sequence SEQ ID NO:9, the HCDR2 of the heavy chain variable region is the same as the HCDR2 of the heavy chain variable region shown in the sequence SEQ ID NO:9 or has one amino acid difference therewith, and the LCDR1, LCDR2 and LCDR3 of the light chain variable region are the same as the LCDR1, LCDR2 and LCDR3 of the light chain variable region shown in the sequence SEQ ID NO:10; iii) the HCDR1, HCDR2 and HCDR3 of the heavy chain variable region are the same as the HCDR1, HCDR2 and HCDR3 of the heavy chain variable region shown in the sequence SEQ ID NO:11, and the LCDR1, LCDR2 and LCDR3 of the light chain variable region are the same as the LCDR1, LCDR2 and LCDR3 of the light chain variable region shown in the sequence SEQ ID NO:12, or iv) the HCDR1 and HCDR3 of the heavy chain variable region are the same as the HCDR1 and HCDR3 of the heavy chain variable region shown in the sequence SEQ ID NO: 13, the HCDR2 of the heavy chain variable region is the same as the HCDR2 of the heavy chain variable region shown in the sequence SEQ ID NO: 13 or has one amino acid difference therebetween, the LCDR1 and LCDR3 of the light chain variable region are the same as the LCDR1 and LCDR3 of the light chain variable region shown in the sequence SEQ ID NO: 14, and the LCDR2 of the light chain variable region is the same as the LCDR2 of the light chain variable region shown in the sequence SEQ ID NO: 14 or has one amino acid difference therebetween; Antibody-drug conjugates. [Aspect 2] The anti-CEA antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, v) the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 set forth in SEQ ID NO:15, SEQ ID NO:16, and SEQ ID NO:17, respectively, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 set forth in SEQ ID NO:18, SEQ ID NO:19, and SEQ ID NO:20, respectively; or the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 set forth in SEQ ID NO:15, SEQ ID NO:38, and SEQ ID NO:17, respectively, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 set forth in SEQ ID NO:18, SEQ ID NO:19, and SEQ ID NO:20, respectively; vi) the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 set forth in SEQ ID NO:21, SEQ ID NO:22, and SEQ ID NO:23, respectively, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 set forth in SEQ ID NO:24, SEQ ID NO:25, and SEQ ID NO:26, respectively; or the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 set forth in SEQ ID NO:21, SEQ ID NO:47, and SEQ ID NO:23, respectively, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 set forth in SEQ ID NO:24, SEQ ID NO:25, and SEQ ID NO:26, respectively; vii) the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 set forth in SEQ ID NO:27, SEQ ID NO:28, and SEQ ID NO:29, respectively, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 set forth in SEQ ID NO:30, SEQ ID NO:31, and SEQ ID NO:32, respectively; or viii) the heavy chain variable region comprises HCDR1 and HCDR3 set forth in SEQ ID NO:33 and SEQ ID NO:34, respectively, and HCDR2 set forth in SEQ ID NO:16 or SEQ ID NO:38, and the light chain variable region comprises LCDR1 and LCDR3 set forth in SEQ ID NO:35 and SEQ ID NO:37, respectively, and LCDR2 set forth in SEQ ID NO:36 or SEQ ID NO:64; 2. The antibody-drug conjugate according to embodiment 1. [Aspect 3] 3. The antibody-drug conjugate according to Aspect 1 or 2, wherein the anti-CEA antibody is a murine antibody, a chimeric antibody, or a humanized antibody. [Aspect 4] The anti-CEA antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, (a) the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO:7 or is at least 90% identical to the amino acid sequence set forth in SEQ ID NO:7, and / or the amino acid sequence of the light chain variable region is set forth in SEQ ID NO:8 or is at least 90% identical to the amino acid sequence set forth in SEQ ID NO:8; or (b) the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO:9 or is at least 90% identical to the amino acid sequence set forth in SEQ ID NO:9, and / or the amino acid sequence of the light chain variable region is set forth in SEQ ID NO:10 or is at least 90% identical to the amino acid sequence set forth in SEQ ID NO:10; or (c) the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO:11 or is at least 90% identical to the amino acid sequence set forth in SEQ ID NO:11, and / or the amino acid sequence of the light chain variable region is set forth in SEQ ID NO:12 or is at least 90% identical to the amino acid sequence set forth in SEQ ID NO:12; or (d) the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO:13 or is at least 90% identical to the amino acid sequence set forth in SEQ ID NO:13, and / or the amino acid sequence of the light chain variable region is set forth in SEQ ID NO:14 or is at least 90% identical to the amino acid sequence set forth in SEQ ID NO:14; The antibody-drug conjugate according to any one of aspects 1 to 3. [Aspect 5] The anti-CEA antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, (e) the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO:39, 40, 41, or 42, or has at least 90% identity to any one of the amino acid sequences set forth in SEQ ID NO:39, 40, 41, or 42, and / or the amino acid sequence of the light chain variable region is set forth in SEQ ID NO:43, 44, 45, or 46, or has at least 90% identity to any one of the amino acid sequences set forth in SEQ ID NO:43, 44, 45, or 46; or (f) the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO:48, 49, 50, 51, or 52, or has at least 90% identity to any one of the amino acid sequences set forth in SEQ ID NO:48, 49, 50, 51, or 52, and / or the amino acid sequence of the light chain variable region is set forth in SEQ ID NO:53, 54, or 55, or has at least 90% identity to any one of the amino acid sequences set forth in SEQ ID NO:53, 54, or 55; or (g) the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO:56, 57, or 58, or has at least 90% identity to any one of the amino acid sequences set forth in SEQ ID NO:56, 57, or 58, and / or the amino acid sequence of the light chain variable region is set forth in SEQ ID NO:59, 60, 61, 62, or 63, or has at least 90% identity to any one of the amino acid sequences set forth in SEQ ID NO:59, 60, 61, 62, or 63; or (h) the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO:65, 66, 67, or 68, or has at least 90% identity to any one of the amino acid sequences set forth in SEQ ID NO:65, 66, 67, or 68, and / or the amino acid sequence of the light chain variable region is set forth in SEQ ID NO:69, 70, 71, 72, 73, 74, 75, or 76, or has at least 90% identity to any one of the amino acid sequences set forth in SEQ ID NO:69, 70, 71, 72, 73, 74, 75, or 76; The antibody-drug conjugate according to any one of aspects 1 to 4. [Aspect 6] the anti-CEA antibody is a humanized antibody, the humanized antibody comprises a framework region derived from a human antibody or a variant thereof, and the variant framework region has at most 10 amino acid backmutations in the light chain framework region and / or the heavy chain framework region of the human antibody, respectively; Preferably, the framework region variant is selected from any one of the following (i) to (l): (i) the framework regions of the light chain variable region comprising LCDR1, LCDR2 and LCDR3, the sequences of which are set forth in SEQ ID NO:18, SEQ ID NO:19 and SEQ ID NO:20, respectively, contain one or more amino acid backmutations selected from 46P, 47W, 49Y, 70S and 71Y; and / or the framework regions of the heavy chain variable region comprising HCDR1, the sequence of which is set forth in SEQ ID NO:15, HCDR2, the sequence of which is set forth in SEQ ID NO:16 or SEQ ID NO:38, and HCDR3, the sequence of which is set forth in SEQ ID NO:17, contain one or more amino acid backmutations selected from 38K or 46K; (j) the framework regions of the light chain variable region, including LCDR1, LCDR2, and LCDR3, whose sequences are set forth in SEQ ID NO:24, SEQ ID NO:25, and SEQ ID NO:26, respectively, contain one or more amino acid backmutations selected from 2V, 42G, 44V, and 71Y; and / or the framework regions of the heavy chain variable region, including HCDR1, whose sequence is set forth in SEQ ID NO:21, HCDR2, whose sequence is set forth in SEQ ID NO:22 or SEQ ID NO:47, and HCDR3, whose sequence is set forth in SEQ ID NO:23, contain one or more amino acid backmutations selected from 66K, 67A, 69L, 71V, 73K, 82F, and 82AR; (k) the framework regions of the light chain variable region, including LCDR1, LCDR2, and LCDR3, whose sequences are set forth in SEQ ID NO:30, SEQ ID NO:31, and SEQ ID NO:32, respectively, contain one or more amino acid backmutations selected from 3V, 43P, and 58V; and / or the framework regions of the heavy chain variable region, including HCDR1, HCDR2, and HCDR3, whose sequences are set forth in SEQ ID NO:27, SEQ ID NO:28, and SEQ ID NO:29, respectively, contain one or more amino acid backmutations selected from 38K, 66K, and 71V; and (l) the framework regions of the light chain variable region, including LCDR1 whose sequence is set forth in SEQ ID NO: 35, LCDR2 whose sequence is set forth in SEQ ID NO: 36 or SEQ ID NO: 64, and LCDR3 whose sequence is set forth in SEQ ID NO: 37, contain one or more amino acid backmutations selected from 4V, 36Y, 43P, 47V, 49E, 70D, and 87I; and / or the framework regions of the heavy chain variable region, including HCDR1 whose sequence is set forth in SEQ ID NO: 33, HCDR2 whose sequence is set forth in SEQ ID NO: 16 or SEQ ID NO: 38, and HCDR3 whose sequence is set forth in SEQ ID NO: 34, contain one or more amino acid backmutations selected from 2I, 38K, and 46K; wherein the reversion sites are numbered according to the Kabat numbering convention; 6. The antibody-drug conjugate according to any one of aspects 1 to 5. [Aspect 7] The anti-CEA antibody or antigen-binding fragment thereof comprises an antibody heavy chain constant region and a light chain constant region, preferably the heavy chain constant region is selected from human IgG1, IgG2, IgG3, and IgG4 constant regions and common variants thereof, and the light chain constant region is selected from human antibody kappa and lambda chain constant regions and common variants thereof, more preferably the antibody comprises a heavy chain constant region having a sequence set forth in SEQ ID NO:77 and a light chain constant region having a sequence set forth in SEQ ID NO:78 or SEQ ID NO:79; Most preferably, the anti-CEA antibody is (m) a heavy chain whose sequence is set forth in SEQ ID NO:80 or has at least 85% identity thereto, and / or a light chain whose sequence is set forth in SEQ ID NO:81 or has at least 85% identity thereto; (n) a heavy chain whose sequence is set forth in SEQ ID NO:82 or has at least 85% identity thereto, and / or a light chain whose sequence is set forth in SEQ ID NO:83 or has at least 85% identity thereto; (o) a heavy chain whose sequence is set forth in SEQ ID NO:84 or has at least 85% identity thereto, and / or a light chain whose sequence is set forth in SEQ ID NO:85 or has at least 85% identity thereto; or (p) a heavy chain whose sequence is set forth in SEQ ID NO: 86 or has at least 85% identity thereto, and / or a light chain whose sequence is set forth in SEQ ID NO: 87 or has at least 85% identity thereto; The antibody-drug conjugate according to any one of aspects 1 to 6. [Aspect 8] An antibody-drug conjugate represented by the general formula (Pc-LYD):

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Claims

1. An antibody-drug conjugate comprising an anti-CEA antibody or an antigen-binding fragment thereof, wherein the anti-CEA antibody or the antigen-binding fragment thereof is bound to a drug via a linker, and comprises an antibody heavy chain variable region and a light chain variable region, wherein: the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 shown in SEQ ID NO: 21, SEQ ID NO: 22 and SEQ ID NO: 23, respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO: 24, SEQ ID NO: 25 and SEQ ID NO: 26, respectively; or the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 shown in SEQ ID NO: 21, SEQ ID NO: 47 and SEQ ID NO: 23, respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO: 24, SEQ ID NO: 25 and SEQ ID NO: 26, respectively. Antibody-drug conjugates.

2. The antibody-drug conjugate of claim 1, wherein the anti-CEA antibody is a murine antibody, a chimeric antibody, or a humanized antibody.

3. 3. The antibody-drug conjugate of claim 1, wherein the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 9 or has at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 9, and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 10 or has at least 90% identity to the amino acid sequence set forth in SEQ ID NO:

10.

4. the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 48, 49, 50, 51 or 52, or has at least 90% identity to any one of the amino acid sequences set forth in SEQ ID NO: 48, 49, 50, 51 or 52, and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 53, 54 or 55, or has at least 90% identity to any one of the amino acid sequences set forth in SEQ ID NO: 53, 54 or 55; 3. The antibody-drug conjugate of claim 1 or 2.

5. The amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 52, and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO:

53.

3. The antibody-drug conjugate of claim 1 or 2.

6. The anti-CEA antibody is a humanized antibody, and the humanized antibody comprises a framework region derived from a human antibody or a variant thereof, and the variant framework region has at most 10 amino acid backmutations in the light chain framework region and / or the heavy chain framework region of the human antibody. The antibody-drug conjugate according to any one of claims 1 to 2 and 4 to 5.

7. the variants in the framework regions of the light chain variable region include one or more amino acid backmutations selected from 2V, 42G, 44V, and 71Y, and / or the variants in the framework regions of the heavy chain variable region include one or more amino acid backmutations selected from 48I, 66K, 67A, 69L, 71V, 73K, 82F, and 82A R; wherein the reversion sites are numbered according to the Kabat numbering convention; 82A R means a mutation that restores the amino acid at position 82A to R according to the Kabat numbering system. The antibody-drug conjugate of claim 6.

8. the anti-CEA antibody or antigen-binding fragment thereof comprises an antibody heavy chain constant region and a light chain constant region, wherein the heavy chain constant region is selected from human IgG1, IgG2, IgG3, and IgG4 constant regions, and the light chain constant region is selected from human antibody κ and λ chain constant regions; The antibody-drug conjugate according to any one of claims 1 to 7.

9. the antibody comprises a heavy chain constant region having the sequence set forth in SEQ ID NO: 77, and a light chain constant region having the sequence set forth in SEQ ID NO: 78 or SEQ ID NO: 79; The antibody-drug conjugate according to any one of claims 1 to 7.

10. The anti-CEA antibody comprises a heavy chain having a sequence set forth in SEQ ID NO: 80 or having at least 85% identity thereto, and a light chain having a sequence set forth in SEQ ID NO: 81 or having at least 85% identity thereto. The antibody-drug conjugate of claim 1.

11. The antibody-drug conjugate of claim 10, wherein the anti-CEA antibody comprises a heavy chain whose sequence is set forth in SEQ ID NO: 80 and a light chain whose sequence is set forth in SEQ ID NO:

81.

12. An antibody-drug conjugate represented by the general formula (Pc-LYD): 【Chemistry 1】 Among them, Y is -O- (CR a R b ) m -CR 1 R 2 -C(O)-, -O-CR 1 R 2 -(CR a R b ) m -, -O-CR 1 R 2 -, -NH- (CR a R b ) m -CR 1 R 2 -C(O)- and -S-(CR a R b ) m -CR 1 R 2 -C(O)-, R a and R b are each independently selected from a hydrogen atom, a deuterium atom, a halogen, an alkyl group, a haloalkyl group, a deuterated alkyl group, an alkoxy group, a hydroxy group, an amino group, a cyano group, a nitro group, a hydroxyalkyl group, a cycloalkyl group, and a heterocyclyl group, or R a and R b together with the carbon atoms to which they are attached form a cycloalkyl group and a heterocyclyl group, R 1 is selected from halogen, haloalkyl, deuterated alkyl, cycloalkyl, cycloalkylalkyl, alkoxyalkyl, heterocyclyl, aryl and heteroaryl groups; R 2 is selected from a hydrogen atom, a halogen, a haloalkyl group, a deuterated alkyl group, a cycloalkyl group, a cycloalkylalkyl group, an alkoxyalkyl group, a heterocyclyl group, an aryl group, and a heteroaryl group, or R 1 and R 2 together with the carbon atoms to which they are attached form a cycloalkyl or heterocyclyl group, Or R a and R 2 together with the carbon atoms to which they are attached form a cycloalkyl or heterocyclyl group, m is an integer from 0 to 4, n is a decimal or integer between 1 and 10, where n represents the average drug loading of the antibody-drug conjugate; L is -L 1 -L 2 -L 3 -L 4 - linker unit, where L 1 -(succinimide-3-yl-N)-WC(O)-, -CH 2 -C(O)-NR 3 -WC(O)- and -C(O)-WC(O)-, where W is C 1-8 Alkyl group, C 1-8 selected from alkyl-cycloalkyl groups and straight chain heteroalkyl groups of 1 to 8 atoms, said heteroalkyl groups containing 1 to 3 heteroatoms selected from N, O and S; L 2 Ha-NR 4 (CH 2 CH 2 O)p 1 CH 2 CH 2 C(O)-, -NR 4 (CH 2 CH 2 O)p 1 CH 2 C(O)-, -S(CH 2 ) p 1 C(O)- and a chemical bond, where p 1 is an integer between 1 and 20, L 3 is a peptide residue consisting of 2 to 7 amino acids, wherein the amino acids are selected from the amino acid residues formed by the amino acids phenylalanine, glycine, valine, lysine, citrulline, serine, glutamic acid and aspartic acid, L 4 is -NR 5 (CR 6 R 7 ) t -, -C(O)NR 5 -, -C(O)NR 5 (CH 2 ) t - and a chemical bond, where t is an integer from 1 to 6; R 3 , R 4 and R 5 are each independently selected from a hydrogen atom, an alkyl group, a haloalkyl group, a deuterated alkyl group, and a hydroxyalkyl group; R 6 and R 7 are each independently selected from a hydrogen atom, a halogen, an alkyl group, a haloalkyl group, a deuterated alkyl group, and a hydroxyalkyl group; Pc is the anti-CEA antibody or antigen-binding fragment thereof according to any one of claims 1 to 11. The antibody-drug conjugate according to any one of claims 1 to 11.

13. The antibody-drug conjugate of claim 12, wherein n is a decimal or integer number from 2 to 8.

14. The antibody-drug conjugate of claim 12, wherein n is a decimal or integer of 4 to 6.

15. Y is -O- (CR a R b ) m -CR 1 R 2 -C(O)-, R a and R b are each independently selected from a hydrogen atom, a deuterium atom, a halogen atom, or an alkyl group; R 1 is a haloalkyl group or C 3-6 is a cycloalkyl group, R 2 represents a hydrogen atom, a haloalkyl group, and C 3-6 cycloalkyl groups, Or R 1 and R 2 are C together with the carbon atoms connected to them. 3-6 forming a cycloalkyl group, m is 0 or 1; The antibody-drug conjugate of claim 12.

16. Y is 【Chemistry 2】 wherein the O-terminus of Y is linked to a linker unit L; 16. The antibody-drug conjugate of claim 15.

17. An antibody-drug conjugate represented by the general formula (Pc-LD): 【Transformation 3】 Among them, L, Pc and n are as defined in claim 12. The antibody-drug conjugate of claim 12.

18. Linker unit -L- is -L 1 -L 2 -L 3 -L 4 - and among them, L 1 teeth 【Chemistry 4】 and s 1 is an integer between 2 and 8, L 2 is a chemical bond, L 3 is a tetrapeptide residue, L 4 Ha-NR 5 (CR 6 R 7 ) t- and R 5 , R 6 or R 7 are each independently a hydrogen atom or an alkyl group, t is 1 or 2, Among them, the above L 1 The end is connected to Pc, and L 4 The end is connected to Y, The antibody-drug conjugate of claim 12. 【Request Item 19】 【Chemistry 5】 Selected from wherein Pc and n are as defined in claim 12; The antibody-drug conjugate of claim 12. 【Request Item 20】 【Chemistry 6】 The antibody-drug conjugate of claim 1, wherein n is a decimal or integer between 1 and 10, where n represents the average drug loading of the antibody-drug conjugate. 、 Hu63-13 is an antibody-drug conjugate comprising a heavy chain whose sequence is set forth in SEQ ID NO:80 and a light chain whose sequence is set forth in SEQ ID NO:

81.

21. A drug composition comprising the antibody-drug conjugate of any one of claims 1 to 20 and one or more pharmaceutically acceptable excipients, diluents or carriers.

22. 22. The pharmaceutical composition of claim 21 for treating a CEA-mediated disease or condition.

23. 22. The pharmaceutical composition according to claim 21, for treating and / or preventing tumors and cancers, wherein the tumors and cancers are head and neck squamous cell carcinoma, head and neck cancer, brain cancer, glioma, glioblastoma multiforme, neuroblastoma, central nervous system cancer, neuroendocrine tumor, pharyngeal cancer, nasopharyngeal cancer, esophageal cancer, thyroid cancer, malignant pleural mesothelioma, lung cancer, breast cancer, liver cancer, hepatic and gallbladder cancer, pancreatic cancer, gastric cancer, gastrointestinal cancer, intestinal cancer, colon cancer, colorectal cancer, renal cancer, clear cell renal cell carcinoma, ovarian cancer, endometrial cancer, cervical cancer, bladder cancer, prostate cancer, testicular cancer, skin cancer, melanoma, leukemia, lymphoma, bone cancer, chondrosarcoma, myeloma, multiple myeloma, myelodysplastic syndrome, Krueckenberg tumor, myeloproliferative neoplasm, squamous cell carcinoma, Ewing sarcoma, systemic light-chain amyloidosis, and Merkel cell carcinoma.

24. The lymphoma is selected from Hodgkin's lymphoma, non-Hodgkin's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, primary mediastinal large B-cell lymphoma, mantle cell lymphoma, small lymphocytic lymphoma, T-cell / histiocyte-rich large B-cell lymphoma, and lymphoplasmacytic lymphoma; the lung cancer is selected from non-small cell lung cancer and small cell lung cancer; and the leukemia is selected from chronic myeloid leukemia, acute myeloid leukemia, lymphocytic leukemia, lymphoblastic leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, and myeloid cell leukemia; 24. The pharmaceutical composition of claim 23.

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