Antibody-drug conjugate targeting folate receptor alpha and method of use

Abstract

Provided are antibody-drug conjugates (ADCs) comprising an antibody construct that specifically binds to human folate receptor alpha (FRα) (anti-FRα antibody construct) conjugated to a camptothecin analog of formula (I), which are useful as therapeutic agents, particularly in the treatment of cancer. TIFF2025510791000395.tif53165

Description

[Technical Field]

[0001] field This disclosure relates to the field of immunotherapy agents, and more particularly to antibody-drug conjugates that target the human folate receptor alpha (hFRα). [Background technology]

[0002] background Folate receptor alpha (FRα) is a glycosylphosphatidylinositol (GPI)-anchored cell surface protein encoded by FOLR1, and is part of the high-affinity folate family, which also includes FRβ (FOLR2), FRγ (FOLR3), and FRδ (FOLR4). FRα is overexpressed in a variety of cancers, including ovarian cancer, triple-negative breast cancer (TNBC), endometrial cancer, mesothelioma, and lung cancer, and is minimally expressed in non-malignant tissues, making it a highly relevant cancer therapy target.

[0003] Several clinical studies are currently underway involving FRα-targeting agents in cancer treatment, including anti-FRα antibodies, faretzumab, and FRα-targeted antibody-drug conjugates (ADCs), such as milbetuximab sorabtansine (ImmunoGen, Inc.), MORAb-202 (Eisai Inc.), and STRO-002 (Sutro Biopharma, Inc.).

[0004] Camptothecin analogs were developed as payloads for antibody-drug conjugates (ADCs). Two such ADCs have been approved for the treatment of cancer: trastuzumab deruxtecan (Enhertu®), in which the camptothecin analog deruxtecan (Dxd) is conjugated with the anti-HER2 antibody trastuzumab via a cleavable tetrapeptide-based linker, and sacituzumab govitecan (Trodelvy®), in which the camptothecin analog SN-38 is conjugated with the anti-Trop-2 antibody sacituzumab via a hydrolyzable pH-sensitive linker.

[0005] Other camptothecin analogs and derivatives, as well as ADCs containing them, have been shown. See, for example, International (PCT) Publication No. WO2019 / 195665 (Patent Document 1), No. WO2019 / 236954 (Patent Document 2), No. WO2020 / 200880 (Patent Document 3), and No. WO2020 / 219287 (Patent Document 4).

[0006] This background information is provided for the purpose of making known any information that the applicant believes may be relevant to this disclosure. None of the foregoing information is necessarily intended to constitute prior art to the claimed invention, nor should it be interpreted as such. [Prior art documents] [Patent Documents]

[0007] [Patent Document 1] International (PCT) Publication No. WO2019 / 195665 [Patent Document 2] International (PCT) Publication No. WO2019 / 236954 [Patent Document 3] International (PCT) Publication No. WO2020 / 200880 [Patent Document 4] International (PCT) Publication No. WO2020 / 219287 [Overview of the project]

[0008] overview This specification describes antibody-drug conjugates (ADCs) targeting human FRα and methods of use. One aspect of this disclosure is formula (X): T-[L-(D) m ] n (X) Regarding antibody-drug conjugates having, m is 1 to 4, n is between 1 and 10. T is an anti-FRα antibody construct comprising an antigen-binding domain that specifically binds to an epitope within human folate receptor alpha (hFRα) comprising the amino acid residues E120, D121, R123, T124, S125, and Y126 of SEQ ID NO: 15, L is a linker, D is a compound of Formula I: TIFF0007877480000001.tif53165 wherein, R 1 is selected from -H, -CH3, -CHF2, -CF3, -F, -Br, -Cl, -OH, -OCH3, -OCF3 and -NH2, R 2 is selected from -H, -CH3, -CF3, -F, -Br, -Cl, -OH, -OCH3, and -OCF3, R 1 when R is -NH2, R is R 3 or R 4 and when R is other than -NH2, R is R 1 where, 4 is, R 3 is selected from -H, -C1-C6 alkyl, -C3-C8 cycloalkyl, -(C1-C6 alkyl)-O-R 5 , TIFF0007877480000002.tif27165-CO2R 8 , -aryl, -heteroaryl, and -(C1-C6 alkyl)-aryl, R 4 is TIFF0007877480000003.tif68165selected from R 5 is selected from -H, -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl, -aryl, and -(C1-C6 alkyl)-aryl, R 6 and R 7 are each independently selected from -H, -C1-C6 alkyl, -C3-C8 cycloalkyl, -(C1-C6 alkyl)-O-R 5 , -C3-C8 heterocycloalkyl, and -C(O)R17 Selected from, R 8 These are selected from -H, -C1~C6 alkyl, -C3~C8 cycloalkyl, and -C3~C8 heterocycloalkyl. Each R 9は Independently selected from -H, -C1~C6 alkyl, -C3~C8 cycloalkyl, -aryl, -heteroaryl, and -(C1~C6 alkyl)-aryl, Each R 10 These are independently -C1~C6 alkyl, -C3~C8 cycloalkyl, and -NR 14 R 14’ Selected from -aryl, -heteroaryl, and -(C1~C6 alkyl)-aryl, R 10’ These are selected from -H, -C1~C6 alkyl, -C3~C8 cycloalkyl, -aryl, -heteroaryl, and -(C1~C6 alkyl)-aryl. R 11 These are selected from -H and -C1~C6 alkyl groups. R 12 -H, -C1~C6 alkyl, -CO2R 8 -aryl, -heteroaryl, -(C1~C6 alkyl)-aryl, -S(O)2R 16 , and Selected from TIFF0007877480000004.tif22165, R 13 These are selected from -H and -C1~C6 alkyl groups. R 14 and R 14’ Each of these is independently selected from -H, C1-C6 alkyl, -C3-C8 cycloalkyl, and -C3-C8 heterocycloalkyl. R 16 These are selected from -C1~C6 alkyl, -C3~C8 cycloalkyl, -aryl, -heteroaryl, and -(C1~C6 alkyl)-aryl. R 17These are selected from -C1~C6 alkyl, -C3~C8 cycloalkyl, -C3~C8 heterocycloalkyl, -(C1~C6 alkyl)-C3~C8 heterocycloalkyl, -aryl, -heteroaryl, and -(C1~C6 alkyl)-aryl. R 18 and R 19 These, along with the N atom to which they are bonded, include halogens, -C1~C6 alkyls, -C3~C8 cycloalkyls, and -(C1~C6 alkyl)-OR 5 Forms a 4, 5, 6, or 7-membered ring having 0 to 3 substituents selected from the above, R 24 , R 25 and R 26 These are each -C1 to C6 alkyl groups, X a and X b Each is independently selected from NH, O, and S. X c It is selected from O, S, and S(O)2, However, the compound is anything other than (S)-9-amino-11-butyl-4-ethyl-4-hydroxy-1,12-dihydro-14H-pyrano[3',4':6,7]indolidino[1,2-b]quinoline-3,14(4H)-dione.

[0009] Another aspect of this disclosure relates to antibody-drug conjugates having a structure selected from the following: TIFF0007877480000005.tif227165TIFF0007877480000006.tif104165In the formula, T is an anti-FRα antibody construct containing two antigen-binding domains functionally linked to the IgG Fc region, each of which is: (a) The VL amino acid sequence described in SEQ ID NO: 39 and the VH amino acid sequence described in SEQ ID NO: 19, or (b) The VL amino acid sequence described in SEQ ID NO: 124 and the VH amino acid sequence described in SEQ ID NO: 91, or (c) The VL amino acid sequence described in Sequence ID No. 64, and (i) The VH amino acid sequence described in Sequence ID No. 50, or (ii) The VH amino acid sequence described in Sequence ID No. 54, or (iii) The VH amino acid sequence described in Sequence ID No. 57, or (iv) The VH amino acid sequence described in Sequence ID No. 61, or (v) The VH amino acid sequence described in Sequence ID No. 76, or (vi) The VH amino acid sequence described in Sequence ID No. 79, or (vii) The VH amino acid sequence described in Sequence ID No. 82, or (viii) The VH amino acid sequence described in Sequence ID No. 85, or (ix) The VH amino acid sequence described in Sequence ID No. 88, or (x) The VH amino acid sequence described in Sequence ID No. 106, or (d) The VL amino acid sequence described in SEQ ID NO: 130, and (i) The VH amino acid sequence described in Sequence ID No. 99, or (ii) The VH amino acid sequence described in Sequence ID No. 106, or (iii) The VH amino acid sequence described in Sequence ID No. 113, or (iv) The VH amino acid sequence described in Sequence ID No. 116, or (v) The VH amino acid sequence described in Sequence ID No. 133, or (vi) The VH amino acid sequence described in SEQ ID NO. 136, or (e) The VL amino acid sequence described in Sequence ID No. 119, and (i) The VH amino acid sequence described in Sequence ID No. 106, or (ii) The VH amino acid sequence described in Sequence ID No. 116, Includes, And n is between 4 and 8.

[0010] Another aspect of this disclosure relates to a pharmaceutical composition comprising an antibody-drug conjugate described herein and a pharmaceutically acceptable carrier or diluent.

[0011] Another aspect of this disclosure relates to a method for inhibiting the proliferation of cancer cells, comprising contacting the cells with an effective amount of an antibody-drug conjugate described herein.

[0012] Another aspect of this disclosure relates to a method for killing cancer cells, comprising contacting cells with an effective amount of an antibody-drug conjugate described herein.

[0013] Another aspect of this disclosure relates to a method for treating cancer in a subject in need, comprising administering to the subject an effective amount of the antibody-drug conjugate described herein.

[0014] Another aspect of this disclosure relates to antibody-drug conjugates described herein for use in therapy.

[0015] Another aspect of this disclosure relates to antibody-drug conjugates described herein for use in the treatment of cancer.

[0016] Another aspect of this disclosure relates to the use of antibody-drug conjugates described herein in the manufacture of drugs for the treatment of cancer. [Invention 1001] Antibody-drug conjugate having formula (X): T-[L-(D) m ] n (X) And in the formula, m is 1 to 4, n is between 1 and 10. T is an anti-FRα antibody construct containing an antigen-binding domain that specifically binds to an epitope within the human folate receptor alpha (hFRα) containing the amino acid residues E120, D121, R123, T124, S125, and Y126 of SEQ ID NO: 15. L is the linker, D is equation I: TIFF0007877480000007.tif53165 It is a compound of the formula, in which, R 1 However, -H, -CH 3 , -CHF2 -CF 3 , -F, -Br, -Cl, -OH, -OCH 3 , -OCF 3 , and -NH 2 Selected from, and R 2 However, -H, -CH 3 -CF 3 , -F, -Br, -Cl, -OH, -OCH 3 , and -OCF 3 Selected from, Furthermore, R 1 ga-NH 2 If that is the case, R is R 3 or R 4 And, R 1 ga-NH 2 If it is anything other than R, R 4 And, R 3 However, -H, -C 1 ~C 6 Alkyl, -C 3 ~C 8 Cycloalkyl, -(C 1 ~C 6 Alkyl)-OR 5 、 TIFF0007877480000008.tif27165 -CO 2 R 8 , -aryl, -heteroaryl, and -(C 1 ~C 6 Selected from alkyl)-aryl, R 4 but, TIFF0007877480000009.tif68165 Selected from, R 5 However, -H, -C 1 ~C 6 Alkyl, -C 3 ~C 8 Cycloalkyl, -aryl, -heteroaryl, -aryl, and -(C 1 ~C 6 Selected from alkyl)-aryl, R 6 and R 7 However, independently of each other, -H and -C 1 ~C 6 Alkyl, -C 3~C 8 Cycloalkyl, -(C 1 ~C 6 Alkyl)-OR 5 、-C 3 ~C 8 Heterocycloalkyl and -C(O)R 17 Selected from, R 8 However, -H, -C 1 ~C 6 Alkyl, -C 3 ~C 8 Cycloalkyl, and -C 3 ~C 8 Selected from heterocycloalkyl groups, Each R 9 However, independently, -H, -C 1 ~C 6 Alkyl, -C 3 ~C 8 Cycloalkyl, -aryl, -heteroaryl, and -(C 1 ~C 6 Selected from alkyl)-aryl, Each R 10 However, independently, -C 1 ~C 6 Alkyl, -C 3 ~C 8 Cycloalkyl, -NR 14 R 14’ , -aryl, -heteroaryl, and -(C 1 ~C 6 Selected from alkyl)-aryl, R 10’ However, -H, -C 1 ~C 6 Alkyl, -C 3 ~C 8 Cycloalkyl, -aryl, -heteroaryl, and -(C 1 ~C 6 Selected from alkyl)-aryl, R 11 However, -H and -C 1 ~C 6 Selected from alkyl groups, R 12 However, -H, -C 1 ~C 6 Alkyl, -CO 2 R 8 -aryl, -heteroaryl, -(C 1 ~C 6 Alkyl)-aryl, -S(O) 2 R 16 , and TIFF0007877480000010.tif22165 Selected from, R 13 However, -H and -C 1 ~C 6 Selected from alkyl groups, R 14 and R 14’ However, independently of each other, -H and C 1 ~C 6 Alkyl, -C 3 ~C 8 Cycloalkyl, and -C 3 ~C 8 Selected from heterocycloalkyl groups, R 16 However, -C 1 ~C 6 Alkyl, -C 3 ~C 8 Cycloalkyl, -aryl, -heteroaryl, and -(C 1 ~C 6 Selected from alkyl)-aryl, R 17 However, -C 1 ~C 6 Alkyl, -C 3 ~C 8 Cycloalkyl, -C 3 ~C 8 Heterocycloalkyl, -(C 1 ~C 6 Alkyl)-C 3 ~C 8 Heterocycloalkyl, -aryl, -heteroaryl, and -(C 1 ~C 6 Selected from alkyl)-aryl, R 18 and R 19 However, along with the N atom to which they bond, halogens, -C 1 ~C 6 Alkyl, -C 3 ~C 8 Cycloalkyl, and -(C 1 ~C 6 Alkyl)-OR 5 Forms a 4, 5, 6, or 7-membered ring having 0 to 3 substituents selected from the above, R 24 、R 25 , and R 26 However, each is -C 1 ~C 6 It is alkyl, Xa and X b However, each is independently selected from NH, O, and S, and X c However, O, S, and S(O) 2 Selected from, However, the compound is other than (S)-9-amino-11-butyl-4-ethyl-4-hydroxy-1,12-dihydro-14H-pyrano[3',4':6,7]indolidino[1,2-b]quinoline-3,14(4H)-dione. The aforementioned antibody-drug conjugate. [Invention 1002] The antibody-drug construct of the present invention 1001, wherein the antigen-binding domain comprises heavy chain CDR amino acid sequences (HCDR1, HCDR2, and HCDR3) containing the sequences described in SEQ ID NOs: 3, 4, and 5, and light chain CDR amino acid sequences (LCDR1, LCDR2, and LCDR3) containing the sequences described in SEQ ID NOs: 6, 7, and 8. [Invention 1003] The antibody-drug conjugate of the present invention 1001, wherein the antigen-binding domain comprises a CDR sequence of a VH domain having the sequence described in any one of SEQ ID NOs: 19, 50, 54, 57, 61, 76, 79, 82, 85, 88, 91, 99, 106, 113, 116, 133, or 136. [Invention 1004] The antibody-drug conjugate of the present invention 1001 or 1003, wherein the antigen-binding domain comprises a CDR sequence of a VL domain having the sequence described in any one of SEQ ID NOs: 39, 64, 119, 124, or 130. [Invention 1005] The antigen-binding domain, (i) an HCDR1 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NOs: 20, 23, 26, 28, 31, 92, 93, 94, 95, or 96; an HCDR2 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NOs: 21, 24, 27, 29, 32, 51, 58, 100, 101, 102, 103, 109, 137, 138, or 139; and an HCDR3 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NOs: 22, 25, 30, 107, 108, or 110; (ii) An LCDR1 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NOs: 40, 43, 45, 65, 125, 126, or 127; an LCDR2 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NOs: 41, 44, or 46; and an LCDR3 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NOs: 42, 47, 120, or 121. The antibody-drug conjugate of the present invention 1001, including the above. [Invention 1006] D is given by equation (IV): TIFF0007877480000011.tif63165 It is a compound of the formula, in which, R 1a However, -H, -CH 3 , -CHF 2 -CF 3 , -F, -Br, -Cl, -OH, -OCH 3 , -OCF 3 , and -NH 2 Selected from, R 2a However, -H, -CH 3 -CF 3 , -F, -Br, -Cl, -OH, -OCH 3 , and -OCF 3 Selected from, X is -O-, -S-, or -NH-, and R 4a but, TIFF0007877480000012.tif68165 Selected from, where * is a connection point with X, and p is 1, 2, 3, or 4, or X is O and R 4a -X- is, TIFF0007877480000013.tif37165 Selected from, R 5a However, -C 1 ~C 6 Alkyl, -C 3 ~C 8 Cycloalkyl, -aryl, -heteroaryl, and -(C 1 ~C 6 Selected from alkyl)-aryl, R 8a However, -C 1 ~C 6 Alkyl, -C 3 ~C 8 Cycloalkyl, and -C 3 ~C 8 Selected from heterocycloalkyl groups, Each R 9a However, independently, -C 1 ~C 6 Alkyl, -C 3 ~C 8 Cycloalkyl, -aryl, -heteroaryl, and -(C 1 ~C 6 Selected from alkyl)-aryl, or R 9a If X does not exist b =X, Each R 10a However, independently, -C 1 ~C 6 Alkyl, -C 3 ~C 8 Cycloalkyl, -aryl, -heteroaryl, -(C 1 ~C 6 Alkyl)-aryl, and TIFF0007877480000014.tif22165 Selected from, Each R 10a’ However, independently, -H, -C 1 ~C 6 Alkyl, -C 3 ~C 8 Cycloalkyl, -aryl, -heteroaryl, and -(C 1 ~C 6 Selected from alkyl)-aryl, Each R 10b However, independently, -C 1 ~C 6 Alkyl, -C 3 ~C 8 Cycloalkyl, -aryl, -heteroaryl, and -(C 1 ~C 6 Selected from alkyl)-aryl, R11a However, it does not exist, or -C 1 ~C 6 It is alkyl, R 12a However, -C 1 ~C 6 Alkyl, -CO 2 R 8a -aryl, -heteroaryl, -(C 1 ~C 6 Alkyl)-aryl, -S(O) 2 R 16a , and TIFF0007877480000015.tif22165 Selected from, R 13a However, -H and -C 1 ~C 6 Selected from alkyl groups, R 14a However, -C 1 ~C 6 Alkyl, -C 3 ~C 8 Cycloalkyl, and -C 3 ~C 8 Selected from heterocycloalkyl groups, R 14a’ However, H, -C 1 ~C 6 Alkyl, -C 3 ~C 8 Cycloalkyl, and -C 3 ~C 8 Selected from heterocycloalkyl groups, R 16a However, -C 1 ~C 6 Alkyl, -C 3 ~C 8 Cycloalkyl, -aryl, -heteroaryl, and -(C 1 ~C 6 Selected from alkyl)-aryl, R 21 However, -C 1 ~C 6 Alkyl, -C 3 ~C 8 Cycloalkyl, and -(C 1 ~C 6 Alkyl)-OR 5a Selected from, R 22 and R23 However, each is independent of -H, -halogen, and -C. 1 ~C 6 Alkyl and -C 3 ~C 8 Selected from cycloalkyl groups, R 24 、R 25 , and R 26 However, each is -C 1 ~C 6 It is alkyl, X a and X b However, each is independently selected from NH, O, and S. X c However, O, S, and S(O) 2 Selected from, and TIFF0007877480000016.tif17165 However, it indicates the connection point with linker L. An antibody-drug conjugate according to any of invention 1001 to 1005. [Invention 1007] R 1a However, -CH 3 -CF 3 ,-OCH 3 , -OCF 3 , and -NH 2 An antibody-drug conjugate of the present invention 1006, selected from the above. [Invention 1008] R 1a However, -CH 3 ,-OCH 3 , and NH 2 An antibody-drug conjugate of the present invention 1006, selected from the above. [Invention 1009] R 2a However, an antibody-drug conjugate of any of the present invention 1006 to 1008, selected from -H, -F, -Br, and -Cl. [Invention 1010] X is -O-, -S-, or -NH-, and R 4a but, TIFF0007877480000017.tif58165 An antibody-drug conjugate selected from any of the present invention 1006 to 1009. [Invention 1011] D is given by equation (V): TIFF0007877480000018.tif53165 It is a compound of the formula, in which, R 2a However, -CH 3 -CF 3 , -F, -Br, -Cl, -OH, -OCH 3 , and -OCF 3 Selected from, R 20a However, -H, -C 1 ~C 6 Alkyl, -C 3 ~C 8 Cycloalkyl, -(C 1 ~C 6 Alkyl)-OR 5 、 TIFF0007877480000019.tif27165 -CO 2 R 8 -aryl, -heteroaryl, -(C 1 ~C 6 Alkyl)-aryl, TIFF0007877480000020.tif68165 Selected from, R 5 However, -H, -C 1 ~C 6 Alkyl, -C 3 ~C 8 Cycloalkyl, -aryl, -heteroaryl, and -(C 1 ~C 6 Selected from alkyl)-aryl, R 6 and R 7 However, independently of each other, -H and -C 1 ~C 6 Alkyl, -C 3 ~C 8 Cycloalkyl, -(C 1 ~C 6 Alkyl)-OR 5 、-C 3 ~C 8 Heterocycloalkyl and -C(O)R 17 Selected from, R 8 However, -H, -C 1 ~C 6 Alkyl, -C 3 ~C 8 Cycloalkyl, and -C 3 ~C 8 Selected from heterocycloalkyl groups, Each R 9 However, independently, -H, -C 1 ~C 6 Alkyl, -C 3 ~C 8 Cycloalkyl, -aryl, -heteroaryl, and -(C 1 ~C 6 Selected from alkyl)-aryl, Each R 10a However, independently, -C 1 ~C 6 Alkyl, -C 3 ~C 8 Cycloalkyl, -aryl, -heteroaryl, -(C 1 ~C 6 Alkyl)-aryl and -NR 14 R 14’ Selected from, Each R 10a’ However, independently, -H, -C 1 ~C 6 Alkyl, -C 3 ~C 8 Cycloalkyl, -aryl, -heteroaryl, and -(C 1 ~C 6 Selected from alkyl)-aryl, R 11 However, -H and -C 1 ~C 6 Selected from alkyl groups, R 12 However, -H, -C 1 ~C 6 Alkyl, -CO 2 R 8 -aryl, -heteroaryl, -(C 1 ~C 6 Alkyl)-aryl, -S(O) 2 R 16 , and TIFF0007877480000021.tif22165 Selected from, R 13 However, -H and -C 1 ~C 6 Selected from alkyl groups, R 14 and R 14’ However, independently of each other, -H and C 1 ~C 6 Alkyl, -C 3 ~C 8 Cycloalkyl, and -C 3 ~C 8 Selected from heterocycloalkyl groups, R 16 However, -C 1 ~C 6 Alkyl, -C 3 ~C 8 Cycloalkyl, -aryl, -heteroaryl, and -(C 1 ~C 6 Selected from alkyl)-aryl, R 17 However, -C 1 ~C 6 Alkyl, -C 3 ~C 8 Cycloalkyl, -C 3 ~C 8 Heterocycloalkyl, -(C 1 ~C 6 Alkyl)-C 3 ~C 8 Heterocycloalkyl, -aryl, -heteroaryl, and -(C 1 ~C 6 Selected from alkyl)-aryl, R 18 and R 19 However, along with the N atom to which they bond, halogens, -C 1 ~C 6 Alkyl, -C 3 ~C 8 Cycloalkyl, and -(C 1 ~C 6 Alkyl)-OR 5 Forms a 4, 5, 6, or 7-membered ring having 0 to 3 substituents selected from the above, R 24 、R 25 , and R 26 However, each is -C 1 ~C 6 It is alkyl, X a and X b However, each is independently selected from NH, O, and S. X c However, O, S, and S(O) 2 Selected from, and TIFF0007877480000022.tif17165 However, it indicates the connection point with linker L. An antibody-drug conjugate according to any of invention 1001 to 1005. [Invention 1012] R2a However, F is the antibody-drug conjugate of the present invention 1011. [Invention 1013] R 20a However, -H, -C 1 ~C 6 Alkyl, -(C 1 ~C 6 Alkyl)-OR 5 、 TIFF0007877480000023.tif27165 -(C 1 ~C 6 Alkyl)-aryl, TIFF0007877480000024.tif58165 An antibody-drug conjugate of the present invention, selected from 1011 or 1012. [Invention 1014] D is given by equation (VI): TIFF0007877480000025.tif63165 It is a compound of the formula, in which, R 2a However, -H, -CH 3 -CF 3 , -F, -Br, -Cl, -OH, -OCH 3 , and -OCF 3 Selected from, X is -O-, -S-, or -NH-, and R 25 However, -C 1 ~C 6 Alkyl, -(C 1 ~C 6 Alkyl)-OR 5a , -CO 2 R 8a -aryl, -heteroaryl, -(C 1 ~C 6 Alkyl)-aryl, TIFF0007877480000026.tif94165 Selected from, where * is a connection point with X, and p is 1, 2, 3, or 4, or X is O and R 25 -X- is, TIFF0007877480000027.tif37165 Selected from, R 5a However, -C 1 ~C 6 Alkyl, -C 3 ~C 8 Cycloalkyl, -aryl, -heteroaryl, and -(C 1 ~C 6 Selected from alkyl)-aryl, R 6a However, -H, -C 1 ~C 6 Alkyl, -C 3 ~C 8 Cycloalkyl, and -C 3 ~C 8 Selected from heterocycloalkyl groups, R 7a However, -C 1 ~C 6 Alkyl, -C 3 ~C 8 Cycloalkyl, -(C 1 ~C 6 Alkyl)-OR 5a 、-C 3 ~C 8 Heterocycloalkyl and -C(O)R 17a Selected from, R 8a However, -C 1 ~C 6 Alkyl, -C 3 ~C 8 Cycloalkyl, and -C 3 ~C 8 Selected from heterocycloalkyl groups, Each R 9a However, independently, -C 1 ~C 6 Alkyl, -C 3 ~C 8 Cycloalkyl, -aryl, -heteroaryl, and -(C 1 ~C 6 Selected from alkyl)-aryl, or R 9a If X does not exist b =X, Each R 10a However, independently, -C 1 ~C 6 Alkyl, -C 3 ~C 8 Cycloalkyl, -aryl, -heteroaryl, -(C 1 ~C 6 Alkyl)-aryl, and TIFF0007877480000028.tif22165 Selected from, Each R 10a’ However, independently, -H, -C 1 ~C 6 Alkyl, -C 3 ~C 8 Cycloalkyl, -aryl, -heteroaryl, and -(C 1 ~C 6 Selected from alkyl)-aryl, Each R 10b However, independently, -C 1 ~C 6 Alkyl, -C 3 ~C 8 Cycloalkyl, -aryl, -heteroaryl, and -(C 1 ~C 6 Selected from alkyl)-aryl, R 11a However, it does not exist, or -C 1 ~C 6 It is alkyl, R 12a However, -C 1 ~C 6 Alkyl, -CO 2 R 8a -aryl, -heteroaryl, -(C 1 ~C 6 Alkyl)-aryl, -S(O) 2 R 16a , and TIFF0007877480000029.tif22165 Selected from, R 13a However, -H and -C 1 ~C 6 Selected from alkyl groups, R 14a However, -C 1 ~C 6 Alkyl, -C 3 ~C 8 Cycloalkyl, and -C 3 ~C 8 Selected from heterocycloalkyl groups, R 14a’ However, H, -C 1 ~C 6 Alkyl, -C 3 ~C 8 Cycloalkyl, and -C 3 ~C 8 Selected from heterocycloalkyl groups, R 16a However, -C 1 ~C 6 Alkyl, -C 3 ~C 8 Cycloalkyl, -aryl, -heteroaryl, and -(C 1 ~C 6 Selected from alkyl)-aryl, R 17a However, -C 1 ~C 6 Alkyl, -C 3 ~C 8 Cycloalkyl, -C 3 ~C 8 Heterocycloalkyl, -(C 1 ~C 6 Alkyl)-C 3 ~C 8 Heterocycloalkyl, -aryl, -heteroaryl, and -(C 1 ~C 6 Selected from alkyl)-aryl, R 21 However, -C 1 ~C 6 Alkyl, -C 3 ~C 8 Cycloalkyl, and -(C 1 ~C 6 Alkyl)-OR 5a Selected from, R 22 and R 23 However, each is independent of -H, -halogen, and -C. 1 ~C 6 Alkyl and -C 3 ~C 8 Selected from cycloalkyl groups, R 24 、R 25 , and R 26 However, each is -C 1 ~C 6 It is alkyl, X a and X b However, each is independently selected from NH, O, and S. X c However, O, S, and S(O) 2 Selected from, and TIFF0007877480000030.tif17165 However, it indicates the connection point with linker L. An antibody-drug conjugate according to any of invention 1001 to 1005. [Invention 1015] R 2a However, -CH 3 -CF 3 , -F, -Br, -Cl, -OH, -OCH 3 , and -OCF 3 An antibody-drug conjugate of the present invention 1014, selected from the above. [Invention 1016] R 2a However, F is the antibody-drug conjugate of the present invention 1014. [Invention 1017] X is -O-, -S-, or -NH-, and R 25 However, -C 1 ~C 6 Alkyl, -(C 1 ~C 6 Alkyl)-OR 5a 、-(C 1 ~C 6 Alkyl)-aryl, TIFF0007877480000031.tif58165 Either X is selected from or X is O and R 25 -X- is, TIFF0007877480000032.tif37165 Selected from, An antibody-drug conjugate according to any of invention 1014 to 1016. [Invention 1018] X is -O-, -S-, or -NH-, and R 25 However, -C 1 ~C 6 Alkyl, -(C 1 ~C 6 Alkyl)-OR 5a 、-(C 1 ~C 6 Alkyl)-aryl, TIFF0007877480000033.tif58165 An antibody-drug conjugate selected from any of the present invention 1014 to 1016. [Invention 1019] An antibody-drug conjugate according to any of the present invention 1001 to 1018, wherein each alkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl group may be substituted with one or more substituents selected from halogen, acyl, acyloxy, alkoxy, carboxy, hydroxy, amino, amide, nitro, cyano, azide, alkylthio, thio, sulfonyl, sulfonamide, alkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl. [Invention 1020] An antibody-drug conjugate according to any of the present invention 1001 to 1018, wherein each alkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl group may be substituted with one or more substituents selected from halogens, acyls, acyloxys, alkoxys, carboxys, hydroxys, aminos, amides, nitros, cyanos, azides, alkylthios, thiosulfonyls, and sulfonamides. [Invention 1021] An antibody-drug conjugate according to any of invention 1001 to 1005, wherein D has the structure of any one of the compounds listed in Table 6 or Table 7. [Invention 1022] An antibody-drug conjugate according to any of inventions 1001 to 1005, wherein D is compound 139 or compound 141. [Invention 1023] An antibody-drug conjugate according to any of invention 1001 to 1022, wherein L is a cleavable linker. [Invention 1024] The antibody-drug conjugate of the present invention 1023, wherein L is a protease-cleavable linker. [Invention 1025] An antibody-drug conjugate according to the present invention 1023 or 1024, wherein L comprises a dipeptide, tripeptide, or tetrapeptide. [Invention 1026] L, (a) Equation (XI) TIFF0007877480000034.tif32165 It has, in the formula, Z is a functional group that can react with the target group on the anti-FRα antibody construct T, Str stands for stretcher, AA 1 and AA 2 However, each is an amino acid independently, AA 1 -[AA 2 ] r However, it forms a protease cleavage site, X is a self-destructing group, q is either 0 or 1, r is 1, 2, or 3, s is 0, 1, or 2, # is a binding site to the anti-FRα antibody construct T, and % is the binding site with camptothecin analog D, or (b) Equation (XII) TIFF0007877480000035.tif32165 It has, in the formula, Z is a functional group that can react with the target group on the anti-FRα antibody construct T, Str stands for stretcher, AA 1 and AA 2 However, each is an amino acid independently, AA 1 -[AA 2 ] r However, it forms a protease cleavage site, Y is -NH-CH 2 -or -NH-CH 2 -C(O)-, q is either 0 or 1, r is 1, 2, or 3, v is either 0 or 1, # is the binding site to the anti-FRα antibody construct T, and % is the binding site with the camptothecin analog D. An antibody-drug conjugate according to any of invention 1023 to 1025. [Invention 1027] An antibody-drug conjugate according to any of the present invention 1001 to 1005, wherein L-(D) in formula (X) has one of the drug linker (DL) structures described in Tables 8 to 10. [Invention 1028] An antibody-drug conjugate according to any of invention 1001 to 1005, wherein L-(D) in formula (X) has one of the drug linker (DL) structures described in Table 8 or Table 9. [Invention 1029] In equation (X), L-(D) is, MT-GGFG-AM-Compound 139 TIFF0007877480000036.tif63165 MC-GGFG-AM-Compound 139 TIFF0007877480000037.tif68165 MT-GGFG-AM-Compound 141 TIFF0007877480000038.tif58165 MC-GGFG-AM-Compound 141 TIFF0007877480000039.tif63165 MT-GGFG-Compound 141 TIFF0007877480000040.tif53165 or MC-GGFG-Compound 141 TIFF0007877480000041.tif42165 The antibody-drug conjugate according to any of the present invention 1001 to 1005. [Invention 1030] An antibody-drug conjugate according to any of the present invention 1001 to 1029, wherein m is 1 to 2. [Invention 1031] An antibody-drug conjugate according to any of Invention 1001 to 1029, wherein m is 1. [Invention 1032] An antibody-drug conjugate according to any of the present invention 1001 to 1031, wherein n is 2 to 8. [Invention 1033] An antibody-drug conjugate according to any of the present invention 1001 to 1031, wherein n is 4 to 8. [Invention 1034] An antibody-drug conjugate according to any one of the present invention 1001 to 1033, wherein the anti-FRα antibody construct further comprises a scaffold, and the antigen-binding domain is functionally linked to the scaffold. [Invention 1035] The aforementioned scaffold is an antibody-drug conjugate of the present invention 1034, comprising an IgG Fc region. [Invention 1036] TIFF0007877480000042.tif227165TIFF0007877480000043.tif104165 An antibody-drug conjugate having a structure selected from, wherein, T is an anti-FRα antibody construct comprising two antigen-binding domains functionally linked to the IgG Fc region, and each of the antigen-binding domains is (a) The VL amino acid sequence described in SEQ ID NO: 39 and the VH amino acid sequence described in SEQ ID NO: 19, or (b) The VL amino acid sequence described in SEQ ID NO: 124 and the VH amino acid sequence described in SEQ ID NO: 91, or (c) The VL amino acid sequence described in Sequence ID No. 64, and (i) The VH amino acid sequence described in Sequence ID No. 50, or (ii) The VH amino acid sequence described in Sequence ID No. 54, or (iii) The VH amino acid sequence described in Sequence ID No. 57, or (iv) The VH amino acid sequence described in Sequence ID No. 61, or (v) The VH amino acid sequence described in Sequence ID No. 76, or (vi) The VH amino acid sequence described in Sequence ID No. 79, or (vii) The VH amino acid sequence described in Sequence ID No. 82, or (viii) The VH amino acid sequence described in Sequence ID No. 85, or (ix) The VH amino acid sequence described in Sequence ID No. 88, or (x) The VH amino acid sequence described in Sequence ID No. 106, or (d) The VL amino acid sequence described in SEQ ID NO: 130, and (i) The VH amino acid sequence described in Sequence ID No. 99, or (ii) The VH amino acid sequence described in Sequence ID No. 106, or (iii) The VH amino acid sequence described in Sequence ID No. 113, or (iv) The VH amino acid sequence described in Sequence ID No. 116, or (v) The VH amino acid sequence described in Sequence ID No. 133, or (vi) The VH amino acid sequence described in SEQ ID NO. 136, or (e) The VL amino acid sequence described in Sequence ID No. 119, and (i) The VH amino acid sequence described in Sequence ID No. 106, or (ii) VH amino acid sequence described in Sequence ID No. 116 including and n is between 4 and 8. The aforementioned antibody-drug conjugate. [Invention 1037] A pharmaceutical composition comprising an antibody-drug conjugate according to any of invention 1001 to 1036 and a pharmaceutically acceptable carrier or diluent. [Invention 1038] A method for suppressing the proliferation of cancer cells, comprising contacting the cells with an effective amount of an antibody-drug conjugate according to any of the present invention 1001 to 1036. [Invention 1039] A method for killing cancer cells, comprising contacting the cells with an effective amount of an antibody-drug conjugate according to any of the present invention 1001 to 1036. [Invention 1040] A method for treating cancer in a subject in need, comprising administering to the subject an effective amount of an antibody-drug conjugate according to any of the present invention 1001 to 1036. [Invention 1041] The method of the present invention 1040, wherein the cancer is breast cancer, ovarian cancer, colorectal cancer, non-small cell lung cancer (NSCLC), pancreatic cancer, or endometrial cancer. [Invention 1042] An antibody-drug conjugate according to any of Invention 1001-1036 for use in therapy. [Invention 1043] An antibody-drug conjugate according to any of Invention 1001-1036 for use in the treatment of cancer. [Invention 1044] An antibody-drug conjugate for use of the present invention 1043, wherein the cancer is breast cancer, ovarian cancer, colorectal cancer, non-small cell lung cancer (NSCLC), pancreatic cancer, or endometrial cancer. [Invention 1045] Use of any antibody-drug conjugate according to invention 1001 to 1036 in the manufacture of a drug for the treatment of cancer. [Invention 1046] Use of Invention 1045, wherein the cancer is breast cancer, ovarian cancer, colorectal cancer, non-small cell lung cancer (NSCLC), pancreatic cancer, or endometrial cancer. [Brief explanation of the drawing]

[0017] [Figure 1] A and B show the sequences of the rabbit heavy chain variable domain CDR of the chimeric anti-FRα antibody v23924 ported to the human VH framework (IGHV3-23*01) (SEQ ID NO: 155) (A) and the rabbit light chain variable domain CDR of the chimeric antibody v23924 ported to the human VL framework (IGKVI-39*01) (SEQ ID NO: 156) (B). The CDRs are assigned according to the AbM definition and are marked in bold italics. [Figure 2A] The profiles of purified parental chimeric anti-FRα antibody v23924 and purified representative humanized variant v30384, analyzed by electrophoresis and ULC-SEC, are shown. A and C show the profiles (C) from electrophoresis under non-reducing (NR) and reducing (R) conditions after preparative SEC purification (after preparative SEC) or protein A purification (after pA) of parental chimeric anti-FRα antibody v23924 (A) and purified representative humanized variant v30384. B and D show the ULC-SEC profiles (D) of parental chimeric anti-FRα antibody v23924 after preparative SEC purification (B) and purified representative humanized variant v30384 after protein A purification. [Figure 2B] Please refer to the explanation in Figure 2A. [Figure 2C] Please refer to the explanation in Figure 2A. [Figure 2D] Please refer to the explanation in Figure 2A. [Figure 3] A and B show biolayer interferometry (BLI) sensograms of the parental chimeric anti-FRα antibody v23924 (A) and the purified representative humanized variant v30384 (B). [Figure 4A]The intact LC / MS profiles of representative humanized variants v30384 (with enlarged views of the main peaks in A and B) and v31422 (with enlarged views of the main peaks in C and D) are shown. [Figure 4B] Please refer to the explanation in Figure 4A. [Figure 4C] Please refer to the explanation in Figure 4A. [Figure 4D] Please refer to the explanation in Figure 4A. [Figure 5] A and B show the receptor-mediated internalization capabilities of the chimeric anti-FRα antibody v23924, a representative humanized variant, v30384, and various concentrations of FRα-targeting antibodies milbetuximab and faretuzumab in the FRα-expressing cell line IGROV-1, as determined by flow cytometry after 6-hour incubation (A) and 24-hour incubation (B). Palivizumab, an anti-RSV antibody, was included as a negative control. [Figure 6] A and B show the receptor-mediated internalization capabilities of the chimeric anti-FRα antibody v23924, a representative humanized variant, v30384, and various concentrations of FRα-targeting antibodies mirbetuximab and faretuzumab in the FRα-expressing cell line OVCAR-3, as determined by flow cytometry after 6-hour incubation (A) and 24-hour incubation (B). Palivizumab, an anti-RSV antibody, was included as a negative control. [Figure 7] This shows the coverage of the hFRα sequence (SEQ ID NO: 15) by peptides produced by pepsin digestion of hFRα. Each bar below the sequence represents a peptide. [Figure 8A] Summary plot (8A) of hydrogen / deuterium exchange mass spectrometry (HDX-MS) kinetics of the peptide produced by pepsin digestion of the hFRα:hFOLR1(hFRα) vs. hFOLR1-v23924 complex is shown. [Figure 8B] The difference plot (8B) of hydrogen / deuterium exchange mass spectrometry (HDX-MS) kinetics of the peptide produced by pepsin digestion of the hFRα:hFOLR1(hFRα) versus hFOLR1-v23924 complex is shown. [Figure 9] A-C show the amide deuteration levels of peptide 119-126 (WEDCRTSY) (SEQ ID NO: 152) after 1 hour of hydrogen / deuterium exchange mass spectrometry (HDX-MS): hFOLR1 (A) vs. hFOLR1-v23924 complex (B), and a difference plot (C). [Figure 10] A and B demonstrate the receptor-mediated internalization capabilities of the parental anti-FRα humanized antibody variant, v30384, and the representative affinity-matured variant, v35356, in FRα-expressing cell lines IGROV-1(A) and JEG-3(B), as determined by flow cytometry after 5-hour and 24-hour incubation periods. Palivizumab was included as a non-targeted control. [Figure 11-1] A table is presented showing the CDR sequences of representative anti-FRα antibodies as defined by IMGT, Chothia, Kabat, Contact, and AbM. [Figure 11-2] Please refer to the explanation in Figure 11-1. [Figure 11-3] Please refer to the explanation in Figure 11-1. [Figure 11-4] Please refer to the explanation in Figure 11-1. [Figure 11-5] Please refer to the explanation in Figure 11-1. [Figure 11-6] Please refer to the explanation in Figure 11-1. [Figure 11-7] Please refer to the explanation in Figure 11-1. [Figure 12-1] A table showing the VH and VL sequences of representative anti-FRα antibodies is provided. [Figure 12-2] Please refer to the explanation in Figure 12-1. [Figure 12-3] Please refer to the explanation in Figure 12-1. [Figure 13-1] Exemplary drug-linker (DL) structures, including a camptothecin analog of formula (I) having a C7 linkage, are shown (Table 8). [Figure 13-2] Please refer to the explanation in Figure 13-1. [Figure 13-3] Please refer to the explanation in Figure 13-1. [Figure 14-1]Exemplary drug-linker (DL) structures, including a camptothecin analog of formula (I) having a C10 bond, are shown (Table 9). [Figure 14-2] Please refer to the explanation in Figure 14-1. [Figure 15-1] Exemplary drug-linker (DL) structures, including camptothecin analogs of formula (I) having a C7 or C10 bond, are shown (Table 10). [Figure 15-2] Please refer to the explanation in Figure 15-1. [Figure 15-3] Please refer to the explanation in Figure 15-1. [Figure 16-1] Exemplary conjugate (DC) structures, including the camptothecin analog of formula (I) having a C7 bond, are shown (Table 11). [Figure 16-2] Please refer to the explanation in Figure 16-1. [Figure 16-3] Please refer to the explanation in Figure 16-1. [Figure 16-4] Please refer to the explanation in Figure 16-1. [Figure 17-1] Exemplary conjugate (DC) structures, including a camptothecin analog of formula (I) having a C10 bond, are shown (Table 12). [Figure 17-2] Please refer to the explanation in Figure 17-1. [Figure 17-3] Please refer to the explanation in Figure 17-1. [Figure 18-1] Exemplary conjugate (DC) structures, including camptothecin analogs of formula (I) having a C7 or C10 bond, are shown (Table 13). [Figure 18-2] Please refer to the explanation in Figure 18-1. [Figure 18-3] Please refer to the explanation in Figure 18-1. [Figure 18-4] Please refer to the explanation in Figure 18-1. [Figure 19A]In the OV90 xenograft model, ADCs containing the anti-FRα humanized antibody variant v30384, conjugated with camptothecin analog compound 139 or compound 141 at DAR8 (A and B), and in the H2110 xenograft model, conjugated with camptothecin analog compound 139, compound 140, compound 141, or compound 148 at DAR8 (C), demonstrate in vivo antitumor activity. ADCs containing palivizumab (v21995) were included as a control. [Figure 19B] Please refer to the explanation in Figure 19A. [Figure 19C] Please refer to the explanation in Figure 19A. [Figure 20] This report describes the pharmacokinetics of four ADCs, including the anti-FRα humanized antibody v36675 and v36675 conjugated to DXd, of camptothecin analog compounds 139 or 141 in DAR8, as evaluated in hFcRn Tg32 mice (n=4). Mean serum concentrations at several time points were calculated using n<4 animals (as some samples were below the detection limit). [Figure 21] This shows the in vivo stability of four ADCs, including the humanized variant v36675 conjugated to DXd or camptothecin analog compound 139 or compound 141, in DAR8 of Tg32 mouse serum. The solid line represents % DAR residue (left axis), and the dotted line represents % maleimide ring opening (right axis). [Figure 22A]The in vivo antitumor activity of ADCs containing the anti-FRα humanized antibody variant v36675 is demonstrated in the OV90 CDX xenograft model, conjugated to camptothecin analog compound 139 or compound 141 at DAR4 or DAR8, respectively (A, B); in the OVCAR3 CDX xenograft model, conjugated to camptothecin analog compound 140 or compound 141 at DAR4 or DAR8, respectively (C); in the GTG-2025 PDX xenograft model, conjugated to camptothecin analog compound 139 at DAR8 (D); and in the GTG-0958 PDX xenograft model, conjugated to camptothecin analog compound 139 at DAR8 (E). [Figure 22B] Please refer to the explanation in Figure 22A. [Figure 22C] Please refer to the explanation in Figure 22A. [Figure 22D] Please refer to the explanation in Figure 22A. [Figure 22E] Please refer to the explanation in Figure 22A. [Figure 23A] In cynomolgus monkey toxicity studies, the total serum antibody concentrations of ADCs containing the anti-FRα humanized antibody v36675 in blood samples collected after the first dose are shown. ADC (A) containing v36675 conjugated to compound 139 or compound 141 at DAR8, administered at 30 mg / kg; ADC (B) containing v36675 conjugated to compound 139 or compound 141 at DAR4, administered at 60 mg / kg; ADC (C) containing v36675 conjugated to compound 139 or compound 141 at DAR8, administered at 80 mg / kg; and ADC (D) containing v36675 conjugated to compound 139 or compound 141 at DAR4 or DAR8, administered at 120 mg / kg. [Figure 23B] Please refer to the explanation in Figure 23A. [Figure 23C] Please refer to the explanation in Figure 23A. [Figure 23D] Please refer to the explanation in Figure 23A. [Figure 24]This study demonstrates the in vitro bystander activity of ADCs containing the anti-FRα humanized antibody variant v30384 conjugated to various camptothecin analogs against the FRα-negative MDA-MB-468 cell line. ADCs v30384-MC-GGFG-AM-DXd1 and v30384-MCvcPABC-MMAE were included as positive controls, while ADCs containing palivizumab (v22277) conjugated to MC-GGFG-AM-DXd1 and MCvcPABC-MMAE were included as negative controls. [Figure 25] A-D show the penetration of the anti-FRα humanized antibody variant v36675 into JEG-3 cell spheroids at 4 hours (25A), 24 hours (25B), 48 hours (25C), and 96 hours (25D), compared to milbetuximab and the negative control palivizumab. [Figure 26] A and B show intracellular (26A) and extracellular (26B) payload release from ADCs containing the anti-FRα humanized antibody variant v36675 (v36675-MC-GGFG-AM-compound 139) conjugated to compound 139 at DAR8, and from ADCs containing the non-targeted control palivizumab (v21995) conjugated to compound 139 at DAR8 in the high-FRα-expressing cell line IGROV-1. [Figure 27A] This shows the in vivo antitumor activity of the anti-FRα humanized antibody variant v36675 (v36675-MC-GGFG-AM-compound139), conjugated to compound 139 at DAR8, and the control ADC, milbetuximab-DM4 DAR4, in a patient-derived xenograft (PDX) model of ovarian cancer when administered at 6 mg / kg. CTG-0703 PDX model (A). [Figure 27B] This shows the in vivo antitumor activity of the anti-FRα humanized antibody variant v36675 (v36675-MC-GGFG-AM-compound139), conjugated to compound 139 at DAR8, and the control ADC, milbetuximab-DM4 DAR4, in a patient-derived xenograft (PDX) model of ovarian cancer when administered at 6 mg / kg. CTG-1301 PDX model (B). [Figure 27C]This shows the in vivo antitumor activity of the anti-FRα humanized antibody variant v36675 (v36675-MC-GGFG-AM-compound139), conjugated to compound 139 at DAR8, and the control ADC, milbetuximab-DM4 DAR4, in a patient-derived xenograft (PDX) model of ovarian cancer when administered at 6 mg / kg. CTG-2025 PDX model (C). [Figure 27D] This shows the in vivo antitumor activity of the anti-FRα humanized antibody variant v36675 (v36675-MC-GGFG-AM-compound139), conjugated to compound 139 at DAR8, and the control ADC, milbetuximab-DM4 DAR4, in a patient-derived xenograft (PDX) model of ovarian cancer when administered at 6 mg / kg. CTG-3383 PDX model (D). [Figure 27E] This shows the in vivo antitumor activity of the anti-FRα humanized antibody variant v36675 (v36675-MC-GGFG-AM-compound139), conjugated to compound 139 at DAR8, and the control ADC, milbetuximab-DM4 DAR4, in a patient-derived xenograft (PDX) model of ovarian cancer when administered at 6 mg / kg. CTG-0947 PDX model (E). [Figure 27F] This shows the in vivo antitumor activity of the anti-FRα humanized antibody variant v36675 (v36675-MC-GGFG-AM-compound139), conjugated to compound 139 at DAR8, and the control ADC, milbetuximab-DM4 DAR4, in a patient-derived xenograft (PDX) model of ovarian cancer at a dose of 6 mg / kg. CTG-0958 PDX model (F). [Figure 27G] This shows the in vivo antitumor activity of the anti-FRα humanized antibody variant v36675 (v36675-MC-GGFG-AM-compound139), conjugated to compound 139 at DAR8, and the control ADC, milbetuximab-DM4 DAR4, in a patient-derived xenograft (PDX) model of ovarian cancer when administered at 6 mg / kg. CTG-3718 PDX model (G). [Figure 27H]This shows the in vivo antitumor activity of the anti-FRα humanized antibody variant v36675 (v36675-MC-GGFG-AM-compound139), conjugated to compound 139 at DAR8, and the control ADC, milbetuximab-DM4 DAR4, in a patient-derived xenograft (PDX) model of ovarian cancer at a dose of 6 mg / kg. CTG-1703 PDX model (H). [Figure 27I] This shows the in vivo antitumor activity of the anti-FRα humanized antibody variant v36675 (v36675-MC-GGFG-AM-compound139), conjugated to compound 139 at DAR8, and the control ADC, milbetuximab-DM4 DAR4, in a patient-derived xenograft (PDX) model of ovarian cancer at a dose of 6 mg / kg. CTG-1602 PDX model (I). [Figure 28A] This image shows fixed cell confirmation screening images from screening for specific off-target binding interactions using Retrogenix Cell Microarray Technology for the 20 μg / mL anti-FRα humanized antibody variant v36675(A). [Figure 28B] This image shows a fixed-cell confirmation screening image from screening for specific off-target binding interactions using Retrogenix Cell Microarray Technology for a 1 μg / mL control antibody (rituximab biosimilar) (B). [Figure 29] This study demonstrates competitive binding between the chimeric anti-FRα antibody v23294 and the anti-FRα antibodies mirbetuximab and faretuzumab, as evaluated in H2110 cells. [Modes for carrying out the invention]

[0018] Detailed explanation This disclosure relates to antibody-drug conjugates (ADCs) comprising an antibody construct (anti-FRα antibody construct) that specifically binds to human folate receptor alpha (FRα), conjugated to a camptothecin analog of formula (I) described herein. In particular, this disclosure relates to formula (X): T-[L-(D) m ] n (X) Regarding an ADC having, in the formula, T is an anti-FRα antibody construct as described herein, L is a linker, D is a camptothecin analog of formula (I) as described herein, m is 1 to 4, and n is between 1 and 10.

[0019] The ADCs of this disclosure may find use as therapeutic agents, for example, particularly in the treatment of cancer.

[0020] definition Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art.

[0021] As used herein, the term "approximately" refers to a variation of approximately ±10% from a given value. It should be understood that such variation is always included in any given value provided herein, whether specifically mentioned or not.

[0022] The use of the words "a" or "an," as used herein in conjunction with the term "including," may mean "one," but also coincides with the meanings of "one or more," "at least one," and "one or more than one."

[0023] As used herein, the terms “comprising,” “having,” “including,” and “containing,” as well as their grammatical variations, are comprehensive, or open-ended, and do not exclude additional elements and / or steps of method that are not enumerated. The term “essentially consisting of,” as used herein in relation to a composition, use, or method, indicates that additional elements and / or steps of method may exist, but these additions do not substantially affect the manner in which the enumerated composition, method, or use functions. The term “consisting of,” as used herein in relation to a composition, use, or method, excludes the existence of additional elements and / or steps of method. A composition, use, or method described herein as containing certain elements and / or steps may also, in certain embodiments, essentially consist of those elements and / or steps, and in other embodiments, whether or not these embodiments are specifically referenced.

[0024] A “complementarity-determining region” or “CDR” is an amino acid sequence that contributes to antigen-binding specificity and affinity. A “framework” region (FR) can help maintain the proper conformation of the CDR, thereby facilitating binding between the antigen-binding region and the antigen. From the N-terminus to the C-terminus, both the light chain variable region (VL) and heavy chain variable region (VH) of an antibody typically contain domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The three heavy chain CDRs are referred to herein as HCDR1, HCDR2, and HCDR3, and the three light chain CDRs are referred to as LCDR1, LCDR2, and LCDR3. CDRs provide the majority of the contact residues necessary for the antibody to bind to an antigen or epitope. Often, three heavy chain CDRs and three light chain CDRs are required for binding to the antigen. However, in some cases, a single variable domain alone can confer binding specificity to the antigen. Furthermore, as is known in the art, in some cases antigen binding may also occur by a combination of at least one CDR selected from the VH and / or VL domains, such as HCDR3.

[0025] Several different definitions of CDR sequences are commonly used, including those described by Kabat et al. (1983, Sequences of Proteins of Immunological Interest, NIH Publication No. 369-847, Bethesda, MD), Chothia et al. (1987, J Mol Biol, 196:901-917), and those described by IMGT, AbM (University of Bath), and Contact (MacCallum, et al., 1996, J Mol Biol, 262(5):732-745). For example, definitions of CDRs according to Kabat, Chothia, IMGT, AbM, and Contact are listed in Table 1 below. Therefore, as will be readily apparent to those skilled in the art, the exact numbering and arrangement of CDRs may vary depending on the numbering system used. However, it should be understood that the VH disclosures herein include disclosures of relevant (inherent) heavy chain CDRs (HCDRs) as defined by any of the known numbering systems. Similarly, the disclosure of VL herein includes the disclosure of the relevant (unique) light chain CDR (LCDR) as defined by one of the known numbering systems.

[0026] [Table 1]

[0027] In the context of two or more polynucleotide or polypeptide sequences, the term “identical” refers to two or more sequences or subsequences that are the same. Sequences are “substantially identical” if, when compared and aligned to achieve the greatest match across a comparison window or a specified region, using one of the commonly used sequence comparison algorithms known to those skilled in the art, or as measured by manual alignment and visual inspection, they have a percentage of identical amino acid residues or nucleotides (e.g., about 80%, 85%, 90%, 95%, or 98% identity across a specified region). For sequence comparison, typically a specified reference sequence and a test sequence are compared. When using a sequence comparison algorithm, the test sequence and reference sequence are entered into a computer, and subsequence coordinates are specified if necessary, along with the parameters of the sequence algorithm program. Default program parameters may be used, or alternative parameters may be specified. The sequence comparison algorithm then calculates the percentage sequence identity of the test sequence compared to the reference sequence based on the program parameters.

[0028] The "comparison window" refers to a segment of a sequence containing consecutive amino acid or nucleotide positions, which may be, for example, about 10 to 600 consecutive amino acid or nucleotide positions, or about 10 to 200 consecutive amino acid or nucleotide positions, or about 10 to 150 consecutive amino acid or nucleotide positions, and the test sequence can be compared to a reference sequence with the same number of consecutive positions after the two sequences have been optimally aligned. Methods for aligning sequences for comparison are known to those skilled in the art. Optimal sequence alignment for comparison can be performed, for example, by the local homology algorithm of Smith & Waterman, 1970, Adv. Appl. Math., 2:482c; by the homology alignment algorithm of Needleman & Wunsch, 1970, J. Mol. Biol., 48:443; by the similarity search method of Pearson & Lipman, 1988, Proc. Natl. Acad. Sci. USA, 85:2444; by computer implementations of these algorithms (e.g., GAP, BESTFIT, FASTA, or TFASTA from Wisconsin Genetics Software Package, Genetics Computer Group, Madison, WI); or by manual alignment and visual inspection (see, for example, Ausubel et al., Current Protocols in Molecular Biology, (1995 supplement), Cold Spring Harbor Laboratory Press). Suitable available algorithms for determining percent sequence identity include the BLAST and BLAST2.0 algorithms, described in Altschul et al., 1997, Nuc. Acids Res., 25:3389-3402, and Altschul et al., 1990, J. Mol. Biol., 215:403-410, respectively. Software for performing BLAST analysis is publicly available from the National Center for Biotechnology Information (NCBI) website.

[0029] The term "acyl," as used herein, refers to the group -C(O)R, where R is hydrogen, alkyl, aryl, heteroaryl, cycloalkyl, or heterocycloalkyl.

[0030] The term "acyloxy" refers to the group -OC(O)R, where R is alkyl.

[0031] The term "alkoxy," as used herein, refers to the group -OR, where R is alkyl, aryl, heteroaryl, cycloalkyl, or cycloheteroalkyl.

[0032] As used herein, the term "alkyl" refers to a linear or branched saturated hydrocarbon group containing a specific number of carbon atoms. Examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, t-butyl, pentyl, isopentyl, t-pentyl, neopentyl, 1-methylbutyl, 2-methylbutyl, and n-hexyl.

[0033] When used herein, the term "alkylaminoaryl" refers to an alkyl group as defined herein, substituted with one aminoaryl group as defined herein.

[0034] When used herein, the term "alkyl heterocycloalkyl" refers to an alkyl group as defined herein, substituted with one heterocycloalkyl group as defined herein.

[0035] As used herein, the term "alkylthio" refers to the group -SR, where R is an alkyl group.

[0036] As used herein, the term "amide" refers to the group -C(O)NRR', where R and R' are independently hydrogen, alkyl, aryl, heteroaryl, cycloalkyl, or heterocycloalkyl.

[0037] The term "amino," as used herein, refers to the group -NRR', where R and R' are independently hydrogen, alkyl, aryl, heteroaryl, cycloalkyl, or heterocycloalkyl.

[0038] When used herein, the term "aminoalkyl" refers to an alkyl group as defined herein, substituted with one or more amino groups, for example, one, two, or three amino groups.

[0039] When used herein, the term "aminoaryl" refers to an aryl group substituted with one amino group, as defined herein.

[0040] As used herein, the term "aryl" refers to a 6- to 12-membered monocyclic or bicyclic hydrocarbon ring system in which at least one ring is aromatic. Examples of aryls include, but are not limited to, phenyl, naphthalenyl, 1,2,3,4-tetrahydro-naphthalenyl, 5,6,7,8-tetrahydro-naphthalenyl, and indanyl.

[0041] The term "carboxy" as used herein refers to the group -C(O)OR, where R is H, alkyl, aryl, heteroaryl, cycloalkyl, or cycloheteroalkyl.

[0042] As used herein, the term "cyano" refers to the group -CN.

[0043] As used herein, the term "cycloalkyl" refers to a monocyclic or bicyclic saturated hydrocarbon containing a specific number of carbon atoms. Examples of cycloalkyls include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptane, bicyclo[2.2.1]heptane, and bicyclo[3.1.1]heptane.

[0044] When used herein, the term "haloalkyl" refers to an alkyl group as defined herein, which is substituted with one or more halogen atoms.

[0045] As used herein, the terms "halogen" and "halo" refer to fluorine (F), bromine (Br), chlorine (Cl), and iodine (I).

[0046] The term "heteroaryl," as used herein, refers to a 6- to 12-membered monocyclic or bicyclic ring system in which at least one ring atom is a heteroatom and at least one ring is aromatic. Examples of heteroatoms include, but are not limited to, O, S, and N. Examples of heteroaryls include, but are not limited to, pyridyl, benzofuranyl, pyrazinyl, pyridadinyl, pyrimidinyl, triazinyl, quinolinyl, benzoxazolyl, benzothiazolyl, isoquinolinyl, quinazolinyl, quinoxalinyl, pyrrolyl, and indolyl.

[0047] The term "heterocycloalkyl," as used herein, refers to a monocyclic or bicyclic non-aromatic ring system containing a specific number of atoms, where at least one ring atom is a heteroatom, e.g., O, S, or N. Heterocyclyl substituents can be bonded via any of their available ring atoms, e.g., ring carbon or ring nitrogen. Examples of heterocycloalkyls include, but are not limited to, azilidinyl, azetidinyl, piperidinyl, morpholinyl, piperazinyl, and pyrrolidinyl.

[0048] The terms "hydroxy" and "hydroxyl" as used herein refer to the group -OH.

[0049] The term "hydroxyalkyl," as used herein, refers to an alkyl group as defined herein, substituted with one or more hydroxyl groups.

[0050] As used herein, the term "nitro" refers to the group -NO2.

[0051] As used herein, the term "sulfonyl" refers to the group -S(O)2R, where R is H, alkyl, or aryl.

[0052] As used herein, the term "sulfonamide" refers to the group -NH-S(O)2R, where R is H, alkyl, or aryl.

[0053] The terms "thio" and "thiol" as used herein refer to the group -SH.

[0054] Unless otherwise specifically stated, any alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl group referred to herein is understood to be "may be substituted," i.e., each such reference includes both unsubstituted and substituted forms of these groups. For example, a reference to "-C1~C6 alkyl" includes both unsubstituted -C1~C6 alkyls and C1~C6 alkyls substituted with one or more substituents. Examples of substituents include, but are not limited to, halogens, acyls, acyloxys, alkoxys, carboxys, hydroxys, aminos, amides, nitros, cyanos, azides, alkylthios, thios, sulfonyls, sulfonamides, alkyls, cycloalkyls, heterocycloalkyls, aryls, or heteroaryls. In certain embodiments, each alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl group referred to herein may optionally be substituted with one or more substituents selected from halogens, acyls, acyloxys, alkoxys, carboxys, hydroxys, aminos, amides, nitros, cyanos, azides, alkylthios, thios, sulfonyls, and sulfonamides.

[0055] The “substituted” chemical groups described herein may include one substituent or multiple substituents up to the maximum valence of the substitution of the group. For example, a methyl group may include one, two, or three substituents, and a phenyl group may include one, two, three, four, or five substituents. When a group is substituted with two or more substituents, the substituents may be the same or different.

[0056] Where used herein, the term “subject” refers to an animal, in some embodiments a mammal, that is the subject of treatment, observation, or experimentation. The animal may be a human, a non-human primate, a pet (e.g., a dog, a cat, etc.), a livestock (e.g., a cattle, a sheep, a pig, a horse, etc.), or a laboratory animal (e.g., a rat, a mouse, a guinea pig, a non-human primate, etc.). In certain embodiments, the subject is a human.

[0057] Any embodiment discussed herein is intended to be implementable with respect to any method, use, or composition disclosed herein, and vice versa.

[0058] Certain features, structures, and / or properties described in conjunction with embodiments disclosed herein can be combined in any preferred manner with features, structures, and / or properties described in conjunction with other embodiments disclosed herein to provide one or more further embodiments.

[0059] It should be understood that the explicit enumeration of features in one embodiment may serve as a basis for excluding features in alternative embodiments. For example, if a list of options is presented for a given embodiment or claim, it should be understood that one or more options may be removed from the list, and the shortened list may form alternative embodiments, whether or not such alternative embodiments are specifically referred to.

[0060] Anti-FRα antibody construct The ADCs of this disclosure include anti-FRα antibody constructs. In this context, the term “antibody construct” means a polypeptide or set of polypeptides comprising one or more antigen-binding domains, each of which binds specifically to an epitope or antigen. If an antibody construct comprises two or more antigen-binding domains, each of the antigen-binding domains may bind to the same epitope or antigen (i.e., the antibody construct is monospecific), or they may bind to different epitopes or antigens (i.e., the antibody construct is bispecific or multispecific). The antibody construct may further comprise a scaffold, and one or more antigen-binding domains may optionally be fused or covalently bound to the scaffold via a linker, as described herein.

[0061] According to this disclosure, an anti-FRα antibody construct comprises at least one antigen-binding domain that specifically binds to human folate receptor (hFRα). “Specifically binding” to hFRα means that the antibody construct binds to hFRα but does not show significant binding to any of the human folate receptors beta (FOLR2), gamma (FOLR3), or delta (FOLR4). In certain embodiments, the anti-FRα antibody construct of this disclosure may bind to one or more non-human species of FRα. In certain embodiments, the anti-FRα antibody construct of this disclosure may bind to cynomolgus monkey FRα.

[0062] Human FRα is also known as "human folate receptor 1" or "FOLR1". Protein sequences of hFRα from various sources are known in the art and readily available from publicly accessible databases such as GenBank or UniProtKB. Examples of hFRα sequences include those provided under NCBI reference numbers P15328, AAX29268.1, AAX37119.1, NP_057937.1, and NP_057936.1. An exemplary hFRα protein sequence is provided in Table 2 as SEQ ID NO: 1 (NCBI reference sequence: NP_057936.1). An exemplary cynomolgus monkey FRα protein sequence is also provided in Table 2 (SEQ ID NO: 2; NCBI reference sequence: XP_005579002.2).

[0063] [Table 2]

[0064] The specific binding of the antigen-binding domain to the target antigen or epitope can be measured by, for example, enzyme-linked immunosorbent assay (ELISA), surface plasmon resonance (SPR) technology (e.g., using the BIAcore instrument) (Liljeblad et al., 2000, Glyco J, 17:323-329), flow cytometry, or conventional binding assays (Heeley, 2002, Endocr Res, 28:217-229). In certain embodiments, specific binding may be defined, for example, by ELISA or flow cytometry, as binding to non-target proteins (such as FOLR2, FOLR3, or FOLR4) being less than approximately 10% of binding to hFRα. In certain embodiments, specific binding of an antibody construct to FRα may be defined as a dissociation constant (K) of ≤1 μM, e.g., ≤500 nM, ≤250 nM, ≤100 nM, ≤50 nM, or ≤10 nM. D ) may be defined by. In certain embodiments, the specific binding of an antibody construct to a particular antigen or epitope is 10 -6 M or less, for example, 10 -7 M or less, or 10 -8 Dissociation constants less than or equal to M (K D ) may be defined by. In some embodiments, the specific binding of an antibody construct to a particular antigen or epitope is 10 -6 M~10 -9 M, for example, 10 -7 M~10 -9 The dissociation constant of M (K D ) can be defined by:

[0065] In certain embodiments, the anti-FRα antibody constructs of this disclosure exhibit higher internalization into FRα-expressing cells than the reference antibodies mirbetuximab (huMov19 or huFR107) and faretuzumab (MORAb-003).

[0066] Antibody internalization can be measured using methods known in the art, for example, by direct internalization following the protocol detailed in Schmidt, M. et al., 2008, Cancer Immunol. Immunother., 57:1879-1890, or by using commercially available fluorescent dyes such as pHAb dyes (Promega Corporation, Madison, WI), pHrodo iFL and Deep Red Dye (ThermoFisher Scientific Corporation, Waltham, MA), and Incucyte® Fabfluor-pH antibody labeling reagent (Sartorius AG, Gottingen, Germany), as well as analytical techniques such as microscopy, FACS, high-concentration imaging, or other plate-based assays.

[0067] In certain embodiments, if the amount of anti-FRα antibody construct internalized into FRα-expressing cells is at least 1.2 times greater than the amount of reference antibody internalized into the same FRα-expressing cells under the same test conditions, the anti-FRα antibody construct is considered to exhibit higher internalization into FRα-expressing cells than the corresponding reference antibody (milbetuximab or faretuzumab). In certain embodiments, the amount of internalized antibody is determined using appropriate fluorescent dyes and high-content imaging. In some embodiments, the amount of internalized antibody is determined in cells expressing high levels of FRα. In some embodiments, the amount of internalized antibody is determined in IGROV-1 cells or cells expressing FRα at similar levels to IGROV-1 cells. In some embodiments, the amount of internalized antibody is determined after a 6-hour incubation period. In some embodiments, the amount of internalized antibody is determined after a 24-hour incubation period.

[0068] In certain embodiments, an anti-FRα antibody construct is considered to exhibit higher internalization to FRα-expressing cells than the corresponding reference antibody (milbetuximab or faretuzumab) if the amount of anti-FRα antibody construct internalized in FRα-expressing cells is at least 1.3 times, at least 1.4 times, at least 1.5 times, 1.6 times, 1.7 times, 1.8 times, 1.9 times, or 2.0 times greater than the amount of reference antibody internalized in the same FRα-expressing cells under the same test conditions. In certain embodiments, the amount of internalized antibody is determined using appropriate fluorescent dyes and high-content imaging. In some embodiments, the amount of internalized antibody is determined in cells expressing FRα at high levels. In some embodiments, the amount of internalized antibody is determined in IGROV-1 cells or cells expressing FRα at similar levels to IGROV-1 cells. In some embodiments, the amount of internalized antibody is determined after a 6-hour incubation period. In some embodiments, the amount of endogenous antibody is determined after a 24-hour incubation period.

[0069] antigen-binding domain The anti-FRα antibody constructs of this disclosure include at least one antigen-binding domain capable of binding to hFRα. The at least one antigen-binding domain capable of binding to hFRα is typically an immunoglobulin-based binding domain, such as an antigen-binding antibody fragment. Examples of antigen-binding antibody fragments include, but are not limited to, Fab fragments, Fab' fragments, single-chain Fab (scFab), single-chain Fv (scFv), and single-domain antibodies (sdAb).

[0070] A "Fab fragment" includes the constant domain (CL) of the light chain and the first constant domain (CH1) of the heavy chain, along with the variable domains (VL and VH, respectively) of the light and heavy chains. A Fab' fragment differs from a Fab fragment in that it has several amino acid residues added to the C-terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. A Fab fragment may also be a single-chain Fab molecule, i.e., a Fab molecule in which the Fab light chain and Fab heavy chain are linked by a peptide linker to form a single peptide chain. For example, the C-terminus of the Fab light chain may be attached to the N-terminus of the Fab heavy chain in a single-chain Fab molecule.

[0071] An "scFv" contains the heavy chain variable domain (VH) and light chain variable domain (VL) of an antibody within a single polypeptide chain. Optionally, an scFv may further contain a polypeptide linker between the VH and VL domains, allowing the scFv to form a desired structure for antigen binding. For example, an scFv may contain a VL connected from its C-terminus to the N-terminus of the VH by a polypeptide linker. Alternatively, an scFv may contain a VH connected via its C-terminus to the N-terminus of the VL by a polypeptide linker (see review article Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994)).

[0072] The "sdAb" format refers to a single immunoglobulin domain. An sdAb may, for example, originate from a camelid antibody. Camelid antibodies lack a light chain, and their antigen-binding site consists of a single domain called "VHH". An sdAb contains three CDR / hypervariable loops that form the antigen-binding site: CDR1, CDR2, and CDR3. sdAbs are fairly stable and readily expressed, for example, as a fusion with the antibody's Fc chain (see, e.g., Harmsen & De Haard, 2007, Appl. Microbiol Biotechnol., 77(1):13-22).

[0073] In embodiments in which the anti-FRα antibody construct comprises two or more antigen-binding domains, each additional antigen-binding domain may independently be an immunoglobulin-based domain, e.g., an antigen-binding antibody fragment, or a non-immunoglobulin-based domain, e.g., a non-immunoglobulin-based antibody mimetic, or another polypeptide or small molecule capable of specifically binding to its target, e.g., a natural or engineered ligand. Non-immunoglobulin-based antibody mimetic formats include, for example, antikalin, finomer, affimer, alpha body, DARPin, and avimer.

[0074] This disclosure describes, in this specification, the identification of an antibody that specifically binds to hFRα (variant v23924), as well as representative humanized versions of this antibody (variants v30384, v30389, v30394, v30399, v31422, v31423, v31424, v31425, and v31426) and representative affinity-matured versions of this antibody (variants v35305, v35342, v35347, v35348, v35350, v35354, v35356, v35358, v36167, v36168, and v36675) (see the table of examples and sequences). Epitope mapping using the hFRα sequence (SEQ ID NO: 15) shown in Figure 7 determined that the epitopes within the hFRα protein bound by variant v23924 include amino acid residues E120, D121, R123, T124, S125, and Y126 of SEQ ID NO: 15 (see Example 13).

[0075] In certain embodiments, at least one antigen-binding domain that binds to hFRα in the anti-FRα antibody construct of the present disclosure binds to an epitope in the hFRα protein comprising the amino acid residues E120, D121, R123, T124, S125, and Y126 of SEQ ID NO: 15. In some embodiments, the hFRα epitope bound by the anti-FRα antibody construct is a nonlinear (or discontinuous) epitope comprising the amino acid residues E120, D121, R123, T124, S125, and Y126 of SEQ ID NO: 15. In certain embodiments, the anti-FRα antibody construct of the present disclosure includes an antigen-binding domain that competes for binding to hFRα with an antibody that binds to an epitope in the hFRα protein comprising the amino acid residues E120, D121, R123, T124, S125, and Y126. In certain embodiments, the anti-FRα antibody construct of this disclosure includes an antigen-binding domain that competes with antibody v23924 described herein for binding to hFRα.

[0076] Using competitive assays known in the art, it is possible to determine whether an antibody construct competes for binding to hFRα with an antibody or antibody v23924 that binds to an epitope in the hFRα protein containing amino acid residues E120, D121, R123, T124, S125, and Y126. For example, an antibody or antibody v23924 (reference antibody) that binds to an epitope in the hFRα protein containing amino acid residues E120, D121, R123, T124, S125, and Y126 is first bound to hFRα under saturated conditions, and then the ability of the test antibody construct to bind to hFRα is measured. If the test antibody construct can bind to hFRα simultaneously with the reference antibody, it is considered that the test antibody construct binds to a different epitope than the reference antibody. Conversely, if the test antibody construct cannot bind to hFRα simultaneously with the reference antibody, it is thought that the test antibody construct will bind to the same epitope, an overlapping epitope, or an epitope adjacent to the epitope bound by the reference antibody. Competitive assays may also be performed by reversing the binding order of the reference antibody and the test antibody, i.e., allowing the test antibody to first bind to hFRα under saturated conditions, and then measuring the ability of the reference antibody construct to bind to hFRα.

[0077] Such competitive assays can be performed using techniques such as ELISA, radioimmunoassay, surface plasmon resonance (SPR), biolayer interference, and flow cytometry. An antibody that "competes with" a reference antibody refers to an antibody that blocks the binding of the reference antibody to its epitope by 50% or more in a competitive assay.

[0078] In certain embodiments, the anti-FRα antibody constructs of this disclosure include at least one antigen-binding domain that specifically binds to hFRα, the antigen-binding domain comprising a set of CDRs based on the CDR of antibody variant v23924 described herein. The CDR sequences of antibody v23924 and representative humanized or affinity-mature versions of this antibody are shown in Figure 11. Analysis of CDR sequences from parental and affinity-mature anti-FRα antibodies identified the minimum amino acid sequence present in each CDR, as defined by one of the IMGT, Chothia, Kabat, Contact, or AbM numbering systems. These amino acid sequences are represented by the minimum consensus CDR sequences provided in Table 3. Extended versions of these CDR consensus sequences based on the CDR sequences, as defined by the AbM numbering system, are shown in Table 4.

[0079] [Table 3]

[0080] [Table 4]

[0081] In certain embodiments, the anti-FRα antibody construct of the present disclosure comprises an antigen-binding domain having heavy chain CDR amino acid sequences (HCDR1, HCDR2, and HCDR3) including the sequences described in SEQ ID NOs: 3, 4, and 5, and light chain CDR amino acid sequences (LCDR1, LCDR2, and LCDR3) including the sequences described in SEQ ID NOs: 6, 7, and 8.

[0082] In certain embodiments, the anti-FRα antibody construct of this disclosure comprises the following antigen-binding domain: (i) The HCDR1 amino acid sequence described in SEQ ID NO: 3, the HCDR2 amino acid sequence described in SEQ ID NO: 4, and the HCDR3 amino acid sequence described in SEQ ID NO: 5 (X 2 L is X 3 is A, or X 2 H is X 3(is P), and (ii) The LCDR1 amino acid sequence described in SEQ ID NO. 6 (X 4 G is X 5 is D, or X 4 is W, and X 5 (is Y), the LCDR2 amino acid sequence described in SEQ ID NO: 7, and the LCDR3 amino acid sequence described in SEQ ID NO: 8 (X 6 is S, X 7 is N, and X 8 is V, X 9 is D, or X 6 is W, and X 7 H is X 8 is I, X 9 (is L).

[0083] In certain embodiments, the anti-FRα antibody construct of the present disclosure comprises an antigen-binding domain having heavy chain CDR amino acid sequences (HCDR1, HCDR2, and HCDR3) including the sequences described in SEQ ID NOs: 9, 10, and 11, and light chain CDR amino acid sequences (LCDR1, LCDR2, and LCDR3) including the sequences described in SEQ ID NOs: 12, 13, and 14.

[0084] In certain embodiments, the anti-FRα antibody construct of this disclosure comprises the following antigen-binding domain: (i) The HCDR1 amino acid sequence described in SEQ ID NO: 9, and the HCDR2 amino acid sequence described in SEQ ID NO: 10 (X 11 is S or A, X 12 is V, or X 11 is S, X 12 (is L), and the HCDR3 amino acid sequence (X) described in SEQ ID NO: 11. 13 L is X 14 is A, or X 13 H is X 14 (is P), and (ii) The LCDR1 amino acid sequence described in Sequence ID No. 12 (X 15 is R or Q, and X 16 G is X 17 is D, or X 15 is R, and X16 is W, and X 17 (is Y), the LCDR2 amino acid sequence described in SEQ ID NO: 13, and the LCDR3 amino acid sequence described in SEQ ID NO: 14 (X 18 is S, X 19 is N, and X 20 is V, X 21 is D, or X 18 is W, and X 19 H is X 20 is I, X 21 (is L).

[0085] In certain embodiments, the anti-FRα antibody construct of this disclosure has the following antigen-binding domain: (i) Variants v23924, v30618, v30384, v30389, v30394, v30399, v31422, v31423, v31424, v31425, v31426, v35305, v35342, v35347, v35348, v35350, v35354, v35356, v35358, v36167, v3616 8. An HCDR1 amino acid sequence selected from any one of the following HCDR1 amino acid sequences, v36675, or variants v23924, v30618, v30384, v30389, v30394, v30399, v31422, v31423, v31424, v31425, v31426, v35305, v35342, v3534 7, an HCDR2 amino acid sequence selected from any one of the following HCDR2 amino acid sequences: v35348, v35350, v35354, v35356, v35358, v36167, v36168, or v36675, and variants v23924, v30618, v30384, v30389, v30394, v30399, v31 An HCDR3 amino acid sequence selected from any one of the following HCDR3 amino acid sequences: 422, v31423, v31424, v31425, v31426, v35305, v35342, v35347, v35348, v35350, v35354, v35356, v35358, v36167, v36168, or v36675, and (ii) Variants v23924, v30618, v30384, v30389, v30394, v30399, v31422, v31423, v31424, v31425, v31426, v35305, v35342, v35347, v35348, v35350, v35354, v35356, v35358, v36167, v3616 8. An LCDR1 amino acid sequence selected from any one of the following LCDR1 amino acid sequences: v23924, v30618, v30384, v30389, v30394, v30399, v31422, v31423, v31424, v31425, v31426, v35305, v35342, v35347. An LCDR2 amino acid sequence selected from any one of the following LCDR2 amino acid sequences: v35348, v35350, v35354, v35356, v35358, v36167, v36168, or v36675, and variants v23924, v30618, v30384, v30389, v30394, v30399, v3142 2, comprising an LCDR3 amino acid sequence selected from any one of the following LCDR3 amino acid sequences: v31423, v31424, v31425, v31426, v35305, v35342, v35347, v35348, v35350, v35354, v35356, v35358, v36167, v36168, or v36675, The CDR amino acid sequence is defined by one of the following numbering systems: IMGT, Chothia, Kabat, Contact, or AbM (see Figure 11).

[0086] In certain embodiments, the anti-FRα antibody construct of the present disclosure is a heavy chain CDR amino acid sequence (HCDR1, HCDR2 and HCD) selected from one of the heavy chain CDR amino acid sequences (HCDR1, HCDR2 and HCD) among variants v23924, v30618, v30384, v30389, v30394, v30399, v31422, v31423, v31424, v31425, v31426, v35305, v35342, v35347, v35348, v35350, v35354, v35356, v35358, v36167, v36168, or v36675, as defined by one of the IMGT, Chothia, Kabat, Contact, or AbM numbering systems. It comprises an antigen-binding domain having a light chain CDR amino acid sequence (LCDR1, LCDR2, and LCDR3) selected from one of the following light chain CDR amino acid sequences (LCDR1, LCDR2, and LCDR3) defined by R3), and any one of the following variants: v23924, v30618, v30384, v30389, v30394, v30399, v31422, v31423, v31424, v31425, v31426, v35305, v35342, v35347, v35348, v35350, v35354, v35356, v35358, v36167, v36168, or v36675.

[0087] In certain embodiments, the anti-FRα antibody constructs of this disclosure are variants v23924, v30618, v30384, v30389, v30394, v30399, v31422, v31423, v31424, v31425, v31426, v3 It contains an antigen-binding domain comprising one heavy chain CDR amino acid sequence (HCDR1, HCDR2, HCDR3) and a light chain CDR amino acid sequence (LCDR1, LCDR2, and LCDR3) from among 5305, v35342, v35347, v35348, v35350, v35354, v35356, v35358, v36167, v36168, or v36675.

[0088] In certain embodiments, the anti-FRα antibody construct of the present disclosure includes an antigen-binding domain containing a CDR sequence of any one of the following VH domains: v23924, v30618, v30384, v30389, v30394, v30399, v31422, v31423, v31424, v31425, v31426, v35305, v35342, v35347, v35348, v35350, v35354, v35356, v35358, v36167, v36168, or v36675. In certain embodiments, the anti-FRα antibody construct of the present disclosure includes an antigen-binding domain containing a CDR sequence of any one of the following VL domains: v23924, v30618, v30384, v30389, v30394, v30399, v31422, v31423, v31424, v31425, v31426, v35305, v35342, v35347, v35348, v35350, v35354, v35356, v35358, v36167, v36168, or v36675. The VH and VL sequences of v23924, v30618, v30384, v30389, v30394, v30399, v31422, v31423, v31424, v31425, v31426, v35305, v35342, v35347, v35348, v35350, v35354, v35356, v35358, v36167, v36168, and v36675 are provided in Figure 12.

[0089] In certain embodiments, the anti-FRα antibody construct of the present disclosure includes an antigen-binding domain comprising a VH amino acid sequence selected from any one of the following VH amino acid sequences: variant v23924, v30618, v30384, v30389, v30394, v30399, v31422, v31423, v31424, v31425, v31426, v35305, v35342, v35347, v35348, v35350, v35354, v35356, v35358, v36167, v36168, or v36675. In certain embodiments, the anti-FRα antibody construct of the present disclosure includes an antigen-binding domain comprising a VL amino acid sequence selected from any one of the following VL amino acid sequences: v23924, v30618, v30384, v30389, v30394, v30399, v31422, v31423, v31424, v31425, v31426, v35305, v35342, v35347, v35348, v35350, v35354, v35356, v35358, v36167, v36168, or v36675.

[0090] In a particular embodiment, the anti-FRα antibody construct of the present disclosure includes an antigen-binding domain comprising a VH amino acid sequence and a VL amino acid sequence selected from any one of the following variants: v23924, v30618, v30384, v30389, v30394, v30399, v31422, v31423, v31424, v31425, v31426, v35305, v35342, v35347, v35348, v35350, v35354, v35356, v35358, v36167, v36168, or v36675.

[0091] Those skilled in the art will understand that a limited number of amino acid substitutions can be introduced into the CDR or VH or VL sequences of known antibodies without causing the antibody to lose its ability to bind to its target. Candidate amino acid substitutions can be identified by computer modeling or by techniques known in the art, such as alanine scanning, and the resulting variants can be tested for binding activity by standard techniques. Therefore, in certain embodiments, the anti-FRα antibody constructs of this disclosure may be variants v23924, v30618, v30384, v30389, v30394, v30399, v31422, v31423, v31424, v31425, v31426, v35305, v35342, v35347, v35348, v35350, v35354, v35356, v35358, v36167, v36168, or v36675. The antigen-binding domain includes an antigen-binding domain comprising a set of CDRs having 90% or greater sequence identity with any one set of CDRs (i.e., heavy chains HCDR1, HCDR2, and HCDR3, and light chains LCDR1, LCDR2, and LCDR3), where % sequence identity is calculated across all six CDRs, and the antigen-binding domain retains the ability to bind to hFRα.

[0092] In certain embodiments, the anti-FRα antibody construct of the present disclosure comprises an antigen-binding domain containing a variant of any one set of CDR sequences from variant v23924, v30618, v30384, v30389, v30394, v30399, v31422, v31423, v31424, v31425, v31426, v35305, v35342, v35347, v35348, v35350, v35354, v35356, v35358, v36167, v36168, or v36675, wherein the variant contains 1 to 10 amino acid substitutions across the set of CDRs (i.e., the CDRs can be modified by up to 10 amino acid substitutions by modifying any combination of 6 CDRs), and the antigen-binding domain retains the ability to bind to hFRα. In some embodiments, the anti-FRα antibody constructs of this disclosure are variants v23924, v30618, v30384, v30389, v30394, v30399, v31422, v31423, v31424, v31425, v31426, v35305, v35342, v35347, v35348, v35350, v35354, v35356, v35358, v36167, v361 The antigen-binding domain comprises an antigen-binding domain containing a variant of one of the sets of CDR sequences, either 68 or v36675, wherein the variant contains 1 to 7 amino acid substitutions, 1 to 5 amino acid substitutions, 1 to 4 amino acid substitutions, 1 to 3 amino acid substitutions, 1 to 2 amino acid substitutions, or 1 amino acid substitution across the set of CDRs, and the antigen-binding domain retains the ability to bind to hFRα.

[0093] In certain embodiments, the anti-FRα antibody constructs of this disclosure include variants v23924, v30618, v30384, v30389, v30394, v30399, v31422, v31423, v31424, v31425, v31426, v35305, v35342, v35347, v35348, v35350, v35354, v35356, v35358, v36167, v36168, or v366 It comprises an antigen-binding domain containing a VH sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of the 75 VH sequences, the antigen-binding domain retaining the ability to bind to hFRα. In certain embodiments, the anti-FRα antibody constructs of this disclosure include variants v23924, v30618, v30384, v30389, v30394, v30399, v31422, v31423, v31424, v31425, v31426, v35305, v35342, v35347, v35348, v35350, v35354, v35356, v35358, v36167, v36168, or v366 It comprises an antigen-binding domain containing a VL sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of the 75 VL sequences, the antigen-binding domain retaining the ability to bind to hFRα.

[0094] In certain embodiments, the anti-FRα antibody construct of this disclosure comprises the following antigen-binding domain: (i) an HCDR1 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NOs: 20, 23, 26, 28, 31, 92, 93, 94, 95, or 96; an HCDR2 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NOs: 21, 24, 27, 29, 32, 51, 58, 100, 101, 102, 103, 109, 137, 138, or 139; and an HCDR3 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NOs: 22, 25, 30, 107, 108, or 110; (ii) An LCDR1 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NOs: 40, 43, 45, 65, 125, 126, or 127; an LCDR2 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NOs: 41, 44, or 46; and an LCDR3 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NOs: 42, 47, 120, or 121.

[0095] In certain embodiments, the anti-FRα antibody construct of the present disclosure includes an antigen-binding domain comprising a CDR sequence of a VH domain having the sequence described in any one of SEQ ID NOs: 19, 50, 54, 57, 61, 76, 79, 82, 85, 88, 91, 99, 106, 113, 116, 133, or 136. In certain embodiments, the anti-FRα antibody construct of the present disclosure includes an antigen-binding domain comprising a CDR sequence of a VL domain having the sequence described in any one of SEQ ID NOs: 39, 64, 119, 124, or 130.

[0096] In certain embodiments, the anti-FRα antibody construct of this disclosure comprises an antigen-binding domain having the following: (a) an LCDR1 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 40, 43, or 45, an LCDR2 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 41, 44, or 46, and an LCDR3 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 42 or 47, and an HCDR1 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 20, 23, 26, 28, or 31, an HCDR2 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 21, 24, 27, 29, or 32, and an HCDR3 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 22, 25, or 30, (b) An LCDR1 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 125, 126, or 127; an LCDR2 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 41, 44, or 46; and an LCDR3 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 42 or 47; and an HCDR1 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 92, 93, 94, 95, or 96; an HCDR2 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 21, 24, 29, 32, or 51; and an HCDR3 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 22, 25, or 30. (c) an LCDR1 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 40, 45, or 65, an LCDR2 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 41, 44, or 46, and an LCDR3 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 42 or 47, and (i) an HCDR1 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NOs: 20, 23, 26, 28, or 31; an HCDR2 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NOs: 21, 24, 29, 32, or 51; an HCDR3 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NOs: 22, 25, or 30; or (ii) an HCDR1 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NOs: 20, 23, 26, 28, or 31; an HCDR2 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NOs: 21, 24, 29, 32, or 58; an HCDR3 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NOs: 22, 25, or 30; or (iii) an HCDR1 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NOs: 92, 93, 94, 95, or 96; an HCDR2 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NOs: 24, 100, 101, 102, or 103; an HCDR3 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NOs: 22, 25, or 30; or (d) an LCDR1 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 125, 126, or 127, an LCDR2 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 41, 44, or 46, and an LCDR3 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 120 or 121, and (i) an HCDR1 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NOs: 92, 93, 94, 95, or 96; an HCDR2 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NOs: 24, 100, 101, 102, or 103; an HCDR3 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NOs: 22, 25, or 30; or (ii) an HCDR1 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NOs: 92, 93, 94, 95, or 96; an HCDR2 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NOs: 21, 24, 29, 32, or 109; an HCDR3 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NOs: 107, 108, or 110; or (iii) an HCDR1 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NOs: 92, 93, 94, 95, or 96; an HCDR2 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NOs: 21, 24, 29, 32, or 109; an HCDR3 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NOs: 22, 25, or 30; or (iv) an HCDR1 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NOs: 20, 23, 26, 28, or 31; an HCDR2 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NOs: 21, 24, 29, 32, or 109; an HCDR3 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NOs: 107, 108, or 110; or (v) An HCDR1 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NOs: 20, 23, 26, 28, or 31; an HCDR2 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NOs: 21, 24, 29, 32, or 109; an HCDR3 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NOs: 22, 25, or 30; or (vi) an HCDR1 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NOs: 92, 93, 94, 95, or 96; an HCDR2 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NOs: 21, 24, 137, 138, or 139; an HCDR3 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NOs: 22, 25, or 30; or (e) an LCDR1 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 40, 45, or 65, an LCDR2 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 41, 44, or 46, and an LCDR3 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 120 or 121, and (i) an HCDR1 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NOs: 92, 93, 94, 95, or 96; an HCDR2 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NOs: 21, 24, 29, 32, or 109; an HCDR3 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NOs: 107, 108, or 110; or (ii) An HCDR1 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NOs: 20, 23, 26, 28, or 31; an HCDR2 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NOs: 21, 24, 29, 32, or 109; and an HCDR3 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NOs: 107, 108, or 110.

[0097] In certain embodiments, the anti-FRα antibody construct of this disclosure comprises an antigen-binding domain having the following: (a) an HCDR1 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 20, 23, 26, 28, or 31; an HCDR2 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 21, 24, 27, 29, or 32; an HCDR3 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 22, 25, or 30; an LCDR1 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 40, 43, or 45; an LCDR2 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 41, 44, or 46; and an LCDR3 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 42 or 47; or (b) an HCDR1 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 20, 23, 26, 28, or 31; an HCDR2 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 21, 24, 29, 32, or 51; an HCDR3 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 22, 25, or 30; an LCDR1 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 40, 45, or 65; an LCDR2 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 41, 44, or 46; and an LCDR3 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 42 or 47; or (c) An HCDR1 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 20, 23, 26, 28, or 31; an HCDR2 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 21, 24, 29, 32, or 58; an HCDR3 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 22, 25, or 30; an LCDR1 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 40, 45, or 65; an LCDR2 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 41, 44, or 46; and an LCDR3 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 42 or 47; or (d) An HCDR1 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 92, 93, 94, 95, or 96; an HCDR2 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 21, 24, 29, 32, or 51; an HCDR3 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 22, 25, or 30; an LCDR1 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 125, 126, or 127; an LCDR2 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 41, 44, or 46; and an LCDR3 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 42 or 47; or (e) an HCDR1 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 92, 93, 94, 95, or 96; an HCDR2 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 24, 100, 101, 102, or 103; an HCDR3 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 22, 25, or 30; an LCDR1 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 125, 126, or 127; an LCDR2 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 41, 44, or 46; and an LCDR3 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 120 or 121; or (f) An HCDR1 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 92, 93, 94, 95, or 96; an HCDR2 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 21, 24, 29, 32, or 109; an HCDR3 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 107, 108, or 110; an LCDR1 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 40, 45, or 65; an LCDR2 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 41, 44, or 46; and an LCDR3 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 42 or 47; or (g) An HCDR1 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 92, 93, 94, 95, or 96; an HCDR2 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 21, 24, 29, 32, or 109; an HCDR3 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 107, 108, or 110; an LCDR1 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 40, 45, or 65; an LCDR2 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 41, 44, or 46; and an LCDR3 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 120 or 121; or (h) An HCDR1 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 92, 93, 94, 95, or 96; an HCDR2 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 21, 24, 29, 32, or 109; an HCDR3 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 107, 108, or 110; an LCDR1 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 125, 126, or 127; an LCDR2 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 41, 44, or 46; and an LCDR3 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 120 or 121; or (i) an HCDR1 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 92, 93, 94, 95, or 96; an HCDR2 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 21, 24, 29, 32, or 109; an HCDR3 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 22, 25, or 30; an LCDR1 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 125, 126, or 127; an LCDR2 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 41, 44, or 46; and an LCDR3 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 120 or 121; or (j) An HCDR1 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 20, 23, 26, 28, or 31; an HCDR2 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 21, 24, 29, 32, or 109; an HCDR3 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 107, 108, or 110; an LCDR1 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 40, 45, or 65; an LCDR2 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 41, 44, or 46; and an LCDR3 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 120 or 121; or (k) An HCDR1 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 20, 23, 26, 28, or 31; an HCDR2 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 21, 24, 29, 32, or 109; an HCDR3 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 107, 108, or 110; an LCDR1 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 125, 126, or 127; an LCDR2 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 41, 44, or 46; and an LCDR3 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 120 or 121; or (l) An HCDR1 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 20, 23, 26, 28, or 31; an HCDR2 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 21, 24, 29, 32, or 109; an HCDR3 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 22, 25, or 30; an LCDR1 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 125, 126, or 127; an LCDR2 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 41, 44, or 46; and an LCDR3 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 120 or 121; or (m) An HCDR1 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 92, 93, 94, 95, or 96; an HCDR2 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 21, 24, 137, 138, or 139; an HCDR3 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 22, 25, or 30; an LCDR1 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 125, 126, or 127; an LCDR2 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 41, 44, or 46; and an LCDR3 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 120 or 121.

[0098] In certain embodiments, the anti-FRα antibody construct of the present disclosure includes an antigen-binding domain comprising a VH amino acid sequence selected from any one of the VH amino acid sequences described in SEQ ID NOs: 19, 50, 54, 57, 61, 76, 79, 82, 85, 88, 91, 99, 106, 113, 116, 133, or 136. In certain embodiments, the anti-FRα antibody construct of the present disclosure includes an antigen-binding domain comprising a VL amino acid sequence selected from any one of the VL amino acid sequences described in SEQ ID NOs: 39, 64, 119, 124, or 130.

[0099] In certain embodiments, the anti-FRα antibody construct of the present disclosure comprises an antigen-binding domain comprising a VH amino acid sequence selected from any one of the VH amino acid sequences described in SEQ ID NOs: 19, 50, 54, 57, 61, 76, 79, 82, 85, 88, 91, 99, 106, 113, 116, 133, or 136, and a VL amino acid sequence selected from any one of the VL amino acid sequences described in SEQ ID NOs: 39, 64, 119, 124, or 130.

[0100] In certain embodiments, the anti-FRα antibody construct of this disclosure comprises an antigen-binding domain including: (a) The VL amino acid sequence described in SEQ ID NO: 39 and the VH amino acid sequence described in SEQ ID NO: 19, or (b) The VL amino acid sequence described in SEQ ID NO: 124 and the VH amino acid sequence described in SEQ ID NO: 91, or (c) The VL amino acid sequence described in Sequence ID No. 64, and (i) The VH amino acid sequence described in Sequence ID No. 50, or (ii) The VH amino acid sequence described in Sequence ID No. 54, or (iii) The VH amino acid sequence described in Sequence ID No. 57, or (iv) The VH amino acid sequence described in Sequence ID No. 61, or (v) The VH amino acid sequence described in Sequence ID No. 76, or (vi) The VH amino acid sequence described in Sequence ID No. 79, or (vii) The VH amino acid sequence described in Sequence ID No. 82, or (viii) The VH amino acid sequence described in Sequence ID No. 85, or (ix) The VH amino acid sequence described in Sequence ID No. 88, or (x) The VH amino acid sequence described in Sequence ID No. 106, or (d) The VL amino acid sequence described in SEQ ID NO: 130, and (i) The VH amino acid sequence described in Sequence ID No. 99, or (ii) The VH amino acid sequence described in Sequence ID No. 106, or (iii) The VH amino acid sequence described in Sequence ID No. 113, or (iv) The VH amino acid sequence described in Sequence ID No. 116, or (v) The VH amino acid sequence described in Sequence ID No. 133, or (vi) The VH amino acid sequence described in SEQ ID NO. 136, or (e) The VL amino acid sequence described in Sequence ID No. 119, and (i) The VH amino acid sequence described in Sequence ID No. 106, or (ii) The VH amino acid sequence described in Sequence ID No. 116.

[0101] In certain embodiments, the anti-FRα antibody construct of this disclosure comprises an antigen-binding domain including: (i) The VH amino acid sequence described in SEQ ID NO: 19 and the VL amino acid sequence described in SEQ ID NO: 39, or (ii) The VH amino acid sequence described in SEQ ID NO: 50 and the VL amino acid sequence described in SEQ ID NO: 64, or (iii) The VH amino acid sequence described in SEQ ID NO: 54, and the VL amino acid sequence described in SEQ ID NO: 64, or (iv) The VH amino acid sequence described in SEQ ID NO: 57 and the VL amino acid sequence described in SEQ ID NO: 64, or (v) The VH amino acid sequence described in SEQ ID NO: 61, and the VL amino acid sequence described in SEQ ID NO: 64, or (vi) The VH amino acid sequence described in SEQ ID NO: 76, and the VL amino acid sequence described in SEQ ID NO: 64, or (vii) The VH amino acid sequence described in SEQ ID NO: 79, and the VL amino acid sequence described in SEQ ID NO: 64, or (viii) The VH amino acid sequence described in SEQ ID NO: 82, and the VL amino acid sequence described in SEQ ID NO: 64, or (ix) The VH amino acid sequence described in SEQ ID NO: 85, and the VL amino acid sequence described in SEQ ID NO: 64, or (x) The VH amino acid sequence described in SEQ ID NO: 88, and the VL amino acid sequence described in SEQ ID NO: 64, or (xi) The VH amino acid sequence described in SEQ ID NO: 91, and the VL amino acid sequence described in SEQ ID NO: 124, or (xii) The VH amino acid sequence described in SEQ ID NO: 99, and the VL amino acid sequence described in SEQ ID NO: 130, or (xiii) The VH amino acid sequence described in SEQ ID NO: 106, and the VL amino acid sequence described in SEQ ID NO: 64, or (xiv) The VH amino acid sequence described in SEQ ID NO: 106, and the VL amino acid sequence described in SEQ ID NO: 119, or (xv) The VH amino acid sequence described in SEQ ID NO: 106, and the VL amino acid sequence described in SEQ ID NO: 130, or (xvi) The VH amino acid sequence described in SEQ ID NO: 113, and the VL amino acid sequence described in SEQ ID NO: 130, or (xvii) The VH amino acid sequence described in SEQ ID NO: 116, and the VL amino acid sequence described in SEQ ID NO: 119, or (xviii) The VH amino acid sequence described in SEQ ID NO: 116, and the VL amino acid sequence described in SEQ ID NO: 130, or (xix) The VH amino acid sequence described in SEQ ID NO: 133, and the VL amino acid sequence described in SEQ ID NO: 130, or (xx) The VH amino acid sequence described in SEQ ID NO: 136, and the VL amino acid sequence described in SEQ ID NO: 130.

[0102] format Anti-FRα antibody constructs can have various formats. The minimum component of an anti-FRα antibody construct is an antigen-binding domain that binds to hFRα. Anti-FRα antibody constructs may further optionally include one or more additional antigen-binding domains and / or scaffolds. In embodiments in which the anti-FRα antibody construct includes two or more antigen-binding domains, each additional antigen-binding domain may bind to the same epitope in hFRα, to a different epitope in hFRα, or to a different antigen. Thus, anti-FRα antibody constructs may be, for example, monospecific, biparatopic, bispecific, or multispecific.

[0103] In certain embodiments, the anti-FRα antibody construct comprises at least one antigen-binding domain and a scaffold that binds to hFRα, wherein the antigen-binding domain is functionally linked to the scaffold. The term "functionally linked," as used herein, means that the components described are in a relationship that enables them to function in the manner they are intended. Examples of preferred scaffolds are described below.

[0104] In certain embodiments, the anti-FRα antibody construct comprises two antigen-binding domains optionally functionally linked to a scaffold. In some embodiments, the anti-FRα antibody construct may comprise three or four antigen-binding domains and optionally a scaffold. In these formats, if a scaffold is included, at least the first antigen-binding domain is functionally linked to the scaffold, and the remaining antigen-binding domains may each be independently functionally linked to the scaffold or the first antigen-binding domain, or, if more than two antigen-binding domains are present, they may be linked to another antigen-binding domain.

[0105] Scaffold-less anti-FRα antibody constructs may contain a single antigen-binding domain in a suitable format such as an sdAb, or they may contain two or more antigen-binding domains optionally functionally linked by one or more linkers. In such anti-FRα antibody constructs, the antigen-binding domain may be in the form of scFv, Fab, sdAb, or a combination thereof. For example, using scFv as the antigen-binding domain, formats such as tandem scFv ((scFv)2 or taFv) can be constructed, where the scFvs are joined together by a flexible linker. scFv can be used to construct a diabody format containing two scFvs linked by a short linker (typically about 5 amino acids in length). The limited length of the linker results in dimerization of scFv in a head-to-tail manner. In either of the preceding formats, scFv may be further stabilized by including an interdomain disulfide bond. For example, a disulfide bond may be introduced between VL and VH by introducing an additional cysteine ​​residue to each chain (e.g., at position 44 of VH and position 100 of VL) (see, e.g., Fitzgerald et al., 1997, Protein Engineering, 10:1221-1225), or a disulfide bond may be introduced between two VHs to provide a construct having the DART format (see, e.g., Johnson et al., 2010, J Mol. Biol., 399:436-449).

[0106] Similarly, in some embodiments, a format comprising two sdAbs, such as VH or VHH, linked together via a suitable linker may be used. Other examples of scaffold-less anti-FRα antibody construct formats include those based on Fab fragments, e.g., Fab2 and F(ab')2 formats, where the Fab fragments are linked via a linker or IgG hinge region.

[0107] By using combinations of antigen-binding domains of different forms, alternative scaffold-less formats can also be constructed. For example, scFv or sdAb can be fused to the C-terminus of either or both the light and heavy chains of a Fab fragment, resulting in a bivalent (Fab-scFv / sdAb) construct.

[0108] In certain embodiments, an anti-FRα antibody construct may be an antibody format based on an immunoglobulin (Ig). In certain embodiments, an anti-FRα antibody construct may be based on an IgG class immunoglobulin, e.g., IgG1, IgG2, IgG3, or IgG4 immunoglobulin. In some embodiments, an anti-FRα antibody construct may be based on IgG1 immunoglobulin. In the context of this disclosure, when an anti-FRα antibody construct is based on a particular immunoglobulin isotype, it means that the anti-FRα antibody construct includes all or part of the constant region of the particular immunoglobulin isotype. For example, an anti-FRα antibody construct based on a given Ig isotype may include at least one antigen-binding domain functionally linked to an Ig scaffold, the scaffold including an Fc region from a given isotype and an Ig hinge region from optionally the same or a different isotype. In some embodiments, it should also be understood that an anti-FRα antibody construct may also include hybrids of isotypes and / or subclasses. Furthermore, it should be understood that the Fc region and / or hinge region may optionally be modified to impart one or more desired functional properties known in the art.

[0109] In some embodiments, the anti-FRα antibody construct may be derived from two or more immunoglobulins from different species; for example, the anti-FRα antibody construct may be a chimeric antibody or a humanized antibody. The terms “chimeric antibody” and “humanized antibody” both generally refer to antibodies that combine immunoglobulin regions or domains from two or more species.

[0110] A "chimeric antibody" typically comprises at least one variable domain from a non-human antibody, such as a rabbit or rodent (e.g., mouse) antibody, and at least one constant domain from a human antibody. The human constant domain of a chimeric antibody does not need to be of the same isotype as the non-human constant domain being replaced. Chimeric antibodies are discussed, for example, in Morrison et al., 1984, Proc. Natl. Acad. Sci. USA, 81:6851-55, and U.S. Patent No. 4,816,567.

[0111] A "humanized antibody" is a type of chimeric antibody that contains a minimal amount of sequences derived from a non-human antibody. Generally, a humanized antibody is a human immunoglobulin (recipient antibody) in which residues from the recipient's hypervariable region (CDR) are replaced with residues from the hypervariable region (CDR) of a non-human species (donor antibody) such as mouse, rat, rabbit, or non-human primate, which have the desired specificity and affinity for the target antigen. The technique for producing these humanized antibodies is often called "CDR grafting."

[0112] In some cases, additional modifications are made to humanized antibodies to further improve their performance. For example, framework region (FR) residues of human immunoglobulins may be replaced with corresponding non-human residues, or residues not found in either the recipient or donor antibody may be included in the humanized antibody. Generally, the variable domain of a humanized antibody includes all or substantially all of the hypervariable region derived from the non-human immunoglobulin and all or substantially all of the FR derived from the human immunoglobulin sequence. Humanized antibodies are described in detail, for example, by Jones, et al., 1986, Nature, 321:522-525, Riechmann, et al., 1988, Nature, 332:323-329, and Presta, 1992, Curr. Op. Struct. Biol., 2:593-596.

[0113] Numerous approaches are known in the art for selecting the optimal human framework for grafting non-human CDRs. Early approaches used a limited subset of well-characterized human antibodies, regardless of sequence identity with the non-human antibody providing the CDR ("fixed framework" approach). More recent approaches employ variable regions that have high amino acid sequence identity with the variable region of the non-human antibody providing the CDR ("homologous matching" or "best-fit" approach). Another approach is to select a fragment of framework sequence within the light or heavy chain variable region derived from several different human antibodies. CDR grafting can, in some cases, partially or completely lose the affinity of the grafted molecule to its target antigen. In such cases, affinity can be restored by reverting some of the human-derived residues to the corresponding non-human-derived residues. Methods for preparing humanized antibodies using these approaches are well known in the art (see, for example, Tsurushita & Vasquez, 2004, Humanization of Monoclonal Antibodies, Molecular Biology of B Cells, 533-545, Elsevier Science (USA); Jones et al., 1986, Nature, 321:522-525; Riechmann et al., 1988, Nature, 332:323-329; Presta et al., 1997, Cancer Res, 57(20):4593-4599).

[0114] In addition to or instead of these conventional approaches, more recent techniques may be employed to further reduce the immunogenicity of CDR-grafted humanized antibodies. For example, a framework based on human germline sequences or consensus sequences may be used as the acceptor human framework, rather than a human framework with somatic mutations (or multiple mutations). Another technique aimed at reducing the potential immunogenicity of non-human CDRs is to graft only specificity-determining residues (SDRs). In this approach, only the minimum number of CDR residues ("SDRs") necessary for antigen-binding activity are grafted onto a human germline framework. This method may help improve the "humanity" (i.e., similarity to human germline sequences) of the humanized antibody and thus reduce the risk of immunogenicity in the variable region. These techniques are described in various publications (see, for example, Almagro & Fransson, 2008, Front Biosci, 13:1619-1633; Tan, et al., 2002, J Immunol, 169:1119-1125; Hwang, et al., 2005, Methods, 36:35-42; Pelat, et al., 2008, J Mol Biol, 384:1400-1407; Tamura, et al., 2000, J Immunol, 164:1432-1441; Gonzales, et al., 2004, Mol Immunol, 1:863-872; and Kashmiri, et al., 2005, Methods, 36:25-34).

[0115] In certain embodiments, the anti-FRα antibody constructs of this disclosure include a humanized antibody sequence, for example, one or more humanized variable domains. In some embodiments, the anti-FRα antibody construct may be a humanized antibody. Non-limiting examples of humanized antibodies based on anti-FRα antibody v23924 are described herein (v30384, v30389, v30394, v30399, v31422, v31423, v31424, v31425, and v31426; see the table of examples and sequences).

[0116] scaffold In certain embodiments, the anti-FRα antibody construct comprises one or more antigen-binding domains functionally linked to a scaffold. The antigen-binding domain(s) may be one or a combination of the above forms (e.g., scFv, Fab, and / or sdAb). Examples of suitable scaffolds, though not limited to those described in more detail below, include immunoglobulin Fc regions, albumins, albumin analogs and derivatives, heterodimerizing peptides (e.g., leucine zipper, heterodimerizing "zipper" peptides derived from Jun and Fos, IgG CH1 and CL domains or burnase burster toxins), cytokines, chemokines, or growth factors. Other examples include antibodies based on DOCK-AND-LOCK® (DNL®) technology developed by IBC Pharmaceuticals, Inc. and Immunomedics, Inc. (see, e.g., Chang, et al., 2007, Clin. Cancer Res., 13:5586s-5591s).

[0117] The scaffold can be a peptide, polypeptide, polymer, nanoparticle, or other chemical substance. If the scaffold is a polypeptide, each antigen-binding domain of the anti-FRα antibody construct can be linked to either the N-terminus or C-terminus of the polypeptide scaffold. Anti-FRα antibody constructs comprising polypeptide scaffolds in which one or more antigen-binding domains are linked to a region other than the N-terminus or C-terminus, for example, via amino acid side chains with or without linkers, are also contemplated in certain embodiments.

[0118] In embodiments where the anti-FRα antibody construct is a peptide or polypeptide scaffold, the antigen-binding domain(s) may be linked to the scaffold by genetic fusion or chemical conjugation. Typically, when the scaffold is a peptide or polypeptide, the antigen-binding domain(s) are linked to the scaffold by genetic fusion. In some embodiments where the scaffold is a polymer or nanoparticle, the antigen-binding domain(s) may be linked to the scaffold by chemical conjugation.

[0119] Several protein domains containing selective pairs of two different polypeptides are known in the art and can be used to form scaffolds. Examples include leucine zipper domains such as Fos and Jun that selectively pair (Kostelny, et al., J Immunol, 148:1547-53 (1992), Wranik, et al., J. Biol. Chem., 287:43331-43339 (2012)). Other examples of selectively pairing molecular pairs include, for example, burnase-burster pairs (Deyev, et al., Nat Biotechnol, 21:1486-1492 (2003)), DNA strand pairs (Chaudri, et al., FEBS Letters, 450(1-2):23-26 (1999)), and split fluorescent protein pairs (International Patent Application Publication WO2011 / 135040).

[0120] Other examples of protein scaffolds include immunoglobulin Fc regions, albumins, albumin analogs and derivatives, toxins, cytokines, chemokines, and growth factors. The use of protein scaffolds in combination with antigen-binding moieties has been described (see, for example, Muller et al., 2007, J. Biol. Chem., 282:12650-12660; McDonaugh et al., 2012, Mol. Cancer Ther., 11:582-593; Vallera et al., 2005, Clin. Cancer Res., 11:3879-3888; Song et al., 2006, Biotech. Appl. Biochem., 45:147-154, and U.S. Patent Application Publication No. 2009 / 0285816).

[0121] For example, fusing antigen-binding moieties such as scFv, diabodies, or single-stranded diabodies to albumin has been shown to improve the serum half-life of the antigen-binding moiety (Muller et al., ibid.). The antigen-binding moiety may optionally be fused to the N-terminus and / or C-terminus of albumin via a linker.

[0122] Derivatives of albumin in the form of heteromultimers are described, comprising two transporter polypeptides obtained by segmentation of an albumin protein, such that the transporter polypeptides self-assemble to form a quasi-natural albumin (see International Patent Application Publications WO2012 / 116453 and WO2014 / 012082). As a result of the segmentation of albumin, the heteromultimer comprises four ends and can therefore optionally fuse to up to four different antigen-binding moieties via a linker.

[0123] In certain embodiments, the anti-FRα antibody construct may include a protein scaffold. In some embodiments, the anti-FRα antibody construct may include a protein scaffold based on an immunoglobulin Fc region, albumin, or an albumin analog or derivative. In some embodiments, the anti-FRα antibody construct may include a protein scaffold based on an immunoglobulin Fc region, for example, an IgG Fc region.

[0124] Fc area The terms “Fc region,” “Fc,” or “Fc domain,” as used herein, refer to the C-terminal region of an immunoglobulin heavy chain, including at least a portion of the constant region. These terms include both the native sequence Fc region and variant Fc regions. Unless otherwise specified herein, the numbering of amino acid residues within the Fc region or constant region follows the EU numbering system, also known as the EU index, as described in Kabat, et al., Sequences of Proteins of Immunological Interest, 5th Ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991).

[0125] In certain embodiments, the anti-FRα antibody construct may include a scaffold based on an immunoglobulin Fc region. The Fc region may be dimer and composed of two Fc polypeptides, or alternatively, the Fc region may consist of a single polypeptide.

[0126] In the context of dimer Fc, "Fc polypeptide" refers to one of the two polypeptides that form the dimer Fc domain, i.e., a polypeptide containing one or more C-terminal constant regions of an immunoglobulin heavy chain capable of stable self-assembly. When referring to polypeptides that form the dimer Fc region, the terms "first Fc polypeptide" and "second Fc polypeptide" may be used interchangeably, provided that the Fc region contains one first Fc polypeptide and one second Fc polypeptide.

[0127] The Fc region may contain a CH3 domain, or it may contain both CH3 and CH2 domains. For example, in certain embodiments, the Fc polypeptide of the dimerized IgG Fc region may contain an IgG CH2 domain sequence and an IgG CH3 domain sequence. In such embodiments, the CH3 domain contains two CH3 sequences, one from each of the two Fc polypeptides of the dimerized Fc region, and the CH2 domain contains two CH2 sequences, one from each of the two Fc polypeptides of the dimerized Fc region.

[0128] In some embodiments, the anti-FRα antibody construct may include a scaffold based on the IgG Fc region. In some embodiments, the anti-FRα antibody construct may include a scaffold based on the human IgG Fc region. In some embodiments, the anti-FRα antibody construct may include a scaffold based on the IgG1 Fc region. In some embodiments, the anti-FRα antibody construct may include a scaffold based on the human IgG1 Fc region.

[0129] In certain embodiments, the anti-FRα antibody construct may include a scaffold based on an IgG Fc region which is a homodimeric Fc region, comprising a first Fc polypeptide and a second Fc polypeptide, wherein the first and second Fc polypeptides each comprise a CH3 sequence and optionally a CH2 sequence, and the amino acid sequences of the first and second Fc polypeptides are identical.

[0130] In certain embodiments, the anti-FRα antibody construct may include a scaffold based on an IgG Fc region which is a heterodimeric Fc region comprising a first Fc polypeptide and a second Fc polypeptide, wherein the first and second Fc polypeptides each comprise a CH3 sequence and optionally a CH2 sequence, and the amino acid sequences of the first and second Fc polypeptides are different. In some embodiments, the anti-FRα antibody construct may include a scaffold based on an Fc region comprising two CH3 sequences, at least one of which comprises one or more amino acid modifications. In some embodiments, the anti-FRα antibody construct may include a scaffold based on an Fc region comprising two CH3 sequences and two CH2 sequences, at least one of which comprises one or more amino acid modifications.

[0131] In some embodiments, the anti-FRα antibody construct may include a heterodimeric Fc region containing a modified CH3 domain, the modified CH3 domain being an asymmetrically modified CH3 domain containing one or more asymmetric amino acid modifications. As used herein, “asymmetric amino acid modification” refers to a modification, such as substitution or insertion, in which an amino acid at a particular position on a first CH3 or CH2 sequence is different from an amino acid at the same position on a second CH3 or CH2 sequence. These asymmetric amino acid modifications may be the result of modification of only one of two amino acids at the same respective amino acid positions on each sequence, or the result of different modifications of both amino acids at the same respective positions on each of the first and second CH3 or CH2 sequences. Each of the first and second CH3 or CH2 sequences of the heterodimeric Fc may contain one or more asymmetric amino acid modifications.

[0132] In some embodiments, the anti-FRα antibody construct may comprise a heterodimer Fc containing a modified CH3 domain, the modified CH3 domain comprising one or more amino acid modifications that facilitate the formation of the heterodimer Fc over the formation of the homodimer Fc. In some embodiments, one or more of the amino acid modifications are asymmetric amino acid modifications.

[0133] Amino acid modifications that may be made to the CH3 domain of Fc to promote the formation of heterodimer Fc are known in the art, for example, those described in International Publication No. WO96 / 027011 ("knobs into holes"), those described in Gunasekaran et al., 2010, J Biol Chem, 285, 19637-46 ("electrostatic steering"), those described in Davis et al., 2010, Prot Eng Des Sel, 23(4):195-202 (strand exchange engineered domain (SEED) technology), and those described in Labrijn et al., 2013, Proc Natl Acad Sci USA, 110(13):5145-50 (Fab arm exchange). Other examples include approaches that combine positive and negative design strategies resulting in asymmetrically modified stable Fc regions, as described in International Publications WO2012 / 058768 and WO2013 / 063702. In certain embodiments, the anti-FRα antibody construct may include a scaffold based on a modified Fc region, as described in International Publications WO2012 / 058768 or WO2013 / 063702.

[0134] Table 5 provides the amino acid sequence of the human IgG1 Fc sequence (SEQ ID NO: 16), corresponding to amino acids 231-447 of the full-length human IgG1 heavy chain. The CH3 sequence contains amino acids 341-447 of the full-length human IgG1 heavy chain. Table 5 also shows CH3 domain amino acid modifications that promote heterodimer Fc formation, as described in International Patent Application Publications WO2012 / 058768 and WO2013 / 063702.

[0135] In certain embodiments, the anti-FRα antibody construct may include a heterodimer Fc scaffold having a modified CH3 domain comprising one of the following modifications: variant 1, variant 2, variant 3, variant 4, or variant 5, as shown in Table 5.

[0136] [Table 5]

[0137] In some embodiments, the anti-FRα antibody construct may include a scaffold based on an Fc region containing two CH3 sequences and two CH2 sequences, at least one of the CH2 sequences containing one or more amino acid modifications. Modifications to the CH2 domain can affect the binding of Fc receptors (FcRs) to Fc, such as receptors of the FcγRI, FcγRII, and FcγRIII subclasses.

[0138] In some embodiments, the anti-FRα antibody construct comprises a scaffold based on IgG Fc having a modified CH2 domain, the modification of the CH2 domain resulting in altered binding to one or more of the FcγRI, FcγRII, and FcγRIII receptors.

[0139] Several amino acid modifications to the CH2 domain that selectively alter the affinity of Fc to different Fcγ receptors are known in the art. Amino acid modifications that result in increased binding and those that result in decreased binding may each be useful in specific indications. For example, increased binding affinity of Fc to FcγRIIIa (activating receptor) may result in increased antibody-dependent cell-mediated cytotoxicity (ADCC), which in turn leads to increased target cell lysis. Similarly, decreased binding to FcγRIIb (inhibitory receptor) may be beneficial in some situations. In certain indications, reduction or elimination of ADCC and complement-mediated cytotoxicity (CDC) may be desirable. In such cases, modified CH2 domains containing amino acid modifications that increase binding to FcγRIIb, or amino acid modifications ("knockout" variants) that reduce or eliminate the binding of the Fc region to all Fcγ receptors, may be useful.

[0140] Examples of amino acid modifications to the CH2 domain that alter Fc binding by the Fcγ receptor include, but are not limited to, the following: S298A / E333A / K334A and S298A / E333A / K334A / K326A (increased affinity for FcγRIIIa) (Lu, et al., 2011, J Immunol Methods, 365(1-2):132-41), F243L / R292P / Y300L / V305I / P396L (increased affinity for FcγRIIIa) (Stavenhagen, et al., 2007, Cancer Res, 67(18):8882-90), F243L / R292P / Y300L / L235V / P396L (increased affinity for FcγRIIIa) (Nordstrom JL, et al.) al., 2011, Breast Cancer Res, 13(6):R123), F243L (increased affinity for FcγRIIIa) (Stewart, et al., 2011, Protein Eng Des Sel., 24(9):671-8), S298A / E333A / K334A (increased affinity for FcγRIIIa) (Shields, et al., 2001, J Biol Chem, 276(9):6591-604), S239D / I332E / A330L and S239D / I332E (increased affinity for FcγRIIIa) (Lazar, et al., 2006, Proc Natl Acad Sci USA, 103(11):4005-10), and S239D / S267E and S267E / L328F (increased affinity for FcγRIIb) (Chu, et al., 2008, Mol Immunol, 45(15):3926-33). Various amino acid modifications to the CH2 domain that alter Fc binding by FcγRIIb are described in international publication WO2021 / 232162. Additional modifications that affect Fc binding to the Fcγ receptor are described in Therapeutic Antibody Engineering (Strohl & Strohl, Woodhead Publishing series in Biomedicine No 11, ISBN 1 907568 37 9, Oct 2012, page 283).

[0141] In certain embodiments, the anti-FRα antibody construct comprises an IgG Fc-based scaffold having a modified CH2 domain, the modified CH2 domain containing one or more amino acid modifications that result in reduced or eliminated binding of all Fc regions to the Fcγ receptor (i.e., a "knockout" variant).

[0142] Various publications describe strategies used to manipulate antibodies to generate "knockout" variants (see, for example, Strohl, 2009, Curr Opin Biotech 20:685-691, and Strohl & Strohl, “Antibody Fc engineering for optimal antibody performance” In Therapeutic Antibody Engineering, Cambridge: Woodhead Publishing, 2012, pp225-249). These strategies include modification of glycosylation, use of IgG2 / IgG4 scaffolds, or reduction of effector function by introducing mutations in the Fc hinge or CH2 domain (see also U.S. Patent Publication 2011 / 0212087, International Publication WO2006 / 105338, U.S. Patent Publication 2012 / 0225058, U.S. Patent Publication 2012 / 0251531, and Strop et al., 2012, J.Mol.Biol., 420:204-219).

[0143] Examples of mutations that can be introduced into the hinge or CH2 domain to generate a "knockout" variant include amino acid modifications L234A / L235A and L234A / L235A / D265S.

[0144] In certain embodiments, the anti-FRα antibody constructs described herein may include a scaffold based on naturally glycosylated IgG Fc. As is known in the art, the glycosylation of Fc may be modified to increase or decrease effector function. For example, mutations of the conserved asparagine residue at position 297 to alanine, glutamine, lysine, or histidine (i.e., N297A, Q, K, or H) result in a non-glycosylated Fc lacking all effector function (Bolt et al., 1993, Eur. J. Immunol., 23:403-411; Tao & Morrison, 1989, J. Immunol., 143:2595-2601).

[0145] Conversely, removal of fucose from heavy-chain N297-linked oligosaccharides has been shown to enhance ADCC based on improved binding to FcγRIIIa (see, for example, Shields et al., 2002, J Biol Chem., 277:26733-26740, and Niwa et al., 2005, J.Immunol.Methods, 306:151-160). Such low-fucose antibodies may be produced, for example, in knockout Chinese hamster ovary (CHO) cells lacking fucosyltransferase (FUT8) (Yamane-Ohnuki et al., 2004, Biotechnol. Bioeng., 87:614-622), in the variant CHO cell line Lec13 with reduced ability to attach fucose to N297-bound carbohydrates (International Publication No. WO03 / 035835), or in other cells that produce non-fucosylated antibodies (see, for example, Li et al., 2006, Nat Biotechnol, 24:210-215; Shields et al., 2002, ibid, and Shinkawa et al., 2003, J. Biol. Chem., 278:3466-3473). In addition, International Publication No. WO2009 / 135181 describes adding a fucose analog to the culture medium during antibody production to inhibit the incorporation of fucose into carbohydrates on antibodies.

[0146] Other methods for producing antibodies with little or no fucose on the Fc glycosylation site (N297) are well known in the art. For example, see the GlymaX® technology (ProBioGen AG) (von Horsten et al., 2010, Glycobiology, 20(12):1607-1618 and U.S. Patent No. 8,409,572).

[0147] Other glycosylated variants include those containing bifid oligosaccharides, for example, variants in which a branched oligosaccharide bound to the antibody's Fc region is bifid by N-acetylglucosamine (GlcNAc). Such glycosylated variants may have reduced fucosylation and / or improved ADCC function (see, e.g., International Publication WO2003 / 011878, U.S. Patent No. 6,602,684, and U.S. Patent Application Publication US2005 / 0123546). Useful glycosylated variants include those having at least one galactose residue in the oligosaccharide bound to the Fc region, which may have improved CDC function (see, e.g., International Publication WO1997 / 030087, WO1998 / 58964, and WO1999 / 22764).

[0148] In certain embodiments, the anti-FRα antibody construct has the format of a full-size antibody (FSA). In some embodiments, the anti-FRα antibody construct has the format of an IgG FSA, e.g., an IgG1 FSA. In some embodiments, the anti-FRα antibody construct is an FSA comprising a first heavy chain sequence (H1), a second heavy chain sequence (H2), a first light chain sequence (L1), and a second light chain sequence (L2). In some embodiments, the anti-FRα antibody construct is a monospecific FSA having a homodimer Fc and comprising H1, H2, L1, and L2 sequences, where H1 and H2 have the same amino acid sequence, and L1 and L2 have the same amino acid sequence. In some embodiments, the anti-FRα antibody construct is a monospecific FSA having a heterodimer Fc and comprising H1, H2, L1, and L2 sequences, where H1 and H2 have different amino acid sequences, and L1 and L2 have the same amino acid sequence. In some embodiments, the anti-FRα antibody construct is a bispecific or biparatopic FSA having a heterodimer Fc, comprising H1, H2, L1, and L2 sequences, where H1 and H2 have different amino acid sequences, and L1 and L2 have different amino acid sequences.

[0149] In a particular embodiment, the anti-FRα antibody construct is an FSA having a set of H1, H2, L1, and L2 sequences, comprising the H1, H2, L1, and L2 amino acid sequences shown in Tables A and B for any one of the variants v23924, v30618, v30384, v30389, v30394, v30399, v31422, v31423, v31424, v31425, v31426, v35305, v35342, v35347, v35348, v35350, v35354, v35356, v35358, v36167, v36168, or v36675. As is known in the art, the expression of an antibody heavy chain sequence from a particular cell line or expression vector may result in the inclusion of a C-terminal lysine residue in one or both of the heavy chains. Accordingly, specific embodiments of the present disclosure relate to anti-FRα antibody constructs which are FSAs having a set of H1, H2, L1, and L2 sequences, including the H1, H2, L1, and L2 amino acid sequences listed in Tables A and B for any one of the variants v23924, v30618, v30384, v30389, v30394, v30399, v31422, v31423, v31424, v31425, v31426, v35305, v35342, v35347, v35348, v35350, v35354, v35356, v35358, v36167, v36168, or v36675, wherein one or both of the H1 and H2 sequences contain C-terminal lysine (see, for example, SEQ ID NO: 157).

[0150] Preparation of anti-FRα antibody constructs The anti-FRα antibody constructs described herein can be produced using standard recombinant methods known in the art (e.g., U.S. Patent No. 4,816,567 and “Antibodies: A Laboratory Manual,” 2 nd See Edition, Ed. Greenfield, Cold Spring Harbor Laboratory Press, New York, 2014.

[0151] Typically, for the recombinant production of an antibody construct, a polynucleotide or set of polynucleotides encoding the anti-FRα antibody construct is generated and inserted into one or more vectors for further cloning and / or expression in host cells. The polynucleotide(s) encoding the anti-FRα antibody construct can be produced by standard methods known in the art (e.g., Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1994 & updated, and “Antibodies: A Laboratory Manual,” 2 nd See Edition, Ed. Greenfield, Cold Spring Harbor Laboratory Press, New York, 2014. As will be understood by those skilled in the art, the number of polynucleotides required for the expression of an anti-FRα antibody construct will depend on the construct's format, including whether the antibody construct contains a scaffold. For example, if the anti-FRα antibody construct is in mAb format with homodimer Fc, two polynucleotides are required, each encoding one polypeptide chain; however, if the anti-FRα antibody construct is in mAb format with heterodimer Fc, three polynucleotides are required, each encoding one polypeptide chain. If multiple polynucleotides are required, they may be incorporated into one vector or into two or more vectors.

[0152] Generally, for expression, a polynucleotide or set of polynucleotides is incorporated into an expression vector (may include more than one) regulatory elements necessary for the efficient transcription of the polynucleotide, such as transcription elements. Examples of such regulatory elements include, but are not limited to, promoters, enhancers, terminators, and polyadenylation signals. Those skilled in the art will understand that the selection of regulatory elements depends on the host cell selected for the expression of the antibody construct, and that such regulatory elements may originate from a variety of sources, including bacterial, fungal, viral, mammalian, or insect genes. The expression vector may optionally further include heterologous nucleic acid sequences that facilitate expression or purification of the expressed protein. Examples include, but are not limited to, signal peptides, as well as affinity tags such as metal affinity tags, histidine tags, avidin / streptavidin coding sequences, glutathione-S-transferase (GST) coding sequences, and biotin coding sequences. The expression vector may be an extrachromosomal vector or an embedded vector.

[0153] Suitable host cells for cloning or expressing anti-FRα antibody constructs include a variety of prokaryotic or eukaryotic cells, as is known in the art. Eukaryotic host cells include, for example, mammalian cells, plant cells, insect cells, and yeast cells (e.g., Saccharomyces or Pichia cells). Prokaryotic host cells include, for example, Escherichia coli (E. coli) cells, A. salmonicida cells, or Bacillus subtilis (B. subtilis) cells.

[0154] In certain embodiments, anti-FRα antibody constructs may be generated in bacteria, particularly when glycosylation and Fc effector function are not required, as described, for example, in U.S. Patents No. 5,648,237, 5,789,199, and 5,840,523, and in Charlton, Methods in Molecular Biology, Vol. 248, pp. 245-254, BKCLo, ed. Humana Press, Totowa, NJ, 2003.

[0155] Eukaryotic microorganisms such as filamentous fungi or yeasts may, in certain embodiments, be suitable expression host cells, particularly fungal and yeast strains whose glycosylation pathways are "humanized," resulting in the production of antibody constructs having a partially or completely human glycosylation pattern (see, for example, Gerngross, 2004, Nat. Biotech. 22:1409-1414, and Li et al., 2006, Nat. Biotech. 24:210-215).

[0156] Suitable host cells for the expression of glycosylated anti-FRα antibody constructs are typically eukaryotic cells. For example, U.S. Patents 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 describe PLANTIBODIES® technology for producing antigen-binding constructs in transgenic plants. Mammalian cell lines adapted for growth in suspension may be particularly useful for the expression of antibody constructs. Examples include SV40(COS-7) transformed monkey kidney CV1 cell line, human embryonic kidney (HEK) cell line 293 or 293 (see, e.g., Graham et al., 1977, J. Gen Virol., 36:59), baby hamster kidney cells (BHK), mouse Sertoli TM4 cells (see, e.g., Mather, 1980, Biol Reprod, 23:243-251), monkey kidney cells (CV1), African green monkey kidney cells (VERO-76), human cervical cancer (HeLa) cells, canine kidney cells (MDCK), buffalo rat hepatocytes (BRL3A), human lung cells (W138), human liver cells (HepG2), mouse mammary tumor cells (MMT060562), and TRI cells (see, e.g., Mather et al., 1982, Annals NY Academia). See Sci, 383:44-68), MRC5 cells, FS4 cells, Chinese hamster ovary (CHO) cells (DHFR - This includes, but is not limited to, CHO cells (see Urlaub et al., 1980, Proc Natl Acad Sci USA, 77:4216) and myeloma cell lines (such as Y0, NS0, and Sp2 / 0). Exemplary mammalian host cell lines suitable for antibody construct production are outlined in Yazaki & Wu, Methods in Molecular Biology, Vol. 248, pp. 255-268 (BKCLo, ed. Humana Press, Totowa, NJ, 2003).

[0157] In certain embodiments, the host cell may be a transient or stable higher eukaryotic cell line, such as a mammalian cell line. In some embodiments, the host cell may be a mammalian HEK293T, CHO, HeLa, NS0, or COS cell line, or a cell line derived from any one of these cell lines. In some embodiments, the host cell may be a stable cell line that allows for the maturation glycosylation of the antibody construct.

[0158] Host cells containing an expression vector (or more) encoding an anti-FRα antibody construct can be cultured using conventional methods for producing the anti-FRα antibody construct. Alternatively, in some embodiments, host cells containing an expression vector (or more) encoding an anti-FRα antibody construct may be used therapeutically or prophylactically to deliver the anti-FRα antibody construct to a target, or polynucleotides or expression vectors may be administered ex vivo to cells from the target, and the cells may then be returned to the target's body.

[0159] Typically, anti-FRα antibody constructs are purified after expression. The protein may be isolated or purified by various methods known to those skilled in the art (e.g., Protein Purification: Principles and Practice, 3). rdSee Ed., Scopes, Springer-Verlag, NY, 1994. Standard purification methods include chromatographic techniques performed at atmospheric pressure or high pressure using systems such as FPLC and HPLC (including ion exchange chromatography, hydrophobic interaction chromatography, affinity chromatography, sizing chromatography, or gel filtration chromatography and reversed-phase chromatography). Further purification methods include electrophoresis, immunological, precipitation, dialysis, and chromatofocusing techniques. Ultrafiltration and diafiltration techniques combined with protein concentration are also useful. As is well known in the art, various native proteins bind to Fc and antibodies, and these proteins can be used to purify specific antibody constructs. For example, bacterial proteins A and G bind to the Fc region. Similarly, bacterial protein L binds to the Fab region of some antibodies. Purification can also be made possible by specific fusion partners. For example, antibodies can be bound to glutathione resin when using GST fusions, or to Ni when using His tags. +2 Affinity chromatography, or, if using flag tagging, purification may be performed using immobilized anti-flag antibodies. The required degree of purification varies depending on the use of the anti-FRα antibody construct. In some cases, purification may not be necessary.

[0160] In certain embodiments, the anti-FRα antibody construct is substantially pure. When used with respect to the anti-FRα antibody constructs described herein, the term “substantially pure” means that the antibody construct is substantially or essentially free from components that normally accompany or interact with proteins, as would be found in its naturally occurring environment, for example, in natural cells, or, in the case of recombinantly produced constructs, in host cells. In certain embodiments, a substantially pure anti-FRα antibody construct is a protein preparation having less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, or less than about 5% (by dry weight) of contaminating proteins.

[0161] Specific embodiments of the present disclosure relate to a method for producing an anti-FRα antibody construct, comprising culturing host cells into which one or more polynucleotides encoding the anti-FRα antibody construct or one or more expression vectors encoding the anti-FRα antibody construct have been introduced under conditions suitable for the expression of the anti-FRα antibody construct, and optionally recovering the anti-FRα antibody construct from the host cells (or host cell culture medium).

[0162] Post-translation modification In certain embodiments, the anti-FRα antibody constructs described herein may include one or more post-translational modifications. Such post-translational modifications may occur in vivo, or they may be carried out in vitro after isolation of the anti-FRα antibody construct from host cells.

[0163] Post-translational modifications include various modifications known in the art (see, for example, Proteins-Structure and Molecular Properties, 2nd Ed., TECreighton, WH Freeman and Company, New York, 1993; Post-Translational Covalent Modification of Proteins, BC Johnson, Ed., Academic Press, New York, pgs. 1-12, 1983; Seifter et al., 1990, Meth. Enzymol., 182:626-646; and Rattan et al., 1992, Ann. NY Acad. Sci., 663:48-62). In embodiments in which an anti-FRα antibody construct contains one or more post-translational modifications, the construct may contain the same type of modification at one or more sites, or different modifications at different sites.

[0164] Examples of post-translational modifications include glycosylation, acetylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, formylation, oxidation, reduction, proteolytic cleavage, or specific chemical cleavage (by cyanide bromide, trypsin, chymotrypsin, papain, V8 protease, or NaBH4).

[0165] Other examples of post-translational modifications include, for example, the addition or removal of N-linked or O-linked glycans, chemical modification of N-linked or O-linked glycans, treatment of the N-terminus or C-terminus, attachment of chemical moieties to the amino acid backbone, and the addition or deletion of N-terminal methionine residues resulting from expression in prokaryotic host cells. Post-translational modifications may also include modifications by detectable labels such as enzymatic labeling, fluorescent labeling, luminescence labeling, isotopic labeling, or affinity labeling to enable detection and isolation of proteins. Suitable examples of enzymatic labeling include, but are not limited to, horseradish peroxidase, alkaline phosphatase, beta-galactosidase, and acetylcholinesterase. Suitable examples of prosthetic group complexes include, but are not limited to, streptavidin / biotin and avidin / biotin. Examples of suitable fluorescent substances include, but are not limited to, umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansilchloride, and phycoerythrin. Examples of luminescent materials include luminol, and bioluminescent materials such as luciferase, luciferin, and aecolin. Examples of suitable radioactive substances include iodine, carbon, sulfur, tritium, indium, technetium, thallium, gallium, palladium, molybdenum, xenon, and fluorine.

[0166] Examples of additional post-translational modifications include transfer RNA-mediated amino acid addition to proteins such as acylation, ADP-ribosylation, amidation, covalent bonding of flavins, covalent bonding of heme moieties, covalent bonding of nucleotides or nucleotide derivatives, covalent bonding of lipids or lipid derivatives, covalent bonding of phosphatidylinositol, crosslinking, cyclization, disulfide bond formation, demethylation, covalent crosslinking, cysteine ​​formation, pyroglutamic acid formation, gamma-carboxylation, GPI anchor formation, hydroxylation, iodization, methylation, myristylation, pegylation, prenylation, racemization, selenoylation, sulfated amino acid addition, and ubiquitination.

[0167] Camptothecin analog The camptothecin analog contained in the ADC of this disclosure is of formula (I): It is a compound having TIFF0007877480000049.tif53165, in which, R 1 It is selected from -H, -CH3, -CHF2, -CF3, -F, -Br, -Cl, -OH, -OCH3, -OCF3 and -NH2, R 2 It is selected from -H, -CH3, -CF3, -F, -Br, -Cl, -OH, -OCH3, and -OCF3. R 1 If is -NH2, then R is R 3 or R 4 And R 1 If R is anything other than -NH2, then R is R 4 And, R 3 These are -H, -C1~C6 alkyl, -C3~C8 cycloalkyl, -(C1~C6 alkyl)-OR 5 , TIFF0007877480000050.tif27165-CO2R 8 Selected from -aryl, -heteroaryl, and -(C1~C6 alkyl)-aryl, R 4 teeth, Selected from TIFF0007877480000051.tif68165, R 5 The elements are selected from -H, -C1~C6 alkyl, -C3~C8 cycloalkyl, -aryl, -heteroaryl, -aryl, and -(C1~C6 alkyl)-aryl. R 6 and R 7 These are, independently, -H, -C1~C6 alkyl, -C3~C8 cycloalkyl, and -(C1~C6 alkyl)-OR 5 -C3~C8 heterocycloalkyl, and -C(O)R 17 Selected from, R 8These are selected from -H, -C1~C6 alkyl, -C3~C8 cycloalkyl, and -C3~C8 heterocycloalkyl. Each R 9 These are independently selected from -H, -C1~C6 alkyl, -C3~C8 cycloalkyl, -aryl, -heteroaryl, and -(C1~C6 alkyl)-aryl, Each R 10 These are independently -C1~C6 alkyl, -C3~C8 cycloalkyl, and -NR 14 R 14’ Selected from -aryl, -heteroaryl and -(C1~C6 alkyl)-aryl, R 10’ The elements are selected from -H, -C1~C6 alkyl, -C3~C8 cycloalkyl, -aryl, -heteroaryl, and -(C1~C6 alkyl)-aryl. R 11 These are selected from -H and -C1~C6 alkyl groups. R 12 -H, -C1~C6 alkyl, -CO2R 8 -aryl, -heteroaryl, -(C1~C6 alkyl)-aryl, -S(O)2R 16 , and Selected from TIFF0007877480000052.tif22165, R 13 These are selected from -H and -C1~C6 alkyl groups. R 14 and R 14’ Each of these is independently selected from -H, C1-C6 alkyl, -C3-C8 cycloalkyl, and -C3-C8 heterocycloalkyl. R 16 These are selected from -C1~C6 alkyl, -C3~C8 cycloalkyl, -aryl, -heteroaryl, and -(C1~C6 alkyl)-aryl. R 17 These are selected from -C1~C6 alkyl, -C3~C8 cycloalkyl, -C3~C8 heterocycloalkyl, -(C1~C6 alkyl)-C3~C8 heterocycloalkyl, -aryl, -heteroaryl, and -(C1~C6 alkyl)-aryl. R 18 and R 19 These, along with the N atom to which they are bonded, include halogens, -C1~C6 alkyl, -C3~C8 cycloalkyl and -(C1~C6 alkyl)-OR 5 Forms a 4, 5, 6, or 7-membered ring having 0 to 3 substituents selected from the above, R 24 , R 25 and R 26 These are each -C1 to C6 alkyl groups, X a and X b Each is independently selected from NH, O, and S. X c It is selected from O, S, and S(O)2, However, the compound is anything other than (S)-9-amino-11-butyl-4-ethyl-4-hydroxy-1,12-dihydro-14H-pyrano[3',4':6,7]indolidino[1,2-b]quinoline-3,14(4H)-dione.

[0168] In some embodiments, the camptothecin analog is a compound of formula (I), however, R 1 If R is NH2, 2 It is anything other than H.

[0169] In some embodiments, in the compound of formula (I), R 1 The ion is selected from -CH3, -CF3, -OCH3, -OCF3, and NH2.

[0170] In some embodiments, in the compound of formula (I), R 1 It is NH2.

[0171] In some embodiments, in the compound of formula (I), R 1 The group is selected from -H, -CH3, -CF3, -F, -Br, -Cl, -OH, -OCH3, and -OCF3.

[0172] In some embodiments, in the compound of formula (I), R 1The appropriate component is selected from -CH3, -CF3, -OCH3, and -OCF3.

[0173] In some embodiments, in the compound of formula (I), R 2 The molecule is selected from -H, -CH3, -CF3, -F, -Cl, -OCH3, and -OCF3.

[0174] In some embodiments, in the compound of formula (I), R 2 The molecule is selected from -CH3, -CF3, -F, -Cl, -OCH3, and -OCF3.

[0175] In some embodiments, in the compound of formula (I), R 2 The element is selected from -H, -F, -Br, and -Cl.

[0176] In some embodiments, in the compound of formula (I), R 2 The ion is selected from -F, -Br, and -Cl.

[0177] In some embodiments, in the compound of formula (I), R 3 -H, unsubstituted -C1~C6 alkyl, -C1~C6 haloalkyl, -C1~C6 hydroxyalkyl, -C3~C8 cycloalkyl, -(C1~C6 alkyl)-OR 5 , TIFF0007877480000053.tif27165-CO2R 8 Selected from unsubstituted-aryl, -aminoaryl, -heteroaryl, and -(C1~C6 alkyl)-aminoaryl.

[0178] In some embodiments, in the compound of formula (I), R 4 teeth, Selected from TIFF0007877480000054.tif58165.

[0179] In some embodiments, in the compound of formula (I), R 5The following are selected: -H, unsubstituted -C1~C6 alkyl, -C1~C6 haloalkyl, -C1~C6 hydroxyalkyl, -C1~C6 aminoalkyl, -C3~C8 cycloalkyl, unsubstituted -aryl, -aminoaryl, -heteroaryl, and -(C1~C6 alkyl)-aminoaryl.

[0180] In some embodiments, in the compound of formula (I), R 6 and R 7 These are, independently, -H, unsubstituted -C1~C6 alkyl, -C1~C6 haloalkyl, -C1~C6 hydroxyalkyl, -C3~C8 cycloalkyl, and -(C1~C6 alkyl)-OR 5 -C3~C8 heterocycloalkyl, and -C(O)R 17 Selected from.

[0181] In some embodiments, in the compound of formula (I), R 8 The group is selected from -H, unsubstituted -C1~C6 alkyl, -C1~C6 haloalkyl, -C1~C6 hydroxyalkyl, -C1~C6 aminoalkyl, -C3~C8 cycloalkyl, and -C3~C8 heterocycloalkyl.

[0182] In some embodiments, in the compound of formula (I), each R 9 These are independently selected from -C1~C6 alkyl, -C3~C8 cycloalkyl, -aryl, and -(C1~C6 alkyl)-aryl.

[0183] In some embodiments, in the compound of formula (I), each R 9 These are independently selected from -C1~C6 alkyl and -(C1~C6 alkyl)-aryl.

[0184] In some embodiments, in the compound of formula (I), each R 9These are independently selected from -H, unsubstituted -C1~C6 alkyl, -C1~C6 haloalkyl, -C1~C6 hydroxyalkyl, -C1~C6 aminoalkyl, -C3~C8 cycloalkyl, unsubstituted -aryl, -aminoaryl, -heteroaryl, and -(C1~C6 alkyl)-aminoaryl.

[0185] In some embodiments, in the compound of formula (I), each R 10 These are independently -C1~C6 alkyl, -C3~C8 cycloalkyl, and -NR 14 R 14’ Selected from -aryl and -(C1~C6 alkyl)-aryl.

[0186] In some embodiments, in the compound of formula (I), each R 10 These are independently -C1~C6 alkyl and -NR 14 R 14’ Selected from aryl and (C1-C6 alkyl)-aryl.

[0187] In some embodiments, in the compound of formula (I), each R 10 These are independently unsubstituted -C1~C6 alkyl, -C1~C6 haloalkyl, -C1~C6 hydroxyalkyl, -C1~C6 aminoalkyl, -C3~C8 cycloalkyl, and -NR. 14 R 14’ Selected from unsubstituted-aryl, -aminoaryl, -heteroaryl, and -(C1~C6 alkyl)-aryl.

[0188] In some embodiments, in the compound of formula (I), R 10’ The elements are selected from -H, unsubstituted -C1~C6 alkyl, -C1~C6 haloalkyl, -C1~C6 hydroxyalkyl, -C1~C6 aminoalkyl, -C3~C8 cycloalkyl, unsubstituted -aryl, -aminoaryl, -heteroaryl, and -(C1~C6 alkyl)-aryl.

[0189] In some embodiments, in the compound of formula (I), R 11The group is selected from -H, unsubstituted -C1~C6 alkyl, -C1~C6 haloalkyl, -C1~C6 hydroxyalkyl, and -C1~C6 aminoalkyl.

[0190] In some embodiments, in the compound of formula (I), each R 12 -H, -C1~C6 alkyl, -CO2R 8 -aryl, -(C1~C6 alkyl)-aryl and -S(O)2R 16 Selected from.

[0191] In some embodiments, in the compound of formula (I), R 12 -H, unsubstituted -C1~C6 alkyl, -C1~C6 haloalkyl, -C1~C6 hydroxyalkyl, -C1~C6 aminoalkyl, -CO2R 8 , unsubstituted-aryl, -aminoaryl, -heteroaryl, -(C1~C6 alkyl)-aminoaryl, -S(O)2R 16 , and Selected from TIFF0007877480000055.tif22165.

[0192] In some embodiments, in the compound of formula (I), R 13 The group is selected from -H, unsubstituted -C1~C6 alkyl, -C1~C6 haloalkyl, -C1~C6 hydroxyalkyl, and -C1~C6 aminoalkyl.

[0193] In some embodiments, in the compound of formula (I), R 14 and R 14’ Each of these is independently selected from -H, unsubstituted -C1~C6 alkyl, -C1~C6 haloalkyl, -C1~C6 hydroxyalkyl, -C1~C6 aminoalkyl, -C3~C8 cycloalkyl, and -C3~C8 heterocycloalkyl.

[0194] In some embodiments, in the compound of formula (I), R 16 The compound is selected from -aryl, -heteroaryl, and -(C1~C6 alkyl)-aryl compounds.

[0195] In some embodiments, in the compound of formula (I), R 16 The following are selected from unsubstituted -C1~C6 alkyl, -C1~C6 haloalkyl, -C1~C6 hydroxyalkyl, -C1~C6 aminoalkyl, -C3~C8 cycloalkyl, unsubstituted aryl, -aminoaryl, -heteroaryl, and -(C1~C6 alkyl)-aminoaryl.

[0196] In some embodiments, in the compound of formula (I), R 17 The following are selected from unsubstituted -C1~C6 alkyl, -C1~C6 hydroxyalkyl, -C3~C8 cycloalkyl, -C3~C8 heterocycloalkyl, -(C1~C6 alkyl)-C3~C8 heterocycloalkyl, unsubstituted aryl, -hydroxyaryl, -aminoaryl, -heteroaryl, and -(C1~C6 alkyl)-aminoaryl.

[0197] In some embodiments, in the compound of formula (I), R 18 and R 19 These, along with the N atom to which they are bonded, include halogens, unsubstituted -C1~C6 alkyls, -C1~C6 haloalkyls, -C1~C6 hydroxyalkyls, -C1~C6 aminoalkyls, -C3~C8 cycloalkyls, and -(C1~C6 alkyl)-OR 5 It forms a 4, 5, 6, or 7-membered ring having 0 to 3 substituents selected from the above.

[0198] In some embodiments, in the compound of formula (I), X a and X b Each of these is independently selected from NH and O.

[0199] Any combination of the aforementioned embodiments relating to the compound of formula (I) is also conceivable, and each combination forms a separate embodiment for the purposes of this disclosure.

[0200] In a particular embodiment, the compound of formula (I) is formula (II): It has TIFF0007877480000056.tif53165, and in the formula, R 2 It is selected from -H, -CH3, -CF3, -F, -Br, -Cl, -OH, -OCH3, and -OCF3. R 20 These are -H, -C1~C6 alkyl, -C3~C8 cycloalkyl, -(C1~C6 alkyl)-OR 5 , TIFF0007877480000057.tif27165-CO2R 8 -aryl, -heteroaryl, -(C1~C6 alkyl)-aryl, Selected from TIFF0007877480000058.tif68165, R 5 These are selected from -H, -C1~C6 alkyl, -C3~C8 cycloalkyl, -aryl, -heteroaryl, and -(C1~C6 alkyl)-aryl. R 6 and R 7 These are, independently, -H, -C1~C6 alkyl, -C3~C8 cycloalkyl, and -(C1~C6 alkyl)-OR 5 -C3~C8 heterocycloalkyl, and -C(O)R 17 Selected from, R 8 These are selected from -H, -C1~C6 alkyl, -C3~C8 cycloalkyl, and -C3~C8 heterocycloalkyl. Each R 9 These are independently selected from -H, -C1~C6 alkyl, -C3~C8 cycloalkyl, -aryl, -heteroaryl, and -(C1~C6 alkyl)-aryl, Each R 10 These are independently -C1~C6 alkyl, -C3~C8 cycloalkyl, and -NR 14 R 14’ Selected from -aryl, -heteroaryl and -(C1~C6 alkyl)-aryl, R 10’The elements are selected from -H, -C1~C6 alkyl, -C3~C8 cycloalkyl, -aryl, -heteroaryl, and -(C1~C6 alkyl)-aryl. R 11 These are selected from -H and -C1~C6 alkyl groups. R 12 -H, -C1~C6 alkyl, -CO2R 8 -aryl, -heteroaryl, -(C1~C6 alkyl)-aryl, -S(O)2R 16 and Selected from TIFF0007877480000059.tif22165, R 13 These are selected from -H and -C1~C6 alkyl groups. R 14 and R 14’ Each of these is independently selected from -H, C1-C6 alkyl, -C3-C8 cycloalkyl, and -C3-C8 heterocycloalkyl. R 16 These are selected from -C1~C6 alkyl, -C3~C8 cycloalkyl, -aryl, -heteroaryl, and -(C1~C6 alkyl)-aryl. R 17 These are selected from -C1~C6 alkyl, -C3~C8 cycloalkyl, -C3~C8 heterocycloalkyl, -(C1~C6 alkyl)-C3~C8 heterocycloalkyl, -aryl, -heteroaryl, and -(C1~C6 alkyl)-aryl. R 18 and R 19 These, along with the N atom to which they are bonded, include halogens, -C1~C6 alkyl, -C3~C8 cycloalkyl and -(C1~C6 alkyl)-OR 5 Forms a 4, 5, 6, or 7-membered ring having 0 to 3 substituents selected from the above, R 24 , R 25 and R 26 These are each -C1 to C6 alkyl groups, X a and X b Each is independently selected from NH, O, and S. Xc It is selected from O, S, and S(O)2, However, the compound is anything other than (S)-9-amino-11-butyl-4-ethyl-4-hydroxy-1,12-dihydro-14H-pyrano[3',4':6,7]indolidino[1,2-b]quinoline-3,14(4H)-dione.

[0201] In some embodiments, in the compound of formula (II), R 2 The group is selected from -CH3, -CF3, -F, -Br, -Cl, -OH, -OCH3, and -OCF3.

[0202] In some embodiments, in the compound of formula (II), R 2 The molecule is selected from -CH3, -CF3, -F, -Cl, -OCH3, and -OCF3.

[0203] In some embodiments, in the compound of formula (II), R 2 This is selected from F and Cl.

[0204] In some embodiments, in the compound of formula (II), R 20 -H, -C1~C6 alkyl, -(C1~C6 alkyl)-OR 5 , TIFF0007877480000060.tif27165-(C1~C6 alkyl)-aryl, Selected from TIFF0007877480000061.tif58165.

[0205] In some embodiments, in the compound of formula (II), R 20 -H, -C1~C6 alkyl, -(C1~C6 alkyl)-OR 5 , TIFF0007877480000062.tif27165-(C1~C6 alkyl)-aryl, Selected from TIFF0007877480000063.tif58165.

[0206] In some embodiments, in the compound of formula (II), R 20 -H, -C1~C6 alkyl, -(C1~C6 alkyl)-OR 5 , Selected from TIFF0007877480000064.tif58165.

[0207] In some embodiments, in the compound of formula (II), R 20 -H, unsubstituted -C1~C6 alkyl, -C1~C6 haloalkyl, -C1~C6 hydroxyalkyl, -C3~C8 cycloalkyl, -(C1~C6 alkyl)-OR 5 , TIFF0007877480000065.tif27165-CO2R 8 , unsubstituted aryl, -aminoaryl, -heteroaryl, -(C1~C6 alkyl)aminoaryl, Selected from TIFF0007877480000066.tif68165.

[0208] In some embodiments, in the compound of formula (II), R 2 It is selected from -CH3, -CF3, -F, -Br, -Cl, -OH, -OCH3, and -OCF3, R 20 -H, -C1~C6 alkyl, -(C1~C6 alkyl)-OR 5 , TIFF0007877480000067.tif27165-(C1~C6 alkyl)-aryl, Selected from TIFF0007877480000068.tif58165.

[0209] In some embodiments, in the compound of formula (II), R 2 It is selected from -CH3, -CF3, -F, -Br, -Cl, -OH, -OCH3, and -OCF3, R 20 -H, -C1~C6 alkyl, -(C1~C6 alkyl)-OR 5 , TIFF0007877480000069.tif27165-(C1~C6 alkyl)-aryl, Selected from TIFF0007877480000070.tif58165.

[0210] In some embodiments, in the compound of formula (II), R 2 It is selected from -CH3, -CF3, -F, -Br, -Cl, -OH, -OCH3, and -OCF3, R 20 -H, -C1~C6 alkyl, -(C1~C6 alkyl)-OR 5 , Selected from TIFF0007877480000071.tif58165.

[0211] In some embodiments, in the compound of formula (II), R 5 The following are selected: -H, unsubstituted -C1~C6 alkyl, -C1~C6 haloalkyl, -C1~C6 hydroxyalkyl, -C1~C6 aminoalkyl, -C3~C8 cycloalkyl, unsubstituted -aryl, -aminoaryl, -heteroaryl, and -(C1~C6 alkyl)-aminoaryl.

[0212] In some embodiments, in the compound of formula (II), R 6 and R 7 These are independently -H, -C1~C6 alkyl, -C3~C8 cycloalkyl, and -C(O)R 17 Selected from.

[0213] In some embodiments, in the compound of formula (II), R 6 H is R 7 These are -H, -C1~C6 alkyl, -C3~C8 cycloalkyl, -(C1~C6 alkyl)-OR 5 -C3~C8 heterocycloalkyl and -C(O)R 17 Selected from.

[0214] In some embodiments, in the compound of formula (II), R 6 H is R7 These are -H, -C1~C6 alkyl, -C3~C8 cycloalkyl and -C(O)R 17 Selected from.

[0215] In some embodiments, in the compound of formula (II), R 6 and R 7 These are, independently, -H, unsubstituted -C1~C6 alkyl, -C1~C6 haloalkyl, -C1~C6 hydroxyalkyl, -C3~C8 cycloalkyl, and -(C1~C6 alkyl)-OR 5 -C3~C8 heterocycloalkyl and -C(O)R 17 Selected from.

[0216] In some embodiments, in the compound of formula (II), R 8 The group is selected from -H, unsubstituted -C1~C6 alkyl, -C1~C6 haloalkyl, -C1~C6 hydroxyalkyl, -C1~C6 aminoalkyl, -C3~C8 cycloalkyl, and -C3~C8 heterocycloalkyl.

[0217] In some embodiments, in the compound of formula (II), each R 9 These are independently selected from -C1~C6 alkyl, -C3~C8 cycloalkyl, -aryl, and -(C1~C6 alkyl)-aryl.

[0218] In some embodiments, in the compound of formula (II), each R 9 These are independently selected from -C1~C6 alkyl and -(C1~C6 alkyl)-aryl.

[0219] In some embodiments, in the compound of formula (II), each R 9 These are independently selected from -H, unsubstituted -C1~C6 alkyl, -C1~C6 haloalkyl, -C1~C6 hydroxyalkyl, -C1~C6 aminoalkyl, -C3~C8 cycloalkyl, unsubstituted -aryl, -aminoaryl, -heteroaryl, and -(C1~C6 alkyl)-aminoaryl.

[0220] In some embodiments, in the compound of formula (II), each R 10 These are independently -C1~C6 alkyl, -C3~C8 cycloalkyl, and -NR 14 R 14’ Selected from -aryl and -(C1~C6 alkyl)-aryl.

[0221] In some embodiments, in the compound of formula (II), each R 10 These are independently -C1~C6 alkyl and -NR 14 R 14’ Selected from -aryl and -(C1~C6 alkyl)-aryl.

[0222] In some embodiments, in the compound of formula (II), each R 10 These are independently unsubstituted -C1~C6 alkyl, -C1~C6 haloalkyl, -C1~C6 hydroxyalkyl, -C1~C6 aminoalkyl, -C3~C8 cycloalkyl, and -NR. 14 R 14’ Selected from unsubstituted-aryl, -aminoaryl, -heteroaryl, and -(C1~C6 alkyl)-aryl.

[0223] In some embodiments, in the compound of formula (II), R 10’ The elements are selected from -H, unsubstituted -C1~C6 alkyl, -C1~C6 haloalkyl, -C1~C6 hydroxyalkyl, -C1~C6 aminoalkyl, -C3~C8 cycloalkyl, unsubstituted -aryl, -aminoaryl, -heteroaryl, and -(C1~C6 alkyl)-aryl.

[0224] In some embodiments, in the compound of formula (II), R 11 The group is selected from -H, unsubstituted -C1~C6 alkyl, -C1~C6 haloalkyl, -C1~C6 hydroxyalkyl, and -C1~C6 aminoalkyl.

[0225] In some embodiments, in the compound of formula (II), R 12-H, -C1~C6 alkyl, -CO2R 8 -aryl, -(C1~C6 alkyl)-aryl and -S(O)2R 16 Selected from.

[0226] In some embodiments, in the compound of formula (II), R 12 -H, unsubstituted -C1~C6 alkyl, -C1~C6 haloalkyl, -C1~C6 hydroxyalkyl, -C1~C6 aminoalkyl, -CO2R 8 , unsubstituted-aryl, -aminoaryl, -heteroaryl, -(C1~C6 alkyl)-aminoaryl, -S(O)2R 16 and Selected from TIFF0007877480000072.tif22165.

[0227] In some embodiments, in the compound of formula (II), R 13 The group is selected from -H, unsubstituted -C1~C6 alkyl, -C1~C6 haloalkyl, -C1~C6 hydroxyalkyl, and -C1~C6 aminoalkyl.

[0228] In some embodiments, in the compound of formula (II), R 14 and R 14’ Each of these is independently selected from -H, unsubstituted -C1~C6 alkyl, -C1~C6 haloalkyl, -C1~C6 hydroxyalkyl, -C1~C6 aminoalkyl, -C3~C8 cycloalkyl, and -C3~C8 heterocycloalkyl.

[0229] In some embodiments, in the compound of formula (II), R 16 The compound is selected from -aryl, -heteroaryl, and -(C1~C6 alkyl)-aryl compounds.

[0230] In some embodiments, in the compound of formula (II), R 16The following are selected from unsubstituted -C1~C6 alkyl, -C1~C6 haloalkyl, -C1~C6 hydroxyalkyl, -C1~C6 aminoalkyl, -C3~C8 cycloalkyl, unsubstituted -aryl, -aminoaryl, -heteroaryl, and -(C1~C6 alkyl)-aminoaryl.

[0231] In some embodiments, in the compound of formula (II), R 17 These are C1-C6 alkyl groups.

[0232] In some embodiments, in the compound of formula (II), R 17 The following are selected from unsubstituted -C1~C6 alkyl, -C1~C6 hydroxyalkyl, -C3~C8 cycloalkyl, -C3~C8 heterocycloalkyl, -(C1~C6 alkyl)-C3~C8 heterocycloalkyl, unsubstituted aryl, -hydroxyaryl, -aminoaryl, -heteroaryl, and -(C1~C6 alkyl)-aminoaryl.

[0233] In some embodiments, in the compound of formula (II), R 18 and R 19 These, along with the N atom to which they are bonded, include halogens, unsubstituted -C1~C6 alkyls, -C1~C6 haloalkyls, -C1~C6 hydroxyalkyls, -C1~C6 aminoalkyls, -C3~C8 cycloalkyls, and -(C1~C6 alkyl)-OR 5 It forms a 4, 5, 6, or 7-membered ring having 0 to 3 substituents selected from the above.

[0234] In some embodiments, in the compound of formula (II), X a and X b Each of these is independently selected from NH and O.

[0235] Any combination of the aforementioned embodiments relating to the compound of formula (II) is also conceivable, and each combination forms a separate embodiment for the purposes of this disclosure.

[0236] In certain embodiments, the compound of formula (I) is formula (III): It has TIFF0007877480000073.tif53165, and in the formula, R 2 It is selected from -H, -CH3, -CF3, -F, -Br, -Cl, -OH, -OCH3, and -OCF3. R 15 It is selected from -H, -CH3, -CHF2, -CF3, -F, -Br, -Cl, -OH, -OCH3, and -OCF3. R 4 teeth, Selected from TIFF0007877480000074.tif68165, R 5 These are selected from -H, -C1~C6 alkyl, -C3~C8 cycloalkyl, -aryl, -heteroaryl, and -(C1~C6 alkyl)-aryl. R 8 These are selected from -H, -C1~C6 alkyl, -C3~C8 cycloalkyl, and -C3~C8 heterocycloalkyl. Each R 9 These are independently selected from -H, -C1~C6 alkyl, -C3~C8 cycloalkyl, -aryl, -heteroaryl and -(C1~C6 alkyl)-aryl, Each R 10 These are independently -C1~C6 alkyl, -C3~C8 cycloalkyl, and -NR 14 R 14’ Selected from -aryl, -heteroaryl and -(C1~C6 alkyl)-aryl, R 10’ The elements are selected from -H, -C1~C6 alkyl, -C3~C8 cycloalkyl, -aryl, -heteroaryl, and -(C1~C6 alkyl)-aryl. R 11 These are selected from -H and -C1~C6 alkyl groups. R 12 -H, -C1~C6 alkyl, -CO2R 8 -aryl, -heteroaryl, -(C1~C6 alkyl)-aryl, -S(O)2R 16 and Selected from TIFF0007877480000075.tif22165, R 13 These are selected from -H and -C1~C6 alkyl groups. R 14 and R 14’ Each of these is independently selected from -H, C1-C6 alkyl, -C3-C8 cycloalkyl, and -C3-C8 heterocycloalkyl. R 16 These are selected from -C1~C6 alkyl, -C3~C8 cycloalkyl, -aryl, -heteroaryl, and -(C1~C6 alkyl)-aryl. R 18 and R 19 These, along with the N atom to which they are bonded, include halogens, -C1~C6 alkyls, -C3~C8 cycloalkyls, and -(C1~C6 alkyl)-OR 5 Forms a 4, 5, 6, or 7-membered ring having 0 to 3 substituents selected from the above, R 24 , R 25 and R 26 These are each -C1 to C6 alkyl groups, X a and X b Each is independently selected from NH, O, and S. X c This is selected from O, S, and S(O)2.

[0237] In some embodiments, in the compound of formula (III), R 2 The molecule is selected from -H, -CH3, -CF3, -F, -Cl, -OCH3, and -OCF3.

[0238] In some embodiments, in the compound of formula (III), R 2 The ion is selected from -H, -F, and -Cl.

[0239] In some embodiments, in the compound of formula (III), R 15 The appropriate component is selected from -CH3, -CF3, -OCH3, and -OCF3.

[0240] In some embodiments, in the compound of formula (III), R 15 The channel is selected from -CH3 and -OCH3.

[0241] In some embodiments, in the compound of formula (III), R 2 is selected from -H, -F, and -Cl, R 15 The appropriate component is selected from -CH3, -CF3, -OCH3, and -OCF3.

[0242] In some embodiments, in the compound of formula (III), R 2 is selected from -H, -F, and -Cl, R 15 The channel is selected from -CH3 and -OCH3.

[0243] In some embodiments, in the compound of formula (III), R 4 teeth, Selected from TIFF0007877480000076.tif58165.

[0244] In some embodiments, in the compound of formula (III), R 5 The following are selected: -H, unsubstituted -C1~C6 alkyl, -C1~C6 haloalkyl, -C1~C6 hydroxyalkyl, -C1~C6 aminoalkyl, -C3~C8 cycloalkyl, unsubstituted -aryl, -aminoaryl, -heteroaryl, and -(C1~C6 alkyl)-aminoaryl.

[0245] In some embodiments, in the compound of formula (III), R 8 The group is selected from -H, unsubstituted -C1~C6 alkyl, -C1~C6 haloalkyl, -C1~C6 hydroxyalkyl, -C1~C6 aminoalkyl, -C3~C8 cycloalkyl, and -C3~C8 heterocycloalkyl.

[0246] In some embodiments, in the compound of formula (III), each R 9These are independently selected from -C1~C6 alkyl, -C3~C8 cycloalkyl, -aryl, and -(C1~C6 alkyl)-aryl.

[0247] In some embodiments, in the compound of formula (III), each R 9 These are independently selected from -C1~C6 alkyl and -(C1~C6 alkyl)-aryl.

[0248] In some embodiments, in the compound of formula (III), each R 9 These are independently selected from -H, unsubstituted -C1~C6 alkyl, -C1~C6 haloalkyl, -C1~C6 hydroxyalkyl, -C1~C6 aminoalkyl, -C3~C8 cycloalkyl, unsubstituted -aryl, -aminoaryl, -heteroaryl, and -(C1~C6 alkyl)-aminoaryl.

[0249] In some embodiments, in the compound of formula (III), each R 10 These are independently -C1~C6 alkyl, -C3~C8 cycloalkyl, and -NR 14 R 14’ Selected from -aryl and -(C1~C6 alkyl)-aryl.

[0250] In some embodiments, in the compound of formula (III), each R 10 These are independently -C1~C6 alkyl and -NR 14 R 14’ Selected from -aryl and -(C1~C6 alkyl)-aryl.

[0251] In some embodiments, in the compound of formula (III), each R 10 These are independently unsubstituted -C1~C6 alkyl, -C1~C6 haloalkyl, -C1~C6 hydroxyalkyl, -C1~C6 aminoalkyl, -C3~C8 cycloalkyl, and -NR. 14 R 14’ Selected from unsubstituted-aryl, -aminoaryl, -heteroaryl, and -(C1~C6 alkyl)-aryl.

[0252] In some embodiments, in the compound of formula (III), R 10’ The elements are selected from -H, unsubstituted -C1~C6 alkyl, -C1~C6 haloalkyl, -C1~C6 hydroxyalkyl, -C1~C6 aminoalkyl, -C3~C8 cycloalkyl, unsubstituted -aryl, -aminoaryl, -heteroaryl, and -(C1~C6 alkyl)-aryl.

[0253] In some embodiments, in the compound of formula (III), R 11 The group is selected from -H, unsubstituted -C1~C6 alkyl, -C1~C6 haloalkyl, -C1~C6 hydroxyalkyl, and -C1~C6 aminoalkyl.

[0254] In some embodiments, in the compound of formula (III), R 12 -H, -C1~C6 alkyl, -CO2R 8 -aryl, -(C1~C6 alkyl)-aryl and -S(O)2R 16 Selected from.

[0255] In some embodiments, in the compound of formula (III), R 12 -H, unsubstituted -C1~C6 alkyl, -C1~C6 haloalkyl, -C1~C6 hydroxyalkyl, -C1~C6 aminoalkyl, -CO2R 8 , unsubstituted-aryl, -aminoaryl, -heteroaryl, -(C1~C6 alkyl)-aminoaryl, -S(O)2R 16 and Selected from TIFF0007877480000077.tif22165.

[0256] In some embodiments, in the compound of formula (III), R 13 The group is selected from -H, unsubstituted -C1~C6 alkyl, -C1~C6 haloalkyl, -C1~C6 hydroxyalkyl, and -C1~C6 aminoalkyl.

[0257] In some embodiments, in the compound of formula (III), R 14 and R 14’ Each of these is independently selected from -H, unsubstituted -C1~C6 alkyl, -C1~C6 haloalkyl, -C1~C6 hydroxyalkyl, -C1~C6 aminoalkyl, -C3~C8 cycloalkyl, and -C3~C8 heterocycloalkyl.

[0258] In some embodiments, in the compound of formula (III), R 16 The compound is selected from -aryl, -heteroaryl, and -(C1~C6 alkyl)-aryl compounds.

[0259] In some embodiments, in the compound of formula (III), R 16 The following are selected from unsubstituted -C1~C6 alkyl, -C1~C6 haloalkyl, -C1~C6 hydroxyalkyl, -C1~C6 aminoalkyl, -C3~C8 cycloalkyl, unsubstituted -aryl, -aminoaryl, -heteroaryl, and -(C1~C6 alkyl)-aminoaryl.

[0260] In some embodiments, in the compound of formula (III), R 18 and R 19 These, along with the N atom to which they are bonded, include halogens, unsubstituted -C1~C6 alkyls, -C1~C6 haloalkyls, -C1~C6 hydroxyalkyls, -C1~C6 aminoalkyls, -C3~C8 cycloalkyls, and -(C1~C6 alkyl)-OR 5 It forms a 4, 5, 6, or 7-membered ring having 0 to 3 substituents selected from the above.

[0261] In some embodiments, in the compound of formula (III), X a and X b Each of these is independently selected from NH and O.

[0262] Any combination of the aforementioned embodiments relating to the compound of formula (III) is also conceivable, and each combination forms a separate embodiment for the purposes of this disclosure.

[0263] In certain embodiments, each alkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl group, as defined in any one of formulas (I), (II), or (III), is optionally substituted with one or more substituents selected from halogens, acyls, acyloxys, alkoxys, carboxys, hydroxys, aminos, amides, nitros, cyanos, azides, alkylthios, thiosulfonyls, sulfonamides, alkyls, cycloalkyls, heterocycloalkyls, aryls, and heteroaryls. In some embodiments, each alkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl group, as defined in any one of formulas (I), (II), or (III), is optionally substituted with one or more substituents selected from halogens, acyls, acyloxys, alkoxys, carboxys, hydroxys, aminos, amides, nitros, cyanos, azides, alkylthios, thiosulfonyls, and sulfonamides.

[0264] In certain embodiments, the camptothecin analog contained in the ADC according to this disclosure is a compound having formula (I) and is selected from the compounds shown in Tables 6 and 7.

[0265] In certain embodiments, the camptothecin analog is a compound having formula (II). In some embodiments, the camptothecin analog is a compound having formula (II), where R 2 F is R 20 H, -(C1~C6)-OR 5 ,or The file is TIFF0007877480000078.tif27165. In some embodiments, the camptothecin analog is a compound having formula (II), where R 2 F is R 20 H, -(C1~C6)-OR 5 or TIFF0007877480000079.tif27165, R 5 H is R 18and R 19 These, together with the N atoms to which they are bonded, form an unsubstituted 4, 5, 6, or 7-membered ring. In some embodiments, the camptothecin analog is a compound having formula (II), where R 2 F is R 20 ha-(C1~C6)-OR 5 And R 5 is H. In certain embodiments, the camptothecin analog is a compound having formula (II) and is selected from the compounds shown in Table 6.

[0266] In certain embodiments, the camptothecin analog is a compound having formula (III). In certain embodiments, the camptothecin analog is a compound having formula (III), where R 2 F is R 15 is -CH3, R 4 teeth TIFF0007877480000080.tif27165, -R 9 It is a C1-C6 hydroxyalkyl group, and X a and X b Each of these is O. In a particular embodiment, the camptothecin analog is a compound having formula (III) and is selected from the compounds shown in Table 7.

[0267] In certain embodiments, the camptothecin analog included in the ADC according to this disclosure is compound 139, compound 140, compound 141, or compound 148. In some embodiments, the camptothecin analog included in the ADC according to this disclosure is compound 139 or compound 141.

[0268] [Table 6] TIFF0007877480000082.tif224165TIFF0007877480000083.tif214165TIFF0007877480000084.tif23016 5TIFF0007877480000085.tif230165TIFF0007877480000086.tif230165TIFF0007877480000087.tif97165

[0269] [Table 7] TIFF0007877480000089.tif230165TIFF0007877480000090.tif219165TIFF0007877480000091.tif218165TIFF0007877480000092.tif21916 5TIFF0007877480000093.tif230165TIFF0007877480000094.tif224165TIFF0007877480000095.tif224165TIFF0007877480000096.tif66165

[0270] Throughout this disclosure, references to compounds of formula (I) should be understood to the same extent as if specific embodiments were enumerated that enumerate each of these formulas or compounds individually, including compounds of formulas (II) and (III), as well as the individual compounds shown in Tables 6 and 7.

[0271] Antibody-drug conjugates This disclosure relates to an antibody-drug conjugate (ADC) comprising an anti-FRα antibody construct conjugated to a camptothecin analog having formula (I). In certain embodiments, the ADC is formula (X): T-[L-(D) m ] n (X) It has, in the formula, T is an anti-FRα antibody construct as described herein, L is a linker, D is a camptothecin analog having formula (I), m is 1 to 4, and n is between 1 and 10.

[0272] In certain embodiments, in the conjugate of equation (X), m is 1 to 2. In some embodiments, m is 1.

[0273] In some embodiments, in the conjugate of equation (X), n is between 1 and 8, for example, 2 to 8. In some embodiments, n is between 4 and 8.

[0274] In some embodiments, in the conjugate of formula (X), m is 1 to 2 and n is 2 to 8 or 4 to 8. In some embodiments, in the conjugate of formula (X), m is 1 and n is 2 to 8 or 4 to 8.

[0275] As reflected above and by the parameters m and n in formula (X), an anti-FRα antibody construct "T" can be conjugated with two or more compounds "D" of formula (I). While any particular anti-FRα antibody construct T is conjugated with an integer number of compounds D, those skilled in the art will understand that analysis of conjugate preparations to determine the ratio of compound D to anti-FRα antibody construct T may yield non-integer results reflecting the statistical mean. This ratio of compound D to the targeted portion T is sometimes commonly referred to as the drug-to-antibody ratio, or "DAR". Conjugate preparations with non-integer DARs are therefore intended to be encompassed by formula (X).

[0276] In certain embodiments, in the conjugate of formula (X), D is a compound of formula (II) or formula (III). In certain embodiments, in the conjugate of formula (X), D is a compound selected from the compounds shown in Tables 6 and 7. In certain embodiments, in the conjugate of formula (X), D is compound 139, compound 140, compound 141, or compound 148. In some embodiments, in the conjugate of formula (X), D is compound 139 or compound 141.

[0277] A particular embodiment of this disclosure relates to an ADC having formula (X), wherein D is formula (IV): The compound is TIFF0007877480000097.tif63165, and in the formula, R 1a It is selected from -H, -CH3, -CHF2, -CF3, -F, -Br, -Cl, -OH, -OCH3, -OCF3 and -NH2, R 2a It is selected from -H, -CH3, -CF3, -F, -Br, -Cl, -OH, -OCH3, and -OCF3. X is -O-, -S-, or -NH-, and R 4a teeth, Selected from TIFF0007877480000098.tif68165, where * is the connection point with X, and p is 1, 2, 3, or 4, or X is O and R 4a -X- is, Selected from TIFF0007877480000099.tif37165, R 5a These are selected from -C1~C6 alkyl, -C3~C8 cycloalkyl, -aryl, -heteroaryl, and -(C1~C6 alkyl)-aryl. R 8a These are selected from -C1~C6 alkyl, -C3~C8 cycloalkyl, and -C3~C8 heterocycloalkyl. Each R 9aThese are independently selected from -C1~C6 alkyl, -C3~C8 cycloalkyl, -aryl, -heteroaryl, and -(C1~C6 alkyl)-aryl, or R 9a If X does not exist b =X, Each R 10a These are independently -C1~C6 alkyl, -C3~C8 cycloalkyl, -aryl, -heteroaryl, -(C1~C6 alkyl)-aryl, and Selected from TIFF0007877480000100.tif22165, Each R 10a’ These are independently selected from -H, -C1~C6 alkyl, -C3~C8 cycloalkyl, -aryl, -heteroaryl, and -(C1~C6 alkyl)-aryl, Each R 10b These are independently selected from -C1~C6 alkyl, -C3~C8 cycloalkyl, -aryl, -heteroaryl, and -(C1~C6 alkyl)-aryl, R 11a It either does not exist or is a -C1~C6 alkyl group. R 12a These are -C1~C6 alkyl, -CO2R 8a -aryl, -heteroaryl, -(C1~C6 alkyl)-aryl, -S(O)2R 16a and Selected from TIFF0007877480000101.tif22165, R 13a These are selected from -H and -C1~C6 alkyl groups. R 14a These are selected from -C1~C6 alkyl, -C3~C8 cycloalkyl, and -C3~C8 heterocycloalkyl. R 14a’ The elements are selected from H, -C1~C6 alkyl, -C3~C8 cycloalkyl, and -C3~C8 heterocycloalkyl. R 16a These are selected from -C1~C6 alkyl, -C3~C8 cycloalkyl, -aryl, -heteroaryl, and -(C1~C6 alkyl)-aryl. R 21 These include -C1~C6 alkyl, -C3~C8 cycloalkyl, and -(C1~C6 alkyl)-OR 5a Selected from, R 22 and R 23 Each of these is independently selected from -H, -halogen, -C1~C6 alkyl, and -C3~C8 cycloalkyl. R 24 , R 25 and R 26 These are each -C1 to C6 alkyl groups, X a and X b Each is independently selected from NH, O, and S. X c is selected from O, S, and S(O)2, and TIFF0007877480000102.tif17165 indicates the connection point with linker L.

[0278] In some embodiments, in the compound of formula (IV), R 1a The ion is selected from -CH3, -CF3, -OCH3, -OCF3, and -NH2.

[0279] In some embodiments, in the compound of formula (IV), R 1a The appropriate component is selected from -CH3, -CF3, -OCH3, and -OCF3.

[0280] In some embodiments, in the compound of formula (IV), R 1a The ion is selected from -CH3, -OCH3, and NH2.

[0281] In some embodiments, in the compound of formula (IV), R 1a The channel is selected from -CH3 and -OCH3.

[0282] In some embodiments, in the compound of formula (IV), R 2a The molecule is selected from -H, -CH3, -CF3, -F, -Cl, -OCH3, and -OCF3.

[0283] In some embodiments, in the compound of formula (IV), R 2a The ion is selected from -H, -F, and -Cl.

[0284] In some embodiments, in the compound of formula (IV), R 2a It is -F.

[0285] In some embodiments, in the compound of formula (IV), X is -O-, -S-, or -NH-, and R 4a teeth, Selected from TIFF0007877480000103.tif58165.

[0286] In some embodiments, in the compound of formula (IV), X is -O- or -NH-.

[0287] In some embodiments, in the compound of formula (IV), each R 9a These are independently selected from -C1~C6 alkyl, -C3~C8 cycloalkyl, -aryl, and -(C1~C6 alkyl)-aryl.

[0288] In some embodiments, in the compound of formula (IV), each R 9a These are independently selected from -C1~C6 alkyl and -(C1~C6 alkyl)-aryl.

[0289] In some embodiments, in the compound of formula (IV), each R 10a These are independently -C1~C6 alkyl, -C3~C8 cycloalkyl, -aryl, -(C1~C6 alkyl)-aryl, and Selected from TIFF0007877480000104.tif22165.

[0290] In some embodiments, in the compound of formula (IV), each R 10a These are independently -C1~C6 alkyl, -aryl, -(C1~C6 alkyl)-aryl, and Selected from TIFF0007877480000105.tif22165.

[0291] In some embodiments, in the compound of formula (IV), R 12a These include -C1~C6 alkyl, -aryl, -(C1~C6 alkyl)-aryl, and -S(O)2R 16 Selected from.

[0292] In some embodiments, in the compound of formula (IV), R 13a The group is selected from -H, unsubstituted -C1~C6 alkyl, -C1~C6 haloalkyl, -C1~C6 hydroxyalkyl, and -C1~C6 aminoalkyl.

[0293] In some embodiments, in the compound of formula (IV), R 14a’ The element is selected from H, unsubstituted -C1~C6 alkyl, -C1~C6 haloalkyl, -C1~C6 hydroxyalkyl, -C1~C6 aminoalkyl, -C3~C8 cycloalkyl, and -C3~C8 heterocycloalkyl.

[0294] In some embodiments, in the compound of formula (IV), R 16a The aryl group is selected from -aryl, -heteroaryl, and -(C1-C6 alkyl)-aryl groups.

[0295] In some embodiments, in the compound of formula (IV), R 22 and R 23 Each of these is independently selected from -H, -halogen, unsubstituted -C1~C6 alkyl, -C1~C6 haloalkyl, -C1~C6 aminoalkyl, -C1~C6 hydroxyalkyl, and -C3~C8 cycloalkyl.

[0296] In some embodiments, in the compound of formula (IV), X a and X b Each of these is independently selected from NH and O.

[0297] In some embodiments, in the compound of formula (IV), X a and X b Each of these is O.

[0298] In some embodiments, in the compound of formula (IV), X is O and R 4a teeth, TIFF0007877480000106.tif27165, X a and X b Each of them is O, and R 9a It is a C1-C6 alkyl group.

[0299] In some embodiments, in the compound of formula (IV), R 1a is -CH3 or -OCH3, X is O, and R 4a teeth, TIFF0007877480000107.tif27165, X a and X b Each of them is O, and R 9a These are C1-C6 alkyl groups.

[0300] In some embodiments, in the compound of formula (IV), R 1a is -CH3 or -OCH3, and R 2a is H or F, X is O and R 4a teeth, TIFF0007877480000108.tif27165, X a and X b These are O and R 9a These are C1-C6 alkyl groups.

[0301] Other combinations of any of the aforementioned embodiments relating to the compound of formula (IV) are also conceivable, each combination forming a separate embodiment for the purposes of this disclosure.

[0302] A particular embodiment of this disclosure relates to an ADC having formula (X), wherein D is formula (V): The compound is TIFF0007877480000109.tif53165, and in the formula, R 2a It is selected from -CH3, -CF3, -F, -Br, -Cl, -OH, -OCH3, and -OCF3. R 20a These are -H, -C1~C6 alkyl, -C3~C8 cycloalkyl, -(C1~C6 alkyl)-OR 5 , TIFF0007877480000110.tif27165-CO2R 8 -aryl, -heteroaryl, -(C1~C6 alkyl)-aryl, Selected from TIFF0007877480000111.tif68165, R 5 These are selected from -H, -C1~C6 alkyl, -C3~C8 cycloalkyl, -aryl, -heteroaryl, and -(C1~C6 alkyl)-aryl. R 6 and R 7 These are, independently, -H, -C1~C6 alkyl, -C3~C8 cycloalkyl, and -(C1~C6 alkyl)-OR 5 -C3~C8 heterocycloalkyl, and -C(O)R 17 Selected from, R 8 These are selected from -H, -C1~C6 alkyl, -C3~C8 cycloalkyl, and -C3~C8 heterocycloalkyl. Each R 9 These are independently selected from -H, -C1~C6 alkyl, -C3~C8 cycloalkyl, -aryl, -heteroaryl and -(C1~C6 alkyl)-aryl, Each R 10 These are independently -C1~C6 alkyl, -C3~C8 cycloalkyl, -aryl, -heteroaryl, -(C1~C6 alkyl)-aryl, and -NR 14 R 14’ Selected from, Each R 10’These are independently selected from -H, -C1~C6 alkyl, -C3~C8 cycloalkyl, -aryl, -heteroaryl and -(C1~C6 alkyl)-aryl, R 11 These are selected from -H and -C1~C6 alkyl groups. R 12 -H, -C1~C6 alkyl, -CO2R 8 -aryl, -heteroaryl, -(C1~C6 alkyl)-aryl, -S(O)2R 16 and Selected from TIFF0007877480000112.tif22165, R 13 These are selected from -H and -C1~C6 alkyl groups. R 14 and R 14’ Each of these is independently selected from -H, C1-C6 alkyl, -C3-C8 cycloalkyl, and -C3-C8 heterocycloalkyl. R 16 These are selected from -C1~C6 alkyl, -C3~C8 cycloalkyl, -aryl, -heteroaryl, and -(C1~C6 alkyl)-aryl. R 17 These are selected from -C1~C6 alkyl, -C3~C8 cycloalkyl, -C3~C8 heterocycloalkyl, -(C1~C6 alkyl)-C3~C8 heterocycloalkyl, -aryl, -heteroaryl, and -(C1~C6 alkyl)-aryl. R 18 and R 19 These, along with the N atom to which they are bonded, include halogens, -C1~C6 alkyl, -C3~C8 cycloalkyl and -(C1~C6 alkyl)-OR 5 Forms a 4, 5, 6, or 7-membered ring having 0 to 3 substituents selected from the above, R 24 , R 25 and R 26 These are each -C1 to C6 alkyl groups, X a and X b Each is independently selected from NH, O, and S. Xc is selected from O, S, and S(O)2, and TIFF0007877480000113.tif17165 indicates the connection point with linker L.

[0303] In some embodiments, in the compound of formula (V), R 2a The molecule is selected from -CH3, -CF3, -F, -Cl, -OCH3, and -OCF3.

[0304] In some embodiments, in the compound of formula (V), R 2a The ion is selected from -CF3, -F, -Cl, and -OCH3.

[0305] In some embodiments, in the compound of formula (V), R 2a It is F.

[0306] In some embodiments, in the compound of formula (V), R 20a These are -H, -C1~C6 alkyl, -C3~C8 cycloalkyl, -(C1~C6 alkyl)-OR 5 , TIFF0007877480000114.tif27165-CO2R 8 -aryl, -heteroaryl, -(C1~C6 alkyl)-aryl, Selected from TIFF0007877480000115.tif58165.

[0307] In some embodiments, in the compound of formula (V), R 20a -H, -C1~C6 alkyl, -(C1~C6 alkyl)-OR 5 , TIFF0007877480000116.tif27165-(C1~C6 alkyl)-aryl, Selected from TIFF0007877480000117.tif58165.

[0308] In some embodiments, in the compound of formula (V), R 20a-H, -C1~C6 alkyl, -(C1~C6 alkyl)-OR 5 , TIFF0007877480000118.tif27165-(C1~C6 alkyl)-aryl, Selected from TIFF0007877480000119.tif58165.

[0309] In some embodiments, in the compound of formula (V), R 20a -H, -C1~C6 alkyl, -(C1~C6 alkyl)-OR 5 , Selected from TIFF0007877480000120.tif58165.

[0310] In some embodiments, in the compound of formula (V), R 20a -H, unsubstituted -C1~C6 alkyl, -C1~C6 haloalkyl, -C1~C6 hydroxyalkyl, -C3~C8 cycloalkyl, -(C1~C6 alkyl)-OR 5 , TIFF0007877480000121.tif27165-CO2R 8 , unsubstituted-aryl, -aminoaryl, -heteroaryl, -(C1~C6 alkyl)-aminoaryl, Selected from TIFF0007877480000122.tif68165.

[0311] In some embodiments, in the compound of formula (V), R 6 and R 7 These are independently -H, -C1~C6 alkyl, -C3~C8 cycloalkyl, and -C(O)R 17 Selected from.

[0312] In some embodiments, in the compound of formula (V), R 6 H is R 7 These are -H, -C1~C6 alkyl, -C3~C8 cycloalkyl, -(C1~C6 alkyl)-OR 5-C3~C8 heterocycloalkyl, and -C(O)R 17 Selected from.

[0313] In some embodiments, in the compound of formula (V), R 6 H is R 7 These are -H, -C1~C6 alkyl, -C3~C8 cycloalkyl and -C(O)R 17 Selected from.

[0314] In some embodiments, in the compound of formula (V), R 6 and R 7 These are, independently, -H, unsubstituted -C1~C6 alkyl, -C1~C6 haloalkyl, -C1~C6 hydroxyalkyl, -C3~C8 cycloalkyl, and -(C1~C6 alkyl)-OR 5 -C3~C8 heterocycloalkyl, and -C(O)R 17 Selected from.

[0315] In some embodiments, in the compound of formula (V), R 8 The group is selected from -H, unsubstituted -C1~C6 alkyl, -C1~C6 haloalkyl, -C1~C6 hydroxyalkyl, -C1~C6 aminoalkyl, -C3~C8 cycloalkyl, and -C3~C8 heterocycloalkyl.

[0316] In some embodiments, in the compound of formula (V), each R 9 These are independently selected from -C1~C6 alkyl, -C3~C8 cycloalkyl, -aryl, and -(C1~C6 alkyl)-aryl.

[0317] In some embodiments, in the compound of formula (V), each R 9 These are independently selected from -C1~C6 alkyl and -(C1~C6 alkyl)-aryl.

[0318] In some embodiments, in the compound of formula (V), each R 9These are independently selected from -H, unsubstituted -C1~C6 alkyl, -C1~C6 haloalkyl, -C1~C6 hydroxyalkyl, -C1~C6 aminoalkyl, -C3~C8 cycloalkyl, unsubstituted -aryl, -aminoaryl, -heteroaryl, and -(C1~C6 alkyl)-aminoaryl.

[0319] In some embodiments, in the compound of formula (V), each R 10 These are independently -C1~C6 alkyl, -C3~C8 cycloalkyl, and -NR 14 R 14’ Selected from aryl and (C1-C6 alkyl)-aryl.

[0320] In some embodiments, in the compound of formula (V), each R 10 These are independently -C1~C6 alkyl and -NR 14 R 14’ Selected from -aryl and -(C1~C6 alkyl)-aryl.

[0321] In some embodiments, in the compound of formula (V), R 11 The group is selected from -H, unsubstituted -C1~C6 alkyl, -C1~C6 haloalkyl, -C1~C6 hydroxyalkyl, and -C1~C6 aminoalkyl.

[0322] In some embodiments, in the compound of formula (V), R 12 These include -H, -C1~C6 alkyl, -aryl, -(C1~C6 alkyl)-aryl, and -S(O)2R 16 Selected from.

[0323] In some embodiments, in the compound of formula (V), R 12 -H, unsubstituted -C1~C6 alkyl, -C1~C6 haloalkyl, -C1~C6 hydroxyalkyl, -C1~C6 aminoalkyl, -CO2R 8 , unsubstituted-aryl, -aminoaryl, -heteroaryl, -(C1~C6 alkyl)-aminoaryl, -S(O)2R 16 and Selected from TIFF0007877480000123.tif22165.

[0324] In some embodiments, in the compound of formula (V), R 13 The group is selected from -H, unsubstituted -C1~C6 alkyl, -C1~C6 haloalkyl, -C1~C6 hydroxyalkyl, and -C1~C6 aminoalkyl.

[0325] In some embodiments, in the compound of formula (V), R 14 and R 14’ Each of these is independently selected from -H, unsubstituted -C1~C6 alkyl, -C1~C6 haloalkyl, -C1~C6 hydroxyalkyl, -C1~C6 aminoalkyl, -C3~C8 cycloalkyl, and -C3~C8 heterocycloalkyl.

[0326] In some embodiments, in the compound of formula (V), R 16 The aryl group is selected from -aryl, -heteroaryl, and -(C1-C6 alkyl)-aryl groups.

[0327] In some embodiments, in the compound of formula (V), R 16 The following are selected from unsubstituted -C1~C6 alkyl, -C1~C6 haloalkyl, -C1~C6 hydroxyalkyl, -C1~C6 aminoalkyl, -C3~C8 cycloalkyl, unsubstituted -aryl, -aminoaryl, -heteroaryl, and -(C1~C6 alkyl)-aminoaryl.

[0328] In some embodiments, in the compound of formula (V), R 17 The following are selected from unsubstituted -C1~C6 alkyl, -C3~C8 cycloalkyl, -C3~C8 heterocycloalkyl, -(C1~C6 alkyl)-C3~C8 heterocycloalkyl, unsubstituted -aryl, -hydroxyaryl, -aminoaryl, -heteroaryl, and -(C1~C6 alkyl)-aminoaryl.

[0329] In some embodiments, in the compound of formula (V), R 18 and R 19 These, along with the N atom to which they are bonded, include halogens, unsubstituted -C1~C6 alkyls, -C1~C6 haloalkyls, -C1~C6 aminoalkyls, -C1~C6 hydroxyalkyls, -C3~C8 cycloalkyls, and -(C1~C6 alkyl)-OR 5 It forms a 4, 5, 6, or 7-membered ring having 0 to 3 substituents selected from the above.

[0330] In some embodiments, in the compound of formula (V), R 17 These are C1-C6 alkyl groups.

[0331] In some embodiments, in a compound of formula (V), X a and X b Each of these is independently selected from NH and O.

[0332] In some embodiments, in a compound of formula (V), X a and X b These are independently O.

[0333] In some embodiments, in the compound of formula (V), R 20a is -(C1~C6 alkyl)-OR 5 That is the case.

[0334] In some embodiments, in the compound of formula (V), R 20a is -(C1~C6 alkyl)-OR 5 And R 5 H is H.

[0335] In some embodiments, in the compound of formula (V), R 2a F is R 20a is -(C1~C6 alkyl)-OR 5 And R 5 H is H.

[0336] Other combinations of any of the aforementioned embodiments relating to the compound of formula (V) are also conceivable, each combination forming a separate embodiment for the purposes of this disclosure.

[0337] A particular embodiment of this disclosure relates to an ADC having formula (X), wherein D is formula (VI): It is a compound of TIFF0007877480000124.tif63165, in which, R 2a It is selected from -H, -CH3, -CF3, -F, -Br, -Cl, -OH, -OCH3, and -OCF3. X is -O-, -S-, or -NH-, and R 25 -C1~C6 alkyl, -(C1~C6 alkyl)-OR 5a , -CO2R 8a -aryl, -heteroaryl, -(C1~C6 alkyl)-aryl, Selected from TIFF0007877480000125.tif94165, where * is the connection point with X, and p is 1, 2, 3, or 4, or X is O and R 25 -X- is, Selected from TIFF0007877480000126.tif37165, R 5a These are selected from -C1~C6 alkyl, -C3~C8 cycloalkyl, -aryl, -heteroaryl, and -(C1~C6 alkyl)-aryl. R 6a These are selected from -H, -C1~C6 alkyl, -C3~C8 cycloalkyl, and -C3~C8 heterocycloalkyl. R 7a -C1~C6 alkyl, -C3~C8 cycloalkyl, -(C1~C6 alkyl)-OR 5a -C3~C8 heterocycloalkyl and -C(O)R 17a Selected from, R 8a These are selected from -C1~C6 alkyl, -C3~C8 cycloalkyl, and -C3~C8 heterocycloalkyl. Each R9a These are independently selected from -C1~C6 alkyl, -C3~C8 cycloalkyl, -aryl, -heteroaryl, and -(C1~C6 alkyl)-aryl, or R 9a It does not exist and X b =X, Each R 10a These are independently -C1~C6 alkyl, -C3~C8 cycloalkyl, -aryl, -heteroaryl, -(C1~C6 alkyl)-aryl, and Selected from TIFF0007877480000127.tif22165, Each R 10a’ These are independently selected from -H, -C1~C6 alkyl, -C3~C8 cycloalkyl, -aryl, -heteroaryl, and -(C1~C6 alkyl)-aryl, Each R 10b These are independently selected from -C1~C6 alkyl, -C3~C8 cycloalkyl, -aryl, -heteroaryl, and -(C1~C6 alkyl)-aryl, R 11a It either does not exist or is a -C1~C6 alkyl group. R 12a These are -C1~C6 alkyl, -CO2R 8a -aryl, -heteroaryl, -(C1~C6 alkyl)-aryl, -S(O)2R 16a , and Selected from TIFF0007877480000128.tif22165, R 13a These are selected from -H and -C1~C6 alkyl groups. R 14a These are selected from -C1~C6 alkyl, -C3~C8 cycloalkyl, and -C3~C8 heterocycloalkyl. R 14a’ The elements are selected from H, -C1~C6 alkyl, -C3~C8 cycloalkyl, and -C3~C8 heterocycloalkyl. R 16a These are selected from -C1~C6 alkyl, -C3~C8 cycloalkyl, -aryl, -heteroaryl, and -(C1~C6 alkyl)-aryl. R 17a These are selected from -C1~C6 alkyl, -C3~C8 cycloalkyl, -C3~C8 heterocycloalkyl, -(C1~C6 alkyl)-C3~C8 heterocycloalkyl, -aryl, -heteroaryl, and -(C1~C6 alkyl)-aryl. R 21 These include -C1~C6 alkyl, -C3~C8 cycloalkyl, and -(C1~C6 alkyl)-OR 5a Selected from, R 22 and R 23 Each of these is independently selected from -H, -halogen, -C1~C6 alkyl, and -C3~C8 cycloalkyl. R 24 , R 25 and R 26 These are each -C1 to C6 alkyl groups, X a and X b Each is independently selected from NH, O, and S. X c is selected from O, S, and S(O)2, and TIFF0007877480000129.tif17165 indicates the connection point with linker L.

[0338] In some embodiments, in the compound of formula (VI), R 2a The group is selected from -CH3, -CF3, -F, -Br, -Cl, -OH, -OCH3, and -OCF3.

[0339] In some embodiments, in the compound of formula (VI), R 2a The molecule is selected from -CH3, -CF3, -F, -Cl, -OCH3, and -OCF3.

[0340] In some embodiments, in the compound of formula (VI), R 2a This is selected from F and Cl.

[0341] In some embodiments, in the compound of formula (VI), R 2aIt is F.

[0342] In some embodiments, in the compound of formula (VI), X is -O-, -S-, or -NH-, and R 25 -C1~C6 alkyl, -(C1~C6 alkyl)-OR 5a , -(C1~C6 alkyl)-aryl, Selected from TIFF0007877480000130.tif58165, or X is O and R 25 -X- is, Selected from TIFF0007877480000131.tif37165.

[0343] In some embodiments, in the compound of formula (VI), X is -O-, -S-, or -NH-, and R 25 -C1~C6 alkyl, -(C1~C6 alkyl)-OR 5a , -(C1~C6 alkyl)-aryl, Selected from TIFF0007877480000132.tif58165.

[0344] In some embodiments, in the compound of formula (VI), X is -O-, -S-, or -NH-, and R 25 -C1~C6 alkyl, -(C1~C6 alkyl)-OR 5a , Selected from TIFF0007877480000133.tif58165.

[0345] In some embodiments, in the compound of formula (VI), X is -O-, -S-, or -NH-, and R 25 teeth, Selected from TIFF0007877480000134.tif58165.

[0346] In some embodiments, in the compound of formula (VI), X is -O- or -NH-.

[0347] In some embodiments, in the compound of formula (VI), R 6a H is H.

[0348] In some embodiments, in the compound of formula (VI), R 6a The group is selected from -H, unsubstituted -C1~C6 alkyl, -C1~C6 haloalkyl, -C1~C6 hydroxyalkyl, -C3~C8 cycloalkyl, and -C3~C8 heterocycloalkyl.

[0349] In some embodiments, in the compound of formula (VI), R 7a These include -C1~C6 alkyl, -C3~C8 cycloalkyl, and -C(O)R 17a Selected from.

[0350] In some embodiments, in the compound of formula (VI), each R 9a These are independently selected from -C1~C6 alkyl, -C3~C8 cycloalkyl, -aryl, and -(C1~C6 alkyl)-aryl.

[0351] In some embodiments, in the compound of formula (VI), each R 9a These are independently selected from -C1~C6 alkyl and -(C1~C6 alkyl)-aryl.

[0352] In some embodiments, in the compound of formula (VI), each R 10a These are independently -C1~C6 alkyl, -C3~C8 cycloalkyl, -aryl, -(C1~C6 alkyl)-aryl, and Selected from TIFF0007877480000135.tif22165.

[0353] In some embodiments, in the compound of formula (VI), each R 10a These are independently -C1~C6 alkyl, -aryl, -(C1~C6 alkyl)-aryl, and Selected from TIFF0007877480000136.tif22165.

[0354] In some embodiments, in the compound of formula (VI), R 12a These include -C1~C6 alkyl, -aryl, -(C1~C6 alkyl)-aryl, and -S(O)2R 16a Selected from.

[0355] In some embodiments, in the compound of formula (VI), R 13a The group is selected from -H, unsubstituted -C1~C6 alkyl, -C1~C6 haloalkyl, -C1~C6 hydroxyalkyl, and -C1~C6 aminoalkyl.

[0356] In some embodiments, in the compound of formula (VI), R 14a’ The element is selected from H, unsubstituted -C1~C6 alkyl, -C1~C6 haloalkyl, -C1~C6 hydroxyalkyl, -C1~C6 aminoalkyl, -C3~C8 cycloalkyl, and -C3~C8 heterocycloalkyl.

[0357] In some embodiments, in the compound of formula (VI), R 16a The aryl group is selected from -aryl, -heteroaryl, and -(C1-C6 alkyl)-aryl groups.

[0358] In some embodiments, in the compound of formula (VI), R 17a These are C1-C6 alkyl groups.

[0359] In some embodiments, in the compound of formula (VI), R 22 and R 23 Each of these is independently selected from -H, -halogen, unsubstituted -C1~C6 alkyl, -C1~C6 haloalkyl, -C1~C6 hydroxyalkyl, -C1~C6 aminoalkyl, and -C3~C8 cycloalkyl.

[0360] In some embodiments, in the compound of formula (VI), X a and X b Each of these is independently selected from NH and O.

[0361] In some embodiments, in the compound of formula (VI), X a and X b Each of these is O.

[0362] In some embodiments, in the compound of formula (VI), X is O and R 25 These are C1-C6 alkyl groups.

[0363] In some embodiments, in the compound of formula (VI), R 2a is F, X is O, and R 25 These are C1-C6 alkyl groups.

[0364] Other combinations of any of the aforementioned embodiments relating to the compound of formula (VI) are also conceivable, each combination forming a separate embodiment for the purposes of this disclosure.

[0365] In certain embodiments, each alkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl group, as defined in any one of formulas (IV), (V), or (VI), is optionally substituted with one or more substituents selected from halogens, acyls, acyloxys, alkoxys, carboxys, hydroxys, aminos, amides, nitros, cyanos, azides, alkylthios, thiosulfonyls, sulfonamides, alkyls, cycloalkyls, heterocycloalkyls, aryls, and heteroaryls. In some embodiments, each alkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl group, as defined in any one of formulas (IV), (V), or (VI), is optionally substituted with one or more substituents selected from halogens, acyls, acyloxys, alkoxys, carboxys, hydroxys, aminos, amides, nitros, cyanos, azides, alkylthios, thiosulfonyls, and sulfonamides.

[0366] In a particular embodiment, in an ADC having formula (X), D is a compound of formula (IV), where R1a is -CH3, R 2a is F. In some embodiments, in an ADC having formula (X), D is a compound of formula (IV), where R 1a is -CH3, R 2a is F, X is -O-, and R 4a teeth TIFF0007877480000137.tif27165, R 9a is a C1-C6 alkyl group, X a and X b Each of these is O.

[0367] In a particular embodiment, in an ADC having formula (X), D is a compound of formula (V), where R 2a F is R 20a H, -(C1~C6)-OR 5 ,or The file is TIFF0007877480000138.tif27165. In some embodiments, in an ADC having formula (X), D is a compound of formula (V), where R 2a F is R 20a H, -(C1~C6)-OR 5 ,or TIFF0007877480000139.tif27165, R 5 H is R 18 and R 19 These, together with the N atoms to which they are bonded, form an unsubstituted 4, 5, 6, or 7-membered ring. In some embodiments, in an ADC having formula (X), D is a compound of formula (V), where R 2a F is R 20a is -(C1~C6)-OR 5 And R 5 H is H.

[0368] In a particular embodiment, in an ADC having formula (X), D is a compound of formula (VI), where R 2a is F, X is -O-, and R 25 It is a C1-C6 alkyl group.

[0369] Linker L The conjugate of formula (X) comprises a linker L, which is a bifunctional or polyfunctional moiety capable of linking one or more camptothecin analogs D to an anti-FRα antibody construct T. A bifunctional (or monovalent) linker L links a single compound D to a single site on the anti-FRα antibody construct T, while a polyfunctional (or polyvalent) linker L links two or more compounds D to a single site on the anti-FRα antibody construct T. A linker linking one compound D to two or more sites on the anti-FRα antibody construct T may also be considered polyfunctional.

[0370] Linker L comprises a functional group capable of reacting with a target group(s) on the anti-FRα antibody construct T, and at least one functional group capable of reacting with a target group on the camptothecin analog D. Suitable functional groups are known in the art and, for example, those described in Bioconjugate Techniques (G.T. Humanson, 2013, Academic Press). Groups on the anti-FRα antibody construct T and camptothecin analog D that can function as target groups for linker binding include, but are not limited to, thiols, hydroxyls, carboxyls, amines, aldehydes, and ketone groups.

[0371] Non-limiting examples of functional groups that can react with thiols include maleimides, haloacetamides, haloacetyls, activated esters (e.g., succinimide esters, 4-nitrophenyl esters, pentafluorophenyl esters, and tetrafluorophenyl esters), anhydrides, acid chlorides, sulfonyl chlorides, isocyanates, and isothiocyanates. In this context, the "self-stabilizing" maleimides described in Lyon et al., 2014, Nat. Biotechnol., 32:1059-1062 are also useful.

[0372] Non-limiting examples of functional groups that can react with amines include activated esters (e.g., N-hydroxysucciniamide (NHS) esters and sulfo-NHS esters), imide esters (e.g., Traut's reagent), isothiocyanates, aldehydes, and acid anhydrides (e.g., diethylenetriaminepentaanhydride (DTPA)). Alternatively, succinimide-1,1,3,3-tetramethyluroniumtetrafluoroboric acid (TSTU) or benzotriazole-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP) may be used to convert the carboxylic acid to an activated ester, which may then be reacted with the amine.

[0373] Non-limiting examples of functional groups that can react with electrophiles such as aldehydes or ketone carbonyl groups include hydrazides, oximes, aminos, hydrazines, thiosemicarbazones, hydrazine carboxylates, and aryl hydrazides.

[0374] In certain embodiments, linker L includes a functional group that enables crosslinking of two interchain cysteine ​​on an anti-FRα antibody construct, such as the ThioBridge® linker (Badescu et al., 2014, Bioconjug. Chem. 25:1124-1136), the dithiomaleimide (DTM) linker (Behrens et al., 2015, Mol. Pharm. 12:3986-3998), the dithioaryl (TCEP) pyridazinedione linker (Lee et al., 2016, Chem. Sci., 7:799-802), or the dibromopyridazinedione linker (Maruani et al., 2015, Nat. Commun., 6:6645).

[0375] Alternatively, the anti-FRα antibody construct T may be modified to include a non-natural reactive group, such as an azide, that enables conjugation with the linker via a complementary reactive group on the linker. For example, the conjugation of the linker to the anti-FRα antibody construct may utilize click chemical reactions such as the azide-alkyne cycloaddition (AAC) reaction, which has been successfully used in the development of antibody-drug conjugates (see, e.g., Chio & Bane, 2020, Methods Mol. Biol., 2078:83-97). The AAC reaction may be a copper-catalyzed AAC (CuAAC) reaction involving the coupling of an azide with a linear alkyne, or a strain-enhanced AAC (SPAAC) reaction involving the coupling of an azide with cyclooctyne.

[0376] Linker L may be a cleavable or incleavable linker. A cleavable linker is one that is easily cleaved under specific conditions, such as intracellular conditions (e.g., within endosomes or lysosomes) or around a target cell (e.g., the tumor microenvironment). Examples include protease-sensitive, acid-sensitive, or reduction-sensitive linkers. In contrast, incleavable linkers generally rely on the degradation of antibodies in the cell, which results in the release of the amino acid-linker-drug moiety.

[0377] Examples of cleavable linkers include, for example, linkers containing amino acid sequences that are protease cleavage recognition sequences. Many such cleavage recognition sequences are known in the art. For conjugates not intended to be internally transported by cells, amino acid sequences that are recognized and cleaved by proteases present in the extracellular matrix surrounding target cells, such as cancer cells, may be used. Examples of extracellular tumor-associated proteases include, for example, plasmin, matrix metalloproteinases (MMPs), elastases, and kallikrein-associated peptidases.

[0378] With respect to conjugates intended to be internally transported by cells, linker L may contain an amino acid sequence that is recognized and cleaved by an endosomal or lysosomal protease. Examples of such proteases include, for example, cathepsins B, C, D, H, L, and S, as well as regmine.

[0379] The cleavage recognition sequence may be, for example, a dipeptide, tripeptide, or tetrapeptide. Non-limiting examples of dipeptide recognition sequences that may be included in the cleavable linker include, but are not limited to, Ala-(D)Asp, Ala-Lys, Ala-Phe, Asn-Lys, Asn-(D)Lys, Asp-Val, His-Val, Ile-Cit, Ile-Pro, Ile-Val, Leu-Cit, Me3Lys-Pro, Met-Lys, Met-(D)Lys, NorVal-(D)Asp, Phe-Arg, Phe-Cit, Phe-Lys, PhenylGly-(D)Lys, Pro-(D)Lys, Trp-Cit, Val-Ala, Val-(D)Asp, Val-Cit, Val-Gly, Val-Gln, and Val-Lys. Examples of tri and tetrapeptide cleavage sequences include, but are not limited to, Ala-Ala-Asn, Ala-Val-Cit, (D)Ala-Phe-Lys, Asp-Val-Ala, Asp-Val-Cit, Gly-Cit-Val, Lys-Val-Ala, Lys-Val-Cit, Met-Cit-Val, (D)Phe-Phe-Lys, Asn-Pro-Val, Ala-Leu-Ala-Leu, Gly-Phe-Leu-Gly, Gly-Gly-Phe-Gly, and Gly-Phe-Gly-Gly.

[0380] Further examples of cleavable linkers include disulfide-containing linkers such as N-succinimidyl-4-(2-pyridyldithio)butanoate (SPDB) and N-succinimidyl-4-(2-pyridyldithio)-2-sulfobutanoate (sulfo-SPDB). Disulfide-containing linkers may optionally include additional groups that introduce steric hindrance adjacent to the disulfide bond, such as geminal dimethyl groups, to improve the extracellular stability of the linker. Other cleavable linkers include linkers that are hydrolyzable at a specific pH or pH range, such as hydrazone linkers. Linkers containing combinations of these functional groups may also be useful; for example, linkers containing both hydrazone and disulfide are known in the art.

[0381] Further examples of cleavable linkers include linkers containing β-glucuronides, which can be cleaved by β-glucuronidase, an enzyme present in lysosomes and tumor stroma (see, for example, De Graaf et al., 2002, Curr. Pharm. Des. 8:1391-1403, and International Patent Publication WO2007 / 011968). β-glucuronides can also function to improve the hydrophilicity of linker L.

[0382] Another example of a linker that is cleaved within a cell to improve hydrophilicity is a linker containing a pyrophosphate diester moiety (see, for example, Kern et al., 2016, J Am Chem Soc., 138:2430-1445).

[0383] In certain embodiments, the linker L contained in the conjugate of formula (X) is a cleavable linker. In some embodiments, linker L includes a cleavage recognition sequence. In some embodiments, linker L may also include an amino acid sequence that is recognized and cleaved by a lysosomal protease.

[0384] The cleavable linker may optionally further contain one or more additional functional groups, such as self-destructing groups, self-detaching groups, stretchers, or hydrophilic moieties.

[0385] Examples of self-destructing and self-detaching groups that find use as linkers include p-aminobenzyl (PAB) and p-aminobenzyloxycarbonyl (PABC) groups, methylated ethylenediamine (MED), and hemiaminal groups. Other examples of self-destructing groups include, but are not limited to, heterocyclic derivatives, such as the 2-aminoimidazole-5-methanol derivative described in U.S. Patent No. 7,375,078, and other aromatic compounds that are electronically similar to PAB or PABC groups. Other examples include groups that cyclize upon hydrolysis of the amide bond, such as substituted and unsubstituted 4-aminobutyric acid amides (Rodrigues et al., 1995, Chemistry Biology 2:223-227) and 2-aminophenylpropionic acid amides (Amsberry, et al., 1990, J. Org. Chem. 55:5867-5877). Self-destructing / self-detaching groups are generally bonded to the amino or hydroxyl group of compound D. Self-destructing / self-detaching groups are often found alone or in combination in peptide-based linkers, but may also be found in other types of linkers.

[0386] Examples of stretchers used as linkers for drug conjugates include alkylene group-based and fatty acid, dibasic acid, amine, or diamine-based stretchers, such as diglycolate, malonate, caproate, and caproamide. Other stretchers include, for example, glycine-based stretchers and polyethylene glycol (PEG) or monomethoxypolyethylene glycol (mPEG) stretchers.

[0387] PEG and mPEG stretchers can also function as hydrophilic portions within the linker. For example, PEG or mPEG can be incorporated into the linker either "inline" or as a pendant group to enhance the linker's hydrophilicity (see, for example, U.S. Patent Application Publication US2016 / 0310612). Various PEG-containing linkers are commercially available from companies such as Quanta BioDesign, Ltd (Plain City, OH). Other hydrophilic groups that may optionally be incorporated into linker L include, for example, β-glucuronides, sulfonates, carboxylates, and pyrophosphate diesters.

[0388] In certain embodiments, the ADC of formula (X) may include a cleavable linker. In certain embodiments, the ADC of formula (X) may include a peptide-containing linker. In certain embodiments, the ADC of formula (X) may include a protease-cleavable linker.

[0389] In some embodiments, in the ADC of formula (X), m is 1 and the linker L is given by formula (XI): A cuttable linker having TIFF0007877480000140.tif32165, in the formula, Z is a functional group that can react with the target group on the anti-FRα antibody construct T. Str stands for stretcher, AA1 and AA2 are amino acids, and AA1-[AA2] r It forms a protease cleavage site, X is a self-destructing group, q is either 0 or 1, r is 1, 2, or 3. s is 0, 1, or 2. # is a binding site to the anti-FRα antibody construct T, and The % indicates the binding site with camptothecin analog D.

[0390] In some embodiments, in the linker of formula (XI), q is 1.

[0391] In some embodiments, s is 1 in the linker of formula (XI). In some embodiments, s is 0 in the ADC of formula (XI).

[0392] In some embodiments, in the linker of formula (XI), r is 1. In some embodiments, in the ADC of formula (XI), r is 3.

[0393] In some embodiments, formula (XI): In the linker, Z is The filename is TIFF0007877480000141.tif32165, where # represents the connection point with T, and * represents the connection point with the rest of the linker.

[0394] In some embodiments, in the linker of formula (XI), Str is Selected from TIFF0007877480000142.tif53165, During the ceremony, R is either H or a C1-C6 alkyl group. t is an integer between 2 and 10. u is an integer between 1 and 10.

[0395] In some embodiments, in the linker of formula (XI), Str is Selected from TIFF0007877480000143.tif22165, During the ceremony, t is an integer between 2 and 10. u is an integer between 1 and 10.

[0396] In some embodiments, in the linker of formula (XI), AA1-[AA2] r is a dipeptide (i.e., r=1). In some embodiments, in the linker of formula (XI), AA1-[AA2] rare Ala-(D)Asp, Ala-Lys, Ala-Phe, Asn-Lys, Asn-(D)Lys, Asp-Val, His-Val, Ile-Cit, Ile-Pro, Ile-Val, Leu-Cit, Me3Lys-Pro, Met-Lys, Met-(D)Lys, NorVa It has a sequence selected from l-(D)Asp, Phe-Arg, Phe-Cit, Phe-Lys, phenylGly-(D)Lys, Pro-(D)Lys, Trp-Cit, Val-Ala, Val-(D)Asp, Val-Cit, Val-Gly, Val-Gln and Val-Lys.

[0397] In some embodiments, in the linker of formula (XI), AA1-[AA2] r is a tripeptide (i.e., r=2). In some embodiments, in the linker of formula (XI), AA1-[AA2] r It has a sequence selected from Ala-Ala-Asn, Ala-Val-Cit, (D)Ala-Phe-Lys, Asp-Val-Ala, Asp-Val-Cit, Gly-Cit-Val, Lys-Val-Ala, Lys-Val-Cit, Met-Cit-Val, (D)Phe-Phe-Lys, and Asn-Pro-Val.

[0398] In some embodiments, in the linker of formula (XI), AA1-[AA2] r is a tetrapeptide (i.e., r=3). In some embodiments, in the linker of formula (XI), AA1-[AA2] r It has a sequence selected from Ala-Leu-Ala-Leu, Gly-Phe-Leu-Gly, Gly-Gly-Phe-Gly, and Gly-Phe-Gly-Gly.

[0399] In a particular embodiment, in the ADC of formula (X), m is 1, and the linker L is given by formula (XII): A cuttable linker having TIFF0007877480000144.tif32165, in the formula, Z is a functional group that can react with the target group on the anti-FRα antibody construct T. Str stands for stretcher, AA1 and AA2 are amino acids, and AA1-[AA2] r It forms a protease cleavage site, Y is -NH-CH2- or -NH-CH2-C(O)-, q is either 0 or 1, r is 1, 2, or 3. v is either 0 or 1. # is a binding site to the anti-FRα antibody construct T, and The % indicates the binding site with camptothecin analog D.

[0400] In some embodiments, in the linker of formula (XII), q is 1.

[0401] In some embodiments, v is 0 in the linker of formula (XII). In some embodiments, v is 1 in the ADC of formula (XII).

[0402] In some embodiments, in the linker of formula (XII), r is 1. In some embodiments, in the ADC of formula (XII), r is 3.

[0403] In some embodiments, formula (XII): In the linker, Z is The filename is TIFF0007877480000145.tif32165, where # represents the connection point with T, and * represents the connection point with the rest of the linker.

[0404] In some embodiments, in the linker of formula (XII), Str is Selected from TIFF0007877480000146.tif53165, During the ceremony, R is either H or a C1-C6 alkyl group. t is an integer between 2 and 10. u is an integer between 1 and 10.

[0405] In some embodiments, in the linker of formula (XII), Str is Selected from TIFF0007877480000147.tif22165, During the ceremony, t is an integer between 2 and 10. u is an integer between 1 and 10.

[0406] In some embodiments, in the linker of formula (XII), AA1-[AA2] r is a dipeptide (i.e., r=1). In some embodiments, in the linker of formula (XII), AA1-[AA2] r are Ala-(D)Asp, Ala-Lys, Ala-Phe, Asn-Lys, Asn-(D)Lys, Asp-Val, His-Val, Ile-Cit, Ile-Pro, Ile-Val, Leu-Cit, Me3Lys-Pro, Met-Lys, Met-(D)Lys, NorVa It has a sequence selected from l-(D)Asp, Phe-Arg, Phe-Cit, Phe-Lys, phenylGly-(D)Lys, Pro-(D)Lys, Trp-Cit, Val-Ala, Val-(D)Asp, Val-Cit, Val-Gly, Val-Gln and Val-Lys.

[0407] In some embodiments, in the linker of formula (XII), AA1-[AA2] r is a tripeptide (i.e., r=2). In some embodiments, in the linker of formula (XII), AA1-[AA2] r It has a sequence selected from Ala-Ala-Asn, Ala-Val-Cit, (D)Ala-Phe-Lys, Asp-Val-Ala, Asp-Val-Cit, Gly-Cit-Val, Lys-Val-Ala, Lys-Val-Cit, Met-Cit-Val, (D)Phe-Phe-Lys, and Asn-Pro-Val.

[0408] In some embodiments, in the linker of formula (XII), AA1-[AA2] r is a tetrapeptide (i.e., r=3). In some embodiments, in the linker of formula (XII), AA1-[AA2] r It has a sequence selected from Ala-Leu-Ala-Leu, Gly-Phe-Leu-Gly, Gly-Gly-Phe-Gly, and Gly-Phe-Gly-Gly.

[0409] In some embodiments, in the linker of formula (XII), Y is -NH-CH2. In some embodiments, in the linker of formula (XII), v is 1 and Y is -NH-CH2.

[0410] In some embodiments, the ADC of formula (X) may include a disulfide-containing linker. In some embodiments, in the ADC of formula (X), m is 1 and the linker L is of formula (XIII): A cuttable linker having TIFF0007877480000148.tif32165, in the formula, Z is a functional group that can react with the target group on the anti-FRα antibody construct T. Q is -(CH2) p -or-(CH2CH2O) q - and p and q are each independent integers from 1 to 10. Each R is independently H or C1-C6 alkyl. n is 1, 2, or 3. # is a binding site to the anti-FRα antibody construct T, and The % indicates the binding site with camptothecin analog D.

[0411] In some embodiments, the ADC of formula (X) may include a β-glucuronide-containing linker.

[0412] Various non-cleavable linkers for linking a drug to a targeting moiety are known in the art and may be useful in certain embodiments for the ADCs of this disclosure. Examples of non-cleavable linkers include linkers having an N-succinimidyl ester or N-sulfosuccinimidyl ester moiety for reaction with anti-FRα antibody constructs, and a maleimide or haloacetyl-based moiety for reaction with camptothecin analogs, or vice versa. An example of such a non-cleavable linker is sulfosuccinimidyl-4-[N-maleimidomethyl]cyclohexane-1-carboxylate (sulfo-SMCC) based. Sulfo-SMCC conjugations typically occur via a maleimide group that reacts with a sulfhydryl (thiol, -SH) on the camptothecin analog, while sulfo-NHS esters are reactive with primary amines (such as lysine and those found at the N-terminus of proteins or peptides) on the anti-FRα antibody constructs. Other non-limiting examples of such linkers include N-succinimidyl 4-(maleimidomethyl)cyclohexanecarboxylate (SMCC), N-succinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxy-(6-amidecaproate) ("long-chain" SMCC or LC-SMCC), κ-maleimidoundecanoate N-succinimidyl ester (KMUA), γ-maleimidobutyrate N-succinimidyl ester (GMBS), and ε-maleimidocaproate Linkers based on N-hydroxysuccinimide ester (EMCS), m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), N-(α-maleimidoacetoxy)-succinimide ester (AMAS), succinimide-6-(β-maleimidopropionamide)hexanoate (SMPH), N-succinimidyl 4-(p-maleimidophenyl)-butyrate (SMPB), and N-(p-maleimidophenyl)isyanate (PMPI) are examples.Other examples include those containing haloacetyl-based functional groups, such as N-succinimidyl-4-(iodoacetyl)-aminobenzoate (SIAB), N-succinimidyliodoacetate (SIA), N-succinimidylbromoacetate (SBA), and N-succinimidyl-3-(bromoacetamide)propionate (SBAP).

[0413] Non-limiting examples of drug-linkers containing the camptothecin analog of formula (I) are shown in Table 8 (Figure 13), Table 9 (Figure 14), and Table 10 (Figure 15). Non-limiting examples of conjugates containing these drug-linkers are shown in Table 11 (Figure 16), Table 12 (Figure 17), and Table 13 (Figure 18). In certain embodiments, the ADC of formula (X) comprises a drug-linker selected from the drug-linkers shown in Tables 8, 9, and 10. In certain embodiments, the ADC of formula (X) is selected from the conjugates shown in Tables 11, 12, and 13, where T is an anti-FRα antibody construct and n is 1 to 10. In some embodiments, the ADC of formula (X) is selected from the conjugates shown in Tables 11, 12, and 13, where T is an anti-FRα antibody construct and n is 2 to 8. In some embodiments, the ADC of formula (X) is selected from the conjugates shown in Tables 11, 12, and 13, where T is an anti-FRα antibody construct and n is 4 to 8.

[0414] In certain embodiments, the ADC of formula (X) is a drug linker (L-(D) selected from MT-GGFG-AM-compound 139, MC-GGFG-AM-compound 139, MT-GGFG-compound 140, MC-GGFG-compound 140, MT-GGFG-AM-compound 141, MC-GGFG-compound 141, MT-GGFG-compound 141, MC-GGFG-compound 148, and MC-GGFG-compound 148. m) comprises n is 4 or 8. In some embodiments, the ADC of formula (X) is a drug linker (L-(D) selected from MT-GGFG-AM-compound 139, MC-GGFG-AM-compound 139, MT-GGFG-compound 140, MC-GGFG-compound 140, MT-GGFG-AM-compound 141, MC-GGFG-compound 141, MT-GGFG-compound 141, MC-GGFG-compound 148, and MC-GGFG-compound 148. m ) is included, and n is 8.

[0415] ADC preparation The ADC of formula (X) can be prepared by standard methods known in the art (see, for example, Bioconjugate Techniques (GT Hermanson, 2013, Academic Press)). Various linkers and linker components are commercially available or can be prepared using standard synthetic organic chemistry techniques (see, for example, March's Advanced Organic Chemistry (Smith & March, 2006, Sixth Ed., Wiley); Toki et al., (2002) J.Org.Chem.67:1866-1872; Frisch et al., (1997) Bioconj.Chem.7:180-186; Bioconjugate Techniques (GT Hermanson, 2013, Academic Press)). In addition, various antibody-drug conjugation services are commercially available from companies such as Lonza Inc. (Allendale, NJ), Abzena PLC (Cambridge, UK), ADC Biotechnology (St. Asaph, UK), Baxter BioPharma Solutions (Baxter Healthcare Corporation, Deerfield, IL), and Piramal Pharma Solutions (Grangemouth, UK).

[0416] Generally, the preparation of an ADC involves first preparing a drug-linker DL comprising one or more camptothecin analogs of formula (I) and linker L, and then conjugating the drug-linker DL with a suitable group on an anti-FRα antibody construct T. However, ligation of the anti-FRα antibody construct-linker TL with one or more camptothecin analogs of formula (I) D, following ligation of linker L with the anti-FRα antibody construct T, remains an available alternative approach in some embodiments.

[0417] Suitable groups on compound D of formula (I) for linking linker L in any of the above approaches include, but are not limited to, thiol groups, amine groups, carboxylic acid groups, and hydroxyl groups. In some embodiments of this disclosure, linker L is linked to the compound via a hydroxyl group or an amine group on compound D of formula (I).

[0418] Suitable groups on the anti-FRα antibody construct T for linker L binding in any of the above approaches include sulfhydryl groups (e.g., on the side chain of a cysteine ​​residue), amino groups (e.g., on the side chain of a lysine residue), carboxylic acid groups (e.g., on the side chain of an aspartic acid or glutamic acid residue), and carbohydrate groups.

[0419] For example, anti-FRα antibody construct T may contain one or more naturally occurring sulfhydryl groups that allow anti-FRα antibody construct T to bind to linker L via the sulfur atom of the sulfhydryl group. Alternatively, anti-FRα antibody construct T may contain one or more lysine residues that are chemically modifiable to introduce one or more sulfhydryl groups. Reagents that can be used to modify lysine residues include, but are not limited to, N-succinimidyl S-acetylthioacetate (SATA), N-succinimidyl-3-(2-pyridyldithio)propionate ("SPDP"), and 2-iminothiolane hydrochloride (Traut reagent). Alternatively, anti-FRα antibody construct T may contain one or more carbohydrate groups that are chemically modifiable to include one or more sulfhydryl groups.

[0420] The carbohydrate group on the anti-FRα antibody construct T may also be oxidized to provide an aldehyde (-CHO) group (see, for example, Laguzza et al., 1989, J. Med. Chem. 32(3):548-55), which may then be reacted with linker L, for example, via a hydrazine or hydroxylamine group on linker L.

[0421] The anti-FRα antibody construct T can also be modified to include additional cysteine ​​residues (see, e.g., U.S. Patents 7,521,541; 8,455,622 and 9,000,130) or non-natural amino acids that provide a reactive handle, such as selenomethionine, p-acetylphenylalanine, formylglycine, or p-azidomethyl-L-phenylalanine (see, e.g., Hofer et al., 2009, Biochemistry, 48:12047-12057; Axup et al., 2012, PNAS, 109:16101-16106; Wu et al., 2009, PNAS, 106:3000-3005; Zimmerman et al., 2014, Bioconj. Chem., 25:351-361) to enable site-specific conjugation. Alternatively, the anti-FRα antibody construct T may be modified to include non-natural reactive groups, such as azides, which enable conjugation with the linker via complementary reactive groups on the linker, for example, through click chemistry (e.g., Chio & Bane, 2020, Methods Mol. Biol., 2078:83-97). A further option is the use of GlycoConnect® technology (Synaffix BV, Nijmegen, Netherlands), which involves enzymatic remodeling of the antibody glycan to enable linker attachment by metal-free click chemistry (see, for example, European Patent No. EP2911699).

[0422] Other protocols for protein modification for linker L binding or association are known in the art, including those described in Colligan et al., Current Protocols in Protein Science, vol.2, John Wiley & Sons (2002).

[0423] Alternatively, ADCs can be prepared using the enzyme transglutaminase, particularly the bacterial transglutaminase (BTG) of Streptomyces mobaraensis (see, e.g., Jeger et al., 2010, Angew. Chem. Int. Ed., 49:9995-9997). BTG forms an amide bond between the glutamine side-chain carboxamide (commonly an amine receptor on an antibody) and an alkylene amino group (commonly an amine donor on a drug-linker), which may be, for example, the ε-amino group or 5-amino-n-pentyl group of lysine. The antibody may also be modified to include a glutamine-containing peptide or "tag" that allows BTG conjugation to be used to conjugate the antibody with a drug-linker (see, e.g., U.S. Patent Application Publication US2013 / 0230543 and International (PCT) Publication WO2016 / 144608).

[0424] A similar conjugation approach utilizes the enzyme saltase A. In this approach, the antibody is typically modified to include a saltase A recognition motif (LPXTG, where X is any native amino acid), and the drug-linker is designed to include an oligoglycine motif (generally GGG) to enable saltase A-mediated peptide transfer (see, e.g., Beerli, et al., 2015, PLos One, 10:e0131177; Chen et al., 2016, Nature: Scientific Reports, 6:31899).

[0425] Once conjugation is complete, the average number of compounds of formula (I) conjugated with the anti-FRα antibody construct T (i.e., the "drug-to-antibody ratio" or DAR) may be determined by standard techniques, e.g., UV / VIS spectroscopy, ELISA-based techniques, or chromatographic techniques, e.g., hydrophobic interaction chromatography (HIC), UV-MALDI mass spectrometry (MS), and MALDI-TOFMS. Furthermore, the distribution of drug-conjugated forms (e.g., the proportion of anti-FRα antibody construct T containing 0, 1, 2, 3, etc., of compounds D of formula (I)) may also be optionally analyzed. Various techniques for evaluating DAR distribution are known in the art, including MS (with or without accompanying chromatographic separation steps), hydrophobic interaction chromatography, reverse-phase HPLC, or isoelectric focusing gel electrophoresis (IEF) (see, e.g., Wakankar et al., 2011, mAbs, 3:161-172).

[0426] Pharmaceutical composition For therapeutic use, the ADCs of this disclosure are typically formulated as pharmaceutical compositions. Accordingly, specific embodiments of this disclosure relate to pharmaceutical compositions comprising the ADCs described herein and pharmaceutically acceptable carriers, diluents, or excipients. Such pharmaceutical compositions can be prepared by known procedures using well-known and readily available components.

[0427] Pharmaceutical compositions may be formulated for administration to a subject by, for example, oral route (e.g., buccal or sublingual route), topical route, parenteral route, rectal or vaginal route, or by inhalation or spray. Parenteral administration may be by subcutaneous injection, or by intradermal, intra-articular, intravenous, intramuscular, intravascular, intrasternal, or intraarachnoid injection or infusion. Pharmaceutical compositions are generally formulated in a form suitable for administration to a subject, such as syrup, elixir, tablet, lozenge, hard capsule, soft capsule, pill, suppository, oily suspension, aqueous suspension, dispersible powder, dispersible granule, emulsion, injection, or solution. Pharmaceutical compositions may also be provided as unit dosage forms.

[0428] In certain embodiments, the pharmaceutical composition containing ADC is formulated for parenteral administration, for example, as a lyophilized preparation or an aqueous solution. Such pharmaceutical compositions may be provided, for example, in an injectable form in unit doses.

[0429] Pharmacokinetically acceptable carriers are generally non-toxic to the recipient at the doses and concentrations used. Examples of such carriers include buffers such as phosphates, citrates, and other organic acids; antioxidants such as ascorbic acid and methionine; preservatives such as octadecyldimethylbenzylammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl alcohol, benzyl alcohol, alkylparabens (such as methyl or propylparaben), catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol; low molecular weight (less than approximately 10 residues) polypeptides; and serum albumin. Examples of such substances include, but are not limited to, proteins such as gelatin; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates such as glucose, mannose, or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes such as Zn-protein complexes; and nonionic surfactants such as polyethylene glycol (PEG).

[0430] In certain embodiments, compositions containing ADC may be in the form of aqueous or oily solutions or suspensions for sterile injection. Such suspensions may be formulated using suitable dispersants or wetting agents and / or suspending agents known in the art. The sterile injection solution or suspension may contain ADC in a non-toxic, parenterally acceptable diluent or carrier. Acceptable diluents and solvents that may be employed include, for example, 1,3-butanediol, water, Ringer's solution, or isotonic sodium chloride solution. Furthermore, sterile non-volatile oils may be used as carriers. For this purpose, a variety of non-irritating fixative oils may be used, including synthetic monoglycerides or diglycerides. In addition, fatty acids such as oleic acid are also used in the preparation of injections. Auxiliaries such as local anesthetics, preservatives, and / or buffers may also be included in the injectable solution or suspension.

[0431] In certain embodiments, compositions comprising ADCs may be formulated for intravenous administration to humans. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. If necessary, the composition may also include a solubilizer and / or a local anesthetic such as a lignocaine to relieve pain at the injection site. Generally, the components are supplied either separately or mixed with each other in a unit dosage form, for example, as lyophilized powders or anhydrous concentrates in sealed containers such as ampoules or sachets indicating the amount of the activator. When the composition is administered by infusion, the composition may be prepared using an infusion bottle containing pharmaceutical-grade sterile water or saline. When the composition is administered by injection, ampoules of sterile water or saline for injection may be provided so that the components can be mixed before administration.

[0432] Other pharmaceutical compositions and methods for preparing such compositions are known in the art and are described, for example, in “Remington: The Science and Practice of Pharmacy” (formerly “Remingtons Pharmaceutical Sciences”) and Gennaro, A., Lippincott, Williams & Wilkins, Philadelphia, PA (2000).

[0433] How to use Certain embodiments of this disclosure relate to the therapeutic use of ADCs described herein. Some embodiments relate to the use of ADCs as therapeutic agents.

[0434] Certain embodiments of this disclosure relate to methods for inhibiting abnormal cancer or tumor cell proliferation, methods for inhibiting cancer or tumor cell proliferation, or methods for treating cancer in a subject, including administering the ADCs described herein. In certain embodiments, the ADCs described herein may be used for the treatment of cancer. Accordingly, some embodiments of this disclosure relate to the use of ADCs as anticancer agents.

[0435] Certain embodiments of this disclosure relate to methods for inhibiting the growth of cancer or tumor cells, comprising contacting cells with an ADC described herein, for example, an ADC of formula (X). Some embodiments relate to methods for killing cancer or tumor cells, comprising contacting cells with an ADC described herein, for example, an ADC of formula (X).

[0436] Some embodiments relate to methods for treating a subject with cancer by administering an ADC described herein, for example, an ADC of formula (X), to the subject. In this context, treating the subject can result in one or more of the following: reduction of tumor size, delay or prevention of tumor size increase, extension of disease-free survival between tumor disappearance or removal and reappearance, prevention of subsequent tumor development (e.g., metastasis), extension of time to progression, reduction of one or more adverse symptoms associated with the tumor, and / or extension of overall survival of the subject with cancer.

[0437] Certain embodiments relate to the use of ADCs described herein, for example, ADC of formula (X), in a method for inhibiting tumor growth in a subject. Some embodiments relate to the use of ADCs described herein, for example, ADC of formula (X), in a method for inhibiting the growth of cancer cells and / or killing cancer cells in vitro. Some embodiments relate to the use of ADCs described herein, for example, ADC of formula (X), in a method for inhibiting the growth of cancer cells and / or killing cancer cells in vivo within a subject having cancer.

[0438] Examples of cancers that can be treated in specific embodiments include carcinomas, melanomas, and sarcomas, including adenocarcinomas and squamous cell carcinomas. Carcinomas and sarcomas are also often referred to as “solid tumors.” Examples of commonly occurring solid tumors that can be treated in specific embodiments include, but are not limited to, brain cancer, breast cancer, cervical cancer, colon cancer, head and neck cancer, kidney cancer, lung cancer, ovarian cancer, pancreatic cancer, prostate cancer, gastric cancer, uterine cancer, non-small cell lung cancer (NSCLC), and colorectal cancer. Various forms of lymphoma can also lead to the formation of solid tumors, and therefore, in certain circumstances, these may also be considered solid tumors. Typically, the cancers treated are FRα-expressing cancers.

[0439] Certain embodiments relate to a method for inhibiting the growth of FRα-positive tumor cells, comprising contacting the cells with an ADC described herein, for example, an ADC of formula (X). The cells may be in vitro or in vivo. In certain embodiments, the ADC may be used in a method for treating a target FRα-positive cancer or tumor.

[0440] Cancers that overexpress FRα are typically solid tumors. Examples include, but are not limited to, ovarian cancer, endometrial cancer, lung cancer (such as non-small cell lung cancer (NSCLC)), mesothelioma, breast cancer (including triple-negative breast cancer (TNBC)), colorectal cancer, biliary tract cancer, pancreatic cancer, and esophageal cancer. Specific embodiments of this disclosure relate to methods for treating FRα-positive cancers, where the cancer is ovarian cancer, endometrial cancer, lung cancer (such as non-small cell lung cancer (NSCLC)), mesothelioma, breast cancer, colorectal cancer, biliary tract cancer, pancreatic cancer, or esophageal cancer, with an ADC described herein, for example, an ADC of formula (X). In some embodiments, an ADC of formula (X) may be useful in treating triple-negative breast cancer (TNBC).

[0441] Specific embodiments of this disclosure relate to a method for treating FRα-positive cancer with an ADC described herein, for example, an ADC of formula (X), wherein the cancer is a solid tumor expressing FRα at high levels (FRα-high solid tumor). Specific embodiments of this disclosure relate to a method for treating FRα-positive cancer with an ADC described herein, for example, an ADC of formula (X), wherein the cancer is a solid tumor expressing FRα at moderate levels (FRα-moderate solid tumor). Specific embodiments of this disclosure relate to a method for treating FRα-positive cancer with an ADC described herein, for example, an ADC of formula (X), wherein the cancer is a solid tumor expressing FRα at moderate to low levels (FRα-moderate / low solid tumor). Specific embodiments of this disclosure relate to a method for treating FRα-positive cancer with an ADC described herein, for example, an ADC of formula (X), wherein the cancer is a solid tumor expressing FRα at low levels (FRα-low solid tumor). In certain embodiments, the solid tumor is breast cancer, ovarian cancer, colorectal cancer, lung cancer (such as NSCLC), pancreatic cancer, or endometrial cancer.

[0442] Medical kit Specific embodiments relate to ADCs described herein, for example, a pharmaceutical kit comprising an ADC of formula (X).

[0443] The kit typically includes a container for holding the ADC and a label and / or accompanying information on or associated with the container. The label or accompanying information includes the explanations customarily included in the market packaging of the therapeutic product and contains information or instructions regarding the indications, use, dosage, administration, contraindications, and / or warnings relating to the use of the therapeutic product. The label or accompanying information may further include a description in a format prescribed by the government agency that has jurisdiction over the manufacture, use, or sale of the pharmaceutical or biological product, which incorporates the government agency's approval for manufacture, use, or sale for human or animal administration. In some embodiments, the container may have a sterile access port. For example, the container may be an intravenous solution bag or vial with a stopper that can be pierced by a subcutaneous injection needle.

[0444] In addition to the container holding the ADC, the kit may optionally include one or more additional containers containing other components of the kit, such as pharmaceutically acceptable buffers (e.g., bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, or dextrose solution), other buffers, or diluents.

[0445] Suitable containers include, for example, bottles, vials, syringes, and intravenous infusion bags. Containers can be formed from a variety of materials, such as glass or plastic. Where appropriate, one or more components of the kit may also be lyophilized or provided in a dry form such as powder or granules, and the kit may further contain a solvent suitable for reconstituting the lyophilized or dried component(s).

[0446] The kit may also include other materials that are desirable from a commercial or user perspective, such as filters, needles, and syringes.

[0447] The following embodiments are provided for illustrative purposes only and are not intended to limit in any way the scope of the claimed invention. [Examples]

[0448] General clauses Chemistry: Examples 1-3 below illustrate various methods for preparing camptothecin analogs of formula (I). Those skilled in the art will understand that these compounds can be produced by similar methods or by combining other methods known in the art. Those skilled in the art will also understand that other compounds of formula (I) not specifically shown below can be produced using the methods or similar methods described below by using appropriate starting components and, if necessary, modifying the parameters of the synthesis. Generally, starting components can be obtained from commercial suppliers such as Sigma Aldrich (Merck KGaA), Alfa Aesar and Maybridge (Thermo Fisher Scientific Inc.), Matrix Scientific, Tokyo Chemical Industry Ltd. (TCI), and Fluorochem Ltd., or synthesized according to materials known to those skilled in the art (see, for example, March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, 7th edition, John Wiley & Sons, Inc., 2013), or prepared as described herein.

[0449] Biological assays: The expression levels of FRα in the cell lines and CDX models used in the examples were evaluated in-house using research-level IHC assays, and relative expression levels (high / medium / low or strong / medium / weak) were assigned. The PDX model was similarly evaluated using archived tumor samples.

[0450] Abbreviation The following abbreviations are used throughout the Examples section: BCA: Bicinchoninic acid, Boc: Di-tert-butyl dicarbonate, CE-SDS: Capillary electrophoretic sodium dodecyl sulfate, DCM: Dichloromethane, DTPA: Diethylenetriaminepentaacetic acid, DIPEA: N,N-Diisopropylethylamine, DMF: Dimethylformamide, DMM™: (4-(4,6-Dimethoxy-1,3,5-Triadin-2-yl)-4-Methylmorpholinium chloride, EDC: 1-Ethyl-3-(3-Dimethylaminopropyl)carbodiimide, Fmoc: Fluorenylmethyloxycarbonyl, HATU: Azabenzotriazoletetramethyluronium hexafluoro Sphaat, HIC: Hydrophobic Interaction Chromatography, HOAt: 1-Hydroxy-7-Azabenzotriazole, HPLC: High-Performance Liquid Chromatography, LC / MS: Liquid Chromatography Mass Spectrometry, MC: Maleimidocaproyl, MT: Maleimidotriethyleneglycolate, NMM: N-Methylmorpholine, PNP: p-Nitrophenol, RP-UPLC-MS: Reverse-Phase Ultrafast Chromatography Mass Spectrometry, SEC: Size Exclusion Chromatography, TCEP: Tris(2-Carboxyethyl)phosphine, Tfp: Tetrafluorophenyl, TLC: Thin-Layer Chromatography, TFA: Trifluoroacetic Acid.

[0451] General chemical procedures General Procedure 1: Conversion of Chloride to Amine A suitable secondary amine (3 equivalents) was added to a stirred solution (0.05-0.1 M) of a chlorine compound in dimethylformamide. Upon completion (measured by LC / MS, generally 1-3 hours), the reaction mixture was purified by reverse-phase HPLC, and the desired product was obtained after lyophilization.

[0452] General Procedure 2: Conversion from Amine to Amide A stirred solution (0.05-0.1 M) of an amine compound in dimethylformamide was to which triethylamine (1.2 equivalents), a suitable carboxylic acid (1.1 equivalents), and then an aqueous solution (1 M) of DMM™ (2 equivalents) was added. Upon completion (measured by LC / MS, generally 16 hours), the reaction mixture was purified by reverse-phase HPLC, and the desired product was obtained after lyophilization.

[0453] General Procedure 3: Conversion from Amine to Sulfonamide To a stirred solution (0.05-0.1 M) of an amine compound in dimethylformamide, DIPEA (3 equivalents) was added, followed by the appropriate sulfonyl chloride. Upon completion (measured by LC / MS, generally 16 hours), the reaction mixture was purified by reverse-phase HPLC, and the desired product was obtained after lyophilization. General Procedure 4: Two-Step Conversion from Amine to Urea (Synthesis Scheme IV; Figure 1D) Step 1: To a stirred solution (0.05-0.1 M) of an amine compound in dichloromethane or dimethylformamide, p-nitrophenyl carbonate (1 equivalent) was added, followed by triethylamine (2 equivalents). Upon completion (measured by LC / MS, generally 1-4 hours), the reaction mixture was concentrated to dryness, purified by reverse-phase HPLC, and lyophilized to obtain the desired PNP-carbamate intermediate. This intermediate may be used to produce a single analogue, or it may be divided into multiple batches to produce multiple analogues in a second step. Step 2: A suitable primary amine (3 equivalents) was added to the PNP-carbamate intermediate (0.1-0.2 M) in dimethylformamide. Upon completion (measured by LC / MS, generally 1 hour), the reaction mixture was purified by reverse-phase HPLC, and the desired product was obtained after lyophilization.

[0454] General Procedure 5: Conversion from Amine to Carbamate To a stirred solution (0.05-0.1 M) of an amine compound in dichloromethane or dimethylformamide, p-nitrophenyl carbonate (1 equivalent) was added, followed by triethylamine (2 equivalents). At completion (measured by LC / MS, generally 1-4 hours), a suitable alcohol was added to the resulting PNP-carbamate intermediate. At completion (measured by LC / MS, generally 1-16 hours), the reaction mixture was purified by reverse-phase HPLC, and the desired product was obtained after lyophilization.

[0455] General procedure 6: Removal of the Boc protecting group TFA (20% by volume) was added to a stirred solution (0.1 M) of a Boc-protected amine compound in dichloromethane. Upon completion (measured by LC / MS, generally 1 hour), the reaction mixture was concentrated under reduced pressure to obtain a crude solid, or purified as described in general procedure 9.

[0456] General Procedure 7: Copper-mediated amide coupling EDC (HCl salt, 3 equivalents) was added to a rapidly stirred solution (0.02 M) of Boc-GGFG-OH (3 equivalents) and HOAt (3 equivalents) in a 10% v / v mixture of dimethylformamide in dichloromethane. After 5 minutes, a solution (0.02 M) of an amine-containing payload (1 equivalent) in a 10% v / v mixture of dimethylformamide in dichloromethane was added, followed immediately by the addition of CuCl2 (4 equivalents). Upon completion (measured by LC / MS, generally 1-16 hours), the reaction mixture was concentrated under reduced pressure to obtain a crude solid, or purified by preparative HPLC, and the desired product was obtained after lyophilization.

[0457] General Procedure 8: Installing MT A stirred solution (approximately 0.02 M) of an amine compound (1 equivalent) in dimethylformamide was mixed with a solution (approximately 0.02 M) of MT-OTfp (1.2-1.5 equivalents) in acetonitrile, followed by the addition of DIPEA (10 μL, 4 equivalents). Upon completion (measured by LC / MS, generally 1-16 hours), the reaction mixture was concentrated under reduced pressure to obtain a crude solid, which was purified by preparative HPLC and lyophilized to obtain the desired product.

[0458] General Procedure 9: Purification of Compounds Flash chromatography: Crude reaction products were purified by elution with a linear gradient of ethyl acetate / hexane or methanol / dichloromethane using Biotage® Snap Ultra columns (10, 25, 50, or 100 g) (Biotage, Charlotte, NC) in a Biotage® Isolera® automated flash system (Biotage, Charlotte, NC). Alternatively, reverse-phase flash purification was performed using Biotage® Snap Ultra C18 columns (12, 30, 60, or 120 g) and elution with a linear gradient of 0.1% TFA in acetonitrile / 0.1% TFA in water. The purified compounds were isolated by either removal of organic solvents using a rotary evaporator or lyophilization of the acetonitrile / water mixture.

[0459] Preparative HPLC: Reverse-phase HPLC of the crude compound was performed using an Agilent 1260 Infinity II preparative LC / MSD system (Agilent Technologies, Inc., Santa Clara, CA) with a Luna® 5-μm C18 100Å (150×30mm) column (Phenomenex, Torrance, CA), eluting with a linear gradient of 0.1% TFA in acetonitrile / 0.1% TFA in water. The purified compound was isolated by lyophilization of the acetonitrile / water mixture.

[0460] General Procedure 10: Analysis of Compounds LC / MS: After monitoring the completion of the reaction, the purified compound was analyzed using an Agilent 1290 HPLC / 6120 single quad LC / MS system (Agilent Technologies, Inc., Santa Clara, CA) with a Kinetex® 2.6-μm C18 100Å (30×3mm) column (Phenomenex, Torrance, CA) by eluting with a 10-100% linear gradient of 0.1% formic acid in acetonitrile / 0.1% formic acid in water.

[0461] NMR: 1 1H NMR spectra were collected using a Bruker AVANCE III 300 Spectrometer (300 MHz) (Bruker Corporation, Billerica, MA). Chemical shifts are reported in parts per million (ppm).

[0462] Example 1: Preparation of a camptothecin analog having a methyl group at the C10 position 1.1:(S)-11-(chloromethyl)-4-ethyl-8-fluoro-4-hydroxy-9-methyl-1,12-dihydro-14H-pyrano[3',4':6,7]indolidino[1,2-b]quinoline-3,14(4H)-dione (compound 1.1) The compound described in the title TIFF0007877480000149.tif42165 was prepared according to the procedure provided in Li, et al., 2019, ACS Med. Chem. Lett., 10(10):1386-1392.

[0463] 1.2:(S)-11-(aminomethyl)-4-ethyl-8-fluoro-4-hydroxy-9-methyl-1,12-dihydro-14H-pyrano[3',4':6,7]indolidino[1,2-b]quinoline-3,14(4H)-dione (compound 1.2) The compound described in the title TIFF0007877480000150.tif42165 was prepared according to the procedure provided in Li, et al., 2019, ACS Med. Chem. Lett., 10(10):1386-1392.

[0464] 1.3:(S)-4-ethyl-8-fluoro-4-hydroxy-9-methyl-11-(morpholinomethyl)-1,12-dihydro-14H-pyrano[3',4':6,7]indolidino[1,2-b]quinoline-3,14(4H)-dione (compound 100) The compound of the title TIFF0007877480000151.tif48165 was prepared according to general procedure 1, starting with compound 1.1 (10 mg) and morpholine. Preparative HPLC purification was performed by elution with a 20 to 60% CH3CN / H2O + 0.1% TFA gradient as described in general procedure 9, to obtain the compound of the title as an off-white solid (TFA salt, 3.6 mg, 26% yield).

[0465] LC / MS:C 26 H 26 Calculated value for FN3O5: m / z = 479.2; measured value: [M+H] + = 480.4.

[0466] 1 H NMR(300 MHz,CDCl3)δ 8.20(d,J=8.0 Hz,1H),7.82(d,J=10.4 Hz,1H),7.67(s,1H),5.77(d,J=16.4 Hz,1H),5.42(s,2H),5.33(d,J=16.4 Hz,1H),4.26(s,2H),3.81(t,J=4.7 Hz,4H),2.82-2.76(m,4H),2.57(d,J=1.7 Hz,3H),1.99-1.82(m,2H),1.06(t,J=7.4 Hz,3H).

[0467] 1.4:((S)-4-ethyl-8-fluoro-4-hydroxy-9-methyl-11-((4-(phenylsulfonyl)piperazine-1-yl)methyl)-1,12-dihydro-14H-pyrano[3',4':6,7]indolidino[1,2-b]quinoline-3,14(4H)-dione (compound 102) The compound of the title TIFF0007877480000152.tif58165 was prepared according to general procedure 1, starting with compound 1.1 (10 mg) and 1-(phenylsulfonyl)piperazine. Preparative HPLC purification was performed by elution with a 20 to 60% CH3CN / H2O + 0.1% TFA gradient as described in general procedure 9, to obtain the compound of the title as an off-white solid (TFA salt, 3.6 mg, 21% yield).

[0468] LC / MS:C 32 H 31 Calculated value for FN4O6: m / z = 618.2; measured value: [M+H] + = 619.4.

[0469] 1 H NMR(300 MHz,CDCl3)δ 8.07(d,J=7.9 Hz,1H),7.88-7.44(m,7H),5.73(d,J=16.4 Hz,1H),5.33(s,2H),5.33-5.26(m,1H),4.19(s,2H),3.12(s,4H),2.80(s,4H),2.54(s,3H),1.90(dt,J=11.6,7.0 Hz,2H),1.04(t,J=7.3 Hz,3H).

[0470] 1.5:(S)-11-((4-((4-aminophenyl)sulfonyl)piperazine-1-yl)methyl)-4-ethyl-8-fluoro-4-hydroxy-9-methyl-1,12-dihydro-14H-pyrano[3',4':6,7]indolidino[1,2-b]quinoline-3,14(4H)-dione (compound 104) The compound of the title TIFF0007877480000153.tif58165 was prepared according to general procedure 1, starting with compound 1.1 (10 mg) and 4-(piperazine-1-ylsulfonyl)aniline. Preparative HPLC purification was performed by eluting with a 20 to 60% CH3CN / H2O + 0.1% TFA gradient as described in general procedure 9, to obtain the compound of the title as an off-white solid (TFA salt, 4.7 mg, 27% yield).

[0471] LC / MS:C 32 H 32 Calculated value for FN5O6: m / z = 633.2; measured value: [M+H] + = 634.4.

[0472] 1 H NMR(300 MHz,MeOD)δ 8.32(d,J=8.0 Hz,1H),7.85(d,J=10.5 Hz,1H),7.65(s,1H),7.46(d,J=8.7 Hz,2H),6.74(d,J=8.7 Hz,2H),5.61(d,J=16.5 Hz,1H),5.44(s,2H),5.41(d,J=16.5 Hz,1H),4.51(s,2H),3.22-3.07(m,8H),2.58(s,3H),2.03-1.93(m,2H),1.02(t,J=7.3 Hz,3H).

[0473] 1.6:(S)-4-ethyl-8-fluoro-4-hydroxy-9-methyl-11-((4-methylpiperazine-1-yl)methyl)-1,12-dihydro-14H-pyrano[3',4':6,7]indolidino[1,2-b]quinoline-3,14(4H)-dione (compound 106) The compound described in the title TIFF0007877480000154.tif48165 was prepared according to general procedure 1, starting with compound 1.1 (10 mg) and N-methylpiperazine. Preparative HPLC purification was performed by elution with a 20 to 50% CH3CN / H2O + 0.1% TFA gradient as described in general procedure 9, to obtain the compound described in the title as an off-white solid (TFA salt, 3.6 mg, 25% yield).

[0474] LC / MS:C 27 H 29 Calculated value for FN4O4: m / z = 492.2, measured value: [M+H] + = 493.4.

[0475] 1.7:(S)-11-((4-(4-aminophenyl)piperazine-1-yl)methyl)-4-ethyl-8-fluoro-4-hydroxy-9-methyl-1,12-dihydro-14H-pyrano[3',4':6,7]indolidino[1,2-b]quinoline-3,14(4H)-dione (compound 108) The compound of the title TIFF0007877480000155.tif58165 was prepared according to general procedure 1, starting with compound 1.1 (10 mg) and 4-(piperazin-1-yl)aniline. Preparative HPLC purification was performed by eluting with a 20 to 50% CH3CN / H2O + 0.1% TFA gradient as described in general procedure 9, to obtain the compound of the title as an off-white solid (TFA salt, 3.7 mg, 23% yield).

[0476] LC / MS:C 32 H 32 Calculated value for FN5O4: m / z = 569.2, measured value: [M+H] + = 570.4.

[0477] 1 H NMR(300 MHz,MeOD)δ 8.39(d,J=8.1 Hz,1H),7.79(d,J=10.6 Hz,1H),7.21(d,J=9.0 Hz,2H),7.14(d,J=9.0 Hz,2H),5.62(d,J=16.4 Hz,1H),5.49(s,2H),5.41(d,J=16.4 Hz,1H),4.45(s,2H),3.44-3.38(m,4H),3.06-3.00(m,4H),2.58(d,J=1.8 Hz,3H),2.00-1.89(m,2H),1.03(t,J=7.3 Hz,3H).

[0478] 1.8:(S)-4-ethyl-8-fluoro-4-hydroxy-9-methyl-11-(piperidine-1-ylmethyl)-1,12-dihydro-14H-pyrano[3',4':6,7]indolidino[1,2-b]quinoline-3,14(4H)-dione (compound 110) The compound of the title TIFF0007877480000156.tif48165 was prepared according to general procedure 1, starting with compound 1.1 (10 mg) and piperidine. Preparative HPLC purification was performed as described in general procedure 9, eluting with a 10 to 60% CH3CN / H2O + 0.1% TFA gradient to obtain the compound of the title as an off-white solid (TFA salt, 1.5 mg, 11% yield).

[0479] LC / MS:C 27 H 28 Calculated value for FN3O4: m / z = 477.2; measured value: [M+H] + = 478.2.

[0480] 1 H NMR(300 MHz,MeOD)δ 8.34(d,J=7.6 Hz,1H),7.94(d,J=10.3 Hz,1H),7.70(s,1H),5.63(d,J=16.4 Hz,1H),5.52(s,2H),5.44(d,J=16.5 Hz,1H),4.99(s,2H),3.73-3.46(m,4H),2.64(s,3H),2.03-1.90(m,2H),1.90-1.84(m,6H),1.03(t,J=7.4 Hz,3H).

[0481] 1.9:tert-butyl(S)-4-((4-ethyl-8-fluoro-4-hydroxy-9-methyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolidino[1,2-b]quinoline-11-yl)methyl)piperazine-1-carboxylate (compound 111) The compound of the title TIFF0007877480000157.tif53165 was prepared according to general procedure 1, starting with compound 1.1 (10 mg) and tert-butylpiperazine-1-carboxylate. Preparative HPLC purification was performed as described in general procedure 9, eluting with a 10 to 60% CH3CN / H2O + 0.1% TFA gradient to obtain the compound of the title as an off-white solid (TFA salt, 6.6 mg, 40% yield).

[0482] LC / MS:C 31 H 35 Calculated value for FN4O6: m / z = 578.2; measured value: [M+H] + = 579.4.

[0483] 1.10:(S)-4-ethyl-8-fluoro-4-hydroxy-9-methyl-11-(piperazine-1-ylmethyl)-1,12-dihydro-14H-pyrano[3',4':6,7]indolidino[1,2-b]quinoline-3,14(4H)-dione (compound 112) The compound in the title TIFF0007877480000158.tif48165 was prepared according to general procedure 6, starting with compound 111 (5.0 mg), to obtain the compound in the title as an off-white solid (TFA salt, 4.4 mg).

[0484] LC / MS:C 26 H 27 Calculated value for FN4O4: m / z = 478.2; measured value: [M+H] + = 479.2.

[0485] 1.11:(S)-4-ethyl-8-fluoro-4-hydroxy-11-(((R)-2-(hydroxymethyl)morpholino)methyl)-9-methyl-1,12-dihydro-14H-pyrano[3',4':6,7]indolidino[1,2-b]quinoline-3,14(4H)-dione (compound 113) The compound of the title TIFF0007877480000159.tif58165 was prepared according to general procedure 1, starting with compound 1.1 (10 mg) and (R)-morpholine-2-ylmethanol. Preparative HPLC purification was performed by elution with a 10 to 60% CH3CN / H2O + 0.1% TFA gradient as described in general procedure 9, to obtain the compound of the title as an off-white solid (TFA salt, 4.6 mg, 32% yield).

[0486] LC / MS:C 27 H 28 Calculated value for FN3O6: m / z = 509.2, measured value: [M+H] + = 510.4.

[0487] 1.12:(4S)-4-ethyl-8-fluoro-4-hydroxy-11-((3-(hydroxymethyl)thiomorpholino)methyl)-9-methyl-1,12-dihydro-14H-pyrano[3',4':6,7]indolidino[1,2-b]quinoline-3,14(4H)-dione (compound 114) The compound of the title TIFF0007877480000160.tif53165 was prepared according to general procedure 1, starting with compound 1.1 (10 mg) and thiomorpholine-3-ylmethanol. Preparative HPLC purification was performed by elution with a 10 to 60% CH3CN / H2O + 0.1% TFA gradient as described in general procedure 9, to obtain the compound of the title as an off-white solid (TFA salt, 1.5 mg, 12% yield).

[0488] LC / MS:C 27 H 28 Calculated value for FN3O5S: m / z = 525.6, measured value: [M+H] + = 526.5.

[0489] 1 H NMR(300 MHz,10%D2O / CD3CN)8.36(d,J=8.1 Hz,1H),7.83(d,J=10.7 Hz,1H),7.50(s,1H),5.57(d,J=16.4 Hz,1H),5.52-5.29(m,3H),5.02(d,J=14.6 Hz,1H),4.71-4.54(m,1H),4.27(dd,J=12.4,5.0 Hz,1H),3.98(dd,J=12.3,3.4 Hz,1H),3.55(s,1H),3.30-3.03(m,4H)2.97-2.72(m,3H),2.62(s,1H),2.55(s,3H),0.95(t,J=7.4 Hz,3H).

[0490] 1.13:(4S)-4-ethyl-8-fluoro-4-hydroxy-11-((4-(hydroxymethyl)-2-oxa-5-azabicyclo[2.2.1]heptan-5-yl)methyl)-9-methyl-1,12-dihydro-14H-pyrano[3',4':6,7]indolidino[1,2-b]quinoline-3,14(4H)-dione (compound 115) The compound of the title TIFF0007877480000161.tif53165 was prepared according to general procedure 1, starting with compound 1.1 (10 mg) and 2-oxa-5-azabicyclo[2.2.1]heptan-4-ylmethanol. Preparative HPLC purification was performed by elution with a 10 to 60% CH3CN / H2O + 0.1% TFA gradient as described in general procedure 9, to obtain the compound of the title as an off-white solid (TFA salt, 3.5 mg, 29% yield).

[0491] LC / MS:C 28 H 28 Calculated value for FN3O6: m / z = 521.5, measured value: [M+H] + = 522.5.

[0492] 1 H NMR(300 MHz,10%D2O / CD3CN)δ 8.36(d,J=7.9 Hz,1H),7.86(dd,J=10.6,5.0 Hz,1H),7.50(d,J=1.8 Hz,1H),5.63-5.49(m,2H),5.37(dd,J=17.8,14.1 Hz,2H),5.05(s,2H),4.63(d,J=2.5 Hz,1H),4.55(d,J=10.7 Hz,1H),4.33(s,2H),3.92(d,J=10.7 Hz,1H),3.36(s,2H),2.57(s,3H),2.41-2.13(m,2H),1.97-1.85(m,2H),0.95(t,J=7.4 Hz,3H).

[0493] 1.14:(4S)-4-ethyl-8-fluoro-4-hydroxy-11-((3-(hydroxymethyl)-1,1-dioxidethiomorpholino)methyl)-9-methyl-1,12-dihydro-14H-pyrano[3',4':6,7]indolidino[1,2-b]quinoline-3,14(4H)-dione (compound 116) The compound in the title TIFF0007877480000162.tif58165 is compound 1.1 (10 mg) and 3-(hydroxymethyl)-1λ 6 Preparation was carried out according to General Procedure 1, starting with -thiomorpholine-1,1-dione. Purification was performed by elution with a 10 to 60% CH3CN / H2O + 0.1% TFA gradient as described in General Procedure 9, to obtain the title compound as an off-white solid (TFA salt, 0.2 mg, 2% yield).

[0494] LC / MS:C 27 H 28 Calculated value for FN3O7S: m / z = 557.6; measured value: [M+H] + = 558.4.

[0495] 1 H NMR(300 MHz,10% D2O / CD3CN)δ 8.44(d,J=8.2 Hz,1H),7.80(d,J=11.0 Hz,1H),7.50(s,1H),5.58(d,J=16.5 Hz,1H),5.45-5.26(m,3H),4.60(d,J=14.9 Hz,1H),4.33(d,J=14.7 Hz,1H),3.88(d,J=4.8 Hz,2H),3.41-2.85(m,4H),2.53(s,2H),2.19(p,J=2.5 Hz,2H),1.74(p,J=2.5 Hz,2H),1.27(s,2H),0.95(t,J=7.4 Hz, 3H).

[0496] 1.15:(4S)-4-ethyl-8-fluoro-4-hydroxy-11-((6-hydroxy-3-azabicyclo[3.1.1]heptan-3-yl)methyl)-9-methyl-1,12-dihydro-14H-pyrano[3',4':6,7]indolidino[1,2-b]quinoline-3,14(4H)-dione (compound 117) The compound of the title TIFF0007877480000163.tif53165 was prepared according to general procedure 1, starting with compound 1.1 (10 mg) and 3-azabicyclo[3.1.1]heptan-6-ol. Preparative HPLC purification was performed by eluting with a 10 to 60% CH3CN / H2O + 0.1% TFA gradient as described in general procedure 9 to obtain the compound of the title as an off-white solid (TFA salt, 1.3 mg, 11% yield).

[0497] LC / MS:C 28 H 28 Calculated value for FN3O5: m / z = 505.5, measured value: [M+H] + = 506.6.

[0498] 1 H NMR(300 MHz,10% D2O / CD3CN)δ 8.25(d,J=7.9 Hz,1H),7.87(d,J=10.6 Hz,1H),7.50(s,1H),5.65-5.27(m,4H),4.98(s,2H),4.24(s,1H),3.83- 3.57(m,4H),2.54(s,5H),2.01-1.86(m,2H),1.70(s,2H),0.95(t,J=7.3 Hz,3H).

[0499] 1.16:(S)-4-ethyl-8-fluoro-11-((3-fluoro-3-(hydroxymethyl)azetidine-1-yl)methyl)-4-hydroxy-9-methyl-1,12-dihydro-14H-pyrano[3',4':6,7]indolidino[1,2-b]quinoline-3,14(4H)-dione (compound 118) The compound of the title TIFF0007877480000164.tif58165 was prepared according to general procedure 1, starting with compound 1.1 (10 mg) and 3-fluoroazetidine-3-ylmethanol. Preparative HPLC purification was performed by elution with a 10 to 60% CH3CN / H2O + 0.1% TFA gradient as described in general procedure 9, to obtain the compound of the title as an off-white solid (TFA salt, 1.4 mg, 12% yield).

[0500] LC / MS:C 26 H 25 Calculated value for F2N3O5: m / z = 497.5; measured value: [M+H] + = 498.4.

[0501] 1 H NMR(300 MHz,10% D2O / CD3CN)δ 8.24(d,J=7.9 Hz,1H),7.85(d,J=10.7 Hz,1H),7.50(s,1H),5.57(d,J=16.5 Hz,1H),5.48-5.28(m,3H),4.98(s,2H),4.44-4.14(m,4H),3.78(d,J=14.9 Hz,2H),2.01-1.86(m,2H),0.95(t,J=7.4 Hz,3H).

[0502] 1.17:(S)-4-ethyl-8-fluoro-4-hydroxy-11-((3-(hydroxymethyl)azetidine-1-yl)methyl)-9-methyl-1,12-dihydro-14H-pyrano[3',4':6,7]indolidino[1,2-b]quinoline-3,14(4H)-dione (compound 119) The compound of the title TIFF0007877480000165.tif58165 was prepared according to general procedure 1, starting with compound 1.1 (10 mg) and azetidine-3-ylmethanol. Preparative HPLC purification was performed by elution with a 10 to 60% CH3CN / H2O + 0.1% TFA gradient as described in general procedure 9, to obtain the compound of the title as an off-white solid (TFA salt, 0.5 mg, 4.5% yield).

[0503] LC / MS:C26 H 26 Calculated value for FN3O5: m / z = 479.5; measured value: [M+H] + = 480.4.

[0504] 1 H NMR(300 MHz,10% D2O / CD3CN)δ 8.23(d,J=7.8 Hz,1H),7.90(d,J=10.6 Hz,1H),7.53(s,1H),5.58(d,J=16.5 Hz,1H),5.50-5.28(m,3H),5.01(s,2H),4.31-4.17(m,2H),4.15-4.00(m,2H),3.62(d,J=3.9 Hz,2H),2.58(s,3H),2.01-1.86(m,2H),0.96(t,J=7.4 Hz,3H).

[0505] 1.18:(4S)-11-((4,4-difluoro-3-(hydroxymethyl)piperidine-1-yl)methyl)-4-ethyl-8-fluoro-4-hydroxy-9-methyl-1,12-dihydro-14H-pyrano[3',4':6,7]indolidino[1,2-b]quinoline-3,14(4H)-dione (compound 120) The compound of the title TIFF0007877480000166.tif63165 was prepared according to general procedure 1, starting with compound 1.1 (10 mg) and 4,4-difluoropiperidine-3-ylmethanol. Preparative HPLC purification was performed by elution with a 10 to 60% CH3CN / H2O + 0.1% TFA gradient as described in general procedure 9, to obtain the compound of the title as an off-white solid (TFA salt, 4 mg, 32% yield).

[0506] LC / MS:C 28 H 28 Calculated value for F3N3O5: m / z = 543.5, measured value: [M+H] + = 544.4.

[0507] 1H NMR(300 MHz,10% D2O / CD3CN)δ 8.25(d,J=8.0 Hz,1H),7.77(dd,J=10.7,1.4 Hz,1H),7.47(s,1H),5.55(d,J=16.5 Hz,1H),5.42-5.25(m,3H),4.66(d,J=3.2 Hz,2H),3.90-3.77(m,1H),3.71-3.45(m,4H),2.24(q,J=11.8,9.2 Hz,2H),2.01-1.86(m,2H),0.94(t,J=7.4 Hz,3H).

[0508] 1.19:(S)-4-ethyl-8-fluoro-4-hydroxy-11-((1-(hydroxymethyl)-7-azabicyclo[2.2.1]heptan-7-yl)methyl)-9-methyl-1,12-dihydro-14H-pyrano[3',4':6,7]indolidino[1,2-b]quinoline-3,14(4H)-dione (compound 121) The compound of the title TIFF0007877480000167.tif53165 was prepared according to general procedure 1, starting with compound 1.1 (10 mg) and 7-azabicyclo[2.2.1]heptan-1-ylmethanol. Preparative HPLC purification was performed by elution with a 10 to 60% CH3CN / H2O + 0.1% TFA gradient as described in general procedure 9, to obtain the compound of the title as an off-white solid (TFA salt, 0.8 mg, 6.6% yield).

[0509] LC / MS:C 29 H 30 Calculated value for FN3O5: m / z = 519.6, measured value: [M+H] + = 520.4.

[0510] 1H NMR(300 MHz,10% D2O / CD3CN)δ 8.22(s,1H),7.92(d,J=10.7 Hz,1H),7.54(s,1H),5.59(dd,J=17.6,7.6 Hz,2H),5.33(t,J=17.4 Hz,2H),4.98-4.81(m,1H),4.67-4.44(m,2H),4.28-3.93(m,4H),2.73(s,2H),2.34-2.03(m,4H),1.91(d,J=14.0 Hz,5H),0.96(t,J=7.4 Hz,3H).

[0511] 1.20:(S)-N-((4-ethyl-8-fluoro-4-hydroxy-9-methyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolidino[1,2-b]quinoline-11-yl)methyl)methanesulfonamide (compound 122) The compound described in the title TIFF0007877480000168.tif53165 was prepared according to general procedure 3, starting with compound 1.2 (10 mg) and methanesulfonyl chloride. Preparative HPLC purification was performed as described in general procedure 9, eluting with a 10 to 50% CH3CN / H2O + 0.1% TFA gradient to obtain the compound described in the title as an off-white solid (0.8 mg, 7% yield).

[0512] LC / MS:C 23 H 22 Calculated value for FN3O6S: m / z = 487.1; measured value: [M+H] + = 488.2.

[0513] 1 H NMR(300 MHz,MeOD)δ 8.33(d,J=8.1 Hz,1H),7.83(d,J=10.8 Hz,1H),7.68(s,1H),5.62(d,J=16.3 Hz,1H),5.52(s,2H),5.42(d,J=16.4 Hz,1H),4.87(s,2H),3.06(s,3H),2.59(s,3H),2.06-1.93(m,2H),1.03(t,J=7.4 Hz,3H).

[0514] 1.21:(S)-N-((4-ethyl-8-fluoro-4-hydroxy-9-methyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolidino[1,2-b]quinoline-11-yl)methyl)-1-(4-nitrophenyl)methanesulfonamide (compound 124) The compound of the title TIFF0007877480000169.tif63165 was prepared according to general procedure 3, starting with compound 1.2 (20 mg) and (4-nitrophenyl)methanesulfonyl chloride. Preparative HPLC purification was performed as described in general procedure 9, eluting with a 10 to 50% CH3CN / H2O + 0.1% TFA gradient to obtain the compound of the title as an off-white solid (5.0 mg, 17% yield).

[0515] LC / MS:C 29 H 25 Calculated value for FN4O8S: m / z = 608.1, measured value: [M+H] + = 609.2.

[0516] 1 H NMR(300 MHz,CDCl3)δ 8.02-7.92(m,3H),7.74(d,J=10.5 Hz,1H),7.65(s,1H),7.33(d,J=8.6 Hz,2H),5.66(d,J=16.8 Hz,1H),5.28(d,J=16.5 Hz,1H),5.14(d,J=5.4 Hz,2H),4.67(s,2H),4.28(d,J=6.3 Hz,2H),3.39(s,3H),2.03-1.83(m,2H),1.04(t,J=7.4 Hz,3H).

[0517] 1.22:(S)-N-((4-ethyl-8-fluoro-4-hydroxy-9-methyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolidino[1,2-b]quinoline-11-yl)methyl)benzenesulfonamide (compound 125) The compound listed in the title TIFF0007877480000170.tif63165 was prepared according to general procedure 3, starting with compound 1.2 (10 mg) and benzenesulfonyl chloride. Preparative HPLC purification was performed as described in general procedure 9, eluting with a 10 to 50% CH3CN / H2O + 0.1% TFA gradient to obtain the compound listed in the title as an off-white solid (9.8 mg, 73% yield).

[0518] LC / MS:C 28 H 24 Calculated value for FN3O6S: m / z = 549.6; measured value: [M+H] + = 550.6.

[0519] 1 H NMR(300 MHz,DMSO-d6)δ 8.60(t,J=6.2 Hz,1H),8.17(d,J=8.1 Hz,1H),7.83(d,J=10.8 Hz,1H),7.71(dd,J=7.1,1.7 Hz,2H),7.66-7.48(m,2H),7.46(dd,J=8.3,6.8 Hz,2H),7.40-7.27(m,2H),7.18(s,1H),7.01(s,1H),5.45(s,2H),5.33(s,2H),4.63(d,J=6.2 Hz,2H),2.48(s,3H),1.98-1.76(m,2H),0.89(t,J=7.3 Hz, 3H).

[0520] 1.23: (S)-N-((4-ethyl-8-fluoro-4-hydroxy-9-methyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolidino[1,2-b]quinoline-11-yl)methyl)-4-nitrobenzenesulfonamide (compound 1.23) TIFF0007877480000171.tif63165 The titled compound was prepared according to General Procedure 3 starting from Compound 1.2 (75 mg) and 4-nitrobenzenesulfonyl chloride. The titled compound was purified as described in General Procedure 9 using a 12 g C18 column and eluting with a 5 to 75% CH3CN / H2O + 0.1% TFA gradient to give the titled compound as an off-white solid (37.8 mg, 47% yield).

[0521] LC / MS:C 28 H 23 Calculated m / z = 594.6 for FN4O8S, found [M+H] + = 595.2.

[0522] 1.24: (S)-4-Amino-N-((4-ethyl-8-fluoro-4-hydroxy-9-methyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-11-yl)methyl)benzenesulfonamide (Compound 127) Platinum 1% vanadium 2% carbon (75 mg) was added to a solution of Compound 1.23 (37.8 mg, 0.064 mmol) in methanol (6.4 mL). The flask was purged with H2 and then stirred at room temperature for 45 minutes under a H2 atmosphere. The mixture was filtered through a pad of celite, washed with DMF, and the filtrate was evaporated to give the titled compound as a pale yellow solid (30 mg, 84% yield).

[0523] LC / MS:C 28 H 24 Calculated m / z = 564.6 for FN4O6S, found [M+H] + = 565.2.

[0524] 1H NMR(300 MHz,DMSO-d6)δ 8.13(d,J=8.2 Hz,1H),8.02(t,J=6.2 Hz,1H),7.88(d,J=10.8 Hz,1H),7.48-7.35(m,2H),7.31(d,J=8.4 Hz,1H),6.63-6.45(m,2H),5.45(s,2H),5.36(s,2H),4.50(d,J=6.3 Hz,2H),1.98-1.75(m,2H),0.89(t,J=7.3 Hz,3H).

[0525] 1.25:(S)-N-((4-ethyl-8-fluoro-4-hydroxy-9-methyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolidino[1,2-b]quinoline-11-yl)methyl)-2-hydroxyethane-1-sulfonamide (compound 129) The compound of the title TIFF0007877480000173.tif53165 was prepared according to general procedure 3, starting with compound 1.2 (20 mg) and 2-hydroxyethanesulfonyl chloride. Preparative HPLC purification was performed as described in general procedure 9, eluting with a 25-50% CH3CN / H2O + 0.1% TFA gradient to obtain the compound of the title as an off-white solid (1.3 mg, 13% yield).

[0526] LC / MS:C 24 H 24 Calculated value for FN3O7S: m / z = 517.1; measured value: [M+H] + = 518.2.

[0527] 1 H NMR(300 MHz,DMSO-d6)δ 8.30(d,J=8.4 Hz,1H),7.91(d,J=10.9 Hz,1H),7.84(t,J=6.3 Hz,1H),7.33(s,1H),5.50-5.33(m,4H),5.07(t,J=5.4 Hz,1H),4.78(d,J=6.0 Hz,2H),4.07(s,3H),3.80(dt,J=6.3 Hz,J=5.8 Hz,2H),1.86(m,2H),0.87(d,J=7.3 Hz,3H).

[0528] 1.26: (S)-N-((4-Ethyl-8-fluoro-4-hydroxy-9-methyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-11-yl)methyl)methanesulfonamide (Compound 131) TIFF0007877480000174.tif53165 tert-Butanol (3 uL) was added to a solution of chlorosulfonyl isocyanate (3 uL) in dichloromethane (1 mL). After stirring this solution for 1 hour, Compound 1.2 (13 mg) dissolved in dichloromethane (1 mL) was added, followed by triethylamine (13 uL). After stirring the reaction for 1 hour, it was concentrated to dryness. The preparative HPLC purification of the intermediate Boc compound was carried out by eluting with a 10 to 50% CH3CN / H2O + 0.1% TFA gradient as described in General Procedure 9. Trifluoroacetic acid (200 uL) was added to the purified solid in dichloromethane (1 mL). After stirring the reaction for 16 hours, it was concentrated to dryness to obtain the title compound as an off-white solid (7.5 mg, 48% yield).

[0529] LC / MS: C 22 H 21 Calculated m / z = 488.1 for FN4O6S, found [M + H] + = 489.0。

[0530] 1 [[ID=२०]]H NMR (300 MHz, MeOD) δ 8.25 (d, J = 8.1 Hz, 1H), 7.73 (d, J = 10.7 Hz, 1H), 7.62 (s, 1H), 5.59 (d, J = 16.4 Hz, 1H), 5.45 (s, 2H), 5.39 (d, J = 16.4 Hz, 1H), 4.81 (s, 2H), 2.55 (d, J = 1.7 Hz, 3H), 2.07 - 1.89 (m, 2H), 1.03 (t, J = 7.4 Hz, 3H).

[0531] 1.27: 4-Nitrophenyl-(S)-((4-ethyl-8-fluoro-4-hydroxy-9-methyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolidino[1,2-b]quinoline-11-yl)methyl)carbamate (compound 1.27) The PNP-carbamate intermediate compound of the title TIFF0007877480000175.tif53165 was prepared according to the first step of general procedure 4, starting with compound 1.2 (24 mg). Purification was performed as described in general procedure 9, by eluting with a 10 to 50% CH3CN / H2O + 0.1% TFA gradient using a 12 g C18 column to obtain the title compound as an off-white solid (14 mg, 53% yield).

[0532] LC / MS:C 29 H 23 Calculated value for FN4O8S: m / z = 574.2; measured value: [M+H] + = 575.2.

[0533] 1.28:(S)-1-((4-ethyl-8-fluoro-4-hydroxy-9-methyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolidino[1,2-b]quinoline-11-yl)methyl)-3-methylurea (compound 132) The compound described in the title TIFF0007877480000176.tif53165 was prepared according to general procedure 4, starting with compound 1.2 (25 mg) and an aqueous solution of methylamine (500 uL, 40 wt% in water) as the primary amine. In this case, the intermediate PNP-carbamate was used unpurified. Preparative HPLC purification was performed by elution with a 10 to 50% CH3CN / H2O + 0.1% TFA gradient as described in general procedure 9, to obtain the compound described in the title as an off-white solid (8.9 mg, 31% yield).

[0534] LC / MS:C 24 H 23 Calculated value for FN4O5: m / z = 466.2, measured value: [M+H] += 467.2.

[0535] 1 H NMR(300 MHz,MeOD)δ 8.26(d,J=8.2 Hz,1H),7.79(d,J=10.7 Hz,1H),7.66(s,1H),5.61(d,J=16.3 Hz,1H),5.48(s,2H),5.41(d,J=16.4 Hz,1H),4.97(s,2H),2.73(s,3H),2.57(s,3H),2.08-1.93(m,2H),1.03(t,J=7.4 Hz,3H).

[0536] 1.29:(S)-1-(4-aminobenzyl)-3-((4-ethyl-8-fluoro-4-hydroxy-9-methyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolidino[1,2-b]quinoline-11-yl)methyl)urea (compound 134) The compound of the title TIFF0007877480000177.tif58165 was prepared according to the second step of general procedure 4, using compound 1.27 (4 mg) as the PNP-carbamate and 4-(aminomethyl)aniline as the primary amine. Preparative HPLC purification was performed by elution with a 20 to 50% CH3CN / H2O + 0.1% TFA gradient as described in general procedure 9 to obtain the compound of the title as an off-white solid (0.6 mg, 12% yield).

[0537] LC / MS:C 30 H 28 Calculated value for FN5O5: m / z = 557.2, measured value: [M+H] + = 558.4.

[0538] 1H NMR(300 MHz,MeOD)δ 8.25(d,J=8.1 Hz,1H),7.80(d,J=10.8 Hz,1H),7.67(s,1H),7.43(d,J=8.2 Hz,2H),7.24(d,J=8.3 Hz,2H),5.63(d,J=16.4 Hz,1H),5.48(s,2H),5.43(d,J=16.4 Hz,1H),5.01(s,2H),4.37(s,2H),2.56(d,J=1.7 Hz,3H),2.05-1.94(m,2H),1.03(t,J=7.3 Hz,3H).

[0539] 1.30:(S)-1-((4-ethyl-8-fluoro-4-hydroxy-9-methyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolidino[1,2-b]quinoline-11-yl)methyl)-3-(2-hydroxyethyl)urea (compound 136) The compound described in the title TIFF0007877480000178.tif53165 was prepared according to the second step of general procedure 4, using compound 1.27 (4 mg) as the PNP-carbamate and hydroxyethylamine as the primary amine. Preparative HPLC purification was performed by elution with a 10 to 50% CH3CN / H2O + 0.1% TFA gradient as described in general procedure 9 to obtain the compound described in the title as an off-white solid (2.4 mg, 66% yield).

[0540] LC / MS:C 25 H 25 Calculated value for FN4O6: m / z = 496.2; measured value: [M+H] + = 497.2.

[0541] 1H NMR(300 MHz,MeOD)δ 8.08(d,J=8.0 Hz,1H),7.74(d,J=10.5 Hz,1H),7.68(s,1H),5.64(d,J=16.4 Hz,1H),5.41(s,2H),5.31(d,J=16.4 Hz,1H),4.96(s,2H),3.63(t,J=5.2 Hz,2H),3.29(t,J=5.3 Hz,2H),2.54(s,3H),1.98-1.87(m,2H),1.01(t,J=7.4 Hz,3H).

[0542] 1.31: Methyl-(S)-((4-ethyl-8-fluoro-4-hydroxy-9-methyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolidino[1,2-b]quinoline-11-yl)methyl)carbamate (compound 138) The compound of the title TIFF0007877480000179.tif53165 was prepared by reacting compound 1.2 (50 mg) with methanol to the intermediate PNP-carbamate, following general procedure 5. Preparative HPLC purification was performed by eluting with a 20-50% CH3CN / H2O + 0.1% TFA gradient as described in general procedure 9, to obtain the compound of the title as an off-white solid (3.5 mg, 6% yield).

[0543] LC / MS:C 24 H 22 Calculated value for FN3O6: m / z = 467.2; measured value: [M+H] + = 468.2.

[0544] 1 H NMR(300 MHz,MeOD)δ 8.17(d,J=8.2 Hz,1H),7.77(d,J=10.5 Hz,1H),7.69(s,1H),5.65(d,J=16.5 Hz,1H),5.48(s,2H),5.33(d,J=16.4 Hz,1H),4.86(d,J=5.6 Hz,2H),3.65(s,3H),2.56(s,3H),2.02-1.89(m,2H),1.02(t,J=7.4 Hz,3H).

[0545] 1.32:2-Hydroxyethyl(S)-((4-ethyl-8-fluoro-4-hydroxy-9-methyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolidino[1,2-b]quinoline-11-yl)methyl)carbamate (compound 139) The compound of the title TIFF0007877480000180.tif58165 was prepared by reacting compound 1.2 (18 mg) with the intermediate PNP-carbamate, starting with compound 1,2-ethanediol, according to general procedure 5. Preparative HPLC purification was performed by eluting with a 10 to 60% CH3CN / H2O + 0.1% TFA gradient as described in general procedure 9, to obtain the compound of the title as an off-white solid (4.2 mg, 19% yield).

[0546] LC / MS:C 25 H 24 Calculated value for FN3O7: m / z = 497.2, measured value: [M+H] + = 498.2.

[0547] 1 H NMR(300 MHz,DMSO)δ 8.23(d,J=8.2 Hz,1H),7.78(d,J=10.7 Hz,1H),7.40(s,1H),5.47(d,J=16.5 Hz,1H),5.42(s,2H),5.34(d,J=16.4 Hz,1H),4.77(s,2H),3.99(t,J=4.9 Hz,2H),3.64-3.38(m,2H),2.48(s,3H),2.02-1.67(m,2H),0.89(t,J=7.3 Hz,3H).

[0548] Example 2: Preparation of a camptothecin analog having methoxy at the C10 position 2.1: 1-(2-amino-4-fluoro-5-methoxyphenyl)-2-chloroethane-1-one (compound 2.1) A solution of 3-fluoro-4-methoxyaniline (10 g, 71 mmol) in DCM (100 mL) was cooled to 0°C. To this solution, 1 M BCl3 in DCM (71 mL, 71 mmol), followed by 1 M chloro(diethyl)alman in DCM (71 mL, 71 mmol), and finally 2-chloroacetonitrile (6.4 g, 85 mmol). The solution was heated under reflux for 3 hours, cooled to room temperature, and quenched by adding 2 M aqueous HCl. The resulting heterogeneous mixture was heated under reflux for 1 hour, cooled to room temperature, and the pH was adjusted to approximately 12 with Na2CO3. The layers were separated, and the aqueous layer was extracted with DCM (3 × 100 mL). The combined organic layers were dried over Na2SO4, concentrated, and flush-purified by elution with 0 to 20% siRNA / hexane as described in general procedure 9 to obtain the title compound (6 g, 28 mmol, 39% yield).

[0549] LC / MS: Calculated value m / z = 217.1 for C9H9ClFNO2, measured value [M+H] + = 218.1.

[0550] 1 H NMR(400 MHz,CDCl3)δ 7.19(d,J=9.2 Hz,1H),6.44(d,J=12.8 Hz,1H),4.59(s,2H),3.86(s,3H)

[0551] 2.2:(S)-11-(chloromethyl)-4-ethyl-8-fluoro-4-hydroxy-9-methoxy-1,12-dihydro-14H-pyrano[3',4':6,7]indolidino[1,2-b]quinoline-3,14(4H)-dione (compound 2.2) To a solution of compound 2.1 (1.65 g, 7.6 mmol) and (S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-f]indolidine-3,6,10(4H)-trione (2 g, 7.6 mmol) in toluene (200 mL), toluene-4-sulfonic acid (157 mg, 0.9 mmol) was added. This solution was heated at 140 °C for 3 hours and then cooled to room temperature. The product was recovered as a yellow precipitate by filtration to obtain the title compound (1.27 g, 2.85 mmol, 37.5% yield).

[0552] LC / MS:C 22 H 18 Calculated value for ClFN2O5: m / z = 445.2; measured value: [M+H] + = 445.1.

[0553] 1 H NMR(400 MHz,DMSO-d6)δ 7.99(d,J =12.0 Hz,1H)7.80(d,J=9.2 Hz,1H)7.27(s,1H),6.50(s,1H),5.45(s,2H),5.41(s,2H),5.33(s,2H)4.08(s,3H),1.87-1.83(m,2H),0.87(t,J=7.2 Hz,3H)

[0554] 2.3:(S)-4-ethyl-8-fluoro-4-hydroxy-9-methoxy-11-(morpholinomethyl)-1,12-dihydro-14H-pyrano[3',4':6,7]indolidino[1,2-b]quinoline-3,14(4H)-dione (compound 101) The compound of the title TIFF0007877480000183.tif53165 was prepared according to general procedure 1, starting with compound 2.2 (10 mg) and morpholine. Preparative HPLC purification was performed by eluting with a 20 to 60% CH3CN / H2O + 0.1% TFA gradient as described in general procedure 9, to obtain the compound of the title as an off-white solid (5.6 mg, 41% yield).

[0555] LC / MS:C26 H 26 Calculated value for FN3O6: m / z = 495.2; measured value: [M+H] + = 496.4.

[0556] 1 H NMR(300 MHz,MeOD)δ 7.84-7.70(m,2H),7.59(s,1H),5.62(d,J=16.3 Hz,1H),5.45-5.36(m,3H),4.29(s,2H),4.12(s,3H),3.58-3.48(m,2H) ,3.28-3.09(m,2H),2.75-2.61(m,2H),2.05-1.91(m,2H),1.02(t,J=7.4 Hz,3H).

[0557] 2.4:(S)-4-ethyl-8-fluoro-4-hydroxy-9-methoxy-11-((4-(phenylsulfonyl)piperazin-1-yl)methyl)-1,12-dihydro-14H-pyrano[3',4':6,7]indolidino[1,2-b]quinoline-3,14(4H)-dione (compound 103) The compound of the title TIFF0007877480000184.tif58165 was prepared according to general procedure 1, starting with compound 2.2 (10 mg) and 1-(phenylsulfonyl)piperazine. Preparative HPLC purification was performed by elution with a 20 to 60% CH3CN / H2O + 0.1% TFA gradient as described in general procedure 9, to obtain the compound of the title as an off-white solid (2.5 mg, 14% yield).

[0558] LC / MS:C 32 H 31 Calculated value for FN4O7S: m / z = 634.2; measured value: [M+H] + = 635.4.

[0559] 2.5:(S)-11-((4-((4-aminophenyl)sulfonyl)piperazine-1-yl)methyl)-4-ethyl-8-fluoro-4-hydroxy-9-methoxy-1,12-dihydro-14H-pyrano[3',4':6,7]indolidino[1,2-b]quinoline-3,14(4H)-dione (compound 105) The compound of the title TIFF0007877480000185.tif63165 was prepared according to general procedure 1, starting with compound 2.2 (10 mg) and 4-(piperazine-1-ylsulfonyl)aniline. Preparative HPLC purification was performed by eluting with a 20 to 60% CH3CN / H2O + 0.1% TFA gradient as described in general procedure 9, to obtain the compound of the title as an off-white solid (4.0 mg, 23% yield).

[0560] LC / MS:C 32 H 32 Calculated value for FN5O7S: m / z = 649.2; Measured value: [M+H] + = 650.4.

[0561] 1 H NMR(300 MHz,DMSO)δ 8.08(s,2H),7.90-7.67(m,2H),7.35(s,1H),7.32-7.26(m,2H),6.67-6.57(m,2H),5.46(d,J=16.5 Hz,1H),5.33 -5.22(m,3H),3.92(s,3H),3.02-2.72(m,4H),2.75-2.58(m,4H),1.97-1.70(m,2H),0.90(t,J=7.3 Hz,3H).

[0562] 2.6:(S)-4-ethyl-8-fluoro-4-hydroxy-9-methoxy-11-((4-methylpiperazine-1-yl)methyl)-1,12-dihydro-14H-pyrano[3',4':6,7]indolidino[1,2-b]quinoline-3,14(4H)-dione (compound 107) The compound listed in the title TIFF0007877480000186.tif53165 was prepared according to general procedure 1, starting with compound 2.2 (10 mg) and N-methylpiperazine. Preparative HPLC purification was performed by elution with a 20 to 60% CH3CN / H2O + 0.1% TFA gradient as described in general procedure 9, to obtain the compound listed in the title as an off-white solid (2.1 mg, 19% yield).

[0563] LC / MS:C27 H 29 Calculated value for FN4O5: m / z = 508.2, measured value: [M+H] + = 509.4.

[0564] 2.7:(S)-11-((4-(4-aminophenyl)piperazin-1-yl)methyl)-4-ethyl-8-fluoro-4-hydroxy-9-methoxy-1,12-dihydro-14H-pyrano[3',4':6,7]indolidino[1,2-b]quinoline-3,14(4H)-dione (compound 109) The compound of the title TIFF0007877480000187.tif58165 was prepared according to general procedure 1, starting with compound 2.2 (10 mg) and 4-(piperazin-1-yl)aniline. Preparative HPLC purification was performed by elution with a 20 to 60% CH3CN / H2O + 0.1% TFA gradient as described in general procedure 9, to obtain the compound of the title as an off-white solid (3.2 mg, 20% yield).

[0565] LC / MS:C 32 H 32 Calculated value for FN5O5: m / z = 585.2, measured value: [M+H] + = 586.4.

[0566] 1 H NMR(300 MHz,MeOD)δ 7.83-7.74(m,2H),7.62(s,1H),7.06(d,J=8.9 Hz,2H),6.98(d,J=8.9 Hz,2H),5.65(d,J=16.4 Hz,1H),5.36(s,2H),5.27(d,J=16.4 Hz,1H),4.13(s,2H),4.06(s,3H),3.26(br s,4H),2.79(br s,4H),1.97-1.83(m,2H),1.00(t,J=7.4 Hz,3H).

[0567] 2.8:(S)-11-(aminomethyl)-4-ethyl-8-fluoro-4-hydroxy-9-methoxy-1,12-dihydro-14H-pyrano[3',4':6,7]indolidino[1,2-b]quinoline-3,14(4H)-dione (compound 2.8) TIFF0007877480000188.tif42165 Compound 2.2 (250 mg, 0.56 mmol) was dissolved in ethanol (7 mL), to which hexamethylenetetramine (236 mg, 1.7 mmol) was added, followed by iPr2NEt (100 μL, 0.56 mmol). This solution was heated under reflux for 5 hours, cooled to room temperature, and quenched with 12 M aqueous HCl (60 μL). This solution was concentrated to approximately half its volume, 1 M aqueous HCl (1.5 mL) was added, stirred for 5 minutes, and then concentrated to obtain a brown residue. Purification was carried out using a 12 g C18 flash column as described in general procedure 9, by elution with a 5 to 40% CH3CN / H2O + 0.1% TFA gradient, to obtain the title compound as a pale yellow solid (179 mg, 75% yield).

[0568] LC / MS:C 22 H 20 Calculated value for FN3O5: m / z = 425.4, measured value: [M+H] + = 426.2.

[0569] 2.9:(S)-N-((4-ethyl-8-fluoro-4-hydroxy-9-methoxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolidino[1,2-b]quinoline-11-yl)methyl)methanesulfonamide (compound 123) The compound described in the title TIFF0007877480000189.tif53165 was prepared according to general procedure 3, starting with compound 2.8 (10 mg) and methanesulfonyl chloride. Preparative HPLC purification was performed as described in general procedure 9, eluting with a 5 to 65% CH3CN / H2O + 0.1% TFA gradient to obtain the compound described in the title as an off-white solid (8.5 mg, 91% yield).

[0570] LC / MS:C 23 H 22 Calculated value for FN3O7S: m / z = 503.1, measured value: [M+H] + = 504.2.

[0571] 1 H NMR(300 MHz,DMSO-d6)δ 7.98(d,J=12.1 Hz,1H),7.89(t,J=6.4 Hz,1H),7.80(d,J=9.1 Hz,1H),7.28(s,1H),5.42(s,2H),5.39(s,2H),4.77(d,J=6.4 Hz,2H),4.06(s,3H),3.06(s,3H),1.95-1.73(m,2H),0.88(d,J=7.3 Hz,3H).

[0572] 2.10:(S)-N-((4-ethyl-8-fluoro-4-hydroxy-9-methoxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolidino[1,2-b]quinoline-11-yl)methyl)benzenesulfonamide (compound 126) The compound described in the title TIFF0007877480000190.tif63165 was prepared according to general procedure 3, starting with compound 2.8 (7.5 mg) and benzenesulfonyl chloride. Preparative HPLC purification was performed as described in general procedure 9, eluting with a 5 to 70% CH3CN / H2O + 0.1% TFA gradient to obtain the compound described in the title as an off-white solid (4.6 mg, 46% yield).

[0573] LC / MS:C 28 H 24 Calculated value for FN3O7S: m / z = 565.6, measured value: [M+H] + = 566.2.

[0574] 11H NMR (300 MHz, DMSO-d6) δ 8.59 (t, J = 6.3 Hz, 1H), 7.94 (d, J = 12.2 Hz, 1H), 7.82 - 7.68 (m, 2H), 7.62 - 7.46 (m, 1H), 7.51 - 7.40 (m, 1H), 7.28 (d, J = 8.3 Hz, 1H), 6.52 (s, 1H), 5.44 (s, 1H), 5.36 (s, 1H), 4.64 (d, J = 6.3 Hz, 1H), 4.09 (s, 2H), 1.95 - 1.81 (m, 1H), 0.89 (t, J = 7.3 Hz, 2H).

[0575] 2.11: (S)-N-((4-Ethyl-8-fluoro-4-hydroxy-9-methoxy-3...

Claims

1. Antibody-drug conjugate having formula (X): T-[L-(D) ｍ ] ｎ (X) And in the formula, m is 1 to 4, n is between 1 and 10, T is an anti-FRα antibody construct containing an antigen-binding domain that specifically binds to an epitope within the human folate receptor alpha (hFRα) consisting of amino acid residues E120, D121, R123, T124, S125, and Y126 of SEQ ID NO:

15. L is the linker, D is, equation I: It is a compound of the formula, in which, R １ is selected from -H, -CH ３ , -CHF ２ , -CF ３ , -F, -Br, -Cl, -OH, -OCH ３ , -OCF ３ , and -NH ２ and is selected from, and R ２ However, -H, -CH ３ , -CF ３ , -F, -Br, -Cl, -OH, -OCH ３ , and -OCF ３ Selected from, Furthermore, R １ ga-NH ２ If that is the case, R is R ３ or R ４ And, R １ ga-NH ２ If it is anything other than R, R ４ And, R ３ However, -H, -C １ ~C ６ Alkyl, -C ３ ~C ８ Cycloalkyl, -(C １ ~C ６ Alkyl)-O-R ５ , -CO ２ R ８ , -aryl, -heteroaryl, and -(C １ ~C ６ Selected from alkyl)-aryl, R ４ but, Selected from, R ５ However, -H, -C １ ~C ６ Alkyl, -C ３ ~C ８ Cycloalkyl, -aryl, -heteroaryl, and -(C １ ~C ６ Selected from alkyl)-aryl, R ６ and R ７ However, independently of each other, -H and -C １ ~C ６ Alkyl, -C ３ ~C ８ Cycloalkyl, -(C １ ~C ６ Alkyl)-O-R ５ , -C ３ ~C ８ Heterocycloalkyl and -C(O)R １７ Selected from, R ８ However, -H, -C １ ~C ６ Alkyl, -C ３ ~C ８ Cycloalkyl and -C ３ ~C ８ Selected from heterocycloalkyl groups, Each R ９ However, independently, -H and -C １ ~C ６ Alkyl, -C ３ ~C ８ Cycloalkyl, -aryl, -heteroaryl, and -(C １ ~C ６ Selected from alkyl)-aryl, Each R １０ However, independently, -C １ ~C ６ Alkyl, -C ３ ~C ８ Cycloalkyl, -NR １４ R １４’ , -aryl, -heteroaryl, and -(C １ ~C ６ Selected from alkyl)-aryl, R １０’ However, -H, -C １ ~C ６ Alkyl, -C ３ ~C ８ Cycloalkyl, -aryl, -heteroaryl, and -(C １ ~C ６ Selected from alkyl)-aryl, R １１ However, -H and -C １ ~C ６ Selected from alkyl groups, R １２ is -H, -C １ ~C ６ alkyl, -CO ２ R ８ , -aryl, -heteroaryl, -(C １ ~C ６ alkyl)-aryl, -S(O) ２ R １６ and Selected from, R １３ is selected from -H and -C １ ~C ６ alkyl, R １４ and R １４’ However, independently of each other, -H and C １ ~C ６ Alkyl, -C ３ ~C ８ Cycloalkyl and -C ３ ~C ８ Selected from heterocycloalkyl groups, R １６ However, -C １ ~C ６ Alkyl, -C ３ ~C ８ Cycloalkyl, -aryl, -heteroaryl, and -(C １ ~C ６ Selected from alkyl)-aryl, R １７ However, -C １ ~C ６ Alkyl, -C ３ ~C ８ Cycloalkyl, -C ３ ~C ８ Heterocycloalkyl, -(C １ ~C ６ Alkyl)-C ３ ~C ８ Heterocycloalkyl, -aryl, -heteroaryl, and -(C １ ~C ６ Selected from alkyl)-aryl, R １８ and R １９ However, along with the N atom to which they bond, halogens, -C １ ~C ６ Alkyl, -C ３ ~C ８ Cycloalkyl and -(C １ ~C ６ Alkyl)-O-R ５ Forming a 4, 5, 6, or 7-membered ring having 0 to 3 substituents selected from, R ２４ , R ２５ , and R ２６ However, each is -C １ ~C ６ It is alkyl, X ａ and X ｂ However, each is independently selected from NH, O, and S, and X ｃ However, O, S, and S(O) ２ Selected from, Here, each alkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl group may be substituted. However, the compound is other than (S)-9-amino-11-butyl-4-ethyl-4-hydroxy-1,12-dihydro-14H-pyrano[3',4':6,7]indolidino[1,2-b]quinoline-3,14(4H)-dione. The aforementioned antibody-drug conjugate.

2. The antibody-drug construct according to claim 1, wherein the antigen-binding domain comprises heavy chain CDR amino acid sequences (HCDR1, HCDR2, and HCDR3) containing the sequences described in SEQ ID NOs: 3, 4, and 5, and light chain CDR amino acid sequences (LCDR1, LCDR2, and LCDR3) containing the sequences described in SEQ ID NOs: 6, 7, and 8.

3. The antigen-binding domain, (i) The CDR sequence of the VH amino acid sequence described in SEQ ID NO: 19, and the CDR sequence of the VL amino acid sequence described in SEQ ID NO: 39, or (ii) The CDR sequence of the VH amino acid sequence described in SEQ ID NO: 50, and the CDR sequence of the VL amino acid sequence described in SEQ ID NO: 64, or (iii) The CDR sequence of the VH amino acid sequence described in SEQ ID NO: 54, and the CDR sequence of the VL amino acid sequence described in SEQ ID NO: 64, or (iv) The CDR sequence of the VH amino acid sequence described in SEQ ID NO: 57, and the CDR sequence of the VL amino acid sequence described in SEQ ID NO: 64, or (v) The CDR sequence of the VH amino acid sequence described in SEQ ID NO: 61, and the CDR sequence of the VL amino acid sequence described in SEQ ID NO: 64, or (vi) The CDR sequence of the VH amino acid sequence described in SEQ ID NO: 76, and the CDR sequence of the VL amino acid sequence described in SEQ ID NO: 64, or (vii) The CDR sequence of the VH amino acid sequence described in SEQ ID NO: 79, and the CDR sequence of the VL amino acid sequence described in SEQ ID NO: 64, or (viiii) The CDR sequence of the VH amino acid sequence described in SEQ ID NO: 82, and the CDR sequence of the VL amino acid sequence described in SEQ ID NO: 64, or (ix) The CDR sequence of the VH amino acid sequence described in SEQ ID NO: 85, and the CDR sequence of the VL amino acid sequence described in SEQ ID NO: 64, or (x) The CDR sequence of the VH amino acid sequence described in SEQ ID NO: 88, and the CDR sequence of the VL amino acid sequence described in SEQ ID NO: 64, or (xi) The CDR sequence of the VH amino acid sequence described in SEQ ID NO: 91, and the CDR sequence of the VL amino acid sequence described in SEQ ID NO: 124, or (xi) The CDR sequence of the VH amino acid sequence described in SEQ ID NO: 99, and the CDR sequence of the VL amino acid sequence described in SEQ ID NO: 130, or (xiii) The CDR sequence of the VH amino acid sequence described in SEQ ID NO: 106, and the CDR sequence of the VL amino acid sequence described in SEQ ID NO: 64, or (xiv) The CDR sequence of the VH amino acid sequence described in SEQ ID NO: 106, and the CDR sequence of the VL amino acid sequence described in SEQ ID NO: 119, or (xv) CDR sequence of the VH amino acid sequence described in SEQ ID NO: 106, and CDR sequence of the VL amino acid sequence described in SEQ ID NO: 130, or (xvi) CDR sequence of the VH amino acid sequence described in SEQ ID NO: 113, and CDR sequence of the VL amino acid sequence described in SEQ ID NO: 130, or (xvii) The CDR sequence of the VH amino acid sequence described in SEQ ID NO: 116, and the CDR sequence of the VL amino acid sequence described in SEQ ID NO: 119, or (xviiii) CDR sequence of the VH amino acid sequence described in SEQ ID NO: 116, and CDR sequence of the VL amino acid sequence described in SEQ ID NO: 130, or (xix) The CDR sequence of the VH amino acid sequence described in SEQ ID NO: 133, and the CDR sequence of the VL amino acid sequence described in SEQ ID NO: 130, or (xx) CDR sequence of the VH amino acid sequence described in SEQ ID NO: 136, and CDR sequence of the VL amino acid sequence described in SEQ ID NO: 130 The antibody-drug conjugate according to claim 1, comprising:

4. The antigen-binding domain, (i) The VH amino acid sequence described in SEQ ID NO: 19, and the VL amino acid sequence described in SEQ ID NO: 39, or (ii) The VH amino acid sequence described in SEQ ID NO: 50, and the VL amino acid sequence described in SEQ ID NO: 64, or (iii) The VH amino acid sequence described in SEQ ID NO: 54, and the VL amino acid sequence described in SEQ ID NO: 64, or (iv) The VH amino acid sequence described in SEQ ID NO: 57, and the VL amino acid sequence described in SEQ ID NO: 64, or (v) The VH amino acid sequence described in SEQ ID NO: 61, and the VL amino acid sequence described in SEQ ID NO: 64, or (vi) The VH amino acid sequence described in SEQ ID NO: 76, and the VL amino acid sequence described in SEQ ID NO: 64, or (vii) The VH amino acid sequence described in SEQ ID NO: 79, and the VL amino acid sequence described in SEQ ID NO: 64, or (viiii) The VH amino acid sequence described in SEQ ID NO: 82, and the VL amino acid sequence described in SEQ ID NO: 64, or (ix) The VH amino acid sequence described in SEQ ID NO: 85, and the VL amino acid sequence described in SEQ ID NO: 64, or (x) The VH amino acid sequence described in SEQ ID NO: 88, and the VL amino acid sequence described in SEQ ID NO: 64, or (xi) The VH amino acid sequence described in SEQ ID NO: 91, and the VL amino acid sequence described in SEQ ID NO: 124, or (xi) The VH amino acid sequence described in SEQ ID NO: 99, and the VL amino acid sequence described in SEQ ID NO: 130, or (xiii) The VH amino acid sequence described in SEQ ID NO: 106, and the VL amino acid sequence described in SEQ ID NO: 64, or (xiv) The VH amino acid sequence described in SEQ ID NO: 106, and the VL amino acid sequence described in SEQ ID NO: 119, or (xv) The VH amino acid sequence described in SEQ ID NO: 106, and the VL amino acid sequence described in SEQ ID NO: 130, or (xvi) The VH amino acid sequence described in SEQ ID NO: 113, and the VL amino acid sequence described in SEQ ID NO: 130, or (xvii) The VH amino acid sequence described in SEQ ID NO: 116, and the VL amino acid sequence described in SEQ ID NO: 119, or (xviiii) The VH amino acid sequence described in SEQ ID NO: 116, and the VL amino acid sequence described in SEQ ID NO: 130, or (xix) The VH amino acid sequence described in SEQ ID NO: 133, and the VL amino acid sequence described in SEQ ID NO: 130, or (xx) VH amino acid sequence described in SEQ ID NO: 136, and VL amino acid sequence described in SEQ ID NO: 130 The antibody-drug conjugate according to claim 1, comprising:

5. D is equation (IV): It is a compound of the formula, in which, R １ａ However, -H, -CH ３ ,-CHF ２ , -CF ３ , -F, -Br, -Cl, -OH, -OCH ３ , -OCF ３ , and -NH ２ Selected from, R ２ａ However, -H, -CH ３ , -CF ３ , -F, -Br, -Cl, -OH, -OCH ３ , and -OCF ３ Selected from, X is -O-, -S-, or -NH-, and R ４ａ but, Selected from, where * is a connection point with X, and p is 1, 2, 3, or 4, or X is O and R ４ａ -X- is, Selected from, R ５ａ However, -C １ ~C ６ Alkyl, -C ３ ~C ８ Cycloalkyl, -aryl, -heteroaryl, and -(C １ ~C ６ Selected from alkyl)-aryl, R ８ａ However, -C １ ~C ６ Alkyl, -C ３ ~C ８ Cycloalkyl and -C ３ ~C ８ Selected from heterocycloalkyl groups, Each R ９ａ However, independently, -C １ ~C ６ Alkyl, -C ３ ~C ８ Cycloalkyl, -aryl, -heteroaryl, and -(C １ ~C ６ Selected from alkyl)-aryl, or R ９ａ If X does not exist ｂ = X, Each R １０ａ However, independently, -C １ ~C ６ Alkyl, -C ３ ~C ８ Cycloalkyl, -aryl, -heteroaryl, -(C １ ~C ６ Alkyl)-aryl, and Selected from, Each R １０ａ’ However, independently, -H and -C １ ~C ６ Alkyl, -C ３ ~C ８ Cycloalkyl, -aryl, -heteroaryl, and -(C １ ~C ６ Selected from alkyl)-aryl, Each R １０ｂ However, independently, -C １ ~C ６ Alkyl, -C ３ ~C ８ Cycloalkyl, -aryl, -heteroaryl, and -(C １ ~C ６ Selected from alkyl)-aryl, R １１ａ However, it does not exist, or -C １ ~C ６ It is alkyl, R １２ａ However, -C １ ~C ６ Alkyl, -CO ２ R ８ａ -aryl, -heteroaryl, -(C １ ~C ６ Alkyl)-aryl, -S(O) ２ R １６ａ , and Selected from, R １３ａ However, -H and -C １ ~C ６ Selected from alkyl groups, R １４ａ However, -C １ ~C ６ Alkyl, -C ３ ~C ８ Cycloalkyl and -C ３ ~C ８ Selected from heterocycloalkyl groups, R １４ａ’ However, H, -C １ ~C ６ Alkyl, -C ３ ~C ８ Cycloalkyl and -C ３ ~C ８ Selected from heterocycloalkyl groups, R １６ａ However, -C １ ~C ６ Alkyl, -C ３ ~C ８ Cycloalkyl, -aryl, -heteroaryl, and -(C １ ~C ６ Selected from alkyl)-aryl, R ２１ However, -C １ ~C ６ Alkyl, -C ３ ~C ８ Cycloalkyl and -(C １ ~C ６ Alkyl)-O-R ５ａ Selected from, R ２２ and R ２３ However, each is independent of -H, -halogen, and -C. １ ~C ６ Alkyl and -C ３ ~C ８ Selected from cycloalkyl groups, R ２４ , R ２５ , and R ２６ However, each is -C １ ~C ６ It is alkyl, X ａ and X ｂ However, each is independently selected from NH, O, and S. X ｃ However, O, S, and S(O) ２ Selected from, and However, it indicates the connection point with linker L. The antibody-drug conjugate according to claim 3.

6. R １ａ However, -CH ３ , -OCH ３ , and NH ２ An antibody-drug conjugate according to claim 5, selected from the above.

7. R ２ａ The antibody-drug conjugate according to claim 5, wherein the conjugate is selected from -H, -F, -Br, and -Cl.

8. X is -O-, -S-, or -NH-, and R ４ａ but, An antibody-drug conjugate according to claim 5, selected from the above.

9. (a) R １ａ However, -CH ３ , -OCH ３ , and NH ２ Selected from, (b) R ２ａ However, it is selected from -H, -F, -Br, and -Cl, (c) X is -O-, -S-, or -NH-, and R ４ａ but, An antibody-drug conjugate according to claim 5, selected from the above.

10. D is equation (V): It is a compound of the formula, in which, R ２ａ However, -CH ３ , -CF ３ , -F, -Br, -Cl, -OH, -OCH ３ , and -OCF ３ Selected from, R ２０ａ However, -H, -C １ ~C ６ Alkyl, -C ３ ~C ８ Cycloalkyl, -(C １ ~C ６ Alkyl)-O-R ５ , -CO ２ R ８ -aryl, -heteroaryl, -(C １ ~C ６ Alkyl)-aryl, Selected from, R ５ However, -H, -C １ ~C ６ Alkyl, -C ３ ~C ８ Cycloalkyl, -aryl, -heteroaryl, and -(C １ ~C ６ Selected from alkyl)-aryl, R ６ and R ７ However, independently of each other, -H and -C １ ~C ６ Alkyl, -C ３ ~C ８ Cycloalkyl, -(C １ ~C ６ Alkyl)-O-R ５ , -C ３ ~C ８ Heterocycloalkyl and -C(O)R １７ Selected from, R ８ However, -H, -C １ ~C ６ Alkyl, -C ３ ~C ８ Cycloalkyl and -C ３ ~C ８ Selected from heterocycloalkyl groups, Each R ９ However, independently, -H and -C １ ~C ６ Alkyl, -C ３ ~C ８ Cycloalkyl, -aryl, -heteroaryl, and -(C １ ~C ６ Selected from alkyl)-aryl, Each R 10 independently, -C １ ~C ６ Alkyl, -C ３ ~C ８ Cycloalkyl, -aryl, -heteroaryl, -(C １ ~C ６ Alkyl)-aryl and -NR １４ R １４’ Selected from, Each R 10' independently has -H and -C １ ~C ６ Alkyl, -C ３ ~C ８ Cycloalkyl, -aryl, -heteroaryl, and -(C １ ~C ６ Selected from alkyl)-aryl, R １１ However, -H and -C １ ~C ６ Selected from alkyl groups, R １２ However, -H, -C １ ~C ６ Alkyl, -CO ２ R ８ -aryl, -heteroaryl, -(C １ ~C ６ Alkyl)-aryl, -S(O) ２ R １６ , and Selected from, R １３ However, -H and -C １ ~C ６ Selected from alkyl groups, R １４ and R １４’ However, independently of each other, -H and C １ ~C ６ Alkyl, -C ３ ~C ８ Cycloalkyl and -C ３ ~C ８ Selected from heterocycloalkyl groups, R １６ However, -C １ ~C ６ Alkyl, -C ３ ~C ８ Cycloalkyl, -aryl, -heteroaryl, and -(C １ ~C ６ Selected from alkyl)-aryl, R １７ However, -C １ ~C ６ Alkyl, -C ３ ~C ８ Cycloalkyl, -C ３ ~C ８ Heterocycloalkyl, -(C １ ~C ６ Alkyl)-C ３ ~C ８ Heterocycloalkyl, -aryl, -heteroaryl, and -(C １ ~C ６ Selected from alkyl)-aryl, R １８ and R １９ However, along with the N atom to which they bond, halogens, -C １ ~C ６ Alkyl, -C ３ ~C ８ Cycloalkyl and -(C １ ~C ６ Alkyl)-O-R ５ Forming a 4, 5, 6, or 7-membered ring having 0 to 3 substituents selected from, R ２４ , R ２５ , and R ２６ However, each is -C １ ~C ６ It is alkyl, X ａ and X ｂ However, each is independently selected from NH, O, and S. X ｃ However, O, S, and S(O) ２ Selected from, and However, it indicates the connection point with linker L. The antibody-drug conjugate according to claim 3.

11. R ２ａ However, F and R ２０ａ However, -H, -C １ ~C ６ Alkyl, -(C １ ~C ６ Alkyl)-O-R ５ , - (C １ ~C ６ Alkyl)-aryl, An antibody-drug conjugate according to claim 10, selected from the above.

12. D is equation (VI): It is a compound of the formula, in which, R ２ａ However, -H, -CH ３ , -CF ３ , -F, -Br, -Cl, -OH, -OCH ３ , and -OCF ３ Selected from, X is -O-, -S-, or -NH-, and R ２５ However, -C １ ~C ６ Alkyl, -(C １ ~C ６ Alkyl)-O-R ５ａ , -CO ２ R ８ａ -aryl, -heteroaryl, -(C １ ~C ６ Alkyl)-aryl, Selected from, where * is a connection point with X, and p is 1, 2, 3, or 4, or X is O and R ２５ -X- is, Selected from, R ５ａ However, -C １ ~C ６ Alkyl, -C ３ ~C ８ Cycloalkyl, -aryl, -heteroaryl, and -(C １ ~C ６ Selected from alkyl)-aryl, R ６ａ However, -H, -C １ ~C ６ Alkyl, -C ３ ~C ８ Cycloalkyl and -C ３ ~C ８ Selected from heterocycloalkyl groups, R ７ａ However, -C １ ~C ６ Alkyl, -C ３ ~C ８ Cycloalkyl, -(C １ ~C ６ Alkyl)-O-R ５ａ , -C ３ ~C ８ Heterocycloalkyl and -C(O)R １７ａ Selected from, R ８ａ However, -C １ ~C ６ Alkyl, -C ３ ~C ８ Cycloalkyl and -C ３ ~C ８ Selected from heterocycloalkyl groups, Each R ９ａ However, independently, -C １ ~C ６ Alkyl, -C ３ ~C ８ Cycloalkyl, -aryl, -heteroaryl, and -(C １ ~C ６ Selected from alkyl)-aryl, or R ９ａ If X does not exist ｂ = X, Each R １０ａ However, independently, -C １ ~C ６ Alkyl, -C ３ ~C ８ Cycloalkyl, -aryl, -heteroaryl, -(C １ ~C ６ Alkyl)-aryl, and Selected from, Each R １０ａ’ However, independently, -H and -C １ ~C ６ Alkyl, -C ３ ~C ８ Cycloalkyl, -aryl, -heteroaryl, and -(C １ ~C ６ Selected from alkyl)-aryl, Each R １０ｂ However, independently, -C １ ~C ６ Alkyl, -C ３ ~C ８ Cycloalkyl, -aryl, -heteroaryl, and -(C １ ~C ６ Selected from alkyl)-aryl, R １１ａ However, it does not exist, or -C １ ~C ６ It is alkyl, R １２ａ However, -C １ ~C ６ Alkyl, -CO ２ R ８ａ -aryl, -heteroaryl, -(C １ ~C ６ Alkyl)-aryl, -S(O) ２ R １６ａ , and Selected from, R １３ａ However, -H and -C １ ~C ６ Selected from alkyl groups, R １４ａ However, -C １ ~C ６ Alkyl, -C ３ ~C ８ Cycloalkyl and -C ３ ~C ８ Selected from heterocycloalkyl groups, R １４ａ’ However, H, -C １ ~C ６ Alkyl, -C ３ ~C ８ Cycloalkyl and -C ３ ~C ８ Selected from heterocycloalkyl groups, R １６ａ However, -C １ ~C ６ Alkyl, -C ３ ~C ８ Cycloalkyl, -aryl, -heteroaryl, and -(C １ ~C ６ Selected from alkyl)-aryl, R １７ａ However, -C １ ~C ６ Alkyl, -C ３ ~C ８ Cycloalkyl, -C ３ ~C ８ Heterocycloalkyl, -(C １ ~C ６ Alkyl)-C ３ ~C ８ Heterocycloalkyl, -aryl, -heteroaryl, and -(C １ ~C ６ Selected from alkyl)-aryl, R ２１ However, -C １ ~C ６ Alkyl, -C ３ ~C ８ Cycloalkyl and -(C １ ~C ６ Alkyl)-O-R ５ａ Selected from, R ２２ and R ２３ However, each is independent of -H, -halogen, and -C. １ ~C ６ Alkyl and -C ３ ~C ８ Selected from cycloalkyl groups, R ２４ , R ２５ , and R ２６ However, each is -C １ ~C ６ It is alkyl, X ａ and X ｂ However, each is independently selected from NH, O, and S. X ｃ However, O, S, and S(O) ２ Selected from, and However, it indicates the connection point with linker L. The antibody-drug conjugate according to claim 3.

13. R ２ａ The antibody-drug conjugate according to claim 12, wherein F is present.

14. X is -O-, -S-, or -NH-, and R ２５ However, -C １ ~C ６ Alkyl, -(C １ ~C ６ Alkyl)-O-R ５ａ , - (C １ ~C ６ Alkyl)-aryl, Either X is selected from or X is O and R ２５ -X- is, Selected from, The antibody-drug conjugate according to claim 12.

15. (a) X is -O- and R ２５ but, And, (b) X ａ and X ｂ However, each is O, and (c) R ９ａ ga-C １ ~C ６ It is alkyl. The antibody-drug conjugate according to claim 12.

16. The antibody-drug conjugate according to any one of claims 1 to 15, wherein each alkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl group may be substituted with one or more substituents selected from halogens, acyl, acyloxy, alkoxy, carboxy, hydroxy, amino, amide, nitro, cyano, azide, alkylthio, thio, sulfonyl, sulfonamide, alkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl.

17. The antibody-drug conjugate according to any one of claims 1 to 15, wherein each alkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl group may be substituted with one or more substituents selected from halogens, acyls, acyloxys, alkoxys, carboxys, hydroxys, aminos, amides, nitros, cyanos, azides, alkylthios, thiosulfonyls, and sulfonamides.

18. D is the following compound: The antibody-drug conjugate according to claim 3, having any one of the structures.

19. D is compound 139 or compound 141 The antibody-drug conjugate according to claim 3.

20. The antibody-drug conjugate according to any one of claims 1 to 15, 18, and 19, wherein L is a cleavable linker.

21. The antibody-drug conjugate according to claim 20, wherein L is a protease-cleavable linker and comprises a dipeptide, tripeptide, or tetrapeptide.

22. L, (a) Equation (XI) It has, in the formula, Z is a functional group that can react with the target group on the anti-FRα antibody construct T, Str stands for stretcher, AA １ and AA ２ However, each is an amino acid independently, AA １ - [AA] ２ ] ｒ However, it forms a protease cleavage site, X is a self-destructing group, q is either 0 or 1, r is 1, 2, or 3, s is 0, 1, or 2, # is a binding site to the anti-FRα antibody construct T, and % is the binding site with camptothecin analog D, or (b) Equation (XII) It has, in the formula, Z is a functional group that can react with the target group on the anti-FRα antibody construct T, Str stands for stretcher, AA １ and AA ２ However, each is an amino acid independently, AA １ - [AA] ２ ] ｒ However, it forms a protease cleavage site, Y is -NH-CH ２ - or -NH-CH ２ -C(O)-, q is either 0 or 1, r is 1, 2, or 3, v is either 0 or 1, # is a binding site to the anti-FRα antibody construct T, and % is the binding site with the camptothecin analog D. The antibody-drug conjugate according to claim 21.

23. In equation (X), L-(D) is the following drug linker (DL): The antibody-drug conjugate according to claim 3, having any one of the structures.

24. In equation (X), L - (D) is MT-GGFG-AM-Compound 139 MC-GGFG-AM-Compound 139 MT-GGFG-AM-Compound 141 MC-GGFG-AM-Compound 141 MT-GGFG-Compound 141 or MC-GGFG-Compound 141 The antibody-drug conjugate according to claim 3.

25. The antibody-drug conjugate according to any one of claims 1 to 15, 18, 19, 23, and 24, wherein m is 1 to 2.

26. The antibody-drug conjugate according to any one of claims 1 to 15, 18, 19, 23, and 24, wherein m is 1.

27. The antibody-drug conjugate according to any one of claims 1 to 15, 18, 19, 23, and 24, wherein n is 2 to 8.

28. The antibody-drug conjugate according to any one of claims 1 to 15, 18, 19, 23, and 24, wherein n is 4 to 8.

29. The antibody-drug conjugate according to any one of claims 1 to 15, 18, 19, 23, and 24, wherein the anti-FRα antibody construct further comprises a scaffold, and the antigen-binding domain is functionally linked to the scaffold.

30. The antibody-drug conjugate according to claim 29, wherein the scaffold includes an IgG Fc region.

31. An antibody-drug conjugate having a structure selected from, wherein, T is an anti-FRα antibody construct comprising two antigen-binding domains functionally linked to the IgG Fc region, and each of the antigen-binding domains is (a) The VL amino acid sequence described in SEQ ID NO: 39, and the VH amino acid sequence described in SEQ ID NO: 19, or (b) The VL amino acid sequence described in SEQ ID NO: 124, and the VH amino acid sequence described in SEQ ID NO: 91, or (c) The VL amino acid sequence described in Sequence ID No. 64, and (i) The VH amino acid sequence described in Sequence ID No. 50, or (ii) The VH amino acid sequence described in Sequence ID No. 54, or (iii) The VH amino acid sequence described in Sequence ID No. 57, or (iv) The VH amino acid sequence described in SEQ ID NO: 61, or (v) The VH amino acid sequence described in SEQ ID NO: 76, or (vi) The VH amino acid sequence described in Sequence ID No. 79, or (vii) The VH amino acid sequence described in Sequence ID No. 82, or (viiii) The VH amino acid sequence described in Sequence ID No. 85, or (ix) The VH amino acid sequence described in Sequence ID No. 88, or (x) The VH amino acid sequence described in Sequence ID No. 106, or (d) The VL amino acid sequence described in Sequence ID No. 130, and (i) The VH amino acid sequence described in Sequence ID No. 99, or (ii) The VH amino acid sequence described in Sequence ID No. 106, or (iii) The VH amino acid sequence described in Sequence ID No. 113, or (iv) The VH amino acid sequence described in SEQ ID NO: 116, or (v) The VH amino acid sequence described in SEQ ID NO: 133, or (vi) The VH amino acid sequence described in SEQ ID NO: 136, or (e) The VL amino acid sequence described in Sequence ID No. 119, and (i) The VH amino acid sequence described in Sequence ID No. 106, or (ii) VH amino acid sequence described in Sequence ID No. 116 including and n is between 4 and 8. The aforementioned antibody-drug conjugate.

32. structure: An antibody-drug conjugate having, in the formula, T is an anti-folate receptor alpha (anti-FRα) antibody construct comprising an antigen-binding domain functionally linked to the IgG Fc region, wherein the antigen-binding domain comprises three heavy chain complementarity-determining regions (CDRs) (HCDR1, HCDR2, and HCDR3) and three light chain CDRs (LCDR1, LCDR2, and LCDR3), where (i) The HCDR1 amino acid sequence includes the sequence described in any one of SEQ ID NOs: 20, 23, 26, 28, or 31; the HCDR2 amino acid sequence includes the sequence described in any one of SEQ ID NOs: 21, 24, 29, 32, or 51; and the HCDR3 amino acid sequence includes the sequence described in any one of SEQ ID NOs: 22, 25, or 30, and (ii) The LCDR1 amino acid sequence includes the sequence described in any one of SEQ ID NOs: 40, 45, or 65; the LCDR2 amino acid sequence includes the sequence described in any one of SEQ ID NOs: 41, 44, or 46; and the LCDR3 amino acid sequence includes the sequence described in any one of SEQ ID NOs: 42 or 47. n is approximately 4 to approximately 8. The aforementioned antibody-drug conjugate.

33. The antibody-drug conjugate according to claim 32, wherein n is approximately 8.

34. A pharmaceutical composition for the treatment of cancer selected from breast cancer, ovarian cancer, non-small cell lung cancer (NSCLC), and endometrial cancer, comprising an antibody-drug conjugate according to any one of claims 1 to 15, 18, 19, 23, 24, 31, 32, and 33, and a pharmaceutically acceptable carrier or diluent.

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