Folate receptor alpha-targeted antibody-drug conjugates and methods of use - Patents.com

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 The present disclosure relates to the field of immunotherapeutics, and in particular 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 a member of a family of high-affinity FRs that also includes FRβ (FOLR2), FRγ (FOLR3), and FRδ (FOLR4). FRα is overexpressed in various cancers, including ovarian cancer, triple-negative breast cancer (TNBC), endometrial cancer, mesothelioma, and lung cancer, and is minimally expressed in non-malignant tissues, identifying it as a highly relevant cancer therapy target.

[0003] Several clinical studies involving FRα-targeted agents in the treatment of cancer are currently underway, including the anti-FRα antibody, farletuzumab, and the FRα-targeted antibody-drug conjugates (ADCs), mirvetuximab soravtansine (ImmunoGen, Inc.), MORAb-202 (Eisai Inc.), and STRO-002 (Sutro Biopharma, Inc.).

[0004] Camptothecin analogs have been 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 to the anti-HER2 antibody trastuzumab via a cleavable tetrapeptide-based linker, and sacituzumab govitecan (Trodelvy™), in which the camptothecin analog SN-38 is conjugated to the anti-Trop-2 antibody sacituzumab via a hydrolyzable, pH-sensitive linker.

[0005] Other camptothecin analogs and derivatives, and ADCs containing them, have been described, see, for example, International (PCT) Publication Nos. WO2019 / 195665 (Patent Document 1), WO2019 / 236954 (Patent Document 2), WO2020 / 200880 (Patent Document 3), and WO2020 / 219287 (Patent Document 4).

[0006] This background information is provided for the purpose of making known information believed by the applicant to be of possible relevance to the present disclosure. No admission is necessarily intended, nor should it be construed, that any of the preceding information constitutes prior art against the claimed invention. [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 Summary of the Invention

[0008] overview Described herein are antibody-drug conjugates (ADCs) that target human FRα and methods of use. One aspect of the disclosure is an ADC having formula (X): T-[L-(D) m ] n (X) and an antibody-drug conjugate comprising: m is 1 to 4; n is 1 to 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 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: TIFF2025510791000002.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 is -NH2, R is R 3 or R 4 and R 1 is other than -NH2, R is R 4 and R 3 is -H, -C1-C6 alkyl, -C3-C8 cycloalkyl, -(C1-C6 alkyl)-OR 5 , TIFF2025510791000003.tif27165-CO2R 8 , -aryl, -heteroaryl, and -(C1-C6 alkyl)-aryl; R 4 teeth, Selected from TIFF2025510791000004.tif68165, 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 -H, -C1-C6 alkyl, -C3-C8 cycloalkyl, -(C1-C6 alkyl)-OR 5 , —C3-C8 heterocycloalkyl, and —C(O)R17 is selected from R 8 is 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 are independently -C1-C6 alkyl, -C3-C8 cycloalkyl, -NR 14 R 14’ , -aryl, -heteroaryl, and -(C1-C6 alkyl)-aryl; R 10’ is selected from -H, -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl, and -(C1-C6 alkyl)-aryl; R 11 is selected from —H and —C1-C6 alkyl; R 12 is -H, -C1-C6 alkyl, -CO2R 8 , -aryl, -heteroaryl, -(C1-C6 alkyl)-aryl, -S(O)2R 16 , and Selected from TIFF2025510791000005.tif22165, R 13 is selected from —H and —C1-C6 alkyl; R 14 and R 14’ are each independently selected from —H, C1-C6 alkyl, —C3-C8 cycloalkyl, and —C3-C8 heterocycloalkyl; R 16 is selected from -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl, and -(C1-C6 alkyl)-aryl; R 17is 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 together with the N atom to which they are attached, represent halogen, -C1-C6 alkyl, -C3-C8 cycloalkyl, and -(C1-C6 alkyl)-OR 5 and forming a 4-, 5-, 6-, or 7-membered ring having 0 to 3 substituents selected from R 24 , R 25 and R 26 are each -C1 to C6 alkyl, X a and X b are each independently selected from NH, O, and S; X c is selected from O, S, and S(O)2; However, the compound is other than (S)-9-amino-11-butyl-4-ethyl-4-hydroxy-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione.

[0009] Another aspect of the present disclosure relates to an antibody-drug conjugate having a structure selected from the following: TIFF2025510791000006.tif227165TIFF2025510791000007.tif104165In the formula, T is an anti-FRα antibody construct comprising two antigen-binding domains operably linked to an IgG Fc region, each of the antigen-binding domains comprising: (a) the VL amino acid sequence set forth in SEQ ID NO: 39 and the VH amino acid sequence set forth in SEQ ID NO: 19, or (b) a VL amino acid sequence set forth in SEQ ID NO: 124 and a VH amino acid sequence set forth in SEQ ID NO: 91; or (c) the VL amino acid sequence set forth in SEQ ID NO: 64, and (i) the VH amino acid sequence set forth in SEQ ID NO: 50, or (ii) the VH amino acid sequence set forth in SEQ ID NO: 54, or (iii) the VH amino acid sequence set forth in SEQ ID NO: 57, or (iv) the VH amino acid sequence set forth in SEQ ID NO: 61, or (v) the VH amino acid sequence set forth in SEQ ID NO: 76, or (vi) the VH amino acid sequence set forth in SEQ ID NO: 79, or (vii) the VH amino acid sequence set forth in SEQ ID NO: 82, or (viii) the VH amino acid sequence set forth in SEQ ID NO: 85; or (ix) the VH amino acid sequence set forth in SEQ ID NO: 88, or (x) a VH amino acid sequence set forth in SEQ ID NO: 106, or (d) the VL amino acid sequence set forth in SEQ ID NO: 130, and (i) the VH amino acid sequence set forth in SEQ ID NO: 99, or (ii) the VH amino acid sequence set forth in SEQ ID NO: 106, or (iii) the VH amino acid sequence set forth in SEQ ID NO: 113, or (iv) the VH amino acid sequence set forth in SEQ ID NO: 116, or (v) the VH amino acid sequence set forth in SEQ ID NO: 133, or (vi) the VH amino acid sequence set forth in SEQ ID NO: 136, or (e) the VL amino acid sequence set forth in SEQ ID NO: 119, and (i) the VH amino acid sequence set forth in SEQ ID NO: 106, or (ii) the VH amino acid sequence set forth in SEQ ID NO: 116; Including, and n is 4 to 8.

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

[0011] Another aspect of the present disclosure relates to a method of 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 the present disclosure relates to a method of killing cancer cells comprising contacting the cells with an effective amount of an antibody-drug conjugate described herein.

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

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

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

[0016] Another aspect of the present disclosure relates to the use of an antibody-drug conjugate described herein in the manufacture of a medicament for the treatment of cancer. [Brief explanation of the drawings]

[0017] [Figure 1] A and B show the rabbit heavy chain variable domain CDR sequences of chimeric anti-FRα antibody v23924 ported onto a human VH framework (IGHV3-23*01) (SEQ ID NO: 155) (A), and the rabbit light chain variable domain CDR sequences of chimeric antibody v23924 ported onto a human VL framework (IGKVI-39*01) (SEQ ID NO: 156) (B). CDRs are assigned using the AbM definition and are marked in bold italics. [Figure 2A]Electrophoretic and UPLC-SEC profiles of purified parent chimeric anti-FRα antibody v23924 and purified representative humanized variant v30384 are shown. A and C show profiles (C) from electrophoresis under non-reducing (NR) and reducing (R) conditions of parent chimeric anti-FRα antibody v23924 (A) and purified representative humanized variant v30384 after preparative SEC purification (after preparative SEC) or Protein A purification (after Protein A). B and D show UPLC-SEC profiles (D) of parent chimeric anti-FRα antibody v23924 after preparative SEC purification (B) and purified representative humanized variant v30384 after Protein A purification. [Figure 2B] See legend to Figure 2A. [Figure 2C] See legend to Figure 2A. [Figure 2D] See legend to Figure 2A. [Figure 3] A and B show biolayer interferometry (BLI) sensorgrams of the parent chimeric anti-FRα antibody v23924 (A) and a purified representative humanized variant v30384 (B). [Figure 4A] Shown are intact LC / MS profiles of representative humanized variants v30384 (A, with a zoom of the main peak in B) and v31422 (C, with a zoom of the main peak in D). [Figure 4B] See legend to Figure 4A. [Figure 4C] See legend to Figure 4A. [Figure 4D] See legend to Figure 4A. [Figure 5] Panels A and B show the receptor-mediated internalization capacity of the chimeric anti-FRα antibody v23924, a representative humanized variant, v30384, and the FRα-targeting antibodies mirvetuximab and farletuzumab at various concentrations in the FRα-expressing cell line IGROV-1, as determined by flow cytometry after 6 hours (A) and 24 hours (B) of incubation. The anti-RSV antibody palivizumab was included as a negative control. [Figure 6](A) and (B) show the receptor-mediated internalization capacity of the chimeric anti-FRα antibody v23924, a representative humanized variant, v30384, and the FRα-targeting antibodies mirvetuximab and farletuzumab at various concentrations in the FRα-expressing cell line OVCAR-3, as determined by flow cytometry after 6 hours (A) and 24 hours (B) of incubation. The anti-RSV antibody, palivizumab, was included as a negative control. [Figure 7] 1 shows coverage of the hFRα sequence (SEQ ID NO: 15) by peptides generated by pepsin digestion of hFRα. Each bar below the sequence represents a peptide. [Figure 8A] Summary plots of hydrogen / deuterium exchange mass spectrometry (HDX-MS) kinetics of peptides generated by pepsin digestion of hFRα:hFOLR1 (hFRα) vs. hFOLR1-v23924 complexes are shown (8A). [Figure 8B] A difference plot of hydrogen / deuterium exchange mass spectrometry (HDX-MS) kinetics of peptides generated by pepsin digestion of hFRα:hFOLR1 (hFRα) vs. hFOLR1-v23924 complexes is shown (8B). [Figure 9] AC show the amide deuteration level 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 the difference plot (C). [Figure 10] A and B show the receptor-mediated internalization capacity of the parent anti-FRα humanized antibody variant, v30384, and a representative affinity-matured variant, v35356, in the FRα-expressing cell lines IGROV-1 (A) and JEG-3 (B) after 5 and 24 hour incubation periods, as determined by flow cytometry. Palivizumab was included as a non-targeting control. [Figure 11-1] A table showing the CDR sequences of representative anti-FRα antibodies as defined by the IMGT, Chothia, Kabat, Contact, and AbM definitions is provided. [Figure 11-2]See the description of Figure 11-1. [Figure 11-3] See the description of Figure 11-1. [Figure 11-4] See the description of Figure 11-1. [Figure 11-5] See the description of Figure 11-1. [Figure 11-6] See the description of Figure 11-1. [Figure 11-7] See the description of Figure 11-1. [Figure 12-1] A table showing the VH and VL sequences of representative anti-FRα antibodies is shown. [Figure 12-2] See the description of Figure 12-1. [Figure 12-3] See the description of Figure 12-1. [Figure 13-1] Exemplary drug-linker (DL) structures are shown (Table 8), including camptothecin analogs of formula (I) with a C7 linkage. [Figure 13-2] See the description of Figure 13-1. [Figure 13-3] See the description of Figure 13-1. [Figure 14-1] Exemplary drug-linker (DL) structures are shown (Table 9), including camptothecin analogs of formula (I) with a C10 bond. [Figure 14-2] See the description of Figure 14-1. [Figure 15-1] Exemplary drug-linker (DL) structures are shown (Table 10), including camptothecin analogs of formula (I) with C7 or C10 linkages. [Figure 15-2] See the description of Figure 15-1. [Figure 15-3] See the description of Figure 15-1. [Figure 16-1] Exemplary conjugate (DC) structures including camptothecin analogs of formula (I) with a C7 linkage are shown (Table 11). [Figure 16-2] See the description of Figure 16-1. [Figure 16-3] See the description of Figure 16-1. [Figure 16-4] See the description of Figure 16-1. [Figure 17-1] Exemplary conjugate (DC) structures including camptothecin analogs of formula (I) with a C10 bond are shown (Table 12). [Figure 17-2] See the description of Figure 17-1. [Figure 17-3] See the description of Figure 17-1. [Figure 18-1] Exemplary conjugate (DC) structures containing camptothecin analogs of formula (I) with C7 or C10 linkages are shown (Table 13). [Figure 18-2] See the description of Figure 18-1. [Figure 18-3] See the description of Figure 18-1. [Figure 18-4] See the description of Figure 18-1. [Figure 19A] Figure 1 shows the in vivo antitumor activity of ADCs containing anti-FRα humanized antibody variant v30384 conjugated to camptothecin analogs Compound 139 or Compound 141 at a DAR of 8 in an OV90 xenograft model (A and B), and to camptothecin analogs Compound 139, Compound 140, Compound 141, or Compound 148 at a DAR of 8 in an H2110 xenograft model (C). An ADC containing palivizumab (v21995) was included as a control. [Figure 19B] See legend to Figure 19A. [Figure 19C] See legend to Figure 19A. [Figure 20] Pharmacokinetics of four ADCs, including anti-FRα humanized antibody v36675 and v36675 conjugated to DXd, camptothecin analogs Compound 139, or Compound 141, evaluated in hFcRn Tg32 mice (n=4) at DAR8. Mean serum concentrations at some time points were calculated using n<4 animals (as some samples were below the detection limit). [Figure 21]Figure 1 shows the in vivo stability of four ADCs containing humanized variant v36675 conjugated to DXd or camptothecin analogs Compound 139 or Compound 141 at a DAR of 8 in Tg32 mouse serum. The solid line indicates % DAR remaining (left axis), and the dotted line indicates % maleimide ring opening (right axis). [Figure 22A] 1 shows the in vivo anti-tumor activity of ADCs containing anti-FRα humanized antibody variant v36675 conjugated to camptothecin analogs Compound 139 or Compound 141 at DAR 4 or DAR 8, respectively, in an OV90 CDX xenograft model (A, B); conjugated to camptothecin analogs Compound 140 or Compound 141 at DAR 4 or DAR 8, respectively, in an OVCAR3 CDX xenograft model (C); conjugated to camptothecin analog Compound 139 at DAR 8, in a GTG-2025 PDX xenograft model (D); and conjugated to camptothecin analog Compound 139 at DAR 8, in a GTG-0958 PDX xenograft model (E). [Figure 22B] See the description of Figure 22A. [Figure 22C] See the description of Figure 22A. [Figure 22D] See the description of Figure 22A. [Figure 22E] See the description of Figure 22A. [Figure 23A]

[0039] Figure 1 shows total antibody serum concentrations of ADCs containing anti-FRα humanized antibody v36675 in blood samples collected after the first dose in a cynomolgus monkey toxicity study. ADC containing v36675 conjugated to Compound 139 or Compound 141 at a DAR of 8 administered at 30 mg / kg (A), ADC containing v36675 conjugated to Compound 139 or Compound 141 at a DAR of 4 administered at 60 mg / kg (B), ADC containing v36675 conjugated to Compound 139 or Compound 141 at a DAR of 8 administered at 80 mg / kg (C), and ADC containing v36675 conjugated to Compound 139 or Compound 141 at a DAR of 4 or DAR of 8 administered at 120 mg / kg (D). [Figure 23B] See the description of Figure 23A. [Figure 23C] See the description of Figure 23A. [Figure 23D] See the description of Figure 23A. [Figure 24]

[0039] Figure 1 shows 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, and an ADC containing palivizumab (v22277) conjugated to MC-GGFG-AM-DXd1 and MCvcPABC-MMAE was included as a negative control. [Figure 25] A to D show the penetration of anti-FRα humanized antibody variant v36675 into JEG-3 cell spheroids compared to mirvetuximab and the negative control palivizumab at 4 hours (25A), 24 hours (25B), 48 hours (25C), and 96 hours (25D). [Figure 26] A and B show intracellular (26A) and extracellular (26B) payload release from an ADC containing the anti-FRα humanized antibody variant v36675 conjugated to Compound 139 at DAR 8 (v36675-MC-GGFG-AM-Compound 139) and an ADC containing the non-targeting control palivizumab (v21995) conjugated to Compound 139 at DAR 8 in the high FRα-expressing cell line IGROV-1. [Figure 27A] Figure 1 shows the in vivo antitumor activity of ADCs in patient-derived xenograft (PDX) models of ovarian cancer when administered at 6 mg / kg, including the anti-FRα humanized antibody variant v36675 conjugated to Compound 139 at a DAR of 8 (v36675-MC-GGFG-AM-Compound 139), and the control ADC, mirvetuximab-DM4 at a DAR of 4. CTG-0703 PDX model (A). [Figure 27B]Figure 1 shows the in vivo antitumor activity of ADCs in patient-derived xenograft (PDX) models of ovarian cancer when administered at 6 mg / kg, including the anti-FRα humanized antibody variant v36675 conjugated to Compound 139 at a DAR of 8 (v36675-MC-GGFG-AM-Compound 139), and the control ADC, mirvetuximab-DM4 at a DAR of 4. (B) CTG-1301 PDX model. [Figure 27C] Figure 1 shows the in vivo antitumor activity of ADCs in a patient-derived xenograft (PDX) model of ovarian cancer when administered at 6 mg / kg, including the anti-FRα humanized antibody variant v36675 conjugated to Compound 139 at a DAR of 8 (v36675-MC-GGFG-AM-Compound 139), and the control ADC, mirvetuximab-DM4 at a DAR of 4. (C) CTG-2025 PDX model. [Figure 27D] (C) shows the in vivo antitumor activity of ADCs in a patient-derived xenograft (PDX) model of ovarian cancer when administered at 6 mg / kg, including the anti-FRα humanized antibody variant v36675 conjugated to Compound 139 at a DAR of 8 (v36675-MC-GGFG-AM-Compound 139), and the control ADC, mirvetuximab-DM4 at a DAR of 4. (D) shows the in vivo antitumor activity of ADCs in a patient-derived xenograft (PDX) model of ovarian cancer when administered at 6 mg / kg, including the CTG-3383 PDX model. [Figure 27E] Figure 1 shows the in vivo antitumor activity of ADCs in a patient-derived xenograft (PDX) model of ovarian cancer when administered at 6 mg / kg, including the anti-FRα humanized antibody variant v36675 conjugated to Compound 139 at a DAR of 8 (v36675-MC-GGFG-AM-Compound 139), and the control ADC, mirvetuximab-DM4 at a DAR of 4. CTG-0947 PDX model (E). [Figure 27F] Figure 1 shows the in vivo antitumor activity of ADCs in a patient-derived xenograft (PDX) model of ovarian cancer when administered at 6 mg / kg, including the anti-FRα humanized antibody variant v36675 conjugated to Compound 139 at a DAR of 8 (v36675-MC-GGFG-AM-Compound 139), and the control ADC, mirvetuximab-DM4 at a DAR of 4. CTG-0958 PDX model (F). [Figure 27G]Figure 1 shows the in vivo antitumor activity of ADCs in patient-derived xenograft (PDX) models of ovarian cancer when administered at 6 mg / kg, including the anti-FRα humanized antibody variant v36675 conjugated to Compound 139 at a DAR of 8 (v36675-MC-GGFG-AM-Compound 139), and the control ADC, mirvetuximab-DM4 at a DAR of 4. CTG-3718 PDX model (G). [Figure 27H] Figure 1 shows the in vivo antitumor activity of ADCs in patient-derived xenograft (PDX) models of ovarian cancer when administered at 6 mg / kg, including the anti-FRα humanized antibody variant v36675 conjugated to Compound 139 at a DAR of 8 (v36675-MC-GGFG-AM-Compound 139), and the control ADC, mirvetuximab-DM4 at a DAR of 4. CTG-1703 PDX model (H). [Figure 27I] Figure 1 shows the in vivo antitumor activity of ADCs in patient-derived xenograft (PDX) models of ovarian cancer when administered at 6 mg / kg, including the anti-FRα humanized antibody variant v36675 conjugated to Compound 139 at a DAR of 8 (v36675-MC-GGFG-AM-Compound 139), and the control ADC, mirvetuximab-DM4 at a DAR of 4. CTG-1602 PDX model (I). [Figure 28A] 1 shows fixed cell confirmation screening images from screening for specific off-target binding interactions using Retrogenix Cell Microarray Technology for anti-FRα humanized antibody variant v36675(A) at 20 μg / mL. [Figure 28B] Fixed cell confirmation screening images from screening for specific off-target binding interactions using Retrogenix Cell Microarray Technology for 1 μg / mL of control antibody (rituximab biosimilar) (B) are shown. [Figure 29] 1 shows competitive binding between the chimeric anti-FRα antibody v23294 and the anti-FRα antibodies mirvetuximab and farletuzumab, assessed in H2110 cells. DETAILED DESCRIPTION OF THE INVENTION

[0018] Detailed Description The present disclosure relates to 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) described herein. In particular, the present disclosure relates to an antibody-drug conjugate (ADC) 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) described herein. T-[L-(D) m ] n (X) For an ADC having the formula: T is an anti-FRα antibody construct 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 1 to 10.

[0019] The ADCs of the present 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 meaning as commonly understood by one of ordinary skill in the art.

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

[0022] The use of the words "a" or "an," when used herein in conjunction with the term "comprising," can mean "one," but is also consistent with the meaning 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 grammatical variations thereof, are inclusive, i.e., open-ended, and do not exclude additional, unrecited elements and / or method steps. The term "consisting essentially of," when used in connection with a composition, use, or method, indicates that additional elements and / or method steps may be present, but that these additions do not materially affect the manner in which the recited composition, method, or use functions. The term "consisting of," when used in connection with a composition, use, or method, excludes the presence of additional elements and / or method steps. A composition, use, or method described herein as including certain elements and / or steps may also consist essentially of those elements and / or steps in certain embodiments, and may consist of those elements and / or steps in other embodiments, whether or not those embodiments are specifically referred to.

[0024] "Complementarity-determining regions" or "CDRs" are amino acid sequences that contribute to antigen-binding specificity and affinity. "Framework" regions (FRs) can help maintain the proper conformation of CDRs to facilitate 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 comprise the 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 contact residues for binding an antibody to an antigen or epitope. In many cases, three heavy chain CDRs and three light chain CDRs are required to bind to an antigen. However, in some cases, even a single variable domain can confer antigen-binding specificity. Furthermore, as is known in the art, in some cases, antigen binding may also occur through a combination of at least one or more CDRs, e.g., HCDR3s, selected from the VH and / or VL domains.

[0025] Several different definitions of CDR sequences are commonly used, including those set forth 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 the definitions of IMGT, AbM (University of Bath), and Contact (MacCallum, et al., 1996, J Mol Biol, 262(5):732-745). By way of example, CDR definitions according to Kabat, Chothia, IMGT, AbM, and Contact are set forth in Table 1 below. Thus, as will be readily apparent to one of skill in the art, the exact numbering and arrangement of CDRs may vary based on the numbering system used. However, it should be understood that the disclosure of a VH herein includes the disclosure of the associated (unique) heavy chain CDRs (HCDRs) defined by any of the known numbering systems. Similarly, the disclosure herein of a VL includes disclosure of the associated (unique) light chain CDR (LCDR), as defined by any known numbering system.

[0026] [Table 1]

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

[0028] A "comparison window" refers to a segment of a sequence that includes contiguous amino acid or nucleotide positions, for example, about 10 to about 600 contiguous amino acid or nucleotide positions, or about 10 to about 200 contiguous amino acid or nucleotide positions, or about 10 to about 150 contiguous amino acid or nucleotide positions, and a test sequence can be compared to a reference sequence over the same number of contiguous positions after the two sequences are 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 search for similarity method of Pearson & Lipman, 1988, Proc. Natl. Acad. Sci. USA, 85:2444, or by computer implementations of these algorithms (e.g., GAP, BESTFIT, FASTA, or TFASTA in the 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). Examples of available algorithms suitable for determining percent sequence identity are the BLAST and BLAST 2.0 algorithms, which are 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 analyses is publicly available from the website of the National Center for Biotechnology Information (NCBI).

[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] The term "alkyl," as used herein, refers to a straight-chain or branched saturated hydrocarbon group containing the specified number of carbon atoms. Examples of alkyl 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, n-hexyl, and the like.

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

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

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

[0036] The term "amide," as used herein, 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] The term "aminoalkyl," as used herein, refers to an alkyl group, as defined herein, substituted with one or more amino groups, for example, 1, 2, or 3 amino groups.

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

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

[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] The term "cyano," as used herein, refers to the group --CN.

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

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

[0045] The terms "halogen" and "halo" as used herein 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, pyridazinyl, pyrimidinyl, triazinyl, quinolinyl, benzoxazolyl, benzothiazolyl, isoquinolinyl, quinazolinyl, quinoxalinyl, pyrrolyl, indolyl, and the like.

[0047] The term "heterocycloalkyl," as used herein, refers to a monocyclic or bicyclic non-aromatic ring system containing the specified number of atoms, wherein at least one ring atom is a heteroatom, such as O, S, or N. The heterocyclyl substituent can be attached via any available ring atom, such as a ring carbon or ring nitrogen. Examples of heterocycloalkyl include, but are not limited to, aziridinyl, azetidinyl, piperidinyl, morpholinyl, piperazinyl, pyrrolidinyl, and the like.

[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 hydroxy groups.

[0050] The term "nitro," as used herein, refers to the group --NO.sub.2.

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

[0052] The term "sulfonamide," as used herein, 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 specifically stated as "unsubstituted," any alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl group referred to herein is understood to be "optionally 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 alkyl and -C1-C6 alkyl substituted with one or more substituents. Examples of substituents include, but are not limited to, halogen, acyl, acyloxy, alkoxy, carboxy, hydroxy, amino, amido, nitro, cyano, azido, alkylthio, thio, sulfonyl, sulfonamido, alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl. In certain embodiments, each alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl group referred to herein is optionally substituted with one or more substituents selected from halogen, acyl, acyloxy, alkoxy, carboxy, hydroxy, amino, amido, nitro, cyano, azido, alkylthio, thio, sulfonyl, and sulfonamido.

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

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

[0057] It is contemplated that any embodiment discussed in this specification can be implemented with respect to any method, use, or composition disclosed herein, and vice versa.

[0058] Particular features, structures and / or characteristics described in connection with an embodiment disclosed herein may be combined in any suitable manner with features, structures and / or characteristics described in connection with other embodiments disclosed herein to provide one or more additional embodiments.

[0059] It should also be understood that the positive enumeration of a feature in one embodiment serves as a basis for excluding features in alternative embodiments. For example, where a list of alternatives is presented for a given embodiment or claim, it should be understood that one or more alternatives may be deleted from the list, and that the shortened list may form an alternative embodiment, whether or not such alternative embodiment is specifically referenced.

[0060] Anti-FRα antibody construct The ADCs of the present disclosure include anti-FRα antibody constructs. In this context, the term "antibody construct" refers to a polypeptide or set of polypeptides comprising one or more antigen-binding domains, each of which specifically binds to an epitope or antigen. When 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 be fused or covalently linked to the scaffold, optionally via a linker, as described herein.

[0061] According to the present disclosure, an anti-FRα antibody construct comprises at least one antigen-binding domain that specifically binds to human FRα (hFRα). "Specifically binds" to hFRα means that the antibody construct binds to hFRα but does not exhibit 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 the present disclosure can bind to FRα from one or more non-human species. In certain embodiments, the anti-FRα antibody construct of the present disclosure can 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. Exemplary 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] Specific binding of an antigen-binding domain to a target antigen or epitope can be measured, for example, by enzyme-linked immunosorbent assay (ELISA), surface plasmon resonance (SPR) technology (e.g., using a 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 can be defined as binding to a non-target protein (such as FOLR2, FOLR3, or FOLR4) being less than about 10% of the binding to hFRα, as measured, for example, by ELISA or flow cytometry. In certain embodiments, the specific binding of an antibody construct to hFRα exhibits a dissociation constant (K) of ≦1 μM, e.g., ≦500 nM, ≦250 nM, ≦100 nM, ≦50 nM, or ≦10 nM. D In certain embodiments, specific binding of an antibody construct to a particular antigen or epitope may be defined by a -6 M or less, e.g., 10 -7 M or less, or 10 -8 The dissociation constant (K D In some embodiments, the specific binding of an antibody construct to a particular antigen or epitope may be defined by a -6 M~10 -9 M, e.g., 10 -7 M~10 -9 Dissociation constant of M (K D )

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

[0066] Antibody internalization can be measured using methods known in the art, for example, by the direct internalization method following the protocol detailed in Schmidt, M. et al., 2008, Cancer Immunol. Immunother., 57:1879-1890, or 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, Göttingen, Germany), and analytical techniques such as microscopy, FACS, high-content imaging, or other plate-based assays.

[0067] In certain embodiments, an anti-FRα antibody construct is considered to exhibit greater internalization into FRα-expressing cells than the corresponding reference antibody (mirvetuximab or farletuzumab) if the amount of the anti-FRα antibody construct internalized into the FRα-expressing cells is at least 1.2-fold greater than the amount of the reference antibody internalized into the same FRα-expressing cells under the same test conditions. In certain embodiments, the amount of internalized antibody is determined using an appropriate fluorescent dye 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 levels similar 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 greater internalization into FRα-expressing cells than the corresponding reference antibody (mirvetuximab or farletuzumab) if the amount of anti-FRα antibody construct internalized into the FRα-expressing cells is at least 1.3-fold greater, at least 1.4-fold greater, at least 1.5-fold greater, 1.6-fold greater, 1.7-fold greater, 1.8-fold greater, 1.9-fold greater, or 2.0-fold greater than the amount of reference antibody internalized into the same FRα-expressing cells under the same test conditions. In certain embodiments, the amount of internalized antibody is determined using an appropriate fluorescent dye 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 levels similar 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.

[0069] antigen-binding domain The anti-FRα antibody constructs of the present disclosure comprise 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 Fabs (scFabs), single-chain Fvs (scFvs), and single-domain antibodies (sdAbs).

[0070] A "Fab fragment" contains the constant domain of the light chain (CL) and the first constant domain of the heavy chain (CH1), along with the variable domains of the light and heavy chains (VL and VH, respectively). Fab' fragments differ from Fab fragments by the addition of a few amino acid residues at the C-terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. Fab fragments may also be single-chain Fab molecules, i.e., Fab molecules in which the Fab light chain and Fab heavy chain are connected by a peptide linker to form a single peptide chain. For example, the C-terminus of a Fab light chain can be connected to the N-terminus of a Fab heavy chain in a single-chain Fab molecule.

[0071] An "scFv" comprises an antibody heavy chain variable domain (VH) and light chain variable domain (VL) within a single polypeptide chain. An scFv may optionally further comprise a polypeptide linker between the VH and VL domains, enabling the scFv to form the desired structure for antigen binding. For example, an scFv can comprise a VL connected from its C-terminus to the N-terminus of the VH by a polypeptide linker. Alternatively, an scFv can comprise a VH connected via its C-terminus to the N-terminus of the VL by a polypeptide linker (see the review in Plückthun 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. sdAbs may, for example, be of camelid origin. Camelid antibodies lack light chains and their antigen binding site consists of a single domain, termed "VHH." sdAbs comprise three CDRs / hypervariable loops that form the antigen binding site: CDR1, CDR2 and CDR3. sdAbs are quite amenable to stable expression, for example as fusions with the Fc chain of an antibody (see, e.g., Harmsen & De Haard, 2007, Appl. Microbiol Biotechnol., 77(1):13-22).

[0073] In those embodiments in which the anti-FRα antibody construct comprises two or more antigen-binding domains, each additional antigen-binding domain can 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 other polypeptide or small molecule, e.g., a natural or engineered ligand, capable of specifically binding to its target. Non-immunoglobulin-based antibody mimetic formats include, for example, anticalins, finomers, affimers, alphabodies, DARPins, and avimers.

[0074] The present disclosure describes herein 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 Examples and Sequence Table). Epitope mapping using the hFRα sequence (sequence number 15) shown in Figure 7 determined that the epitope within the hFRα protein bound by variant v23924 includes amino acid residues E120, D121, R123, T124, S125, and Y126 of sequence number 15 (see Example 13).

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

[0076] Competition assays known in the art can be used to determine whether an antibody construct competes for binding to hFRα with an antibody that binds to an epitope in the hFRα protein containing amino acid residues E120, D121, R123, T124, S125, and Y126, or antibody v23924. For example, an antibody that binds to an epitope in the hFRα protein containing amino acid residues E120, D121, R123, T124, S125, and Y126 (reference antibody) or antibody v23924 is first allowed to bind to hFRα under saturating 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, the test antibody construct is considered to bind to a different epitope from the reference antibody. Conversely, if the test antibody construct is unable to bind to hFRα simultaneously with the reference antibody, the test antibody construct is considered to bind to the same epitope, an overlapping epitope, or an epitope adjacent to the epitope bound by the reference antibody. A competition assay may also be performed in which the binding order of the reference and test antibodies is reversed, i.e., the test antibody is first allowed to bind to hFRα under saturating conditions, and then the ability of the reference antibody construct to bind to hFRα is measured.

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

[0078] In certain embodiments, an anti-FRα antibody construct of the present disclosure comprises at least one antigen-binding domain that specifically binds hFRα, wherein the antigen-binding domain comprises a set of CDRs based on the CDRs of antibody variant v23924 described herein. The CDR sequences of antibody v23924 and representative humanized or affinity-matured versions of this antibody are shown in Figure 11. Analysis of the CDR sequences from the parental and affinity-matured anti-FRα antibodies identified the minimal amino acid sequences present in each CDR, as defined by any one of the IMGT, Chothia, Kabat, Contact, or AbM numbering systems. These amino acid sequences are represented by the minimal consensus CDR sequences provided in Table 3. Expanded versions of these CDR consensus sequences, based on the CDR sequences 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) comprising the sequences set forth in SEQ ID NOs: 3, 4, and 5, and light chain CDR amino acid sequences (LCDR1, LCDR2, and LCDR3) comprising the sequences set forth in SEQ ID NOs: 6, 7, and 8.

[0082] In certain embodiments, the anti-FRα antibody construct of the present disclosure comprises the following antigen-binding domain: (i) the HCDR1 amino acid sequence set forth in SEQ ID NO: 3, the HCDR2 amino acid sequence set forth in SEQ ID NO: 4, and the HCDR3 amino acid sequence set forth in SEQ ID NO: 5 (X 2 is L and X 3 is A or X 2 is H and X 3is P), and (ii) the LCDR1 amino acid sequence set forth in SEQ ID NO: 6 (X 4 is G and X 5 is D or X 4 is W and X 5 is Y), the LCDR2 amino acid sequence set forth in SEQ ID NO: 7, and the LCDR3 amino acid sequence set forth in SEQ ID NO: 8 (X 6 is S and X 7 is N and X 8 is V and X 9 is D or X 6 is W and X 7 is H and X 8 is I and 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) comprising the sequences set forth in SEQ ID NOs: 9, 10, and 11, and light chain CDR amino acid sequences (LCDR1, LCDR2, and LCDR3) comprising the sequences set forth in SEQ ID NOs: 12, 13, and 14.

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

[0085] In certain embodiments, the anti-FRα antibody construct of the present disclosure comprises 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, or v36675, variants v23924, v30618, v30384, v30389, v30394, v30399, v31422, v31423, v31424, v31425, v31426, v35305, v35342, v3534 HCDR2 amino acid sequences selected from the HCDR2 amino acid sequences of any one of v23924, v30618, v30384, v30389, v30394, v30399, v31 an HCDR3 amino acid sequence selected from the HCDR3 amino acid sequences of any one of v31422, 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, or v36675, variants v23924, v30618, v30384, v30389, v30394, v30399, v31422, v31423, v31424, v31425, v31426, v35305, v35342, v35347 , v35348, v35350, v35354, v35356, v35358, v36167, v36168, or v36675, and variants v23924, v30618, v30384, v30389, v30394, v30399, v3142 v35354, v35356, v35358, v36167, v36168, or v36675; The CDR amino acid sequences are as defined by any one of the IMGT, Chothia, Kabat, Contact or AbM numbering systems (see Figure 11).

[0086] In certain embodiments, the anti-FRα antibody constructs of the present disclosure comprise heavy chain CDR amino acid sequences (HCDR1, HCDR2, and HCDR3) selected from the heavy chain CDR amino acid sequences (HCDR1, HCDR2, and HCDR3) of any one of 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 any one of the IMGT, Chothia, Kabat, Contact, or AbM numbering systems. R3), and the light chain CDR amino acid sequences (LCDR1, LCDR2, and LCDR3) of any one of 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 any one of the IMGT, Chothia, Kabat, Contact, or AbM numbering systems.

[0087] In certain embodiments, the anti-FRα antibody constructs of the present disclosure comprise variants v23924, v30618, v30384, v30389, v30394, v30399, v31422, v31423, v31424, v31425, v31426, v31427, v31428, v31429, v31430, v31431, v31432, v31433, v31434, v31435, v31436, v31437, v31438, v31439, v31440, v31441, v31442, v31443, v31444, v31445, v31446, v31447, v31448, v31449, v31450, v31451, v31452, v31453, v31454, v31455, v31456, v31457, v31458, v31459, v31460, v31461, v31462, v31463, v31464, v31465, v31466, v31467, v31468, v31469, v31470, v31471, v31472, v31473, v31474, v31475, v31476, v31477, v31478, v31479, v31480, v31481, v31482, v31483, v31484, v31485, v3 and a light chain CDR amino acid sequence (LCDR1, LCDR2, and LCDR3) of any one of v35305, v35342, v35347, v35348, v35350, v35354, v35356, v35358, v36167, v36168, or v36675.

[0088] In certain embodiments, an anti-FRα antibody construct of the present disclosure comprises an antigen-binding domain comprising the CDR sequences of the VH domain of any one of variants 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, an anti-FRα antibody construct of the present disclosure comprises an antigen-binding domain comprising the CDR sequences of the VL domain of any one of 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 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, an anti-FRα antibody construct of the present disclosure comprises an antigen-binding domain comprising a VH amino acid sequence selected from the VH amino acid sequences of any one of variants 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, an anti-FRα antibody construct of the present disclosure comprises an antigen-binding domain comprising a VL amino acid sequence selected from the VL amino acid sequences of any one of variants 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 certain embodiments, an anti-FRα antibody construct of the present disclosure comprises an antigen-binding domain comprising a VH amino acid sequence and a VL amino acid sequence selected from the VH amino acid sequence and the VL amino acid sequence of any one of 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 sequences or VH or VL sequences of a known antibody without losing the ability of the antibody 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 are tested for binding activity by standard techniques. Thus, in certain embodiments, the anti-FRα antibody constructs of the present disclosure comprise variants v23924, v30618, v30384, v30389, v30394, v30399, v31422, v31423, v31424, v31425, v31426, v35305, v35342, v35347, v35348, v35350, v35354, v35356, v35358, v36167, v36168, or v36675. and an antigen-binding domain comprising a set of CDRs (i.e., heavy chain HCDR1, HCDR2, and HCDR3, and light chain LCDR1, LCDR2, and LCDR3) that have 90% or more, 95% or more, 98% or more, 99% or more, or 100% sequence identity to any one of the sets of CDRs, where the % sequence identity is calculated across all six CDRs, and wherein the antigen-binding domain retains the ability to bind to hFRα.

[0092] In certain embodiments, an anti-FRα antibody construct of the present disclosure comprises an antigen-binding domain comprising a variant of the set of CDR sequences of any one of 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 the variant comprises 1 to 10 amino acid substitutions across the set of CDRs (i.e., the CDRs may be modified by up to 10 amino acid substitutions by modifying any combination of the six CDRs), and wherein the antigen-binding domain retains the ability to bind to hFRα. In some embodiments, the anti-FRα antibody constructs of the present disclosure comprise variants v23924, v30618, v30384, v30389, v30394, v30399, v31422, v31423, v31424, v31425, v31426, v35305, v35342, v35347, v35348, v35350, v35354, v35356, v35358, v36167, v361 68, or v36675, wherein the variant comprises 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 wherein the antigen-binding domain retains the ability to bind to hFRα.

[0093] In certain embodiments, the anti-FRα antibody constructs of the disclosure comprise variants v23924, v30618, v30384, v30389, v30394, v30399, v31422, v31423, v31424, v31425, v31426, v35305, v35342, v35347, v35348, v35350, v35354, v35356, v35358, v36167, v36168, or v366 75, wherein the antigen-binding domain comprises 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 the VH sequence of any one of the VH sequences ... In certain embodiments, the anti-FRα antibody constructs of the disclosure comprise variants v23924, v30618, v30384, v30389, v30394, v30399, v31422, v31423, v31424, v31425, v31426, v35305, v35342, v35347, v35348, v35350, v35354, v35356, v35358, v36167, v36168, or v366 75, wherein the antigen-binding domain comprises 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 the VL sequence of any one of the VL sequences ...

[0094] In certain embodiments, the anti-FRα antibody construct of the present disclosure comprises the following antigen-binding domain: (i) an HCDR1 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 20, 23, 26, 28, 31, 92, 93, 94, 95, or 96; an HCDR2 amino acid sequence selected from the amino acid sequences set forth in any one of 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 the amino acid sequences set forth in any one of SEQ ID NOs: 22, 25, 30, 107, 108, or 110; and (ii) an LCDR1 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 40, 43, 45, 65, 125, 126, or 127; an LCDR2 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 41, 44, or 46; and an LCDR3 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 42, 47, 120, or 121.

[0095] In certain embodiments, an anti-FRα antibody construct of the present disclosure comprises an antigen binding domain comprising the CDR sequences of a VH domain having the sequence set forth 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, an anti-FRα antibody construct of the present disclosure comprises an antigen binding domain comprising the CDR sequences of a VL domain having the sequence set forth in any one of SEQ ID NOs: 39, 64, 119, 124, or 130.

[0096] In certain embodiments, the anti-FRα antibody constructs of the present disclosure comprise an antigen-binding domain having: (a) an LCDR1 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 40, 43, or 45, an LCDR2 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 41, 44, or 46, and an LCDR3 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 42 or 47; and an HCDR1 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 20, 23, 26, 28, or 31, an HCDR2 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 21, 24, 27, 29, or 32, and an HCDR3 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 22, 25, or 30; (b) an LCDR1 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 125, 126, or 127; an LCDR2 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 41, 44, or 46; and an LCDR3 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 42 or 47; and an HCDR1 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 92, 93, 94, 95, or 96; an HCDR2 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 21, 24, 29, 32, or 51; and an HCDR3 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 22, 25, or 30; (c) an LCDR1 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 40, 45, or 65, an LCDR2 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 41, 44, or 46, and an LCDR3 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 42 or 47; and (i) an HCDR1 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 20, 23, 26, 28, or 31; an HCDR2 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 21, 24, 29, 32, or 51; an HCDR3 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 22, 25, or 30; or (ii) an HCDR1 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 20, 23, 26, 28, or 31; an HCDR2 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 21, 24, 29, 32, or 58; or an HCDR3 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 22, 25, or 30; or (iii) an HCDR1 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 92, 93, 94, 95, or 96; an HCDR2 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 24, 100, 101, 102, or 103; or an HCDR3 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 22, 25, or 30; or (d) an LCDR1 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 125, 126, or 127, an LCDR2 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 41, 44, or 46, and an LCDR3 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 120 or 121; and (i) an HCDR1 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 92, 93, 94, 95, or 96; an HCDR2 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 24, 100, 101, 102, or 103; or an HCDR3 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 22, 25, or 30; or (ii) an HCDR1 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 92, 93, 94, 95, or 96; an HCDR2 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 21, 24, 29, 32, or 109; or an HCDR3 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 107, 108, or 110; or (iii) an HCDR1 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 92, 93, 94, 95, or 96; an HCDR2 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 21, 24, 29, 32, or 109; or an HCDR3 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 22, 25, or 30; or (iv) an HCDR1 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 20, 23, 26, 28, or 31; an HCDR2 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 21, 24, 29, 32, or 109; or an HCDR3 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 107, 108, or 110; or (v) an HCDR1 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 20, 23, 26, 28, or 31; an HCDR2 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 21, 24, 29, 32, or 109; or an HCDR3 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 22, 25, or 30; or (vi) an HCDR1 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 92, 93, 94, 95, or 96; an HCDR2 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 21, 24, 137, 138, or 139; or an HCDR3 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 22, 25, or 30; or (e) an LCDR1 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 40, 45, or 65, an LCDR2 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 41, 44, or 46, and an LCDR3 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 120 or 121; and (i) an HCDR1 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 92, 93, 94, 95, or 96; an HCDR2 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 21, 24, 29, 32, or 109; an HCDR3 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 107, 108, or 110; or (ii) an HCDR1 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 20, 23, 26, 28, or 31; an HCDR2 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 21, 24, 29, 32, or 109; and an HCDR3 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 107, 108, or 110.

[0097] In certain embodiments, the anti-FRα antibody constructs of the present disclosure comprise an antigen-binding domain having: (a) an HCDR1 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 20, 23, 26, 28, or 31; an HCDR2 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 21, 24, 27, 29, or 32; an HCDR3 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 22, 25, or 30; an LCDR1 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 40, 43, or 45; an LCDR2 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 41, 44, or 46; and an LCDR3 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 42 or 47; or (b) an HCDR1 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 20, 23, 26, 28, or 31; an HCDR2 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 21, 24, 29, 32, or 51; an HCDR3 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 22, 25, or 30; an LCDR1 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 40, 45, or 65; an LCDR2 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 41, 44, or 46; and an LCDR3 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 42 or 47; or (c) an HCDR1 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 20, 23, 26, 28, or 31; an HCDR2 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 21, 24, 29, 32, or 58; an HCDR3 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 22, 25, or 30; an LCDR1 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 40, 45, or 65; an LCDR2 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 41, 44, or 46; and an LCDR3 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 42 or 47; or (d) an HCDR1 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 92, 93, 94, 95, or 96; an HCDR2 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 21, 24, 29, 32, or 51; an HCDR3 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 22, 25, or 30; an LCDR1 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 125, 126, or 127; an LCDR2 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 41, 44, or 46; and an LCDR3 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 42 or 47; or (e) an HCDR1 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 92, 93, 94, 95, or 96; an HCDR2 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 24, 100, 101, 102, or 103; an HCDR3 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 22, 25, or 30; an LCDR1 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 125, 126, or 127; an LCDR2 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 41, 44, or 46; and an LCDR3 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 120 or 121; or (f) an HCDR1 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 92, 93, 94, 95, or 96; an HCDR2 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 21, 24, 29, 32, or 109; an HCDR3 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 107, 108, or 110; an LCDR1 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 40, 45, or 65; an LCDR2 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 41, 44, or 46; and an LCDR3 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 42 or 47; or (g) an HCDR1 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 92, 93, 94, 95, or 96; an HCDR2 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 21, 24, 29, 32, or 109; an HCDR3 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 107, 108, or 110; an LCDR1 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 40, 45, or 65; an LCDR2 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 41, 44, or 46; and an LCDR3 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 120 or 121; or (h) an HCDR1 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 92, 93, 94, 95, or 96; an HCDR2 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 21, 24, 29, 32, or 109; an HCDR3 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 107, 108, or 110; an LCDR1 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 125, 126, or 127; an LCDR2 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 41, 44, or 46; and an LCDR3 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 120 or 121; or (i) an HCDR1 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 92, 93, 94, 95, or 96; an HCDR2 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 21, 24, 29, 32, or 109; an HCDR3 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 22, 25, or 30; an LCDR1 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 125, 126, or 127; an LCDR2 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 41, 44, or 46; and an LCDR3 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 120 or 121; or (j) an HCDR1 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 20, 23, 26, 28, or 31; an HCDR2 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 21, 24, 29, 32, or 109; an HCDR3 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 107, 108, or 110; an LCDR1 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 40, 45, or 65; an LCDR2 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 41, 44, or 46; and an LCDR3 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 120 or 121; or (k) an HCDR1 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 20, 23, 26, 28, or 31; an HCDR2 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 21, 24, 29, 32, or 109; an HCDR3 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 107, 108, or 110; an LCDR1 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 125, 126, or 127; an LCDR2 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 41, 44, or 46; and an LCDR3 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 120 or 121; or (l) an HCDR1 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 20, 23, 26, 28, or 31; an HCDR2 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 21, 24, 29, 32, or 109; an HCDR3 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 22, 25, or 30; an LCDR1 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 125, 126, or 127; an LCDR2 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 41, 44, or 46; and an LCDR3 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 120 or 121; or (m) an HCDR1 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 92, 93, 94, 95, or 96; an HCDR2 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 21, 24, 137, 138, or 139; an HCDR3 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 22, 25, or 30; an LCDR1 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 125, 126, or 127; an LCDR2 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 41, 44, or 46; and an LCDR3 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 120 or 121.

[0098] In certain embodiments, an anti-FRα antibody construct of the present disclosure comprises an antigen-binding domain comprising a VH amino acid sequence selected from the VH amino acid sequences set forth 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, an anti-FRα antibody construct of the present disclosure comprises an antigen-binding domain comprising a VL amino acid sequence selected from the VL amino acid sequences set forth in any one of SEQ ID NOs: 39, 64, 119, 124, or 130.

[0099] In certain embodiments, an anti-FRα antibody construct of the present disclosure comprises an antigen-binding domain comprising a VH amino acid sequence selected from the VH amino acid sequences set forth 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, and a VL amino acid sequence selected from the VL amino acid sequences set forth in any one of SEQ ID NOs: 39, 64, 119, 124, or 130.

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

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

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

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

[0104] In certain embodiments, the anti-FRα antibody construct comprises two antigen-binding domains operably linked, optionally, 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, when a scaffold is included, at least the first antigen-binding domain is operably linked to the scaffold, and the remaining antigen-binding domain(s) may each independently be operably linked to the scaffold or the first antigen-binding domain, or, if more than two antigen-binding domains are present, 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 operably linked by one or more linkers. In such anti-FRα antibody constructs, the antigen-binding domain may be in the form of an scFv, Fab, sdAb, or a combination thereof. For example, using scFvs as antigen-binding domains allows for the construction of formats such as tandem scFvs ((scFv)2 or taFv), in which scFvs are connected together by a flexible linker. scFvs can also be used to construct diabody formats containing two scFvs connected by a short linker (usually about 5 amino acids in length). The limited length of the linker results in dimerization of the scFvs in a head-to-tail manner. In any of the preceding formats, the scFvs can be further stabilized by including an interdomain disulfide bond. For example, a disulfide bond may be introduced between the VL and VH by introducing an additional cysteine ​​residue in each chain (e.g., at position 44 in the VH and position 100 in the 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 with a 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 VHs or VHHs, connected together via a suitable linker may be used. Other examples of anti-FRα antibody construct formats lacking a scaffold include those based on Fab fragments, e.g., Fab2 and F(ab')2 formats, where the Fab fragments are connected via a linker or an IgG hinge region.

[0107] Combinations of different forms of antigen-binding domains can also be used to create alternative scaffold-less formats, for example, an 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, the anti-FRα antibody construct can be an immunoglobulin (Ig)-based antibody format. In certain embodiments, the anti-FRα antibody construct can be based on an IgG class immunoglobulin, e.g., an IgG1, IgG2, IgG3, or IgG4 immunoglobulin. In some embodiments, the anti-FRα antibody construct can be based on an IgG1 immunoglobulin. In the context of the present disclosure, when an anti-FRα antibody construct is based on a particular immunoglobulin isotype, it means that the anti-FRα antibody construct comprises all or a portion of the constant region of the particular immunoglobulin isotype. For example, an anti-FRα antibody construct based on a given Ig isotype can comprise at least one antigen-binding domain operably linked to an Ig scaffold, the scaffold comprising an Fc region from the given isotype and, optionally, an Ig hinge region from the same or a different isotype. It should also be understood that in some embodiments, the anti-FRα antibody construct can also comprise a hybrid of isotypes and / or subclasses. It is also understood that the Fc region and / or hinge region may optionally be modified to impart one or more desired functional properties as known in the art.

[0109] In some embodiments, an anti-FRα antibody construct may be derived from two or more immunoglobulins from different species, e.g., 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., murine) antibody, and at least one constant domain from a human antibody. The human constant domains of a chimeric antibody need not be of the same isotype as the non-human constant domains they replace. Chimeric antibodies are discussed, for example, in Morrison et al., 1984, Proc. Natl. Acad. Sci. USA, 81:6851-55, and U.S. Pat. No. 4,816,567.

[0111] A "humanized antibody" is a type of chimeric antibody that contains minimal sequence derived from a non-human antibody. In general, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region (CDR) of the recipient are replaced by residues from a hypervariable region (CDR) of a non-human species (donor antibody) such as mouse, rat, rabbit, or non-human primate having the desired specificity and affinity for the target antigen. The technique for producing this humanized antibody is often referred to as "CDR grafting."

[0112] In some cases, additional modifications are made to humanized antibodies to further refine antibody performance. For example, framework region (FR) residues of the human immunoglobulin may be replaced by corresponding non-human residues, or the humanized antibody may contain residues that are not found in either the recipient antibody or the donor antibody. Generally, the variable domains of humanized antibodies will contain all or substantially all of the hypervariable regions from the non-human immunoglobulin and all or substantially all of the FRs 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 for selecting optimal human frameworks for grafting nonhuman CDRs are known in the art. Early approaches used a limited subset of well-characterized human antibodies, regardless of their sequence identity with the nonhuman antibody providing the CDRs (the "fixed framework" approach). More recent approaches employ variable regions with high amino acid sequence identity with the variable region of the nonhuman antibody providing the CDRs (the "homology matching" or "best-fit" approach). Another approach is to select fragments of framework sequences within the light or heavy chain variable regions from several different human antibodies. CDR grafting can, in some cases, partially or completely lose the affinity of the grafted molecule for its target antigen. In such cases, affinity can be restored by backmutating some of the human-derived residues to the corresponding nonhuman-derived residues. Methods for preparing humanized antibodies by these approaches are well known in the art (see, e.g., 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] Alternatively or in addition to such traditional approaches, more recent techniques can be employed to further reduce the immunogenicity of CDR-grafted humanized antibodies. For example, instead of a human framework with somatic mutation(s), a framework based on a human germline sequence or consensus sequence can be used as the acceptor human framework. Another technique aimed at reducing the potential immunogenicity of non-human CDRs is to graft only the specificity-determining residues (SDRs). In this approach, only the minimum CDR residues ("SDRs") required for antigen-binding activity are grafted onto a human germline framework. This method can help improve the "humanity" of humanized antibodies (i.e., similarity to human germline sequences) and thus reduce the risk of immunogenicity of the variable regions. These techniques are described in various publications (see, e.g., 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 the present disclosure comprise humanized antibody sequences, e.g., one or more humanized variable domains. In some embodiments, the anti-FRα antibody constructs can be humanized antibodies. 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 Examples and Sequence Table).

[0116] scaffold In certain embodiments, the anti-FRα antibody construct comprises one or more antigen-binding domains operably linked to a scaffold. The antigen-binding domain(s) can be in one or a combination of the above forms (e.g., scFv, Fab, and / or sdAb). Examples of suitable scaffolds are described in more detail below and include, but are not limited to, immunoglobulin Fc regions, albumin, albumin analogs and derivatives, heterodimerizing peptides (e.g., leucine zippers, heterodimerizing "zipper" peptides derived from Jun and Fos, IgG CH1 and CL domains, or barnase barstar toxin), 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.When 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.In certain embodiments, the anti-FRα antibody construct also includes a polypeptide scaffold in which one or more antigen binding domains are linked to a region other than the N-terminus or C-terminus, for example, via an amino acid side chain with or without a linker.

[0118] In embodiments in which the anti-FRα antibody construct comprises a peptide or polypeptide scaffold, the antigen-binding domain(s) can 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 in which the scaffold is a polymer or nanoparticle, the antigen-binding domain(s) can 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. For example, leucine zipper domains such as Fos and Jun selectively pair (Kostelny, et al., J Immunol, 148:1547-53 (1992); Wranik, et al., J. Biol. Chem., 287:43331-43339 (2012)). Other selectively paired molecular pairs include, for example, Barnase-Barstar 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 No. WO2011 / 135040).

[0120] Other examples of protein scaffolds include immunoglobulin Fc regions, albumin, 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, e.g., 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 an antigen-binding moiety, such as an scFv, diabody, or single-chain diabody, to albumin has been shown to improve the serum half-life of the antigen-binding moiety (Muller et al., ibid.). The antigen-binding moiety can be fused at the N-terminus and / or C-terminus of albumin, optionally via a linker.

[0122] Derivatives of albumin in the form of heteromultimers have been described, which contain two transporter polypeptides obtained by segmentation of the albumin protein, such that the transporter polypeptides self-assemble to form quasi-native albumin (see International Patent Applications WO 2012 / 116453 and WO 2014 / 012082). As a result of the segmentation of albumin, the heteromultimer contains four termini and can therefore be fused, optionally via linkers, to up to four different antigen-binding moieties.

[0123] In certain embodiments, the anti-FRα antibody construct may comprise a protein scaffold. In some embodiments, the anti-FRα antibody construct may comprise 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 comprise 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 that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. Unless otherwise specified herein, the numbering of amino acid residues within an Fc region or constant region is according to 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 comprise a scaffold based on an immunoglobulin Fc region. The Fc region may be dimeric and comprised of two Fc polypeptides, or alternatively, the Fc region may be comprised of a single polypeptide.

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

[0127] The Fc region may comprise a CH3 domain, or may comprise both a CH3 and a CH2 domain. For example, in certain embodiments, the Fc polypeptides of a dimeric IgG Fc region may comprise an IgG CH2 domain sequence and an IgG CH3 domain sequence. In such embodiments, the CH3 domain comprises two CH3 sequences, one from each of the two Fc polypeptides of the dimeric Fc region, and the CH2 domain comprises two CH2 sequences, one from each of the two Fc polypeptides of the dimeric Fc region.

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

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

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

[0131] In some embodiments, an anti-FRα antibody construct can include a heterodimeric Fc region comprising a modified CH3 domain, where the modified CH3 domain is 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 a substitution or insertion, in which an amino acid at a particular position on a first CH3 or CH2 sequence is different from the amino acid at the same position on a second CH3 or CH2 sequence. These asymmetric amino acid modifications can be the result of modification of only one of the two amino acids at the same respective amino acid position on each sequence, or different modifications of both amino acids at the same respective position 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 can contain one or more asymmetric amino acid modifications.

[0132] In some embodiments, the anti-FRα antibody construct can comprise a heterodimeric Fc comprising a modified CH3 domain, wherein the modified CH3 domain comprises one or more amino acid modifications that promote the formation of a heterodimeric Fc over the formation of a homodimeric 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 an Fc to promote heterodimeric Fc formation are known in the art and include, for example, those described in International Publication No. WO 96 / 027011 ("knobs into holes"), Gunasekaran et al., 2010, J Biol Chem, 285, 19637-46 ("electrostatic steering"), Davis et al., 2010, Prot Eng Des Sel, 23(4):195-202 (strand exchange engineered domain (SEED) technology), and 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 to result in asymmetrically modified stable Fc regions, as described in International Publication Nos. WO2012 / 058768 and WO2013 / 063702. In certain embodiments, anti-FRα antibody constructs can include scaffolds based on modified Fc regions as described in International Publication Nos. WO2012 / 058768 or WO2013 / 063702.

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

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

[0136] [Table 5]

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

[0138] In some embodiments, the anti-FRα antibody construct comprises an IgG Fc-based scaffold with a modified CH2 domain, wherein the modification of the CH2 domain results 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 for different Fcγ receptors are known in the art. Amino acid modifications that result in increased binding and decreased binding may each be useful in certain applications. For example, increasing the binding affinity of Fc to FcγRIIIa (an activating receptor) can result in increased antibody-dependent cell-mediated cytotoxicity (ADCC), which in turn leads to increased target cell lysis. Similarly, decreased binding to FcγRIIb (an inhibitory receptor) may be beneficial in some situations. In certain applications, reducing or eliminating 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 that reduce or eliminate binding of the Fc region to all Fcγ receptors ("knockout" variants) may be useful.

[0140] Examples of amino acid modifications to the CH2 domain that alter Fc binding by Fcγ receptors include, but are not limited to, 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 No. WO2021 / 232162. Additional modifications that affect Fc binding to Fcγ receptors are described in Therapeutic Antibody Engineering (Strohl & Strohl, Woodhead Publishing series in Biomedicine No. 11, ISBN 1 907568 37 9, October 2012, page 283).

[0141] In certain embodiments, the anti-FRα antibody construct comprises an IgG Fc-based scaffold with a modified CH2 domain, which contains one or more amino acid modifications that reduce or eliminate binding of the entire Fc region to Fcγ receptors (i.e., a "knockout" variant).

[0142] Various publications describe strategies used to engineer antibodies to generate "knockout" variants (see, e.g., 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 modifying glycosylation, using an IgG2 / IgG4 scaffold, or reducing effector function by introducing mutations in the hinge or CH2 domain of the Fc (see also U.S. Patent Publication No. 2011 / 0212087, International Publication No. WO2006 / 105338, U.S. Patent Publication No. 2012 / 0225058, U.S. Patent Publication No. 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 the amino acid modifications L234A / L235A and L234A / L235A / D265S.

[0144] In certain embodiments, the anti-FRα antibody constructs described herein may comprise a scaffold based on an IgG Fc with modified native glycosylation. As is known in the art, glycosylation of Fc may be altered to increase or decrease effector function. For example, mutation of the conserved asparagine residue at position 297 to alanine, glutamine, lysine, or histidine (i.e., N297A, Q, K, or H) results in an aglycosylated 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 due to improved binding to FcγRIIIa (see, e.g., 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 can 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 the N297-linked carbohydrate (International Publication No. WO 03 / 035835), or in other cells that produce non-fucosylated antibodies (see, e.g., 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, WO2009 / 135181 describes the addition of fucose analogues to the culture medium during antibody production to inhibit the incorporation of fucose into the carbohydrate on the antibody.

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

[0147] Other glycosylation variants include those containing bisected oligosaccharides, such as variants in which biantennary oligosaccharides attached to the Fc region of an antibody are bisected by N-acetylglucosamine (GlcNAc). Such glycosylation variants may have reduced fucosylation and / or improved ADCC function (see, e.g., International Publication No. WO2003 / 011878, U.S. Patent No. 6,602,684, and U.S. Patent Application Publication No. US2005 / 0123546). Useful glycosylation variants also include variants containing at least one galactose residue in the oligosaccharide attached to the Fc region, which may have improved CDC function (see, e.g., International Publication Nos. WO1997 / 030087, WO1998 / 58964, and WO1999 / 22764).

[0148] In certain embodiments, the anti-FRα antibody construct has a full-size antibody (FSA) format. In some embodiments, the anti-FRα antibody construct has an IgG FSA format, 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 with a homodimeric Fc, comprising H1, H2, L1, and L2 sequences, wherein 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 with a heterodimeric Fc, comprising H1, H2, L1, and L2 sequences, wherein 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 with a heterodimeric Fc and comprises H1, H2, L1 and L2 sequences, wherein H1 and H2 have different amino acid sequences and L1 and L2 have different amino acid sequences.

[0149] In certain embodiments, 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 set forth in Tables A and B for any one of 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, expression of antibody heavy chain sequences from particular cell lines or particular expression vectors can result in one or both of the heavy chains containing a C-terminal lysine residue. Accordingly, certain embodiments of the present disclosure relate to anti-FRα antibody constructs that are FSAs having a set of H1, H2, L1, and L2 sequences that include the H1, H2, L1, and L2 amino acid sequences set forth in Tables A and B for any one of 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 includes a C-terminal lysine (see, e.g., 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 (see, e.g., U.S. Pat. No. 4,816,567 and "Antibodies: A Laboratory Manual," 2002). nd Edition, Ed. Greenfield, Cold Spring Harbor Laboratory Press, New York, 2014).

[0151] Typically, for 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 a host cell. 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 & update, and "Antibodies: A Laboratory Manual," 2002). nd (See, for example, "Anti-FRα Antibody Constructs: A Guide to the Development of Anti-FRα Antibody Constructs," 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 expression of an anti-FRα antibody construct will depend on the format of the construct, including whether the antibody construct includes a scaffold. For example, if the anti-FRα antibody construct is in a mAb format with a homodimeric Fc, two polynucleotides each encoding one polypeptide chain will be required, whereas if the anti-FRα antibody construct is in a mAb format with a heterodimeric Fc, three polynucleotides each encoding one polypeptide chain will be required. When multiple polynucleotides are required, they can 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(s) along with one or more regulatory elements, e.g., transcription elements, necessary for efficient transcription of the polynucleotide. 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 choice of regulatory element will depend on the host cell selected for expression of the antibody construct, and that such regulatory elements may be derived from a variety of sources, including bacterial, fungal, viral, mammalian, or insect genes. The expression vector may optionally further comprise a heterologous nucleic acid sequence that facilitates expression or purification of the expressed protein. Examples include, but are not limited to, signal peptides and 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 or integrating vector.

[0153] Suitable host cells for cloning or expressing anti-FRα antibody constructs include various prokaryotic or eukaryotic cells, as 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, E. coli cells, A. salmonicida cells, or B. subtilis cells.

[0154] In certain embodiments, anti-FRα antibody constructs can be produced in bacteria, particularly when glycosylation and Fc effector functions are not required, as described, for example, in U.S. Pat. Nos. 5,648,237, 5,789,199, and 5,840,523, and Charlton, Methods in Molecular Biology, Vol. 248, pp. 245-254, BKC Lo, ed. Humana Press, Totowa, NJ, 2003.

[0155] Eukaryotic microbes such as filamentous fungi or yeast may be suitable expression host cells in certain embodiments, particularly fungal and yeast strains in which the glycosylation pathway has been "humanized," resulting in the production of antibody constructs with partially or fully human glycosylation patterns (see, e.g., Gerngross, 2004, Nat. Biotech. 22:1409-1414, and Li et al., 2006, Nat. Biotech. 24:210-215).

[0156] Host cells suitable for expressing glycosylated anti-FRα antibody constructs are usually eukaryotic cells. For example, U.S. Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 describe the PLANTIBODIES™ technology for producing antigen-binding constructs in transgenic plants. Mammalian cell lines adapted to grow in suspension can be particularly useful for expressing antibody constructs. Examples include SV40 (COS-7) transformed monkey kidney CV1 line, human embryonic kidney (HEK) line 293 or 293 cells (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 carcinoma (HeLa) cells, canine kidney cells (MDCK), buffalo rat hepatocytes (BRL3A), human lung cells (W138), human liver cells (HepG2), mouse mammary tumor (MMT060562), TRI cells (see, e.g., Mather et al., 1982, Annals of NY Acad. Sci. USA, 2004, 14:147-148), and the like. Sci, 383: 44-68), MRC5 cells, FS4 cells, Chinese hamster ovary (CHO) cells (DHFR - Exemplary mammalian host cell lines suitable for the production of antibody constructs include, but are 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 the production of antibody constructs are reviewed in Yazaki & Wu, Methods in Molecular Biology, Vol. 248, pp. 255-268 (BKC Lo, 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 mature glycosylation of the antibody construct.

[0158] Host cells containing an expression vector(s) encoding an anti-FRα antibody construct can be cultured using conventional methods to produce the anti-FRα antibody construct. Alternatively, in some embodiments, host cells containing an expression vector(s) encoding an anti-FRα antibody construct can be used therapeutically or prophylactically to deliver the anti-FRα antibody construct to a subject, or the polynucleotide or expression vector can be administered ex vivo to cells from a subject, which can then be returned to the subject's body.

[0159] Typically, the anti-FRα antibody construct is purified after expression. Proteins may be isolated or purified by various methods known to those skilled in the art (e.g., Protein Purification: Principles and Practice, 3). rd(See, Ed., Scopes, Springer-Verlag, NY, 1994). Standard purification methods include chromatographic techniques (including ion-exchange, hydrophobic interaction, affinity, sizing or gel filtration, and reverse-phase chromatography) performed at atmospheric or elevated pressure using systems such as FPLC and HPLC. Additional purification methods include electrophoretic, 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 natural 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 enabled by specific fusion partners. For example, antibodies can be purified using glutathione resins when GST fusions are used, or Ni-tags when His-tags are used. +2 Purification may be performed using affinity chromatography or, if a Flag tag is used, using an immobilized anti-Flag antibody. The degree of purification required 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. The term "substantially pure" (or "substantially purified"), when used with reference to the anti-FRα antibody constructs described herein, means that the antibody construct is substantially or essentially free from components that normally accompany or interact with the protein as found in its naturally occurring environment, e.g., 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 protein.

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

[0162] Post-translational modifications In certain embodiments, the anti-FRα antibody constructs described herein may contain one or more post-translational modifications. Such post-translational modifications may occur in vivo or may be performed in vitro after isolation of the anti-FRα antibody construct from a host cell.

[0163] Post-translational modifications include various modifications known in the art (see, for example, Proteins - Structure and Molecular Properties, 2nd Ed., TECreighton, W.H. Freeman and Company, New York, 1993; Post-Translational Covalent Modification of Proteins, B.C. Johnson, Ed., Academic Press, New York, pp. 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 may contain 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 cyanogen bromide, trypsin, chymotrypsin, papain, V8 protease or NaBH4).

[0165] Other examples of post-translational modifications include, for example, the addition or removal of N- or O-linked glycans, chemical modification of N- or O-linked glycans, N- or C-terminal processing, 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 can also include modification with a detectable label, such as an enzyme label, a fluorescent label, a luminescent label, an isotopic label, or an affinity label, to enable detection and isolation of the protein. Examples of suitable enzyme labels include, but are not limited to, horseradish peroxidase, alkaline phosphatase, beta-galactosidase, and acetylcholinesterase. Examples of suitable prosthetic group complexes include, but are not limited to, streptavidin / biotin and avidin / biotin. Examples of suitable fluorescent materials include, but are not limited to, umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, and phycoerythrin. Examples of luminescent materials include luminol and bioluminescent materials such as luciferase, luciferin, and aecolin. Examples of suitable radioactive materials include iodine, carbon, sulfur, tritium, indium, technetium, thallium, gallium, palladium, molybdenum, xenon, and fluorine.

[0166] Additional examples of post-translational modifications include acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphatidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, gamma-carboxylation, GPI anchor formation, hydroxylation, iodination, methylation, myristylation, pegylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA-mediated addition of amino acids to proteins such as arginylation, and ubiquitination.

[0167] Camptothecin analogues Camptothecin analogs encompassed by the ADCs of the present disclosure have formula (I): TIFF2025510791000013.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 is -NH2, R is R 3 or R 4 and R 1 is other than -NH2, R is R 4 and R 3 is -H, -C1-C6 alkyl, -C3-C8 cycloalkyl, -(C1-C6 alkyl)-OR 5 , TIFF2025510791000014.tif27165-CO2R 8 , -aryl, -heteroaryl, and -(C1-C6 alkyl)-aryl; R 4 teeth, Selected from TIFF2025510791000015.tif68165, 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 -H, -C1-C6 alkyl, -C3-C8 cycloalkyl, -(C1-C6 alkyl)-OR 5 , —C3-C8 heterocycloalkyl, and —C(O)R 17 is selected from R 8is selected from —H, —C1-C6 alkyl, —C3-C8 cycloalkyl, and —C3-C8 heterocycloalkyl; Each R 9 is independently selected from -H, -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl, and -(C1-C6 alkyl)-aryl; Each R 10 are independently -C1-C6 alkyl, -C3-C8 cycloalkyl, -NR 14 R 14’ , -aryl, -heteroaryl and -(C1-C6 alkyl)-aryl; R 10’ is selected from -H, -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl, and -(C1-C6 alkyl)-aryl; R 11 is selected from —H and —C1-C6 alkyl; R 12 is -H, -C1-C6 alkyl, -CO2R 8 , -aryl, -heteroaryl, -(C1-C6 alkyl)-aryl, -S(O)2R 16 , and Selected from TIFF2025510791000016.tif22165, R 13 is selected from —H and —C1-C6 alkyl; R 14 and R 14’ are each independently selected from —H, C1-C6 alkyl, —C3-C8 cycloalkyl, and —C3-C8 heterocycloalkyl; R 16 is selected from -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl, and -(C1-C6 alkyl)-aryl; R 17 is 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 together with the N atom to which they are attached, include halogen, -C1-C6 alkyl, -C3-C8 cycloalkyl, and -(C1-C6 alkyl)-OR 5 and forming a 4-, 5-, 6-, or 7-membered ring having 0 to 3 substituents selected from R 24 , R 25 and R 26 are each -C1 to C6 alkyl, X a and X b are each independently selected from NH, O, and S; X c is selected from O, S, and S(O)2; However, the compound is other than (S)-9-amino-11-butyl-4-ethyl-4-hydroxy-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione.

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

[0169] In some embodiments, in the compound of Formula (I), R 1 is selected from —CH 3 , —CF 3 , —OCH 3 , —OCF 3 and NH 2 .

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

[0171] In some embodiments, in the compound of Formula (I), R 1 is selected from —H, —CH 3 , —CF 3 , —F, —Br, —Cl, —OH, —OCH 3 , and —OCF 3 .

[0172] In some embodiments, in the compound of Formula (I), R 1is selected from —CH 3 , —CF 3 , —OCH 3 , and —OCF 3 .

[0173] In some embodiments, in the compound of Formula (I), R 2 is selected from —H, —CH 3 , —CF 3 , —F, —Cl, —OCH 3 and —OCF 3 .

[0174] In some embodiments, in the compound of Formula (I), R 2 is selected from —CH 3 , —CF 3 , —F, —Cl, —OCH 3 and —OCF 3 .

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

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

[0177] In some embodiments, in the compound of Formula (I), R 3 is -H, unsubstituted -C1-C6 alkyl, -C1-C6 haloalkyl, -C1-C6 hydroxyalkyl, -C3-C8 cycloalkyl, -(C1-C6 alkyl)-OR 5 , TIFF2025510791000017.tif27165-CO2R 8 , unsubstituted -aryl, -aminoaryl, -heteroaryl and -(C1-C6 alkyl)-aminoaryl.

[0178] In some embodiments, in the compound of Formula (I), R 4 teeth, Selected from TIFF2025510791000018.tif58165.

[0179] In some embodiments, in the compound of Formula (I), R 5is 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.

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

[0181] In some embodiments, in the compound of Formula (I), R 8 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 is 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 is independently selected from -C1-C6 alkyl and -(C1-C6 alkyl)-aryl.

[0184] In some embodiments, in the compound of Formula (I), each R 9are 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 are independently -C1-C6 alkyl, -C3-C8 cycloalkyl, -NR 14 R 14’ , -aryl and -(C1-C6 alkyl)-aryl.

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

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

[0188] In some embodiments, in the compound of Formula (I), R 10’ is 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 11is 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 is -H, -C1-C6 alkyl, -CO2R 8 , -aryl, -(C1-C6 alkyl)-aryl and -S(O)R 16 is selected from.

[0191] In some embodiments, in the compound of Formula (I), R 12 is -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 TIFF2025510791000019.tif22165.

[0192] In some embodiments, in the compound of Formula (I), R 13 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’ are each 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 is selected from -aryl, -heteroaryl and -(C1-C6 alkyl)-aryl.

[0195] In some embodiments, in the compound of Formula (I), R 16 is 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 is 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 together with the N atom to which they are attached, represent halogen, unsubstituted -C1-C6 alkyl, -C1-C6 haloalkyl, -C1-C6 hydroxyalkyl, -C1-C6 aminoalkyl, -C3-C8 cycloalkyl, and -(C1-C6 alkyl)-OR 5 and forming a 4-, 5-, 6-, or 7-membered ring having 0 to 3 substituents selected from:

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

[0199] Combinations of any of the foregoing embodiments for compounds of formula (I) are also envisioned, with each combination forming a separate embodiment for purposes of this disclosure.

[0200] In certain embodiments, the compound of formula (I) has formula (II): TIFF2025510791000020.tif53165, wherein R 2 is selected from —H, —CH3, —CF3, —F, —Br, —Cl, —OH, —OCH3, and —OCF3; R 20 is -H, -C1-C6 alkyl, -C3-C8 cycloalkyl, -(C1-C6 alkyl)-OR 5 , TIFF2025510791000021.tif27165-CO2R 8 , -aryl, -heteroaryl, -(C1-C6 alkyl)-aryl, Selected from TIFF2025510791000022.tif68165, R 5 is selected from -H, -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl, and -(C1-C6 alkyl)-aryl; R 6 and R 7 are each independently -H, -C1-C6 alkyl, -C3-C8 cycloalkyl, -(C1-C6 alkyl)-OR 5 , —C3-C8 heterocycloalkyl, and —C(O)R 17 is selected from R 8 is selected from —H, —C1-C6 alkyl, —C3-C8 cycloalkyl, and —C3-C8 heterocycloalkyl; Each R 9 is independently selected from -H, -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl, and -(C1-C6 alkyl)-aryl; Each R 10 are independently -C1-C6 alkyl, -C3-C8 cycloalkyl, -NR 14 R 14’ , -aryl, -heteroaryl and -(C1-C6 alkyl)-aryl; R 10’is selected from -H, -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl, and -(C1-C6 alkyl)-aryl; R 11 is selected from —H and —C1-C6 alkyl; R 12 is -H, -C1-C6 alkyl, -CO2R 8 , -aryl, -heteroaryl, -(C1-C6 alkyl)-aryl, -S(O)2R 16 and Selected from TIFF2025510791000023.tif22165, R 13 is selected from —H and —C1-C6 alkyl; R 14 and R 14’ are each independently selected from —H, C1-C6 alkyl, —C3-C8 cycloalkyl, and —C3-C8 heterocycloalkyl; R 16 is selected from -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl, and -(C1-C6 alkyl)-aryl; R 17 is 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 together with the N atom to which they are attached, include halogen, -C1-C6 alkyl, -C3-C8 cycloalkyl, and -(C1-C6 alkyl)-OR 5 and forming a 4-, 5-, 6-, or 7-membered ring having 0 to 3 substituents selected from R 24 , R 25 and R 26 are each -C1 to C6 alkyl, X a and X b are each independently selected from NH, O, and S; Xc is selected from O, S, and S(O)2; However, the compound is other than (S)-9-amino-11-butyl-4-ethyl-4-hydroxy-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione.

[0201] In some embodiments, in the compound of Formula (II), R 2 is selected from —CH 3 , —CF 3 , —F, —Br, —Cl, —OH, —OCH 3 , and —OCF 3 .

[0202] In some embodiments, in the compound of Formula (II), R 2 is selected from —CH 3 , —CF 3 , —F, —Cl, —OCH 3 and —OCF 3 .

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

[0204] In some embodiments, in the compound of Formula (II), R 20 is -H, -C1-C6 alkyl, -(C1-C6 alkyl)-OR 5 , TIFF2025510791000024.tif27165-(C1-C6 alkyl)-aryl, Selected from TIFF2025510791000025.tif58165.

[0205] In some embodiments, in the compound of Formula (II), R 20 is -H, -C1-C6 alkyl, -(C1-C6 alkyl)-OR 5 , TIFF2025510791000026.tif27165-(C1-C6 alkyl)-aryl, Selected from TIFF2025510791000027.tif58165.

[0206] In some embodiments, in the compound of Formula (II), R 20 is -H, -C1-C6 alkyl, -(C1-C6 alkyl)-OR 5 , Selected from TIFF2025510791000028.tif58165.

[0207] In some embodiments, in the compound of Formula (II), R 20 is -H, unsubstituted -C1-C6 alkyl, -C1-C6 haloalkyl, -C1-C6 hydroxyalkyl, -C3-C8 cycloalkyl, -(C1-C6 alkyl)-OR 5 , TIFF2025510791000029.tif27165-CO2R 8 , unsubstituted aryl, -aminoaryl, -heteroaryl, -(C1-C6 alkyl)aminoaryl, Selected from TIFF2025510791000030.tif68165.

[0208] In some embodiments, in the compound of Formula (II), R 2 is selected from —CH3, —CF3, —F, —Br, —Cl, —OH, —OCH3, and —OCF3; R 20 is -H, -C1-C6 alkyl, -(C1-C6 alkyl)-OR 5 , TIFF2025510791000031.tif27165-(C1-C6 alkyl)-aryl, Selected from TIFF2025510791000032.tif58165.

[0209] In some embodiments, in the compound of Formula (II), R 2 is selected from —CH3, —CF3, —F, —Br, —Cl, —OH, —OCH3, and —OCF3; R 20 is -H, -C1-C6 alkyl, -(C1-C6 alkyl)-OR 5 , TIFF2025510791000033.tif27165-(C1-C6 alkyl)-aryl, Selected from TIFF2025510791000034.tif58165.

[0210] In some embodiments, in the compound of Formula (II), R 2 is selected from —CH3, —CF3, —F, —Br, —Cl, —OH, —OCH3, and —OCF3; R 20 is -H, -C1-C6 alkyl, -(C1-C6 alkyl)-OR 5 , Selected from TIFF2025510791000035.tif58165.

[0211] In some embodiments, in the compound of Formula (II), R 5 is 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.

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

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

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

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

[0216] In some embodiments, in the compound of Formula (II), R 8 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 is 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 is independently selected from -C1-C6 alkyl and -(C1-C6 alkyl)-aryl.

[0219] In some embodiments, in the compound of Formula (II), each R 9 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 are independently -C1-C6 alkyl, -C3-C8 cycloalkyl, -NR 14 R 14’ , -aryl and -(C1-C6 alkyl)-aryl.

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

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

[0223] In some embodiments, in the compound of Formula (II), R 10’ is 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 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 12is -H, -C1-C6 alkyl, -CO2R 8 , -aryl, -(C1-C6 alkyl)-aryl and -S(O)R 16 is selected from.

[0226] In some embodiments, in the compound of Formula (II), R 12 is -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 TIFF2025510791000036.tif22165.

[0227] In some embodiments, in the compound of Formula (II), R 13 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’ are each 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 is selected from -aryl, -heteroaryl and -(C1-C6 alkyl)-aryl.

[0230] In some embodiments, in the compound of Formula (II), R 16is 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 is —C1 to C6 alkyl.

[0232] In some embodiments, in the compound of Formula (II), R 17 is 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 together with the N atom to which they are attached, represent halogen, unsubstituted -C1-C6 alkyl, -C1-C6 haloalkyl, -C1-C6 hydroxyalkyl, -C1-C6 aminoalkyl, -C3-C8 cycloalkyl, and -(C1-C6 alkyl)-OR 5 and forming a 4-, 5-, 6-, or 7-membered ring having 0 to 3 substituents selected from:

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

[0235] Combinations of any of the foregoing embodiments for compounds of formula (II) are also contemplated, with each combination forming a separate embodiment for purposes of this disclosure.

[0236] In certain embodiments, the compound of formula (I) has formula (III): TIFF2025510791000037.tif53165, wherein R 2 is selected from —H, —CH3, —CF3, —F, —Br, —Cl, —OH, —OCH3, and —OCF3; R 15 is selected from —H, —CH3, —CHF2, —CF3, —F, —Br, —Cl, —OH, —OCH3, and —OCF3; R 4 teeth, Selected from TIFF2025510791000038.tif68165, R 5 is selected from -H, -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl, and -(C1-C6 alkyl)-aryl; R 8 is selected from —H, —C1-C6 alkyl, —C3-C8 cycloalkyl, and —C3-C8 heterocycloalkyl; Each R 9 are independently selected from -H, -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl, and -(C1-C6 alkyl)-aryl; Each R 10 are independently -C1-C6 alkyl, -C3-C8 cycloalkyl, -NR 14 R 14’ , -aryl, -heteroaryl and -(C1-C6 alkyl)-aryl; R 10’ is selected from -H, -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl, and -(C1-C6 alkyl)-aryl; R 11 is selected from —H and —C1-C6 alkyl; R 12 is -H, -C1-C6 alkyl, -CO2R 8 , -aryl, -heteroaryl, -(C1-C6 alkyl)-aryl, -S(O)2R 16 and Selected from TIFF2025510791000039.tif22165, R 13 is selected from —H and —C1-C6 alkyl; R 14 and R 14’ are each independently selected from —H, C1-C6 alkyl, —C3-C8 cycloalkyl, and —C3-C8 heterocycloalkyl; R 16 is selected from -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl, and -(C1-C6 alkyl)-aryl; R 18 and R 19 together with the N atom to which they are attached, represent halogen, -C1-C6 alkyl, -C3-C8 cycloalkyl, and -(C1-C6 alkyl)-OR 5 and forming a 4-, 5-, 6-, or 7-membered ring having 0 to 3 substituents selected from R 24 , R 25 and R 26 are each -C1 to C6 alkyl, X a and X b are each independently selected from NH, O, and S; X c is selected from O, S, and S(O)2.

[0237] In some embodiments, in the compound of Formula (III), R 2 is selected from —H, —CH 3 , —CF 3 , —F, —Cl, —OCH 3 and —OCF 3 .

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

[0239] In some embodiments, in the compound of Formula (III), R 15 is selected from —CH 3 , —CF 3 , —OCH 3 , and —OCF 3 .

[0240] In some embodiments, in the compound of Formula (III), R 15 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 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 is selected from —CH3 and —OCH3.

[0243] In some embodiments, in the compound of Formula (III), R 4 teeth, Selected from TIFF2025510791000040.tif58165.

[0244] In some embodiments, in the compound of Formula (III), R 5 is 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.

[0245] In some embodiments, in the compound of Formula (III), R 8 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 9is 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 is independently selected from -C1-C6 alkyl and -(C1-C6 alkyl)-aryl.

[0248] In some embodiments, in the compound of Formula (III), each R 9 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 are independently -C1-C6 alkyl, -C3-C8 cycloalkyl, -NR 14 R 14’ , -aryl and -(C1-C6 alkyl)-aryl.

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

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

[0252] In some embodiments, in the compound of Formula (III), R 10’ is 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 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 is -H, -C1-C6 alkyl, -CO2R 8 , -aryl, -(C1-C6 alkyl)-aryl and -S(O)R 16 is selected from.

[0255] In some embodiments, in the compound of Formula (III), R 12 is -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 TIFF2025510791000041.tif22165.

[0256] In some embodiments, in the compound of Formula (III), R 13 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’ are each 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 is selected from -aryl, -heteroaryl and -(C1-C6 alkyl)-aryl.

[0259] In some embodiments, in the compound of Formula (III), R 16 is 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 together with the N atom to which they are attached, represent halogen, unsubstituted -C1-C6 alkyl, -C1-C6 haloalkyl, -C1-C6 hydroxyalkyl, -C1-C6 aminoalkyl, -C3-C8 cycloalkyl, and -(C1-C6 alkyl)-OR 5 and forming a 4-, 5-, 6-, or 7-membered ring having 0 to 3 substituents selected from:

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

[0262] Combinations of any of the foregoing embodiments for compounds of formula (III) are also envisioned, with each combination forming a separate embodiment for 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 halogen, acyl, acyloxy, alkoxy, carboxy, hydroxy, amino, amido, nitro, cyano, azido, alkylthio, thio, sulfonyl, sulfonamido, alkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl. 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 halogen, acyl, acyloxy, alkoxy, carboxy, hydroxy, amino, amido, nitro, cyano, azido, alkylthio, thio, sulfonyl, and sulfonamido.

[0264] In certain embodiments, the camptothecin analog included in an ADC according to the present 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), wherein R 2 is F and R 20 is H, -(C1~C6)-OR 5 ,or TIFF2025510791000042.tif27165. In some embodiments, the camptothecin analog is a compound having formula (II), wherein R 2 is F and R 20 is H, -(C1~C6)-OR 5 or TIFF2025510791000043.tif27165, R 5 is H and R 18and R 19 together with the N atom to which they are attached form an unsubstituted 4-, 5-, 6-, or 7-membered ring. In some embodiments, the camptothecin analog is a compound having formula (II), wherein R 2 is F and 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 the formula (III): In certain embodiments, the camptothecin analog is a compound having the formula (III), wherein R 2 is F and R 15 is -CH3 and R 4 teeth TIFF2025510791000044.tif27165, -R 9 is -C1-C6 hydroxyalkyl, and X a and X b are each O. In certain embodiments, 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 an ADC according to the disclosure is Compound 139, Compound 140, Compound 141, or Compound 148. In some embodiments, the camptothecin analog included in an ADC according to the disclosure is Compound 139 or Compound 141.

[0268] [Table 6] TIFF2025510791000046.tif224165TIFF2025510791000047.tif214165TIFF2025510791000048.tif23016 5TIFF2025510791000049.tif230165TIFF2025510791000050.tif230165TIFF2025510791000051.tif97165

[0269] [Table 7] TIFF2025510791000053.tif230165TIFF2025510791000054.tif219165TIFF2025510791000055.tif218165TIFF2025510791000056.tif21916 5TIFF2025510791000057.tif230165TIFF2025510791000058.tif224165TIFF2025510791000059.tif224165TIFF2025510791000060.tif66165

[0270] It will be understood that throughout this disclosure, references to compounds of formula (I) include, in various embodiments, compounds of formula (II) and formula (III), as well as the individual compounds set forth in Tables 6 and 7, to the same extent as if the embodiments individually reciting each of these formulas or compounds were specifically recited.

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

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

[0273] In some embodiments, in the conjugate of formula (X), n is 1 to 8, for example, 2 to 8. In some embodiments, n is 4 to 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 discussed above and reflected by the parameters m and n in formula (X), an anti-FRα antibody construct "T" can be conjugated to two or more compounds "D" of formula (I). While any particular anti-FRα antibody construct T is conjugated to an integer number of compounds D, those skilled in the art will understand that analysis of a conjugate preparation to determine the ratio of compound D to anti-FRα antibody construct T may yield a non-integer result reflecting a statistical average. This ratio of compound D to targeting moiety T is sometimes commonly referred to as the drug-to-antibody ratio, or "DAR." Thus, conjugate preparations having a non-integer DAR are 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] Certain embodiments of the present disclosure relate to ADCs having formula (X), wherein D is a group represented by formula (IV): TIFF2025510791000061.tif63165, wherein R 1a is selected from —H, —CH3, —CHF2, —CF3, —F, —Br, —Cl, —OH, —OCH3, —OCF3, and —NH2; R 2a is selected from —H, —CH3, —CF3, —F, —Br, —Cl, —OH, —OCH3, and —OCF3; X is -O-, -S-, or -NH-, and R 4a teeth, TIFF2025510791000062.tif68165, wherein * is the point of attachment to X and p is 1, 2, 3, or 4; or X is O and R 4a -X- is Selected from TIFF2025510791000063.tif37165, R 5a is selected from -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl, and -(C1-C6 alkyl)-aryl; R 8a is selected from -C1-C6 alkyl, -C3-C8 cycloalkyl, and -C3-C8 heterocycloalkyl; Each R 9aare independently selected from -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl, and -(C1-C6 alkyl)-aryl, or R 9a does not exist and X b =X, Each R 10a are independently -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl, -(C1-C6 alkyl)-aryl, and Selected from TIFF2025510791000064.tif22165, Each R 10a’ is independently selected from -H, -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl, and -(C1-C6 alkyl)-aryl; Each R 10b is independently selected from -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl, and -(C1-C6 alkyl)-aryl; R 11a is absent or is -C1-C6 alkyl, R 12a is -C1 to C6 alkyl, -CO2R 8a , -aryl, -heteroaryl, -(C1-C6 alkyl)-aryl, -S(O)2R 16a and Selected from TIFF2025510791000065.tif22165, R 13a is selected from —H and —C1-C6 alkyl; R 14a is selected from -C1-C6 alkyl, -C3-C8 cycloalkyl, and -C3-C8 heterocycloalkyl; R 14a’ is selected from H, —C1-C6 alkyl, —C3-C8 cycloalkyl, and —C3-C8 heterocycloalkyl; R 16a is selected from -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl, and -(C1-C6 alkyl)-aryl; R 21 is -C1-C6 alkyl, -C3-C8 cycloalkyl, and -(C1-C6 alkyl)-OR 5a is selected from R 22 and R 23 are each independently selected from -H, -halogen, -C1-C6 alkyl, and -C3-C8 cycloalkyl; R 24 , R 25 and R 26 are each -C1 to C6 alkyl, X a and X b are each independently selected from NH, O, and S; X c is selected from O, S, and S(O)2, and TIFF2025510791000066.tif17165 shows the attachment point to the linker L.

[0278] In some embodiments, in the compound of formula (IV), R 1a is selected from —CH 3 , —CF 3 , —OCH 3 , —OCF 3 and —NH 2 .

[0279] In some embodiments, in the compound of formula (IV), R 1a is selected from —CH 3 , —CF 3 , —OCH 3 , and —OCF 3 .

[0280] In some embodiments, in the compound of formula (IV), R 1a is selected from —CH 3 , —OCH 3 and NH 2 .

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

[0282] In some embodiments, in the compound of formula (IV), R 2a is selected from —H, —CH 3 , —CF 3 , —F, —Cl, —OCH 3 and —OCF 3 .

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

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

[0285] In some embodiments, in the compound of formula (IV), X is —O—, —S—, or —NH—, and R 4a teeth, Selected from TIFF2025510791000067.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 is 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 is independently selected from -C1-C6 alkyl, and -(C1-C6 alkyl)-aryl.

[0289] In some embodiments, in the compound of Formula (IV), each R 10a are independently -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -(C1-C6 alkyl)-aryl, and Selected from TIFF2025510791000068.tif22165.

[0290] In some embodiments, in the compound of Formula (IV), each R 10a are independently -C1-C6 alkyl, -aryl, -(C1-C6 alkyl)-aryl, and Selected from TIFF2025510791000069.tif22165.

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

[0292] In some embodiments, in the compound of formula (IV), R 13a 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’ 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 is selected from -aryl, -heteroaryl, and -(C1-C6 alkyl)-aryl.

[0295] In some embodiments, in the compound of formula (IV), R 22 and R 23 are each 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 are each independently selected from NH and O.

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

[0298] In some embodiments, in the compound of Formula (IV), X is O and R 4a teeth, TIFF2025510791000070.tif27165, X a and X b are O and R 9a is -C1 to C6 alkyl.

[0299] In some embodiments, in the compound of formula (IV), R 1a is -CH3 or -OCH3, X is O, and R 4a teeth, TIFF2025510791000071.tif27165, X a and X b are O and R 9a is —C1 to C6 alkyl.

[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, TIFF2025510791000072.tif27165, X a and X b are O and R, respectively. 9a is —C1 to C6 alkyl.

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

[0302] Certain embodiments of the present disclosure relate to ADCs having formula (X), wherein D is a group represented by formula (V): TIFF2025510791000073.tif53165, wherein R 2a is selected from —CH3, —CF3, —F, —Br, —Cl, —OH, —OCH3, and —OCF3; R 20a is -H, -C1-C6 alkyl, -C3-C8 cycloalkyl, -(C1-C6 alkyl)-OR 5 , TIFF2025510791000074.tif27165-CO2R 8 , -aryl, -heteroaryl, -(C1-C6 alkyl)-aryl, Selected from TIFF2025510791000075.tif68165, R 5 is selected from -H, -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl, and -(C1-C6 alkyl)-aryl; R 6 and R 7 are each independently -H, -C1-C6 alkyl, -C3-C8 cycloalkyl, -(C1-C6 alkyl)-OR 5 , —C3-C8 heterocycloalkyl, and —C(O)R 17 is selected from R 8 is selected from —H, —C1-C6 alkyl, —C3-C8 cycloalkyl, and —C3-C8 heterocycloalkyl; Each R 9 are independently selected from -H, -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl, and -(C1-C6 alkyl)-aryl; Each R 10 are independently -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl, -(C1-C6 alkyl)-aryl, and -NR 14 R 14’ is selected from Each R 10’are independently selected from -H, -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl, and -(C1-C6 alkyl)-aryl; R 11 is selected from —H and —C1-C6 alkyl; R 12 is -H, -C1-C6 alkyl, -CO2R 8 , -aryl, -heteroaryl, -(C1-C6 alkyl)-aryl, -S(O)2R 16 and Selected from TIFF2025510791000076.tif22165, R 13 is selected from —H and —C1-C6 alkyl; R 14 and R 14’ are each independently selected from —H, C1-C6 alkyl, —C3-C8 cycloalkyl, and —C3-C8 heterocycloalkyl; R 16 is selected from -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl, and -(C1-C6 alkyl)-aryl; R 17 is 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 together with the N atom to which they are attached, include halogen, -C1-C6 alkyl, -C3-C8 cycloalkyl, and -(C1-C6 alkyl)-OR 5 and forming a 4-, 5-, 6-, or 7-membered ring having 0 to 3 substituents selected from R 24 , R 25 and R 26 are each -C1 to C6 alkyl, X a and X b are each independently selected from NH, O, and S; Xc is selected from O, S, and S(O)2, and TIFF2025510791000077.tif17165 shows the attachment point to the linker L.

[0303] In some embodiments, in the compound of Formula (V), R 2a is selected from —CH 3 , —CF 3 , —F, —Cl, —OCH 3 and —OCF 3 .

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

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

[0306] In some embodiments, in the compound of Formula (V), R 20a is -H, -C1-C6 alkyl, -C3-C8 cycloalkyl, -(C1-C6 alkyl)-OR 5 , TIFF2025510791000078.tif27165-CO2R 8 , -aryl, -heteroaryl, -(C1-C6 alkyl)-aryl, Selected from TIFF2025510791000079.tif58165.

[0307] In some embodiments, in the compound of Formula (V), R 20a is -H, -C1-C6 alkyl, -(C1-C6 alkyl)-OR 5 , TIFF2025510791000080.tif27165-(C1-C6 alkyl)-aryl, Selected from TIFF2025510791000081.tif58165.

[0308] In some embodiments, in the compound of Formula (V), R 20ais -H, -C1-C6 alkyl, -(C1-C6 alkyl)-OR 5 , TIFF2025510791000082.tif27165-(C1-C6 alkyl)-aryl, Selected from TIFF2025510791000083.tif58165.

[0309] In some embodiments, in the compound of Formula (V), R 20a is -H, -C1-C6 alkyl, -(C1-C6 alkyl)-OR 5 , Selected from TIFF2025510791000084.tif58165.

[0310] In some embodiments, in the compound of Formula (V), R 20a is -H, unsubstituted -C1-C6 alkyl, -C1-C6 haloalkyl, -C1-C6 hydroxyalkyl, -C3-C8 cycloalkyl, -(C1-C6 alkyl)-OR 5 , TIFF2025510791000085.tif27165-CO2R 8 , unsubstituted aryl, aminoaryl, heteroaryl, (C1-C6 alkyl) aminoaryl, Selected from TIFF2025510791000086.tif68165.

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

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

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

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

[0315] In some embodiments, in the compound of Formula (V), R 8 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 is 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 is independently selected from -C1-C6 alkyl, and -(C1-C6 alkyl)-aryl.

[0318] In some embodiments, in the compound of Formula (V), each R 9is 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 are independently -C1-C6 alkyl, -C3-C8 cycloalkyl, -NR 14 R 14’ , -aryl, and -(C1-C6 alkyl)-aryl.

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

[0321] In some embodiments, in the compound of Formula (V), R 11 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 is -H, -C1-C6 alkyl, -aryl, -(C1-C6 alkyl)-aryl, and -S(O)2R 16 is selected from.

[0323] In some embodiments, in the compound of Formula (V), R 12 is -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 TIFF2025510791000087.tif22165.

[0324] In some embodiments, in the compound of Formula (V), R 13 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’ are each 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 is selected from -aryl, -heteroaryl, and -(C1-C6 alkyl)-aryl.

[0327] In some embodiments, in the compound of Formula (V), R 16 is 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 is 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 together with the N atom to which they are attached, represent halogen, unsubstituted -C1-C6 alkyl, -C1-C6 haloalkyl, -C1-C6 aminoalkyl, -C1-C6 hydroxyalkyl, -C3-C8 cycloalkyl, and -(C1-C6 alkyl)-OR 5 and forming a 4-, 5-, 6-, or 7-membered ring having 0 to 3 substituents selected from:

[0330] In some embodiments, in the compound of Formula (V), R 17 is —C1 to C6 alkyl.

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

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

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

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

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

[0336] Other combinations of any of the foregoing embodiments for compounds of formula (V) are also envisioned, with each combination forming a separate embodiment for purposes of this disclosure.

[0337] Certain embodiments of the present disclosure relate to ADCs having formula (X), wherein D is a group represented by formula (VI): TIFF2025510791000088.tif63165, wherein: R 2a is selected from —H, —CH3, —CF3, —F, —Br, —Cl, —OH, —OCH3, and —OCF3; X is -O-, -S-, or -NH-, and R 25 is -C1-C6 alkyl, -(C1-C6 alkyl)-OR 5a , -CO2R 8a , -aryl, -heteroaryl, -(C1-C6 alkyl)-aryl, TIFF2025510791000089.tif94165, wherein * is the point of attachment to X and p is 1, 2, 3, or 4; or X is O and R 25 -X- is Selected from TIFF2025510791000090.tif37165, R 5a is selected from -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl, and -(C1-C6 alkyl)-aryl; R 6a is selected from —H, —C1-C6 alkyl, —C3-C8 cycloalkyl, and —C3-C8 heterocycloalkyl; R 7a is -C1-C6 alkyl, -C3-C8 cycloalkyl, -(C1-C6 alkyl)-OR 5a , —C3-C8 heterocycloalkyl and —C(O)R 17a is selected from R 8a is selected from -C1-C6 alkyl, -C3-C8 cycloalkyl, and -C3-C8 heterocycloalkyl; Each R9a are independently selected from -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl, and -(C1-C6 alkyl)-aryl, or R 9a does not exist and X b =X, Each R 10a are independently -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl, -(C1-C6 alkyl)-aryl, and Selected from TIFF2025510791000091.tif22165, Each R 10a’ is independently selected from -H, -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl, and -(C1-C6 alkyl)-aryl; Each R 10b is independently selected from -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl, and -(C1-C6 alkyl)-aryl; R 11a is absent or is -C1-C6 alkyl, R 12a is -C1 to C6 alkyl, -CO2R 8a , -aryl, -heteroaryl, -(C1-C6 alkyl)-aryl, -S(O)2R 16a , and Selected from TIFF2025510791000092.tif22165, R 13a is selected from —H and —C1-C6 alkyl; R 14a is selected from -C1-C6 alkyl, -C3-C8 cycloalkyl, and -C3-C8 heterocycloalkyl; R 14a’ is selected from H, —C1-C6 alkyl, —C3-C8 cycloalkyl, and —C3-C8 heterocycloalkyl; R 16a is selected from -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl, and -(C1-C6 alkyl)-aryl; R 17a is 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 is -C1-C6 alkyl, -C3-C8 cycloalkyl, and -(C1-C6 alkyl)-OR 5a is selected from R 22 and R 23 are each independently selected from -H, -halogen, -C1-C6 alkyl, and -C3-C8 cycloalkyl; R 24 , R 25 and R 26 are each -C1 to C6 alkyl, X a and X b are each independently selected from NH, O, and S; X c is selected from O, S, and S(O)2, and TIFF2025510791000093.tif17165 shows the attachment point to the linker L.

[0338] In some embodiments, in the compound of formula (VI), R 2a is selected from —CH 3 , —CF 3 , —F, —Br, —Cl, —OH, —OCH 3 , and —OCF 3 .

[0339] In some embodiments, in the compound of formula (VI), R 2a is selected from —CH 3 , —CF 3 , —F, —Cl, —OCH 3 and —OCF 3 .

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

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

[0342] In some embodiments, in the compound of Formula (VI), X is —O—, —S—, or —NH—, and R 25 is -C1-C6 alkyl, -(C1-C6 alkyl)-OR 5a , -(C1-C6 alkyl)-aryl, TIFF2025510791000094.tif58165, or X is O and R 25 -X- is Selected from TIFF2025510791000095.tif37165.

[0343] In some embodiments, in the compound of Formula (VI), X is —O—, —S—, or —NH—, and R 25 is -C1-C6 alkyl, -(C1-C6 alkyl)-OR 5a , -(C1-C6 alkyl)-aryl, Selected from TIFF2025510791000096.tif58165.

[0344] In some embodiments, in the compound of Formula (VI), X is —O—, —S—, or —NH—, and R 25 is -C1-C6 alkyl, -(C1-C6 alkyl)-OR 5a , Selected from TIFF2025510791000097.tif58165.

[0345] In some embodiments, in the compound of Formula (VI), X is —O—, —S—, or —NH—, and R 25 teeth, Selected from TIFF2025510791000098.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 is H.

[0348] In some embodiments, in the compound of formula (VI), R 6a 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 is -C1-C6 alkyl, -C3-C8 cycloalkyl and -C(O)R 17a is selected from.

[0350] In some embodiments, in the compound of Formula (VI), each R 9a is 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 is independently selected from -C1-C6 alkyl, and -(C1-C6 alkyl)-aryl.

[0352] In some embodiments, in the compound of Formula (VI), each R 10a are independently -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -(C1-C6 alkyl)-aryl, and Selected from TIFF2025510791000099.tif22165.

[0353] In some embodiments, in the compound of Formula (VI), each R 10a are independently -C1-C6 alkyl, -aryl, -(C1-C6 alkyl)-aryl, and Selected from TIFF2025510791000100.tif22165.

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

[0355] In some embodiments, in the compound of formula (VI), R 13a 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’ 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 is selected from -aryl, -heteroaryl, and -(C1-C6 alkyl)-aryl.

[0358] In some embodiments, in the compound of formula (VI), R 17a is —C1 to C6 alkyl.

[0359] In some embodiments, in the compound of formula (VI), R 22 and R 23 are each 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 are each independently selected from NH and O.

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

[0362] In some embodiments, in the compound of Formula (VI), X is O and R 25 is —C1 to C6 alkyl.

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

[0364] Other combinations of any of the foregoing embodiments relating to compounds of formula (VI) are also envisioned, with each combination forming a separate embodiment for 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 halogen, acyl, acyloxy, alkoxy, carboxy, hydroxy, amino, amido, nitro, cyano, azido, alkylthio, thio, sulfonyl, sulfonamido, alkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl. 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 halogen, acyl, acyloxy, alkoxy, carboxy, hydroxy, amino, amido, nitro, cyano, azido, alkylthio, thio, sulfonyl, and sulfonamido.

[0366] In certain embodiments, in the ADC having formula (X), D is a compound of formula (IV), wherein R1a is -CH3 and R 2a is F. In some embodiments, in the ADC having formula (X), D is a compound of formula (IV), wherein R 1a is -CH3 and R 2a is F, X is -O-, and R 4a teeth TIFF2025510791000101.tif27165, R 9a is -C1-C6 alkyl, and X a and X b are O respectively.

[0367] In certain embodiments, in the ADC having formula (X), D is a compound of formula (V), wherein R 2a is F and R 20a is H, -(C1~C6)-OR 5 ,or TIFF2025510791000102.tif27165. In some embodiments, in the ADC having Formula (X), D is a compound of Formula (V), wherein R 2a is F and R 20a is H, -(C1~C6)-OR 5 ,or TIFF2025510791000103.tif27165, R 5 is H and R 18 and R 19 are taken together with the N atom to which they are attached to form an unsubstituted 4-, 5-, 6-, or 7-membered ring. In some embodiments, in the ADC having Formula (X), D is a compound of Formula (V), wherein R 2a is F and R 20a -(C1~C6)-OR 5 and R 5 is H.

[0368] In certain embodiments, in the ADC having formula (X), D is a compound of formula (VI), wherein R 2a is F, X is -O-, and R 25 is -C1 to C6 alkyl.

[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, whereas 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 that links one compound D to two or more sites on the anti-FRα antibody construct T can also be considered polyfunctional.

[0370] The linker L comprises a functional group capable of reacting with a targeting group(s) on the anti-FRα antibody construct T and at least one functional group capable of reacting with a targeting group on the camptothecin analog D. Suitable functional groups are known in the art and include, for example, those described in Bioconjugate Techniques (G.T. Hermanson, 2013, Academic Press). Groups on the anti-FRα antibody construct T and the camptothecin analog D that can function as targeting groups for linker attachment include, but are not limited to, thiol, hydroxyl, carboxyl, amine, aldehyde, 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. Also useful in this context are the "self-stabilizing" maleimides described in Lyon et al., 2014, Nat. Biotechnol., 32:1059-1062.

[0372] Non-limiting examples of functional groups that can react with amines include activated esters (e.g., N-hydroxysuccinamide (NHS) esters and sulfo-NHS esters), imidoesters (e.g., Traut's reagent), isothiocyanates, aldehydes, and acid anhydrides (e.g., diethylenetriaminepentaacetic anhydride (DTPA)). As another example, succinimido-1,1,3,3-tetra-methyluronium tetrafluoroborate (TSTU) or benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP) may be used to convert a carboxylic acid to an activated ester, which can then be reacted with an amine.

[0373] Non-limiting examples of functional groups that can react with electrophilic groups such as aldehyde or ketone carbonyl groups include hydrazide, oxime, amino, hydrazine, thiosemicarbazone, hydrazine carboxylate, and arylhydrazide.

[0374] In certain embodiments, the linker L comprises a functional group that enables cross-linking of two interchain cysteines on the anti-FRα antibody construct, such as a ThioBridge™ linker (Badescu et al., 2014, Bioconjug. Chem. 25:1124-1136), a dithiomaleimide (DTM) linker (Behrens et al., 2015, Mol. Pharm. 12:3986-3998), a dithioaryl (TCEP) pyridazinedione-based linker (Lee et al., 2016, Chem. Sci., 7:799-802), or a dibromopyridazinedione-based 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 allows conjugation with a linker via a complementary reactive group on the linker. For example, conjugation of a linker to the anti-FRα antibody construct may utilize a click chemistry reaction, such as the azide-alkyne cycloaddition (AAC) reaction, which has been successfully used in the development of antibody-drug conjugates (see, for example, Chio & Bane, 2020, Methods Mol. Biol., 2078:83-97). The AAC reaction may be a copper-catalyzed AAC (CuAAC) reaction, which involves coupling an azide with a linear alkyne, or a strain-promoted AAC (SPAAC) reaction, which involves coupling an azide with a cyclooctyne.

[0376] The linker L may be a cleavable linker or a non-cleavable linker. A cleavable linker is a linker that is easily cleaved under certain conditions, such as intracellular conditions (e.g., in endosomes or lysosomes) or in the vicinity of target cells (e.g., tumor microenvironment). Examples include protease-sensitive, acid-sensitive, or reduction-sensitive linkers. In contrast, non-cleavable linkers generally rely on the degradation of the antibody in cells, which results in the release of the amino acid-linker-drug moiety.

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

[0378] For conjugates intended to be internalized by cells, the linker L may comprise an amino acid sequence that is recognized and cleaved by an endosomal or lysosomal protease, examples of which include cathepsins B, C, D, H, L, and S, and legumain.

[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 contain additional groups that provide 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 within a pH range, such as hydrazone linkers. Linkers containing a combination of these functional groups may also be useful; for example, linkers containing both hydrazones and disulfides are known in the art.

[0381] Further examples of cleavable linkers are linkers containing β-glucuronides that can be cleaved by β-glucuronidase, an enzyme present in lysosomes and tumor stroma (see, e.g., De Graaf et al., 2002, Curr. Pharm. Des. 8:1391-1403, and International Patent Publication No. WO 2007 / 011968). The β-glucuronide can also function to increase the hydrophilicity of the linker L.

[0382] Another example of a linker that is cleaved intracellularly to increase hydrophilicity is a linker that contains a pyrophosphate diester moiety (see, e.g., Kern et al., 2016, J Am Chem Soc., 138:2430-1445).

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

[0384] The cleavable linker may optionally further comprise one or more additional functional groups, such as a self-immolative group, a self-leaving group, a stretcher, or a hydrophilic moiety.

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

[0386] Stretchers used in linkers for drug conjugates include, for example, alkylene groups 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 moieties within a linker. For example, PEG or mPEG can be included in a linker either "in-line" or as a pendant group to increase the hydrophilicity of the linker (see, e.g., U.S. Patent Application Publication No. 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 the linker L include, for example, β-glucuronide, sulfonate, carboxylate, and pyrophosphate diester.

[0388] In certain embodiments, an ADC of Formula (X) may comprise a cleavable linker. In certain embodiments, an ADC of Formula (X) may comprise a peptide-containing linker. In certain embodiments, an ADC of Formula (X) may comprise a protease-cleavable linker.

[0389] In some embodiments, in the ADC of formula (X), m is 1 and the linker L is of formula (XI): TIFF2025510791000104.tif32165, wherein Z is a functional group capable of reacting with a targeting group on the anti-FRα antibody construct T. Str is a stretcher AA1 and AA2 are each independently an amino acid, and AA1-[AA2] r forms a protease cleavage site, X is a self-immolative group, q is 0 or 1, r is 1, 2 or 3; s is 0, 1 or 2; # is the point of attachment to the anti-FRα antibody construct T, and % is the point of attachment to camptothecin analogue D.

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

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

[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, a compound of formula (XI): In the linker, Z is TIFF2025510791000105.tif32165, where # is the point of attachment to T and * is the point of attachment to the rest of the linker.

[0394] In some embodiments, in the linker of formula (XI), Str is Selected from TIFF2025510791000106.tif53165, During the ceremony, R is H or C1-C6 alkyl; t is an integer from 2 to 10, u is an integer from 1 to 10.

[0395] In some embodiments, in the linker of formula (XI), Str is Selected from TIFF2025510791000107.tif22165, During the ceremony, t is an integer from 2 to 10, u is an integer from 1 to 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 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 has a sequence selected from Ala-Leu-Ala-Leu, Gly-Phe-Leu-Gly, Gly-Gly-Phe-Gly and Gly-Phe-Gly-Gly.

[0399] In certain embodiments, in the ADC of formula (X), m is 1 and the linker L is of formula (XII): TIFF2025510791000108.tif32165, wherein Z is a functional group capable of reacting with a targeting group on the anti-FRα antibody construct T; Str is a stretcher AA1 and AA2 are each independently an amino acid, and AA1-[AA2] r forms a protease cleavage site, Y is -NH-CH2- or -NH-CH2-C(O)-; q is 0 or 1, r is 1, 2 or 3; v is 0 or 1, # is the point of attachment to the anti-FRα antibody construct T, and % is the point of attachment to camptothecin analogue D.

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

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

[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, a compound of formula (XII): In the linker, Z is TIFF2025510791000109.tif32165, where # is the point of attachment to T and * is the point of attachment to the rest of the linker.

[0404] In some embodiments, in the linker of formula (XII), Str is Selected from TIFF2025510791000110.tif53165, During the ceremony, R is H or C1-C6 alkyl; t is an integer from 2 to 10, u is an integer from 1 to 10.

[0405] In some embodiments, in the linker of formula (XII), Str is Selected from TIFF2025510791000111.tif22165, During the ceremony, t is an integer from 2 to 10, u is an integer from 1 to 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 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 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-CH. In some embodiments, in the linker of formula (XII), v is 1 and Y is -NH-CH.

[0410] In some embodiments, the ADC of formula (X) may comprise a disulfide-containing linker. In some embodiments, in the ADC of formula (X), m is 1 and the linker L is of formula (XIII): TIFF2025510791000112.tif32165, wherein Z is a functional group capable of reacting with a targeting group on the anti-FRα antibody construct T; Q is -(CH2) p -or-(CH2CH2O) q -, and p and q are each independently an integer of 1 to 10; each R is independently H or C1-C6 alkyl; n is 1, 2 or 3; # is the point of attachment to the anti-FRα antibody construct T, and % is the point of attachment to camptothecin analogue D.

[0411] In some embodiments, an ADC of Formula (X) may comprise a β-glucuronide-containing linker.

[0412] Various non-cleavable linkers for linking drugs to targeting moieties are known in the art and may be useful in certain embodiments for the ADCs of the present 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 a sulfosuccinimidyl-4-[N-maleimidomethyl]cyclohexane-1-carboxylate (sulfo-SMCC)-based linker. Sulfo-SMCC conjugation typically occurs via a maleimide group that reacts with sulfhydryls (thiols, -SH) on camptothecin analogs, while sulfo-NHS esters are reactive with primary amines (such as those found on lysines and the N-termini of proteins or peptides) on 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-amidocaproate) ("long chain" SMCC or LC-SMCC), κ-maleimidoundecanoic acid N-succinimidyl ester (KMUA), γ-maleimidobutyric acid N-succinimidyl ester (GMBS), ε-maleimidocaproic acid These include linkers based on N-hydroxysuccinimide ester (EMCS), m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), N-(α-maleimidoacetoxy)-succinimide ester (AMAS), succinimide-6-(β-maleimidopropionamido)hexanoate (SMPH), N-succinimidyl 4-(p-maleimidophenyl)-butyrate (SMPB), and N-(p-maleimidophenyl)isocyanate (PMPI).Other examples include those containing haloacetyl-based functional groups such as N-succinimidyl-4-(iodoacetyl)-aminobenzoate (SIAB), N-succinimidyl iodoacetate (SIA), N-succinimidyl bromoacetate (SBA), and N-succinimidyl 3-(bromoacetamido)propionate (SBAP).

[0413] Non-limiting examples of drug-linkers comprising camptothecin analogs of Formula (I) are shown in Table 8 (Figure 13), Table 9 (Figure 14), and Table 10 (Figure 15). Non-limiting examples of conjugates comprising these drug-linkers are shown in Table 11 (Figure 16), Table 12 (Figure 17), and Table 13 (Figure 18). In certain embodiments, an ADC of Formula (X) comprises a drug-linker selected from those shown in Tables 8, 9, and 10. In certain embodiments, an 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, an 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) comprises 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-AM-Compound 141, MT-GGFG-Compound 141, MC-GGFG-Compound 141, MT-GGFG-Compound 148, and MC-GGFG-Compound 148. m), wherein n is 4 or 8. In some embodiments, the ADC of Formula (X) comprises 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-AM-Compound 141, MT-GGFG-Compound 141, MC-GGFG-Compound 141, MT-GGFG-Compound 148, and MC-GGFG-Compound 148. m ), where n is 8.

[0415] Preparation of ADCs ADCs of formula (X) can be prepared by standard methods known in the art (see, for example, Bioconjugate Techniques (G.T. 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 (G.T. 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] In general, preparation of an ADC involves first preparing a drug-linker DL comprising one or more camptothecin analogs of Formula (I) and a linker L, and then conjugating the drug-linker DL to a suitable group on an anti-FRα antibody construct T. However, ligation of the linker L to the anti-FRα antibody construct T, followed by ligation of the anti-FRα antibody construct-linker TL to one or more camptothecin analogs D of Formula (I), remains an alternative approach available in some embodiments.

[0417] Suitable groups on compound D of formula (I) for attachment of 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 the present disclosure, linker L is attached to the compound through a hydroxyl or amine group on compound D of formula (I).

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

[0419] For example, the anti-FRα antibody construct T may contain one or more naturally occurring sulfhydryl groups that allow the anti-FRα antibody construct T to be bound to the linker L via the sulfhydryl group's sulfur atom. Alternatively, the anti-FRα antibody construct T may contain one or more lysine residues that can be chemically modified 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's reagent). Alternatively, the anti-FRα antibody construct T may contain one or more carbohydrate groups that can be chemically modified to contain one or more sulfhydryl groups.

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

[0421] The anti-FRα antibody construct T can also be modified to include additional cysteine ​​residues (see, e.g., U.S. Patent Nos. 7,521,541; 8,455,622; and 9,000,130) or unnatural amino acids that provide reactive handles, 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 can be modified to include a non-natural reactive group, such as an azide, that allows for conjugation with a linker via a complementary reactive group on the linker, e.g., click chemistry (e.g., Chio & Bane, 2020, Methods Mol. Biol., 2078:83-97). Another option is the use of GlycoConnect™ technology (Synaffix BV, Nijmegen, Netherlands), which involves enzymatic remodeling of antibody glycans to allow for attachment of a linker via metal-free click chemistry (see, e.g., European Patent No. EP2911699).

[0422] Other protocols for modifying proteins for attachment or association of a linker L are known in the art, including those described in Coligan et al., Current Protocols in Protein Science, vol. 2, John Wiley & Sons (2002).

[0423] Alternatively, ADCs can be prepared using the enzyme transglutaminase, particularly bacterial transglutaminase (BTG) from Streptomyces mobaraensis (see, e.g., Jeger et al., 2010, Angew. Chem. Int. Ed., 49:9995-9997). BTG forms an amide bond between the side chain carboxamide of glutamine (typically an amine acceptor on an antibody) and an alkylene amino group (typically an amine donor on a drug-linker), which can be, for example, the ε-amino group of lysine or a 5-amino-n-pentyl group. Antibodies can also be modified to include a glutamine-containing peptide, or "tag," that enables BTG conjugation to be used to conjugate the antibody with a drug-linker (see, e.g., U.S. Patent Application Publication No. US2013 / 0230543 and International (PCT) Publication No. WO2016 / 144608).

[0424] A similar conjugation approach utilizes the enzyme sortase A. In this approach, an antibody is typically engineered to contain a sortase A recognition motif (LPXTG, where X is any naturally occurring amino acid), and the drug-linker is designed to contain an oligoglycine motif (typically GGG) to enable sortase A-mediated transpeptidation (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 to the anti-FRα antibody construct T (i.e., the "drug-to-antibody ratio" or DAR) can be determined by standard techniques, such as UV / VIS spectroscopy, ELISA-based techniques, chromatographic techniques, such as hydrophobic interaction chromatography (HIC), UV-MALDI mass spectrometry (MS), and MALDI-TOF MS. Additionally, the distribution of drug-linked forms (e.g., the proportion of anti-FRα antibody construct T containing 0, 1, 2, 3, etc. compounds of Formula (I) D) can also be optionally analyzed. Various techniques for assessing DAR distribution are known in the art, including MS (with or without an accompanying chromatographic separation step), 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 the present disclosure are typically formulated as pharmaceutical compositions. Accordingly, certain embodiments of the present disclosure relate to pharmaceutical compositions comprising the ADCs described herein and a pharmaceutically acceptable carrier, diluent, or excipient. Such pharmaceutical compositions can be prepared by known procedures using well-known and readily available ingredients.

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

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

[0429] Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed. Examples of such carriers include buffers such as phosphate, citrate, 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 about 10 residues) polypeptides; serum albumin or Examples of suitable surfactants 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 non-ionic surfactants such as polyethylene glycol (PEG).

[0430] In certain embodiments, compositions containing the ADC may be in the form of a sterile injectable aqueous or oily solution or suspension. Such suspensions may be formulated using suitable dispersing or wetting agents and / or suspending agents known in the art. The sterile injectable solution or suspension may contain the 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. Additionally, sterile, fixed oils may be employed as carriers. For this purpose, various bland fixed oils may be used, including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid may also be used in the preparation of injectable solutions. Adjuvants such as local anesthetics, preservatives, and / or buffers may also be included in the injectable solution or suspension.

[0431] In certain embodiments, a composition comprising an ADC 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 solubilizing agent and / or a local anesthetic such as lignocaine to ease pain at the injection site. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a lyophilized powder or water-free concentrate in a hermetically sealed container such as an ampoule or sachet indicating the quantity of active agent. When the composition is administered by infusion, it can be dispensed using an infusion bottle containing pharmaceutical-grade sterile water or saline. When the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients can be mixed prior to administration.

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

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

[0434] Certain embodiments of the present disclosure relate to methods of inhibiting abnormal cancer or tumor cell proliferation, methods of inhibiting cancer or tumor cell proliferation, or methods of treating cancer in a subject, comprising administering an ADC described herein. In certain embodiments, the ADC described herein can be used to treat cancer. Accordingly, some embodiments of the present disclosure relate to the use of an ADC as an anticancer agent.

[0435] Certain embodiments of the present disclosure relate to methods of inhibiting the proliferation of cancer or tumor cells, comprising contacting the cells with an ADC described herein, e.g., an ADC of Formula (X). Some embodiments relate to methods of killing cancer or tumor cells, comprising contacting the cells with an ADC described herein, e.g., an ADC of Formula (X).

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

[0437] Certain embodiments relate to the use of an ADC described herein, e.g., an ADC of Formula (X), in a method of inhibiting tumor growth in a subject. Some embodiments relate to the use of an ADC described herein, e.g., an ADC of Formula (X), in a method of inhibiting cancer cell growth and / or killing cancer cells in vitro. Some embodiments relate to the use of an ADC described herein, e.g., an ADC of Formula (X), in a method of inhibiting cancer cell growth and / or killing cancer cells in vivo in a subject with cancer.

[0438] Examples of cancers that can be treated in certain 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 certain embodiments include, but are not limited to, brain cancer, breast cancer, cervical cancer, colon cancer, head and neck cancer, renal 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, they may also be considered solid tumors. Typically, the cancer to be treated is a FRα-expressing cancer.

[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, e.g., 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 an FRα-positive cancer or tumor in a subject.

[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. Certain embodiments of the present disclosure relate to methods of treating FRα-positive cancer with an ADC described herein, e.g., an ADC of Formula (X), 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. In some embodiments, an ADC of Formula (X) may be useful for treating triple-negative breast cancer (TNBC).

[0441] Certain embodiments of the present disclosure relate to methods of treating FRα-positive cancer with an ADC described herein, e.g., an ADC of Formula (X), wherein the cancer is a solid tumor that expresses FRα at high levels (FRα-high solid tumor). Certain embodiments of the present disclosure relate to methods of treating FRα-positive cancer with an ADC described herein, e.g., an ADC of Formula (X), wherein the cancer is a solid tumor that expresses FRα at intermediate levels (FRα-medium solid tumor). Certain embodiments of the present disclosure relate to methods of treating FRα-positive cancer with an ADC described herein, e.g., an ADC of Formula (X), wherein the cancer is a solid tumor that expresses FRα at intermediate to low levels (FRα-medium / low solid tumor). Certain embodiments of the present disclosure relate to methods of treating FRα-positive cancer with an ADC described herein, e.g., an ADC of Formula (X), wherein the cancer is a solid tumor that expresses 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] Medicine Kit Certain embodiments relate to pharmaceutical kits that include an ADC described herein, e.g., an ADC of Formula (X).

[0443] The kit typically includes a container holding the ADC and a label and / or package insert on or associated with the container. The label or package insert includes instructions customarily included in commercial packaging for therapeutic products, providing information or instructions regarding the indications, use, dosage, administration, contraindications, and / or warnings for use of the therapeutic product. The label or package insert may further include instructions in a format prescribed by a government agency having jurisdiction over the manufacture, use, or sale of pharmaceuticals or biological products, which instructions incorporate approval by the government agency 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 having a stopper that can be pierced by a hypodermic injection needle.

[0444] In addition to the container that holds the ADC, the kit may optionally include one or more additional containers that contain other components of the kit, such as a pharmaceutically acceptable buffer (such as 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, intravenous infusion bags, etc. The containers can be formed from a variety of materials, such as glass or plastic. If appropriate, one or more components of the kit can also be lyophilized or provided in a dry form, such as a powder or granules, and the kit can further include a solvent suitable for reconstituting the lyophilized or dried component(s).

[0446] The kit may also include other materials desirable from a commercial or user standpoint, including filters, needles, and syringes.

[0447] The following examples are offered for illustrative purposes and are not intended to limit in any way the scope of the claimed invention. [Example]

[0448] General Provisions Chemistry: Examples 1-3 below illustrate various methods for preparing camptothecin analogs of Formula (I). It is understood that those skilled in the art can produce these compounds by similar methods or by combining other methods known in the art. It is also understood that those skilled in the art can produce other compounds of Formula (I) not specifically shown below using the methods described below or similar methods by using appropriate starting components and modifying the synthetic parameters as needed. In general, the starting components can be obtained from commercial sources 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 can be 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 can be prepared as described herein.

[0449] Biological Assays: Expression levels of FRα in the cell lines and CDX models used in the Examples were assessed in-house using a research-grade IHC assay and assigned relative expression levels (high / medium / low or strong / medium / weak). PDX models were similarly assessed using archival tumor samples.

[0450] Abbreviation The following abbreviations are used throughout the Examples section: BCA: bicinchoninic acid, Boc: di-tert-butyl dicarbonate, CE-SDS: capillary electrophoresis sodium dodecyl sulfate, DCM: dichloromethane, DTPA: diethylenetriaminepentaacetic acid, DIPEA: N,N-diisopropylethylamine, DMF: dimethylformamide, DMM™: (4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methyl-morpholinium chloride, EDC: 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, Fmoc: fluorenylmethyloxycarbonyl, HATU: azabenzotriazole tetramethyluronium hexafluorophosphate sulfate, HIC: hydrophobic interaction chromatography, HOAt: 1-hydroxy-7-azabenzotriazole, HPLC: high-performance liquid chromatography, LC / MS: liquid chromatography mass spectrometry, MC: maleimidocaproyl, MT: maleimidotriethylene glycolate, NMM: N-methylmorpholine, PNP: p-nitrophenol, RP-UPLC-MS: reversed-phase ultra-performance liquid chromatography mass spectrometry, SEC: size exclusion chromatography, TCEP: tris(2-carboxyethyl)phosphine, Tfp: tetrafluorophenyl, TLC: thin-layer chromatography, TFA: trifluoroacetic acid.

[0451] General Chemistry Procedures General Procedure 1: Conversion of Chlorides to Amine To a stirred solution of the chlorinated compound in dimethylformamide (0.05-0.1 M) was added the appropriate secondary amine (3 equiv.). Upon completion (as determined by LC / MS, typically 1-3 h), the reaction mixture was purified by reverse-phase HPLC to give the desired product after lyophilization.

[0452] General Procedure 2: Conversion of amines to amides To a stirred solution of the amine compound in dimethylformamide (0.05-0.1 M) was added triethylamine (1.2 equiv.), the appropriate carboxylic acid (1.1 equiv.), followed by a 1 M solution of DMMTM (2 equiv.). Upon completion (as determined by LC / MS, typically 16 h), the reaction mixture was purified by reverse-phase HPLC to afford the desired product after lyophilization.

[0453] General Procedure 3: Conversion of amines to sulfonamides To a stirred solution of the amine compound in dimethylformamide (0.05-0.1 M) was added DIPEA (3 equiv.), followed by the appropriate sulfonyl chloride. Upon completion (as determined by LC / MS, typically 16 h), the reaction mixture was purified by reverse-phase HPLC to give the desired product after lyophilization. General Procedure 4: Two-Step Conversion of Amines to Ureas (Synthetic Scheme IV; Figure 1D) Step 1: To a stirred solution (0.05-0.1 M) of the amine compound in dichloromethane or dimethylformamide was added p-nitrophenyl carbonate (1 equivalent), followed by triethylamine (2 equivalents). Upon completion (as determined by LC / MS, typically 1-4 hours), the reaction mixture was concentrated to dryness and then purified by reverse-phase HPLC to yield the desired PNP-carbamate intermediate after lyophilization. This intermediate may be used to generate a single analog or may be split into multiple batches to generate multiple analogs in a second step. Step 2: To the PNP-carbamate intermediate (0.1-0.2 M) in dimethylformamide was added the appropriate primary amine (3 equiv.). Upon completion (as determined by LC / MS, typically 1 h), the reaction mixture was purified by reverse-phase HPLC to afford the desired product after lyophilization.

[0454] General Procedure 5: Conversion of amines to carbamates To a stirred solution (0.05-0.1 M) of the amine compound in dichloromethane or dimethylformamide, p-nitrophenyl carbonate (1 equivalent) was added, followed by triethylamine (2 equivalents). Upon completion (as determined by LC / MS, typically 1-4 hours), the appropriate alcohol was added to the resulting PNP-carbamate intermediate. Upon completion (as determined by LC / MS, typically 1-16 hours), the reaction mixture was purified by reverse-phase HPLC to afford the desired product after lyophilization.

[0455] General Procedure 6: Removal of the Boc protecting group To a stirred solution of the Boc-protected amine compound in dichloromethane (0.1 M) was added TFA (20% by volume). Upon completion (as determined by LC / MS, typically 1 h), the reaction mixture was concentrated in vacuo to give a crude solid, or purified as described in General Procedure 9.

[0456] General Procedure 7: Copper-Mediated Amide Coupling To a rapidly stirred solution (0.02 M) of Boc-GGFG-OH (3 equiv.) and HOAt (3 equiv.) in a 10% v / v mixture of dimethylformamide in dichloromethane was added EDC (HCl salt, 3 equiv.). After 5 min, a solution (0.02 M) of the amine-containing payload (1 equiv.) in a 10% v / v mixture of dimethylformamide in dichloromethane was added, followed immediately by CuCl (4 equiv.). Upon completion (as determined by LC / MS, typically 1-16 h), the reaction mixture was concentrated in vacuo to give a crude solid or purified by preparative HPLC to give the desired product after lyophilization.

[0457] General Step 8: Install MT To a stirred solution (approximately 0.02 M) of the amine compound (1 equivalent) in dimethylformamide was added a solution (approximately 0.02 M) of MT-OTfp (1.2-1.5 equivalents) in acetonitrile, followed by DIPEA (10 μL, 4 equivalents). Upon completion (as determined by LC / MS, typically 1-16 hours), the reaction mixture was concentrated in vacuo to give a crude solid, which was purified by preparative HPLC to give the desired product after lyophilization.

[0458] General Procedure 9: Compound Purification Flash chromatography: Crude reaction products were purified using Biotage® Snap Ultra columns (10, 25, 50, or 100 g) (Biotage, Charlotte, NC) eluting with a linear gradient of ethyl acetate / hexane or methanol / dichloromethane on 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) eluting with a linear gradient of 0.1% TFA in acetonitrile / 0.1% TFA in water. Purified compounds were isolated either by removal of organic solvents by rotary evaporation or by lyophilization of the acetonitrile / water mixture.

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

[0460] General Procedure 10: Compound Analysis LC / MS: Reaction completion was monitored, and purified compounds were analyzed on an Agilent 1290 HPLC / 6120 Single Quad LC / MS system (Agilent Technologies, Inc., Santa Clara, CA) using a Kinetex® 2.6-μm C18 100 Å (30 × 3 mm) column (Phenomenex, Torrance, CA) eluted with a 10–100% linear gradient of 0.1% formic acid in acetonitrile / 0.1% formic acid in water.

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

[0462] Example 1: Preparation of camptothecin analogs bearing methyl at C10 position 1.1: (S)-11-(chloromethyl)-4-ethyl-8-fluoro-4-hydroxy-9-methyl-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (compound 1.1) TIFF2025510791000113.tif42165 The title compound 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]indolizino[1,2-b]quinoline-3,14(4H)-dione (compound 1.2) TIFF2025510791000114.tif42165 The title compound 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]indolizino[1,2-b]quinoline-3,14(4H)-dione (compound 100) The title compound was prepared according to General Procedure 1, starting from compound 1.1 (10 mg) and morpholine. Preparative HPLC purification was performed as described in General Procedure 9, eluting with a 20 to 60% CHCN / HO + 0.1% TFA gradient to afford the title compound as an off-white solid (TFA salt, 3.6 mg, 26% yield).

[0465] LC / MS:C 26 H 26 Calculated m / z for FN3O5 = 479.2, observed [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)piperazin-1-yl)methyl)-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (compound 102) The title compound was prepared according to general procedure 1, starting from compound 1.1 (10 mg) and 1-(phenylsulfonyl)piperazine. Preparative HPLC purification was performed as described in general procedure 9, eluting with a 20 to 60% CHCN / HO + 0.1% TFA gradient to afford the title compound as an off-white solid (TFA salt, 3.6 mg, 21% yield).

[0468] LC / MS:C 32 H 31 Calculated m / z for FN4O6 = 618.2, observed [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)piperazin-1-yl)methyl)-4-ethyl-8-fluoro-4-hydroxy-9-methyl-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (compound 104) The title compound was prepared according to general procedure 1, starting from compound 1.1 (10 mg) and 4-(piperazin-1-ylsulfonyl)aniline. Preparative HPLC purification was performed as described in general procedure 9, eluting with a 20 to 60% CHCN / HO + 0.1% TFA gradient to afford the title compound as an off-white solid (TFA salt, 4.7 mg, 27% yield).

[0471] LC / MS:C 32 H 32 Calculated m / z for FN5O6 = 633.2, found [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-methylpiperazin-1-yl)methyl)-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (compound 106) The title compound was prepared according to general procedure 1, starting from compound 1.1 (10 mg) and N-methylpiperazine. Preparative HPLC purification was performed as described in general procedure 9, eluting with a 20 to 50% CHCN / HO + 0.1% TFA gradient to afford the title compound as an off-white solid (TFA salt, 3.6 mg, 25% yield).

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

[0475] 1.7: (S)-11-((4-(4-aminophenyl)piperazin-1-yl)methyl)-4-ethyl-8-fluoro-4-hydroxy-9-methyl-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (compound 108) TIFF2025510791000119.tif58165 The title compound was prepared according to General Procedure 1 starting from compound 1.1 (10 mg) and 4-(piperazin-1-yl)aniline. Preparative HPLC purification was performed as described in General Procedure 9, eluting with a 20 to 50% CHCN / HO + 0.1% TFA gradient to afford the title compound as an off-white solid (TFA salt, 3.7 mg, 23% yield).

[0476] LC / MS:C 32 H 32 Calculated m / z for FN5O4 = 569.2, observed [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-(piperidin-1-ylmethyl)-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (compound 110) The title compound was prepared according to General Procedure 1, starting from compound 1.1 (10 mg) and piperidine. Preparative HPLC purification was performed as described in General Procedure 9, eluting with a 10 to 60% CHCN / HO + 0.1% TFA gradient to afford the title compound as an off-white solid (TFA salt, 1.5 mg, 11% yield).

[0479] LC / MS:C 27 H 28 Calculated m / z for FN3O4 = 477.2, observed [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]indolizino[1,2-b]quinolin-11-yl)methyl)piperazine-1-carboxylate (compound 111) TIFF2025510791000121.tif53165 The title compound was prepared according to general procedure 1 starting from compound 1.1 (10 mg) and tert-butyl piperazine-1-carboxylate. Preparative HPLC purification was performed as described in general procedure 9, eluting with a 10 to 60% CHCN / HO + 0.1% TFA gradient to afford the title compound as an off-white solid (TFA salt, 6.6 mg, 40% yield).

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

[0483] 1.10: (S)-4-Ethyl-8-fluoro-4-hydroxy-9-methyl-11-(piperazin-1-ylmethyl)-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (compound 112) TIFF2025510791000122.tif48165 The title compound was prepared according to general procedure 6 starting from compound 111 (5.0 mg) to afford the title compound as an off-white solid (TFA salt, 4.4 mg).

[0484] LC / MS:C 26 H 27 Calculated m / z for FN4O4 = 478.2, observed [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]indolizino[1,2-b]quinoline-3,14(4H)-dione (compound 113) The title compound was prepared according to general procedure 1, starting from compound 1.1 (10 mg) and (R)-morpholin-2-ylmethanol. Preparative HPLC purification was performed as described in general procedure 9, eluting with a 10 to 60% CHCN / HO + 0.1% TFA gradient, to afford the title compound as an off-white solid (TFA salt, 4.6 mg, 32% yield).

[0486] LC / MS:C 27 H 28 Calculated m / z for FN3O6 = 509.2, found [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]indolizino[1,2-b]quinoline-3,14(4H)-dione (compound 114) The title compound was prepared according to general procedure 1, starting from compound 1.1 (10 mg) and thiomorpholin-3-ylmethanol. Preparative HPLC purification was performed as described in general procedure 9, eluting with a 10 to 60% CHCN / HO + 0.1% TFA gradient, to afford the title compound as an off-white solid (TFA salt, 1.5 mg, 12% yield).

[0488] LC / MS:C 27 H 28 Calculated m / z for FN3O5S = 525.6, observed [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]indolizino[1,2-b]quinoline-3,14(4H)-dione (compound 115) The title compound was prepared according to general procedure 1, starting from compound 1.1 (10 mg) and 2-oxa-5-azabicyclo[2.2.1]heptan-4-ylmethanol. Preparative HPLC purification was performed as described in general procedure 9, eluting with a 10 to 60% CHCN / HO + 0.1% TFA gradient, to afford the title compound as an off-white solid (TFA salt, 3.5 mg, 29% yield).

[0491] LC / MS:C 28 H 28 Calculated m / z for FN3O6 = 521.5, observed [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]indolizino[1,2-b]quinoline-3,14(4H)-dione (compound 116) TIFF2025510791000126.tif58165 The title compound was prepared by dissolving compound 1.1 (10 mg) and 3-(hydroxymethyl)-1λ 6 Prepared according to general procedure 1 starting from -thiomorpholine-1,1-dione. Purification was carried out as described in general procedure 9, eluting with a 10 to 60% CHCN / HO + 0.1% TFA gradient to afford the title compound as an off-white solid (TFA salt, 0.2 mg, 2% yield).

[0494] LC / MS:C 27 H 28 Calculated m / z for FN3O7S = 557.6, found [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]indolizino[1,2-b]quinoline-3,14(4H)-dione (compound 117) The title compound was prepared according to general procedure 1, starting from compound 1.1 (10 mg) and 3-azabicyclo[3.1.1]heptan-6-ol. Preparative HPLC purification was performed as described in general procedure 9, eluting with a 10 to 60% CHCN / HO + 0.1% TFA gradient, to afford the title compound as an off-white solid (TFA salt, 1.3 mg, 11% yield).

[0497] LC / MS:C 28 H 28 Calculated m / z for FN3O5 = 505.5, observed [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)azetidin-1-yl)methyl)-4-hydroxy-9-methyl-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (compound 118) The title compound was prepared according to general procedure 1, starting from compound 1.1 (10 mg) and 3-fluoroazetidin-3-ylmethanol. Preparative HPLC purification was performed as described in general procedure 9, eluting with a 10 to 60% CHCN / HO + 0.1% TFA gradient, to afford the title compound as an off-white solid (TFA salt, 1.4 mg, 12% yield).

[0500] LC / MS:C 26 H 25 Calculated m / z for F2N3O5 = 497.5, observed [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)azetidin-1-yl)methyl)-9-methyl-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (compound 119) The title compound was prepared according to general procedure 1, starting from compound 1.1 (10 mg) and azetidin-3-ylmethanol. Preparative HPLC purification was performed as described in general procedure 9, eluting with a 10 to 60% CHCN / HO + 0.1% TFA gradient, to afford the title compound as an off-white solid (TFA salt, 0.5 mg, 4.5% yield).

[0503] LC / MS:C26 H 26 Calculated m / z for FN3O5 = 479.5, observed [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)piperidin-1-yl)methyl)-4-ethyl-8-fluoro-4-hydroxy-9-methyl-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (compound 120) The title compound was prepared according to general procedure 1, starting from compound 1.1 (10 mg) and 4,4-difluoropiperidin-3-ylmethanol. Preparative HPLC purification was performed as described in general procedure 9, eluting with a 10 to 60% CHCN / HO+0.1% TFA gradient to afford the title compound as an off-white solid (TFA salt, 4 mg, 32% yield).

[0506] LC / MS:C 28 H 28 Calculated m / z for F3N3O5 = 543.5, observed [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]indolizino[1,2-b]quinoline-3,14(4H)-dione (compound 121) The title compound was prepared according to general procedure 1, starting from compound 1.1 (10 mg) and 7-azabicyclo[2.2.1]heptan-1-ylmethanol. Preparative HPLC purification was performed as described in general procedure 9, eluting with a 10 to 60% CHCN / HO + 0.1% TFA gradient, to afford the title compound as an off-white solid (TFA salt, 0.8 mg, 6.6% yield).

[0509] LC / MS:C 29 H 30 Calculated m / z for FN3O5 = 519.6, observed [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]indolizino[1,2-b]quinolin-11-yl)methyl)methanesulfonamide (compound 122) The title compound was prepared according to general procedure 3, starting from 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% CHCN / HO+0.1% TFA gradient to afford the title compound as an off-white solid (0.8 mg, 7% yield).

[0512] LC / MS:C 23 H 22 Calculated m / z for FN3O6S = 487.1, observed [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]indolizino[1,2-b]quinolin-11-yl)methyl)-1-(4-nitrophenyl)methanesulfonamide (compound 124) The title compound was prepared according to general procedure 3, starting from 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% CHCN / HO+0.1% TFA gradient to afford the title compound as an off-white solid (5.0 mg, 17% yield).

[0515] LC / MS:C 29 H 25 Calculated m / z for FN4O8S = 608.1, observed [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]indolizino[1,2-b]quinolin-11-yl)methyl)benzenesulfonamide (compound 125) The title compound was prepared according to general procedure 3, starting from 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% CHCN / HO+0.1% TFA gradient to afford the title compound as an off-white solid (9.8 mg, 73% yield).

[0518] LC / MS:C 28 H 24 Calculated m / z for FN3O6S = 549.6, observed [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]indolizino[1,2-b]quinolin-11-yl)methyl)-4-nitrobenzenesulfonamide (compound 1.23) The title compound was prepared according to general procedure 3 starting from compound 1.2 (75 mg) and 4-nitrobenzenesulfonyl chloride. Purification of the title compound was carried out as described in general procedure 9 using a 12 g C18 column eluted with a 5 to 75% CH3CN / HO + 0.1% TFA gradient to afford the title compound as an off-white solid (37.8 mg, 47% yield).

[0521] LC / MS:C 28 H 23 Calculated m / z for FN4O8S = 594.6, observed [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) TIFF2025510791000136.tif63165 To a solution of compound 1.23 (37.8 mg, 0.064 mmol) in methanol (6.4 mL) was added platinum 1% vanadium 2% carbon (75 mg). The flask was purged with H2 and then stirred under an H2 atmosphere at room temperature for 45 minutes. The mixture was filtered through a pad of Celite, washed with DMF, and the filtrate was evaporated to give the title compound as a pale yellow solid (30 mg, 84% yield).

[0523] LC / MS:C 28 H 24 Calculated m / z for FN4O6S = 564.6, observed [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]indolizino[1,2-b]quinolin-11-yl)methyl)-2-hydroxyethane-1-sulfonamide (compound 129) The title compound was prepared according to general procedure 3, starting from compound 1.2 (20 mg) and 2-hydroxyethanesulfonyl chloride. Preparative HPLC purification was performed as described in general procedure 9, eluting with a 25 to 50% CHCN / HO+0.1% TFA gradient to afford the title compound as an off-white solid (1.3 mg, 13% yield).

[0526] LC / MS:C 24 H 24 Calculated m / z for FN3O7S = 517.1, observed [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)methanesulfamide (Compound 131) TIFF2025510791000138.tif53165 To a solution of chlorosulfonyl isocyanate (3 uL) in dichloromethane (1 mL) was added tert-butanol (3 uL). This solution was stirred for 1 hour, after which compound 1.2 (13 mg) dissolved in dichloromethane (1 mL) was added, followed by triethylamine (13 uL). The reaction was stirred for 1 hour and then concentrated to dryness. Preparative HPLC purification of the intermediate Boc compound was performed as described in General Procedure 9, eluting with a 10-50% CH3CN / HO + 0.1% TFA gradient. To the purified solid in dichloromethane (1 mL) was added trifluoroacetic acid (200 uL). The reaction was stirred for 16 hours and then concentrated to dryness to give the title compound as an off-white solid (7.5 mg, 48% yield).

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

[0530] 1 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]indolizino[1,2-b]quinolin-11-yl)methyl)carbamate (compound 1.27) The title PNP-carbamate intermediate compound was prepared according to the first step of General Procedure 4, starting from compound 1.2 (24 mg). Purification was carried out as described in General Procedure 9 using a 12 g C18 column eluted with a 10 to 50% CHCN / HO + 0.1% TFA gradient to afford the title compound as an off-white solid (14 mg, 53% yield).

[0532] LC / MS:C 29 H 23 Calculated m / z for FN4O8S = 574.2, observed [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]indolizino[1,2-b]quinolin-11-yl)methyl)-3-methylurea (compound 132) The title compound was prepared according to general procedure 4, starting with compound 1.2 (25 mg) and aqueous methylamine (500 μL, 40 wt % in water) as the primary amine. In this case, the intermediate PNP-carbamate was used crude. Preparative HPLC purification was performed as described in general procedure 9, eluting with a 10 to 50% CHCN / HO + 0.1% TFA gradient, to afford the title compound as an off-white solid (8.9 mg, 31% yield).

[0534] LC / MS:C 24 H 23 Calculated m / z for FN4O5 = 466.2, observed [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]indolizino[1,2-b]quinolin-11-yl)methyl)urea (compound 134) The title compound 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 as described in General Procedure 9, eluting with a 20 to 50% CHCN / HO+0.1% TFA gradient to afford the title compound as an off-white solid (0.6 mg, 12% yield).

[0537] LC / MS:C 30 H 28 Calculated m / z for FN5O5 = 557.2, found [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]indolizino[1,2-b]quinolin-11-yl)methyl)-3-(2-hydroxyethyl)urea (compound 136) The title compound 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 as described in General Procedure 9, eluting with a 10 to 50% CHCN / HO + 0.1% TFA gradient to afford the title compound as an off-white solid (2.4 mg, 66% yield).

[0540] LC / MS:C 25 H 25 Calculated m / z for FN4O6 = 496.2, observed [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]indolizino[1,2-b]quinolin-11-yl)methyl)carbamate (compound 138) The title compound was prepared according to general procedure 5, starting from compound 1.2 (50 mg) and reacting methanol with the intermediate PNP-carbamate. Preparative HPLC purification was performed as described in general procedure 9, eluting with a 20 to 50% CHCN / HO + 0.1% TFA gradient to afford the title compound as an off-white solid (3.5 mg, 6% yield).

[0543] LC / MS:C 24 H 22 Calculated m / z for FN3O6 = 467.2, observed [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]indolizino[1,2-b]quinolin-11-yl)methyl)carbamate (compound 139) The title compound was prepared according to general procedure 5, starting from compound 1.2 (18 mg) by reacting 1,2-ethanediol with the intermediate PNP-carbamate. Preparative HPLC purification was performed as described in general procedure 9, eluting with a 10 to 60% CHCN / HO + 0.1% TFA gradient to afford the title compound as an off-white solid (4.2 mg, 19% yield).

[0546] LC / MS:C 25 H 24 Calculated m / z for FN3O7 = 497.2, observed [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 camptothecin analogs with methoxy at C10 position 2.1: 1-(2-amino-4-fluoro-5-methoxyphenyl)-2-chloroethan-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 was added first 1 M BCl in DCM (71 mL, 71 mmol), followed by 1 M chloro(diethyl)almane in DCM (71 mL, 71 mmol), and finally 2-chloroacetonitrile (6.4 g, 85 mmol). The solution was heated at reflux for 3 h, cooled to room temperature, and quenched by the addition of 2 M aqueous HCl. The resulting heterogeneous mixture was heated at reflux for 1 h, cooled to room temperature, and then the pH was adjusted to approximately 12 with NaCO. 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 flash purified as described in general procedure 9, eluting with 0 to 20% EtOAc / hexanes, to give the title compound (6 g, 28 mmol, 39% yield).

[0549] LC / MS: m / z calculated for C9H9ClFNO2 = 217.1, found [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]indolizino[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]indolizine-3,6,10(4H)-trione (2 g, 7.6 mmol) in toluene (200 mL) was added toluene-4-sulfonic acid (157 mg, 0.9 mmol). The solution was heated at 140° C. for 3 hours and then cooled to room temperature. The product was collected by filtration as a yellow precipitate to give the title compound (1.27 g, 2.85 mmol, 37.5% yield).

[0552] LC / MS:C 22 H 18 Calculated m / z for ClFN2O5 = 445.2, found [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]indolizino[1,2-b]quinoline-3,14(4H)-dione (compound 101) The title compound was prepared according to General Procedure 1, starting from compound 2.2 (10 mg) and morpholine. Preparative HPLC purification was performed as described in General Procedure 9, eluting with a 20 to 60% CHCN / HO+0.1% TFA gradient to afford the title compound as an off-white solid (5.6 mg, 41% yield).

[0555] LC / MS:C26 H 26 Calculated m / z for FN3O6 = 495.2, observed [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]indolizino[1,2-b]quinoline-3,14(4H)-dione (compound 103) The title compound was prepared according to general procedure 1, starting from compound 2.2 (10 mg) and 1-(phenylsulfonyl)piperazine. Preparative HPLC purification was performed as described in general procedure 9, eluting with a 20 to 60% CHCN / HO+0.1% TFA gradient to afford the title compound as an off-white solid (2.5 mg, 14% yield).

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

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

[0560] LC / MS:C 32 H 32 Calculated m / z for FN5O7S = 649.2, observed [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-methylpiperazin-1-yl)methyl)-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (compound 107) The title compound was prepared according to general procedure 1, starting from compound 2.2 (10 mg) and N-methylpiperazine. Preparative HPLC purification was performed as described in general procedure 9, eluting with a 20 to 60% CHCN / HO+0.1% TFA gradient to afford the title compound as an off-white solid (2.1 mg, 19% yield).

[0563] LC / MS:C27 H 29 Calculated m / z for FN4O5 = 508.2, observed [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]indolizino[1,2-b]quinoline-3,14(4H)-dione (compound 109) The title compound was prepared according to general procedure 1, starting from compound 2.2 (10 mg) and 4-(piperazin-1-yl)aniline. Preparative HPLC purification was performed as described in general procedure 9, eluting with a 20 to 60% CHCN / HO+0.1% TFA gradient to afford the title compound as an off-white solid (3.2 mg, 20% yield).

[0565] LC / MS:C 32 H 32 Calculated m / z for FN5O5 = 585.2, observed [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]indolizino[1,2-b]quinoline-3,14(4H)-dione (compound 2.8) To a solution of compound 2.2 (250 mg, 0.56 mmol) in ethanol (7 mL) was added hexamethylenetetramine (236 mg, 1.7 mmol), followed by iPrNEt (100 μL, 0.56 mmol). The solution was heated to reflux for 5 h, cooled to room temperature, and quenched with 12 M aqueous HCl (60 μL). The solution was concentrated to approximately half its volume, and 1 M aqueous HCl (1.5 mL) was added, stirred for 5 min, and then concentrated to give a brown residue. Purification was carried out as described in General Procedure 9 using a 12 g C18 flash column eluting with a 5 to 40% CH3CN / HO + 0.1% TFA gradient to give the title compound as a pale yellow solid (179 mg, 75% yield).

[0568] LC / MS:C 22 H 20 Calculated m / z for FN3O5 = 425.4, observed [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]indolizino[1,2-b]quinolin-11-yl)methyl)methanesulfonamide (compound 123) The title compound was prepared according to general procedure 3, starting from 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% CHCN / HO + 0.1% TFA gradient to afford the title compound as an off-white solid (8.5 mg, 91% yield).

[0570] LC / MS:C 23 H 22 Calculated m / z for FN3O7S = 503.1, observed [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]indolizino[1,2-b]quinolin-11-yl)methyl)benzenesulfonamide (compound 126) The title compound was prepared according to general procedure 3, starting from 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% CHCN / HO + 0.1% TFA gradient to afford the title compound as an off-white solid (4.6 mg, 46% yield).

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

[0574] 1H 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-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)methyl)-4-nitrobenzenesulfonamide (compound 2.11) The title compound was prepared according to general procedure 3 starting from compound 2.8 (12 mg) and 4-nitrobenzenesulfonyl chloride. Purification was carried out as described in general procedure 9 using a 12 g C18 flash column eluted with a 5 to 75% CH3CN / HO + 0.1% TFA gradient to afford the title compound as a pale yellow solid (9.7 mg, 71% yield).

[0576] LC / MS:C 28 H 23 Calculated m / z for FN4O9S = 610.6, observed [M+H] + =611.5.

[0577] 2.12: (S)-4-amino-N-((4-ethyl-8-fluoro-4-hydroxy-9-methoxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)methyl)benzenesulfonamide (compound 128) TIFF2025510791000156.tif63165 To a solution of compound 2.11 (9.7 mg, 0.016 mmol) in methanol (1.6 mL) was added platinum 1% vanadium 2% carbon (15 mg). The flask was purged with H2 and then stirred under an H2 atmosphere at room temperature for 45 minutes. The mixture was filtered through a pad of Celite, washed with DMF, and the filtrate was evaporated to give the title compound as a pale yellow solid (1.5 mg, 16% yield).

[0578] LC / MS:C 28 H 25 Calculated m / z for FN4O7S = 580.6, observed [M+H] + =581.4.

[0579] 1 H NMR(300 MHz,MeOD)δ 7.77(d,J=11.0 Hz,1H),7.58(s,1H),7.48(d,J=8.6 Hz,1H),6.61(d,J=8.6 Hz,1H),5.59(d,J=16.3 Hz,1H),5.39(d,J=16.4 Hz,1H),5.30(s,1H),4.56(s,1H),4.10(d,J=3.7 Hz,3H),2.04-1.91(m,2H),1.31(s,1H),1.02(t,J=7.3 Hz,3H),0.90(s,1H).

[0580] 2.13: (S)-N-((4-ethyl-8-fluoro-4-hydroxy-9-methoxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)methyl)-2-hydroxyethane-1-sulfonamide (compound 130) The title compound was prepared according to general procedure 3, starting from compound 2.8 (8 mg) and 2-hydroxyethanesulfonyl chloride. Preparative HPLC purification was performed as described in general procedure 9, eluting with a 15 to 50% CHCN / HO + 0.1% TFA gradient to afford the title compound as an off-white solid (2.2 mg, 22% yield).

[0581] LC / MS:C 24 H 24 Calculated m / z for FN3O8S = 533.1, observed [M+H] + =534.2.

[0582] 1 H NMR(300 MHz,DMSO-d6)δ 7.99(d,J=12.2 Hz,1H),7.89-7.79(m,2H),7.29(s,1H),5.43(s,2H),5.40(s,2H),4.76(d,J=6.4 Hz,2H),4.06(s,3H),3.81(t,J=6.3 Hz,2H),3.34(t,J=6.3 Hz,2H),1.94-1.75(m,2H),0.87(d,J=7.4 Hz,3H).

[0583] 2.14: 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]indolizino[1,2-b]quinolin-11-yl)methyl)carbamate (compound 2.14) The title PNP-carbamate intermediate compound was prepared according to the first step of General Procedure 4, starting with compound 2.8 (65 mg) and using a 1:1 mixture of dimethylformamide and dichloromethane as the solvent. Flash purification was performed as described in General Procedure 9 using a 12 g C12 column eluted with a 10 to 50% CH3CN / HO + 0.1% TFA gradient to afford the title compound as an off-white solid (61 mg, 86% yield). This intermediate was resolved and used to generate the next compound.

[0584] LC / MS:C 29 H 23 Calculated m / z for FN4O9 = 590.1, observed [M+H] + =591.2.

[0585] 2.15: (S)-1-((4-ethyl-8-fluoro-4-hydroxy-9-methoxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)methyl)-3-methylurea (compound 133) The title compound was prepared according to the second step of General Procedure 4 using compound 2.14 (15 mg) as the PNP-carbamate and aqueous methylamine (500 uL, 40 wt% in water) as the primary amine. Preparative HPLC purification was performed as described in General Procedure 9, eluting with a 20 to 60% CHCN / HO + 0.1% TFA gradient to afford the title compound as an off-white solid (5.8 mg, 47% yield).

[0586] LC / MS:C 24 H 23 Calculated m / z for FN4O6 = 482.2, observed [M+H] + =483.2.

[0587] 1 H NMR(300 MHz,DMSO-d6)δ 8.00-7.87(m,2H),7.31(s,1H),5.48-5.39(m,3H),4.81(s,3H),2.56(s,3H),1.93-1.81(m,2H),0.89(t,J=7.3 Hz,3H).

[0588] 2.16: (S)-1-(4-aminobenzyl)-3-((4-ethyl-8-fluoro-4-hydroxy-9-methoxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)methyl)urea (compound 135) The title compound was prepared according to the second step of General Procedure 4 using compound 2.14 (15 mg) as the PNP-carbamate and 4-(aminomethyl)aniline as the primary amine. Preparative HPLC purification was performed as described in General Procedure 9, eluting with a 20 to 60% CHCN / HO + 0.1% TFA gradient to afford the title compound as an off-white solid (TFA salt, 2.1 mg, 12% yield).

[0589] LC / MS:C 30 H 28 Calculated m / z for FN5O6 = 573.2, found [M+H] + =574.2.

[0590] 1 H NMR(300 MHz,MeOD)δ 7.79(d,J=11.9 Hz,1H),7.74(d,J=9.0 Hz,1H),7.59(s,1H),7.43(d,J=8.2 Hz,2H),7.25(d,J=8.2 Hz,2H),5.61(d,J=16.3 Hz,1H),5.52-5.35(m,3H),4.98(s,2H),4.39(s,2H),4.01(s,3H),2.03-1.93(m,2H),1.03(t,J=7.4 Hz,3H).

[0591] 2.17: (S)-1-((4-ethyl-8-fluoro-4-hydroxy-9-methoxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)methyl)-3-(2-hydroxyethyl)urea (compound 137) The title compound was prepared according to the second step of General Procedure 4 using compound 2.14 (15 mg) as the PNP-carbamate and hydroxyethylamine as the primary amine. Preparative HPLC purification was performed as described in General Procedure 9, eluting with a 20 to 60% CHCN / HO + 0.1% TFA gradient to afford the title compound as an off-white solid (1.5 mg, 12% yield).

[0592] LC / MS:C 25 H 25 Calculated m / z for FN4O7 = 512.2, observed [M+H] + =513.2.

[0593] 1 H NMR(300 MHz,MeOD)δ 7.93(d,J=12.1 Hz,1H),7.88(d,J=9.2 Hz,1H),7.56(s,1H),5.62(d,J=16.2 Hz,1H),5.52(s,2H),5.45(d,J=16.3 Hz,1H),4.98(s,2H),4.17(s,3H),3.59(t,J=5.6 Hz,2H),3.28(t,J=5.6 Hz,2H),2.10-1.91(m,2H),1.05(t,J=7.3 Hz,3H).

[0594] Example 3: Preparation of camptothecin analogs with an amino at the C10 position 3.1: 5-Bromo-4-fluoro-2-nitrobenzaldehyde (compound 3.1) TIFF2025510791000162.tif2716To a stirred solution of HNO3 (121.2 mL, 67% purity, 2.0 equiv.) in H2SO4 (500 mL) at 50 °C was added 3-bromo-4-fluorobenzaldehyde (180 g, 1.0 equiv.). After the addition was complete, the ice bath was removed and the reaction was stirred at 25 °C for 5 h. The mixture was poured onto ice (5 L), filtered, and then dried under reduced pressure. The title compound was obtained as a yellow solid (219 g).

[0595] 1H NMR(400 MHz,CDCl3)δ 10.39(s,1H),8.23(d,J=6.8 Hz,1H),7.91(d,J=7.6 Hz,1H).

[0596] 3.2: tert-Butyl (2-fluoro-5-formyl-4-nitrophenyl)carbamate (compound 3.2) A mixture of compound 3.1 (219 g, 1.0 equiv.), tert-butyl carbamate (124 g, 1.2 equiv.), CsCO (575 g, 2.0 equiv.), Pd(dba) (40 g, 0.05 equiv.), and XPhos (84 g, 0.2 equiv.) in toluene (2000 mL) was degassed and purged with N for three cycles. The mixture was then stirred at 90 °C under a N atmosphere for 15 h. The reaction mixture was diluted with H2O (800 mL) and extracted with EtOAc (300 mL × 2). The combined organic layers were washed with brine (200 mL × 2), dried over sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (SiO2, petroleum ether:ethyl acetate = 100:1 to 20:1) to give the title compound as a yellow solid (140 g, 56% yield).

[0597] 1 H NMR(400 MHz,DMSO-d6)δ 10.24(s,1H),9.94(s,1H),8.42(d,J=7.6 Hz,1H),8.16(d,J=10.8 Hz,1H),1.50(s,9H)

[0598] 3.3: tert-Butyl (4-amino-2-fluoro-5-formylphenyl)carbamate (compound 3.3) TIFF2025510791000164.tif27165To a solution of compound 3.2 (100 g, 1.0 equiv.) in HO (300 mL) and EtOH (1200 mL) was added NH4Cl (30.5 g, 1.62 equiv.). Iron (78.6 g, 4.0 equiv.) was added portionwise at 80 °C. The mixture was stirred at 80 °C for 6 h. The mixture was filtered, water was added to the filtrate, and the resulting mixture was extracted with ethyl acetate. The organic layer was washed with brine, dried over sodium sulfate, and concentrated under reduced pressure. The residue was purified by column chromatography (SiO2, petroleum ether:ethyl acetate = 1:0 to 0:1) and TLC (petroleum ether) to give the title compound as a yellow solid (19.0 g, 21% yield).

[0599] LC / MS:C 12 H 15 Calculated m / z for FN2O3 = 254.1, observed [M+H] + =255.0.

[0600] 1 H NMR(400 MHz,DMSO-d6)δ 9.73(s,1 H),8.57(s,1 H),7.58(d,J=4.8 Hz,1 H),7.21(s,2 H),6.53(d,J=12.8 Hz,1 H),1.43(s,9 H).

[0601] 3.4: tert-Butyl (S)-(4-ethyl-8-fluoro-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl)carbamate (compound 3.4) A mixture of compound 3.3 (4.20 g, 1.2 equiv.), (S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-f]indolizine-3,6,10(4H)-trione (3.5 g, 1 equiv.), and TsOH (monohydrate, 253 mg, 0.1 equiv.) in toluene (350 mL) was stirred at 110 °C for 2 h. The reaction solution was cooled to 25 °C, and the solid was washed with methyl t-butyl ether (30 mL) and then dried under reduced pressure. The title compound was obtained as a yellow solid (4.5 g, 62% yield).

[0602] LC / MS:C 25 H 24 Calculated m / z for FN3O6 = 481.2, observed [M+H] + =482.1.

[0603] 1 H NMR(400 MHz,DMSO-d6)δ 9.49(s,1H),8.65(s,1H),8.43(d,J =8.4 Hz,1H),7.95(d,J=12.0 Hz,1H),7.30(s,1H),6.51(s,1H),5.42(s,2H),5.25(s,2H),1.80-1.92(m,2H),1.52(s,9H),0.88(t,J=7.2 Hz,3H)

[0604] 3.5: tert-Butyl (S)-(4-ethyl-8-fluoro-4-hydroxy-11-(hydroxymethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl)carbamate (compound 3.5) TIFF2025510791000166.tif42165To a mixture of compound 3.4 (4.00 g) in MeOH (360 mL), a solution of FeSO4 (heptahydrate, 1.2 g) in HO (4 mL) and H2SO4 (280 mL) was added. The reaction mixture was heated to 65 °C while HO2 (24 mL, 30% purity) was added dropwise over 30 min and then stirred for 0.5 h. The reaction solution was cooled to 25 °C and filtered to give the title compound as a yellow solid (1.53 g, 33.2% yield). HO (400 mL) was added to the filtrate, which was then quenched with saturated aqueous Na2SO3. The pH was adjusted to 7-8 with saturated aqueous Na2CO3, and the solution was concentrated and filtered. The solid was triturated with MeOH (30 mL) at 55° C. for 1 h and then filtered to give a second batch of the title compound as a brown solid (1.09 g, 26% yield).

[0605] LC / MS:C 26 H 26 Calculated m / z for FN3O7 = 511.2, observed [M+H] + =512.2.

[0606] 1 H NMR(300 MHz,d6-DMSO)δ 9.47(s,1H),8.47(d,J =7.6 Hz,1H),7.94(d,J =12.0 Hz,1H),7.29(d,J =1.6 Hz,1H),6.49(s,1H),5.86-5.76(m,1H),5.42(s,2H),5.38(s,2H),5.16(d,J =4.4 Hz,2H),1.90-1.83(m,2H),1.52(s,9H),0.88(t,J=6.4 Hz,3H).

[0607] 3.6: tert-Butyl (S)-(4-ethyl-8-fluoro-11-formyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl)carbamate (compound 3.6) To a 50 mL round-bottom flask containing compound 3.5 (150 mg, 0.293 mmol), DCM (2.9 mL) was added, followed by Dess-Martin periodinane (0.56 g, 1.32 mmol) and water (15.8 μL, 0.88 mmol). The solution was stirred at room temperature for 18 h, then diluted with DCM and washed with saturated aqueous NaHCO3 and brine. The layers were separated, and the combined organic layer was evaporated onto Celite. Flash purification was performed as described in General Procedure 9 using a 10 g silica column eluting with 0 to 10% DCM / MeOH to afford the title product as an orange powder (42.5 mg, 28%).

[0608] LC / MS:C 26 H 24 Calculated m / z for FN3O7 = 509.2, found [M+H] + =510.4.

[0609] 1 H NMR(300 MHz,acetone-d6)δ 11.10(s,1H),9.68(d,J =8.6 Hz,1H),8.81(s,1H),8.04(d,J =11.9 Hz,1H),7.63(s,1H),5.73(s,2H),5.69(d,J =16.2 Hz,1H),5.42(d,J =16.2 Hz,1H),2.02-1.95(m,2H),8.47(d,J =7.6 Hz,1H),7.94(d,J =12.0 Hz,1H),7.29(d,J =1.6 Hz,1H),6.49(s,1H),5.86-5.76(m,1H),5.42(s,2H),5.38(s,2H),5.16(d,J =4.4 Hz,2H),1.90-1.83(m,2H),1.52(s,9H),0.88(t,J=6.4 Hz,3H).

[0610] 3.7: (S)-9-Amino-4-ethyl-8-fluoro-4-hydroxy-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (compound 140) TIFF2025510791000168.tif37165 The title compound was prepared according to general procedure 6 starting from compound 3.4 (40 mg) to afford the title compound as a red solid (TFA salt, 36 mg, 87% yield).

[0611] LC / MS:C 20 H 16 Calculated m / z for FN3O4 = 381.1, observed [M+H] + =382.2.

[0612] 1 H NMR(300 MHz,DMSO)δ 8.28(s,1H),7.72(d,J=12.5 Hz,1H),7.21(d,J=7.3 Hz,1H),5.43(d,J=16.2 Hz,1H),5.34(d,J=16.2 Hz,1H),5.17(s,2H),1.92-1.74(m,2H),0.88(t,J=7.3 Hz,3H).

[0613] 3.8: (S)-9-Amino-4-ethyl-8-fluoro-4-hydroxy-11-(hydroxymethyl)-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (compound 141) TIFF2025510791000169.tif42165 The title compound was prepared according to general procedure 6 starting from compound 3.5 (5 mg) to afford the title compound as a red solid (TFA salt, 4.1 mg, 78% yield).

[0614] LC / MS:C 21 H 18 Calculated m / z for FN3O5 = 411.2, observed [M+H] + =412.2.

[0615] 1H NMR(300 MHz,MeOD)δ 7.71(d,J=12.2 Hz,1H),7.60(s,1H),7.29(d,J=9.5 Hz,1H),5.61(d,J=16.3 Hz,1H),5.47(s,2H),5.40(d,J=16.3 Hz,1H),5.25(s,2H),2.03-1.94(m,2H),1.03(t,J=7.4 Hz,3H).

[0616] 3.9: tert-Butyl (S)-(11-(chloromethyl)-4-ethyl-8-fluoro-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl)carbamate (compound 3.9) To a stirred solution of compound 3.5 (100 mg) in dichloromethane (5 mL) was added a solution of thionyl chloride (14 μL) in dichloromethane (0.1 mL). After 1 hour, additional thionyl chloride (14 μL) in dichloromethane (0.1 mL) was added. After an additional hour, the reaction was diluted with dichloromethane (10 mL) and toluene (1 mL) and then concentrated in vacuo to give the title compound as a red solid, which was used in the next reaction without further purification.

[0617] LC / MS:C 26 H 25 Calculated m / z for ClFN3O6 = 529.1, found [M+H] + =530.2.

[0618] 3.10: tert-Butyl (S)-(11-(aminomethyl)-4-ethyl-8-fluoro-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl)carbamate (compound 3.10) TIFF2025510791000171.tif42165 To compound 3.9 (100 mg) in ethanol (500 μL) was added hexamethylenetetramine (79 mg), followed by DIPEA (99 μL). The solution was heated at 60° C. for 16 hours and then concentrated to dryness in vacuo. Flash purification was performed as described in General Procedure 9 using a 12 g C18 column eluted with a 10-50% CH3CN / HO + 0.1% TFA gradient to afford the title compound as an off-white solid (TFA salt, 29 mg, 24% yield).

[0619] LC / MS:C 26 H 27 Calculated m / z for FN4O6 = 510.2, observed [M+H] + =511.4.

[0620] 1 H NMR(300 MHz,MeOD)δ 8.88(d,J=8.2 Hz,1H),7.96(d,J=11.9 Hz,1H),7.62(s,1H),5.60(d,J=16.4 Hz,1H),5.48(s,2H),5.41(d,J=16.4 Hz,1H),4.80(s,2H),2.07-1.89(m,2H),1.64(s,9H),1.02(t,J=7.3 Hz,3H).

[0621] 3.11: (S)-9-Amino-11-(aminomethyl)-4-ethyl-8-fluoro-4-hydroxy-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (compound 145) TIFF2025510791000172.tif53165 The title compound was prepared according to general procedure 6 starting from compound 3.10 (2.1 mg) to afford the title compound as a red solid (TFA salt, 1.8 mg, 100% yield).

[0622] LC / MS:C 21 H 19 Calculated m / z for FN4O4 = 410.1, found [M+H] + =411.2.

[0623] 1 H NMR(300 MHz,MeOD)δ 7.82(d,J=12.1 Hz,1H),7.60(s,1H),7.37(d,J=9.1 Hz,1H),5.61(d,J=16.3 Hz,1H),5.42(s,2H),5.41(d,J=16.3 Hz,1H),4.69(s,2H),2.08-1.94(m,2H),1.03(t,J=7.4 Hz,3H).

[0624] Example 3.12: (S)-9-Amino-4-ethyl-8-fluoro-4-hydroxy-11-(morpholinomethyl)-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (Compound 3.12) The title compound was prepared according to General Procedure 1, starting from compound 3.9 (150 mg) and morpholine. Preparative HPLC purification was performed as described in General Procedure 9, eluting with a 10 to 60% CHCN / HO + 0.1% TFA gradient to afford the title compound as a red solid (TFA salt, 103 mg, 52% yield).

[0625] LC / MS:C 30 H 33 Calculated m / z for FN4O7 = 580.2, observed [M+H] + =581.4.

[0626] 1 H NMR(300 MHz,MeOD)δ 9.06(d,J=8.3 Hz,1H),7.93(d,J=12.0 Hz,1H),7.66(s,1H),5.63(d,J=16.3 Hz,1H),5.51(s,2H),5.43(d,J=16.4 Hz,1H),4.92(s,2H),3.84(s,4H),3.10(s,4H),1.99(d,J=5.5 Hz,2H),1.63(s,9H),1.03(t,J=7.4 Hz,3H).

[0627] 3.13: (S)-9-Amino-4-ethyl-8-fluoro-4-hydroxy-11-(morpholinomethyl)-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (compound 142) TIFF2025510791000174.tif53165 The title compound was prepared according to general procedure 6 starting from compound 3.12 (45 mg) to afford the title compound as a red solid (TFA salt, 37 mg, 99% yield).

[0628] LC / MS:C 25 H 25 Calculated m / z for FN4O5 = 480.2, observed [M+H] + =481.4.

[0629] 1 H NMR(300 MHz,MeOD)δ 7.73(d,J=12.0 Hz,1H),7.54(s,1H),7.48(d,J=9.2 Hz,1H),5.60(d,J=16.3 Hz,1H),5.47-5.34(m,3H),4.65(s,2H),3.91-3.85(m,4H),3.30-3.24(m,4H),2.08-1.91(m,2H),1.02(t,J=7.3 Hz,3H).

[0630] 3.14: (S)-9-Amino-4-ethyl-8-fluoro-4-hydroxy-11-(piperidin-1-ylmethyl)-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (compound 148) To a 5 mL flask containing compound 3.6 (37 mg, 0.067 mmol), dichloromethane (1.45 mL) was added, followed by acetic acid (18.69 μL, 0.327 mmol), piperidine (21.52 μL, 0.218 mmol), and sodium triacetoxyborohydride (23.0 mg, 0.109 mmol). The solution was then stirred at room temperature for 2 h, quenched by the addition of water + 0.1% TFA and DMF (1:1, 1.0 mL), and partially evaporated. Purification was carried out as described in General Procedure 9 using a 12 g C18 flash column eluted with a 5 to 40% CH3CN / HO + 0.1% TFA gradient to give the Boc-protected intermediate as a yellow powder. This intermediate was then deprotected according to general procedure 6 to give the title compound as a yellow solid (TFA salt, 32.5 mg, 98% yield).

[0631] LC / MS:C 26 H 27 Calculated m / z for FN4O4 = 478.2, observed [M+H] + =479.4.

[0632] 1 H NMR(300 MHz,MeOD)δ 7.78(d,J=12.1 Hz,1H),7.56(s,1H),7.41(d,J=9.1 Hz,1H),5.60(d,J=16.4 Hz,1H),5.47-5.35(m,3H),4.86(s,2H),3.80-3.68(m,2H),3.28-3.19(m,2H),2.02-1.68(m,8H),1.01(t,J=7.4 Hz,3H).

[0633] 3.15: (S)-9-Amino-4-ethyl-8-fluoro-4-hydroxy-11-((4-methylpiperazin-1-yl)methyl)-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (compound 149) To a 2 mL vial containing compound 3.6 (15 mg, 0.029 mmol) was added dichloromethane (0.59 mL), acetic acid (7.58 μL, 0.132 mmol), and N-methylpiperazine (4.90 μL, 0.044 mmol). The solution was stirred at room temperature for 4 h, followed by the addition of sodium triacetoxyborohydride (7.8 mg, 0.037 mmol) and stirring for an additional 45 min. The excess hydride was then quenched by the addition of 0.1% aqueous TFA (0.5 mL). Purification was carried out as described in General Procedure 9 using a 12 g C18 flash column eluted with a 5 to 40% CH3CN / HO + 0.1% TFA gradient to give the Boc-protected intermediate as a yellow powder. This intermediate was deprotected according to general procedure 6 to give the title product as a yellow solid (TFA salt, 1.5 mg, 7.1% yield).

[0634] LC / MS:C 26 H 28 Calculated m / z for FN5O4 = 493.2, observed [M+H] + =494.4.

[0635] 1 H NMR(300 MHz,MeOD)δ 7.68(d,J=12.2 Hz,1H),7.56(s,1H),7.53(d,J=9.5 Hz,1H),5.60(d,J=16.3 Hz,1H),5.45-5.30(m,3H),4.15(s,2H),3.55-3.44(m,2H),3.18-3.07( m,2H),2.93(s,3H),2.70-2.51(m,2H),2.03-1.89(m,2H),1.02(t,J=7.4 Hz,3H).

[0636] 3.16: (S)-9-Amino-4-ethyl-8-fluoro-4-hydroxy-11-((4-(phenylsulfonyl)piperazin-1-yl)methyl)-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (compound 153) The Boc-protected precursor of the title compound was prepared according to General Procedure 1, starting from compound 3.9 (10 mg) and 1-(phenylsulfonyl)piperazine. Preparative HPLC was performed as described in General Procedure 9, eluting with a 35 to 44% CHCN / HO + 0.1% TFA gradient, to afford the Boc-protected intermediate as a yellow powder. This intermediate was then deprotected according to General Procedure 6 to afford the title compound (TFA salt, 2.4 mg, 17% yield over two steps).

[0637] LC / MS:C 31 H 30 Calculated m / z for FN5O6S = 619.2, observed [M+H] + =520.4.

[0638] 1 H NMR(300 MHz,MeOD)δ 7.81-7.60(m,7H),7.34(s,1H),5.51(d,J=16.4 Hz,1H),5.35(d,J=16.4 Hz,1H),5.22(s,2H),4.10(s,2H),3.15-3.02(m,4H),2.79-2.71(m,4H),2.00-1.93(m,2H),1.00(t,J=7.4 Hz,3H).

[0639] 3.17: (S)—N-((9-amino-4-ethyl-8-fluoro-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)methyl)acetamide (compound 147) The title compound was prepared according to General Procedure 2 followed by General Procedure 6 starting from compound 3.10 (8 mg) and acetic acid. Preparative HPLC purification of the intermediate Boc-protected compound was performed as described in General Procedure 9, eluting with a 10 to 60% CHCN / HO + 0.1% TFA gradient. The title compound was obtained as a red solid (4.0 mg, 56% yield).

[0640] LC / MS:C 23 H 21 Calculated m / z for FN4O5 = 452.2, observed [M+H] + =453.2.

[0641] 1 H NMR(300 MHz,MeOD)δ 7.69(d,J=12.1 Hz,1H),7.56(s,1H),7.38(d,J=9.3 Hz,1H),5.59(d,J=16.3 Hz,1H),5.44-5.33(m,3H),4.85(s,3H),2.03(s,3H),2.00-1.84(m,2H),1.03(t,J=7.4 Hz,3H).

[0642] 3.18: (S)—N-((9-amino-4-ethyl-8-fluoro-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-11-yl)methyl)methanesulfonamide (compound 146) The title compound was prepared according to general procedure 3 followed by general procedure 6 starting from compound 3.10 (8 mg) and methanesulfonyl chloride. Preparative HPLC purification of the intermediate Boc-protected compound was performed as described in general procedure 9, eluting with a 10 to 60% CHCN / HO + 0.1% TFA gradient. The title compound was obtained as a red solid (4.4 mg, 57% yield).

[0643] LC / MS:C 22 H 21 Calculated m / z for FN4O6S = 488.1, observed [M+H] + =489.2.

[0644] 1H NMR(300 MHz,MeOD)δ 7.74(d,J=12.2 Hz,1H),7.60(s,1H),7.49(d,J=9.3 Hz,1H),5.61(d,J=16.2 Hz,1H),5.45(s,2H),5.40(d,J=16.2 Hz,1H),4.78(s,2H),3.05(s,3H),2.08-1.94(m,2H),1.03(t,J=7.4 Hz,3H).

[0645] 3.19: (S)—N-((9-amino-4-ethyl-8-fluoro-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-11-yl)methyl)-2-hydroxyethane-1-sulfonamide (compound 150) The title compound was prepared according to General Procedure 3, followed by General Procedure 6, starting from compound 3.10 (6 mg) and 2-hydroxyethanesulfonyl chloride. Preparative HPLC purification of the intermediate Boc-protected compound was performed as described in General Procedure 9, eluting with a 10 to 60% CHCN / HO + 0.1% TFA gradient. The title compound was obtained as a red solid (1 mg, 16% yield).

[0646] LC / MS:C 23 H 23 Calculated m / z for FN4O7S = 518.5, observed [M+H] + =519.5.

[0647] 1 H NMR(300 MHz,10% D2O / CD3CN)δ 7.77-7.61(m,1H),7.48-7.30(m,2H),5.53(d,J=16.3 Hz,1H),5.31(d,J=15.4 Hz,3H),4.69(s,2H),3.97(dd,J=6.6,4.9 Hz,2H),3.39(t,J=5.8 Hz,2H),2.93(s,1H),1.99-1.83(m,2H),0.94(t,J=7.3 Hz,3H).

[0648] 3.20: 4-Nitrophenyl (S)-((9-amino-4-ethyl-8-fluoro-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)methyl)carbamate (compound 3.20) To a solution of compound 3.10 (10 mg, 0.02 mmol) in DMF (400 μL, 0.05 M) was added 4-nitrophenyl carbonate (12 mg, 0.04 mmol) and diisopropylethylamine (6.8 μL, 0.04 mmol). The solution was stirred at room temperature for approximately 30 minutes and then used directly in the next reaction.

[0649] 3.21: Methyl (S)-((9-amino-4-ethyl-8-fluoro-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)methyl)carbamate (compound 143) The title compound was prepared by adding MeOH (100 uL) to a solution of 200 uL of compound 3.20. The solution was stirred at room temperature for 30 minutes. Preparative HPLC purification of the intermediate Boc-protected compound was performed as described in General Procedure 9, eluting with a 10-60% CHCN / HO + 0.1% TFA gradient. The title compound was obtained as a red solid (2.1 mg, 47% yield) following General Procedure 6.

[0650] LC / MS:C 23 H 21 Calculated m / z for FN4O6 = 468.4, observed [M+H] + =468.3.

[0651] 1H NMR(300 MHz,10% D2O / CD3CN)δ 7.72(d,J=12.2 Hz,1H),7.41(d,J=18.1 Hz,1H),6.96(s,1H),5.52(d,J=3.6 Hz,1H),5.39-5.23(m,3H),4.82(s,1H),4.73(s,1H),3.63(d,J=1.2 Hz,3H),1.56(s,3H),1.27(s,2H),0.94(t,J=7.4 Hz,3H).

[0652] 3.22: (S)-1-((9-amino-4-ethyl-8-fluoro-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)methyl)-3-methylurea (compound 144) The title compound was prepared by adding methylamine hydrochloride (10 mg) to a 200 μL solution of compound 3.20, followed by the addition of iPrNEt (5 μL). The solution was stirred at room temperature for 30 minutes. Preparative HPLC purification of the intermediate Boc-protected compound was performed as described in General Procedure 9, eluting with a 10-60% CHCN / HO + 0.1% TFA gradient. The title compound was obtained as a red solid (2.9 mg, 64.5% yield) following General Procedure 6.

[0653] LC / MS:C 23 H 21 Calculated m / z for FN5O5 = 467.5, observed [M+H] + =468.5.

[0654] 1H NMR(300 MHz,10% D2O / CD3CN)δ 8.13(d,J=9.2 Hz,1H),7.92(s,1H),7.73(d,J=12.3 Hz,1H),7.52-7.35(m,2H),6.94(d,J=9.2 Hz,2H),5.55(d,J=16.5 Hz,2H),5.44-5.27(m,4H),4.85(s,2H),4.78(s,1H),1.56(d,J=2.5 Hz,3H),1.27(s,2H),0.93(q,J=11.7,9.5 Hz,3H).

[0655] 3.23: (S)-1-((9-amino-4-ethyl-8-fluoro-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)methyl)-3-(2-hydroxyethyl)urea (compound 151) The title compound was prepared by adding ethanolamine (100 uL) to a solution of 200 uL of compound 3.20. The solution was stirred at room temperature for 30 minutes. Preparative HPLC purification of the intermediate Boc-protected compound was performed as described in General Procedure 9, eluting with a 10-60% CHCN / HO + 0.1% TFA gradient. The title compound was obtained as a red solid (0.5 mg, 8.5% yield) following General Procedure 6.

[0656] LC / MS:C 24 H 24 Calculated m / z for FN5O6 = 497.5, observed [M+H] + =498.5.

[0657] 1H NMR(300 MHz,10% D2O / CD3CN)δ 7.77-7.61(m,1H),7.48-7.30(m,2H),5.53(d,J=16.3 Hz,1H),5.31(d,J=15.4 Hz,1H),5.19(s,2H),4.69(s,2H),3.97(dd,J=6.6,4.9 Hz,2H),3.39(t,J=5.8 Hz,2H),2.93(s,1H),2.01-1.83(m,2H),0.94(t,J=7.3 Hz,3H).

[0658] 3.24: (S)-9-Amino-11-(azidomethyl)-4-ethyl-8-fluoro-4-hydroxy-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (compound 152) To a stirred solution of compound 3.5 (100 mg) in 2 mL of dichloromethane was added thionyl chloride (35 μL, 2.5 equiv.). The solution was stirred at room temperature for 20 minutes, after which additional thionyl chloride (35 μL, 2.5 equiv.) was added. After 20 minutes, toluene (1 mL) was added, and the reaction mixture was concentrated in vacuo. The crude solid was suspended in DMSO (1 mL), and sodium azide (19 mg, 1.5 equiv.) was added. The solution was stirred at room temperature for 16 hours. Purification was carried out as described in General Procedure 9, eluting with a 5-50% CH3CN / HO + 0.1% TFA gradient, to afford the title compound as an off-white solid (20 mg, 23% yield).

[0659] LC / MS:C 21 H 17 Calculated m / z for FN6O4 = 436.1, observed [M+H] + =437.2.

[0660] 1H NMR(300 MHz,MeOD)δ 7.75(d,J=12.2 Hz,1H),7.60(s,1H),7.38(d,J=9.3 Hz,1H),5.61(d,J=16.3 Hz,1H),5.46-5.35(m,3H),5.07(s,2H),2.03-1.97(m,2H),1.03(t,J=7.3 Hz,3H).

[0661] 3.25: (S)-N-((9-amino-4-ethyl-8-fluoro-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)methyl)acetamide (compound 164) The title compound was prepared according to general procedure 2, starting from compound 145 (10 mg) and glycolic acid. Preparative HPLC purification was performed as described in general procedure 9, eluting with a 10 to 45% CHCN / HO + 0.1% TFA gradient. The title compound was obtained as a yellow solid (6.9 mg, 60% yield).

[0662] LC / MS:C 23 H 21 Calculated m / z for FN4O6 = 468.1, observed [M+H] + =469.2.

[0663] 1 H NMR(300 MHz,MeOD)7.70(d,J=12.2 Hz,1H),7.60(s,1H),7.42(d,J=9.4 Hz,1H),5.62(d,J=16.3 Hz,1H),5.43(s,2H),5.36(d,J=16.2 Hz,1H),4.95(d,J=5.9 Hz,2H),4.08(s,2H),2.04-1.90(m,1H),1.03(t,J=7.4 Hz,3H).

[0664] 3.26: (S)-1-((9-amino-4-ethyl-8-fluoro-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)methyl)-3-methylthiourea (compound 161) To a solution of compound 145 (9 mg, 1.0 equiv.) in DMF (1 mL) was added thiocarbonyldiimidazole (6 mg, 1.5 equiv.), followed by DIPEA (8 μL, 2.0 equiv.). The resulting solution was stirred at 25° C. for 2 h, after which complete conversion to the isothiocyanate intermediate was confirmed. Methylammonium chloride (3 mg, 2.0 equiv.) was then added, and the reaction mixture was heated at 60° C. for 30 min. Preparative HPLC purification was performed as described in General Procedure 9, eluting with a 10 to 45% CH CN / HO + 0.1% TFA gradient. The title compound was obtained as a yellow solid (2.3 mg, 22% yield).

[0665] LC / MS:C 23 H 22 Calculated m / z for FN5O4S = 483.1, observed [M+H] + =484.2.

[0666] 1 H NMR(300 MHz,MeOD)δ 7.70(d,J=12.0 Hz,1H),7.60(s,1H),7.38(d,J=9.3 Hz,1H),5.62(d,J=16.2 Hz,1H),5.36(s,2H),5.31(d,J=16.2 Hz,1H),5.30(s,2H),3.04(s,3H),1.99-1.90(m,2H),1.02(t,J=7.4 Hz,3H).

[0667] 3.27: S-(2-Hydroxyethyl)-(S)-((9-amino-4-ethyl-8-fluoro-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)methyl)carbamothioate (compound 160) The title compound was prepared according to general procedure 5, starting from compound 145 (10 mg) and 2-mercaptoethanol. Preparative HPLC purification was performed as described in general procedure 9, eluting with a 10 to 45% CHCN / HO + 0.1% TFA gradient. The title compound was obtained as a yellow solid (4.2 mg, 43% yield).

[0668] LC / MS:C 24 H 23 Calculated m / z for FN4O6S = 514.1, observed [M+H] + =515.2.

[0669] 1 H NMR(300 MHz,MeOD)δ 7.71(d,J=12.1 Hz,1H),7.60(s,1H),7.36(d,J=9.4 Hz,1H),5.62(d,J=16.3 Hz,1H),5.42(s,2H),5.35(d,J=16.2 Hz,1H),4.88(d,J=4.6 Hz,2H),3.68(t,J=6.4 Hz,2H),3.03(t,J=6.5 Hz,2H),2.04-1.92(m,2H),1.03(t,J=7.4 Hz,3H).

[0670] 3.28: (S)-9-Amino-4,11-diethyl-8-fluoro-4-hydroxy-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (compound 154) TIFF2025510791000189.tif42165 To a 5 mL flask containing compound 140 (50 mg), water (0.72 mL), FeSO4 (heptahydrate, 11.0 mg), and propionaldehyde (74 μL) were added. The resulting suspension was cooled to −15 °C using an ice-salt water bath, and then sulfuric acid (0.40 mL) was added dropwise. Hydrogen peroxide (95 μL) was then added dropwise. The mixture was stirred at −15 °C for 10 minutes, then warmed to room temperature, and stirred for 2 hours. The reaction mixture was diluted with water (30 mL), and the resulting suspension was extracted with DCM (3 × 30 mL). The organic phase was then evaporated to dryness. Preparative HPLC purification was performed as described in General Procedure 9, eluting with a 25 to 70% CH3CN / H2O+0.1% TFA gradient to afford the title compound as a deep orange solid (2.4 mg, 4.4% yield).

[0671] LC / MS:C 22 H 20 Calculated m / z for FN3O4 = 410.1, found [M+H] + =410.2.

[0672] 1 H NMR(300 MHz,MeOD)δ 7.63(d,J=12.3 Hz,1H),7.55(s,1H),7.36(d,J=9.4 Hz,1H),5.57(d,J=16.4 Hz,1H),5.37(d,J=16.4 Hz,1H),5.21(s,2H),3.13(q,J=7.7 Hz,2H),2.02-1.90(m,2H),1.38(t,J=7.7 Hz,3H),1.01(t,J=7.3 Hz,3H).

[0673] 3.29: tert-Butyl-(S)-(11-((carbamoyloxy)methyl)-4-ethyl-8-fluoro-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl)carbamate (compound 3.29) Compound 3.5 (15 mg) was added to a 5 mL Erlenmeyer flask containing a solution of chlorosulfonyl isocyanate (7.7 μL) in dimethylformamide (0.29 mL) at −20° C. The resulting suspension was stirred at −20° C. for 5 minutes. Water (59 μL) was added, and the reaction mixture was warmed to room temperature and stirred for 2 hours, then heated ...

Claims

1. An antibody-drug conjugate having the formula (X): T-[L-(D) ｍ ] ｎ (X) wherein m is 1 to 4; n is 1 to 10; an anti-FRα antibody construct comprising an antigen binding domain that specifically binds to an epitope within human folate receptor alpha (hFRα) comprising amino acid residues E120, D121, R123, T124, S125, and Y126 of SEQ ID NO: 15; L is a linker, D is of formula I: is a compound of the formula R １ is -H, -CH ３ , -CHF ２ , -CF ３ , -F, -Br, -Cl, -OH, -OCH ３ , -OCF ３ , and -NH ２ and R ２ is -H, -CH ３ , -CF ３ , -F, -Br, -Cl, -OH, -OCH ３ , and -OCF ３ is selected from And R １ Ga-NH ２ R is R ３ Or R ４ And R １ Ga-NH ２ If other than, R is R ４ and R ３ But -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 alkyl)-aryl; R ４ but, is selected from R ５ But -H, -C １ ~C ６ Alkyl, -C ３ ~C ８ Cycloalkyl, -aryl, -heteroaryl, -aryl, and -(C １ ~C ６ alkyl)-aryl; R ６ and R ７ are each independently -H, -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 is selected from R ８ But -H, -C １ ~C ６ Alkyl, -C ３ ~C ８ Cycloalkyl, and -C ３ ~C ８ heterocycloalkyl; Each R ９ are independently -H, -C １ ~C ６ Alkyl, -C ３ ~C ８ Cycloalkyl, -aryl, -heteroaryl, and -(C １ ~C ６ alkyl)-aryl; Each R １０ But independently, -C １ ~C ６ Alkyl, -C ３ ~C ８ Cycloalkyl, -NR １４ R １４’ , -aryl, -heteroaryl, and -(C １ ~C ６ alkyl)-aryl; R １０’ But -H, -C １ ~C ６ Alkyl, -C ３ ~C ８ Cycloalkyl, -aryl, -heteroaryl, and -(C １ ~C ６ alkyl)-aryl; R １１ is -H and -C １ ~C ６ alkyl, R １２ But -H, -C １ ~C ６ Alkyl, -CO ２ R ８ , -aryl, -heteroaryl, -(C １ ~C ６ alkyl)-aryl, -S(O) ２ R １６ , and is selected from R １３ is -H and -C １ ~C ６ alkyl, R １４ and R １４’ are each independently -H, C １ ~C ６ Alkyl, -C ３ ~C ８ Cycloalkyl, and -C ３ ~C ８ heterocycloalkyl; R １６ But, -C １ ~C ６ Alkyl, -C ３ ~C ８ Cycloalkyl, -aryl, -heteroaryl, and -(C １ ~C ６ alkyl)-aryl; R １７ But, -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 alkyl)-aryl; R １８ and R １９ together with the N atom to which they are attached, 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 are -C １ ~C ６ is alkyl, X ａ and X ｂ are each independently selected from NH, O, and S; and X ｃ is O, S, and S(O) ２ is selected from With the proviso that the compound is other than (S)-9-amino-11-butyl-4-ethyl-4-hydroxy-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione; The antibody-drug conjugate.

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

3. The antibody-drug conjugate of claim 1, wherein the antigen binding domain comprises a CDR sequence of a VH domain having a sequence set forth 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.

4. The antibody-drug conjugate of claim 1 or 3, wherein the antigen-binding domain comprises a CDR sequence of a VL domain having a sequence set forth in any one of SEQ ID NOs: 39, 64, 119, 124, or 130.

5. The antigen-binding domain comprises: (i) an HCDR1 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 20, 23, 26, 28, 31, 92, 93, 94, 95, or 96; an HCDR2 amino acid sequence selected from the amino acid sequences set forth in any one of 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 the amino acid sequences set forth in any one of SEQ ID NOs: 22, 25, 30, 107, 108, or 110; (ii) an LCDR1 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 40, 43, 45, 65, 125, 126, or 127; an LCDR2 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 41, 44, or 46; and an LCDR3 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 42, 47, 120, or 121. The antibody-drug conjugate of claim 1, comprising:

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

7. R １ａ But -CH ３ , -CF ３ , -OCH ３ , -OCF ３ , and -NH ２ The antibody-drug conjugate of claim 6, selected from the group consisting of

8. R １ａ But -CH ３ , -OCH ３ , and N.H. ２ The antibody-drug conjugate of claim 6, selected from the group consisting of

9. R ２ａ The antibody-drug conjugate of any one of claims 6 to 8, wherein is selected from -H, -F, -Br, and -Cl.

10. X is -O-, -S-, or -NH-, and R ４ａ but, The antibody-drug conjugate according to any one of claims 6 to 9, selected from the group consisting of

11. D is a compound of formula (V): is a compound of the formula R ２ａ But -CH ３ , -CF ３ , -F, -Br, -Cl, -OH, -OCH ３ , and -OCF ３ is selected from R ２０ａ But -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, is selected from R ５ But -H, -C １ ~C ６ Alkyl, -C ３ ~C ８ Cycloalkyl, -aryl, -heteroaryl, and -(C １ ~C ６ alkyl)-aryl; R ６ and R ７ are each independently -H, -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 is selected from R ８ But -H, -C １ ~C ６ Alkyl, -C ３ ~C ８ Cycloalkyl, and -C ３ ~C ８ heterocycloalkyl; Each R ９ are independently -H, -C １ ~C ６ Alkyl, -C ３ ~C ８ Cycloalkyl, -aryl, -heteroaryl, and -(C １ ~C ６ alkyl)-aryl; Each R １０ａ But independently, -C １ ~C ６ Alkyl, -C ３ ~C ８ Cycloalkyl, -aryl, -heteroaryl, -(C １ ~C ６ alkyl)-aryl, and -NR １４ R １４’ is selected from Each R １０ａ’ are independently -H, -C １ ~C ６ Alkyl, -C ３ ~C ８ Cycloalkyl, -aryl, -heteroaryl, and -(C １ ~C ６ alkyl)-aryl; R １１ is -H and -C １ ~C ６ alkyl, R １２ But -H, -C １ ~C ６ Alkyl, -CO ２ R ８ , -aryl, -heteroaryl, -(C １ ~C ６ alkyl)-aryl, -S(O) ２ R １６ , and is selected from R １３ is -H and -C １ ~C ６ alkyl, R １４ and R １４’ are each independently -H, C １ ~C ６ Alkyl, -C ３ ~C ８ Cycloalkyl, and -C ３ ~C ８ heterocycloalkyl; R １６ But, -C １ ~C ６ Alkyl, -C ３ ~C ８ Cycloalkyl, -aryl, -heteroaryl, and -(C １ ~C ６ alkyl)-aryl; R １７ But, -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 alkyl)-aryl; R １８ and R １９ together with the N atom to which they are attached, 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 are -C １ ~C ６ is alkyl, X ａ and X ｂ are each independently selected from NH, O, and S; X ｃ is O, S, and S(O) ２ and indicates the point of attachment to the linker L; The antibody-drug conjugate according to any one of claims 1 to 5.

12. R ２ａ The antibody-drug conjugate of claim 11, wherein is F.

13. R ２０ａ But -H, -C １ ~C ６ Alkyl, -(C １ ~C ６ alkyl)-O-R ５ , - (C １ ~C ６ alkyl)-aryl, The antibody-drug conjugate of claim 11 or 12, selected from the group consisting of

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

15. R ２ａ But -CH ３ , -CF ３ , -F, -Br, -Cl, -OH, -OCH ３ , and -OCF ３ The antibody-drug conjugate of claim 14, selected from

16. R ２ａ The antibody-drug conjugate of claim 14, wherein is F.

17. X is -O-, -S-, or -NH-, and R ２５ But, -C １ ~C ６ Alkyl, -(C １ ~C ６ alkyl)-O-R ５ａ , -(C １ ~C ６ alkyl)-aryl, or X is O and R ２５ -X- is Selected from: The antibody-drug conjugate according to any one of claims 14 to 16.

18. X is -O-, -S-, or -NH-, and R ２５ But, -C １ ~C ６ Alkyl, -(C １ ~C ６ alkyl)-O-R ５ａ , -(C １ ~C ６ alkyl)-aryl, The antibody-drug conjugate according to any one of claims 14 to 16, selected from the group consisting of

19. The antibody-drug conjugate of any one of claims 1 to 18, wherein each of the alkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl groups is optionally substituted with one or more substituents selected from halogen, acyl, acyloxy, alkoxy, carboxy, hydroxy, amino, amido, nitro, cyano, azido, alkylthio, thio, sulfonyl, sulfonamido, alkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl.

20. The antibody-drug conjugate of any one of claims 1 to 18, wherein each of the alkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl groups is optionally substituted with one or more substituents selected from halogen, acyl, acyloxy, alkoxy, carboxy, hydroxy, amino, amido, nitro, cyano, azido, alkylthio, thio, sulfonyl, and sulfonamido.

21. The antibody-drug conjugate of any one of claims 1 to 5, wherein D has the structure of any one of the compounds listed in Table 6 or Table 7.

22. The antibody-drug conjugate of any one of claims 1 to 5, wherein D is compound 139 or compound 141.

23. The antibody-drug conjugate of any one of claims 1 to 22, wherein L is a cleavable linker.

24. The antibody-drug conjugate of claim 23, wherein L is a protease cleavable linker.

25. The antibody-drug conjugate of claim 23 or 24, wherein L comprises a dipeptide, tripeptide, or tetrapeptide.

26. L, (a) a compound represented by formula (XI) wherein Z is a functional group capable of reacting with a targeting group on the anti-FRα antibody construct T; Str is a stretcher, A.A. １ and A.A. ２ are each independently an amino acid, １ - [AA ２ ] ｒ forms a protease cleavage site, X is a self-immolative group; q is 0 or 1; r is 1, 2, or 3; s is 0, 1, or 2; # is the point of attachment to the anti-FRα antibody construct T; and % are points of attachment to camptothecin analogue D, or (b) Formula (XII) wherein Z is a functional group capable of reacting with a targeting group on the anti-FRα antibody construct T; Str is a stretcher, A.A. １ and A.A. ２ are each independently an amino acid, １ - [AA ２ ] ｒ forms a protease cleavage site, Y is -NH-CH ２ - or -NH-CH ２ -C(O)-, q is 0 or 1; r is 1, 2, or 3; v is 0 or 1; # is the point of attachment to the anti-FRα antibody construct T; and % are points of attachment to the camptothecin analog D; The antibody-drug conjugate according to any one of claims 23 to 25.

27. The antibody-drug conjugate according to any one of claims 1 to 5, wherein L-(D) in formula (X) has any one of the structures of the drug linkers (DL) listed in Tables 8 to 10.

28. The antibody-drug conjugate according to any one of claims 1 to 5, wherein L-(D) in formula (X) has any one of the structures of the drug linkers (DL) listed in Table 8 or Table 9.

29. In the formula (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 any one of claims 1 to 5,

30. The antibody-drug conjugate according to any one of claims 1 to 29, wherein m is 1 to 2.

31. The antibody-drug conjugate of any one of claims 1 to 29, wherein m is 1.

32. The antibody-drug conjugate of any one of claims 1 to 31, wherein n is 2 to 8.

33. The antibody-drug conjugate of any one of claims 1 to 31, wherein n is 4 to 8.

34. The antibody-drug conjugate of any one of claims 1 to 33, wherein the anti-FRα antibody construct further comprises a scaffold, and the antigen-binding domain is operably linked to the scaffold.

35. The antibody-drug conjugate of claim 34 , wherein the scaffold comprises an IgG Fc region.

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

37. A pharmaceutical composition comprising the antibody-drug conjugate of any one of claims 1 to 36 and a pharma- ceutically acceptable carrier or diluent.

38. A method for inhibiting the proliferation of cancer cells, the method comprising contacting the cells with an effective amount of the antibody-drug conjugate of any one of claims 1 to 36.

39. A method of killing cancer cells, comprising contacting said cells with an effective amount of an antibody-drug conjugate according to any one of claims 1 to 36.

40. 37. A method of treating cancer in a subject in need thereof, comprising administering to the subject an effective amount of an antibody-drug conjugate of any one of claims 1 to 36.

41. 41. The method of claim 40, wherein the cancer is breast cancer, ovarian cancer, colorectal cancer, non-small cell lung cancer (NSCLC), pancreatic cancer, or endometrial cancer.

42. An antibody-drug conjugate according to any one of claims 1 to 36 for use in therapy.

43. The antibody-drug conjugate of any one of claims 1 to 36 for use in the treatment of cancer.

44. 44. The antibody-drug conjugate for use according to claim 43, wherein the cancer is breast cancer, ovarian cancer, colorectal cancer, non-small cell lung cancer (NSCLC), pancreatic cancer, or endometrial cancer.

45. Use of the antibody-drug conjugate of any one of claims 1 to 36 in the manufacture of a medicament for the treatment of cancer.

46. 46. ​​The use of claim 45, wherein the cancer is breast cancer, ovarian cancer, colorectal cancer, non-small cell lung cancer (NSCLC), pancreatic cancer, or endometrial cancer.

Images