CN118922447A - Folate receptor alpha-targeting antibody-drug conjugates and methods of use - Google Patents

Abstract

An antibody-drug conjugate (ADC) comprising an antibody construct (anti-fra antibody construct) that specifically binds to human folate receptor alpha (fra) conjugated to a camptothecin analog of formula (I). The ADC is useful as a therapeutic agent, particularly in the treatment of cancer.

Description

Antibody-drug conjugates targeting folate receptor alpha and methods of use

Technical Field

The present disclosure relates to the field of immunotherapeutics, and in particular to antibody-drug conjugates targeting human folate receptor alpha (hFRα).

Background Art

Folate receptor α (FRα) is a glycosylphosphatidylinositol (GPI)-anchored cell surface protein encoded by FOLR1 and is a member of the high-affinity FR family, which also includes FRβ (FOLR2), FRg (FOLR3), and FRd (FOLR4). FRα has been identified as a highly relevant cancer therapeutic target because it is overexpressed in a variety of cancers, including ovarian cancer, triple-negative breast cancer (TNBC), endometrial cancer, mesothelioma, and lung cancer, with minimal expression in non-malignant tissues.

Several clinical studies involving FRα-targeted agents in cancer treatment 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.).

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.

Other camptothecin analogs and derivatives, and ADCs comprising them, have been described. See, for example, International (PCT) Publication Nos. WO 2019/195665, WO 2019/236954, WO 2020/200880, and WO 2020/219287.

This background information is provided for the purpose of making known information believed by the applicant to be potentially relevant to the present disclosure. It is not necessarily intended to be, nor should it be construed as, an admission that any of the previous information constitutes prior art to the claimed invention.

Summary of the invention

Described herein are antibody-drug conjugates (ADCs) targeting human FRα and methods of use. One aspect of the disclosure relates to an antibody-drug conjugate having formula (X):

T-[L-(D) _m ] _n

(X)

in:

m is between 1 and 4;

n is between 1 and 10;

T is an anti-FRα antibody construct comprising an antigen binding domain that specifically binds to an epitope within human folate receptor α (hFRα) comprising amino acid residues E120, D121, R123, T124, S125, and Y126 of SEQ ID NO: 15;

L is a connector, and

D is a compound of formula I:

in:

^R1 is selected from: -H, _-CH3 , _-CHF2 , _-CF3 , -F, -Br, -Cl, -OH, _-OCH3 , _-OCF3 and _-NH2 , and

R ² is selected from: -H, -CH ₃ , -CF ₃ , -F, -Br, -Cl, -OH, -OCH ₃ and -OCF ₃ ,

And among them:

When R ¹ is -NH ₂ , then R is R ³ or R ⁴ , and when R ¹ is not -NH ₂ , then R is R ⁴ ;

R ³ is selected from: -H, -C ₁ -C ₆ alkyl, -C ₃ -C ₈ cycloalkyl, -(C ₁ -C ₆ alkyl)-OR ⁵ , -CO ₂ R ⁸ , -aryl, -heteroaryl and -(C ₁ -C ₆ alkyl)-aryl;

^R4 is selected from:

R ⁵ is selected from: -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 selected from: -H, -C ₁ -C ₆ alkyl, -C ₃ -C ₈ cycloalkyl, -(C ₁ -C ₆ alkyl)-OR ⁵ , -C ₃ -C ₈ heterocycloalkyl and -C(O)R ¹⁷ ;

R ⁸ is selected from: -H, -C ₁ -C ₆ alkyl, -C ₃ -C ₈ cycloalkyl and -C ₃ -C ₈ heterocycloalkyl;

Each R ⁹ is independently selected from: -H, -C ₁ -C ₆ alkyl, -C ₃ -C ₈ cycloalkyl, -aryl, -heteroaryl, and -(C ₁ -C ₆ alkyl)-aryl;

Each R ¹⁰ is independently selected from: -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′ is selected from: -H, -C ₁ -C ₆ alkyl, -C ₃ -C ₈ cycloalkyl, -aryl, -heteroaryl and -(C ₁ -C ₆ alkyl)-aryl;

R ¹¹ is selected from: -H and -C ₁ -C ₆ alkyl;

R ¹² is selected from: -H, -C ₁ -C ₆ alkyl, -CO ₂ R ⁸ , -aryl, -heteroaryl, -(C ₁ -C ₆ alkyl)-aryl, -S(O) ₂ R ¹⁶ and

R ¹³ is selected from: -H and -C ₁ -C ₆ alkyl;

R ¹⁴ and R ^14' are each independently selected from: -H, C ₁ -C ₆ alkyl, -C ₃ -C ₈ cycloalkyl and -C ₃ -C ₈ heterocycloalkyl;

R ¹⁶ is selected from: -C ₁ -C ₆ alkyl, -C ₃ -C ₈ cycloalkyl, -aryl, -heteroaryl and -(C ₁ -C ₆ alkyl)-aryl;

R ¹⁷ is selected from: -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 bound form a 4-, 5-, 6- or 7-membered ring having 0 to 3 substituents selected from the group consisting of halogen, -C ₁ -C ₆ alkyl, -C ₃ -C ₈ cycloalkyl and -(C ₁ -C ₆ alkyl)-OR ⁵ ;

R ²⁴ , R ²⁵ and R ²⁶ are each -C ₁ -C ₆ alkyl;

^Xa and ^Xb are each independently selected from: NH, O and S, and

^Xc is selected from the group consisting of O, S and S(O) ₂ ,

Provided that the compound is not (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.

Another aspect of the present disclosure relates to an antibody-drug conjugate having a structure selected from:

in:

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 as shown in SEQ ID NO:39 and the VH amino acid sequence as shown in SEQ ID NO:19; or

(b) the VL amino acid sequence as shown in SEQ ID NO: 124 and the VH amino acid sequence as shown in SEQ ID NO: 91; or

(c) the VL amino acid sequence as shown in SEQ ID NO: 64 and

(i) the VH amino acid sequence as shown in SEQ ID NO: 50, or

(ii) the VH amino acid sequence as shown in SEQ ID NO: 54, or

(iii) the VH amino acid sequence as shown in SEQ ID NO: 57, or

(iv) the VH amino acid sequence as shown in SEQ ID NO: 61, or

(v) the VH amino acid sequence as shown in SEQ ID NO: 76, or

(vi) the VH amino acid sequence as shown in SEQ ID NO: 79, or

(vii) the VH amino acid sequence as shown in SEQ ID NO: 82, or

(viii) the VH amino acid sequence as shown in SEQ ID NO: 85, or

(ix) the VH amino acid sequence as shown in SEQ ID NO: 88, or

(x) the VH amino acid sequence as shown in SEQ ID NO: 106; or

(d) the VL amino acid sequence as shown in SEQ ID NO: 130 and

(i) the VH amino acid sequence as shown in SEQ ID NO: 99, or

(ii) the VH amino acid sequence as shown in SEQ ID NO: 106, or

(iii) the VH amino acid sequence as shown in SEQ ID NO: 113, or

(iv) the VH amino acid sequence as shown in SEQ ID NO: 116, or

(v) the VH amino acid sequence as shown in SEQ ID NO: 133, or

(vi) the VH amino acid sequence as shown in SEQ ID NO: 136; or

(e) the VL amino acid sequence as shown in SEQ ID NO: 119 and

(i) the VH amino acid sequence as shown in SEQ ID NO: 106, or

(ii) the VH amino acid sequence as shown in SEQ ID NO: 116,

And wherein n is between 4 and 8.

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

Another aspect of the present disclosure relates to a method of inhibiting proliferation of cancer cells, comprising contacting the cells with an effective amount of an antibody-drug conjugate as described herein.

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 as described herein.

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 as described herein.

Another aspect of the present disclosure relates to an antibody-drug conjugate as described herein for use in therapy.

Another aspect of the present disclosure relates to an antibody-drug conjugate as described herein for use in the treatment of cancer.

Another aspect of the present disclosure relates to the use of an antibody-drug conjugate as described herein in the manufacture of a medicament for the treatment of cancer.

BRIEF DESCRIPTION OF THE DRAWINGS

Figures 1A and 1B show the sequence of the rabbit heavy chain variable domain CDR of the chimeric anti-FRα antibody v23924 grafted onto a human VH framework (IGHV3-23*01) (SEQ ID NO: 155) (1A), and the sequence of the rabbit light chain variable domain CDR of the chimeric antibody v23924 grafted onto a human VL framework (IGKVI-39*01) (SEQ ID NO: 156) (1B). The CDRs are designated by the AbM definition and marked in bold italic font.

Figures 2A-2D show the profiles of purified parental chimeric anti-FRα antibody v23924 and purified representative humanized variant v30384 as analyzed by electrophoresis and UPLC-SEC. Figures 2A and 2C show the electrophoretic profiles of parental chimeric anti-FRα antibody v23924 (2A) and purified representative humanized variant v30384 (2C) after preparative SEC purification (prep-SEC post) or after protein A purification (pA post) under non-reducing (NR) and reducing (R) conditions, and Figures 2B and 2D show the UPLC-SEC profiles of parental chimeric anti-FRα antibody v23924 (2B) after preparative SEC purification and purified representative humanized variant v30384 (2D) after protein A purification.

Figures 3A and 3B depict biolayer interferometry (BLI) sensorgrams of parental chimeric anti-FRα antibody v23924 (3A) and purified representative humanized variant v30384 (3B).

Figures 4A-4D depict complete LC/MS spectra of representative humanized variants v30384 (4A, expanded view of main peak in 4B) and v31422 (4C, expanded view of main peak in 4D).

Figures 5A and 5B show the receptor-mediated internalization capacity of chimeric anti-FRα antibody v23924, representative humanized variant v30384, and antibodies targeting FRα, Sumituximab and Fatuzumab at various concentrations in the FRα-expressing cell line IGROV-1 after 6 hours of incubation (5A) and 24 hours of incubation (5B) as determined by flow cytometry. Anti-RSV antibody palivizumab was included as a negative control.

Figures 6A and 6B show the receptor-mediated internalization capacity of chimeric anti-FRα antibody v23924, representative humanized variant v30384, and antibodies targeting FRα, Sumituximab and Fatuzumab at various concentrations in the FRα-expressing cell line OVCAR-3 as determined by flow cytometry after 6 hours (6A) and 24 hours (6B). Anti-RSV antibody Palivizumab was included as a negative control.

Figure 7 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.

Figures 8A and 8B show summary (8A) and difference (8B) graphs of hydrogen/deuterium exchange mass spectrometry (HDX-MS) kinetics of peptides generated by pepsin digestion of hFRα: hFOLR1 (hFRα) versus hFOLR1-v23924 complex.

9A-9C show the amide deuteration level of peptide 119-126 (WEDCRTSY) (SEQ ID NO: 152) after 1 hour hydrogen/deuterium exchange mass spectrometry (HDX-MS): hFOLR1 (9A) versus hFOLR1-v23924 complex (9B), and the difference graph (9C).

Figures 10A and 10B show the receptor-mediated internalization capacity of the parental anti-FRα humanized antibody variant v30384 and the representative affinity matured variant v35356 in the FRα expressing cell lines IGROV-1 (10A) and JEG-3 (10B) as determined by flow cytometry after 5 and 24 hour incubation periods. Palivizumab was included as a non-targeting control.

FIG11 presents a table showing the CDR sequences of representative anti-FRα antibodies as defined by the IMGT, Chothia, Kabat, Contact, and AbM definitions.

FIG12 presents a table showing the VH and VL sequences of representative anti-FRα antibodies.

FIG. 13 shows exemplary drug-linker (DL) structures comprising camptothecin analogs of Formula (I) having a C7 bond (Table 8).

FIG. 14 shows exemplary drug-linker (DL) structures comprising camptothecin analogs of Formula (I) having a C10 bond (Table 9).

FIG. 15 shows exemplary drug-linker (DL) structures comprising camptothecin analogs of Formula (I) having a C7 or C10 bond (Table 10).

FIG. 16 shows exemplary conjugate (DC) structures comprising camptothecin analogs of Formula (I) having a C7 bond (Table 11).

FIG. 17 shows exemplary conjugate (DC) structures comprising camptothecin analogs of Formula (I) having a C10 bond (Table 12).

FIG. 18 shows exemplary conjugate (DC) structures comprising camptothecin analogs of Formula (I) having a C7 or C10 bond (Table 13).

Figures 19A-19C show the in vivo anti-tumor activity of ADCs comprising anti-FRα humanized antibody variant v30384 conjugated to camptothecin analogs Compound 139 or Compound 141 (19A and 19B) in the OV90 xenograft model at DAR 8, and conjugated to camptothecin analogs Compound 139, Compound 140, Compound 141, or Compound 148 (19C) in the H2110 xenograft model at DAR 8. An ADC comprising palivizumab (v21995) was included as a control.

Figure 20 shows the pharmacokinetics of anti-FRα humanized antibody v36675 and four ADCs comprising v36675 conjugated with DAR8 to DXd or camptothecin analogs Compound 139 or Compound 141 evaluated in hFcRn Tg32 mice (n=4). Mean serum concentrations at some time points were calculated using n<4 animals (as some samples were below the limit of detection).

21 shows the in vivo stability of four ADCs in Tg32 mouse serum, comprising humanized variant v36675 conjugated with DAR8 to DXd or camptothecin analog compound 139 or compound 141. The solid line shows the remaining DAR% (left axis) and the dashed line shows the maleimide ring opening% (right axis).

Figures 22A-22E show the in vivo anti-tumor activity of ADCs comprising anti-FRα humanized antibody variant v36675 conjugated to camptothecin analog compound 139 or compound 141 at DAR 4 or DAR 8, respectively, in the OV90 CDX xenograft model (22A, 22B); conjugated to camptothecin analog compound 140 or compound 141 at DAR 4 or DAR 8, respectively, in the OVCAR3 CDX xenograft model (22C); conjugated to camptothecin analog compound 139 at DAR 8 in the GTG-2025 PDX xenograft model (22D), and conjugated to camptothecin analog compound 139 at DAR 8 in the GTG-0958 PDX xenograft model (22E).

Figures 23A-23D show the total antibody serum concentration of ADCs comprising anti-FRα humanized antibody v36675 in blood samples collected after the first dose in cynomolgus monkey toxicity studies; ADCs comprising v36675 conjugated to compound 139 or compound 141 with DAR 8 were administered at 30 mg/kg (23A), ADCs comprising v36675 conjugated to compound 139 or compound 141 with DAR 4 were administered at 60 mg/kg (23B), ADCs comprising v36675 conjugated to compound 139 or compound 141 with DAR 8 were administered at 80 mg/kg (23C), and ADCs comprising v36675 conjugated to compound 139 or compound 141 with DAR 4 or DAR 8 were administered at 120 mg/kg (23D).

Figure 24 shows the in vitro bystander activity of ADCs comprising 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 comprising palivizumab (v22277) conjugated to MC-GGFG-AM-DXd1 and MCvcPABC-MMAE was included as a negative control.

25A-25D show the penetration of anti-FRα humanized antibody variant v36675 into JEG-3 cell spheroids at 4 hours (25A), 24 hours (25B), 48 hours (25C), and 96 hours (25D) compared to sometuximab and negative control palivizumab.

Figures 26A and 26B show intracellular (26A) and extracellular (26B) payload release from the following ADCs in the high FRα expressing cell line IGROV-1: ADC comprising the anti-FRα humanized antibody variant v36675 conjugated to compound 139 with DAR 8 (v36675-MC-GGFG-AM-Compound 139) and ADC comprising the non-targeting control palivizumab (v21995) conjugated to compound 139 with DAR 8.

Figures 27A-I show the in vivo anti-tumor activity of ADCs comprising the anti-FRα humanized antibody variant v36675 conjugated to Compound 139 with DAR 8 (v36675-MC-GGFG-AM-Compound 139) and a control ADC, Sumetuximab-DM4 DAR4, in patient-derived ovarian cancer xenograft (PDX) models when dosed at 6 mg/kg: CTG-0703 PDX model (27A), CTG-1301 PDX model (27B), CTG-2025 PDX model (27C), CTG-3383 PDX model (27D), CTG-0947 PDX model (27E), CTG-0958 PDX model (27F), CTG-3718 PDX model (27G), CTG-1703 PDX model (27H), and CTG-1602 PDX model (27I).

Figures 28A and 28B show fixed cell confirmation screening images of 20 mg/mL of anti-FRα humanized antibody variant v36675 (28A) and 1 mg/mL of control antibody (rituximab biosimilar) (28B) using Retrogenix cell microarray technology to screen for specific off-target binding interactions.

FIG. 29 shows competition binding between chimeric anti-FRα antibody v23294 and anti-FRα antibodies Sometuximab and Fatuzumab assessed in H2110 cells.

DETAILED DESCRIPTION

The present disclosure relates to antibody-drug conjugates (ADCs) comprising an antibody construct (anti-FRα antibody construct) that specifically binds to human folate receptor α (FRα) conjugated to a camptothecin analog of formula (I) as described herein. Specifically, the present disclosure relates to an ADC having formula (X):

T-[L-(D) _m ] _n

(X)

in:

T is an anti-FRα antibody construct as described herein;

L is the connector;

D is a camptothecin analog of formula (I) as described herein;

m is between 1 and 4, and

n is between 1 and 10.

The ADCs of the present disclosure can be used, for example, as therapeutic agents, especially in the treatment of cancer.

definition

Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art.

As used herein, the term "about" refers to a variation of approximately +/- 10% from a given value. It should be understood that such variations are always included in any given value provided herein, whether or not specifically mentioned.

When used in conjunction with the term "comprising" in this document, the use of the word "a/kind" may mean "one/kind", but it also has the meaning of "one/kind or more/kinds", "at least one/kind" and "one/kind or more than one/kind".

As used herein, the terms "comprising," "having," "including," and "containing," and grammatical variations thereof, are inclusive or open-ended and do not exclude additional, unlisted elements and/or method steps. When used in conjunction with a composition, use, or method herein, the term "consisting essentially of means that additional elements and/or method steps may be present, but these additions do not substantially affect the manner in which the enumerated composition, method, or use functions. The term "consisting of" does not include the presence of additional elements and/or method steps when used in combination with a composition, use, or method herein. A composition, use, or method described herein as comprising certain elements and/or steps may also consist essentially of those elements and/or steps in certain embodiments, and consist of those elements and/or steps in other embodiments, whether or not these embodiments are specifically mentioned.

"Complementarity determining region" or "CDR" is an amino acid sequence that contributes to antigen binding specificity and affinity. "Framework" region (FR) can help maintain the correct conformation of CDR to promote the binding between antigen binding region and antigen. From N-terminus to C-terminus, the light chain variable region (VL) and heavy chain variable region (VH) of an antibody generally include domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The three heavy chain CDRs are referred to as HCDR1, HCDR2 and HCDR3 in this article, and the three light chain CDRs are referred to as LCDR1, LCDR2 and LCDR3. CDR provides most of the contact residues for the binding of an antibody to an antigen or epitope. Generally, three heavy chain CDRs and three light chain CDRs are required to bind antigen. However, in some cases, even a single variable domain can also confer antigen binding specificity. In addition, as known in the art, in some cases, antigen binding can also occur by a combination of at least one or more CDRs (e.g., HCDR3) selected from VH and/or VL domains.

Many different definitions of CDR sequences are commonly used, including those described by Kabat et al. (1983, Sequences of Proteins of Immunological Interest, NIH Publication No. 369-847, Bethesda, MD), Chothia et al. (1987, J Mol Biol, 196: 901-917), and IMGT, AbM (University of Bath) and Contact (MacCallum et al., 1996, J Mol Biol, 262 (5): 732-745). For example, the definitions of CDRs according to Kabat, Chothia, IMGT, AbM and Contact are provided in Table 1 below. Therefore, it will be apparent to one skilled in the art that the exact numbering and placement of CDRs may differ based on the numbering system employed. However, it should be understood that the disclosure of VH herein includes disclosure of the associated (intrinsic) heavy chain CDRs (HCDRs) as defined by any known numbering system. Similarly, disclosure of a VL herein includes disclosure of the associated (intrinsic) light chain CDRs (LCDRs) as defined by any known numbering system.

Table 1: Common CDR ^definitions1

¹ Either the Kabat or Chothia numbering system may be used for all defined HCDR2, HCDR3 and light chain CDRs except Contact which use Chothia numbering.

² Using kabat numbering. The positions in the Kabat numbering scheme that delineate the ends of the Chothia and IMGT CDR-H1 loops vary depending on the length of the loop, as Kabat places insertions outside of those CDR definitions at positions 35A and 35B. However, the IMGT and Chothia CDR-H1 loops can be unambiguously defined using Chothia numbering. CDR-H1 definitions using Chothia numbering: Kabat H31-H35, Chothia H26-H32, AbM H26-H35, IMGT H26-H33, Contact H30-H35.

In the context of two or more polynucleotides or polypeptide sequences, the term "identical" refers to two or more identical sequences or subsequences. When sequences are compared and aligned to obtain the maximum consistency measured as one of the conventional sequence comparison algorithms known to those of ordinary skill in the art or by manual alignment and visual inspection on a comparison window or in a specified region, if the sequence has a certain percentage of identical amino acid residues or nucleotides (e.g., about 80%, about 85%, about 90%, about 95% or about 98% identity in a specified region), the sequence is "substantially identical". For sequence comparison, a test sequence is usually compared with a specified reference sequence. When using a sequence comparison algorithm, the test sequence and the reference sequence are input into a computer, and if necessary, subsequence coordinates are specified, and sequence algorithm program parameters are specified. Default program parameters can be used, or alternative parameters can be specified. The sequence comparison algorithm then calculates the sequence identity percentage of the test sequence relative to the reference sequence based on the program parameters.

A "comparison window" refers to a segment of a sequence comprising contiguous amino acid or nucleotide positions, which can be, for example, about 10 to 600 contiguous amino acid or nucleotide positions, or about 10 to about 200, or about 10 to about 150 contiguous amino acid or nucleotide positions, over which a test sequence can be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods of sequence alignment for comparison are known to those of ordinary skill in the art. Optimal alignment of sequences for comparison can be performed, for example, by the local homology algorithm of Smith & Waterman, 1970, Adv. Appl. Math., 2:482c; the homology alignment algorithm of Needleman & Wunsch, 1970, J. Mol. Biol., 48:443; the similarity search method of Pearson & Lipman, 1988, Proc. Natl. Acad. Sci. USA, 85:2444; or by computerized 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, e.g., Ausubel et al., Current Protocols in Molecular Biology, (1995 supplement), Cold Spring Harbor Laboratory Press). Examples of available algorithms suitable for determining percentage of sequence identity are 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 analysis is publicly available through the website of the National Center for Biotechnology Information (NCBI).

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

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

As used herein, the term "alkoxy" refers to the group -OR, where R is alkyl, aryl, heteroaryl, cycloalkyl, or heterocycloalkyl.

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

As used herein, the term "alkylaminoaryl" refers to an alkyl group, as defined herein, substituted with an aminoaryl group, as defined herein.

As used herein, the term "alkylheterocycloalkyl" refers to an alkyl group, as defined herein, substituted with a heterocycloalkyl group, as defined herein.

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

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

As used herein, the term "amino" refers to the group -NRR', wherein R and R' are independently hydrogen, alkyl, aryl, heteroaryl, cycloalkyl, or heterocycloalkyl.

As used herein, the term "aminoalkyl" refers to an alkyl group, as defined herein, substituted with one or more amino groups (eg, one, two or three amino groups).

As used herein, the term "aminoaryl" refers to an aryl group, as defined herein, substituted with an amino group.

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

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

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

As used herein, the term "cycloalkyl" 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.

As used herein, the term "haloalkyl" refers to an alkyl group, as defined herein, substituted with one or more halogen atoms.

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

As used herein, the term "heteroaryl" 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 heteroaryl include, but are not limited to, pyridyl, benzofuranyl, pyrazinyl, pyridazinyl, pyrimidinyl, triazinyl, quinolyl, benzoxazolyl, benzothiazolyl, isoquinolyl, quinazolinyl, quinoxalinyl, pyrrolyl, indolyl, and the like.

As used herein, the term "heterocycloalkyl" refers to a monocyclic or bicyclic non-aromatic ring system containing a specified number of atoms and at least one of the ring atoms being a heteroatom (e.g., O, S, or N). The heterocyclyl substituent may be attached via any of its available ring atoms (e.g., ring carbon or ring nitrogen). Examples of heterocycloalkyl include, but are not limited to, aziridinyl, azetidinyl, piperidinyl, morpholinyl, piperazinyl, pyrrolidinyl, etc.

As used herein, the terms "hydroxy" and "hydroxyl" refer to the group -OH.

As used herein, the term "hydroxyalkyl" refers to an alkyl group, as defined herein, substituted with one or more hydroxy groups.

As used herein, the term "nitro" refers to the group _-NO2 .

As used herein, the term "sulfonyl" refers to the group -S(O) _2R , where R is H, alkyl, or aryl.

As used herein, the term "sulfonylamino" refers to the group -NH-S(O) _2R , wherein R is H, alkyl, or aryl.

As used herein, the terms "sulfenyl" and "thiol" refer to the group -SH.

Unless expressly indicated as "unsubstituted", any alkyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl group mentioned herein should be understood as "optionally substituted", i.e., each such reference includes unsubstituted and substituted forms of these groups. For example, reference to "-C ₁ -C ₆ alkyl" includes unsubstituted -C ₁ -C ₆ alkyl and -C ₁ -C ₆ alkyl substituted with one or more substituents. Examples of substituents include, but are not limited to, halogen, acyl, acyloxy, alkoxy, carboxyl, hydroxy, amino, amido, nitro, cyano, azido, alkylthio, sulfonyl, sulfonamido, alkyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl. In certain embodiments, each alkyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl group mentioned herein is optionally substituted with one or more substituents selected from the group consisting of halogen, acyl, acyloxy, alkoxy, carboxyl, hydroxy, amino, amido, nitro, cyano, azido, alkylthio, thio, sulfonyl and sulfonamido.

A chemical group described as "substituted" herein may include one substituent or multiple substituents, up to the full valence of the substitution of the group. For example, a methyl group may include 1, 2, or 3 substituents, and a phenyl group may include 1, 2, 3, 4, or 5 substituents. When a group is substituted with more than one substituent, the substituents may be the same or they may be different.

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

It is contemplated that any embodiment discussed herein can be implemented by any method, use, or composition disclosed herein, and vice versa.

Certain features, structures, and/or characteristics described in connection with one embodiment disclosed herein may be combined with features, structures, and/or characteristics described in connection with another embodiment disclosed herein in any suitable manner to provide one or more further embodiments.

It should also be understood that the positive recitation of a feature in one embodiment is a basis for excluding that feature in another embodiment. For example, where a list of options is presented for a given embodiment or claim, it should be understood that one or more options may be deleted from the list and the shortened list may form an alternative embodiment, regardless of whether such an alternative embodiment is specifically mentioned.

Anti-FRα antibody constructs

The ADC of the present disclosure comprises an anti-FRα antibody construct. In this context, the term "antibody construct" refers to a polypeptide or a group of polypeptides comprising one or more antigen binding domains, wherein each of the one or more antigen binding domains specifically binds to an epitope or antigen. When the antibody construct comprises two or more antigen binding domains, each antigen binding domain can bind to the same epitope or antigen (i.e., the antibody construct is monospecific) or they can bind to different epitopes or antigens (i.e., the antibody construct is bispecific or multispecific). The antibody construct may also include a scaffold, and the one or more antigen binding domains may be fused or covalently attached to the scaffold, optionally connected via a linker, as described herein.

According to the present disclosure, the anti-FRα antibody construct comprises at least one antigen binding domain that specifically binds to human FRα (hFRα). "Specific binding" to hFRα means that the antibody construct binds to hFRα but does not exhibit significant binding to any of the human folate receptors β (FOLR2), γ (FOLR3), or δ (FOLR4). In certain embodiments, the anti-FRα antibody construct of the present disclosure is capable of binding to FRα from one or more non-human species. In certain embodiments, the anti-FRα antibody construct of the present disclosure is capable of binding to cynomolgus monkey FRα.

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 are readily available from publicly accessible databases such as GenBank or UniProtKB. Examples of hFRα sequences include, for example, 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 (SEQ ID NO: 2; NCBI reference sequence: XP_005579002.2) is also provided in Table 2.

Table 2: Human and cynomolgus monkey FRα protein sequences

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 (using, for example, a BIAcore instrument) (Liljeblad et al., 2000, Glyco J, 17:323-329), flow cytometry, or traditional binding assays (Heeley, 2002, Endocr Res, 28:217-229). In certain embodiments, specific binding can be defined as, for example, less than about 10% of the binding to hFRα as measured by ELISA or flow cytometry to a non-target protein such as FOLR2, FOLR3, or FOLR4. In certain embodiments, specific binding of an antibody construct to FRα can be defined by a dissociation constant ( _KD ) ≤ 1 μM, e.g., ≤ 500 nM, ≤ 250 nM, ≤ 100 nM, ≤ 50 nM, or ≤ 10 nM. In certain embodiments, specific binding of an antibody construct to a particular antigen or epitope may be defined by a dissociation constant ( _KD ) of ^10-6 M or less, such as ^10-7 M or less or ^10-8 M. In some embodiments, specific binding of an antibody construct to a particular antigen or epitope may be defined by a dissociation constant ( _KD ) of between ^10-6 M and ^10-9 M, such as between ^10-7 M and ^10-9 M.

In certain embodiments, the anti-FRα antibody constructs of the present disclosure show higher internalization into FRα expressing cells than the reference antibodies somituximab (huMov19 or huFR107) and faetuzumab (MORAb-003).

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

In certain embodiments, when the amount of anti-FRα antibody construct internalized into FRα-expressing cells under the same test conditions is at least 1.2 times the amount of reference antibody internalized into the same FRα-expressing cells, the anti-FRα antibody construct is considered to exhibit higher internalization into FRα-expressing cells than the corresponding reference antibody (somituximab or fatuzumab). In certain embodiments, the amount of internalized antibody is determined using appropriate fluorescent dyes and high-content imaging. In some embodiments, the amount of internalized antibody in cells expressing FRα at high levels is determined. In some embodiments, the amount of internalized antibody in IGROV-1 cells or cells expressing FRα at a level similar to that of IGROV-1 cells is determined. 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.

In certain embodiments, when the amount of anti-FRα antibody construct internalized into FRα expressing cells under the same test conditions is at least 1.3 times, at least 1.4 times, at least 1.5 times, 1.6 times, 1.7 times, 1.8 times, 1.9 times or 2.0 times the amount of reference antibody internalized into the same FRα expressing cells, the anti-FRα antibody construct is considered to be higher than the corresponding reference antibody (somituximab or fatuzumab) in internalization into FRα expressing cells. In certain embodiments, the amount of internalized antibody is determined using appropriate fluorescent dyes and high-content imaging. In some embodiments, the amount of internalized antibody in cells expressing FRα at high levels is determined. In some embodiments, the amount of internalized antibody in IGROV-1 cells or cells expressing FRα at a level similar to that of IGROV-1 cells is determined. 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.

Antigen binding domain

The anti-FRα antibody constructs disclosed herein 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 Fab (scFab), single-chain Fv (scFv), and single-domain antibodies (sdAb).

A "Fab fragment" contains the constant domain (CL) of the light chain and the first constant domain (CH1) of the heavy chain, as well as the variable domains of the light and heavy chains (VL and VH, respectively). A Fab' fragment differs from a Fab fragment in that it has several amino acid residues added to the carboxyl terminus of the heavy chain CH1 domain, including one or more cysteines from the hinge region of the antibody. A Fab fragment can also be a single-chain Fab molecule, i.e., a Fab molecule in which a Fab light chain and a 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.

"scFv" comprises the heavy chain variable domain (VH) and light chain variable domain (VL) of an antibody in a single polypeptide chain. ScFv may optionally further comprise a polypeptide linker between the VH and VL domains so that the scFv can form a structure required for antigen binding. For example, scFv may include a VL connected to the N-terminus of VH from the C-terminus via a polypeptide linker. Alternatively, scFv may comprise a VH connected to the N-terminus of VL via its C-terminus via a polypeptide linker (see the review in Pluckthun in The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg and Moore, eds., Springer-Verlag, New York, pp. 269-315 (1994)).

The "sdAb" format refers to a single immunoglobulin domain. sdAb can be, for example, of camel origin. Camel antibodies lack light chains, and their antigen binding sites consist of a single domain called "VHH". sdAb contains three CDR/hypervariable loops CDR1, CDR2, and CDR3 that form the antigen binding site. sdAb is quite stable and easy to express, for example, expressed as a fusion with an antibody Fc chain (see, e.g., Harmsen & De Haard, 2007, Appl. Microbiol Biotechnol., 77 (1): 13-22).

In those embodiments where the anti-FRα antibody construct comprises two or more antigen binding domains, each additional antigen binding domain can independently be an immunoglobulin-based domain (such as an antigen-binding antibody fragment) or a non-immunoglobulin-based domain (such as a non-immunoglobulin-based antibody mimetic), or other polypeptides or small molecules that are capable of specifically binding to their targets (e.g., natural or engineered ligands). Non-immunoglobulin-based antibody mimetic forms include, for example, anticalins, fynomers, affimers, alphabodies, DARPins, and avimers.

The present disclosure describes herein the identification of an antibody that specifically binds hFRα (variant v23924), as well as representative humanized versions of the antibody (variants v30384, v30389, v30394, v30399, v31422, v31423, v31424, v31425, and v31426), and representative affinity matured versions of the antibody (variants v35305, v35342, v35347, v35348, v35350, v35354, v35356, v35358, v36167, v36168, and v36675) (see the Examples and Sequence Listing). Epitope mapping was performed using the hFRα sequence shown in Figure 7 (SEQ ID NO: 15), and it was determined that the epitope within the hFRα protein to which variant v23924 binds comprises amino acid residues E120, D121, R123, T124, S125 and Y126 of SEQ ID NO: 15 (see Example 13).

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

Competition assays known in the art can be used to determine whether an antibody construct competes with an antibody that binds to an epitope comprising amino acid residues E120, D121, R123, T124, S125 and Y126 within the hFRα protein or with antibody v23924 for binding to hFRα. For example, an antibody that binds to an epitope comprising amino acid residues E120, D121, R123, T124, S125 and Y126 within the hFRα protein or antibody v23924 (reference antibody) is first allowed to bind to hFRα under saturation conditions, and then the ability of the test antibody construct to bind to hFRα is measured. If the test antibody construct is able to bind to hFRα simultaneously with the reference antibody, it is considered that the test antibody construct binds to a different epitope than the reference antibody. Conversely, if the test antibody construct cannot bind to hFRα simultaneously with the reference antibody, it is considered that the test antibody construct binds to an epitope that is identical to the epitope bound to the reference antibody, an overlapping epitope, or an epitope that is closely adjacent to the epitope bound to the reference antibody. Competition assays can also be run in which the order of binding of the reference and test antibodies is reversed, ie, the test antibody is first allowed to bind hFRα under saturating conditions and the ability of the reference antibody construct to bind hFRα is then measured.

Such competition 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 the binding of the reference antibody to its epitope in a competition assay by 50% or more.

In certain embodiments, the anti-FRα antibody constructs of the present disclosure comprise at least one antigen binding domain that specifically binds to 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 the antibody are shown in Figure 11. Analysis of the CDR sequences from parental and affinity matured anti-FRα antibodies identified the minimum amino acid sequence present in each CDR as defined by any of the IMGT, Chothia, Kabat, Contact or AbM numbering systems. These amino acid sequences are represented by the minimum consensus CDR sequences provided in Table 3. Extended versions of these CDR consensus sequences based on the CDR sequences defined by the AbM numbering system are shown in Table 4.

Table 3: Minimal CDR consensus sequences of anti-FRo antibodies

Table 4: CDR consensus sequences of anti-FRα antibodies based on the AbM numbering system

In certain embodiments, the anti-FRα antibody constructs of the present disclosure comprise an antigen binding domain having a heavy chain CDR amino acid sequence (HCDR1, HCDR2, and HCDR3) comprising the sequences set forth in SEQ ID NOs: 3, 4, and 5, and a light chain CDR amino acid sequence (LCDR1, LCDR2, and LCDR3) comprising the sequences set forth in SEQ ID NOs: 6, 7, and 8.

In certain embodiments, the anti-FRα antibody constructs of the present disclosure comprise an antigen binding domain having:

(i) the HCDR1 amino acid sequence as shown in SEQ ID NO:3; the HCDR2 amino acid sequence as shown in SEQ ID NO:4; and the HCDR3 amino acid sequence as shown in SEQ ID NO:5, wherein ^X2 is L and ^X3 is A, or ^X2 is H and ^X3 is P, and

(ii) the LCDR1 amino acid sequence as shown in SEQ ID NO:6, wherein ^X4 is G and ^X5 is D, or ^X4 is W and ^X5 is Y; the LCDR2 amino acid sequence as shown in SEQ ID NO:7; and the LCDR3 amino acid sequence as shown in SEQ ID NO:8, wherein ^X6 is S, ^X7 is N, ^X8 is V and ^X9 is D, or ^X6 is W, ^X7 is H, ^X8 is I and ^X9 is L.

In certain embodiments, the anti-FRα antibody constructs of the present disclosure comprise an antigen binding domain having a heavy chain CDR amino acid sequence (HCDR1, HCDR2, and HCDR3) comprising the sequences set forth in SEQ ID NOs: 9, 10, and 11, and a light chain CDR amino acid sequence (LCDR1, LCDR2, and LCDR3) comprising the sequences set forth in SEQ ID NOs: 12, 13, and 14.

In certain embodiments, the anti-FRα antibody constructs of the present disclosure comprise an antigen binding domain having:

(i) the HCDR1 amino acid sequence as shown in SEQ ID NO:9; the HCDR2 amino acid sequence as shown in SEQ ID NO:10, wherein ^X11 is S or A and ^X12 is V, or ^X11 is S and ^X12 is L; and the HCDR3 amino acid sequence as shown in SEQ ID NO:11, wherein ^X13 is L and ^X14 is A, or ^X13 is H and ^X14 is P, and

(ii) the LCDR1 amino acid sequence as shown in SEQ ID NO: 12, wherein ^X15 is R or Q, ^X16 is G and ^X17 is D, or ^X15 is R, ^X16 is W and ^X17 is Y; the LCDR2 amino acid sequence as shown in SEQ ID NO: 13; and the LCDR3 amino acid sequence as shown in SEQ ID NO: 14, wherein ^X18 is S, ^X19 is N, ^X20 is V and ^X21 is D, or ^X18 is W, ^X19 is H, ^X20 is I and ^X21 is L.

In certain embodiments, the anti-FRα antibody constructs of the present disclosure comprise an antigen binding domain having:

(i) a HCDR1 amino acid sequence selected from the group consisting of variants v23924, v30618, v30384, v30389, v30394, v30399, v31422, v31423, v31424, v31425, v31426, v35305, v35342, v35347, v35348, v35350, v35354, v3 a HCDR1 amino acid sequence selected from 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 a HCDR3 amino acid sequence selected from variants v23924, v30618, v30384, v30389, v3 the HCDR3 amino acid sequence of any one of v30394, v30399, v31422, v31423, v31424, v31425, v31426, v35305, v35342, v35347, v35348, v35350, v35354, v35356, v35358, v36167, v36168, or v36675, and

(ii) a LCDR1 amino acid sequence selected from the group consisting of variants v23924, v30618, v30384, v30389, v30394, v30399, v31422, v31423, v31424, v31425, v31426, v35305, v35342, v35347, v35348, v35350, v35354, v35355 a LCDR1 amino acid sequence selected from variants v23924, v30618, v30384, v30389, v30394, v30399, v31422, v31423, v31424, v31425, v31426, v31427, v31428, v31429, v3142A, v3142B, v3142C, v3142D, v3142E, v3142F, v3142A, v3142A, v3142A, v3142B, v3142C, v3142D, v3142E, v3142A, v3142A, v3142A 6, a LCDR2 amino acid sequence of any one of v35305, v35342, v35347, v35348, v35350, v35354, v35356, v35358, v36167, v36168, or v36675; and a LCDR3 amino acid sequence selected from variants v23924, v30618, v30384, v30389, v35391, v35410, v35411, v35413, v35414, v35415 the LCDR3 amino acid sequence of any one of v30394, v30399, v31422, v31423, v31424, v31425, v31426, v35305, v35342, v35347, v35348, v35350, v35354, v35356, v35358, v36167, v36168, or v36675,

The CDR amino acid sequences are defined in any of the IMGT, Chothia, Kabat, Contact or AbM numbering systems (see Figure 11).

In certain embodiments, the anti-FRα antibody constructs of the present disclosure comprise an antigen binding domain having a heavy chain CDR amino acid sequence (HCDR1, HCDR2, and HCDR3) selected from variants v23924, v30618, v30384, v30389, v30394, v30399, v31422, v31423, v31424, v31425, v31426, v35305, v35342, v35347, v35348, v35350, v35354, v35356, v35357, v35358, v35359, v35361, v35362, v35363, v35364, v35365, v35366, v35367, v35368, v35369, v35370, v35371, v35372, v35373, v35374, v35375 The heavy chain CDR amino acid sequence (HCDR1, HCDR2 and HCDR3) of any one of v6167, v36168 or v36675, and the light chain CDR amino acid sequence is selected from variants v23924, v30618, v30384, v30389, v30394, v33967, v33968, v33969, v34081, v34083, v34084, v34085, v34086, v34087, v34088, v34089, v34083, v34087, v34088, v34089, v34081 The light chain CDR amino acid sequences (LCDR1, LCDR2, and LCDR3) of any one of: V399, v31422, v31423, v31424, v31425, v31426, v35305, v35342, v35347, v35348, v35350, v35354, v35356, v35358, v36167, v36168, or v36675.

In certain embodiments, the anti-FRα antibody constructs of the present disclosure comprise an antigen binding domain comprising variants v23924, v30618, v30384, v30389, v30394, v30399, v31422, v31423, v31424, as defined by any of the IMGT, Chothia, Kabat, Contact, or AbM numbering systems. The heavy chain CDR amino acid sequence (HCDR1, HCDR2, and HCDR3) and light chain CDR amino acid sequence (LCDR1, LCDR2, and LCDR3) of any one of 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 VH domain of any 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 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. Figure 12 provides 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.

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 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. 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 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.

In certain embodiments, the anti-FRα antibody constructs of the present disclosure comprise an antigen binding domain comprising a VH amino acid sequence and a VL amino acid sequence selected from the VH and 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.

Those skilled in the art will appreciate 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 the antibody losing its ability to bind to its target. Candidate amino acid substitutions can be identified by computer modeling or by techniques known in the art such as alanine scanning, and the binding activity of the resulting variants tested by standard techniques. Thus, in certain embodiments, the anti-FRα antibody constructs of the present disclosure comprise an antigen binding domain comprising a variant v23924, v30618, v30384, v30389, v30394, v30399, v31422, v31423, v31424, v31425, v31426, v35305, v35342, v35347, v35348, v35350, v35354, v35355 A set of CDRs of any one of v56, v35358, v36167, v36168 or v36675 has a set of CDRs (i.e., heavy chain HCDR1, HCDR2 and HCDR3 and light chain LCDR1, LCDR2 and LCDR3) with 90% or more, 95% or more, 98% or more, 99% or more or 100% sequence identity, wherein the % sequence identity is calculated in all six CDRs, and wherein the antigen binding domain retains the ability to bind to hFRα.

In certain embodiments, an anti-FRα antibody construct of the present disclosure comprises an antigen binding domain comprising a variant of a 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 between the set of CDRs (i.e., the CDRs can be modified by up to 10 amino acid substitutions, wherein any combination of the 6 CDRs are modified), 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 an antigen binding domain comprising variants v23924, v30618, v30384, v30389, v30394, v30399, v31422, v31423, v31424, v31425, v31426, v35305, v35342, v35347, v35348, v353 The invention relates to a variant of a set of CDR sequences of any one of v50, v35354, v35356, v35358, v36167, v36168 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 in the set of CDRs, and wherein the antigen binding domain retains the ability to bind to hFRα.

In certain embodiments, the anti-FRα antibody constructs of the present disclosure comprise an antigen binding domain comprising a polypeptide that is identical to variants v23924, v30618, v30384, v30389, v30394, v30399, v31422, v31423, v31424, v31425, v31426, v35305, v35342, v35347, v35348, v35350, The VH sequence of any one of v35354, v35356, v35358, v36167, v36168 or v36675 has 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, wherein the antigen binding domain retains the ability to bind to hFRα. In certain embodiments, the anti-FRα antibody constructs of the present disclosure comprise an antigen binding domain comprising a polypeptide that is identical to variants v23924, v30618, v30384, v30389, v30394, v30399, v31422, v31423, v31424, v31425, v31426, v35305, v35342, v35347, v35348, v35350, The VL sequence of any one of v35354, v35356, v35358, v36167, v36168 or v36675 has 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, wherein the antigen binding domain retains the ability to bind to hFRα.

In certain embodiments, the anti-FRα antibody constructs of the present disclosure comprise an antigen binding domain having:

(i) a HCDR1 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 20, 23, 26, 28, 31, 92, 93, 94, 95 or 96; a HCDR2 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 21, 24, 27, 29, 32, 51, 58, 100, 101, 102, 103, 109, 137, 138 or 139; and a HCDR3 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 22, 25, 30, 107, 108 or 110, and

(ii) a LCDR1 amino acid sequence selected from the group consisting of an amino acid sequence as shown in any one of SEQ ID NOs: 40, 43, 45, 65, 125, 126 or 127; a LCDR2 amino acid sequence selected from the group consisting of an amino acid sequence as shown in any one of SEQ ID NOs: 41, 44 or 46; and a LCDR3 amino acid sequence selected from the group consisting of an amino acid sequence as shown in any one of SEQ ID NOs: 42, 47, 120 or 121.

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

In certain embodiments, the anti-FRα antibody constructs of the present disclosure comprise an antigen binding domain having:

(a) a LCDR1 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 40, 43 or 45; a LCDR2 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 41, 44 or 46; and a LCDR3 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 42 or 47; and a HCDR1 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 20, 23, 26, 28 or 31; a HCDR2 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 21, 24, 27, 29 or 32; and a HCDR3 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 22, 25 or 30;

(b) a LCDR1 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 125, 126 or 127; a LCDR2 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 41, 44 or 46; and a LCDR3 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 42 or 47; and a HCDR1 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 92, 93, 94, 95 or 96; a HCDR2 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 21, 24, 29, 32 or 51; and a HCDR3 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 22, 25 or 30;

(c) a LCDR1 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 40, 45 or 65; a LCDR2 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 41, 44 or 46; and a LCDR3 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 42 or 47; and

(i) a HCDR1 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 20, 23, 26, 28 or 31; a HCDR2 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 21, 24, 29, 32 or 51; a HCDR3 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 22, 25 or 30; or

(ii) a HCDR1 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 20, 23, 26, 28 or 31; a HCDR2 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 21, 24, 29, 32 or 58; a HCDR3 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 22, 25 or 30; or

(iii) a HCDR1 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 92, 93, 94, 95 or 96; a HCDR2 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 24, 100, 101, 102 or 103; a HCDR3 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 22, 25 or 30; or

(d) a LCDR1 amino acid sequence selected from the group consisting of an amino acid sequence as shown in any one of SEQ ID NOs: 125, 126 or 127; a LCDR2 amino acid sequence selected from the group consisting of an amino acid sequence as shown in any one of SEQ ID NOs: 41, 44 or 46; and a LCDR3 amino acid sequence selected from the group consisting of an amino acid sequence as shown in any one of SEQ ID NOs: 120 or 121; and

(i) a HCDR1 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 92, 93, 94, 95 or 96; a HCDR2 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 24, 100, 101, 102 or 103; a HCDR3 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 22, 25 or 30; or

(ii) a HCDR1 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 92, 93, 94, 95 or 96; a HCDR2 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 21, 24, 29, 32 or 109; a HCDR3 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 107, 108 or 110; or

(iii) a HCDR1 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 92, 93, 94, 95 or 96; a HCDR2 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 21, 24, 29, 32 or 109; a HCDR3 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 22, 25 or 30; or

(iv) a HCDR1 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 20, 23, 26, 28 or 31; a HCDR2 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 21, 24, 29, 32 or 109; a HCDR3 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 107, 108 or 110; or

(v) a HCDR1 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 20, 23, 26, 28 or 31; a HCDR2 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 21, 24, 29, 32 or 109; a HCDR3 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 22, 25 or 30; or

(vi) a HCDR1 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 92, 93, 94, 95 or 96; a HCDR2 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 21, 24, 137, 138 or 139; a HCDR3 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 22, 25 or 30; or

(e) a LCDR1 amino acid sequence selected from the group consisting of an amino acid sequence as shown in any one of SEQ ID NOs: 40, 45 or 65; a LCDR2 amino acid sequence selected from the group consisting of an amino acid sequence as shown in any one of SEQ ID NOs: 41, 44 or 46; and a LCDR3 amino acid sequence selected from the group consisting of an amino acid sequence as shown in any one of SEQ ID NOs: 120 or 121; and

(i) a HCDR1 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 92, 93, 94, 95 or 96; a HCDR2 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 21, 24, 29, 32 or 109; a HCDR3 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 107, 108 or 110; or

(ii) a HCDR1 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 20, 23, 26, 28 or 31; a HCDR2 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 21, 24, 29, 32 or 109; and a HCDR3 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 107, 108 or 110.

In certain embodiments, the anti-FRα antibody constructs of the present disclosure comprise an antigen binding domain having:

(a) a HCDR1 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 20, 23, 26, 28 or 31; a HCDR2 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 21, 24, 27, 29 or 32; a HCDR3 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 22, 25 or 30; a LCDR1 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 40, 43 or 45; a LCDR2 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 41, 44 or 46; and a LCDR3 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 42 or 47; or

(b) a HCDR1 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 20, 23, 26, 28 or 31; a HCDR2 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 21, 24, 29, 32 or 51; a HCDR3 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 22, 25 or 30; a LCDR1 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 40, 45 or 65; a LCDR2 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 41, 44 or 46; and a LCDR3 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 42 or 47; or

(c) a HCDR1 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 20, 23, 26, 28 or 31; a HCDR2 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 21, 24, 29, 32 or 58; a HCDR3 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 22, 25 or 30; a LCDR1 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 40, 45 or 65; a LCDR2 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 41, 44 or 46; and a LCDR3 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 42 or 47; or

(d) a HCDR1 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 92, 93, 94, 95 or 96; a HCDR2 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 21, 24, 29, 32 or 51; a HCDR3 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 22, 25 or 30; a LCDR1 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 125, 126 or 127; a LCDR2 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 41, 44 or 46; and a LCDR3 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 42 or 47; or

(e) a HCDR1 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 92, 93, 94, 95 or 96; a HCDR2 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 24, 100, 101, 102 or 103; a HCDR3 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 22, 25 or 30; a LCDR1 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 125, 126 or 127; a LCDR2 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 41, 44 or 46; and a LCDR3 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 120 or 121; or

(f) a HCDR1 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 92, 93, 94, 95 or 96; a HCDR2 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 21, 24, 29, 32 or 109; a HCDR3 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 107, 108 or 110; a LCDR1 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 40, 45 or 65; a LCDR2 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 41, 44 or 46; and a LCDR3 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 42 or 47; or

(g) a HCDR1 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 92, 93, 94, 95 or 96; a HCDR2 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 21, 24, 29, 32 or 109; a HCDR3 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 107, 108 or 110; a LCDR1 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 40, 45 or 65; a LCDR2 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 41, 44 or 46; and a LCDR3 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 120 or 121; or

(h) a HCDR1 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 92, 93, 94, 95 or 96; a HCDR2 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 21, 24, 29, 32 or 109; a HCDR3 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 107, 108 or 110; a LCDR1 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 125, 126 or 127; a LCDR2 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 41, 44 or 46; and a LCDR3 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 120 or 121; or

(i) a HCDR1 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 92, 93, 94, 95 or 96; a HCDR2 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 21, 24, 29, 32 or 109; a HCDR3 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 22, 25 or 30; a LCDR1 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 125, 126 or 127; a LCDR2 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 41, 44 or 46; and a LCDR3 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 120 or 121; or

(j) a HCDR1 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 20, 23, 26, 28 or 31; a HCDR2 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 21, 24, 29, 32 or 109; a HCDR3 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 107, 108 or 110; a LCDR1 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 40, 45 or 65; a LCDR2 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 41, 44 or 46; and a LCDR3 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 120 or 121; or

(k) a HCDR1 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 20, 23, 26, 28 or 31; a HCDR2 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 21, 24, 29, 32 or 109; a HCDR3 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 107, 108 or 110; a LCDR1 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 125, 126 or 127; a LCDR2 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 41, 44 or 46; and a LCDR3 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 120 or 121; or

(l) a HCDR1 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 20, 23, 26, 28 or 31; a HCDR2 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 21, 24, 29, 32 or 109; a HCDR3 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 22, 25 or 30; a LCDR1 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 125, 126 or 127; a LCDR2 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 41, 44 or 46; and a LCDR3 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 120 or 121; or

(m) a HCDR1 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 92, 93, 94, 95 or 96; a HCDR2 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 21, 24, 137, 138 or 139; a HCDR3 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 22, 25 or 30; a LCDR1 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 125, 126 or 127; a LCDR2 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 41, 44 or 46; and a LCDR3 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 120 or 121.

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

In certain embodiments, the anti-FRα antibody constructs of the present disclosure comprise an antigen binding domain comprising a VH amino acid sequence selected from the VH amino acid sequence shown 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 sequence shown in any one of SEQ ID NOs: 39, 64, 119, 124, or 130.

In certain embodiments, the anti-FRα antibody constructs of the present disclosure comprise an antigen binding domain comprising:

(a) the VL amino acid sequence as shown in SEQ ID NO:39 and the VH amino acid sequence as shown in SEQ ID NO:19; or

(b) the VL amino acid sequence as shown in SEQ ID NO: 124 and the VH amino acid sequence as shown in SEQ ID NO: 91; or

(c) the VL amino acid sequence as shown in SEQ ID NO: 64 and

(i) the VH amino acid sequence as shown in SEQ ID NO: 50, or

(ii) the VH amino acid sequence as shown in SEQ ID NO: 54, or

(iii) the VH amino acid sequence as shown in SEQ ID NO: 57, or

(iv) the VH amino acid sequence as shown in SEQ ID NO: 61, or

(v) the VH amino acid sequence as shown in SEQ ID NO: 76, or

(vi) the VH amino acid sequence as shown in SEQ ID NO: 79, or

(vii) the VH amino acid sequence as shown in SEQ ID NO: 82, or

(viii) the VH amino acid sequence as shown in SEQ ID NO: 85, or

(ix) the VH amino acid sequence as shown in SEQ ID NO: 88, or

(x) the VH amino acid sequence as shown in SEQ ID NO: 106; or

(d) the VL amino acid sequence as shown in SEQ ID NO: 130 and

(i) the VH amino acid sequence as shown in SEQ ID NO: 99, or

(ii) the VH amino acid sequence as shown in SEQ ID NO: 106, or

(iii) the VH amino acid sequence as shown in SEQ ID NO: 113, or

(iv) the VH amino acid sequence as shown in SEQ ID NO: 116, or

(v) the VH amino acid sequence as shown in SEQ ID NO: 133, or

(vi) the VH amino acid sequence as shown in SEQ ID NO: 136; or

(e) the VL amino acid sequence as shown in SEQ ID NO: 119 and

(i) the VH amino acid sequence as shown in SEQ ID NO: 106, or

(ii) the VH amino acid sequence as shown in SEQ ID NO:116.

In certain embodiments, the anti-FRα antibody constructs of the present disclosure comprise an antigen binding domain comprising:

(i) the VH amino acid sequence shown in SEQ ID NO: 19 and the VL amino acid sequence shown in SEQ ID NO: 39, or

(ii) the VH amino acid sequence shown in SEQ ID NO:50 and the VL amino acid sequence shown in SEQ ID NO:64, or

(iii) the VH amino acid sequence shown in SEQ ID NO:54 and the VL amino acid sequence shown in SEQ ID NO:64, or

(iv) the VH amino acid sequence shown in SEQ ID NO:57 and the VL amino acid sequence shown in SEQ ID NO:64, or

(v) the VH amino acid sequence shown in SEQ ID NO:61 and the VL amino acid sequence shown in SEQ ID NO:64, or

(vi) the VH amino acid sequence shown in SEQ ID NO:76 and the VL amino acid sequence shown in SEQ ID NO:64, or

(vii) the VH amino acid sequence shown in SEQ ID NO:79 and the VL amino acid sequence shown in SEQ ID NO:64, or

(viii) the VH amino acid sequence shown in SEQ ID NO: 82 and the VL amino acid sequence shown in SEQ ID NO: 64, or

(ix) the VH amino acid sequence shown in SEQ ID NO: 85 and the VL amino acid sequence shown in SEQ ID NO: 64, or

(x) the VH amino acid sequence shown in SEQ ID NO: 88 and the VL amino acid sequence shown in SEQ ID NO: 64, or

(xi) the VH amino acid sequence shown in SEQ ID NO:91 and the VL amino acid sequence shown in SEQ ID NO:124, or

(xii) the VH amino acid sequence shown in SEQ ID NO:99 and the VL amino acid sequence shown in SEQ ID NO:130, or

(xiii) the VH amino acid sequence shown in SEQ ID NO: 106 and the VL amino acid sequence shown in SEQ ID NO: 64, or

(xiv) the VH amino acid sequence shown in SEQ ID NO: 106 and the VL amino acid sequence shown in SEQ ID NO: 119, or

(xv) the VH amino acid sequence shown in SEQ ID NO: 106 and the VL amino acid sequence shown in SEQ ID NO: 130, or

(xvi) the VH amino acid sequence shown in SEQ ID NO: 113 and the VL amino acid sequence shown in SEQ ID NO: 130, or

(xvii) the VH amino acid sequence shown in SEQ ID NO: 116 and the VL amino acid sequence shown in SEQ ID NO: 119, or

(xviii) the VH amino acid sequence shown in SEQ ID NO: 116 and the VL amino acid sequence shown in SEQ ID NO: 130, or

(xix) the VH amino acid sequence shown in SEQ ID NO: 133 and the VL amino acid sequence shown in SEQ ID NO: 130, or

(xx) the VH amino acid sequence shown in SEQ ID NO:136 and the VL amino acid sequence shown in SEQ ID NO:130.

Format

Anti-FRα antibody constructs can have various formats. The minimum component of an anti-FRα antibody construct is an antigen binding domain that binds hFRα. The anti-FRα antibody construct may also optionally include 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, may bind to different epitopes within hFRα, or may bind to different antigens. Therefore, the anti-FRα antibody construct may be, for example, monospecific, biparatopic, bispecific, or multispecific.

In certain embodiments, the anti-FRα antibody construct comprises at least one antigen binding domain that binds 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.

In certain embodiments, the anti-FRα antibody construct comprises two antigen binding domains that are optionally operably linked to a scaffold. In some embodiments, the anti-FRα antibody construct may comprise three or four antigen binding domains and optionally a scaffold. In these formats, when a scaffold is included, at least the first antigen binding domain is operably linked to the scaffold, and the remaining antigen binding domains can each be independently operably linked to the scaffold or the first antigen binding domain, or when there are more than two antigen binding domains, operably linked to another antigen binding domain.

Anti-FRα antibody constructs lacking a scaffold may include a single antigen binding domain in an appropriate format, such as an sdAb, or they may include two or more antigen binding domains optionally operably connected by one or more joints. In such anti-FRα antibody constructs, the antigen binding domain may be in the format of scFv, Fab, sdAb, or a combination thereof. For example, using scFv as an antigen binding domain, a format such as a tandem scFv ((scFv) ₂ or taFv) may be constructed, wherein the scFvs are connected together by a flexible joint. ScFv can also be used to construct a double antibody format, which includes two scFvs connected by a short joint (usually about 5 amino acids in length). The restricted joint length causes the scFv to dimerize in a head-to-tail manner. In any of the aforementioned forms, the scFv can be further stabilized by including an interdomain disulfide bond. For example, a disulfide bond can be introduced between VL and VH by introducing an additional cysteine residue in each chain (e.g., at position 44 of VH and position 100 of VL) (see, e.g., Fitzgerald et al., 1997, Protein Engineering, 10: 1221-1225), or a disulfide bond can be introduced between the two VHs to provide a construct with a DART format (see, e.g., Johnson et al., 2010, J Mol. Biol., 399: 436-449).

Similarly, in some embodiments, a format comprising two sdAbs (such as VH or VHH) linked together by a suitable linker may be employed. Other examples of anti-FRα antibody construct formats lacking a scaffold include those based on Fab fragments, such as Fab ₂ and F(ab') ₂ formats, in which the Fab fragments are linked by a linker or an IgG hinge region.

Combinations of antigen binding domains of varying sizes can also be employed to generate alternative scaffold-free formats. For example, a scFv or sdAb can be fused to the C-terminus of one or both of the light and heavy chains of a Fab fragment, thereby generating a bivalent (Fab-scFv/sdAb) construct.

In certain embodiments, the anti-FRα antibody construct may be an antibody format based on an immunoglobulin (Ig). In certain embodiments, the anti-FRα antibody construct may be based on an IgG class immunoglobulin, such as an IgG1, IgG2, IgG3, or IgG4 immunoglobulin. In some embodiments, the anti-FRα antibody construct may be based on an IgG1 immunoglobulin. In the context of the present disclosure, when the anti-FRα antibody construct is based on a specified immunoglobulin isotype, it means that the anti-FRα antibody construct comprises all or part of the constant region of the specified immunoglobulin isotype. For example, an anti-FRα antibody construct based on a given Ig isotype may comprise at least one antigen binding domain operably linked to an Ig scaffold, wherein the scaffold comprises an Fc region from a given isotype and optionally an Ig hinge region from the same or different isotypes. It should be understood that in some embodiments, the anti-FRα antibody construct may also comprise a hybrid of an isotype and/or subclass. It should also be understood that the Fc region and/or hinge region may be optionally modified to confer one or more desired functional properties known in the art.

In some embodiments, the anti-FRα antibody construct can be derived from two or more immunoglobulins from different species, for example, the anti-FRα antibody construct can be a chimeric antibody or a humanized antibody. The terms "chimeric antibody" and "humanized antibody" generally refer to antibodies that combine immunoglobulin regions or domains from more than one species.

"Chimeric antibodies" typically comprise 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 domain of a chimeric antibody need not have the same isotype as the non-human constant domain it replaces. 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.

"Humanized antibodies" are a class of chimeric antibodies that contain minimal sequences derived from non-human antibodies. Typically, humanized antibodies are human immunoglobulins (recipient antibodies) in which residues from the hypervariable regions (CDRs) of the recipient are replaced with residues from hypervariable regions (CDRs) of non-human species (donor antibodies) that have the desired specificity and affinity for the target antigen, such as mice, rats, rabbits, or non-human primates. This technique for creating humanized antibodies is often referred to as "CDR transplantation."

In some cases, additional modifications may be made to humanized antibodies to further improve antibody performance. For example, the framework region (FR) residues of human immunoglobulins are replaced by corresponding non-human residues, or humanized antibodies may include residues not found in recipient antibodies or donor antibodies. In general, the variable domains in humanized antibodies will include all or nearly all hypervariable regions from non-human immunoglobulins and all or nearly all FRs from human immunoglobulin sequences. Humanized antibodies are described in more detail in, for example, 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.

Many methods are known in the art for selecting the most appropriate human framework for transplanting non-human CDR therein. Early methods use a limited subset of fully characterized human antibodies, independent of the sequence identity of the non-human antibodies providing CDR ("fixed framework" method). The most recent method has adopted a variable region with high amino acid sequence identity to the variable region of the non-human antibodies providing CDR ("homologous matching" or "best fit" method). An alternative method is to select a fragment of framework sequence from each light chain or heavy chain variable region from several different human antibodies. In some cases, CDR transplantation may cause the affinity of the transplanted molecule to its target antigen to be partially or completely lost. In such cases, affinity can be restored by backmutating some of the residues in some human sources to corresponding non-human residues. Methods for preparing humanized antibodies by these methods 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).

Alternatively, or in addition to these traditional methods, newer technology can be used to further reduce the immunogenicity of the humanized antibody of CDR transplantation.For example, a framework based on human germline sequence or consensus sequence can be used as a receptor human framework rather than a human framework with somatic mutations.Another technology intended to reduce the potential immunogenicity of non-human CDR is to transplant specificity determining residues (SDR) only.In this method, only the minimum CDR residues (" SDR ") required for antigen binding activity are transplanted into the human germline framework.This method improves the "humanity" (i.e., similarity to human germline sequence) of humanized antibodies, and therefore can contribute to reducing the immunogenicity risk of variable regions. These techniques have been 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).

In certain embodiments, the anti-FRα antibody constructs of the present disclosure comprise humanized antibody sequences, such as one or more humanized variable domains. In some embodiments, the anti-FRα antibody constructs may be humanized antibodies. Non-limiting examples of humanized antibodies based on the anti-FRα antibody v23924 are described herein (v30384, v30389, v30394, v30399, v31422, v31423, v31424, v31425, and v31426; see Examples and Sequence Listing).

Bracket

In certain embodiments, the anti-FRα antibody construct comprises one or more antigen binding domains operably linked to a scaffold. The antigen binding domain may be 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, heterodimerized peptides (such as leucine zippers, "zipper" peptides derived from Jun and Fos forming heterodimers, IgG CH1 and CL domains, or barnase-barstar toxins), cytokines, chemokines, or growth factors. Other examples include antibodies based on DOCK-AND-LOCK ^™ (DNL ^™ ) technology developed by IBC Pharmaceuticals, Inc. and Immunomedics, Inc. (see, e.g., Chang et al., 2007, Clin. Cancer Res., 13: 5586s-5591s).

The scaffold can be a peptide, polypeptide, polymer, nanoparticle or other chemical entity. When the scaffold is a polypeptide, each antigen binding domain of the anti-FRα antibody construct can be connected to the N-terminus or C-terminus of the polypeptide scaffold. Anti-FRα antibody constructs comprising polypeptide scaffolds are also contemplated in certain embodiments, wherein one or more antigen binding domains are connected to regions other than the N-terminus or C-terminus, for example, via amino acid side chains with or without a linker.

In embodiments where the anti-FRα antibody construct comprises a scaffold that is a peptide or polypeptide, the antigen binding domain can be attached to the scaffold by genetic fusion or chemical conjugation. Typically, when the scaffold is a peptide or polypeptide, the antigen binding domain is attached to the scaffold by genetic fusion. In some embodiments, where the scaffold is a polymer or nanoparticle, the antigen binding domain can be attached to the scaffold by chemical conjugation.

Many protein domains are known in the art that contain selective pairing of two different polypeptides and can be used to form a scaffold. Examples are leucine zipper domains that are selectively paired together, such as Fos and Jun (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. WO 2011/135040).

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., Müller 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).

For example, it has been demonstrated that fusing an antigen-binding moiety such as scFv, diabody or single-chain diabody to albumin can improve the serum half-life of the antigen-binding moiety (Müller et al., supra). The antigen-binding moiety may be fused to the N-terminus and/or C-terminus of albumin, optionally via a linker.

Albumin derivatives in the form of heteromultimers have been described, which comprise two transporter polypeptides obtained by fragmenting albumin, such that the transporter polypeptides self-assemble to form a natural albumin-like structure (see International Patent Application Publication Nos. WO 2012/116453 and WO 2014/012082). Due to the fragmentation of albumin, the heteromultimer comprises four termini and can therefore optionally be fused to up to four different antigen-binding moieties via a linker.

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 (e.g., an IgG Fc region).

Fc region

As used herein, the term "Fc region," "Fc" or "Fc domain" refers to the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of a constant region. The term includes native sequence Fc regions and variant Fc regions. Unless otherwise indicated herein, the numbering of amino acid residues in 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).

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 consist of two Fc polypeptides, or alternatively, the Fc region may consist of a single polypeptide.

In the case of dimeric Fc, "Fc polypeptide" refers to one of the two polypeptides forming the dimeric Fc domain, i.e., a polypeptide comprising one or more C-terminal constant regions of immunoglobulin heavy chains capable of stable self-association. When referring to polypeptides forming the dimeric Fc region, the terms "first Fc polypeptide" and "second Fc polypeptide" can be used interchangeably, provided that the Fc region comprises one first Fc polypeptide and one second Fc polypeptide.

The Fc region may comprise a CH3 domain or it may comprise both a CH3 and a CH2 domain. For example, in certain embodiments, the Fc polypeptide 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.

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.

In certain embodiments, the anti-FRα antibody construct may comprise a scaffold based on an IgG Fc region, which 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 sequence of the first and second Fc polypeptides are identical.

In certain embodiments, the anti-FRα antibody construct may include a scaffold based on an IgG Fc region, the IgG Fc region being a heterodimeric 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 different. In some embodiments, the anti-FRα antibody construct may include a scaffold based on an Fc region, the Fc region comprising two CH3 sequences, wherein at least one CH3 sequence comprises one or more amino acid modifications. In some embodiments, the anti-FRα antibody construct may include a scaffold based on an Fc region, the Fc region comprising two CH3 sequences and two CH2 sequences, wherein at least one CH2 sequence comprises one or more amino acid modifications.

In some embodiments, the anti-FRα antibody construct may comprise a heterodimeric Fc region comprising a modified CH3 domain, wherein 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 the amino acid at a specific position on the first CH3 or CH2 sequence is different from the amino acid at the same position on the second CH3 or CH2 sequence. These asymmetric amino acid modifications may be the result of modification of only one of the two amino acids at the same corresponding amino acid position on each sequence, or different modifications of the two amino acids at the same corresponding 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 may comprise one or more asymmetric amino acid modifications.

In some embodiments, the anti-FRα antibody construct may comprise a heterodimeric Fc containing a modified CH3 domain, wherein the modified CH3 domain comprises one or more amino acid modifications that promote heterodimeric Fc formation over homodimeric Fc formation. In some embodiments, one or more of the amino acid modifications are asymmetric amino acid modifications.

Amino acid modifications that can be made to the CH3 domain of Fc to promote the formation of heterodimeric Fc are known in the art and include, for example, those described in International Publication No. WO 96/027011 ("knob-mortise"), Gunasekaran et al., 2010, J Biol Chem, 285, 19637-46 ("electrostatic manipulation"), Davis et al., 2010, Prot Eng Des Sel, 23(4): 195-202 (chain 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 methods of combining positive and negative design strategies to generate stable asymmetric modified Fc regions, such as those described in International Publication Nos. WO 2012/058768 and WO 2013/063702. In certain embodiments, an anti-FRα antibody construct may comprise a modified Fc region based scaffold as described in International Publication Nos. WO 2012/058768 or WO 2013/063702.

Table 5 provides the amino acid sequence of a human IgG1 Fc sequence (SEQ ID NO: 16), which corresponds to amino acids 231 to 447 of a full-length human IgG1 heavy chain. The CH3 sequence comprises amino acids 341-447 of a full-length human IgG1 heavy chain. Also shown in Table 5 are CH3 domain amino acid modifications that promote heterodimeric Fc formation, as described in International Patent Application Publication Nos. WO 2012/058768 and WO 2013/063702.

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

Table 5: Human IgG1 Fc sequence and CH3 domain amino acid modifications that promote heterodimer formation

¹ Sequence at position 231-447 (EU numbering)

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

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.

A variety of amino acid modifications that selectively change the affinity of Fc for different Fcγ receptors to the CH2 domain are known in the art. Amino acid modifications that lead to increased binding and amino acid modifications that lead to reduced binding can each be used for certain indications. For example, increasing the binding affinity of Fc to FcγRIIIa (an activating receptor) can cause an increase in antibody-dependent cell-mediated cytotoxicity (ADCC), which in turn causes an increase in the lysis of target cells. Reducing the binding to FcγRIIb (an inhibitory receptor) may also be beneficial in some cases. In certain indications, it may be necessary to reduce or eliminate ADCC and complement-mediated cytotoxicity (CDC). In such cases, a modified CH2 domain ("knockout" variant) comprising an amino acid modification that leads to an increase in binding to FcγRIIb or reduces or eliminates the binding of the Fc region to all Fcγ receptors may be useful.

Examples of amino acid modifications to the CH2 domain that alter the binding of Fcγ receptors to Fc include, but are not limited to, the following: S298A/E333A/K334A and S298A/E333A/K334A/K326A (increased affinity for FcγRIIIa) (Lu, et al., 2011, J Immunol Methods, 365(1-2):132-41); F243L/R292P/Y300L/V305I/P396L (increased affinity for FcγRIIIa) (Stavenha gen, et al., 2007, Cancer Res, 67(18):8882-90); F243L/R292P/Y300L/L235V/P396L (increased affinity for FcγRIIIa) (Nordstrom JL, et 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 change the binding of FcγRIIb to Fc are described in International Publication No. WO 2021/232162. Additional modifications that affect the binding of Fc 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).

In certain embodiments, the anti-FRα antibody construct comprises an IgG Fc based scaffold with a modified CH2 domain comprising one or more amino acid modifications that result in reduced or eliminated binding of the Fc region to all Fcγ receptors (ie, a "knockout" variant).

Various publications describe strategies that have been used to engineer antibodies to produce "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, pp. 225-249). These strategies include reducing effector function by glycosylation modification, using IgG2/IgG4 scaffolds, or introducing mutations in the hinge or CH2 domains of Fc (see also U.S. Patent Publication No. 2011/0212087, International Publication No. WO 2006/105338, 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).

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

In certain embodiments, the anti-FRα antibody constructs described herein may comprise an IgG Fc-based scaffold in which the native glycosylation has been modified. As is known in the art, the sugar groups of Fc can be modified 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 the production of aglycosylated Fc lacking all effector functions (Bolt et al., 1993, Eur. J. Immunol., 23: 403-411; Tao & Morrison, 1989, J. Immunol., 143: 2595-2601).

In contrast, removal of fucose from heavy chain N297-linked oligosaccharides has been shown to enhance ADCC based on 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 that lack fucosyltransferase (FUT8) (Yamane-Ohnuki et al., 2004, Biotechnol. Bioeng., 87:614-622); in a variant CHO cell line Lec 13 with reduced ability to attach fucose to N297-linked carbohydrates (International Publication No. WO 03/035835), or other cells that produce afucosylated 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). Additionally, International Publication No. WO 2009/135181 describes the addition of a fucose analog to the culture medium during antibody production to inhibit the incorporation of fucose into carbohydrates on the antibody.

Other methods for producing antibodies containing little or no fucose at the Fc glycosylation site (N297) are well known in the art. For example, Technology (ProBioGen AG) (see von Horsten et al., 2010, Glycobiology, 20(12): 1607-1618 and US Pat. No. 8,409,572).

Other glycosylation variants include those with bisected oligosaccharide variants, for example, wherein the biantennary oligosaccharide connected to the Fc region of the antibody is bisected by N-acetylglucosamine (GlcNAc) variants. Such glycosylation variants can have reduced fucosylation and/or improved ADCC function (see, e.g., International Publication No. WO 2003/011878, U.S. Patent No. 6,602,684 and U.S. Patent Application Publication No. US 2005/0123546). Useful glycosylation variants also include those variants having at least one galactose residue in the oligosaccharide connected to the Fc region, which can have improved CDC function (see, e.g., International Publication No. WO 1997/030087, WO 1998/58964 and WO 1999/22764).

In certain embodiments, the anti-FRα antibody construct has the format of a full-size antibody (FSA). In some embodiments, the anti-FRα antibody construct has an IgG FSA format, such as 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 and comprises 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 and comprises 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.

In certain embodiments, the anti-FRα antibody construct is a FSA having a set of H1, H2, L1, and L2 sequences comprising the H1, H2, L1, and L2 amino acid sequences as shown in Tables A and B for any 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 in certain cell lines or from certain expression vectors can result in the inclusion of a C-terminal lysine residue on one or both heavy chains. Thus, 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 comprising the H1, H2, L1, and L2 amino acid sequences as shown in Tables A and B for any 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 comprise a C-terminal lysine (see, e.g., SEQ ID NO:157).

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,” 2nd edition, Greenfield, ed., Cold Spring Harbor Laboratory Press, New York, 2014).

Typically, in order to recombinantly produce an antibody construct, a polynucleotide or a set of polynucleotides encoding an anti-FRα antibody construct is produced and inserted into one or more vectors for further cloning and/or expression in a host cell. Polynucleotides encoding anti-FRα antibody constructs can be produced by standard methods known in the art (see, e.g., Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1994 and updates, and "Antibodies: A Laboratory Manual," 2nd edition, Greenfield ed., Cold Spring Harbor Laboratory Press, New York, 2014). As will be appreciated by those skilled in the art, the number of polynucleotides required to express an anti-FRα antibody construct will depend on the format of the construct, including whether the antibody construct contains a scaffold. For example, when the anti-FRα antibody construct is in the mAb format with a homodimer Fc, two polynucleotides encoding each polypeptide chain will be required, and when the anti-FRα antibody construct is in the mAb format with a heterodimer Fc, three polynucleotides encoding each polypeptide chain will be required. When multiple polynucleotides are desired, they can be incorporated into one vector or more than one vector.

Typically, for expression, a polynucleotide or a group of polynucleotides is incorporated into one or more expression vectors together with one or more regulatory elements, such as transcription elements, which are required for the effective transcription of the polynucleotides. Examples of such regulatory elements include, but are not limited to, promoters, enhancers, terminators, and polyadenylation signals. It will be appreciated by those skilled in the art that the selection of regulatory elements depends on the host cell selected for expressing the antibody construct, and such regulatory elements may be derived from a variety of sources, including bacteria, fungi, viruses, mammals, or insect genes. The expression vector may optionally further contain a heterologous nucleic acid sequence, which 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 vector or an integration vector.

Suitable host cells for cloning or expressing anti-FRα antibody constructs include various prokaryotic or eukaryotic cells known in the art. Eukaryotic host cells include, for example, mammalian cells, plant cells, insect cells, and yeast cells (such as Saccharomyces or Pichia cells). Prokaryotic host cells include, for example, Escherichia coli, Aeromonas salmonicida, or Bacillus subtilis cells.

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

In certain embodiments, eukaryotic microorganisms such as filamentous fungi or yeast may be suitable expression host cells, particularly fungi and yeast strains whose glycosylation pathways have been "humanized" to result 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).

Suitable host cells for expressing glycosylated anti-FRα antibody constructs are generally eukaryotic cells. For example, U.S. Pat. Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978 and 6,417,429 describe PLANTIBODIES ^™ technology for producing antigen-binding constructs in transgenic plants. Mammalian cell lines adapted for growth in suspension are particularly useful for expressing antibody constructs. Examples include, but are not limited to, monkey kidney CV1 line transformed by SV40 (COS-7), 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 liver cells (BRL 3A), human lung cells (W138), human liver cells (Hep G2), mouse mammary tumor (MMT 060562), TRI cells (see, e.g., Mather et al., 1982, Annals NY Acad Sci, 383:44-68), MRC 5 cells, FS4 cells, Chinese hamster ovary (CHO) cells (including DHFR ^- 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 producing antibody constructs are reviewed in Yazaki and Wu, Methods in Molecular Biology, Vol. 248, pp. 255-268 (BKCLo ed., Humana Press, Totowa, NJ, 2003).

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

Host cells containing expression vectors encoding anti-FRα antibody constructs can be cultured using conventional methods to produce anti-FRα antibody constructs. Alternatively, in some embodiments, host cells containing expression vectors encoding anti-FRα antibody constructs can be used therapeutically or prophylactically to deliver anti-FRα antibody constructs to subjects, or polynucleotides or expression vectors can be administered ex vivo to cells from subjects and then the cells are returned to the subject's body.

Typically, anti-FRα antibody constructs are purified after expression. Proteins can be isolated or purified in a variety of ways known to those skilled in the art (see, for example, Protein Purification: Principles and Practice, 3rd Edition, Scopes, Springer-Verlag, NY, 1994). Standard purification methods include chromatographic techniques, including ion exchange chromatography, hydrophobic interaction chromatography, affinity chromatography, fractionated chromatography or gel filtration and reverse phase chromatography, performed at atmospheric pressure or high pressure using systems such as FPLC and HPLC. Additional purification methods include electrophoresis, immunization, precipitation, dialysis and chromatofocusing techniques. Ultrafiltration and diafiltration techniques combined with protein concentration are also useful. As is well known in the art, a variety of natural proteins bind to Fc and antibodies, and these proteins can be used to purify certain antibody constructs. For example, bacterial proteins A and G bind to the Fc region. Similarly, the bacterial protein L binds to the Fab region of some antibodies. Purification can also be performed by a specific fusion partner. For example, if a GST fusion is used, the antibody can be purified using glutathione resin, if a His tag is used, the antibody can be purified using Ni ⁺² affinity chromatography, or if a Flag tag is used, the antibody can be purified using an immobilized anti-Flag antibody. The degree of purification necessary will vary depending on the anti-FRα antibody construct used. In some cases, purification may not be necessary.

In certain embodiments, the anti-FRα antibody construct is substantially pure. The term "substantially pure" (or "substantially purified"), when used in reference to the anti-FRα antibody constructs described herein, means that the antibody construct is substantially or essentially free of components that normally accompany or interact with the protein as found in its naturally occurring environment (such as a natural cell, or a host cell in the case of a recombinantly produced construct). In certain embodiments, a substantially pure anti-FRα antibody construct is a protein preparation having less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, or less than about 5% (by dry weight) of contaminating proteins.

Certain embodiments of the present disclosure relate to methods for preparing an anti-FRα antibody construct, the methods comprising culturing a host cell into which has been introduced one or more polynucleotides encoding the anti-FRα antibody construct or one or more expression vectors encoding the anti-FRα antibody construct under conditions suitable for expression of the anti-FRα antibody construct, and optionally recovering the anti-FRα antibody construct from the host cell (or from the host cell culture medium).

Post-translational modification

In certain embodiments, the anti-FRα antibody constructs described herein may comprise one or more post-translational modifications. Such post-translational modifications may occur in vivo, or in vitro after the anti-FRα antibody construct is isolated from a host cell.

Post-translational modifications include various modifications as known in the art (see, e.g., Proteins-Structure and Molecular Properties, 2nd Edition, T.E.Creighton, 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.N.Y.Acad.Sci., 663:48-62). In those embodiments where the anti-FRα antibody construct comprises one or more post-translational modifications, the construct may comprise the same type of modification at one or several sites, or it may comprise different modifications at different sites.

Examples of post-translational modifications include glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, formylation, oxidation, reduction, proteolytic cleavage, or specific chemical cleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8 protease, or _NaBH4 .

Other examples of post-translational modifications include, for example, the addition or removal of N-linked or O-linked carbohydrate chains, chemical modifications of N-linked or O-linked carbohydrate chains, treatment of the N-terminus or C-terminus, attachment of chemical moieties to the amino acid backbone, and addition or deletion of N-terminal methionine residues produced by prokaryotic host cell expression. Post-translational modifications may also include modification with a detectable label (such as an enzyme label, a fluorescent label, a luminescent label, an isotope label, or an affinity label) to allow detection and separation of proteins. Examples of suitable enzyme labels include, but are not limited to, horseradish peroxidase, alkaline phosphatase, β-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, dichlorotriazineamine fluorescein, dansyl chloride, and phycoerythrin. Examples of luminescent materials include luminol and bioluminescent materials such as luciferase, luciferin, and aequorin. Examples of suitable radioactive materials include iodine, carbon, sulfur, tritium, indium, technetium, thallium, gallium, palladium, molybdenum, xenon, and fluorine.

Additional examples of post-translational modifications include acetylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or a nucleotide derivative, covalent attachment of a lipid or a 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.

Camptothecin analogs

The camptothecin analogs included in the ADC of the present disclosure are compounds having formula (I):

in:

^R1 is selected from: -H, _-CH3 , _-CHF2 , _-CF3 , -F, -Br, -Cl, -OH, _-OCH3 , _-OCF3 and _-NH2 , and

R ² is selected from: -H, -CH ₃ , -CF ₃ , -F, -Br, -Cl, -OH, -OCH ₃ and -OCF ₃ ,

And among them:

When R ¹ is -NH ₂ , then R is R ³ or R ⁴ , and when R ¹ is not -NH ₂ , then R is R ⁴ ;

R ³ is selected from: -H, -C ₁ -C ₆ alkyl, -C ₃ -C ₈ cycloalkyl, -(C ₁ -C ₆ alkyl)-OR ⁵ , -CO ₂ R ⁸ , -aryl, -heteroaryl and -(C ₁ -C ₆ alkyl)-aryl;

^R4 is selected from:

R ⁵ is selected from: -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 selected from: -H, -C ₁ -C ₆ alkyl, -C ₃ -C ₈ cycloalkyl, -(C ₁ -C ₆ alkyl)-OR ⁵ , -C ₃ -C ₈ heterocycloalkyl and -C(O)R ¹⁷ ;

R ⁸ is selected from: -H, -C ₁ -C ₆ alkyl, -C ₃ -C ₈ cycloalkyl and -C ₃ -C ₈ heterocycloalkyl;

Each R ⁹ is independently selected from: -H, -C ₁ -C ₆ alkyl, -C ₃ -C ₈ cycloalkyl, -aryl, -heteroaryl, and -(C ₁ -C ₆ alkyl)-aryl;

Each R ¹⁰ is independently selected from: -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′ is selected from: -H, -C ₁ -C ₆ alkyl, -C ₃ -C ₈ cycloalkyl, -aryl, -heteroaryl and -(C ₁ -C ₆ alkyl)-aryl;

R ¹¹ is selected from: -H and -C ₁ -C ₆ alkyl;

R ¹² is selected from: -H, -C ₁ -C ₆ alkyl, -CO ₂ R ⁸ , -aryl, -heteroaryl, -(C ₁ -C ₆ alkyl)-aryl, -S(O) ₂ R ¹⁶ and

R ¹³ is selected from: -H and -C ₁ -C ₆ alkyl;

R ¹⁴ and R ^14' are each independently selected from: -H, C ₁ -C ₆ alkyl, -C ₃ -C ₈ cycloalkyl and -C ₃ -C ₈ heterocycloalkyl;

R ¹⁶ is selected from: -C ₁ -C ₆ alkyl, -C ₃ -C ₈ cycloalkyl, -aryl, -heteroaryl and -(C ₁ -C ₆ alkyl)-aryl;

R ¹⁷ is selected from: -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 bound form a 4-, 5-, 6- or 7-membered ring having 0 to 3 substituents selected from the group consisting of halogen, -C ₁ -C ₆ alkyl, -C ₃ -C ₈ cycloalkyl and -(C ₁ -C ₆ alkyl)-OR ⁵ ;

R ²⁴ , R ²⁵ and R ²⁶ are each -C ₁ -C ₆ alkyl;

^Xa and ^Xb are each independently selected from: NH, O and S, and

^Xc is selected from the group consisting of O, S and S(O) ₂ ,

Provided that the compound is not (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.

In some embodiments, the camptothecin analog is a compound of formula (I), with the proviso that when R ¹ is NH ₂ , R ² is not H.

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 .

In some embodiments, in the compound of Formula (I), R ¹ is NH ₂ .

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 .

In some embodiments, in the compound of formula (I), R ¹ is selected from: -CH ₃ , -CF ₃ , -OCH ₃ , and -OCF ₃ .

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 .

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 .

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

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

In some embodiments, in the compound of formula (I), R ³ is selected from: -H, unsubstituted -C ₁ -C ₆ alkyl, -C ₁ -C ₆ haloalkyl, -C ₁ -C ₆ hydroxyalkyl, -C ₃ -C ₈ cycloalkyl, -(C ₁ -C ₆ alkyl)-OR ⁵ , -CO ₂ R ⁸ , unsubstituted -aryl, -aminoaryl, -heteroaryl and -(C ₁ -C ₆ alkyl)-aminoaryl.

In some embodiments, in the compound of formula (I), R ⁴ is selected from:

In some embodiments, in the compound of formula (I), R ⁵ is selected from: -H, unsubstituted -C ₁ -C ₆ alkyl, -C ₁ -C 6 haloalkyl, -C ₁ -C ₆ hydroxyalkyl, -C ₁ -C ₆ aminoalkyl, -C ₃ -C ₈ cycloalkyl, unsubstituted _-aryl , -aminoaryl, -heteroaryl and -(C ₁ -C ₆ alkyl)-aminoaryl.

In some embodiments, in the compound of formula (I), R ⁶ and R ⁷ are each independently selected from: -H, unsubstituted -C ₁ -C ₆ alkyl, -C ₁ -C ₆ haloalkyl, -C ₁ -C ₆ hydroxyalkyl, -C ₃ -C ₈ cycloalkyl, -(C ₁ -C ₆ alkyl)-OR ⁵ , -C ₃ -C ₈ heterocycloalkyl and -C(O)R ¹⁷ .

In some embodiments, in the compound of formula (I), R ⁸ is selected from: -H, unsubstituted -C ₁ -C ₆ alkyl, -C ₁ -C ₆ haloalkyl, -C ₁ -C ₆ hydroxyalkyl, -C ₁ -C ₆ aminoalkyl, -C ₃ -C ₈ cycloalkyl and -C ₃ -C ₈ heterocycloalkyl.

In some embodiments, in the compound of formula (I), each R ⁹ is independently selected from: -C ₁ -C ₆ alkyl, -C ₃ -C ₈ cycloalkyl, -aryl, and -(C ₁ -C ₆ alkyl)-aryl.

In some embodiments, in the compound of formula (I), each R ⁹ is independently selected from: -C ₁ -C ₆ alkyl and -(C ₁ -C ₆ alkyl)-aryl.

In some embodiments, in the compound of formula (I), each R ⁹ is independently selected from: -H, unsubstituted -C ₁ -C ₆ alkyl, -C ₁ -C ₆ haloalkyl, -C 1 -C ₆ hydroxyalkyl, -C ₁ -C ₆ aminoalkyl, -C ₃ -C ₈ cycloalkyl, unsubstituted -aryl, -aminoaryl, -heteroaryl and -(C ₁ -C ₆ alkyl ₎ -aminoaryl.

In some embodiments, in the compound of formula (I), each R ¹⁰ is independently selected from: -C ₁ -C ₆ alkyl, -C ₃ -C ₈ cycloalkyl, -NR ¹⁴ R ^14' , -aryl, and -(C ₁ -C ₆ alkyl)-aryl.

In some embodiments, in the compound of formula (I), each R ¹⁰ is independently selected from: -C ₁ -C ₆ alkyl, -NR ¹⁴ R ^14' , -aryl, and -( ₁ -C ₆ alkyl)-aryl.

In some embodiments, in the compound of formula (I), each R ¹⁰ is independently selected from: unsubstituted -C ₁ -C ₆ alkyl, -C ₁ -C ₆ haloalkyl, -C ₁ -C ₆ hydroxyalkyl, -C ₁ -C ₆ aminoalkyl, -C ₃ -C ₈ cycloalkyl, -NR ¹⁴ R ^14' , unsubstituted -aryl, -aminoaryl, -heteroaryl and -(C ₁ -C ₆ alkyl)-aryl.

In some embodiments, in the compound of formula (I), R ^10′ is selected from: -H, unsubstituted -C 1 -C ₆ alkyl, -C ₁ -C ₆ haloalkyl, -C ₁ -C ₆ hydroxyalkyl, -C ₁ -C ₆ aminoalkyl, -C ₃ -C ₈ cycloalkyl, unsubstituted -aryl, -aminoaryl, -heteroaryl and -(C _{1 -C 6} alkyl)-aryl.

In some embodiments, in the compound of formula (I), R ¹¹ is selected from: -H, unsubstituted -C ₁ -C ₆ alkyl, -C ₁ -C ₆ haloalkyl, -C ₁ -C ₆ hydroxyalkyl, and -C ₁ -C ₆ aminoalkyl.

In some embodiments, in the compound of formula (I), R ¹² is selected from: -H, -C ₁ -C ₆ alkyl, -CO ₂ R ⁸ , -aryl, -(C ₁ -C ₆ alkyl)-aryl, and -S(O) ₂ R ¹⁶ .

In some embodiments, in the compound of formula (I), R ¹² is selected from: -H, unsubstituted -C ₁ -C ₆ alkyl, -C ₁ -C ₆ haloalkyl, -C ₁ -C ₆ hydroxyalkyl, -C ₁ -C ₆ aminoalkyl, -CO ₂ R ⁸ , unsubstituted -aryl, -aminoaryl, -heteroaryl, -(C ₁ -C ₆ alkyl)-aminoaryl, -S(O) ₂ R ¹⁶ and

In some embodiments, in the compound of formula (I), R ¹³ is selected from: -H, unsubstituted -C ₁ -C ₆ alkyl, -C ₁ -C ₆ haloalkyl, -C ₁ -C ₆ hydroxyalkyl, and -C ₁ -C ₆ aminoalkyl.

In some embodiments, in the compound of formula (I), R ¹⁴ and R ^14′ are each independently selected from: -H, unsubstituted -C ₁ -C ₆ alkyl, _{-C 1} -C 6 haloalkyl, -C ₁ -C ₆ hydroxyalkyl, -C ₁ -C ₆ aminoalkyl, -C ₃ -C ₈ cycloalkyl and -C ₃ -C ₈ heterocycloalkyl.

In some embodiments, in the compound of formula (I), R ¹⁶ is selected from: -aryl, -heteroaryl, and -(C ₁ -C ₆ alkyl)-aryl.

In some embodiments, in the compound of formula (I), R ¹⁶ is selected from: unsubstituted -C ₁ -C ₆ alkyl, -C 1 -C 6 haloalkyl, -C ₁ -C ₆ hydroxyalkyl, -C ₁ -C ₆ aminoalkyl, -C ₃ -C ₈ cycloalkyl, unsubstituted _{-aryl, -aminoaryl, -heteroaryl and -(C 1 -C 6 alkyl ) -aminoaryl} .

In some embodiments, in the compound of formula (I), R ¹⁷ is selected from: unsubstituted C ₁ -C ₆ alkyl, -C ₁ -C ₆ hydroxyalkyl, -C ₃ -C ₈ cycloalkyl, -C ₃ -C ₈ heterocycloalkyl, -(C ₁ -C ₆ alkyl)-C ₃ -C ₈ heterocycloalkyl, unsubstituted aryl, -hydroxyaryl, -aminoaryl, -heteroaryl and -(C ₁ -C ₆ alkyl)-aminoaryl.

In some embodiments, in the compound of formula (I), R ¹⁸ and R ^19, together with the N atom to which they are bound, form a 4-, 5-, 6-, or 7-membered ring having 0 to 3 substituents selected from the group consisting of halogen, unsubstituted C ₁ -C ₆ alkyl, -C ₁ -C ₆ haloalkyl, -C ₁ -C ₆ hydroxyalkyl, -C ₁ -C ₆ aminoalkyl, -C ₃ -C ₈ cycloalkyl, and -(C ₁ -C ₆ alkyl)-OR ⁵ .

In some embodiments, in the compound of Formula (I), ^Xa and ^Xb are each independently selected from: NH and O.

Combinations of any of the foregoing embodiments of compounds of formula (I) are also contemplated, and each combination forms a separate embodiment for the purposes of this disclosure.

In certain embodiments, the compound of formula (I) has formula (II):

in:

R ² is selected from: -H, -CH ₃ , -CF ₃ , -F, -Br, -Cl, -OH, -OCH ₃ and -OCF ₃ ;

R ²⁰ is selected from: -H, -C ₁ -C ₆ alkyl, -C ₃ -C ₈ cycloalkyl, -(C ₁ -C ₆ alkyl)-OR ⁵ , -CO ₂ R ⁸ , -aryl, -heteroaryl, -(C ₁ -C ₆ alkyl)-aryl,

R ⁵ is selected from: -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 selected from: -H, -C ₁ -C ₆ alkyl, -C ₃ -C ₈ cycloalkyl, -(C ₁ -C ₆ alkyl)-OR ⁵ , -C ₃ -C ₈ heterocycloalkyl and -C(O)R ¹⁷ ;

R ⁸ is selected from: -H, -C ₁ -C ₆ alkyl, -C ₃ -C ₈ cycloalkyl and -C ₃ -C ₈ heterocycloalkyl;

Each R ⁹ is independently selected from: -H, -C ₁ -C ₆ alkyl, -C ₃ -C ₈ cycloalkyl, -aryl, -heteroaryl, and -(C ₁ -C ₆ alkyl)-aryl;

Each R ¹⁰ is independently selected from: -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′ is selected from: -H, -C ₁ -C ₆ alkyl, -C ₃ -C ₈ cycloalkyl, -aryl, -heteroaryl and -(C ₁ -C ₆ alkyl)-aryl;

R ¹¹ is selected from: -H and -C ₁ -C ₆ alkyl;

R ¹² is selected from: -H, -C ₁ -C ₆ alkyl, -CO ₂ R ⁸ , -aryl, -heteroaryl, -(C ₁ -C ₆ alkyl)-aryl, -S(O) ₂ R ¹⁶ and

R ¹³ is selected from: -H and -C ₁ -C ₆ alkyl;

R ¹⁴ and R ^14' are each independently selected from: -H, C ₁ -C ₆ alkyl, -C ₃ -C ₈ cycloalkyl and -C ₃ -C ₈ heterocycloalkyl;

R ¹⁶ is selected from: -C ₁ -C ₆ alkyl, -C ₃ -C ₈ cycloalkyl, -aryl, -heteroaryl and -(C ₁ -C ₆ alkyl)-aryl;

R ¹⁷ is selected from: -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 bound form a 4-, 5-, 6- or 7-membered ring having 0 to 3 substituents selected from the group consisting of halogen, -C ₁ -C ₆ alkyl, -C ₃ -C ₈ cycloalkyl and -(C ₁ -C ₆ alkyl)-OR ⁵ ;

R ²⁴ , R ²⁵ and R ²⁶ are each -C ₁ -C ₆ alkyl;

^Xa and ^Xb are each independently selected from: NH, O and S, and

^Xc is selected from the group consisting of O, S and S(O) ₂ ,

Provided that the compound is not (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.

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 .

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 .

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

In some embodiments, in the compound of formula (II), R ²⁰ is selected from: -H, -C ₁ -C ₆ alkyl, -(C ₁ -C ₆ alkyl)-OR ⁵ , –(C ₁ -C ₆ alkyl)-aryl,

In some embodiments, in the compound of formula (II), R ²⁰ is selected from: -H, -C ₁ -C ₆ alkyl, -(C ₁ -C ₆ alkyl)-OR ⁵ , –(C ₁ -C ₆ alkyl)-aryl,

In some embodiments, in the compound of formula (II), R ²⁰ is selected from: -H, -C ₁ -C ₆ alkyl, -(C ₁ -C ₆ alkyl)-OR ⁵ ,

In some embodiments, in the compound of formula (II), R ²⁰ is selected from: -H, unsubstituted -C ₁ -C ₆ alkyl, -C ₁ -C ₆ haloalkyl, -C ₁ -C ₆ hydroxyalkyl, -C ₃ -C ₈ cycloalkyl, -(C ₁ -C ₆ alkyl)-OR ⁵ , -CO ₂ R ⁸ , unsubstituted aryl, -aminoaryl, -heteroaryl, -(C ₁ -C ₆ alkyl)-aminoaryl,

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 , and R ²⁰ is selected from: -H, -C ₁ -C ₆ alkyl, -(C ₁ -C ₆ alkyl)-OR ⁵ , –(C ₁ -C ₆ alkyl)-aryl,

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 , and R ²⁰ is selected from: -H, -C ₁ -C ₆ alkyl, -(C ₁ -C ₆ alkyl)-OR ⁵ , –(C ₁ -C ₆ alkyl)-aryl,

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 , and R ²⁰ is selected from: -H, -C ₁ -C ₆ alkyl, -(C ₁ -C ₆ alkyl)-OR ⁵ ,

In some embodiments, in the compound of formula (II), R ⁵ is selected from: -H, unsubstituted -C ₁ -C ₆ alkyl, -C ₁ -C 6 haloalkyl, -C ₁ -C ₆ hydroxyalkyl, -C ₁ -C ₆ aminoalkyl, -C ₃ -C ₈ cycloalkyl, unsubstituted _-aryl , -aminoaryl, -heteroaryl and -(C ₁ -C ₆ alkyl)-aminoaryl.

In some embodiments, in the compound of formula (II), R ⁶ and R ⁷ are independently selected from: -H, -C ₁ -C ₆ alkyl, -C ₃ -C ₈ cycloalkyl, and -C(O)R ¹⁷ .

In some embodiments, in the compound of formula (II), R ⁶ is H, and R ⁷ is selected from: -H, -C ₁ -C ₆ alkyl, -C ₃ -C ₈ cycloalkyl, -(C ₁ -C ₆ alkyl)-OR ⁵ , -C ₃ -C ₈ heterocycloalkyl, and -C(O)R ¹⁷ .

In some embodiments, in the compound of formula (II), R ⁶ is H, and R ⁷ is selected from: -H, -C ₁ -C ₆ alkyl, -C ₃ -C ₈ cycloalkyl, and -C(O)R ¹⁷ .

In some embodiments, in the compound of formula (II), R ⁶ and R ⁷ are each independently selected from: -H, unsubstituted -C ₁ -C ₆ alkyl, -C ₁ -C ₆ haloalkyl, -C ₁ -C ₆ hydroxyalkyl, -C ₃ -C ₈ cycloalkyl, -(C ₁ -C ₆ alkyl)-OR ⁵ , -C ₃ -C ₈ heterocycloalkyl and -C(O)R ¹⁷ .

In some embodiments, in the compound of formula (II), R ⁸ is selected from: -H, unsubstituted -C ₁ -C ₆ alkyl, -C ₁ -C ₆ haloalkyl, -C ₁ -C ₆ hydroxyalkyl, -C ₁ -C ₆ aminoalkyl, -C ₃ -C ₈ cycloalkyl and -C ₃ -C ₈ heterocycloalkyl.

In some embodiments, in the compound of formula (II), each R ⁹ is independently selected from: -C ₁ -C ₆ alkyl, -C ₃ -C ₈ cycloalkyl, -aryl, and -(C ₁ -C ₆ alkyl)-aryl.

In some embodiments, in the compound of formula (II), each R ⁹ is independently selected from: -C ₁ -C ₆ alkyl and -(C ₁ -C ₆ alkyl)-aryl.

In some embodiments, in the compound of formula (II), each R ⁹ is independently selected from: -H, unsubstituted -C 1 -C ₆ alkyl, -C ₁ -C ₆ haloalkyl, -C 1 -C ₆ hydroxyalkyl, -C ₁ -C ₆ aminoalkyl, -C ₃ -C ₈ cycloalkyl, unsubstituted -aryl, -aminoaryl, -heteroaryl and -(C _{1 -C 6} alkyl ₎ -aminoaryl.

In some embodiments, in the compound of formula (II), each R ¹⁰ is independently selected from: -C ₁ -C ₆ alkyl, -C ₃ -C ₈ cycloalkyl, -NR ¹⁴ R ^14' , -aryl, and -(C ₁ -C ₆ alkyl)-aryl.

In some embodiments, in the compound of formula (II), each R ¹⁰ is independently selected from: -C ₁ -C ₆ alkyl, -NR ¹⁴ R ^14' , -aryl, and -( ₁ -C ₆ alkyl)-aryl.

In some embodiments, in the compound of formula (II), each R ¹⁰ is independently selected from: unsubstituted -C ₁ -C ₆ alkyl, -C ₁ -C ₆ haloalkyl, -C ₁ -C ₆ hydroxyalkyl, -C ₁ -C ₆ aminoalkyl, -C ₃ -C ₈ cycloalkyl, -NR ¹⁴ R ^14' , unsubstituted -aryl, -aminoaryl, -heteroaryl and -(C ₁ -C ₆ alkyl)-aryl.

In some embodiments, in the compound of formula (II), R ^10′ is selected from: -H, unsubstituted -C 1 -C ₆ alkyl, -C ₁ -C ₆ haloalkyl, -C ₁ -C ₆ hydroxyalkyl, -C ₁ -C ₆ aminoalkyl, -C ₃ -C ₈ cycloalkyl, unsubstituted -aryl, -aminoaryl, -heteroaryl and -(C _{1 -C 6} alkyl)-aryl.

In some embodiments, in the compound of formula (II), R ¹¹ is selected from: -H, unsubstituted -C ₁ -C ₆ alkyl, -C ₁ -C ₆ haloalkyl, -C ₁ -C ₆ hydroxyalkyl, and -C ₁ -C ₆ aminoalkyl.

In some embodiments, in the compound of formula (II), R ¹² is selected from: -H, -C ₁ -C ₆ alkyl, -CO ₂ R ⁸ , -aryl, -(C ₁ -C ₆ alkyl)-aryl, and -S(O) ₂ R ¹⁶ .

In some embodiments, in the compound of formula (II), R ¹² is selected from: -H, unsubstituted -C ₁ -C ₆ alkyl, -C ₁ -C ₆ haloalkyl, -C ₁ -C ₆ hydroxyalkyl, -C ₁ -C ₆ aminoalkyl, -CO ₂ R ⁸ , unsubstituted -aryl, -aminoaryl, -heteroaryl, -(C ₁ -C ₆ alkyl)-aminoaryl, -S(O) ₂ R ¹⁶ and

In some embodiments, in the compound of formula (II), R ¹³ is selected from: -H, unsubstituted -C ₁ -C ₆ alkyl, -C ₁ -C ₆ haloalkyl, -C ₁ -C ₆ hydroxyalkyl, and -C ₁ -C ₆ aminoalkyl.

In some embodiments, in the compound of formula (II), R ¹⁴ and R ^14′ are each independently selected from: -H, unsubstituted -C ₁ -C ₆ alkyl, _{-C 1} -C 6 haloalkyl, -C ₁ -C ₆ hydroxyalkyl, -C ₁ -C ₆ aminoalkyl, -C ₃ -C ₈ cycloalkyl and -C ₃ -C ₈ heterocycloalkyl.

In some embodiments, in the compound of formula (II), R ¹⁶ is selected from: -aryl, -heteroaryl, and -(C ₁ -C ₆ alkyl)-aryl.

In some embodiments, in the compound of formula (II), R ¹⁶ is selected from: unsubstituted -C ₁ -C ₆ alkyl, -C 1 -C 6 haloalkyl, -C ₁ -C ₆ hydroxyalkyl, -C ₁ -C ₆ aminoalkyl, -C ₃ -C ₈ cycloalkyl, unsubstituted _{-aryl, -aminoaryl, -heteroaryl and -(C 1 -C 6 alkyl ) -aminoaryl} .

In some embodiments, in the compound of formula (II), R ¹⁷ is -C ₁ -C ₆ alkyl.

In some embodiments, in the compound of formula (II), R ¹⁷ is selected from: unsubstituted C ₁ -C ₆ alkyl, -C ₁ -C ₆ hydroxyalkyl, -C ₃ -C ₈ cycloalkyl, -C ₃ -C ₈ heterocycloalkyl, -(C ₁ -C ₆ alkyl)-C ₃ -C ₈ heterocycloalkyl, unsubstituted aryl, -hydroxyaryl, -aminoaryl, -heteroaryl and -(C ₁ -C ₆ alkyl)-aminoaryl.

In some embodiments, in the compound of formula (II), R ¹⁸ and R ^19, together with the N atom to which they are bound, form a 4-, 5-, 6-, or 7-membered ring having 0 to 3 substituents selected from the group consisting of halogen, unsubstituted -C ₁ -C ₆ alkyl, -C ₁ -C ₆ haloalkyl, -C ₁ -C ₆ hydroxyalkyl, -C ₁ -C ₆ aminoalkyl, -C ₃ -C ₈ cycloalkyl, and -(C ₁ -C ₆ alkyl)-OR ⁵ .

In some embodiments, in the compound of formula (II), ^Xa and ^Xb are each independently selected from: NH and O.

Combinations of any of the foregoing embodiments of compounds of formula (II) are also contemplated, and each combination forms a separate embodiment for the purposes of this disclosure.

In certain embodiments, the compound of formula (I) has formula (III):

in:

R ² is selected from: -H, -CH ₃ , -CF ₃ , -F, -Br, -Cl, -OH, -OCH ₃ and -OCF ₃ ;

R ¹⁵ is selected from: -H, -CH ₃ , -CHF ₂ , -CF ₃ , -F, -Br, -Cl, -OH, -OCH ₃ and -OCF ₃ ;

^R4 is selected from:

R ⁵ is selected from: -H, -C ₁ -C ₆ alkyl, -C ₃ -C ₈ cycloalkyl, -aryl, -heteroaryl and -(C ₁ -C ₆ alkyl)-aryl;

R ⁸ is selected from: -H, -C ₁ -C ₆ alkyl, -C ₃ -C ₈ cycloalkyl and -C ₃ -C ₈ heterocycloalkyl;

Each R ⁹ is independently selected from: -H, -C ₁ -C ₆ alkyl, -C ₃ -C ₈ cycloalkyl, -aryl, -heteroaryl, and -(C ₁ -C ₆ alkyl)-aryl;

Each R ¹⁰ is independently selected from: -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′ is selected from: -H, -C ₁ -C ₆ alkyl, -C ₃ -C ₈ cycloalkyl, -aryl, -heteroaryl and -(C ₁ -C ₆ alkyl)-aryl;

R ¹¹ is selected from: -H and -C ₁ -C ₆ alkyl;

R ¹² is selected from: -H, -C ₁ -C ₆ alkyl, -CO ₂ R ⁸ , -aryl, -heteroaryl, -(C ₁ -C ₆ alkyl)-aryl, -S(O) ₂ R ¹⁶ and

R ¹³ is selected from: -H and -C ₁ -C ₆ alkyl;

R ¹⁴ and R ^14' are each independently selected from: -H, C ₁ -C ₆ alkyl, -C ₃ -C ₈ cycloalkyl and -C ₃ -C ₈ heterocycloalkyl;

R ¹⁶ is selected from: -C ₁ -C ₆ alkyl, -C ₃ -C ₈ cycloalkyl, -aryl, -heteroaryl and -(C ₁ -C ₆ alkyl)-aryl;

R ¹⁸ and R ¹⁹ together with the N atom to which they are bound form a 4-, 5-, 6- or 7-membered ring having 0 to 3 substituents selected from the group consisting of halogen, -C ₁ -C ₆ alkyl, -C ₃ -C ₈ cycloalkyl and -(C ₁ -C ₆ alkyl)-OR ⁵ ;

R ²⁴ , R ²⁵ and R ²⁶ are each -C ₁ -C ₆ alkyl;

^Xa and ^Xb are each independently selected from: NH, O and S, and

X ^c is selected from the group consisting of: O, S and S(O) ₂ .

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 .

In some embodiments, in the compound of formula (III), R ² is selected from: -H, -F and -Cl.

In some embodiments, in the compound of formula (III), R ¹⁵ is selected from: -CH ₃ , -CF ₃ , -OCH ₃ , and -OCF ₃ .

In some embodiments, in the compound of formula (III), R ¹⁵ is selected from: -CH ₃ and -OCH ₃ .

In some embodiments, in the compound of formula (III), R ² is selected from: -H, -F, and -Cl, and R ¹⁵ is selected from: -CH ₃ , -CF ₃ , -OCH ₃ , and -OCF ₃ .

In some embodiments, in the compound of formula (III), R ² is selected from: -H, -F, and -Cl, and R ¹⁵ is selected from: -CH ₃ and -OCH ₃ .

In some embodiments, in the compound of formula (III), R ⁴ is selected from:

In some embodiments, in the compound of formula (III), R ⁵ is selected from: -H, unsubstituted -C ₁ -C ₆ alkyl, -C ₁ -C 6 haloalkyl, -C ₁ -C ₆ hydroxyalkyl, -C ₁ -C ₆ aminoalkyl, -C ₃ -C ₈ cycloalkyl, unsubstituted _-aryl , -aminoaryl, -heteroaryl and -(C ₁ -C ₆ alkyl)-aminoaryl.

In some embodiments, in the compound of formula (III), R ⁸ is selected from: -H, unsubstituted -C ₁ -C ₆ alkyl, -C ₁ -C ₆ haloalkyl, -C ₁ -C ₆ hydroxyalkyl, -C ₁ -C ₆ aminoalkyl, -C ₃ -C ₈ cycloalkyl and -C ₃ -C ₈ heterocycloalkyl.

In some embodiments, in the compound of formula (III), each R ⁹ is independently selected from: -C ₁ -C ₆ alkyl, -C ₃ -C ₈ cycloalkyl, -aryl, and -(C ₁ -C ₆ alkyl)-aryl.

In some embodiments, in the compound of formula (III), each R ⁹ is independently selected from: -C ₁ -C ₆ alkyl and -(C ₁ -C ₆ alkyl)-aryl.

In some embodiments, in the compound of formula (III), each R ⁹ is independently selected from: -H, unsubstituted -C 1 -C ₆ alkyl, -C ₁ -C ₆ haloalkyl, -C 1 -C ₆ hydroxyalkyl, -C ₁ -C ₆ aminoalkyl, -C ₃ -C ₈ cycloalkyl, unsubstituted -aryl, -aminoaryl, -heteroaryl and -(C _{1 -C 6} alkyl ₎ -aminoaryl.

In some embodiments, in the compound of formula (III), each R ¹⁰ is independently selected from: -C ₁ -C ₆ alkyl, -C ₃ -C ₈ cycloalkyl, -NR ¹⁴ R ^14' , -aryl, and -(C ₁ -C ₆ alkyl)-aryl.

In some embodiments, in the compound of formula (III), each R ¹⁰ is independently selected from: -C ₁ -C ₆ alkyl, -NR ¹⁴ R ^14' , -aryl, and -( ₁ -C ₆ alkyl)-aryl.

In some embodiments, in the compound of formula (III), each R ¹⁰ is independently selected from: unsubstituted -C ₁ -C ₆ alkyl, -C ₁ -C ₆ haloalkyl, -C ₁ -C ₆ hydroxyalkyl, -C ₁ -C ₆ aminoalkyl, -C ₃ -C ₈ cycloalkyl, -NR ¹⁴ R ^14' , unsubstituted -aryl, -aminoaryl, -heteroaryl and -(C ₁ -C ₆ alkyl)-aryl.

In some embodiments, in the compound of formula (III), R ¹⁰ ′ is selected from: -H, unsubstituted -C 1 -C ₆ alkyl, -C ₁ -C ₆ haloalkyl, -C ₁ -C ₆ hydroxyalkyl, -C ₁ -C ₆ aminoalkyl, -C ₃ -C ₈ cycloalkyl, unsubstituted -aryl, -aminoaryl, -heteroaryl and -(C _{1 -C 6} alkyl)-aryl.

In some embodiments, in the compound of formula (III), R ¹¹ is selected from: -H, unsubstituted -C ₁ -C ₆ alkyl, -C ₁ -C ₆ haloalkyl, -C ₁ -C ₆ hydroxyalkyl, and -C ₁ -C ₆ aminoalkyl.

In some embodiments, in the compound of formula (III), R ¹² is selected from: -H, -C ₁ -C ₆ alkyl, -CO ₂ R ⁸ , -aryl, -(C ₁ -C ₆ alkyl)-aryl, and -S(O) ₂ R ¹⁶ .

In some embodiments, in the compound of formula (III), R ¹² is selected from: -H, unsubstituted -C ₁ -C ₆ alkyl, -C ₁ -C ₆ haloalkyl, -C ₁ -C ₆ hydroxyalkyl, -C ₁ -C ₆ aminoalkyl, -CO ₂ R ⁸ , unsubstituted -aryl, -aminoaryl, -heteroaryl, -(C ₁ -C ₆ alkyl)-aminoaryl, -S(O) ₂ R ¹⁶ and

In some embodiments, in the compound of formula (III), R ¹³ is selected from: -H, unsubstituted -C ₁ -C ₆ alkyl, -C ₁ -C ₆ haloalkyl, -C ₁ -C ₆ hydroxyalkyl, and -C ₁ -C ₆ aminoalkyl.

In some embodiments, in the compound of formula (III), R ¹⁴ and R ^14′ are each independently selected from: -H, unsubstituted -C ₁ -C ₆ alkyl, _{-C 1} -C 6 haloalkyl, -C ₁ -C ₆ hydroxyalkyl, -C ₁ -C ₆ aminoalkyl, -C ₃ -C ₈ cycloalkyl and -C ₃ -C ₈ heterocycloalkyl.

In some embodiments, in the compound of formula (III), R ¹⁶ is selected from: -aryl, -heteroaryl, and -(C ₁ -C ₆ alkyl)-aryl.

In some embodiments, in the compound of formula (III), R ¹⁶ is selected from: unsubstituted -C ₁ -C ₆ alkyl, -C 1 -C 6 haloalkyl, -C ₁ -C ₆ hydroxyalkyl, -C ₁ -C ₆ aminoalkyl, -C ₃ -C ₈ cycloalkyl, unsubstituted _{-aryl, -aminoaryl, -heteroaryl and -(C 1 -C 6 alkyl ) -aminoaryl} .

In some embodiments, in the compound of formula (III), R ¹⁸ and R ^19, together with the N atom to which they are bound, form a 4-, 5-, 6-, or 7-membered ring having 0 to 3 substituents selected from the group consisting of halogen, unsubstituted C ₁ -C ₆ alkyl, -C ₁ -C ₆ haloalkyl, -C ₁ -C ₆ hydroxyalkyl, -C ₁ -C ₆ aminoalkyl, -C ₃ -C ₈ cycloalkyl, and -(C ₁ -C ₆ alkyl)-OR ⁵ .

In some embodiments, in the compound of formula (III), ^Xa and ^Xb are each independently selected from: NH and O.

Combinations of any of the foregoing embodiments of compounds of formula (III) are also contemplated, and each combination forms a separate embodiment for the purposes of this disclosure.

In certain embodiments, each alkyl, cycloalkyl, heterocycloalkyl, aryl and heteroaryl as defined in any one of formula (I), (II) or (III) is optionally substituted by one or more substituents selected from the group consisting of halogen, acyl, acyloxy, alkoxy, carboxyl, hydroxyl, amino, amido, nitro, cyano, azido, alkylthio, sulfo, sulfonyl, sulfonamido, alkyl, cycloalkyl, heterocycloalkyl, aryl and heteroaryl. In some embodiments, each alkyl, cycloalkyl, heterocycloalkyl, aryl and heteroaryl as defined in any one of formula (I), (II) or (III) is optionally substituted by one or more substituents selected from the group consisting of halogen, acyl, acyloxy, alkoxy, carboxyl, hydroxyl, amino, amido, nitro, cyano, azido, alkylthio, sulfo, sulfonyl and sulfonamido.

In certain embodiments, the camptothecin analog comprised by the ADC according to the present disclosure is a compound having formula (I) and is selected from the compounds shown in Table 6 and Table 7.

In certain embodiments, the camptothecin analog is a compound having formula (II). In certain embodiments, the camptothecin analog is a compound having formula (II), wherein R ² is F, and R ²⁰ is H, -(C ₁ -C ₆ )-OR ⁵ or In some embodiments, the camptothecin analog is a compound having formula (II), wherein R ² is F; R ²⁰ is H, -(C ₁ -C ₆ )-OR ⁵ or R ⁵ is H, and R ¹⁸ and R ¹⁹ together with the N atom to which they are bound form an unsubstituted 4-membered, 5-membered, 6-membered or 7-membered ring. In some embodiments, the camptothecin analog is a compound of formula (II), wherein R ² is F; R ²⁰ is -(C ₁ -C ₆ )-OR ⁵ , and R ⁵ is H. In certain embodiments, the camptothecin analog is a compound of formula (II) and is selected from the compounds shown in Table 6.

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; R ¹⁵ is -CH ₃ ; R ⁴ is R ⁹ is -C ₁ -C ₆ 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.

In certain embodiments, the camptothecin analog comprised by the ADC according to the present disclosure is Compound 139, Compound 140, Compound 141, or Compound 148. In some embodiments, the camptothecin analog comprised by the ADC according to the present disclosure is Compound 139 or Compound 141.

Table 6: Exemplary Camptothecin Analogs of Formula (II)

Table 7: Exemplary Camptothecin Analogs of Formula (II)

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

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)

in:

T is an anti-FRα antibody construct as described herein;

L is the connector;

D is a camptothecin analog having formula (I);

m is between 1 and 4, and

n is between 1 and 10.

In certain embodiments, in the conjugate of formula (X), m is between 1 and 2. In some embodiments, m is 1.

In some embodiments, in the conjugate of formula (X), n is between 1 and 8, such as between 2 and 8. In some embodiments, n is between 4 and 8.

In certain embodiments, in the conjugate of formula (X), m is between 1 and 2, and n is between 2 and 8, or between 4 and 8. In some embodiments, in the conjugate of formula (X), m is 1, and n is between 2 and 8, or between 4 and 8.

As shown above and reflected by the parameters m and n in formula (X), the anti-FRα antibody construct "T" can be conjugated to more than one compound "D" of formula (I). One skilled in the art will appreciate that while any particular anti-FRα antibody construct T is conjugated to an integer number of compounds D, analysis of preparations of conjugates to determine the ratio of compounds D to anti-FRα antibody constructs T may yield non-integer results, reflecting a statistical average. This ratio of compound D to targeting moiety T may generally be referred to as the drug to antibody ratio or "DAR". Thus, conjugate preparations having non-integer DARs are intended to be encompassed by formula (X).

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 Table 6 and Table 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.

Certain embodiments of the present disclosure relate to ADCs having formula (X), wherein D is a compound of formula (IV):

in:

R ^1a is selected from: -H, -CH ₃ , -CHF ₂ , -CF ₃ , -F, -Br, -Cl, -OH, -OCH ₃ , -OCF ₃ and -NH ₂ ;

R ^2a is selected from: -H, -CH ₃ , -CF ₃ , -F, -Br, -Cl, -OH, -OCH ₃ and -OCF ₃ ;

X is -O-, -S- or -NH-, and R ^4a is selected from:

wherein * is the point of attachment to X, and wherein p is 1, 2, 3 or 4; or

X is O, and R ^4a -X- is selected from:

R ^5a is selected from: -C ₁ -C ₆ alkyl, -C ₃ -C ₈ cycloalkyl, -aryl, -heteroaryl and -(C ₁ -C ₆ alkyl)-aryl;

R ^8a is selected from: -C ₁ -C ₆ alkyl, -C ₃ -C ₈ cycloalkyl and -C ₃ -C ₈ heterocycloalkyl;

Each R ^9a is independently selected from: -C ₁ -C ₆ alkyl, -C ₃ -C ₈ cycloalkyl, -aryl, -heteroaryl, and -(C ₁ -C ₆ alkyl)-aryl; or R ^9a is absent, and X ^b ＝X;

Each R ^10a is independently selected from: -C ₁ -C ₆ alkyl, -C ₃ -C ₈ cycloalkyl, -aryl, -heteroaryl, -(C ₁ -C ₆ alkyl)-aryl and

Each R ^10a' is independently selected from: -H, -C ₁ -C ₆ alkyl, -C ₃ -C ₈ cycloalkyl, -aryl, -heteroaryl, and -(C ₁ -C ₆ alkyl)-aryl;

Each R ^10b is independently selected from: -C ₁ -C ₆ alkyl, -C ₃ -C ₈ cycloalkyl, -aryl, -heteroaryl, and -(C ₁ -C ₆ alkyl)-aryl;

R ^11a is absent or is -C ₁ -C ₆ alkyl;

R ^12a is selected from: -C ₁ -C ₆ alkyl, -CO ₂ R ^8a , -aryl, -heteroaryl, -(C ₁ -C ₆ alkyl)-aryl, -S(O) ₂ R ^16a and

R ^13a is selected from: -H and -C ₁ -C ₆ alkyl;

R ^14a is selected from: -C ₁ -C ₆ alkyl, -C ₃ -C ₈ cycloalkyl and -C ₃ -C ₈ heterocycloalkyl;

R ^14a' is selected from the group consisting of: H, -C ₁ -C ₆ alkyl, -C ₃ -C ₈ cycloalkyl, and -C ₃ -C ₈ heterocycloalkyl;

R ^16a is selected from: -C ₁ -C ₆ alkyl, -C ₃ -C ₈ cycloalkyl, -aryl, -heteroaryl and -(C ₁ -C ₆ alkyl)-aryl;

R ²¹ is selected from: -C ₁ -C ₆ alkyl, -C ₃ -C ₈ cycloalkyl and -(C ₁ -C ₆ alkyl)-OR ^5a ;

R ²² and R ²³ are each independently selected from: -H, -halogen, -C ₁ -C ₆ alkyl and -C ₃ -C ₈ cycloalkyl;

R ²⁴ , R ²⁵ and R ²⁶ are each -C ₁ -C ₆ alkyl;

^Xa and ^Xb are each independently selected from: NH, O and S;

^Xc is selected from the group consisting of: O, S and S(O) ₂ , and

Indicates the connection point with the linker L.

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 .

In some embodiments, in the compound of formula (IV), R ^1a is selected from: -CH ₃ , -CF ₃ , -OCH ₃ , and -OCF ₃ .

In some embodiments, in the compound of formula (IV), R ^1a is selected from: -CH ₃ , -OCH ₃ , and NH ₂ .

In some embodiments, in the compound of formula (IV), R ^1a is selected from: -CH ₃ and -OCH ₃ .

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 .

In some embodiments, in the compound of Formula (IV), R ^2a is selected from: -H, -F and -Cl.

In some embodiments, in the compound of Formula (IV), R ^2a is -F.

In some embodiments, in the compound of formula (IV), X is -O-, -S- or -NH-, and R ^4a is selected from:

In some embodiments, in the compound of Formula (IV), X is -O- or -NH-.

In some embodiments, in the compound of formula (IV), each R ^9a is independently selected from: -C ₁ -C ₆ alkyl, -C ₃ -C ₈ cycloalkyl, -aryl, and -(C ₁ -C ₆ alkyl)-aryl.

In some embodiments, in the compound of formula (IV), each R ^9a is independently selected from: -C ₁ -C ₆ alkyl and -(C ₁ -C ₆ alkyl)-aryl.

In some embodiments, in the compound of formula (IV), each R ^10a is independently selected from: -C ₁ -C ₆ alkyl, -C ₃ -C ₈ cycloalkyl, -aryl, -(C ₁ -C ₆ alkyl)-aryl and

In some embodiments, in the compound of formula (IV), each R ^10a is independently selected from: -C ₁ -C ₆ alkyl, -aryl, -( ₁ -C ₆ alkyl)-aryl and

In some embodiments, in the compound of formula (IV), R ^12a is selected from: -C ₁ -C ₆ alkyl, -aryl, -(C ₁ -C ₆ alkyl)-aryl, and -S(O) ₂ R ¹⁶ .

In some embodiments, in the compound of formula (IV), R ^13a is selected from: -H, unsubstituted -C ₁ -C ₆ alkyl, -C ₁ -C ₆ haloalkyl, -C ₁ -C ₆ hydroxyalkyl, and -C ₁ -C ₆ aminoalkyl.

In some embodiments, in the compound of formula (IV), R ^14a′ is selected from: H, unsubstituted -C ₁ -C ₆ alkyl, -C ₁ -C ₆ haloalkyl, -C ₁ -C ₆ hydroxyalkyl, -C ₁ -C ₆ aminoalkyl, -C ₃ -C ₈ cycloalkyl, and -C ₃ -C ₈ heterocycloalkyl.

In some embodiments, in the compound of formula (IV), R ^16a is selected from: -aryl, -heteroaryl, and -(C ₁ -C ₆ alkyl)-aryl.

In some embodiments, in the compound of formula (IV), R ²² and R ²³ are each independently selected from: -H, -halogen, unsubstituted -C ₁ -C ₆ alkyl, -C ₁ -C ₆ haloalkyl, -C _{1 -C 6 aminoalkyl, -C 1 -C 6 hydroxyalkyl} and -C ₃ -C ₈ cycloalkyl.

In some embodiments, in the compound of formula (IV), ^Xa and ^Xb are each independently selected from: NH and O.

In some embodiments, in the compound of Formula (IV), ^Xa and ^Xb are each O. and ^Xb are each O, and R ^9a is -C ₁ -C ₆ alkyl.

In some embodiments, in the compound of formula (IV), X is O; R ^4a is ^X

In some embodiments, in the compound of formula (IV), R ^1a is -CH ₃ or -OCH ₃ ; X is O; R ^4a is ^Xa and ^Xb are each O; and ^R9a is _-C1 - _C6 alkyl.

In some embodiments, in the compound of formula (IV), R ^1a is -CH ₃ or -OCH ₃ ; R ^2a is H or F; X is O; R ^4a is ^Xa and ^Xb are each O; and ^R9a is _-C1 - _C6 alkyl.

Other combinations of any of the foregoing embodiments of compounds of formula (IV) are also contemplated, and each combination forms a separate embodiment for the purposes of this disclosure.

Certain embodiments of the present disclosure relate to ADCs having formula (X), wherein D is a compound of formula (V):

in:

R ^2a is selected from: -CH ₃ , -CF ₃ , -F, -Br, -Cl, -OH, -OCH ₃ and -OCF ₃ ;

R ^20a is selected from: -H, -C ₁ -C ₆ alkyl, -C ₃ -C ₈ cycloalkyl, -(C ₁ -C ₆ alkyl)-OR ⁵ , -CO ₂ R ⁸ , -aryl, -heteroaryl, -(C ₁ -C ₆ alkyl)-aryl,

R ⁵ is selected from: -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 selected from: -H, -C ₁ -C ₆ alkyl, -C ₃ -C ₈ cycloalkyl, -(C ₁ -C ₆ alkyl)-OR ⁵ , -C ₃ -C ₈ heterocycloalkyl and -C(O)R ¹⁷ ;

R ⁸ is selected from: -H, -C ₁ -C ₆ alkyl, -C ₃ -C ₈ cycloalkyl and -C ₃ -C ₈ heterocycloalkyl;

Each R ⁹ is independently selected from: -H, -C ₁ -C ₆ alkyl, -C ₃ -C ₈ cycloalkyl, -aryl, -heteroaryl, and -(C ₁ -C ₆ alkyl)-aryl;

Each R ¹⁰ is independently selected from: -C ₁ -C ₆ alkyl, -C ₃ -C ₈ cycloalkyl, -aryl, -heteroaryl, -(C ₁ -C ₆ alkyl)-aryl, and -NR ¹⁴ R ^14' ;

Each R ^10′ is independently selected from: -H, -C ₁ -C ₆ alkyl, -C ₃ -C ₈ cycloalkyl, -aryl, -heteroaryl, and -(C ₁ -C ₆ alkyl)-aryl;

R ¹¹ is selected from: -H and -C ₁ -C ₆ alkyl;

R ¹² is selected from: -H, -C ₁ -C ₆ alkyl, -CO ₂ R ⁸ , -aryl, -heteroaryl, -(C ₁ -C ₆ alkyl)-aryl, -S(O) ₂ R ¹⁶ and

R ¹³ is selected from: -H and -C ₁ -C ₆ alkyl;

R ¹⁴ and R ^14' are each independently selected from: -H, C ₁ -C ₆ alkyl, -C ₃ -C ₈ cycloalkyl and -C ₃ -C ₈ heterocycloalkyl;

R ¹⁶ is selected from: -C ₁ -C ₆ alkyl, -C ₃ -C ₈ cycloalkyl, -aryl, -heteroaryl and -(C ₁ -C ₆ alkyl)-aryl;

R ¹⁷ is selected from: -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 bound form a 4-, 5-, 6- or 7-membered ring having 0 to 3 substituents selected from the group consisting of halogen, -C ₁ -C ₆ alkyl, -C ₃ -C ₈ cycloalkyl and -(C ₁ -C ₆ alkyl)-OR ⁵ ;

R ²⁴ , R ²⁵ and R ²⁶ are each -C ₁ -C ₆ alkyl;

^Xa and ^Xb are each independently selected from: NH, O and S;

^Xc is selected from the group consisting of: O, S and S(O) ₂ , and

Indicates the connection point with the linker L.

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 .

In some embodiments, in the compound of Formula (V), R ^2a is selected from: -CF ₃ , -F, -Cl, and -OCH ₃ .

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

In some embodiments, in the compound of formula (V), R ^20a is selected from: -H, -C ₁ -C ₆ alkyl, -C ₃ -C ₈ cycloalkyl, -(C ₁ -C ₆ alkyl)-OR ⁵ , -CO ₂ R ⁸ , -aryl, -heteroaryl, -(C ₁ -C ₆ alkyl)-aryl,

In some embodiments, in the compound of formula (V), R ^20a is selected from: -H, -C ₁ -C ₆ alkyl, -(C ₁ -C ₆ alkyl)-OR ⁵ , -(C ₁ -C ₆ alkyl)-aryl,

In some embodiments, in the compound of formula (V), R ^20a is selected from: -H, -C ₁ -C ₆ alkyl, -(C ₁ -C ₆ alkyl)-OR ⁵ , -(C ₁ -C ₆ alkyl)-aryl,

In some embodiments, in the compound of formula (V), R ^20a is selected from: -H, -C ₁ -C ₆ alkyl, -(C ₁ -C ₆ alkyl)-OR ⁵ ,

In some embodiments, in the compound of formula (V), R ^20a is selected from: -H, unsubstituted -C ₁ -C ₆ alkyl, -C ₁ -C ₆ haloalkyl, -C ₁ -C ₆ hydroxyalkyl, -C ₃ -C ₈ cycloalkyl, -(C ₁ -C ₆ alkyl)-OR ⁵ , -CO ₂ R ⁸ , unsubstituted -aryl, -aminoaryl, -heteroaryl, -(C ₁ -C ₆ alkyl)-aminoaryl,

In some embodiments, in the compound of formula (V), R ⁶ and R ⁷ are independently selected from: -H, -C ₁ -C ₆ alkyl, -C ₃ -C ₈ cycloalkyl, and -C(O)R ¹⁷ .

In some embodiments, in the compound of formula (V), R ⁶ is H, and R ⁷ is selected from: -H, -C ₁ -C ₆ alkyl, -C ₃ -C ₈ cycloalkyl, -(C ₁ -C ₆ alkyl)-OR ⁵ , -C ₃ -C ₈ heterocycloalkyl, and -C(O)R ¹⁷ .

In some embodiments, in the compound of formula (V), R ⁶ is H, and R ⁷ is selected from: -H, -C ₁ -C ₆ alkyl, -C ₃ -C ₈ cycloalkyl, and -C(O)R ¹⁷ .

In some embodiments, in the compound of formula (V), R ⁶ and R ⁷ are each independently selected from: -H, unsubstituted -C ₁ -C ₆ alkyl, -C ₁ -C ₆ haloalkyl, -C ₁ -C ₆ hydroxyalkyl, -C ₃ -C ₈ cycloalkyl, -(C ₁ -C ₆ alkyl)-OR ⁵ , -C ₃ -C ₈ heterocycloalkyl and -C(O)R ¹⁷ .

In some embodiments, in the compound of formula (V), R ⁸ is selected from: -H, unsubstituted -C ₁ -C ₆ alkyl, -C ₁ -C ₆ haloalkyl, -C ₁ -C ₆ hydroxyalkyl, -C ₁ -C ₆ aminoalkyl, -C ₃ -C ₈ cycloalkyl and -C ₃ -C ₈ heterocycloalkyl.

In some embodiments, in the compound of formula (V), each R ⁹ is independently selected from: -C ₁ -C ₆ alkyl, -C ₃ -C ₈ cycloalkyl, -aryl, and -(C ₁ -C ₆ alkyl)-aryl.

In some embodiments, in the compound of Formula (V), each R ⁹ is independently selected from: -C ₁ -C ₆ alkyl and -(C ₁ -C ₆ alkyl)-aryl.

In some embodiments, in the compound of formula (V), each R ⁹ is independently selected from: -H, unsubstituted -C 1 -C ₆ alkyl, -C ₁ -C ₆ haloalkyl, -C 1 -C ₆ hydroxyalkyl, -C ₁ -C ₆ aminoalkyl, -C ₃ -C ₈ cycloalkyl, unsubstituted -aryl, -aminoaryl, -heteroaryl and -(C _{1 -C 6} alkyl ₎ -aminoaryl.

In some embodiments, in the compound of formula (V), each R ¹⁰ is independently selected from: -C ₁ -C ₆ alkyl, -C ₃ -C ₈ cycloalkyl, -NR ¹⁴ R ^14' , -aryl, and -(C ₁ -C ₆ alkyl)-aryl.

In some embodiments, in the compound of formula (V), each R ¹⁰ is independently selected from: -C ₁ -C ₆ alkyl, -NR ¹⁴ R ^14' , -aryl, and -( ₁ -C ₆ alkyl)-aryl.

In some embodiments, in the compound of formula (V), R ¹¹ is selected from: -H, unsubstituted -C ₁ -C ₆ alkyl, -C ₁ -C ₆ haloalkyl, -C ₁ -C ₆ hydroxyalkyl, and -C ₁ -C ₆ aminoalkyl.

In some embodiments, in the compound of Formula (V), R ¹² is selected from: -H, -C ₁ -C ₆ alkyl, -aryl, -(C ₁ -C ₆ alkyl)-aryl, and -S(O) ₂ R ¹⁶ .

In some embodiments, in the compound of formula (V), R ¹² is selected from: -H, unsubstituted -C ₁ -C ₆ alkyl, -C ₁ -C ₆ haloalkyl, -C ₁ -C ₆ hydroxyalkyl, -C ₁ -C ₆ aminoalkyl, -CO ₂ R ⁸ , unsubstituted -aryl, -aminoaryl, -heteroaryl, -(C ₁ -C ₆ alkyl)-aminoaryl, -S(O) ₂ R ¹⁶ and

In some embodiments, in the compound of formula (V), R ¹³ is selected from: -H, unsubstituted -C ₁ -C ₆ alkyl, -C ₁ -C ₆ haloalkyl, -C ₁ -C ₆ hydroxyalkyl, and -C ₁ -C ₆ aminoalkyl.

In some embodiments, in the compound of formula (V), R ¹⁴ and R ^14′ are each independently selected from: -H, unsubstituted -C ₁ -C ₆ alkyl, _{-C 1} -C 6 haloalkyl, -C ₁ -C ₆ hydroxyalkyl, -C ₁ -C ₆ aminoalkyl, -C ₃ -C ₈ cycloalkyl and -C ₃ -C ₈ heterocycloalkyl.

In some embodiments, in the compound of Formula (V), R ¹⁶ is selected from: -aryl, -heteroaryl, and -(C ₁ -C ₆ alkyl)-aryl.

In some embodiments, in the compound of formula (V), R ¹⁶ is selected from: unsubstituted -C ₁ -C ₆ alkyl, -C 1 -C 6 haloalkyl, -C ₁ -C ₆ hydroxyalkyl, -C ₁ -C ₆ aminoalkyl, -C ₃ -C ₈ cycloalkyl, unsubstituted _{-aryl, -aminoaryl, -heteroaryl and -(C 1 -C 6 alkyl ) -aminoaryl} .

In some embodiments, in the compound of formula (V), R ¹⁷ is selected from: unsubstituted -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, unsubstituted -aryl, -hydroxyaryl, -aminoaryl, -heteroaryl and -(C 1 -C 6 alkyl ) -aminoaryl .}

In some embodiments, in the compound of formula (V), R ¹⁸ and R ^19, together with the N atom to which they are bound, form a 4-, 5-, 6-, or 7-membered ring having 0 to 3 substituents selected from the group consisting of halogen, unsubstituted -C ₁ -C ₆ alkyl, -C ₁ -C ₆ haloalkyl, -C ₁ -C ₆ aminoalkyl, -C ₁ -C ₆ hydroxyalkyl, -C ₃ -C ₈ cycloalkyl, and -(C ₁ -C ₆ alkyl)-OR ⁵ .

In some embodiments, in the compound of Formula (V), R ¹⁷ is -C ₁ -C ₆ alkyl.

In some embodiments, in the compound of Formula (V), ^Xa and ^Xb are each independently selected from: NH and O.

In some embodiments, in the compound of Formula (V), ^Xa and ^Xb are each O.

In some embodiments, in the compound of Formula (V), R ^20a is —(C ₁ -C ₆ alkyl)-OR ⁵ .

In some embodiments, in the compound of Formula (V), R ^20a is —(C ₁ -C ₆ alkyl)-OR ⁵ , and R ⁵ is H.

In some embodiments, in the compound of Formula (V), R ^2a is F; R ^20a is —(C ₁ -C ₆ alkyl)-OR ⁵ , and R ⁵ is H.

Other combinations of any of the foregoing embodiments of compounds of formula (V) are also contemplated, and each combination forms a separate embodiment for the purposes of this disclosure.

Certain embodiments of the present disclosure relate to ADCs having formula (X), wherein D is a compound of formula (VI):

in:

R ^2a is selected from: -H, -CH ₃ , -CF ₃ , -F, -Br, -Cl, -OH, -OCH ₃ and -OCF ₃ ;

X is -O-, -S- or -NH-, and R ²⁵ is selected from: -C ₁ -C ₆ alkyl, -(C ₁ -C ₆ alkyl)-OR ^5a , -CO ₂ R ^8a , -aryl, -heteroaryl, -(C ₁ -C ₆ alkyl)-aryl,

wherein * is the point of attachment to X, and wherein p is 1, 2, 3 or 4; or

X is O, and R ²⁵ -X- is selected from:

R ^5a is selected from: -C ₁ -C ₆ alkyl, -C ₃ -C ₈ cycloalkyl, -aryl, -heteroaryl and -(C ₁ -C ₆ alkyl)-aryl;

R ^6a is selected from: -H, -C ₁ -C ₆ alkyl, -C ₃ -C ₈ cycloalkyl and -C ₃ -C ₈ heterocycloalkyl;

R ^7a is selected from: -C ₁ -C ₆ alkyl, -C ₃ -C ₈ cycloalkyl, -(C ₁ -C ₆ alkyl)-OR ^5a , -C ₃ -C ₈ heterocycloalkyl and -C(O)R ^17a ;

R ^8a is selected from: -C ₁ -C ₆ alkyl, -C ₃ -C ₈ cycloalkyl and -C ₃ -C ₈ heterocycloalkyl;

Each R ^9a is independently selected from: -C ₁ -C ₆ alkyl, -C ₃ -C ₈ cycloalkyl, -aryl, -heteroaryl, and -(C ₁ -C ₆ alkyl)-aryl; or R ^9a is absent, and X ^b ＝X;

Each R ^10a is independently selected from: -C ₁ -C ₆ alkyl, -C ₃ -C ₈ cycloalkyl, -aryl, -heteroaryl, -(C ₁ -C ₆ alkyl)-aryl and

Each R ^10a' is independently selected from: -H, -C ₁ -C ₆ alkyl, -C ₃ -C ₈ cycloalkyl, -aryl, -heteroaryl, and -(C ₁ -C ₆ alkyl)-aryl;

Each R ^10b is independently selected from: -C ₁ -C ₆ alkyl, -C ₃ -C ₈ cycloalkyl, -aryl, -heteroaryl, and -(C ₁ -C ₆ alkyl)-aryl;

R ^11a is absent or is -C ₁ -C ₆ alkyl;

R ^12a is selected from: -C ₁ -C ₆ alkyl, -CO ₂ R ^8a , -aryl, -heteroaryl, -(C ₁ -C ₆ alkyl)-aryl, -S(O) ₂ R ^16a and

R ^13a is selected from: -H and -C ₁ -C ₆ alkyl;

R ^14a is selected from: -C ₁ -C ₆ alkyl, -C ₃ -C ₈ cycloalkyl and -C ₃ -C ₈ heterocycloalkyl;

R ^14a' is selected from the group consisting of: H, -C ₁ -C ₆ alkyl, -C ₃ -C ₈ cycloalkyl, and -C ₃ -C ₈ heterocycloalkyl;

R ^16a is selected from: -C ₁ -C ₆ alkyl, -C ₃ -C ₈ cycloalkyl, -aryl, -heteroaryl and -(C ₁ -C ₆ alkyl)-aryl;

R ^17a is selected from: -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 is selected from: -C ₁ -C ₆ alkyl, -C ₃ -C ₈ cycloalkyl and -(C ₁ -C ₆ alkyl)-OR ^5a ;

R ²² and R ²³ are each independently selected from: -H, -halogen, -C ₁ -C ₆ alkyl and -C ₃ -C ₈ cycloalkyl;

R ²⁴ , R ²⁵ and R ²⁶ are each -C ₁ -C ₆ alkyl;

^Xa and ^Xb are each independently selected from: NH, O and S;

^Xc is selected from the group consisting of: O, S and S(O) ₂ , and

Indicates the connection point with the linker L.

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 .

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 .

In some embodiments, in the compound of Formula (VI), R ^2a is selected from: F and Cl.

In some embodiments, in the compound of Formula (VI), R ^2a is F.

In some embodiments, in the compound of formula (VI), X is -O-, -S- or -NH-, and R ²⁵ is selected from: -C ₁ -C ₆ alkyl, -(C ₁ -C ₆ alkyl)-OR ^5a , -(C ₁ -C ₆ alkyl)-aryl,

or X is O, and R ²⁵ -X- is selected from:

In some embodiments, in the compound of formula (VI), X is -O-, -S- or -NH-, and R ²⁵ is selected from: -C ₁ -C ₆ alkyl, -(C ₁ -C ₆ alkyl)-OR ^5a , -(C ₁ -C ₆ alkyl)-aryl,

In some embodiments, in the compound of formula (VI), X is -O-, -S- or -NH-, and R ²⁵ is selected from: -C ₁ -C ₆ alkyl, -(C ₁ -C ₆ alkyl)-OR ^5a ,

In some embodiments, in the compound of formula (VI), X is -O-, -S- or -NH-, and R ²⁵ is selected from:

In some embodiments, in the compound of Formula (VI), X is -O- or -NH-.

In some embodiments, in the compound of Formula (VI), R ^6a is H.

In some embodiments, in the compound of formula (VI), R ^6a is selected from: -H, unsubstituted -C ₁ -C ₆ alkyl, -C ₁ -C ₆ haloalkyl, -C ₁ -C ₆ hydroxyalkyl, -C ₃ -C ₈ cycloalkyl and -C ₃ -C ₈ heterocycloalkyl.

In some embodiments, in the compound of formula (VI), R ^7a is selected from: -C ₁ -C ₆ alkyl, -C ₃ -C ₈ cycloalkyl, and -C(O)R ^17a .

In some embodiments, in the compound of formula (VI), each R ^9a is independently selected from: -C ₁ -C ₆ alkyl, -C ₃ -C ₈ cycloalkyl, -aryl, and -(C ₁ -C ₆ alkyl)-aryl.

In some embodiments, in the compound of Formula (VI), each R ^9a is independently selected from: -C ₁ -C ₆ alkyl and -(C ₁ -C ₆ alkyl)-aryl.

In some embodiments, in the compound of formula (VI), each R ^10a is independently selected from: -C ₁ -C ₆ alkyl, -C ₃ -C ₈ cycloalkyl, -aryl, -(C ₁ -C ₆ alkyl)-aryl and

In some embodiments, in the compound of formula (VI), each R ^10a is independently selected from: -C ₁ -C ₆ alkyl, -aryl, -( ₁ -C ₆ alkyl)-aryl and

In some embodiments, in the compound of formula (VI), R ^12a is selected from: -C ₁ -C ₆ alkyl, -aryl, -(C ₁ -C ₆ alkyl)-aryl, and -S(O) ₂ R ^16a .

In some embodiments, in the compound of formula (VI), R ^13a is selected from: -H, unsubstituted -C ₁ -C ₆ alkyl, -C ₁ -C ₆ haloalkyl, -C ₁ -C ₆ hydroxyalkyl, and -C ₁ -C ₆ aminoalkyl.

In some embodiments, in the compound of formula (VI), R ^14a′ is selected from: H, unsubstituted -C ₁ -C ₆ alkyl, -C ₁ -C ₆ haloalkyl, -C ₁ -C ₆ hydroxyalkyl, -C ₁ -C ₆ aminoalkyl, -C ₃ -C ₈ cycloalkyl, and -C ₃ -C ₈ heterocycloalkyl.

In some embodiments, in the compound of Formula (VI), R ^16a is selected from: -aryl, -heteroaryl, and -(C ₁ -C ₆ alkyl)-aryl.

In some embodiments, in the compound of Formula (VI), R ^17a is -C ₁ -C ₆ alkyl.

In some embodiments, in the compound of formula (VI), R ²² and R ²³ are each independently selected from: -H, -halogen, unsubstituted -C ₁ -C ₆ alkyl, -C ₁ -C ₆ haloalkyl, -C ₁ -C ₆ hydroxyalkyl, -C ₁ -C ₆ aminoalkyl and -C ₃ -C ₈ cycloalkyl.

In some embodiments, in the compound of Formula (VI), ^Xa and ^Xb are each independently selected from: NH and O.

In some embodiments, in the compound of Formula (VI), ^Xa and ^Xb are each O.

In some embodiments, in the compound of Formula (VI), X is O, and R ²⁵ is -C ₁ -C ₆ alkyl.

In some embodiments, in the compound of Formula (VI), R ^2a is F; X is O, and R ²⁵ is -C ₁ -C ₆ alkyl.

Other combinations of any of the foregoing embodiments of compounds of formula (VI) are also contemplated, and each combination forms a separate embodiment for purposes of this disclosure.

In certain embodiments, each alkyl, cycloalkyl, heterocycloalkyl, aryl and heteroaryl as defined in any one of formula (IV), (V) or (VI) is optionally substituted with one or more substituents selected from the group consisting of halogen, acyl, acyloxy, alkoxy, carboxyl, hydroxyl, amino, amido, nitro, cyano, azido, alkylthio, sulfo, sulfonyl, sulfonamido, alkyl, cycloalkyl, heterocycloalkyl, aryl and heteroaryl. In some embodiments, each alkyl, cycloalkyl, heterocycloalkyl, aryl and heteroaryl as defined in any one of formula (IV), (V) or (VI) is optionally substituted with one or more substituents selected from the group consisting of halogen, acyl, acyloxy, alkoxy, carboxyl, hydroxyl, amino, amido, nitro, cyano, azido, alkylthio, sulfo, sulfonyl and sulfonamido.

In certain embodiments, in the ADC having formula (X), D is a compound of formula (IV), wherein R ^1a is -CH ₃ , 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 -CH ₃ ; R ^2a is F; X is -O-; R ^4a is R ^9a is -C ₁ -C ₆ alkyl, and X ^a and X ^b are each O.

In certain embodiments, in an ADC having formula (X), D is a compound of formula (V), wherein R ^2a is F, and R ^20a is H, -(C ₁ -C ₆ )-OR ⁵ or In some embodiments, in the ADC having formula (X), D is a compound of formula (V), wherein R ^2a is F; R ^20a is H, -(C ₁ -C ₆ )-OR ⁵ or ^R5 is H, and ^R18 and ^R19 together with the N atom to which they are bound 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 ^R2a is F; ^R20a is -( _C1 - _C6 ) ^-OR5 , and ^R5 is H.

In certain embodiments, in an ADC having formula (X), D is a compound of formula (VI), wherein R ^2a is F; X is -O-, and R ²⁵ is -C ₁ -C ₆ alkyl.

Connector L

The conjugate of formula (X) includes a linker L, which is a bifunctional or multifunctional moiety capable of linking one or more camptothecin analogs D to the anti-FRα antibody construct T. A bifunctional (or monovalent) linker L links a single compound D to a single site on the anti-FRα antibody construct T, while a multifunctional (or multivalent) linker L links more than one compound D to a single site on the anti-FRα antibody construct T. Linkers that link one compound D to more than one site on the anti-FRα antibody construct T can also be considered multifunctional.

The linker L includes a functional group capable of reacting with one or more target groups on the anti-FRα antibody construct T, and at least one functional group capable of reacting with a target group on the camptothecin analog D. Suitable functional groups are known in the art, and 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 be used as target groups for linker connection include, but are not limited to, thiol, hydroxyl, carboxyl, amine, aldehyde, and ketone groups.

Non-limiting examples of functional groups capable of reacting with thiols include maleimide, haloacetamide, haloacetyl, activated esters (such as succinimidyl esters, 4-nitrophenyl esters, pentafluorophenyl esters and tetrafluorophenyl esters), anhydrides, acyl chlorides, sulfonyl chlorides, isocyanates and isothiocyanates. In this case, "self-stabilizing" maleimides as described in Lyon et al., 2014, Nat. Biotechnol., 32: 1059-1062 can also be used.

Non-limiting examples of functional groups capable of reacting with amines include activated esters such as N-hydroxysuccinamide (NHS) esters and sulfo-NHS esters, imidoesters such as Traut's reagent, isothiocyanates, aldehydes, and anhydrides such as diethylenetriaminepentaacetic anhydride (DTPA). Other examples include the use of succinimidyl-1,1,3,3-tetramethyluronium tetrafluoroborate (TSTU) or benzotriazol-1-yl-oxytripyrrolephosphonium hexafluorophosphate (PyBOP) to convert carboxylic acids into activated esters, which can then react with amines.

Non-limiting examples of functional groups capable of reacting with an electrophilic group such as an aldehyde or keto carbonyl group include hydrazide, oxime, amino, hydrazine, thiosemicarbazone, hydrazine carboxylate, and arylhydrazide.

In certain embodiments, the linker L may comprise a functional group that allows for bridging of two interchain cysteines on the anti-FRα antibody construct, such as a ThioBridge ^TM 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).

Alternatively, the anti-FRα antibody construct T may be modified to include a non-natural reactive group, such as an azide, which allows conjugation to the joint through a complementary reactive group on the joint. For example, the conjugation of the joint to the anti-FRα antibody construct can utilize click chemistry (see, for example, Chio & Bane, 2020, Methods Mol. Biol., 2078: 83-97), such as azide-alkyne cycloaddition (AAC) reaction, which has been successfully used to develop antibody-drug conjugates. The AAC reaction can be a copper-catalyzed AAC (CuAAC) reaction, which involves the conjugation of azide to straight-chain alkynes; or a strain-promoted AAC (SPAAC) reaction, which involves the conjugation of azide to cyclooctyne.

The linker L can be a cleavable or non-cleavable linker. A cleavable linker is a linker that is easily cleaved under specific conditions, for example, under intracellular conditions (such as in endosomes or lysosomes) or near target cells (such as in tumor microenvironments). Examples include protease-sensitive, acid-sensitive or reduction-sensitive linkers. In contrast, non-cleavable linkers rely on degradation of the antibody in the cell, which generally results in the release of the amino acid-linker-drug moiety.

The example of cleavable linker includes, for example, a linker comprising an amino acid sequence as a cleavage recognition sequence of a protease. Many such cleavage recognition sequences are known in the art. For conjugates that are not intended to be internalized by cells, for example, an amino acid sequence recognized and cleaved by a protease present in the extracellular matrix near target cells such as cancer cells can be used. The example of extracellular tumor-associated proteases includes, for example, plasmin, matrix metalloproteinases (MMPs), elastases, and kallikrein-related peptidases.

For conjugates intended to be internalized by cells, the linker L may comprise an amino acid sequence that is recognized and cleaved by endosomal or lysosomal proteases. Examples of such proteases include, for example, cathepsins B, C, D, H, L, and S, and legumin.

The cleavage recognition sequence can be, for example, a dipeptide, a tripeptide or a tetrapeptide. Non-limiting examples of dipeptide recognition sequences that can 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, Me ₃ Lys-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 tripeptide 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.

Other examples of cleavable linkers include linkers containing disulfide bonds, such as N-succinimidyl-4-(2-pyridyldithio)butyrate (SPDB) and N-succinimidyl-4-(2-pyridyldithio)-2-sulfobutyrate (sulfo-SPDB). Linkers containing disulfide bonds may optionally include additional groups to provide steric hindrance near the disulfide bond to improve the extracellular stability of the linker, for example, including a geminal dimethyl group. Other cleavable linkers include linkers that are hydrolyzable at a specific pH or pH range, such as hydrazone linkers. Linkers containing combinations of these functionalities may also be useful, for example, linkers containing both hydrazone and disulfide bonds are known in the art.

Another example of a cleavable linker is a linker comprising β-glucuronide, which 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). β-glucuronide can also be used to improve the hydrophilicity of the linker L.

Another example of a linker that is cleaved internally within the cell and improves hydrophilicity is a linker comprising a pyrophosphate diester moiety (see, eg, Kern et al., 2016, J Am Chem Soc., 138:2430-1445).

In certain embodiments, the linker L comprised by 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 recognized and cleaved by a lysosomal protease.

The cleavable linker may optionally further comprise one or more additional functional groups, such as self-immolative and self-immolative groups, extending groups or hydrophilic moieties.

Self-decomposition and self-eliminating groups that can be used for joints include, for example, p-aminobenzyl (PAB) and p-aminobenzyloxycarbonyl (PABC) groups, methylated ethylenediamine (MED) and hemiacetal groups. Other examples of self-decomposition groups include, but are not limited to, aromatic compounds similar to PAB or PABC group electronics, such as heterocyclic derivatives, such as 2-aminoimidazole-5-methanol derivatives described in U.S. Patent No. 7,375,078. Other examples include groups that are cyclized when amide bonds are hydrolyzed, such as substituted and unsubstituted 4-aminobutyric acid amides (Rodrigues et al., 1995, Chemistry Biology 2: 223-227) and 2-aminophenylpropionic acid amides (Amsberry et al., 1990, J.Org.Chem.55: 5867-5877). Self-decomposition/self-eliminating groups are generally connected to amino or hydroxyl groups on compound D. Self-decomposition/self-eliminating groups are generally included in peptide-based joints alone or in combination, but may also be included in other types of joints.

Extenders that can be used in the linker of the drug conjugate include, for example, alkylene groups and extenders based on aliphatic acids, diacids, amines or diamines, such as diglycolates, malonates, caproates and caproamides. Other extenders include, for example, glycine-based extenders and polyethylene glycol (PEG) or monomethoxy polyethylene glycol (mPEG) extenders.

PEG and mPEG extenders can also be used as hydrophilic moieties in joints. For example, PEG or mPEG can be "directly inserted" or included in the joint as a side group to increase the hydrophilicity of the joint (see, for example, U.S. Patent Application Publication No. US2016/0310612). Various PEG-containing joints are commercially available from companies such as Quanta BioDesign, Ltd (Plain City, OH). Other hydrophilic groups that may be optionally incorporated into joint L include, for example, β-glucuronide, sulfonic acid groups, carboxylic acid groups, and pyrophosphate diesters.

In certain embodiments, the ADC of formula (X) may comprise a cleavable linker. In some embodiments, the ADC of formula (X) may comprise a peptide-containing linker. In some embodiments, the ADC of formula (X) may comprise a protease-cleavable linker.

In some embodiments, in the ADC of formula (X), m is 1, and the linker L is a cleavable linker having formula (XI):

in:

Z is a functional group capable of reacting with a target group on the anti-FRα antibody construct T;

Str is the extension segment;

AA ₁ and AA ₂ are each independently an amino acid, wherein AA ₁ -[AA ₂ ] _r forms a protease cleavage site;

X is a self-decomposing group;

q is 0 or 1;

r is 1, 2, or 3;

s is 0, 1, or 2;

# is the connection point with the anti-FRα antibody construct T, and

% is the point of attachment to camptothecin analog D.

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

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

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

In some embodiments, in the linker of formula (XI):

Z is Where # is the point of connection to T and * is the point of connection to the rest of the linker.

In some embodiments, in the linker of formula (XI), Str is selected from:

in:

R is H or C ₁ -C ₆ alkyl;

t is an integer between 2 and 10, and

u is an integer between 1 and 10.

In some embodiments, in the linker of formula (XI), Str is selected from:

in:

t is an integer between 2 and 10, and

u is an integer between 1 and 10.

In some embodiments, in the linker of Formula (XI), AA ₁ -[AA ₂ ] _r is a dipeptide (ie, r=1). In some embodiments, in the linker of Formula (XI), AA ₁ -[AA ₂ ] _r has a sequence selected from the group consisting of Ala-(D)Asp, Ala-Lys, Ala-Phe, Asn-Lys, Asn-(D)Lys, Asp-Val, His-Val, Ile-Cit, Ile-Pro, Ile-Val, Leu-Cit, Me ₃ Lys-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.

In some embodiments, in the linker of formula (XI), AA ₁ -[AA ₂ ] _r is a tripeptide (ie, r=2). In some embodiments, in the linker of formula (XI), AA ₁ -[AA ₂ ] _r has a sequence selected from the group consisting of 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.

In some embodiments, in the linker of formula (XI), AA ₁ -[AA ₂ ] _r is a tetrapeptide (ie, r=3). In some embodiments, in the linker of formula (XI), AA ₁ -[AA ₂ ] _r has a sequence selected from the group consisting of Ala-Leu-Ala-Leu, Gly-Phe-Leu-Gly, Gly-Gly-Phe-Gly, and Gly-Phe-Gly-Gly.

In certain embodiments, in the ADC of formula (X), m is 1, and the linker L is a cleavable linker having formula (XII):

in:

Z is a functional group capable of reacting with a target group on the anti-FRα antibody construct T;

Str is the extension segment;

AA ₁ and AA ₂ are each independently an amino acid, wherein AA 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 connection point with the anti-FRα antibody construct T, and

% is the point of attachment to camptothecin analog D.

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

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

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

In some embodiments, in the linker of formula (XII):

Z is Where # is the point of connection to T and * is the point of connection to the rest of the linker.

In some embodiments, in the linker of formula (XII), Str is selected from:

in:

R is H or C ₁ -C ₆ alkyl;

t is an integer between 2 and 10, and

u is an integer between 1 and 10.

In some embodiments, in the linker of formula (XII), Str is selected from:

in:

t is an integer between 2 and 10, and

u is an integer between 1 and 10.

In some embodiments, in the linker of Formula (XII), AA ₁ -[AA ₂ ] _r is a dipeptide (ie, r=1). In some embodiments, in the linker of Formula (XII), AA ₁ -[AA ₂ ] _r has a sequence selected from the group consisting of Ala-(D)Asp, Ala-Lys, Ala-Phe, Asn-Lys, Asn-(D)Lys, Asp-Val, His-Val, Ile-Cit, Ile-Pro, Ile-Val, Leu-Cit, Me ₃ Lys-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.

In some embodiments, in the linker of formula (XII), AA ₁ -[AA ₂ ] _r is a tripeptide (ie, r=2). In some embodiments, in the linker of formula (XII), AA ₁ -[AA ₂ ] _r has a sequence selected from the group consisting of 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.

In some embodiments, in the linker of formula (XII), AA ₁ -[AA ₂ ] _r is a tetrapeptide (ie, r=3). In some embodiments, in the linker of formula (XII), AA ₁ -[AA ₂ ] _r has a sequence selected from the group consisting of Ala-Leu-Ala-Leu, Gly-Phe-Leu-Gly, Gly-Gly-Phe-Gly, and Gly-Phe-Gly-Gly.

In some embodiments, in the linker of formula (XII), Y is -NH-CH _2. In some embodiments, in the linker of formula (XII), v is 1 and Y is -NH-CH ₂ .

In some embodiments, the ADC of formula (X) may comprise a disulfide bond-containing linker. In some embodiments, in the ADC of formula (X), m is 1, and the linker L is a cleavable linker having formula (XIII):

in:

Z is a functional group capable of reacting with a target group on the anti-FRα antibody construct T;

Q is -(CH ₂ ) _p - or -(CH ₂ CH ₂ O) _q -, wherein p and q are each independently an integer between 1 and 10;

Each R is independently H or C ₁ -C ₆ alkyl;

n is 1, 2 or 3;

# is the connection point with the anti-FRα antibody construct T, and

% is the point of attachment to camptothecin analog D.

In some embodiments, the ADC of formula (X) may comprise a b-glucuronide-containing linker.

Various non-cleavable linkers are known in the art for connecting drugs to targeting moieties, and can be used in the ADC of the present disclosure in certain embodiments. Examples of non-cleavable linkers include N-succinimidyl esters or N-sulfosuccinimidyl esters for reacting with anti-FRα antibody constructs, and maleimido or haloacetyl-based linkers for reacting with camptothecin analogs, or vice versa. Examples of such non-cleavable linkers are based on sulfosuccinimidyl-4-[N-maleimidomethyl]cyclohexane-1-carboxylates (sulfoSMCC). Sulfo-SMCC conjugation usually occurs via maleimide groups, which react with sulfhydryls (thiols, -SH) on camptothecin analogs, while sulfo-NHS esters are reactive to primary amines on anti-FRα antibody constructs (such as found in lysine and at the N-terminus of proteins or peptides). Other non-limiting examples of such linkers include those based on N-succinimidyl 4-(maleimidomethyl)cyclohexanecarboxylate (SMCC), N-succinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxy-(6-aminohexanoate) ("long chain" SMCC or LC-SMCC), kappa-maleimidoundecanoic acid N-succinimidyl ester (KMUA), gamma-maleimidobutyric acid N-succinimidyl ester (GMBS), epsilon-maleimidobutyric acid N-succinimidyl ester (EMB), and succinimidyl-succinimidyl ester (SMB), succinimidyl-succinimidyl ester (SMB), and succinimidyl-succinimidyl ester (SMB). Examples include imidocaproic acid N-hydroxysuccinimide ester (EMCS), m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), N-(α-maleimidoacetoxy)-succinimide ester (AMAS), succinimidyl-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).

Non-limiting examples of drug-linkers comprising camptothecin analogs of Formula (I) are shown in Table 8 (FIG. 13), Table 9 (FIG. 14), and Table 10 (FIG. 15). Non-limiting examples of conjugates comprising these drug-linkers are shown in Table 11 (FIG. 16), Table 12 (FIG. 17), and Table 13 (FIG. 18). In certain embodiments, the ADC of Formula (X) comprises a drug-linker selected from the drug-linkers shown in Table 8, Table 9, and Table 10. In certain embodiments, the ADC of Formula (X) is selected from the conjugates shown in Table 11, Table 12, and Table 13, wherein T is an anti-FRα antibody construct and n is between 1 and 10. In some embodiments, the ADC of Formula (X) is selected from the conjugates shown in Table 11, Table 12, and Table 13, wherein T is an anti-FRα antibody construct and n is between 2 and 8. In some embodiments, the ADC of formula (X) is selected from the conjugates shown in Table 11, Table 12, and Table 13, wherein T is an anti-FRα antibody construct and n is between 4 and 8.

In certain embodiments, the ADC of formula (X) comprises a drug-linker (L-(D) _m ) selected from the group consisting of 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, and n is 4 or 8. In some embodiments, the ADC of formula (X) comprises a drug-linker (L-(D) _m ) selected from the group consisting of 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, and n is 8.

Preparation of ADC

ADC of formula (X) can be prepared by standard methods known in the art (see, e.g., 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, e.g., 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).

Typically, the preparation of ADCs involves first preparing a drug-linker D-L comprising one or more camptothecin analogs of Formula (I) and a linker L, and then conjugating the drug-linker D-L to an appropriate group on an anti-FRα antibody construct T. However, the connection of the linker L to the anti-FRα antibody construct T and the subsequent connection of the anti-FRα antibody construct-linker T-L to one or more camptothecin analogs D of Formula (I) is still an alternative method that can be used in some embodiments.

In any of the above methods, suitable groups on the compound D of formula (I) for connecting the linker L include, but are not limited to, thiol groups, amine groups, carboxylic acid groups, and hydroxyl groups. In some embodiments of the present disclosure, the linker L is connected to the compound D of formula (I) via a hydroxyl or amine group on the compound.

In any of the above methods, suitable groups on the anti-FRα antibody construct T for attachment to the linker L include sulfhydryl groups (e.g., on the side chain of a cysteine residue), amino groups (e.g., on the side chain of a lysine residue), carboxylic acid groups (e.g., on the side chain of an aspartic acid or glutamic acid residue), and carbohydrate groups.

For example, the anti-FRα antibody construct T may include one or more naturally occurring sulfhydryl groups that allow the anti-FRα antibody construct T to bond to the linker L through the sulfur atom of the sulfhydryl group. Alternatively, the anti-FRα antibody construct T may include one or more lysine residues that may 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 include one or more carbohydrate groups that may be chemically modified to include one or more sulfhydryl groups.

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

Anti-FRα antibody construct T can also be modified to include additional cysteine residues (see, e.g., U.S. Pat. Nos. 7,521,541; 8,455,622 and 9,000,130) or unnatural amino acids that provide a reactive handle, such as selenomethionine, p-acetylphenylalanine, formylglycine, or p-azidomethyl-L-phenylalanine (see, e.g., Hofer et al., 2009, Biochemistry, 48:12047-12057; Axup et al., 2012, PNAS, 109:16101-16106; Wu et al., 2009, PNAS, 106:3000-3005; Zimmerman et al., 2014, Bioconj. Chem., 25:351-361), to allow site-specific conjugation. Alternatively, the anti-FRα antibody construct T can be modified to include a non-natural reactive group, such as an azide, which allows conjugation to the linker through a complementary reactive group on the linker, for example, by click chemistry (see, e.g., Chio & Bane, 2020, Methods Mol. Biol., 2078: 83-97). Another option is to use GlycoConnect ^™ technology (Synaffix BV, Nijmegen, Netherlands), which involves enzymatic remodeling of antibody glycans to allow the linker to be connected by metal-free click chemistry (see, e.g., European Patent No. EP 2 911 699).

Other protocols for modifying proteins to attach or associate a linker L are known in the art and include those described in Coligan et al., Current Protocols in Protein Science, Vol. 2, John Wiley & Sons (2002).

Alternatively, ADCs can be prepared using transglutaminases, especially bacterial transglutaminases (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 (amine acceptor, usually on antibodies) and an alkyleneamino group (amine donor, usually on drug-linkers), 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 peptide or "tag" containing glutamine, which allows the antibody to be conjugated to a drug-linker using BTG conjugation (see, e.g., U.S. Patent Application Publication No. US2013/0230543 and International (PCT) Publication No. WO 2016/144608).

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

Once conjugation is complete, the average number of compounds of formula (I) conjugated to anti-FRα antibody constructs T (i.e., the "drug-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. In addition, the distribution of drug-linked forms (e.g., the fraction of anti-FRα antibody constructs T containing 0, 1, 2, 3, etc. compounds D of formula (I)) can also be optionally analyzed. Various techniques for measuring 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).

Pharmaceutical composition

For therapeutic use, the ADC of the present disclosure is generally formulated as a pharmaceutical composition. Therefore, certain embodiments of the present disclosure relate to a pharmaceutical composition comprising an ADC as 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.

The pharmaceutical composition can be formulated for administration to a subject, for example, by oral (including, for example, buccal or sublingual), topical, parenteral, rectal or vaginal routes, or by inhalation or spraying. "Parenteral" administration can be subcutaneous injection or intradermal, intraarticular, intravenous, intramuscular, intravascular, intrasternal, intrathecal injection or infusion. The pharmaceutical composition will generally be formulated into a form suitable for administration to a subject, such as a syrup, elixir, tablet, lozenge, pastille, hard or soft capsule, pill, suppository, oily or aqueous suspension, dispersible powder or granules, emulsion, injection or solution. The pharmaceutical composition can be provided as a unit dose formulation.

In certain embodiments, the pharmaceutical composition comprising the ADC is formulated for parenteral administration, for example, in the form of a lyophilized formulation or an aqueous solution. Such pharmaceutical compositions can be provided, for example, in a unit dose injectable form.

Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed. Examples of such carriers include, but are not limited to: buffers such as phosphate, citrate and other organic acids; antioxidants such as ascorbic acid and methionine; preservatives such as octadecyldimethylbenzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl alcohol, benzyl alcohol, alkyl parabens (such as methyl paraben or propyl paraben), catechol, resorcinol, cyclohexanol, 3-pentanol and m-cresol; low molecular weight (less than about 10 residues) polypeptides; Proteins such as serum albumin or 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 ions; metal complexes such as Zn-protein complexes; and non-ionic surfactants such as polyethylene glycol (PEG).

In certain embodiments, the composition comprising ADC can be in the form of a sterile injectable aqueous or oily solution or suspension. Such suspensions can be prepared using suitable dispersants or wetting agents and/or suspending agents known in the art. Sterile injectable solutions or suspensions can contain ADC in non-toxic parenteral acceptable diluents or carriers. Acceptable diluents and carriers that can be used include, for example, 1,3-butanediol, water, Ringer's solution, or isotonic sodium chloride solution. In addition, sterile fixed oils can be used as carriers. To this end, various mild fixed oils can be used, including synthetic monoglycerides or diglycerides. In addition, fatty acids such as oleic acid can be used to prepare injections. Adjuvants, such as local anesthetics, preservatives, and/or buffers can also be included in injectable solutions or suspensions.

In certain embodiments, the composition comprising ADC can be formulated for intravenous administration to humans. Typically, the composition for intravenous administration is a solution in a sterile isotonic aqueous buffer. If necessary, the composition may also include a solubilizing agent and/or a local anesthetic, such as lidocaine, to relieve pain at the injection site. Typically, the components are provided separately or mixed together in unit dosage form, for example, as a dried lyophilized powder or anhydrous concentrate in a sealed container (such as an ampoule or a sachet) indicating the amount of the active agent. When the composition needs to be administered by infusion, it can be distributed with an infusion bottle containing sterile pharmaceutical grade 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 components can be mixed before administration.

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

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 ADCs as therapeutic agents.

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

Certain embodiments of the present disclosure relate to methods of inhibiting proliferation of cancer or tumor cells, comprising contacting the cells with an ADC as 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 as described herein, e.g., an ADC of formula (X).

Some embodiments relate to methods of treating a subject with cancer by administering an ADC as described herein, e.g., an ADC of Formula (X), to the subject. In this case, treating the subject may result in one or more of the following: a reduction in tumor size, a slowing or prevention of an increase in tumor size, a prolonged disease-free survival time between the disappearance or removal of a tumor and its reappearance, prevention of subsequent occurrences of a tumor (e.g., metastasis), a prolonged time to progression, a reduction in one or more adverse symptoms associated with a tumor, and/or a prolonged overall survival time of a subject with cancer.

Certain embodiments relate to the use of an ADC as 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 as described herein, e.g., an ADC of formula (X), in a method of inhibiting cancer cell proliferation and/or killing cancer cells in vitro. Some embodiments relate to the use of an ADC as described herein, e.g., an ADC of formula (X), in a method of inhibiting cancer cell proliferation and/or killing cancer cells in a subject with cancer.

Examples of cancers that can be treated in certain embodiments are carcinomas, including adenocarcinomas and squamous cell carcinomas; melanomas and sarcomas. Carcinomas and sarcomas are also commonly referred to as "solid tumors". Examples of common 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, kidney cancer, lung cancer, ovarian cancer, pancreatic cancer, prostate cancer, gastric cancer, uterine cancer, non-small cell lung cancer (NSCLC) and colorectal cancer. Various forms of lymphoma can also lead to the formation of solid tumors and therefore can also be considered solid tumors in certain cases. Typically, the cancer to be treated is a cancer that expresses FRα.

Certain embodiments relate to methods of inhibiting the growth of FRα-positive tumor cells, comprising contacting the cells with an ADC as 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 of treating a FRα-positive cancer or tumor in a subject.

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 for treating FRα-positive cancers with an ADC as described herein, such as an ADC of formula (X), wherein 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, the ADC of formula (X) can be used to treat triple-negative breast cancer (TNBC).

Certain embodiments of the present disclosure relate to methods for treating FRα-positive cancers with an ADC as described herein, such as an ADC of formula (X), wherein the cancer is a solid tumor expressing FRα at high levels (high FRα solid tumors). Certain embodiments of the present disclosure relate to methods for treating FRα-positive cancers with an ADC as described herein, such as an ADC of formula (X), wherein the cancer is a solid tumor expressing FRα at moderate levels (medium FRα solid tumors). Certain embodiments of the present disclosure relate to methods for treating FRα-positive cancers with an ADC as described herein, such as an ADC of formula (X), wherein the cancer is a solid tumor expressing FRα at moderate to low levels (medium/low FRα solid tumors). Certain embodiments of the present disclosure relate to methods for treating FRα-positive cancers with an ADC as described herein, such as an ADC of formula (X), wherein the cancer is a solid tumor expressing FRα at low levels (low FRα solid tumors). In certain embodiments, the solid tumor is breast cancer, ovarian cancer, colorectal cancer, lung cancer (such as NSCLC), pancreatic cancer, or endometrial cancer.

Drug kit

Certain embodiments relate to a pharmaceutical kit comprising an ADC as described herein, eg, an ADC of Formula (X).

The kit will typically include a container for containing the ADC and a label and/or package insert on or accompanying the container. The label or package insert contains instructions typically included in the commercial packaging of the therapeutic product, providing information about the indications, usage, dosage, administration, contraindications and/or warnings for using such therapeutic products. The label or package insert may also include a notice in the form of a governmental agency regulation for the manufacture, use or sale of regulated drugs or biological products, which reflects the approval of the use or sale of the manufacturing agency for human or animal administration. In some embodiments, the container may have a sterile inlet. For example, the container may be an intravenous solution bag or a vial with a stopper pierceable by a hypodermic needle.

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

Suitable containers include, for example, bottles, vials, syringes, and intravenous solution bags, etc. The container may be made of various materials such as glass or plastic. Where appropriate, one or more components of the kit may be lyophilized or provided in a dry form (such as a powder or granules), and the kit may additionally contain a suitable solvent for reconstituting the lyophilized or dried component.

The kit may also include other materials desirable from a commercial and user standpoint, such as filters, needles, and syringes.

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

Example

Overview

Chemistry: Examples 1 to 3 below illustrate various methods for preparing camptothecin analogs of formula (I). It should be understood that those skilled in the art can prepare these compounds by similar methods or by combining other methods known in the art. It should also be understood that those skilled in the art will be able to use the methods described below or similar methods to prepare other compounds of formula (I) not specifically described below by using appropriate starting components and modifying synthesis parameters as needed. Typically, starting components can be obtained from commercial sources, such as Sigma Aldrich (Merck KGaA), Alfa Aesar and Maybridge (ThermoFisher Scientific Inc.), Matrix Scientific, Tokyo Chemical Industry Ltd. (TCI) and Fluorochem Ltd., or synthesized according to sources known to those skilled in the art (see, for example, March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, 7th edition, John Wiley & Sons, Inc., 2013) or prepared as described herein.

Bioassays: Expression levels of FRα in the cell lines and CDX models employed 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 archived tumor samples.

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; DMMTM: (4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methyl-morpholinium chloride; EDC: 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; Fmoc: fluorenylmethoxycarbonyl; HATU: Tetramethyluronium azabenzotriazole hexafluorophosphate; HIC: hydrophobic interaction chromatography; HOAt: 1-hydroxy-7-azabenzotriazole; HPLC: high performance liquid chromatography; LC/MS: liquid chromatography-mass spectrometry; MC: maleimidocaproyl; MT: maleimidotriglycolate; NMM: N-methylmorpholine; PNP: p-nitrophenol; RP-UPLC-MS: reversed-phase ultra-performance chromatography-mass spectrometry; SEC: size exclusion chromatography; TCEP: tris(2-carboxyethyl)phosphine; Tfp: tetrafluorophenyl; TLC: thin layer chromatography; TFA: trifluoroacetic acid.

General Chemistry Procedures

General Procedure 1: Conversion of Chlorides to Amines

To a stirred solution of the chloride compound in dimethylformamide (0.05M to 0.1M) is added the appropriate secondary amine (3 equiv.) Upon completion (typically 1 to 3 hours as determined by LC/MS), the reaction mixture is purified by reverse phase HPLC to afford the desired product after lyophilization.

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 eq), the appropriate carboxylic acid (1.1 eq), followed by a solution of DMMTM (2 eq) in water (1 M). Upon completion (determined by LC/MS, typically 16 h), the reaction mixture was purified by reverse phase HPLC to provide the desired product after lyophilization.

General Procedure 3: Conversion of amines to sulfonamides

To a stirred solution of the amine compound in dimethylformamide (0.05M to 0.1M) was added DIPEA (3 equiv.) followed by the appropriate sulfonyl chloride. Upon completion (typically 16 hours as determined by LC/MS), the reaction mixture was purified by reverse phase HPLC to provide the desired product after lyophilization.

General Procedure 4: 2-step conversion of amines to ureas (Synthetic Scheme IV; Figure 1D)

Step 1: Add p-nitrophenyl carbonate (1 equivalent) to a stirred solution of the amine compound in dichloromethane or dimethylformamide (0.05M to 0.1M), followed by triethylamine (2 equivalents). After completion (determined by LC/MS, typically 1 to 4 hours), the reaction mixture is concentrated to dryness and then purified by reverse phase HPLC to provide the desired PNP-carbamate intermediate after lyophilization. This intermediate can be used to generate a single analog or divided into multiple batches to generate multiple analogs in the second step. Step 2: Add appropriate primary amine (3 equivalents) to the PNP-carbamate intermediate (0.1M to 0.2M) in dimethylformamide. After completion (determined by LC/MS, typically 1 hour), the reaction mixture is purified by reverse phase HPLC to provide the desired product after lyophilization.

General Procedure 5 Conversion of Amines to Carbamates

To a stirred solution of the amine compound in dichloromethane or dimethylformamide (0.05M to 0.1M) is added p-nitrophenyl carbonate (1 equivalent) followed by triethylamine (2 equivalents). After completion (determined by LC/MS, typically 1 to 4 hours), the appropriate alcohol is added to the resulting PNP-carbamate intermediate. After completion (determined by LC/MS, typically 1 to 16 hours), the reaction mixture is purified by reverse phase HPLC to provide the desired product after lyophilization.

General Procedure 6: Removal of Boc Protecting Group

To a stirred solution of the Boc-protected amine compound in dichloromethane (0.1 M) was added TFA (20 vol%). After completion (typically 1 hour as determined by LC/MS), the reaction mixture was concentrated in vacuo to afford a crude solid or purified as described in General Procedure 9.

General Procedure 7: Copper-mediated amide coupling

To a rapidly stirred solution (0.02 M) of Boc-GGFG-OH (3 eq) and HOAt (3 eq) in a 10% v/v mixture of dimethylformamide in dichloromethane was added EDC (HCl salt, 3 eq). After 5 minutes, a solution (0.02 M) of the amine-containing payload (1 eq) in a 10% v/v mixture of dimethylformamide in dichloromethane was added, followed immediately by the addition of _CuCl2 (4 eq). Upon completion (determined by LC/MS, typically 1 to 16 hours), the reaction mixture was concentrated in vacuo to afford a crude solid or purified by preparative HPLC to afford the desired product after lyophilization.

General Procedure 8: MT Placement

To a stirred solution of the amine compound (1 eq.) in dimethylformamide (about 0.02 M) was added a solution of MT-OTfp (1.2 eq. to 1.5 eq.) in acetonitrile (about 0.02 M), followed by DIPEA (10 uL, 4 eq.) Upon completion (determined by LC/MS, typically 1 to 16 hours), the reaction mixture was concentrated in vacuo to provide a crude solid, which was purified by preparative HPLC to provide the desired product after lyophilization.

General Procedure 9 Compound Purification

Flash chromatography : The crude reaction product was Snap Ultra columns (10 g, 25 g, 50 g or 100 g) (Biotage, Charlotte, NC) were used for purification. The elution was performed on an Isolera ^™ automated rapid system (Biotage, Charlotte, NC) using a linear gradient of ethyl acetate/hexane or methanol/dichloromethane. Reverse phase flash purification was performed on a Snap Ultra C18 column (12 g, 30 g, 60 g or 120 g) eluting with a linear gradient of 0.1% TFA in acetonitrile/0.1% TFA in water. The purified compound was isolated by removing the organic solvent by rotary evaporation or lyophilizing the acetonitrile/water mixture.

Preparative HPLC: Reverse phase HPLC of crude compounds using 5-μm C18 The HPLC-MS/MS analysis was performed on an Agilent 1260 Infinity II preparative LC/MSD system (Agilent Technologies, Inc., Santa Clara, CA) with a linear gradient of 0.1% TFA in acetonitrile/0.1% TFA in water. The purified compound was isolated by lyophilization of the acetonitrile/water mixture.

General Procedure 10: Compound Analysis

LC/MS: Monitor reaction completion and use 2.6-μm C18 The purified compound was analyzed on an Agilent 1290 HPLC/6120 single quadrupole LC/MS system (Agilent Technologies, Inc., Santa Clara, CA) (30×3 mm) column (Phenomenex, Torrance, CA) eluted with a 10% to 100% linear gradient of 0.1% formic acid/acetonitrile/0.1% formic acid/water.

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

Example 1: Preparation of Camptothecin Analogues Having a Methyl Group at the C10 Position

1.1: (S)-11-(Chloromethyl)-4-ethyl-8-fluoro-4-hydroxy-9-methyl-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (Compound 1.1)

The title compound was prepared according to the procedure provided in Li et al., 2019, ACS Med. Chem. Lett., 10(10): 1386-1392.

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)

The title compound was prepared according to the procedure provided in Li et al., 2019, ACS Med. Chem. Lett., 10(10): 1386-1392.

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 starting from compound 1.1 (10 mg) and morpholine according to General Procedure 1. Preparative HPLC purification was accomplished as described in General Procedure 9 eluting with a gradient of 20% to 60% _CH3CN / _H2O + 0.1% TFA to afford the title compound as an off-white solid (TFA salt, 3.6 mg, 26% yield).

LC _/ MS: m/ _z calcd for _C26H26FN3O5 = 479.2, found [M ₊ H] ⁺ = 480.4.

¹ H NMR (300MHz, CDCl ₃ ) δ8.20 (d, J = 8.0Hz, 1H), 7.82 (d, J = 10.4Hz, 1H), 7.67 (s, 1H), 5.77 (d, J = 16.4Hz ,1H),5.42(s,2H),5.33(d,J＝16.4Hz,1H),4.26(s,2H),3.81(t,J＝4.7Hz,4H),2.82–2.76(m,4H) ,2.57(d,J＝1.7Hz,3H),1.99–1.82(m,2H),1.06(t,J＝7.4Hz,3H).

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 starting from compound 1.1 (10 mg) and 1-(phenylsulfonyl)piperazine according to General Procedure 1. Preparative HPLC purification was accomplished as described in General Procedure 9 eluting with a gradient of 20% to 60% _CH3CN / _H2O + 0.1% TFA to afford the title compound as an off-white solid (TFA salt, 3.6 mg, 21% yield).

LC _/ MS: m/ _z calcd for _C32H31FN4O6 = 618.2, found [M ₊ H] ⁺ = 619.4.

¹ H NMR (300MHz, CDCl ₃ ) δ8.07(d,J＝7.9Hz,1H),7.88–7.44(m,7H),5.73(d,J＝16.4Hz,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＝11.6,7.0Hz,2H ), 1.04 (t, J = 7.3Hz, 3H).

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 starting from compound 1.1 (10 mg) and 4-(piperazin-1-ylsulfonyl)aniline according to General Procedure 1. Preparative HPLC purification was accomplished as described in General Procedure 9 eluting with a gradient of 20% to 60% _CH3CN / _H2O + 0.1% TFA to afford the title compound as an off-white solid (TFA salt, 4.7 mg, 27% yield).

LC _{/MS: m/z calcd for C32H32FN5O6 = 633.2} , found [M ₊ H] ⁺ = 634.4.

¹ H NMR (300MHz, MeOD) δ8.32 (d, J = 8.0Hz, 1H), 7.85 (d, J = 10.5Hz, 1H), 7.65 (s, 1H), 7.46 (d, J = 8.7Hz, 2H),6.74(d,J＝8.7Hz,2H),5.61(d,J＝16.5Hz,1H),5.44(s,2H),5.41(d,J＝16.5Hz,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 = 7.3Hz, 3H).

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 starting from compound 1.1 (10 mg) and N-methylpiperazine according to General Procedure 1. Preparative HPLC purification was accomplished as described in General Procedure 9 eluting with a gradient of 20% to 50% _CH3CN / _H2O + 0.1% TFA to afford the title compound as an off-white solid (TFA salt, 3.6 mg, 25% yield).

LC _/ MS: m/ _z calcd for _C27H29FN4O4 = 492.2, found [M ₊ H] ⁺ = 493.4.

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)

The title compound was prepared starting from compound 1.1 (10 mg) and 4-(piperazin-1-yl)aniline according to General Procedure 1. Preparative HPLC purification was accomplished as described in General Procedure 9 eluting with a gradient of 20% to 50% _CH3CN / _H2O + 0.1% TFA to afford the title compound as an off-white solid (TFA salt, 3.7 mg, 23% yield).

LC _{/MS: m/z calcd for C32H32FN5O4 = 569.2} , found [M ₊ H] ⁺ = 570.4.

¹ H NMR (300MHz, MeOD) δ8.39 (d, J = 8.1Hz, 1H), 7.79 (d, J = 10.6Hz, 1H), 7.21 (d, J = 9.0Hz, 2H), 7.14 (d, J＝9.0Hz,2H),5.62(d,J＝16.4Hz,1H),5.49(s,2H),5.41(d,J＝16.4Hz,1H),4.45(s,2H),3.44–3.38( m,4H),3.06–3.00(m,4H),2.58(d,J＝1.8Hz,3H),2.00–1.89(m,2H),1.03(t,J＝7.3Hz,3H).

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 starting from compound 1.1 (10 mg) and piperidine according to General Procedure 1. Preparative HPLC purification was accomplished as described in General Procedure 9, eluting with a gradient of 10% to 60% _CH3CN / _H2O + 0.1% TFA to afford the title compound as an off-white solid (TFA salt, 1.5 mg, 11% yield).

LC _/ MS: m/ _z calcd for _C27H28FN3O4 = 477.2, found [M ₊ H] ⁺ = 478.2.

¹ H NMR (300MHz, MeOD) δ8.34 (d, J = 7.6Hz, 1H), 7.94 (d, J = 10.3Hz, 1H), 7.70 (s, 1H), 5.63 (d, J = 16.4Hz, 1H),5.52(s,2H),5.44(d,J＝16.5Hz,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＝7.4Hz,3H).

1.9: (S)-tert-butyl 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)

The title compound was prepared starting from compound 1.1 (10 mg) and tert-butyl piperazine-1-carboxylate according to General Procedure 1. Preparative HPLC purification was accomplished as described in General Procedure 9 eluting with a gradient of 10% to 60% _CH3CN / _H2O + 0.1% TFA to afford the title compound as an off-white solid (TFA salt, 6.6 mg, 40% yield).

LC _/ MS: m/ _z calcd for _C31H35FN4O6 = 578.2, found [M ₊ H] ⁺ = 579.4.

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)

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).

LC/MS: m/ _z calcd for _C26H27FN4O4 = 478.2, found [M _{+ H} ] ⁺ = 479.2.

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 starting from compound 1.1 (10 mg) and (R)-morpholin-2-ylmethanol according to General Procedure 1. Preparative HPLC purification was accomplished as described in General Procedure 9 eluting with a gradient of 10% to 60% _CH3CN / _H2O + 0.1% TFA to afford the title compound as an off-white solid (TFA salt, 4.6 mg, 32% yield).

LC _/ MS: m/ _z calcd for _C27H28FN3O6 = 509.2, found [M ₊ H] ⁺ = 510.4.

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 starting from compound 1.1 (10 mg) and thiomorpholin-3-ylmethanol according to General Procedure 1. Preparative HPLC purification was accomplished as described in General Procedure 9 eluting with a gradient of 10% to 60% _CH3CN / _H2O + 0.1% TFA to afford the title compound as an off-white solid (TFA salt, 1.5 mg, 12% yield).

LC/MS: calculated m/z = 525.6 (C ₂₇ H ₂₈ FN ₃ O ₅ S), found [M+H] ⁺ = 526.5.

¹ H NMR (300MHz, 10% D ₂ O/CD ₃ CN)8.36(d,J＝8.1Hz,1H),7.83(d,J＝10.7Hz,1H),7.50(s,1H),5.57(d,J＝16.4Hz,1H),5.52–5.29(m ,3H),5.02(d,J＝14.6Hz,1H),4.71–4.54(m,1H),4.27(dd,J＝12.4,5.0Hz,1H),3.98(dd,J＝12.3,3.4Hz, 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＝7.4Hz, 3H).

1.13: (4S)-4-ethyl-8-fluoro-4-hydroxy-11-((4-(hydroxymethyl)-2-oxa-5-azabicyclo[2.2.1]hept-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 starting from compound 1.1 (10 mg) and 2-oxa-5-azabicyclo[2.2.1]heptan-4-ylmethanol according to General Procedure 1. Preparative HPLC purification was accomplished as described in General Procedure 9 eluting with a gradient of 10% to 60% _CH3CN / _H2O + 0.1% TFA to afford the title compound as an off-white solid (TFA salt, 3.5 mg, 29% yield).

LC _/ MS: m/ _z calcd for _C28H28FN3O6 = 521.5, found [M ₊ H] ⁺ = 522.5.

¹ H NMR (300MHz, 10% D ₂ O/CD ₃ CN)δ8.36(d,J＝7.9Hz,1H),7.86(dd,J＝10.6,5.0Hz,1H),7.50(d,J＝1.8Hz,1H),5.63–5.49(m,2H) ,5.37(dd,J＝17.8,14.1Hz,2H),5.05(s,2H),4.63(d,J＝2.5Hz,1H),4.55(d,J＝10.7Hz,1H),4.33(s, 2H),3.92(d,J＝10.7Hz,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=7.4Hz,3H).

1.14: (4S)-4-ethyl-8-fluoro-4-hydroxy-11-((3-(hydroxymethyl)-1,1-dioxothiomorpholino)methyl)-9-methyl-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (Compound 116)

The title compound was prepared starting from compound 1.1 (10 mg) and 3-(hydroxymethyl)-1λ ⁶ -thiomorpholine-1,1-dione according to General Procedure 1. Purification was accomplished as described in General Procedure 9, eluting with a gradient of 10% to 60% CH ₃ CN/H ₂ O + 0.1% TFA to afford the title compound as an off-white solid (TFA salt, 0.2 mg, 2% yield).

LC/MS: calculated m/z = 557.6 (C ₂₇ H ₂₈ FN ₃ O ₇ S), found [M+H] ⁺ = 558.4.

¹ H NMR (300MHz, 10% D ₂ O/CD ₃ CN) δ8.44 (d, J = 8.2 Hz, 1H), 7.80 (d, J = 11.0 Hz, 1H), 7.50 (s, 1H), 5.58 (d,J＝16.5Hz,1H),5.45–5.26(m,3H),4.60(d,J＝14.9Hz,1H),4.33(d,J＝14.7Hz,1H),3.88(d,J＝ 4.8Hz,2H),3.41-2.85(m,4H),2.53(s,2H),2.19(p,J＝2.5Hz,2H),1.74(p,J＝2.5Hz,2H),1.27(s, 2H), 0.95 (t, J = 7.4Hz, 3H).

1.15: (4S)-4-ethyl-8-fluoro-4-hydroxy-11-((6-hydroxy-3-azabicyclo[3.1.1]hept-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 starting from compound 1.1 (10 mg) and 3-azabicyclo[3.1.1]heptan-6-ol according to General Procedure 1. Preparative HPLC purification was accomplished as described in General Procedure 9 eluting with a gradient of 10% to 60% _CH3CN / _H2O + 0.1% TFA to afford the title compound as an off-white solid (TFA salt, 1.3 mg, 11% yield).

LC _/ MS: m/ _z calcd for _C28H28FN3O5 = 505.5, found [M ₊ H] ⁺ = 506.6.

¹ H NMR (300MHz, 10% D ₂ O/CD ₃ CN) δ8.25 (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=7.3Hz,3H).

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 starting from compound 1.1 (10 mg) and 3-fluoroazetidin-3-ylmethanol according to General Procedure 1. Preparative HPLC purification was accomplished as described in General Procedure 9 eluting with a gradient of 10% to 60% _CH3CN / _H2O + 0.1% TFA to afford the title compound as an off-white solid (TFA salt, 1.4 mg, 12% yield).

LC/MS: calculated m/z = 497.5 (C ₂₆ H ₂₅ F ₂ N ₃ O ₅ ), found [M+H] ⁺ = 498.4.

¹ H NMR (300MHz, 10% D ₂ O/CD ₃ CN) δ8.24 (d, J = 7.9 Hz, 1H), 7.85 (d, J = 10.7 Hz, 1H), 7.50 (s, 1H), 5.57 (d,J＝16.5Hz,1H),5.48–5.28(m,3H),4.98(s,2H),4.44–4.14(m,4H),3.78(d,J＝14.9Hz,2H),2.01- 1.86(m,2H),0.95(t,J＝7.4Hz,3H).

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 starting from compound 1.1 (10 mg) and azetidin-3-ylmethanol according to General Procedure 1. Preparative HPLC purification was accomplished as described in General Procedure 9 eluting with a gradient of 10% to 60% _CH3CN / _H2O + 0.1% TFA to afford the title compound as an off-white solid (TFA salt, 0.5 mg, 4.5% yield).

LC _/ MS: m/ _z calcd for _C26H26FN3O5 = 479.5, found [M ₊ H] ⁺ = 480.4.

¹ H NMR (300MHz, 10% D ₂ O/CD ₃ CN) δ8.23 (d, J = 7.8 Hz, 1H), 7.90 (d, J = 10.6 Hz, 1H), 7.53 (s, 1H), 5.58 (d,J＝16.5Hz,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 =3.9Hz,2H),2.58(s,3H),2.01-1.86(m,2H),0.96(t,J=7.4Hz,3H).

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 starting from compound 1.1 (10 mg) and 4,4-difluoropiperidin-3-ylmethanol according to General Procedure 1. Preparative HPLC purification was accomplished as described in General Procedure 9 eluting with a gradient of 10% to 60% _CH3CN / _H2O + 0.1% TFA to afford the title compound as an off-white solid (TFA salt, 4 mg, 32% yield).

LC/MS: calculated m/z = 543.5 (C ₂₈ H ₂₈ F ₃ N ₃ O ₅ ), found [M+H] ⁺ = 544.4.

¹ H NMR (300MHz, 10% D ₂ O/CD ₃ CN) δ8.25 (d, J = 8.0 Hz, 1H), 7.77 (dd, J = 10.7, 1.4 Hz, 1H), 7.47 (s, 1H) ,5.55(d,J＝16.5Hz,1H),5.42–5.25(m,3H),4.66(d,J＝3.2Hz,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).

1.19: (S)-4-ethyl-8-fluoro-4-hydroxy-11-((1-(hydroxymethyl)-7-azabicyclo[2.2.1]hept-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 starting from compound 1.1 (10 mg) and 7-azabicyclo[2.2.1]heptan-1-ylmethanol according to General Procedure 1. Preparative HPLC purification was accomplished as described in General Procedure 9, eluting with a gradient of 10% to 60% _CH3CN / _H2O + 0.1% TFA to afford the title compound as an off-white solid (TFA salt, 0.8 mg, 6.6% yield).

LC/MS: m/ _z calcd for _C29H30FN3O5 = 519.6, found [M _{+ H} ] ⁺ = 520.4.

¹ H NMR (300MHz, 10% D ₂ O/CD ₃ CN) δ8.22 (s, 1H), 7.92 (d, J = 10.7 Hz, 1H), 7.54 (s, 1H), 5.59 (dd, J = 17.6,7.6Hz,2H),5.33(t,J＝17.4Hz,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＝14.0Hz,5H),0.96(t,J＝7.4Hz,3H).

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 starting from compound 1.2 (10 mg) and methanesulfonyl chloride according to General Procedure 3. Preparative HPLC purification was accomplished as described in General Procedure 9 eluting with a gradient of 10% to 50% _CH3CN / _H2O + 0.1% TFA to afford the title compound as an off-white solid (0.8 mg, 7% yield).

LC _/ MS: m/ _{z calcd for C23H22FN3O6S =} 487.1, found [M ₊ H] ⁺ = 488.2.

¹ H NMR (300MHz, MeOD) δ8.33 (d, J = 8.1Hz, 1H), 7.83 (d, J = 10.8Hz, 1H), 7.68 (s, 1H), 5.62 (d, J = 16.3Hz, 1H),5.52(s,2H),5.42(d,J＝16.4Hz,1H),4.87(s,2H),3.06(s,3H),2.59(s,3H),2.06-1.93(m,2H ), 1.03 (t, J = 7.4Hz, 3H).

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 starting from compound 1.2 (20 mg) and (4-nitrophenyl)methanesulfonyl chloride according to General Procedure 3. Preparative HPLC purification was accomplished as described in General Procedure 9 eluting with a gradient of 10% to 50% _CH3CN / _H2O + 0.1% TFA to afford the title compound as an off-white solid (5.0 mg, 17% yield).

LC _/ MS: m/ _{z calcd for C29H25FN4O8S =} 608.1, found [M ₊ H] ⁺ = 609.2.

¹ H NMR (300MHz, CDCl ₃ ) δ8.02–7.92(m,3H),7.74(d,J＝10.5Hz,1H),7.65(s,1H),7.33(d,J＝8.6Hz,2H) ,5.66(d,J＝16.8Hz,1H),5.28(d,J＝16.5Hz,1H),5.14(d,J＝5.4Hz,2H),4.67(s,2H),4.28(d,J＝ 6.3Hz, 2H), 3.39 (s, 3H), 2.03–1.83 (m, 2H), 1.04 (t, J = 7.4Hz, 3H).

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 starting from compound 1.2 (10 mg) and benzenesulfonyl chloride according to General Procedure 3. Preparative HPLC purification was accomplished as described in General Procedure 9 eluting with a gradient of 10% to 50% _CH3CN / _H2O + 0.1% TFA to afford the title compound as an off-white solid (9.8 mg, 73% yield).

LC _/ MS: m/ _{z calcd for C28H24FN3O6S =} 549.6, found [M ₊ H] ⁺ = 550.6.

¹ H NMR (300MHz, DMSO-d6) δ8.60(t,J＝6.2Hz,1H),8.17(d,J＝8.1Hz,1H),7.83(d,J＝10.8Hz,1H),7.71( dd,J＝7.1,1.7Hz,2H),7.66–7.48(m,2H),7.46(dd,J＝8.3,6.8Hz,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＝6.2Hz,2H),2.48(s,3H),1.98–1.76(m,2H), 0.89(t,J=7.3Hz,3H).

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 starting from compound 1.2 (75 mg) and 4-nitrobenzenesulfonyl chloride according to General Procedure 3. Purification of the title compound was accomplished as described in General Procedure 9 using a 12 g C18 column and gradient elution from 5% to 75% _CH3CN / _H2O + 0.1% TFA to afford the title compound as an off-white solid (37.8 mg, 47% yield).

LC _/ MS: m/ _{z calcd for C28H23FN4O8S =} 594.6, found [M ₊ H] ⁺ = 595.2.

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)

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 _H and then stirred at room temperature under _H atmosphere 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 (30 mg, 84% yield) as a light yellow solid.

LC _/ MS: m/ _{z calcd for C28H24FN4O6S =} 564.6, found [M ₊ H] ⁺ = 565.2.

¹ H NMR (300MHz, DMSO-d6) δ8.13(d,J＝8.2Hz,1H),8.02(t,J＝6.2Hz,1H),7.88(d,J＝10.8Hz,1H),7.48– 7.35(m,2H),7.31(d,J＝8.4Hz,1H),6.63–6.45(m,2H),5.45(s,2H),5.36(s,2H),4.50(d,J＝6.3Hz ,2H),1.98–1.75(m,2H),0.89(t,J=7.3Hz,3H).

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 starting from compound 1.2 (20 mg) and 2-hydroxyethanesulfonyl chloride according to General Procedure 3. Preparative HPLC purification was accomplished as described in General Procedure 9 eluting with a gradient of 25% to 50% _CH3CN / _H2O + 0.1% TFA to afford the title compound as an off-white solid (1.3 mg, 13% yield).

LC _/ MS: m/ _{z calcd for C24H24FN3O7S =} 517.1, found [M ₊ H] ⁺ = 518.2.

¹ H NMR (300MHz, DMSO-d6) δ8.30(d,J＝8.4Hz,1H),7.91(d,J＝10.9Hz,1H),7.84(t,J＝6.3Hz,1H),7.33( s,1H),5.50-5.33(m,4H),5.07(t,J＝5.4Hz,1H),4.78(d,J＝6.0Hz,2H),4.07(s,3H),3.80(dt,J =6.3Hz, J=5.8Hz, 2H), 1.86 (m, 2H), 0.87 (d, J=7.3Hz, 3H).

1.26: (S)-N-((4-ethyl-8-fluoro-4-hydroxy-9-methyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)methyl)methanesulfonamide (Compound 131)

To a solution of chlorosulfonyl isocyanate (3uL) in dichloromethane (1mL) was added tert-butyl alcohol (3uL). The solution was stirred for 1 hour, then compound 1.2 (13mg) dissolved in dichloromethane (1mL) was added, followed by triethylamine (13uL). The reaction was stirred for 1 hour, then concentrated to dryness. Preparative HPLC purification of the intermediate Boc compound was completed as described in General Procedure 9, with a 10% to 50% CH ₃ CN/H ₂ O+0.1% TFA gradient elution. Trifluoroacetic acid (200uL) was added to the purified solid in dichloromethane (1mL). The reaction was stirred for 16 hours, then concentrated to dryness to give the title compound (7.5mg, 48% yield) as an off-white solid.

LC/MS: m _{/ z calcd for C22H21FN4O6S =} 488.1, found [M ₊ H] ⁺ = 489.0.

¹ H NMR (300MHz, MeOD) δ8.25 (d, J = 8.1Hz, 1H), 7.73 (d, J = 10.7Hz, 1H), 7.62 (s, 1H), 5.59 (d, J = 16.4Hz, 1H),5.45(s,2H),5.39(d,J＝16.4Hz,1H),4.81(s,2H),2.55(d,J＝1.7Hz,3H),2.07–1.89(m,2H), 1.03(t,J=7.4Hz,3H).

1.27: (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)carbamic acid 4-nitrophenyl ester (Compound 1.27)

The title PNP-carbamate intermediate compound was prepared starting from compound 1.2 (24 mg) according to the first step of General Procedure 4. Purification was accomplished as described in General Procedure 9 using a 12 g column C18 column eluting with a gradient of 10% to 50% _CH3CN / _H2O + 0.1% TFA to afford the title compound as an off-white solid (14 mg, 53% yield).

LC/MS: Calcd. for C ₂₉ H ₂₃ FN ₄ O ₈ S m/z = 574.2, found [M+H] ⁺ = 575.2

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 starting from compound 1.2 (25 mg) and aqueous methylamine as the primary amine (500 uL, 40 wt% in water) according to General Procedure 4. In this case, the crude intermediate PNP-carbamate was used. Preparative HPLC purification was accomplished as described in General Procedure 9, eluting with a gradient of 10% to 50% _CH3CN / _H2O + 0.1% TFA to afford the title compound as an off-white solid (8.9 mg, 31% yield).

LC _/ MS: m/ _z calcd for _C24H23FN4O5 = 466.2, found [M ₊ H] ⁺ = 467.2.

¹ H NMR (300MHz, MeOD) δ8.26 (d, J = 8.2Hz, 1H), 7.79 (d, J = 10.7Hz, 1H), 7.66 (s, 1H), 5.61 (d, J = 16.3Hz, 1H),5.48(s,2H),5.41(d,J＝16.4Hz,1H),4.97(s,2H),2.73(s,3H),2.57(s,3H),2.08–1.93(m,2H ), 1.03 (t, J = 7.4Hz, 3H).

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 accomplished as described in General Procedure 9, eluting with a gradient of 12% to 50% _CH3CN / _H2O + 0.1% TFA to afford the title compound as an off-white solid (0.6 mg, 20% yield).

LC/MS: m/ _z calcd for _C30H28FN5O5 = 557.2, found [M _{+ H} ] ⁺ = 558.4.

¹ H NMR (300MHz, MeOD) δ8.25 (d, J = 8.1Hz, 1H), 7.80 (d, J = 10.8Hz, 1H), 7.67 (s, 1H), 7.43 (d, J = 8.2Hz, 2H),7.24(d,J＝8.3Hz,2H),5.63(d,J＝16.4Hz,1H),5.48(s,2H),5.43(d,J＝16.4Hz,1H),5.01(s, 2H), 4.37 (s, 2H), 2.56 (d, J = 1.7Hz, 3H), 2.05–1.94 (m, 2H), 1.03 (t, J = 7.3Hz, 3H).

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 accomplished as described in General Procedure 9 eluting with a gradient of 10% to 50% _CH3CN / _H2O + 0.1% TFA to afford the title compound as an off-white solid (2.4 mg, 66% yield).

LC _{/MS: m/z calcd for C25H25FN4O6 = 496.2} , found [M ₊ H] ⁺ = 497.2.

¹ H NMR (300MHz, MeOD) δ8.08(d,J＝8.0Hz,1H),7.74(d,J＝10.5Hz,1H),7.68(s,1H),5.64(d,J＝16.4Hz, 1H),5.41(s,2H),5.31(d,J＝16.4Hz,1H),4.96(s,2H),3.63(t,J＝5.2Hz,2H),3.29(t,J＝5.3Hz, 2H), 2.54 (s, 3H), 1.98–1.87 (m, 2H), 1.01 (t, J = 7.4Hz, 3H).

1.31: (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 accomplished as described in General Procedure 9 eluting with a gradient of 20% to 50% _CH3CN / _H2O + 0.1% TFA to afford the title compound as an off-white solid (3.5 mg, 6% yield).

LC _/ MS: m/ _z calcd for _C24H22FN3O6 = 467.2, found [M ₊ H] ⁺ = 468.2.

¹ H NMR (300MHz, MeOD) δ8.17 (d, J = 8.2Hz, 1H), 7.77 (d, J = 10.5Hz, 1H), 7.69 (s, 1H), 5.65 (d, J = 16.5Hz, 1H),5.48(s,2H),5.33(d,J＝16.4Hz,1H),4.86(d,J＝5.6Hz,2H),3.65(s,3H),2.56(s,3H),2.02– 1.89(m,2H),1.02(t,J=7.4Hz,3H).

1.32: (S)-2-Hydroxyethyl ((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) and reacting 1,2-ethanediol with the intermediate PNP-carbamate. Preparative HPLC purification was accomplished as described in General Procedure 9 eluting with a gradient of 10% to 60% _CH3CN / _H2O + 0.1% TFA to afford the title compound as an off-white solid (4.2 mg, 19% yield).

LC _/ MS: m/ _z calcd for _C25H24FN3O7 = 497.2, found [M ₊ H] ⁺ = 498.2.

¹ H NMR (300MHz, DMSO) δ8.23 (d, J = 8.2Hz, 1H), 7.78 (d, J = 10.7Hz, 1H), 7.40 (s, 1H), 5.47 (d, J = 16.5Hz, 1H),5.42(s,2H),5.34(d,J＝16.4Hz,1H),4.77(s,2H),3.99(t,J＝4.9Hz,2H),3.64–3.38(m,2H), 2.48(s,3H),2.02–1.67(m,2H),0.89(t,J=7.3Hz,3H).

Example 2: Preparation of Camptothecin Analogs Having a Methoxy Group at the C10 Position

2.1: 1-(2-amino-4-fluoro-5-methoxyphenyl)-2-chloroethane-1-one (Compound 2.1)

A solution of 3-fluoro-4-methoxyaniline (10 g, 71 mmol) in DCM (100 mL) was cooled to 0 °C. To this solution was first added 1 M BCl ₃ in DCM (71 mL, 71 mmol), followed by 1 M chloro(diethyl)aluminate 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 about 12 with Na ₂ CO _3. The layers were separated and the aqueous layer was extracted with DCM (3×100 mL). The combined organic layers were dried over Na ₂ SO ₄ , 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).

LC/MS: m/z calcd for C ₉ H ₉ ClFNO ₂ = 217.1, found [M+H] ⁺ = 218.1.

¹ H NMR (400MHz, CDCl ₃ ) δ7.19 (d, J = 9.2Hz, 1H), 6.44 (d, J = 12.8Hz, 1H), 4.59 (s, 2H), 3.86 (s, 3H)

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)

Toluene-4-sulfonic acid (157 mg, 0.9 mmol) was added to a solution (200 mL) of compound 2.1 (1.65 g, 7.6 mmol) and (S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyrans [3,4-f] indolizine-3,6,10 (4H)-trione (2 g, 7.6 mmol) in toluene. The solution was heated at 140 ° C for 3 hours and then cooled to room temperature. The product as a yellow precipitate was collected by filtration to obtain the title compound (1.27 g, 2.85 mmol, 37.5% yield).

LC _{/MS: m/z calcd for C22H18ClFN2O5 = 445.2} , found [M ₊ H] ⁺ = 445.1.

¹ H NMR (400MHz, DMSO-d6) δ7.99 (d, J = 12.0Hz, 1H) 7.80 (d, J = 9.2Hz, 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＝7.2Hz,3H)

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 starting from compound 2.2 (10 mg) and morpholine according to General Procedure 1. Preparative HPLC purification was accomplished as described in General Procedure 9, eluting with a gradient of 41% to 60% _CH3CN / _H2O + 0.1% TFA to afford the title compound as an off-white solid (5.6 mg, 20% yield).

LC _/ MS: m/ _z calcd for _C26H26FN3O6 = 495.2, found [M ₊ H] ⁺ = 496.4.

¹ H NMR (300MHz, MeOD) δ7.84–7.70 (m, 2H), 7.59 (s, 1H), 5.62 (d, J = 16.3Hz, 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=7.4Hz,3H).

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 starting from compound 2.2 (10 mg) and 1-(phenylsulfonyl)piperazine according to General Procedure 1. Preparative HPLC purification was accomplished as described in General Procedure 9, eluting with a gradient of 20% to 60% _CH3CN / _H2O + 0.1% TFA to afford the title compound as an off-white solid (2.5 mg, 14% yield).

LC _/ MS: m/ _{z calcd for C32H31FN4O7S =} 634.2, found [M ₊ H] ⁺ = 635.4.

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 starting from compound 2.2 (10 mg) and 4-(piperazin-1-ylsulfonyl)aniline according to General Procedure 1. Preparative HPLC purification was accomplished as described in General Procedure 9 eluting with a gradient of 20% to 60% _CH3CN / _H2O + 0.1% TFA to afford the title compound as an off-white solid (4.0 mg, 23% yield).

LC _/ MS: m/ _{z calcd for C32H32FN5O7S =} 649.2, found [M ₊ H] ⁺ = 650.4.

¹ H NMR (300MHz, DMSO) δ8.08(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＝16.5Hz,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=7.3Hz,3H).

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 starting from compound 2.2 (10 mg) and N-methylpiperazine according to General Procedure 1. Preparative HPLC purification was accomplished as described in General Procedure 9, eluting with a gradient of 20% to 60% _CH3CN / _H2O + 0.1% TFA to afford the title compound as an off-white solid (2.1 mg, 19% yield).

LC _/ MS: m/ _z calcd for _C27H29FN4O5 = 508.2, found [M ₊ H] ⁺ = 509.4.

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 starting from compound 2.2 (10 mg) and 4-(piperazin-1-yl)aniline according to General Procedure 1. Preparative HPLC purification was accomplished as described in General Procedure 9 eluting with a gradient of 20% to 60% _CH3CN / _H2O + 0.1% TFA to afford the title compound as an off-white solid (3.2 mg, 20% yield).

LC/MS: m/ _z calcd for _C32H32FN5O5 = 585.2, found [M _{+ H} ] ⁺ = 586.4.

¹ H NMR (300MHz, MeOD) δ7.83–7.74(m,2H),7.62(s,1H),7.06(d,J＝8.9Hz,2H),6.98(d,J＝8.9Hz,2H), 5.65(d,J＝16.4Hz,1H),5.36(s,2H),5.27(d,J＝16.4Hz,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 = 7.4Hz, 3H).

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 iPr ₂ NEt (100 uL, 0.56 mmol). The solution was heated to reflux for 5 h, cooled to room temperature and quenched with 12 M aqueous HCl (60 uL). The solution was concentrated to about 1/2 volume and 1 M aqueous HCl (1.5 mL) was added, stirred for 5 min, then concentrated to give a brown residue. Purification was accomplished as described in General Procedure 9 using a 12 g C18 flash column eluting with a 5% to 40% CH ₃ CN/H ₂ O+0.1% TFA gradient to give the title compound (179 mg, 75% yield) as a light yellow solid.

LC/MS: Calculated for C ₂₂ H ₂₀ FN ₃ O ₅ m/z = 425.4, found [M+H] ⁺ = 426.2

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 starting from compound 2.8 (10 mg) and methanesulfonyl chloride according to General Procedure 3. Preparative HPLC purification was accomplished as described in General Procedure 9, eluting with a gradient of 5% to 65% _CH3CN / _H2O + 0.1% TFA to afford the title compound as an off-white solid (8.5 mg, 91% yield).

LC _/ MS: m/ _{z calcd for C23H22FN3O7S =} 503.1, found [M ₊ H] ⁺ = 504.2.

¹ H NMR (300MHz, DMSO-d6) δ7.98(d,J＝12.1Hz,1H),7.89(t,J＝6.4Hz,1H),7.80(d,J＝9.1Hz,1H),7.28( s,1H),5.42(s,2H),5.39(s,2H),4.77(d,J＝6.4Hz,2H),4.06(s,3H),3.06(s,3H),1.95-1.73(m ,2H),0.88(d,J=7.3Hz,3H).

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 starting from compound 2.8 (7.5 mg) and benzenesulfonyl chloride according to General Procedure 3. Preparative HPLC purification was accomplished as described in General Procedure 9 eluting with a gradient of 5% to 70% _CH3CN / _H2O + 0.1% TFA to afford the title compound as an off-white solid (4.6 mg, 46% yield).

LC _/ MS: m/ _{z calcd for C28H24FN3O7S =} 565.6, found [M ₊ H] ⁺ = 566.2.

¹ H NMR (300MHz, DMSO-d6) δ8.59(t,J＝6.3Hz,1H),7.94(d,J＝12.2Hz,1H),7.82–7.68(m,2H),7.62–7.46(m ,1H),7.51–7.40(m,1H),7.28(d,J＝8.3Hz,1H),6.52(s,1H),5.44(s,1H),5.36(s,1H),4.64(d, J＝6.3Hz,1H),4.09(s,2H),1.95–1.81(m,1H),0.89(t,J＝7.3Hz,2H).

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 starting from compound 2.8 (12 mg) and 4-nitrobenzenesulfonyl chloride according to General Procedure 3. Purification was accomplished as described in General Procedure 9 using a 12 g C18 flash column and gradient elution from 5% to 75% _CH3CN / _H2O + 0.1% TFA to afford the title compound as a light yellow solid (9.7 mg, 71% yield).

LC _/ MS: m/ _{z calcd for C28H23FN4O9S =} 610.6, found [M ₊ H] ⁺ = 611.5.

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)

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 _H and then stirred at room temperature under _H atmosphere 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 (1.5 mg, 16% yield) as a light yellow solid.

LC _/ MS: m/ _{z calcd for C28H25FN4O7S =} 580.6, found [M ₊ H] ⁺ = 581.4.

¹ H NMR (300MHz, MeOD) δ7.77(d,J＝11.0Hz,1H),7.58(s,1H),7.48(d,J＝8.6Hz,1H),6.61(d,J＝8.6Hz, 1H),5.59(d,J＝16.3Hz,1H),5.39(d,J＝16.4Hz,1H),5.30(s,1H),4.56(s,1H),4.10(d,J＝3.7Hz, 3H), 2.04–1.91 (m, 2H), 1.31 (s, 1H), 1.02 (t, J = 7.3Hz, 3H), 0.90 (s, 1H).

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]indolizine[1,2-b]quinolin-11-yl)methyl)-2-hydroxyethane-1-sulfonamide (Compound 130)

The title compound was prepared starting from compound 2.8 (8 mg) and 2-hydroxyethanesulfonyl chloride according to General Procedure 3. Preparative HPLC purification was accomplished as described in General Procedure 9 eluting with a gradient of 15% to 50% _CH3CN / _H2O + 0.1% TFA to afford the title compound as an off-white solid (2.2 mg, 22% yield).

LC _/ MS: m/ _{z calcd for C24H24FN3O8S =} 533.1, found [M ₊ H] ⁺ = 534.2.

¹ H NMR (300MHz, DMSO-d6) δ7.99 (d, J = 12.2Hz, 1H), 7.89-7.79 (m, 2H), 7.29 (s, 1H), 5.43 (s, 2H), 5.40 (s ,2H),4.76(d,J＝6.4Hz,2H),4.06(s,3H),3.81(t,J＝6.3Hz,2H),3.34(t,J＝6.3Hz,2H),1.94-1.75 (m,2H),0.87(d,J=7.4Hz,3H).

2.14: (S)-4-nitrophenyl ((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 from compound 2.8 (65 mg) and using a 1:1 mixture of dimethylformamide and dichloromethane as solvent. Flash purification was accomplished as described in General Procedure 9 using a 12 g C12 column eluting with a gradient of 10% to 50% CH ₃ CN/H ₂ O + 0.1% TFA to afford the title compound as an off-white solid (61 mg, 86% yield). This intermediate was isolated and used to generate the following compounds.

LC _/ MS: m/ _z calcd for _C29H23FN4O9 = 590.1, found [M ₊ H] ⁺ = 591.2.

2.15: (S)-1-((4-ethyl-8-fluoro-4-hydroxy-9-methoxy-3,14-dioxo-3,4,12,14-methylhydro-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 accomplished as described in General Procedure 9, eluting with a gradient of 47% to 60% _CH3CN / _H2O + 0.1% TFA to afford the title compound as an off-white solid (5.8 mg, 20% yield).

LC/MS: m/ _z calcd for _C24H23FN4O6 = 482.2, found [M _{+ H} ] ⁺ = 483.2.

¹ H NMR (300MHz, DMSO-d6) δ8.00–7.87(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=7.3Hz,3H).

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 accomplished as described in General Procedure 9, eluting with a gradient of 20% to 60% _CH3CN / _H2O + 0.1% TFA to afford the title compound as an off-white solid (TFA salt, 2.1 mg, 12% yield).

LC _{/MS: m/z calcd for C30H28FN5O6 = 573.2} , found [M ₊ H] ⁺ = 574.2.

¹ H NMR (300MHz, MeOD) δ7.79 (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＝8.2Hz,2H),5.61(d,J＝16.3Hz,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＝7.4Hz,3H).

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 accomplished as described in General Procedure 9, eluting with a gradient of 12% to 60% _CH3CN / _H2O + 0.1% TFA to afford the title compound as an off-white solid (1.5 mg, 20% yield).

LC _/ MS: m/ _z calcd for _C25H25FN4O7 = 512.2, found [M ₊ H] ⁺ = 513.2.

¹ H NMR (300MHz, MeOD) δ7.93 (d, J = 12.1Hz, 1H), 7.88 (d, J = 9.2Hz, 1H), 7.56 (s, 1H), 5.62 (d, J = 16.2Hz, 1H),5.52(s,2H),5.45(d,J＝16.3Hz,1H),4.98(s,2H),4.17(s,3H),3.59(t,J＝5.6Hz,2H),3.28( t,J＝5.6Hz,2H),2.10–1.91(m,2H),1.05(t,J＝7.3Hz,3H).

Example 3: Preparation of Camptothecin Analogs Having an Amino Group at the C10 Position

3.1: 5-Bromo-4-fluoro-2-nitrobenzaldehyde (Compound 3.1)

To a stirred solution of HNO ₃ (121.2 mL, 67% purity, 2.0 eq) in H ₂ SO ₄ (500 mL) at 0° C. was added 3-bromo-4-fluorobenzaldehyde (180 g, 1.0 eq). After the addition was complete, the ice bath was removed and the reaction was stirred at 25° C. for 5 hours. The mixture was poured into ice (5 L), filtered, and then dried in vacuo. The title compound (219 g) was obtained as a yellow solid.

¹ H NMR (400MHz, CDCl ₃ ) δ 10.39 (s, 1H), 8.23 (d, J = 6.8 Hz, 1H), 7.91 (d, J = 7.6 Hz, 1H).

3.2: (2-Fluoro-5-formyl-4-nitrophenyl)carbamic acid tert-butyric acid (Compound 3.2)

A mixture of compound 3.1 (219 g, 1.0 eq.), tert-butyl carbamate (124 g, 1.2 eq.), Cs ₂ CO ₃ (575 g, 2.0 eq.), Pd ₂ (dba) ₃ (40 g, 0.05 eq.) and XPhos (84 g, 0.2 eq.) in toluene (2000 mL) was degassed and purged with N ₂ for three cycles. The mixture was then stirred at 90 ° C. and under N ₂ atmosphere for 15 hours. The reaction mixture was diluted with H ₂ O (800 mL) and extracted with EtOAc (300 mL×2). The combined organic layers were washed with brine (200 mL×2), then dried over sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by column chromatography (SiO ₂ , petroleum ether:ethyl acetate=100:1 to 20:1) to give the title compound (140 g, 56% yield) as a yellow solid.

¹ H NMR (400MHz, DMSO-d6) δ10.24(s,1H),9.94(s,1H),8.42(d,J＝7.6Hz,1H),8.16(d,J＝10.8Hz,1H), 1.50(s,9H)

3.3: tert-Butyl (4-amino-2-fluoro-5-formylphenyl)carbamate (Compound 3.3)

To a solution of compound 3.2 (100 g, 1.0 eq.) in H ₂ O (300 mL) and EtOH (1200 mL) was added NH ₄ Cl (30.5 g, 1.62 eq.). Iron (78.6 g, 4.0 eq.) was added in portions at 80° C. The mixture was stirred at 80° C. for 6 hours. 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 vacuum. The residue was purified by column chromatography (SiO ₂ , petroleum ether:ethyl acetate=1:0 to 0:1), TLC (petroleum ether) to give the title compound (19.0 g, 21% yield) as a yellow solid.

LC _/ MS: m/ _z calcd for _C12H15FN2O3 = 254.1, found [M ₊ H] ⁺ = 255.0.

¹ H NMR (400MHz, DMSO-d6) δ9.73 (s, 1H), 8.57 (s, 1H), 7.58 (d, J = 4.8Hz, 1H), 7.21 (s, 2H), 6.53 (d, J =12.8Hz,1H),1.43(s,9H).

3.4: (S)-tert-butyl (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 eq.), (S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-f]indolizine-3,6,10(4H)-trione (3.5 g, 1 eq.) and TsOH (monohydrate, 253 mg, 0.1 eq.) in toluene (350 mL) was stirred for 2 hours at 110 ° C. The reaction solution was cooled to 25 ° C, filtered, and the solid was washed with methyl tert-butyl ether (30 mL) and then dried in vacuo. The title compound (4.5 g, 62% yield) was obtained as a yellow solid.

LC _/ MS: m/ _z calcd for _C25H24FN3O6 = 481.2, found [M ₊ H] ⁺ = 482.1.

¹ H NMR (400MHz, DMSO-d6) δ9.49 (s, 1H), 8.65 (s, 1H), 8.43 (d, J = 8.4Hz, 1H), 7.95 (d, J = 12.0Hz, 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＝ 7.2Hz,3H)

3.5: (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)carbamic acid tert-butyl ester (Compound 3.5)

To a mixture of compound 3.4 (4.00 g) in MeOH (360 mL) was added a solution of FeSO ₄ (heptahydrate, 1.2 g), H ₂ SO ₄ (280 mL) in H ₂ O (4 mL). The reaction mixture was heated at 65 ° C while H ₂ O ₂ (24 mL, 30% purity) was added dropwise over 30 minutes and then stirred for 0.5 hours. The reaction solution was cooled to 25 ° C and then filtered to give the title compound as a yellow solid (1.53 g, 33.2% yield). H ₂ O (400 mL) was added to the filtrate and then quenched with a saturated aqueous solution of Na ₂ S ₂ O _3. The pH was adjusted to 7 to 8 with a saturated aqueous solution of Na ₂ CO ₃ , and the solution was then concentrated and filtered. The solid was ground with MeOH (30 mL) at 55 ° C for 1 hour and then filtered to give a second batch of the title compound as a brown solid (1.09 g, 26% yield).

LC _/ MS: m/ _z calcd for _C26H26FN3O7 = 511.2, found [M ₊ H] ⁺ = 512.2.

¹ H NMR (300MHz, d6-DMSO) δ9.47 (s, 1H), 8.47 (d, J = 7.6Hz, 1H), 7.94 (d, J = 12.0Hz, 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＝4.4Hz,2H),1.90-1.83 (m, 2H), 1.52 (s, 9H), 0.88 (t, J = 6.4Hz, 3H).

3.6: (S)-(4-ethyl-8-fluoro-11-formyl-4-hydroxy-3,14-dioxo-3,4,12,14-methylhydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl)carbamic acid tert-butyl ester (Compound 3.6)

DCM (2.9 mL) was added to a 50 mL round-bottom flask containing compound 3.5 (150 mg, 0.293 mmol), 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 hours, then diluted with DCM, washed with saturated aqueous NaHCO ₃ and brine. The layers were separated and the combined organic layers were evaporated onto celite. Rapid purification was accomplished as described in General Procedure 9 using a 10 g silica gel column eluted with 0% to 10% DCM/MeOH to give the title product (42.5 mg, 28%) as an orange powder.

LC _/ MS: m/ _z calcd for _C26H24FN3O7 = 509.2, found [M ₊ H] ⁺ = 510.4.

¹ H NMR (300 MHz, acetone-d6) δ11.10 (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＝7.6Hz,1H),7.94(d,J＝12.0Hz,1H),7.29(d,J＝1.6Hz,1H),6.49(s,1H),5.86-5.76(m,1H),5.42(s,2H),5.38(s,2H),5.16(d,J＝4.4Hz,2H),1.90-1.8 3(m,2H),1.52(s,9H),0.88(t,J=6.4Hz,3H).

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)

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).

LC _/ MS: m/ _z calcd for _C20H16FN3O4 = 381.1, found [M ₊ H] ⁺ = 382.2.

¹ H NMR (300MHz, DMSO) δ8.28 (s, 1H), 7.72 (d, J = 12.5Hz, 1H), 7.21 (d, J = 7.3Hz, 1H), 5.43 (d, J = 16.2Hz, 1H), 5.34 (d, J = 16.2Hz, 1H), 5.17 (s, 2H), 1.92–1.74 (m, 2H), 0.88 (t, J = 7.3Hz, 3H).

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)

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).

LC _/ MS: m/ _z calcd for _C21H18FN3O5 = 411.2, found [M ₊ H] ⁺ = 412.2.

¹ H NMR (300MHz, MeOD) δ7.71 (d, J = 12.2Hz, 1H), 7.60 (s, 1H), 7.29 (d, J = 9.5Hz, 1H), 5.61 (d, J = 16.3Hz, 1H), 5.47 (s, 2H), 5.40 (d, J = 16.3Hz, 1H), 5.25 (s, 2H), 2.03–1.94 (m, 2H), 1.03 (t, J = 7.4Hz, 3H).

3.9: (S)-tert-butyl (11-(chloromethyl)-4-ethyl-8-fluoro-4-hydroxy-3,14-dioxo-3,4,12,14-methyl-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 uL) in dichloromethane (0.1 mL). After 1 hour, a solution of thionyl chloride (14 uL) in dichloromethane (0.1 mL) was added. After another hour, the reactant 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 subsequent reactions without further purification.

LC _/ MS: m/ _z calcd for _{C26H25ClFN3O6} = 529.1, found [M ₊ H] ⁺ = 530.2.

3.10: (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)aminomethyl tert-butyl ester (Compound 3.10)

To a solution of compound 3.9 (100 mg) in ethanol (500 uL) was added hexamethylenetetramine (79 mg) followed by DIPEA (99 uL). The solution was heated at 60 °C for 16 hours and then concentrated to dryness in vacuo. Flash purification was accomplished as described in General Procedure 9 using a 12 g C18 column eluting with a 10% to 50% CH ₃ CN/H ₂ O + 0.1% TFA gradient to afford the title compound as an off-white solid (TFA salt, 29 mg, 24% yield).

LC _/ MS: m/ _z calcd for _C26H27FN4O6 = 510.2, found [M ₊ H] ⁺ = 511.4.

¹ H NMR (300MHz, MeOD) δ8.88 (d, J = 8.2Hz, 1H), 7.96 (d, J = 11.9Hz, 1H), 7.62 (s, 1H), 5.60 (d, J = 16.4Hz, 1H),5.48(s,2H),5.41(d,J＝16.4Hz,1H),4.80(s,2H),2.07–1.89(m,2H),1.64(s,9H),1.02(t,J =7.3Hz,3H).

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)

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).

LC _/ MS: m/ _z calcd for _C21H19FN4O4 = 410.1, found [M ₊ H] ⁺ = 411.2.

¹ H NMR (300MHz, MeOD) δ7.82 (d, J = 12.1Hz, 1H), 7.60 (s, 1H), 7.37 (d, J = 9.1Hz, 1H), 5.61 (d, J = 16.3Hz, 1H), 5.42 (s, 2H), 5.41 (d, J = 16.3Hz, 1H), 4.69 (s, 2H), 2.08–1.94 (m, 2H), 1.03 (t, J = 7.4Hz, 3H).

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 starting from compound 3.9 (150 mg) and morpholine according to General Procedure 1. Preparative HPLC purification was accomplished as described in General Procedure 9, eluting with a gradient of 10% to 60% _CH3CN / _H2O + 0.1% TFA to afford the title compound as a red solid (TFA salt, 103 mg, 52% yield).

LC/MS: m/ _z calcd for _C30H33FN4O7 = 580.2, found [M _{+ H} ] ⁺ = 581.4.

¹ H NMR (300MHz, MeOD) δ9.06 (d, J = 8.3Hz, 1H), 7.93 (d, J = 12.0Hz, 1H), 7.66 (s, 1H), 5.63 (d, J = 16.3Hz, 1H),5.51(s,2H),5.43(d,J＝16.4Hz,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 = 7.4Hz, 3H).

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)

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).

LC _/ MS: m/ _z calcd for _C25H25FN4O5 = 480.2, found [M ₊ H] ⁺ = 481.4.

¹ H NMR (300MHz, MeOD) δ7.73 (d, J = 12.0Hz, 1H), 7.54 (s, 1H), 7.48 (d, J = 9.2Hz, 1H), 5.60 (d, J = 16.3Hz, 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=7.3Hz,3H).

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) was added dichloromethane (1.45 mL) 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 hours, quenched by the addition of water + 0.1% TFA and DMF (1: 1, 1.0 mL), and partially evaporated. Purification was accomplished as described in General Procedure 9 using a 12 g C18 flash column and eluting with a 5% to 40% CH ₃ CN / H ₂ O + 0.1% TFA gradient to afford the Boc-protected intermediate as a yellow powder. The intermediate was then deprotected according to General Procedure 6 to afford the title compound as a yellow solid (TFA salt, 32.5 mg, 98% yield).

LC/MS: m/ _z calcd for _C26H27FN4O4 = 478.2, found [M _{+ H} ] ⁺ = 479.4.

¹ H NMR (300MHz, MeOD) δ7.78 (d, J = 12.1Hz, 1H), 7.56 (s, 1H), 7.41 (d, J = 9.1Hz, 1H), 5.60 (d, J = 16.4Hz, 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=7.4Hz,3H).

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 hours, then sodium triacetoxyborohydride (7.8 mg, 0.037 mmol) was added and stirred for another 45 minutes. Excess hydride was then quenched by the addition of 0.1% aqueous TFA (0.5 mL). Purification was accomplished as described in General Procedure 9 using a 12 g C18 flash column and eluting with a 5% to 40% CH ₃ CN/H ₂ O + 0.1% TFA gradient to afford the Boc protected intermediate as a yellow powder. The intermediate was deprotected according to General Procedure 6 to afford the title product as a yellow solid (TFA salt, 1.5 mg, 7.1% yield).

LC _/ MS: m/ _z calcd for _C26H28FN5O4 = 493.2, found [M ₊ H] ⁺ = 494.4.

¹ H NMR (300MHz, MeOD) δ7.68 (d, J = 12.2Hz, 1H), 7.56 (s, 1H), 7.53 (d, J = 9.5Hz, 1H), 5.60 (d, J = 16.3Hz, 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 = 7.4Hz, 3H).

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 starting from compound 3.9 (10 mg) and 1-(phenylsulfonyl)piperazine according to General Procedure 1. Preparative HPLC was performed as described in General Procedure 9, eluting with a gradient of 35% to 44% _CH3CN / _H2O + 0.1% TFA 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 2 steps).

LC _/ MS: m/ _{z calcd for C31H30FN5O6S =} 619.2, found [M ₊ H] ⁺ = 520.4.

¹ H NMR (300MHz, MeOD) δ7.81-7.60 (m, 7H), 7.34 (s, 1H), 5.51 (d, J = 16.4Hz, 1H), 5.35 (d, J = 16.4Hz, 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＝7.4Hz, 3H).

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 starting from compound 3.10 (8 mg) and acetic acid according to General Procedure 2 followed by General Procedure 6. Preparative HPLC purification of the intermediate Boc protected compound was accomplished as described in General Procedure 9, eluting with a gradient of 10% to 60% _CH3CN / _H2O + 0.1% TFA. The title compound was obtained as a red solid (4.0 mg, 56% yield).

LC _/ MS: m/ _z calcd for _C23H21FN4O5 = 452.2, found [M ₊ H] ⁺ = 453.2.

¹ H NMR (300MHz, MeOD) δ7.69 (d, J = 12.1Hz, 1H), 7.56 (s, 1H), 7.38 (d, J = 9.3Hz, 1H), 5.59 (d, J = 16.3Hz, 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 = 7.4Hz, 3H).

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 starting from compound 3.10 (8 mg) and methanesulfonyl chloride according to General Procedure 3 followed by General Procedure 6. Preparative HPLC purification of the intermediate Boc protected compound was accomplished as described in General Procedure 9, eluting with a 10% to 60% _CH3CN / _H2O + 0.1% TFA gradient. The title compound was obtained as a red solid (4.4 mg, 57% yield).

LC/MS: m _{/ z calcd for C22H21FN4O6S =} 488.1, found [M ₊ H] ⁺ = 489.2.

¹ H NMR (300MHz, MeOD) δ7.74(d,J＝12.2Hz,1H),7.60(s,1H),7.49(d,J＝9.3Hz,1H),5.61(d,J＝16.2Hz, 1H),5.45(s,2H),5.40(d,J＝16.2Hz,1H),4.78(s,2H),3.05(s,3H),2.08–1.94(m,2H),1.03(t,J =7.4Hz,3H).

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 starting from compound 3.10 (6 mg) and 2-hydroxyethanesulfonyl chloride according to General Procedure 3 followed by General Procedure 6. Preparative HPLC purification of the intermediate Boc protected compound was accomplished as described in General Procedure 9, eluting with a gradient of 10% to 60% _CH3CN / _H2O + 0.1% TFA. The title compound was obtained as a red solid (1 mg, 16% yield).

LC _/ MS: m/ _{z calcd for C23H23FN4O7S =} 518.5, found [M ₊ H] ⁺ = 519.5.

¹ H NMR (300MHz, 10% D ₂ O/CD ₃ CN) δ7.77–7.61 (m, 1H), 7.48–7.30 (m, 2H), 5.53 (d, J = 16.3Hz, 1H), 5.31 ( d,J＝15.4Hz,3H),4.69(s,2H),3.97(dd,J＝6.6,4.9Hz,2H),3.39(t,J＝5.8Hz,2H),2.93(s,1H), 1.99-1.83(m,2H),0.94(t,J=7.3Hz,3H).

3.20: (S)-4-nitrophenyl ((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 uL, 0.05 M) was added 4-nitrophenyl carbonate (12 mg, 0.04 mmol) and diisopropylethylamine (6.8 uL, 0.04 mmol). The solution was stirred at room temperature for about 30 minutes and then used directly in the subsequent reaction.

3.21: (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 200 ul of a solution of compound 3.20. The solution was stirred at room temperature for 30 minutes. Preparative HPLC purification of the intermediate Boc protected compound was accomplished as described in General Procedure 9, eluting with a 10% to 60% CH ₃ CN/H ₂ O + 0.1% TFA gradient. The title compound was obtained as a red solid according to General Procedure 6 (2.1 mg, 47% yield).

LC _{/MS: m/z calcd for C23H21FN4O6 = 468.4} , found [M ₊ H] ⁺ = 468.3.

¹ H NMR (300MHz, 10% D ₂ O/CD ₃ CN) δ7.72 (d, J = 12.2 Hz, 1H), 7.41 (d, J = 18.1 Hz, 1H), 6.96 (s, 1H), 5.52 (d,J＝3.6Hz,1H),5.39–5.23(m,3H),4.82(s,1H),4.73(s,1H),3.63(d,J＝1.2Hz,3H),1.56(s, 3H), 1.27 (s, 2H), 0.94 (t, J = 7.4Hz, 3H).

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 ul solution of compound 3.20 followed by iPr ₂ NEt (5 uL). The solution was stirred at room temperature for 30 minutes. Preparative HPLC purification of the intermediate Boc protected compound was accomplished as described in General Procedure 9 using a 10% to 60% CH ₃ CN/H ₂ O + 0.1% TFA gradient. The title compound was obtained as a red solid according to General Procedure 6 (2.9 mg, 64.5% yield).

LC _/ MS: m/ _z calcd for _C23H21FN5O5 = 467.5, found [M ₊ H] ⁺ = 468.5.

¹ H NMR (300MHz, 10% D ₂ O/CD ₃ CN) δ8.13 (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＝9.2Hz,2H),5.55(d,J＝16.5Hz,2H),5.44–5.27(m,4H),4.85(s,2H),4.78( s, 1H), 1.56 (d, J = 2.5Hz, 3H), 1.27 (s, 2H), 0.93 (q, J = 11.7, 9.5Hz, 3H).

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 200 ul solution of compound 3.20. The solution was stirred at room temperature for 30 minutes. Preparative HPLC purification of the intermediate Boc protected compound was accomplished as described in General Procedure 9, eluting with a 10% to 60% CH ₃ CN/H ₂ O + 0.1% TFA gradient. The title compound was obtained as a red solid according to General Procedure 6 (0.5 mg, 8.5% yield).

LC _/ MS: m/ _z calcd for _C24H24FN5O6 = 497.5, found [M ₊ H] ⁺ = 498.5.

¹ H NMR (300MHz, 10% D ₂ O/CD ₃ CN) δ7.77–7.61 (m, 1H), 7.48–7.30 (m, 2H), 5.53 (d, J = 16.3Hz, 1H), 5.31 ( d,J＝15.4Hz,1H),5.19(s,2H),4.69(s,2H),3.97(dd,J＝6.6,4.9Hz,2H),3.39(t,J＝5.8Hz,2H), 2.93(s,1H),2.01-1.83(m,2H),0.94(t,J=7.3Hz,3H).

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 mL, 2.5 equiv). The solution was stirred at room temperature for 20 minutes before additional thionyl chloride (35 mL, 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 accomplished as described in General Procedure 9 with a gradient elution of 5% to 50% CH ₃ CN/H ₂ O + 0.1% TFA to afford the title compound (20 mg, 23% yield) as an off-white solid.

LC _/ MS: m/ _z calcd for _C21H17FN6O4 = 436.1, found [M ₊ H] ⁺ = 437.2.

¹ H NMR (300MHz, MeOD) δ7.75 (d, J = 12.2Hz, 1H), 7.60 (s, 1H), 7.38 (d, J = 9.3Hz, 1H), 5.61 (d, J = 16.3Hz, 1H), 5.46–5.35 (m, 3H), 5.07 (s, 2H), 2.03–1.97 (m, 2H), 1.03 (t, J = 7.3Hz, 3H).

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 starting from compound 145 (10 mg) and glycolic acid according to General Procedure 2. Preparative HPLC purification was accomplished as described in General Procedure 9, eluting with a gradient of 10% to 45% CH ₃ CN/H ₂ O + 0.1% TFA. The title compound was obtained as a yellow solid (6.9 mg, 60% yield).

LC _{/MS: m/z calcd for C23H21FN4O6 = 468.1} , found [M ₊ H] ⁺ = 469.2.

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

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 eq.) in DMF (1 mL) was added thiocarbonyldiimidazole (6 mg, 1.5 eq.) followed by DIPEA (8 μL, 2.0 eq.). The resulting solution was stirred at 25 °C for 2 hours after which complete conversion to the isothiocyanate intermediate was observed. Methylammonium chloride (3 mg, 2.0 eq.) was then added and the reaction mixture was heated at 60 °C for 30 minutes. Preparative HPLC purification was accomplished as described in General Procedure 9 with a 10% to 45% CH ₃ CN/H ₂ O+0.1% TFA gradient elution. The title compound (2.3 mg, 22% yield) was obtained as a yellow solid.

LC _/ MS: m _{/z calcd for C23H22FN5O4S =} 483.1, found [M ₊ H] ⁺ = 484.2.

¹ H NMR (300MHz, MeOD) δ7.70 (d, J = 12.0Hz, 1H), 7.60 (s, 1H), 7.38 (d, J = 9.3Hz, 1H), 5.62 (d, J = 16.2Hz, 1H),5.36(s,2H),5.31(d,J＝16.2Hz,1H),5.30(s,2H),3.04(s,3H),1.99–1.90(m,2H),1.02(t,J =7.4Hz,3H).

3.27: (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)thiocarbamic acid S-(2-hydroxyethyl) ester (Compound 160)

The title compound was prepared starting from compound 145 (10 mg) and 2-mercaptoethanol according to General Procedure 5. Preparative HPLC purification was accomplished as described in General Procedure 9, eluting with a gradient of 10% to 45% _CH3CN / _H2O + 0.1% TFA. The title compound was obtained as a yellow solid (4.2 mg, 43% yield).

LC _/ MS: m/ _{z calcd for C24H23FN4O6S =} 514.1, found [M ₊ H] ⁺ = 515.2.

¹ H NMR (300MHz, MeOD) δ7.71 (d, J = 12.1Hz, 1H), 7.60 (s, 1H), 7.36 (d, J = 9.4Hz, 1H), 5.62 (d, J = 16.3Hz, 1H),5.42(s,2H),5.35(d,J＝16.2Hz,1H),4.88(d,J＝4.6Hz,2H),3.68(t,J＝6.4Hz,2H),3.03(t, J＝6.5Hz,2H),2.04–1.92(m,2H),1.03(t,J＝7.4Hz,3H).

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)

To a 5 mL flask containing compound 140 (50 mg) was added water (0.72 mL), FeSO ₄ (heptahydrate, 11.0 mg) and propionaldehyde (74 μL). The resulting suspension was cooled to -15 °C using an ice-salt bath, and sulfuric acid (0.40 mL) was then 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 completed as described in General Procedure 9, with a gradient elution of 25% to 70% CH ₃ CN/H ₂ O+0.1% TFA to give the title compound (2.4 mg, 4.4% yield) as a dark orange solid.

LC _/ MS: m/ _z calcd for _C22H20FN3O4 = 410.1, found [M ₊ H] ⁺ = 410.2.

¹ H NMR (300MHz, MeOD) δ7.63 (d, J = 12.3Hz, 1H), 7.55 (s, 1H), 7.36 (d, J = 9.4Hz, 1H), 5.57 (d, J = 16.4Hz, 1H),5.37(d,J＝16.4Hz,1H),5.21(s,2H),3.13(q,J＝7.7Hz,2H),2.02–1.90(m,2H),1.38(t,J＝7.7 Hz, 3H), 1.01 (t, J = 7.3Hz, 3H).

3.29: (S)-tert-butyl (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 conical 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 allowed to warm to room temperature and stirred for 2 hours, then heated at 70°C for 1 hour. The reaction mixture was cooled to room temperature and partially evaporated. Preparative HPLC purification was completed as described in General Procedure 9, with a gradient elution of 40% to 55% CH ₃ CN/H ₂ O+0.1% TFA to give the title compound (5.1 mg, 31% yield) as a dark orange solid.

LC _/ MS: m/ _z calcd for _C27H27FN4O8 = 555.2, found [M ₊ H] ⁺ = 555.2.

¹ H NMR (300MHz, DMSO-d6) δ9.53 (s, 1H), 8.56 (d, J = 8.5Hz, 1H), 8.00 (d, J = 12.0Hz, 1H), 7.31 (s, 1H), 7.11-6.62(m,2H),6.52(s,1H),5.58(s,2H),5.49-5.27(m,4H),1.94-1.77(m,2H),1.52(s,9H),1.38( t,J＝7.7Hz,3H),0.87(t,J＝7.2Hz,3H).

3.30: (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)carbamic acid methyl ester (Compound 169)

The title compound was prepared according to General Procedure 6 starting from compound 3.29 (5.1 mg) to afford the title compound as a yellow powder (TFA salt, 3.8 mg, 73% yield).

LC _/ MS: m/ _z calcd for _C22H19FN4O6 = 455.1, found [M ₊ H] ⁺ = 455.2.

¹ H NMR (300MHz, DMSO-d6) δ7.79 (d, J = 12.4Hz, 1H), 7.29 (d, J = 9.7Hz, 1H), 7.21 (s, 1H), 7.0-6.50 (m, 2H ), 5.45 (s, 2H), 5.40 (s, 2H), 5.33 (s, 2H), 1.95-1.77 (m, 2H), 0.87 (t, J = 7.3Hz, 3H).

3.31: ((S)-9-amino-4-ethyl-8-fluoro-4-hydroxy-11-(methoxymethyl)-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (Compound 155)

In a 50 mL flask containing compound 3.5 (30 mg) was added MeOH/dioxane (1:1) (9.8 mL) and sulfuric acid (0.73 mL). The reaction mixture was then stirred at reflux for 24 hours. The reaction mixture was concentrated, poured into water (30 mL), and extracted with DCM (3×50 mL). The organic phases were combined and dried over MgSO _4. Preparative HPLC purification was accomplished as described in General Procedure 9, eluting with a gradient of 25% to 40% CH ₃ CN/H ₂ O+0.1% TFA to afford the title compound (5.1 mg, 16% yield) as a dark orange solid.

LC _/ MS: m/ _z calcd for _C22H20FN3O5 = 426.1, found [M ₊ H] ⁺ = 426.2.

¹ H NMR (300MHz, DMSO-d6) δ7.75 (d, J＝12.3Hz, 1H), 7.24 (d, J＝9.9Hz, 1H), 7.20 (s, 1H), 6.47 (s, 1H), 6.30-5.92(brs,2H),5.40(s,2H),5.24(s,2H),4.93(s,2H),3.43(s,3H),1.95-1.75(m,2H),0.87(t, J=7.3Hz,3H).

3.32: (4S)-9-amino-4-ethyl-8-fluoro-4-hydroxy-11-(((1R,5S)-6-hydroxy-3-azabicyclo[3.1.1]hept-3-yl)methyl)-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (Compound 158)

In a 5 mL conical flask containing compound 3.6 (15 mg) was added dichloromethane (0.6 mL) followed by 3-azabicyclo[3.1.1]heptan-6-ol (10 mg) and acetic acid (7.6 μL). The reaction was stirred at room temperature and sodium triacetoxyborohydride (9.4 mg) was added. After 1 hour at room temperature, the reaction was quenched by adding water + 0.1% TFA and diluted with DMF. The reaction mixture was then partially evaporated. Preparative HPLC purification was completed as described in General Procedure 9, with a gradient elution of 20% to 50% CH ₃ CN / H ₂ O + 0.1% TFA to give the Boc-protected title compound as a yellow powder. Deprotection was performed according to General Procedure 6 and the resulting residue was purified by preparative HPLC purification as described in General Procedure 9, with a gradient elution of 20% to 50% CH ₃ CN / H ₂ O + 0.1% TFA to give the title compound as a yellow powder (TFA salt, 7.1 mg, 39% yield).

LC _/ MS: m/ _z calcd for _C27H27FN4O5 = 507.2, found [M ₊ H] ⁺ = 507.4.

¹ H NMR (300MHz, DMSO-d6) δ7.85 (d, J = 12.1Hz, 1H), 7.46 (d, J = 9.4Hz, 1H), 7.23 (s, 1H), 6.64-5.85 (m, 3H ),5.60-5.25(m,4H),4.85(s,1H),4.10-3.95(m,1H),3.68(s,2H),2.45-2.33(m,2H),1.96-1.72(m,2H ),0.87(t,J=7.3Hz,3H).

3.33: (S)-9-amino-4-ethyl-8-fluoro-11-((3-fluoro-3-(hydroxymethyl)azetidin-1-yl)methyl)-4-hydroxy-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (Compound 159)

To a 5 mL conical flask containing compound 3.6 (15 mg) was added dichloromethane (0.6 mL), followed by (3-fluoroazetidin-3-yl)methanol (9.3 mg) and acetic acid (7.6 μL). The reaction was stirred at room temperature and sodium triacetoxyborohydride (9.4 mg) was added. After 1 hour at room temperature, the reaction was quenched by the addition of water + 0.1% TFA, diluted with DMF, and then partially evaporated. Preparative HPLC purification was completed as described in General Procedure 9, with a gradient elution of 20% to 50% CH ₃ CN / H ₂ O + 0.1% TFA to give the Boc-protected title compound as a yellow powder. Deprotection was then performed according to General Procedure 6. The resulting residue was purified by preparative HPLC purification as described in General Procedure 9, with a gradient elution of 20% to 50% CH ₃ CN / H ₂ O + 0.1% TFA to give the title compound as a yellow powder (TFA salt, 1.8 mg, 10% yield).

LC _{/MS: m/z calcd for C25H24F2N4O5 = 499.2} , found [ _{M +} H] ⁺ = 499.4.

¹ H NMR (300MHz, DMSO-d6) δ7.82 (d, J = 12.4Hz, 1H), 7.45 (d, J = 9.5Hz, 1H), 7.21 (s, 1H), 5.45-5.33 (m, 4H ), 3.75-3.61 (m, 2H), 1.93-1.78 (m, 2H), 0.87 (t, J = 7.3Hz, 3H).

3.34: tert-Butyl-(S)-(4-ethyl-8-fluoro-4-hydroxy-11-((methylamino)methyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl)carbamate (Compound 3.34)

To a stirred solution of compound 3.9 (210 mg) in DMF (5 mL) was added sodium iodide (5.9 mg) followed by methylammonium chloride (107 mg). The reaction mixture was then stirred at room temperature overnight. Reverse phase purification was accomplished as described in General Procedure 9 using a 30 g C18 column eluting with a 10% to 65% CH ₃ CN/H ₂ O + 0.1% TFA gradient to afford the title compound (15.0 mg, 7.2% yield) as a yellow solid.

LC _/ MS: m/ _z calcd for _C27H29FN4O6 = 524.2, found [M ₊ H] ⁺ = 525.4.

3.35: (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-hydroxy-N-methylacetamide (Compound 165)

The Boc-protected form of the title compound was prepared starting from compound 3.34 (6.4 mg) and glycolic acid according to General Procedure 2. Preparative HPLC purification was accomplished as described in General Procedure 9, eluting with a gradient of 20% to 50% _CH3CN / _H2O + 0.1% TFA. Deprotection was then performed according to General Procedure 6 to afford the title compound as a yellow powder (TFA salt, 2.0 mg, 28% yield).

LC _/ MS: m/ _z calcd for _C24H23FN4O6 = 482.2, found [M ₊ H] ⁺ = 483.2.

¹ H NMR (300MHz, DMSO-d6) δ7.79(d,J＝12.3Hz,1H),7.27(d,J＝9.5Hz,1H),7.22(s,1H),6.48(s,1H), 6.28-6.02(m,2H),5.40(s,2H),5.21(s,2H),5.06-4.93(m,2H),4.18(s,2H),2.80(s,3H),1.92-1.78( m, 2H), 0.87 (t, J = 7.3Hz, 3H).

3.36: (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)-N-methylmethanesulfonamide (Compound 166)

The Boc-protected form of the title compound was prepared starting from compound 3.34 (8.0 mg) and methanesulfonyl chloride according to General Procedure 3. Preparative HPLC purification was accomplished as described in General Procedure 9, eluting with a gradient of 10% to 50% _CH3CN / _H2O + 0.1% TFA. Deprotection was then performed according to General Procedure 6 to afford the title compound as a yellow powder (TFA salt, 2.6 mg, 34% yield).

LC _/ MS: m/ _{z calcd for C23H23FN4O6S =} 502.1, found [M ₊ H] ⁺ = 503.2.

¹ H NMR (300MHz, DMSO-d6) δ7.81(d,J＝12.3Hz,1H),7.41(d,J＝9.4Hz,1H),7.23(s,1H),6.63-5.84(m,2H ),5.42(s,2H),5.29(s,2H),4.81-4.64(m,2H),3.14(s,3H),2.67(s,3H),1.96-1.76(m,2H),0.88( t,J=7.3Hz,3H).

3.37: (S)-9-Amino-4-ethyl-8-fluoro-4-hydroxy-11-(2-methoxyethyl)-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (Compound 170)

To a 10 mL round bottom flask containing compound 3.4 (62.0 mg) was added water (0.89 mL), FeSO ₄ (heptahydrate, 18.0 mg) and 3-methoxypropanal (113.0 mg). To the resulting suspension was added sulfuric acid (0.495 mL) dropwise while stirring in an ice-salt bath at -15°C. Hydrogen peroxide (0.118 mL) was then added dropwise. The mixture was stirred at -15°C for 10 minutes and then allowed to warm to room temperature and stirred for 1 hour. The reaction mixture was then diluted with water (30 mL) and the resulting suspension was extracted with DCM (3×30 mL). The organic phase was evaporated to dryness. Preparative HPLC purification was accomplished as described in General Procedure 9 with a gradient elution of 25% to 45% CH ₃ CN/H ₂ O+0.1% TFA to afford the title compound as a dark orange solid (TFA salt, 3.1 mg, 4.4% yield).

LC _/ MS: m/ _z calcd for _C23H22FN3O5 = 440.2, found [M ₊ H] ⁺ = 440.2.

¹ H NMR (300MHz, DMSO-d6) δ7.75 (d, J = 12.4Hz, 1H), 7.33 (d, J = 9.4Hz, 1H), 7.20 (s, 1H), 6.60-6.42 (m, 2H ),5.40(s,2H),5.25(s,2H),3.69(t,J＝6.5Hz,2H),3.24(s,3H),3.23(t,J＝6.5Hz,2H),1.96-1.76 (m,2H),0.88(t,J=7.3Hz,3H).

3.38: (S)-N-(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)acetamide (Compound 171)

To a 25 mL round bottom flask containing acetic acid (0.071 mL) in dimethylformamide (0.69 mL) was added N-methylmorpholine (0.343 mL), HOAt (0.142 g) and HATU (0.435 g). After stirring at room temperature for 5 min, the solution was added to a 10 mL conical flask containing compound 140 (0.127 g). The solution was stirred at room temperature for 24 h and then directly purified by preparative HPLC as described in General Procedure 9, eluting with a gradient of 25% to 45% CH ₃ CN/H ₂ O + 0.1% TFA to give the title compound as a bright yellow powder (43.0 mg, 38% yield).

LC _/ MS: m/ _z calcd for _C22H18FN3O5 = 424.1, found [M ₊ H] ⁺ = 424.2.

¹ H NMR (300MHz, DMSO-d6) δ10.13(s,1H),8.73(d,J＝8.5Hz,1H),8.61(s,1H),7.96(d,J＝912.1Hz,1H), 7.29(s,1H),6.60-6.42(m,2H),5.41(s,2H),5.21(s,2H),2.20(s,3H),1.96-1.76(m,2H),0.88(t, J=7.3Hz,3H).

3.39: tert-Butyl (5-formyl-2-methoxy-4-nitrophenyl)carbamate (Compound 3.39)

To a solution of compound 3.2 (1.3 g, 1.0 eq.) in MeOH (12 mL) was added sodium methoxide (0.74 g, 3.0 eq.) at 0 °C. After the addition was complete, the ice bath was removed and the resulting solution was stirred at room temperature for 72 hours. The reaction was then quenched with ice water (50 mL) and extracted with DCM (3×100 mL). The combined organic layers were washed with brine (50 mL), dried over sodium sulfate, filtered and concentrated in vacuo to give the title compound (1.2 g, 89% yield) as an orange solid.

LC _/ MS: m/ _{z calculated for C13H16N2O6 =} 296.10, found [M ₊ H] ⁺ = 297.1.

¹ H NMR(300MHz,MeOD)δ10.29(s,1H),8.61(s,1H),7.73(s,1H),4.08(s,3H),1.57(s,9H)

3.40: tert-Butyl (4-amino-5-formyl-2-methoxyphenyl)carbamate (Compound 3.40)

To a solution of compound 3.39 (500 mg, 1 eq.) in MeOH (10 mL) and H ₂ O (1 mL) was added B ₂ (OH) ₄ (454 mg, 3 eq.). The resulting mixture was cooled to 0 °C and 5 M aqueous NaOH (2.75 mL) was added over 10 min with stirring. The reaction mixture was stirred for an additional 5 min and then quenched by pouring the solution into ice (40 mL). The resulting mixture was extracted with DCM (3×50 mL), dried over sodium sulfate, filtered, and concentrated in vacuo. Flash purification was accomplished as described in General Procedure 9 using a 25 g silica gel column and eluting with 10% to 50% hexanes/EtOAc to afford the title compound (386 mg, 86%) as an orange solid.

LC _/ MS: m/ _{z calculated for C13H18N2O4 =} 266.1, found [M ₊ H] ⁺ = 297.2.

3.41: (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 168)

At 110 ° C, the mixture of compound 3.40 (385 mg, 1.0 equivalent) and (S) -4- ethyl -4- hydroxy -7,8- dihydro -1H- pyrans [3,4-f] indolizine -3,6,10 (4H) -trione (362 mg, 0.95 equivalent), TsOH (monohydrate, 25 mg, 0.1 equivalent) and toluene (30 mL) in a 250 mL round-bottom flask equipped with a Dean-Stark apparatus was stirred for 2 hours. The reaction mixture was then cooled to 25 ° C and concentrated in vacuo. Purification was completed as described in General Procedure 9, using a 25 g silica gel column and eluted with a 0% to 50% DCM/MeOH gradient to obtain a red solid Boc protected intermediate. This material was then deprotected according to General Procedure 6 and subsequently purified by preparative HPLC as described in General Procedure 9 eluting with a gradient of 20% to 65% _CH3CN / _H2O + 0.1% TFA to afford the title compound as a red solid (TFA salt, 300 mg, 53% yield).

LC _{/MS: m/z calcd for C21H19N3O5 = 393.2} , found [M ₊ H] ⁺ = 393.2.

¹ H NMR (300MHz, MeOD) δ8.27 (s, 1H), 7.62 (s, 1H), 7.42 (s, 1H), 7.11 (s, 1H), 5.61 (d, J = 16.2Hz, 1H), 5.38(d,J＝16.2Hz,1H),5.24(s,2H),4.11(s,3H),2.06–1.91(m,2H),1.04(t,J＝7.4Hz,3H).

3.42: 5-Bromo-2-nitro-4-(trifluoromethyl)benzaldehyde (Compound 3.42)

To a stirred solution of HNO ₃ (2.0 g, 1.4 mL, 67% purity, 2 eq) in H ₂ SO ₄ (8 mL) at 0° C. was added 3-bromo-4-(trifluoromethyl)benzaldehyde (4 g, 1 eq). After the addition was complete, the ice bath was removed and the reaction was stirred at room temperature for 5 hours. The mixture was poured into ice (100 mL) and the precipitate was extracted with DCM (3×100 mL). The combined organic fractions were then washed with brine (50 mL), dried over Na ₂ SO ₄ , and concentrated in vacuo to give the title compound (4.4 g, 93% yield) as a yellow solid.

LC/MS: m/z calcd for C ₈ H ₃ BrF ₃ NO ₃ = 296.90, found [M+H] ⁺ = 298.0.

¹ H NMR (300MHz, MeOD) δ 10.35 (s, 1H), 8.29 (s, 1H), 8.23 (s, 1H).

3.43: tert-Butyl (5-formyl-4-nitro-2-(trifluoromethyl)phenyl)carbamate (Compound 3.43)

A mixture of compound 3.42 (800 mg, 1 eq.), tert-butyl carbamate (378 mg, 1.2 eq.), Cs ₂ CO ₃ (1.7 g, 2 eq.), Pd ₂ (dba) ₃ (122 mg, 0.05 eq.), and dicyclohexyl [2', 4', 6'-tri (propan-2-yl) [1, 1'-biphenyl] -2-yl] phosphane (XPhos) (256 mg, 0.2 eq.) in toluene (5 mL) was degassed and purged with N ₂ for three cycles. The mixture was then stirred at 90 ° C. under N ₂ atmosphere for 15 hours. The reaction mixture was diluted with H ₂ O (25 mL) and extracted with EtOAc (3×50 mL). The combined organic layers were washed with brine (2×25 mL), dried over sodium sulfate, filtered, and concentrated under reduced pressure. Flash purification was accomplished according to General Procedure 9 using a 25 g silica column and eluting with 0% to 25% DCM/MeOH to afford the title compound as an orange solid (750 mg, 84% yield).

LC/MS: m/z calculated for C ₁₃ H ₁₃ FN ₂ O ₅ = 334.1, found [MH] ⁻ = 333.1.

3.44: tert-Butyl (4-amino-5-formyl-2-(trifluoromethyl)phenyl)carbamate (Compound 3.44)

To a solution of compound 3.43 (750 mg, 1 eq.) in MeOH (16 mL) and H ₂ O (1.6 mL) was added B ₂ (OH) ₄ (603 mg, 3 eq.). The resulting mixture was cooled to 0 °C and 5 M aqueous NaOH (2.75 mL) was added over 10 min with stirring. The reaction mixture was stirred for an additional 5 min and then quenched by pouring the solution into ice (50 mL). The resulting mixture was extracted with DCM (3×75 mL), dried over sodium sulfate, filtered, and concentrated in vacuo. Flash purification was accomplished as described in General Procedure 9 using a 25 g silica gel column and eluting with 10% to 50% hexanes/EtOAc to afford the title compound (460 mg, 67%) as an orange solid.

LC/MS: Calculated for C ₁₃ H ₁₅ F ₃ N ₂ O _3, m/z = 304.1, found [M+H] ⁺ = 305.2

3.45: (S)-9-amino-4-ethyl-4-hydroxy-8-(trifluoromethyl)-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (Compound 167)

At 110 ° C, a mixture of compound 3.44 (460 mg, 1 equivalent) and (S) -4- ethyl -4- hydroxy -7,8- dihydro -1H- pyrans [3,4-f] indolizine -3,6,10 (4H) -trione (378 mg, 0.95 equivalent), TsOH (monohydrate, 26 mg, 0.1 equivalent) and toluene (35 mL) in a 250 mL round-bottom flask equipped with a Dean-Stark apparatus was stirred for 2 hours. The reaction mixture was then cooled to 25 ° C and concentrated in vacuo. Purification was completed as described in General Procedure 9, using a 25 g silica gel column and eluted with a 0% to 50% DCM/MeOH gradient to obtain a red solid Boc protected intermediate. This material was then deprotected according to General Procedure 6 and subsequently purified by preparative HPLC as described in General Procedure 9 eluting with a gradient of 20% to 65% _CH3CN / _H2O + 0.1% TFA to afford the title compound as a yellow solid (6.2 mg, 48%).

LC/MS: m/ _z calcd for _C21H16F3N3O4 = 431.1, found [M _{+ H} ] ₊ ⁼ 432.2.

¹ H NMR (300MHz, MeOD) δ8.29 (s, 1H), 8.27 (s, 1H), 7.59 (s, 1H), 7.24 (s, 1H), 5.59 (d, J = 16.3Hz, 1H), 5.39(d,J＝16.3Hz,1H),5.28(s,2H),2.00–1.89(m,2H),1.03(t,J＝7.4Hz,3H).

Example 4: Preparation of drug-linker

4.1: (((9H-fluoren-9-yl)methoxy)carbonyl)glycylglycine 2,5-dioxopyrrolidin-1-yl ester (Compound 4.1)

The title compound was prepared according to the procedure described in Chinese Patent Publication No. CN105218644.

4.2: (((9H-fluoren-9-yl)methoxy)carbonyl)glycylglycyl-L-phenylalanine (Fmoc-GGF-OH; compound 4.2)

To a solution of L-phenylalanine (965 mg) in acetonitrile (10 mL) and dimethylformamide (0.5 mL) was added DIPEA (1.51 mL) followed by compound 4.1 (1.3 g). After 1 hour, the reaction was concentrated to dryness. Flash purification was accomplished as described in General Procedure 9 with a gradient elution of 10% to 50% CH ₃ CN/H ₂ O + 0.1% TFA to afford the title compound (430 mg, 30% yield) as a white solid.

LC _/ MS: m/ _z calcd for _C28H71N3O6S = 501.2, found [M ₊ H] ⁺ = 502.4.

¹ H NMR (300MHz, DMSO) δ8.16 (d, J = 8.1Hz, 1H), 8.04 (t, J = 5.8Hz, 1H), 7.90 (d, J = 7.5Hz, 2H), 7.72 (d, J＝7.4Hz,2H),7.59(t,J＝6.0Hz,1H),7.54–7.39(m,2H),7.33(t,J＝7.6Hz,2H),7.28–7.13(m,5H), 4.44(td,J＝8.5,5.1Hz,1H),4.33–4.13(m,3H),3.83–3.59(m,4H),3.06(dd,J＝13.7,5.1Hz,1H),2.88(dd, J＝13.8,9.0Hz,1H).

4.3: 2,3,5,6-tetrafluorophenyl 3-(2-(2-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)ethoxy)ethoxy)propanoate (MT-OTfp; compound 4.3)

The title compound was prepared according to the procedure described in International Patent Publication No. WO 2017/054080.

4.4: (3-(2-(2-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)ethoxy)ethoxy)propionyl)glycylglycyl-L-phenylalanine (Compound 4.4)

To a solution of compound 4.3 (1.61 g, 3.58 mmol) in DMF (35 mL) was added Gly-Gly-Phe (1 g, 3.58 mmol) in one portion followed by iPr ₂ NEt (1.25 mL, 7.2 mmol). The solution was stirred at room temperature for 1 hour and then evaporated to dryness. Purification was accomplished as described in General Procedure 9 using a 30 g C18 flash column eluting with a 10% to 90% CH ₃ CN/H ₂ O + 0.1% TFA gradient to afford the title compound (400 mg, 20% yield) as a white solid.

LC/MS: m/z calculated for C ₂₆ H ₃₄ N ₄ O ₁₀ = 562.6, found [MH] ⁻ = 561.5.

¹ H NMR (300MHz, CDCl ₃ ) δ7.60 (t, J＝5.6Hz, 2H), 7.41 (d, J＝7.7Hz, 1H), 7.32–7.07 (m, 5H), 6.70 (s, 2H) ,6.33–6.07(m,3H),4.72(td,J＝7.6,5.3Hz,1H),4.12–3.78(m,4H),3.72(ddd,J＝15.2,6.9,4.8Hz,5H),3.60 (dd, J = 11.6, 6.1 Hz, 10H), 3.12 (ddd, J = 48.2, 14.0, 6.5 Hz, 2H), 2.52 (d, J = 11.7 Hz, 2H).

4.5: (S)-11-Benzyl-1-(9H-fluoren-9-yl)-3,6,9,12,15-pentaoxo-2-oxa-4,7,10,13,16-pentaazaheptadecan-17-yl acetate (Compound 4.5)

The title compound was prepared according to the procedure described in US Patent Publication No. US2017/021031.

4.6: (S)-11-Benzyl-1-(9H-fluoren-9-yl)-3,6,9,12,15-pentaoxo-2-oxa-4,7,10,13,16-pentaazaheptadecan-17-yl acetate (Compound 4.6)

The title compound was prepared according to the procedure described in US Patent Publication No. US2017/021031 using Fmoc-GGFG G-OH as the starting peptide.

4.7: tert-butyl (2-((2-(((S)-1-((2-((4-((4-(((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)piperazin-1-yl)sulfonyl)phenyl)amino)-2-oxoethyl)amino)-1-oxo-3-phenylpropan-2-yl)amino)-2-oxoethyl)amino)-2-oxoethyl)carbamate (Compound 4.7)

The title compound was prepared starting from compound 104 (20 mg) according to General Procedure 7. Preparative HPLC purification was accomplished as described in General Procedure 9 eluting with a gradient of 10% to 60% _CH3CN / _H2O + 0.1% TFA to afford the title compound as a white solid (14 mg, 42% yield).

LC/MS: m/z calcd for C ₅₂ H ₅₈ N ₉ O ₁₂ S = 1051.4, found [M+H] ⁺ = 1052.6.

4.8: (S)-2-(1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-12,15-dioxo-3,6,9-trioxa-13,16-diazaoctadecane-18-amido)-N-(2-((4-((4-(((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)piperazin-1-yl)sulfonyl)phenyl)amino)-2-oxoethyl)-3-phenylpropanamide (MT-GGFG-Compound 104)

The title compound was prepared starting from compound 4.7 (14 mg) according to Procedure 6 followed by Procedure 8. Preparative HPLC purification was accomplished as described in General Procedure 9 eluting with a gradient of 10% to 60% _CH3CN / _H2O + 0.1% TFA to afford the title compound as a white solid (9.1 mg, 56% yield).

LC/MS: m/ _z calcd for _{C60H67FN10O16S} = 1234.4, found [M _{+ H} ] ⁺ = 1235.8.

4.9: tert-butyl (2-((2-(((S)-1-((2-((4-(4-(((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)piperazin-1-yl)phenyl)amino)-2-oxoethyl)amino)-1-oxo-3-phenylpropan-2-yl)amino)-2-oxoethyl)amino)-2-oxoethyl)carbamate (Compound 4.9)

The title compound was prepared starting from compound 108 (12 mg) according to General Procedure 7. Preparative HPLC purification was accomplished as described in General Procedure 9 eluting with a gradient of 10% to 60% _CH3CN / _H2O + 0.1% TFA to afford the title compound as a white solid (13 mg, 62% yield).

LC/MS: m/z calcd for C ₅₂ H ₅₈ N ₉ O ₁₀ = 987.4, found [M+H] ⁺ = 988.6.

4.10: (S)-2-(1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-12,15-dioxo-3,6,9-trioxa-13,16-diazaoctadecane-18-amido)-N-(2-((4-(4-(((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)piperazin-1-yl)phenyl)amino)-2-oxoethyl)-3-phenylpropanamide (MT-GGFG-Compound 108)

The title compound was prepared starting from compound 4.9 (13 mg) according to Procedure 6 followed by Procedure 8. Preparative HPLC purification was accomplished as described in General Procedure 9 eluting with a gradient of 10% to 50% _CH3CN / _H2O + 0.1% TFA to afford the title compound as a white solid (3.1 mg, 20% yield).

LC/MS: m/ _z calcd for _{C60H67FN10O14} = 1170.5, found [M _{+ H} ] ⁺ = 1171.6.

4.11: (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)-3,10-dioxo-7-oxa-2,4,9-triazaundecane-11-yl)carbamic acid (9H-fluoren-9-yl)methyl ester (Compound 4.11)

To a solution of compound 1.2 (31 mg, 0.076 mmol) in DMF (750 uL) was added (9H-fluoren-9-yl)methyl (2-(((2-(((4-nitrophenoxy)carbonyl)amino)ethoxy)methyl)amino)-2-oxoethyl)carbamate (41 mg, 0.076 mmol) followed by _iPr2NEt (26 uL, 0.15 mmol). The solution was stirred at room temperature for 2 hours and then applied directly to a 12 g C18 column. Purification was accomplished as described in General Procedure 9, eluting with a 10% to 100% _CH3CN / _H2O + 0.1% TFA gradient to afford the title compound (21 mg, 35% yield) as a white solid.

LC _{/MS: m/z calcd for C43H41FN6O9 = 804.87} , found [M ₊ H] ⁺ = 805.6.

4.12: (S)-2-(1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-12,15-dioxo-3,6,9-trioxa-13,16-diazaoctadecane-18-amido)-N-(1-((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)-3,10-dioxo-7-oxa-2,4,9-triazaundecane-11-yl)-3-phenylpropanamide (MT-GGFG-AM-Compound 136)

Compound 4.11 (21 mg, 0.026 mmol) was dissolved in a 10% solution of piperidine in DMF (1 mL) and stirred for 10 minutes. The piperidine solution was evaporated and the resulting residue was redissolved in DMF (5 mL) and evaporated to dryness again. To this residue was added DMF (50 uL) and DCM (450 uL) followed by compound 4.4 (15 mg, 0.026 mmol), NMM (10 uL) and HATU (10 mg, 0.026 mmol). Preparative HPLC purification was accomplished as described in General Procedure 9 with a gradient elution of 30% to 60% CH ₃ CN/H ₂ O + 0.1% TFA to afford the title compound (7.6 mg, 26% yield) as a yellow solid.

LC/MS: m/ _z calcd for _{C54H63FN10O16} = 1127.1, found [M _{+ H} ] ⁺ = 1128.2.

4.13: (9H-fluoren-9-yl)methyl (2-(((2-(chlorosulfonyl)ethoxy)methyl)amino)-2-oxoethyl)carbamate (Compound 4.13)

To a solution of compound 4.5 (50 mg, 0.14 mmol) in DCM (800 uL) was added 2-hydroxyethane-1-sulfonyl chloride (100 mg, 0.7 mmol) followed by TFA (200 uL). The solution was stirred at room temperature for 30 minutes and then evaporated to dryness. Purification was accomplished as described in General Procedure 9 using a 10 g silica gel column and eluting with a 10% to 100% EtOAc/hexane gradient to give the title compound (31 mg, 50% yield) as a clear film.

LC/MS: Calcd. for C ₂₀ H ₂₁ ClN ₂ O ₆ S m/z = 452.1, found [M+Na] ⁺ = 472.9

4.14: (S)-(2-(((2-(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)sulfamoyl)ethoxy)methyl)amino)-2-oxoethyl)carbamic acid (9H-fluoren-9-yl)methyl ester (Compound 4.14)

The title compound was prepared using compound 1.2 (28 mg, 0.07 mmol) and compound 4.13 (31 mg, 0.07 mmol) as described in General Procedure 3. Purification was accomplished as described in General Procedure 9 using a 12 g C18 column eluting with a 10% to 100% _CH3CN / _H2O + 0.1% TFA gradient to afford the title compound (22 mg, 39% yield) as a yellow solid.

LC/MS: m/ _{z calcd for C42H40FN5O10S =} 825.9, found [M _{+ H} ] ⁺ = 826.7.

4.15: (S)-2-(1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-12,15-dioxo-3,6,9-trioxa-13,16-diazaoctadecane-18-amido)-N-(2-(((2-(N-(((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)sulfamoyl)ethoxy)methyl)amino)-2-oxoethyl)-3-phenylpropanamide (MT-GGFG-AM-Compound 129)

Compound 4.14 (22 mg, 0.027 mmol) was dissolved in 10% piperidine in DMF (1 mL) and stirred for 10 min. The piperidine solution was evaporated and the resulting residue was redissolved in DMF (5 mL) and evaporated to dryness again. To this residue was added DMF (50 uL) and DCM (450 uL) followed by compound 4.4 (30 mg, 0.053 mmol), NMM (10 uL) and HATU (18 mg, 0.048 mmol). Preparative HPLC purification was accomplished as described in General Procedure 9 with a gradient elution of 30% to 60% CH ₃ CN/H ₂ O + 0.1% TFA to afford the title compound (6.4 mg, 21% yield) as a yellow solid.

LC _/ MS: m _{/z calcd for C53H62FN9O17S = 1148.2} , found [M+H] ⁺ = 1148.6.

¹ H NMR (300MHz, MeOD) δ8.60(t,J＝6.5Hz,1H),8.36(t,J＝8.6Hz,2H),8.13(d,J＝6.6Hz,1H),7.77(d, J＝10.6Hz,1H),7.65(d,J＝4.8Hz,1H),7.28–7.00(m,6H),6.80(s,2H),5.69–5.50(m,3H),5 .45–5.33(m,2H),4.44(dd,J＝8.7,5.7Hz,1H),3.96(t,J＝5.3Hz,2H),3.90–3.76(m,5H),3.76–3.57(m ,7H),3.09–2.81(m,3H),2.61–2.45(m,5H),2.04–1.90(m,2H),1.03(t,J＝7.3Hz,3H).

4.16: (S)-(9H-fluoren-9-yl)methyl (2-(((morpholin-2-ylmethoxy)methyl)amino)-2-oxoethyl)carbamate (Compound 4.16)

To a solution of compound 4.5 (100 mg, 0.27 mmol) in DCM (800 uL) was added (S)-morpholin-2-ylmethanol (160 mg, 1.36 mmol) followed by TFA (200 uL). The solution was stirred at room temperature for 1 hour and then evaporated to dryness. Purification was accomplished as described in General Procedure 9 using a 12 g C18 flash column eluting with a 10% to 90% CH ₃ CN/H ₂ O + 0.1% TFA gradient to afford the title compound as a yellow solid (TFA salt, 105 mg, 72% yield).

LC _/ MS: m/ _z calcd for _C23H27N3O5 = 425.2, found [M ₊ Na] ⁺ = 448.0.

4.17: (9H-fluoren-9-yl)methyl (2-(((((S)-4-ethyl-8-fluoro-4-hydroxy-9-methyl-3,14-dioxo-3,4,12,14-methylhydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)methyl)morpholin-2-yl)methoxy)methyl)amino)-2-oxoethyl)carbamate (Compound 4.17)

The title compound was prepared starting from compound 1.1 (50 mg, 0.117 mmol) and compound 4.16 (63 mg, 0.117 mmol) according to General Procedure 1. Preparative HPLC purification was accomplished as described in General Procedure 9 eluting with a gradient of 10% to 100% _CH3CN / _H2O + 0.1% TFA to afford the title compound as an off-white solid (TFA salt, 33 mg, 35% yield).

LC _{/MS: m/z calcd for C45H44FN5O9 = 817.9} , found [M ₊ H] ⁺ = 818.7.

4.18: (S)-2-(1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-12,15-dioxo-3,6,9-trioxa-13,16-diazaoctadecane-18-amido)-N-(2-(((((S)-4-(((S)-4-ethyl-8-fluoro-4-hydroxy-9-methyl-3,14-dioxo-3,4,12,14-methylhydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)methyl)morpholin-2-yl)methoxy)methyl)amino)-2-oxoethyl)-3-phenylpropanamide (MT-GGFG-AM-Compound 113)

Compound 4.17 (33 mg, 0.04 mmol) was dissolved in 10% piperidine in DMF (1 mL) and stirred for 10 min. The piperidine solution was evaporated and the resulting residue was redissolved in DMF (5 mL) and evaporated to dryness again. To this residue was added DMF (100 uL) and DCM (900 uL) followed by compound 4.4 (45 mg, 0.08 mmol), NMM (20 uL) and HATU (28 mg, 0.073 mmol). Preparative HPLC purification was accomplished as described in General Procedure 9 with a gradient elution of 30% to 60% CH ₃ CN/H ₂ O + 0.1% TFA to afford the title compound (22 mg, 48% yield) as a yellow solid.

LC _{/MS: m/z calcd for C56H66FN9O16 = 1140.2} , found [M ₊ H] ⁺ = 1141.1.

¹ H NMR (300MHz, MeOD) δ8.35 (d, J = 7.5Hz, 2H), 7.74–7.61 (m, 1H), 7.53 (s, 1H), 7.34–7.10 (m, 6H), 6.81 (s ,2H),5.65–5.30(m,4H),4.64(t,J＝3.4Hz,2H),4.42(tt,J＝6.3,2.5Hz ,1H),4.09(d,J＝12.3Hz,1H),3.98–3.76(m,8H),3.72(t,J＝6.0Hz,2H),3.69–3.44(m,17H),3.21–2.85( m,3H),2.64–2.42(m,5H),2.03–1.84(m,2H),0.98(t,J＝7.3Hz,3H).

4.19: (S)-(2-((((9-amino-4-ethyl-8-fluoro-4-hydroxy-3,14-dioxo-3,4,12,14-methylhydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)methoxy)methyl)amino)-2-oxoethyl)carbamic acid (9H-fluoren-9-yl)methyl ester (Compound 4.19)

Compound 3.5 (55 mg, 0.11 mmol) was dissolved in TFA (500 uL) and stirred at room temperature for 20 minutes, then hexafluoroisopropanol (2 mL) was added, followed by compound 4.5 (40 mg, 0.11 mmol). The solution was stirred at room temperature for about 16 hours, then concentrated to dryness. Purification was accomplished as described in General Procedure 9 using a 12 g C18 flash column eluting with a gradient of 10% to 50% CH ₃ CN/H ₂ O + 0.1% TFA to afford the title compound (11 mg, 14% yield) as a yellow solid.

LC _{/MS: m/z calcd for C39H34FN5O8 = 719.7} , found [M ₊ H] ⁺ = 720.6.

4.20: (S)-N-(2-(((((S)-9-amino-4-ethyl-8-fluoro-4-hydroxy-3,14-dioxo-3,4,12,14-methylenehydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)methoxy)methyl)amino)-2-oxoethyl)-2-(1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-12,15-dioxo-3,6,9-trioxa-13,16-diazaoctadecane-18-amido)-3-phenylpropanamide (MT-GGFG-AM-Compound 141)

Compound 4.19 (11 mg, 0.015 mmol) was dissolved in 10% piperidine in DMF (1 mL) and stirred for 10 min. The piperidine solution was evaporated and the resulting residue was redissolved in DMF (5 mL) and evaporated to dryness again. To this residue was added DMF (50 uL) and DCM (450 uL) followed by compound 4.4 (26 mg, 0.045 mmol), NMM (5 uL) and HATU (18 mg, 0.045 mmol). Preparative HPLC purification was accomplished as described in General Procedure 9 with a gradient elution of 32% to 45% CH ₃ CN/H ₂ O + 0.1% TFA to afford the title compound (4.6 mg, 29% yield) as a yellow solid.

LC _{/MS: m/z calcd for C50H56FN9O15 = 1142.0} , found [M ₊ H] ⁺ = 1143.1.

¹ H NMR (300MHz, MeOD) δ8.36 (s, 1H), 8.28 (d, J = 6.1Hz, 1H), 8.16 (dd, J = 20.1, 6.8Hz, 3H), 7.59–7.44 (m, 2H ),7.31–7.08(m,6H),6.79(s,2H),5.58(d,J＝16.1Hz,1H),5.37(d,J＝16.1Hz,1H),5.30–5.16(m,3H) ,4.56–4.39(m,1H),4.07–3.90(m ,2H),3.85(dt,J＝11.5,5.4Hz,4H),3.79–3.67(m,4H),3.67–3.55(m,7H),3.54(d,J＝6.5Hz,8H),3.10( dd,J＝14.0,6.1Hz,1H),2.92(dd,J＝13.9,9.1Hz,1H),2.53(t,J＝6.0Hz,2H),1.98(q,J＝7.2Hz,2H), 1.31(s,1H),1.04(t,J=7.3Hz,3H).

4.21: N-((S)-1-((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)-9-benzyl-5,8,11,14-methoxy-2-oxa-4,7,10,13-tetraazapentadecan-15-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide (MC-GGFG-AM-Compound 141)

Compound 4.19 (25 mg, 0.035 mmol) was dissolved in 10% piperidine in DMF (1 mL) and stirred for 10 min. The piperidine solution was evaporated and the resulting residue was redissolved in DMF (5 mL) and evaporated to dryness again. To this residue was added DMF (50 uL) and DCM (450 uL), followed by MC-GGF-OH (33 mg, 0.07 mmol), NMM (20 uL) and HATU (25 mg, 0.066 mmol). Preparative HPLC purification was accomplished as described in General Procedure 9, eluting with a 30% to 50% CH ₃ CN/H ₂ O + 0.1% TFA gradient to give the title compound (4.3 mg, 13% yield) as a yellow solid.

LC _{/MS: m/z calcd for C47H50FN9O12 = 952.0} , found [M ₊ H] ⁺ = 952.9.

¹ H NMR (300MHz, CD ₃ CN) δ7.96–7.72 (m, 1H), 7.39–7.07 (m, 8H), 6.94 (d, J = 9.1Hz, 1H), 6.73 (s, 2H), 5.44 (d,J＝16.2Hz,1H),5.25(d,J＝16.2Hz,1H),5.06(d,J＝4.4Hz,2H),4.81(d,J＝26.1Hz,4 H),4.61(s,1H),3.96(s,1H),3.77(d,J＝8.1Hz,7H),3.02(d,J＝5.6Hz,5H),2.19(t,J＝7.7Hz, 3H), 1.50 (dp, J＝14.8, 7.4Hz, 6H), 1.32–1.12 (m, 3H), 0.96 (t, J＝7.2Hz, 3H).

4.22: tert-butyl (2-((2-(((S)-1-((2-(((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)amino)-2-oxoethyl)amino)-1-oxo-3-phenylpropan-2-yl)amino)-2-oxoethyl)amino)-2-oxoethyl)carbamate (Compound 4.22)

The title compound was prepared starting from compound 140 (28 mg) according to Procedure 7. Preparative HPLC purification was accomplished as described in General Procedure 9, eluting with a gradient of 10% to 50% _CH3CN / _H2O + 0.1% TFA to afford the title compound as a white solid (10 mg, 17% yield).

LC/MS: m _{/z calcd for C40H42N7O10 = 799.3} , found [M ₊ H] ⁺ = 800.6.

4.23: (S)-2-(1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-12,15-dioxo-3,6,9-trioxa-13,16-diazaoctadecane-18-amido)-N-(2-(((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)amino)-2-oxoethyl)-3-phenylpropanamide (MT-GGFG-Compound 140)

The title compound was prepared starting from compound 4.22 (10 mg) according to General Procedure 6 followed by General Procedure 8. Preparative HPLC purification was accomplished as described in General Procedure 9 eluting with a gradient of 10% to 50% _CH3CN / _H2O + 0.1% TFA to afford the title compound as a white solid (6.8 mg, 55% yield).

LC _{/MS: m/z calcd for C48H51FN8O14 = 982.4} , found [M ₊ H] ⁺ = 983.6.

4.24: tert-butyl (2-((2-(((S)-1-((2-((((S)-4-ethyl-8-fluoro-4-hydroxy-11-(morpholinomethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl)amino)-2-oxoethyl)amino)-1-oxo-3-phenylpropan-2-yl)amino)-2-oxoethyl)amino)-2-oxoethyl)carbamate (Compound 4.24)

The title compound was prepared starting from compound 142 (TFA salt, 45 mg) according to General Procedure 7. Purification was accomplished as described in General Procedure 9 using a 12 g C18 flash column eluting with a 10% to 50% _CH3CN / _H2O + 0.1% TFA gradient to afford the title compound as a white solid (13 mg, 22% yield).

LC _{/MS: m/z calcd for C45H51N8O11 = 898.4} , found [M ₊ H] ⁺ = 899.6.

4.25: (S)-2-(1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-12,15-dioxo-3,6,9-trioxa-13,16-diazaoctadecane-18-amido)-N-(2-(((S)-4-ethyl-8-fluoro-4-hydroxy-11-(morpholinomethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl)amino)-2-oxoethyl)-3-phenylpropanamide (MT-GGFG-Compound 142)

The title compound was prepared starting from compound 4.24 (13 mg) according to General Procedure 6 followed by General Procedure 8. Preparative HPLC purification was accomplished as described in General Procedure 9 eluting with a gradient of 10% to 50% _CH3CN / _H2O + 0.1% TFA to afford the title compound as a white solid (2.6 mg, 17% yield).

LC _{/MS: m/z calcd for C53H60FN9O15 = 1081.4} , found [M ₊ H] ⁺ = 1082.6.

¹ H NMR(300MHz,MeOD)δ9.34(d,J＝8.5Hz,1H),7.87(d,J＝11.8Hz,1H),7.62(s,1H),7.33–7.19(m,5H), 6.80(s,2H),5.62(d,J＝16.3Hz,1H),5.51(s,2H),5.47–5.35(m,3H),4.73(dd,J＝9.6,5.1Hz,1H),4.61 (s,3H), 4.30–4.15(m,2H),4.11(s,2H),4.00–3.82(m,4H),3.82–3.70(m,7H),3.70–3.50(m,13H),3.18–3.04(m,1H ),2.88(s,1H),2.64(d,J＝5.8Hz,4H),2.54(t,J＝6.0Hz,2H),2.09–1.92(m,2H),1.03(t,J＝7.3Hz ,3H).

4.26: (S)-(12-Benzyl-1-(4-nitrophenoxy)-1,8,11,14,17-pentaoxo-2,5-dioxa-7,10,13,16-tetrahydrooctadecane-18-yl)carbamic acid (9H-fluoren-9-yl)methyl ester (Compound 4.26)

Ethylene glycol (100uL) was added to the stirred solution of compound 4.6 (60mg) in dichloromethane (2mL), followed by trifluoroacetic acid (0.4mL). After 30 minutes, the reactant was concentrated in vacuo. The purification of the intermediate compound was completed as described in General Procedure 9, using 10g quick columns and eluting with a 0% to 20% dichloromethane/methanol gradient. Bis-nitrophenol carbonate (58mg) was added to the purified intermediate in tetrahydrofuran (0.5mL), followed by DIPEA (50uL). The solution was stirred for 16 hours, quenched with acetic acid (about 100uL), and then concentrated to dryness. Purification was completed as described in General Procedure 9, using 10g quick columns and eluting with a 0% to 20% dichloromethane/methanol gradient to obtain the title compound (40mg, 53% yield from compound 4.6) as a white solid.

LC _/ MS: m/ _z calcd for _C40H40N6O12 = 796.3, found [M ₊ Na] ⁺ = 819.4.

4.27: (S)-16-amino-10-benzyl-6,9,12,15-tetraoxo-3-oxa-5,8,11,14-tetraazahexadecyl (((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 4.27)

To a solution of compound 4.26 (40 mg) in dimethylformamide (1 mL) was added DIPEA (26 uL) followed by a solution of compound 1.2 (24 mg) in dimethylformamide (0.5 mL). The solution was stirred at room temperature for 4 hours and then quenched with 20% piperidine in dimethylformamide (0.5 mL) and stirred for an additional 20 minutes. Purification was accomplished as described in General Procedure 9 using a 12 g C18 flash column and eluting with a gradient of 10% to 50% CH ₃ CN/H ₂ O + 0.1% TFA to afford the title compound as a white solid (TFA salt, 19 mg, 39% yield).

LC _{/MS: m/z calcd for C41H45FN8O11 = 844.3} , found [M ₊ H] ⁺ = 845.6.

4.28: (S)-10-Benzyl-29-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-6,9,12,15,18-pentaoxo-3,21,24,27-tetraoxa-5,8,11,14,17-pentaazaheneicosyl (((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 (MT-GGFG-AM-Compound 139)

The title compound was prepared starting from compound 4.27 (10 mg) according to General Procedure 8. Preparative HPLC purification was accomplished as described in General Procedure 9 eluting with a gradient of 10% to 50% _CH3CN / _H2O + 0.1% TFA to afford the title compound as a white solid (8.8 mg, 75% yield).

LC _{/MS: m/z calcd for C54H62FN9O17 = 1127.4} , found [M ₊ H] ⁺ = 1128.6.

¹ H NMR(300MHz,MeOD)δ8.27(d,J＝8.1Hz,1H),7.81(d,J＝10.7Hz,1H),7.65(s,1H),7.32–7.16(m,5H), 6.81(s,2H),5.62(d,J＝16.4Hz,1H),5.53(s,2H),5.42(d,J＝16.4Hz,1H),4.93(s,2H),4.67(s,1H ),4.51 (dd,J＝9.3,5.6Hz,1H),4.18(t,J＝4.7Hz,2H),4.01–3.44(m,19H),3.17(dd,J＝13.9,5.8Hz,1H),2.97( dd,J＝13.9,9.0Hz,1H),2.57(s,3H),2.52(t,J＝6.0Hz,2H),2.03–1.91(m,2H),1.03(t,J＝7.4Hz,3H ).

4.29: (S)-2-amino-N-(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)acetamide (Compound 4.29)

To a stirred solution of Fmoc-glycine (217 mg) in dimethylformamide (2.5 mL) was added HATU (254 mg), HOAt (83 mg) and then NMM (188 uL). The solution was stirred for 10 minutes and then compound 141 (50 mg) was added and the reaction was stirred at room temperature for 16 hours. Lithium hydroxide (2.5 mL, 1 M in water) was added and the reaction mixture was stirred for 2 hours. The solution was partially concentrated and then a solution of 20% piperidine in dimethylformamide (0.5 mL) was added and stirred for an additional 20 minutes. The reaction was then evaporated onto celite and purification was accomplished as described in General Procedure 9 using a 12 g C18 flash column and gradient elution from 0% to 40% CH ₃ CN/H ₂ O + 0.1% TFA to afford the title compound as a white solid (TFA salt, 44 mg, 62% yield).

LC _{/MS: m/z calcd for C23H21FN4O6 = 468.1} , found [M ₊ H] ⁺ = 469.4.

¹ H NMR (300MHz, MeOD) δ8.99(d,J＝8.3Hz,1H),7.99(s,1H),7.87(d,J＝12.0Hz,1H),7.55(s,1H),5.60( d,J＝16.3Hz,1H),5.46–5.35(m,3H),5.30(s,2H),3.53–3.45(m,1H),3.43–3.38(m,1H),2.03–1.87(m, 2H), 1.02 (t, J = 7.3Hz, 3H).

4.30: (S)-2-(1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-12,15-dioxo-3,6,9-trioxa-13,16-diazaoctadecane-18-amido)-N-(2-(((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)amino)-2-oxoethyl)-3-phenylpropanamide (MT-GGFG-Compound 141)

To a stirred solution of compound 4.4 (23 mg) in a mixture of dimethylformamide (0.1 mL) and dichloromethane (0.9 mL) was added HATU (14 mg), a solution of compound 4.29 (20 mg) in dimethylformamide (0.1 mL) and dichloromethane (0.9 mL) and DIPEA (24 uL). The mixture was stirred for 15 minutes and then the reaction was partially concentrated. Preparative HPLC purification was accomplished as described in General Procedure 9 with a gradient elution of 0% to 40% CH ₃ CN/H ₂ O + 0.1% TFA to give the title compound (7.1 mg, 20% yield) as a white solid.

LC _/ MS: m/ _z calcd for _C49H53FN8O15 = 1012.4, found [M ₊ H] ⁺ = 1013.6.

¹ H NMR (300MHz, MeOD) δ9.89 (s, 1H), 8.75 (d, J = 8.3Hz, 1H), 8.44–8.32 (m, 1H), 8.27–8.14 (m, 2H), 7.78 (d ,J＝11.9Hz,1H),7.53(s,1H),7.39–7.20(m,5H),6.82(s,2H),5.57(d,J＝16.3Hz,1H),5.39(d,J＝ 16.3Hz,1H),5.34–5.25(m,2H ),5.22(s,2H),4.32–4.09(m,2H),3.96–3.83(m,3H),3.76(t,J＝6.0Hz,2H),3.69–3.62(m,2H),3.62– 3.47(m,9H),3.40–3.33(m,1H),3.08(dd,J＝14.0,9.6Hz,1H),2.56(t,J＝6.1Hz,2H),2.03–1.91(m,2H) ,1.04(t,J=7.3Hz,3H).

4.31: (S)-tert-butyl ((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 4.31)

To a stirred solution of compound 145 (32 mg) in dichloromethane (2 mL) and acetonitrile (0.5 mL) was added di-tert-butyl dicarbonate (20 uL) followed by DIPEA (42 uL). The reaction mixture was stirred at room temperature for 3 hours and then concentrated to dryness to afford the title compound (34 mg, 87%) as a red solid.

LC _/ MS: m/ _z calcd for _C26H27FN4O6 = 510.2, found [M ₊ H] ⁺ = 511.2.

4.32: (S)-tert-butyl ((9-(2-aminoacetamido)-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 4.32)

To a stirred solution of Fmoc-glycine (98 mg) in dimethylformamide (1 mL) was added HATU (115 mg), HOAt (37 mg), and then NMM (85 mL). The solution was stirred for 10 minutes, and then compound 4.31 (28 mg) was added. The reaction was stirred at room temperature for 16 hours, and then quenched with 20% piperidine in dimethylformamide (1 mL) and stirred for another 20 minutes. Purification was accomplished as described in General Procedure 9 using a 12 g C18 flash column eluting with a gradient of 5% to 40% CH ₃ CN/H ₂ O + 0.1% TFA to give the title compound as a white solid (TFA salt, 25 mg, 67% yield).

LC _{/MS: m/z calcd for C28H30FN6O7 = 567.2} , found [M ₊ H] ⁺ = 568.4.

¹ H NMR (300MHz, MeOD) δ9.01 (d, J = 8.3 Hz, 1H), 7.83 (d, J = 11.9 Hz, 1H), 7.52 (s, 1H), 5.57 (d, J = 16.4 Hz, 1H),5.38(d,J＝16.3Hz,1H),5.27(d,J＝3.1Hz,2H),4.80(s,2H),4.10(s,2H),1.97(q,J＝7.4Hz, 2H), 1.50 (s, 9H), 1.02 (t, J = 7.3Hz, 3H).

4.33: tert-butyl (((S)-9-(2-((S)-2-(2-(2-aminoacetamido)acetamido)-3-phenylpropionamido)acetamido)-4-ethyl-8-fluoro-4-hydroxy-3,14-dioxo-3,4,12,14-methylhydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)methyl)carbamate (Compound 4.33)

To a stirred solution of Fmoc-GGF-OH (28 mg) and HATU (20 mg) in a mixture of DMF (0.2 mL) and dichloromethane (1.8 mL) was added compound 4.32 (25 mg) followed by DIPEA (32 uL). The solution was stirred at room temperature for 15 minutes, quenched with a solution of 20% piperidine in dimethylformamide (0.250 mL), stirred for an additional 20 minutes, and then partially concentrated in vacuo. Purification was accomplished as described in General Procedure 9 using a 12 g C18 flash column eluting with a gradient of 10% to 45% CH ₃ CN/H ₂ O + 0.1% TFA to afford the title compound as a white solid (TFA salt, 22 mg, 64% yield).

LC _{/MS: m/z calcd for C41H45FN8O10 = 828.3} , found [M ₊ H] ⁺ = 829.6.

4.34: (S)-N-(2-(((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)amino)-2-oxoethyl)-2-(1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-12,15-dioxo-3,6,9-trioxa-13,16-diazaoctadecane-18-amido)-3-phenylpropanamide (MT-GGFG-Compound 145)

The title compound was prepared starting from compound 4.33 (15 mg) according to Procedure 6 followed by Procedure 8. Preparative HPLC purification of the intermediate Boc-protected compound was accomplished as described in General Procedure 9, eluting with a 10% to 50% _CH3CN / _H2O + 0.1% TFA gradient. The title compound was obtained after Boc-deprotection as a white solid (TFA salt, 8.5 mg, 52% yield).

LC _{/MS: m/z calcd for C49H53FN8O15 = 1011.4} , found [M ₊ H] ⁺ = 1012.6.

¹ H NMR (300MHz, MeOD) δ9.04(d,J＝8.0Hz,1H),8.40(d,J＝5.7Hz,1H),8.21(d,J＝7.7Hz,1H),8.05(d, J＝11.5Hz,1H),7.67(s,1H),7.42–7.03(m,5H),6.81(s,2H),5.63(d,J＝16.4Hz,1H),5.51(s,1H), 5.43(d,J＝16.5Hz,1 H),4.81(s,2H),4.75–4.58(m,1H),4.29–4.10(m,2H),3.98–3.81(m,4H),3.78–3.71(m,2H),3.71–3.63( m,2H),3.62–3.53(m,9H),3.14–2.98(m,1H),2.54(t,J＝6.0Hz,2H),2.08–1.93(m,2H),1.03(t,J＝ 7.3Hz,3H).

4.35: (S)-(2-((4-ethyl-8-fluoro-4-hydroxy-3,14-dioxo-11-(piperidin-1-ylmethyl)-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl)amino)-2-oxoethyl)carbamic acid (9H-fluoren-9-yl)methyl ester (Compound 4.35)

To a solution of Fmoc-Gly-OH (100.9 mg, 0.34 mmol) in dimethylformamide (550 uL) was added NMM (0.112 mL, 1.02 mmol) and HATU (0.103 g, 0.272 mmol). The solution was stirred at room temperature for 20 minutes, then a solution of compound 148 (32.5 mg, 0.068 mmol) in DMF (250 uL) was added and the reaction mixture was stirred for 16 hours. Purification was accomplished as described in General Procedure 9 using a 12 g C18 flash column eluting with a 5% to 40% CH ₃ CN/H ₂ O + 0.1% TFA gradient. The resulting residue was repurified according to General Procedure 9 using a 10 g flash column eluting with a 0% to 10% MeOH/DCM gradient to give the title compound (15.3 mg, 30% yield) as a yellow powder.

LC _{/MS: m/z calcd for C43H40FN5O7 = 757.3} , found [M ₊ H] ⁺ = 758.6.

4.36: (S)-2-(1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-12,15-dioxo-3,6,9-trioxa-13,16-diazaoctadecane-18-amido)-N-(2-(((S)-4-ethyl-8-fluoro-4-hydroxy-3,14-dioxo-11-(piperidin-1-ylmethyl)-3,4,12,14-methylhydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl)amino)-2-oxoethyl)-3-phenylpropanamide (MT-GGFG-Compound 148)

To a 50 mL flask containing compound 4.35 (15.3 mg, 0.02 mmol) was added a solution of 20% piperidine in DMF (2.0 mL). The solution was stirred at room temperature for 5 minutes and then evaporated to dryness. The resulting residue was then dissolved in 10% DMF/DCM (1.0 mL) and NMM (5.50 μL, 0.05 mmol), compound 4.4 (11.2 mg, 0.02 mmol) and HATU (8.7 mg, 0.02 mmol) were then added. The solution was stirred for 45 minutes and then partially evaporated. Preparative HPLC purification was accomplished as described in General Procedure 9 with a gradient elution of 15% to 45% CH ₃ CN/H ₂ O + 0.1% TFA to give the title product as a yellow powder (7.8 mg, 33% yield).

LC _{/MS: m/z calcd for C54H62FN9O14 = 1079.4} , found [M ₊ H] ⁺ = 1080.8.

¹ H NMR (300MHz, MeOD) δ8.99(d,J＝8.2Hz,1H),7.52(d,J＝12.3Hz,1H),7.39–7.25(m,5H),7.25–7.17(m,1H ),6.79(s,2H),5.53(d,J＝16.4Hz,1H),5.33(d,J＝16.5Hz,1H),4.80–4.72(m,1H),4.32–4.11(m,2H) ,3.98–3.79(m,6H),3.76(t,J＝6.0Hz,2H),3.66–3.60(m,2H),3.62–3.49(m,10H),3.15–3.03(m,1H),2.65 –2.47(m,6H),1.96(q,J＝7.4Hz,2H),1.72–1.57(m,4H),1.57–1.42(m,2H),1.03(t,J＝7.4Hz,3H).

4.37: tert-butyl (2-((2-(((S)-1-((2-((4-(N-(((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)sulfamoyl)phenyl)amino)-2-oxoethyl)amino)-1-oxo-3-phenylpropan-2-yl)amino)-2-oxoethyl)amino)-2-oxoethyl)carbamate (Compound 4.37)

The title compound was prepared starting from compound 127 (46 mg) according to General Procedure 7. Preparative HPLC purification was accomplished as described in General Procedure 9 eluting with a gradient of 10% to 60% _CH3CN / _H2O + 0.1% TFA to afford the title compound as a white solid (7.2 mg, 21% yield).

LC _/ MS: m/ _{z calcd for C48H51N8O12S =} 983.0, found [M ₊ H] ⁺ = 983.9.

4.38: (S)-2-(1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-12,15-dioxo-3,6,9-trioxa-13,16-diazaoctadecane-18-amido)-N-(2-((4-(N-(((S)-4-ethyl-8-fluoro-4-hydroxy-9-methyl-3,14-dioxo-3,4,12,14-methylhydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)methyl)sulfamoyl)phenyl)amino)-2-oxoethyl)-3-phenylpropanamide (MT-GGFG-Compound 127)

The title compound was prepared starting from compound 4.37 (7.2 mg) according to Procedure 6 followed by Procedure 8. Preparative HPLC purification was accomplished as described in General Procedure 9 eluting with a gradient of 10% to 50% _CH3CN / _H2O + 0.1% TFA to afford the title compound as a white solid (1 mg, 12% yield).

LC/MS: Calculated for C ₅₆ H ₆₀ FN ₉ O ₁₆ m/z = 1166.2, found [M+H] ⁺ = 1167.1

4.39: ((7S)-1-((3-azabicyclo[3.1.1]hept-6-yl)oxy)-7-benzyl-3,6,9,12-tetraoxo-2,5,8,11-tetraazatridec-13-yl)carbamic acid (9H-fluoren-9-yl)methyl ester (Compound 4.39)

To a stirred solution of compound 4.6 (44 mg) in dichloromethane (2 mL) was added 3-azabicyclo[3.1.1]heptan-6-ol (5.3 mg) followed by trifluoroacetic acid (0.4 mL). After 30 minutes, the reaction was concentrated in vacuo. Purification was accomplished as described in General Procedure 9 using a 10 g flash column and eluting with a 0% to 20% dichloromethane/methanol gradient to afford the title compound (14.7 mg, 46% yield) as a white solid.

LC/MS: m/ _z calcd for _C37H42N6O7 = 682.8, found [M _{+ H} ] ⁺ = 683.6.

4.40: (2S)-2-(2-(2-aminoacetamido)acetamido)-N-(2-((((3-(((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)-3-azabicyclo[3.1.1]hept-6-yl)oxy)methyl)amino)-2-oxoethyl)-3-phenylpropanamide (Compound 4.40)

The title compound was prepared according to General Procedure 1 starting from compound 1.1 (3 mg, 0.007 mmol) and compound 4.39 (14.7 mg, 0.022 mmol) using 200 uL DMF. After complete consumption of compound 1.1, a solution of 20% piperidine in DMF (200 uL) was added and the solution was stirred at room temperature for 10 minutes. Preparative HPLC purification was accomplished as described in General Procedure 9 using a gradient elution of 20% to 37% _CH3CN / _H2O + 0.1% TFA to afford the title compound as an off-white solid (TFA salt, 1.8 mg, 29% yield).

LC _{/MS: m/z calcd for C44H49FN8O9 = 852.9} , found [M ₊ H] ⁺ = 853.7.

4.41: (2S)-2-(1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-12,15-dioxo-3,6,9-trioxa-13,16-diazaoctadecane-18-amido)-N-(2-((((3-(((S)-4-ethyl-8-fluoro-4-hydroxy-9-methyl-3,14-dioxo-3,4,12,14-methylhydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)methyl)-3-azabicyclo[3.1.1]hept-6-yl)oxy)methyl)amino)-2-oxoethyl)-3-phenylpropanamide (MT-GGFG-AM-Compound 117)

The title compound was prepared starting from compound 4.40 (1.8 mg) according to Procedure 8. Preparative HPLC purification was accomplished as described in General Procedure 9, eluting with a gradient of 25% to 45% _CH3CN / _H2O + 0.1% TFA to afford the title compound as a white solid (TFA salt, 0.5 mg, 22% yield).

LC _{/MS: m/z calcd for C57H66FN9O15 = 1135.5} , found [M ₊ H] ⁺ = 1136.3.

4.42: (S)-(9-Benzyl-1-(3-fluoroazetidin-3-yl)-5,8,11,14-tetraoxo-2-oxa-4,7,10,13-tetraazapentadecan-15-yl)carbamic acid (9H-fluoren-9-yl)methyl ester (Compound 4.42)

To a stirred solution of compound 4.6 (144 mg) in dichloromethane (2 mL) was added (3-fluoroazetidine-3-yl)methanol (16 mg) followed by trifluoroacetic acid (0.4 mL). After 30 minutes, the reaction was concentrated in vacuo. Purification was accomplished as described in General Procedure 9 using a 10 g flash column and eluting with a 0% to 20% dichloromethane/methanol gradient to afford the title compound (55 mg, 54% yield) as a white solid.

LC/MS: _{Calcd . for C35H39N6FO7 ,} m/ _z = 674.7, found [M+H] ⁺ = 675.6.

4.43: (S)-2-(2-(2-aminoacetamido)acetamido)-N-(2-((((1-(((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)-3-fluoroazetidin-3-yl)methoxy)methyl)amino)-2-oxoethyl)-3-phenylpropanamide (Compound 4.43)

The title compound was prepared according to General Procedure 1 starting from compound 1.1 (11.6 mg, 0.027 mmol) and compound 4.42 (55 mg, 0.082 mmol) using 500 uL DMF. After complete consumption of compound 1.1, a solution of 20% piperidine in DMF (500 uL) was added and the solution was stirred at room temperature for 10 minutes. Preparative HPLC purification was accomplished as described in General Procedure 9 using a gradient elution of 25% to 32% _CH3CN / _H2O + 0.1% TFA to afford the title compound as an off-white solid (TFA salt, 8.1 mg, 28% yield).

LC/MS: calculated value m/z = 844.3 (C ₄₂ H ₄₆ F ₂ N ₈ O ₉ ), found value [M+H] ⁺ = 845.3

4.44: (S)-2-(1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-12,15-dioxo-3,6,9-trioxa-13,16-diazaoctadecane-18-amido)-N-(2-((((1-(((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)-3-fluoroazetidin-3-yl)methoxy)methyl)amino)-2-oxoethyl)-3-phenylpropanamide (MT-GGFG-AM-Compound 118)

The title compound was prepared starting from compound 4.43 (8.1 mg) according to Procedure 8. Preparative HPLC purification was accomplished as described in General Procedure 9, eluting with a gradient of 25% to 45% _CH3CN / _H2O + 0.1% TFA to afford the title compound as a white solid (TFA salt, 2.9 mg, 28% yield).

LC _/ MS: m/ _z calcd for _{C55H63F2N9O15} = 1127.4, found [M _{+ H} ] ⁺ = 1128.8.

4.45: (S)-10-Benzyl-23-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-6,9,12,15,18-pentaoxo-3-oxa-5,8,11,14,17-pentaazaeicosyl (((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 (MC-GGFG-AM-Compound 139)

To compound 4.27 (450 mg) was added a solution of 2,5-dioxopyrrolidin-1-yl)hexanoate (130 mg) and N-ethyldiisopropylamine (250 uL) in DMF (10 mL). The solution was stirred at room temperature for 30 minutes and then concentrated to a volume of about 1 mL. Purification was accomplished as described in General Procedure 9, first using a 60 g C18 flash column, eluting with a 10% to 60% CH ₃ CN/H ₂ O+0.1% TFA gradient, then preparative HPLC of the impure fractions using a 20% to 50% CH ₃ CN/H ₂ O+0.1% TFA gradient elution to afford the title compound (320 mg, 66% yield) as a white solid.

LC _{/MS: m/z calcd for C51H56FN9O14 = 1037.4} , found [M ₊ H] ⁺ = 1038.6.

¹ H NMR (300MHz, MeOD) δ8.10 (d, J = 8.1Hz, 2H), 8.01 (s, 1H), 7.95 (d, J = 7.0Hz, 1H), 7.74 (d, J = 10.4Hz, 1H),7.66(s,1H),7.56(s,1H),7.32–7.10(m,5H),6.69(s,2H),5.63(d,J＝16.4Hz,1H),5.46(s,2H ),5.32(s,1H),5.28(d,J＝16.5Hz,1H),4.88(s,2H),4.67(d,J＝6.4Hz,2H),4.48(d,J＝7.1Hz,2H ) ,4.15(t,J＝4.2Hz,2H),3.92(dd,J＝17.1,6.2Hz,2H),3.83–3.57(m,6H),3.46(t,J＝7.1Hz,2H),3.16( dd,J＝14.0,5.9Hz,1H),2.95(dd,J＝13.9,8.9Hz,1H),2.53(s,3H),2.21(t,J＝7.6Hz,2H),1.97–1.79(m ,2H),1.58(dp,J＝15.0,7.6Hz,4H),1.29(dd,J＝16.6,9.3Hz,3H),1.01(t,J＝7.3Hz,3H).

4.46: (S)-tert-butyl (2-((4-ethyl-8-fluoro-4-hydroxy-3,14-dioxo-3,4,12,14-methanehydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl)amino)-2-oxoethyl)carbamate (Compound 4.46)

A solution of compound 140 (860 mg, 1.7 mmol, TFA salt), Boc-Gly-OH (760 mg, 4.3 mmol), HATU (1.6 g, 4.1 mmol) and N-ethyldiisopropylamine (0.6 mL) in DMF (4 mL) was stirred at room temperature for 24 h and then poured into water (50 mL). The resulting solid was collected by filtration, redissolved in 10% MeOH/DCM, and purified as described in General Procedure 9 using a 30 g silica gel column and eluting with 0% to 10% MeOH/DCM to give the title compound (750 mg, 80% yield) as a yellow solid.

LC _/ MS: m/ _z calcd for _C27H27FN4O7 = 538.5, found [M ₊ H] ⁺ = 539.4.

¹ H NMR (300MHz, MeOD) δ8.84(d,J＝8.4Hz,1H),8.52(s,1H),8.00(s,1H),7.87(d,J＝12.1Hz,1H),7.62( s,1H),5.60(d,J＝16.3Hz,1H),5.40(d,J＝16.4Hz,1H),5.27(s,2H),4.02(s,2H),1.99(dt,J＝8.7 ,6.7Hz,2H),1.52(s,9H),1.03(t,J＝7.4Hz,3H).

4.47: (S)-1-((2-(((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)amino)-2-oxoethyl)amino)-1-oxo-3-phenylpropane-2-ammonium (Compound 4.47)

The title compound was prepared in three steps by compound 4.46 (750 mg). The Boc protecting group was cleaved in pure TFA (2 mL) and then precipitated in _Et2O (50 mL). The solid was collected by filtration and added to a solution of (2S)-2-[(tert-butoxycarbonyl)amino]-3-phenylpropionic acid 2,5-dioxopyrrolidin-1-yl ester (340 mg, 1.1 equivalents) and N-ethyldiisopropylamine (300 uL) in DMF (1.7 mL). The solution was stirred at room temperature for 30 minutes and then pipetted into _Et2O (50 mL). The precipitate was collected by filtration, dried in vacuo, and then dissolved in pure TFA (2 mL). After 20 minutes, _Et2O (50 mL) was added and the precipitate was collected by filtration to obtain the title compound (531 mg, 54% yield) as a yellow solid.

LC _{/MS: m/z calcd for C31H28FN5O6 = 585.2} , found [M ₊ H] ⁺ = 586.1.

4.48: 2-((2-(((S)-1-((2-(((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)amino)-2-oxoethyl)amino)-1-oxo-3-phenylpropan-2-yl)amino)-2-oxoethyl)amino)-2-oxoethane-1-ammonium (Compound 4.48)

To compound 4.47 (490 mg) was added a solution of Boc-gly-gly-NHS (250 mg, 1.1 equiv) and N-ethyldiisopropylamine (250 uL) in DMF (3 mL). The solution was stirred at room temperature for 30 minutes and then pipetted into _Et2O (50 mL). The precipitate was collected by filtration and then dissolved in neat TFA (2 mL). After 20 minutes, _Et2O (50 mL) was added and the precipitate was collected by filtration to give the title compound (500 mg, 88% yield) as a yellow solid.

LC _{/MS: m/z calcd for C35H34FN7O8 = 699.2} , found [M ₊ H] ⁺ = 700.4.

4.49: 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-(2-((2-(((S)-1-((2-(((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)amino)-2-oxoethyl)amino)-1-oxo-3-phenylpropan-2-yl)amino)-2-oxoethyl)amino)-2-oxoethyl)hexanamide (MC-GGFG-Compound 140)

To compound 4.48 (500 mg) was added a solution of 2,5-dioxocyclopentyl 6-(2,5-dioxopyrrolidin-1-yl)hexanoate (210 mg, 1.1 equiv) and N-ethyldiisopropylamine (215 uL) in DMF (4 mL). The solution was stirred at room temperature for 30 min and then pipetted into _Et2O (50 mL). The precipitate was collected by filtration and then dissolved in DMF (2 mL). Preparative HPLC purification was accomplished as described in General Procedure 9 with a gradient elution of 24% to 38% _CH3CN / _H2O + 0.1% TFA to afford the title compound (190 mg, 40% yield) as a yellow solid.

LC _{/MS: m/z calcd for C45H45FN8O11 = 892.9} , found [M ₊ H] ⁺ = 893.6.

¹ H NMR (300MHz, CD ₃ CN) δ8.67(d,J＝8.4Hz,1H),8.44(s,1H),7.78(d,J＝12.1Hz,1H),7.41(s,1H), 7.30(d,J＝4.3Hz,4H),7.26–7.16(m,1H),6.72(s,2H),5.52(d,J＝16.4Hz,1H),5.31(d,J＝16.4Hz,1H ),5.12(s,2H),4.64(dd,J＝9.7 ,5.0Hz,1H),4.11(d,J＝3.2Hz,2H),3.87–3.68(m,4H),3.37(t,J＝7.1Hz,2H),3.00(dd,J＝14.0,9.7Hz ,1H),2.20(t,J＝7.6Hz,2H),1.49(dq,J＝19.5,7.4Hz,4H),1.22(p,J＝7.6,7.1Hz,2H),0.94(t,J＝ 7.3Hz,3H).

4.50: (S)-tert-butyl (2-((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)amino)-2-oxoethyl)carbamate (Compound 4.50)

A solution of compound 4.46 (1.8 g), iron (II) sulfate heptahydrate (1.4 g, 1.5 eq.) and sulfuric acid (450 uL, 2.5 eq.) in MeOH (33 mL) was heated to 60 °C and hydrogen peroxide (1.25 mL, 12 eq.) was added dropwise over 10 min. The solution was heated for another 20 min, then cooled to room temperature and poured into ice water (about 200 mL). The precipitate was collected by filtration and the filtrate was quenched _with saturated _{aqueous Na2S2O3 .} MeOH was evaporated and the solution was allowed to stand for 2 h while a second brown precipitate was formed. The precipitate was collected by filtration and the combined precipitate was purified using a 50 g silica gel column and a 0% to 15% MeOH/DCM gradient elution as described in General Procedure 9 to give the title compound (860 mg, 45% yield) as a yellow solid.

LC _/ MS: m/ _z calcd for _C28H29FN4O8 = 568.5, found [M ₊ H] ⁺ = 569.7.

4.51: (S)-1-((2-(((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)amino)-2-oxoethyl)amino)-1-oxo-3-phenylpropan-2-ammonium (Compound 4.51)

The title compound was prepared in three steps by compound 4.50 (750 mg). The Boc protecting group was cleaved in pure TFA (2 mL), and then precipitated in _Et2O (100 mL). The solid was collected by filtration and added to a solution of (2S)-2-[(tert-butoxycarbonyl)amino]-3-phenylpropionic acid 2,5-dioxopyrrolidin-1-yl ester (600 mg, 1.1 equivalents) and N-ethyldiisopropylamine (300 uL) in DMF (7 mL). The solution was stirred at room temperature for 30 minutes, and then pipetted into _Et2O (100 mL). The precipitate was collected by filtration, dried in vacuo, and then dissolved in pure TFA (2 mL). After 20 minutes, _Et2O (100 mL) was added, and the precipitate was collected by filtration to obtain the title compound (756 mg, 78% yield) as a yellow solid.

LC _{/MS: m/z calcd for C32H30FN5O7 = 615.2} , found [M ₊ H] ⁺ = 616.3.

4.52: 2-((2-(((S)-1-((2-(((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)amino)-2-oxoethyl)amino)-1-oxo-3-phenylpropan-2-yl)amino)-2-oxoethyl)amino)-2-oxoethane-1-ammonium (Compound 4.52)

To compound 4.51 (756 mg) was added a solution of Boc-gly-gly-NHS (375 mg, 1.1 equiv) and N-ethyldiisopropylamine (400 uL) in DMF (5 mL). The solution was stirred at room temperature for 30 minutes and then pipetted into _Et2O (75 mL). The precipitate was collected by filtration and then dissolved in neat TFA (4 mL). After 20 minutes, _Et2O (100 mL) was added and the precipitate was collected by filtration to give the title compound (826 mg, 95% yield) as a yellow solid.

LC _{/MS: m/z calcd for C36H36FN7O9 = 729.2} , found [M ₊ H] ⁺ = 730.2.

4.53: 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-(2-((2-(((S)-1-((2-(((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)amino)-2-oxoethyl)amino)-1-oxo-3-phenylpropan-2-yl)amino)-2-oxoethyl)amino)-2-oxoethyl)hexanamide (MC-GGFG-Compound 141)

To compound 4.52 (826 mg) was added a solution of 2,5-dioxocyclopentyl 6-(2,5-dioxopyrrolidin-1-yl)hexanoate (382 mg, 1.1 equiv) and N-ethyldiisopropylamine (300 uL) in DMF (5.5 mL). The solution was stirred at room temperature for 30 minutes and then pipetted into _Et2O (100 mL). The precipitate was collected by filtration and then dissolved in DMF (2 mL). Preparative HPLC purification was accomplished as described in General Procedure 9 with a gradient elution of 25% to 40% _CH3CN / _H2O + 0.1% TFA to afford the title compound (370 mg, 35% yield) as a yellow solid.

LC _{/MS: m/z calcd for C46H47FN8O12 = 922.9} , found [M ₊ H] ⁺ = 923.8.

¹ H NMR (300MHz, CD ₃ CN) δ8.63(d,J＝8.4Hz,1H),7.67(d,J＝11.9Hz,1H),7.38–7.27(m,5H),7.24(d,J ＝4.3Hz,1H),6.72(s,2H),5.48(d,J＝16.4Hz,1H),5.28(d,J＝16.3Hz,1H),5.24–5.01(m,4H),4.65(dd ,J＝9.7,4.9H z,1H),4.13(s,2H),3.85–3.75(m,3H),3.37(t,J＝7.1Hz,2H),3.00(dd,J＝14.0,9.8Hz,1H),2.21(t ,J＝7.6Hz,2H),1.51(dp,J＝22.0,7.4Hz,4H),1.22(p,J＝7.4,7.0Hz,2H),0.94(t,J＝7.3Hz,3H).

4.54: tert-Butyl ((S)-1-(((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)amino)-1-oxopropan-2-yl)carbamate (Compound 4.54)

To compound 3.4 (500 mg, 1.0 mmol) was added TFA (4 mL) and the solution was allowed to stand at room temperature for 1 hour, followed by the addition of _Et2O (100 mL) and the precipitate was collected by filtration. The solid was dissolved in DMF (3.4 mL), and Boc-Ala-OH (590 mg, 3.1 mmol, 3 equiv) and HATU (1.2 g, 3.1 mmol, 3 equiv) were added, followed by the addition of N-ethyldiisopropylamine (0.9 mL, 5.2 mmol, 5 equiv). The solution was stirred at room temperature for 3 days, then poured into ice water (50 mL), and the precipitate was collected by filtration to give the title compound (125 mg, 22% yield) as a brown solid.

LC _/ MS: m/ _z calcd for _C28H29FN4O7 = 552.6, found [M ₊ H] ⁺ = 553.7.

4.55: (S)-2-amino-N-((S)-1-(((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)amino)-1-oxopropan-2-yl)-3-methylbutanamide (Compound 4.55)

To compound 4.54 (125 mg, 0.225 mmol) in a 100 mL round-bottom flask, TFA (2 mL) was added. The solution was allowed to stand for 10 minutes, then Et ₂ O (50 mL) was added, and the precipitate was collected by filtration. The resulting orange solid was added to a solution of Boc-Val-NHS (78 mg, 0.25 mmol, 1.1 equivalents) and N-ethyldiisopropylamine (80 uL, 0.45 mmol, 2 equivalents) in DMF (2 mL). The solution was stirred at room temperature for 30 minutes, then pipetted into Et ₂ O (40 mL) in a 50 mL falcon tube, and the precipitate was collected by centrifugation and decanting Et ₂ O. The precipitate was dissolved in TFA (2 mL) and allowed to stand for 10 minutes, then Et ₂ O (40 mL) was added. The precipitate was collected by centrifugation and decanting Et ₂ O. The precipitate was dried under high vacuum to afford the title compound as an orange solid (135 mg, 90% yield over 3 steps).

LC _{/MS: m/z calcd for C28H30FN5O6 = 551.2} , found [M ₊ H] ⁺ = 552.2.

4.56: 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-((S)-1-(((S)-1-(((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)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)hexanamide (MC-VA-Compound 140)

To compound 4.55 (20 mg, 0.03 mmol) was added a solution of 2,5-dioxopyrrolidin-1-yl 6-(2,5-dioxopyrrolidin-1-yl)hexanoate (11 mg, 0.036 mmol) and N-ethyldiisopropylamine (10 uL) in DMF (1 mL). The solution was stirred at room temperature for 30 minutes and then purified directly. Preparative HPLC purification was accomplished as described in General Procedure 9, eluting with a gradient of 20% to 60% CH ₃ CN/H ₂ O + 0.1% TFA to afford the title compound (8.8 mg, 40% yield) as a yellow solid.

LC _/ MS: m/ _z calcd for _C38H41FN6O9 = 744.8, found [M ₊ H] ⁺ = 745.6.

¹ H NMR (300MHz, 10% D ₂ O/CD ₃ CN) δ8.60 (d, J = 8.5 Hz, 1H), 8.31 (s, 1H), 7.96 (d, J = 6.5 Hz, 1H), 7.65 (d,J＝12.0Hz,1H),7.37–7.26(m,2H),6.75(s,2H),5.45(d,J＝16.6Hz,1H),5.25(d,J＝16.3Hz,1H) ,5.04(d,J＝4.0Hz,2H),4.78–4.58 (m,1H),4.30–4.13(m,1H),2.32–2.16(m,2H),2.10(dt,J＝13.6,6.8Hz,1H),1.88(q,J＝7.4Hz,2H), 1.57(dq,J＝15.5,7.6Hz,4H), 1.45(d,J＝7.1Hz,3H), 1.26(tt,J＝10.1,6.1Hz,2H), 1.05–0.83(m,9H).

4.57: 2,5-dioxopyrrolidin-1-yl 6-(((S)-1-(((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)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)amino)-6-oxohexanoic acid ester (NHC-C-VA-Compound 140)

To compound 4.55 (20 mg, 0.03 mmol) was added a solution of bis(2,5-dioxopyrrolidin-1-yl)adipate (30 mg, 0.09 mmol, 3 equiv) and N-ethyldiisopropylamine (10 uL) in DMF (1 mL). The solution was stirred at room temperature for 30 min and then purified directly. Preparative HPLC purification was accomplished as described in General Procedure 9 with a gradient elution of 25% to 35% CH ₃ CN/H ₂ O + 0.1% TFA to afford the title compound (4.1 mg, 18% yield) as a yellow solid.

LC _/ MS: m/ _z calcd for _C38H41FN6O11 = 776.8, found [M ₊ H] ⁺ = 777.6.

¹ H NMR (300MHz, 10% D ₂ O/CD ₃ CN) δ8.65 (dd, J＝8.4, 2.3Hz, 1H), 8.38 (s, 1H), 7.95 (d, J＝6.5Hz, 1H) ,7.72(d,J＝12.0Hz,1H),7.37(d,J＝11.8Hz,2H),5.58–5.19(m,2H),5.10(s,2H),4.78–4.56(m,1H), 4.23(dd ,J＝8.4,7.0Hz,1H),2.80(s,4H),2.65(t,J＝6.9Hz,2H),2.39–2.22(m,2H),2.11(q,J＝6.8Hz,1H) ,1.94–1.81(m,2H),1.79–1.57(m,4H),1.45(d,J＝7.1Hz,3H),1.10–0.78(m,9H).

4.58: (S)-2-(3,2-Azido-5-oxo-3,9,12,15,18,21,24,27,30-nonaoxa-6-azatriacontanoylamino)-N-((S)-1-(((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)amino)-1-oxopropan-2-yl)-3-methylbutanamide (2-((azido-PEG8-carbamoyl)methoxy)acetamido-VA-Compound 140)

A solution of compound 4.55 (20 mg, 0.03 mmol), 3,2-azido-5-oxo-3,9,12,15,18,21,24,27,30-nonaoxa-6-azatriacontanoic acid (17 mg, 0.03 mmol) and HATU (13 mg, 0.03 mmol) in DMF (300 uL) was cooled to 0 °C and N-ethyldiisopropylamine (16 uL, 0.09 mmol) was added. The solution was stirred for 30 minutes and purified directly. Preparative HPLC purification was accomplished as described in General Procedure 9 with a gradient elution of 20% to 50% CH ₃ CN/H ₂ O + 0.1% TFA to give the title compound (13.7 mg, 42% yield) as a yellow solid.

LC _{/MS: m/z calcd for C50H70FN9O17 = 1088.2} , found [M ₊ H] ⁺ = 1088.8.

¹ H NMR (300MHz, 10% D ₂ O/CD ₃ CN)δ8.62(d,J＝8.5Hz,1H),8.33(s,1H),7.66(d,J＝12.2Hz,1H),7.35(s,1H),5.52–5.18(m,2H) ,5.04(s,2H),4.72(q,J＝7.1Hz,1H),4.32(d,J＝7.3Hz,1H),4.15–3.98(m,4H),3.68–3.48(m,35H), 3.41–3.34(m,6H),2.18(h,J＝6.8Hz,1H),1.88(q,J＝7.4Hz,2H),1.47(d,J＝7.1Hz,3H),1.13–0.84(m ,9H).

4.58: tert-Butyl (2-(pyridin-2-yldisulfanyl)ethyl)carbamate (Compound 4.58)

The title compound was prepared as described in Wang et al., Nano Lett., 2014, 14(10):5577–5583.

4.59: tert-Butyl (2-((2-hydroxyethyl)disulfanyl)ethyl)carbamate (Compound 4.59)

To a solution of compound 4.58 (200 mg, 0.7 mmol) in DCM (1.4 mL) was added β-mercaptoethanol (50 μL, 0.7 mmol) and the solution was stirred at room temperature for 5 hours. The solution was diluted with DCM (10 mL), washed with water (3×10 mL), dried over Na ₂ SO ₄ and concentrated to an oil. Purification was accomplished as described in General Procedure 9 using a 10 g silica column and eluting with 0% to 10% MeOH/DCM to give the title compound (212 mg, 82% yield) as a colorless solid.

LC _{/MS: m/z calcd for C11H23NO3S2 = 253.1} , found [M+H, -Boc] ₊ ⁼ 154.0.

¹ H NMR (300 MHz, CHLOROFORM-d) δ 4.94 (s, 1H), 3.91 (t, J = 5.7 Hz, 2H), 3.49 (q, J = 6.4 Hz, 2H), 2.86 (dt, J = 23.7, 6.1 Hz, 4H), 2.15 (s, 2H), 1.47 (s, 9H).

4.60: 2-((2-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propionamido)ethyl)disulfanyl)ethyl(4-nitrophenyl)carbonate (Compound 4.60)

4M HCl/ dioxane solution (5mL) is added to compound 4.59 (212mg, 0.837mmol) in a 25mL round-bottom flask and the solution is stirred at room temperature for 30 minutes, then evaporated to dryness. The residue is suspended in EtOAc (10mL) and evaporated to dryness to obtain amine, which is HCl salt and a white powder. A solution of 3- (2,5- dioxopyrrolidin-1-yl) propionic acid 2,5- dioxopyrrolidin-1-yl ester (245mg, 0.92mmol, 1.1 equivalents) and N- ethyldiisopropylamine (0.438mL, 2.51mmol) in DMF (1.7mL) is added to the solid. The solution is stirred at room temperature for 20 minutes, then 4- nitrophenyl carbonate (280mg, 0.92mmol) is added, and the reactant is then stirred overnight. Purification of the crude reaction mixture was accomplished as described in General Scheme 9 using a 12 g C18 flash column and eluting with a 10% to 100% _CH3CN / _H2O + 0.1% TFA gradient to afford the title compound as a white solid (141 mg, 36% yield).

LC _/ MS: m/ _z calcd for _C18H19N3O8S2 = 469.5, found [M ₊ H] ₊ ⁼ 470.2.

¹ H NMR (300MHz, chloroform-d) δ8.37–8.25 (m, 2H), 7.46–7.35 (m, 2H), 6.71 (d, J = 2.1Hz, 2H), 6.32 (s, 1H), 4.55 (t,J＝6.6Hz,2H),3.83(t,J＝7.0Hz,2H),3.65–3.50(m,2H),3.09–2.99(m,2H),2.84(q,J＝6.1Hz, 2H), 2.52 (td, J=7.1, 3.1Hz, 2H).

4.61: (S)-2-((2-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propionamido)ethyl)disulfanyl)ethyl(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 (DiS-Compound 145)

A solution of compound 4.60 (18 mg, 0.038 mmol) and N-ethyldiisopropylamine (15 uL, 0.087 mmol) in DMF (300 uL) was added to compound 145 (13 mg, 0.029 mmol) and the solution was stirred at room temperature for 20 minutes. The solution was acidified with 1 M aqueous HCl (100 uL) and purified directly. Preparative HPLC purification was accomplished as described in General Procedure 9 with a gradient elution of 20% to 45% CH ₃ CN/H ₂ O + 0.1% TFA to afford the title compound (6.8 mg, 32% yield) as a yellow solid.

LC _{/MS: m/z calcd for C33H33FN6O9S2 = 740.8} , found [ _{M +} H] ⁺ = 741.5.

¹ H NMR (300MHz, 10% D ₂ O/CD ₃ CN)δ7.63(d,J＝12.1Hz,1H),7.39–7.22(m,2H),6.74(d,J＝6.7Hz,2H),5.50(d,J＝16.2Hz,1H),5.26 (d,J＝16.2Hz,1H),5.20(s,2H)4.69(s,2H),4.28(t,J＝6.3Hz,2H),3.62(t,J＝7.0Hz,2H),3.31( t,J＝6.6Hz,2H),2.74–2.64(m,2H),2.35(t,J＝7.0Hz,2H),1.90(dd,J＝15.5,8.1Hz,2H),1.23–1.04(m ,6H),0.93(t,J=7.4Hz,3H).

Example 5: In vitro cytotoxicity of camptothecin analogs

The cytotoxicity of camptothecin analogs was evaluated in vitro as follows.

In vitro potency was evaluated on multiple cancer cell lines: SK-BR-3 (breast cancer), SKOV-3 (ovarian cancer), Calu-3 (lung cancer), ZR-75-1 (breast cancer), and MDA-MB-468 (breast cancer). Serial dilutions of camptothecin analogs were prepared in RPMI 1640 + 10% FBS, and 20uL of each dilution was added to a 384-well plate. Cells cultured in logarithmic phase growth were detached by brief incubation in 0.05% trypsin and resuspended in the corresponding culture medium at 20,000 cells/mL (except ZR-75 cells, which were resuspended at 10,000 cells/mL). 50uL of the cell suspension was then added to the plate containing the test article. The cells were incubated with the test article at 37°C for 4 days (except ZR-75 cells, which were incubated for 5 days). Cells were cultured in 384-well plates using the CellTiter- Growth inhibition was assessed using ELISA (Promega Corporation, Madison, WI) and luminescence was measured on a microplate reader. IC50 values were determined using GraphPad Prism (GraphPad Software, San Diego, CA).

The results are shown in Table 5.1.

Table 5.1: In vitro cytotoxicity (pIC50) of camptothecin analogs

*ND = Not Determined

Example 6: Preparation of anti-folate receptor alpha antibodies

Antibodies that specifically bind to folate receptor alpha (FRa) were generated by immunizing rabbits with human FRa antigen, isolated and sequenced as described below.

6.1 Immunization

Antibodies to FRa were generated in rabbits immunized with soluble HIS-labeled human folate receptor 1 antigen (FRa-HIS) (ACROBiosystems, Newark, DE; Catalog No. FO1-H82E2). Briefly, two New Zealand white rabbits were immunized with a primary boost consisting of 200 μg of FRa-HIS antigen mixed with alum (5 mg/injection)/CpG (10 μg/injection), administered subcutaneously at 3 sites along the rabbit's dorsal body (0.333 mL/site). These were followed by 4 immunizations with 100 μg of FRa-HIS antigen mixed with alum (5 mg/injection)/CpG (10 μg/injection). Each immunization was separated by 14 days. Animals were bled for serological analysis before the fourth immunization.

6.2 Selection of animals for harvesting

Anti-human FRa antibody titers were determined by flow cytometry using CHO cells expressing human FRa. In brief, CHO cells were transiently transfected with a pTT5-based expression plasmid (National Research Council of Canada) encoding human FRa according to the manufacturer's instructions for Lipofectamine ^™ 2000 (Thermo Fisher Scientific Corp., Waltham, MA). Dilutions of immune rabbit serum starting at 1:400 and serially diluted 11 points at 1:2 were incubated with 50,000 CHO cells transiently expressing human FRa for 30 minutes. The samples were then washed and antibody binding was detected by flow cytometry using a goat anti-rabbit secondary antibody (Jackson Immuno Research Labs, West Grove, PA) conjugated to Alexa Fluor-647. Titers were determined by identifying the highest dilution sample that showed a fluorescence signal at least 2 times that of the background.

6.3 Recovery of B cells and discovery of anti-human FRa antibodies

The rabbits immunized with a required titer higher than 100,000 were killed, and the spleen was harvested. Lymphoid cells were dissociated by grinding in FACS buffer (PBS, 2% v/v FBS) to release cells from tissues. The cells were precipitated and then suspended in 5 mL of Pharm Lyse ^TM (Becton, Dickinson & Co., Franklin Lakes, NJ) for 1 minute to lyse red blood cells. An equal volume of FACS buffer was added to neutralize Pharm Lyse ^TM and the resulting lymphocyte samples were precipitated and resuspended in FACS buffer.

The lymphocyte suspension was then stained with goat anti-rabbit IgG Alexa Fluor-647 (Jackson Immuno Research Labs, West Grove, PA) to identify IgG+B cells. After staining for 30 minutes, IgG+B cells were sorted and counted on FACSAria ^TM (Becton, Dickinson & Co., Franklin Lakes, NJ). Using the selected lymphocyte antibody method (SLAM) (Babcook et al., 1996, Proc Natl Acad Sci USA, 93 (15): 7843–7848), B cells were seeded in 384-well plates at different densities from a single cell to 50 cells, amplified in culture for 7 days, and the supernatant was harvested for detection of anti-human FRa antibodies. The 384-well plates were stored at -80 °C.

Human FRa-specific monoclonal antibodies in the supernatant were screened by ELISA. 384-well ELISA plates were coated with 25 μL/well of human FRa-HIS (2 μg/mL) in PBS and then incubated overnight at 4°C. After incubation, the plates were washed twice with water, 90 μL/well blocking buffer (2% skim milk, PBS) was added and the plates were incubated at room temperature for 1 hour. After incubation, the plates were washed and 12.5 μL/well of antibody-containing supernatant + 12.5 μL blocking buffer and positive and negative controls were added, and the plates were incubated at room temperature for 2 hours.

After incubation, wash the plate, add 25 μL of 0.4 μg/mL goat anti-rabbit IgG Fc-HRP detection antibody (Jackson Immuno Research Labs, West Grove, PA) to each well, and incubate the plate at room temperature for 1 hour. After incubation, wash the plate and add 25 μL of tetramethylbenzidine (TMB) to allow the plate to develop for about 10 minutes (until the negative control wells begin to show signals). Then add 25 μL of stop solution (1N HCL) to each well and read the plate at a wavelength of 450 nm on a Synergy ^TM H1 microplate (BioTek Instruments, Winooski, VT).

6.4 Sequencing of Anti-human FRa Antibodies

Total RNA was extracted from the wells containing antibodies with the desired characteristics using RNeasy (Qiagen, Hilden, Germany) according to the manufacturer's protocol. The total RNA obtained was used as a template to transcribe cDNA from mRNA using SuperScript ^TM III (Thermo Fisher Scientific Corp., Waltham, MA) and oligo-dT20 (Integrated DNA Technologies, Inc., Coralville, IA). The cDNA was subsequently treated with RNase H (New England Biolabs, Ipswich, MA). Using primers and methods modified from Babcook et al., 1996, Proc Natl Acad Sci USA, 93 (15): 7843–7848 and von Boehmer et al., 2016, Nat Protoc., 11 (10): 1908, initial PCR of heavy and light chain antibody coding sequences was performed using cDNA as a nucleic acid template. The PCR product was cloned into pCRTOPO4 using the Zero Blunt ^™ TOPO ^™ PCR Cloning Kit (ThermoFisher Scientific Corp., Waltham, MA) and transformed into Cells (Lucigen Corporation, Middleton, WI). Antibiotic-resistant clones were subjected to Sanger sequencing and analyzed for unique antibody coding sequences.

Subsequent PCR reactions were then performed on these unique sequences using V segment family and J segment family specific primers. The resulting amplicons were cloned into pTT5-based expression plasmids (National Research Council of Canada). Unique heavy and light chain sequences from single-well samples were co-expressed in all possible combinations in HEK293-6E cells (National Research Council of Canada) to determine the correct heavy and light chain pairing. The generated antibodies were assayed for binding to antigens transiently expressed on HEK293 cells.

6.5 Production of chimeric antibodies

The coding sequences of the antibody variable regions were cloned in-frame into the huIgG1 expression vector and the huCK expression vector (based on the pTT5 vector). The huIgG1 constant region starts at alanine Kabat-118, and the huCK constant region starts at arginine Kabat-108. The activity of the resulting recombinant chimeric antibodies was confirmed in a specific binding assay and found to be equivalent to the parental antibody.

Example 7: Humanization of anti-FRα antibody

One of the chimeric anti-human FRα (anti-hFRα) generated as described in Example 6, variant v23924, was selected for humanization. The CDR sequences of v23924 are provided in Table 7.1 and the VH and VL sequences are provided in Table 7.2. Humanization was performed as described below.

Table 7.1: CDR sequences of anti-hFRα antibody v23924

Table 7.2: VH and VL sequences of anti-hFRα antibody v23924

7.1 Humanization

The rabbit VH and VL sequences of v23924 were aligned with the corresponding human germline sequences, and IGHV3-23*01 and IGKVI-39*01 were identified as the closest and most common human germline sequences. The CDR sequences defined according to AbM (see Table 2.1) were transplanted onto the framework of these selected human germline sequences, as shown in Figure 1. Back mutations to rabbit residues at positions in the resulting sequence that were judged to be important for maintaining binding affinity to the antigen hFRα were included to generate several humanized sequences, where the generated sequences were largely built on the previous sequence and where the first humanized sequence contained the minimum number of back mutations. No variant modified the CDRs of the parent antibody as defined by the AbM method.

The process is carried out in two cycles, wherein the first cycle ("cycle 1") produces six variable heavy chain humanized sequences and five variable light chain humanized sequences. The second cycle ("cycle 2") is expanded to another 5 variable heavy chain humanized sequences, in pursuit of a closer parent-like affinity of humanized antibodies to hFRα. Complete heavy chain sequences containing humanized heavy chain variable domains (VH) and hIgG1 heavy chain constant domains (CH1, hinge, CH2, CH3), and complete light chain sequences containing humanized light chain variable domains (VL) and human κ light chain constant domains (κCL) are assembled. Monoclonal antibody (mAb) variants are then assembled so that in cycle 1, each humanized heavy chain is paired with each humanized light chain to provide 30 humanized variants, and in cycle 2, additional humanized heavy chains are paired with selected humanized light chains to obtain another 15 humanized variants, with a total of 45 humanized variants to be evaluated using experimental methods.

7.2 Generation of humanized antibodies

Each of the 45 humanized mAb constructs, as well as the parental v23924 mAb construct, was produced in a full-size antibody (FSA) format containing two identical full-length heavy chains (parental v23924 and 15 humanized variants), resulting in a homodimeric Fc region (HomoFc), or containing a heterodimeric full-length heavy chain (30 humanized variants) in the CH3 region that contained complementary mutations to drive unique heavy chain pairing, resulting in a heterodimeric Fc region (HetFc). A version of v23924 containing HetFc instead of HomoFc was also produced (variant v30618). All constructs included two identical kappa light chains.

The two identical full-length heavy chains contained in the HomoFc region contain the human CH1-hinge-CH2-CH3 domain sequence of IGHG1*01 (SEQ ID NO: 146; see Table 7.3). The heterodimeric full-length heavy chains (HetFc-A and HetFc-B) contained in the HetFc region contain the human CH1-hinge-CH2-CH3 domain sequence of IGHG1*01, and have the following mutations in the Fc region:

HetFc-A：T350V_L351Y_F405A_Y407V

HetFc-B：T350V_T366L_K392L_T394W

The sequences of HetFc-A (SEQ ID NO: 148) and HetFc-B (SEQ ID NO: 149) are provided in Table 7.3. For all constructs, the human kappa CL sequence of IGKC*01 (SEQ ID NO: 147; see Table 7.3) was used.

Table 7.3: HomoFc and HetFc sequences

Each humanized VH domain sequence from cycle 1 was appended to the human CH1-hinge-CH2-CH3 (HetFc-A and HetFc-B) domain sequence of IGHG1*01 to provide twelve humanized full heavy chain sequences (six humanized VH x2). Rabbit VH and each of the five additional humanized VH domain sequences from cycle 2 were appended to the human CH1-hinge-CH2-CH3 domain sequence of IGHG1*01 to provide the parent rabbit-human chimeric full heavy chain sequence and five additional humanized full heavy chain sequences. Each VL domain sequence was appended to the human κCL sequence of IGKC*01 to provide five humanized light chain sequences. All sequences were reverse translated into DNA, codon optimized for mammalian expression, and gene synthesized.

The heavy chain vector insert containing the signal peptide (artificially designed sequence: MRPTWAWWLFLVLLLALWAPARG (SEQ ID NO: 150) (Barash et al., 2002, Biochem and Bio phys Res. Comm., 294: 835–842)) and the heavy chain clone terminating at residue G446 (EU numbering) of the CH3 domain were ligated into the pTT5 vector to generate a heavy chain expression vector. The light chain vector insert containing the same signal peptide was ligated into the pTT5 vector to generate a light chain expression vector. The resulting heavy and light chain expression vectors were sequenced to confirm the correct reading frame and sequence of the encoding DNA.

The heavy and light chains of each humanized antibody variant were expressed in 200 ml CHO-3E7 cell cultures. Briefly, CHO-3E7 cells were cultured at 37°C in FreeStyle ^™ F17 medium (ThermoFisher Scientific, Waltham, MA) supplemented with 4 mM glutamine (GE Life Sciences, Marlborough, MA) and 0.1% Pluronica F-68 (Gibco/Thermo Fisher Scientific, Waltham, MA) at a density of 1.7-2 x 10 ⁶ cells/ml with a viability of >95%. PEI- (Polyscience, Inc., Philadelphia, PA), a total of 200ug DNA (100ug antibody DNA and 100ug GFP/AKT/filler DNA) was used to transfect CHO-3E7 cells in a total volume of 200ml at a DNA:PEI ratio of 1:4 (w/w) + 1x antibiotic/antimycotic (GE Life Sciences, Marlborough, MA). Twenty-four hours after adding the DNA-PEI mixture, 0.5mM valproic acid (final concentration) + 1% w/v tryptone (final concentration) was added to the cells, which were then transferred to 32°C and incubated for another 6 days before harvesting.

Protein A purification was performed in batch mode or using 1 mL HiTrap ^TM MabSelect ^TM SuRe ^TM column (Cytiva, Marlborough, MA). In batch mode, the clarified supernatant samples were incubated with mAb Select SuRe ^TM resin (GE Healthcare, Chicago, IL) in batches, cleaned in place (CIP'd) with NaOH and balanced in Dulbecco's PBS (DPBS). The resin was poured into the CIP'd column and the column was washed with DPBS. In both purification modes, the protein was eluted with 100 mM sodium citrate buffer (pH 3.0). The eluted fraction was pH adjusted by adding 10% (v/v) 1 M HEPES (pH about 10.6-10.7) to obtain a final pH of 6-7. The sample buffer was exchanged into DPBS. Protein was quantified based on the absorbance at 280 nm (A280 nm). After protein A purification, the parental rabbit-human antibody chimeric variants (v23924 and v30618) were further purified by preparative SEC chromatography on a Superdex200 Increase 10/30 column (GE Healthcare, Chicago, IL) in DPBS mobile phase.

After purification, high-throughput protein expression assays and Caliper The purity of the samples was assessed by electrophoresis under non-reducing and reducing conditions using a GXII or GXII Touch HT (Perkin Elmer, Waltham, MA). The procedure was followed according to the User Guide, Version 2, with the following modifications. 2 μl or 5 μl of antibody sample (concentration range 5-2000 ng/μl) was added to individual wells in a 96-well plate (BioRad, Hercules, CA) along with 7 μl of HT protein expression sample buffer (Perkin Elmer, catalog number 760328). The antibody samples were then denatured at 70°C for 15 minutes. The instrument was operated using the HT Protein Express Chip (Perkin Elmer, Waltham, MA) and the Ab-200 assay setup.

The yields of 45 humanized antibody variants and the parent chimeric antibody v23924 (after protein A purification) ranged from about 9-17 mg (or 45-85 mg/L). Figures 2A and 2C show the Caliper electrophoresis results of the parent chimeric antibody v23924 and the representative humanized variant v30384. As can be seen from Figure 2, for the representative humanized antibody samples, the non-reduced (NR) and reduced (R) Calipers reflect a single species corresponding to the full-size antibody and the complete heavy and light chains. This is also the case for other humanized variants.

7.3 Quality Assessment of Humanized Antibodies

The species homogeneity of the humanized antibody variants was assessed by UPLC-SEC after Protein A purification and after preparative SEC purification of the parental chimeric antibody v23924.

UPLC-SEC was performed using a Waters Acquity BEH200 SEC column (2.5 mL, 4.6 x 150 mm, stainless steel, 1.7 μm particles) (Waters LTD, Mississauga, ON) set to 30°C and mounted on a Waters Acquity UPLC ^™ H-Class Bio system with a photodiode array (PDA) detector. The mobile phase was Dulbecco's phosphate buffered saline (DPBS) (pH 7.4) containing 0.02% Tween 20, and the flow rate was 0.4 ml/min. The total run time for each injection was 7 minutes, and the total mobile phase volume was 2.8 mL. Elution was monitored by UV absorbance in the range of 210-500 nm, and chromatograms were extracted at 280 nm. Using a Waters 3 software, using Apex Track ^TM for peak integration and shoulder feature detection.

Figure 2B and Figure 2D show the UPLC-SEC profiles of the parent chimeric antibody v23924 (after SEC purification) and the representative humanized antibody v30384 (after protein A purification), respectively. The UPLC-SEC profiles of the representative humanized antibody samples reflect the same high species homogeneity as the parent chimeric antibody samples. Samples from the remaining humanized antibody variants have similar characteristics as those shown in the representative humanized antibody samples.

Example 8: Binding of humanized antibodies to hFRα

8.1 Evaluation of affinity of humanized antibodies for hFRα

To determine whether the humanization process affected the affinity of the humanized variants for their target, the ability of 45 purified humanized antibody variants to bind to the hFRα antigen was assessed by biolayer interferometry (BLI) as follows.

use The RED96 system (ForteBio, Fremont, CA) was used to assess hFRα antigen binding by cycling the following steps: loading the antibody (0.9 μg/mL) onto an anti-human IgG Fc capture (AHC) biosensor in 200 seconds; baseline stabilization for 60 seconds; association with recombinant His-tagged human FRα (ACROBiosystems, Newark, DE) at multiple relevant concentrations spanning the expected _KD for 400-500 seconds; dissociation was recorded for 500-1000 seconds; regeneration was performed by cycling 3 times between 10 mM glycine pH 1.5 (15 seconds) and assay buffer (1 second) before proceeding to the next antibody. The assay buffer used was KB buffer (kinetic buffer, composed of PBS (pH 7.4), 0.1% BSA, 0.02% Tween 20, 0.05% sodium azide) supplemented with 0.06% Tween 20 and in some cases 1% BSA. The experiment was carried out at 30°C with a shaking speed of 1000 rpm.

Data analysis was performed using Data Analysis Software 9.0 (ForteBio, Fremont CA). Reference-subtracted binding curves were globally fit to a 1:1 interaction model to generate binding kinetic parameters _kon , _koff , and dissociation constant _KD .

Nine of the 45 humanized antibody variants were found to bind hFRα with K _values ranging from about 63 nM to 210 nM (see Table 8.1). The _K of the parent chimeric antibody (v23924) was determined to be 27 nM. The humanized antibody variants that exhibit binding to hFRα are characterized by a decrease in affinity by about 2-fold to about 8-fold compared to the parent chimeric antibody. As can be seen from Table 8.1, all successful humanized variants share the same variable light chain (4L), but differ in variable heavy chain composition. The light and heavy chain sequences of the successful humanized variants are provided in Table 8.2. Figure 3 shows the BLI sensorgrams of the parent chimeric antibody v23924 and the representative humanized antibody v30384.

Table 8.1: Pass Antigen binding assessment of humanized variants

¹ HetFc = heterodimeric Fc region; HomoFc = homodimeric Fc region (see Example 2)

² n＝2

Table 8.2: Amino acid sequences of humanized VH and VL chains

8.2 Evaluation of the affinity of humanized antibodies for hFRα

The affinity of antigen binding of the parental and selected humanized variants in FSA format was assessed by surface plasmon resonance (SPR) as described below.

SPR assays for determining hFRα affinity and avidity of the parent chimeric antibody (v23924) and humanized variants were performed at 25°C on a Biacore ^™ T200 SPR system with PBS-T (PBS + 0.05% (v/v) Tween 20) running buffer (supplemented with 0.5M EDTA stock solution to reach a final concentration of 3.4mM). CM5 Series S sensor chips, Biacore ^™ amine coupling kit (NHS, EDC and 1M ethanolamine) and 10mM sodium acetate buffer were purchased from GE Healthcare Life Science (Mississauga, ON, Canada). PBS running buffer (PBST) with 0.05% Tween 20 was purchased from Teknova Inc. (Hollister, CA). Recombinant human FRα was purchased from ACRObiosystems (Newark, DE).

Screening for variants that bind to the hFRα antigen was performed in two steps: hFRα was captured onto the surface, followed by injection of 3 to 5 concentrations of variants. The hFRα surface was prepared on a CM5 Series S sensor chip by a standard amine coupling method as described by the manufacturer (GE Healthcare LifeScience, Mississauga, ON, Canada). For resonance units (RU) ranging from 5 to 200 RU, hFRα was immobilized using the Biacore ^™ T200 immobilization guide and amine coupling methods. Using multi-cycle kinetics, samples of a two-fold dilution series of 3 to 5 concentrations starting at 300 nM were injected at 50 uL/min for 180 seconds with a 600 second dissociation phase against blank buffer, producing a set of sensorgrams with a buffer blank reference. The hFRα surface was regenerated by pulsing 10 mM glycine/HCl (pH 1.5) once at 30 uL/min for 30 seconds to prepare for the next injection cycle. The blank-subtracted sensorgrams were analyzed using Biacore ^™ T200 Evaluation Software v3.0. The blank-subtracted sensorgrams were then fit to a 1:1 Langmuir binding model.

The results are shown in Table 8.3. The parent antibody (v23924) and two humanized variants (v30384 and v30399) of the conventional bivalent antibody format (FSA) all exhibited affinity for binding to the hFRα antigen. That is, compared with the one-armed antibody (OAA) format at medium-low antigen density, the _K values obtained in FSA were about 1/17 in the case of the parent sample and about 1/27-1/39 in the case of the humanized sample, and further decreased to about 1/108 and about 1/116-1/181, respectively, at high antigen density.

Table 8.3: Affinity assessment of selected humanized variants by SPR

¹ OAA = one-armed antibody, FSA = full-sized antibody; OAA antibody samples are equivalent to the corresponding FSA samples and were produced in a similar manner as the FSA samples described in Example 2.

² n＝2

Example 9: Purity of humanized anti-FRα antibody

The apparent purity of the humanized antibody variants from Example 8 was assessed using mass spectrometry following Protein A purification (Example 7) and native deglycosylation.

Since the antibody variant samples only contained Fc N-linked glycans, the samples were treated with N-glycosidase F (PNGase-F) only. The purified samples were deglycosylated with PNGase F as follows: 0.1 U PNGase F/μg antibody in 50 mM Tris-HCl (pH 7.0), incubated overnight at 37°C, with a final protein concentration of 0.48 mg/mL. After deglycosylation, the samples were stored at 4°C before LC-MS analysis.

Deglycosylated protein samples were analyzed by intact LC-MS using an Agilent 1100 HPLC system (adjusted for optimal detection of larger proteins (>50 kDa)) coupled to a LTQ-Orbitrap ^™ XL mass spectrometer (ThermoFisher, Waltham, MA) via an Ion Max electrospray source. Samples were injected onto a 2.1 x 30 mm Poros R2 reverse phase column (Applied Biosystems Corp., Waltham, MA) and resolved using a linear gradient of 0.1% formic acid in water/acetonitrile (degassed) consisting of increasing concentrations (20%-90%) of acetonitrile. The column was heated to 82.5°C and the solvent was heated to 80°C before the column to improve protein peak shape. The cone voltage (source fragmentation setting) was approximately 40 V, the FT resolution setting was 7,500 and the scan range was m/z 400-4,000. The LC-MS system was evaluated for IgG sample analysis using deglycosylated IgG standards (Waters IgG standards) and deglycosylated mAb standard mixtures (half-size: full-size antibodies at 25:75). For each LC-MS analysis, the mass spectra obtained across the antibody peak (usually 3.6-4.1 minutes) were summed, and the entire multi-charged ion envelope (m/z 1,200-4,000) was deconvoluted into a molecular weight spectrum using the MaxEnt 1 module of MassLynx ^TM data analysis software (Waters, Milford, MA). The apparent amount of every kind of antibody in each sample was determined by the peak height in the resulting molecular weight spectrum.

The results are shown in Table 9.1. Almost all humanized variants are highly pure, ranging from about 89%-100% of the desired species, with only v30389 showing a lower purity (82.6%). The purity of the four variants v30384, v30389, v30394 and v30399 is slightly lower than that of the remaining variants because these variants contain heterodimeric CH3 regions that result in the presence of some half antibodies (see Example 7). Figure 4 depicts the LC/MS spectra of two representative humanized variants v30384 and v31422. In the LC/MS spectra of all samples, a side peak of approximately +266Da was observed, which may be an artifact of the analysis.

Table 9.1: Purity of humanized variants determined by LC/MS

Variants Required type% v30384 93.2 v30389 82.6 v30394 89.1 v30399 91.8 v31422 100 v31423 100 v31424 100 v31425 100 v31426 100

Example 10: Thermal stability of humanized anti-FRα antibodies

The thermal stability of the humanized antibody variants was assessed by differential scanning calorimetry (DSC) as described below.

400mL purified samples mainly at 0.4mg/mL concentration in PBS are used to perform DSC analysis using VP-capillary DSC (GE Healthcare, Chicago, IL). At the beginning of each DSC run, 5 buffer blank injections are performed to stabilize the baseline, and buffer injections are arranged before each sample injection for reference. Utilizing low feedback, 8 seconds filter, 3 minutes pre-scan thermostat and 70psi nitrogen pressure, each sample is scanned from 20°C to 100°C at a rate of 60°C/h. The resulting thermal spectra are referenced and analyzed using Origin 7 software (OriginLab Corporation, Northampton, MA), to determine the melting temperature (Tm) as a thermal stability indicator.

The Fab Tm values determined for the humanized variants are shown in Table 10.1. All humanized variants exhibited increased thermal stability compared to the parent antibody v23924 (Fab Tm of about 72°C), with Fab Tm values ranging from about 81-84°C. Among the humanized variants, v30394 showed the highest thermal stability.

Table 10.1: Thermal stability of humanized variants

Variants Fab Tm(℃) v23924 (parent) 72.1 v30384 81.7 v30389 80.7 v30394 84.4 v30399 83.4 v31422 82.6 v31423 83.0 v31424 81.3 v31425 82.4 v31426 81.9

Example 11: Determination of isoelectric point of humanized anti-FRα antibody

The isoelectric points of the humanized antibody variants were determined by capillary isoelectric focusing (cIEF) as described below.

cIEF was performed using a CE-UV Agilent 7100 capillary electrophoresis (CE) system. 5ug (or maximum 2.5uL) sample was applied to the capillary (ampholyte range of 3.0-10.0). A pI standard mixture, 4.1, 4.22, 5.5, 7.0 and 10.0 were used for system suitability tests, and 4.1 and 10.0 were used for sample analysis. For all CE runs, an Agilent 7100 CE system equipped with an external water bath set to 6°C, a detector filter assembly (280nm) and an external pressure of 9 bar was used. Neutral coated capillaries (fluorocarbons) were cut at a distance of 8.5cm and 24.5cm from the detection window at both ends, respectively, equipped with a green alignment interface and assembled into an Agilent capillary box. Once a day, capillaries were conditioned as follows: high pressure rinsed with 350mM acetic acid at 3.5 bar for 5 minutes, rinsed with water for 2 minutes and rinsed with cIEF gel for 5 minutes. Before each operation, the capillary was adjusted as follows: high pressure washing at 3.5 bar for 3 minutes with 4.3M urea solution and rinsed with water for 2 minutes. By applying 2 bar high pressure for 100 seconds, the inlet electrode and outlet electrode water soaking were then carried out to inject the sample. Focusing was performed for 10 minutes at 25kV using 200mM phosphoric acid as anolyte and 300mM NaOH as catholyte. Using chemical mobilization, the outlet bottle was replaced with 350mM acetic acid and 30kV was applied for 30 minutes. After each operation, high pressure washing was performed with water at 3.5 bar for 2 minutes. Manual integration of peak RT and electrophoretogram was obtained using Agilent OpenLAB Intelligent Reporting A.01.06.111 software. Raw data (signal and retention time) were exported to CVS files and the pI (based on internal pI standards) of main isoform pI, pI range and mass center were calculated in Microsoft Excel.

The results are shown in Table 11.1. The pI values determined for the major isoforms of most humanized variants ranged from 7.78 to 7.97, with one variant having a pI of 8.25 (v31426), all falling within the typical range for therapeutic antibodies and relatively comparable to the pI value of the parent chimeric antibody v23924 (pI of 7.65).

Table 11.1: Isoelectric points of humanized variants

Variants pI (major isoform) v23924 (parent) 7.65 v30384 7.79 v30389 7.78 v30394 7.89 v30399 7.89 v31422 7.93 v31423 7.94 v31424 7.97 v31425 7.93 v31426 8.25

Example 12: Chromatographic Analysis of Anti-FRα Antibodies

Parental and humanized variants were analyzed by hydrophobic interaction chromatography (HIC) and size exclusion chromatography (SEC) as described below.

12.1 HIC analysis

The hydrophilicity/hydrophobicity of the antibody was assessed by HIC as described in Antibody Drug Conjugates, Methods in Molecular Biology, 2013, Vol. 1045, pp. 275-284, edited by L. Ducry. The antibody was pre-equilibrated with 5 column volumes of buffer A (1.5 M (NH ₄ ) ₂ SO ₄ , 25 mM NaH ₂ PO ₄ , pH = 6.95) at room temperature. Butyl-NPR column (2.5 μm, 4.6x35 mm, TOSOH Bioscience GmbH, Griesheim, Germany) was used for the experiment on Agilent Infinity II 1290 HPLC. Typically, 20-30 mg of sample at a concentration of 2-3 mg/mL was loaded onto a column with 95% buffer A and 5% buffer B (75% 25 mM PO ₄ ^3- plus 25% isopropanol, pH 6.95) and run at 0.5 mL/min for 15 minutes using the gradient shown in Table 12.1. The HIC chromatogram was integrated using appropriate parameters that provided a complete baseline-baseline integration.

Table 12.1: HIC solvent gradient

Time (minutes) Buffer A% Buffer B% 0 95 5 0.1 95 5 5 80 20 9.5 65 35 11.5 50 50 12.5 5 95 13.5 5 95 12.6 95 5 15 95 5

12.2 SEC Analysis

At room temperature, SEC was performed using an Advance Bio SEC column ( Analytical SEC was performed on an Agilent Infinity II 1260 HPLC (2.7 μm, 7.8×150 mm), the column being balanced with 5 column volumes of mobile phase A (150 mM Na ₂ PO ₄ , pH 6.95). Typically, 20-30 mg of a sample at a concentration of 2-3 mg/mL was eluted isostatically at 1 mL/min for 7 minutes and the absorbance was monitored at A280. The chromatogram was integrated to provide a complete baseline-baseline integration of each peak, with reasonable separation positions between partially separated peaks. Based on the SEC spectrum of the control trastuzumab, the peak corresponding to the main component of IgG (approximately retention time 3.3 minutes) was reported as a monomer. Any peak appearing before 3.3 minutes was designated as a high molecular weight species (HMWS), and any peak appearing after 3.3 minutes was designated as a low molecular weight species (LMWS), excluding solvent peaks (over 5.2 minutes).

12.3 Results

A summary of the HIC retention time (HIC-RT) and SEC monomer % of the parent and humanized variants is provided in Table 12.2. In summary, HIC and SEC show favorable biophysical behavior of all humanized variants. The parent chimeric antibody v23924 eluted at 6.5 minutes on the HIC gradient, while all humanized variants eluted between 6.0-6.7 minutes. The SEC profile shows>90% monomer for all humanized variants, with variant v30389 having the lowest monomer % (94%). All of these variants have <5% HMWS and <5% LMWS.

Table 12.2: HIC and SEC analysis of parental and humanized anti-FRα antibodies

*No prior Prep-SEC

Example 13: Additional stability studies

To further investigate the stability of the humanized antibody variants, 40°C stability and acid stability studies were performed, wherein samples were characterized at specific time points by Caliper and UPLC-SEC as described in Example 7, and in the case of the 40°C stability studies additionally by cIEF and Octet antigen binding as described in Example 11 and Example 8, respectively. Trastuzumab was used as a control in these studies.

The selected humanized variants (v30389, v30394, v30399, v31423 and v31424) were subjected to a 40°C stability study for 14 days in PBS pH 7.4 buffer at a sample concentration of about 1 mg/ml. Samples were characterized at time points 0, 5, 7, 11 and 14 days. Acid stability studies were performed after the sample buffer was exchanged for an acetate buffer of pH 3.6 at 25°C for 1 hour at different sample concentrations, wherein the time points were characterized at 0, 15, 30 and 60 minutes. In addition, freeze-thaw (from -80°C to room temperature) was performed in PBS (pH 7.4) at a sample concentration of about 1 mg/ml, and freeze-thaw constituted three cycles of 30 minutes per cycle.

The results are shown in Table 13.1. No significant issues with the stability of the humanized variants were identified in the study. Freeze-thaw and acid stability studies did not reveal any changes in sample composition during the study as determined by Caliper and UPLC-SEC. The 40°C stability study showed minor changes in the Caliper and UPLC-SEC profiles as the study proceeded (specifically, some amounts of low molecular weight species appeared, ranging from about 10%-17%). No changes in antigen binding affinity were observed. cIEF revealed an expected small increase in more acidic species relative to the major isoform.

Table 13.1: Stability assessment of humanized variants at 40°C, acid stability and freeze-thaw studies

^1Trastuzumab control

² Err = Error baseline

Example 14: Functional Characterization of Anti-FRα Antibodies—FRα Specificity

The parental chimeric antibody v23924 was evaluated for binding cross-reactivity to FRα (FOLR1), FOLR2, FOLR3, and FOLR4 by flow cytometry and ELISA. FRα, FOLR2, and FOLR4 binding were evaluated by flow cytometry using HEK293 transfected cells. FOLR3 binding was evaluated by ELISA because FOLR3 is a soluble protein. A control anti-FOLR2 (mouse anti-human FOLR2; Nordic BioSite AB, Sweden; Catalog No. AFC-4544-2), anti-FOLR3 (mouse anti-human FOLR3; LS Bio, Seattle, WA; Catalog No. LS-C125621), and anti-FOLR4 (mouse anti-His DyLight ^™ 650; Novus Biologicals, Littleton, CO; Catalog No. NBP2-31055C) antibodies were included in these experiments.

FRα, FOLR2 and FOLR4 binding : Briefly, HEK293-6e cells were transfected for approximately 24 hours to transiently express human FRα (Cat. No. 13420), FOLR2 (Cat. No. 13481) and FOLR4 (Cat. No. 13483) (all from GenScript Biotech, Piscataway, NJ) with 1 ug DNA per 1 million cells. After transfection, 50,000 cells were seeded in a V-bottom 96-well plate and incubated with 50 nM primary antibody for 45 minutes under standard culture conditions. After incubation, cells were washed and stained with anti-human IgG FcAF647 conjugate (Jackson Immuno Research Labs, West Grove, PA; Cat. No. 109-605-098) at room temperature (RT) for 45 minutes. After incubation and washing, fluorescence was detected by flow cytometry on a BD LSRFortessa ^™ cell analyzer (BD Biosciences, Franklin Lake, NJ), with a minimum of 1,000 events collected per well.

FOLR3 binding : ELISA 96-well plates were coated with commercially purified FOLR3 protein (R&D Systems, Inc., Minneapolis, MN; Catalog No. 5319-FR) at 37°C for 1 hour. The plates were blocked with 1% milk in PBS (pH 7.4) for 1 hour at room temperature. After blocking, primary antibodies were added at 7 nM for 1 hour at room temperature. HRP-conjugated secondary antibodies (Jackson Immuno Research Labs, West Grove, PA; Catalog No. 109-035-098) were then added at 0.4 μg/ml at room temperature for 1 hour. The plates were developed using tetramethylbenzidine (TMB) and the reaction was terminated using HCl. The absorbance was read at 450 nm using a Synergy ^TM H1 microplate reader (BioTek Instruments, Winooski, VT).

result

The results are shown in Table 14.1 and Table 14.2. By flow cytometry or ELISA, anti-FRα, anti-FOLR2, anti-FOLR3 and anti-FOLR4 control antibodies showed expected binding to the corresponding target proteins. By flow cytometry, FlowJo ^™ v8 software (BD Biosciences, Franklin Lake, NJ) was used to set gates on the live single cell population and determine the AF647 GeoMean and positive binding % for each antibody in the population. For ELISA, the positive binding % of anti-FOLR3 and v23924 antibodies was determined using the raw absorbance values compared to the negative control absorbance signal. v23924 showed expected binding to human FRα, while showing no binding cross-reactivity with FOLR2, FOLR3 or FOLR4, indicating FRα specificity.

Table 14.1: Binding to FRα, FOLR2 and FOLR4 assessed by flow cytometry

Table 14.2: Binding to FOLR3 assessed by ELISA

Antibody Absorbance 450nm Combined % v23924 0.29 8.9 Anti-FRα control 0.24 7.4 Anti-FOLR3 control 3.27 100.0 Human IgG 0.15 4.5

Example 15: Functional Characterization of Anti-FRα Antibodies - Binding to Cynomolgus Monkey FRα

The parental chimeric antibody v23924 was evaluated for cross-reactivity with human and cynomolgus monkey FRα by flow cytometry using transfected CHO-S cells as described below. Palivizumab (anti-RSV) (v22277) was used as a negative control.

In brief, CHO-S cells were transfected for about 24 hours to transiently express human or cynomolgus monkey FRα, 1 ug DNA per 1 million cells. After transfection, cells were seeded in V-bottom 96-well plates at 50,000 cells/well and treated with antibodies for 24 hours at 4°C to prevent internalization. After incubation, cells were washed and stained with anti-human IgG Fc AF647 conjugate (Jackson ImmunoResearch Labs, West Grove, PA; Catalog No. 109-605-098) at 4°C for 30 minutes. After incubation and washing, fluorescence was detected by flow cytometry on a BD LSRFortessa ^TM cell analyzer (BD Biosciences, Franklin Lake, NJ).

The results are shown in Table 15.1. v23924 showed equivalent binding to human and cynomolgus FRα on CHO-S transfected cells, with apparent Kd values of 83.89 pM and 121.60 pM on human FRα and cynomolgus FRα transfected cells, respectively. As expected, no binding was observed for the control v22277.

Table 15.1: Binding to cynomolgus monkey FRα

Example 16: Functional Characterization of Anti-FRα Antibodies - Cell Binding

The on-cell binding abilities of the parental chimeric antibody v23924 and the representative humanized variant v30384 were assessed by flow cytometry on JEG-3 and HEC-1-A endogenous FRα expressing cell lines as described below.

In brief, cells were seeded in V-bottom 96-well plates at 50,000 cells/well and treated with antibodies for 24 hours at 4°C to prevent internalization. After incubation, cells were washed and stained with anti-human IgG Fc AF647 conjugate (Jackson ImmunoResearch Labs, West Grove, PA; Catalog No. 109-605-098) at 4°C for 30 minutes. After incubation and washing, fluorescence was detected by flow cytometry on a BD LSRFortessa ^TM cell analyzer (BD Biosciences, Franklin Lake, NJ), and 1,000 minimum events were collected in each well. GraphPad Prism version 9 (GraphPad Software, San Diego, CA) was used to plot AF647/APC-A GeoMean (fluorescence signal geometric mean, proportional to anti-human AF647 binding) in live cell populations.

The results are shown in Table 16.1. Both the chimeric (v23924) and humanized (v30384) antibodies produced equivalent apparent Kd and Bmax values in both JEG-3 and HEC-1-A cell lines (high and moderate endogenous FRα expression, respectively).

Table 16.1: Cell Binding

Example 17: Functional Characterization of Anti-FRα Antibodies - Internalization

The receptor-mediated internalization capacity of chimeric antibody v23924 and representative humanized variant v30384 in FRα-expressing cell lines (IGROV-1 and OVCAR-3) was determined by high-content imaging as described below. Antibodies targeting FRα, somituzumab and fatuzumab, were used as positive controls, and palivizumab (anti-RSV) (v22277) was used as a negative control.

In brief, the antibodies were fluorescently labeled by coupling with anti-human IgG Fc Fab fragment pHAb dye conjugate (Promega Corporation, Madison, WI; Catalog No. G9841) (about 3 dye molecules per Fab fragment) at 5: 1 molar excess at 4 ° C for 24 hours. Cells were seeded in 96-well plates and incubated overnight at 37 ° C in 5% CO _2. The coupled antibodies were added to the cells the next day and incubated at 37 ° C for 6-24 hours to allow internalization. After incubation, cells were stained with dye cycle violet (Dye Cycle Violet) (ThermoFisher Scientific Corporation, Waltham, MA; Catalog No. V35003) for live cell identification and analyzed by high-content imaging of internalized fluorescence in live cells using CellInsight ^TM CX5 high-content screening (HCS) platform (Thermo Fisher Scientific Corporation, Waltham, MA). The fold-over fluorescence of the chimeric antibody v23924 was plotted using GraphPad Prism version 9 (GraphPad Software, San Diego, CA).

result

The results are shown in Figures 5A and 5B (IGROV-1 cells) and Figures 6A and 6B (OVCAR-3 cells). Chimeric antibody v23924 showed equivalent internalization levels to humanized variant v30384 in both IGROV-1 cells (high FRα) and OVCAR-3 cells (medium FRα). In IGROV-1 and OVCAR cells, chimeric antibody v23924 and humanized variant v30384 showed increased internalization compared to the positive controls of sometuximab and facilitation at all tested concentrations (25 to 1 nM) and time points (6-24 hours). For example, after 6 hours of incubation in IGROV-1 cells, the humanized variant v30384 showed an increase of 3.3-fold and 6.1-fold in internalized fluorescence compared to Suomi and Fatuzumab at 25 nM, respectively, and an increase of 2.6-fold and 19.1-fold in internalized fluorescence compared to Suomi and Fatuzumab at 5 nM, respectively (Figure 5A). Similarly, after 24 hours of incubation in IGROV-1 cells, the humanized variant v30384 showed an increase of 2.1-fold and 3.9-fold in internalized fluorescence compared to Suomi and Fatuzumab at 25 nM, respectively, and an increase of 1.9-fold and 4.7-fold in internalized fluorescence compared to Suomi and Fatuzumab at 5 nM, respectively (Figure 5B).

Example 18: Epitope Mapping

High-resolution epitope mapping of the parental chimeric antibody v23924 on the human FRα antigen (hFRα) was performed by hydrogen/deuterium exchange mass spectrometry (HDX-MS) at NovoAb Bioanalytics Inc. (Victoria, BC. Canada) as described below.

18.1 Sample Preparation for HDX-MS

Lyophilized hFRα was purchased from ACROBiosystems (Newark, DE; catalog number: FO1-H5229) and dissolved to a concentration of 2.5 mg/ml. Antigen-antibody complexes were prepared by mixing hFRα with the parent chimeric antibody v23924 at a molar ratio of 2:1. All samples had a pH of 7.4 and were clear (no precipitation was observed). For peptide identification, hFRα at a concentration of 10 μM was reduced with 100 mM tris-(2-carboxyethyl)phosphine (TCEP) in the presence of 2 M guanidine at pH 2.4, and then digested with pepsin at an enzyme to protein molar ratio of 1:1. HDX was initiated by mixing the protein sample with D ₂ O buffer at a ratio of 2:8 (v/v). The resulting solution was incubated at 26° C., and aliquots were removed at 20 seconds, 7 minutes, 1 hour, and 4 hours and immediately quenched by adding a 200 mM TCEP solution containing 4 M guanidine. These samples were flash frozen in liquid nitrogen and stored at -80°C. During LC-MS experiments, protein aliquots were rapidly thawed and kept on ice for 2 min of reduction followed by pepsin digestion at 0°C for 2 min.

18.2LC-MS and LC-MS/MS

In the LC-MS experiments, a 20 μL aliquot of each sample was immediately injected into a C18 analytical column and separated by reversed phase liquid chromatography using a Dionex UHPLC system (Thermo Fisher Scientific, Bremen, Germany) at a flow rate of 100 μL/min. The UHPLC system was coupled to a ThermoScientific Orbitrap Fusion ^TM mass spectrometer equipped with a heated electrospray ionization (HESI) II source. The column, accessories, syringes, and solvent delivery lines were embedded in an ice bucket to minimize H/D back exchange. The syringes used for injection were cooled on ice. The mobile phases were 0.1% formic acid (A) and 100% acetonitrile/0.1% formic acid (B), and the peptides were separated by a 13-minute gradient. MS survey scans were performed in the range of m/z 300-1600 with a mass resolution of 120,000 FWHM. The Orbitrap detector was calibrated to an error of <3 ppm by using a calibration mixture (Calmix; ThermoFisher Scientific Corporation, Waltham, MA). In the electron transfer dissociation (ETD) experiment, the fluoranthene radical anion was introduced into the ion trap within 50 milliseconds. Collision induced dissociation (CID) and ETD fragment ions were detected in the Orbitrap using a scan range of 150-2000 m/z.

For data analysis, the bottom-up LC-MS/MS raw data were processed using the Proteome Discoverer ^™ software suite (Thermo Fisher Scientific). The generated peak list was submitted to an in-house Mascot 2.2 server and searched against the sequence of hFRα. The peptides thus identified were used for HDX data analysis. ETD data were processed using Xcalibur ^™ software (ThermoFisher Scientific Corporation, Waltham, MA), and the resulting ETD peak list was searched against the sequence of hFRα using ProteinProspector (available online from the University of California, San Francisco website; http://prospector.ucsf.edu). Matched ions were also checked and confirmed by manual inspection. The mass shift of the peptides and the deuteration state of each amide were determined based on their center of mass m/z values before and after H/D exchange. All HDX data were normalized to 100% D ₂ O content (80% D exchange in buffer at all time points). The percent deuterium incorporation value was obtained by comparing the number of deuterium picked up to the total number of amide hydrogens contained in each peptide.Amide level deuteration information was calculated based on deuterium uptake by the ETD fragments.

18.3 Results

Protein sequence coverage and peptide identification

The presence of protein disulfide bonds has a significant impact on the pepsin digestion pattern and efficiency in peptide-based HDX-MS analysis. Since both hFRα and antibody v23924 contain multiple disulfide bridges, an optimized protocol for rapid protein disulfide reduction and pepsin digestion was first developed. The reduction time and digestion time were each optimized to 2 minutes, and these conditions were applied to both hFRα and the hFRα-v23924 complex. The peptides so identified covered 100% of the antigen sequence (see Figure 7).

Comparison of HDX at the peptide level

The deuterium incorporation levels of peptides from the hFRα and v23924 complex were plotted versus HDX time (20 seconds, 7 minutes, 1 hour, and 4 hours). The results are summarized and shown in FIG8 . Most peptides had the same deuterium uptake behavior before and after antibody binding ( FIG8A ), indicating that the binding site (epitope) of the v23924 antibody is quite localized. In the difference map shown in FIG8B , three peptides (Nos. 14, 15, and 16) showed a significant decrease in deuterium uptake after v23924 binding, indicating that they are in the epitope region. The sequences of these peptides are WWEDCRTSY (118-126) (SEQ ID NO: 151), WEDCRTSY (119-126) (SEQ ID NO: 152), and WEDCRTSYTCKSNWHKGWNWTSGF (119-142) (SEQ ID NO: 153), respectively.

Epitope determination at the amino acid level

Although the three epitope peptides differ in sequence, their HDX differences are identical ( FIG. 8 ), and they all contain the sequence WEDCRTSY (119-126) (SEQ ID NO: 152). This indicates that all epitope residues are included in the shortest peptide 119-126. Multiple differential peptides were observed in the same region, providing further confirmation that this region of the protein is the binding site for the v23924 antibody. To further localize the HDX differences and pinpoint the epitope to a single amino acid, MSMS was performed on peptide 119-126 using ETD. The 1 hour HDX time point was selected because this produced the greatest difference. ETD fragments provide single residue resolution. The deuteration level of each amino acid between the hFRα and v23924 complexes was calculated and compared ( FIG. 9 ). Based on the results of the difference map, the epitope residues were determined to be E120, D121, R123, T124, S125, and Y126 of SEQ ID NO: 15 (i.e., the epitope sequence is: EDRTSY; SEQ ID NO: 154).

Example 19: Affinity maturation of anti-FRα antibodies

The humanized antibody v30384 (see Example 8) was affinity matured using the HuTarg ^™ system (Innovative Targeting Solutions, Vancouver, BC, Canada).Genetic recombination was applied to the variable regions of the humanized variant v30384, and high affinity mutants were identified using next generation sequencing (NGS).

19.1 Design of the plasmid library

The CDR loops of the humanized variant v30384 variable domains are interspersed with RAG1/2 recombination signal sequences (RSS).These variable regions were synthesized at Integrated DNA Technologies, Inc. (Coralville, IA) and cloned into plasmid E951 (Innovative Targeting Solutions, Vancouver, BC, Canada).

19.2 HuTarg ^TM Library Generation

The following steps were performed according to the protocol of the Innovative Targeting System. The E951-based plasmid library was integrated into HuTarg ^™ cells and RAG1/2 expression was induced for 48 hours. HuTarg ^™ cells that showed successful recombinant antibodies after staining with PE-conjugated goat anti-human kappa light chain antibody (Bio-Rad Laboratories, Hercules, CA; catalog number 206009) were selected for further study.

19.3 FACS-based selection of affinity mutants

HuTarg ^™ cells were subjected to multiple rounds of FACS-based sorting on a BD FACSAria ^™ flow cytometer (BD Biosciences, Franklin Lakes, NJ), each round using decreasing amounts of biotinylated soluble HIS-tagged FRα antigen (FRα-HIS, ACROBiosystems Newark, DE; catalog number FO1-H82E2) and detected with streptavidin conjugated to AlexaFluor-647 (Thermo Fisher Scientific Corp., Waltham, MA; catalog number S11223). HuTarg ^™ cells that showed increased binding to biotinylated FRα-HIS were sorted directly into RNAzol RT (Sigma-Aldrich, St. Louis, MI; catalog number R4533) in preparation for next-generation sequencing.

19.4 Next-generation sequencing of affinity mutants

Total RNA was isolated from cells lysed in RNAzol according to the manufacturer's instructions. RNA was then digested with ezDNase ^™ (Thermo Fisher Scientific Corp., Waltham, MA; Catalog No. 11766051) and cDNA was transcribed using Superscript ^™ IV (Thermo Fisher Scientific Corp., Waltham, MA; Catalog No. 18090010) and gene-specific primers. The VH and VK domains were targeted for PCR amplification and expressed using Molecular barcoding was performed using the 500-cycle Ultra ^™ DNA library preparation kit (New England Biolabs, Ipswich, MA; catalog number E7370L). Samples were pooled and run on an Illumina MiSeq ^™ sequencer using a 500-cycle kit using v2 chemistry (Illumina, San Diego, CA; catalog number MS-102-2003). Sequence analysis was performed to identify mutations within the VH and VK sequences that showed a high likelihood of conferring increased affinity.

19.5 Recombinant Expression of Affinity Mutants

DNA sequences encoding mutant VH and VK domains were synthesized as "MiniGenes" (Integrated Technologies, Inc., Coralville, IA) and cloned into expression vectors to provide expression plasmids encoding complete human IgG1 heavy chains and human kappa light chains, respectively. The expression plasmids were matrixed to each other so that each heavy chain plasmid was paired with each light chain plasmid. The matrix was recombinantly expressed in Expi293 ^TM cells (Thermo Fisher Scientific Corp., Waltham, MA; Catalog No. A14635) according to the manufacturer's instructions to generate 64 samples.

19.6 Evaluation of affinity mutants

Protein G particles (Spherotech Inc., Lake Forest, IL) were coated with normalized concentrations of humanized variant v30384 or affinity matured antibodies. Soluble human FRα was diluted to the limiting antigen concentration and incubated with antibody-coated beads. AlexaFluor-647-conjugated streptavidin and AlexaFluor-488-conjugated goat anti-human IgG Fcy (both from Jackson Laboratories, Bar Harbor, ME) were used to detect FRα antigen binding and antibody capture, respectively. Samples were analyzed by flow cytometry on a BD LSRFortessa ^TM cell analyzer (BD Biosciences, Franklin Lakes, NJ). The geometric mean of FRα binding and antibody capture for each sample was analyzed. Antibody capture was normalized to FRα binding to the affinity grade humanized variant v30384 for affinity matured antibodies.

Single point affinity ranking was performed by measuring the ratio of antibody captured on the beads to the amount of antigen captured by the antibody. Using a human FRα concentration of 1.9 nM, variant v30384 binding was minimal (3x background), while most mutant variants showed higher binding ratios. Of the 64 mutant variants, 3 variants showed a 4-fold increase in binding ratio, and 10 variants showed equivalent binding ratios.

Example 20: Affinity assessment of affinity matured antibodies for FRα

The 10 affinity matured variants described in Example 19 were produced in full-size antibody (FSA) format at WuXi Biologics (Hong Kong) Limited via transient transfection and affinity capture purification in CHO-K1 cells, with subsequent polishing steps (if necessary) mainly involving preparative SEC or CEX chromatography, resulting in a sample purity of greater than 97% by HPLC-SEC. The FSA format is similar to the parent humanized variant v30384, except that these variants contain HomoFc instead of HetFc. The FSA format was used as described in Example 8. The RED96 system characterizes the binding of 10 affinity matured variants to hFRα.

result

The results are shown in Table 20.1. Affinity maturation was successful in obtaining humanized variants with significantly higher affinities for hFRα than the parental humanized antibody v30384 ( _KD 1.27E-07M), with affinity improvements ranging from about 12 to 58 fold. The lowest _KD of 2.2E-09M was observed for variant v35348. It was determined that the increase in affinity was primarily achieved by reducing the dissociation constant.

Table 20.1: Affinity evaluation of affinity matured variants

*n=2

Example 21: Chromatographic Analysis of Affinity Matured Antibodies

The 10 affinity matured variants from Example 20 were analyzed by hydrophobic interaction chromatography (HIC) and size exclusion chromatography (SEC) as described in Example 12. The results are shown in Table 21.1. Affinity maturation of the antibodies resulted in changes in hydrophobicity/hydrophilicity, as evidenced by variable HIC-RT. The antibody monomer content was above 97% in all cases and was independent of HIC-RT.

Table 21.1: HIC and SEC analysis of affinity matured anti-FRα antibodies

Example 22: Functional Characterization of Affinity Matured Antibodies - Cell Binding

As described in Example 16, the on-cell binding ability of the representative affinity matured variant v35356 was assessed by flow cytometry on IGROV-1 and JEG-3 endogenous FRα expressing cell lines.

result

The results are shown in Table 22.1. Both the parental humanized variant v30384 and the affinity matured variant v35356 produced equivalent apparent Kd and Bmax values in both IGROV-1 and JEG-3 cell lines (high and medium endogenous FRα expression, respectively).

Table 22.1: Cell Binding

Example 23: Functional Characterization of Affinity Matured Antibodies - Internalization

The receptor-mediated internalization capacity of the parental humanized variant v30384 and the representative affinity matured variant v35356 in FRα expressing cell lines (IGROV-1 and JEG-3) was determined by flow cytometry as described below. Palivizumab (anti-RSV) (v22277) was used as a negative control.

In brief, the antibody was fluorescently labeled by coupling with Fab-AF488 anti-human IgG Fc labeling reagent (Jackson ImmunoResearch Labs, West Grove, PA; Catalog No. 109-547-008) at a 1: 1 molar ratio at 4 ° C for 24 hours. Cells were seeded in 48-well plates and incubated overnight at 37 ° C in 5% CO _2. The coupled antibody was added to the cells the next day and incubated at 37 ° C for 24 hours to allow internalization. After incubation, the cells were dissociated, washed and incubated at 4 ° C for 45 minutes using 100nM anti-488 antibody to quench the surface AF488 fluorescence. The quenched AF488 fluorescence (internalized fluorescence) of all samples was analyzed by flow cytometry on a BD LSRFortessa ^TM cell analyzer (BD Biosciences, Franklin Lake, NJ), and 1,000 minimum events were collected per well. AF488/FITC-AGeoMean in live cell populations was plotted using GraphPad Prism version 9 (GraphPad Software, San Diego, CA).

result

The results are shown in Figure 10. The parental humanized variant v30384 and the affinity matured variant v35356 showed equivalent internalization in IGROV-1 (Figure 10(A)) and JEG-3 (Figure 10(B)) cells when administered at 20 nM at 5 and 24 hours of exposure.

Example 24: Preparation of Antibody-Drug Conjugates

The antibody-drug conjugates shown in Table 24.1 were prepared. An exemplary scheme is provided below. Variant v36675 comprises the same paratope as variant v30384, but has a HomoFc Fc region instead of a HetFc region.

v36675-MC-GGFG-AM-DXd1 : A solution of humanized variant v36675 (1.5 g) in PBS (83.5 mL) (pH 7.4) was reduced by adding 5 mM diethylenetriaminepentaacetic acid (DTPA) (24 mL PBS solution, pH adjusted to 7.4) and 10 mM tris(2-carboxyethyl)phosphine (TCEP) aqueous solution (12.5 mL, 12 equivalents). After 3 hours at 37°C, the reduced antibody was diluted to 125 mL with PBS and used XL ultrafiltration module (Ultracel ^30kDa 0.005m2; MilliporeSigma, Burlington, MA; PXC030C50) was purified with approximately 5 diavolumes of 10mM NaOAc (pH 5.5). The purified antibody (1133mg) was diluted to a final volume of 211mL using 10mM NaOAc (pH 5.5). 6.4mL DMSO and excess drug-linker MC-GGFG-AM-DXd1 (9.43mL; 12 equivalents) from a 10mM DMSO stock solution were added to the antibody solution. The conjugation reaction was carried out at room temperature and under mixing for 75 minutes. An excess of 10mM N-acetyl-L-cysteine solution (4.72mL, 6 equivalents) was added to quench the conjugation reaction.

v36675-MC-GGFG-AM-Compound 141, v36675-MC-GGFG-AM-Compound 139 and v36675-MC- GGFG-Compound 141 : A solution of humanized variant v36675 (60 mg) in PBS (pH 7.4) (2.95 mL) was reduced by adding 5 mM diethylenetriaminepentaacetic acid (DTPA) (0.96 mL PBS solution, pH adjusted to 7.4) and 1 mM tris(2-carboxyethyl)phosphine (TCEP) aqueous solution (0.9 mL, 2.15 equiv.). After 100 min at 37° C., 1.6 mL of the reduced antibody was diluted with 0.92 mL PBS (pH 7.4) and 1.08 mL 100 mM NaOAc (pH 5.5). 289uL DMSO and excess MC-GGFG-AM-Compound 141, MC-GGFG-AM-Compound 139 or MC-GGFG-Compound 141 (111uL; 8 equivalents) from a 10mM DMSO stock solution were added to the antibody solution. The conjugation reaction was allowed to proceed for 60 minutes at room temperature with mixing. An excess of 10mM cysteamine-HCl solution (444uL, 32 equivalents) was added to quench each conjugation reaction.

Table 24.1: Antibody-drug conjugates

¹ See the structures in Tables 8 and 9

^2Palivizumab (anti-RSV)

³ The structure is shown below. The abbreviations DXd1 and DXd are used interchangeably to refer to the same payload.

MC-GGFG-AM-DXd1:

MT-GGFG-AM-DXd1:

Example 25: Purification and characterization of antibody-drug conjugates

use ADC prepared on a large scale as described in Example 24 was purified using a XL ultrafiltration module (MilliporeSigma, Burlington, MA) and sterile filtered (0.22 mm). An exemplary protocol is provided below.

The quenched ADC solution from Example 24 was diluted to approximately 5 mg/mL with 10 mM NaOAc (pH 5.5) and used Purification was performed using an Ultracel XL ultrafiltration module (Ultracel 30 kDa 0.005 ^m2 ; MilliporeSigma, Burlington, MA; PXC030C50) with 11 diafiltration volumes of 10 mM NaOAc, pH 4.5, followed by 4 diafiltration volumes of 10 mM NaOAc, pH 4.5 with 9% (v/v) sucrose. The purified ADC was then sterile filtered (0.2 mm).

ADC prepared on a small scale as described in Example 24 was purified on an AKTA ^™ Pure Chromatography System (Cytiva Life Sciences, Marlborough, MA) using a 53 mL HiPrep 26/10 Desalting column (Cytiva Life Sciences, Marlborough, MA) and a mobile phase consisting of 10 mM NaOAc (pH 4.5) and 150 mM NaCl and a flow rate of 10 mL/min.

After purification, the concentration of the ADC was determined by BCA assay with reference to a standard curve generated using the humanized variant v36675. Alternatively, the concentration was estimated by measuring the absorption at 280 nm using an extinction coefficient taken from the literature (European Patent No. 3342785 for MC-GGFG-AM-DXd1) or experimentally determined (for the remaining drug-linkers). The ADC was also characterized by hydrophobic interaction chromatography (HIC) and size exclusion chromatography (SEC) as described below.

25.1 Hydrophobic Interaction Chromatography

Antibodies and ADCs were analyzed by HIC to estimate drug-antibody ratio (DAR). Chromatography was performed on a Butyl-NPR column (2.5 μm, 4.6×35 mm; TOSOH Bioscience GmbH, Griesheim, Germany) with a gradient from 95/5% MPA/MPB to 5/95% MPA/MPB in 12 min at a flow rate of 0.5 mL/min (MPA=1.5 M (NH ₄ ) ₂ SO ₄ , 25 mM Na x PO ₄ , pH 7, and MPB=75% 25 mM Na _x PO ₄ , pH 7, 25% isopropanol). Detection was by absorbance at 280 nm.

25.22 Size Exclusion Chromatography

The aggregation degree of antibodies and ADC (about 15 g to 150 μg, 5 μL injection volume) was evaluated by SEC on an Agilent Infinity II 1260 HPLC (Agilent Technologies, Santa Clara, CA) using an AdvanceBio SEC column (300 angstroms, 2.7 μm, 7.8×150 mm) (Agilent, Santa Clara, California) and a mobile phase consisting of 150 mM phosphate (pH 6.95) and a flow rate of 1 mL/min. Detection was performed by absorbance at 280 nm.

result

The individual contributions of DAR0, DAR2, DAR4, DAR6 and DAR8 species to the average DAR of the purified ADC were assessed by integration of the HPLC-HIC chromatogram. The average drug-antibody ratio (DAR) for each ADC was determined by the weighted average of each DAR species. When rounded to the nearest integer, the average DAR for each ADC was the same as the target DAR shown in Table 25.1.

Aggregation and monomer content were assessed by integration of HPLC-SEC chromatograms. The monomer peak for each ADC was identified as the peak with the same retention time as the non-conjugated antibody from which each ADC was produced. All peaks with earlier retention times relative to the monomer species were determined to be aggregate species. The percentage of monomer species determined for each ADC is shown in Table 25.1. All ADC preparations showed >95% monomer species.

Table 25.1

Example 26: In vitro cytotoxicity of antibody-drug conjugates - 2D monolayer

As described below, the cell growth inhibition (cytotoxicity) ability of humanized variant v30384 conjugated to various drug-linkers was evaluated in a panel of FRα-expressing cell lines. The cell lines used were KB-HeLa (endocervical carcinoma), JEG-3 (choriocarcinoma), T-47D (breast cancer), and MDA-MB-468 (breast cancer; FRα-negative). An ADC containing the antibody palivizumab (v21995) was used as a non-targeting control.

Briefly, cells were seeded in 384-well plates and treated with test article titrations generated in cell growth medium. Cells were incubated for 4 days under standard culture conditions. Reagent (Promega Corporation, Madison, WI) was added to all wells, and the luminescence corresponding to the ATP present in each well was measured using a Synergy ^™ H1 microplate reader (BioTek Instruments, Winooski, VT). Based on blank wells (no test article added), % cytotoxicity values were calculated using GraphPad Prism 9 software (GraphPad Software, San Diego, CA) and plotted against test article concentration.

result

The results are shown in Table 26.1. All v30384 ADCs showed significant cytotoxicity in FRα-expressing cell lines KB-HeLa, JEG-3, and T-47D, producing single-digit nM or lower EC50 values after 4 days of treatment. In the FRα-negative cell line MDA-MB-468, the ADCs did not show target-dependent cytotoxicity. v30384 and control (palivizumab) ADCs showed equivalent potency in this cell line.

Table 26.1: In vitro cytotoxicity - 2D monolayer

*Incomplete curve

Example 27: In vitro cytotoxicity of antibody-drug conjugates - 3D spheroids

As described below, the 3D cytotoxicity capacity of humanized variants v30384 conjugated to various drug-linkers as shown in Table 27.1 was evaluated in a panel of FRα-expressing cell line spheroids. Spheroids provide a three-dimensional organization of cells with different cell population stratification and the formation of different gradients from the outer to the inner regions. Cell signaling is more complex in spheroids than in two-dimensional cell cultures. Due to these features, spheroids have the potential to reproduce drug resistance and metabolic adaptation.

The cell lines used were IGROV-1 (ovarian adenocarcinoma), T-47D (breast cancer), OVCAR-3 (ovarian adenocarcinoma), HEC-1-A (uterine adenocarcinoma) and EBC-1 (lung cancer; FRα-negative). An ADC comprising the antibody palivizumab (v21995) was used as a non-targeting control.

Briefly, cells were plated in Ultra-Low Attachment 384-well plates, centrifuged and incubated under standard culture conditions to allow spheroid formation and growth. Monocultured cell line spheroids were then treated with test article titrations generated in cell growth medium. Spheroids were incubated under standard culture conditions for 6 days. 3D reagent (Promega Corporation, Madison, WI) was incorporated into all wells. The plate was incubated in the dark for 1 hour at room temperature and luminescence was quantified using a BioTek Cytation 5 cell imaging multi-mode reader (Agilent Technologies, Inc., Santa Clara, CA). Based on blank wells (no test article added), GraphPad Prism 9 software (GraphPad Software, San Diego, CA) was used to calculate the cytotoxicity percentage value and plot a curve for the test article concentration.

result

The results are shown in Table 27.1. All v30384 ADCs showed significant cytotoxicity in FRα-expressing monoculture spheroids (IGROV-1, T-47D, OVCAR-3, and HEC-1-A), producing single-digit nM EC50 values in spheroids after 6 days of treatment. In FRα-negative cell line spheroids, EBC-1, v30384 ADC did not show target-dependent cytotoxicity. v30384 and control (palivizumab) ADCs showed equivalent potency in this cell line spheroid.

Table 27.1: In vitro cytotoxicity - 3D spheroids

Example 28: In vivo efficacy study #1

The in vivo anti-tumor activity of humanized variants v30384 conjugated to various drug-linkers was evaluated in a number of xenograft models as described below. In some models, an ADC comprising the antibody palivizumab (v21995) was used as a non-targeting control.

The ADCs, xenograft models, doses, and study durations used in each xenograft study are summarized in Table 28.1. All ADCs were DAR 8. For each xenograft study, tumor volume and body weight of the animals were measured twice weekly.

Table 28.1: Study parameters

For OV90 model studies #1 and #2, tumor cell suspension (0.1 ml 50% 1x10 ^{7 cells in 5% paraformaldehyde (1x10 7} cells) were implanted subcutaneously into female CB.17 SCID mice. When the mean tumor volume reached 100 to 150 mm ³ , the animals were randomized into groups (n=6 per group for Study #1 and n=8 per group for Study #2) and treated with a single IV dose of the test article on Study Day 1 as shown in Table 28.1. Serum was collected at multiple time points for PK analysis.

For NCI-H2110 CDX model studies, tumor cell suspension (0.1 ml 50% 1x10 ⁷ cells in 5% paraformaldehyde (1x10 7 cells) were implanted subcutaneously into CB.17 SCID mice. When the mean tumor volume reached approximately 140 mm ³ , the animals were randomized into groups (n=6 per group) and treated with a single IV dose of the test article on study day 0, as shown in Table 28.1.

result

The results are shown in Figure 19. In OV90 model study #1 (see Figure 19A), when dosed at 3 mg/kg, all ADCs resulted in a statistically significant reduction in tumor growth rate compared to control (p<0.02). Compared to v30384-MC-GGFG-AM-DXd, ADCs v30384-MT-GGFG-AM-Compound 139, v30384-MT-GGFG-AM-Compound 141, and v30384-MT-GGFG-Compound 141 all resulted in a stronger inhibition of tumor growth rate (p<0.01). Similarly, in OV90 model study #2 (see FIG. 19B ), v30384-MT-GGFG-Compound 140, v30384-MT-GGFG-AM-Compound 141, and v30384-MC-GGFG-AM-Compound 141 all resulted in tumor regression when dosed at 3 mg/kg, whereas v30384-MC-GGFG-AM-DXd had a marginal effect on tumor growth compared to control. The non-targeted v21995 ADC did not substantially affect tumor growth.

In the NCI-H2110 CDX model study (see Figure 19C), when dosed at 6 mg/kg, v30384-MT-GGFG-Compound 140, v30384-MC-GGFG-Compound 140, and v30384-MT-GGFG-Compound 148 all resulted in tumor growth arrest for approximately 2 weeks after dosing, representing a statistically significant inhibition of tumor growth rate compared to each of the control, v30384-MC-GGFG-AM-DXd, and the non-targeted v21995 ADC (p<0.01). v30384-GGFG-AM-DXd, v30384-MC-GGFG-AM-Compound 141, v30384-MT-GGFG-AM-Compound 141, and v30384-MT-GGFG-AM-Compound 139 did not result in significant inhibition of tumor growth rate in this model.

Example 29: Pharmacokinetics of ADC in an in vivo efficacy model

Serum was collected from the xenograft study described in Example 28 and analyzed for ADC pharmacokinetics (PK) as described below. An ADC comprising the antibody palivizumab (v21995) was included as a non-targeting control.

The test article concentration in mouse serum was measured by sandwich ELISA using anti-human IgG1 Fc capture antibody (Jackson Immuno Research Labs, West Grove, PA; Catalog No. 709-005-098) and HRP-conjugated anti-IgG1 Fab detection antibody (Jackson Immuno Research Labs; Catalog No. 109-035-097) to detect total IgG levels. The absorbance at 450 nM was measured using Synergy ^TM H1 Hybrid multi-mode microplate reader (BioTek Instruments, Winooski, VT). Pharmacokinetic parameters were calculated by non-compartmental analysis using Phoenix WinNonlin ^TM software (Certara, Princeton, NJ).

In summary, the OV90 studies in immunocompromised tumor-bearing mice demonstrated that v30384 ADCs utilizing payloads Compound 141, Compound 139, and Compound 140 had favorable PK properties, as indicated by longer or equivalent elimination half-lives compared to v30384-MC-GGFG-AM-DXd1 (control). Shorter elimination half-lives were observed for all v30384 ADCs (including the DXd1 control) in the NCI-H2110 model compared to the OV90 model. The elimination half-lives of the non-targeted control v21995 ADC were equivalent in both the OV90 and NCI-H2110 models (Table 29.1).

Table 29.1: Elimination half-lives of ADCs

Example 30: Mouse tolerance study

The tolerability of ADCs comprising humanized variants of v30384 conjugated to various drug-linkers as shown in Table 30.1 was evaluated in mice at single doses of 60 and 200 mg/kg as described below.

The test article was administered to mice (Balb/c, female, 6-8 weeks old, approximately 20 g) via 20 ml/kg intraperitoneal injection at 60 mg/kg and 200 mg/kg. From each dose group, 3 mice were planned to be observed for 3 weeks after administration. Another 3 mice were planned to be observed for 1 week after administration, followed by termination and examination of formalin-fixed, paraffin-embedded organs. If the body weight decreased by ≥20% from the pre-dose level, the mice were euthanized. It was planned to collect serum from all mice 24 hours and 7 days after administration for pharmacokinetic analysis.

Table 30.1: ADCs, doses, and unplanned deaths in mouse tolerance studies

result

ADC 30384-MC-GGFG-AM-DXd1, v30384-MT-GGFG-AM-Compound 139, v30384-MT-GGFG-AM-Compound 141, v30384-MT-GGFG-Compound 141, and v30384-MC-GGFG-Compound 141 were well tolerated at 60 mg/kg and 200 mg/kg doses, with no significant weight loss observed over 21 days, similar to mice administered vehicle controls. ADC v30384-MT-GGFG-Compound 140, v30384-MT-GGFG-Compound 148, and v30384-MC-GGFG-Compound 140 caused rapid weight loss, death, or sacrifice due to moribundity between 3-6 days after dosing (see Table 30.1).

No treatment-related macroscopic changes were observed in mice treated with ADC 30384-MC-GGFG-AM-DXd1, v30384-MT-GGFG-AM-Compound 139, v30384-MT-GGFG-AM-Compound 141, v30384-MT-GGFG-Compound 141, and v30384-MC-GGFG-Compound 141 at 60 mg/kg or 200 mg/kg. There were macroscopic changes thought to be related to the ADC in pre-mortem animals treated with 60 mg/kg and/or 200 mg/kg of v30384-MT-GGFG-Compound 140, v30384-MT-GGFG-Compound 148, and v30384-MC-GGFG-Compound 140. Different macroscopic findings included watery/reduced/discolored intestinal contents, reduced thymus and spleen size. Watery intestinal contents are microscopically associated with degeneration/necrosis of the crypt/glandular epithelium and associated atrophy of the villi or mucosa of the small and large intestine. Reduced thymus and spleen size are microscopically associated with reduced cellularity in these organs.

There were no treatment-related microscopic findings in mice administered ADCs v30384-MC-GGFG-AM-DXd1, v30384-MT-GGFG-AM-Compound 139, and v30384-MC-GGFG-Compound 141. Microscopic changes believed to be associated with administration of ADCs v30384-MT-GGFG-Compound 140, v30384-MT-GGFG-Compound 148, and v30384-MC-GGFG-Compound 140 at a dose of 360 mg/kg were present in the intestine, bone marrow, thymus, spleen, and mesenteric lymph nodes.

Example 31: Pharmacokinetic study in Tg32 mice

The pharmacokinetics of humanized antibody v36675 and four ADCs comprising v36675 were evaluated in hFcRn Tg32 mice as described below. The ADCs were: v36675-MC-GGFG-AM-Dxd (control); v36675-MC-GGFG-AM-Compound 141; v36675-MC-GGFG-Compound 141, and v36675-MC-GGFG-AM-Compound 139. All ADCs were DAR 8.

Each of the antibody v36675 and the four ADCs was administered to hFcRn Tg32 mice (The Jackson Laboratory, Bar Harbor, ME; stock number 014565) at 5 mg/kg by intravenous injection. For each test article, blood was collected from n=4 animals by retro-orbital bleeding at 1, 3, and 6 hours and 1, 3, 7, 10, 14, and 21 days after dosing. The blood was processed into serum and stored frozen at -80°C in 96-well storage plates prior to pharmacokinetic analysis.

The test article concentration in mouse serum was measured by sandwich ELISA using anti-human IgG1 Fc capture antibody (Jackson Immuno Research Labs, West Grove, PA; Catalog No. 709-005-098) and HRP-conjugated anti-IgG1 Fab detection antibody (Jackson Immuno Research Labs; Catalog No. 109-035-097) to detect total IgG levels. The absorbance at 450 nM was measured using Synergy ^TM H1 Hybrid multi-mode microplate reader (BioTek Instruments, Winooski, VT). Pharmacokinetic parameters were calculated by non-compartmental analysis using Phoenix WinNonlin ^TM software (Certara, Princeton, NJ).

result

The results are shown in Figure 20. Analysis in hFcRn Tg32 mice demonstrated that the total IgG PK of the four ADCs was equivalent to that of naked antibodies, with typical antibody-like long-term exposure. By non-compartmental analysis, the elimination half-life of the v36675 antibody was determined to be 5.2 days; the elimination half-life of the ADCs was determined to be 4.36 days for v36675-MC-GGFG-AM-Dxd (control), 4.61 days for v36675-MC-GGFG-AM-Compound 141, 6.20 days for v36675-MC-GGFG-Compound 141, and 4.09 days for v36675-MC-GGFG-AM-Compound 139.

Example 32: In vivo stability

The in vivo stability of four ADCs comprising the humanized variant v36675 in Tg32 mice was evaluated using immunoprecipitation/mass spectrometry as described below. The ADCs evaluated were: v36675-MC-GGFG-AM-Dxd (control); v36675-MC-GGFG-AM-Compound 141; v36675-MC-GGFG-Compound 141, and v36675-MC-GGFG-AM-Compound 139. All ADCs were DAR 8. Serum samples taken from Tg32 mice at different time points in the circulation (1 hour to 10 days) as described in Example 31 were used for immunoprecipitation/mass spectrometry. For all groups, mouse serum samples from each time point (1 hour to 10 days after dosing) were pooled between animals (n=4) and tested.

In brief, biotinylated anti-human IgG F(ab')2 antibodies were coupled to streptavidin-coated magnetic beads (15ug antibody per sample) at room temperature for 30 minutes. After coupling, the beads were incubated with the sample at room temperature for 1.5 hours to allow immune capture. After incubation and washing with DynaMag ^™ -2 magnets (ThermoFisher Scientific Corporation, Waltham, MA), the immune-captured samples were reduced at room temperature for 1 hour using dithiothreitol (DTT) (pH 7.4) in PBS. After reduction, the samples were eluted by incubating with pH 3.0 buffer (distilled water containing 20% acetonitrile and 1% formic acid) for 1 hour at room temperature. The purified ADC samples were then analyzed by mass spectrometry to quantify DAR or drug loading, or kept frozen at -80°C until further analysis.

The drug loading and % maleimide ring opening of the purified ADC samples were evaluated using an Agilent 1290 Infinity II HPLC system coupled to an Agilent 6545 quadrupole time-of-flight mass spectrometer (Agilent Technologies, Santa Clara, CA). The extent of drug loading and % maleimide ring opening at each time point was plotted using GraphPad Prism software (GraphPad Software, San Diego, CA).

result

The results are shown in Figure 21 and Table 32.1. In summary, all four ADCs showed highly similar results for DAR loss and maleimide ring opening over time. All ADCs showed 48-55% DAR loss and 51-54% maleimide ring opening within 10 days. No significant linker drug decomposition was observed in any of the tested ADCs.

Table 32.1: DAR loss and maleimide ring opening % (change over 10 days)

ADC DAR Loss % Maleimide ring opening % v36675-MC-GGFG-AM-DXd1 52 52 v36675-MC-GGFG-AM-Compound 141 47 53 v36675-MC-GGFG-Compound 141 45 51 v36675-MC-GGFG-AM-Compound 139 50 54

Example 33: Rat toxicity study

A 2-dose (once every 3 weeks) intravenous exploratory toxicity and toxicokinetics study of the ADC shown in Table 33.1 was conducted in Sprague Dawley rats as described below. The purpose of this study is to determine the potential toxicity of the ADC when administered twice to female Sprague Dawley (SD) rats, as well as to assess the reversibility, persistence or delayed occurrence of toxic effects after a 4-week recovery period, and to determine the toxicokinetics (TK) of the ADC after the first dose. Sprague Dawley rats were selected as non-cross-reactive rodent species to evaluate the target-independent toxic effects of these ADCs.

In this study, as shown in Table 33.1, at the 1st and 22nd day, in 10 minutes, the vehicle or test article ADC was administered via slow intravenous injection at the dosage level of 30, 60 and 200 mg/kg (6 animals/dose level). The morbidity/mortality rate, clinical signs, body weight, food consumption, ophthalmological examination, clinical pathology (hematology, serum chemistry, coagulation and urinalysis), change of organ weight and macroscopic and microscopic changes of organs/tissues of all animals were evaluated. Blood samples were collected after the first dose for toxicokinetic analysis of total antibodies. The test article concentration in all dosage formulations was analyzed using bicinchoninic acid (BCA) determination. A planned complete autopsy was performed at the end of the administration phase (study the 29th day) and the recovery phase (study the 51st day). The research design is presented in Table 33.1.

Table 33.1: Study Design

Conc.＝Concentration

result

v36675-MC-GGFG-AM-DXd1 (Control) : After the first dose of v36675-MC-GGFG-AM-Dxd, systemic exposure increased approximately in proportion to the dose as the dose increased from 30 to 200 mg/kg/dose.

There were no deaths; no test article-related changes in clinical signs, ophthalmic examinations, or coagulation/serum chemistry parameters; macroscopic examinations were performed at terminal and/or recovery sacrifice.

At the time of terminal sacrifice, test article-related hematological changes included a decrease in lymphocyte count (≥60 mg/kg/dose) and a decrease in red blood cell count (200 mg/kg/dose). These changes were reversed during the recovery phase. At the end of the dosing and recovery phases, the presence of urinary protein was noted in one animal at 200 mg/kg/dose. At the time of terminal sacrifice, test article-related microscopic changes were present in lymphoid organs (≥60 mg/kg/dose) and bone marrow (200 mg/kg/dose). These changes were reversed during the recovery phase.

v36675-MC-GGFG-AM-Compound 141 : After the first dose of v36675-MC-GGFG-AM-Compound 141, systemic exposure increased approximately in proportion to the dose as the dose increased from 30 to 200 mg/kg/dose.

Test article-related death was noted in one animal on Day 7 at 200 mg/kg/dose. The cause of the moribund state was attributed to mucosal atrophy and/or crypt dilation in the gastrointestinal (GI) tract. Clinical signs of the moribund animal included dishevelled, decreased activity, emaciation (associated with 31% weight loss between Days -1 and 7), hunched posture, and yellow stained fur. Prior to death, changes in hematology and clinical chemistry parameters were noted. Test article-related microscopic findings were observed in the gastrointestinal tract, lymphoid organs, pancreas, salivary glands, and ovaries.

There were no test article-related changes in ophthalmological examinations, coagulation/serum chemistry parameters at terminal and/or recovery sacrifice.

There were only clinical signs related to the test article at 200 mg/kg/dose (reduced activity, soiled fur, hunched posture, thinness and dishevelled), significant weight loss (12% or 14% weight loss in the first week after each dose), and reduced food consumption (up to 44.1%). At the terminal sacrifice, test article-related hematological changes included increased neutrophil/monocyte counts and decreased lymphocyte counts at 200 mg/kg/dose. These changes were reversed during the recovery phase. At the end of the dosing and/or recovery phase, the presence of urinary protein was noted in one animal at ≥60 mg/kg/dose. At the final sacrifice, test article-related changes included reduced thymus size (200 mg/kg/dose), reduced thymus weight (≥60 mg/kg/dose), and microscopic changes in the bone marrow (≥60 mg/kg/dose), gastrointestinal tract (≥30 mg/kg/dose), and lymphoid organs (≥30 mg/kg/dose). All of these changes were reversed during the recovery phase.

v36675-MC-GGFG-Compound 140 : Test article-related deaths were noted in all animals on Day 5 or 6 at ≥60 mg/kg/dose. The cause of the moribund state was attributed to mucosal atrophy and/or crypt dilation in the gastrointestinal tract. Clinical signs of moribund animals included soiled fur, hunched posture, emaciation (associated with up to 23% weight loss after one dose), dishevelled hair, red/brown material around the nose, mucoid and soft stools, pale ear skin, and yellow teeth. In moribund animals, test article-related microscopic findings were observed in the bone marrow, gastrointestinal tract, lymphoid organs, pancreas, salivary glands, ovaries, and adrenal glands.

There were no test article-related changes in ophthalmologic examinations, coagulation/serum chemistry/urinalysis parameters, and macroscopic examinations at terminal and/or recovery period sacrifice in surviving 30 mg/kg/dose animals.

There were test article-related clinical signs (thinness and unkemptness), weight loss (9% or 8% weight loss in the first week after each dose), and decreased food consumption (up to 31.7%↓) at 30 mg/kg/dose. At terminal sacrifice, test article-related hematologic changes included decreases in red blood cell counts, hemoglobin, hematocrit, and reticulocyte counts. These changes were reversed during the recovery phase. At terminal sacrifice, test article-related changes at 30 mg/kg/dose included decreased thymus/ovarian weights; microscopic changes in bone marrow, gastrointestinal tract, and lymphoid organs. All of these changes were reversed during the recovery phase.

v36675-MC-GGFG-Compound 141 : After the first dose of v36675-MC-GGFG-Compound 141, systemic exposure increased in a dose-proportional manner as the dose increased from 30 mg/kg/dose to 200 mg/kg/dose.

Test article-related mortality was noted in one animal on Day 6. The cause of the moribund state was attributed to mucosal atrophy and/or crypt dilation in the gastrointestinal tract. Clinical signs in animals in the days prior to death included emaciation, soiled fur, hunched posture, and abnormal gait. In animals found dead, spleen/thymus size was reduced and test article-related microscopic findings were present in the bone marrow, gastrointestinal tract, lymphoid organs, pancreas, salivary glands, and adrenal glands.

There were no test article-related changes in ophthalmological examinations, coagulation/serum chemistry parameters at terminal sacrifice and/or recovery sacrifice.

There were test article-related clinical signs (hunched posture, thinness and dishevelledness), weight loss (3.3% or 2.3% weight loss in the first week after each dose), and decreased food consumption (up to 36.3% reduction) at 200 mg/kg/dose. At terminal sacrifice, test article-related hematological changes included decreased lymphocyte counts (60 and 200 mg/kg/dose) and increased neutrophil/reticulocyte/platelet counts (200 mg/kg/dose). These changes were reversed during the recovery phase. Urinary protein was noted in one animal at ≥60 mg/kg/dose at the end of the dosing and/or recovery phase. At terminal sacrifice, test article-related changes included decreased thymus size (200 mg/kg/dose); decreased thymus weight (≥60 mg/kg/dose); microscopic changes in bone marrow (200 mg/kg/dose), lymphoid organs (≥60 mg/kg/dose), and pancreas/salivary glands (≥60 mg/kg/dose). All of these changes were reversed during the recovery phase.

v36675-MC-GGFG-AM-Compound 139 : After the first dose of v36675-MC-GGFG-AM-Compound 139, systemic exposure increased approximately in proportion to the dose as the dose increased from 30 to 200 mg/kg/dose.

There were no deaths and no test article-related changes in clinical signs, ophthalmological examinations, clinical pathological parameters, and macroscopic examinations at the terminal and/or recovery period sacrifices.

At 200 mg/kg/dose, there was a test article-related weight loss (1.35% or 6.15%) and a reduction in food consumption (up to 11.8%) in the first week after each dose. At terminal sacrifice, there were test article-related microscopic changes in lymphoid organs at ≥30 mg/kg/dose and these changes were reversed during the recovery phase.

in conclusion

This study demonstrated that two intravenous injections of v36675-MC-GGFG-AM-Dxd1 (control) and v36675-MC-GGFG-AM-Compound 139 at 30, 60 and 200 mg/kg/dose on days 1 and 22 to female SD rats, followed by 4 weeks of recovery, were well tolerated, resulting in a maximum tolerated dose (MTD) of 200 mg/kg.

Female SD rats were injected intravenously twice with v36675-MC-GGFG-AM-Compound 141 and v36675-MC-GGFG-Compound 141 at 30, 60 and 200 mg/kg/dose on days 1 and 22, followed by 4 weeks of recovery, causing death at 200 mg/kg/dose, resulting in an MTD of 60 mg/kg.

Female SD rats were injected twice intravenously with v36675-MC-GGFG-Compound 140 at 30, 60 and 200 mg/kg/dose on days 1 and 22, followed by 4 weeks of recovery, causing mortality at 60 and 200 mg/kg/dose, yielding an MTD of 30 mg/kg.

Example 34: In vivo efficacy study #2

The in vivo anti-tumor activity of humanized variants v36675 conjugated to various drug-linkers with DAR 4 or DAR 8 was evaluated in a number of cell line-derived xenograft (CDX) and patient-derived xenograft (PDX) models as described below. In some models, ADCs including v32603 (mituximab-DM4), v32385 (MORAb-202) and v36675 conjugated to DXd were evaluated for comparison. The OVCAR-3 model included unconjugated v36675 mAb for comparison.

The ADCs, DARs, xenograft models, and doses used in each xenograft study are summarized in Table 34.1. For each xenograft study, tumor volume and body weight of the animals were measured twice weekly.

Table 34.1: Study parameters

For OV90 CDX model study #3, tumor cell suspension ( ^1x107 cells in 0.1 ml 50% For OVCAR3CDX model studies, tumor fragments (approximately 1 mm ³ ) were implanted subcutaneously into female CB.17 SCID mice. When the mean tumor volume reached 100-150 mm ³ , animals were randomized into groups (n=6 per group in each study) and treated on study day 1 with a single IV dose of the test article as shown in Table 34.1.

For the ovarian cancer PDX models CTG-2025 and CTG-0958, tumor fragments were implanted subcutaneously into female athymic nude Foxn1nu mice. When the mean tumor volume reached approximately 240 ^mm3 (for CTG-2025) or approximately 220 ^mm3 (for CTG-0958), animals were randomized (n=3 per group in each study) and treated on study day 1 with a single IV dose of test article as shown in Table 34.1.

result

The results are summarized in Figure 22. In the OV90 CDX model, v36675-MC-GGFG-AM-Compound 139DAR8 caused moderate inhibition of tumor growth when dosed at 3 mg/kg, similar to v36675-MC-GGFG-AM-DXd (Figure 22A). When dosed at 6 mg/kg, v36675-MC-GG FG-AM-Compound 139 caused moderate and strong inhibition of tumor growth as DAR4 and DAR8 ADCs, respectively (Figure 22B). At 3 mg/kg, v36675-MC-GGFG-AM-Compound 141 caused moderate and strong inhibition of tumor growth as DAR4 and DAR8 ADCs, respectively (Figure 22A). When dosed at 6 mg/kg, v36675-MC-GGFG-AM-Compound 141 caused strong tumor growth inhibition as a DAR4 ADC and caused excellent activity as a DAR8 ADC (Figure 22B).

In the OVCAR3 CDX model (see FIG. 22C ), both v36675-MC-GGFG-AM-Compound 139 and v36675-MC-GGFG-AM-Compound 141 showed a trend of superior tumor growth inhibition as DAR8 ADCs than as DAR4 ADCs when dosed at 0.75 mg/kg. At both DARs, v36675-MC-GGFG-AM-Compound 141 showed a trend of tumor growth inhibition superior to v36675-MC-GGF G-AM-Compound 139. v36675-MC-GGFG-AM-Compound 141 DAR8 caused equivalent tumor growth inhibition to the v36675-MC-GGFG-AM-DXd control ADC. When dosed at 0.75 mg/kg, the control ADCs Mituximab-DM4 and MORAb-202 were both inactive.

In the CTG-2025 PDX model (see FIG. 22D ), both v36675-MC-GGFG-AM-Compound 139DAR8 and Sumetuximab-DM4 caused robust inhibition of tumor growth when dosed at 6 mg/kg.

In the CTG-0958 PDX model (see FIG. 22E ), v36675-MC-GGFG-AM-Compound 139DAR8 caused strong inhibition of tumor growth when dosed at 6 mg/kg, whereas Sumetuximab-DM4 was inactive.

Example 35: Cynomolgus Monkey Toxicity Study

The purpose of this study is to determine the maximum tolerated dose (MTD) and potential toxicity of 4 ADCs containing humanized variants v36675 conjugated to various drug-linkers when administered to male cynomolgus monkeys twice. Cynomolgus monkeys were selected as cross-reactive species to evaluate the toxic effects of these ADCs. The ADCs tested were v36675-MC-GGFG-AM-Compound 139 (DAR8), v36675-MC-GGFG-AM-Compound 141 (DAR8), v36675-MC-GGFG-AM-Compound 139 (DAR4) and v36675-MC-GGFG-AM-Compound 141 (DAR4), as well as ADCs containing variant v36675 conjugated to Dxd (v36675-MC-GGFG-AM-DXd, DAR8).

Materials and methods

In this study, vehicle and test article ADC were administered to male cynomolgus monkeys (n=2/group) via slow intravenous injection over 3 minutes on the 1st and 22nd days. The study design, dose level and dose volume details are summarized in Table 35.1. All animals were evaluated for moribund/mortality, clinical signs, body weight, food consumption, clinical pathology (hematology, serum chemistry and coagulation), changes in organ weights, and macroscopic and microscopic changes in organs/tissues. Blood samples were collected after the first dose for toxicokinetic analysis (see Example 36). The test article concentrations in all dose formulations were analyzed using UV-Vis or bicinchoninic acid (BCA) assays. Planned autopsies were performed on the 29th day of the study.

Table 35.1: Study Design

result

v36675-MC-GGFG-AM-Compound 139, DAR8 : Test article-related deaths were noted in one animal at 80 mg/kg/dose (Day 7) and in 2 animals at 120 mg/kg/dose (Days 8 and 10). Animals treated at 30 mg/kg/dose and animals treated at 80 mg/kg/dose all survived to terminal necropsy.

Pre-terminal animals: Compared with their own baseline, significant increases in the values of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), total bilirubin (TBIL), blood urea (BU), and glucose (GLU) were observed; significant decreases in reticulocyte (ABRETIC) and lymphocyte (ABLYMP) counts; and significant decreases in prothrombin time (PT).

Terminal animals: Compared to the mean values of the control group and their own baseline values, at 30 and 80 mg/kg/dose, a significant increase in ALT, AST, and BU values was observed; a significant decrease in ABRETIC counts and relatively low PT values. At 80 mg/kg/dose, a significant decrease in ABLYMP counts was observed. Microscopic findings were present in the thymus, stomach, kidney, meninges, and testes.

v36675-MC-GGFG-AM-Compound 141, DAR8 : Test article-related deaths were noted in one animal at 80 mg/kg/dose (Day 11) and in 2 animals at 120 mg/kg/dose (Days 7 and 8). Animals treated at 30 mg/kg/dose and animals treated at 80 mg/kg/dose all survived to terminal necropsy.

Pre-terminal animals: Compared with their own baseline, significant increases in the values of ALT, AST, creatine kinase (CK), lactate dehydrogenase (LDH), TBIL, BU, creatinine (CRE), and GLU were observed; significant decreases in ABRETIC and ABLYMP counts; and significant increases in fibrinogen (FIB) levels.

Terminal animals: Compared to the mean values of the control group and their own baseline values, at 30 and 80 mg/kg/dose, a substantial increase in the values of ALT, AST, CK, LDH and TBIL, as well as a substantial decrease in ABRETIC and ABLYMP counts were observed. Animals administered at 80 mg/kg/day showed low activated partial thromboplastin time (APTT), low PT and relatively high FIB levels. Microscopic findings were present in the thymus, liver, spleen, pancreas, gastrointestinal tract, adrenal glands, kidneys, meninges and mesenteric lymph nodes.

v36675-MC-GGFG-AM-Compound 139, DAR4 : There were no deaths up to 120 mg/kg/dose, and all animals survived until terminal necropsy.

Terminal animals: At 60 and 120 mg/kg/dose, there were no significant changes in serum chemistry parameters; significant decreases in ABRETIC and neutrophil (ABNEUT) counts were observed. Microscopic findings were present in the thymus, spleen, stomach, adrenal glands, testes, prostate, meninges, and mesenteric lymph nodes.

v36675-MC-GGFG-AM-Compound 141, DAR4 : Test article-related deaths were observed in two animals at 120 mg/kg/dose (Day 7 and Day 8). All animals treated at 60 mg/kg/dose survived to terminal necropsy.

Pre-terminal animals: Compared with their own baseline, a significant increase in the values of AST, CK, LDH, TBIL, BU, CRE, GLU, phosphorus (P), and BUN/C was observed; a significant decrease in the albumin/globulin ratio (A/G); and a significant decrease in the ABRETIC count were observed.

Terminal animals: At 60 mg/kg/dose, significant and dose-dependent increases in AST, CK, and LDH were observed compared to the mean values of the control group and their own baseline values; ABRETIC and white blood cell (WBC) counts were significantly reduced. Microscopic findings were present in the thymus, kidney, prostate, meninges, and mesenteric lymph nodes.

v36675-MC-GGFG-AM-DXd, DAR8 : Test article-related death was observed in one animal (Day 8) at 80 mg/kg/dose. Animals treated at 30 mg/kg/dose and animals treated at 80 mg/kg/dose all survived to terminal necropsy.

Pre-terminal animals: Compared with baseline, a significant increase in ALT, CK, LDH, TBIL, CRE, BUN/C, and BU values was observed; a significant decrease in ABRETIC counts; and a significant shortening of PT. Target organs in pre-terminal animals included the thymus, esophagus, liver, spleen, gastrointestinal tract, pancreas, adrenal glands, kidneys, mesenteric and mandibular lymph nodes, bone marrow, skin, and femur.

Terminal animals: At 80 mg/kg/dose, substantial increases in ALT, CK, TBIL, and LDH were observed compared to the mean values of the control group and their own baseline values. Substantial decreases in ABRETIC, WBC, and ABLYMP counts were observed at 30 and 80 mg/kg/dose. Relatively high APTT was observed at 30 mg/kg/dose. Microscopic findings were present in the thymus, spleen, large intestine, kidney, testis, meninges, mandibular and mesenteric lymph nodes, sternal bone marrow, and femur.

in conclusion

The study demonstrated that in male cynomolgus monkeys, two intravenous administrations (day 1 and day 22) of ADC v36675-MC-GGFG-AM-Compound 139 (DAR4) were well tolerated up to 120 mg/kg/dose; ADC v36675-MC-GGFG-AM-Compound 141 (DAR4) was well tolerated at 60 mg/kg/dose; and ADCs v36675-MC-GGFG-AM-Compound 139 (DAR8), v36675-MC-GGFG-AM-Compound 141 (DAR8), and v36675-MC-GGFG-AM-DXd (DAR8) were well tolerated at 30 mg/kg/dose.

ADCs v36675-MC-GGFG-AM-Compound 139 (DAR8) and v36675-MC-GGFG-AM-Compound 141 (DAR8) at 80 and 120 mg/kg/dose, ADC v36675-MC-GGFG-AM-Compound 141 (DAR4) at 120 mg/kg/dose, and ADC v36675-MC-GGFG-AM-DXd (DAR8) at 80 mg/kg/dose caused moribundity/death in one or two monkeys. The cause of moribundity/death in these monkeys was related to test article administration.

Example 36: Toxicokinetic Analysis

ELISA was used to analyze the total antibody serum concentration of ADC in blood samples collected after the first dose in the toxicity study described in Example 35. ADC from the serum was captured on a 384-well plate coated with a mouse anti-human IgG Fc antibody (Abcam plc, Cambridge, UK; catalog number ab124055). Total anti-IgG antibodies were detected with a secondary goat anti-human IgG (H+L) horseradish peroxidase (HRP) antibody (Novus Biologicals, LLC, Centennial, CO; NB7489). After adding 3,3',5,5"-tetramethylbenzidine (TMB) substrate and quenching the reaction by adding 1N hydrochloric acid solution, the absorbance at 450 nm was read. The ADC was detected using Sample data were analyzed using Molecular Devices Pro 7.1 (Molecular Devices, San Jose, CA).

result

The results are shown in Figure 23A-D. All ADCs showed typical antibody-like exposure, with similar dose ratios at the administered dose levels. The pharmacokinetic characteristics between different ADCs were equivalent at each dose level.

Example 37: Bystander activity of antibody drug conjugates

The ability of humanized variants v30384 conjugated to various drug-linkers to exert bystander killing effects on cancer cells was evaluated according to the following method. Bystander killing most often occurs after target-specific uptake of the ADC into antigen-positive cells. The transport and degradation of the ADC leads to the release of the active catabolite free drug, which then crosses the cell membrane of nearby cells to trigger cell death.

The following ADCs were tested in JEG-3 cells (high FRα expression) and MDA-MB-468 cells (negative FRα expression): v30384-MT-GGFG-AM-Compound 139, v30384-MT-GGFG-Compound 141, v30384-MT-GGFG-AM-Compound 141, v30384-MT-GGFG-Compound 140, v30384-MT-GGFG-Compound 148 (all DAR8). Its drug linkers are the positive controls v30384-MC-GGFG-AM-DXd1 (DAR 8) and v30384-MCvcPABC-MMAE (DAR 4) known to have bystander activity, and the negative control palivizumab (anti-RSV) (v22277) conjugated to MC-GGFG-AM-DXd1 (DAR 8) and MCvcPABC-MMAE (DAR 4) is included.

FRα-positive JEG-3 and FRα-negative MDA-MB-468 cells were seeded as monocultures or cocultures at 20,000 cells and 10,000 cells, respectively, in 100 μL of assay medium (minimal essential medium supplemented with 10% fetal bovine serum (both from Thermo Fisher Scientific, Waltham, MA)) in 48-well plates. ADCs were diluted to 4 nM in assay medium and 100 μL were added to the plates containing cells (2 nM final ADC concentration). Cells were incubated with ADCs under standard culture conditions (37°C/5% CO ₂ ) for 4 days. After incubation, cells were dissociated, washed, and stained with a viability dye. (ThermoFisher Scientific, Waltham, MA) and conjugated to Alexa 647 was stained with the anti-FRα antibody Sumituximab (v17716) at 4°C for 20 minutes. After incubation, cells were washed in FACS buffer (phosphate-buffered saline (pH 7.4) supplemented with 2% FBS (ThermoFisher Scientific, Waltham, MA)), resuspended in 70 μL of FACS buffer per well, and 35 μL/well was analyzed on a BD LSRFortessa ^TM cell analyzer (BD Biosciences, Franklin Lake, NJ). The number of JEG-3 and MDA-MB-468 cells was determined by Alexa Fluor 647 positive (FRα positive) and Alexa The percentage of viability was calculated as the number of cells in the treated condition divided by the number of cells in the untreated condition.

Knot

The results are provided in Table 37.1 below and Figure 24. The bystander effect was calculated by comparing the viability of FRα-negative MDA-MB-468 cells (black bars) treated as a single culture with cells treated as a co-culture with FRα-positive JEG-3 cells (grey bars) (see Figure 24). A greater reduction in viability in the co-culture indicates a higher bystander effect compared to the single culture. The camptothecin analog ADCs targeting FRα all showed varying degrees of bystander ability. As expected, the positive controls v30384-MC-GGFG-AM-DXd1 and v30384-MCvcPABC-MMAE showed positive bystander activity. As expected, the negative controls palivizumab MC-GGFG-AM-DXd1 and palivizumab MCvcPABC-MMAE showed negative bystander activity.

Table 37.1: Bystander activity of anti-FRα ADCs against FRα-negative MDA-MB-468 cells

* relative to unhandled whitespace

Example 38: Penetration of anti-FRα antibodies in multicellular tumor spheroids

The ability of anti-FRα antibodies to penetrate spheroids of FRα-expressing cell lines was evaluated according to the following method. Spheroids provide a three-dimensional organization of cells with different cell population stratification and the formation of different gradients from the outer to the inner regions. Cell signaling is more complex in spheroids than in two-dimensional cell cultures. Due to these characteristics, spheroids have the potential to reproduce drug resistance and metabolic adaptation.

The spheroid penetration capacity of humanized variant v36675 was compared with that of Sumetuximab (v17716) and the non-FRα-targeted control Palivizumab (anti-RSV) (v22277). Variant v36675 is identical to variant v30384 but contains HomoFc instead of HetFc. The cell line used was high FRα-expressing JEG-3 (placental choriocarcinoma).

The antibodies were fluorescently labeled by coupling with Fab fragment AF488 conjugate targeting anti-human IgG Fc (Jackson ImmunoResearch Labs, West Grove, PA; catalog number 109-547-008) at a 1:1 molar ratio in PBS (pH 7.4) (ThermoFisher Scientific, Waltham, MA; catalog number 10010-023) at 4°C for 24 hours.

JEG-3 cells were detached from the culture vessel using TrypLE ^™ Express Enzyme (1X) (Thermo Fisher Scientific, Waltham, MA) and expressed using MX high-throughput automated cell counter (Nexcelom Bioscience LLC, Lawrence, MA) was used for counting. Cells were diluted in complete growth medium (minimal essential medium supplemented with 10% fetal bovine serum (both from Thermo Fisher Scientific, Waltham, MA)) and seeded at 3,000 cells/well into 96-well CellCarrier Spheroid ultra-low attachment plates (Perkin Elmer, Waltham, MA), centrifuged and incubated under standard culture conditions for 3 days to allow spheroid formation and growth.

After spheroid formation, Fab-AF488 conjugate antibody was added to the spheroid at a final concentration of 25 nM and incubated for 4-96 hours under standard culture conditions. After incubation, excess antibody was removed by adding 100 μL of complete growth medium and removing 100 μL of medium from the wells, washing three times in total. Spheroids were treated with a solution of 1 μM Hoechst 33342 (Thermo Fisher Scientific, Waltham, MA) and 100 nM anti-Alexa Fluor 488 antibody (Thermo Fisher Scientific, Waltham, MA; Catalog No. A-11094) and incubated for 2 hours at 37°C/5% _CO2 .

Imaging was performed using an Operetta CLS ^™ High Content Analysis System (Perkin Elmer, Waltham, MA) with confocal acquisition and a 10× magnification air objective. Z-stacks of 15 planes spaced 15 μm apart were acquired, and the slice with the largest diameter representing the central slice of the spheroid was selected for 2D analysis. Image analysis was performed using 4.5 software (PerkinElmer, Waltham, MA). In brief, spheroid identification was performed by applying a mask to one spheroid per well around Hoechst 33342-positive objects. The spheroid region was divided into subregions of concentric bands, each subregion accounting for 10% of the spheroid region. The average AF488 fluorescence within each subregion band was quantified, corrected by subtracting the internal 10% average AF488 fluorescence, and plotted using GraphPad Prism version 9 (GraphPad Software, San Diego, CA).

result

The results are summarized in Table 38.1 and Figure 25. The humanized antibody variant v36675 showed a greater degree of penetration into the JEG-3 spheroids than Sumituximab, as measured by the AF488 intensity at each subregional band and by the distance from the edge of the spheroid to where AF488 fluorescence was detectable at all time points evaluated. The non-binding control palivizumab showed less AF488 signal throughout the spheroids than both anti-FRα antibodies at all time points evaluated.

Example 39: Release and quantification of intracellular payload

The intracellular and extracellular payload delivery capabilities of humanized variant v36675 conjugated to Compound 139 with DAR 8 (v36675-MC-GGFG-AM-Compound 139; v36789) were evaluated using mass spectrometry as follows. Palivizumab (v21995) conjugated to Compound 139 with DAR 8 was used as a non-targeting control.

In brief, adherent IGROV-1 tumor cells were seeded in 12-well plates with 200,000 cells/well and treated with ADC for 24, 48 and 72 hours under standard culture conditions. After incubation, the supernatant was transferred to a 96-well plate for quantification of extracellular payload. The remaining cells were washed with PBS (pH 7.4), collected, counted, transferred to a separate 96-well plate and frozen at -80 ° C for quantification of intracellular payload. After thawing, pure acetonitrile was used to lyse the cells. The initial supernatant and cell lysate supernatant were injected into LC/MS instruments (Agilent 6545 quadrupole time of flight (Q-TOF) liquid chromatography/mass spectrometer, Agilent Technologies, Santa Clara, CA), 15 μL/injection, and the free compound 139 in the sample was quantified using a known concentration of compound 139 standard. Deconvolution and data analysis were performed using MassHunter software (Agilent Technologies, Santa Clara, CA).

result

The results are shown in Figure 26. The humanized variant v36675 (v36789 ADC) conjugated to compound 139 showed a high degree of intracellular payload (compound 139) delivery in the high FRα expressing cell line IGROV-1. Intracellular payload delivery is time-dependent, showing an increase from 24 hours to 72 hours (Figure 26A). The extracellular payload release from ADC is much lower than the intracellular concentration, but it is also time-dependent, also showing an increase from 24 hours to 72 hours (Figure 26B). The non-targeted control palivizumab (v21995) conjugated to compound 139 did not show intracellular payload delivery (Figure 26A), and produced a low level of extracellular payload release (Figure 26B).

Example 40: In vivo efficacy study #3

As described below, the in vivo antitumor activity of the humanized variant v36675 conjugated to compound 139 as DAR 8 (v36789) was evaluated in a number of patient-derived ovarian cancer xenograft (PDX) models. ADC v32603 (somituximab-DM4 DAR 4) was evaluated for comparison.

The ADCs, DARs, xenograft models, and doses used in each xenograft study are summarized in Table 40.1. For each xenograft study, tumor volume and body weight of the animals were measured twice weekly.

Table 40.1: Study parameters

Tumor fragments were implanted subcutaneously into female athymic nude Foxn1nu mice. When the mean tumor volume reached approximately 200-250 ^mm3 , animals were divided into groups (n=3 per group) and treated on study day 1 with a single IV dose of the test article as shown in Table 40.1.

result

The results are shown in Figures 27A-I. In CTG-0703, CTG-1301, CTG-2025, and CTG-3383 PDX models (Figures 27A, 27B, 27C, and 27D, respectively), both v36675-MC-GGFG-AM-Compound 139DAR8 and Sumetuximab-DM4 caused strong inhibition of tumor growth when administered at 6 mg/kg.

In the CTG-0947 PDX model ( FIG. 27E ), v36675-MC-GGFG-AM-Compound 139DAR8 caused strong inhibition of tumor growth, while Sumituximab-DM4 caused moderate inhibition of tumor growth when dosed at 6 mg/kg.

In the CTG-0958, CTG-3718, and CTG-1703 PDX models (Figure 27F, Figure 27G, and Figure 27H, respectively), v36675-MC-GGFG-AM-Compound 139DAR8 caused strong inhibition of tumor growth when dosed at 6 mg/kg, while Sumituximab-DM4 was inactive.

In the CTG-1602 PDX model ( FIG. 27I ), both v36675-MC-GGFG-AM-Compound 139 DAR8 and Sumetuximab-DM4 were inactive when dosed at 6 mg/kg.

In summary, these data demonstrate that while both v36675-MC-GGFG-AM-Compound 139DAR8 and Sumituximab-DM4 are active in PDX models assessed to express strong levels of FRα, only v36675-MC-GGFG-AM-Compound 139DAR8 has strong activity in PDX models expressing moderate/weak levels of FRα.

Example 41: Specificity evaluation of anti-FRα antibodies

The following anti-FRα antibodies were screened for any specific off-target binding interactions using Retrogenix cellular microarray technology (Charles River Laboratories, Wilmington, MA): humanized variant v36675 and affinity matured variant v35356.

Retrogenix Cell Microarray technology identifies interactions with cell surface receptors and secreted proteins by screening cDNA clone libraries representing more than 6,300 human proteins for bound test ligands. These proteins include plasma membrane monomers, heterodimers (formed by co-expression of individual subunits), and secreted proteins (expressed with inert cell membrane tethers). Each cDNA is spotted in duplicate onto a specialized slide and covered with HEK293 cells. These cells become reverse transfected, generating cell clusters that each overexpress a different single protein (or heterodimeric complex).

The study was conducted at Charles River Laboratories and consisted of three phases: prescreening, library screening, and confirmation screening. Prescreening was first performed to determine the appropriate concentration of the test antibody for library screening (i.e., the concentration at which low levels of background binding to fixed, untransfected HEK293 cells and strong binding to cells overexpressing FRα were observed). In the library screening phase, the test antibodies were screened as a library against fixed HEK293 cells expressing approximately 6,300 human proteins alone. In the confirmation screening phase, each library hit was re-expressed and re-tested with each test antibody alone using fixed and live HEK293 cells.

In all three stages, slides were spotted individually with expression vectors encoding ZsGreen1 (for assessment of transfection efficiency) and (1) in the pre-screening stage: human FRa or control receptors (EGFR and CD20); (2) in the library screening stage: the above protein libraries arrayed in duplicate on multiple microarray slides ("slide sets"), where two replicate slides were screened for each slide set; or (3) in the confirmatory screening stage: protein hits identified in the library screening or control receptors (EGFR and CD20) arrayed in duplicate.

In the prescreening phase, 2, 5, or 20 mg/mL of each selected antibody, 1 mg/mL of a control antibody (a CD20-binding rituximab biosimilar), or PBS was added to the slides after cell fixation. In the library screening phase, a pool of two antibodies (variant v36675 at 20 mg/mL and variant v35356 at 5 mg/mL) was added to each slide after cell fixation. In the confirmatory screening phase, slides were treated with the antibodies alone without fixation (live cells; n = 1 slide per treatment) and after cell fixation (n = 2 slides per treatment): 20 mg/mL of variant v36675, 5 mg/mL of variant v35356, or 1 mg/mL of a control antibody (a rituximab biosimilar), or no test article.

use The binding was detected by anti-human IgG H+L (AF647 anti-hIgG H+L) labeled with 647, followed by fluorescence imaging. ImageQuant ^™ software (version 8.2; GE Healthcare, Chicago, IL) was used to analyze the fluorescence images and quantify. Protein hits were defined as repeated spots showing increased signals compared to background levels, and were identified by visual inspection using images gridded on ImageQuant ^™ software. Hits were classified as strong, medium, weak or very weak by visual inspection based on the intensity of repeated spots by two experienced scientists.

result

Most of the initial hits identified in the library screen (spot intensities ranged from very weak to strong) were confirmed as hits in the confirmation screen for variant v36675 and variant v35356. However, in addition to the hits reflecting the expected strong interaction with FRa, other hits were considered nonspecific because these hits 1) were also observed for the control antibody (rituximab biosimilar) because they included FcgR receptors (signaling due to Fc domain-mediated interactions) or 2) included various immunoglobulins recognized by the test antibody.

For the humanized variant v36675, no other interactions were identified (see Figure 28), indicating that variant v36675 has high specificity for its primary target FRa. The weak interaction observed for this variant with the heterodimer FCGR3A+CD247 was not observed in the control (compare Figure 28A and Figure 28B), and was considered insufficiently strong to be a hit.

For affinity matured variant v35356, a weak intensity signal for protein COL6A2 isoform 2C2A was detected in the library screen. The "hits" were confirmed in the fixed cell screen but not in the live cell screen, indicating that the observed inconsistency may be caused by fixation artifacts.

Example 42: Competition Assay

Competition binding to the FRα target between the parental chimeric antibody v23924 and the anti-FRα antibodies sometuximab (v17716) and fatuzumab (v31629) was assessed by flow cytometry in the moderate FRα expressing tumor cell line H2110 as follows.

In brief, the antibodies were fluorescently labeled with Alexa Fluor 647 (AF647; ThermoFisher Scientific, Waltham, MA; Catalog No. A20006) according to the manufacturer's instructions before cell treatment. Tumor cells were seeded in V-bottom 96-well plates at 50,000 cells/well and treated with unlabeled antibodies for 1 hour at 4°C to prevent internalization. After incubation, the cells were washed and fluorescently labeled antibodies were added for 1 hour at 4°C. After incubation and washing, fluorescence was detected by flow cytometry on a BD LSRFortessa ^TM cell analyzer (BD Biosciences, Franklin Lake, NJ), with 1,000 minimum events collected per well. Competitive binding % relative to untreated controls was calculated using AF647/APC-AGeoMean (fluorescence signal geometric mean, proportional to anti-human AF647 binding) in the live cell population, and data were plotted using GraphPadPrism version 9 (GraphPad Software, San Diego, CA).

result

The results are shown in Figure 29 and Table 42.1. It was found that the chimeric antibody v23924 competed with fatuzumab for FRα binding in the H2110 cell line, producing close to 100% competitive binding in both binding directions (primary antibody fatuzumab and fluorescently labeled secondary antibody variant v23924, and primary antibody variant v23924 and fluorescently labeled secondary antibody fatuzumab). It was found that the chimeric antibody v23924 did not compete with sometuximab for FRα binding in the H2110 cell line. These antibodies showed low levels of competition % in both binding directions. In contrast, fatuzumab showed competitive binding with sometuximab. Therefore, the chimeric antibody v23924 exhibited binding characteristics different from those of sometuximab and fatuzumab.

Table 42.1: Competition binding with facilitation and somituximab on H2110 cells

The disclosures of all patents, patent applications, publications, and database entries cited in this specification are hereby expressly incorporated by reference in their entirety to the same extent as if each such individual patent, patent application, publication, and database entry was specifically and individually indicated to be incorporated by reference.

Modifications of the specific embodiments described herein which are apparent to those skilled in the art are intended to be within the scope of the following claims.

Sequence Listing

Table A: Clone numbers of variants

Table B: Cloned sequences

Claims (46)

1. An antibody-drug conjugate having the formula (X):

T-[L-(D)_m]_n

(X)

Wherein:

m is between 1 and 4;

n is between 1 and 10;

t is an anti-FR alpha antibody construct comprising an antigen binding domain that specifically binds an epitope within human folate receptor alpha (hFR alpha) that comprises amino acid residues E120, D121, R123, T124, S125, and Y126 of SEQ ID NO 15;

L is a linker, and

D is a compound of formula I:

Wherein:

R ¹ is selected from: -H, -CH ₃、-CHF₂、-CF₃、-F、-Br、-Cl、-OH、-OCH₃、-OCF₃, and-NH ₂, and

R ² is selected from: -H, -CH ₃、-CF₃、-F、-Br、-Cl、-OH、-OCH₃ and-OCF ₃,

And wherein:

When R ¹ is-NH ₂, then R is R ³ or R ⁴, and when R ¹ is not-NH ₂ then R is R ⁴;

R ³ is selected from: -H, -C ₁-C₆ alkyl, -C ₃-C₈ cycloalkyl, - (C ₁-C₆ alkyl) -O-R ⁵, -CO ₂R⁸, -aryl, -heteroaryl, and- (C ₁-C₆ alkyl) -aryl;

R ⁴ is selected from:

R ⁵ is selected from: -H, -C ₁-C₆ alkyl, -C ₃-C₈ cycloalkyl, -aryl, -heteroaryl, -aryl, and- (C ₁-C₆ alkyl) -aryl;

R ⁶ and R ⁷ are each independently selected from: -H, -C ₁-C₆ alkyl, -C ₃-C₈ cycloalkyl, - (C ₁-C₆ alkyl) -O-R ⁵、-C₃-C₈ heterocycloalkyl, and-C (O) R ¹⁷;

R ⁸ is selected from: -H, -C ₁-C₆ alkyl, -C ₃-C₈ cycloalkyl and-C ₃-C₈ heterocycloalkyl;

Each R ⁹ is independently selected from: -H, -C ₁-C₆ alkyl, -C ₃-C₈ cycloalkyl, -aryl, -heteroaryl, and- (C ₁-C₆ alkyl) -aryl;

each R ¹⁰ is independently selected from: -C ₁-C₆ alkyl, -C ₃-C₈ cycloalkyl, -NR ¹⁴R^14', -aryl, -heteroaryl and- (C ₁-C₆ alkyl) -aryl;

R ^10' is selected from: -H, -C ₁-C₆ alkyl, -C ₃-C₈ cycloalkyl, -aryl, -heteroaryl, and- (C ₁-C₆ alkyl) -aryl;

r ¹¹ is selected from: -H and-C ₁-C₆ alkyl;

R ¹² is selected from: -H, -C ₁-C₆ alkyl, -CO ₂R⁸, -aryl, -heteroaryl, - (C ₁-C₆ alkyl) -aryl, -S (O) ₂R¹⁶ and

R ¹³ is selected from: -H and-C ₁-C₆ alkyl;

R ¹⁴ and R ^14' are each independently selected from: -H, C ₁-C₆ alkyl, -C ₃-C₈ cycloalkyl and-C ₃-C₈ heterocycloalkyl;

R ¹⁶ is selected from: -C ₁-C₆ alkyl, -C ₃-C₈ cycloalkyl, -aryl, -heteroaryl and- (C ₁-C₆ alkyl) -aryl;

R ¹⁷ is selected from: -C ₁-C₆ alkyl, -C ₃-C₈ cycloalkyl, -C ₃-C₈ heterocycloalkyl, - (C ₁-C₆ alkyl) -C ₃-C₈ heterocycloalkyl, -aryl, -heteroaryl, and- (C ₁-C₆ alkyl) -aryl;

R ¹⁸ and R ¹⁹ together with the N atom to which they are bound form a 4-, 5-, 6-or 7-membered ring having 0 to 3 substituents selected from: halogen, -C ₁-C₆ alkyl, -C ₃-C₈ cycloalkyl and- (C ₁-C₆ alkyl) -O-R ⁵;

R ²⁴、R²⁵ and R ²⁶ are each-C ₁-C₆ alkyl;

X ^a and X ^b are each independently selected from: NH, O and S, and

X ^c is selected from: o, S and S (O) ₂,

With the proviso that the compound is not (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.

2. The antibody-drug construct of claim 1, wherein the antigen binding domain comprises heavy chain CDR amino acid sequences (HCDR 1, HCDR2, and HCDR 3) comprising sequences as set forth in SEQ ID NOs 3,4, and 5 and light chain CDR amino acid sequences (LCDR 1, LCDR2, and LCDR 3) comprising sequences as 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 as 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 as set forth in any one of SEQ ID NOs 39, 64, 119, 124 or 130.

5. The antibody-drug conjugate of claim 1, wherein the antigen binding domain comprises:

(i) An HCDR1 amino acid sequence selected from the group consisting of amino acid sequences shown as 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 group consisting of amino acid sequences as shown 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 group consisting of 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 group consisting of the amino acid sequences shown as any one of SEQ ID NOs 40, 43, 45, 65, 125, 126 or 127; an LCDR2 amino acid sequence selected from the group consisting of the amino acid sequences shown 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.

6. The antibody-drug conjugate of any one of claims 1 to 5, wherein D is a compound of formula (IV):

Wherein:

R ^1a is selected from: -H, -CH ₃、-CHF₂、-CF₃、-F、-Br、-Cl、-OH、-OCH₃、-OCF₃ and-NH ₂;

R ^2a is selected from: -H, -CH ₃、-CF₃、-F、-Br、-Cl、-OH、-OCH₃, and-OCF ₃;

x is-O- -S-or-NH-, and R ^4a is selected from: Wherein is the point of attachment to X, and wherein p is 1,2, 3 or 4; or (b)

X is O and R ^4a -X-is selected from:

R ^5a is selected from: -C ₁-C₆ alkyl, -C ₃-C₈ cycloalkyl, -aryl, -heteroaryl and- (C ₁-C₆ alkyl) -aryl;

R ^8a is selected from: -C ₁-C₆ alkyl, -C ₃-C₈ cycloalkyl and-C ₃-C₈ heterocycloalkyl;

Each R ^9a is independently selected from: -C ₁-C₆ alkyl, -C ₃-C₈ cycloalkyl, -aryl, -heteroaryl and- (C ₁-C₆ alkyl) -aryl; or R ^9a is absent, and X ^b = X;

Each R ^10a is independently selected from: -C ₁-C₆ alkyl, -C ₃-C₈ cycloalkyl, -aryl, -heteroaryl, - (C ₁-C₆ alkyl) -aryl and

Each R ^10a' is independently selected from: -H, -C ₁-C₆ alkyl, -C ₃-C₈ cycloalkyl, -aryl, -heteroaryl, and- (C ₁-C₆ alkyl) -aryl;

Each R ^10b is independently selected from: -C ₁-C₆ alkyl, -C ₃-C₈ cycloalkyl, -aryl, -heteroaryl and- (C ₁-C₆ alkyl) -aryl;

R ^11a is absent or is-C ₁-C₆ alkyl;

R ^12a is selected from: -C ₁-C₆ alkyl, -CO ₂R^8a, -aryl, -heteroaryl, - (C ₁-C₆ alkyl) -aryl, -S (O) ₂R^16a and

R ^13a is selected from: -H and-C ₁-C₆ alkyl;

R ^14a is selected from: -C ₁-C₆ alkyl, -C ₃-C₈ cycloalkyl and-C ₃-C₈ heterocycloalkyl;

R ^14a' is selected from: H. -C ₁-C₆ alkyl, -C ₃-C₈ cycloalkyl and-C ₃-C₈ heterocycloalkyl;

r ^16a is selected from: -C ₁-C₆ alkyl, -C ₃-C₈ cycloalkyl, -aryl, -heteroaryl and- (C ₁-C₆ alkyl) -aryl;

R ²¹ is selected from: -C ₁-C₆ alkyl, -C ₃-C₈ cycloalkyl and- (C ₁-C₆ alkyl) -O-R ^5a;

R ²² and R ²³ are each independently selected from: -H, -halogen, -C ₁-C₆ alkyl, and-C ₃-C₈ cycloalkyl;

R ²⁴、R²⁵ and R ²⁶ are each-C ₁-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

Indicating the point of connection to the joint L.

7. The antibody-drug conjugate of claim 6, wherein R ^1a is selected from the group consisting of: -CH ₃、-CF₃、-OCH₃、-OCF₃ and-NH ₂.

8. The antibody-drug conjugate of claim 6, wherein R ^1a is selected from the group consisting of: -CH ₃、-OCH₃ and NH ₂.

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

10. The antibody-drug conjugate of any one of claims 6 to 9, wherein X is-O-, -S-or-NH-, and R ^4a is selected from:

11. the antibody-drug conjugate of any one of claims 1 to 5, wherein D is a compound of formula (V):

Wherein:

r ^2a is selected from: -CH ₃、-CF₃、-F、-Br、-Cl、-OH、-OCH₃ and-OCF ₃;

R ^20a is selected from: -H, -C ₁-C₆ alkyl, -C ₃-C₈ cycloalkyl, - (C ₁-C₆ alkyl) -O-R ⁵, -CO ₂R⁸, -aryl, -heteroaryl, - (C ₁-C₆ alkyl) -aryl,

R ⁵ is selected from: -H, -C ₁-C₆ alkyl, -C ₃-C₈ cycloalkyl, -aryl, -heteroaryl, and- (C ₁-C₆ alkyl) -aryl;

R ⁶ and R ⁷ are each independently selected from: -H, -C ₁-C₆ alkyl, -C ₃-C₈ cycloalkyl, - (C ₁-C₆ alkyl) -O-R ⁵、-C₃-C₈ heterocycloalkyl, and-C (O) R ¹⁷;

R ⁸ is selected from: -H, -C ₁-C₆ alkyl, -C ₃-C₈ cycloalkyl and-C ₃-C₈ heterocycloalkyl;

Each R ⁹ is independently selected from: -H, -C ₁-C₆ alkyl, -C ₃-C₈ cycloalkyl, -aryl, -heteroaryl, and- (C ₁-C₆ alkyl) -aryl;

Each R ^10a is independently selected from: -C ₁-C₆ alkyl, -C ₃-C₈ cycloalkyl, -aryl, -heteroaryl, - (C ₁-C₆ alkyl) -aryl and-NR ¹⁴R^14';

Each R ^10a' is independently selected from: -H, -C ₁-C₆ alkyl, -C ₃-C₈ cycloalkyl, -aryl, -heteroaryl, and- (C ₁-C₆ alkyl) -aryl;

r ¹¹ is selected from: -H and-C ₁-C₆ alkyl;

R ¹² is selected from: -H, -C ₁-C₆ alkyl, -CO ₂R⁸, -aryl, -heteroaryl, - (C ₁-C₆ alkyl) -aryl, -S (O) ₂R¹⁶ and

R ¹³ is selected from: -H and-C ₁-C₆ alkyl;

R ¹⁴ and R ^14' are each independently selected from: -H, C ₁-C₆ alkyl, -C ₃-C₈ cycloalkyl and-C ₃-C₈ heterocycloalkyl;

R ¹⁶ is selected from: -C ₁-C₆ alkyl, -C ₃-C₈ cycloalkyl, -aryl, -heteroaryl and- (C ₁-C₆ alkyl) -aryl;

R ¹⁷ is selected from: -C ₁-C₆ alkyl, -C ₃-C₈ cycloalkyl, -C ₃-C₈ heterocycloalkyl, - (C ₁-C₆ alkyl) -C ₃-C₈ heterocycloalkyl, -aryl, -heteroaryl, and- (C ₁-C₆ alkyl) -aryl;

R ¹⁸ and R ¹⁹ together with the N atom to which they are bound form a 4-, 5-, 6-or 7-membered ring having 0 to 3 substituents selected from: halogen, -C ₁-C₆ alkyl, -C ₃-C₈ cycloalkyl and- (C ₁-C₆ alkyl) -O-R ⁵;

R ²⁴、R²⁵ and R ²⁶ are each-C ₁-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

Indicating the point of connection to the joint L.

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

13. The antibody-drug conjugate of claim 11 or 12, wherein R ^20a is selected from: -H, -C ₁-C₆ alkyl, - (C ₁-C₆ alkyl) -O-R ⁵,- (C ₁-C₆ alkyl) -aryl,

14. The antibody-drug conjugate of any one of claims 1 to 5, wherein D is a compound of formula (VI):

Wherein:

R ^2a is selected from: -H, -CH ₃、-CF₃、-F、-Br、-Cl、-OH、-OCH₃, and-OCF ₃;

X is-O-, -S-or-NH-, and R ²⁵ is selected from: -C ₁-C₆ alkyl, - (C ₁-C₆ alkyl) -O-R ^5a、-CO₂R^8a, -aryl, -heteroaryl, - (C ₁-C₆ alkyl) -aryl, Wherein is the point of attachment to X, and wherein p is 1,2,3 or 4; or (b)

X is O and R ²⁵ -X-is selected from:

R ^5a is selected from: -C ₁-C₆ alkyl, -C ₃-C₈ cycloalkyl, -aryl, -heteroaryl and- (C ₁-C₆ alkyl) -aryl;

R ^6a is selected from: -H, -C ₁-C₆ alkyl, -C ₃-C₈ cycloalkyl and-C ₃-C₈ heterocycloalkyl;

R ^7a is selected from: -C ₁-C₆ alkyl, -C ₃-C₈ cycloalkyl, - (C ₁-C₆ alkyl) -O-R ^5a、-C₃-C₈ heterocycloalkyl and-C (O) R ^17a;

R ^8a is selected from: -C ₁-C₆ alkyl, -C ₃-C₈ cycloalkyl and-C ₃-C₈ heterocycloalkyl;

Each R ^9a is independently selected from: -C ₁-C₆ alkyl, -C ₃-C₈ cycloalkyl, -aryl, -heteroaryl and- (C ₁-C₆ alkyl) -aryl; or R ^9a is absent, and X ^b = X;

Each R ^10a is independently selected from: -C ₁-C₆ alkyl, -C ₃-C₈ cycloalkyl, -aryl, -heteroaryl, - (C ₁-C₆ alkyl) -aryl and

Each R ^10a' is independently selected from: -H, -C ₁-C₆ alkyl, -C ₃-C₈ cycloalkyl, -aryl, -heteroaryl, and- (C ₁-C₆ alkyl) -aryl;

Each R ^10b is independently selected from: -C ₁-C₆ alkyl, -C ₃-C₈ cycloalkyl, -aryl, -heteroaryl and- (C ₁-C₆ alkyl) -aryl;

R ^11a is absent or is-C ₁-C₆ alkyl;

R ^12a is selected from: -C ₁-C₆ alkyl, -CO ₂R^8a, -aryl, -heteroaryl, - (C ₁-C₆ alkyl) -aryl, -S (O) ₂R^16a and

R ^13a is selected from: -H and-C ₁-C₆ alkyl;

R ^14a is selected from: -C ₁-C₆ alkyl, -C ₃-C₈ cycloalkyl and-C ₃-C₈ heterocycloalkyl;

R ^14a' is selected from: H. -C ₁-C₆ alkyl, -C ₃-C₈ cycloalkyl and-C ₃-C₈ heterocycloalkyl;

r ^16a is selected from: -C ₁-C₆ alkyl, -C ₃-C₈ cycloalkyl, -aryl, -heteroaryl and- (C ₁-C₆ alkyl) -aryl;

r ^17a is selected from: -C ₁-C₆ alkyl, -C ₃-C₈ cycloalkyl, -C ₃-C₈ heterocycloalkyl, - (C ₁-C₆ alkyl) -C ₃-C₈ heterocycloalkyl, -aryl, -heteroaryl, and- (C ₁-C₆ alkyl) -aryl;

R ²¹ is selected from: -C ₁-C₆ alkyl, -C ₃-C₈ cycloalkyl and- (C ₁-C₆ alkyl) -O-R ^5a;

R ²² and R ²³ are each independently selected from: -H, -halogen, -C ₁-C₆ alkyl, and-C ₃-C₈ cycloalkyl;

R ²⁴、R²⁵ and R ²⁶ are each-C ₁-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

Indicating the point of connection to the joint L.

15. The antibody-drug conjugate of claim 14, wherein R ^2a is selected from the group consisting of: -CH ₃、-CF₃、-F、-Br、-Cl、-OH、-OCH₃ and-OCF ₃.

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

17. The antibody-drug conjugate of any one of claims 14 to 16, wherein X is-O-, -S-or-NH-, and R ²⁵ is selected from: -C ₁-C₆ alkyl, - (C ₁-C₆ alkyl) -O-R ^5a、–(C₁-C₆ alkyl) -aryl, Or X is O and R ²⁵ -X-is selected from:

18. The antibody-drug conjugate of any one of claims 14 to 16, wherein X is-O-, -S-or-NH-, and R ²⁵ is selected from: -C ₁-C₆ alkyl, - (C ₁-C₆ alkyl) -O-R ^5a、-(C₁-C₆ alkyl) -aryl,

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

21. The antibody-drug conjugate of any one of claims 1 to 5, wherein D has the structure of any one of the compounds as shown 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. The antibody-drug conjugate of any one of claims 23 to 25, wherein L has:

(a) Formula (XI)

Wherein:

z is a functional group capable of reacting with a target group on the anti-FR alpha antibody construct T;

Str is an extension segment;

AA ₁ and AA ₂ are each independently amino acids, wherein AA ₁-[AA₂]_r forms a protease cleavage site;

x is a self-cleaving 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-FR alpha antibody construct T, and

% Is the point of attachment to the camptothecin analog D, or

(B) (XII)

Wherein:

z is a functional group capable of reacting with a target group on the anti-FR alpha antibody construct T;

Str is an extension segment;

AA ₁ and AA ₂ are each independently amino acids, wherein AA ₁-[AA₂]_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-FR alpha antibody construct T, and

% Is the point of attachment to the camptothecin analog D.

27. The antibody-drug conjugate of any one of claims 1 to 5, wherein L- (D) in formula (X) has the structure of any one of the drug-linkers (DL) as shown in tables 8-10.

28. The antibody-drug conjugate of any one of claims 1 to 5, wherein L- (D) in formula (X) has the structure of any one of the drug-linkers (DL) as shown in table 8 or table 9.

29. The antibody-drug conjugate of any one of claims 1 to 5, wherein L- (D) in formula (X) 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

30. The antibody-drug conjugate of any one of claims 1 to 29, wherein m is between 1 and 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 between 2 and 8.

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

34. The antibody-drug conjugate of any one of claims 1 to 33, wherein the anti-fra antibody construct further comprises a scaffold, and wherein 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 group consisting of:

Wherein:

T is an anti-fra antibody construct comprising two antigen binding domains operably linked to an IgG Fc region, each of the antigen binding domains comprising:

(a) A VL amino acid sequence as shown in SEQ ID NO. 39 and a VH amino acid sequence as shown in SEQ ID NO. 19; or (b)

(B) A VL amino acid sequence as set forth in SEQ ID NO. 124 and a VH amino acid sequence as set forth in SEQ ID NO. 91; or (b)

(C) VL amino acid sequence shown in SEQ ID NO. 64 and

(I) A VH amino acid sequence as shown in SEQ ID NO 50, or

(Ii) A VH amino acid sequence as shown in SEQ ID NO 54, or

(Iii) A VH amino acid sequence as shown in SEQ ID NO 57, or

(Iv) A VH amino acid sequence as shown in SEQ ID NO. 61, or

(V) A VH amino acid sequence as shown in SEQ ID NO 76, or

(Vi) A VH amino acid sequence as shown in SEQ ID NO 79, or

(Vii) A VH amino acid sequence as shown in SEQ ID NO 82, or

(Viii) A VH amino acid sequence as shown in SEQ ID NO. 85, or

(Ix) A VH amino acid sequence as shown in SEQ ID NO 88, or

(X) A VH amino acid sequence as set forth in SEQ ID No. 106; or (b)

(D) VL amino acid sequence shown in SEQ ID NO. 130 and

(I) A VH amino acid sequence as shown in SEQ ID NO 99, or

(Ii) A VH amino acid sequence as shown in SEQ ID NO 106, or

(Iii) A VH amino acid sequence as shown in SEQ ID NO 113, or

(Iv) A VH amino acid sequence as shown in SEQ ID NO 116, or

(V) A VH amino acid sequence as shown in SEQ ID NO 133, or

(Vi) A VH amino acid sequence as set forth in SEQ ID No. 136; or (b)

(E) VL amino acid sequence shown in SEQ ID NO 119 and

(I) A VH amino acid sequence as shown in SEQ ID NO 106, or

(Ii) The VH amino acid sequence shown in SEQ ID NO. 116,

And wherein n is between 4 and 8.

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

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

39. A method of killing a cancer cell comprising contacting the cell with an effective amount of the antibody-drug conjugate of any one of claims 1 to 36.

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

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

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.