Protein tyrosine kinase 7 antibodies and antibody-drug conjugates

Abstract

To provide PTK-7 antibody drug conjugates and pharmaceutical compositions thereof, and methods of using for the treatment of cancer.SOLUTION: Provided is an antibody-drug conjugate (ADC) comprising an antibody conjugated to a cytotoxic agent, the antibody binding human PTK-7 and comprising a heavy chain variable region (VH) and a light chain variable region (VL), the VH comprising heavy chain complementarity determining regions (HCDR) HCDR1, HCDR2, and HCDR3 of specific sequences and the VL comprising light chain complementarity determining regions (LCDR) LCDR1, LCDR2, and LCDR3 of specific sequences.SELECTED DRAWING: None

Description

[Technical Field]

[0001] (Reference to sequence listing) This application has been submitted with a Sequence Listing in ST.26 XML format. The Sequence Listing is provided as a file titled "30976_WO," created on January 30, 2025, and is 63 kilobytes in size. The Sequence Listing information in ST.26 XML format is incorporated herein by reference in its entirety.

[0002] FIELD OF THE INVENTION The present disclosure relates to the field of medicine. More particularly, the present disclosure relates to protein tyrosine kinase 7 (PTK-7) antibodies, antibody-drug conjugates, and pharmaceutical compositions thereof, and their use in the treatment of cancer. [Background technology]

[0003] PTK-7 belongs to the family of Wnt-related pseudokinases and is overexpressed in multiple tumor types, including triple-negative breast cancer, non-small cell lung cancer, colon cancer, gastric cancer, esophageal cancer, and ovarian cancer.

[0004] Antibody-drug conjugates (ADCs) for use as tumor treatments contain tumor-targeting antibodies conjugated to payloads designed to kill tumor cells upon entry. Certain PTK-7 antibodies have been used to generate ADCs with MMAE payloads (WO2015168019) and camptothecin analogs (Kong et al. Mol Cancer Ther 22(10):1128).

[0005] ADCs for use in oncology are very challenging compounds to design because they must balance multiple aspects of the molecule, such as sufficient specificity for tumor targets over healthy cells, maintaining the desired activity against bystander tumor cells while maintaining acceptable toxicity, and a labile payload that maintains good physical and chemical stability while allowing intracellular delivery. Summary of the Invention

[0006] There remains a need for PTK-7 ADCs for treating cancer. In particular, there remains a need for PTK-7 ADCs with a sufficient therapeutic index based on better tolerability and / or better efficacy to support a sufficiently high dose that not only effectively kills tumor cells but is also well-tolerated by patients. In particular, there remains a need for PTK-7 ADCs with effector-null antibodies, fully human antibodies, and topoisomerase I payloads. In particular, there remains a need for PTK-7 ADCs with enhanced bystander activity against PTK-7-low tumors. In particular, there remains a need for PTK-7 ADCs with enhanced bystander activity against tumors with heterogeneous PTK-7 expression. In particular, there remains a need for PTK-7 ADCs with low immunogenicity, stable in vivo pharmacokinetics, and appropriate chemical and physical stability. Additionally, there remains a need for PTK-7 ADCs that have one or more of the following characteristics: better anti-tumor activity as measured in specific tumor models, enhanced bystander activity against PTK-7-low tumors, lower immunogenicity, no measurable antibody effector function, no Fcγ receptor binding, and / or better physical and chemical stability. The ADCs provided herein address one or more of these needs.

[0007] Provided herein are certain PTK-7 ADCs and compositions comprising PTK-7 ADCs. Also provided herein are methods of using the PTK-7 ADCs or compositions comprising PTK-7 ADCs for cancer in a subject.

[0008]

[0010] In one aspect, provided herein is an antibody that binds to human PTK-7, the antibody comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises heavy chain complementarity determining regions (HCDRs) HCDR1, HCDR2, and HCDR3, and the VL comprises light chain complementarity determining regions (LCDRs) LCDR1, LCDR2, and LCDR3; a) HCDR1 comprises SEQ ID NO: 4, HCDR2 comprises SEQ ID NO: 5, HCDR3 comprises SEQ ID NO: 6, LCDR1 comprises SEQ ID NO: 7, LCDR2 comprises SEQ ID NO: 8, and LCDR3 comprises SEQ ID NO: 9; b) HCDR1 comprises SEQ ID NO: 14, HCDR2 comprises SEQ ID NO: 15, HCDR3 comprises SEQ ID NO: 16, LCDR1 comprises SEQ ID NO: 17, LCDR2 comprises SEQ ID NO: 18, and LCDR3 comprises SEQ ID NO: 19; c) HCDR1 comprises SEQ ID NO: 24, HCDR2 comprises SEQ ID NO: 25, HCDR3 comprises SEQ ID NO: 26, LCDR1 comprises SEQ ID NO: 27, LCDR2 comprises SEQ ID NO: 28, and LCDR3 comprises SEQ ID NO: 29, or d) HCDR1 comprises SEQ ID NO: 34, HCDR2 comprises SEQ ID NO: 35, HCDR3 comprises SEQ ID NO: 36, LCDR1 comprises SEQ ID NO: 37, LCDR2 comprises SEQ ID NO: 18, and LCDR3 comprises SEQ ID NO: 38.

[0009] In a further aspect, provided herein is an antibody that binds to human PTK-7, the antibody comprising a heavy chain variable region (VH) and a light chain variable region (VL): a) the VH comprises SEQ ID NO: 10 and the VL comprises SEQ ID NO: 11; b) the VH comprises SEQ ID NO: 20 and the VL comprises SEQ ID NO: 21; c) the VH comprises SEQ ID NO: 30 and the VL comprises SEQ ID NO: 31; or d) VH comprises SEQ ID NO:39 and VL comprises SEQ ID NO:40.

[0010] In another aspect, provided herein is an antibody that binds to human PTK-7, the antibody comprising a heavy chain (HC) and a light chain (LC): a) HC comprises amino acids 2 to 441 of SEQ ID NO: 2 and LC comprises SEQ ID NO: 3; b) HC comprises amino acids 2 to 448 of SEQ ID NO: 12, and LC comprises amino acids 2 to 215 of SEQ ID NO: 13; c) HC comprises amino acids 2 to 447 of SEQ ID NO: 22 and LC comprises amino acids 2 to 215 of SEQ ID NO: 23; d) HC comprises amino acids 2 to 444 of SEQ ID NO: 32, and LC comprises amino acids 2 to 215 of SEQ ID NO: 33.

[0011] In another aspect, provided herein is an antibody-drug conjugate (ADC) comprising a PTK-7 antibody disclosed herein conjugated directly or via a linker to a cytotoxic agent.

[0012] In a further aspect, provided herein is an ADC, wherein the cytotoxic agent is a camptothecin analog comprising Formula I:

[0013] [ka]

[0014] In another aspect, there is provided herein an ADC of Formula II:

[0015] [ka] where Ab is a PTK-7 antibody disclosed herein and n is from about 1 to about 16.

[0016] In another aspect, there is provided herein an ADC of Formula III:

[0017] [ka] where Ab is a PTK-7 antibody disclosed herein and n is from about 1 to about 16.

[0018] In another aspect, provided herein is a pharmaceutical composition comprising a PTK-7 antibody disclosed herein and one or more pharmaceutically acceptable carriers, diluents, or excipients. In another aspect, provided herein is a pharmaceutical composition comprising a PTK-7 ADC disclosed herein and one or more pharmaceutically acceptable carriers, diluents, or excipients.

[0019] In another aspect, provided herein is a method of treating cancer comprising administering to a patient in need thereof an effective amount of a PTK-7 ADC disclosed herein. In a further aspect, provided herein is a method of treating cancer comprising administering to a patient in need thereof an effective amount of a PTK-7 ADC disclosed herein, wherein the cancer is ovarian cancer, lung cancer, breast cancer, gastric cancer, kidney cancer, prostate cancer, liver cancer, or colorectal cancer. DETAILED DESCRIPTION OF THE INVENTION

[0020] PTK-7 As used herein, "human PTK-7" refers to human protein tyrosine kinase 7, also known as colon cancer kinase-4 (CCK-4). The amino acid sequence of human PTK-7 can be found in NP_002812, including the signal peptide provided in SEQ ID NO:1.

[0021] PTK-7 antibodies (also known as anti-PTK-7 antibodies) As used herein, the term "antibody" refers to an immunoglobulin molecule that binds to an antigen. The antibody may be of any class (e.g., IgG, IgE, IgM, IgD, IgA) and any subclass (e.g., IgG1, IgG2, IgG3, IgG4).

[0022] An exemplary antibody of the present disclosure is an immunoglobulin G (IgG)-type antibody composed of four polypeptide chains: two heavy chains (HC) and two light chains (LC) cross-linked via interchain disulfide bonds. The amino-terminal portion of each of the four polypeptide chains contains a variable region of about 100 to 125 amino acids or more that is primarily responsible for antigen recognition. The carboxy-terminal portion of each of the four polypeptide chains contains a constant region that is primarily responsible for effector function. Each heavy chain is composed of a heavy chain variable region (VH) and a heavy chain constant region. Each light chain is composed of a light chain variable region (VL) and a light chain constant region. IgG isotypes may be further divided into subclasses (e.g., IgG1, IgG2, IgG3, and IgG4).

[0023] The VH and VL regions can be further subdivided into hypervariable regions, termed complementarity determining regions (CDRs), interspersed with more conserved regions, termed framework regions (FRs). The CDRs are exposed on the surface of the protein and are critical regions of the antibody for antigen-binding specificity. Each VH and VL is composed of three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. Herein, the three CDRs of the heavy chain are referred to as "HCDR1, HCDR2, and HCDR3," and the three CDRs of the light chain are referred to as "LCDR1, LCDR2, and LCDR3." The CDRs contain most of the residues that form specific interactions with the antigen.The assignment of amino acid residues to CDRs can be performed using the methods of Kabat (Kabat et al., "Sequences of Proteins of Immunological Interest," National Institutes of Health, Bethesda, Md. (1991)), Chothia (Chothia et al., "Canonical structures for the hypervariable regions of immunoglobulins," Journal of Molecular Biology, 196, 901-917 (1987); Al-Lazikani et al., "Standard conformations for the canonical structures of immunoglobulins," Journal of Molecular Biology, 273, 927-948 (1997)), North (North et al., "A New Clustering of Antibody CDR Loop Conformations," Journal of Molecular Biology, 406, 228-256 (2011)), or IMGT (the international ImMunoGeneTics database, available at www.imgt.org; see Lefranc et al., Nucleic Acids Res. 1999;27:209-212). The CDRs of the present disclosure are determined according to North.

[0024] Certain antibodies described herein contain an IgG1 Fc region or an Fc region derived from human IgG1, e.g., an altered IgG1 Fc region with altered Fc effector function. IgG1 is known to induce antibody-dependent cell cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). Some antibodies of the present disclosure have amino acid substitutions introduced into the IgG1 Fc region to alter effector function. According to some disclosures herein, mutations are introduced at positions 234 and 235 (according to EU index numbering) of the Fc region. According to some disclosures herein, mutations are introduced at positions 234, 235, and 265 (according to EU index numbering) of the Fc region. In some aspects, the PTK-7 antibodies of the disclosure comprise a modified human IgG1 Fc region (also referred to as a hIgG1 effector null or hIgG1EN Fc region, according to EU index numbering) comprising alanines at residues 234 and 235 and a serine at position 265. In further aspects, some antibodies have additional mutations in the Fc region including a glutamine, alanine, or glycine at position 297, an alanine or glutamine at position 322, an alanine or glycine at position 329, and / or an alanine or serine at position 331 (according to EU index numbering). In some aspects, these antibody mutations are an alanine at position 234, a glutamic acid at position 235, an alanine at position 237, a serine at position 330, and a serine at position 331 (according to EU index numbering). In further aspects, these amino acid substitutions introduced into the IgG1 Fc region reduced or eliminated measurable antibody effector function.

[0025] In certain embodiments of the disclosure, the PTK-7 antibody has a modified human IgG1 or human IgG4 constant domain containing one or more engineered cysteine ​​residues. In further embodiments, the antibody contains an engineered cysteine ​​at one or more sites within heavy chain constant domain 1 (CH1), heavy chain constant domain 2 (CH2), and / or heavy chain constant domain 3 (CH3).

[0026] Mammalian antibody expression typically results in glycosylation. Glycosylation of antibodies is typically either N-linked or O-linked. N-linked glycosylation refers to the attachment of a carbohydrate moiety to the side chain of an asparagine residue. O-linked glycosylation refers to the attachment of a sugar, such as N-acetylgalactosamine, galactose, or xylose, to a hydroxyamino acid. Typically, glycosylation occurs in the Fc region of antibodies at a highly conserved N-glycosylation site (e.g., position 297 in IgG1 according to the IMGT or EU index numbering). Glycosylation sites can be modified to alter glycosylation (e.g., to block or reduce glycosylation, or to alter the amino acid sequence to generate additional or diverse glycosylation).

[0027] Expression of antibodies from the IgG subclass in a mammal can result in the truncation of C-terminal amino acids from one or both heavy chains; for example, in the case of IgG1 antibodies, one or two C-terminal amino acids may be removed. For IgG1 antibodies, the C-terminal lysine, if present, may be truncated or trimmed from the heavy chain during expression. Additionally, the penultimate glycine may be similarly truncated or trimmed from the heavy chain.

[0028] Expression of an antibody in a mammal may also result in modification of the N-terminal amino acid. For example, if the most N-terminal amino acid of a heavy or light chain is glutamine or glutamic acid, it may be changed to pyroglutamic acid. For example, if the most C-terminal amino acid of a heavy or light chain is lysine or glycine, it may be removed.

[0029] The terms "nucleic acid" or "polynucleotide," as used interchangeably herein, refer to a polymer of nucleotides, including single- and / or double-stranded nucleotide-containing molecules, such as DNA, cDNA, and RNA molecules, that incorporate naturally occurring nucleotides, modified nucleotides, and / or nucleotide analogs. A polynucleotide of the present disclosure can also include substrates incorporated therein, for example, by a DNA or RNA polymerase or a synthetic reaction.

[0030] The polynucleotides of the present disclosure can be expressed in host cells, for example, after the polynucleotide is operably linked to an expression control sequence. Expression control sequences capable of expressing an operably linked polynucleotide are well known in the art. For example, an expression vector can include a sequence encoding one or more signal peptides that facilitate secretion of the polypeptide from a host cell. The signal peptide can be, for example, an immunoglobulin signal peptide or a heterologous signal peptide. An expression vector containing a polynucleotide of interest (e.g., a polynucleotide encoding an antibody polypeptide) can be transferred into host cells by well-known methods. Additionally, the expression vector can include one or more selectable markers, such as, for example, tetracycline, neomycin, and dihydrofolate reductase, to facilitate detection of host cells transformed with the desired polynucleotide sequence.

[0031] Host cells include cells stably or transiently transfected, transformed, transduced, or infected with one or more expression vectors expressing all or a portion of an antibody of the present disclosure. According to some embodiments, host cells can be stably or transiently transfected, transformed, transduced, or infected with an expression vector expressing the HC polypeptide and an expression vector expressing the LC polypeptide of an antibody of the present disclosure. In some embodiments, host cells can be stably or transiently transfected, transformed, transduced, or infected with expression vectors expressing the HC and LC polypeptides of an antibody of the present disclosure. Antibodies of the present disclosure can be produced in mammalian cells, such as CHO, NS0, HEK293, or COS cells, according to techniques well known in the art.

[0032] The medium into which the antibodies of the present disclosure are secreted can be purified by conventional techniques, such as mixed-mode methods of ion exchange and hydrophobic interaction chromatography. For example, the medium can be applied to and eluted from a Protein A or Protein G column using conventional methods; mixed-mode methods of ion exchange and hydrophobic interaction chromatography can also be used. Soluble aggregates and multimers can be effectively removed by common techniques, including size exclusion, hydrophobic interaction, ion exchange, or hydroxyapatite chromatography. The product can be immediately frozen, for example, at -70°C, refrigerated, or lyophilized. Various methods of protein purification can be used, and such methods are known in the art and are described, for example, in Deutscher, Methods in Enzymology 182:83-89 (1990), and Scopes, Protein Purification: Principles and Practice, 3rd Edition, Springer, NY (1994).

[0033] In one aspect, provided herein is an antibody that binds to human PTK-7, the antibody comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises heavy chain complementarity determining regions (HCDRs) HCDR1, HCDR2, and HCDR3, and the VL comprises light chain complementarity determining regions (LCDRs) LCDR1, LCDR2, and LCDR3, wherein HCDR1 comprises SEQ ID NO: 4, HCDR2 comprises SEQ ID NO: 5, HCDR3 comprises SEQ ID NO: 6, LCDR1 comprises SEQ ID NO: 7, LCDR2 comprises SEQ ID NO: 8, and LCDR3 comprises SEQ ID NO: 9. In another aspect, provided herein is an antibody that binds to human PTK-7, wherein HCDR1 comprises SEQ ID NO: 14, HCDR2 comprises SEQ ID NO: 15, HCDR3 comprises SEQ ID NO: 16, LCDR1 comprises SEQ ID NO: 17, LCDR2 comprises SEQ ID NO: 18, and LCDR3 comprises SEQ ID NO: 19. In another aspect, provided herein is an antibody that binds to human PTK-7, wherein HCDR1 comprises SEQ ID NO: 24, HCDR2 comprises SEQ ID NO: 25, HCDR3 comprises SEQ ID NO: 26, LCDR1 comprises SEQ ID NO: 27, LCDR2 comprises SEQ ID NO: 28, and LCDR3 comprises SEQ ID NO: 29. In another aspect, provided herein is an antibody that binds to human PTK-7, wherein HCDR1 comprises SEQ ID NO: 34, HCDR2 comprises SEQ ID NO: 35, HCDR3 comprises SEQ ID NO: 36, LCDR1 comprises SEQ ID NO: 37, LCDR2 comprises SEQ ID NO: 18, and LCDR3 comprises SEQ ID NO: 38.

[0034] In one aspect, provided herein is an antibody that binds to human PTK-7, wherein the antibody comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the antibody comprises a VH comprising SEQ ID NO: 10 and a VL comprising SEQ ID NO: 11. In another aspect, provided herein is an antibody that binds to human PTK-7, wherein the antibody comprises a VH comprising SEQ ID NO: 20 and a VL comprising SEQ ID NO: 21. In another aspect, provided herein is an antibody that binds to human PTK-7, wherein the antibody comprises a VH comprising SEQ ID NO: 30 and a VL comprising SEQ ID NO: 31. In another aspect, provided herein is an antibody that binds to human PTK-7, wherein the antibody comprises a VH comprising SEQ ID NO: 39 and a VL comprising SEQ ID NO: 40.

[0035] In a further aspect, provided herein is an antibody that binds to human PTK-7, the antibody comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the antibody has a human IgG1 or IgG4 isotype. In a further aspect, the antibody has a human IgG1 isotype. In a further aspect, the antibody comprises alanine at residues 234 and 235 (according to EU index numbering). In a further aspect, the antibody further comprises a serine at position 265 (according to EU index numbering). In another aspect, the antibody has a human IgG4 isotype.

[0036] In one aspect, provided herein is an antibody that binds to human PTK-7, the antibody comprising a heavy chain (HC) and a light chain (LC): a) HC comprises amino acids 2 to 441 of SEQ ID NO: 2, and LC consists of SEQ ID NO: 3; b) HC comprises amino acids 2 to 448 of SEQ ID NO: 12, and LC comprises amino acids 2 to 215 of SEQ ID NO: 13; c) HC comprises amino acids 2 to 447 of SEQ ID NO: 22 and LC comprises amino acids 2 to 215 of SEQ ID NO: 23; d) HC comprises amino acids 2 to 444 of SEQ ID NO: 32, and LC comprises amino acids 2 to 215 of SEQ ID NO: 33.

[0037] In one aspect, provided herein is an antibody that binds to human PTK-7, the antibody comprising a heavy chain (HC) and a light chain (LC), wherein the HC comprises amino acids 2-441 of SEQ ID NO: 2, and the LC comprises SEQ ID NO: 3. In one aspect, provided herein is an antibody that binds to human PTK-7, the antibody comprising a heavy chain (HC) and a light chain (LC), wherein the HC comprises amino acids 2-448 of SEQ ID NO: 12, and the LC comprises amino acids 2-215 of SEQ ID NO: 13. In one aspect, provided herein is an antibody that binds to human PTK-7, the antibody comprising a heavy chain (HC) and a light chain (LC), wherein the HC comprises amino acids 2-447 of SEQ ID NO: 22, and the LC comprises amino acids 2-215 of SEQ ID NO: 23. In one aspect, provided herein is an antibody that binds to human PTK-7, the antibody comprising a heavy chain (HC) and a light chain (LC), wherein the HC comprises amino acids 2-444 of SEQ ID NO: 32, and the LC comprises amino acids 2-215 of SEQ ID NO: 33.

[0038] In a further aspect, provided herein is an antibody that binds to human PTK-7, wherein the HC consists of SEQ ID NO: 2 and the LC consists of SEQ ID NO: 3. In another aspect, provided herein is an antibody that binds to human PTK-7, wherein the HC consists of SEQ ID NO: 12 and the LC consists of SEQ ID NO: 13. In another aspect, provided herein is an antibody that binds to human PTK-7, wherein the HC consists of SEQ ID NO: 22 and the LC consists of SEQ ID NO: 23. In another aspect, provided herein is an antibody that binds to human PTK-7, wherein the HC consists of SEQ ID NO: 32 and the LC consists of SEQ ID NO: 33.

[0039] In another aspect, provided herein are different mammalian cells comprising a DNA molecule comprising a polynucleotide sequence encoding a polypeptide having the amino acid sequence of SEQ ID NO:2 and SEQ ID NO:3, SEQ ID NO:12 and SEQ ID NO:13, SEQ ID NO:22 and SEQ ID NO:23, SEQ ID NO:32 and SEQ ID NO:33, wherein the cell is capable of expressing a PTK-7 antibody disclosed herein.

[0040] In another aspect, provided herein is a mammalian cell comprising a first DNA molecule and a second DNA molecule, wherein the first DNA molecule comprises a polynucleotide sequence encoding a polypeptide having an amino acid sequence as follows:

[0041] [Table 1]

[0042] In another aspect, provided herein is a process for producing a PTK-7 antibody, comprising culturing one of the mammalian cells disclosed herein under conditions such that the antibody is expressed, and recovering the expressed antibody.

[0043] In another aspect, provided herein are antibodies produced by culturing mammalian cells containing a DNA molecule comprising a polynucleotide sequence encoding a polypeptide having the amino acid sequence of SEQ ID NO:2 and SEQ ID NO:3, SEQ ID NO:12 and SEQ ID NO:13, SEQ ID NO:22 and SEQ ID NO:23, SEQ ID NO:32 and SEQ ID NO:33 under conditions such that the antibody is expressed, and recovering the expressed antibody.

[0044] In another aspect, provided herein is an antibody produced by culturing a mammalian cell comprising a first DNA molecule and a second DNA molecule, wherein the first DNA molecule comprises a polynucleotide sequence encoding a polypeptide having the following amino acid sequence:

[0045] [Table 2]

[0046] As used herein, the term "cofetuzumab" refers to a humanized anti-PTK-7 IgG1 antibody (hu6M024) expressed and purified using standard conditions and having the sequence disclosed in WO2012112943. The term "cofetuzumab peridotin" refers to an ADC having a cleavable valine-citrulline linker, auristatin-0101, as the payload, with a drug-to-antibody ratio (DAR) of 4.

[0047] payload The PTK-7 antibodies of the present disclosure can be conjugated to a variety of payloads (including pharmaceutically acceptable salts thereof) to form antibody drug conjugates (ADCs). Moieties suitable for conjugation to the PTK-7 antibodies disclosed herein include cytotoxic agents (e.g., chemotherapeutic agents), prodrug-converting enzymes, radioisotopes or compounds, toxins, and other payloads known in the art.

[0048] Exemplary ADCs herein utilize camptothecin-based payloads (e.g., camptothecin analogs). Camptothecin analogs are topoisomerase I (TOPO1) inhibitors that have been shown to have anticancer activity. Camptothecin and its analogs bind to the TOPO1 / DNA complex, preventing the reannealing of partially cleaved DNA, the accumulation of which leads to cell death. Other camptothecin analogs known in the art, such as topotecan, irinotecan, SN-38, belotecan, exatecan, deruxtecan (Dxd), and salts thereof, such as exatecan mesylate, can be used as payloads.

[0049] In some embodiments, provided herein are ADCs wherein the camptothecin analog is exatecan. In some embodiments, provided herein are ADCs wherein exatecan is represented by Formula IV, V, or VI, respectively:

[0050] [ka] It contains one of the compounds:

[0051] Other payloads for ADCs known in the art include, for example, maytansinoids (e.g., DM1 and DM4), pyrrolobenzodiazepines (e.g., PBD dimers), auristatin peptides (e.g., MMAE and MMAF), duocarmycins, calicheamicins, DNA minor groove binders (e.g., enediynes and lexitropsins), and taxanes (e.g., paclitaxel and docetaxel).

[0052] Self-Immolative Unit Self-immolation, or self-removal, of a portion of an ADC can be designed into the overall structure of the ADC. Self-immolation typically involves activation of a trigger group, leading to a spontaneous chemical and / or biological reaction that causes elimination of the group itself, i.e., the self-immolative unit. The self-immolative unit can provide favorable properties to the ADC, such as providing space to reduce steric hindrance for cellular proteases reaching the peptide cleavage site within the ADC.

[0053] In some embodiments of the present disclosure, the ADCs described herein contain a self-immolative unit. If present, the self-immolative unit on the ADC is exposed and triggered after cleavage of the ADC linker, such as a peptide unit by a cellular protease or a sugar-cleaving unit by a cellular enzyme. In further embodiments, the self-immolative unit is para-aminobenzyloxycarbonyl (PABC), ortho-aminobenzyl carbonate (OABC), or another self-immolative unit known in the art.

[0054] In some aspects of the disclosure, the ADCs described herein comprise a self-immolative unit of formula VII.

[0055] [ka]

[0056] In some aspects of the disclosure, the ADCs described herein comprise a self-immolative unit of formula VIII.

[0057] [ka]

[0058] In some embodiments of the present disclosure, the ADCs described herein do not contain a self-immolative unit. In these embodiments, the camptothecin analogs described herein are linked directly to a peptide unit or a glycocleaving unit.

[0059] It is understood that the payloads described herein can exist as stereoisomers, and embodiments of the present disclosure include all enantiomers, diastereomers, and mixtures thereof.

[0060] Drug-to-Antibody Ratio (DAR) In this disclosure, the average drug loading, when referring to a composition comprising a population of ADCs, is the drug-to-antibody ratio, or DAR. The average number of drugs per antibody in a preparation can be characterized by conventional means, such as mass spectrometry, HIC, ELISA, and HPLC. A higher DAR can produce more potent ADCs, but a higher DAR can also result in destabilization, aggregation, increased off-target toxicity, and enhanced drug clearance from the systemic circulation.

[0061] In some embodiments, the DAR is 1 to about 16, about 2 to about 14, or about 2 to about 10. In some embodiments, the DAR is about 2 to about 5. In further embodiments, the DAR averages 4. In other embodiments, the DAR is about 6 to about 10. In further embodiments, the DAR averages 8.

[0062] In some embodiments, at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% of the ADCs disclosed herein have a DAR of 4. In some embodiments, at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% of the ADCs disclosed herein have a DAR of 8. In some embodiments, the ADCs disclosed herein are present in a composition, and the ADCs present in the composition have an average DAR of greater than about 1, 2, 3, 4, 5, 6, 7, or 8.

[0063] Linker As disclosed herein, a payload can be conjugated to a PTK-7 antibody by methods understood by one of skill in the art to form a PTK-7 ADC described herein. One example of such a conjugate includes connecting a payload described herein to a PTK-7 antibody described herein via a linker.

[0064] The linker used in the ADC is designed for stability in plasma to allow time for the ADC to localize to target cells. Premature release of the payload reduces the therapeutic index of the ADC by damaging all types of non-target tissue. Once the ADC is internalized into the target cells, the linker must provide a mechanism for payload release so that the payload can function as designed.

[0065] Linkers known to those of skill in the art include, for example, cleavable and non-cleavable moieties. Accordingly, provided herein are ADCs in which a payload, e.g., a camptothecin analog, is conjugated to an antibody via a linker having a cleavable moiety or a linker having a non-cleavable moiety.

[0066] Any suitable linker known in the art can be used in preparing the ADCs of the disclosure. In certain aspects, the linker comprises reactive groups that can conjugate both the antibody of the disclosure and the drug or cytotoxic agent. Examples include N-succinimidyl-4-(N-maleimidomethyl)-cyclohexane-l-carboxylate (SMCC), N-succinimidyl-4-(iodoacetyl)-aminobenzoate (SIAB), bis-maleimide polyethylene glycol (BMPEO), BM(PEO)2, BM(PEO)3, N-(b-maleimidopropyloxy)succinimide ester (BMPS), g-maleimidobutyric acid N-succinimidyl ester (GMBS), e-maleimidocaproic acid N-hydroxysuccinimide ester (EMCS), 5-maleimidovaleric acid NHS ester, N-succinimidyl-4-(N-maleimidomethyl)-cyclohexane-l-carboxy-(6-amidocaproate), m-maleimidobenzoate, yl-N-hydroxysuccinimide ester (MBS), 4-(4-N-maleimidophenyl)-butyric acid hydrazide or HCl salt (MPBH), N-succinimidyl 3-(bromoacetamido)propionate (SBAP), N-succinimidyl iodoacetate (SIA), k-maleimidoundecanoic acid N-succinimidyl ester (KMUA), N-succinimidyl 4-(p-maleimidophenyl)-butyrate (SMPB), succinimidyl-6-(-maleimidopropionamido)hexanoate (SMPH), succinimidyl-(4-vinylsulfonyl)benzoate (SVSB), dithiobis-maleimidoethane (DTME), l,4-bis-maleimidobutane (BMB), l,4-bismaleimidyl-2,3-Dihydroxybutane (BMDB), bis-maleimidohexane (BMH), bis-maleimidoethane (BMOE), sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-l-carboxylate (sulfo-SMCC), sulfosuccinimidyl (4-iodo-acetyl)aminobenzoate (sulfo-SIAB), m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester (sulfo-MBS), N-(y-maleimidobutyryloxy)sulfosuccinimide ester (sulfo-GMBS or sGMBS), N-(e-maleimidocaproyloxy)sulfosuccinimide ester (sulfo-EMCS), N-(K-maleimidoundecanoyloxy)sulfosuccinimide ester (sulfo-KMUS), and sulfosuccinimidyl 4-(p-maleimidophenyl) butyrate (sulfo-SMPB), succinimidyl 6-hydrazinonicotinamide acetone hydrazone (SANH), succinimidyl 4-hydrazide terephthalate hydrochloride (SHTH), succinimidyl hydrazinium nicotinate hydrochloride (SHNH), succinimidyl-p-formylbenzoate (SFB), and succinimidyl-p-formylphenoxyacetate (S FPA), V-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), V-succinimidyl-4-(2-pyridyldithio)pentanoate (SPP), N-succinimidyl-4-(2-pyridyldithio)butanoate (SPDB), and V-succinimidyl-4-(2-pyridyldithio)2-sulfobutanoate (sulfo-SPDB).

[0067] In certain aspects of the present disclosure, the ADCs comprise a cleavable linker. Different mechanisms employed in linkers to release the drug or cytotoxic agent are known in the art. These mechanisms include (1) utilizing a protease or other enzymatic cleavage site in the linker that is cleaved by cellular enzymes (e.g., cathepsin B or β-glucuronidase), (2) using the lower pH of lysosomes to cause hydrolysis of acid-labile units in the linker, or (3) utilizing higher intracellular levels of glutathione to reduce disulfide bridges in the linker.

[0068] In some aspects of the disclosure, the ADCs described herein comprise a linker comprising a peptide unit that provides a site for protease cleavage.In some embodiments, the peptide units are -Gly-Gly-Gly-, -Ala-Val-, -Val-Ala-, -Val-Cit-, -Val-Lys-, -Lys-Val-, -Phe-Lys-, -Lys-Phe-, -Lys-Lys-, -Ala-L ys-, -Lys-Ala-, -Phe-Cit-, -Cit-Phe,-Leu-Cit-, -Cit-Leu-, -Ile-Cit-, -Phe-Ala-, -Ala-Phe-, -Phe-Phe-Lys-, -Lys-Phe-Phe-, -Gly-Phe -Lys-, -Lys-Phe-Gly-, -Leu-Ala-Leu-, -Ile-Ala-Leu-, -Leu-Ala-Ile-, -Val-Ala-Val-, -Ala-Leu-Ala-Leu- (SEQ ID NO: 49), -Leu-Ala-Leu-Ala -(SEQ ID NO:50), -Gly-Phe-Leu-Gly-(SEQ ID NO:51), -Gly-Leu-Phe-Gly-(SEQ ID NO:52), -Val-Arg-, -Arg-Val-, -Arg-Arg-, -Ala-Ala-, -Ala-Met-, -Met-Al a-, -Thr-Thr-, -Thr-Met-, -Met-Thr-, -Leu-Ala-, -Ala-Leu-, -Cit-Val-, -Gln-Val-, -Val-Gln-, -Ser-Val-, -Val-Ser-, -Ser-Ala-, -Ser- Gly-, -Ala-Ser-, -Gly-Ser-, -Leu-Gln-, -Gln-Leu-, -Phe-Arg-, -Arg-Phe-, -Tyr-Arg-, -Arg-Tyr-, -Phe-Gln-, -Gln-Phe-, -Val-Thr-, -Thr -Val-, -Met-Tyr-, -Tyr-Met-, -Ala-Ala-, -Ala-Ala-Ala-, -Ala-Ala-Ala-Ala-(SEQ ID NO: 53), -Gly-Ala-Gly-Gly-(SEQ ID NO: 54), -Gly-Gly-Ala-Gly-(SEQ ID NO: 55), -Gly-Val-Gly-Gly-(SEQ ID NO: 56), -Gly-Gly-Val-Gly-(SEQ ID NO: 57), -Gly-Phe-Gly-Gly-(SEQ ID NO: 58), or -Gly-Gly-Phe-Gly-(SEQ ID NO: 59). Citrulline is represented by Cit.

[0069] In embodiments of the present disclosure, the peptide units contain all naturally occurring amino acids in L-amino acid form. In further embodiments, the peptide units may include all D-amino acids or L-amino acids, or combinations thereof. Thus, the peptide units may include, for example, D-Val-D-Ala, L-Val-L-Ala, D-Val-L-Ala, or L-Val-D-Ala.

[0070] As used herein, the term "sugar-cleaving unit" refers to a site in a linker that contains a sugar moiety that can be cleaved by an enzyme, such as a lysosomal acid hydrolase, such as β-glucuronidase. In some aspects of the disclosure, the ADCs provided herein include a linker that includes a sugar-cleaving unit that provides a site for cleavage. In further aspects, the sugar-cleaving unit is a glucuronide or galactoside, such as a β-glucuronide or β-galactoside moiety. In further aspects, the sugar-cleaving unit includes a β-glucuronide of Formula IX:

[0071] [ka]

[0072] As used herein, the term "hydrophobic masking group" refers to a group capable of reducing the apparent hydrophobicity of a compound. In some embodiments of the present disclosure, the ADCs described herein, particularly the linkers described herein, contain a polysarcosine, polyethylene glycol, or chitooligosaccharide hydrophobic masking entity. In further embodiments, the hydrophobic masking group comprising polysarcosine, polyethylene glycol, or chitooligosaccharide is in a branched configuration rather than being connected linearly on the linker. Examples of such branched configurations for hydrophobic masking groups are found in Formulas XXVI and XXI. The number of ethylene glycol or sarcosine moieties can vary over a wide range. For example, the number of ethylene glycol or sarcosine moieties in the hydrophobic masking entity can be 2 to 500, 5 to 100, or 5 to 25. In further embodiments, the ADCs provided herein have a polysarcosine hydrophobic masking entity containing 2 to 50 sarcosine moieties, 4 to 30 sarcosine moieties, 6 to 24 sarcosine moieties, or 10 to 12 sarcosine moieties. The number of chitosans in the chitooligosaccharide can vary between 2 and 20, or between 2 and 8.

[0073] In embodiments of the present disclosure, the ADCs comprise a linker comprising a connecting unit that connects the linker-payload to the antibody. In further embodiments, the ADCs provided herein have a connecting unit that connects a cysteine ​​of an antibody disclosed herein to a linker and / or payload described herein. Some chemistries used in the art for connecting cysteines include maleimide, succinimide, or bromoacetamide compounds and can be utilized in the ADCs of the present disclosure. In further embodiments, a spacer, such as a CH2 and / or PEG (e.g., 2-PEG or 3-PEG) chain, is utilized as part of the linking unit. In further embodiments of the present disclosure, a maleimide-type connecting unit, such as maleimidocaproyl (mc) or maleimidomethylcyclohexane-1-carboxylate, is used.

[0074] In embodiments of the present disclosure, maleimide-containing linking units that are conjugated to antibodies via Michael addition with cysteine ​​thiols to create succinimide bonds to form ADCs can exist or be slowly converted in vivo to open forms by hydrolytic cleavage of the succinimide.

[0075] In some aspects of the disclosure, provided herein are ADCs having a connecting unit of formula X:

[0076] [ka] In the formula, z is 1 to 5.

[0077] In some aspects of the disclosure, provided herein are ADCs having a connecting unit of formula XI:

[0078] [ka] In the formula, z is 1 to 5.

[0079] In some aspects of the disclosure, provided herein are ADCs having a connecting unit of formula XII:

[0080] [ka] In the formula, z is 1 to 5.

[0081] In some aspects of the disclosure, provided herein are ADCs having a connecting unit of formula XIII:

[0082] [ka] In the formula, z is 1 to 5.

[0083] In another aspect, provided herein is an ADC, wherein the ADC is of Formula II, IIa, or IIb:

[0084] [ka] where Ab is a PTK-7 antibody disclosed herein and n is from about 1 to about 16.

[0085] In another aspect, provided herein is an ADC, wherein the ADC is of formula III, IIIa, or IIIb:

[0086] [ka]

[0087] [ka] where Ab is a PTK-7 antibody disclosed herein and n is from about 1 to about 16.

[0088] In a further embodiment, n is from about 2 to about 12. In another embodiment, n is from about 2 to about 8. In another embodiment, n is from about 4 to about 8. In another embodiment, n is from about 8 to about 12. In another embodiment, n is about 2. In another embodiment, n is about 4. In another embodiment, n is about 6. In another embodiment, n is about 8. In another embodiment, n is about 10. In another embodiment, n is about 12.

[0089] In certain embodiments disclosed herein, the Ab comprises an HC comprising amino acids 2-441 of SEQ ID NO:2 and an LC comprising SEQ ID NO:3, where n is about 8. In certain embodiments disclosed herein, the Ab comprises an HC comprising amino acids 2-441 of SEQ ID NO:2 and an LC comprising SEQ ID NO:3, where n is about 4. In further embodiments disclosed herein, the Ab comprises an HC consisting of SEQ ID NO:2 and an LC consisting of SEQ ID NO:3.

[0090] In certain embodiments disclosed herein, the Ab comprises an HC comprising amino acids 2-448 of SEQ ID NO: 12 and an LC comprising amino acids 2-215 of SEQ ID NO: 13, where n is about 8. In certain embodiments disclosed herein, the Ab comprises an HC comprising amino acids 2-448 of SEQ ID NO: 12 and an LC comprising amino acids 2-215 of SEQ ID NO: 13, where n is about 4. In further embodiments disclosed herein, the Ab comprises an HC consisting of SEQ ID NO: 12 and an LC consisting of SEQ ID NO: 13.

[0091] In certain embodiments disclosed herein, the Ab comprises an HC comprising amino acids 2-447 of SEQ ID NO: 22 and an LC comprising amino acids 2-215 of SEQ ID NO: 23, where n is about 8. In certain embodiments disclosed herein, the Ab comprises an HC comprising amino acids 2-447 of SEQ ID NO: 22 and an LC comprising amino acids 2-215 of SEQ ID NO: 23, where n is about 4. In further embodiments disclosed herein, the Ab comprises an HC consisting of SEQ ID NO: 22 and an LC consisting of SEQ ID NO: 23.

[0092] In certain embodiments disclosed herein, the Ab comprises an HC comprising amino acids 2-444 of SEQ ID NO: 32 and an LC comprising amino acids 2-215 of SEQ ID NO: 33, where n is about 8. In certain embodiments disclosed herein, the Ab comprises an HC comprising amino acids 2-444 of SEQ ID NO: 32 and an LC comprising amino acids 2-215 of SEQ ID NO: 33, where n is about 4. In further embodiments disclosed herein, the Ab comprises an HC consisting of SEQ ID NO: 32 and an LC consisting of SEQ ID NO: 33.

[0093] It is understood that the linkers and linker-payloads described herein can exist as stereoisomers, and embodiments of the present disclosure include all enantiomers, diastereomers, and mixtures thereof.

[0094] Conjugates to anti-PTK-7 antibodies Methods for conjugating the antibodies disclosed herein to the payloads and linker-payloads disclosed herein are known in the art. In some methods, the antibody is conjugated to the linker in a first reaction, and then the antibody and linker are conjugated to the payload in a second reaction. In some methods, the antibody is conjugated to the payload or payload / linker in a single reaction.

[0095] In some embodiments of the present disclosure, the PTK-7 antibody described herein is covalently linked to the camptothecin analog described herein via the thiol group of one or more cysteine ​​residues located on the PTK-7 antibody. In further embodiments, the cysteine ​​residues used in the conjugate are each interchain disulfide cysteine ​​residues. Methods for controlling the reduction of interchain disulfides to allow conjugation to the participating cysteines are known in the art. In other embodiments, the cysteine ​​residues used in the conjugate are engineered into the antibody separately from those used in the interchain disulfides.

[0096] In some embodiments of the ADCs disclosed herein, the Ab is conjugated to the linker-payload via the thiol group of one or more cysteine ​​residues. In some embodiments, the Ab is conjugated via the thiol group of one or more cysteine ​​residues in the HC of the Ab disclosed herein. In some embodiments, the Ab is conjugated via the thiol group of one or more cysteine ​​residues, and the cysteines are selected from C219, C225, and C228 of the heavy chain and C215 of the light chain, or a combination thereof.

[0097] In another aspect of the disclosure, the PTK-7 antibodies described herein are covalently linked to a camptothecin analog described herein through the amino group of one or more lysine residues located on the PTK-7 antibody. In another aspect of the disclosure, the PTK-7 antibodies described herein are covalently linked to a camptothecin analog described herein through the amino group of one or more glutamine residues located on the PTK-7 antibody.

[0098] In certain embodiments, provided herein are ADCs wherein the attachment of the cytotoxic agent, cytotoxic agent-self-immolative spacer, or cytotoxic agent-self-immolative spacer-linker to the antibody occurs via a thiol group on one or more cysteines of the antibody. In further embodiments, each of the one or more cysteines is a naturally occurring cysteine ​​in the hinge region of the antibody.

[0099] In certain aspects, ADCs are provided herein, wherein the linker-payload used in conjugation to a PTK7 antibody described herein is of Formula XIV or XV:

[0100] [ka]

[0101] The present disclosure provides a method of producing an ADC, the method comprising: (a) reducing a PTK-7 antibody disclosed herein with a reducing agent to produce a reduced PTK-7 antibody; (b) contacting the reduced PTK-7 antibody with a compound of the disclosure to produce a conjugate, wherein the compound comprises a linker-payload disclosed herein, such as Formula XIV or XV. In a further embodiment, the reducing agent is DTT or TCEP.

[0102] The conjugates of the present disclosure or salts thereof can be readily prepared by a variety of procedures known to those skilled in the art, some of which are illustrated in the preparations and examples below. Those skilled in the art will recognize that the specific synthetic steps for each of the described routes can be combined in different ways or steps from different schemes can be combined to prepare the conjugates of the present disclosure or salts thereof. The products of each step can be recovered by conventional methods well known in the art, including extraction, evaporation, precipitation, chromatography, filtration, trituration, and crystallization. All substituents are as previously defined unless otherwise indicated. Reagents and starting materials are readily available to those skilled in the art. The following preparations, examples, and assays further illustrate the present disclosure but should in no way be construed as limiting the scope of the disclosure.

[0103] therapeutic use In another aspect, provided herein is a method of treating cancer comprising administering to a patient in need thereof an effective amount of a PTK-7 ADC or pharmaceutical composition described herein. In a further aspect, provided herein is a method of treating cancer comprising administering to a patient in need thereof an effective amount of an ADC or pharmaceutical composition described herein, wherein the cancer is ovarian cancer, endometrial cancer, lung cancer, head and neck cancer, thyroid cancer, breast cancer, gastric cancer, kidney cancer, prostate cancer, liver cancer, pancreatic cancer, or colorectal cancer.

[0104] In a further aspect, a method of treating cancer is provided, wherein the cancer is ovarian cancer. In a further aspect, a method of treating cancer is provided, wherein the cancer is endometrial cancer. In a further aspect, a method of treating cancer is provided, wherein the cancer is lung cancer. In a further aspect, a method of treating cancer is provided, wherein the cancer is head and neck cancer. In a further aspect, a method of treating cancer is provided, wherein the cancer is thyroid cancer. In a further aspect, a method of treating cancer is provided, wherein the cancer is breast cancer. In a further aspect, a method of treating cancer is provided, wherein the cancer is gastric cancer. In a further aspect, a method of treating cancer is provided, wherein the cancer is kidney cancer. In a further aspect, a method of treating cancer is provided, wherein the cancer is prostate cancer. In a further aspect, a method of treating cancer is provided, wherein the cancer is liver cancer. In a further aspect, a method of treating cancer is provided, wherein the cancer is pancreatic cancer. In a further aspect, a method of treating cancer is provided, wherein the cancer is colon cancer.

[0105] In a further aspect, the patient being treated with an ADC or pharmaceutical composition described herein has previously received a programmed death receptor-1 (PD-1) or programmed death-ligand 1 (PD-L1) inhibitor, with or without platinum-containing chemotherapy, in the neoadjuvant / adjuvant, locally advanced, or metastatic setting.

[0106] In a further aspect, methods are provided that comprise administering an effective amount of an ADC or pharmaceutical composition described herein in simultaneous, separate, or sequential combination with one or more anti-tumor agents. In a further aspect, methods are provided that comprise administering an effective amount of an ADC or pharmaceutical composition described herein in simultaneous, separate, or sequential combination with a PD-1 inhibitor or a PD-L1 inhibitor.

[0107] In another aspect, provided herein is a PTK-7 ADC or pharmaceutical composition described herein for use in therapy. In a further aspect, provided is an ADC or pharmaceutical composition described herein for use in treating cancer. In a further aspect, the cancer is ovarian cancer, endometrial cancer, lung cancer, head and neck cancer, thyroid cancer, breast cancer, gastric cancer, kidney cancer, prostate cancer, liver cancer, pancreatic cancer, or colorectal cancer.

[0108] In a further aspect, provided herein is an ADC or pharmaceutical composition described herein for use in the treatment of ovarian cancer. In a further aspect, provided herein is an ADC or pharmaceutical composition described herein for use in the treatment of endometrial cancer. In a further aspect, provided herein is an ADC or pharmaceutical composition described herein for use in the treatment of lung cancer. In a further aspect, provided herein is an ADC or pharmaceutical composition described herein for use in the treatment of head and neck cancer. In a further aspect, provided herein is an ADC or pharmaceutical composition described herein for use in the treatment of thyroid cancer. In a further aspect, provided herein is an ADC or pharmaceutical composition described herein for use in the treatment of breast cancer. In a further aspect, provided herein is an ADC or pharmaceutical composition described herein for use in the treatment of gastric cancer. In a further aspect, provided herein is an ADC or pharmaceutical composition described herein for use in the treatment of kidney cancer. In a further aspect, provided herein is an ADC or pharmaceutical composition described herein for use in the treatment of prostate cancer. In a further aspect, provided herein is an ADC or pharmaceutical composition described herein for use in the treatment of liver cancer. In a further aspect, provided herein is an ADC or pharmaceutical composition described herein for use in the treatment of pancreatic cancer. In a further aspect, provided herein is an ADC or pharmaceutical composition described herein for use in the treatment of colorectal cancer.

[0109] In a further aspect, provided herein is an ADC or pharmaceutical composition described herein for use in the treatment of cancer, wherein the prior use occurs to a PD-1 or PD-L1 inhibitor with or without platinum-containing chemotherapy in the neoadjuvant / adjuvant, locally advanced, or metastatic setting.

[0110] In a further aspect, provided herein is an ADC or pharmaceutical composition described herein in simultaneous, separate, or sequential combination with one or more anti-tumor agents for use in the treatment of cancer. In a further aspect, the anti-tumor agent is a PD-1 inhibitor or a PD-L1 inhibitor.

[0111] In another aspect, provided herein is the use of a PTK-7 ADC or pharmaceutical composition described herein for the manufacture of a medicament for the treatment of cancer. In a further aspect, provided herein is the use of an ADC or pharmaceutical composition described herein for the manufacture of a medicament for the treatment of cancer, wherein the cancer is ovarian cancer, endometrial cancer, lung cancer, head and neck cancer, thyroid cancer, breast cancer, gastric cancer, kidney cancer, prostate cancer, liver cancer, pancreatic cancer, or colorectal cancer.

[0112] In a further aspect, provided herein is the use of an ADC or pharmaceutical composition described herein in the manufacture of a medicament for the treatment of cancer, where the prior use of a PD-1 or PD-L1 inhibitor, with or without platinum-containing chemotherapy, in the neoadjuvant / adjuvant, locally advanced, or metastatic setting.

[0113] In a further aspect, provided herein is the use of an ADC or pharmaceutical composition described herein in the manufacture of a medicament for the treatment of cancer, wherein the medicament is administered simultaneously, separately, or sequentially with one or more anti-tumor agents. In a further aspect, provided herein is the use of an ADC or pharmaceutical composition described herein in the manufacture of a medicament for the treatment of cancer, wherein the medicament is administered simultaneously, separately, or sequentially with a PD-1 inhibitor or a PD-L1 inhibitor.

[0114] In a further embodiment, the breast cancer is HR-positive, HER2-negative, or triple-negative breast cancer (TNBC). In a further embodiment, the breast cancer is ductal or lobular. In a further embodiment, the lung cancer is squamous non-small cell lung cancer (NSCLC) or non-squamous NSCLC. In a further embodiment, the NSCLC is metastatic or advanced. In a further embodiment, the lung cancer is squamous, adenocarcinoma, or small cell carcinoma. In a further embodiment, the lung cancer is small cell lung cancer (SCLC). In a further embodiment, the small cell lung cancer is metastatic or advanced. In a further embodiment, the gastric cancer is esophageal cancer or gastroesophageal junction cancer. In a further embodiment, the ovarian cancer is metastatic or advanced ovarian cancer. In a further embodiment, the ovarian cancer is serous or mucinous. In a further embodiment, the ovarian cancer is fallopian tube or peritoneal. In a further aspect, the ovarian cancer is fallopian tube or epithelial. In a further aspect, the ovarian cancer patient has recurrent or refractory disease after treatment with one or a combination of paclitaxel, carboplatin, platinum-based chemotherapy, and mirvetuximab soravtansine.

[0115] In a further aspect, the patient or cancer has relapsed or become refractory to one or more of the following standard therapies: carboplatin and etoposide with or without atezolizumab (or durvalumab), cisplatin and etoposide with or without atezolizumab (or durvalumab), pembrolizumab, carboplatin and pemetrexed with or without pembrolizumab, docetaxel with or without ramucirumab, carboplatin and paclitaxel with or without bevacizumab.

[0116] In further embodiments, the anti-tumor agent may be a chemotherapy treatment agent, including platinum-containing chemotherapy, and / or may include cisplatin, carboplatin, dacarbazine, liposomal doxorubicin, docetaxel, cyclophosphamide and doxorubicin, navelbine, eribulin, paclitaxel, paclitaxel protein-bound particle injectable suspension, ixabepilone, capecitabine, FOLFOX (leucovorin, fluorouracil, and oxaliplatin), FOLFIRI (leucovorin, fluorouracil, and irinotecan), gemcitabine, topotecan, liposomal irinotecan, pemetrexed, and cetuximab. In a further aspect, the anti-tumor agent can be an immuno-oncology agent, including one selected from the group consisting of nivolumab, ipilimumab, pidilizumab, pembrolizumab, tremelimumab, urelumab, lirilumab, atezolizumab, epacadostat, and durvalumab. In a further aspect, the anti-tumor agent can be carboplatin and etoposide with or without atezolizumab (or durvalumab), cisplatin and etoposide with or without atezolizumab (or durvalumab), pembrolizumab, pembrolizumab with or without carboplatin and pemetrexed, docetaxel with or without ramucirumab, carboplatin and paclitaxel with or without bevacizumab.

[0117] Pharmaceutical compositions and methods of administration The antibodies or ADCs described herein can be formulated as pharmaceutical compositions, which are administered by any route that makes the antibodies or ADCs bioavailable, such as, for example, oral, topical, or subcutaneous administration.

[0118] Also provided herein are pharmaceutical compositions comprising an antibody or ADC provided herein and one or more agents selected from the group consisting of physiologically acceptable carriers, diluents, excipients, and adjuvants.

[0119] The antibodies or ADCs of the present disclosure, or pharmaceutical compositions comprising same, can be administered parenterally (e.g., subcutaneously and intravenously). The antibodies or ADCs of the present disclosure can be administered alone to a patient in a single dose or multiple doses together with a pharmaceutically acceptable carrier, diluent, or excipient. The pharmaceutical compositions described herein can be prepared by methods well known in the art (e.g., Remington: The Science and Practice of Pharmacy, 22nd ed. (2012), A. Loyd et al., Pharmaceutical Press) and comprise an antibody or ADC disclosed herein and one or more pharmaceutically acceptable carriers, diluents, or excipients.

[0120] Disclosed herein in certain aspects are pharmaceutical compositions comprising an antibody disclosed herein and one or more pharmaceutically acceptable carriers, diluents, or excipients. Disclosed herein in certain aspects are pharmaceutical compositions comprising an ADC disclosed herein and one or more pharmaceutically acceptable carriers, diluents, or excipients.

[0121] definition As used herein, the terms "a," "an," "the," and similar terms as used in the context of this disclosure (particularly in the context of the claims) should be construed to cover both the singular and the plural, unless otherwise specified herein or clearly contradicted by context.

[0122] The term "bind," as used herein, unless otherwise specified, is intended to mean the ability of a protein or molecule to form a chemical bond or attractive interaction with another protein or molecule, resulting in proximity of the two proteins or molecules as determined by common methods known in the art.

[0123] As used herein, the term "effective amount" refers to the amount (duration and means of administration) necessary to achieve the desired therapeutic result. The effective amount of a protein or conjugate may vary depending on factors such as the individual's medical condition, age, sex, and weight, and the ability of the protein or conjugate to elicit a desired response in the individual. An effective amount is also one in which any toxic or detrimental effects of the protein or conjugate are outweighed by the therapeutically beneficial effects.

[0124] As used herein, the terms "treat," "treatment," or "treating" refer to any process that may slow, control, retard, or stop the progression of a disorder or disease disclosed herein, or that may ameliorate the disorder or disease symptoms, but does not necessarily indicate the complete disappearance of all disorder or disease symptoms.

[0125] As used herein, the term "patient" refers to a human patient.

[0126] Certain abbreviations are defined as follows: "ACN" refers to acetonitrile, "AEEA" refers to 2-aminoethoxy-2-ethoxyacetic acid, "CTC" refers to 2-chlorotrityl chloride, "DCM" refers to dichloromethane, "DEA" refers to diethylamine, "DIC" refers to N,N'-diisopropylcarbodiimide, "DIPEA" refers to N,N-diisopropylethylamine, and "DBU" refers to 1,8-diazabicyclo[5.4 0.0]undec-7-ene, "DMF" refers to N,N-dimethylformamide, "DTT" refers to dithiothreitol, "EtOAc" refers to ethyl acetate, "EDTA" refers to ethylenediaminetetraacetic acid, "EDCI" refers to 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, "FA" refers to formic acid, "h" refers to hour, "Fmoc" refers to fluorenylmethyloxycarbonyl, and "HEPES" refers to (N-2-hydroxybenzoates). "HATU" refers to hexafluorophosphate azabenzotriazole tetramethyluronium; "HBTU" refers to hexafluorophosphate benzotriazole tetramethyluronium; "HOBt" refers to hydroxybenzotriazole; "HOPO" refers to 2-hydroxypyridine-1-oxide; "MeOH" refers to methanol; and "MTBE" refers to methyl tert -butyl ether, "MWCO" refers to molecular weight cut off, "NHS" refers to N-hydroxysuccinimide, "NMP" refers to (N-methyl-2-pyrrolidone), "OAll" refers to allyloxy, "Sar" refers to sarcosine, "Su" refers to succinimide, "THF" refers to tetrahydrofuran, "TsOH" refers to p-toluenesulfonic acid, and "TCEP" refers to (tris(2-carboxyethyl)phosphine).

[0127] Embodiment Embodiments of the present disclosure are contemplated, including, but not limited to, the following. 1. An antibody that binds to human PTK-7, wherein the antibody comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises heavy chain complementarity determining regions (HCDRs) HCDR1, HCDR2, and HCDR3, and the VL comprises light chain complementarity determining regions (LCDRs) LCDR1, LCDR2, and LCDR3; a) HCDR1 comprises SEQ ID NO: 4, HCDR2 comprises SEQ ID NO: 5, HCDR3 comprises SEQ ID NO: 6, LCDR1 comprises SEQ ID NO: 7, LCDR2 comprises SEQ ID NO: 8, and LCDR3 comprises SEQ ID NO: 9; b) HCDR1 comprises SEQ ID NO: 14, HCDR2 comprises SEQ ID NO: 15, HCDR3 comprises SEQ ID NO: 16, LCDR1 comprises SEQ ID NO: 17, LCDR2 comprises SEQ ID NO: 18, and LCDR3 comprises SEQ ID NO: 19; c) HCDR1 comprises SEQ ID NO: 24, HCDR2 comprises SEQ ID NO: 25, HCDR3 comprises SEQ ID NO: 26, LCDR1 comprises SEQ ID NO: 27, LCDR2 comprises SEQ ID NO: 28, and LCDR3 comprises SEQ ID NO: 29, or d) An antibody wherein HCDR1 comprises SEQ ID NO: 34, HCDR2 comprises SEQ ID NO: 35, HCDR3 comprises SEQ ID NO: 36, LCDR1 comprises SEQ ID NO: 37, LCDR2 comprises SEQ ID NO: 18, and LCDR3 comprises SEQ ID NO: 38. 2. The antibody of embodiment 1, wherein HCDR1 comprises SEQ ID NO: 4, HCDR2 comprises SEQ ID NO: 5, HCDR3 comprises SEQ ID NO: 6, LCDR1 comprises SEQ ID NO: 7, LCDR2 comprises SEQ ID NO: 8, and LCDR3 comprises SEQ ID NO: 9. 3. The antibody of embodiment 1, wherein HCDR1 comprises SEQ ID NO: 14, HCDR2 comprises SEQ ID NO: 15, HCDR3 comprises SEQ ID NO: 16, LCDR1 comprises SEQ ID NO: 17, LCDR2 comprises SEQ ID NO: 18, and LCDR3 comprises SEQ ID NO: 19. 4. The antibody of embodiment 1, wherein HCDR1 comprises SEQ ID NO: 24, HCDR2 comprises SEQ ID NO: 25, HCDR3 comprises SEQ ID NO: 26, LCDR1 comprises SEQ ID NO: 27, LCDR2 comprises SEQ ID NO: 28, and LCDR3 comprises SEQ ID NO: 29. 5. The antibody of embodiment 1, wherein HCDR1 comprises SEQ ID NO: 34, HCDR2 comprises SEQ ID NO: 35, HCDR3 comprises SEQ ID NO: 36, LCDR1 comprises SEQ ID NO: 37, LCDR2 comprises SEQ ID NO: 18, and LCDR3 comprises SEQ ID NO: 38. 6. a) the VH comprises SEQ ID NO: 10 and the VL comprises SEQ ID NO: 11; b) the VH comprises SEQ ID NO: 20 and the VL comprises SEQ ID NO: 21; c) the VH comprises SEQ ID NO: 30 and the VL comprises SEQ ID NO: 31; d) The antibody of embodiment 1, wherein the VH comprises SEQ ID NO: 39 and the VL comprises SEQ ID NO: 40. 7. The antibody of embodiment 6, wherein the antibody comprises a VH comprising SEQ ID NO: 10 and a VL comprising SEQ ID NO: 11. 8. The antibody of embodiment 6, wherein the antibody comprises a VH comprising SEQ ID NO: 20 and a VL comprising SEQ ID NO: 21. 9. The antibody of embodiment 6, wherein the antibody comprises a VH comprising SEQ ID NO: 30 and a VL comprising SEQ ID NO: 31. 10. The antibody of embodiment 6, wherein the antibody comprises a VH comprising SEQ ID NO: 39 and a VL comprising SEQ ID NO: 40. 11. The antibody of any one of embodiments 1 to 10, wherein the antibody has a human IgG1 or IgG4 isotype. 12. The antibody of embodiment 11, wherein the antibody has a human IgG1 isotype. 13. The antibody of embodiment 12, comprising alanines at residues 234 and 235 (according to EU index numbering). 14. The antibody of embodiment 13, further comprising a serine at position 265 (according to EU index numbering). 15. The antibody comprises a heavy chain (HC) and a light chain (LC), a) HC comprises amino acids 2 to 441 of SEQ ID NO: 2 and LC comprises SEQ ID NO: 3; b) HC comprises amino acids 2 to 448 of SEQ ID NO: 12, and LC comprises amino acids 2 to 215 of SEQ ID NO: 13; c) HC comprises amino acids 2 to 447 of SEQ ID NO: 22 and LC comprises amino acids 2 to 215 of SEQ ID NO: 23, or d) The antibody of embodiment 1, wherein the HC comprises amino acids 2 to 444 of SEQ ID NO: 32 and the LC comprises amino acids 2 to 215 of SEQ ID NO: 33. 16. a) HC consists of SEQ ID NO: 2 and LC consists of SEQ ID NO: 3; b) HC consists of SEQ ID NO: 12 and LC consists of SEQ ID NO: 13; c) HC consists of SEQ ID NO: 22 and LC consists of SEQ ID NO: 23, or d) The antibody of embodiment 15, wherein the HC consists of SEQ ID NO: 32 and the LC consists of SEQ ID NO: 33. 17. The antibody of embodiment 15 or 16, wherein the HC consists of SEQ ID NO: 2 and the LC consists of SEQ ID NO: 3. 18. The antibody of embodiment 15 or 16, wherein HC consists of SEQ ID NO: 12 and LC consists of SEQ ID NO: 13. 19. The antibody of embodiment 15 or 16, wherein the HC consists of SEQ ID NO: 22 and the LC consists of SEQ ID NO: 23. 20. The antibody of embodiment 15 or 16, wherein HC consists of SEQ ID NO: 32 and LC consists of SEQ ID NO: 33. 21. An antibody-drug conjugate (ADC) comprising the antibody of any one of embodiments 1 to 20 conjugated to a cytotoxic agent. 22. The ADC of embodiment 21, wherein the cytotoxic agent is selected from the group consisting of a microtubule inhibitor, a topoisomerase I inhibitor, a DNA damaging agent, a DNA alkylating agent, and a DNA minor groove binder. 23. The ADC of embodiment 21 or 22, wherein the cytotoxic agent is a topoisomerase I inhibitor. 24. The ADC of embodiment 23, wherein the topoisomerase I inhibitor is a camptothecin analogue. 25. A camptothecin analog having the formula:

[0128] [ka] 25. The ADC of embodiment 24, comprising the compound: 26. A camptothecin analogue having the formula:

[0129] [ka] 26. The ADC of embodiment 24 or 25, comprising one of the compounds 27. The ADC of any one of embodiments 21-26, wherein the ADC further comprises a linker connecting the antibody to the cytotoxic agent. 28. The ADC of embodiment 27, wherein the linker comprises a peptide unit or a glycocleaving unit. 29. The ADC of embodiment 28, wherein the peptide unit comprises Val-Ala, Val-Cit, Phe-Lys, or Ala-Ala-Asn. 30. The ADC of embodiment 29, wherein the peptide unit comprises Val-Ala. 31. The ADC of embodiment 29, wherein the peptide unit comprises Val-Cit. 32. ADC is expressed by the formula:

[0130] [ka] 31. The ADC of any one of embodiments 21-30, comprising a compound of: 33. A sugar-cleaving unit having the formula:

[0131] [ka] 29. The ADC of embodiment 28, comprising the compound: 34. ADC is a compound having the formula:

[0132] [ka] 34. The ADC of embodiment 33, comprising the compound: 35. The ADC of any one of embodiments 21-34, wherein the linker further comprises a hydrophobic masking group. 36. The ADC of embodiment 35, wherein the hydrophobic masking group is selected from polysarcosine or polyethylene glycol. 37. The ADC of embodiment 35 or 36, wherein the hydrophobic masking groups are in a branched arrangement on the linker. 38. The hydrophobic masking group has the formula:

[0133] [ka] wherein k is an integer from 6 to 12, and X1 is H, OH, or NH2. 39. The ADC of embodiment 38, wherein k is 10. 40. The ADC of embodiment 38, wherein k is 12. 41. The ADC of any one of embodiments 27-40, wherein the linker further comprises a connecting unit. 42. The connection unit is the formula:

[0134] [ka] wherein z is 1 to 5. 43. The connection unit is the formula:

[0135] [ka] wherein z is 1 to 5. 44. The connection unit is the formula:

[0136] [ka] wherein z is 1 to 5. 45. The connection unit is the formula:

[0137] [ka] wherein z is 1 to 5. 46. ​​An antibody-drug conjugate (ADC), wherein the ADC has the formula:

[0138] [ka] is one of the compounds During the ceremony, Ab is an antibody according to any one of embodiments 1 to 21; n is about 1 to 16, ADC. 47. The ADC of embodiment 46, wherein n is about 2 to 12. 48. The ADC of embodiment 46, wherein n is about 2 to 8. 49. ADC is a compound having the formula:

[0139] [ka] 49. The ADC of any one of embodiments 46-48, wherein the ADC is a compound of the formula: 50.ADC is expressed as:

[0140] [ka] 49. The ADC of any one of embodiments 46-48, wherein the ADC is a compound of the formula: 51. The ADC of any one of embodiments 46-50, wherein n is about 2. 52. The ADC of any one of embodiments 46-50, wherein n is about 4. 53. The ADC of any one of embodiments 46-50, wherein n is about 6. 54. The ADC of any one of embodiments 46-50, wherein n is about 8. 55. The ADC of any one of embodiments 46-54, wherein the connection to the antibody occurs via a thiol group of one or more cysteines of the antibody. 56. The ADC of embodiment 55, wherein the one or more cysteines are each a naturally occurring cysteine ​​in the hinge region of the antibody. 57. The ADC of any one of embodiments 46-56, wherein the Ab comprises an HC comprising amino acids 2-441 of SEQ ID NO: 2 and an LC comprising SEQ ID NO: 3, and n is about 8. 58. The ADC of embodiment 57, wherein the Ab comprises an HC consisting of SEQ ID NO:2 and an LC consisting of SEQ ID NO:3. 59. The ADC of any one of embodiments 46-56, wherein the Ab comprises an HC comprising amino acids 2-448 of SEQ ID NO: 12 and an LC comprising amino acids 2-215 of SEQ ID NO: 13, and n is about 8. 60. The ADC of embodiment 59, wherein the Ab comprises an HC consisting of SEQ ID NO: 12 and an LC consisting of SEQ ID NO: 13. 61. The ADC of any one of embodiments 46-56, wherein the Ab comprises an HC comprising amino acids 2-447 of SEQ ID NO: 22 and an LC comprising amino acids 2-215 of SEQ ID NO: 23, and n is about 8. 62. The ADC of embodiment 61, wherein the Ab comprises an HC consisting of SEQ ID NO: 22 and an LC consisting of SEQ ID NO: 23. 63. The ADC of any one of embodiments 46-56, wherein the Ab comprises amino acids 2-444 of SEQ ID NO: 32 and an LC comprising amino acids 2-215 of SEQ ID NO: 33, and n is about 8. 64. The ADC of embodiment 63, wherein the Ab comprises an HC consisting of SEQ ID NO: 32 and an LC consisting of SEQ ID NO: 33. 65. A pharmaceutical composition comprising an antibody according to any one of embodiments 1 to 20 and one or more pharmaceutically acceptable carriers, diluents or excipients. 66. A pharmaceutical composition comprising an ADC according to any one of embodiments 21 to 64, and one or more pharmaceutically acceptable carriers, diluents, or excipients. 67. A method of treating cancer, comprising administering to a patient in need thereof an effective amount of an ADC of any one of embodiments 21-64. 68. The method of embodiment 67, wherein the cancer is ovarian cancer, lung cancer, breast cancer, stomach cancer, kidney cancer, prostate cancer, liver cancer, or colon cancer. 69. The method of embodiment 67 or 68, further comprising administering simultaneously, separately or sequentially a PD-1 inhibitor or a PD-L1 inhibitor. 70. An ADC according to any one of embodiments 21-64, for use in therapy. 71. An ADC according to any one of embodiments 21 to 64 for use in the treatment of cancer. 72. The ADC for use according to embodiment 71, wherein the cancer is ovarian cancer, lung cancer, breast cancer, gastric cancer, kidney cancer, prostate cancer, liver cancer, or colon cancer. 73. The ADC for use according to any one of embodiments 21 to 64, wherein the ADC is administered in combination simultaneously, separately or sequentially with a PD-1 inhibitor or a PD-L1 inhibitor. 74. A pharmaceutical composition for use in the treatment of cancer, comprising an effective amount of an ADC of any one of embodiments 21-64. 75. The composition for use according to embodiment 74, wherein the cancer is ovarian cancer, lung cancer, breast cancer, stomach cancer, kidney cancer, prostate cancer, liver cancer, or colon cancer. 76. The composition according to embodiment 74 or 75, which is administered in combination with a PD-1 inhibitor or a PD-L1 inhibitor simultaneously, separately or sequentially. 77. Use of an ADC according to any one of embodiments 21 to 64 for the manufacture of a medicament for the treatment of cancer. 78. The use according to embodiment 77, wherein the cancer is ovarian cancer, lung cancer, breast cancer, stomach cancer, kidney cancer, prostate cancer, liver cancer, or colon cancer. 79. The use according to embodiment 77 or 78, wherein the medicament further comprises a PD-1 inhibitor or a PD-L1 inhibitor. 80. A method for preparing an ADC, comprising conjugating an antibody according to any one of embodiments 1 to 20 to a linker-payload. 81. The linker-payload has the formula:

[0141] [ka] 81. The method of embodiment 80, comprising the compound of formula (I). 82. The linker-payload has the formula:

[0142] [ka] 81. The method of embodiment 80, comprising the compound of formula (I). 83. A method for producing an ADC, comprising administering an antibody according to any one of embodiments 1 to 20 to a compound of the formula:

[0143] [ka] with a compound of formula (I). 84. The method of embodiment 83, further comprising, prior to the contacting, reducing the antibody with a reducing agent to produce a reduced PTK-7 antibody. [Example]

[0144] Example 1: Generation of PTK-7 antibodies The amino acid sequences of the CDRs, variable regions, complete heavy and light chains, and the nucleotide sequences encoding them of Antibodies 1-4 are listed below in the section entitled "Amino Acid and Nucleotide Sequences." Additionally, SEQ ID NOs for the CDRs, light chains, heavy chains, light chain variable regions, and heavy chain variable regions of Antibodies 1-4 are set forth in Tables 1 and 2.

[0145] Anti-PTK-7 antibodies of the present disclosure, including but not limited to antibodies 1-4, can be expressed and purified essentially as follows: Antibodies 1-4 are fully human antibodies generated from immunization in transgenic systems.

[0146] The antibody was synthesized and purified by well-known methods. Genes encoding the antibody heavy and light chain variable regions were cloned into PBv2-pKlight and pPB-huIgG1(AAS) vectors for mAb expression and purification according to standard procedures. The light chain is human kappa. The heavy chain is human IgG1-AAS:L234A / L235A / D265S based on the EU index number.

[0147] Suitable host cells, such as Chinese hamster ovary cells (CHO), can be transiently or stably transfected with an expression system to secrete antibodies using a predetermined HC:LC ratio when two vectors are used, or using a single vector system encoding both the heavy and light chains. The clarified medium into which the antibody is secreted can be purified using commonly used techniques. Initial capture utilizes Protein A column chromatography using conventional methods and buffer systems, followed by ion exchange and / or hydrophobic interaction chromatography as a polishing step to remove host cell proteins and high molecular weight species. The product can be immediately frozen, for example, at -80°C, or stored at 2-8°C for several months.

[0148] [Table 3]

[0149] [Table 4]

[0150] [Table 5]

[0151] Example 2: Generation of PTK-7 ADC Synthesis of PSAR-Glucuronide-Exatecan-Containing Linker-Payload of Formula XIV

[0152] [ka]

[0153] To prepare for conjugation to the PTK antibodies of the present disclosure, linker-payloads of Formula XVI were synthesized from precursors or intermediates including polysarcosine (PSAR) compounds, 4-beta-glucuronide-3-nitro-octapamine compounds, exatecan compounds, and maleimido-proprionyl compounds. Linker-payloads can be synthesized using methods disclosed in WO 2019081455 and WO 2022207699.

[0154] PSAR intermediate: FmocNH-PEG2-Glu(Su)-PSAR10-NH 2

[0155] [ka]

[0156] Scheme 1

[0157] [ka]

[0158] The PSAR intermediate can be prepared using Scheme 1. On-resin synthesis of polysarcosine is carried out using an iterative submonomer synthesis procedure for Rink amide with the commercially available Fmoc-Sar-Sar-OH dipeptoid building block. Unless otherwise specified, all reactions are carried out at room temperature.

[0159] The starting material is Rink amide preloaded with the first Fmoc-sarcosine residue. The Rink amide preloaded with Fmoc-sarcosine is treated with 20% piperidine in DMF (1 mL per 100 mg of resin) twice for 15 minutes at room temperature. The resin is then washed with DMF (4 times) and DCM (4 times). A solution of Fmoc-Sar-Sar-OH (3 equivalents), HATU (2.9 equivalents), and DIPEA (6 equivalents) in DMF (1 mL per 100 mg of resin) is added to the resin. The reaction vessel is agitated for 2 hours, and the resin is washed with DMF (4 times) and DCM (4 times). The resin is treated with 20% piperidine in DMF (1 mL per 100 mg of resin) twice for 15 minutes at room temperature. The resin is then washed with DMF (4x) and DCM (4x) to obtain Rink resin with n = 3 polysarcosine oligomers.

[0160] The polysarcosine oligomer (n = 3) is elongated using a submonomer synthesis procedure by alternating bromoacetylation and amine substitution steps until the desired length is achieved. The bromoacetylation step is carried out by adding 10 equivalents of bromoacetic acid and 13 equivalents of diisopropylcarbodiimide in DMF (2 mL per 100 mg of resin). The mixture is stirred for 30 minutes, drained, and washed with DMF (4 times). For the amine substitution step, 40% (wt) methylamine in water (1.5 mL per 100 mg of resin) is added, and the vessel is shaken for 30 minutes, drained, and washed with DMF (4 times) and DCM (4 times).

[0161] Once the desired polysarcosine oligomer length is reached (e.g., PSAR 10-mer), orthogonal chemical functionalization is performed. This may be followed by final capping with an Fmoc-protected amino acid group or other group. The Fmoc-protecting group may be removed before or after resin cleavage.

[0162] Polysarcosine can be functionalized with glutamic acid and amino-3,6-dioxaoctanoic acid as follows: Fmoc-Glu(OAll)-OH (3 equivalents), HATU (2.9 equivalents), and DIPEA (6 equivalents) in DMF (1 mL per 100 mg of resin) are added to Rink resin. The reaction vessel is agitated for 90 minutes, and the resin is washed with DMF (4 times) and DCM (4 times). The resin is then treated twice with 20% piperidine in DMF (1 mL per 100 mg of resin) for 15 minutes at room temperature. The resin is washed with DMF (4 times) and DCM (4 times), followed by coupling with Fmoc-amino-3,6-dioxaoctanoic acid (3 equivalents), HATU (2.9 equivalents), and DIPEA (6 equivalents) in DMF (1 mL per 100 mg of resin) for 1 hour. The resin is washed with DMF (4 times) and DCM (4 times). The alloc protecting group is removed by two 30-minute treatments with a DCM solution containing 0.25 equivalents of Pd(PPh3)4 and 20 equivalents of phenylsilane (gently stirred under an argon stream). The resin is then washed with DMF (5 times) and DCM (5 times). The N-hydroxysuccinimide (NHS) ester is introduced onto the carboxylic acid side chain of the final polysarcosine compound by treatment with a DMF solution containing 50 equivalents of DIC and 60 equivalents of N-hydroxysuccinimide (1.5 mL per 100 mg of resin) for 90 minutes. The resin is then washed with DMF (4 times) and DCM (4 times). The final polysarcosine compound is then cleaved from the resin (100% TFA, 2 times, 30 minutes).

[0163] 4-beta-glucuronide-3 nitro-octopamine intermediate: (2S,3R,4S,5S,6S)-2-(4-(2-((tert-butoxycarbonyl)amino)-1-(((4-nitrophenoxy)carbonyl)oxy)ethyl)-2-nitrophenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate

[0164] [ka]

[0165] Scheme 2

[0166] [ka]

[0167] 4-beta-glucuronide-3-nitro-octopamine intermediate 4-beta-glucuronide-3-nitro-octopamine intermediate: (2S,3R,4S,5S,6S)-2-(4-(2-((tert-butoxycarbonyl)amino)-1-(((4-nitrophenoxy)carbonyl)oxy)ethyl)-2-nitrophenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate can be prepared using Scheme 2.

[0168] The octopamine or octopamine intermediate utilized in the present method can be obtained as a racemic mixture or as an enantiopure compound. The racemic mixture can be subjected to chiral separation as known in the art to obtain the enantiopure compound.

[0169] Octopamine (±) hydrochloride (1690 mg, 11 mmol) is suspended in 4 mL of distilled water. The flask is cooled to 0 °C and 4 mL of pre-chilled 65% nitric acid solution is slowly added. The reaction is maintained at 0 °C for 20 min, and mononitration is evaluated by HPLC. The mononitrated octopamine precursor is transferred to a pre-chilled 250 mL Erlenmeyer flask and slowly neutralized with saturated NaHCO3 solution at 0 °C until a pH value of 8-9 is reached. 30 mL of dioxane is then added, followed by Boc2O (7202 mg / 13.2 mmol). The reaction is allowed to reach room temperature and stirred overnight. The reaction is then diluted with EtOAc and washed three times with saturated citric acid solution and once with saturated NaCl solution. The organic phase is dried over MgSO, filtered, and evaporated under vacuum to give the crude product, which is purified by chromatography on silica gel (petroleum ether / EtOAc, 70:30 to 20:80 gradient) to give the mononitrated octopamine precursor: tert-butyl (2-hydroxy-2-(4-hydroxy-3-nitrophenyl)ethyl)carbamate. The resulting mononitrated octopamine precursor is then subjected to chiral separation before further synthetic steps are carried out.

[0170] Chiral separation of racemic tert-butyl (2-hydroxy-2-(4-hydroxy-3-nitrophenyl)ethyl)carbamate was carried out using an MPLC (medium pressure liquid chromatography) column with a mobile phase of DCM ± 0.2% (v / v) EtOH (isocratic gradient) and a sample solvent of DCM ± 0.2% (v / v) EtOH. To determine the absolute configuration, the phenolic position of both enantiomers was esterified with 1.2 molar equivalents of 4-nitrobenzoyl chloride and 2 molar equivalents of triethylamine in anhydrous THF. The compound was purified by chromatography on silica gel (petroleum ether / EtOAc, gradient 90:10 to 10:90) to give 4-(2-((tert-butoxycarbonyl)amino)-1-hydroxyethyl)-2-nitrophenol 4-nitrobenzoate. The absolute configuration of the enantiomers (previously dissolved in a 1:1 mixture of heptane / dichloromethane and allowed to slowly evaporate for 3 weeks to induce crystal formation) is confirmed by X-ray crystallography.

[0171] In a round-bottom flash reactor, AgCO (1500 mg, 5.4 mmol) and 1,1,4,7,10,10-hexamethyltriethylenetetramine (251 mg, 1.1 mmol) were suspended in 4 mL of anhydrous acetonitrile and stirred at room temperature for 2 h. Enantiopure tert-butyl (2-hydroxy-2-(4-hydroxy-3-nitrophenyl)ethyl)carbamate (292 mg, 0.98 mmol) and 1-bromo-2,3,4-tri-O-acetyl-α-D-glucuronide methyl ester (583 mg, 1.46 mmol) were added at 0 °C, and the solution mixture was stirred at room temperature for 4 h. The reaction mixture was then filtered through diatomaceous earth, diluted with EtOAc, and washed three times with saturated citric acid solution and once with saturated NaCl solution. The organic phase is dried over MgSO, filtered, and evaporated in vacuo to give the crude product, which is purified by chromatography on silica gel (petroleum ether / EtOAc, 70:30 to 30:70 gradient) to give (2S,3R,4S,5S,6S)-2-(4-(2-((tert-butoxycarbonyl)amino)-1-hydroxyethyl)-2-nitrophenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate.

[0172] (2S,3R,4S,5S,6S)-2-(4-(2-((tert-butoxycarbonyl)amino)-1-hydroxyethyl)-2-nitrophenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (334 mg / 0.54 mmol) and 4-nitrophenyl chloroformate (219 mg / 1.09 mmol) are dissolved in 6 mL of dry DCM at 0° C. Anhydrous pyridine (112 mg, 1.41 mmol) is added and the mixture is stirred at room temperature for 30 minutes. The reaction is filtered through a 0.45 μm PTFE filter and purified by chromatography on silica gel (petroleum ether / EtOAc, 85:15 to 30:70 gradient) to give (2S,3R,4S,5S,6S)-2-(4-(2-((tert-butoxycarbonyl)amino)-1-(((4-nitrophenoxy)carbonyl)oxy)ethyl)-2-nitrophenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate.

[0173] 4-beta-Glucuronide-3-nitro-octopamine-exatecan intermediate: (2S,3R,4S,5S,6S)-2-(4-(2-amino-1-((((1R,9R)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':]6,7]indolizino[1,2-b]quinolin-1-yl)carbamoyl)oxy)ethyl)-2-nitrophenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid

[0174] [ka]

[0175] Scheme 3

[0176] [ka]

[0177] The 4-beta-glucuronide-3-nitro-octopamine intermediate can be conjugated to exatecan using Scheme 3 to provide the 4-beta-glucuronide-3-nitro-octopamine-exatecan intermediate: (2S,3R,4S,5S,6S)-2-(4-(2-amino-1-((((1R,9R)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamoyl)oxy)ethyl)-2-nitrophenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid.

[0178] 101 mg (0.13 mmol) of 4-beta-glucuronide-3-nitro-octopamine intermediate (2S,3R,4S,5S,6S)-2-(4-(2-((tert-butoxycarbonyl)amino)-1-(((4-nitrophenoxy)carbonyl)oxy)ethyl)-2-nitrophenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate, 0.14 mmol of exatecan mesylate, and 18 mg (0.13 mmol) of HOBt are dissolved in 1 mL of an 85:15 (v / v) mixture of anhydrous DMF / pyridine. 16.7 mg (0.13 mmol) of DIPEA are added. The reaction is stirred at 40°C for 2 hours and the volatiles are evaporated under reduced pressure. The crude residue is purified by chromatography on silica gel (DCM / MeOH gradient 99:1 to 95:5) to give the intermediate compound, which is then subjected to deprotection.

[0179] 144 mg (0.106 mmol) of this intermediate compound is dissolved in 3 mL of MeOH (75:25) at 0 °C. LiOH monohydrate (44.5 mg, 1.06 mmol) is dissolved in water (0.4 mL) and added to the reaction vessel. After stirring, the mixture is neutralized with acetic acid (83 mg, 1.4 mmol) and concentrated under reduced pressure. The resulting crude product is redissolved in a TFA / DCM solution and stirred. The volatiles are evaporated under reduced pressure and the crude residue is collected and purified using HPLC to give (2S,3R,4S,5S,6S)-2-(4-(2-amino-1-((((1R,9R)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':]6,7]indolizino[1,2-b]quinolin-1-yl)carbamoyl)oxy)ethyl)-2-nitrophenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid.

[0180] Conjugation of PSAR intermediate and 4-beta-glucuronide-3-nitro-octopamine-exatecan intermediate to obtain functionalized PSAR-4-beta-glucuronide-3-nitro-octopamine-exatecan intermediate

[0181] [ka]

[0182] Scheme 4

[0183] [ka]

[0184] The PSAR intermediate and 4-beta-glucuronide-3-nitro-octopamine-exatecan intermediate can be conjugated using Scheme 4 to provide a functionalized PSAR-4-beta-glucuronide-3-nitro-octopamine-exatecan intermediate.

[0185] Dissolve 100 mg (0.081 mmol) of PSAR intermediate and 51 mg (0.061 mmol) of 4-beta-glucuronide-3-nitro-octopamine-exatecan intermediate in anhydrous DMF. Add 41 mg (0.405 mmol) of triethylamine and stir the reaction at room temperature for 30 minutes. After complete conversion of the reaction is assessed by HPLC, add piperidine directly to the reaction vial to achieve an 8% (v / v) piperidine solution in DMF. Then, stir the reaction at room temperature for 5–10 minutes until complete Fmoc deprotection is observed by HPLC. Slowly neutralize the reaction with a 10% TFA solution in 1:1 water / ACN (v / v) and purify using HPLC to obtain the functionalized PSAR-4-beta-glucuronide-3-nitro-octopamine-exatecan intermediate.

[0186] Linker-payload of formula XIV: (2S,3S,4S,5R,6S)-6-(4-((3S,9S)-40-amino-9-(2-(2-(2-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)ethoxy)ethoxy)acetamido)-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′, 4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-11,14,17,20,23,26,29,32,35,38-decamethyl-1,6,10,13,16,19,22,25,28,31,34,37,40-tridecaoxo-2-oxa-5,11,14,17,20,23,26,29,32,35,38-undecaazatetracontan-3-yl)-2-nitrophenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid

[0187] [ka]

[0188] Scheme 5

[0189] [ka]

[0190] The linker-payload of formula XIV can be prepared by conjugating maleimidopropionic acid N-hydroxysuccinimide ester and a functionalized-PSAR-4-beta-glucuronide-3-nitro-octopamine-exatecan intermediate using Scheme 5.

[0191] Maleimidopropionic acid N-hydroxysuccinimide ester and functionalized-PSAR-4-beta-glucuronide-3-nitro-octopamine-exatecan intermediate were dissolved in anhydrous DMF (0.1M concentration of maleimide compound). 1.56 mg (0.015) of triethylamine was added, and the reaction was stirred for 2 hours until complete conversion of the reaction was observed by HPLC. The reaction mixture was then diluted with 1% aqueous TFA / ACN 1:1 (v / v) and purified using HPLC preparative method 6 to obtain the linker-payload of formula XIV.

[0192] Synthesis of PSAR-Val-Ala-Exatecan-Containing Linker-Payload of Formula XV

[0193] [ka]

[0194] To prepare for conjugation to the PTK antibodies of the present disclosure, linker-payloads of formula XV were synthesized from precursors or intermediates including polysarcosine (PSAR) compounds, valine-alanine compounds, exatecan compounds, and maleimide compounds. The linker-payloads can be synthesized using the methods disclosed in WO2019081455 and WO2022207699.

[0195] (S)-32-(3-(((S)-2-(4-((S)-2-((S)-2-acetamido-3-methylbutanamido)propanamido)phenyl)-2-((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamoyl (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-4,7,10,13,16,19,22,25,28,31,34,43-dodecaoxo-36,39-dioxa-3,6,9,12,15,18,21,24,27,30,33,42-dodecaazapentatetracontanoic acid

[0196] Scheme 6

[0197] [ka]

[0198] Step 6.1a

[0199] [ka]

[0200] tert-Butyl ((S)-2-(4-((S)-2-aminopropanamido)phenyl)-2-hydroxyethyl)carbamate Fmoc-Ala-OH (473.5 g, 1.52 mmol, 1.3 equiv) was solubilized in DCM (3000 mL). The solution was cooled to 10 °C. HOPO (154.2 g, 1.39 mmol, 1.6 equiv) and EDCI (291.3 g, 1.88 mmol, 1.2 equiv) were added under N2. After stirring at 10 °C for 0.5 h, tert-butyl (S)-(2-(4-aminophenyl)-2-hydroxyethyl)carbamate (295.0 g, 1.17 mmol, 1 equiv) was added and stirred at 15 °C for 1 h. DEA (1.67 kg, 19.5 equiv) was added dropwise over 1 h. After the addition was complete, the mixture was stirred at 15 °C for 1 h and then at 0 °C for 14 h. The mixture was concentrated under vacuum (35 °C). Chromatography: eluent: n-heptane / DCM=10 / 1 to 1 / 10, DCM / THF=10 / 1 to 1 / 10, eluent: THF / MeOH=100 / 1 to 10 / 1. After evaporation under reduced pressure, the title compound (270.0 g, 71%) was obtained.

[0201] Step 6.1b

[0202] [ka]

[0203] 2,5-Dioxopyrrolidin-1-ylacetyl-L-valinate Ac-Val-OH (60, 0 g, 1 equiv) was solubilized in DCM (1200 mL), then NHS (52.1 g, 1.2 equiv) was added and stirred at 0° C. for 18 h. DCC (93.3 g, 1.2 equiv) was added in one portion and stirred at 0° C. for 18 h. Filtered, washed three times with DCM, and evaporated to half volume (40° C.). Cooled to 30° C. and stirred for 1 h. Filtered, washed cake with 1:2 DCM / MTBE (50 mL) and dried under vacuum (30° C.) for 22 h. Obtained the title compound (86.7 g, 89.8%) as a solid.

[0204] Process 6.2

[0205] [ka]

[0206] tert-Butyl ((S)-2-(4-((S)-2-((S)-2-acetamido-3-methylbutanamido)propanamido)phenyl)-2-hydroxyethyl)carbamate tert-Butyl ((S)-2-(4-((S)-2-aminopropanamido)phenyl)-2-hydroxyethyl)carbamate (17.0 g, 52.6 mmol, 1 equiv.) was solubilized in THF (250 mL). The temperature was adjusted to 10-20 °C. 2,5-Dioxopyrrolidin-1-ylacetyl-L-valinate (13.47 g, 52.6 mmol, 1.0 equiv.) was added and stirred at 10-20 °C for 3 h. The temperature was adjusted to -5-5 °C. The reaction mixture was filtered, and the filter cake was dried under a stream of N at 10-20 °C for 14 h. The filter cake was taken up in THF (100 mL), and HO (350 mL) was added. The temperature was adjusted to 10-20 °C and stirred for 13 h. The mixture was filtered, and the filter cake was washed with HO (60 mL). The filter cake was dried for 54 hours at 40-50° C. The title compound (21 g, 86%) was obtained as a solid.

[0207] Process 6.3

[0208] [ka]

[0209] tert-Butyl ((S)-2-(4-((S)-2-((S)-2-acetamido-3-methylbutanamido)propanamido)phenyl)-2-(((4-nitrophenoxy)carbonyl)oxy)ethyl)carbamate Under N2, tert-butyl ((S)-2-(4-((S)-2-((S)-2-acetamido-3-methylbutanamido)propanamido)phenyl)-2-hydroxyethyl)carbamate (100.0 g, 215 mmol, 1 equiv.) was dissolved in anhydrous DMF (900 g). The temperature was adjusted to 5-15 °C and stirred for 5-15 minutes. Bis(4-nitrophenyl)carbonate (132.0 g, 434 mmol, 2.0 equiv.) was added. DIPEA (83.5 g, 3 equiv.) was added dropwise at 5-15 °C under N2. The temperature was adjusted to 15-25 °C and stirred for 4-6 hours. Bis(4-nitrophenyl)carbonate (6.5 g, 21 mmol, 0.10 equiv.) was then added. The mixture was stirred under N2 at 15-25 °C for 1-2 hours. EtOAc (4400 g) and H2O (4500 g) were added. The mixture was stirred at 15-25°C for 10-30 minutes. The mixture was allowed to stand for 10-30 minutes. The organic layer was separated and saved for later use. EtOAc (4400 g) was added to the aqueous layer and stirred at 15-25°C for 10-30 minutes. The mixture was allowed to stand for 10-30 minutes. The previous organic layer was added again, followed by 10% aqueous NaCl (4000 g). The mixture was stirred at 15-25°C for 10-30 minutes. The mixture was allowed to stand for 10-30 minutes. The aqueous layer was separated. Anhydrous Na2SO4 (200 g) was added to the organic layer. The temperature was adjusted to 15-25°C and the mixture was allowed to stand for 10-30 minutes. The mixture was filtered, and the filter cake was washed with EtOAc (200 g). The organic layer was collected and concentrated to approximately half its volume at less than 40°C. The temperature was adjusted to 30-40°C and stirred for 10-30 minutes. n-Heptane (1400 g) was added dropwise at 30-40°C over 0.5 hours. The temperature was adjusted to 10-20°C and stirred for 3-6 hours. The mixture was filtered and the filter cake was washed with 1:2 EtOAc / n-heptane (500 g). The filter cake was triturated with EtOAc (9000 g) under N2, the temperature was adjusted to 30-40°C, and stirred for 10-30 minutes. n-Heptane (1400 g) was added dropwise at 30-40°C over 0.5 hours. The temperature was adjusted to 10-20°C and stirred for 3-6 hours. The mixture was filtered and the filter cake was washed with 1:2 EtOAc / n-heptane (500 g). The filter cake was dried at 30-40°C for 20-30 hours. The title compound (119 g, 88%) was obtained as a solid.

[0210] Process 6.4

[0211]

change

[0212] (S)-1-(4-((S)-2-((S)-2-acetamido-3-methylbutanamido)propanamido)phenyl)-2-aminoethyl ((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamate To a suspension of exatecan mesylate (100.0 g, 188.1 mmol, 1 equiv.) in DMF (1504 g) and pyridine (392 g) under N2 at 10-20 °C, tert-butyl ((S)-2-(4-((S)-2-((S)-2-acetamido-3-methylbutanamido)propanamido)phenyl)-2-(((4-nitrophenoxy)carbonyl)oxy)ethyl)carbamate (142.2 g, 225.8 mmol, 1.2 equiv.), HOBt (25.42 g, 188.1 mmol, 1.0 equiv.), and DIPEA (48.63 g, 376.3 mmol, 2.0 equiv.) were added. The mixture was stirred under N2 at 10-20 °C for 4-6 h. The temperature was adjusted to 15-25 °C. DCM (8000 g) and 10% aqueous Na2CO3 were added and stirred for 10–30 minutes. After standing for 10–30 minutes, the aqueous layer was separated. DMF (450 g), H2O (1000 g), and 10% aqueous citric acid (150.0 g) were added to the organic layer and the pH was adjusted to 6–8. The mixture was stirred at 10–20°C for 5–10 minutes. After standing for 5–30 minutes under N2, the aqueous layer was separated. Anhydrous Na2SO4 (500 g) was added to the organic layer and stirred at 10–20°C for 10–30 minutes. The mixture was filtered and washed with DCM (500 g). The filtrate was concentrated at ≤30°C. This mixture was added dropwise to MTBE (3000 g) and the temperature was adjusted to 10–20°C. The mixture was stirred under N2 at 10–20°C for 0.5–1 hour. The mixture was filtered and washed with MTBE (800 g). DCM was added to the filter cake under N2, the temperature was adjusted to -10 to 0 °C, and the mixture was stirred for 5 to 15 minutes. TFA (459 g) was added dropwise over 0.5 hours. The mixture was stirred under N2 at -10 to 0 °C for 1 to 4 hours. TFA (30 g) was then added dropwise. The mixture was stirred under N2 at -10 to 0 °C for 0.5 to 2 hours. TFA (30 g) was then added dropwise. The mixture was stirred under N2 at -10 to 0 °C for 0.5 to 2 hours. The reaction solution was added dropwise to a solution of MTBE (14,000 g) and n-heptane (3,415 g) over 0.5 hours and cooled to -10 to 5 °C. The reaction mixture was filtered and washed with MTBE (800 g). The filter cake was dried at 25 to 35 °C for 10 to 15 hours. The title compound (139 g, 90%) was obtained as a solid.

[0213] Scheme 7

[0214] [ka]

[0215] Process 7.1

[0216] [ka]

[0217] (S)-14-(3-((2,5-dioxopyrrolidin-1-yl)oxy)-3-oxopropyl)-1-(9H-fluoren-9-yl)-16,19,22,25,28,31,34,37,40,43-decamethyl-3,12,15,18,21,24,27,30,33,36,39,42-dodecaoxo-2,7,10-trioxa-4,13,16,19,22,25,28,31,34,37,40,43-dodecaazapentatetracontan-45-oic acid 1) DCM was added to CTC resin (12.80 g, 10.00 mmol, 0.78 mmol / g) and Fmoc-Sar-OH (2.48 g, 8.00 mmol, 0.80 equiv.) with N bubbling. 2) DIPEA (4.00 equivalents) was added dropwise and mixed for 2 hours. 3) MeOH (13 mL) was added and mixed for 30 minutes. 4) Drain and wash with DMF five times. 5) 20% piperidine / DMF was added and reacted for 30 minutes. 6) Drain and wash with DMF five times. 7) The Fmoc-amino acid solution was added and mixed for 30 seconds, after which the activation buffer was added and N2 was bubbled through for about 1 hour. 8) Steps 4 to 7 above were repeated for the coupling of the following amino acids:

[0218] [Table 6]

[0219] The coupling reaction was monitored by the ninhydrin test and the resin was washed five times with DMF.

[0220] Peptide cleavage and purification: 1) Cleavage buffer (1% TFA, 99% DCM) was added to the flask containing the side-chain protected peptide at room temperature. 2) Filter and collect the filtrate. 3) The solvent was removed under vacuum to give the crude peptide. 4) The crude peptide was purified by preparative HPLC (A: 0.075% TFA in H2O, B: ACN) to give the title compound (3.10 g, purity 90.80%, yield 23.44%).

[0221] Purification conditions: Dissolution conditions: Dissolved in ACN / H2O Equipment: Hanbon DAC-100 Mobile phase: A: H2O (0.1% TFA in H2O), B: 0.1% TFA in ACN Gradient: 18-48% 42 minutes Holding time: 30 minutes Column: Luna 250*100mm, C18, 10μm, 100Å Flow rate: 250mL / min Wavelength: 220 / 254nm Oven temperature: Room temperature

[0222] Process 7.2

[0223] [ka]

[0224] (S)-32-(3-(((S)-2-(4-((S)-2-((S)-2-acetamido-3-methylbutanamido)propanamido)phenyl)-2-((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2- b]quinolin-1-yl)carbamoyl)oxy)ethyl)amino)-3-oxopropyl)-41-amino-3,6,9,12,15,18,21,24,27,30-decamethyl-4,7,10,13,16,19,22,25,28,31,34-undecaoxo-36,39-dioxa-3,6,9,12,15,18,21,24,27,30,33-undecaazahentetracontanoic acid (S)-1-(4-((S)-2-((S)-2-acetamido-3-methylbutanamido)propanamido)phenyl)-2-aminoethyl ((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamate (100.0 g, 1 equivalent) and DMF (400 g) were dissolved in (S)-14-(3-((2,5-dioxopyrrolidine A mixture of (121.68 g) of (3-oxopropyl)-1-(9H-fluoren-9-yl)-16,19,22,25,28,31,34,37,40,43-decamethyl-3,12,15,18,21,24,27,30,33,36,39,42-dodecaoxo-2,7,10-trioxa-4,13,16,19,22,25,28,31,34,37,40,43-dodecaazapentatetracontan-45-oic acid (DMF) was added to the mixture, cooled to -5 to 5 °C under N2, and stirred for 5 to 15 minutes. TEA (49 g, 4.0 equiv.) was added over 15 minutes, and the mixture was stirred for 1 to 2 hours at -5 to 5 °C under N2. (S)-1-(4-((S)-2-((S)-2-acetamido-3-methylbutanamido)propanamido)phenyl)-2-aminoethyl ((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamate (1.0 g, 0.01 equiv.) was added and stirred for 1-2 hours. DEA (35.4 g) was added dropwise over 0.5 hours. The mixture was stirred for 12-22 hours under N2 at -5 to 5°C. The reaction solution was transferred dropwise to MTBE (14,800 g) over 0.5 h, cooled to 0–10°C, and stirred under N2 for 0.5–1 h. The reaction mixture was filtered and washed with MTBE (1,200 g). The filter cake was dried under a stream of N2 at 5–15°C for 5–12 h. The title compound (105.2 g, 48%) was obtained as a solid.

[0225] Process 7.3

[0226] [ka]

[0227] (S)-32-(3-(((S)-2-(4-((S)-2-((S)-2-acetamido-3-methylbutanamido)propanamido)phenyl)-2-((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamoyl (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-4,7,10,13,16,19,22,25,28,31,34,43-dodecaoxo-36,39-dioxa-3,6,9,12,15,18,21,24,27,30,33,42-dodecaazapentatetracontanoic acid 3-Maleimidopropionic acid N-hydroxysuccinimide ester (16.8 g, 63.1 mmol) and DMF (100 g) were dissolved in (S)-32-(3-(((S)-2-(4-((S)-2-((S)-2-acetamido-3-methylbutanamido)propanamido)phenyl)-2-((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizidine) The mixture was added to a solution of (1,2-b)-2,3-diamino-3,6,9,12,15,18,21,24,27,30-decamethyl-4,7,10,13,16,19,22,25,28,31,34-undecaoxo-36,39-dioxa-3,6,9,12,15,18,21,24,27,30,33-undecaazahenetetracontanoic acid (100.0 g, 55.2 mmol) and DMF (900 g), cooled to -15 to -5 °C, and stirred at -15 to -5 °C for 5 to 15 minutes under N2. TEA (5.59 g) was added dropwise and stirred under N2 at -15 to -5°C for 1 to 4 hours. TEA (0.559 g) was further added dropwise and stirred under N2 at -15 to -5°C for 0.5 to 2 hours. MTBE (7400 g) was added and the temperature was adjusted to -5 to 5°C. The reaction mixture was filtered and washed with MTBE (600 g). The filter cake was dried under a stream of N2 at 5 to 15°C for 5 to 12 hours. The title compound (73.6 g, 68%) was obtained as a solid.

[0228] Part I. Preparation of ADCs with maleimide linker-payload To prepare antibody-drug conjugates with eight drugs per antibody (DAR8), an IgG1 antibody is fully reduced using 6–8 molar equivalents of a reducing reagent, such as DTT or TCEP, at 37°C for 2 hours. The reduced antibody is then buffer-exchanged using a PD-10 desalting column containing 50 mM HEPES (pH 7.0) with 2 mM EDTA, and the eluate is adjusted to a protein concentration of 5–10 mg / mL with HEPES buffer. An excess of linker-payload (e.g., 10 molar equivalents) is added over 1 hour, and the conjugation reaction can be quenched by the addition of a substantial excess of L-cysteine ​​(e.g., 6 molar equivalents). The resulting ADC mixture can be purified using a PD-10 desalting column equilibrated with 25 mM histidine, 9% sucrose (pH 5.5), followed by three centrifugation cycles using a 30 kDa MWCO centrifuge unit to remove unreacted linker-payload-related species. Finally, the resulting ADC can be sterile filtered through a 0.2 μM filter and stored at 4° C. or −80° C. for future use.

[0229] Part II. Preparation of ADCs with bromoacetyl linker-payloads To prepare antibody-drug conjugates with eight drugs per antibody, IgG1 antibodies are fully reduced using 6-8 molar equivalents of a reducing reagent such as DTT or TCEP at 37°C for 2 hours. The reduced antibody is then buffer-exchanged using a PD-10 desalting column containing 50 mM HEPES (pH 7.4) with 2 mM EDTA, and the eluate is adjusted to a protein concentration of 5-10 mg / mL with HEPES buffer. Excess linker-payload (e.g., 12 molar equivalents) is added over 2-3 hours, and the conjugation reaction can be quenched by adding a substantial excess of L-cysteine ​​(e.g., 10 molar equivalents). The resulting ADC mixture can be purified using a PD-10 desalting column equilibrated with 25 mM histidine, 9% sucrose (pH 5.5), followed by three centrifugation cycles using a 30 kDa MWCO centrifuge unit to remove unreacted linker-payload-related species. Finally, the resulting ADC can be sterile filtered through a 0.2 μM filter and stored at 4° C. or −80° C. for future use.

[0230] Example 3: Antibody Binding Affinity, Cross-Reactivity, and Selectivity Characterization of human and cross-species binding of PTK-7 ADCs by surface plasmon resonance A Biacore 8K+ instrument (Cytiva, Marlborough, MA) was used to determine the kinetic and affinity parameters of the binding interaction of the ADC binding to recombinant human PTK7 (Acro Biosystems catalog number PT7-H52H3, Newark, DE).

[0231] The anti-human Fc sensor surface was prepared by amine coupling of goat anti-human IgG Fc (Southern Biotech, catalog number 2014-01, Birmingham, AL) to a Biacore Series S C1 (Cytiva, catalog number BR-100535) sensor surface at 25 °C. A running buffer of 10 mM HEPES, 150 mM NaCl, and 0.05% Surfactant P20, pH 7.4, was used for immobilization. All eight channels of flow cells 1 and 2 were activated with a 1:1 (v / v) mixture of 400 mM 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and 100 mM N-hydroxysuccinimide (NHS) at a flow rate of 10 μL / min for 7 min. Anti-human IgG Fc capture reagent was then bound to the sensor surface (diluted to 50 μg / mL in 10 mM acetate, pH 4.5 buffer) by injecting it into all flow cells and channels at a flow rate of 10 μL / min for 7 min. Remaining active groups were blocked by injecting 100 mM ethylenediamine (in 200 mM borate buffer, pH 8.5) into all flow cells and channels at a flow rate of 10 μL / min for 7 min. All channels and flow cells were then preconditioned with three consecutive 1-min injections of 75 mM phosphoric acid at 10 μL / min.

[0232] For kinetic and affinity analysis, the running buffer and sample dilution buffer were 10 mM HEPES, 150 mM NaCl, 0.05% Surfactant P20, pH 7.4, 1 mg / mL bovine serum albumin (BSA), and the analysis temperature was 37°C.

[0233] In each analysis cycle, a different ADC was captured on flow cell 2 of each channel by injecting a 5 μg / mL solution at 10 μL / min for 2 minutes. After capture, the same analyte was injected into flow cells 1 and 2 of all eight channels at 30 μL / min for 2 minutes, and dissociation was monitored for 10 minutes. After dissociation, all surfaces were regenerated by three consecutive 1-minute injections of 75 mM phosphoric acid at 10 μL / min. The ADC capture and analyte cycles were repeated to obtain human PTK7 analyte binding for each ADC at analyte concentrations of 0, 2.47, 7.41, 22.2, 66.7, 200, and 600 nM.

[0234] As shown in Table 4, the sensorgram data were reference subtracted, blank subtracted, and then globally fitted using the default 1:1 binding model in Biacore Insight Evaluation Software v3.0.12.15655.

[0235] Certain antibodies of the present disclosure, including Ab1-4, have K in the nM range in cynomolgus monkeys. D Combine with.

[0236] [Table 7] Kinetic and affinity parameters of the ADC / human PTK7 interaction at 37°C. a is the binding rate constant, and k d is the dissociation rate constant, and K D is the equilibrium dissociation constant (K D =k d / k a (Calculated using

[0237] Characterization of cell surface binding of PTK-7 antibodies in cell lines expressing the PTK-7 receptor Certain PTK-7 antibodies of the present disclosure were tested for cell surface binding to three PTK-7-expressing tumor cell lines. Cell lines were selected to represent high, medium, and low receptor densities. OV90-PTK-7 is an overexpressing engineered ovarian tumor line exhibiting high expression, NCI-H446 human lung cancer cells exhibiting medium endogenous expression, and OVCAR-3 human ovarian cancer cells exhibiting low endogenous expression. OV90-PTK-7, NCI-H446, and OVCAR3 cells were determined to have antibody binding capacities of ~500,000, ~40,000, and ~20,000, respectively (using the MESF quantitation kit, Bangs Laboratories). OV90 parental cells and Namalwa-luc-GFP cells were selected as PTK-7-negative cell lines. Antibody binding was quantified by flow cytometry, and the EC values ​​of the binding curves were calculated. 50 Both the maximum binding MFI and the maximum binding MFI for each antibody were recorded.

[0238] Cells were dissociated using non-enzymatic dissociation buffer for 5 min at 37°C. Cells were counted and plated at 10 wells in a V-bottom polypropylene 96-well plate. 5Cells were dispensed at 0.1% per well. Cells were centrifuged at 1800 rpm for 3 minutes, and the supernatant was discarded. An 11-point antibody dilution series was prepared in assay buffer (1x PBS containing 5% FBS), starting at 300 nM and diluted 1:4. The dilution series was added to the cells at 100 μL per well and mixed by pipetting. Several untreated control wells per cell line were prepared with assay buffer only. The cells and antibody were incubated for 1 hour at 4°C on an orbital shaker. After incubation, the assay plate was centrifuged and washed twice with 300 μL per well of assay buffer. The cell pellet was then stained with a 1:500 dilution of Alexa647-conjugated mouse anti-human IgG secondary antibody in assay buffer (100 μL per well). The assay plate was incubated in the dark at 4°C for 1 hour with shaking. After incubation, the plate was centrifuged, and the cells were washed twice with 300 μL per well of assay buffer. A 1:2000 dilution of Zombie Green live / dead marker in 1x PBS was dispensed onto the cells at 100 μL / well, and the plate was incubated at room temperature with shaking in the dark for 10 minutes. The cell pellet was then washed once with assay buffer and fixed with 200 μL / well of 4% paraformaldehyde in 1x PBS for 15 minutes at room temperature in the dark. The cells were centrifuged, washed, and then resuspended in 100 μL of assay buffer and acquired on an Attune Cytpix Cytometer.

[0239] Cells were acquired using an Attune Cytpix Cytometer, and FCS files were generated using Attune™ Cytometric Software (v.6.0.1). The FCS files were then analyzed using FlowJo (v10.8.1). Debris was excluded from the analysis by forward scatter (FSC) versus side scatter (SSC) gating, and single cells were selected by forward scatter area (FSC-A) versus height (FSC-H) gating. Finally, dead cells stained positive with Zombie Green were excluded, and the MFI of Alexa647-positive live cells was quantified. Data were graphed and analyzed using GraphPad Prism (v10.1.1). EC 50was determined via agonist versus response-variable slope (four parameter) curve fitting, and % maximum binding of cofetuzumab was calculated by setting the mean of maximum cofetuzumab MFI in each cell line to 100%.

[0240] Certain PTK7 antibodies of the present disclosure did not bind to the PTK-7-negative cell lines OV90 parental and namalwa-luc-GFP, as expected. The four antibodies shown in Table 5 bound to all three PTK-7-expressing tumor lines.

[0241] [Table 8] MFI = Mean Fluorescence Intensity

[0242] Example 4: ADC Binding and Internalization Characterization of the internalization of PTK-7 antibodies and ADCs in human PTK-7-positive cells using fluorescence imaging Single-clonal HeLa PTK-7-eGFP-overexpressing engineered cells were seeded at 5000 cells per well in a 384-well plate (PhenoPlate 384-well, black, optically clear, flat-bottom, tissue culture-treated, PerkinElmer 6057308) in complete culture medium (DMEM, 10% FBS, Glutamax, Pen / Strep). The following day, cells were treated with 6 μg / mL (40 nM) ADC or antibody and 6 μg / mL (120 nM) pHrodo labeling reagent (Zenon™ pHrodo™ iFL IgG Labeling Reagent, Catalog No. Z25612, Invitrogen™). The ADC or antibody was mixed with the pHrodo labeling reagent for at least 10 minutes before addition to the cells. Cells were then imaged every 90 minutes for up to 24 hours using the Opera Phenix Plus High-Content Screening System. Data were processed and analyzed with Harmony and Microsoft Excel and graphed with GraphPad Prism.

[0243] As shown in Table 6, the PTK-7 antibody and PTK-7 ADC demonstrate superior lysosomal trafficking (approximately 2-fold) compared to cofetuzumab peridotin.

[0244] [Table 9]

[0245] Example 5: ADC cytotoxicity and bystander activity Characterization of the activity of PTK-7 ADCs in low (OVCAR-3, ABC = ~20,000) and high (OV90-PTK-7, ABC = ~500,000) PTK-7 expressing cell lines OVCAR3 cells were seeded in culture medium (OVCAR3: ATCC modified RPMI 1640 + 1x GlutaMax + 20% heat-inactivated fetal bovine serum + 10 μg / mL bovine insulin) in white, clear-bottom 96-well tissue culture plates. Cells were incubated overnight at 37°C, 5% CO2. The following day, ADCs were added at a 1:4 serial dilution in culture medium starting from a final working concentration of 200 nM. Plates were covered with Breathe-Easy® sealing membranes and incubated at 37°C, 5% CO2. After 7 days of treatment, plates were read using the CellTiter-Glo Luminescent Cell Viability Assay. 100 μL / well of CellTiter-Glo reagent was incubated in the plate for 10 minutes at room temperature. Luminescence was read using a SpectraMax M5e. RLU (relative luminescence units) were obtained using SoftMax Pro 5.4. Percent cell death was calculated relative to 0% untreated. Data were graphed and analyzed using Graphpad Prism version v10.1.1. IC 50 was determined by log(inhibitor) vs. response-variable slope (four parameter) curve fitting.

[0246] The OV90-PTK-7 engineered cell line was seeded in culture medium (1:1 MCDB105: Medium 199 + 15% heat-inactivated fetal bovine serum + 1x Glutamax). ADCs were added at a final working concentration of 200 nM, serially diluted 1:4 in culture medium, for 5 days.

[0247] Certain PTK-7 ADCs of the present disclosure demonstrated potent maximal cell killing similar to that of cofetuzumab-peridotin conjugates in PTK-7 cell lines with differential expression levels. 50 The values ​​suggest similar potency across all ADCs in OV90-PTK-7 cells and variable potency among the ADCs tested in OVCAR3 cells. No nonspecific cytotoxicity was observed in the OV90 parental (PTK-7 negative cell line).

[0248] [Table 10]

[0249] Characterization of the bystander effect of PTK-7 ADC in Namalwa-Luc-GFP cells (a PTK-7 negative cell line) co-cultured with OV90-PTK-7 and NCI-H446 (PTK-7 positive cell lines with different expression levels) OV90-PTK-7:Namalwa-Luc-GFP clone cells were mixed and seeded in a 2:1 ratio in culture medium (1:1 MCDB105:Medium 199 + 15% heat-inactivated fetal bovine serum + 1x Glutamax) in a clear, flat-bottom 96-well plate at a total of 3000 cells / well / 100 μL. Namalwa-Luc-GFP cells were negative for PTK-7 expression. The plate was incubated overnight at 37°C and 5% CO2. The next day, ADC was added in a 1:4 serial dilution in culture medium starting from a final working concentration of 50 nM. The plate was covered with a Breathe-Easy® sealing membrane. After 5 days of treatment, the plate was read using the ONE-Glo™ Luciferase Assay System. 100 μL / well of ONE-Glo™ Assay Reagent was incubated in the plate at room temperature for 10 minutes. Luminescence was read on a SpectraMax M5e. RLU (relative luminescence units) were obtained using SoftMax Pro 5.4. Percentage of Namalwa-Luc-GFP death was calculated relative to 0% untreated. Data were graphed and analyzed using Graphpad Prism version v10.1.1. IC 50was determined by log(inhibitor) vs. response (3-parameter) curve fitting for ADC cytotoxicity.

[0250] NCI-H446:Namalwa-Luc-GFP clone cells were mixed and seeded in a 9:1 ratio in culture medium (RPM1 1640 + 10% heat-inactivated fetal bovine serum + 1x Glutamax + 1 mM sodium pyruvate) in a clear flat-bottom 96-well plate at a total of 5000 cells / well / 100 μL. The plate was incubated overnight at 37°C and 5% CO2. The next day, ADC was added at a 1:4 serial dilution in culture medium starting from a final working concentration of 300 nM. The plate was covered with a Breathe-Easy® sealing membrane. After 7 days of treatment, the plate was read using the ONE-Glo™ Luciferase Assay System.

[0251] As shown in Table 8, certain PTK-7 ADCs of the present disclosure exhibited a better bystander effect than cofetuzumab peridotin in Namalwa-Luc-GFP cells with OV90-PTK-7 as the positive cell line, and a similar bystander effect in Namalwa-Luc-GFP cells with NCI-H446 as the positive cell line.

[0252] [Table 11]

[0253] Example 6: ADCC, ADCP, and / or CDC Assays In vitro antibody-dependent cell-mediated cytotoxicity (ADCC) assay for PTK-7 antibodies The OV90 cell line was engineered to express high levels of human PTK-7 with an antibody binding capacity of 500,000 (using the MESF quantification kit, Bangs Laboratories). Target cells, OV90-human PTK-7, were added to a clear tissue culture 96-well plate in test medium (IMDM + 1x GlutaMax + 10% heat-inactivated fetal bovine serum + Pen-Strep 100 U / mL to 100 μg / mL) and incubated overnight at 37°C and 5% CO2. The following day, 40 μL / well of antibody was added, serially diluted 1:3 in test medium from a final working concentration of 200 nM. The antibody was incubated for 1 hour at 37°C and 5% CO2. Effector cells, Jurkat-Lucia NFAT-CD16, were then added at 150,000 / 80 μL / well and incubated at 37°C and 5% CO2. After 24 hours, 20 μL of supernatant and 50 μL of pre-prepared QUANTI-Luc per well were mixed in a white opaque plate. Luminescence was read using a SpectraMax M5e. RLU (relative luminescence units) were obtained using SoftMax Pro 5.4 and plotted on the Y-axis against compound concentration on the X-axis using GraphPad Prism version 9.5.1.

[0254] Unlike the cofetuzumab antibody, which has wild-type IgG1 Fc, PTK7 Abs 1 to 4 of the present disclosure are effector-null antibodies and did not induce antibody-dependent cellular cytotoxicity.

[0255] In vitro antibody-dependent cellular phagocytosis (ADCP) assay for PTK-7 antibodies The OV90 cell line was engineered to express high levels of human PTK-7 with an antibody binding capacity of 500,000 (using the MESF quantification kit, Bangs Laboratories). Target cells, OV90-human PTK-7, were seeded in test medium (IMDM + 1x GlutaMax + 10% heat-inactivated fetal bovine serum + Pen-Strep 100 U / mL to 100 μg / mL) in clear tissue culture 96-well plates and incubated overnight at 37°C, 5% CO2. The following day, 40 μL / well of mAb was added, serially diluted 1:3 in test medium from a final working concentration of 200 nM, and incubated for 1 hour at 37°C, 5% CO2. Effector cells, Jurkat-Lucia NFAT-CD32, were then added at 200,000 / 80 μL / well and incubated at 37°C, 5% CO2. After 24 hours, 20 μL of supernatant and 50 μL of pre-prepared QUANTI-Luc per well were mixed in a white opaque plate. Luminescence was read using a SpectraMax M5e. RLU (relative luminescence units) were obtained using SoftMax Pro 5.4 and plotted on the Y-axis against compound concentration on the X-axis using GraphPad Prism version 9.5.1.

[0256] Unlike the cofetuzumab antibody, which has wild-type IgG1 Fc, PTK-7 Abs 1 to 4 of the present disclosure are effector-null antibodies and did not induce antibody-dependent cellular phagocytosis.

[0257] In vitro complement-dependent cytotoxicity (CDC) assay of PTK-7 antibodies The OV90 cell line was engineered to express high levels of human PTK7 with an antibody binding capacity of 500,000 (using the MESF quantification kit, Bangs Laboratories). OV90-human PTK7 cells were added to white, clear-bottom, 96-well tissue culture plates in assay medium (1:1 MCDB105 / Medium199 + 1x GlutaMax + 15% heat-inactivated fetal bovine serum) and incubated overnight at 37°C in a 5% CO2 incubator. The following day, 50 μL / well of antibody was added, serially diluted 1:3 in assay medium from a final working concentration of 200 nM, and incubated for 1 hour at 37°C in 5% CO2. Diluted human serum complement (1:3) was then added at 50 μL / well in assay medium and incubated for 3 hours at 37°C in 5% CO2. Plates were read using the CellTiter-Glo Luminescent Cell Viability Assay. 100 μL / well of CellTiter-Glo reagent was incubated in the plate for 10 minutes at room temperature. Luminescence was read on a SpectraMax M5e. RLU (relative luminescence units) were obtained using SoftMax Pro 5.4. Percent cell death was calculated relative to untreated. Data were graphed and analyzed using GraphPad Prism version 9.5.1.

[0258] As a positive control, Jeko-1 cells were treated as above with anti-CD20 antibody in assay medium (RPMI1640 + 1x GlutaMAX + 10% heat-inactivated fetal bovine serum).

[0259] Similar to cofetuzumab, PTK-7 Abs 1-4 of the present disclosure did not exhibit CDC capability in the PTK-7-expressing OV90 cell line. An anti-CD20 control antibody produced CDC activity and was used as a positive control in CD20-expressing Jeko-1 cells.

[0260] Example 7: Efficacy of PTK-7 ADCs in tumor xenograft models Envigo female athymic nude mice, 5-7 weeks old and weighing 18-20 grams, were implanted subcutaneously in the right flank with 5x10^6 OVCAR3, NCI-H446, or OV90-PTK7 cells in 50% Matrigel in Hank's balanced salt solution. Tumors grew to approximately 150-250 mm. 3 When tumor volume reached 100 μg / kg, animals were divided into treatment or control groups based on tumor volume, and dosing was initiated (day 0, n = 5-8 per group). A single dose of test substance (2 mg / kg for OVCAR3 and NCI-H446, 0.5 mg / kg for OV90-PTK7) formulated with 5% dextrose was intravenously injected. Tumors were measured biweekly using digital calipers.

[0261] As shown in Table 8, treatment with the PTK-7 ADC using Abs 1-4 resulted in more effective tumor growth inhibition in the OVCAR3 cell line than the benchmark cofetuzumab-peridotin ADC.

[0262] [Table 12] SEM = standard error of the mean

[0263] Example 8: Stability and Tolerability The stability of the ADC with the PTK7 antibody of the present disclosure with PSAR10-VA-exatecan was evaluated in rat and human plasma (100 μg / mL). There was minimal loss of DAR (~2) over 7 days.

[0264] The tolerability of the ADC of the present disclosure using the PTK7 antibody of the present disclosure (together with PSAR10-VA-exatecan) was tested in 2-year-old male cynomolgus monkeys. Cynomolgus monkeys received a slow bolus IV injection of 50 mg / kg on days 1 and 22. Samples were collected pre-dose and at ~0.5 (30 minutes), 1, 4, 8, 24, 48, 96, and 168 hours, and (on day 1 only) 240, 336, and 504 hours post-dose. Bioanalysis was performed by LCMS using qualified methods. No ADAs were detected. There were no deaths, hematopoietic (BM, thymus, spleen, hematology), or GI (minimal to moderate histopathological changes in the GI tract). T1 / 2 was approximately 8 days.

[0265] Amino acid and nucleotide sequences SEQ ID NO: 1 (human PTK-7)

[0266] SEQ ID NO: 2 (HC of Ab1) EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYIMNWVRQAPGKGLEWVSSISSSSTFIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKGFDYWGQGTLVT VSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHT CPCPPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVSVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI SKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0267] SEQ ID NO: 3 (LC of Ab1) DIQLTQSPSFLSASVGDRVTITCRASQDISSYLVWYQQKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQLNTYPRTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0268] SEQ ID NO: 4 (HCDR1 of Ab1) AASGFTFSSYIMN

[0269] SEQ ID NO: 5 (HCDR2 of Ab1) SISSSSTFIY

[0270] SEQ ID NO: 6 (HCDR3 of Ab1) AKGFDY

[0271] SEQ ID NO: 7 (LCDR1 of Ab1) RASQDISSYLV

[0272] SEQ ID NO: 8 (LCDR2 of Ab1) YAASTLQS

[0273] SEQ ID NO: 9 (LCDR3 of Ab1) QQLNTYPRT

[0274] SEQ ID NO: 10 (VH of Ab1) EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYIMNWVRQAPGKGLEWVSSISSSSTFIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKGFDYWGQGTLVTVSS

[0275] SEQ ID NO: 11 (VL of Ab1) DIQLTQSPSFLSASVGDRVTITCRASQDISSYLVWYQQKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQLNTYPRTFGQGTKVEIK

[0276] SEQ ID NO: 12 (HC of Ab2) QVQLQESGPGLVKPSETLSLTCTVSGGSISTYYWSWIRQPAGKGLEWIGRIYTSGLTNYNPSLKSRVTMSVDTSKNQFSLKLSSVTAADTAVYYCARDPHYYDGSGFDYWGQ GTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDK THTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVSVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0277] SEQ ID NO: 13 (LC of Ab2) EIVLTQSPGTLSLSPGERATLSCRASQSVSDNYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPFTFGGPGTKVDI KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0278] SEQ ID NO: 14 (HCDR1 of Ab2) TVSGGSISTYYWS

[0279] SEQ ID NO: 15 (HCDR2 of Ab2) RIYTSGLTN

[0280] SEQ ID NO: 16 (HCDR3 of Ab2) ARDPHYYDGSGFDY

[0281] SEQ ID NO: 17 (LCDR1 of Ab2) RASQSVSDNYLA

[0282] SEQ ID NO: 18 (LCDR2 of Ab2 and Ab4) YGASSRAT

[0283] SEQ ID NO: 19 (LCDR3 of Ab2) QQYGSSPFT

[0284] SEQ ID NO: 20 (VH of Ab2) QVQLQESGPGLVKPSETLSLTCTVSGGSISTYYWSWIRQPAGKGLEWIGRIYTSGLTNYNPSLKSRVTMSVDTSKNQFSLKLSSVTAADTAVYYCARDPHYYDGSGFDYWGQGTLVTVSS

[0285] SEQ ID NO: 21 (VL of Ab2) EIVLTQSPGTLSLSPGERATLSCRASQSVSDNYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPFTFGGPGTKVDIK

[0286] SEQ ID NO: 22 (HC of Ab3) QVQLQESGPGLVKPSETLSLTCTVSGGSISTYYWNWIRQPAGKGLEWIGRIYSSGSTNYNPSLKSRVTMSVDTSKNQFSLKLSSVTAADTAVYYCARDPLYSGSLFDIWGQG TMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDK THTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVSVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0287] SEQ ID NO: 23 (LC of Ab3) EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASIRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPYTFGQGTKLEI KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0288] SEQ ID NO: 24 (HCDR1 of Ab3) TVSGGSISTYYWN

[0289] SEQ ID NO: 25 (HCDR2 of Ab3) RIYSSGSTN

[0290] SEQ ID NO: 26 (HCDR3 of Ab3) ARDPLYSGSLFDI

[0291] SEQ ID NO: 27 (LCDR1 of Ab3) RASQSVSSSYLA

[0292] SEQ ID NO: 28 (LCDR2 of Ab3) YGASIRAT

[0293] SEQ ID NO: 29 (LCDR3 of Ab3) QQYGSSPYT

[0294] SEQ ID NO: 30 (VH of Ab3) QVQLQESGPGLVKPSETLSLTCTVSGGSISTYYWNWIRQPAGKGLEWIGRIYSSGSTNYNPSLKSRVTMSVDTSKNQFSLKLSSVTAADTAVYYCARDPLYSGSLFDIWGQGTMVTVSS

[0295] SEQ ID NO: 31 (VL of Ab3) EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASIRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPYTFGQGTKLEIK

[0296] SEQ ID NO: 32 (HC of Ab4) EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSSIDSSSSFIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGTGLFDFWGQGTL VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTH TCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVSVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0297] SEQ ID NO: 33 (LC of Ab4) EIVLTQSPGTLSLSPGERATLSCRASQSFSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGRTPWTFGQGTKVEI KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0298] SEQ ID NO: 34 (HCDR1 of Ab4) AASGFTFSSYSMN

[0299] SEQ ID NO: 35 (HCDR2 of Ab4) SIDSSSSFIY

[0300] SEQ ID NO: 36 (HCDR3 of Ab4) ARGTGLFDF

[0301] SEQ ID NO: 37 (LCDR1 of Ab4) RASQSFSSSYLA

[0302] SEQ ID NO: 38 (LCDR3 of Ab4) QQYGRTPWT

[0303] SEQ ID NO: 39 (VH of Ab4) EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSSIDSSSSFIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGTGLFDFWGQGTLVTVSS

[0304] SEQ ID NO: 40 (VL of Ab4) EIVLTQSPGTLSLSPGERATLSCRASQSFSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGRTPWTFGQGTKVEIK

[0305] SEQ ID NO: 41 (DNA of HC against Ab1)

[0306] SEQ ID NO: 42 (DNA of LC for Ab1) GACATCCAGTTGACCCAGTCTCCATCCTTCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCCAGTCAGGACATTAGCAGTTATTTAGTCTGGTATCAGCAAAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCACTTTGCAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGAATTCACTCTCACAATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCAACAACTTAATACTTACCCTCGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAAagaactgtggcggcgccatctgtcttcatcttcccgccatctgatgagcagttgaaatccggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgctaa

[0307] SEQ ID NO: 43 (DNA of HC for Ab2)

[0308] SEQ ID NO: 44 (DNA of LC against Ab2) GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCGACAACTACTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTACTACTGTCAGCAGTATGGTAGTTCACCATTCACTTTCGGCCCTGGGACCAAGGTGGATATCAAAagaactgtggcggcgccatctgtcttcatcttcccgccatctgatgagcagttgaaatccggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgctaa

[0309] SEQ ID NO: 45 (DNA of HC against Ab3)

[0310] SEQ ID NO: 46 (DNA of LC against Ab3) GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCCTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCATCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAGTATGGTAGCTCACCGTACACTTTTGGCCAGGGGACCAAGCTGGAGATCAAAagaactgtggcggcgccatctgtcttcatcttcccgccatctgatgagcagttgaaatccggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgctaa

[0311] SEQ ID NO: 47 (DNA of HC against Ab4)

[0312] SEQ ID NO: 48 (DNA of LC against Ab4) GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTTTTAGCAGCAGCTACTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCAGC AGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGTATGGTAGGACACCGTGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAA agaactgtggcggcgccatctgtcttcatcttcccgccatctgatgagcagttgaaatccggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggag agtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgctaa

[0313] SEQ ID NO: 49 - Ala-Leu-Ala-Leu- (also known as ALAL in single letter notation)

[0314] SEQ ID NO: 50 -Leu-Ala-Leu-Ala- (also known as the one-letter code LALA)

[0315] SEQ ID NO: 51 -Gly-Phe-Leu-Gly- (also known as GFLG in the one-letter code)

[0316] SEQ ID NO: 52 - Gly-Leu-Phe-Gly- (also known as GLFG in single letter code)

[0317] SEQ ID NO: 53 - Ala-Ala-Ala-Ala- (also known as AAAA in single letter notation)

[0318] SEQ ID NO: 54 - Gly-Ala-Gly-Gly- (also known as GAGG in single letter code)

[0319] SEQ ID NO: 55 - Gly-Gly-Ala-Gly- (also known as the one-letter code GGAG)

[0320] SEQ ID NO: 56 - Gly-Val-Gly-Gly- (also known as GVGG in single letter code)

[0321] SEQ ID NO: 57 - Gly-Gly-Val-Gly- (also known as the one-letter code GGVG)

[0322] SEQ ID NO: 58 - Gly-Phe-Gly-Gly- (also known as the one-letter code GFGG)

[0323] SEQ ID NO: 59 - Gly-Gly-Phe-Gly- (also known as GGFG in single letter code)

Claims

1. An antibody-drug conjugate (ADC) comprising an antibody conjugated to a cytotoxic agent, wherein the antibody binds to human PTK-7 and comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises heavy chain complementarity determining regions (HCDRs) HCDR1, HCDR2, and HCDR3, and the VL comprises light chain complementarity determining regions (LCDRs) LCDR1, LCDR2, and LCDR3; a) the HCDR1 comprises SEQ ID NO: 4, the HCDR2 comprises SEQ ID NO: 5, the HCDR3 comprises SEQ ID NO: 6, the LCDR1 comprises SEQ ID NO: 7, the LCDR2 comprises SEQ ID NO: 8, and the LCDR3 comprises SEQ ID NO: 9; b) the HCDR1 comprises SEQ ID NO: 14, the HCDR2 comprises SEQ ID NO: 15, the HCDR3 comprises SEQ ID NO: 16, the LCDR1 comprises SEQ ID NO: 17, the LCDR2 comprises SEQ ID NO: 18, and the LCDR3 comprises SEQ ID NO: 19; c) the HCDR1 comprises SEQ ID NO: 24, the HCDR2 comprises SEQ ID NO: 25, the HCDR3 comprises SEQ ID NO: 26, the LCDR1 comprises SEQ ID NO: 27, the LCDR2 comprises SEQ ID NO: 28, and the LCDR3 comprises SEQ ID NO: 29; or d) An ADC, wherein the HCDR1 comprises SEQ ID NO: 34, the HCDR2 comprises SEQ ID NO: 35, the HCDR3 comprises SEQ ID NO: 36, the LCDR1 comprises SEQ ID NO: 37, the LCDR2 comprises SEQ ID NO: 18, and the LCDR3 comprises SEQ ID NO:

38.

2. a) the VH comprises SEQ ID NO: 10 and the VL comprises SEQ ID NO: 11; b) the VH comprises SEQ ID NO: 20 and the VL comprises SEQ ID NO: 21; c) the VH comprises SEQ ID NO: 30 and the VL comprises SEQ ID NO: 31; or d) The ADC of claim 1, wherein the VH comprises SEQ ID NO: 39 and the VL comprises SEQ ID NO:

40.

3. the antibody comprises a heavy chain (HC) and a light chain (LC); a) the HC comprises amino acids 2-441 of SEQ ID NO:2 and the LC comprises SEQ ID NO:3; b) the HC comprises amino acids 2-448 of SEQ ID NO: 12 and the LC comprises amino acids 2-215 of SEQ ID NO: 13; c) the HC comprises amino acids 2-447 of SEQ ID NO: 22 and the LC comprises amino acids 2-215 of SEQ ID NO: 23; or d) The ADC of claim 1, wherein the HC comprises amino acids 2-444 of SEQ ID NO: 32 and the LC comprises amino acids 2-215 of SEQ ID NO:

33.

4. a) the HC consists of SEQ ID NO: 2 and the LC consists of SEQ ID NO: 3; b) the HC consists of SEQ ID NO: 12 and the LC consists of SEQ ID NO: 13; c) the HC consists of SEQ ID NO: 22 and the LC consists of SEQ ID NO: 23; or d) The ADC of claim 3, wherein the HC consists of SEQ ID NO: 32 and the LC consists of SEQ ID NO:

33.

5. 5. The ADC of claims 1-4, wherein the cytotoxic agent is selected from the group consisting of a microtubule inhibitor, a topoisomerase I inhibitor, a DNA damaging agent, a DNA alkylating agent, and a DNA minor groove binder.

6. 6. The ADC of claim 5, wherein the cytotoxic agent is a topoisomerase I inhibitor.

7. 7. The ADC of claim 6, wherein the topoisomerase I inhibitor is a camptothecin analog.

8. The camptothecin analog has the formula: 【Chemistry 1】 8. The ADC of claim 7, comprising the compound of formula:

9. The ADC has the formula: 【Chemistry 2】 9. The ADC of claim 8, comprising the compound of formula:

10. The ADC of any one of claims 1 to 9, wherein the ADC further comprises a linker connecting the antibody to the cytotoxic agent.

11. 11. The ADC of claim 10, wherein the linker comprises a peptide unit or a glycocleaving unit.

12. 12. The ADC of claim 11, wherein the peptide unit comprises Val-Ala, Val-Cit, Phe-Lys, or Ala-Ala-Asn.

13. The ADC has the formula: 【Transformation 3】 13. The ADC of any one of claims 1 to 12, comprising a compound of formula:

14. The sugar-cleaving unit has the formula: 【Chemistry 4】 12. The ADC of claim 11, comprising the compound of formula:

15. The ADC has the formula: 【Transformation 5】 15. The ADC of claim 14, comprising the compound of formula:

16. 16. The ADC of any one of claims 10 to 15, wherein the linker further comprises a hydrophobic masking group.

17. 17. The ADC of claim 16, wherein the hydrophobic masking group is selected from polysarcosine or polyethylene glycol.

18. 18. The ADC of claim 16 or 17, wherein the hydrophobic masking groups are in a branched configuration on the linker.

19. The hydrophobic masking group has the formula: 【Transformation 6】 is polysarcosine of In the formula, k is an integer from 6 to 12, and X １ is H, OH or NH ２ 19. The ADC of claim 17 or 18, wherein

20. 20. The ADC of any one of claims 10 to 19, wherein the linker further comprises a connecting unit.

21. The connecting unit has the formula: 【Transformation 7】 is one of the compounds 21. The ADC of claim 20, wherein z is 1 to 5.

22. The ADC has the formula: 【Transformation 8】 is one of the compounds During the ceremony, Ab is the antibody; a) the HC comprises amino acids 2-441 of SEQ ID NO:2 and the LC comprises SEQ ID NO:3; b) the HC comprises amino acids 2-448 of SEQ ID NO: 12 and the LC comprises amino acids 2-215 of SEQ ID NO: 13; c) the HC comprises amino acids 2-447 of SEQ ID NO: 22 and the LC comprises amino acids 2-215 of SEQ ID NO: 23; or d) the HC comprises amino acids 2-444 of SEQ ID NO: 32 and the LC comprises amino acids 2-215 of SEQ ID NO: 33; and 22. The ADC of any one of claims 1 to 21, wherein n is about 1 to 16.

23. The ADC has the formula: 【Chemistry 9】 23. The ADC of claim 22, which is a compound of the formula:

24. The ADC has the formula: 【Chemistry 10】 23. The ADC of claim 22, which is a compound of the formula:

25. 25. The ADC of any one of claims 22 to 24, wherein the attachment to the antibody occurs via a thiol group of one or more cysteines of the antibody.

26. 26. The ADC of claim 25, wherein the one or more cysteines are each a native cysteine ​​in the hinge region of the antibody.

27. 27. The ADC of any one of claims 22-26, wherein the Ab comprises a HC comprising amino acids 2-441 of SEQ ID NO:2 and a LC comprising SEQ ID NO:3, and n is about 8.

28. 27. The ADC of any one of claims 22-26, wherein the Ab comprises a HC comprising amino acids 2-448 of SEQ ID NO: 12 and a LC comprising amino acids 2-215 of SEQ ID NO: 13, and n is about 8.

29. 27. The ADC of any one of claims 22-26, wherein the Ab comprises a HC comprising amino acids 2-447 of SEQ ID NO: 22 and a LC comprising amino acids 2-215 of SEQ ID NO: 23, and n is about 8.

30. 27. The ADC of any one of claims 22-26, wherein the Ab comprises amino acids 2-444 of SEQ ID NO: 32 and an LC comprising amino acids 2-215 of SEQ ID NO: 33, and n is about 8.

31. An antibody that binds to human PTK-7, the antibody comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises heavy chain complementarity determining regions (HCDRs) HCDR1, HCDR2, and HCDR3, and the VL comprises light chain complementarity determining regions (LCDRs) LCDR1, LCDR2, and LCDR3; a) the HCDR1 comprises SEQ ID NO: 4, the HCDR2 comprises SEQ ID NO: 5, the HCDR3 comprises SEQ ID NO: 6, the LCDR1 comprises SEQ ID NO: 7, the LCDR2 comprises SEQ ID NO: 8, and the LCDR3 comprises SEQ ID NO: 9; b) the HCDR1 comprises SEQ ID NO: 14, the HCDR2 comprises SEQ ID NO: 15, the HCDR3 comprises SEQ ID NO: 16, the LCDR1 comprises SEQ ID NO: 17, the LCDR2 comprises SEQ ID NO: 18, and the LCDR3 comprises SEQ ID NO: 19; c) the HCDR1 comprises SEQ ID NO: 24, the HCDR2 comprises SEQ ID NO: 25, the HCDR3 comprises SEQ ID NO: 26, the LCDR1 comprises SEQ ID NO: 27, the LCDR2 comprises SEQ ID NO: 28, and the LCDR3 comprises SEQ ID NO: 29; or d) An antibody wherein the HCDR1 comprises SEQ ID NO: 34, the HCDR2 comprises SEQ ID NO: 35, the HCDR3 comprises SEQ ID NO: 36, the LCDR1 comprises SEQ ID NO: 37, the LCDR2 comprises SEQ ID NO: 18, and the LCDR3 comprises SEQ ID NO:

38.

32. a) the VH comprises SEQ ID NO: 10 and the VL comprises SEQ ID NO: 11; b) the VH comprises SEQ ID NO: 20 and the VL comprises SEQ ID NO: 21; c) the VH comprises SEQ ID NO: 30 and the VL comprises SEQ ID NO: 31; or d) The antibody of claim 31, wherein the VH comprises SEQ ID NO: 39 and the VL comprises SEQ ID NO:

40.

33. the antibody comprises a heavy chain (HC) and a light chain (LC); a) the HC comprises amino acids 2-441 of SEQ ID NO:2 and the LC comprises SEQ ID NO:3; b) the HC comprises amino acids 2-448 of SEQ ID NO: 12 and the LC comprises amino acids 2-215 of SEQ ID NO: 13; c) the HC comprises amino acids 2-447 of SEQ ID NO: 22 and the LC comprises amino acids 2-215 of SEQ ID NO: 23; or d) The antibody of claim 31, wherein the HC comprises amino acids 2 to 444 of SEQ ID NO: 32 and the LC comprises amino acids 2 to 215 of SEQ ID NO:

33.

34. a) the HC consists of SEQ ID NO: 2 and the LC consists of SEQ ID NO: 3; b) the HC consists of SEQ ID NO: 12 and the LC consists of SEQ ID NO: 13; c) the HC consists of SEQ ID NO: 22 and the LC consists of SEQ ID NO: 23; or d) The antibody of claim 33, wherein the HC consists of SEQ ID NO: 32 and the LC consists of SEQ ID NO:

33.

35. 31. A pharmaceutical composition comprising the ADC of any one of claims 1 to 30 and one or more pharmaceutically acceptable carriers, diluents, or excipients.

36. A method of treating cancer, comprising administering to a patient in need thereof an effective amount of the ADC of any one of claims 1 to 30.

37. 37. The method of claim 36, wherein the cancer is ovarian cancer, lung cancer, breast cancer, stomach cancer, kidney cancer, prostate cancer, liver cancer, or colon cancer.

38. 38. The method of claim 36 or 37, further comprising administering simultaneously, separately, or sequentially a PD-1 inhibitor or a PD-L1 inhibitor.

39. 31. The ADC of any one of claims 1 to 30 for use in therapy.

40. 31. The ADC of any one of claims 1 to 30 for use in the treatment of cancer.

41. 41. The ADC for use according to claim 40, wherein the cancer is ovarian cancer, lung cancer, breast cancer, gastric cancer, kidney cancer, prostate cancer, liver cancer, or colon cancer.

42. The ADC for use according to any one of claims 1 to 30, wherein the ADC is administered in combination with a PD-1 inhibitor or a PD-L1 inhibitor simultaneously, separately, or sequentially.

43. 31. A pharmaceutical composition for use in the treatment of cancer, comprising an effective amount of the ADC of any one of claims 1 to 30.

44. 44. The composition for use of claim 43, wherein the cancer is ovarian cancer, lung cancer, breast cancer, stomach cancer, kidney cancer, prostate cancer, liver cancer, or colon cancer.

45. 31. Use of the ADC of any one of claims 1 to 30 for the manufacture of a medicament for the treatment of cancer.

46. 46. ​​The use of claim 45, wherein the cancer is ovarian cancer, lung cancer, breast cancer, stomach cancer, kidney cancer, prostate cancer, liver cancer, or colon cancer.

47. 35. A method of preparing an ADC comprising conjugating the antibody of any one of claims 31 to 34 to a linker-payload, wherein the linker-payload has the formula: 【Chemistry 11】 The method of claim 1, wherein the compound is one of

48. A method for producing an ADC, comprising administering to a subject an antibody of any one of claims 31 to 34 a compound of the formula: 【Chemistry 12】 with a compound of formula (I).

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