WO2025223455A1

WO2025223455A1 - Anti-ptk7/b7h3 antibodies and uses thereof

Info

Publication number: WO2025223455A1
Application number: PCT/CN2025/090662
Authority: WO
Inventors: Peiran LI; Yanan GUO
Original assignee: Biocytogen Pharmaceuticals Beijing Co Ltd
Current assignee: Biocytogen Pharmaceuticals Beijing Co Ltd
Priority date: 2024-04-24
Filing date: 2025-04-23
Publication date: 2025-10-30
Anticipated expiration: 2026-10-24

Abstract

This disclosure relates to anti-PTK7/B7H3 antibodies, and antibody drug conjugates derived therefrom, wherein the antibodies specifically bind to at least two different antigens PTK7 and B7H3. Also disclosed are pharmaceutical compositions comprising such antibodies and antibody drug conjugates, methods of using and methods of making such antibodies and antibody drug conjugates.

Description

ANTI-PTK7/B7H3 ANTIBODIES AND USES THEREOF

CLAIM OF PRIORITY

This application claims the benefit of PCT Application No. PCT/CN2024/089487, filed on April 24, 2024. The entire content of the foregoing is incorporated herein by reference.

TECHNICAL FIELD

This disclosure relates to multi-specific anti-PTK7/B7H3 antibodies (e.g., bispecific antibodies or antigen-binding fragments thereof) , and antibody drug conjugates derived therefrom.

BACKGROUND

A bispecific antibody is an artificial protein that can simultaneously bind to two different types of antigens or two different epitopes. This dual specificity opens up a wide range of applications, including redirecting T cells to tumor cells, dual targeting of different disease mediators, and delivering payloads to targeted sites. The approval of catumaxomab (anti-EpCAM and anti-CD3) and blinatumomab (anti-CD19 and anti-CD3) has become a major milestone in the development of bispecific antibodies.

As bispecific antibodies have various applications, there is a need to continue to develop various therapeutics based on bispecific antibodies.

SUMMARY

This disclosure relates to anti-PTK7/B7H3 antibodies or antigen-binding fragments thereof, wherein the antibodies or antigen-binding fragments thereof specifically bind to PTK7 and B7H3. In some embodiments, the antibodies or antigen-binding fragments thereof have identical light chain variable regions. In some embodiments, the antibodies or antigen-binding fragments thereof have a common light chain. The disclosure also relates to antibody drug conjugates derived from these anti-PTK7/B7H3 antibodies.

In one aspect, the disclosure provides an anti-PTK7/B7H3 antibody or antigen-binding fragment thereof, comprising: a first antigen-binding domain that specifically binds to PTK7; and a second antigen-binding domain that specifically binds to B7H3.

In some embodiments, the first antigen-binding domain comprises a first heavy chain variable region (VH1) and a first light chain variable region (VL1) ; and the second antigen-binding domain comprises a second heavy chain variable region (VH2) and a second light chain variable region (VL2) . In some embodiments, the first heavy chain variable region (VH1) comprises complementarity determining regions (CDRs) 1, 2, and 3, wherein the VH1 CDR1 region comprises an amino acid sequence that is at least 80%identical to a selected VH1 CDR1 amino acid sequence, the VH1 CDR2 region comprises an amino acid sequence that is at least 80%identical to a selected VH1 CDR2 amino acid sequence, and the VH1 CDR3 region comprises an amino acid sequence that is at least 80%identical to a selected VH1 CDR3 amino acid sequence; and the first light chain variable region (VL1) comprises CDRs 1, 2, and 3, wherein the VL1 CDR1 region comprises an amino acid sequence that is at least 80%identical to a selected VL1 CDR1 amino acid sequence, the VL1 CDR2 region comprises an amino acid sequence that is at least 80%identical to a selected VL1 CDR2 amino acid sequence, and the VL1 CDR3 region comprises an amino acid sequence that is at least 80%identical to a selected VL1 CDR3 amino acid sequence, wherein the selected VH1 CDRs 1, 2, and 3 amino acid sequences, the selected VL1 CDRs 1, 2, and 3 amino acid sequences are one of the following:

(1) the selected VH1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 4-6, respectively, and the selected VL1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 1-3, respectively;

(2) the selected VH1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 7-9, respectively, and the selected VL1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 1-3, respectively;

(3) the selected VH1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 10-12, respectively, and the selected VL1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 1-3, respectively; and

(4) the selected VH1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 13-15, respectively, and the selected VL1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 1-3, respectively.

In some embodiments, the second heavy chain variable region (VH2) comprises CDRs 1, 2, and 3, wherein the VH2 CDR1 region comprises an amino acid sequence that is at least 80%identical to a selected VH2 CDR1 amino acid sequence, the VH2 CDR2 region comprises an amino acid sequence that is at least 80%identical to a selected VH2 CDR2 amino acid sequence, and the VH2 CDR3 region comprises an amino acid sequence that is at least 80%identical to a selected VH2 CDR3 amino acid sequence; and the second light chain variable region (VL2) comprises CDRs 1, 2, and 3, wherein the VL2 CDR1 region comprises an amino acid sequence that is at least 80%identical to a selected VL2 CDR1 amino acid sequence, the VL2 CDR2 region comprises an amino acid sequence that is at least 80%identical to a selected VL2 CDR2 amino acid sequence, and the VL2 CDR3 region comprises an amino acid sequence that is at least 80%identical to a selected VL2 CDR3 amino acid sequence, wherein the selected VH2 CDRs 1, 2, and 3 amino acid sequences, and the selected VL2 CDRs 1, 2, and 3 amino acid sequences are one of the following:

(1) the selected VH2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 16-18, respectively, and the selected VL2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 1-3, respectively;

(2) the selected VH2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 19-21, respectively, and the selected VL2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 1-3, respectively;

(3) the selected VH2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 22-24, respectively, and the selected VL2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 1-3, respectively; and

(4) the selected VH2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 25-27, respectively, and the selected VL2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 1-3, respectively.

In some embodiments, the first antigen-binding domain comprises a first heavy chain variable region (VH1) and a first light chain variable region (VL1) ; and the second antigen-binding domain comprises a second heavy chain variable region (VH2) and a second light chain variable region (VL2) . In some embodiments, the first heavy chain variable region (VH1) comprises complementarity determining regions (CDRs) 1, 2, and 3, with no more than one amino acid substitution relative to a selected VH1 CDR1 amino acid sequence, a selected VH1 CDR2 amino acid sequence, and/or a selected VH1 CDR3 amino acid sequence; and the first light chain variable region (VL1) comprises CDRs 1, 2, and 3, with no more than one amino acid substitution relative to a selected VL1 CDR1 amino acid sequence, a selected VL1 CDR2 amino acid sequence, and/or a selected VL1 CDR3 amino acid sequence, wherein the selected VH1 CDRs 1, 2, and 3 amino acid sequences, the selected VL1 CDRs 1, 2, and 3 amino acid sequences are one of the following:

In some embodiments, the second heavy chain variable region (VH2) comprises CDRs 1, 2, and 3, with no more than one amino acid substitution relative to a selected VH2 CDR1 amino acid sequence, aselected VH2 CDR2 amino acid sequence, and/or a selected VH2 CDR3 amino acid sequence; and the second light chain variable region (VL2) comprises CDRs 1, 2, and 3, with no more than one amino acid substitution relative to a selected VL2 CDR1 amino acid sequence, a selected VL2 CDR2 amino acid sequence, and/or a selected VL2 CDR3 amino acid sequence, wherein the selected VH2 CDRs 1, 2, and 3 amino acid sequences, and the selected VL2 CDRs 1, 2, and 3 amino acid sequences are one of the following:

In some embodiments, the selected VH1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 4-6, respectively, and the selected VL1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 1-3, respectively, and the selected VH2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 16-18, respectively, and the selected VL2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 1-3, respectively.

In some embodiments, the selected VH1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 4-6, respectively, and the selected VL1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 1-3, respectively, and the selected VH2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 22-24, respectively, and the selected VL2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 1-3, respectively.

In some embodiments, the selected VH1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 10-12, respectively, and the selected VL1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 1-3, respectively, and the selected VH2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 16-18, respectively, and the selected VL2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 1-3, respectively.

In some embodiments, the selected VH1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 10-12, respectively, and the selected VL1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 1-3, respectively, and the selected VH2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 22-24, respectively, and the selected VL2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 1-3, respectively.

In some embodiments, the selected VH1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 7-9, respectively, and the selected VL1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 1-3, respectively, and the selected VH2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 19-21, respectively, and the selected VL2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 1-3, respectively.

In some embodiments, the selected VH1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 7-9, respectively, and the selected VL1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 1-3, respectively, and the selected VH2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 25-27, respectively, and the selected VL2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 1-3, respectively.

In some embodiments, the selected VH1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 13-15, respectively, and the selected VL1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 1-3, respectively, and the selected VH2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 19-21, respectively, and the selected VL2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 1-3, respectively.

In some embodiments, the selected VH1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 13-15, respectively, and the selected VL1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 1-3, respectively, and the selected VH2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 25-27, respectively, and the selected VL2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 1-3, respectively.

In some embodiments, the first heavy chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100%identical to SEQ ID NO: 29, the first light chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100%identical to SEQ ID NO: 28, the second heavy chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100%identical to SEQ ID NO: 31, and the second light chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100%identical to SEQ ID NO: 28.

In some embodiments, the first heavy chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100%identical to SEQ ID NO: 29, the first light chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100%identical to SEQ ID NO: 28, the second heavy chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100%identical to SEQ ID NO: 32, and the second light chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100%identical to SEQ ID NO: 28.

In some embodiments, the first heavy chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100%identical to SEQ ID NO: 30, the first light chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100%identical to SEQ ID NO: 28, the second heavy chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100%identical to SEQ ID NO: 31, and the second light chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100%identical to SEQ ID NO: 28.

In some embodiments, the first heavy chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100%identical to SEQ ID NO: 30, the first light chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100%identical to SEQ ID NO: 28, the second heavy chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100%identical to SEQ ID NO: 32, and the second light chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100%identical to SEQ ID NO: 28.

In some embodiments, the VH1 comprises an amino acid sequence that is at least 90%identical to a selected VH sequence, and the VL1 comprises an amino acid sequence that is at least 90%identical to a selected VL sequence, wherein the selected VH sequence and the selected VL sequence are one of the following:

(1) the selected VH sequence is SEQ ID NO: 29, and the selected VL sequence is SEQ ID NO: 28; and

(2) the selected VH sequence is SEQ ID NO: 30, and the selected VL sequence is SEQ ID NO: 28.

In some embodiments, the VH1 comprises VH CDR1, VH CDR2, and VH CDR3 that are identical to VH CDR1, VH CDR2, and VH CDR3 of a selected VH sequence; and the VL1 comprising VL CDR1, VL CDR2, and VL CDR3 that are identical to VL CDR1, VL CDR2, and VL CDR3 of a selected VL sequence, wherein the selected VH sequence and the selected VL sequence are one of the following:

In some embodiments, the VH2 comprises an amino acid sequence that is at least 90%identical to a selected VH sequence, and the VL2 comprises an amino acid sequence that is at least 90%identical to a selected VL sequence, wherein the selected VH sequence and the selected VL sequence are one of the following:

(1) the selected VH sequence is SEQ ID NO: 31, and the selected VL sequence is SEQ ID NO: 28; and

(2) the selected VH sequence is SEQ ID NO: 32, and the selected VL sequence is SEQ ID NO: 28.

In some embodiments, the VH2 comprises VH CDR1, VH CDR2, and VH CDR3 that are identical to VH CDR1, VH CDR2, and VH CDR3 of a selected VH sequence; and the VL2 comprising VL CDR1, VL CDR2, and VL CDR3 that are identical to VL CDR1, VL CDR2, and VL CDR3 of a selected VL sequence, wherein the selected VH sequence and the selected VL sequence are one of the following:

In some embodiments, the VH1 comprises the sequence of SEQ ID NO: 29 and the VL1 comprises the sequence of SEQ ID NO: 28.

In some embodiments, the VH1 comprises the sequence of SEQ ID NO: 30 and the VL1 comprises the sequence of SEQ ID NO: 28.

In some embodiments, the VH2 comprises the sequence of SEQ ID NO: 31 and the VL2 comprises the sequence of SEQ ID NO: 28.

In some embodiments, the VH2 comprises the sequence of SEQ ID NO: 32 and the VL2 comprises the sequence of SEQ ID NO: 28.

In some embodiments, the VH1 comprises the sequence of SEQ ID NO: 29 and the VL1 comprises the sequence of SEQ ID NO: 28, and the VH2 comprises the sequence of SEQ ID NO: 31 and the VL2 comprises the sequence of SEQ ID NO: 28.

In some embodiments, the VH1 comprises the sequence of SEQ ID NO: 30 and the VL1 comprises the sequence of SEQ ID NO: 28, and the VH2 comprises the sequence of SEQ ID NO: 32 and the VL2 comprises the sequence of SEQ ID NO: 28.

In one aspect, the disclosure provides an anti-PTK7/B7H3 antibody or antigen-binding fragment thereof, comprising: a first antigen-binding domain that specifically binds to PTK7 comprising a VH1 and a VL1; and a second antigen-binding domain that specifically binds to B7H3 comprising a VH2 and a VL2; wherein:

A (1) the VH1 comprises CDRs 1, 2, 3 amino acid sequences as set forth in SEQ ID NOs: 4-6, respectively, and the VL1 comprises CDRs 1, 2, 3 amino acid sequences as set forth in SEQ ID NOs: 1-3, respectively; or

A (2) the VH1 comprises CDRs 1, 2, 3 amino acid sequences as set forth in SEQ ID NOs: 7-9, respectively, and the VL1 comprises CDRs 1, 2, 3 amino acid sequences as set forth in SEQ ID NOs: 1-3, respectively;

and

B (1) the VH2 comprises CDRs 1, 2, 3 amino acid sequences as set forth in SEQ ID NOs: 16-18, respectively, and the VL2 comprises CDRs 1, 2, 3 amino acid sequences as set forth in SEQ ID NOs: 1-3, respectively; or

B (2) the VH2 comprises CDRs 1, 2, 3 amino acid sequences as set forth in SEQ ID NOs: 19-21, respectively, and the VL2 comprises CDRs 1, 2, 3 amino acid sequences as set forth in SEQ ID NOs: 1-3, respectively.

A (1) the VH1 comprises CDRs 1, 2, 3 amino acid sequences as set forth in SEQ ID NOs: 10-12, respectively, and the VL1 comprises CDRs 1, 2, 3 amino acid sequences as set forth in SEQ ID NOs: 1-3, respectively; or

A (2) the VH1 comprises CDRs 1, 2, 3 amino acid sequences as set forth in SEQ ID NOs: 13-15, respectively, and the VL1 comprises CDRs 1, 2, 3 amino acid sequences as set forth in SEQ ID NOs: 1-3, respectively;

and

B (1) the VH2 comprises CDRs 1, 2, 3 amino acid sequences as set forth in SEQ ID NOs: 22-24, respectively, and the VL2 comprises CDRs 1, 2, 3 amino acid sequences as set forth in SEQ ID NOs: 1-3, respectively; or

B (2) the VH2 comprises CDRs 1, 2, 3 amino acid sequences as set forth in SEQ ID NOs: 25-27, respectively, and the VL2 comprises CDRs 1, 2, 3 amino acid sequences as set forth in SEQ ID NOs: 1-3, respectively.

In some embodiments, the first antigen-binding domain specifically binds to human or monkey PTK7; and/or the second antigen-binding domain specifically binds to human or monkey B7H3.

In some embodiments, the first antigen-binding domain is human or humanized; and/or the second antigen-binding domain is human or humanized.

In some embodiments, the anti-PTK7/B7H3 antibody is a multi-specific antibody (e.g., abispecific antibody) .

In some embodiments, the first antigen-binding domain is a single-chain variable fragment (scFv) ; and/or the second antigen-binding domain is a scFv.

In some embodiments, the first light chain variable region and the second light chain variable region are identical.

In one aspect, the disclosure provides an anti-PTK7/B7H3 antibody or antigen-binding fragment thereof that cross-competes with the anti-PTK7/B7H3 antibody or antigen-binding fragment thereof as described herein.

In one aspect, the disclosure provides a nucleic acid comprising a polynucleotide encoding the anti-PTK7/B7H3 antibody or antigen-binding fragment thereof as described herein.

In one aspect, the disclosure provides a vector comprising the nucleic acid as described herein.

In one aspect, the disclosure provides a cell comprising the vector as described herein. In some embodiments, the cell is a CHO cell.

In one aspect, the disclosure provides a cell comprising the nucleic acid as described herein.

In one aspect, the disclosure provides a composition comprising a first vector encoding the VH1, a second vector encoding the VH2, and a third vector encoding the VL1/VL2, of the anti-PTK7/B7H3 antibody or antigen-binding fragment thereof as described herein.

In one aspect, the disclosure provides a cell comprising the composition as described herein. In some embodiments, the cell is a CHO-S cell.

In one aspect, the disclosure provides a method of producing an anti-PTK7/B7H3 antibody or an antigen-binding fragment thereof, the method comprising:

(a) culturing the cell as described herein under conditions sufficient for the cell to produce the anti-PTK7/B7H3 antibody or the antigen-binding fragment thereof; and

(b) collecting the anti-PTK7/B7H3 antibody or the antigen-binding fragment thereof produced by the cell.

In one aspect, the disclosure provides an anti-PTK7/B7H3 antibody-drug conjugate (ADC) comprising a therapeutic agent covalently bound to the anti-PTK7/B7H3 antibody or antigen-binding fragment thereof as described herein. In some embodiments, the therapeutic agent is a cytotoxic or cytostatic agent. In some embodiments, the therapeutic agent is MMAE or MMAF.

In some embodiments, the therapeutic agent is selected from

In some embodiments, the therapeutic agent is linked to the antibody or antigen-binding fragment thereof via a linker. In some embodiments, the linker has a structure of:

In some embodiments, the antibody-drug conjugate has a structure of:

in some embodiments, n=1-8; in some embodiments, “Ab” represents the antibody or antigen-binding fragment thereof.

In some embodiments, the drug-to-antibody ratio (DAR) is about 2 to 8. In some embodiments, the drug-to-antibody ratio (DAR) is about 4. In some embodiments, the drug-to-antibody ratio (DAR) is about 8.

In one aspect, the disclosure provides an anti-PTK7/B7H3 ADC comprising:

(i) an anti-PTK7/B7H3 antibody or antigen-binding fragment thereof comprising: a first antigen-binding domain that specifically binds to PTK7 comprising a VH1 and a VL1; and a second antigen-binding domain that specifically binds to B7H3 comprising a VH2 and a VL2; wherein:

and

B (2) the VH2 comprises CDRs 1, 2, 3 amino acid sequences as set forth in SEQ ID NOs: 19-21, respectively, and the VL2 comprises CDRs 1, 2, 3 amino acid sequences as set forth in SEQ ID NOs: 1-3, respectively, and

(ii)

covalently attached to the antibody or antigen-binding fragment thereof via a linker.

In some embodiments, the linker is

In one aspect, the disclosure provides a method of preparing an antibody-drug conjugate, the method comprising:

(a) providing the anti-PTK7/B7H3 antibody or antigen-binding fragment thereof as described herein;

(b) conjugating a therapeutic agent to the anti-PTK7/B7H3 antibody or antigen-binding fragment thereof via a linker to form the antibody-drug conjugate.

In some embodiments, the method further comprises treating the anti-PTK7/B7H3 antibody or antigen-binding fragment thereof with a reductant to generate one or more thiol groups before step (b) .

In some embodiments, the conjugating step comprises reacting one or more thiol groups of the antibody or antigen-binding fragment with a maleimide-functionalized linker that is linked to the therapeutic agent.

In some embodiments, the method further comprises purifying the antibody-drug conjugate.

In some embodiments, the drug-to-antibody ratio (DAR) is about 2～8.

In some embodiments, the therapeutic agent is a cytotoxic or cytostatic agent. In some embodiments, the therapeutic agent is selected from

In some embodiments, the therapeutic agent is linked to the anti-PTK7/B7H3 antibody or antigen-binding fragment thereof via a linker. In some embodiments, the linker has a structure of:

In some embodiments, the antibody-drug conjugate has a structure of:

wherein n=1-8; wherein “Ab” represents the anti-PTK7/B7H3 antibody or antigen-binding fragment thereof as described herein.

In some embodiments, the antibody-drug conjugate has a structure of:

In one aspect, the disclosure provides a method of treating a subject having cancer, the method comprising administering a therapeutically effective amount of a composition comprising the anti-PTK7/B7H3 antibody or antigen-binding fragment thereof as described herein, or the anti-PTK7/B7H3 antibody-drug conjugate as described herein, to the subject. In some embodiments, the subject has a cancer expressing PTK7 and/or B7H3.

In some embodiments, the cancer is a solid tumor, lung cancer (e.g., non-small cell lung cancer, small cell lung cancer, lung adenocarcinoma, or lung carcinoma) , gastric cancer (e.g., gastric carcinoma) , colorectal cancer, breast cancer, pancreatic cancer, head and neck cancer, ovarian cancer, prostate cancer, thyroid cancer, liver cancer, nasopharynx cancer, brain cancer, bladder cancer, cervical cancer, or oesophageal cancer.

In some embodiments, the subject is a human.

In some embodiments, the method further comprises administering an anti-PD1 antibody to the subject.

In some embodiments, the method further comprises administering a chemotherapy to the subject.

In one aspect, the disclosure provides a method of decreasing the rate of tumor growth, the method comprising contacting a tumor cell with an effective amount of a composition comprising the anti-PTK7/B7H3 antibody or antigen-binding fragment thereof one as described herein, or the anti-PTK7/B7H3 antibody-drug conjugate one as described herein.

In one aspect, the disclosure provides a method of killing a tumor cell, the method comprising contacting a tumor cell with an effective amount of a composition comprising the anti-PTK7/B7H3 antibody or antigen-binding fragment thereof as described herein, or the anti-PTK7/B7H3 antibody-drug conjugate as described herein.

In one aspect, the disclosure provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and (a) the anti-PTK7/B7H3 antibody or antigen-binding fragment thereof as described herein, and/or (b) the anti-PTK7/B7H3 antibody-drug conjugate as described herein.

In one aspect, the disclosure provides an anti-PTK7/B7H3 antibody-drug conjugate (ADC) comprising a therapeutic agent covalently bound to a bispecific antibody or antigen-binding fragment thereof comprising: a first antigen-binding domain that specifically binds to PTK7; and a second antigen-binding domain that specifically binds to B7H3. In some embodiments, the drug-to-antibody ratio (DAR) is about 2 to 8. In some embodiments, the drug-to-antibody ratio (DAR) is about 8. In some embodiments, the drug-to-antibody ratio (DAR) is about 4.

As used herein, the term “antigen-binding domain” refers to one or more protein domain (s) (e.g., formed from amino acids from a single polypeptide or formed from amino acids from two or more polypeptides (e.g., the same or different polypeptides) ) that is capable of specifically binding to one or more different antigen (s) (e.g., an effector antigen or control antigen) . In some examples, an antigen-binding domain can bind to an antigen or epitope with specificity and affinity similar to that of naturally-occurring antibodies. In some embodiments, the antigen-binding domain can be an antibody or a fragment thereof. One example of an antigen-binding domain is an antigen-binding domain formed by a VH-VL dimer. In some embodiments, an antigen-binding domain can include an alternative scaffold. In some embodiments, the antigen-binding domain is a VHH. Non-limiting examples of antigen-binding domains are described herein. Additional examples of antigen-binding domains are known in the art. In some examples, an antigen-binding domain can bind to a single antigen (e.g., one of an effector antigen and a control antigen) . In other examples, an antigen-binding domain can bind to two different antigens (e.g., an effector antigen and a control antigen) .

The term “antibody” is used herein in its broadest sense and includes certain types of immunoglobulin molecules that include one or more antigen-binding domains that specifically bind to an antigen or epitope. An antibody specifically includes, e.g., intact antibodies (e.g., intact immunoglobulins) , antibody fragments, bispecific antibodies, and multi-specific antibodies. One example of an antibody is a protein complex that includes two heavy chains and two light chains. Additional examples of an antibody are described herein.

As used herein, the term “multispecific antibody” is an antibody that includes two or more different antigen-binding domains that collectively specifically bind two or more different epitopes. The two or more different epitopes may be epitopes on the same antigen (e.g., a single polypeptide present on the surface of a cell) or on different antigens (e.g., different proteins present on the surface of the same cell or present on the surface of different cells) . In some aspects, a multi-specific antibody binds two different epitopes (i.e., a “bispecific antibody” ) . In some aspects, a multi-specific antibody binds three different epitopes (i.e., a “trispecific antibody” ) . In some aspects, a multi-specific antibody binds four different epitopes (i.e., a “quadspecific antibody” ) . In some aspects, a multi-specific antibody binds five different epitopes (i.e., a “quintspecific antibody” ) . Each binding specificity may be present in any suitable valency. Non-limiting examples of multi-specific antibodies are described herein.

As used herein, the term “bispecific antibody” refers to an antibody that binds to two different epitopes. The epitopes can be on the same antigen or on different antigens.

As used herein, the term “common light chain” refers to a light chain that can interact with two or more different heavy chains, forming different antigen-binding sites, wherein these different antigen-binding sites can specifically bind to different antigens or epitopes. Similarly, the term “common light chain variable region” refers to a light chain variable region that can interact with two or more different heavy chain variable regions, forming different antigen-binding sites, wherein these different antigen-binding sites can specifically bind to different antigens or epitopes. In some embodiments, the antibody or antigen-binding fragment thereof can have a common light chain. In some embodiments, the anti-PTK7/B7H3 antibody or antigen-binding fragment thereof can have a common light chain variable region.

As used herein, the term “anti-PTK7/B7H3 antibody or antigen-binding fragment thereof” refers to an antibody or antigen-binding fragment that binds to both PTK7 and B7H3.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials are described herein for use in the present invention; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

Other features and advantages of the invention will be apparent from the following detailed description and figures, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1 shows the binding activities of antibodies to tumor cells NCI-H520 (A) and SHP-77 (B) .

FIG. 2 shows the binding activities of antibodies to tumor cells NCI-H69 (A) and NCI-N87 (B) .

FIG. 3 shows endocytosis activities of antibodies in NCI-H520 cells (A) and HCC70 cells (B) .

FIG. 4 shows the average tumor volume in different groups of B-NDG mice that were injected with colorectal cancer patient-derived tumor fragments, and were treated with PBS or ADCs.

FIG. 5 shows the average tumor volume in different groups of B-NDG mice that were injected with lung cancer patient-derived tumor fragments, and were treated with PBS or ADCs.

FIG. 6 shows the average tumor volume in different groups of B-NDG mice that were injected with lung cancer patient-derived tumor fragments, and were treated with PBS or ADCs.

FIG. 7 shows the average tumor volume in different groups of B-NDG mice that were injected with lung cancer patient-derived tumor fragments, and were treated with PBS or ADCs.

FIG. 8 shows the average tumor volume in different groups of B-NDG mice that were injected with pancreatic cancer patient-derived tumor fragments, and were treated with PBS or ADCs.

FIG. 9 shows the average tumor volume in different groups of B-NDG mice that were injected with breast cancer patient-derived tumor fragments, and were treated with PBS or ADCs.

FIG. 10 shows the average tumor volume in different groups of B-NDG mice that were injected with breast cancer patient-derived tumor fragments, and were treated with PBS or ADCs.

FIG. 11 shows the average tumor volume in different groups of B-NDG mice that were injected with breast cancer patient-derived tumor fragments, and were treated with PBS or ADCs.

FIG. 12 lists heavy chain variable region and light chain variable region CDR sequences of anti-PTK7 antigen binding domain (3C4 and 3E6) and anti-B7H3 antigen binding domain (21A9 and 20H8) in anti-PTK7/B7H3 antibodies as defined by Kabat numbering.

FIG. 13 lists heavy chain variable region and light chain variable region CDR sequences of anti-PTK7 antigen binding domain (3C4 and 3E6) and anti-B7H3 antigen binding domain (21A9 and 20H8) in anti-PTK7/B7H3 antibodies as defined by Chothia numbering.

FIG. 14 lists additional amino acid sequences discussed in the disclosure.

FIG. 15 shows the average tumor volume in different groups of B-NDG mice that were injected with colorectal cancer patient-derived tumor fragments, and were treated with PBS or ADCs.

FIG. 16 shows the average tumor volume in different groups of B-NDG mice that were injected with lung cancer patient-derived tumor fragments, and were treated with PBS or ADCs.

FIG. 17 shows the average tumor volume in different groups of B-NDG mice that were injected with breast cancer patient-derived tumor fragments, and were treated with PBS or ADCs.

FIG. 18 shows the average tumor volume in different groups of B-NDG mice that were injected with breast cancer patient-derived tumor fragments, and were treated with PBS or ADCs.

FIG. 19 shows the free payload levels after 14 days of incubation in the humans, monkeys, rats, and mice plasma.

DETAILED DESCRIPTION

A bispecific antibody or antigen-binding fragment thereof is an artificial protein that can simultaneously bind to two different epitopes (e.g., on two different antigens) . In some embodiments, abispecific antibody or antigen-binding fragment thereof can have two arms. Each arm can have one heavy chain variable region and one light chain variable region, forming an antigen-binding domain (or an antigen-binding region) . In some embodiments, the bispecific antibody has a common light chain.

The present disclosure relates to anti-PTK7/B7H3 antibodies (e.g., bispecific antibodies or antigen-binding fragments thereof) that specifically bind to B7H3 and PTK7, and antibody drug conjugates derived from these anti-PTK7/B7H3 antibodies.

Anti-PTK7/B7H3 Antibody

Protein tyrosine kinase 7 (PTK7) is a highly conserved member of the pseudokinase family of receptor tyrosine kinases. The lack of observable kinase activity across species is likely explained by substitutions at residues that are typically conserved in kinase domains. Genetic and biochemical studies have demonstrated a key function for PTK7 in noncanonical Wnt signaling, and PTK7-deficient embryos exhibit severe developmental defects in planar cell polarity. There is also evidence for additional, possibly context-dependent functions of PTK7 in the vascular endothelial growth factor (VEGF) , semaphorin/plexin, and canonical Wnt signaling pathways. Oncogenic functions of PTK7 have been documented in colon cancer, lung cancer, and esophageal cancer, and PTK7 promotes cell survival and resistance to chemotherapy in acute myeloid leukemia.

PTK7 is highly expressed in breast cancer and correlates with worse prognosis and associates with tumor metastasis and progression in TNBC. Co-expression analysis and gain-or loss-of-function of PTK7 in TNBC cell lines revealed that PTK7 participates in EGFR/Akt signaling regulation and associated with extracellular matrix organization and migration genes in breast cancer, including COL1A1, FN1, WNT5B, MMP11, MMP14 and SDC1. Gain-or loss-of-function experiments of PTK7 suggested that PTK7 promotes proliferation and migration in TNBC cell lines. PTK7 knockdown cell bearing mouse model further demonstrated that PTK7-deficiency inhibits TNBC tumor progression in vivo.

A detailed review of PTK7 and its functions can be found in Damelin M, et al., A PTK7-targeted antibody-drug conjugate reduces tumor-initiating cells and induces sustained tumor regressions. Sci Transl Med. 2017 Jan 11; 9 (372) : eaag2611. doi: 10.1126/scitranslmed. aag2611. PMID: 28077676; Xiang et al., Biomater Res. 2022 Dec 5; 26 (1) : 74. doi: 10.1186/s40824-022-00328-9; Cui et al., Front. Oncol., 22 July 2021, Sec. Breast Cancer, Volume 11-2021; and Kim et al., Int J Mol Sci. 2022 Oct 13; 23 (20) : 12195. doi: 10.3390/ijms232012195, each of which is incorporated by reference in its entirety.

B7 Homolog 3 (B7H3; also known as CD276, Cluster of Differentiation 276, B7RP-2, or 4Ig-B7H3) is a type I transmembrane protein encoded by chromosome 9 in mice and chromosome 15 in humans. The extracellular domain is composed of a single pair of immunoglobulin variable domain and immunoglobulin constant domain in mice (2Ig-B7H3 isoform) and two identical pairs in human (4Ig-B7H3 isoform) due to exon duplication. The intracellular tail of B7H3 is short and has no known signaling motif. B7H3 was first described in humans and then in mice, but is universally expressed among species. A soluble form, cleaved from the surface of activated T cell, monocyte, or DCs by a matrix metallopeptidase MMP or produced through alternative splicing of the intron, is also detectable in human sera. Soluble B7H3 can activate the NF-κB signaling pathway to enhance the invasion and metastasis of pancreatic cancer cells. In addition, the level of soluble B7H3 in the pleural effusion of patients with non-small cell lung cancer is significantly higher than that of healthy people. Thus, B7H3 can be used as a diagnostic and prognostic indicator for related tumors.

B7H3 is expressed on many tissues and cell types. At the mRNA level, it is ubiquitously found in non-lymphoid and lymphoid organs such as liver, heart, prostate, spleen and thymus. Despite broad mRNA expression, protein expression is limited at steady state, suggesting the presence of an important post-transcriptional control mechanism. B7H3 is constitutively found on non-immune resting fibroblasts, endothelial cells (EC) , osteoblasts, and amniotic fluid stem cells. Moreover, B7H3 expression is induced on immune cells, specifically antigen-presenting cells. In particular, coculture with regulatory T cells (Treg) , IFN-γ, lipopolysaccharide (LPS) , or anti-CD40 in vitro stimulation all induce the expression of B7H3 on dendritic cells (DCs) . Monocytes and monocytes-derived DCs upregulate B7H3 after LPS stimulation or cytokine-induced differentiation respectively. Additionally, B7H3 is also detected on natural killer (NK) cells, B cells, and a minor population of T cells following PMA/ionomycin stimulation.

The B7H3 pathway has a dual role in contributing to the regulation of innate immune responses. One study found that neuroblastoma cells express B7H3 on their cell surface, which protects them from NK cell-mediated lysis. Another group argues that B7H3 co-stimulates innate immunity by augmenting pro-inflammatory cytokines release from LPS-stimulated monocytes/macrophages, in both a Toll-like receptor 4-and 2-dependent manner.

B7H3 plays an important role in T cell-mediated adaptive immunity, although the nature of its signaling remains controversial. A co-stimulatory role of B7H3 on human T cells was initially reported in vitro. Murine studies showing B7H3 worsens experimental autoimmune encephalomyelitis (EAE) , arthritis, bacterial meningitis and chronic allograft rejection supported this claim. However, subsequent studies have mostly shown that B7H3 acts as a T cell co-inhibitor. B7H3 inhibits polyclonal or allogeneic CD4+and CD8+T cell activation, proliferation and effector cytokine production (IFN-γand IL-2) in mice and humans. This negative regulation of T cells is associated with diminished NFAT, NF-kB and AP-1 transcriptional factor activity. Independent studies utilizing either protein blockade or gene-knockout mice have reported that B7H3 ameliorates graft-versus-host-disease, prolongs cardiac allograft survival, reduces airway hypersensitivity, and delays EAE onset, especially by down-regulating Th1 responses. These examples lend more credence to the co-inhibitory nature of B7H3.

The crystal structure of mouse B7H3 reveals that its receptor engagement on T cells involves the particular segment connecting F and G strands (the FG loop) of the immunoglobulin variable domain of B7H3. Moreover, B7H3 crystallizes as a glycosylated monomer but also undergoes an unusual dimerization in vitro. Altogether, the nature of the receptor (s) , differences in cellular context, and various disease models certainly account for the discrepancies in the function of the B7H3 pathway in regulating both innate and adaptive immunity during homeostasis and inflammation.

Beyond the immune system, the B7H3 pathway has a non-immunological role in promoting osteoblastic differentiation and bone mineralization in mice, ensuring normal bone formation. Indeed, B7H3 knockout mice had reduced bone mineral density and were more susceptible to bone fractures compared to wild-type mice. Furthermore, similar to other immune checkpoints of the B7-CD28 pathways, B7H3 is also expressed in human cancers and participates in tumorigenesis through modulation of both immune and non-immune related pathways.

A detailed description of B7H3 and its function can be found, e.g., in Picarda, E. et al., "Molecular pathways: targeting B7-H3 (CD276) for human cancer immunotherapy. " Clinical Cancer Research 22.14 (2016) : 3425-3431; Collins, M. et al., "The B7 family of immune-regulatory ligands. " Genome Biology 6.6 (2005) : 1-7; Castellanos, J. R. et al., "B7-H3 role in the immune landscape of cancer. " American Journal of Clinical andExperimental Immunology 6.4 (2017) : 66; and Yang, S. et al., "B7-H3, a checkpoint molecule, as a target for cancer immunotherapy. " International Journal of Biological Sciences 16.11 (2020) : 1767; each of which is incorporated by reference in its entirety.

In some embodiments, the bispecific anti-PTK7/B7H3 antibody described herein can be designed to have an IgG1 subtype structure with knobs-into-holes (KIH) mutations, which can promote heterodimerization and avoid mispairing between the two heavy chains. In some embodiments, the bispecific anti-PTK7/B7H3 antibody has a higher endocytosis rate than the corresponding monoclonal antibodies or the positive control antibodies.

In some embodiments, the bispecific anti-PTK7/B7H3 antibody described herein can be conjugated with a therapeutic agent, forming an antibody drug conjugate (ADC) . In some embodiments, the drug-to-antibody ratio (DAR) of the ADCs described herein is about 3.8, about 3.9, about 4.0, about 4.1, about 4.2, about 4.3, about 4.4, about 4.5, about 4.6, or about 4.7. In some embodiments, the DAR of the ADCs described herein is about 3.5 to about 4.5. In some embodiments, the DAR of the ADCs described herein is about 7.5 to about 8.5, about 7.6 to about 8.5, about 7.7 to about 8.5, about 7.8 to about 8.5, about 7.9 to about 8.5, about 8.0 to about 8.5, about 8.1 to about 8.5, about 8.2 to about 8.5, about 8.3 to about 8.5, about 8.4 to about 8.5.

In some embodiments, the anti-PTK7/B7H3 ADC described herein can effectively inhibit in vitro cancer cell growth at a concentration of less than 10μg/mL, less than 3.33μg/mL, less than 1.11μg/mL, less than 0.37μg/mL, less than 0.12μg/mL, less than 0.04μg/mL, or less than 0.01μg/mL. In some embodiments, the anti-PTK7/B7H3 ADC described herein can inhibit in vivo cancer cell growth (e.g., colorectal cancer, lung cancer, pancreatic cancer, gastric cancer, or breast cancer) in a xenograft mouse model at a dose level of less than 30 mg/kg, 25 mg/kg, 20 mg/kg, 15 mg/kg, 10 mg/kg, 9 mg/kg, 8 mg/kg, 7 mg/kg, 6 mg/kg, 5 mg/kg, 4 mg/kg, 3 mg/kg, 2 mg/kg, or 1 mg/kg.

In some embodiments, the anti-PTK7/B7H3 antibody described herein has a common light chain. In some embodiments, the anti-PTK7/B7H3 antibody includes an anti-PTK7 antigen-binding domain (e.g., 3C4 and 3E6) or an anti-B7H3 antigen-binding domain (e.g., 21A9 and 20H8) . In some embodiments, the anti-PTK7/B7H3 antibodies have a heavy chain variable region targeting B7H3 (e.g., any one of the VH targeting B7H3 described herein) , a heavy chain variable region targeting PTK7 (e.g., any one of the VH targeting PTK7 described herein) , and two identical common light chain variable regions.

The CDR sequences for 3C4 antigen-binding domain include CDRs of the heavy chain variable domain, SEQ ID NOs: 4-6, and CDRs of the light chain variable domain, SEQ ID NOs: 1-3 as defined by Kabat numbering. The CDRs can also be defined by Chothia system. Under the Chothia numbering, the CDR sequences of the heavy chain variable domain are set forth in SEQ ID NOs: 7-9, and CDR sequences of the light chain variable domain are set forth in SEQ ID NOs: 1-3. The human heavy chain variable region and human light chain variable region for 3C4 are shown in SEQ ID NO: 29 and SEQ ID NO: 28, respectively.

The CDR sequences for 3E6 antigen-binding domain include CDRs of the heavy chain variable domain, SEQ ID NOs: 10-12, and CDRs of the light chain variable domain, SEQ ID NOs: 1-3, as defined by Kabat numbering. Under Chothia numbering, the CDR sequences of the heavy chain variable domain are set forth in SEQ ID NOs: 13-15, and CDRs of the light chain variable domain are set forth in SEQ ID NOs: 1-3. The human heavy chain variable region and human light chain variable region for 3E6 are shown in SEQ ID NO: 30 and SEQ ID NO: 28, respectively.

The CDR sequences for 21A9 antigen-binding domain include CDRs of the heavy chain variable domain, SEQ ID NOs: 16-18, and CDRs of the light chain variable domain, SEQ ID NOs: 1-3 as defined by Kabat numbering. The CDRs can also be defined by Chothia system. Under the Chothia numbering, the CDR sequences of the heavy chain variable domain are set forth in SEQ ID NOs: 19-21, and CDR sequences of the light chain variable domain are set forth in SEQ ID NOs: 1-3. The human heavy chain variable region and human light chain variable region for 21A9 are shown in SEQ ID NO: 31 and SEQ ID NO: 28, respectively.

The CDR sequences for 20H8 antigen-binding domain include CDRs of the heavy chain variable domain, SEQ ID NOs: 22-24, and CDRs of the light chain variable domain, SEQ ID NOs: 1-3, as defined by Kabat numbering. Under Chothia numbering, the CDR sequences of the heavy chain variable domain are set forth in SEQ ID NOs: 25-27, and CDRs of the light chain variable domain are set forth in SEQ ID NOs: 1-3. The human heavy chain variable region and human light chain variable region for 20H8 are shown in SEQ ID NO: 32 and SEQ ID NO: 28, respectively.

In some embodiments, the anti-PTK7/B7H3 antibodies described herein can contain one, two, or three heavy chain variable region CDRs selected from the group of SEQ ID NOs: 4-6, SEQ ID NOs: 7-9, SEQ ID NOs: 10-12, SEQ ID NOs: 13-15, SEQ ID NOs: 16-18, SEQ ID NOs: 19-21, SEQ ID NOs: 22-24, and SEQ ID NOs: 25-27; and/or one, two, or three light chain variable region CDRs selected from the group of SEQ ID NOs: 1-3.

In some embodiments, the anti-PTK7/B7H3 antibodies or antigen-binding fragments thereof can have a heavy chain variable region (VH) comprising complementarity determining regions (CDRs) 1, 2, 3, wherein the CDR1 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to a selected VH CDR1 amino acid sequence, the CDR2 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to a selected VH CDR2 amino acid sequence, and the CDR3 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to a selected VH CDR3 amino acid sequence, and a light chain variable region (VL) comprising CDRs 1, 2, 3, wherein the CDR1 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to a selected VL CDR1 amino acid sequence, the CDR2 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to a selected VL CDR2 amino acid sequence, and the CDR3 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to a selected VL CDR3 amino acid sequence. The selected VH CDRs 1, 2, 3 amino acid sequences and the selected VL CDRs, 1, 2, 3 amino acid sequences are shown in FIGs. 12-13.

In some embodiments, the anti-PTK7/B7H3 antibody or antigen-binding fragment described herein can contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 4 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 5 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 6 with zero, one or two amino acid insertions, deletions, or substitutions.

In some embodiments, the anti-PTK7/B7H3 antibody or antigen-binding fragment described herein can contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 7 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 8 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 9 with zero, one or two amino acid insertions, deletions, or substitutions.

In some embodiments, the anti-PTK7/B7H3 antibody or antigen-binding fragment described herein can contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 10 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 11 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 12 with zero, one or two amino acid insertions, deletions, or substitutions.

In some embodiments, the anti-PTK7/B7H3 antibody or antigen-binding fragment described herein can contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 13 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 14 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 15 with zero, one or two amino acid insertions, deletions, or substitutions.

In some embodiments, the anti-PTK7/B7H3 antibody or antigen-binding fragment described herein can contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 16 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 17 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 18 with zero, one or two amino acid insertions, deletions, or substitutions.

In some embodiments, the anti-PTK7/B7H3 antibody or antigen-binding fragment described herein can contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 19 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 20 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 21 with zero, one or two amino acid insertions, deletions, or substitutions.

In some embodiments, the anti-PTK7/B7H3 antibody or antigen-binding fragment described herein can contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 22 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 23 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 24 with zero, one or two amino acid insertions, deletions, or substitutions.

In some embodiments, the anti-PTK7/B7H3 antibody or antigen-binding fragment described herein can contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 25 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 26 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 27 with zero, one or two amino acid insertions, deletions, or substitutions.

In some embodiments, the anti-PTK7/B7H3 antibody or antigen-binding fragment described herein can contain a light chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 1 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 2 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 3 with zero, one or two amino acid insertions, deletions, or substitutions.

The insertions, deletions, and substitutions can be within the CDR sequence, or at one or both terminal ends of the CDR sequence.

In some embodiments, the anti-PTK7/B7H3 antibodies contain a heavy chain variable region (VH) comprising or consisting of an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to a selected VH sequence, and a light chain variable region (VL) comprising or consisting of an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to a selected VL sequence. In some embodiments, the selected VH sequence is SEQ ID NOs: 29, 30, 31 or 32, and the selected VL sequence is SEQ ID NO: 28.

In some embodiments, the anti-PTK7/B7H3 antibody or antigen-binding fragment can have 3 VH CDRs that are identical to the CDRs of any VH sequences as described herein. In some embodiments, the anti-PTK7/B7H3 antibody or antigen-binding fragment can have 3 VL CDRs that are identical to the CDRs of any VL sequences as described herein.

The disclosure also provides nucleic acid sequences comprising a polynucleotide encoding an anti-PTK7/B7H3 antibody. The immunoglobulin heavy chain or immunoglobulin light chain in the anti-PTK7/B7H3 antibody comprises CDRs as shown in FIGs. 12-13, or have sequences as shown in FIG. 14. When the polypeptides are paired with corresponding polypeptide (e.g., a corresponding heavy chain variable region or a corresponding light chain variable region) , the paired polypeptides bind to PTK7 and/or B7H3.

Due to the degeneracy of the genetic code, each of the polypeptide sequences depicted herein can be encoded by a large number of polynucleotide sequences. One of ordinary skill in the art will appreciate that the present application thus provides adequate written description and enablement for degenerate nucleic acid sequences encoding each antigen binding protein, e.g., each of the CDRsequences, the FR sequences, and the heavy and light chain sequences.

The anti-PTK7/B7H3 antibodies can also be anti-PTK7/B7H3 antibody variants (including derivatives and conjugates) of anti-PTK7/B7H3 antibodies or antibody fragments. Additional anti-PTK7/B7H3 antibodies provided herein are polyclonal, monoclonal, multi-specific (multimeric, e.g., bispecific) , human antibodies, chimeric antibodies (e.g., human-mouse chimera) , single-chain antibodies, intracellularly-made antibodies (i.e., intrabodies) , and antigen-binding fragments thereof. The anti-PTK7/B7H3 antibodies can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY) , class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) , or subclass. In some embodiments, the anti-PTK7/B7H3 antibody or antigen-binding fragment is an IgG (e.g., IgG1) antibody or antigen-binding fragment thereof.

Fragments of anti-PTK7/B7H3 antibodies are suitable for use in the methods provided so long as they retain the desired affinity and specificity to both PTK7 and B7H3. Thus, a fragment of an anti-PTK7/B7H3 antibody will retain an ability to bind to PTK7 and B7H3.

Antibodies and Antigen Binding Fragments thereof

In some embodiments, the multi-specific anti-PTK7/B7H3 antibody (e.g., bispecific antibody) includes an antigen-binding domain that is derived from an anti-PTK7 antibody, and an antigen-binding domain that is derived from an anti-B7H3 antibody. These anti-PTK7/B7H3 antibodies and antigen-binding fragments thereof can have various forms.

In general, antibodies (also called immunoglobulins) can be made up of two classes of polypeptide chains, light chains and heavy chains. A non-limiting anti-PTK7/B7H3 antibody of the present disclosure can be an intact, four immunoglobulin chain antibody comprising two heavy chains and two light chains. The heavy chain of the anti-PTK7/B7H3 antibody can be of any isotype including IgM, IgG, IgE, IgA, or IgD or sub-isotype including IgG1, IgG2, IgG2a, IgG2b, IgG3, IgG4, IgE1, IgE2, etc. The light chain can be a kappa light chain or a lambda light chain.

The hypervariable regions, known as the complementary determining regions (CDRs) , form loops that comprise the principle antigen binding surface of the antibody. The four framework regions largely adopt a beta-sheet conformation and the CDRs form loops connecting, and in some cases forming part of, the beta-sheet structure. The CDRs in each chain are held in close proximity by the framework regions and, with the CDRs from the other chain, contribute to the formation of the antigen-binding domain.

Methods for identifying the CDR regions of an antibody by analyzing the amino acid sequence of the antibody are well known, and a number of definitions of the CDRs are commonly used. The Kabat definition is based on sequence variability, and the Chothia definition is based on the location of the structural loop regions. These methods and definitions are described in, e.g., Martin, "Protein sequence and structure analysis of antibody variable domains, " Antibody engineering, Springer Berlin Heidelberg, 2001. 422-439; Abhinandan, et al. "Analysis and improvements to Kabat and structurally correct numbering of antibody variable domains, " Molecular immunology 45.14 (2008) : 3832-3839; Wu, T. T. and Kabat, E. A. (1970) J. Exp. Med. 132: 211-250; Martin et al., Methods Enzymol. 203: 121-53 (1991) ; Morea et al., Biophys Chem. 68 (1-3) : 9-16 (Oct. 1997) ; Morea et al., J Mol Biol. 275 (2) : 269-94 (Jan. 1998) ; Chothia et al., Nature 342 (6252) : 877-83 (Dec. 1989) ; Ponomarenko and Bourne, BMC Structural Biology 7: 64 (2007) ; each of which is incorporated herein by reference in its entirety.

The CDRs are important for recognizing an epitope of an antigen. As used herein, an “epitope” is the smallest portion of a target molecule capable of being specifically bound by the antigen-binding domain of an antibody. The minimal size of an epitope may be about three, four, five, six, or seven amino acids, but these amino acids need not be in a consecutive linear sequence of the antigen’s primary structure, as the epitope may depend on an antigen’s three-dimensional configuration based on the antigen’s secondary and tertiary structure.

In some embodiments, the anti-PTK7/B7H3 antibody is an intact immunoglobulin molecule (e.g., IgG1, IgG2a, IgG2b, IgG3, IgM, IgD, IgE, IgA) . The IgG subclasses (IgG1, IgG2, IgG3, and IgG4) are highly conserved, differ in their constant region, particularly in their hinges and upper CH2 domains. The sequences and differences of the IgG subclasses are known in the art, and are described, e.g., in Vidarsson, et al, "IgG subclasses and allotypes: from structure to effector functions. " Frontiers in immunology 5 (2014) ; Irani, et al. "Molecular properties of human IgG subclasses and their implications for designing therapeutic monoclonal antibodies against infectious diseases. " Molecular immunology 67.2 (2015) : 171-182; Shakib, Farouk, ed. The human IgG subclasses: molecular analysis of structure, function and regulation. Elsevier, 2016; each of which is incorporated herein by reference in its entirety.

The anti-PTK7/B7H3 antibody can also be an immunoglobulin molecule that is derived from any species (e.g., human, rodent, mouse, rat, camelid) . The antigen-binding domain or antigen binding fragment is a portion of an antibody that retains specific binding activity of the intact antibody, i.e., any portion of an antibody that is capable of specific binding to an epitope on the intact antibody’s target molecule. It includes, e.g., Fab, Fab’, F (ab’) ₂, and variants of these fragments. Thus, in some embodiments, an anti-PTK7/B7H3 antibody or antigen binding fragment thereof can comprise e.g., a scFv, a Fv, a Fd, a dAb, a bispecific antibody, a bispecific scFv, a diabody, a linear antibody, a single-chain antibody molecule, a multi-specific antibody formed from antibody fragments, and any polypeptide that includes a binding domain which is, or is homologous to, an antibody binding domain. Non-limiting examples of antigen-binding domains include, e.g., the heavy chain and/or light chain CDRs of an intact antibody, the heavy and/or light chain variable regions of an intact antibody, full length heavy or light chains of an intact antibody, or an individual CDR from either the heavy chain or the light chain of an intact antibody.

In some embodiments, the scFv in an anti-PTK7/B7H3 antibody has two heavy chain variable domains, and two light chain variable domains. In some embodiments, the anti-PTK7/B7H3 scFv has two antigen binding regions, and the two antigen binding regions can bind to the respective target antigens with different affinities.

In some embodiments, the anti-PTK7/B7H3 antibodies or antigen-binding fragments thereof can comprise one, two, or three heavy chain variable region CDRs selected from FIGs. 12-13. In some embodiments, the anti-PTK7/B7H3 antibodies or antigen-binding fragments thereof can comprises one, two, or three light chain variable region CDRs selected from FIGs. 12-13.

In some embodiments, the anti-PTK7/B7H3 antibodies described herein can be conjugated to a therapeutic agent. The anti-PTK7/B7H3 antibody-drug conjugate comprising the antibody or antigen-binding fragment thereof can covalently or non-covalently bind to a therapeutic agent. In some embodiments, the therapeutic agent is a cytotoxic or cytostatic agent (e.g., monomethyl auristatin E, monomethyl auristatin F, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin, maytansinoids such as DM-1 and DM-4, dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, epirubicin, and cyclophosphamide and analogs) . In some embodiments, the therapeutic agent is MMAE or MMAF. In some embodiments, the therapeutic agent is conjugated via a linker, e.g., a VC linker. Details of the linkers used for ADCs can be found, e.g., in Su, Z. et al. "Antibody-drug conjugates: Recent advances in linker chemistry. " Acta Pharmaceutica Sinica B (2021) , which is incorporated herein by reference in its entirety.

In some embodiments, the anti-PTK7/B7H3 antibody is a bispecific antibody. Bispecific antibodies can be made by engineering the interface between a pair of antibody molecules to maximize the percentage of heterodimers that are recovered from recombinant cell culture. For example, the interface can contain at least a part of the CH3 domain of an antibody constant domain. In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan) . Compensatory “cavities” of identical or similar size to the large side chain (s) are created on the interface of the second antibody molecule by replacing large amino acid side chains with smaller ones (e.g., alanine or threonine) . This provides a mechanism for increasing the yield of the heterodimer over other unwanted end-products such as homodimers. This method is described, e.g., in WO 96/27011, which is incorporated by reference in its entirety.

Any of the anti-PTK7/B7H3 antibodies or antigen-binding fragments thereof described herein may be conjugated to a stabilizing molecule (e.g., a molecule that increases the half-life of the antibody or antigen-binding fragment thereof in a subject or in solution) . Non-limiting examples of stabilizing molecules include: a polymer (e.g., a polyethylene glycol) or a protein (e.g., serum albumin, such as human serum albumin) . The conjugation of a stabilizing molecule can increase the half-life or extend the biological activity of an anti-PTK7/B7H3 antibody or an antigen-binding fragment in vitro (e.g., in tissue culture or when stored as a pharmaceutical composition) or in vivo (e.g., in a human) .

The anti-PTK7/B7H3 antibodies or antigen-binding fragments thereof can also have various forms. Many different formats of bispecific antibodies or antigen-binding fragments thereof are known in the art, and are described e.g., in Suurs, et al. "A review of bispecific antibodies and antibody constructs in oncology and clinical challenges, " Pharmacology&therapeutics (2019) , which is incorporated herein by reference in the entirety.

In some embodiments, the anti-PTK7/B7H3 antibody is a BiTe, a (scFv) ₂, a nanobody, a nanobody-HSA, a DART, a TandAb, a scDiabody, a scDiabody-CH3, scFv-CH-CL-scFv, a HSAbody, scDiabody-HAS, or a tandem-scFv. In some embodiments, the anti-PTK7/B7H3 antibody is a VHH-scAb, a VHH-Fab, a Dual scFab, a F (ab’) ₂, a diabody, a crossMab, a DAF (two-in-one) , a DAF (four-in-one) , aDutaMab, a DT-IgG, a knobs-in-holes common light chain, a knobs-in-holes assembly, a charge pair, aFab-arm exchange, a SEEDbody, a LUZ-Y, a Fcab, aκλ-body, an orthogonal Fab, a DVD-IgG, a IgG (H) -scFv, a scFv- (H) IgG, IgG (L) -scFv, scFv- (L) IgG, IgG (L, H) -Fv, IgG (H) -V, V (H) -IgG, IgG (L) -V, V (L) -IgG, KIH IgG-scFab, 2scFv-IgG, IgG-2scFv, scFv4-Ig, Zybody, DVI-IgG, Diabody-CH3, a triple body, aminiantibody, a minibody, a TriBi minibody, scFv-CH3 KIH, Fab-scFv, a F (ab’) ₂-scFv2, a scFv-KIH, aFab-scFv-Fc, a tetravalent HCAb, a scDiabody-Fc, a Diabody-Fc, a tandem scFv-Fc, an Intrabody, a dock and lock, a lmmTAC, an IgG-IgG conjugate, a Cov-X-Body, or a scFv1-PEG-scFv2.

In some embodiments, the anti-PTK7/B7H3 antibody can be a TrioMab. In a TrioMab, the two heavy chains are from different species, wherein different sequences restrict the heavy-light chain pairing.

In some embodiments, the anti-PTK7/B7H3 antibody has two different heavy chains and one common light chain. Heterodimerization of heavy chains can be based on the knobs-into-holes or some other heavy chain pairing technique.

In some embodiments, CrossMAb technique can be used to produce bispecific anti-PTK7/B7H3 antibodies. CrossMAb technique can be used to enforce correct light chain association in bispecific heterodimeric IgG antibodies, this technique allows the generation of various bispecific antibody formats, including bi- (1+1) , tri- (2+1) and tetra- (2+2) valent bispecific antibodies, as well as non-Fc tandem antigen-binding fragment (Fab) -based antibodies. These formats can be derived from any existing antibody pair using domain crossover, without the need for the identification of common light chains, post-translational processing/in vitro chemical assembly or the introduction of a set of mutations enforcing correct light chain association. The method is described in Klein et al., "The use of CrossMAb technology for the generation of bi-and multi-specific antibodies. " MAbs. Vol. 8. No. 6. Taylor&Francis, 2016, which is incorporated by reference in its entirety. In some embodiments, the CH1 in the heavy chain and the CL domain in the light chain are swapped.

The anti-PTK7/B7H3 antibody can be a Duobody. The Fab-exchange mechanism naturally occurring in IgG4 antibodies is mimicked in a controlled matter in IgG1 antibodies, a mechanism called controlled Fab exchange. This format can ensure specific pairing between the heavy-light chains.

In Dual-variable-domain antibody (DVD-Ig) , additional VH and variable light chain (VL) domain are added to each N-terminus for bispecific targeting. This format resembles the IgG-scFv, but the added binding domains are bound individually to their respective N-termini instead of a scFv to each heavy chain N-terminus.

In scFv-IgG, the two scFv are connected to the C-terminus of the heavy chain (CH3) . The scFv-IgG format has two different bivalent binding sites and is consequently also called tetravalent. There are no heavy-chain and light-chain pairing problem in the scFv-IgG.

In some embodiments, the anti-PTK7/B7H3 antibody can have an IgG-IgG format. Two intact IgG antibodies are conjugated by chemically linking the C-terminals of the heavy chains.

The anti-PTK7/B7H3 antibody can also have a Fab-scFv-Fc format. In Fab-scFv-Fc format, alight chain, heavy chain and a third chain containing the Fc region and the scFv are assembled. It can ensure efficient manufacturing and purification.

In some embodiments, the anti-PTK7/B7H3 antibody can be a TF. Three Fab fragments are linked by disulfide bridges. Two fragments target the tumor associated antigen (TAA) and one fragment targets a hapten. The TF format does not have an Fc region.

ADAPTIR has two scFvs bound to each side of an Fc region. It abandons the intact IgG as a basis for its construct, but conserves the Fc region to extend the half-life and facilitate purification.

Dual affinity retargeting (DART) has two peptide chains connecting the opposite fragments, thus VLA with VHB and VLB with VHA, and a sulfur bond at their C-termini fusing them together. In DART, the sulfur bond can improve stability over BiTEs.

In DART-Fc, an Fc region is attached to the DART structure. It can be generated by assembling three chains, two via a disulfide bond, as with the DART. One chain contains half of the Fc region which will dimerize with the third chain, only expressing the Fc region. The addition of Fc region enhances half-life leading to longer effective concentrations, avoiding continuous IV.

In tetravalent DART, four peptide chains are assembled. Basically, two DART molecules are created with half an Fc region and will dimerize. This format has bivalent binding to both targets, thus it is a tetravalent molecule.

Tandem diabody (TandAb) comprises two diabodies. Each diabody consists of an VHA and VLB fragment and a VHA and VLB fragment that are covalently associated. The two diabodies are linked with a peptide chain. It can improve stability over the diabody consisting of two scFvs. It has two bivalent binding sites.

The scFv-scFv-toxin includes toxin and two scFv with a stabilizing linker. It can be used for specific delivery of payload.

In some embodiments, the anti-PTK7/B7H3 antibody is a bispecific antibody. In some embodiments, the bispecific antibody in present disclosure is designed to be 1+1 (monovalent for each target) and has an IgG1 subtype structure. This can reduce the avidity to cells with low expression levels of B7H3 and PTK7, and increase the avidity to cells that co-express B7H3 and PTK7, to achieve enhanced targeting function.

In some embodiments, the anti-PTK7/B7H3 antibodies or antigen-binding fragments thereof have a light chain constant region that is at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%identical to SEQ ID NO: 33, and a heavy chain constant region that is at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%identical to any one of SEQ ID NOs: 34 and 35.

In some embodiments, the anti-PTK7/B7H3 antibodies include KIH mutations. In some embodiments, the anti-PTK7/B7H3 antibody includes a first antigen-binding domain that specifically binds to PTK7, and a second antigen-binding domain that specifically binds to B7H3. In some embodiments, the first antigen-binding domain includes a heavy chain that includes one or more knob mutations (a knob heavy chain) , and the second antigen-binding domain includes a heavy chain including one or more hole mutations (a hole heavy chain) . In some embodiments, the first antigen-binding domain includes a heavy chain that includes one or more hole mutations (a hole heavy chain) , and the second antigen-binding domain includes a heavy chain including one or more knob mutations (a knob heavy chain) . In some embodiments, the anti-PTK7/B7H3 antibody includes a knob heavy chain comprising a constant region that is at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%identical to SEQ ID NO: 34. In some embodiments, the anti-PTK7/B7H3 antibody includes a hole heavy chain comprising a constant region that is at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%identical to SEQ ID NO: 35.

Antibody and ADC Characteristics

The anti-PTK7/B7H3 antibodies can include an anti-PTK7 antigen-binding domain and any anti-B7H3 antigen-binding domain as described herein.

The disclosure provides anti-PTK7/B7H3 antibodies and antigen-binding fragments thereof that can specifically bind to PTK7. These anti-PTK7/B7H3 antibodies can be agonists or antagonists. General techniques can be used to measure the affinity of an antibody for an antigen include, e.g., ELISA, RIA, and surface plasmon resonance (SPR) . Affinities can be deduced from the quotient of the kinetic rate constants (KD=koff/kon) . In some implementations, the anti-PTK7/B7H3 antibodies or antigen-binding fragments thereof can bind to PTK7 (e.g., human PTK7, monkey PTK7, mouse PTK7, and/or chimeric PTK7) with a dissociation rate (koff) of less than 0.1 s^-1, less than 0.01 s^-1, less than 0.001 s^-1, less than 0.0001 s^-1, or less than 0.00001 s^-1. In some embodiments, the dissociation rate (koff) is greater than 0.01 s^-1, greater than 0.001 s^-1, greater than 0.0001 s^-1, greater than 0.00001 s^-1, or greater than 0.000001 s^-1.

In some embodiments, kinetic association rate (kon) is greater than 1×10²/Ms, greater than 1×10³/Ms, greater than 1×10⁴/Ms, greater than 1×10⁵/Ms, or greater than 1×10⁶/Ms. In some embodiments, kinetic association rate (kon) is less than 1×10⁵/Ms, less than 1×10⁶/Ms, or less than 1×10⁷/Ms.

In some embodiments, the anti-PTK7/B7H3 antibodies or antigen-binding fragments thereof can bind to PTK7 (e.g., human PTK7, monkey PTK7, mouse PTK7, and/or chimeric PTK7) with a KD of less than 1×10^-6M, less than 1×10^-7M, less than 1×10^-8 M, less than 1×10^-9 M, or less than 1×10^-10 M. In some embodiments, the KD is less than 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM,5 nM, 4 nM, 3 nM, 2 nM, or 1 nM. In some embodiments, KD is greater than 1×10^-7M, greater than 1×10^-8M, greater than 1×10^-9 M, or greater than 1×10^-10 M.

The anti-PTK7/B7H3 antibodies or antigen-binding fragments thereof can also include an antigen-binding domain that can specifically bind to B7H3. In some embodiments, the anti-PTK7/B7H3 antibodies as described herein are B7H3 agonist. In some embodiments, the anti-PTK7/B7H3 antibodies are B7H3 antagonist.

In some implementations, the anti-PTK7/B7H3 antibodies or antigen-binding fragments thereof can bind to B7H3 (e.g., human B7H3, monkey B7H3, mouse B7H3, and/or chimeric B7H3) with a dissociation rate (koff) of less than 0.1 s^-1, less than 0.01 s^-1, less than 0.001 s^-1, less than 0.0001 s^-1, or less than 0.00001 s^-1. In some embodiments, the dissociation rate (koff) is greater than 0.01 s^-1, greater than 0.001 s^-1, greater than 0.0001 s^-1, greater than 0.00001 s^-1, or greater than 0.000001 s^-1.

Affinities can be deduced from the quotient of the kinetic rate constants (KD=koff/kon) . In some embodiments, KD is less than 1×10^-6M, less than 1×10^-7M, less than 1×10^-8 M, less than 1×10^-9 M, or less than 1×10^-10 M. In some embodiments, the KD is less than 50 nM, 40 nM, 30 nM, 20 nM, 15 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, or 1 nM. In some embodiments, KD is greater than 1×10^-7M, greater than 1×10^-8M, greater than 1×10^-9 M, or greater than 1×10^-10 M.

Because the anti-PTK7/B7H3 antibody (e.g., bispecific antibody) binds to both PTK7 and B7H3, for cells that express both PTK7 and B7H3, the antibody has a higher binding affinity to these cells. Avidity can be used to measure the binding affinity of an antibody to these cells. Avidity is the accumulated strength of multiple affinities of individual non-covalent binding interactions.

In some embodiments, the anti-PTK7/B7H3 antibody or ADC described herein can bind to cells expressing PTK7 and/or B7H3 (e.g., NCI-H520 cells, HSP-77 cells, NCI-H69 or NCI-N87 cells) with a EC50 value that is less than 3 nM, less than 2.5 nM, less than 2 nM, less than 1.9 nM, less than 1.8 nM, less than 1.7 nM, less than 1.6 nM, or less than 1.5 nM.

Thermal stabilities can also be determined. The anti-PTK7/B7H3 antibodies or antigen-binding fragments thereof as described herein can have a Tm greater than 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95℃. As IgG can be described as a multi-domain protein, the melting curve sometimes shows two transitions, with a first denaturation temperature, Tm D1, and a second denaturation temperature Tm D2. The presence of these two peaks of ten indicate the denaturation of the Fc domains (Tm D1) and Fab domains (Tm D2) , respectively. When there are two peaks, Tm usually refers to Tm D2. Thus, in some embodiments, the anti-PTK7/B7H3 antibodies or antigen-binding fragments thereof as described herein has a Tm D1 greater than 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95℃. In some embodiments, the anti-PTK7/B7H3 antibodies or antigen-binding fragments thereof as described herein has a Tm D2 greater than 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95℃. In some embodiments, Tm, Tm D1, Tm D2 are less than 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95℃.

In some embodiments, the anti-PTK7/B7H3 antibodies or antigen-binding fragments thereof can bind to human B7H3 or monkey B7H3. In some embodiments, the anti-PTK7/B7H3 antibodies or antigen-binding fragments thereof cannot bind to human B7H3 or monkey B7H3. In some embodiments, the anti-PTK7/B7H3 antibodies or antigen-binding fragments thereof can bind to human PTK7 or monkey PTK7. In some embodiments, the anti-PTK7/B7H3 antibodies or antigen-binding fragments thereof cannot bind to human PTK7 or monkey PTK7.

In some embodiments, the anti-PTK7/B7H3 antibody, antigen-binding fragment, or ADC has a purity that is greater than 30%, 40%, 50%, 60%, 70%, 72.5%, 75%, 77.5%, 80%, 82.5%, 85%, 87.5%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, e.g., as measured by HPLC. In some embodiments, the purity is less than 30%, 40%, 50%, 60%, 70%, 72.5%, 75%, 77.5%, 80%, 82.5%, 85%, 87.5%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, e.g., as measured by HPLC.

In some embodiments, the anti-PTK7/B7H3 antibody, antigen-binding fragment, or ADC has a tumor growth inhibition rate or percentage (TGI%) that is greater than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, or 200%. In some embodiments, the anti-PTK7/B7H3 antibody, antigen-binding fragment, or ADC has a tumor growth inhibition percentage that is less than 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, or 150%. The TGI (%) can be determined, e.g., at 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, or 41 days after the treatment starts. As used herein, the tumor growth inhibition rate or percentage (TGI%) is calculated using the following formula:
TGI (%) = [1- (Ti-T0) / (Vi-V0) ] ×100%

Ti is the average tumor volume in the treatment group on day i. T0 is the average tumor volume in the treatment group on day zero. Vi is the average tumor volume in the control group on day i. V0 is the average tumor volume in the control group on day zero.

In some embodiments, the anti-PTK7/B7H3 antibody, antigen-binding fragment, or ADC has a functional Fc region. In some embodiments, effector function of a functional Fc region is antibody-dependent cell-mediated cytotoxicity (ADCC) . In some embodiments, effector function of a functional Fc region is phagocytosis. In some embodiments, effector function of a functional Fc region is ADCC and phagocytosis. In some embodiments, the Fc region is human IgG1, human IgG2, human IgG3, or human IgG4.

In some embodiments, the anti-PTK7/B7H3 antibody, antigen-binding fragment, or ADC does not have a functional Fc region. For example, the anti-PTK7/B7H3 antibodies or antigen-binding fragments thereof are Fab, Fab’, F (ab’) ₂, and Fv fragments. In some embodiments, the anti-PTK7/B7H3 antibodies or antigen-binding fragments thereof as described herein have an Fc region without effector function. In some embodiments, the Fc is a human IgG4 Fc. In some embodiments, the Fc does not have a functional Fc region. For example, the Fc region has LALA mutations (L234A and L235A mutations in EU numbering) , or LALA-PG mutations (L234A, L235A, P329G mutations in EU numbering) .

Some other modifications to the Fc region can be made. For example, a cysteine residue (s) can be introduced into the Fc region, thereby allowing interchain disulfide bond formation in this region. The homodimeric fusion protein thus generated may have any increased half-life in vitro and/or in vivo.

In some embodiments, the IgG4 has S228P mutation (EU numbering) . The S228P mutation prevents in vivo and in vitro IgG4 Fab-arm exchange.

In some embodiments, Fc regions are provided having a carbohydrate structure that lacks fucose attached (directly or indirectly) to an Fc region. For example, the amount of fucose in such Fc region composition may be from 1%to 80%, from 1%to 65%, from 5%to 65%or from 20%to 40%. The amount of fucose is determined by calculating the average amount of fucose within the sugar chain at Asn297, relative to the sum of all glycostructures attached to Asn 297 (e.g. complex, hybrid and high mannose structures) as measured by MALDI-TOF mass spectrometry, as described in WO 2008/077546, for example. Asn297 refers to the asparagine residue located at about position 297 in the Fc region (Eu numbering of Fc region residues; or position 314 in Kabat numbering) ; however, Asn297 may also be located about±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300, due to minor sequence variations in Fc region sequences. Such fucosylation variants may have improved ADCC function. In some embodiments, to reduce glycan heterogeneity, the Fc region can be further engineered to replace the Asparagine at position 297 with Alanine (N297A) .

In some embodiments, the main peak of HPLC-SEC accounts for at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 100%of the protein complex described herein after purification by protein A-based affinity chromatography and/or size-exclusive chromatography.

In some embodiments, the anti-PTK7/B7H3 ADC described herein has an IC50 for in vitro killing of cancer cells (e.g., NCI-H520 or NCI-N87) of less than 5μg/mL, less than 4.5μg/mL, less than 4 μg/mL, less than 3.5μg/mL, less than 3μg/mL, less than 2.5μg/mL, less than 2μg/mL, less than 1.5 μg/mL, less than 1μg/mL, less than 0.9μg/mL, less than 0.8μg/mL, less than 0.7μg/mL, less than 0.6 μg/mL, less than 0.5μg/mL, less than 0.4μg/mL, less than 0.3μg/mL, less than 0.2μg/mL, less than 0.1 μg/mL, less than 0.05μg/mL, less than 0.025μg/mL, less than 0.0125μg/mL, less than 0.005μg/mL, or less than 0.0025μg/mL. In some embodiments, the anti-PTK7/B7H3 ADC described herein has an IC50 for in vitro killing of cancer cells (e.g., NCI-H520 or NCI-N87) of less than 15μg/mL, less than 10 μg/mL, less than 5μg/mL, less than 1μg/mL, less than 0.9μg/mL, less than 0.8μg/mL, less than 0.7 μg/mL, less than 0.6μg/mL, or less than 0.5μg/mL.

In some embodiments, the anti-PTK7/B7H3 antibody or ADC described herein has a higher endocytosis rate than the corresponding monoclonal antibodies and/or control bispecific antibodies described herein. In some embodiments, the anti-PTK7/B7H3 antibody or ADC described herein has a higher endocytosis rate than Ref1. In some embodiments, the anti-PTK7/B7H3 antibody or ADC described herein has a higher endocytosis rate than Ref2, Ref3, and/or Ref4. In some embodiments, the anti-PTK7/B7H3 antibodies described herein has a higher endocytosis rate than an isotype control.

Antibody Drug Conjugates (ADC)

The anti-PTK7/B7H3 antibodies or antigen-binding fragments thereof described herein can be conjugated to a therapeutic agent (a drug) . The therapeutic agent can be covalently or non-covalently bind to the anti-PTK7/B7H3 antibody. In some embodiments, the anti-PTK7/B7H3 antibody is an anti-PTK7/B7H3 bispecific antibody. In some embodiments, the bispecific antibody has a common light chain.

In some embodiments, the therapeutic agent is a cytotoxic or cytostatic agent (e.g., monomethyl auristatin E, monomethyl auristatin F, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin, maytansinoids such as DM-1 and DM-4, dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, epirubicin, and cyclophosphamide and analogs) . Useful classes of cytotoxic, cytostatic, or immunomodulatory agents include, for example, antitubulin agents, DNA minor groove binders, DNA replication inhibitors, and alkylating agents.

In some embodiments, the therapeutic agent can include, but not limited to, cytotoxic reagents, such as chemo-therapeutic agents, immunotherapeutic agents and the like, antiviral agents or antimicrobial agents. In some embodiments, the therapeutic agent to be conjugated can be selected from, but not limited to, MMAE (monomethyl auristatin E) , MMAD (monomethyl auristatin D) , or MMAF (monomethyl auristatin F) .

Definitions of specific functional groups and chemical terms are described in more detail below. For purpose of this invention, the chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75^th Edition, inside cover, and specific functional groups are generally defined as described therein. Additionally, general principles of organic chemistry, as well as specific functional moieties and reactivity, are described in Organic Chemistry, Thomas Sorrell, University Science Books, Sausalito, 1999; Smith and March, March’s Advanced Organic Chemistry, 5^th Edition, John Wiley&Sons, Inc., New York, 2001; Larock, Comprehensive Organic Transformations, VCH Publishers, Inc., New York, 1989; Carruthers, Some Modem Methods of Organic Synthesis, 3^rd Edition, Cambridge University Press, Cambridge, 1987.

All ranges cited herein are inclusive, unless expressly stated to the contrary. When a range of values is listed, it is intended to encompass each value and sub-range within the range. For example, “C_1- ₆” is intended to encompass, C₁, C₂, C₃, C₄, C₅, C₆, C_1-6, C_1-5, C_1-4, C_1-3, C_1-2, C_2-6, C_2-5, C_2-4, C_2-3, C_3-6, C_3-5, C_3-4, C_4-6, C_4-5, and C_5-6.

The compounds or any formula depicting and describing the compounds of the present disclosure may have one or more chiral (asymmetric) centers. The present invention encompasses all stereoisomeric forms of the compounds or any formula depicting and describing the compounds of the present invention. Centers of asymmetry that are present in the compounds or any formula depicting and describing the compounds of the present invention can all independently of one another have (R) or (S) configuration. When bonds to a chiral carbon are depicted as straight lines in the structural formulas, or when a compound name is recited without an (R) or (S) chiral designation for a chiral carbon, it is understood that both the (R) and (S) configurations of each such chiral carbon, and hence each enantiomer or diastereomer and mixtures thereof, are embraced within the formula or by the name.

The disclosure includes all possible enantiomers and diastereomers and mixtures of two or more stereoisomers, for example mixtures of enantiomers and/or diastereomers, in all ratios. Thus, enantiomers are a subject of the disclosure in enantiomerically pure form, both as levorotatory and as dextrorotatory antipodes, in the form of racemates and in the form of mixtures of the two enantiomers in all ratios. In the case of a cis/trans isomerism the disclosure includes both the cis form and the trans form as well as mixtures of these forms in all ratios. The preparation of individual stereoisomers can be carried out, if desired, by separation of a mixture by customary methods, for example by chromatography or crystallization, by the use of stereochemically uniform starting materials for the synthesis or by stereoselective synthesis. Optionally a derivatization can be carried out before a separation of stereoisomers. The separation of a mixture of stereoisomers can be carried out at an intermediate step during the synthesis of a compound or it can be done on a final racemic product. Absolute stereochemistry may be determined by X-ray crystallography of crystalline products or crystalline intermediates which are derivatized, if necessary, with a reagent containing a stereogenic center of known configuration. Alternatively, absolute stereochemistry may be determined by Vibrational Circular Dichroism (VCD) spectroscopy analysis.

Unless otherwise stated, the structures depicted herein are also meant to include the compounds that differ only in the presence of one or more isotopically enriched atoms, in other words, the compounds wherein one or more atoms are replaced by atoms having the same atomic number, but an atomic mass or mass number different from the atomic mass or mass number which predominates in nature. Such compounds are referred to as a “isotopic variant” . The present disclosure is intended to include all pharmaceutically acceptable isotopic variants of the compounds or any formula depicting and describing the compounds of the present invention. Examples of isotopes suitable for inclusion in the compounds of the present invention include, but not limited to, isotopes of hydrogen, such as ²H (i.e., D) and³H; carbon, such as ¹¹C, ¹³C, and ¹⁴C; chlorine, such as ³⁶Cl; fluorine, such as ¹⁸F; iodine, such as ¹²³I and ¹²⁵I; nitrogen, such as ¹³N and ¹⁵N; oxygen, such as ¹⁵O, ¹⁷O, and ¹⁸O; phosphorus, such as ³²P; and sulfur, such as ³⁵S. Certain isotopic variants of the compounds or any formula depicting and describing the compounds of the present disclosure, for example those incorporating a radioactive isotope, may be useful in drug and/or substrate tissue distribution studies. Particularly, compounds having the depicted structures that differ only in the replacement with heavier isotopes, such as the replacement of hydrogen by deuterium (²H, or D) , can afford certain therapeutic advantages, for example, resulting from greater metabolic stability, increased in vivo half-life, or reduced dosage requirements and, hence, may be utilized in some particular circumstances. Isotopic variants of compounds or any formula depicting and describing the compounds of the present disclosure can generally be prepared by techniques known to those skilled in the art or by processes analogous to those described in the accompanying examples and synthesis using an appropriate isotopically-labeled reagent in place of the non-labeled reagent previously employed.

The compounds as provided herein are described with reference to both generic formulas and specific compounds. In addition, the compounds of the present disclosure may exist in a number of different forms or derivatives, all within the scope of the disclosure. These include, for example, pharmaceutically acceptable salts, tautomers, stereoisomers, racemic mixtures, regioisomers, prodrugs, solvated forms, different crystal forms or polymorphs, and active metabolites, etc.

As used herein, the term “pharmaceutically acceptable salt” , unless otherwise stated, includes salts that retain the biological effectiveness of the free acid/base form of the specified compound and that are not biologically or otherwise undesirable. Pharmaceutically acceptable salts may include salts formed with inorganic bases or acids and organic bases or acids. In cases where the compounds of the present disclosure contain one or more acidic or basic groups, the disclosure also comprises their corresponding pharmaceutically acceptable salts. Thus, the compounds of the present invention which contain acidic groups, such as carboxyl groups, can be present in salt form, and can be used according to the invention, for example, as alkali metal salts, alkaline earth metal salts, aluminum salts or as ammonium salts. More non-limiting examples of such salts include lithium salts, sodium salts, potassium salts, calcium salts, magnesium salts, barium salts, or salts with ammonia or organic amines such as ethylamine, ethanolamine, diethanolamine, triethanolamine, piperidine, N-methylglutamine, or amino acids. These salts are readily available, for instance, by reacting the compound having an acidic group with a suitable base, e.g., lithium hydroxide, sodium hydroxide, sodium propoxide, potassium hydroxide, potassium ethoxide, magnesium hydroxide, calcium hydroxide, or barium hydroxide. Other base salts of compounds of the present disclosure include but are not limited to copper (I) , copper (II) , iron (II) , iron (III) , manganese (II) , and zinc salts. Compounds of the present disclosure which contain one or more basic groups, e.g., groups which can be protonated, can be present in salt form, and can be used according to the disclosure in the form of their addition salts with inorganic or organic acids. Examples of suitable acids include hydrogen chloride, hydrogen bromide, hydrogen iodide, phosphoric acid, sulfuric acid, nitric acid, methanesulfonic acid, p-toluenesulfonic acid, naphthalenedisulfonic acid, sulfoacetic acid, trifluoroacetic acid, oxalic acid, acetic acid, tartaric acid, lactic acid, salicylic acid, benzoic acid, carbonic acid, formic acid, propionic acid, pivalic acid, diethylacetic acid, malonic acid, succinic acid, pimelic acid, fumaric acid, maleic acid, malic acid, embonic acid, mandelic acid, sulfaminic acid, phenylpropionic acid, gluconic acid, ascorbic acid, isonicotinic acid, citric acid, adipic acid, taurocholic acid, glutaric acid, stearic acid, glutamic acid, or aspartic acid, and other acids known to those skilled in the art. The salts which are formed are, inter alia, hydrochlorides, chlorides, hydrobromides, bromides, iodides, sulfates, phosphates, methanesulfonates (mesylates) , tosylates, carbonates, bicarbonates, formates, acetates, sulfoacetates, triflates, oxalates, malonates, maleates, succinates, tartrates, malates, embonates, mandelates, fumarates, lactates, citrates, glutarates, stearates, aspartates, and glutamates. The stoichiometry of the salts formed from the compounds of the disclosure may moreover be an integral or non-integral multiple of one.

Compounds of the present disclosure which contain basic nitrogen-containing groups can be quaternized using agents such as C_1-4alkyl halides, for example, methyl, ethyl, isopropyl, and tert-butyl chloride, bromide, and iodide; diC_1-4alkyl sulfates, for example, dimethyl, diethyl, and diamyl sulfate; C_10- ₁₈alkyl halides, for example, decyl, dodecyl, lauryl, myristyl, and stearyl chloride, bromide, and iodide; and arylC_1-4alkyl halides, for example, benzyl chloride and phenethyl bromide.

If the compounds of the present disclosure simultaneously contain acidic and basic groups in the molecule, the disclosure also includes, in addition to the salt forms mentioned, inner salts or betaines (zwitterions) . The respective salts can be obtained by customary methods which are known to those skilled in the art, for example by contacting these with an organic or inorganic acid or base in a solvent or dispersant, or by anion exchange or cation exchange with other salts. The present disclosure also includes all salts of the compounds of the present disclosure which, owing to low physiological compatibility, are not directly suitable for use in pharmaceuticals but which can be used, for example, as intermediates for chemical reactions or for the preparation of pharmaceutically acceptable salts. For a review on more suitable salts, see Stahl and Wermuth, Handbook of Pharmaceutical Salts: Properties, Selection, and Use (Wiley-VCH, 2002) .

The compound or any formula depicting and describing the compounds of the present disclosure and pharmaceutically acceptable salts thereof may exist in unsolvated and solvated forms. As used herein, the term “solvate” refers to a molecular complex comprising the compound of Formula (I) , or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable solvent molecules. For example, the term “hydrate” is employed when the solvent is water.

Pharmaceutically acceptable solvates in accordance with the present disclosure may include those wherein the solvent of crystallization may be isotopically substituted, e.g., D₂O, d₆-acetone, d₆-DMSO.

Linker (linking agent compound)

In some embodiments, the therapeutic agent is conjugated via a linker (or a linking agent compound) . As used herein, the term “linker” or “linking agent compound” refers to a compound that can connect a ligand (e.g., the antibodies or the antigen-binding fragments thereof described herein) and a therapeutic agent (e.g., any of the therapeutic agents described herein) together to form a ligand-drug conjugate by reacting with a group of the ligand compound and the therapeutic agent compound respectively by, for example, a coupling reaction.

In some embodiments, the linker described herein is a compound having the following formula:
Q-L
Formula (I) ,

or a pharmaceutically acceptable salt, solvate, stereoisomer, or isotopic variant thereof, wherein Q denotes to a junction moiety capable of being coupled to a ligand via a bond selected from the group consisting of carbonyl, thioether, amide, disulfide and hydrazone bond; L denotes to a linker moiety capable of connecting Q to a therapeutic agent.

In some embodiments, the junction moiety (Q in Formula (I) ) has the following structure:

In some embodiments, the linker moiety (L in Formula (I) ) has the following formula:

where L₁ is a polypeptide residue consisting of three to eight amino acid residues which comprises at least one amino acid residue with a side chain carboxyl group, for example, glutamic acid residue or aspartic acid residue, where “-COOH” denotes carboxyl group of an amino acid residue at C-terminal of the polypeptide residue;

L₂ is absent or a monodentate, bidentate or tridentate hydrophilic group attached to the side chain carboxyl group on the amino acid residue of the polypeptide residue L₁, and L₂ has a structure of-NHC (R^L2a) (R^L2b) (R^L2c) , where R^L2a, R^L2b, and R^L2c are each independently selected from the group consisting of H, - (CH₂O) (CH₂CH₂O) _m (CH₂) _pC (O) OH, and- (CH₂O) (CH₂CH₂O) _m (CH₂) _pC (O) NHR^L2d, R^L2d is H or C_1-6 alkyl optionally substituted with 1 to 6 hydroxy groups, each m is independently an integer from 0 to 10, preferably 0 to 4, for example 0, 1, 2, 3, or 4, especially preferably m is 0, and each p is independent an integer from 1 to 4, for example, 1, 2, 3, or 4; and

denotes to the N-terminal side of the polypeptide residue covalently attached to thejunction moiety Q.

In some embodiments, the polypeptide residue L₁ is ^NH-Glu-Val-Ala-^COOH. In some embodiments, the hydrophilic group L₂has the following structure:

wherein “*” denotes the site covalently attached to polypeptide residue L₁, e.g., side chain of the Glu residue in ^NH-Glu-Val-Ala-^COOH.

In some embodiments, the hydrophilic group L₂has the following structure:

In some embodiments, the linker described herein is a compound having the following structure:

In some embodiments, the linker is a VC linker. Details of the linkers used for ADCs can be found, e.g., in Su, Z. et al. "Antibody–drug conjugates: Recent advances in linker chemistry. " Acta Pharmaceutica Sinica B (2021) , which is incorporated herein by reference in its entirety.

Therapeutic agent

In some embodiments, the therapeutic agent that is conjugated to the antibodies or the antigen-binding fragments thereof described herein is discussed as follows.

In some embodiments, the therapeutic agent described herein is a cytotoxic agent. In some embodiments, the cytotoxic agent is a camptothecin compound, an analogue or a derivative thereof. In some preferred embodiments, the camptothecin compound is a compound having the following structure:

wherein X is selected from the group consisting of-CH2-, O and S; Y is selected from the group consisting of H, D, and F.

In some embodiments, the therapeutic agent is (S) -4-amino-9-ethyl-9-hydroxy-1, 9, 12, 15-tetrahydro-13H-pyrano [3', 4': 6, 7] indolizino [1, 2-b] thiopyrano [4, 3, 2-de] quinoline-10, 13 (2H) -dione) (CPT-1) . The structure of CPT-1 is shown below:

In some embodiments, the therapeutic agent is (S) -4-amino-9-ethyl-9-hydroxy-1, 9, 12, 15-tetrahydro-13H-pyrano [4, 3, 2-de] pyrano [3', 4': 6, 7] indolizino [1, 2-b] quinoline-10, 13 (2H) -dione (CPT-2) . The structure of CPT-2 is shown below:

In some embodiments, the therapeutic agent is CPT-3. The structure of CPT-3 is shown below:

In some embodiments, the therapeutic agent is (S) -4-amino-9-ethyl-5-fluoro-9-hydroxy-1, 9, 12, 15-tetrahydro-13H-pyrano [4, 3, 2-de] pyrano [3', 4': 6, 7] indolizino [1, 2-b] quinoline-10, 13 (2H) -dione (CPT-4) . The structure of CPT-4 is shown below:

In some embodiments, the therapeutic agent is an auristatin, such as auristatin E (also known in the art as a derivative of dolastatin-10) or a derivative thereof. The auristatin can be, for example, an ester formed between auristatin E and a keto acid. For example, auristatin E can be reacted with paraacetyl benzoic acid or benzoylvaleric acid to produce AEB and AEVB, respectively. Other typical auristatins include AFP, MMAF, and MMAE. The synthesis and structure of exemplary auristatins are described in U.S. Patent Application Publication No. 2003-0083263; International Patent Publication No. WO 04/010957, International Patent Publication No. WO 02/088172, and U.S. Pat. Nos. 7,498,298, 6,884,869, 6,323,315; 6,239,104; 6,034,065; 5,780,588; 5,665,860; 5,663,149; 5,635,483; 5,599,902; 5,554,725; 5,530,097; 5,521,284; 5,504,191; 5,410,024; 5,138,036; 5,076,973; 4,986,988; 4,978,744; 4,879,278; 4,816,444; and 4,486,414, each of which is incorporated by reference herein in its entirety and for all purposes.

Auristatins have been shown to interfere with microtubule dynamics and nuclear and cellular division and have anticancer activity. Auristatins bind tubulin and can exert a cytotoxic or cytostatic effect on cancer cell. There are a number of different assays, known in the art, which can be used for determining whether an auristatin or resultant antibody-drug conjugate exerts a cytostatic or cytotoxic effect on a desired cell.

In some embodiments, the therapeutic agent is a chemotherapeutic agent. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclosphosphamide (CYTOXAN^TM) ; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphaoramide and trimethylolomelamine; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics such as aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, calicheamicin, carabicin, carminomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5-fluorouracil (5-FU) ; folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, 5-FU; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elfornithine; elliptinium acetate; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK7; razoxane; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2’, 2’, 2’-trichlorotriethylamine; urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ( “Ara-C” ) ; cyclophosphamide; taxanes, e.g. paclitaxel (Bristol-Myers Squibb Oncology, Princeton, N.J. ) and doxetaxel (Rhone-Poulenc Rorer, Antony, France) ; chlorambucil; gemcitabine; 6-thioguanine; platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16) ; ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO) ; retinoic acid; esperamicins; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the above. Also included in this definition are anti-hormonal agents that act to regulate or inhibit hormone action on tumors such as anti-estrogens including for example tamoxifen, raloxifene, aromatase inhibiting 4 (5) -imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and toremifene (Fareston) ; and anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and pharmaceutically acceptable salts, acids or derivatives of any of the above. A detailed description of the chemotherapeutic agents can be found in, e.g., US20180193477A1, which is incorporated by reference in its entirety.

Linker-Therapeutic agent compound

In some embodiments, a linker (e.g., any of the linkers described herein) and a therapeutic agent (e.g., any of the therapeutic agents described herein) can be linked to form a “linker-therapeutic agent” compound.

In some embodiments, the linker-therapeutic agent compound has the following structure:

In some embodiments, an antibody ( “Ab” ) , e.g., any of the antibodies or the antigen-binding fragments thereof described herein, can be linked to a linker-therapeutic agent compound (e.g., any of the linker-therapeutic agent compounds described herein) to generate an antibody-drug conjugate. In some embodiments, the antibody-drug conjugate has the following structure:

wherein n=1-8. In some embodiments, n=1-8. In some embodiments, n is about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8. In some embodiments, n is about 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 2-8, 2-7, 2-6, 2-5, 2-4, 2-3, 3-8, 3-7, 3-6, 3-5, 3-4, 4-8, 4-7, 4-6, 4-5, 5-8, 5-7, 5-6, 6-8, 6-7, or 7-8. In some embodiments, n is an integral or non-integral multiple of one.

In some embodiments, the anti-PTK7/B7H3 antibody is coupled to the drug via a cleavable linker e.g. a SPBD linker or a maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl (VC) linker. In some embodiments, the anti-PTK7/B7H3 antibody is coupled to the drug via a non-cleavable linker e.g. a MCC linker formed using SMCC or sulfo-SMCC. Selection of an appropriate linker for a given ADC can be readily made by the skilled person having knowledge of the art and taking into account relevant factors, such as the site of attachment to the anti-PTK7/B7H3 antibody, any structural constraints of the drug and the hydrophobicity of the drug (see, for example, review in Nolting, Chapter 5, Antibody-Drug Conjugates: Methods in Molecular Biology, 2013, Ducry (Ed. ) , Springer) . A number of specific linker-toxin combinations have been described and may be used with the anti-PTK7/B7H3 antibodies or antigen-binding fragments thereof described herein to prepare ADCs in certain embodiments. Examples include, but are not limited to, cleavable peptide-based linkers with auristatins such as MMAE and MMAF, camptothecins such as SN-38, duocarmycins and PBD dimers; non-cleavable MC-based linkers with auristatins MMAF and MMAE; acid-labile hydrazone-based linkers with calicheamicins and doxorubicin; disulfide-based linkers with maytansinoids such as DM1 and DM4, and bis-maleimido-trioxyethylene glycol (BMPEO) -based linkers with maytansinoid DM1. Some these therapeutic agents and linkers are described, e.g., in Peters&Brown, (2015) Biosci. Rep. e00225; Dosio et al., (2014) Recent Patents on Anti-Cancer Drug Discovery 9: 35-65; US Patent Publication No. US 2015/0374847, and US20180193477A1; which are incorporated herein by reference in the entirety.

Depending on the desired drug and selected linker, those skilled in the art can select suitable method for coupling them together. For example, some conventional coupling methods, such as amine coupling methods, can be used to form the desired drug-linker complex which still contains reactive groups for conjugating to the anti-PTK7/B7H3 antibodies or antigen-binding fragments thereof through covalent linkage. In some embodiments, a drug-maleimide complex (i.e., maleimide linking drug) can be used for the payload bearing reactive group in the present disclosure. Most common reactive group capable of bonding to thiol group in ADC preparation is maleimide. Additionally, organic bromides, iodides also are frequently used.

The anti-PTK7/B7H3 ADC can be prepared by one of several routes known in the art, employing organic chemistry reactions, conditions, and reagents known to those skilled in the art (see, for example, Bioconjugate Techniques (G. T. Hermanson, 2013, Academic Press) . For example, conjugation can be achieved by (1) reaction of a nucleophilic group or an electrophilic group of an antibody with a bivalent linker reagent, to form antibody-linker intermediate Ab-L, via a covalent bond, followed by reaction with an activated drug moiety D; or (2) reaction of a nucleophilic group or an electrophilic group of a drug moiety with a linker reagent, to form drug-linker intermediate D-L, via a covalent bond, followed by reaction with the nucleophilic group or an electrophilic group of an antibody. Conjugation methods (1) and (2) can be employed with a variety of antibodies, drug moieties, and linkers to prepare the anti-PTK7/B7H3 ADCs described here. Various prepared linkers, linker components and toxins are commercially available or may be prepared using standard synthetic organic chemistry techniques. These methods are described e.g., in March’s Advanced Organic Chemistry (Smith&March, 2006, Sixth Ed., Wiley) ; Toki et al., (2002) J. Org. Chem. 67: 1866-1872; Frisch et al., (1997) Bioconj. Chem. 7: 180-186; Bioconjugate Techniques (G.T. Hermanson, 2013, Academic Press) ; US20210379193A1, and US20180193477A1, which are incorporated herein by reference in the entirety. In addition, a number of pre-formed drug-linkers suitable for reaction with a selected anti-PTK7/B7H3 antibody or antigen-binding fragment are also available commercially, for example, linker-toxins comprising DM1, DM4, MMAE, MMAF or Duocarmycin SA are available from Creative BioLabs (Shirley, N.Y. ) .

Several specific examples of methods of preparing anti-PTK7/B7H3 ADCs are known in the art and are described in U.S. Pat. No. 8,624,003 (pot method) , U.S. Pat. No. 8,163,888 (one-step) , and U.S. Pat. No. 5,208,020 (two-step method) , and US20180193477A1, which are incorporated herein by reference in the entirety. Other methods are known in the art and include those described in Antibody-Drug Conjugates: Methods in Molecular Biology, 2013, Ducry (Ed. ) , Springer.

Drug loading is represented by the number of drug moieties per antibody in a molecule of ADC. For some antibody-drug conjugates, the drug loading may be limited by the number of attachment sites on the antibody. For example, where the attachment is a cysteine thiol, as in certain exemplary embodiments described herein, the drug loading may range from 0 to 8 drug moieties per antibody. In certain embodiments, higher drug loading, e.g. p≥5, may cause aggregation, insolubility, toxicity, or loss of cellular permeability of certain antibody-drug conjugates. In certain embodiments, the average drug loading for an anti-PTK7/B7H3 antibody-drug conjugate ranges from 1 to about 8; from about 2 to about 6; or from about 3 to about 5. Indeed, it has been shown that for certain antibody-drug conjugates, the optimal ratio of drug moieties per antibody can be around 4. In some embodiments, the DAR for an anti-PTK7/B7H3 ADC composition is about or at least 1, 2, 3, 4, 5, 6, 7, or 8. In some embodiments, the average DAR in the anti-PTK7/B7H3 ADC composition is about 1～about 2, about 2～about 3, about 3～about 4, about 3～about 5, about 4～about 5, about 5～about 6, about 6～about 7, or about 7～about 8.

In some embodiments, anti-PTK7/B7H3 antibody variants are provided having a carbohydrate structure that lacks fucose attached (directly or indirectly) to an Fc region. For example, the amount of fucose in such antibody may be from 1%to 80%, from 1%to 65%, from 5%to 65%or from 20%to 40%. The amount of fucose is determined by calculating the average amount of fucose within the sugar chain at Asn297, relative to the sum of all glycostructures attached to Asn 297 (e.g. complex, hybrid and high mannose structures) as measured by MALDI-TOF mass spectrometry, as described in WO 2008/077546, for example. Asn297 refers to the asparagine residue located at about position 297 in the Fc region (Eu numbering of Fc region residues; or position 314 in Kabat numbering) ; however, Asn297 may also be located about±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300, due to minor sequence variations in antibodies. Such fucosylation variants may have improved ADCC function. In some embodiments, to reduce glycan heterogeneity, the Fc region of the anti-PTK7/B7H3 antibody can be further engineered to replace the Asparagine at position 297 with Alanine (N297A) .

In some embodiments, to facilitate production efficiency by avoiding Fab-arm exchange, the Fc region of the anti-PTK7/B7H3 antibodies or antigen-binding fragments thereof was further engineered to replace the serine at position 228 (EU numbering) of IgG4 with proline (S228P) . A detailed description regarding S228 mutation is described, e.g., in Silva et al. "The S228P mutation prevents in vivo and in vitro IgG4 Fab-arm exchange as demonstrated using a combination of novel quantitative immunoassays and physiological matrix preparation. " Journal of Biological Chemistry 290.9 (2015) : 5462-5469, which is incorporated by reference in its entirety.

In some embodiments, the methods described here are designed to make a bispecific anti-PTK7/B7H3 antibody. Bispecific anti-PTK7/B7H3 antibodies can be made by engineering the interface between a pair of antibody molecules to maximize the percentage of heterodimers that are recovered from recombinant cell culture. For example, the interface can contain at least a part of the CH3 domain of an antibody constant domain. In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan) . Compensatory “cavities” of identical or similar size to the large side chain (s) are created on the interface of the second antibody molecule by replacing large amino acid side chains with smaller ones (e.g., alanine or threonine) . This provides a mechanism for increasing the yield of the heterodimer over other unwanted end-products such as homodimers. This method is described, e.g., in WO 96/27011, which is incorporated by reference in its entirety.

In some embodiments, knobs-into-holes (KIH) technology can be used, which involves engineering CH3 domains to create either a “knob” or a “hole” in each heavy chain to promote heterodimerization. The KIH technique is described e.g., in Xu, Yiren, et al. "Production of bispecific antibodies in ‘knobs-into-holes’ using a cell-free expression system. " MAbs. Vol. 7. No. 1. Taylor&Francis, 2015, which is incorporated by reference in its entirety. In some embodiments, one heavy chain has a T366W, and/or S354C (knob) substitution (EU numbering) , and the other heavy chain has an Y349C, T366S, L368A, and/or Y407V (hole) substitution (EU numbering) . In some embodiments, one heavy chain has one or more of the following substitutions Y349C and T366W (EU numbering) . The other heavy chain can have one or more the following substitutions E356C, T366S, L368A, and Y407V (EU numbering) . Furthermore, a substitution (-ppcpScp->-ppcpPcp-) can also be introduced at the hinge regions of both substituted IgG.

Recombinant Vectors

The present disclosure also provides recombinant vectors (e.g., expression vectors) that include an isolated polynucleotide disclosed herein (e.g., a polynucleotide that encodes a polypeptide disclosed herein) , host cells into which are introduced the recombinant vectors (i.e., such that the host cells contain the polynucleotide and/or a vector comprising the polynucleotide) , and the production of anti-PTK7/B7H3 antibody polypeptides or fragments thereof by recombinant techniques.

As used herein, a “vector” is any construct capable of delivering one or more polynucleotide (s) of interest to a host cell when the vector is introduced to the host cell. An “expression vector” is capable of delivering and expressing the one or more polynucleotide (s) of interest as an encoded polypeptide in a host cell into which the expression vector has been introduced. Thus, in an expression vector, the polynucleotide of interest is positioned for expression in the vector by being operably linked with regulatory elements such as a promoter, enhancer, and/or a poly-A tail, either within the vector or in the genome of the host cell at or near or flanking the integration site of the polynucleotide of interest such that the polynucleotide of interest will be translated in the host cell introduced with the expression vector.

A vector can be introduced into the host cell by methods known in the art, e.g., electroporation, chemical transfection (e.g., DEAE-dextran) , transformation, transfection, and infection and/or transduction (e.g., with recombinant virus) . Thus, non-limiting examples of vectors include viral vectors (which can be used to generate recombinant virus) , naked DNA or RNA, plasmids, cosmids, phage vectors, and DNA or RNA expression vectors associated with cationic condensing agents.

In some implementations, a polynucleotide disclosed herein (e.g., a polynucleotide that encodes a polypeptide disclosed herein) is introduced using a viral expression system (e.g., vaccinia or other pox virus, retrovirus, or adenovirus) , which may involve the use of a non-pathogenic (defective) , replication competent virus, or may use a replication defective virus. In the latter case, viral propagation generally will occur only in complementing virus packaging cells. Suitable systems are disclosed, for example, in Fisher-Hoch et al., 1989, Proc. Natl. Acad. Sci. USA 86: 317-321; Flexner et al., 1989, Ann. N.Y. Acad Sci. 569: 86-103; Flexner et al., 1990, Vaccine, 8: 17-21; U.S. Pat. Nos. 4,603,112, 4,769,330, and 5,017,487; WO 89/01973; U.S. Pat. No. 4,777,127; GB 2,200,651; EP 0,345,242; WO 91/02805; Berkner-Biotechniques, 6: 616-627, 1988; Rosenfeld et al., 1991, Science, 252: 431-434; Kolls et al., 1994, Proc. Natl. Acad. Sci. USA, 91: 215-219; Kass-Eisler et al., 1993, Proc. Natl. Acad. Sci. USA, 90: 11498-11502; Guzman et al., 1993, Circulation, 88: 2838-2848; and Guzman et al., 1993, Cir. Res., 73: 1202-1207. Techniques for incorporating DNA into such expression systems are well known to those of ordinary skill in the art. The DNA may also be “naked, ” as described, for example, in Ulmer et al., 1993, Science, 259: 1745-1749, and Cohen, 1993, Science, 259: 1691-1692. The uptake of naked DNA may be increased by coating the DNA onto biodegradable beads that are efficiently transported into the cells.

For expression, the DNA insert comprising a polypeptide-encoding polynucleotide disclosed herein can be operatively linked to an appropriate promoter (e.g., a heterologous promoter) , such as the phage lambda PL promoter, the E. coli lac, trp and tac promoters, the SV40 early and late promoters and promoters of retroviral LTRs, to name a few. Other suitable promoters are known to the skilled artisan. The expression constructs can further contain sites for transcription initiation, termination and, in the transcribed region, a ribosome binding site for translation. The coding portion of the mature transcripts expressed by the constructs may include a translation initiating at the beginning and a termination codon (UAA, UGA, or UAG) appropriately positioned at the end of the polypeptide to be translated.

As indicated, the expression vectors can include at least one selectable marker. Such markers include dihydrofolate reductase or neomycin resistance for eukaryotic cell culture and tetracycline or ampicillin resistance genes for culturing in E. coli and other bacteria. Representative examples of appropriate hosts include, but are not limited to, bacterial cells, such as E. coli, Streptomyces, and Salmonella typhimurium cells; fungal cells, such as yeast cells; insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS, Bowes melanoma, and HK 293 cells; and plant cells. Appropriate culture mediums and conditions for the host cells described herein are known in the art.

Non-limiting vectors for use in bacteria include pQE70, pQE60 and pQE-9, available from Qiagen; pBS vectors, Phagescript vectors, Bluescript vectors, pNH8A, pNH16a, pNH18A, pNH46A, available from Stratagene; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 available from Pharmacia. Non-limiting eukaryotic vectors include pWLNEO, pSV2CAT, pOG44, pXT1 and pSG available from Stratagene; and pSVK3, pBPV, pMSG and pSVL available from Pharmacia. Other suitable vectors will be readily apparent to the skilled artisan.

Non-limiting bacterial promoters suitable for use include the E. coli lacI and lacZ promoters, the T3 and T7 promoters, the gpt promoter, the lambda PR and PL promoters and the trp promoter. Suitable eukaryotic promoters include the CMV immediate early promoter, the HSV thymidine kinase promoter, the early and late SV40 promoters, the promoters of retroviral LTRs, such as those of the Rous sarcoma virus (RSV) , and metallothionein promoters, such as the mouse metallothionein-I promoter.

In the yeast Saccharomyces cerevisiae, a number of vectors containing constitutive or inducible promoters such as alpha factor, alcohol oxidase, and PGH may be used. For reviews, see Ausubel et al. (1989) Current Protocols in Molecular Biology, John Wiley&Sons, New York, N.Y, and Grant et al., Methods Enzymol., 153: 516-544 (1997) .

Introduction of the construct into the host cell can be effected by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection or other methods. Such methods are described in many standard laboratory manuals, such as Davis et al., Basic Methods In Molecular Biology (1986) , which is incorporated herein by reference in its entirety.

Transcription of DNA encoding an anti-PTK7/B7H3 antibody of the present disclosure by higher eukaryotes may be increased by inserting an enhancer sequence into the vector. Enhancers are cis-acting elements of DNA, usually about from 10 to 300 bp that act to increase transcriptional activity of a promoter in a given host cell-type. Examples of enhancers include the SV40 enhancer, which is located on the late side of the replication origin at base pairs 100 to 270, the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.

For secretion of the translated protein into the lumen of the endoplasmic reticulum, into the periplasmic space or into the extracellular environment, appropriate secretion signals may be incorporated into the expressed polypeptide. The signals may be endogenous to the polypeptide or they may be heterologous signals.

The polypeptide (e.g., an anti-PTK7/B7H3 antibody) can be expressed in a modified form, such as a fusion protein (e.g., a GST-fusion) or with a histidine-tag, and may include not only secretion signals, but also additional heterologous functional regions. For instance, a region of additional amino acids, particularly charged amino acids, may be added to the N-terminus of the polypeptide to improve stability and persistence in the host cell, during purification, or during subsequent handling and storage. Also, peptide moieties can be added to the polypeptide to facilitate purification. Such regions can be removed prior to final preparation of the polypeptide. The addition of peptide moieties to polypeptides to engender secretion or excretion, to improve stability and to facilitate purification, among others, are familiar and routine techniques in the art.

The disclosure also provides a nucleic acid sequence that is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%identical to any nucleotide sequence as described herein, and an amino acid sequence that is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%identical to any amino acid sequence as described herein.

The disclosure also provides a nucleic acid sequence that has a homology of at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%to any nucleotide sequence as described herein, and an amino acid sequence that has a homology of at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%to any amino acid sequence as described herein.

In some embodiments, the disclosure relates to nucleotide sequences encoding any peptides that are described herein, or any amino acid sequences that are encoded by any nucleotide sequences as described herein. In some embodiments, the nucleic acid sequence is less than 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 150, 200, 250, 300, 350, 400, 500, or 600 nucleotides. In some embodiments, the amino acid sequence is less than 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, or 400 amino acid residues.

In some embodiments, the amino acid sequence (i) comprises an amino acid sequence; or (ii) consists of an amino acid sequence, wherein the amino acid sequence is any one of the sequences as described herein.

In some embodiments, the nucleic acid sequence (i) comprises a nucleic acid sequence; or (ii) consists of a nucleic acid sequence, wherein the nucleic acid sequence is any one of the sequences as described herein.

To determine the percent identity of two amino acid sequences, or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes) . The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein amino acid or nucleic acid “identity” is equivalent to amino acid or nucleic acid “homology” ) . The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. For example, the comparison of sequences and determination of percent identity between two sequences can be accomplished using a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.

The percentage of sequence homology (e.g., amino acid sequence homology or nucleic acid homology) can also be determined. How to determine percentage of sequence homology is known in the art. In some embodiments, amino acid residues conserved with similar physicochemical properties (percent homology) , e.g. leucine and isoleucine, can be used to measure sequence similarity. Families of amino acid residues having similar physicochemical properties have been defined in the art. These families include e.g., amino acids with basic side chains (e.g., lysine, arginine, histidine) , acidic side chains (e.g., aspartic acid, glutamic acid) , uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine) , nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan) , beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine) . The homology percentage, in many cases, is higher than the identity percentage.

The disclosure provides one or more nucleic acid encoding any of the polypeptides as described herein. In some embodiments, the nucleic acid (e.g., cDNA) includes a polynucleotide encoding a polypeptide of a heavy chain as described herein. In some embodiments, the nucleic acid includes a polynucleotide encoding a polypeptide of a light chain as described herein. In some embodiments, the nucleic acid includes a polynucleotide encoding a scFv polypeptide as described herein.

In some embodiments, the vector can have two of the nucleic acids as described herein, wherein the vector encodes the VL region and the VH region that together bind to B7H3. In some embodiments, apair of vectors is provided, wherein each vector comprises one of the nucleic acids as described herein, wherein together the pair of vectors encodes the VL region and the VH region that together bind to B7H3.

In some embodiments, the vector includes two of the nucleic acids as described herein, wherein the vector encodes the VL region and the VH region that together bind to PTK7. In some embodiments, apair of vectors is provided, wherein each vector comprises one of the nucleic acids as described herein, wherein together the pair of vectors encodes the VL region and the VH region that together bind to PTK7.

Methods of Treatment

The methods described herein include methods for the treatment of disorders associated with cancer. Generally, the methods include administering a therapeutically effective amount of anti-PTK7/B7H3 antibodies or anti-PTK7/B7H3 antibody-drug conjugates as described herein, to a subject who is in need of, or who has been determined to be in need of, such treatment.

As used in this context, to “treat” means to ameliorate at least one symptom of the disorder associated with cancer. Often, cancer results in death; thus, a treatment can result in an increased life expectancy (e.g., by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months, or by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 years) . Administration of a therapeutically effective amount of an agent described herein for the treatment of a condition associated with cancer will result in decreased number of cancer cells and/or alleviated symptoms.

As used herein, the term “cancer” refers to cells having the capacity for autonomous growth, i.e., an abnormal state or condition characterized by rapidly proliferating cell growth. The term is meant to include all types of cancerous growths or oncogenic processes, metastatic tissues or malignantly transformed cells, tissues, or organs, irrespective of histopathologic type or stage of invasiveness. The term “tumor” as used herein refers to cancerous cells, e.g., a mass of cancerous cells. Cancers that can be treated or diagnosed using the methods described herein include malignancies of the various organ systems, such as affecting lung, breast, thyroid, lymphoid, gastrointestinal, and genito-urinary tract, as well as adenocarcinomas which include malignancies such as most colon cancers, renal-cell carcinoma, prostate cancer and/or testicular tumors, non-small cell carcinoma of the lung, cancer of the small intestine and cancer of the esophagus. In some embodiments, the agents described herein are designed for treating or diagnosing a carcinoma in a subject. The term “carcinoma” is art recognized and refers to malignancies of epithelial or endocrine tissues including respiratory system carcinomas, gastrointestinal system carcinomas, genitourinary system carcinomas, testicular carcinomas, breast carcinomas, prostatic carcinomas, endocrine system carcinomas, and melanomas. In some embodiments, the cancer is renal carcinoma or melanoma. Exemplary carcinomas include those forming from tissue of the cervix, lung, prostate, breast, head and neck, colon and ovary. The term also includes carcinosarcomas, e.g., which include malignant tumors composed of carcinomatous and sarcomatous tissues. An “adenocarcinoma” refers to a carcinoma derived from glandular tissue or in which the tumor cells form recognizable glandular structures. The term “sarcoma” is art recognized and refers to malignant tumors of mesenchymal derivation.

In some embodiments, the cancer is a chemotherapy resistant cancer.

In one aspect, the disclosure also provides methods for treating a cancer in a subject, methods of reducing the rate of the increase of volume of a tumor in a subject over time, methods of reducing the risk of developing a metastasis, or methods of reducing the risk of developing an additional metastasis in a subject. In some embodiments, the treatment can halt, slow, retard, or inhibit progression of a cancer. In some embodiments, the treatment can result in the reduction of in the number, severity, and/or duration of one or more symptoms of the cancer in a subject.

In one aspect, the disclosure features methods that include administering a therapeutically effective amount of anti-PTK7/B7H3 antibodies or an anti-PTK7/B7H3 antibody drug conjugates disclosed herein to a subject in need thereof, e.g., a subject having, or identified or diagnosed as having, acancer, e.g., solid tumor, lung cancer (e.g., non-small cell lung cancer, lung adenocarcinoma, or lung carcinoma) , gastric cancer (e.g., gastric carcinoma) , skin cancer (e.g., skin carcinoma) , colorectal cancer, breast cancer, head and neck cancer, ovarian cancer, prostate cancer, thyroid cancer, pancreatic cancer, CNS cancer, liver cancer, nasopharynx cancer, brain cancer, colon cancer, bladder cancer, oral squamous cell carcinoma, cervical cancer, or oesophageal cancer.

As used herein, the terms “subject” and “patient” are used interchangeably throughout the specification and describe an animal, human or non-human, to whom treatment according to the methods of the present invention is provided. Veterinary and non-veterinary applications are contemplated by the present invention. Human patients can be adult humans orjuvenile humans (e.g., humans below the age of 18 years old) . In addition to humans, patients include but are not limited to mice, rats, hamsters, guinea-pigs, rabbits, ferrets, cats, dogs, and primates. Included are, for example, non-human primates (e.g., monkey, chimpanzee, gorilla, and the like) , rodents (e.g., rats, mice, gerbils, hamsters, ferrets, rabbits) , lagomorphs, swine (e.g., pig, miniature pig) , equine, canine, feline, bovine, and other domestic, farm, and zoo animals.

In some embodiments, the compositions and methods disclosed herein can be used for treatment of patients at risk for a cancer. Patients with cancer can be identified with various methods known in the art.

As used herein, by an “effective amount” is meant an amount or dosage sufficient to effect beneficial or desired results including halting, slowing, retarding, or inhibiting progression of a disease, e.g., a cancer. An effective amount will vary depending upon, e.g., an age and a body weight of a subject to which the anti-PTK7/B7H3 antibody, anti-PTK7/B7H3 antigen binding fragment, anti-PTK7/B7H3 antibody-drug conjugates, anti-PTK7/B7H3 antibody-encoding polynucleotide, vector comprising the polynucleotide, and/or compositions thereof is to be administered, a severity of symptoms and a route of administration, and thus administration can be determined on an individual basis.

An effective amount can be administered in one or more administrations. By way of example, an effective amount of an anti-PTK7/B7H3 antibody, an anti-PTK7/B7H3 antigen binding fragment, or an anti-PTK7/B7H3 antibody-drug conjugate is an amount sufficient to ameliorate, stop, stabilize, reverse, inhibit, slow and/or delay progression of an autoimmune disease or a cancer in a patient or is an amount sufficient to ameliorate, stop, stabilize, reverse, slow and/or delay proliferation of a cell (e.g., a biopsied cell, any of the cancer cells described herein, or cell line (e.g., a cancer cell line) ) in vitro. As is understood in the art, an effective amount of an anti-PTK7/B7H3 antibody, anti-PTK7/B7H3 antigen binding fragment, or anti-PTK7/B7H3 antibody-drug conjugate may vary, depending on, inter alia, patient history as well as other factors such as the type (and/or dosage) of the agent used.

Effective amounts and schedules for administering the anti-PTK7/B7H3 antibodies, anti-PTK7/B7H3 antigen-binding fragments thereof, anti-PTK7/B7H3 antibody-encoding polynucleotides, anti-PTK7/B7H3 antibody-drug conjugates, and/or compositions disclosed herein may be determined empirically, and making such determinations is within the skill in the art. Those skilled in the art will understand that the dosage that must be administered will vary depending on, for example, the mammal that will receive the anti-PTK7/B7H3 antibodies, anti-PTK7/B7H3 antigen-binding fragments thereof, anti-PTK7/B7H3 antibody-encoding polynucleotides, anti-PTK7/B7H3 antibody-drug conjugates, and/or compositions disclosed herein, the route of administration, the particular type of the agent or compositions disclosed herein used and other drugs being administered to the mammal.

A typical daily dosage of an effective amount of an anti-PTK7/B7H3 antibody or anti-PTK7/B7H3 ADC is 0.01 mg/kg to 100 mg/kg. In some embodiments, the dosage can be less than 100 mg/kg, 30 mg/kg, 20 mg/kg, 10 mg/kg, 9 mg/kg, 8 mg/kg, 7 mg/kg, 6 mg/kg, 5 mg/kg, 4 mg/kg, 3 mg/kg, 2 mg/kg, 1 mg/kg, 0.5 mg/kg, or 0.1 mg/kg. In some embodiments, the dosage can be greater than 10 mg/kg, 9 mg/kg, 8 mg/kg, 7 mg/kg, 6 mg/kg, 5 mg/kg, 4 mg/kg, 3 mg/kg, 2 mg/kg, 1 mg/kg, 0.5 mg/kg, 0.1 mg/kg, 0.05 mg/kg, or 0.01 mg/kg. In some embodiments, the dosage is about or at least 10 mg/kg, 9 mg/kg, 8 mg/kg, 7 mg/kg, 6 mg/kg, 5 mg/kg, 4 mg/kg, 3 mg/kg, 2 mg/kg, 1 mg/kg, 0.9 mg/kg, 0.8 mg/kg, 0.7 mg/kg, 0.6 mg/kg, 0.5 mg/kg, 0.4 mg/kg, 0.3 mg/kg, 0.2 mg/kg, or 0.1 mg/kg.

In any of the methods described herein, the at least one anti-PTK7/B7H3 antibody, the anti-PTK7/B7H3 antigen-binding fragment thereof, anti-PTK7/B7H3 antibody-drug conjugates, or pharmaceutical composition (e.g., comprising any of the anti-PTK7/B7H3 antibodies, anti-PTK7/B7H3 antigen-binding antibody fragments, or anti-PTK7/B7H3 ADC) and, optionally, at least one additional therapeutic agent can be administered to the subject (e.g., once a week, twice a week, three times a week, four times a week, once a day, twice a day, or three times a day) .

In some embodiments, the one or more additional therapeutic agents can be administered to the subject prior to, or after administering the at least one anti-PTK7/B7H3 antibody, anti-PTK7/B7H3 antigen-binding antibody fragment, anti-PTK7/B7H3 antibody-drug conjugate, or pharmaceutical composition (e.g., comprising any of the anti-PTK7/B7H3 antibodies, anti-PTK7/B7H3 antigen-binding antibody fragments, or anti-PTK7/B7H3 ADC) . In some embodiments, the one or more additional therapeutic agents and the at least one anti-PTK7/B7H3 antibody, anti-PTK7/B7H3 antigen-binding antibody fragment, or anti-PTK7/B7H3 antibody-drug conjugate are administered to the subject such that there is an overlap in the bioactive period of the one or more additional therapeutic agents and the at least one anti-PTK7/B7H3 antibody, anti-PTK7/B7H3 antigen-binding fragment, or anti-PTK7/B7H3 ADC in the subject.

In some embodiments, the subject can be administered the at least one anti-PTK7/B7H3 antibody, anti-PTK7/B7H3 antigen-binding antibody fragment, anti-PTK7/B7H3 antibody-drug conjugate, or pharmaceutical composition (e.g., comprising any of the anti-PTK7/B7H3 antibodies, anti-PTK7/B7H3 antigen-binding antibody fragments, or anti-PTK7/B7H3 ADC) over an extended period of time (e.g., over a period of at least 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 1 year, 2 years, 3 years, 4 years, or 5 years) . A skilled medical professional may determine the length of the treatment period using any of the methods described herein for diagnosing or following the effectiveness of treatment (e.g., the observation of at least one symptom of cancer) . As described herein, a skilled medical professional can also change the identity and number (e.g., increase or decrease) of anti-PTK7/B7H3 antibodies or anti-PTK7/B7H3 antigen-binding antibody fragments, anti-PTK7/B7H3 antibody-drug conjugates (and/or one or more additional therapeutic agents) administered to the subject and can also adjust (e.g., increase or decrease) the dosage or frequency of administration of at least one anti-PTK7/B7H3 antibody, anti-PTK7/B7H3 antigen-binding antibody fragment, or anti-PTK7/B7H3 ADC (and/or one or more additional therapeutic agents) to the subject based on an assessment of the effectiveness of the treatment (e.g., using any of the methods described herein and known in the art) .

In some embodiments, one or more additional therapeutic agents can be administered to the subject. The additional therapeutic agent can comprise one or more inhibitors selected from the group consisting of an inhibitor of B-Raf, an inhibitor of a MEK, an inhibitor of ERK, an inhibitor of K-Ras, an inhibitor of c-Met, an inhibitor of EGFR, an inhibitor of anaplastic lymphoma kinase (ALK) , an inhibitor of a phosphatidylinositol 3-kinase (PI3K) , an inhibitor of an Akt, an inhibitor of mTOR, a dual PI3K/mTOR inhibitor, an inhibitor of Bruton's tyrosine kinase (BTK) , and an inhibitor of Isocitrate dehydrogenase 1 (IDH1) and/or Isocitrate dehydrogenase 2 (IDH2) . In some embodiments, the additional therapeutic agent is an inhibitor of indoleamine 2, 3-dioxygenase (IDO1) (e.g., epacadostat) .

In some embodiments, the additional therapeutic agent can comprise one or more inhibitors selected from the group consisting of an inhibitor of HER3, an inhibitor of LSD1, an inhibitor of MDM2, an inhibitor of BCL2, an inhibitor of CHK1, an inhibitor of activated hedgehog signaling pathway, and an agent that selectively degrades the estrogen receptor.

In some embodiments, the additional therapeutic agent can comprise one or more therapeutic agents selected from the group consisting of Trabectedin, nab-paclitaxel, Trebananib, Pazopanib, Cediranib, Palbociclib, everolimus, fluoropyrimidine, IFL, regorafenib, Reolysin, Alimta, Zykadia, Sutent, temsirolimus, axitinib, sorafenib, Votrient, IMA-901, AGS-003, cabozantinib, Vinflunine, an Hsp90 inhibitor, Ad-GM-CSF, Temazolomide, IL-2, IFNa, vinblastine, Thalomid, dacarbazine, cyclophosphamide, lenalidomide, azacytidine, bortezomid, amrubicine, carfilzomib, pralatrexate, and enzastaurin.

In some embodiments, the additional therapeutic agent can comprise one or more therapeutic agents selected from the group consisting of an adjuvant, a TLR agonist, tumor necrosis factor (TNF) alpha, IL-1, HMGB1, an IL-10 antagonist, an IL-4 antagonist, an IL-13 antagonist, an IL-17 antagonist, an HVEM antagonist, an ICOS agonist, a treatment targeting CX3CL1, a treatment targeting CXCL9, atreatment targeting CXCL10, a treatment targeting CCL5, an LFA-1 agonist, an ICAM1 agonist, and a Selectin agonist.

In some embodiments, carboplatin, nab-paclitaxel, paclitaxel, cisplatin, pemetrexed, gemcitabine, FOLFOX, or FOLFIRI are administered to the subject.

In some embodiments, the additional therapeutic agent is an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-CTLA4 antibody, an anti-CD40 antibody, an anti-OX40 antibody, and/or an anti-41BB antibody.

Pharmaceutical Compositions and Routes of Administration

Also provided herein are pharmaceutical compositions that contain at least one (e.g., one, two, three, or four) of the anti-PTK7/B7H3 antibodies (e.g., bispecific antibodies) , anti-PTK7/B7H3 antigen-binding fragments, or anti-PTK7/B7H3 antibody-drug conjugates described herein. The pharmaceutical compositions may be formulated in any manner known in the art.

Pharmaceutical compositions are formulated to be compatible with their intended route of administration (e.g., intravenous, intraarterial, intramuscular, intradermal, subcutaneous, or intraperitoneal) . The compositions can include a sterile diluent (e.g., sterile water or saline) , a fixed oil, polyethylene glycol, glycerine, propylene glycol or other synthetic solvents, antibacterial or antifungal agents, such as benzyl alcohol or methyl parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like, antioxidants, such as ascorbic acid or sodium bisulfite, chelating agents, such as ethylenediaminetetraacetic acid, buffers, such as acetates, citrates, or phosphates, and isotonic agents, such as sugars (e.g., dextrose) , polyalcohols (e.g., mannitol or sorbitol) , or salts (e.g., sodium chloride) , or any combination thereof. Liposomal suspensions can also be used as pharmaceutically acceptable carriers (see, e.g., U.S. Patent No. 4,522,811) . Preparations of the compositions can be formulated and enclosed in ampules, disposable syringes, or multiple dose vials. Where required (as in, for example, injectable formulations) , proper fluidity can be maintained by, for example, the use of a coating, such as lecithin, or a surfactant. Absorption of the anti-PTK7/B7H3 antibody, anti-PTK7/B7H3 antigen-binding fragment thereof, or the anti-PTK7/B7H3 ADC can be prolonged by including an agent that delays absorption (e.g., aluminum monostearate and gelatin) . Alternatively, controlled release can be achieved by implants and microencapsulated delivery systems, which can include biodegradable, biocompatible polymers (e.g., ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid; Alza Corporation and Nova Pharmaceutical, Inc. ) .

Compositions containing one or more of any of the anti-PTK7/B7H3 antibodies, anti-PTK7/B7H3 antigen-binding fragments, anti-PTK7/B7H3 antibody-drug conjugates described herein can be formulated for parenteral (e.g., intravenous, intraarterial, intramuscular, intradermal, subcutaneous, or intraperitoneal) administration in dosage unit form (i.e., physically discrete units containing a predetermined quantity of active compound for ease of administration and uniformity of dosage) .

Toxicity and therapeutic efficacy of compositions can be determined by standard pharmaceutical procedures in cell cultures or experimental animals (e.g., monkeys) . One can determine the LD50 (the dose lethal to 50%of the population) and the ED50 (the dose therapeutically effective in 50%of the population) : the therapeutic index being the ratio of LD50: ED50. Agents that exhibit high therapeutic indices are preferred. Where an agent exhibits an undesirable side effect, care should be taken to minimize potential damage (i.e., reduce unwanted side effects) . Toxicity and therapeutic efficacy can be determined by other standard pharmaceutical procedures.

Data obtained from cell culture assays and animal studies can be used in formulating an appropriate dosage of any given agent for use in a subject (e.g., a human) . A therapeutically effective amount of the anti-PTK7/B7H3 antibodies, an anti-PTK7/B7H3 antigen-binding fragment thereof, or an anti-PTK7/B7H3 ADC will be an amount that treats the disease (e.g., kills cancer cells) in a subject (e.g., a human subject identified as having cancer) , or a subject identified as being at risk of developing the disease (e.g., a subject who has previously developed cancer but now has been cured) , decreases the severity, frequency, and/or duration of one or more symptoms of a disease in a subject (e.g., a human) . The effectiveness and dosing of any of the anti-PTK7/B7H3 antibodies, the anti-PTK7/B7H3 antigen-binding fragment thereof, or the anti-PTK7/B7H3 ADC described herein can be determined by a health care professional or veterinary professional using methods known in the art, as well as by the observation of one or more symptoms of disease in a subject (e.g., a human) . Certain factors may influence the dosage and timing required to effectively treat a subject (e.g., the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and the presence of other diseases) .

Exemplary doses include milligram or microgram amounts of any of the anti-PTK7/B7H3 antibodies, the anti-PTK7/B7H3 antigen-binding fragments thereof, or the anti-PTK7/B7H3 ADCs described herein per kilogram of the subject’s weight (e.g., about 1μg/kg to about 500 mg/kg; about 100 μg/kg to about 500 mg/kg; about 100μg/kg to about 50 mg/kg; about 10μg/kg to about 5 mg/kg; about 10μg/kg to about 0.5 mg/kg; or about 0.1 mg/kg to about 0.5 mg/kg) . In some embodiments, the dose level is between 5-30 mg/kg, 5-25 mg/kg, 5-20 mg/kg, 5-15 mg/kg, 5-10 mg/kg, 10-30 mg/kg, 10-25 mg/kg, 10-20 mg/kg, 10-15 mg/kg, 15-30 mg/kg, 15-25 mg/kg, 15-20 mg/kg, 20-30 mg/kg, 20-25 mg/kg, or 25-30 mg/kg. In some embodiments, the dose level is about 5 mg/kg, 6 mg/kg, 7 mg/kg, 8 mg/kg, 9 mg/kg, 10 mg/kg, 11 mg/kg, 12 mg/kg, 13 mg/kg, 14 mg/kg, 15 mg/kg, 16 mg/kg, 17 mg/kg, 18 mg/kg, 19 mg/kg, 20 mg/kg, 21 mg/kg, 22 mg/kg, 23 mg/kg, 24 mg/kg, 25 mg/kg, 26 mg/kg, 27 mg/kg, 28 mg/kg, 29 mg/kg, or 30 mg/kg. In some embodiments, the dose levels described herein do not induce severe toxic effects to the subject. While these doses cover a broad range, one of ordinary skill in the art will understand that therapeutic agents vary in their potency, and effective amounts can be determined by methods known in the art. Typically, relatively low doses are administered at first, and the attending health care professional or veterinary professional (in the case of therapeutic application) or a researcher (when still working at the development stage) can subsequently and gradually increase the dose until an appropriate response is obtained. In addition, it is understood that the specific dose level for any particular subject will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, gender, and diet of the subject, the time of administration, the route of administration, the rate of excretion, and the half-life of the therapeutic agent in vivo.

The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration. The disclosure also provides methods of manufacturing the anti-PTK7/B7H3 antibodies, the anti-PTK7/B7H3 antigen-binding fragment thereof, or the anti-PTK7/B7H3 ADC for various uses as described herein.

EXAMPLES

The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

Example 1. Preparation of anti-PTK7/B7H3 antibodies

Provided herein are bispecific antigen-binding molecules targeting PTK7 and B7H3. These antigen-binding molecules are also referred to as anti-PTK7/B7H3 bispecific antibodies below.

Anti-PTK7/B7H3 bispecific antibodies can have anti-PTK7 antigen binding domains (3C4, VH: SEQ ID NO: 29, VL: SEQ ID NO: 28; or 3E6, VH: SEQ ID NO: 30, VL: SEQ ID NO: 28) and anti-B7H3 antigen binding domains (21A9, VH: SEQ ID NO: 31, VL: SEQ ID NO: 28; or20H8, VH: SEQ ID NO: 32, VL: SEQ ID NO: 28) . Vectors encoding the light chain and heavy chain of the anti-PTK7/B7H3 antibodies were constructed. CHO-S cells were co-transduced with three vectors, including a first vector encoding the heavy chain of an anti-PTK7 binding arm, a second vector encoding the heavy chain of an anti-B7H3 binding arm, and a third vector encoding the common light chain. After 14 days of culture, the cell supernatant was collected and purified by Protein A affinity chromatography.

Various methods can be used to reduce the chance of mispairing between the two heavy chains. For example, knobs-into-holes mutations were introduced in the Fc regions of the anti-PTK7 arm heavy chain and the anti-B7H3 arm heavy chain. Exemplary bispecific antibodies obtained include 21A9-3C4, 20H8-3C4, 21A9-3E6 and 20H8-3E6. In 21A9-3C4, the heavy chain constant region of 21A9 includes knob mutations, and the heavy chain constant region of 3C4 includes hole mutations. In 20H8-3C4, the heavy chain constant region of 20H8 includes knob mutations, and the heavy chain constant region of 3C4 includes hole mutations. And so on.

The sequences of the light chain constant region, the heavy chain constant region with knob mutations, and the heavy chain constant region with hole mutations are shown in SEQ ID NO: 33, SEQ ID NO: 34 and SEQ ID NO: 35, respectively.

One reference antibody with specificity for PTK7 (designated Ref1) and three different antibodies with specificity for B7H3 (designated Ref2, Ref3 and Ref4) , synthesized from published amino acid sequence information, were used in the following experiments. Specifically, The VH and VL sequences set forth in SEQ ID NOs: 36-37, SEQ ID NOs: 38-39, SEQ ID NOs: 40-41, and SEQ ID NOs: 42-43 were linked to the human IgG1 constant region respectively, to form Ref1, Ref2, Ref3 and Ref4.

Example 2. Cross-species binding activity of anti-PTK7/B7H3 antibodies

CHO-hB7H3 (4Ig) cells, CHO-hB7H3 (2Ig) cells, or CHO-fasB7H3 cells were transferred to a 96-well plate at a density of 1×10⁵ cells/well respectively. 30μL of purified anti-B7-H3 antibodies (2.5 μg/mL) were added to each well of the 96-well plate, and the plate was then incubated at 4℃ for 30 minutes. Then, after washing with PBS, the cells were incubated with the secondary antibody anti-hIgG-Fc-Alexa647 (RL1-H) (Jackson ImmunoResearch Laboratories, Inc., Cat#: 109-606-170) at 4℃in the dark for 15 minutes before flow cytometry analysis. Human IgG1 protein was used as an isotype control (ISO) .

CHO-hB7H3 (4Ig) cells, CHO-hB7H3 (2Ig) cells and CHO-fasB7H3 cells were obtained by transfecting CHO-S cells with vectors expressing human B7H3 (4Ig) amino acid sequence (hB7-H3 (4Ig) , SEQ ID NO: 44) , human B7H3 (2Ig) amino acid sequence (hB7H3 (2Ig) , SEQ ID NO: 45) and monkey B7H3 amino acid sequence (fasB7H3, SEQ ID NO: 46) , respectively.

The test results are shown in the table below, which showed that the four anti-PTK7/B7H3 antibodies 21A9-3C4, 21A9-3E6, 20H8-3C4, and 20H8-3E6 can bind to both human B7H3 and monkey B7H3.

Table 1

Example 3. Binding activity of anti-PTK7/B7H3 antibodies to B7H3 family proteins

Experiments were performed to test the binding activity of anti-PTK7/B7H3 antibodies to B7H3 family proteins, e.g., CD80, CD86, PD-L1, PD-L2, B7H4, B7H5, B7H6 and B7H7. Specifically, CHO-hCD80 cells, CHO-hCD86 cells, CHO-hPD-L1 cells, CHO-hPD-L2 cells, CHO-hB7H4 cells, CHO-hB7H5 cells, CHO-hB7H6 cells, or CHO-hB7H7 cells were transferred to a 96-well plate at a density of 5 ×10⁴ cells/well, respectively. The sample anti-B7H3 antibody (2.5μg/mL) was added to the 96-well plate, and the plate was incubated at 4℃ for 30 minutes. Then, the cells were incubated with the secondary antibody anti-hIgG-Fc-Alexa647 (RL1-H) at 4℃ in the dark for 15 minutes before flow cytometry analysis.

CHO-hCD80 cells, CHO-hCD86 cells, CHO-hPD-L1, CHO-hPD-L2, CHO-hB7H4 cells, CHO-hB7H5 cells, CHO-hB7H6 cells, and CHO-hB7H7 cells were obtained by transfecting CHO-S cells with vectors encoding human CD80 (hCD80, SEQ ID NO: 47) , human CD86 (hCD86, SEQ ID NO: 48) , human PD-L1 (hPD-L1, SEQ ID NO: 49) , human PD-L2 (hPD-L2, SEQ ID NO: 50) , human B7H4 (hB7H4, SEQ ID NO: 51) , human B7H5 (hB7H5, SEQ ID NO: 52) , and human B7H6 (hB7H6, SEQ ID NO: 53) , respectively.

The results are summarized in the table below, which indicated that none of 21A9-3C4, 21A9-3E6, 20H8-3C4 and 20H8-3E6 can bind to human CD80, CD86, PD-L1, PD-L2, B7H4, B7H5 and B7H6, while the positive control Ref2 showed evident binding to the above B7H3 family proteins, demonstrating the specific binding activity of the anti-PTK7/B7H3 antibodies.

Table 2

Example 4. Binding affinities of anti-PTK7/B7H3 bispecific antibodies

The binding affinities of anti-PTK7/B7H3 bispecific antibodies to human PTK7, human B7H3, monkey PTK7, and monkey B7H3 were verified by surface plasmon resonance (SPR) using Biacore^TM(Biacore, Inc., Piscataway N. J. ) 8K biosensor equipped with pre-immobilized Protein A sensor chips.

Specifically, His-tagged human PTK7 protein (hPTK7-His, Sino Biological, Cat#: 19399-H08H) , monkey PTK7 protein (fasPTK7-His, ACRO Biosystems, Cat#: PT7-C52H3) , human B7H3 (hB7H3 (4Ig) -His, ACRO Biosystems, Cat#: B7B-H52E7; and hB7H3 (2Ig) -His, ACRO Biosystems, Cat#: B73-M52H4) and monkey B7H3 protein (fasB7H3-His, ACRO Biosystems, Cat#: B73-C52Ha) were diluted to 200 nM with 1×HBS-EP+buffer (pH 7.4) . Purified antibodies were injected into the Biacore^TM8K biosensor at 10 mL/min for about 50 seconds to achieve a desired protein density (e.g., about 100 response units (RU) ) and the diluted antigen protein at a concentration of 200 nM was then injected at 30 mL/min for 180 seconds. Dissociation was monitored for 400 seconds. The chip was regenerated after the last injection of each titration with a glycine solution (pH 1.5) at 30 mL/min for 30 seconds.

Kinetic association rates (kon) and dissociation rates (koff) were obtained simultaneously by fitting the data globally to a 1: 1 Langmuir binding model (Karlsson, R. Roos, H. Fagerstam, L. Petersson, B., 1994. Methods Enzymology 6. 99-110) using Biacore^TM8K Evaluation Software 3.0. Affinities were deduced from the quotient of the kinetic rate constants (KD=koff/kon) . As a person of ordinary skill in the art would understand, the same method with appropriate adjustments for parameters (e.g., antibody concentration) was performed for each tested antibody.

The results for the tested antibodies are summarized in the table below, which showed that all the four anti-PTK7/B7H3 antibodies bind to human PTK7, monkey PTK7, human B7H3, and monkey B7H3 with high affinity.

Table 3

“/” means no binding

Example 5. Binding activity of anti-PTK7/B7H3 antibodies to tumor cells

The binding activity of anti-PTK7/B7H3 antibodies to cancer cell lines was verified by flow cytometry. Briefly, the human lung cancer cells NCI-H520 or SHP-77 were plated in a 96-well plate at a density of 1×10⁵ cells/well respectively. Series diluted antibodies (highest concentration: 50μg/mL, 3-fold dilution) were added to each well and incubated at 4℃ for 30 minutes. Then, after one wash with PBS, the cells were incubated with the secondary antibody Alexa647 anti-human IgG Fcγ(Jackson ImmunoResearch Laboratories, Inc., Cat#: 109-606-170) at 4℃ for 15 minutes before flow cytometry analysis. The cells were collected, and the mean fluorescence intensity (MFI) was determined. Human IgG1 was used as an ISO control.

The results are shown in FIG. 1, which indicated that 21A9-3C4 and 20H8-3C4 exhibited better binding activities than the positive controls (Ref1, Ref2, Ref3) and the corresponding parent monoclonal antibodies (21A9, 20H8, 3C4) to NCI-H520 cells (FIG. 1 (A) ) and SHP-77 cells (FIG. 1 (B) ) .

In a similar experiment, the binding activity of 21A9-3C4 to human small cell lung cancer cells NCI-H69 was verified by flow cytometry. As shown in FIG. 2 (A) , compared to the positive controls Ref1, Ref2 and Ref3, 21A9-3C4 showed superior binding activity.

In another similar experiment, the binding activity of antibodies to human gastric cancer cells NCI-N87 was verified. As shown in FIG. 2 (B) , 21A9-3C4 and 20H8-3C4 demonstrated better binding activities than the positive controls (Ref1, Ref3) and the corresponding parent monoclonal antibodies (21A9, 20H8, 3C4) . In addition, the binding activity of 20H8-3C4 is more effective than that of the positive control Ref2.

Example 6. Internalization of anti-PTK7/B7H3 antibodies in tumor cells

Anti-PTK7 antibodies, anti-B7H3 antibodies, or anti-PTK7/B7H3 antibodies with a concentration of 2.5μg/mL together with the pHAb-Goat anti-human IgG secondary antibody were added to human lung cancer cells NCI-H520 or human breast cancer cells HCC70 respectively, and incubated for 24 hours. The cells were centrifuged and washed with FACS buffer, and then measured using a flow cytometer. For isotype control (ISO) , human IgG1 protein was used. The results are shown in FIG. 3.

21A9-3C4 showed higher endocytosis rate than the positive controls Ref1, Ref3 and Ref4 both in NCI-H520 cells (FIG. 3 (A) ) and HCC70 cells (FIG. 3 (B) ) . In addition, compared with the corresponding parent monoclonal antibodies 21A9 and 3C4, the bispecific antibody 21A9-3C4 exhibited higher endocytosis efficiency with a synergistic effect.

Example 7. Biophysical properties of anti-PTK7/B7H3 antibodies

Anti-PTK7/B7H3 bispecific antibodies were diluted using a buffer (3 mg/mL histidine, 80 mg/mL sucrose, and 0.2 mg/mL Tween^TM80) at pH 6.0. The diluted antibodies were kept in sealed Eppendorf tubes at 40±3℃ (hereinafter referred to as 40℃) for 7 days or 14 days, and their thermal stability was evaluated. Alternatively, the bispecific antibodies were also incubated at low pH conditions. Specifically, the antibodies were incubated in 1 mol/L acetic acid at pH 3.5 for 0 hour, 6 hours or 24 hours to determine its stability in acidic conditions.

After the above treatments, the following tests were performed: (1) observing the solution appearance and presence of visible non-soluble objects; (2) detecting the purity changes of antibodies by Size-Exclusion Ultra Performance Liquid Chromatography (SEC-UPLC) (indicated as the percentage of the main peak area to the sum of all peak areas (Purity, %) ) ; (3) detecting changes in the apparent hydrophobicity of the antibodies using the Hydrophobic Interaction Chromatography-High Performance Liquid Chromatography (HIC-HPLC) method (indicated as the retention time of the main peak (HIC, min) ) ; (4) detecting the purity changes of antibodies by capillary electrophoresis-sodium dodecyl sulfate (CE-SDS) under non-reducing (CE-SDS (NR) ) conditions (indicated as the percentage of the main peak area to the sum of all peak areas (Purity, %) ) ; (5) detecting charge variants in the antibodies by the Capillary Isoelectric Focusing (cIEF) method (indicated as the percentages of the main component, acidic component, and alkaline component) .

In the SEC-UPLC experiments, the antibody samples were diluted to 1 mg/mL with purified water and an Agilent 1290 chromatography system (connected with Xbridge^TM Protein BEH SEC column (Waters Corporation) ) was used. The following parameters were used: mobile phase: 100 mmol/L phosphate buffer ( “PB” ) (pH 7.4) +0.2 mol/L NaCl+10%acetonitrile; flow rate: 1.8 mL/min; column temperature: 25℃; detection wavelength: 280 nm; injection volume: 10 mL; sample tray temperature: about 6℃; and running time: 7 minutes.

In the HIC-HPLC experiments, an Agilent 1260 chromatography system (connected with ProPac^TM HIC-10 column (4.6×250 mm, Thermo Scientific) ) was used, and samples were diluted using mobile phase A to 0.5 mg/mL. The following parameters were used: mobile phase A: 1.0 M PB, 10%acetonitrile pH 6.5; mobile phase B: 0.1 M PB, 10%acetonitrile pH 6.5; flow rate: 0.8 mL/min; gradient: 0 min 100%A, 2 min 100%A, 32 min 100%B, 34 min 100%B, 35 min 100%A, and45 min 100%A; column temperature: 30℃; detection wavelength: 280 nm; injection volume: 10 mL; sample tray temperature: about 6℃; and running time: 45 minutes.

In the cIEF experiments, a Maurice cIEF Method Development Kit (Protein Simple, Cat#: PS-MDK01-C) was used for sample preparation. Specifically, 40μg protein sample was mixed with the following reagents in the kit: 1 mL Maurice cIEF Pi Marker-4.05, 1 mL Maurice CiefPi Marker-9.99, 35 mL 1%Methyl Cellulose Solution, 2 mL Maurice Cief500 mM Arginine, 4 mL Ampholytes (Pharmalyte pH ranges 3-10) , and water (added to make a final volume of 100 mL) . On the Maurice analyzer (Protein Simple, Santa Clara, CA) , Maurice cIEF Cartridges (PS-MC02-C) were used to generate imaging capillary isoelectric focusing spectra. The sample was focused for a total of 10 minutes. The analysis software installed on the instrument was used to integrate the absorbance of the 280 nm-focused protein.

In the CE-SDS (NR) experiments, Maurice (Protein simple, Maurice^TM) and Maurice CE-SDS Size Application Kit (Protein simple, Cat#: PS-MAK02-S) were used. 54 mL Sample Buffer, 6 mL antibody sample, 2.4 mL 25×internal standard, 3 mL 250 nM Iodoacetamide (SIGMA, Cat#: 16125) were add to a microcentrifuge tube, followed by centrifugation at 3000 rpm for 1 minute and heating in a 70℃ water bath for 10 minutes. The samples were then cooled to room temperature followed by centrifugation at 10000 rpm for3 minutes. Supernatant sample preparations were then transferred to a 96-well plate and tested in Maurice. The following parameters were used: injection voltage: 4.6 kV; injection time: 20 seconds; separation voltage: 5.75 kV; and separation time: 40 minutes.

Detailed results of anti-PTK7/B7H3 bispecific antibodies are shown in the table below. The results showed that 21A9-3C4 and 20H8-3C4 had good stability as well as physical and chemical properties.

Table 4

Example 8. Antibody Drug Conjugates

Each purified antibody (3C4, 3E6, 21A9, 20H8, 21A9-3C4, 20H8-3C4, 21A9-3E6, or 20H8-3E6) was coupled with CPT-1, CPT-2, CPT-3 or CPT-4, through CPT-L linker. For the names of antibody-drug conjugates, CPTx is added directly after the antibody name. For example, if 21A9-3C4 is coupled to CPT-1, it is named as 21A9-3C4-CPT1. For another example, if 21A9-3C4 is coupled to CPT-2, it is named as 21A9-3C4-CPT2. For isotype control, human IgG1 was coupled to CPT-2 to form ISO-CPT2. MS (Mass Spectrometry) was used to detect the coupling of antibodies with drug molecules. The MS detection results showed that the drug-to-antibody ratio (DAR) of the ADCs was about 8.

In addition, low payload ADCs with a DAR of about 4 was constructed. For the names of low payload ADCs, DAR4 is added directly after the ADCs name. For example, if 21A9-3C4-CPT2 has low payload with a DAR of about 4, it is named as 21A9-3C4-CPT2-DAR4. For clarity, ifthere is no DAR value marked in the ADCs name, it indicates that the DAR of ADCs is about 8.

Taking 21A9-3C4-CPT2 as an example, the exemplary ADC preparation process is as follows:

Reduction of the antibody: The purified antibody was diluted with PBS to a concentration of 5 mg/mL. The antibody solution was mixed with 1 mM TCEP, and the disulfide bonds at the hinge region of the antibody was reduced by incubating at 25℃for 2.5 hours.

Conjugation between antibody and drug linker: Following the melting of the CPT-L-CPT2 solution for 30 minutes at room temperature, a5 mmol/L CPT-L-CPT2 solution was added to the reduced antibody solution at the programmed molar ratio. The mixture was incubated at 25℃for 1.5 hours to facilitate the conjugation of the drug linker to the antibody. Subsequently, a solution containing four times the molar ratio of cysteine was added to the mixture to terminate the reaction. This was incubated at 25℃ for an additional 30 minutes.

Purification: The above solution was added to the desalting column for desalting. The desalted solution was then transferred to an ultrafiltration tube and exchanged into 25 mM HIS buffer. The concentration was adjusted to approximately 5 mg/mL using the HIS buffer. Finally, the purified solution was filtered using a 0.22μM filter membrane.

Physicochemical characterization: Free payload quantification, purity, DAR value and drug load distribution were analyzed using RP-HPLC, SEC-HPLC and LC-MS methods.

Antibody concentration: The absorbance at 280 nm (A280) and 377 nm (A377) of the ADC solution were measured using a microplate reader. The corrected concentration was calculated using the following formula: C= (A280-A377*1.35) /protein extinction coefficient (1.57 (mg/mL) ^-1) /light range (cm^-1) .

For comparative purposes, the reference antibody Ref3 was also coupled with Dxd through GGFG linker to form the ADC with a DAR of about 4 (designated as Ref3-Dxd-DAR4) . Human IgG1 was also coupled to Dxd to form ISO-Dxd-DAR4 for isotype control.

Example 9. Anti-tumor activity in human colorectal cancer PDX model

The ADCs were tested for the effect in human colorectal cancer patient-derived xenograft (PDX) model. Co-expression of PTK7 and B7H3 was tested in the patient-derived colorectal tumor tissue by Immunohistochemistry (IHC) assessment, with H-score of 28.57 and 28.05 respectively. B-NDG mice (Biocytogen Pharmaceuticals (Beijing) Co., Ltd., Cat#B-CM-002) were engrafted in the right flank with the patient-derived tumor fragment (2 mm×2 mm×2 mm) . When the tumor volume reached about 200 mm³, the mice were randomly placed into different groups based on the tumor volume. The mice were then injected with phosphate buffer saline (PBS) or ADCs by intravenous (i.v. ) administration. Details of the administration scheme are shown in the table below.

Table 5

The tumor volumes were measured twice a week and body weights of the mice were recorded as well. Euthanasia was performed when tumor volume of a mouse reached 2000 mm³.

The length of the long axis and the short axis of the tumor were measured and the volume of the tumor was calculated as 0.5× (long axis) × (short axis) ². The weight of the mice was also measured twice a week. The tumor growth inhibition percentage (TGI%) was calculated using the following formula: TGI (%)= [1- (Ti-T0) / (Vi-V0) ] ×100%. Ti is the average tumor volume in the treatment group on day i. T0 is the average tumor volume in the treatment group on day zero. Vi is the average tumor volume in the control group on day i. V0 is the average tumor volume in the control group on day zero. T-test was performed for statistical analysis. A TGI%higher than 60%indicates clear suppression of tumor growth. P<0.05 is a threshold to indicate significant difference.

As shown in FIG. 4, 21A9-3C4-CPT2 and 20H8-3C4-CPT2 inhibited tumor growth in a dose-dependent manner (G2 to G5) , and induced significant tumor growth inhibition at 10 mg/kg with a higher TGI% (e.g., on Day 42) than that of the positive control Ref3-CPT2 (G4 to G6) .

In another experiment, patient-derived colorectal tumor fragments (2 mm×2 mm×2 mm) were engrafted in the right flank of B-NDG mice. When the tumor volume reached about 200-300 mm³, the mice were randomly placed into different groups based on tumor volumes. The mice were then injected with PBS or ADCs by i.v. administration on day 0 (D0) and day 16 (D16) . Details of the administration scheme are shown in the table below. The tumor volumes were measured twice a week.

Table 6

As shown in FIG. 15, both 21A9-3C4-CPT2-DAR4 (G6 and G7) and 21A9-3C4-CPT2 (G8 and G9) exhibited better tumor inhibition activities than the positive controls Ref1-CPT2-DAR4 and Ref3-Dxd-DAR4 (G10 and G11) , with a dose-dependent manner. In addition, 21A9-3C4-CPT2-DAR4 (G6) exhibited significant synergistic effects on tumor inhibition compared with the parent monoclonal ADC 21A9-CPT2-DAR4 and 3C4-CPT2-DAR4 (G4 and G5) .

Example 10. Anti-tumor activity in lung cancer PDX model

The ADCs were tested for their effects on tumor growth in vivo in a xenograft model of lung cancer. Specifically, tumor fragments derived from patient with lung cancer were inoculated subcutaneously in B-NDG mice. The expression levels (H-score) of PTK7 and B7H3 were 98.49 and 2.38 respectively by IHC assessment. When the tumors in the mice reached a volume of about 250 mm³, the mice were randomly placed into different groups based on tumor volume. The mice were then injected with PBS or ADCs by i.v. administration. The frequency of administration was once a week (1 administration in total) . Details are shown in the table below.

Table 7

During the experimental period, there was no difference observed in the body weight of mice across the groups. The tumor volume of mice in different groups are shown in FIGs. 5-6. FIG. 5 indicated that the bispecific ADCs 21A9-3C4-CPT2, 21A9-3E6-CPT2, and 20H8-3C4-CPT2 inhibited tumor growth with a higher TGI% (e.g., on Day 21) than that of the positive controls Ref2-CPT2 and Ref3-CPT2. FIG. 6 demonstrated that the bispecific ADCs (G7 to G9) exhibited significant synergistic effects on tumor inhibition compared with the parent monoclonal ADCs (G3 to G6) .

In a similar experiment, tumor fragments derived from patient with lung cancer were inoculated subcutaneously in B-NDG mice. H-scores of PTK7 and B7H3 in the tumor fragments were 102.97 and 6.41 respectively by IHC assessment. When the tumors in the mice reached a volume of about 250 mm³, the mice were randomly placed into different groups based on tumor volume. The mice were then injected with PBS (G1) , 21A9-3C4-CPT2 (G2) , 20H8-3C4-CPT2 (G3) , or Ref2-CPT2 (G4) with a concentration of 6 mg/kg by i.v. administration. The frequency of administration was once a week (1 administration in total) .

The tumor volume of mice in different groups treated with the PBS or ADCs are shown in FIG. 7. Compared with the positive control Ref2-CPT2, the bispecific ADCs 21A9-3C4-CPT2 and 20H8-3C4-CPT2 exhibited better tumor inhibitory effects with higher TGI%.

In another experiment, lung cancer PDX model with PTK7/B7H3 H-scores of 102.97/6.41 was used. The lung tumor fragments (2 mm×2 mm×2 mm) were engrafted in the right flank of B-NDG mice. When the tumor volume reached about 200-300 mm³, the mice were randomly placed into different groups based on tumor volumes. The mice were then injected with PBS or ADCs by i.v. administration. Details of the administration scheme are shown in the table below.

Table 8

As shown in FIG. 16, both 21A9-3C4-CPT2-DAR4 and 21A9-3C4-CPT2 (G6 to G9) inhibited tumor growth in a dose-dependent manner and had a higher TGI% (e.g., on Day 16) than that of the positive controls Ref1-CPT2-DAR4 (G11) and Ref3-Dxd (G10) . In addition, 21A9-3C4-CPT2-DAR4 (G6) exhibited synergistic effects on tumor inhibition compared with the parent monoclonal ADCs 21A9-CPT2-DAR4 and 3C4-CPT2-DAR4 (G4 and G5) .

Example 11. Anti-tumor activity in pancreatic cancer PDX Model

The ADCs were tested for their effects on tumor growth in vivo in a xenograft model of pancreatic cancer. Specifically, tumor fragments (H-score of PTK7/B7H3: 159.69/31.29) derived from patient with pancreatic cancer were inoculated subcutaneously in B-NDG mice. When the tumors in the mice reached a volume of about 230 mm³, the mice were randomly placed into different groups based on tumor volume. The mice were then injected with PBS or ADCs by i.v. administration. Details are shown in the table below.

Table 9

The tumor volume of mice in different groups are shown in FIG. 8, in which the bispecific ADCs 21A9-3C4-CPT2, 21A9-3E6-CPT2 and 20H8-3C4-CPT2 inhibited tumor growth with a dose-dependent manner.

Example 12. Anti-tumor activity in human breast cancer PDX model

The ADCs were tested for the effect in human breast cancer patient-derived xenograft models. B-NDG mice were engrafted in the right flank with the patient-derived tumor fragment (2 mm×2 mm×2 mm) (H-score of PTK7/B7H3: 190.06/73.79) . When the tumors in the mice reached a volume of about 250 mm³, the mice were randomly placed into different groups based on the volume of the tumor. The mice were then injected with PBS or ADCs by i.v. administration. Details of the administration scheme are shown in the table below.

Table 10

As shown in FIG. 9, all the three anti-PTK7/B7H3 bispecific ADCs 21A9-3C4-CPT2, 21A9-3E6-CPT2 and 20H8-3C4-CPT2 exhibited better tumor inhibitory effects than the positive controls Ref2-CPT2 and Ref3-CPT2. In particular, 21A9-3C4-CPT2 obtained the best anti-tumor activity among the bispecific ADCs. In addition, compared with the parent monoclonal ADCs (G2 to G5) , the bispecific ADCs (G6 to G8) exhibited significant synergistic effects on tumor inhibition (FIG. 10) .

In another similar experiment, B-NDG mice were engrafted with another breast cancer patient-derived tumor fragment (2 mm×2 mm×2 mm) . The H-score of PTK7 and B7H3 were 127.39 and 106.57 respectively by IHC test. When the tumors in the mice reached a volume of about 200 mm³, the mice were randomly placed into different groups based on the volume of the tumor. The mice were then injected with PBS or ADCs by i.v. administration. Details of the administration scheme are shown in the table below.

Table 11

As shown in FIG. 11, compared with the positive controls Ref2-CPT2, Ref3-CPT2, and Ref4-CPT2, the bispecific ADCs 21A9-3C4-CPT2 and 20H8-3C4-CPT2 exhibited better tumor inhibitory effects at 1 mg/kg dose level. With regard to 3 mg/kg dosage, both 21A9-3C4-CPT2 and 20H8-3C4-CPT2 persisted and regressed PTK7/B7H3 co-expressing xenograft tumors in mice. In addition, the bispecific ADCs inhibited tumor growth with a dose-dependent manner, with higher doses led to better therapeutic effects.

The mouse survival was also monitored. On Day 18, all mice died in group G1 due to reaching the euthanasia criteria. On Day 28, all mice died in group G8. At the end of the experiment on Day 39, 5 mice all survived in groups G2, G4 and G5, with tumor disappeared in 1 mouse in G5 group. This indicates that the anti-PTK7/B7H3 ADCs possess significant and sustained anti-tumor activity in vivo.

In another experiment, breast cancer PDX model with PTK7/B7H3 H-scores of 190.06/73.79 was used. The breast tumor fragments (2 mm×2 mm×2 mm) were engrafted in the right flank of B-NDG mice. When the tumor volume reached about 200-300 mm³, the mice were randomly placed into different groups based on tumor volumes. The mice were then injected with PBS or ADCs by i.v. administration. Details of the administration scheme are shown in the table below.

Table 12

As shown in FIG. 17, both 21A9-3C4-CPT2-DAR4 and 21A9-3C4-CPT2 (G5 to G8) inhibited tumor growth in a dose-dependent manner and had a higher TGI% (e.g., on Day 21) than that of the positive controls Ref1-CPT2-DAR4 (G10) and Ref3-Dxd (G9) .

In another experiment, breast cancer PDX model with PTK7/B7H3 H-scores of 127.39/106.57 was used. The breast tumor fragments (2 mm×2 mm×2 mm) were engrafted in the right flank of B-NDG mice. When the tumor volume reached about 200-300 mm³, the mice were randomly placed into different groups based on tumor volumes. The mice were then injected with PBS or ADCs by i.v. administration. Details of the administration scheme are shown in the table below.

Table 13

As shown in FIG. 18, both 21A9-3C4-CPT2-DAR4 and 21A9-3C4-CPT2 (G6 to G9) inhibited tumor growth in a dose-dependent manner and had better tumor growth inhibition than that of the positive controls Ref1-CPT2-DAR4 (G11) and Ref3-Dxd (G10) . 21A9-3C4-CPT2-DAR4 (G6) exhibited significant synergistic effects on tumor inhibition compared with the parent monoclonal ADCs (G4 and G5) .

Example 13. Proliferation inhibitory activity of anti-PTK7/B7H3 ADC

SHP-77 cells, NCI-H1781 cells, NCI-H358 cells, NCI-H520 cells, NCI-H460 cells, or Calu-6 cells were transferred to a 96-well plate and incubated at 37℃, 5%CO₂ overnight. Serially diluted ADCs were added to the 96-well plate respectively, and then the plate was incubated at 37℃, 5%CO₂ for 7 days. The plate was centrifuged at 500 g for 5 minutes, and the supernatant was discarded. 100μL Promega Luminescent Cell Viability Assay reagent (Promega, Cat#: G7571) was added to the corresponding well and incubated at room temperature in the dark for 10 minutes. The luminescence signal value was read by the microplate reader and the cell viability was calculated.

The cell viability was used to generate a fitting curve with respect to the antibody concentrations. More specifically, logarithm of antibody concentrations was calculated and was used as the X-axis variable, while the cell viability was used as the Y-axis variable. The IC₅₀ value was determined. The test results are shown in the table below. 21A9-3C4-CPT2 exhibited better proliferation inhibitory activity than the positive control (Ref1-CPT2, Ref2-CPT2, and/or Ref3-CPT2) on SHP-77 cells, NCI-H1781 cells, NCI-H520 cells, NCI-H460 cells, NCI-H358 cells, and Calu-6 cells.

Table 14

Example 14. Stability of anti-PTK7/B7H3 ADC in plasma

Stability of anti-PTK7/B7H3 ADC in plasma was tested. Specifically, 21A9-3C4-CPT2 with a final concentration of 100μg/mL was added to Human plasma (Oricells, Cat#: FTS-P-100) , Monkey plasma (Iphase, Cat#: 032B13.22) , Rat plasma (Iphase, Cat#: 032D13.22) , Mouse plasma (Iphase, Cat#: 032E13.22) , or 0.5%BSA, and incubated at 37℃for 14 days.

The contents of free payload (CPT-2) on 1 day, 3 days, 7 days, and 14 days were measured using LC-MS/MS. The results are shown in FIG. 19, which indicated that 21A9-3C4-CPT2 exhibited a high stability in human, monkey, rat, and mouse plasma, with a concentration of free CPT2 no more than 6 ng/mL.

OTHER EMBODIMENTS

It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

An anti-PTK7/B7H3 antibody or antigen-binding fragment thereof, comprising: a first antigen-binding domain that specifically binds to PTK7; and a second antigen-binding domain that specifically binds to B7H3.
The anti-PTK7/B7H3 antibody or antigen-binding fragment thereof of claim 1, wherein the first antigen-binding domain comprises a first heavy chain variable region (VH1) and a first light chain variable region (VL1) ; and the second antigen-binding domain comprises a second heavy chain variable region (VH2) and a second light chain variable region (VL2) .
The anti-PTK7/B7H3 antibody or antigen-binding fragment thereof of claim 2, wherein the first heavy chain variable region (VH1) comprises complementarity determining regions (CDRs) 1, 2, and 3, wherein the VH1 CDR1 region comprises an amino acid sequence that is at least 80%identical to a selected VH1 CDR1 amino acid sequence, the VH1 CDR2 region comprises an amino acid sequence that is at least 80%identical to a selected VH1 CDR2 amino acid sequence, and the VH1 CDR3 region comprises an amino acid sequence that is at least 80%identical to a selected VH1 CDR3 amino acid sequence; and the first light chain variable region (VL1) comprises CDRs 1, 2, and 3, wherein the VL1 CDR1 region comprises an amino acid sequence that is at least 80%identical to a selected VL1 CDR1 amino acid sequence, the VL1 CDR2 region comprises an amino acid sequence that is at least 80%identical to a selected VL1 CDR2 amino acid sequence, and the VL1 CDR3 region comprises an amino acid sequence that is at least 80%identical to a selected VL1 CDR3 amino acid sequence, wherein the selected VH1 CDRs 1, 2, and 3 amino acid sequences, the selected VL1 CDRs 1, 2, and 3 amino acid sequences are one of the following:

(1) the selected VH1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 4-6, respectively, and the selected VL1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 1-3, respectively;

(2) the selected VH1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 7-9, respectively, and the selected VL1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 1-3, respectively;

(3) the selected VH1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 10-12, respectively, and the selected VL1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 1-3, respectively; and

(4) the selected VH1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 13-15, respectively, and the selected VL1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 1-3, respectively.
The anti-PTK7/B7H3 antibody or antigen-binding fragment thereof of claim 2, wherein the first heavy chain variable region (VH1) comprises complementarity determining regions (CDRs) 1, 2, and 3, with no more than one amino acid substitution relative to a selected VH1 CDR1 amino acid sequence, a selected VH1 CDR2 amino acid sequence, and/or a selected VH1 CDR3 amino acid sequence; and the first light chain variable region (VL1) comprises CDRs 1, 2, and 3, with no more than one amino acid substitution relative to a selected VL1 CDR1 amino acid sequence, a selected VL1 CDR2 amino acid sequence, and/or a selected VL1 CDR3 amino acid sequence, wherein the selected VH1 CDRs 1, 2, and 3 amino acid sequences and the selected VL1 CDRs 1, 2, and 3 amino acid sequences are one of the following:

(1) the selected VH1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 4-6, respectively, and the selected VL1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 1-3, respectively;

(2) the selected VH1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 7-9, respectively, and the selected VL1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 1-3, respectively;

(3) the selected VH1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 10-12, respectively, and the selected VL1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 1-3, respectively; and

(4) the selected VH1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 13-15, respectively, and the selected VL1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 1-3, respectively.
The anti-PTK7/B7H3 antibody or antigen-binding fragment thereof of any one of claims 2-4, wherein the second heavy chain variable region (VH2) comprises CDRs 1, 2, and 3, wherein the VH2 CDR1 region comprises an amino acid sequence that is at least 80%identical to a selected VH2 CDR1 amino acid sequence, the VH2 CDR2 region comprises an amino acid sequence that is at least 80%identical to a selected VH2 CDR2 amino acid sequence, and the VH2 CDR3 region comprises an amino acid sequence that is at least 80%identical to a selected VH2 CDR3 amino acid sequence; and the second light chain variable region (VL2) comprises CDRs 1, 2, and 3, wherein the VL2 CDR1 region comprises an amino acid sequence that is at least 80%identical to a selected VL2 CDR1 amino acid sequence, the VL2 CDR2 region comprises an amino acid sequence that is at least 80%identical to a selected VL2 CDR2 amino acid sequence, and the VL2 CDR3 region comprises an amino acid sequence that is at least 80%identical to a selected VL2 CDR3 amino acid sequence, wherein the selected VH2 CDRs 1, 2, and 3 amino acid sequences, and the selected VL2 CDRs 1, 2, and 3 amino acid sequences are one of the following:

(1) the selected VH2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 16-18, respectively, and the selected VL2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 1-3, respectively;

(2) the selected VH2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 19-21, respectively, and the selected VL2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 1-3, respectively;

(3) the selected VH2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 22-24, respectively, and the selected VL2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 1-3, respectively; and

(4) the selected VH2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 25-27, respectively, and the selected VL2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 1-3, respectively.
The anti-PTK7/B7H3 antibody or antigen-binding fragment thereof of any one of claims 2-4, wherein the second heavy chain variable region (VH2) comprises CDRs 1, 2, and 3, with no more than one amino acid substitution relative to a selected VH2 CDR1 amino acid sequence, a selected VH2 CDR2 amino acid sequence, and/or a selected VH2 CDR3 amino acid sequence; and the second light chain variable region (VL2) comprises CDRs 1, 2, and 3, with no more than one amino acid substitution relative to a selected VL2 CDR1 amino acid sequence, a selected VL2 CDR2 amino acid sequence, and/or a selected VL2 CDR3 amino acid sequence, wherein the selected VH2 CDRs 1, 2, and 3 amino acid sequences, and the selected VL2 CDRs 1, 2, and 3 amino acid sequences are one of the following:

(1) the selected VH2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 16-18, respectively, and the selected VL2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 1-3, respectively;

(2) the selected VH2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 19-21, respectively, and the selected VL2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 1-3, respectively;

(3) the selected VH2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 22-24, respectively, and the selected VL2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 1-3, respectively; and

(4) the selected VH2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 25-27, respectively, and the selected VL2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 1-3, respectively.
The anti-PTK7/B7H3 antibody or antigen-binding fragment thereof of any one of claims 2-6, wherein

(1)the selected VH1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 4-6, respectively, and the selected VL1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 1-3, respectively, and the selected VH2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 16-18, respectively, and the selected VL2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 1-3, respectively;

(2) the selected VH1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 4-6, respectively, and the selected VL1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 1-3, respectively, and the selected VH2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 22-24, respectively, and the selected VL2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 1-3, respectively;

(3) the selected VH1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 10-12, respectively, and the selected VL1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 1-3, respectively, and the selected VH2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 16-18, respectively, and the selected VL2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 1-3, respectively;

(4) the selected VH1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 10-12, respectively, and the selected VL1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 1-3, respectively, and the selected VH2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 22-24, respectively, and the selected VL2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 1-3, respectively;

(5) the selected VH1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 7-9, respectively, and the selected VL1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 1-3, respectively, and the selected VH2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 19-21, respectively, and the selected VL2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 1-3, respectively;

(6) the selected VH1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 7-9, respectively, and the selected VL1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 1-3, respectively, and the selected VH2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 25-27, respectively, and the selected VL2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 1-3, respectively;

(7) the selected VH1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 13-15, respectively, and the selected VL1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 1-3, respectively, and the selected VH2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 19-21, respectively, and the selected VL2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 1-3, respectively; or

(8) the selected VH1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 13-15, respectively, and the selected VL1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 1-3, respectively, and the selected VH2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 25-27, respectively, and the selected VL2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 1-3, respectively.
The anti-PTK7/B7H3 antibody or antigen-binding fragment thereof of any one of claims 2-7, wherein the first heavy chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100%identical to SEQ ID NO: 29, the first light chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100%identical to SEQ ID NO: 28, the second heavy chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100%identical to SEQ ID NO: 31, and the second light chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100%identical to SEQ ID NO: 28.
The anti-PTK7/B7H3 antibody or antigen-binding fragment thereof of any one of claims 2-7, wherein the first heavy chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100%identical to SEQ ID NO: 29, the first light chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100%identical to SEQ ID NO: 28, the second heavy chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100%identical to SEQ ID NO: 32, and the second light chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100%identical to SEQ ID NO: 28.
The anti-PTK7/B7H3 antibody or antigen-binding fragment thereof of any one of claims 2-7, wherein the first heavy chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100%identical to SEQ ID NO: 30, the first light chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100%identical to SEQ ID NO: 28, the second heavy chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100%identical to SEQ ID NO: 31, and the second light chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100%identical to SEQ ID NO: 28.
The anti-PTK7/B7H3 antibody or antigen-binding fragment thereof of any one of claims 2-7, wherein the first heavy chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100%identical to SEQ ID NO: 30, the first light chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100%identical to SEQ ID NO: 28, the second heavy chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100%identical to SEQ ID NO: 32, and the second light chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100%identical to SEQ ID NO: 28.
The anti-PTK7/B7H3 antibody or antigen-binding fragment thereof of any one of claims 2-11, wherein the VH1 comprises an amino acid sequence that is at least 90%identical to a selected VH sequence, and the VL1 comprises an amino acid sequence that is at least 90%identical to a selected VL sequence, wherein the selected VH sequence and the selected VL sequence are one of the following:

(1) the selected VH sequence is SEQ ID NO: 29, and the selected VL sequence is SEQ ID NO: 28; and

(2) the selected VH sequence is SEQ ID NO: 30, and the selected VL sequence is SEQ ID NO: 28.
The anti-PTK7/B7H3 antibody or antigen-binding fragment thereof of any one of claims 2-12, wherein the VH1 comprises VH CDR1, VH CDR2, and VH CDR3 that are identical to VH CDR1, VH CDR2, and VH CDR3 of a selected VH sequence; and the VL1 comprising VL CDR1, VL CDR2, and VL CDR3 that are identical to VL CDR1, VL CDR2, and VL CDR3 of a selected VL sequence, wherein the selected VH sequence and the selected VL sequence are one of the following:

(1) the selected VH sequence is SEQ ID NO: 29, and the selected VL sequence is SEQ ID NO: 28; and

(2) the selected VH sequence is SEQ ID NO: 30, and the selected VL sequence is SEQ ID NO: 28.
The anti-PTK7/B7H3 antibody or antigen-binding fragment thereof of any one of claims 2-13, wherein the VH2 comprises an amino acid sequence that is at least 90%identical to a selected VH sequence, and the VL2 comprises an amino acid sequence that is at least 90%identical to a selected VL sequence, wherein the selected VH sequence and the selected VL sequence are one of the following:

(1) the selected VH sequence is SEQ ID NO: 31, and the selected VL sequence is SEQ ID NO: 28; and

(2) the selected VH sequence is SEQ ID NO: 32, and the selected VL sequence is SEQ ID NO: 28.
The anti-PTK7/B7H3 antibody or antigen-binding fragment thereof of any one of claims 2-14, wherein the VH2 comprises VH CDR1, VH CDR2, and VH CDR3 that are identical to VH CDR1, VH CDR2, and VH CDR3 of a selected VH sequence; and the VL2 comprising VL CDR1, VL CDR2, and VL CDR3 that are identical to VL CDR1, VL CDR2, and VL CDR3 of a selected VL sequence, wherein the selected VH sequence and the selected VL sequence are one of the following:

(1) the selected VH sequence is SEQ ID NO: 31, and the selected VL sequence is SEQ ID NO: 28; and

(2)the selected VH sequence is SEQ ID NO: 32, and the selected VL sequence is SEQ ID NO: 28.
The anti-PTK7/B7H3 antibody or antigen-binding fragment thereof of any one of claims 2-15, wherein the VH1 comprises the sequence of SEQ ID NO: 29 and the VL1 comprises the sequence of SEQ ID NO: 28.
The anti-PTK7/B7H3 antibody or antigen-binding fragment thereof of any one of claims 2-15, wherein the VH1 comprises the sequence of SEQ ID NO: 30 and the VL1 comprises the sequence of SEQ ID NO: 28.
The anti-PTK7/B7H3 antibody or antigen-binding fragment thereof of any one of claims 2-15, wherein the VH2 comprises the sequence of SEQ ID NO: 31 and the VL2 comprises the sequence of SEQ ID NO: 28.
The anti-PTK7/B7H3 antibody or antigen-binding fragment thereof of any one of claims 2-15, wherein the VH2 comprises the sequence of SEQ ID NO: 32 and the VL2 comprises the sequence of SEQ ID NO: 28.
The anti-PTK7/B7H3 antibody or antigen-binding fragment thereof of any one of claims 2-15, wherein the VH1 comprises the sequence of SEQ ID NO: 29 and the VL1 comprises the sequence of SEQ ID NO: 28, and the VH2 comprises the sequence of SEQ ID NO: 31 and the VL2 comprises the sequence of SEQ ID NO: 28.
The anti-PTK7/B7H3 antibody or antigen-binding fragment thereof of any one of claims 2-15, wherein the VH1 comprises the sequence of SEQ ID NO: 30 and the VL1 comprises the sequence of SEQ ID NO: 28, and the VH2 comprises the sequence of SEQ ID NO: 32 and the VL2 comprises the sequence of SEQ ID NO: 28.
An anti-PTK7/B7H3 antibody or antigen-binding fragment thereof, comprising: a first antigen-binding domain that specifically binds to PTK7 comprising a VH1 and a VL1; and a second antigen-binding domain that specifically binds to B7H3 comprising a VH2 and a VL2; wherein:

A(1) the VH1 comprises CDRs 1, 2, 3 amino acid sequences as set forth in SEQ ID NOs: 4-6, respectively, and the VL1 comprises CDRs 1, 2, 3 amino acid sequences as set forth in SEQ ID NOs: 1-3, respectively; or

A(2) the VH1 comprises CDRs 1, 2, 3 amino acid sequences as set forth in SEQ ID NOs: 7-9, respectively, and the VL1 comprises CDRs 1, 2, 3 amino acid sequences as set forth in SEQ ID NOs: 1-3, respectively;

and

B (1) the VH2 comprises CDRs 1, 2, 3 amino acid sequences as set forth in SEQ ID NOs: 16-18, respectively, and the VL2 comprises CDRs 1, 2, 3 amino acid sequences as set forth in SEQ ID NOs: 1-3, respectively; or

B (2) the VH2 comprises CDRs 1, 2, 3 amino acid sequences as set forth in SEQ ID NOs: 19-21, respectively, and the VL2 comprises CDRs 1, 2, 3 amino acid sequences as set forth in SEQ ID NOs: 1-3, respectively.
An anti-PTK7/B7H3 antibody or antigen-binding fragment thereof, comprising: a first antigen-binding domain that specifically binds to PTK7 comprising a VH1 and a VL1; and a second antigen-binding domain that specifically binds to B7H3 comprising a VH2 and a VL2; wherein:

A (1) the VH1 comprises CDRs 1, 2, 3 amino acid sequences as set forth in SEQ ID NOs: 10-12, respectively, and the VL1 comprises CDRs 1, 2, 3 amino acid sequences as set forth in SEQ ID NOs: 1-3, respectively; or

A (2) the VH1 comprises CDRs 1, 2, 3 amino acid sequences as set forth in SEQ ID NOs: 13-15, respectively, and the VL1 comprises CDRs 1, 2, 3 amino acid sequences as set forth in SEQ ID NOs: 1-3, respectively;

and

B (1) the VH2 comprises CDRs 1, 2, 3 amino acid sequences as set forth in SEQ ID NOs: 22-24, respectively, and the VL2 comprises CDRs 1, 2, 3 amino acid sequences as set forth in SEQ ID NOs: 1-3, respectively; or

B (2) the VH2 comprises CDRs 1, 2, 3 amino acid sequences as set forth in SEQ ID NOs: 25-27, respectively, and the VL2 comprises CDRs 1, 2, 3 amino acid sequences as set forth in SEQ ID NOs: 1-3, respectively.
The anti-PTK7/B7H3 antibody or antigen-binding fragment thereof of any one of claims 1-23, wherein the first antigen-binding domain specifically binds to human or monkey PTK7; and/or the second antigen-binding domain specifically binds to human or monkey B7H3.
The anti-PTK7/B7H3 antibody or antigen-binding fragment thereof of any one of claims 1-24, wherein the first antigen-binding domain is human or humanized; and/or the second antigen-binding domain is human or humanized.
The anti-PTK7/B7H3 antibody or antigen-binding fragment thereof of any one of claims 1-25, wherein the antibody is a multi-specific antibody (e.g., a bispecific antibody) .
The anti-PTK7/B7H3 antibody or antigen-binding fragment thereof of any one of claims 1-26, wherein the first antigen-binding domain is a single-chain variable fragment (scFv) ; and/or the second antigen-binding domain is a scFv.
The anti-PTK7/B7H3 antibody or antigen-binding fragment thereof of any one of claims 1-27, wherein the first light chain variable region and the second light chain variable region are identical.
An anti-PTK7/B7H3 antibody or antigen-binding fragment thereofthat cross-competes with the anti-PTK7/B7H3 antibody or antigen-binding fragment thereof of any one of claims 1-28.
A nucleic acid comprising a polynucleotide encoding the anti-PTK7/B7H3 antibody or antigen-binding fragment thereof of any one of claims 1-28.
A vector comprising the nucleic acid of claim 30.
A cell comprising the vector of claim 31.
The cell of claim 32, wherein the cell is a CHO cell.
A cell comprising the nucleic acid of claim 30.
A method of producing an anti-PTK7/B7H3 antibody or an antigen-binding fragment thereof, the method comprising

(a) culturing the cell of any one of claims 32-34 under conditions sufficient for the cell to produce the anti-PTK7/B7H3 antibody or the antigen-binding fragment thereof; and

(b) collecting the anti-PTK7/B7H3 antibody or the antigen-binding fragment thereof produced by the cell.
A composition comprising:

a first vector encoding the VH1,

a second vector encoding the VH2,

and a third vector encoding the VL1/VL2,

of the anti-PTK7/B7H3 antibody or antigen-binding fragment thereof of any one of claims 1-28.
A cell comprising the composition of claim 36.
A cell of claim 37, which is a CHO-S cell.
A method ofproducing an anti-PTK7/B7H3 antibody or an antigen-binding fragment thereof, the method comprising

(a) culturing the cell of claim 37 or 38 under conditions sufficient for the cell to produce the anti-PTK7/B7H3 antibody or the antigen-binding fragment thereof; and

(b) collecting the anti-PTK7/B7H3 antibody or the antigen-binding fragment thereof produced by the cell.
An anti-PTK7/B7H3 antibody-drug conjugate (ADC) comprising a therapeutic agent covalently bound to the anti-PTK7/B7H3 antibody or antigen-binding fragment thereof of any one of claims 1-28.
The anti-PTK7/B7H3 antibody drug conjugate of claim 40, wherein the therapeutic agent is a cytotoxic or cytostatic agent.
The antibody-drug conjugate of claim 40 or 41, wherein the therapeutic agent is selected from
The antibody-drug conjugate of any one of claims 40-42, wherein the therapeutic agent is linked to the antibody or antigen-binding fragment thereof via a linker.
The antibody-drug conjugate of claim 43, wherein the linker has a structure of:
The antibody-drug conjugate of any one of claims 40-44, wherein the antibody-drug conjugate has a structure of:

wherein n=1-8; wherein “Ab” represents the antibody or antigen-binding fragment thereof.
The antibody-drug conjugate of any one of claims 40-45, wherein the antibody-drug conjugate has a structure of:

wherein n=1-8; wherein “Ab” represents the antibody or antigen-binding fragment thereof.
An anti-PTK7/B7H3 ADC comprising:

(i) an anti-PTK7/B7H3 antibody or antigen-binding fragment thereof comprising: a first antigen-binding domain that specifically binds to PTK7 comprising a VH1 and a VL1; and a second antigen-binding domain that specifically binds to B7H3 comprising a VH2 and a VL2; wherein:

A (1) the VH1 comprises CDRs 1, 2, 3 amino acid sequences as set forth in SEQ ID NOs: 4-6, respectively, and the VL1 comprises CDRs 1, 2, 3 amino acid sequences as set forth in SEQ ID NOs: 1-3, respectively; or

A (2) the VH1 comprises CDRs 1, 2, 3 amino acid sequences as set forth in SEQ ID NOs: 7-9, respectively, and the VL1 comprises CDRs 1, 2, 3 amino acid sequences as set forth in SEQ ID NOs: 1-3, respectively;

and

B (1) the VH2 comprises CDRs 1, 2, 3 amino acid sequences as set forth in SEQ ID NOs: 16-18, respectively, and the VL2 comprises CDRs 1, 2, 3 amino acid sequences as set forth in SEQ ID NOs: 1-3, respectively; or

B (2) the VH2 comprises CDRs 1, 2, 3 amino acid sequences as set forth in SEQ ID NOs: 19-21, respectively, and the VL2 comprises CDRs 1, 2, 3 amino acid sequences as set forth in SEQ ID NOs: 1-3, respectively, and

(ii)

covalently attached to the antibody or antigen-binding fragment thereof via a linker.
The anti-PTK7/B7H3 ADC of claim 47, wherein the linker is
A method of preparing an antibody-drug conjugate, the method comprising:

(a) providing the anti-PTK7/B7H3 antibody or antigen-binding fragment thereof of any one of claims 1-28;

(b) conjugating a therapeutic agent to the anti-PTK7/B7H3 antibody or antigen-binding fragment thereof via a linker to form the antibody-drug conjugate.
The method of claim 49, wherein the method further comprises treating the anti-PTK7/B7H3 antibody or antigen-binding fragment thereof with a reductant to generate one or more thiol groups before step (b) .
The method of claim 49 or 50, wherein the conjugating step comprises reacting one or more thiol groups of the antibody or antigen-binding fragment with a maleimide-functionalized linker that is linked to the therapeutic agent.
The method of any one of claims 49-51, further comprising purifying the antibody-drug conjugate.
The method of any one of claims 49-52, wherein the drug-to-antibody ratio (DAR) is about 2～8.
The method of any one of claims 49-53, wherein the therapeutic agent is a cytotoxic or cytostatic agent.
The method of any one of claims 49-54, wherein the therapeutic agent is selected from
The method of any one of claims 49-55, wherein the therapeutic agent is linked to the anti-PTK7/B7H3 antibody or antigen-binding fragment thereof via a linker.
The method of any one of claims 49-56, wherein the linker has a structure of:
The method of any one of claims 49-57, wherein the antibody-drug conjugate has a structure of:

wherein n=1-8; wherein “Ab” represents the anti-PTK7/B7H3 antibody or antigen-binding fragment thereof.
The antibody-drug conjugate of any one of claims 49-58, wherein the antibody-drug conjugate has a structure of:

wherein n=1-8; wherein “Ab” represents the antibody or antigen-binding fragment thereof.
A method of treating a subject having cancer, the method comprising administering a therapeutically effective amount of a composition comprising the anti-PTK7/B7H3 antibody or antigen-binding fragment thereof of any one of claims 1-28, or the anti-PTK7/B7H3 antibody-drug conjugate of any one of claims 40-48, to the subject.
The method of claim 60, wherein the subject has a cancer expressing PTK7 and/or B7H3.
The method of claim 60 or 61, wherein the cancer is a solid tumor, lung cancer (e.g., non-small cell lung cancer, small cell lung cancer, lung adenocarcinoma, or lung carcinoma) , gastric cancer (e.g., gastric carcinoma) , colorectal cancer, breast cancer, pancreatic cancer, head and neck cancer, ovarian cancer, prostate cancer, thyroid cancer, liver cancer, nasopharynx cancer, brain cancer, bladder cancer, cervical cancer, or oesophageal cancer.
The method of any one of claims 60-62, wherein the subject is a human.
The method of any one of claims 60-63, wherein the method further comprises administering an anti-PD1 antibody to the subject.
The method of any one of claims 60-64, wherein the method further comprises administering a chemotherapy to the subject.
A method of decreasing the rate of tumor growth, the method comprising contacting a tumor cell with an effective amount of a composition comprising the anti-PTK7/B7H3 antibody or antigen-binding fragment thereof of any one of claims 1-28, or the anti-PTK7/B7H3 antibody-drug conjugate of any one of claims 40-48.
A method of killing a tumor cell, the method comprising contacting a tumor cell with an effective amount of a composition comprising the anti-PTK7/B7H3 antibody or antigen-binding fragment thereof of any one of claims 1-28, or the anti-PTK7/B7H3 antibody-drug conjugate of any one of claims 40-48.
A pharmaceutical composition comprising a pharmaceutically acceptable carrier and

(a) the anti-PTK7/B7H3 antibody or antigen-binding fragment thereof of any one of claims 1-28, and/or

(b) the anti-PTK7/B7H3 antibody-drug conjugate of any one of claims 40-48.
An anti-PTK7/B7H3 ADC comprising a therapeutic agent covalently bound to a bispecific antibody or antigen-binding fragment thereof comprising: a first antigen-binding domain that specifically binds to PTK7; and a second antigen-binding domain that specifically binds to B7H3.
The anti-PTK7/B7H3 ADC of any one of claims 40-48 and 69, wherein the drug-to-antibody ratio (DAR) is about 2 to 8.