Antibody-drug conjugates targeting B7-H4 for treatment of cancer

Abstract

Disclosed herein are dosing regimens of antibody-drug conjugates targeting B7-H4 for the treatment of cancer.

Description

[0001] Related Applications

[0002] This application claims priority to and the benefit of U.S. Provisional Application No. 63 / 342,657, filed May 17, 2022, and U.S. Provisional Application No. 63 / 377,437, filed September 28, 2022. The contents of each of these applications are hereby incorporated by reference in their entirety.

[0003] Incorporation by Reference into the Sequence Listing

[0004] The sequence listing XML associated with this application is provided electronically in xml file format and is hereby incorporated by reference into this specification. The name of the xml file containing the sequence listing is MRSN_038_001WO_SeqList_ST26.xml. The xml file is 13,132 bytes, created on May 12, 2023 and electronically submitted via the USPTO Patent Center. Technical Field

[0005] The present disclosure generally relates to dosing regimens for administering antibody-drug conjugates targeting B7-H4 for treating cancer. Background Art

[0006] B7-H4, also known as B7-H4, B7x, B7S1, B7-S1 and VTCN1, is a type I transmembrane protein and a member of the B7 protein superfamily that provides a co-signal that cooperates with the T cell receptor antigen signal. B7-H4 is a negative regulator of T cell function, and ligation of T cells inhibits their growth, cytokine secretion and cytotoxicity. Elimination of B7-H4 in mice does not affect immune cell homeostasis and shows no signs of autoimmunity. The receptor for B7-H4 is unknown and unidentified.

[0007] Human B7-H4 is mapped to chromosome 1 and contains six exons and five introns spanning 66 kb, of which exon 6 is used for alternative splicing to generate two different transcripts. It is a 282 amino acid protein (including the amino-terminal signal sequence), of which 227 amino acids are predicted to be in the extracellular space after cleavage of the amino-terminal signal sequence. B7-H4 contains an Ig-like V domain, an Ig-like C domain, a transmembrane domain, and a short cytoplasmic tail.

[0008] Although B7-H4 expression in healthy tissues is relatively limited at the protein level, B7-H4 is consistently overexpressed in several solid tumors, such as gynecological cancers of the breast, ovary, and endometrium. Expression of B7-H4 in tumors tends to correlate with poor prognosis. The receptor for B7-H4 is unknown, but it is believed to be expressed on T cells. B7-H4 is believed to directly inhibit T cell activity.

[0009] A wide variety of therapeutic modalities are available for the treatment of advanced cancer, including radiation therapy, conventional chemotherapy with cytotoxic anti-tumor agents, hormone therapy (aromatase inhibitors, luteinizing hormone releasing hormone analogs), bisphosphonates, and signal-transduction inhibitors. Unfortunately, however, many patients respond poorly or not at all to any of these treatment modalities. Therefore, there is a need to identify new therapeutic agents that target the biological activity of B7-H4.

[0010] Therefore, there is a need for therapeutics that target the biological activity of B7-H4. Summary of the invention

[0011] The present disclosure provides methods for treating subjects with TNBC, HR+ / HER2- breast cancer, endometrial cancer, ovarian cancer, fallopian tube cancer, or primary peritoneal cancer, or solid tumors, the methods comprising administering about 7.0 mg / m 2 on the first day of treatment and every three weeks (i.e., 21-day cycles) or every four weeks (i.e., 28-day cycles) thereafter. 2 About 90 mg / m 2 The subject is administered an antibody-drug conjugate targeting B7-H4 (XMT-1660) by infusion at a dose of:

[0012]

[0013] in

[0014] id 13 is about 2;

[0015] ii. The antibody binds to B7-H4 and comprises: comprising the amino acid sequence GFIVSRNY (SEQ ID

[0016] NO:2) of the variable heavy chain complementary determining region 1 (CDRH1), comprising the amino acid sequence IYGSGRT (SEQ

[0017] ID NO:3), a variable heavy chain complementary determining region 2 (CDRH2) comprising the amino acid sequence ARDADYGLDV (SEQ ID NO:4), a variable light chain complementary determining region 1 (CDRL1) comprising the amino acid sequence QSVSSSY (SEQ ID NO:5), a variable light chain complementary determining region 2 (CDRL2) comprising the amino acid sequence GAS (SEQ ID NO:6), and a variable light chain complementary determining region 3 (CDRL3) comprising the amino acid sequence QQYGSSPLYT (SEQ ID NO:7).

[0018] iii. The drug is attached to the

[0019] Asparagine of the heavy chain of the antibody;

[0020] iv. ■ is GlcNAc; Δ is Fuc; and □ is GalNAc.

[0021] In some embodiments, GlcNAc refers to N-acetylglucosamine (i.e., β-D-(acetylamino)-2-deoxy-glucopyranose or N-acetyl-D-glucosamine); Fuc refers to fucose (i.e., (2S,3R,4R,5S)-6-methyltetrahydro-2H-pyran-2,3,4,5-tetrol or 6-deoxy-1-galactose); and GalNAc refers to N-acetylgalactosamine (i.e., 2-(acetylamino)-2-deoxy-D-galactose, 2-acetamido-2-deoxy-D-galactose, N-acetylchondrosamine, 2-acetamido-2-deoxy-D-galactopyranose or N-acetyl-D-galactosamine).

[0022] In some embodiments, the antibody-drug conjugate targeting B7-H4 is administered to the subject by intravenous infusion once every 3 weeks (i.e., 21-day cycle) or once every 4 weeks (i.e., 28-day cycle) at a dose of about 6.7 mg / m 2 To about 7.7mg / m 2 , 13.9mg / m 2 To about 14.9mg / m 2 , 21.1mg / m 2 To about 22.1mg / m 2 , 28.2mg / m 2 To about 29.2mg / m 2 , 37.6mg / m 2 To about 38.6mg / m 2 , 50.2mg / m 2 To about 51.2mg / m 2 , 66.9mg / m 2 To about 67.9mg / m 2 or 87.1 mg / m 2 To about 88.1mg / m 2 In some embodiments, the antibody-drug conjugate targeting B7-H4 is administered to the subject by intravenous infusion once every 3 weeks (i.e., 21-day cycle) or once every 4 weeks (i.e., 28-day cycle) at a dose of about 7.2 mg / m 2 , about 14.4mg / m 2 , about 21.6mg / m 2 , about 28.7mg / m 2 , about 38.1mg / m2 , about 50.7mg / m 2 , about 67.4mg / m 2 or about 87.6 mg / m 2 In some embodiments, the conjugate dose is about 7.2 mg / m 2 In some embodiments, the conjugate dose is about 14.4 mg / m 2 In some embodiments, the conjugate dose is about 21.6 mg / m 2 In some embodiments, the conjugate dose is about 28.7 mg / m 2 In some embodiments, the conjugate dose is about 38.1 mg / m 2 In some embodiments, the conjugate dose is about 50.7 mg / m 2 In some embodiments, the conjugate dose is about 67.4 mg / m 2 In some embodiments, the conjugate dose is about 87.6 mg / m 2 .

[0023] In some embodiments, the antibody-drug targeting B7-H4 is administered to the subject until disease progression, death, unacceptable toxicity, or voluntary withdrawal, whichever comes first.

[0024] In some embodiments, the subject with unresectable or recurrent / metastatic TNBC has received at least 2 lines of systemic therapy in the setting of locally advanced or metastatic breast cancer.

[0025] In some embodiments, subjects with HR+ / HER2- breast cancer following CDK 4 / 6 inhibitor and endocrine-based therapy in the setting of advanced or metastatic breast cancer have received at least 1 line of systemic therapy, which must include a CDK4 / 6 inhibitor and endocrine therapy (ET).

[0026] In some embodiments, the subject with endometrial cancer has received at least 1 line of systemic therapy for advanced or metastatic disease that included platinum-based chemotherapy.

[0027] In some embodiments, the subject with ovarian cancer, fallopian tube cancer, or primary peritoneal cancer has received at least 2 lines of systemic therapy for advanced or metastatic disease, which should include platinum-based chemotherapy.

[0028] In some embodiments, the cancer is B7-H4 positive.

[0029] In some embodiments, the subject is a human.

[0030] Other features and advantages of the invention will be apparent from the following detailed description, and from the claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 is a graph showing the anti-tumor efficacy of an antibody-drug conjugate targeting B7-H4 (XMT-1660) (4.68 / 0.150 mg / kg) and a rituximab isotype control conjugate (4.60 / 0.150 mg / kg) (all doses given as antibody / payload) in a patient-derived endometrial cancer xenograft mouse model.

[0032] Figure 2 is a graph showing the anti-tumor efficacy of an antibody-drug conjugate targeting B7-H4 (XMT-1660) (4.61 / 0.150 mg / kg) and a palivizumab isotype control (4.54 / 0.150 mg / kg) (all doses given as antibody / payload) in a patient-derived ovarian cancer xenograft mouse model.

[0033] Figure 3 is a graph showing the anti-tumor efficacy of an antibody-drug conjugate targeting B7-H4 (XMT-1660) (2.30 / 0.075 or 4.60 / 0.150 mg / kg, DAR 5.96) and a palivizumab isotype control ADC (2.27 / 0.075 or 4.54 / 0.150 mg / kg, DAR 5.97) (all doses are given as antibody / payload) in the CTG-1692 patient-derived ovarian cancer model.

[0034] Figure 4 is a graph showing the anti-tumor efficacy of palivizumab isotype control conjugate (1.51 / 0.050 or 4.54 / 0.150 mg / kg), antibody-drug conjugate targeting B7-H4 (XMT-1660) (1.53 / 0.050 or 4.60 / 0.150 mg / kg), anti-PD-1 (10.0 / 0 mg / kg) and XMT-1660+anti-PD-1 (1.53 / 0.050 mg / kg+10.0 / 0 mg / kg) (all doses are given as antibody / payload) in the mBR9013 syngeneic breast cancer mouse model. DETAILED DESCRIPTION

[0035] The present disclosure provides a method for treating a subject with TNBC, HR+ / HER2- breast cancer, endometrial cancer, ovarian cancer, fallopian tube cancer, or primary peritoneal cancer, or a solid tumor thereof, the method comprising administering to the subject an antibody-drug conjugate targeting B7-H4 that specifically binds to the extracellular region of B7-H4 (XMT-1660). Specifically, the present invention provides a dosing regimen for treating cancer or solid tumors expressing B7-H4 by administration as an intravenous infusion. XMT-1660 is an antibody-drug conjugate targeting B7-H4, comprising a B7-H4 modified antibody and two molecules of a fully synthetic macromolecular linker-payload that carries three copies of the microtubule inhibitor auristatin F-hydroxypropylamide (AF-HPA). The fully synthetic macromolecular linker-payload is conjugated to the modified B7-H4-antibody in a site-specific manner, which is achieved by performing glycan reconstruction of the antibody at the asparagine group of the antibody. In some aspects, the asparagine is in the conserved Fc region of the antibody. In some aspects, the asparagine group is N297 according to EU numbering. The average drug to antibody ratio (DAR) of the antibody-drug conjugates targeting B7-H4 is about 6.

[0036] XMT-1660 is administered intravenously to patients with TNBC, HR+ / HER2- breast cancer, endometrial cancer, ovarian cancer, fallopian tube cancer, or primary peritoneal cancer once every 3 weeks (i.e., 21-day cycle) or once every 4 weeks (i.e., 28-day cycle). Therefore, the present invention is characterized by a method for treating TNBC, HR+ / HER2- breast cancer, endometrial cancer, ovarian cancer, fallopian tube cancer, or primary peritoneal cancer by administering the following XMT-1660 infusion doses to subjects (i.e., humans) once every 3 weeks (i.e., 21-day cycle) or once every 4 weeks (i.e., 28-day cycle) in a dose escalation study, wherein the infusion dose of XMT-1660 is 7.2 mg / m 2 , 14.4mg / m 2 , 21.6mg / m 2 , 28.7mg / m 2 , 38.1mg / m 2 , 50.7mg / m 2 , 67.4mg / m 2 or 87.6 mg / m 2 .

[0037] In some embodiments, the subject has a cancer that expresses B7-H4. In some aspects, the subject has a tumor that expresses B7-H4.

[0038] In some embodiments, the subject is identified as having B7-H4 expression. In some embodiments, B7-H4 expression is in the form of a tumor expressing B7-H4. B7-H4 expression is detected by methods known in the art. For example, by RNA expression analysis of B7-H4 transcripts or other genes associated with cancer measured in tumor samples or fluorescence in situ hybridization (FISH) assays, immunohistochemistry (IHC) analysis. Blood-based biomarkers, which can include serum cytokines, circulating immune cells, and circulating tumor cells, can also be used to determine B7-H4 expression levels.

[0039] B7-H4 Antibody

[0040] The present disclosure provides isolated antibodies that bind to B7-H4, which is a type I transmembrane protein found, for example, on the surface of antigen presenting cells (APCs). B7-H4 antibodies include, but are not limited to, humanized antibodies, chimeric antibodies, mouse antibodies, human antibodies, and antibodies comprising the heavy chain and / or light chain CDRs discussed herein.

[0041] In some embodiments, the B7-H4 antibodies of the present disclosure specifically bind to an epitope of a full-length human B7-H4 protein comprising the following amino acid sequence:

[0042] MASLGQILFWSIISIIIILAGAIALIIGFGISGRHSITVTTVASAGNIGEDGILSCTFEPDIKLSDIVIQWLKEGVLGLVHEFKEGKDELSEQDEMFRGRTAVFADQVIVGNASLRLKNVQLTDAGTYKCYIITSKGKGNANL EYKTGAFSMPEVNVDYNASSETLRCEAPRWFPQPTVVWASQVDQGANFSEVSNTSFELNSENVTMKVVSVLYNVTINNTYSCMIENDIAKATGDIKVTESEIKRRSHLQLLNSKASLCVSSFFAISWALLPLSPYLMLK(SEQ ID NO:1).

[0043] In some embodiments, the B7-H4 antibody is a human antibody. In some embodiments, the B7-H4 antibody modulates B7-H4 activity. In some embodiments, the antibody is an antibody that induces an antibody-dependent cellular cytotoxicity (ADCC) reaction in a subject receiving the antibody. In some embodiments, the B7-H4 antibody does not inhibit the T cell inhibitory activity of B7-H4. The B7-H4 antibody of the present disclosure can be, for example, a full-length antibody. Alternatively, the B7-H4 antibody can be an antibody fragment such as a Fab, Fab' or Fab'2 fragment or a single-chain antibody (e.g., scFv). In some embodiments, the antibody is an IgG1 antibody.

[0044] In some embodiments, the B7-H4 antibody comprises a heavy chain variable region and a light chain variable region. In some embodiments, the B7-H4 antibody comprises at least one heavy chain and at least one light chain, the heavy chain comprising a heavy chain variable region and at least a portion of a heavy chain constant region, and the light chain comprising a light chain variable region and at least a portion of a light chain constant region. In some embodiments, the B7-H4 antibody comprises two heavy chains and two light chains, wherein each heavy chain comprises a heavy chain variable region and at least a portion of a heavy chain constant region, wherein each light chain comprises a light chain variable region and at least a portion of a light chain constant region.

[0045] In some embodiments, human B7-H4 antibodies include one or more human constant regions. In some embodiments, the human heavy chain constant region belongs to an isotype selected from IgA, IgG (e.g., IgG1, IgG2, IgG3, or IgG4) and IgD. In some embodiments, the human light chain constant region belongs to an isotype selected from κ and λ. In some embodiments, the human antibodies described herein include human IgG constant regions. In some embodiments, the human antibodies described herein include human IgG4 heavy chain constant regions. In some embodiments, the human antibodies described herein include human IgG4 constant regions and human κ light chains.

[0046] In some embodiments, when the effector function is as expected, a human B7-H4 antibody comprising a human IgG1 heavy chain constant region or a human IgG3 heavy chain constant region is selected. In some embodiments, when the effector function is not as expected, a human B7-H4 antibody comprising a human IgG4 or IgG2 heavy chain constant region is selected.

[0047] In some embodiments, the antibodies of the present disclosure are characterized by a particular functional characteristic or property of the antibody. For example, the antibody specifically binds to human B7-H4. Typically, the antibodies of the present disclosure bind to B7-H4 with high affinity, for example, with an affinity of 1×10 -7 M or lower K D Binding to B7-H4. The anti-B7-H4 antibodies of the present disclosure typically exhibit one or more of the following characteristics:

[0048] (a) 1×10 -7 M or lower K D binds to human B7-H4; and / or

[0049] (b) Binding to human CHO cells transfected with B7-H4 (e.g., human B7-H4).

[0050] In some embodiments, the antibody is expressed at 5×10 -8 M or lower K D Combined with human B7-H4, 2×10 -8 M or lower K D Combined with human B7-H4, 5×10 -9 M or lower K D Combined with human B7-H4, 4×10 -9 M or lower K D Combined with human B7-H4, 3×10 -9 M or lower K D Combined with human B7-H4, 2×10 -9 M or lower K D Combined with human B7-H4 or 1×10 -9 M or lower K D Binds human B7-H4.

[0051] Standard assays for evaluating the ability of antibodies to bind to B7-H4 are known in the art, including, for example, ELISA, Western blot, RIA, and flow cytometry analysis. The binding kinetics (e.g., binding affinity) of the antibodies can also be assessed by standard assays known in the art, such as by ELISA, Scatchard and System analysis to evaluate.

[0052] Potential therapeutic mAbs must not only bind their targets, but must also be free of "developability issues," such as poor stability or high levels of aggregation. We describe guiding values ​​for five metrics that are thought to implicate poor developability: total length of the complementarity determining regions (CDRs), degree and magnitude of surface hydrophobicity, positive and negative charges in the CDRs, and asymmetry of net heavy and light chain surface charge. Guiding cutoffs for each property, derived from values ​​observed in the CST, and a flagging system are proposed to identify noncompliant candidates.

[0053] Provided below are nucleic acid and amino acid sequences of monoclonal B7-H4 antibodies of the present disclosure. The complementarity determining regions (CDRs) of the heavy and light chains are underlined in the amino acid sequences presented below. The amino acids encompassing the complementarity determining regions (CDRs) shown below are numbered according to the IMGT numbering system (see International ImMunoGeneTics information system . Available online: http: / / www.imgt.org / ) definitions.

[0054] B7-H4 variable region

[0055] In some embodiments, a B7-H4 antibody of the present disclosure comprises a light chain variable region comprising or consisting of the amino acid sequence of SEQ ID NO:8.

[0056] B7-H 4_ V L (SEQ ID NO: 8) L The chain comprises or consists of the following amino acid sequence:

[0057] EIVLTQSPGTLSLSPGERATLSCRAS QSVSSSY LAWYQQKPGQAPRLLIY GAS S RATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYC QQYGSSPLYT FGQGTKLEIK (SEQ ID NO: 8).

[0058] The VH chain of B7-H4 (SEQ ID NO: 44) (also referred to herein as XMT-1604 VH) comprises or consists of the following amino acid sequence:

[0059] EVQLVESGGGLIQPGGSLRLSCAAS GFIVSRNY MNWVRQAPGKGLEWVSV IYG DSADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC ARDADYGLD V WGQGTTVTVSS (SEQ ID NO: 9).

[0060] The B7-H4 variable heavy chain is also referred to herein as the XMT-1604 variable heavy chain.

[0061] In some embodiments, the B7-H4 antibody (XMT-1604) comprises the following: a variable heavy chain complementary determining region 1 (CDRH1) comprising the amino acid sequence GFIVSRNY (SEQ ID NO:2), a variable heavy chain complementary determining region 2 (CDRH2) comprising the amino acid sequence IYGSGRT (SEQ ID NO:3), and a variable heavy chain complementary determining region 3 (CDRH3) comprising the amino acid sequence ARDADYGLDV (SEQ ID NO:4).

[0062] In some embodiments, the B7-H4 antibody (XMT-1604) comprises the following: a variable light chain complementary determining region 1 (CDRL1) comprising the amino acid sequence QSVSSSY (SEQ ID NO:5), a variable light chain complementary determining region 2 (CDRL2) comprising the amino acid sequence GAS (SEQ ID NO:6), and a variable light chain complementary determining region 3 (CDRL3) comprising the amino acid sequence QQYGSSPLYT (SEQ ID NO:7).

[0063] In some embodiments, the B7-H4 antibodies of the present disclosure have a light chain constant region comprising or consisting of the following amino acid sequence:

[0064] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 10).

[0065] The B7-H4 light chain constant region (SEQ ID NO: 10) is also referred to herein as B7-H4 LC.

[0066] In some embodiments, the antibody of the present disclosure comprises a light chain comprising or consisting of a light chain variable region amino acid sequence and a light chain constant region amino acid sequence. The antibody light chain (variable region and constant region) of the present disclosure comprises or consists of the amino acid sequence of SEQ ID NO: 12.

[0067] The B7-H4 antibody of the present disclosure has an IgG1 heavy chain constant region comprising or consisting of the following amino acid sequence:

[0068] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG(SEQ ID NO:11). The B7-H4 IgG1 heavy chain constant region (SEQ ID NO: 11) is also referred to herein as B7-H4 HC. In some embodiments, the IgG1 heavy chain constant region comprising or consisting of SEQ ID NO: 11 further comprises one or more amino acids at the N-terminus or C-terminus. In some embodiments, the IgG1 heavy chain constant region comprises a C-terminal lysine.

[0069] In some embodiments, an antibody of the present disclosure comprises a heavy chain set forth in SEQ ID NO: 13, which comprises or consists of a heavy chain variable region amino acid sequence and a heavy chain constant region amino acid sequence.

[0070] In yet another embodiment, the B7-H4 antibodies of the present disclosure comprise heavy and light chain variable regions comprising amino acid sequences homologous to the amino acid sequences of the antibodies described herein, and wherein the antibodies retain the desired functional properties of the anti-B7-H4 antibodies of the present disclosure. For example, the present disclosure provides an isolated monoclonal antibody, or antigen-binding portion thereof, comprising a heavy chain variable region and a light chain variable region, wherein:

[0071] (a) the heavy chain variable region comprises an amino acid sequence that is at least 80% homologous to an amino acid sequence selected from SEQ ID NO: 9;

[0072] (b) the light chain variable region comprises an amino acid sequence that is at least 80% homologous to the amino acid sequence of SEQ ID NO: 8;

[0073] (c) Antibodies were measured at 1×10 -7 M or lower K D binds human B7-H4; and

[0074] (d) Antibody binding to human CHO cells transfected with B7-H4.

[0075] In various embodiments, the antibody can be, for example, a human antibody, a humanized antibody, or a chimeric antibody.

[0076] In various embodiments, the VH and / or VL amino acid sequences can be 85%, 90%, 95%, 96%, 97%, 98% or 99% homologous to the sequences shown above. B7-H4 antibodies having VH and VL regions with high (i.e., 80% or greater) homology to the VH and VL regions of the sequences shown above can be obtained by mutagenizing (e.g., site-directed mutagenesis or PCR-mediated mutagenesis) a nucleic acid molecule encoding the amino acid sequence shown in SEQ ID NO: 9, followed by testing the retained function of the encoded altered antibody using the functional assays described herein (i.e., the functions shown in (c) and (d) above).

[0077] In a preferred embodiment, the heavy chain variable region CDR2 sequence comprises the amino acid sequence of SEQ ID NO:3 and conservative modifications thereof; and the light chain variable region CDR2 sequence comprises the amino acid sequence of SEQ ID NO:6 and conservative modifications thereof. In another preferred embodiment, the heavy chain variable region CDR1 sequence comprises the amino acid sequence of SEQ ID NO:2 and conservative modifications thereof; and the light chain variable region CDR1 sequence comprises the amino acid sequence of SEQ ID NO:5 and conservative modifications thereof. In another preferred embodiment, the heavy chain variable region CDR3 sequence comprises the amino acid sequence of SEQ ID NO:4 and conservative modifications thereof; and the light chain variable region CDR3 sequence comprises the amino acid sequence of SEQ ID NO:7 and conservative modifications thereof.

[0078] In various embodiments, the antibody can be, for example, a human antibody, a humanized antibody, or a chimeric antibody.

[0079] In some embodiments, the antibody binds to the same epitope of human B7-H4 as any of the B7-H4 monoclonal antibodies of the disclosure (i.e., an antibody that has the ability to cross-compete with any of the monoclonal antibodies of the disclosure for binding to B7-H4).

[0080] Accordingly, another embodiment of the present disclosure is directed to an isolated monoclonal antibody, or antigen-binding portion thereof, comprising a heavy chain variable region comprising CDR1, CDR2, and CDR3 sequences comprising the amino acid sequence of SEQ ID NO: 9, respectively, and a light chain variable region comprising CDR1, CDR2, and CDR3 sequences comprising the amino acid sequence of SEQ ID NO: 5, 6, and 7, respectively.

[0081] Thus, in another embodiment, the disclosure provides an isolated anti-B7-H4 monoclonal antibody, or antigen-binding portion thereof, comprising a heavy chain variable region comprising: (a) a VH CDR1 region comprising: an amino acid sequence comprising SEQ ID NO:2; or an amino acid sequence having one, two, three, four or five amino acid substitutions, deletions or additions compared to SEQ ID NO:2; (b) a VH CDR2 region comprising: an amino acid sequence comprising SEQ ID NO:3; or an amino acid sequence having one, two, three, four or five amino acid substitutions, deletions or additions compared to SEQ ID NO:3; (c) a VH CDR3 region comprising: an amino acid sequence selected from SEQ ID NO:4 or an amino acid sequence having one, two, three, four or five amino acid substitutions, deletions or additions compared to SEQ ID NO:4.

[0082] In some embodiments, the antibodies disclosed herein contain a heavy chain having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO:13, and a light chain having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO:12.

[0083] In some embodiments, the antibodies disclosed herein contain a combination of heavy and light chain amino acid sequences comprising a heavy chain amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of SEQ ID NO:13 and a light chain amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of SEQ ID NO:12.

[0084] In some embodiments, the antibodies disclosed herein comprise a heavy chain amino acid sequence and a light chain amino acid sequence, wherein the heavy chain amino acid sequence is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of SEQ ID NO:13, and the light chain amino acid sequence is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of SEQ ID NO:12.

[0085] In some embodiments, the antibodies disclosed herein comprise a heavy chain having an amino acid sequence consisting of SEQ ID NO:13 and a light chain having an amino acid sequence of SEQ ID NO:12.

[0086] In some embodiments, the antibodies disclosed herein contain a combination of heavy chain and light chain amino acid sequences of the heavy chain amino acid sequence of SEQ ID NO:13 and the light chain amino acid sequence of SEQ ID NO:12.

[0087] In some embodiments, the antibodies disclosed herein contain a heavy chain amino acid sequence of SEQ ID NO:13 and a light chain amino acid sequence of SEQ ID NO:12.

[0088] The antibodies disclosed herein comprise a heavy chain variable region having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO:9 and a light chain variable region having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to a sequence consisting of SEQ ID NO:8.

[0089] In some embodiments, the three heavy chain CDRs of the antibodies disclosed herein include: a heavy chain complementary determining region 1 (CDRH1) comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to a sequence comprising SEQ ID NO: 2; a heavy chain complementary determining region 2 (CDRH2) comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to a sequence comprising SEQ ID NO: 3; and a heavy chain complementary determining region 3 (CDRH3) comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to a sequence comprising SEQ ID NO: 4; and NO:13 has a heavy chain amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical.

[0090] The three light chain CDRs of the antibodies disclosed herein include: a light chain complementary determining region 1 (CDRL1), which includes an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to a sequence comprising SEQ ID NO:5; a light chain complementary determining region 2 (CDRL2), which includes an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to a sequence comprising SEQ ID NO:6; and a light chain complementary determining region 3 (CDRL3), which includes an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to a sequence comprising SEQ ID NO:7.

[0091] The antibody comprises a combination of heavy chain CDR and light chain CDR sequences comprising: CDRH1 comprising an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to a sequence comprising SEQ ID NO:2; CDRH2 comprising an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to a sequence comprising SEQ ID NO:3; CDRH3 comprising an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to a sequence comprising SEQ ID NO:4; CDRL1 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:5; NO:5 having an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the sequence of SEQ ID NO:5; CDRL2, which includes an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the sequence of SEQ ID NO:6; and CDRL3, which includes an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO:7; and a heavy chain amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of SEQ ID NO:45.

[0092] The three heavy chain CDRs of the antibodies disclosed herein include: CDRH1, which includes an amino acid sequence selected from SEQ ID NO: 2; CDRH2, which includes an amino acid sequence comprising SEQ ID NO: 3; and CDRH3, which includes an amino acid sequence comprising SEQ ID NO: 4; and a heavy chain amino acid sequence comprising SEQ ID NO: 13.

[0093] The three light chain CDRs of the antibodies disclosed herein include: CDRL1, which has an amino acid sequence of SEQ ID NO: 5; CDRL2, which has an amino acid sequence of SEQ ID NO: 6; and CDRL3, which has an amino acid sequence of SEQ ID NO: 7. The antibodies disclosed herein include a combination of heavy chain CDR and light chain CDR sequences, which include: CDHR1, which includes a selected amino acid sequence comprising SEQ ID NO: 2; CDRH2, which includes an amino acid sequence comprising SEQ ID NO: 3; CDRH3, which includes an amino acid sequence comprising SEQ ID NO: 4; CDRL1, which has an amino acid sequence of SEQ ID NO: 5; CDRL2, which has an amino acid sequence of SEQ ID NO: 6; and CDRL3, which has an amino acid sequence of SEQ ID NO: 7; and a heavy chain amino acid sequence of SEQ ID NO: 13. The antibodies disclosed herein contain a combination of heavy chain complementary determining region and light chain complementary determining region amino acid sequences selected from (i) the CDRH1 amino acid sequence of SEQ ID NO:2, the CDRH2 amino acid sequence of SEQ ID NO:3, the CDRH3 amino acid sequence of SEQ ID NO:4, the CDRL1 amino acid sequence of SEQ ID NO:5, the CDRL2 amino acid sequence of SEQ ID NO:6, the CDRL3 amino acid sequence of SEQ ID NO:7 and the heavy chain amino acid sequence of SEQ ID NO:13.

[0094] In some embodiments, the antibodies disclosed herein comprise a CDRH1 amino acid sequence of SEQ ID NO:2, a CDRH2 amino acid sequence of SEQ ID NO:3, a CDRH3 amino acid sequence of SEQ ID NO:4, a CDRL1 amino acid sequence of SEQ ID NO:5, a CDRL2 amino acid sequence of SEQ ID NO:6, a CDRL3 amino acid sequence of SEQ ID NO:7, and a heavy chain amino acid sequence of SEQ ID NO:13.

[0095] Modified B7-H4 Antibody

[0096] In some embodiments, the B7-H4 antibody is a modified B7-H4 antibody. As used herein, the term "modified B7-H4 antibody" can be used to describe a B7-H4 antibody of the present disclosure having a modification including a mutation, deletion, or substitution. As used herein, the term "modified B7-H4 antibody" can be used to describe a B7-H4 antibody having a modification to the amino acids of the antibody, the modification including the covalent attachment of a chemical moiety to the amino acid side. In some embodiments, the "modified B7-H4 antibody" can have a sugar or sugar derivative moiety covalently attached to the amino acids of the antibody. In some embodiments, the "modified B7-H4 antibody" can have a sugar or sugar derivative moiety replaced by a different sugar or sugar derivative moiety.

[0097] In some embodiments of the modified B7-H4 antibodies, * indicates grounded or indirectly attached to the rest of the modified B7-H4 antibody. In some embodiments, S" is a sugar or a derivatized sugar. In some embodiments, A" is a functional group capable of forming a covalent bond with a functional group of a macromolecular linker-payload (i.e., a drug or linker-drug moiety).

[0098] In some embodiments, the modified B7-H4 antibody comprises a sugar-derivative moiety of *-S"-A" prior to conjugation.

[0099] In some embodiments, the modified B7-H4 antibody comprises an asparagine group in the constant region of the antibody. The constant region of IgG antibodies is very conservative, and each amino acid can be determined by EU numbering. See Edelman, GM et al., Proc.Natl.Acad.USA, 63, 78-85 (1969). In some aspects, the asparagine group is in the 290-305 region (e.g., at N297; EU numbering). Asparagine 297 is found in the conserved Fc region of the heavy chain of IgG antibodies. In some embodiments, the sugar-derivative portion is directly or indirectly attached to the asparagine group (e.g., at N297).

[0100] In some embodiments, an asparagine in the B7-H4 antibody heavy chain constant region shown in SEQ ID NO: 11 is modified. In some embodiments, the asparagine to be modified (N297 according to EU numbering) corresponds to position 180 (N180) of SEQ ID NO: 11. In some aspects, the macromolecular linker-payload (e.g., a drug or linker-drug moiety) of the B7-H4 ADC is attached to N180 of SEQ NO: 11.

[0101] In some embodiments, an asparagine of the B7-H4 antibody heavy chain shown in SEQ ID NO: 13 is modified. In some embodiments, the asparagine to be modified (N297 according to EU numbering) corresponds to position 296 (N296) of SEQ ID NO: 13. In some aspects, the macromolecular linker-payload (e.g., a drug or linker-drug moiety) of the B7-H4 ADC is attached to N296 of SEQ NO: 13.

[0102] In some embodiments, the modified B7-H4 antibody comprises a modified GlcNAc moiety prior to conjugation, *-GlcNAc-S"-A", wherein GlcNAc is N-acetylglucosamine.

[0103] In some embodiments, the modified GlcNAc moiety is linked to the remainder of the modified B7-H4 antibody via the C1 position of the GlcNAc. In some embodiments, the modified GlcNAc moiety further comprises fucose.

[0104] In some embodiments, the modified GlcNAc moiety is attached directly or indirectly to the asparagine group (eg, at N297).

[0105] In some embodiments, the modified B7-H4 antibody is conjugated to the drug via a linker moiety (i.e., a macromolecular linker-payload or linker-drug moiety) via a covalent bond formed between A" and a functional group of the linker-drug moiety.

[0106] In some embodiments, the modified B7-H4 antibody of the present disclosure is obtained by a method comprising:

[0107] (a) contacting a glycoprotein comprising a B7-H4 antibody and a core-GlcNAc portion (e.g., a B7-H4 antibody glycan) with an endoglycosidase, thereby forming an intermediate antibody comprising the antibody and a terminal-GlcNAc portion, and optionally the terminal-GlcNAc portion further comprises fucose; and

[0108] (b) contacting the intermediate antibody with a compound having the structure P″-S″-A″ in the presence of a glycosyltransferase, thereby forming a modified B7-H4 antibody comprising the antibody and a modified GlcNAc moiety *-GlcNAc-S″-A″, and optionally the modified GlcNAc moiety is attached to the remainder of the modified B7-H4 antibody at the C1 position of the GlcNAc; wherein

[0109] GlcNAc is N-acetylglucosamine;

[0110] S” is a sugar or a derived sugar;

[0111] A" is azido, keto or alkynyl; and

[0112] P" is uridine diphosphate (UDP), guanosine diphosphate (GDP), or cytidine diphosphate (CDP).

[0113] In some embodiments, steps (a) and (b) are performed sequentially. In some embodiments, steps (a) and (b) are performed simultaneously.

[0114] In some embodiments, the B7-H4 antibody is an IgG1 antibody.

[0115] In some embodiments, the antibody is a full length antibody and the antibody glycan comprises one or more core-GlcNAc moieties.

[0116] In some embodiments, the antibody is a full-length antibody and the antibody glycan comprises one or more core-GlcNAc moieties attached to each heavy chain of the antibody.

[0117] In some embodiments, the core-GlcNAc moiety further comprises fucose.

[0118] In some embodiments, the antibody is a full length antibody and the antibody glycan comprises two or more core-GlcNAc moieties attached to the full length antibody.

[0119] In some embodiments, the antibody is a full length antibody and the antibody glycan comprises two core-GlcNAc moieties attached to the full length antibody.

[0120] In some embodiments, at least one of the two or more core-GlcNAc moieties further comprises fucose.

[0121] In some embodiments, the two or more core-GlcNAc moieties each further comprise fucose.

[0122] In some embodiments, the Core-GlcNAc moiety is attached to a position of the antibody, wherein the Core-GlcNAc moiety does not substantially hinder the antigen binding site of the antibody.

[0123] In some embodiments, the core-GlcNAc portion is connected to the Fc fragment of the antibody. In some embodiments, the core-GlcNAc portion is connected to the CH domain. In some embodiments, the core-GlcNAc portion is connected to the Fab or Fc fragment of the antibody. In some embodiments, the core-GlcNAc portion is connected to the antibody via the N-glycosidic bonding of the amide nitrogen atom in the side chain of the asparagine amino acid of the antibody. In some embodiments, the core-GlcNAc portion is connected to the native N-glycosylation site of the antibody.

[0124] In some embodiments, the antibody is an IgG antibody and the core-GlcNAc moiety is linked to the native N-glycosylation site of the IgG.

[0125] In some embodiments, the antibody is an IgG antibody, and the core-GlcNAc portion is linked to a native N-glycosylation site of IgG (e.g., N297 N-glycosylation site of IgG, EU numbering). In some embodiments, the N297 N-glycosylation site is present in the conserved Fc region of the heavy chain of an IgG antibody at an asparagine in the 290-305 region (e.g., at N297).

[0126] In some embodiments, the intermediate antibody is an antibody of formula (XXII):

[0127] in:

[0128] Ab is the B7-H4 antibody; GlcNAc is N-acetylglucosamine; Fuc is fucose; u3 is 0 or 1; and u4 is an integer ranging from 1 to 16.

[0129] In some embodiments, u4 is an integer in the range of 1 to 10. In some embodiments, u4 is an integer in the range of 1 to 4. In some embodiments, u4 is 1, 2, 3, 4, 5, 6, 7, or 8. In some embodiments, u4 is 1, 2, 3, 4, 5, or 6. In some embodiments, u4 is 1, 2, 3, or 4. In some embodiments, u4 is 2 or 4. In some embodiments, u4 is 1 or 2. In some embodiments, u4 is 1. In some embodiments, u4 is 2.

[0130] In some embodiments, the antibody comprises one Core-GlcNAc moiety (eg, u4 is 1). In some embodiments, the antibody comprises two Core-GlcNAc moieties (eg, u4 is 2).

[0131] In some embodiments, the modified B7-H4 antibody is obtained by the method outlined in Scheme 1. As shown below, an intermediate antibody of Formula (XXIII) comprising a terminal-GlcNAc moiety is contacted with a compound having the structure P"-S"-A" in the presence of a glycosyltransferase to provide a modified B7-H4 antibody comprising a modified GlcNAc moiety (e.g., a modified B7-H4 antibody of Formula (XXIIIa)).

[0132] In some embodiments, a modified B7-H4 antibody is obtained by contacting an intermediate antibody of formula (XXIV) comprising two terminal -GlcNAc moieties with a compound having the structure P"-S"-A" in the presence of a glycosyltransferase, providing a modified B7-H4 antibody comprising two modified GlcNAc moieties (e.g., a modified B7-H4 antibody of formula (XXIVa)).

[0133] Solution 1

[0134]

[0135] wherein u3, Ab, S", A" and P" are as defined herein.

[0136] In some embodiments, the antibody glycan to be modified in the methods according to the present disclosure comprises a glycan comprising a core-GlcNAc moiety, i.e., a GlcNAc moiety present at the non-reducing end of the glycan. In some embodiments, the glycan comprises one or more sugar moieties and may be linear or branched.

[0137] In some embodiments, upon reaction with an endoglycosidase, an intermediate antibody comprising a terminal GlcNAc moiety (eg, an intermediate antibody of formula (XXIII) or (XXIV)) may be formed.

[0138] In some embodiments, step (a) of the method (deglycosylation or trimming) is wherein a mixture of antibody glycoforms G2F, GIF, G0F, G2, Gl, G0 and M5 is converted into an intermediate antibody comprising a terminal GlcNAc moiety, wherein the terminal GlcNAc moiety optionally comprises fucose (e.g., u3 is 0 or 1).

[0139] In some embodiments, the endoglycosidase is Endo S or Endo SH or a combination thereof. In some embodiments, the endoglycosidase is Endo SH.

[0140] In some embodiments, step (b) of the method (formation of a modified B7-H4 antibody), wherein the intermediate antibody comprises a monoclonal antibody (mAb) and a terminal GlcNAc moiety (which optionally comprises fucose (e.g., u3 is 0 or 1)) on each heavy chain of the monoclonal antibody (mAb).

[0141] In some embodiments, the P"-S"-A" compound is GalNAz-UDP (e.g., 4-AzGalNAc-UDP). In some embodiments, the terminal -GlcNAc moiety is *-GlcNAc-GalNAz or *-GlcNAc(Fuc)-GalNAz, where * indicates attachment to the remainder of the modified B7-H4 antibody.

[0142] In some embodiments, the deglycosylation / trimming step and the step of forming the modified B7-H4 antibody are performed sequentially.

[0143] In some embodiments, the deglycosylation / trimming step and the step of forming the modified B7-H4 antibody are performed simultaneously.

[0144] In some embodiments, the method of preparing a modified B7-H4 antibody is performed in a suitable buffer solution, such as a buffered saline (e.g., phosphate buffered saline, Tris buffered saline), citrate, HEPES, Tris, and glycine. In some embodiments, the buffer solution is phosphate buffered saline (PBS) or Tris buffered saline. In some embodiments, the buffer solution is phosphate buffered saline (PBS).

[0145] In some embodiments, the method is carried out at a temperature ranging from about 4°C to about 50°C. In some embodiments, the method is carried out at a temperature ranging from about 10°C to about 45°C. In some embodiments, the method is carried out at a temperature ranging from about 20°C to about 40°C. In some embodiments, the method is carried out at a temperature ranging from about 30°C to about 37°C. In some embodiments, the method is carried out at a temperature of about 30°C. In some embodiments, the method is carried out at a temperature of 30°C.

[0146] In some embodiments, the method is performed at a pH ranging from about 5 to about 9, such as about 5.5 to about 8.5, about 6 to about 8, or about 7 to about 8. In some embodiments, the method is performed at a pH of about 7.4.

[0147] In some embodiments, a method of preparing a modified B7-H4 antibody comprises:

[0148] (a) contacting a glycoprotein (e.g., a B7-H4 antibody glycan) comprising a B7-H4 antibody and a core-GlcNAc moiety linked to position N297 (according to EU numbering) of the antibody with an endoglycosidase Endo SH, thereby forming an intermediate antibody comprising a terminal GlcNAc moiety; and

[0149] (b) contacting the intermediate antibody with 4-AzGalNAc-UDP in the presence of β-(l,4)-GalNAcT enzyme to form a modified B7-H4 antibody comprising a modified GlcNAc portion;

[0150] Wherein steps (a) and (b) are carried out simultaneously.

[0151] In some embodiments, the endoglycosidase is Endo SH, a fusion between two endoglycosidases, Endo S and EndoH, connected by a Gly-rich spacer, comprising an internal 6xHis tag resulting in a total molecular weight of 139 kDa.

[0152] In some embodiments, the β-(1,4)-GalNAcT enzyme comprises an N-terminal 6xHis tag and has a total molecular weight of 45.7 kDa. In some embodiments, the β-(1,4)-GalNAcT enzyme comprising an N-terminal 6xHis tag is derived from Trichopulsia ni.

[0153] In some embodiments, the method is performed in PBS buffer at a pH of about 7.4 and at a temperature of about 30°C.

[0154] Antibody-drug conjugates targeting B7-H4

[0155] The present invention relates to therapies involving immunoconjugates comprising an antibody conjugated via site-specific conjugation to a cytotoxic agent, such as a toxin (eg, an enzymatically active toxin of bacterial, fungal, plant or animal origin, or fragments thereof).

[0156] The conjugates described herein are antibody-drug conjugates targeting B7-H4, comprising a B7-H4 modified antibody and two molecules of a fully synthetic macromolecular linker-payload carrying three copies of the microtubule inhibitor auristatin F-hydroxypropylamide (AF-HPA). The fully synthetic macromolecular linker-payload is conjugated to the modified B7-H4 antibody in a site-specific manner, which is achieved by glycan remodeling of the antibody at the asparagine group at position 297 of the antibody according to EU numbering.

[0157] The average drug-to-antibody ratio (DAR) of the antibody-drug conjugates targeting B7-H4 is about 6. In some embodiments, the DAR of the antibody-drug conjugates targeting B7-H4 is about 2 to about 8. In some embodiments, the DAR of the antibody-drug conjugates targeting B7-H4 is about 5 to about 7. In some embodiments, the DAR of the antibody-drug conjugates targeting B7-H4 is about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, or about 6.5, or any value therebetween.

[0158] In some embodiments, the B7-H4 antibody-drug conjugate is a conjugate of formula (I):

[0159]

[0160] in

[0161] id 13 It is about 2;

[0162] ii. the antibody binds to B7-H4 and comprises: a variable heavy chain complementary determining region 1 (CDRH1) comprising the amino acid sequence of GFIVSRNY (SEQ ID NO:2), a variable heavy chain complementary determining region 2 (CDRH2) comprising the amino acid sequence of IYGSGRT (SEQ ID NO:3), a variable heavy chain complementary determining region 3 (CDRH3) comprising the amino acid sequence of ARDADYGLDV (SEQ ID NO:4), a variable light chain complementary determining region 1 (CDRL1) comprising the amino acid sequence of QSVSSSY (SEQ ID NO:5), a variable light chain complementary determining region 2 (CDRL2) comprising the amino acid sequence of GAS (SEQ ID NO:6), and a variable light chain complementary determining region 3 (CDRL3) comprising the amino acid sequence of QQYGSSPLYT (SEQ ID NO:7);

[0163] iii. The drug is attached to the

[0164] Antibody;

[0165] iv. ■ is GlcNAc; Δ is Fuc; and □ is GalNAc.

[0166] In some embodiments, d 13 It can be from about 1 to about 3. In some embodiments, d 13 It can be about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, about 2.0, about 2.1, about 2.2, about 2.3, about 2.4 or about 2.5.

[0167] GlcNAc refers to N-acetylglucosamine (ie, β-D-(acetylamino)-2-deoxy-glucopyranose or N-acetyl-D-glucosamine).

[0168] Fuc refers to fucose (ie, (2S,3R,4R,5S)-6-methyltetrahydro-2H-pyran-2,3,4,5-tetrol or 6-deoxy-l-galactose).

[0169] GalNAc refers to N-acetylgalactosamine (ie, 2-(acetylamino)-2-deoxy-D-galactose, 2-acetamido-2-deoxy-D-galactose, N-acetylchondrosamine, 2-acetamido-2-deoxy-D-galactopyranose, or N-acetyl-D-galactosamine).

[0170] In some embodiments, the GlcNAc is bonded to the conjugate through the reactive moiety of the GlcNAc.

[0171] In some embodiments, Fuc is bonded to the conjugate via a reactive moiety of Fuc.

[0172] In some embodiments, the GalNAc is bonded to the conjugate through a reactive moiety of the GalNAc.

[0173] In some embodiments, the drug is attached to the antibody at position N180 of SEQ NO: 11, the position corresponding to N297 (as numbered by EU numbering).

[0174] In some embodiments, the drug is attached to the antibody at position N296 of SEQ NO: 13 (the position corresponding to N297 as numbered by EU numbering).

[0175] In some embodiments, a method of preparing a modified B7-H4 antibody comprises:

[0176] (a) contacting a glycoprotein (e.g., a B7-H4 antibody glycan) comprising a B7-H4 antibody and a core-GlcNAc moiety linked to position N297 (according to EU numbering) of the antibody with an endoglycosidase Endo SH, thereby forming an intermediate antibody comprising a terminal GlcNAc moiety; and

[0177] (b) contacting the intermediate antibody with 4-AzGalNAc-UDP in the presence of β-(l,4)-GalNAcT enzyme to form a modified B7-H4 antibody comprising a modified GlcNAc portion;

[0178] Wherein steps (a) and (b) are carried out simultaneously.

[0179] In some embodiments, the endoglycosidase is Endo SH, a fusion between two endoglycosidases, Endo S and EndoH, connected by a Gly-rich spacer, comprising an internal 6xHis tag resulting in a total molecular weight of 139 kDa.

[0180] In some embodiments, the β-(1,4)-GalNAcT enzyme comprises an N-terminal 6xHis tag and has a total molecular weight of 45.7 kDa. In some embodiments, the β-(1,4)-GalNAcT enzyme comprising an N-terminal 6xHis tag is derived from Trichoplusia ni.

[0181] In some embodiments, the method is performed in PBS buffer at a pH of about 7.4 and at a temperature of about 30°C.

[0182] B7-H4 antibody-drug conjugates (e.g., XMT-1660) suitable for use in the methods disclosed herein can be produced and purified by well-known techniques, such as WO 2018098269 and US 17 / 568,378, each of which is incorporated herein by reference in its entirety.

[0183] In some embodiments, the B7-H4 antibody-drug conjugate of formula (I) is a conjugate of formula (Ia):

[0184]

[0185] wherein □ is GalNAc; and

[0186] d 13 It is about 2.

[0187] In some embodiments, the B7-H4 antibody-drug conjugate of formula (I) is a conjugate of formula (Ib):

[0188]

[0189] in is GlcNAc; and

[0190] d 13 It is about 2.

[0191] In some embodiments, the B7-H4 antibody-drug conjugate of formula (I) is a conjugate of formula (Ic):

[0192]

[0193] where Δ is Fuc; and

[0194] d 13 It is about 2.

[0195] In some embodiments, the B7-H4 antibody-drug conjugate is a conjugate of Formula (IA):

[0196]

[0197] Dosage and Administration

[0198] The cancer therapies provided herein containing antibody-drug conjugates targeting B7-H4 are administered in an amount sufficient to exert a therapeutically useful effect. Typically, the active agent is administered in an amount that does not cause undesirable side effects in the patient being treated or in an amount that minimizes or reduces observed side effects. Cancers expressing B7-H4 include, for example, breast cancer, endometrial cancer, ovarian cancer, non-small cell lung cancer (e.g., squamous cell carcinoma), pancreatic cancer, thyroid cancer, kidney cancer (e.g., renal cell carcinoma), bladder cancer (e.g., urothelial cell carcinoma), colon cancer, head and neck cancer, small cell lung cancer, gastric cancer, melanoma, bile duct cancer, uterine cancer, and cholangiocarcinoma.

[0199] It should be understood that the precise dosage and duration of treatment are functions of the tissue or tumor being treated, and can be determined empirically using known test protocols or by extrapolation from in vivo or in vitro test data, and / or can be determined from known dosing regimens of specific agents. It should be noted that concentrations and dosage values ​​can also vary with the age of the individual being treated, the individual's weight, route of administration, and / or the extent or severity of the disease, as well as other factors considered within the level of a skilled medical practitioner. In general, the dosage regimen is selected to limit toxicity. It should be noted that the attending physician will know how to terminate, interrupt or adjust therapy and when to terminate, interrupt or adjust therapy to reduce the dose, due to toxicity or dysfunction of the bone marrow, liver or kidney or other tissues. On the contrary, the attending physician will also know how to adjust the treatment to a higher level and when to adjust the treatment to a higher level (excluding toxic side effects) if the clinical response is insufficient. It should be further understood that for any particular subject, the specific dosage regimen should be adjusted over time according to the professional judgment of the personnel administering or supervising the administration of the preparation, and the concentration ranges shown herein are exemplary only and are not intended to limit their scope. For example, an antibody-drug conjugate targeting B7-H4 is administered in a therapeutically effective amount to reduce tumor volume.

[0200] Antibody-drug conjugates targeting B7-H4 (eg, XMT-1660) are administered to patients with a variety of solid tumors, including breast cancer, ovarian cancer, and endometrial cancer, in amounts sufficient to exert a therapeutically useful effect.

[0201] In some embodiments, the tumor or cancer is positive for B7-H4 expression.

[0202] In some embodiments, the subject suffers from triple negative breast cancer (TNBC). In some embodiments, the subject suffers from HR+ / HER2- breast cancer. In some embodiments, the subject suffers from endometrial cancer. In other embodiments, the subject suffers from ovarian cancer, fallopian tube cancer, or primary peritoneal cancer.

[0203] In some embodiments, the subject has unresectable or recurrent / metastatic TNBC after standard chemotherapy. In some embodiments, the subject with TNBC has received at least 2 lines of systemic therapy in the context of locally advanced or metastatic breast cancer. In some embodiments, the patient has a histologically or cytologically confirmed diagnosis of breast cancer with evidence of metastatic or locally advanced disease. In some embodiments, based on local testing of the most recent tumor biopsy, the patient has a tumor that is estrogen receptor (ER) negative, progesterone receptor (PR) negative, and HER2 negative. In some embodiments, the patient has received 1-3 lines of chemotherapy for a metastatic background. In some embodiments, patients with BRCA mutations have received prior treatment with PARP inhibitors.

[0204] In some embodiments, the subject has HR+ / HER2- breast cancer after CDK 4 / 6 inhibitor and endocrine-based therapy. In some embodiments, in the context of advanced or metastatic breast cancer, subjects with HR+ / HER2- breast cancer have received at least 1 line of systemic therapy, which must have included CDK 4 / 6 inhibitors and endocrine therapy (ET). In some embodiments, subjects with HR+ / HER2- breast cancer have a histologically or cytologically confirmed diagnosis of breast cancer, with evidence of metastatic or locally advanced disease. In some embodiments, based on local testing of the most recent tumor biopsy, the subject has ER-positive and / or PR-positive tumors and HER2-negative tumors.

[0205] In some embodiments, the subject with endometrial cancer has received at least 1 line of systemic therapy for advanced or metastatic disease, including platinum-based chemotherapy. In some embodiments, the subject has a diagnosis of endometrial cancer confirmed by histology or cytology, with evidence of metastatic or locally advanced disease, the subject does not have satisfactory treatment options and experimental therapy is suitable for the subject. In some embodiments, the subject has received at least 1 line of platinum-doublet chemotherapy for recurrent or metastatic cancer and no more than 3 lines of systemic therapy, excluding hormone therapy. In some embodiments, the subject with endometrial cancer must not have endometrial sarcoma or carcinosarcoma.

[0206] In some embodiments, subjects with ovarian cancer, fallopian tube cancer or primary peritoneal cancer have received at least 2 lines of systemic therapy for advanced or metastatic disease, which should include platinum-based chemotherapy. In some embodiments, the subject has a histological diagnosis of high-grade serous ovarian cancer, including metastatic or recurrent fallopian tube cancer or primary peritoneal cancer. In some embodiments, the subject must have platinum-resistant recurrent disease, which is defined as completing 4 or more cycles of platinum-based therapy and having radiographic progression within 6 months of the last platinum-based therapy. In some embodiments, subjects who have received the first line of chemotherapy must have achieved complete or partial remission for the therapy, and their disease progresses between 3 to 6 months after the last dose of platinum in the first-line background. In some embodiments, subjects who have received 2 to 4 lines of chemotherapy in advance have disease progression within less than or equal to 6 months after the last dose of platinum in the most recent chemotherapy. In some embodiments, the subject has received 1 to 4 prior lines of systemic therapy for ovarian cancer, including at least 1 prior line of a platinum-containing regimen.

[0207] In some embodiments, an antibody-drug conjugate targeting B7-H4 (e.g., XMT-1660) is administered by infusion to subjects with TNBC, HR+ / HER2- breast cancer, endometrial cancer, ovarian cancer, fallopian tube cancer, or primary peritoneal cancer in a dose amount based on body surface area (BSA). The BSA-adjusted dose is calculated using the Mosteller formula when possible. The starting dose is calculated based on height and weight collected within 14 days of the first dose (which can be collected on the day of the first dose). For weight changes of ≥5% from the most recent dose calculation, dose calculations in subsequent cycles are performed. Dose adjustments are made based on subsequent weight measurements. Obtain additional weight measurements and confirmed or changed BSA before dosing in the second cycle and every 2 cycles thereafter.

[0208] In some embodiments, the antibody-drug conjugate targeting B7-H4 (e.g., XMT-1660) is administered via infusion. The method of infusion can include any method known in the art for infusing a therapeutic agent into a subject. In some embodiments, the infusion is an intravenous (IV) infusion.

[0209] In some embodiments, the infusion of the antibody-drug conjugate targeting B7-H4 (e.g., XMT-1660) occurs over a duration of at least 1 minute, at least 5 minutes, at least 10 minutes, at least 15 minutes, at least 20 minutes, at least 25 minutes, at least 30 minutes, at least 35 minutes, at least 45 minutes, at least 50 minutes, at least 55 minutes, at least 60 minutes, at least 65 minutes, at least 70 minutes, at least 75 minutes, at least 80 minutes, at least 85 minutes, at least 90 minutes, at least 95 minutes, at least 100 minutes, at least 105 minutes, at least 110 minutes, at least 115 minutes, at least 120 minutes, or any number of minutes therebetween. In some embodiments, the duration of the infusion can vary from the first infusion to the second and subsequent infusions.

[0210] In some embodiments, for each subject, the initial dose of the antibody-drug conjugate targeting B7-H4 (eg, XMT-1660) is administered over 90±10 minutes. If no infusion-related reactions occur, all subsequent doses may be administered over 60±10 minutes.

[0211] In some embodiments, about 5 mg / m 2 About 100 mg / m 2 An antibody-drug conjugate targeting B7-H4 (e.g., XMT-1660) is administered by infusion to subjects with TNBC, HR+ / HER2- breast cancer, endometrial cancer, ovarian cancer, fallopian tube cancer, or primary peritoneal cancer in a dosage amount of

[0212] In some embodiments, the dose of the antibody-drug conjugate targeting B7-H4 is about 5 mg / m 2 About 50 mg / m 2 or about 50 mg / m 2 About 100 mg / m 2 .

[0213] In some embodiments, the dose is about 5 mg / m 2 About 10 mg / m 2 , about 10mg / m 2 About 15 mg / m 2 , about 15mg / m 2 About 20 mg / m 2 , about 20mg / m 2 About 25 mg / m 2 , about 25mg / m 2 About 30 mg / m 2 , about 30mg / m 2 To about 35mg / m 2 , about 35mg / m 2Up to about 40 mg / m 2 , about 40mg / m 2 Up to about 45 mg / m 2 , about 45mg / m 2 About 50 mg / m 2 , about 50mg / m 2 To about 55mg / m 2 , about 55mg / m 2 About 60 mg / m 2 , about 60mg / m 2 Up to about 65 mg / m 2 , about 65mg / m 2 Up to about 70 mg / m 2 , about 75mg / m 2 About 80 mg / m 2 , about 85mg / m 2 About 90 mg / m 2 , about 90mg / m 2 To about 95mg / m 2 or about 95 mg / m 2 About 100 mg / m 2 .

[0214] In some embodiments, the dose of the antibody-drug conjugate targeting B7-H4 is about 6.0 mg / m 2 To about 8.0mg / m 2 In some embodiments, the dose of the antibody-drug conjugate targeting B7-H4 is about 6.7 mg / m 2 To about 7.7mg / m 2 In some aspects, the dose is about 6.0 mg / m 2 , about 6.1mg / m 2 , about 6.2mg / m 2 , about 6.3mg / m 2 , about 6.4mg / m 2 , about 6.5mg / m 2 , about 6.6mg / m 2 , about 6.7mg / m 2 , about 6.8mg / m 2 , about 6.9mg / m 2 , about 7.0mg / m 2 , about 7.1mg / m 2 , about 7.2mg / m 2 , about 7.3mg / m 2 , about 7.4mg / m 2 , about 7.5mg / m 2 , about 7.6mg / m 2, about 7.7mg / m 2 , about 7.8mg / m 2 , about 7.9mg / m 2 or about 8.0 mg / m 2 In some embodiments, the dose of the antibody-drug conjugate targeting B7-H4 is about 7.2 mg / m 2 .

[0215] In some embodiments, the dose of the antibody-drug conjugate targeting B7-H4 is about 12.0 mg / m 2 To about 15.0mg / m 2 In some embodiments, the dose of the antibody-drug conjugate targeting B7-H4 is about 13.9 mg / m 2 To about 14.9mg / m 2 In some aspects, the dose is about 13.0 mg / m 2 , about 13.1mg / m 2 , about 13.2mg / m 2 , about 13.3mg / m 2 , about 13.4mg / m 2 , about 13.5mg / m 2 , about 13.6mg / m 2 , about 13.7mg / m 2 , about 13.8mg / m 2 , about 13.9mg / m 2 , about 14.0mg / m 2 , about 14.1mg / m 2 , about 14.2mg / m 2 , about 14.3mg / m 2 , about 14.4mg / m 2 , about 14.5mg / m 2 , about 14.6mg / m 2 , about 14.7mg / m 2 , about 14.8mg / m 2 , about 14.9mg / m 2 or about 15.0 mg / m 2 In some embodiments, the dose of the antibody-drug conjugate targeting B7-H4 is about 14.4 mg / m 2 .

[0216] In some embodiments, the dose of the antibody-drug conjugate targeting B7-H4 is about 20.0 mg / m 2 To about 23.0mg / m 2 In some embodiments, the dose of the antibody-drug conjugate targeting B7-H4 is about 21.1 mg / m2 To about 22.1mg / m 2 In some aspects, the dose is about 20.0 mg / m 2 , about 20.1mg / m 2 , about 20.2mg / m 2 , about 20.3mg / m 2 , about 20.4mg / m 2 , about 20.5mg / m 2 , about 20.6mg / m 2 , about 20.7mg / m 2 , about 20.8mg / m 2 , about 20.9mg / m 2 , about 21.0mg / m 2 , about 21.1mg / m 2 , about 21.2mg / m 2 , about 21.3mg / m 2 , about 21.4mg / m 2 , about 21.5mg / m 2 , about 21.6mg / m 2 , about 21.7mg / m 2 , about 21.8mg / m 2 , about 21.9mg / m 2 , about 22.0mg / m 2 , about 22.1mg / m 2 , about 22.2mg / m 2 , about 22.3mg / m 2 , about 22.4mg / m 2 , about 22.5mg / m 2 , about 22.6mg / m 2 , about 22.7mg / m 2 , about 22.8mg / m 2 , about 22.9mg / m 2 or about 23.0 mg / m 2 In some embodiments, the dose of the antibody-drug conjugate targeting B7-H4 is about 21.6 mg / m 2 .

[0217] In some embodiments, the dose of the antibody-drug conjugate targeting B7-H4 is about 27.0 mg / m 2 To about 30.0mg / m 2 In some embodiments, the dose of the antibody-drug conjugate targeting B7-H4 is about 28.2 mg / m 2 To about 29.2mg / m 2In some aspects, the dose is about 27.0 mg / m 2 , about 27.1mg / m 2 , about 27.2mg / m 2 , about 27.3mg / m 2 , about 27.4mg / m 2 , about 27.5mg / m 2 , about 27.6mg / m 2 , about 27.7mg / m 2 , about 27.8mg / m 2 , about 27.9mg / m 2 , about 28.0mg / m 2 , about 28.1mg / m 2 , about 28.2mg / m 2 , about 28.3mg / m 2 , about 28.4mg / m 2 , about 28.5mg / m 2 , about 28.6mg / m 2 , about 28.7mg / m 2 , about 28.8mg / m 2 , about 28.9mg / m 2 , about 29.0mg / m 2 , about 29.1mg / m 2 , about 29.2mg / m 2 , about 29.3mg / m 2 , about 29.4mg / m 2 , about 29.5mg / m 2 , about 29.6mg / m 2 , about 29.7mg / m 2 , about 29.8mg / m 2 , about 29.9mg / m 2 or about 30.0 mg / m 2 In some embodiments, the dose of the antibody-drug conjugate targeting B7-H4 is about 28.7 mg / m 2 .

[0218] In some embodiments, the dose of the antibody-drug conjugate targeting B7-H4 is about 36.0 mg / m 2 To about 40.0mg / m 2 In some embodiments, the dose of the antibody-drug conjugate targeting B7-H4 is about 37.6 mg / m 2 To about 38.6mg / m 2 In some aspects, the dose is about 36.0 mg / m 2 , about 36.1mg / m2 , about 36.2mg / m 2 , about 36.3mg / m 2 , about 36.4mg / m 2 , about 36.5mg / m 2 , about 36.6mg / m 2 , about 36.7mg / m 2 , about 36.8mg / m 2 , about 36.9mg / m 2 , about 37.0mg / m 2 , about 37.1mg / m 2 , about 37.2mg / m 2 , about 37.3mg / m 2 , about 37.4mg / m 2 , about 37.5mg / m 2 , about 37.6mg / m 2 , about 37.7mg / m 2 , about 37.8mg / m 2 , about 37.9mg / m 2 , about 38.0mg / m 2 , about 38.1mg / m 2 , about 38.2mg / m 2 , about 38.3mg / m 2 , about 38.4mg / m 2 , about 38.5mg / m 2 , about 38.6mg / m 2 , about 38.7mg / m 2 , about 38.8mg / m 2 , about 38.9mg / m 2 , about 39.0mg / m 2 , about 39.1mg / m 2 , about 39.2mg / m 2 , about 39.3mg / m 2 , about 39.4mg / m 2 , about 39.5mg / m 2 , about 39.6mg / m 2 , about 39.7mg / m 2 , about 39.8mg / m 2 , about 39.9mg / m 2 , about 40.0mg / m 2 In some embodiments, the dose of the antibody-drug conjugate targeting B7-H4 is about 38.1 mg / m 2 .

[0219] In some embodiments, the dose of the antibody-drug conjugate targeting B7-H4 is about 49.0 mg / m 2 To about 52.0mg / m 2 In some embodiments, the dose of the antibody-drug conjugate targeting B7-H4 is about 50.2 mg / m 2 To about 51.2mg / m 2 In some aspects, the dose is about 49.0 mg / m 2 , about 49.1mg / m 2 , about 49.2mg / m 2 , about 49.3mg / m 2 , about 49.4mg / m 2 , about 49.5mg / m 2 , about 49.6mg / m 2 , about 49.7mg / m 2 , about 49.8mg / m 2 , about 49.9mg / m 2 , about 50.0mg / m 2 , about 50.1mg / m 2 , about 50.2mg / m 2 , about 50.3mg / m 2 , about 50.4mg / m 2 , about 50.5mg / m 2 , about 50.6mg / m 2 , about 50.7mg / m 2 , about 50.8mg / m 2 , about 50.9mg / m 2 , about 51.0mg / m 2 , about 51.1mg / m 2 , about 51.2mg / m 2 , about 51.3mg / m 2 , about 51.4mg / m 2 , about 51.5mg / m 2 , about 51.6mg / m 2 , about 51.7mg / m 2 , about 51.8mg / m 2 , about 51.9mg / m 2 or about 52.0 mg / m 2 In some embodiments, the dose of the antibody-drug conjugate targeting B7-H4 is about 50.7 mg / m 2 .

[0220] In some embodiments, the dose of the antibody-drug conjugate targeting B7-H4 is about 66.0 mg / m 2To about 69.0mg / m 2 In some embodiments, the dose of the antibody-drug conjugate targeting B7-H4 is about 66.9 mg / m 2 To about 67.9mg / m 2 In some aspects, the dose is about 66.0 mg / m 2 , about 66.1mg / m 2 , about 66.2mg / m 2 , about 66.3mg / m 2 , about 66.4mg / m 2 , about 66.5mg / m 2 , about 66.6mg / m 2 , about 66.7mg / m 2 , about 66.8mg / m 2 , about 66.9mg / m 2 , about 67.0mg / m 2 , about 67.1mg / m 2 , about 67.2mg / m 2 , about 67.3mg / m 2 , about 67.4mg / m 2 , about 67.5mg / m 2 , about 67.6mg / m 2 , about 67.7mg / m 2 , about 67.8mg / m 2 , about 67.9mg / m 2 , about 68.0mg / m 2 , about 68.1mg / m 2 , about 68.2mg / m 2 , about 68.3mg / m 2 , about 68.4mg / m 2 , about 68.5mg / m 2 , about 68.6mg / m 2 , about 68.7mg / m 2 , about 68.8mg / m 2 , about 68.9mg / m 2 or about 69.0 mg / m 2 In some embodiments, the dose of the antibody-drug conjugate targeting B7-H4 is about 67.4 mg / m 2 .

[0221] In some embodiments, the dose of the antibody-drug conjugate targeting B7-H4 is about 86.0 mg / m 2 To about 89.0mg / m 2In some embodiments, the dose of the antibody-drug conjugate targeting B7-H4 is about 87.1 mg / m 2 To about 88.1mg / m 2 In some aspects, the dose is about 86.0 mg / m 2 , about 86.1mg / m 2 , about 86.2mg / m 2 , about 86.3mg / m 2 , about 86.4mg / m 2 , about 86.5mg / m 2 , about 86.6mg / m 2 , about 86.7mg / m 2 , about 86.8mg / m 2 , about 86.9mg / m 2 , about 87.0mg / m 2 , about 87.1mg / m 2 , about 87.2mg / m 2 , about 87.3mg / m 2 , about 87.4mg / m 2 , about 87.5mg / m 2 , about 87.6mg / m 2 , about 87.7mg / m 2 , about 87.8mg / m 2 , about 87.9mg / m 2 , about 88.0mg / m 2 , about 88.1mg / m 2 , about 88.2mg / m 2 , about 88.3mg / m 2 , about 8.4mg / m 2 , about 88.5mg / m 2 , about 88.6mg / m 2 , about 88.7mg / m 2 , about 88.8mg / m 2 , about 88.9mg / m 2 or about 89.0 mg / m 2 In some embodiments, the dose of the antibody-drug conjugate targeting B7-H4 is about 87.6 mg / m 2 In some embodiments, the dose of the antibody-drug conjugate targeting B7-H4 is about 1.0 mg / m 2 , about 2.0mg / m 2 , about 3.0mg / m 2 , about 4.0mg / m 2 , about 5.0mg / m 2 , about 6.0mg / m2 , about 7.0mg / m 2 , about 8.0mg / m 2 , about 9.0mg / m 2 , about 10.0mg / m 2 , about 11.0mg / m 2 , about 12.0mg / m 2 , about 13.0mg / m 2 , about 14.0mg / m 2 , about 15.0mg / m 2 , about 16.0mg / m 2 , about 17.0mg / m 2 , about 18.0mg / m 2 , about 19.0mg / m 2 , about 20.0mg / m 2 , about 21.0mg / m 2 , about 22.0mg / m 2 , about 23.0mg / m 2 , about 24.0mg / m 2 , about 25.0mg / m 2 , about 26.0mg / m 2 , about 27.0mg / m 2 , about 28.0mg / m 2 , about 29.0mg / m 2 , about 30.0mg / m 2 , about 31.0mg / m 2 , about 32.0mg / m 2 , about 33.0mg / m 2 , about 34.0mg / m 2 , about 35.0mg / m 2 , about 36.0mg / m 2 , about 37.0mg / m 2 , about 38.0mg / m 2 , about 39.0mg / m 2 , about 40.0mg / m 2 , about 41.0mg / m 2 , about 42.0mg / m 2 , about 43.0mg / m 2 , about 44.0mg / m 2 , about 45.0mg / m 2 , about 46.0mg / m 2 , about 47.0mg / m 2 , about 48.0mg / m 2 , about 49.0mg / m 2, about 50.0mg / m 2 , about 51.0mg / m 2 , about 52.0mg / m 2 , about 53.0mg / m 2 , about 54.0mg / m 2 , about 55.0mg / m 2 , about 56.0mg / m 2 , about 57.0mg / m 2 , about 58.0mg / m 2 , about 59.0mg / m 2 , about 60.0mg / m 2 , about 61.0mg / m 2 , about 62.0mg / m 2 , about 63.0mg / m 2 , about 64.0mg / m 2 , about 65.0mg / m 2 , about 66.0mg / m 2 , about 67.0mg / m 2 , about 68.0mg / m 2 , about 69.0mg / m 2 , about 70.0mg / m 2 , about 71.0mg / m 2 , about 72.0mg / m 2 , about 73.0mg / m 2 , about 74.0mg / m 2 , about 75.0mg / m 2 , about 76.0mg / m 2 , about 77.0mg / m 2 , about 78.0mg / m 2 , about 79.0mg / m 2 , about 80.0mg / m 2 , about 81.0mg / m 2 , about 82.0mg / m 2 , about 83.0mg / m 2 , about 84.0mg / m 2 , about 85.0mg / m 2 , about 86.0mg / m 2 , about 87.0mg / m 2 , about 88.0mg / m 2 , about 89.0mg / m 2 , about 90.0mg / m 2 , about 91.0mg / m 2 , about 92.0mg / m 2 , about 93.0mg / m2 , about 94.0mg / m 2 , about 95.0mg / m 2 , about 96.0mg / m 2 , about 97.0mg / m 2 , about 98.0mg / m 2 , about 99.0mg / m 2 or about 100.0 mg / m 2 .

[0222] In some embodiments, at about 7.2 mg / m 2 To about 87.6mg / m 2 In some embodiments, the dose of the antibody-drug conjugate targeting B7-H4 (e.g., XMT-1660) is about 7.2 mg / m 2 In some embodiments, the dose of the antibody-drug conjugate targeting B7-H4 (e.g., XMT-1660) is about 14.4 mg / m 2 In some embodiments, the dose of the antibody-drug conjugate targeting B7-H4 (e.g., XMT-1660) is about 21.6 mg / m 2 In other embodiments, the dose of the antibody-drug conjugate targeting B7-H4 (e.g., XMT-1660) is about 28.7 mg / m 2 In some embodiments, the dose of the antibody-drug conjugate targeting B7-H4 (e.g., XMT-1660) is about 38.1 mg / m 2 In other embodiments, the dose of the antibody-drug conjugate targeting B7-H4 (e.g., XMT-1660) is about 50.7 mg / m 2 In some embodiments, the dose of the antibody-drug conjugate targeting B7-H4 (e.g., XMT-1660) is about 67.4 mg / m 2 In some embodiments, the dose of the antibody-drug conjugate targeting B7-H4 (e.g., XMT-1660) is about 87.6 mg / m 2 .

[0223] In some embodiments, the dosage amount is administered intravenously once a week (ie, a 7 day cycle), once every two weeks (ie, a 14 day cycle), once every three weeks (ie, a 21 day cycle), or once every four weeks (ie, a 28 day cycle).

[0224] In some embodiments, a dosage amount of an antibody-drug conjugate targeting B7-H4 is administered every week (i.e., a 7-day cycle), every two weeks (i.e., a 14-day cycle), every three weeks (i.e., a 21-day cycle), every four weeks (i.e., a 28-day cycle), every five weeks (i.e., a 35-day cycle), every six weeks (i.e., a 42-day cycle), every seven weeks (i.e., a 49-day cycle), or every eight weeks (i.e., a 56-day cycle).

[0225] In some embodiments, an antibody-drug conjugate targeting B7-H4 (e.g., XMT-1660) is infused at about 7.2 mg / m 2 To about 87.6mg / m 2 The dosage amount is administered as an infusion as follows: every week (i.e., a 7-day cycle), every two weeks (i.e., a 14-day cycle), every three weeks (i.e., a 21-day cycle), every four weeks (i.e., a 28-day cycle), every five weeks (i.e., a 35-day cycle), every six weeks (i.e., a 42-day cycle), every seven weeks (i.e., a 49-day cycle), or every eight weeks (i.e., a 56-day cycle).

[0226] In some embodiments, at about 7.2 mg / m 2 To about 87.6mg / m 2 The subject is administered an antibody-drug conjugate targeting B7-H4 (e.g., XMT-1660) at a dosage of about 7.2 mg / m 2 To about 87.6mg / m 2 XMT-1660 was administered to subjects once every 4 weeks (ie, 28-day cycles) at a dosage of

[0227] In some embodiments, at about 7.2 mg / m 2 To about 87.6mg / m 2 The subject is administered an antibody-drug conjugate targeting B7-H4 (eg, XMT-1660) in a dosage amount of 1:1, with the first infusion being over 90 minutes, followed by subsequent infusions once every 3 weeks (ie, a 21-day cycle) being over 90 minutes.

[0228] In some embodiments, at about 7.2 mg / m 2 To about 87.6mg / m 2 The subject is administered an antibody-drug conjugate targeting B7-H4 (eg, XMT-1660) in a dosage amount of 1:1, with the first infusion being over 90 minutes, followed by subsequent infusions once every 3 weeks (ie, a 21-day cycle) being over 60 minutes.

[0229] In some embodiments, at about 7.2 mg / m 2 To about 87.6mg / m 2The subject is administered an antibody-drug conjugate targeting B7-H4 (e.g., XMT-1660) in a dosage amount of 1:1, with the first infusion being over 90 minutes, and then subsequent infusions being once every 4 weeks (i.e., a 28-day cycle) being over 30 minutes.

[0230] In some embodiments, at about 7.2 mg / m 2 The subject is administered an antibody-drug conjugate targeting B7-H4 (e.g., XMT-1660) at a dose of 1:1, with the first infusion being over 90 minutes, and then subsequent infusions once every 3 weeks (i.e., a 21-day cycle) being over 60 minutes or 90 minutes.

[0231] In some embodiments, at about 14.4 mg / m 2 The subject is administered an antibody-drug conjugate targeting B7-H4 (e.g., XMT-1660) at a dose of 1:1, with the first infusion over 90 minutes, followed by subsequent infusions over 60 minutes or 90 minutes once every 3 weeks.

[0232] In some embodiments, at about 21.6 mg / m 2 The subject is administered an antibody-drug conjugate targeting B7-H4 (e.g., XMT-1660) at a dose of 1:1, with the first infusion being over 90 minutes, followed by subsequent infusions once every 3 weeks over 60 minutes or 90 minutes.

[0233] In some embodiments, at about 28.7 mg / m 2 The subject is administered an antibody-drug conjugate targeting B7-H4 (e.g., XMT-1660) at a dose of 1:1, with the first infusion being over 90 minutes, followed by subsequent infusions once every 3 weeks over 60 minutes or 90 minutes.

[0234] In some embodiments, at about 38.1 mg / m 2 The subject is administered an antibody-drug conjugate targeting B7-H4 (e.g., XMT-1660) at a dose of 1:1, with the first infusion being over 90 minutes, followed by subsequent infusions once every 3 weeks over 60 minutes or 90 minutes.

[0235] In some embodiments, at about 50.7 mg / m 2 The subject is administered an antibody-drug conjugate targeting B7-H4 (e.g., XMT-1660) at a dose of 1:1, with the first infusion being over 90 minutes, followed by subsequent infusions once every 3 weeks over 60 minutes or 90 minutes.

[0236] In some embodiments, at about 67.4 mg / m 2 The subject is administered an antibody-drug conjugate targeting B7-H4 (e.g., XMT-1660) at a dose of 1:1, with the first infusion being over 90 minutes, followed by subsequent infusions once every 3 weeks over 60 minutes or 90 minutes.

[0237] In some embodiments, at about 87.6 mg / m 2 XMT-1660 was administered to subjects at a dose of 100 mg / kg, with the first infusion being over 90 minutes, followed by subsequent infusions over either 60 minutes or 90 minutes every 3 weeks.

[0238] In some embodiments, 7.2 mg / m 2 The subject is administered an antibody-drug conjugate targeting B7-H4 (e.g., XMT-1660) at a dose of 1:1, with the first infusion being over 90 minutes, followed by subsequent infusions once every 4 weeks over 60 minutes or 90 minutes.

[0239] In some embodiments, at about 14.4 mg / m 2 The subject is administered an antibody-drug conjugate targeting B7-H4 (e.g., XMT-1660) at a dose of 1:1, with the first infusion being over 90 minutes, followed by subsequent infusions once every 4 weeks over 60 minutes or 90 minutes.

[0240] In some embodiments, at about 21.6 mg / m 2 The subject is administered an antibody-drug conjugate targeting B7-H4 (e.g., XMT-1660) at a dose of 1:1, with the first infusion being over 90 minutes, followed by subsequent infusions once every 4 weeks over 60 minutes or 90 minutes.

[0241] In some embodiments, at about 28.7 mg / m 2 The subject is administered an antibody-drug conjugate targeting B7-H4 (e.g., XMT-1660) at a dose of 1:1, with the first infusion being over 90 minutes, followed by subsequent infusions once every 4 weeks over 60 minutes or 90 minutes.

[0242] In some embodiments, at about 38.1 mg / m 2 The subject is administered an antibody-drug conjugate targeting B7-H4 (e.g., XMT-1660) at a dose of 1:1, with the first infusion being over 90 minutes, followed by subsequent infusions once every 4 weeks over 60 minutes or 90 minutes.

[0243] In some embodiments, at about 50.7 mg / m 2 The subject is administered an antibody-drug conjugate targeting B7-H4 (e.g., XMT-1660) at a dose of 1:1, with the first infusion being over 90 minutes, followed by subsequent infusions once every 4 weeks over 60 minutes or 90 minutes.

[0244] In some embodiments, at about 67.4 mg / m 2The subject is administered an antibody-drug conjugate targeting B7-H4 (e.g., XMT-1660) at a dose of 1:1, with the first infusion being over 90 minutes, followed by subsequent infusions once every 4 weeks over 60 minutes or 90 minutes.

[0245] In some embodiments, at about 87.6 mg / m 2 The subject is administered an antibody-drug conjugate targeting B7-H4 (e.g., XMT-1660) at a dose of 1:1, with the first infusion being over 90 minutes, followed by subsequent infusions once every 4 weeks over 60 minutes or 90 minutes.

[0246] The frequency and time of administration and the dosage amount can be administered periodically within the administration cycle to maintain the continuous and / or long-term effect of the active agent for the desired length of time. The provided B7-H4-targeting antibody-drug conjugate compositions can be administered hourly, daily, weekly, monthly, annually, or once. The length of the administration cycle can be determined empirically and depends on the disease to be treated, the severity of the disease, the particular patient, and other considerations within the skill level of the treating physician. The length of time for treatment with the combination therapy provided herein can be one week, two weeks, one month, several months, one year, several years, or longer.

[0247] For example, the frequency of administration of an antibody-drug conjugate targeting B7-H4 is once a day, once every other day, twice a week, once a week, once every 2 weeks, once every 3 weeks, or once every 4 weeks. During the course of treatment, the dosage can be divided into multiple administration cycles. For example, an antibody-drug conjugate targeting B7-H4 can be administered at a frequency over a period of about one month, 2 months, 3 months, 4 months, 5 months, 6 months, one year, or longer. The frequency of administration can be the same or different over the period of the entire cycle. For example, an exemplary dosage frequency is twice a week, at least for the first week of the administration cycle. After the first week, the frequency can continue twice a week, can be increased to more than twice a week, or can be reduced to no more than once a week. It is within the level of the skilled person to determine the specific dosage frequency and administration cycle based on the specific dose administered, the disease or condition being treated, the severity of the disease or condition, the age of the subject, and other similar factors.

[0248] If disease symptoms persist in the absence of cessation of treatment, treatment can be continued for an additional length of time.During the course of treatment, evidence of disease and / or treatment-related toxicity or side effects can be monitored.

[0249] The administration cycle of the antibody-drug conjugate targeting B7-H4 can be customized to add a period of cessation of treatment to provide a rest period from exposure to the agent. The length of time that treatment is stopped can last for a predetermined time or can be determined empirically depending on how the patient responds or depending on observed side effects. For example, treatment can be stopped for one week, two weeks, three weeks, one month, or several months. In general, the period of cessation of treatment is built into the cycle of the patient's dosing regimen.

[0250] An exemplary dosing regimen is a 21-day or 28-day treatment cycle or administration cycle. Preferably, the dosing regimen is a treatment cycle or administration cycle of 28 days. The antibody-drug conjugate targeting B7-H4 disclosed herein is administered on day 1, followed by 20 days of no administration or on day 1, followed by 27 days of no administration. Determining the exact administration cycle and dosing schedule is within the level of those skilled in the art.

[0251] As noted above, the administration cycle can continue for any desired length of time. Therefore, a 21-day administration cycle or a 28-day administration cycle can be repeated for any length of time. For example, a 21-day administration cycle or a 28-day administration cycle can be repeated for 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 1.5 years, 2 years, 2.5 years, 3 years or longer. It is within the technical level of the treating physician to adopt an administration cycle and a dosage regimen that meet the patient's needs depending on individual considerations specific to the patient and the disease to be treated.

[0252] In some embodiments, an antibody-drug conjugate targeting B7-H4 (e.g., XMT-1660) is administered to a subject until disease progression, death, unacceptable toxicity, or voluntary withdrawal, whichever comes first.

[0253] Dose reductions and delays

[0254] In some cases, toxicity or adverse reactions may occur in response to an initial dose of an antibody-drug conjugate targeting B7-H4 (e.g., XMT-1660) at a dose described herein. Subsequent doses may be reduced or the time between doses may be delayed or extended. In some embodiments, toxicity or adverse reactions that may cause dose reduction or delay include, but are not limited to, elevated ALT or AST levels, hepatotoxicity, interstitial lung disease (ILD), pneumonia, hematotoxicity, proteinuria, fatigue, nausea, other clinically significant non-laboratory toxicities, other clinically significant adverse events.

[0255] Measurement of B7-H4 expression

[0256] In various embodiments, the invention provides methods for identifying cancer patients suitable for B7-H targeted therapy or monitoring treatment regimens by measuring the status of B7-H4 expression in a tumor sample obtained from the patient.

[0257] In some embodiments, a B7-H4 diagnostic test can be used to identify subjects for treatment with a drug conjugate targeting B7-H4.

[0258] The sample is derived from a subject with cancer. A sample of cancer cells is stripped from a tissue removed or obtained from a subject. In some embodiments, the sample is a fresh, frozen or archived biopsy sample.

[0259] In some embodiments, the test cell population is derived from fresh, unfrozen tissue from a biopsy sample. In other embodiments, the test cell population is derived from a primary or metastatic site. In some embodiments, the test cell population is derived from fresh or frozen tissue or ascites or pleural fluid from a biopsy or surgical sample. In some embodiments, the test cell population is derived from a fixed tissue (e.g., formalin-fixed or formalin-fixed paraffin-embedded (FFPE)) from a biopsy or surgical sample or a cell block derived from a fluid sample. The tissue sample can be frozen or fresh.

[0260] The required level of B7-H4 expression can be a level identified by any method known in the art and more specifically by the methods described herein. For example, the level of B7-H4 expression can be measured by performing a known immunoassay for protein expression using an antibody that specifically recognizes B7-H4, such as an enzyme immunoassay, a radioimmunoassay, a competitive immunoassay, a double antibody sandwich assay, a fluorescent immunoassay, an ELISA, a Western blot technique, an agglutination assay, a cell fluorescence assay (e.g., flow cytometry), a fluorescent in situ hybridization (FISH), a colorimetric or immunohistochemical staining assay (IHC). For example, cell-based assays such as flow cytometry (FC), immunohistochemistry (IHC), RNA expression analysis, or immunofluorescence (IF) are particularly desirable in determining B7-H4 expression status because such assay formats are clinically appropriate.

[0261] Flow cytometry (FC) can be used to determine the cell surface expression of B7-H4 in tumor samples before, during, and after treatment with a drug. For example, if desired, tumor cells can be analyzed by flow cytometry for B7-H4 expression, as well as markers for identifying cancer cell types, etc. Flow cytometry can be performed according to standard methods. See, for example, Chow et al., Cytometry (Communications in Clinical Cytometry) 46:72-78 (2001). Briefly and by way of example, the following protocol for cytometry analysis can be used: cells are fixed with 2% paraformaldehyde for 10 minutes at 37°C, followed by permeabilization in 90% methanol on ice for 30 minutes. Cells can then be stained with B7-H4-specific antibodies, washed, and labeled with fluorescently labeled secondary antibodies. Cells are then analyzed on a flow cytometer (e.g., Beckman Coulter FC500) according to the specific protocol of the instrument used. Such analysis identifies the level of B7-H4 expressed in the tumor.

[0262] Immunohistochemical (IHC) staining can also be used to determine the expression of B7-H4 in tumor samples before, during, and after treatment with the drug. IHC can be performed according to well-known techniques. See, for example, ANTIBODIES; A LABORATORY MANUAL, Chapter 10, Harlow & Lane, ed., Cold Spring Harbor Laboratory (1988). Briefly, and by way of example, paraffin-embedded tissue (e.g., tumor tissue from a biopsy) is prepared for immunohistochemical staining by: deparaffinizing tissue sections with xylene followed by ethanol; hydrating in water and then in PBS; exposing the antigen by heating the slides in sodium citrate buffer; incubating the sections in hydrogen peroxide; blocking in blocking solution; incubating the slides in primary polypeptide antibodies and secondary antibodies; and finally detecting using the ABC avidin / biotin method according to the manufacturer's instructions.

[0263] Immunofluorescence (IF) assays can also be used to determine the expression of B7-H4 in tumor samples before, during, and after treatment with a drug. IF can be performed according to well-known techniques. See, for example, JM Polac and S. Van Noorden (1997) INTRODUCTION TO IMMUNOCYTO CHEMISTRY, 2nd Edition; ROYAL MICROSCOPYSOCIETY MICROSCOPY HANDBOOK 37, BioScientific / Springer-Verlag. Briefly, and by way of example, patient samples can be fixed in paraformaldehyde followed by methanol, blocked with a blocking solution such as horse serum, incubated with a primary antibody against the polypeptide followed by a secondary antibody labeled with a fluorescent dye such as Alexa 488, and analyzed using an epifluorescence microscope.

[0264] The antibodies employed in the assays described above can advantageously be conjugated to fluorescent dyes (e.g., Alexa 488, PE) or other labels (e.g., quantum dots) together with other signaling (phospho-AKT, phosphorylated Erk 1 / 2) and / or cellular marker (cytokeratin) antibodies for multiparameter analysis.

[0265] In one embodiment, the expression of B7-H4 in samples from tumors is determined immunohistochemically using a system, such as the Leica Bond III automated stainer (BOND III) system.

[0266] Alternatively, the assay may include preparing RNA from the sample, optionally for PCR (polymerase chain reaction) or other analytical methods. The PCR method is optionally, for example, RT-PCR (reverse transcription-PCR) or quantitative PCR, such as real-time RT-PCR, RNAseq, etc. Alternatively, the assay may be performed using an array, such as a microarray known in the relevant art, such as, for example, nanostring technology.

[0267] The patient is identified as responding to treatment, wherein the treatment is monitored or the cancer is detected by detecting and / or measuring the expression level of B7-H4 in tumor cells in the sample.

[0268] The detection / measurement of the expression level of B7-H4 is determined by calculating a B7-H4 score. The B7-H4 score is quantitative or semi-quantitative. For example, the detection is pathologically scored to obtain a pathological score. It is contemplated that any scoring method known in the art can be used in the methods of the present invention. In particular, any histological scoring method known in the art.

[0269] Methods for evaluating the measurement results obtained by immunohistochemical staining assays include, for example, the H score method, TPS (tumor proportion score) or PS2+ (percentage score) score. The H score (Am J Clin Pathol. 1988; 90 (3): 233-9), TPS score and PS2+ score are determined by the following calculation formula. H score = ((% at 0) x 0) + ((% at 1+) x 1) + ((% at 2+) x 2) + ((% at 3+) x 3), TPS score = (% at 1+) + (% at 2+) + (% at 3+); and PS2+ score = (% at 2+) + (% at 3+); wherein staining intensity 0 is unstained; staining intensity 1 is weak staining; staining intensity 2 is moderate staining; and staining intensity 3 is strong staining. In some embodiments, when TPS is scored as tumor cell membrane reactivity, subjects with a TPS ≥ 75 are considered B7-H4 positive (high) in this assay, while subjects with a TPS < 75 are considered B7-H4 negative (low).

[0270] In the evaluation by the scoring method, only the cancer cell part is used. For negative or positive control of staining intensity, formalin-fixed paraffin-embedded cell lines or xenografts (lines whose protein expression levels are known in advance) can be used. When there is no control sample, multiple samples are evaluated simultaneously to confirm the overall distribution of the staining intensity of the samples, and then the staining intensity can be set.

[0271] In addition to the scoring methods mentioned above, other scoring methods known in the art may also be used, such as the Allred method (Harvey et al., Journal of Clinical Oncology 17, No. 5 (May 1999) 1474-1474). A cutoff value needs to be set in each method. Allred score = score of positive cell percentage + staining intensity score.

[0272] The present disclosure also provides kits and / or methods for identifying or otherwise refining (e.g., stratifying) a patient population suitable for therapeutic administration of an antibody-drug conjugate targeting B7-H4 disclosed herein by identifying a subject's B7-H4 score prior to treatment with an antibody-drug conjugate targeting B7-H4 disclosed herein. In some embodiments, the test cell population is derived from fresh, unfrozen tissue from a biopsy sample. In some embodiments, the test cell population is derived from a primary or metastatic site. In some embodiments, the test cell population is derived from frozen tissue or ascites or pleural fluid from a biopsy or surgical sample. In some embodiments, the test cell population is derived from fixed tissue (e.g., formalin fixed) from a biopsy or surgical sample. The IHC test measures the amount of B7-H4 receptor protein on the surface of cells in a cancer tissue sample.

[0273] definition

[0274] As utilized in accordance with this disclosure, unless otherwise indicated, the following terms shall be understood to have the following meanings:

[0275] As used herein, the terms "anti-B7-H4 antibody," "B7-H4 antibody," and "antibody that binds B7-H4" refer to an antibody that is capable of binding B7-H4 with sufficient affinity such that the antibody can be used as a diagnostic and / or therapeutic agent targeting B7-H4.

[0276] As used herein, the term "B7-H4" refers to any natural mature B7-H4 that is produced by processing of a B7-H4 precursor protein in a cell. The term includes B7-H4 from any vertebrate source, including mammals such as primates (e.g., humans and cynomolgus monkeys) and rodents (e.g., mice and rats), unless otherwise indicated. The term also includes naturally occurring variants of B7-H4, such as splice variants or allelic variants.

[0277] The term "B7-H4 positive cancer" refers to a cancer that contains cells that express B7-H4 on their surface. In some embodiments, the expression of B7-H4 on the cell surface is determined, for example, using antibodies against B7-H4 in methods such as immunohistochemistry, FACS, etc.

[0278] Alternatively, B7-H4 mRNA expression is considered to correlate with B7-H4 expression on the cell surface and can be determined by a method selected from in situ hybridization and RT-PCR (including quantitative RT-PCR).

[0279] The term "B7-H4 positive cells" refers to cells that express B7-H4 on their surface.

[0280] As used herein, the term "antibody" is used in the broadest sense and covers various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, as long as they exhibit the desired antigen binding activity. Various methods for numbering the amino acid sequence of antibodies and identifying complementary determining regions are known in the art. For example, the Kabat numbering system (see Kabat, EA et al., Sequences of Protein of immunological interest, 5th edition, US Department of Health and Human Services, US Government Printing Office (1991)) or the IMGT numbering system (see International Immunogenetics Information Available online: http: / / www.imgt.org / ). The IMGT numbering system is routinely used in the art and is accepted as a reliable and accurate system to determine amino acid positions in coding sequences, align alleles, and easily compare sequences in immunoglobulins (IG) and T-cell receptors (TR) from all vertebrate species. The accuracy and consistency of IMGT data is based on IMGT-ONTOLOGY, the first and so far unique ontology of immunogenetics and immunoinformatics (see Lefranc. MP et al., Biomolecules, December 2014; 4(4), 1102-1139). IMGT tools and databases operate against the IMGT reference catalog, which is built from a large set of sequence libraries. In the IMGT system, exon delimitation is taken into account whenever appropriate, and IG V domains and IG C domains are delimited. Therefore, with the availability of more sequences in the IMGT database, those skilled in the art can use and "are using" the IMGT exon numbering system to reliably determine amino acid positions in coding sequences and for allele alignment. In addition, the correspondence between IMGT unique numbers and other numbers (i.e., Kabat) can be obtained in the IMGT scientific chart (see Lefranc. MP et al., Biomolecules, December 2014; 4 (4), 1102-1139). The EU numbering system can also be used (see Edelman, GM et al., Proc. Natl. Acad. USA, 63, 78-85 (1969)). In some aspects, the EU numbering can be used to number the amino acid positions of antibodies. In some aspects, the EU numbering system can be used to determine the position of the constant region of the heavy chain or light chain of an antibody.

[0281] The term "antibody fragment" refers to a molecule other than an intact antibody that contains a portion of an intact antibody and binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments.

[0282] As used herein, the term "antibody that binds to the same epitope" as a reference antibody refers to an antibody that blocks the binding of the reference antibody to its antigen by 50% or more in a competition assay, and conversely, the reference antibody blocks the binding of the antibody to its antigen by 50% or more in a competition assay. Exemplary competition assays are provided herein.

[0283] The term "class" of an antibody refers to the type of constant domain or constant region possessed by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these can be further divided into subclasses (isotypes), such as IgGi, IgG2, IgG3, IgG4, IgAi, and IgA2. The heavy chain constant domains corresponding to the different classes of immunoglobulins are referred to as α, δ, ε, γ, and μ, respectively.

[0284] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous antibody population, i.e., the individual antibodies that make up the population are identical and / or bind to the same epitope, except for possible variant antibodies, such as variant antibodies containing naturally occurring mutations or produced during the production of monoclonal antibody preparations, such variants are usually present in trace amounts. Contrary to polyclonal antibody preparations that typically include different antibodies for different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed to a single determinant on an antigen. Therefore, the modifier "monoclonal" indicates that the antibody is characterized in that it is obtained from a substantially homogeneous antibody population and should not be interpreted as having to produce antibodies by any particular method. For example, the monoclonal antibody to be used according to the present invention can be prepared by various techniques, including but not limited to hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing all or part of transgenic animals containing human immunoglobulin loci, such methods and other exemplary methods for preparing monoclonal antibodies are described herein.

[0285] The term "naked antibody" refers to an antibody that is not conjugated to a heterologous moiety (eg, a cytotoxic moiety or a STING agonist drug moiety). Naked antibodies can be present in a pharmaceutical formulation.

[0286] The term "natural antibody" refers to naturally occurring immunoglobulin molecules with different structures. For example, natural IgG antibodies are heterotetrameric glycoproteins of about 150,000 daltons, which are composed of two identical light chains and two identical heavy chains bonded by disulfide bonds. From N-terminal to C-terminal, each heavy chain has a variable region (VH), also referred to as a variable heavy domain or a heavy chain variable domain, followed by three constant domains (CH1, CH2 and CH3). Similarly, from - to C-terminal, each light chain has a variable region (VL), also referred to as a variable light domain or a light chain variable domain, followed by a constant light (CL) domain. The light chain of an antibody can be assigned to one of two types called κ and λ based on the amino acid sequence of its constant domain.

[0287] An "isolated antibody" is an antibody that has been separated from a component of its natural environment. In some embodiments, the antibody is purified to greater than 95% or 99% purity as determined by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reversed-phase HPLC). For a review of methods for assessing antibody purity, see, e.g., Flatman et al., J. Chromatogr. B 848:79-87 (2007).

[0288] The term "epitope" refers to the specific site on an antigen molecule to which an antibody binds.

[0289] The term "humanized antibody" of an antibody refers to an antibody derived from a non-human antibody (e.g., murine) that retains or substantially retains the antigen-binding properties of the parent antibody but is less immunogenic in humans. Humanization as used herein is intended to include deimmunized antibodies.

[0290] The term "humanized form" of an antibody (eg, a non-human antibody) refers to an antibody that has undergone humanization.

[0291] When used in the context of two or more antibodies herein, the term "competes with" or "cross-competes with" indicates that the two or more antibodies compete for binding to B7-H4, e.g., compete for binding of B7-H4. An antibody "blocks" or "cross-blocks" binding of one or more other antibodies to B7-H4 if the antibody competes with the other antibody by 25% or more, where 25%-74% represents "partial blocking" and 75%-400% represents "complete blocking." Unless otherwise defined or negated by the context, the terms "compete with," "cross-compete with," "block," or "cross-block" when used herein are also intended to cover such antibody pairs.

[0292] As used herein, an antibody that "specifically binds to human B7-H4" is intended to refer to an antibody that has such a KD An antibody that binds to human B7-H4, the K D 1×10 -7 or lower, more typically 5×10 -8 M or lower, more typically 3×10 -8 M or lower, more typically 1×10 -9 M or lower, even more typically 5×10 -9 M or lower.

[0293] As used herein, the term "does not substantially bind" a protein or cell means not binding to the protein or cell or not binding to the protein or cell with high affinity, i.e., with such a K D Binding proteins or cells, the K D 1×10 -8 M or more, more preferably 1×10 -5 M or more, more preferably 1×10 -4 M or more, more preferably 1×10 -3 M or more, even more preferably 1×10 -2 M or higher.

[0294] The term "sugar" refers to monosaccharides such as glucose (Glc), galactose (Gal), mannose (Man) and fucose (Fuc). The term "sugar derivative" refers to a derivative of a monosaccharide, i.e., a monosaccharide that includes substituents and / or functional groups. Examples of sugar derivatives include, but are not limited to, amino sugars and sugar acids. Examples of sugar derivatives also include denoted as S'(F') X1 A compound wherein S' is a sugar or a sugar derivative, F' is a functional group, and x1 indicates the number of the functional groups.

[0295] As used herein, the term "core-GlcNAc moiety" refers to a monosaccharide, polysaccharide or oligosaccharide moiety comprising GlcNAc (e.g., core-GlcNAc) attached to an antibody (e.g., via the C1 position of GlcNAc). In some embodiments, GlcNAc is attached to the antibody via an N-glycosidic bond to an amide nitrogen atom in the side chain of an asparagine amino acid of the antibody. In some embodiments, the core-GlcNAc moiety is present in the native glycosylation site of the antibody or is introduced at a different site of the antibody. In some embodiments, the core-GlcNAc moiety is a monosaccharide (e.g., the core-GlcNAc moiety is also a terminal-GlcNAc moiety). In some embodiments, the core-GlcNAc moiety further comprises fucose, such as the core-GlcNAc moiety is a disaccharide core-GlcNAc-(α1-6-Fuc) moiety (which may be referred to as GlcNAc(Fuc)). Thus, when the antibody comprises a core-GlcNAc moiety, the antibody may comprise a monosaccharide or a disaccharide core-GlcNAc moiety, and the core-GlcNAc moiety may further comprise fucose (e.g., a disaccharide core-GlcNAc(Fuc) moiety). If the core-GlcNAc moiety further comprises fucose, the fucose may be attached to the O-6 of the core-GlcNAc moiety at the α-1,6 position. The core-GlcNAc moiety further comprising fucose may be referred to as core-GlcNAc(Fuc).

[0296] The term "core-GlcNAc" refers to an internal GlcNAc that is part of a polysaccharide or oligosaccharide, wherein the polysaccharide or oligosaccharide is attached to the antibody via the internal GlcNAc.

[0297] As used herein, the term "terminal-GlcNAc moiety" refers to a moiety comprising GlcNAc attached to an antibody and having a terminal functional group available for further modification (e.g., with a P"-S"-A" compound). In some embodiments, the terminal-GlcNAc moiety further comprises fucose. In some embodiments, the terminal-GlcNAc moiety is formed by reacting a core-GlcNAc moiety of a glycoprotein (e.g., an antibody glycan) with an endoglycosidase.

[0298] The term "nucleotide" is used in its normal scientific sense and refers to a molecule consisting of a nucleobase, a five-carbon sugar (ribose or 2-deoxyribose) and one, two or three phosphate groups. In the absence of a phosphate group, the nucleobase and the sugar constitute a nucleoside. Therefore, a nucleotide may also be referred to as a nucleoside monophosphate, a nucleoside diphosphate or a nucleoside triphosphate. The nucleobase may be adenine, guanine, cytosine, uracil or thymine.

[0299] The term "protein" is used in its normal scientific sense and includes polypeptides comprising about 10 or more amino acids. Proteins may comprise natural or unnatural amino acids.

[0300] The term "glycoprotein" is used herein in its normal scientific sense and refers to a protein comprising one or more monosaccharide or oligosaccharide chains ("glycans") covalently bonded to a protein. Glycans can be attached to hydroxyl groups on a protein (O-linked glycans), to amide functional groups on a protein (N-glycoproteins), or to carbons on a protein (C-glycoproteins). A glycoprotein can contain more than one glycan, a combination of one or more monosaccharides and one or more oligosaccharide glycans, and a combination of N-linked, O-linked, and C-linked glycans. It is estimated that more than 50% of all proteins have some form of glycosylation and are therefore qualified as glycoproteins.

[0301] The term "glycan" is used herein with its normal scientific meaning and refers to a monosaccharide or oligosaccharide chain connected to a protein. Therefore, a glycan refers to the carbohydrate portion of a glycoprotein. A glycan is attached to a protein via the C-1 carbon of a sugar, which may not have further substitutions (monosaccharides) or may be further substituted (oligosaccharides) at one or more of its hydroxyl groups. Naturally occurring glycans typically contain 1 to about 10 sugar moieties. However, when a longer sugar chain is connected to a protein, the sugar chain is also considered to be a glycan. The glycan of a glycoprotein may be a monosaccharide. A glycan may also be an oligosaccharide. The oligosaccharide chain of a glycoprotein may be linear or branched. In oligosaccharides, the sugar directly attached to a protein is referred to as a core sugar. In oligosaccharides, a sugar that is not directly attached to a protein but is attached to at least two other sugars is referred to as an endosugar. In oligosaccharides, a sugar that is not directly attached to a protein but is attached to a single other sugar, i.e., a sugar that does not carry an additional sugar substituent at one or more of its other hydroxyl groups, is referred to as a terminal sugar. For the avoidance of doubt, there may be multiple terminal sugars but only one core sugar in the oligosaccharides of a glycoprotein. Glycans may be O-linked glycans, N-linked glycans or C-linked glycans. In an O-linked glycan, a monosaccharide or oligosaccharide glycan is bonded to a C atom in an amino acid of a protein.

[0302] The term "glycosyltransferase" refers to a superfamily of enzymes involved in the synthesis of complex carbohydrates present on glycoproteins and glycolipids.

[0303] The term "N-acetylgalactosaminyltransferase" (GalNAc-T) is an N-acetyl-D-galactosamine transferase that catalyzes the addition of N-acetyl-D-galactosamine to proteins.

[0304] As used herein, the term "independently" means that where more than one substituent is selected from a number of possible substituents, those substituents may be the same or different.

[0305] As used herein, the term "pharmaceutically acceptable" refers to those compounds, conjugates, materials, compositions and dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and animals without excessive toxicity, irritation, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0306] As used herein, the term "treating" or "treatment" describes the management and care of a patient for the purpose of combating a disease, condition, or disorder, and includes the administration of a compound of the disclosure, or a pharmaceutically acceptable salt, polymorph, or solvate thereof, to alleviate the symptoms or complications of a disease, condition, or disorder, or to eliminate the disease, condition, or disorder. The term "treatment" may also include treating cells in vitro or in an animal model.

[0307] As used herein, the terms "preventing," "prevent," or "protecting against" describe the reduction or elimination of the onset of symptoms or complications of such disease, condition, or disorder.

[0308] A therapeutically "effective amount" is intended to mean an amount of the conjugate, as defined herein, sufficient to be effective for treatment or prevention when administered to a patient in need of such treatment. The amount of a given conjugate corresponding to such an amount varies depending on factors such as the specific conjugate (e.g., potency (pICso) of the specific conjugate, efficacy (EC 50 ) and biological half-life), the disease condition and its severity, the identity of the patient in need of treatment (e.g., age, size and weight), etc., but nonetheless can be routinely determined by one skilled in the art. Likewise, the duration of treatment with the conjugate and the time period of administration (time period between doses and timing of doses, e.g., before / with / after meals) vary depending on the identity of the mammal in need of treatment (e.g., weight), the specific conjugate and its properties (e.g., pharmacokinetic properties), the disease or disorder and its severity, and the specific composition and method used, but nonetheless can be determined by one skilled in the art.

[0309] The term "composition" refers to a product comprising the specified ingredients in therapeutically effective amounts, as well as any product which results, directly or indirectly, from combination of the specified ingredients in the specified amounts.

[0310] As used herein, the term "pharmaceutically acceptable excipient" means an excipient useful in preparing a pharmaceutical composition that is generally safe, non-toxic, and not biologically or otherwise undesirable, and includes excipients that are acceptable for veterinary use as well as human pharmaceutical use. "Pharmaceutically acceptable excipient" as used in the specification and claims includes one and more than one such excipient.

[0311] As used herein, the term "conjugate(s) of the disclosure" or "conjugate(s) of the present disclosure" means a conjugate as defined herein in any form, i.e. any tautomeric form, any isomeric form, any salt or non-salt form (e.g. as a free acid or base form or as a salt, in particular a pharmaceutically acceptable salt thereof), and any physical form thereof (e.g. including non-solid forms (e.g. liquid or semi-solid forms) and solid forms (e.g. amorphous or crystalline forms, specific polymorphic forms, solvate forms, including hydrate forms (e.g. monohydrates, dihydrates and hemihydrates)) and mixtures of various forms.

[0312] Thus, the present disclosure includes conjugates as disclosed herein in any salt or non-salt form and any physical form thereof, as well as mixtures of the various forms. While these are included in the present disclosure, it is understood that the conjugates of the present disclosure in any salt or non-salt form and in any physical form thereof may have different levels of activity, different bioavailability, and different handling properties for formulation purposes.

[0313] It should be understood that throughout the specification, when a composition is described as having, including or comprising a specific component, it is contemplated that the composition also consists essentially of or consists of the component. Similarly, when a method or process is described as having, including or comprising a specific process step, the process also consists essentially of or consists of the process step. In addition, it should be understood that as long as the present invention remains operable, the order of the steps or the order in which certain actions are performed is insignificant. In addition, two or more steps or actions may be performed simultaneously.

[0314] When the term "competes with" or "cross-competes with" is used in the context of two or more antibodies herein, the term means that the two or more antibodies compete for binding to B7-H4, for example, for binding to B7-H4 in the assay described in Example 5 or 8. An antibody "blocks" or "cross-blocks" binding of one or more other antibodies to B7-H4 if the antibody competes with the other antibody by 25% or more, wherein 25%-74% represents "partial blocking" and 75%-400% represents "complete blocking", preferably as determined using the assays of Examples 5 and 8. For the same antibody pair, competition or blocking in the assays of Example 5 or 8 is observed only when one antibody is coated on the plate and the other is used for competition, but not vice versa. Unless otherwise defined or negated by the context, the terms "compete with", "cross-compete with", "block" or "cross-block" when used herein are also intended to cover such antibody pairs.

[0315] As used herein, "dosage regimen" or "dosage regimen" refers to the amount of an agent, such as a composition containing an antibody-drug conjugate targeting B7-H4, administered and the frequency of administration. The dosing regimen is a function of the disease or condition to be treated and can vary accordingly.

[0316] As used herein, the "frequency" of administration refers to the time between successive treatment administrations. For example, the frequency can be days, weeks, or months. For example, the frequency can be more than once a week, such as twice a week, three times a week, four times a week, five times a week, six times a week, or every day. The frequency can also be one week, two weeks, three weeks, or four weeks. The specific frequency is a function of the specific disease or condition being treated. The frequency is typically more than once a week and typically twice a week.

[0317] As used herein, "administration cycle" refers to the repetitive time schedule of the administration regimen of the administration of the enzyme and / or the second agent repeated within the successive administration. For example, an exemplary administration cycle is a 28-day cycle, wherein twice a week is administered for three weeks, followed by a week of stopping administration. A preferred administration cycle is a 7-day cycle administered once every 7 days, a 14-day cycle administered once every 14 days (i.e., 2 weeks), a 21-day cycle administered once every 21 days (i.e., 3 weeks), or a 28-day cycle administered once every 28 days (i.e., 4 weeks).

[0318] As used herein, when referring to dosages based on mg / kg of a subject, the average human subject is considered to have a mass of approximately 70 kg-75 kg (e.g., 70 kg) and a body weight of 1.73 m 2 of body surface area (BSA).

[0319] As used herein, amelioration of the symptoms of a particular disease or condition by treatment, such as by administration of a pharmaceutical composition or other therapeutic agent, refers to any alleviation of the symptoms of the condition or an adverse effect, whether the alleviation is permanent or temporary, long-lasting or transient, such as alleviation of an adverse effect associated with or occurring upon administration of an antibody-drug conjugate targeting B7-H4.

[0320] As used herein, when referring to a body surface area (BSA; m 2 ), the "body surface area" is the measured or calculated body surface area of ​​the human body. For many clinical purposes, BSA is a better indicator of metabolic quality than body weight because it is less affected by abnormal fat mass. Various calculations have been published to obtain BSA without direct measurement. In the following formula, BSA is expressed in m 2BSA = 0.007184 × W, W is the mass in kg, and H is the height in cm. The most widely used is Du Bois, DuBois formula: BSA = 0.007184 × W 0.425 ×H 0.725 Other methods for determining BSA include, for example, the Mosteller, Haycock, Gehan and George, Boyd, Fujimoto, Takahira, Shuter and Aslani or Schlich formulas.

[0321] "Subject" includes mammals. The mammal can be, for example, any mammal, such as a human, a primate, a bird, a mouse, a rat, poultry, a dog, a cat, a cow, a horse, a goat, a camel, a sheep or a pig. Preferably, the mammal is a human.

[0322] As used herein, "unit dose form" or "unit dosage form" refers to physically discrete units suitable for human and animal subjects and packaged individually as known in the art.

[0323] As used herein, a single-dose formulation refers to a formulation that is intended for a single dose.

[0324] As used herein, "time proximity" refers to the administration of one therapeutic agent (e.g., an antibody-drug conjugate targeting B7-H4 disclosed herein) occurring within a time period before or after the administration of another therapeutic agent (e.g., an immune checkpoint inhibitor disclosed herein), so that the therapeutic effect of one therapeutic agent overlaps with the therapeutic effect of another therapeutic agent. In some embodiments, the therapeutic effect of one therapeutic agent completely overlaps with the therapeutic effect of another therapeutic agent. In some embodiments, "time proximity" means that the administration of one therapeutic agent occurs within a time period before or after the administration of another therapeutic agent, so that there is a synergistic effect between one therapeutic agent and another therapeutic agent. "Time proximity" can vary according to various factors, including but not limited to the age, sex, weight, genetic background, medical condition, disease history, and treatment history of the subject to whom the therapeutic agent is to be administered; the disease or condition to be treated or to be improved; the treatment outcome to be achieved; the dosage, frequency of administration, and duration of administration of the therapeutic agent; the pharmacokinetics and pharmacodynamics of the therapeutic agent; and the route by which the therapeutic agent is administered. In some embodiments, "temporal proximity" means within 15 minutes, within 30 minutes, within one hour, within two hours, within four hours, within six hours, within eight hours, within 12 hours, within 18 hours, within 24 hours, within 36 hours, within 2 days, within 3 days, within 4 days, within 5 days, within 6 days, within one week, within 2 weeks, within 3 weeks, within 4 weeks, within 6 weeks, or within 8 weeks. In some embodiments, multiple administrations of one therapeutic agent can occur in temporal proximity to a single administration of another therapeutic agent. In some embodiments, temporal proximity can change during a treatment cycle or within a dosing regimen.

[0325] As used herein, "kit" refers to a combination of components, such as a composition herein and another item for purposes including, but not limited to, reconstitution, activation, and an instrument / device for delivery, administration, diagnosis, and assessment of biological activity or properties. The kit optionally includes instructions for use.

[0326] As used herein, "prior line platinum-containing regimens" include:

[0327] i. Adjuvant ± neoadjuvant is considered as 1st line of therapy as long as they are the same regimen (eg, 4 cycles of platinum / taxane before surgery followed by 4 cycles of platinum / taxane after surgery).

[0328] ii. Maintenance therapy (e.g. bevacizumab, PARPi or endocrine therapy) was considered part of the previous line of therapy. Substitution to a different platinum agent or taxane was not counted as a new line.

[0329] iii. Therapy given for only 1 cycle in the absence of progression and stopped due to toxicity will not be counted as a line of therapy. Substitution to a different platinum agent or taxane will not be counted as a new line.

[0330] iv. Hormonal therapy (eg, tamoxifen, letrozole) counts as a single line of therapy unless it is given as maintenance.

[0331] As used herein, "recurrent disease" refers to a subject with disease progression after a partial or complete remission to one or more therapeutic agents. In some embodiments, recurrence may be confined to the original site of disease (i.e., one or more ovaries) or at a distant or metastatic location.

[0332] The present disclosure is intended to include all isotopes of atoms present in the present compounds. Isotopes include those atoms having the same atomic number but different mass numbers. By way of general example and not limitation, isotopes of hydrogen include tritium and deuterium. Isotopes of carbon include C-13 and C-14.

[0333] The present disclosure is intended to include all isomers of the compounds, which refers to and includes optical isomers and tautomers, wherein optical isomers include enantiomers and diastereomers, chiral isomers and non-chiral isomers, and optical isomers include isolated optical isomers and mixtures of optical isomers, including racemic and non-racemic mixtures; wherein the isomers may be in isolated form or in a mixture with one or more other isomers.

[0334] Unless otherwise indicated, all percentages and ratios used herein are by body weight. Other features and advantages of the present disclosure are apparent from the various examples. The examples provided illustrate the different components and methods that can be used to practice the present disclosure. The examples do not limit the disclosure claimed. Based on the present disclosure, the skilled person can identify and adopt other components and methods that can be used to practice the present disclosure.

[0335] Other Implementations

[0336] All publications and patent documents cited herein are incorporated herein by reference, as if each such publication or document was specifically and individually indicated to be incorporated herein by reference. The citation of publications and patent documents is not intended as an admission that any publication and patent document is relevant prior art, nor does it constitute any admission as to the contents or date of the publications and patent documents. Having now described the invention by written description, those skilled in the art will recognize that the invention can be implemented in various embodiments, and the foregoing description and the following examples are for the purpose of illustrating, not limiting, the appended claims.

[0337] Example

[0338] The following examples are illustrative and are not intended to be limiting, and one skilled in the art will readily appreciate that other reagents or methods may be utilized.

[0339] abbreviation

[0340] The following abbreviations are used in the following reaction schemes and synthetic examples. This list is not meant to be an all-inclusive list of abbreviations used in this application, as additional standard abbreviations readily understood by one skilled in the art of organic synthesis may also be used in the synthetic schemes and examples.

[0341]

[0342]

[0343]

[0344] General Information

[0345] Antibody-drug conjugates targeting B7-H4 (eg, XMT-1660) were prepared as described in U.S. Patent Application No. 17 / 568,376.

[0346] CDRs are identified by the Kabat numbering scheme.

[0347] Complementarity determining regions (CDRs) are defined according to the IMGT numbering system (see International Immunogenetics Information Available online: http: / / www.imgt.org / ).

[0348] Example 1: Study Design and Methods

[0349] The single-arm study presented herein is a first-in-human study of an antibody-drug conjugate targeting B7-H4 (XMT-1660) in a Phase 1b open-label trial in previously treated participants with metastatic TNBC, HR+ / HER2- breast cancer, endometrial or ovarian cancer, fallopian tube cancer, or primary peritoneal cancer. The study consists of 2 parts: a dose escalation (DES) part and an expansion (EXP) part. The DES part of the study is a dose finding cohort to evaluate the tolerability and safety of XMT-1660 and determine the maximum tolerated dose (MTD) and / or the recommended dose for Phase 2 (RP2D). The EXP part of the study further evaluates the preliminary efficacy and safety of XMT-1660 at the MTD and / or RP2D in participants with advanced / metastatic (1) TNBC; (2) HR+ / HER2- breast cancer; and (3) endometrial cancer, ovarian cancer, fallopian tube cancer, or primary peritoneal cancer.

[0350] The Bayesian optimal interval (BOIN) design is used to determine the MTD in the dose escalation part of the study, wherein the target DLT rate is set at 30%. Seven dose levels are planned to be studied, but the intermediate dose level can be added based on the emerging data. Dose escalation is completed according to the dose escalation scheme in Table 1. Initially, XMT-1660 is intravenously administered every 3 weeks, but, based on the available data, an alternative cycle length (such as Q4W) can be introduced under the recommendation of the sponsor and the Safety Review Committee. The same period group of a single participant is used for the first dose level and for dose level 1.5 (if it is open). All the same period groups of subsequent dose levels have 3 participants.

[0351] Table 1

[0352] Dosage Levels <![CDATA[XMT-1660 dose (mg / m 2 )]]> <![CDATA[1 a,b (N=1)]]> 7.2 2(N＝3) 14.4 3 21.6 4 28.7 5 38.1 6 50.7 7 67.4 8 87.6

[0353] a: For DL1, if non-DLT drug-related grade ≥2 AEs (excluding grade 2 nausea or fatigue that resolved within 7 days) were observed during the dose-limiting toxicity (DLT) period, the dose level for the next cohort was increased by 50% (DL1.5) instead of 100% (DL2).

[0354] b: Single participant enrolled in DL1 and DL1.5 (if open). However, if DLT is observed, 2 additional participants are added at the same dose level.

[0355] Eight dose levels are planned to be studied, but intermediate dose levels may be added based on observed data.

[0356] Once the MTD dose is determined, additional participants are enrolled in the MTD and MTD DL-1 cohorts, for a total of approximately 15 participants in each cohort.

[0357] The SRC meets throughout the study to discuss ongoing dose escalation. The SRC reviews the data outlined in the charter, including but not limited to safety, PK, and efficacy at the MTD and MTD DL-1 cohorts to determine the RP2D.

[0358] Approximately 30 or 35 participants were enrolled in each cohort. Based on emerging data, the sample size of each cohort may be increased to enroll up to approximately 100 participants.

[0359] RP2D is determined based on the totality of clinical data including safety, preliminary anti-tumor effect, PK and relevant biomarker data. 30 or 35 participants are enrolled in each expansion cohort participant population. Additional participants may be enrolled in each of these populations based on emerging data.

[0360] In both the DES and EXP parts, blood sampling was performed to determine the levels of total antibody (XMT-1604), conjugated and free AF-HPA, and primary metabolites of AF-HPA (AF). Pharmacokinetic parameters were then determined from these analytes. Tests for anti-drug antibodies (ADA) and neutralizing antibodies (nAb) were performed.

[0361] If emerging data suggest that q3w dosing results in excessive toxicity, an alternative schedule may be evaluated. If an alternative schedule is evaluated, the starting dose is the dose that does not exceed the maximum tolerated dose (MTD) using the q3w dosing schedule, and dose escalation continues using the guidelines listed in Table 1.

[0362] Duration of treatment:

[0363] Treatment with XMT-1660 administered intravenously every 3 weeks can continue indefinitely unless one of the following occurs:

[0364] Disease progression

[0365] Unacceptable adverse events

[0366] ● Participants withdraw consent to participate in the study

[0367] - A general or specific change in the participant's condition renders the participant unacceptable for further treatment.

[0368] Number of participants

[0369] In the DES part of the study, eight dose levels are planned to be evaluated, which requires a maximum sample size of 42 participants to determine the MTD. However, the sample size can be increased based on the actual number of dose levels of emerging data and evaluation. Once the MTD is determined, other participants are enrolled under the MTD and MTD DL-1 so that these dose levels each have about 15 participants.

[0370] In the expansion (EXP) portion of the trial, 30, 35, and 35 participants with advanced / metastatic (1) TNBC; (2) HR+ / HER2- breast cancer; and (3) endometrial, ovarian, fallopian tube, or primary peritoneal cancer were enrolled and treated at the MTD and / or RP2D determined in the dose escalation portion of the study, respectively. Based on emerging data, the sample size for each cancer population may be increased to enroll up to approximately 100 participants. Further decisions to enroll additional participants are made based on the full data.

[0371] Further enrollment with different dosing schedules, biomarker enrichment, or subpopulations may be considered. Additional expansion cohorts may also be opened based on emerging data.

[0372] Target

[0373] The primary and secondary objectives for dose escalation and dose expansion are given below.

[0374] The primary goals of dose escalation are:

[0375] ●Determine the maximum tolerated dose (MTD) and / or recommended phase 2 dose (RP2D) of XMT-1660

[0376] ●Evaluate the safety and tolerability of XMT-1660

[0377] Secondary objectives of dose escalation:

[0378] ●Evaluate the preliminary anti-tumor activity of XMT-1660

[0379] Primary goal of dose expansion

[0380] ● Further evaluate the safety and tolerability of XMT-1660 at the MTD and / or RP2D

[0381] ●Evaluate the preliminary anti-tumor activity of XMT-1660 at the MTD and / or RP2D by measuring ORR and DOR based on RECIST 1.1

[0382] Secondary objectives of dose expansion:

[0383] ●Evaluate the PK of XMT-1660, its release products and selected metabolites

[0384] ● Evaluate the development of anti-drug antibodies (ADA) and neutralizing antibodies (nAb) targeting XMT-1660

[0385] Exploratory objectives:

[0386] ●Evaluate tumor expression of B7-H4 and PD-L1 by IHC, as determined by tumor type

[0387] ●Assess the correlation between the level of B7-H4 expression and the anti-tumor activity of XMT-1660

[0388] ●Evaluate the relationship between the expression of other markers related to cancer or inflammation and clinical outcomes

[0389] ● Evaluate the population pharmacokinetic (pop PK) characteristics of XMT-1660 and determine any pop PK exposure / response relationships

[0390] Eligibility

[0391] To be eligible for enrollment in this study, all participants must meet all inclusion criteria as defined below and not meet any exclusion criteria:

[0392] Main criteria for eligibility:

[0393] The general inclusion criteria for dose escalation and dose expansion are the following. Participants are eligible for the study if all of the following criteria are met:

[0394] 1. Participants have confirmed recurrent or advanced solid tumors and have disease progression after treatment with available anticancer therapies known to confer benefit or are intolerant to treatment.

[0395] 2. Participants must be at least 18 years old.

[0396] 3. Participants must have an ECOG performance status of 0 or 1.

[0397] 4. Participants must be able to understand the research procedures and agree to participate in the study by providing written informed consent.

[0398] 5. Participants must have a cardiac left ventricular ejection fraction (LVEF) of ≥50% or ≥ the institution's lower limit of normal by ECHO or MUGA scan.

[0399] 6. Participants with toxicity from prior therapy or surgery must have recovered to ≤ Grade 1. Participants with alopecia, Grade 2 peripheral sensory neuropathy, stable immune-related toxicity (e.g., hypothyroidism on hormone replacement therapy), or adrenal insufficiency treated with ≤10 mg of prednisone (or equivalent) per day are exceptions to this criterion and may be eligible for this study.

[0400] 7. Participants must have adequate organ function within 14 days prior to Cycle 1 Day 1, as defined by the following criteria:

[0401]

[0402] 8. During the study, female study participants of childbearing potential (WOCBP) must use a contraceptive method that is highly effective (failure rate <1% / year when used correctly and consistently) during study participation and for 6 months after receiving the last dose of XMT-1660.

[0403] 9. Male study participants must use barrier contraception (condoms) and refrain from sperm donation for the duration of study drug treatment and for at least 6 months after the last dose of study drug. Spouses of male study participants considered WOCBP must use highly effective contraceptive methods for the same duration.

[0404] 10. Participants must have at least one measurable lesion as defined by RECIST version 1.1.

[0405] 11. If medically feasible, participants must be willing to undergo a minimally invasive tumor biopsy to obtain tumor tissue for local testing prior to C1D1. If the investigator feels a biopsy is not medically feasible, a waiver request must be submitted to the study medical monitor for approval. In addition, if available, archived tissue samples should be submitted to a central laboratory to enable longitudinal analysis of tumor biomarkers. Note: Confirmation of B7-H4 expression is not required for study enrollment.

[0406] The general exclusion criteria for dose escalation and expansion are as follows. Participants are ineligible for the study if any of the following criteria are met:

[0407] 1. Participants have received prior treatment with another ADC containing an auristatin or maytansinoid payload (e.g., mirvetuximab).

[0408] Note: Prior treatment with another ADC containing other payloads (non-auristatin or non-maytansinoid) (e.g., sacituzumab govitecan, fam-trastuzumab deruxtecan-nxki) was allowed.

[0409] 2. Participants have undergone major surgery within 28 days of starting study treatment, received systemic anticancer therapy within 28 days or 5 half-lives of the previous therapy (14 days or 5 half-lives for small molecule targeted therapy) (whichever is smaller) before starting study treatment, or received palliative radiotherapy within 14 days of starting study treatment.

[0410] 3. A diagnosis of another malignancy requiring active treatment (including surgery, systemic therapy, and radiation) within 2 years prior to screening, except for adequately treated basal cell or squamous cell skin cancer or carcinoma in situ of the breast or cervix.

[0411] 4. Participants have untreated hepatitis B virus (HBV) or hepatitis C virus (HCV). In addition, negative serology is required during screening (baseline) for HBV and HCV:

[0412] ●HBV: Eligible participants with HBV infection must have HBsAG(-) and HBcab(+) serology with viral load below the limit of quantification.

[0413] ●HCV: Participants with a history of HCV infection should have completed curative antiviral therapy and have an HCV viral load below the limit of quantification.

[0414] 5. Participants with human immunodeficiency virus (HIV) infection.

[0415] 6. Participants have a history of cirrhosis, liver fibrosis, esophageal or gastric varices, or other clinically significant liver disease. Liver function tests beyond laboratory studies otherwise defined in the eligibility criteria were performed to diagnose potential clinically significant liver disease based on risk factors such as hepatic steatosis or history of excessive alcohol intake, based on the investigator's clinical judgment.

[0416] 7. Participants may not receive medications associated with hepatotoxicity while on XMT-1660. Participants may receive acetaminophen / paracetamol for a limited time but the total daily dose is ≤2 g / day. Use of NSAIDs or steroids to treat fever is encouraged.

[0417] 8. Participants have current severe uncontrolled systemic illness (e.g., clinically significant cardiovascular, pulmonary, or metabolic disease) or intercurrent illness that may interfere with per-protocol assessments. In addition, participants with the following characteristics are excluded:

[0418] ● Significant baseline prolongation of the QT / QTcF interval (CTCAE grade >1): Repeated demonstration of a QTcF interval >480 milliseconds (ms) using Fridericia's QT correction formula.

[0419] ●History of additional risk factors for Torsades de Pointes (eg, family history of long QT syndrome).

[0420] 9. Participants are receiving concurrent anti-cancer therapy (e.g., chemotherapy, radiotherapy, biological therapy, immunotherapy, hormone therapy, investigational therapy).

[0421] 10. The participant is unable or unlikely to comply with the dosing regimen and study assessments.

[0422] 11. Participants have a history of pneumonia or interstitial lung disease or are suspected of having pneumonia or interstitial lung disease.

[0423] 12. Participants are currently using continuous or intermittent supplemental oxygen therapy.

[0424] 13. The participant's oxygen saturation is < 93% on room air.

[0425] 14. Participants are currently taking strong CYP450 3A inhibitors or inducers that cannot be discontinued while receiving study treatment. Participants currently taking these medications must stop within 14 days of the first dose of study drug.

[0426] 15. Participants have a history of untreated CNS metastases (including new and progressive brain metastases), leptomeningeal metastases, or carcinomatous meningitis.

[0427] a. Participants were eligible if CNS metastases were adequately treated and participants were neurologically stable for at least 2 weeks prior to enrollment.

[0428] b. In addition, participants must be off corticosteroids or on a stable / reduced dose of ≤ 10 mg per day of prednisone (or equivalent). Anticonvulsants are permitted except for those associated with hepatotoxicity.

[0429] Disease-specific inclusion criteria for dose escalation are summarized in Table 2 .

[0430] Table 2

[0431]

[0432]

[0433] For the dose expansion cohort, the eligibility criteria for hormone receptor-positive (HR+), human epidermal growth factor receptor 2-negative (HER2-) breast cancer (HR+ / HER2-BC) were:

[0434] 1. Participants have a histologically or cytologically confirmed diagnosis of breast cancer with evidence of advanced or metastatic disease for whom the experimental therapy is appropriate, as determined by the treating physician.

[0435] 2. Participants had documentation of ER-positive and / or PR-positive breast cancer and HER2-negative tumors based on local testing of a recent tumor biopsy as defined in the relevant American Sociely of Clinical Oncology (ASCO) / College of Amencan Pathologists (CAP) guidelines.

[0436] 3. Participants were required to have received prior therapy with a combined CDK 4 / 6 inhibitor and endocrine therapy in any setting.

[0437] 4. Participants have not received more than 2 lines of prior chemotherapy for advanced or metastatic disease. For participants with BRCA mutations, treatment with PARP inhibitors is allowed and does not count as chemotherapy.

[0438] Eligibility criteria for the triple-negative breast cancer (TNBC) dose expansion cohort were:

[0439] 1. Participants have a histologically or cytologically confirmed diagnosis of breast cancer with evidence of locally advanced or metastatic disease for whom the experimental therapy is appropriate, as determined by the treating physician.

[0440] 2. Participants had documentation of estrogen receptor (ER)-negative, progesterone receptor (PR)-negative, and HER2-negative breast cancer based on local testing of a recent tumor biopsy as defined in the relevant American Society of Clinical Oncology (ASCO) / College of American Pathologists (CAP) guidelines.

[0441] 3. Participants have received 1-3 prior lines of chemotherapy for the metastatic setting. For participants with BRCA mutations, treatment with PARP inhibitors is allowed and chemotherapy is not counted.

[0442] There were no disease-specific exclusion criteria for the HR+ / HER2- BC or TNBC cohorts.

[0443] Eligibility criteria for the endometrial cancer dose-expansion cohort were:

[0444] 1. Participants with a histologically or cytologically confirmed diagnosis of endometrial carcinoma with evidence of metastatic or locally advanced disease who have no satisfactory treatment options and for whom an experimental therapy is appropriate as determined by the treating physician.

[0445] 2. Received at least 1 prior line of platinum-based doublet combination chemotherapy and no more than 3 prior lines of systemic therapy for recurrent or metastatic disease, excluding hormonal therapy.

[0446] Participants enrolled in the endometrial cancer cohort had to be free of endometrial sarcoma or carcinosarcoma.

[0447] Inclusion criteria for the ovarian, fallopian tube, or primary peritoneal cancer dose expansion cohort were:

[0448] 1. Histological diagnosis of high-grade serous ovarian cancer, including metastatic or recurrent fallopian tube cancer or primary peritoneal cancer.

[0449] 2. Participants must have platinum-resistant relapsed disease, defined as completion of 4 or more cycles of platinum-based therapy and radiographic progression within 6 months of the last platinum-based therapy.

[0450] a. For participants with 1 prior line of chemotherapy, they must have achieved a complete or partial response to that therapy and had their disease progress between 3 months and 6 months after the last dose of a platinum-based setting in the first-line setting.

[0451] b. For participants with 2 to 4 prior lines of chemotherapy, disease progression within less than or equal to 6 months after the last dose of platinum in the most recent chemotherapy.

[0452] 3. 1 to 4 prior lines of systemic therapy for ovarian cancer, including at least 1 prior line of platinum-containing regimen.

[0453] ●Definition of prior line of therapy:

[0454] i. Adjuvant ± neoadjuvant is considered 1st line therapy as long as they are the same regimen (eg, 4 cycles of platinum / taxane before surgery followed by 4 cycles of platinum / taxane after surgery).

[0455] ii. Maintenance therapy (e.g. bevacizumab, PARPi or endocrine therapy) was considered part of the prior line of therapy. Substitution to a different platinum agent or taxane was not counted as a new line.

[0456] iii. Therapy given for only 1 cycle in the absence of progression and stopped due to toxicity will not be counted as a line of therapy. Substitution to a different platinum agent or taxane will not be counted as a new line.

[0457] iv. Hormonal therapy (eg, tamoxifen, letrozole) counts as a single line of therapy unless it is given as maintenance.

[0458] Exclusion criteria for the ovarian, fallopian tube, or primary peritoneal cancer dose expansion cohort were:

[0459] 1. Participants to be included in the ovarian cancer cohort must not have low-grade, clear cell, endometrioid, mucinous, carcinosarcoma, germ cell, or stromal tumors.

[0460] 2. Participants to be included in the ovarian cancer cohort must not have primary platinum-resistant disease, defined as lack of response or progression within 3 months after completion of first-line platinum-containing therapy.

[0461] Evaluation criteria:

[0462] Security

[0463] The frequency and grade of AEs and SAEs were based on the National Cancer Institute (NCI) Common Terminology Criteria version 5.0 (CTCAE v5.0).

[0464] Changes in clinical laboratory parameters (hematology, chemistry, urinalysis), vital signs, ECOG performance status, ECG parameters, physical examination, and concomitant medications.

[0465] Clinical activities

[0466] Tumor response (ORR, DOR) and disease control rate (DCR) were assessed by the investigator at the end of cycle 2 and every 2 cycles thereafter using Response Evaluation Criteria in Solid Tumors (RECIST), version 1.1.

[0467] Pharmacokinetics

[0468] Pharmacokinetics of XMT-1660 (C max , C trough ,t max , AUC, AUC last , AUC ∞ ,t 1 / 2, CL and V ss )

[0469] - In both the DES and EXP fractions, blood sampling was performed to determine the levels of total antibody (XMT-1604), conjugated and free XMT-1267, and the primary metabolite of XMT-1267 (XMT-1521). Pharmacokinetic parameters were then determined from these analytes.

[0470] ●Tests for anti-drug antibodies (ADA) and neutralizing antibodies (nAb) are performed.

[0471] • Population pharmacokinetic and population PK exposure / response analyses were also performed.

[0472] Dose-limiting toxicity (DLT)

[0473] The DLT observation period is the time between the start of the Cycle 1 infusion of XMT-1660 and the completion of Cycle 1 (e.g., 21 days for Q3W or 28 days for Q4W) and should include the pre-dose assessment prior to receiving a dose of Cycle 2. Participants considered evaluable for tolerability must have received at least 80% of their planned dose and completed Cycle 1 unless the dose reduction, interruption, or discontinuation was the result of a DLT.

[0474] A DLT was defined as any toxicity occurring during the DLT observation period that was not attributable to disease progression or another clearly identifiable cause detailed in the protocol.

[0475] Rationale for the study:

[0476] Despite advances in novel therapies that confer benefits in progression-free survival (PFS) and overall survival (OS), most patients with unresectable or recurrent / metastatic breast cancer eventually acquire resistance to these therapies and die from their disease. Therefore, unresectable or recurrent / metastatic TNBC and HR+ / HER2- breast cancer remain serious and life-threatening with unmet medical needs.

[0477] Notably, B7-H4 protein expression is observed in various human tumors, such as breast cancer, endometrial cancer, and ovarian cancer. Furthermore, B7-H4 expression in tumor cells of these malignancies appears to correlate with disease progression and poor prognosis. B7-H4 is expressed in ˜77% of TNBC and ˜60% of HR+ and HER2+ tumors. B7-H4 expression is high in ˜45% of breast cancers and is independent of ER / HER2 status. Furthermore, it is reported to be widely expressed in endometrial and ovarian cancers.

[0478] The hypothesis is that by targeting B7-H4, Dolasynthen ADC (XMT-1660) confers clinical benefit to patients with unresectable or recurrent / metastatic TNBC following standard chemotherapy and HR+ / HER2- following CDK 4 / 6 inhibitors and endocrine-based therapies. To test this hypothesis, a first-in-human study with XMT-1660, a B7-H4-directed antibody conjugated to an auristatin payload, was initiated in tumors that are likely to express B7-H4. The primary objective is to evaluate the safety and tolerability of XMT-1660 administered intravenously as a single agent until disease progression or unacceptable toxicity in previously treated participants with TNBC, HR+ / HER2- breast cancer, endometrial and ovarian cancer, fallopian tube cancer, or primary peritoneal cancer. Secondary objectives are to obtain pharmacokinetics, immunogenicity, and preliminary efficacy for this dosing regimen. Exploratory objectives will be to explore the association of target expression in tumors and other markers associated with cancer or inflammation.

[0479] Example 2: Administration of XMT-1660

[0480] Antibody-drug conjugates targeting B7-H4 (e.g., XMT-1660) are administered based on body surface area (BSA). BSA-adjusted doses will be calculated following the standard practice of each institution. Mosteller formula is used when possible. The starting dose is calculated based on height and weight collected within 14 days of the first dose (which may be collected on the day of the first dose). For weight changes of ≥5% from the most recent dose calculation, dose calculations should be performed in subsequent cycles. Dose adjustments based on subsequent weight measurements should be made according to the standard practice of each institution. If the institution does not have a standard practice, additional weight measurements and confirmed or changed BSA will be obtained prior to dosing in Cycle 2 and every 2 cycles thereafter.

[0481] XMT-1660 is administered intravenously via an antecubital or indwelling intravenous catheter. The initial dose for each participant is administered over 90 minutes. If no IRR occurs, all subsequent doses can be administered over 60 (± 10) minutes at the discretion of the investigator. Subsequent infusions can be administered on an outpatient basis as long as tolerated, but during cycles 1-3, participants are monitored for safety assessments and PK sampling approximately 6 hours after XMT-1660 administration.

[0482] In the EXP portion of the study, once a participant completes Cycle 1, dose adjustments may be made at the investigator's discretion if the participant experiences toxicity but would benefit from further treatment with XMT-1660.

[0483] Example 3: Analysis

[0484] The safety analysis set included all participants who received any amount of XMT-1660 dose (partial or complete) in DES and EXP. All safety analyses were performed in the safety analysis set, except for the assessment of tolerability. Participants considered evaluable for tolerability must have received at least 80% of their planned dose of XMT-1660 and completed Cycle 1, unless dose reduction, interruption, or discontinuation was the result of a DLT.

[0485] Enrolled participants who received any amount of their assigned dose of XMT-1660 and had a sufficient number of concentration measurements to allow PK calculations included the PK analysis set. Analytical results for PK concentration data per dose level, per indication, and per participant are provided.

[0486] The Efficacy Response Evaluable Analysis Set (EFREAS) included a subset of the Efficacy Analysis Set for whom a baseline response assessment and at least one post-baseline response assessment were available.

[0487] Security Analysis

[0488] At the end of the DES phase of the trial, the MTD was chosen as the dose for which the isosmotic estimate of the probability of DLT was closest to the target DLT rate.The isosmotic estimate of the probability of DLT was obtained using a pooled adjacent violators algorithm.

[0489] The number and percentage of participants who experienced DLTs are reported by dose level and across all dose levels in the DES phase of the study.

[0490] Participants who completed Cycle 1 without DLT were considered tolerating the XMT-1660 regimen. The number and percentage of participants who tolerated the XMT-1660 regimen were reported by dose level and across all dose levels in the subgroup of participants who were evaluable for tolerability in the DES phase of the study.

[0491] In the DES phase of the study, all AEs and adverse events of clinical interest (AECI) were reported by dose level and across all dose levels. In the extension phase, all AEs and AECI were reported by cancer population. The relationship of the severity, CTCAE severity grading and investigator-reported adverse events to the study drug was summarized and listed. Safety laboratory data were summarized and listed.

[0492] Preliminary efficacy analysis

[0493] Both DES and EXP data were used for preliminary efficacy analysis. The primary endpoint was the ORR reviewed by the investigator radiology, and it was defined as the proportion of participants achieving a PR or CR confirmed according to RECIST v1.1. The secondary endpoint was DCR, which was defined as the proportion of participants achieving complete remission, partial remission and / or stable disease of any duration according to RECIST v1.1. The number and percentage of participants achieving remission or clinical benefit were summarized, and an exact 95% confidence interval (CI) was provided.

[0494] Analyses of other efficacy endpoints including DOR were also reported whenever possible according to standard response criteria. Median and quartile Kaplan-Meier estimates with 95% CI were reported for these statistics. Efficacy endpoints were analyzed using both EFAS and EFREAS in each cancer type cohort.

[0495] Pharmacokinetic analysis

[0496] The PK characteristics of the active moiety / released products / metabolites of XMT-1660 for each participant were determined by non-compartmental analysis using validated PK software (i.e., Phoenix WinNonlin). The PK parameters for each participant included the time to maximum observed concentration (t max ), maximum concentration (C max ) and the area under the concentration curve (AUC 0-last When the terminal elimination phase can be identified, additional parameters are determined, such as terminal elimination (t 1 / 2), clearance (CL) and volume of distribution (Vss).

[0497] Details on the handling of missing concentration and covariate data, outliers, and values ​​below the limit of quantitation, as well as modeling of dose-response and PK parameter-response relationships were provided in a separate clinical pharmacology analysis plan.

[0498] Objective response rate and measurement of B7-H4 expression

[0499] B7-H4 expression status was confirmed by central laboratory testing. ORR and DOR (and associated CI) are reported per participant stratified by B7-H4 status.

[0500] Example 4: Tumor Growth Response to Administration of XMT-1660 in the CTG-1280 Patient-Derived Endometrial Cancer Xenograft Mouse Model

[0501] Female athymic nude mice were implanted subcutaneously with CTG-1280 patient-derived endometrial cancer tumor fragments. 3 (Average value = 221mm 3 Animals were randomly divided into treatment groups at 1:10 / group (n=10 / group). Vehicle, rituximab isotype control conjugate (prepared as described in U.S. Patent Application No. 2022 / 0233707A1, 4.60 / 0.150 mg / kg), XMT-1660 (4.68 / 0.150 mg / kg, DAR 6) were administered intravenously on day 1 (all doses are given by antibody / payload). No significant weight loss or clinical observations were noted.

[0502] Figure 1 Results are provided for tumor volume of mice bearing CTG-1280 tumors treated with isotype control conjugates and XMT-1660. On day 17, treatment with 4.60 / 0.150 mg / kg of isotype control conjugate had no significant effect on tumor volume compared to vehicle control (14% TGI, adjusted p=0.5839). On day 17, treatment with 4.68 / 0.150 mg / kg of XMT-1660 caused a significant reduction in tumor volume compared to the vehicle control group (73% TGI, adjusted p<0.00001).

[0503] Example 5: Tumor Growth Response to Administration of XMT-1660 in the OV2423 Patient-Derived Ovarian Cancer Xenograft Mouse Model

[0504] OV2423 patient-derived ovarian cancer tumor fragments (~2-3 mm) were implanted subcutaneously in female BALB / c nude mice. 3 / mouse). When the tumor volume was 101-230mm 3 (Average value = 155mm 3Animals were randomly divided into treatment groups at 1:1 (n=8 / group). Vehicle, palivizumab isotype control conjugate (prepared as described in U.S. Patent Application No. 2022 / 0233707A1, 4.54 / 0.150 mg / kg) and XMT-1660 (4.61 / 0.150 mg / kg, DAR 6) were administered intravenously on day 1 (all doses are given by antibody / payload). No significant weight loss or clinical observations were noted.

[0505] Figure 2 Results of tumor volume of mice bearing OV2423 tumors treated with isotype control and XMT-1660 are provided. On day 22, treatment with palivizumab isotype control conjugate (4.54 / 0.150 mg / kg) had no significant effect on tumor volume compared to vehicle control (24.8% TGI, adjusted p=0.977). On day 22, XMT-1660 administered at 4.61 / 0.150 mg / kg caused a significant reduction in tumor volume (124.05% TGI, adjusted p<0.001) and caused 1PR and 7TFS compared to the vehicle control group.

[0506] Example 6: Tumor Growth Response to Administration of XMT-1660 Cytotoxic Drug Conjugate in the CTG-1692 Patient-Derived Ovarian Cancer Xenograft Mouse Model

[0507] Female athymic nude mice were implanted subcutaneously with CTG-1692 patient-derived ovarian cancer tumor fragments. 3 (Average value = 229mm 3 Animals were randomly divided into treatment groups at 1:100 (n = 8 / group). Vehicle, palivizumab isotype control conjugate (2.27 / 0.075 or 4.54 / 0.150 mg / kg, DAR 6) or XMT-1660 (2.30 / 0.075 or 4.60 / 0.150 mg / kg, DAR 6) were administered intravenously on day 1 (all doses are given by antibody / payload), which were prepared as described in U.S. Patent Application No. 2022 / 0233707A1. No significant weight loss or clinical observations were noted.

[0508] Figure 3Results are provided for tumor volume of mice bearing CTG-1692 tumors treated with isotype control conjugates or XMT-1660. At day 49, treatment with 2.27 / 0.075 mg / kg or 4.54 / 0.150 mg / kg of isotype control conjugates had no significant effect on tumor volume compared to vehicle control (TGI = -48%, adjusted p = 0.2638, or TGI = 8%, adjusted p = 0.9973, respectively). At day 49, treatment with 2.30 / 0.075 mg / kg of XMT-1660 resulted in a significant reduction in tumor volume (TGI = 99%, adjusted p = 0.0023) and a 1PR compared to the vehicle control group. Treatment with 4.60 / 0.150 mg / kg of XMT-1660 resulted in a significant reduction in tumor volume compared to the vehicle control group on day 49 (TGI = 92%, adjusted p = 0.0058). Treatment with both dose levels of the isotype control conjugate at 4.60 / 0.150 mg / kg and XMT-1660 resulted in no CR, PR or TFS.

[0509] Example 7: Tumor Growth Response to Administration of XMT-1660 Cytotoxic Drug Conjugate in the mBR9013 Syngeneic Breast Cancer Mouse Model

[0510] Female FVB / NJ mice were implanted subcutaneously with mBR9013 tumor fragments (2-3 mm 3 ). When the tumor volume is 96-277mm 3 (Average value = 148 mm 3 Animals were randomly divided into treatment groups when the subjects received 100 mg / kg of palivizumab isotype control conjugates (1.51 / 0.050 or 4.54 / 0.150 mg / kg), XMT-1660 (1.53 / 0.050 or 4.60 / 0.150 mg / kg, DAR 6) (prepared as described in U.S. Patent Application No. 2022 / 0233707A1), anti-PD-1 (10.0 / 0 mg / kg) and XMT-1660+anti-PD-1 (1.53 / 0.050 mg / kg+10.0 / 0 mg / kg) (all doses are given by antibody / payload) on day 1. Vehicle, isotype control conjugates and XMT-1660 were administered intravenously. Starting from day 1, anti-PD-1 was given as an intraperitoneal dose twice a week for 3 weeks. No significant weight loss or clinical observations were noted.

[0511] Figure 4Results of tumor volume of mice bearing mBR9013 tumors treated with isotype control conjugates, XMT-1660 and anti-PD-1 are provided. On day 15, treatment with 1.51 / 0.050mg / kg or 4.54 / 0.150mg / kg of palivizumab isotype control conjugate had no significant effect on tumor volume compared to vehicle control (TGI=-7.91%, adjusted p=0.999 or TGI=32.02%, adjusted p=0.857, respectively). On day 15, treatment with 1.53 / 0.050mg / kg or 4.60 / 0.150mg / kg of XMT-1660 caused a significant reduction in tumor volume compared to vehicle control group (TGI=92.69%, adjusted p=0.000276 or TGI=119.29%, adjusted p=1.45e-08, respectively). On day 15, treatment with 10.0 / 0 mg / kg of anti-PD-1 had no significant effect on tumor volume compared to vehicle control (TGI = -9.10%, adjusted p = 0.999). On day 15, combined treatment with 1.53 / 0.050 + 10.0 / 0 mg / kg of XMT-1660 + anti-PD-1 caused a significant reduction in tumor volume compared to vehicle control (TGI = 100.99%, adjusted p = 2.34e-05).

[0512] Other Implementations

[0513] 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 embodiments, advantages, and modifications are within the scope of the appended claims.

Claims

1. A method of treating triple-negative breast cancer (TNBC), hormone receptor-positive / HER2-negative (HR+ / HER2-) breast cancer, endometrial cancer, ovarian cancer, fallopian tube cancer, or primary peritoneal cancer in a subject, the method comprising administering about 7.0 mg / m 2 on the first day of treatment and every three weeks thereafter. 2 About 90 mg / m 2 administering an antibody-drug conjugate targeting B7-H4 to a subject by infusion at a dose of The antibody-drug conjugate targeting B7-H4 is a conjugate of formula (I): in id 13 is about 2; ii. the antibody binds to B7-H4 and comprises: a variable heavy chain complementary determining region 1 (CDRH1) comprising the amino acid sequence GFIVSRNY (SEQ ID NO:2), a variable heavy chain complementary determining region 2 (CDRH2) comprising the amino acid sequence IYGSGRT (SEQ ID NO:3), a variable heavy chain complementary determining region 3 (CDRH3) comprising the amino acid sequence ARDADYGLDV (SEQ ID NO:4), a variable light chain complementary determining region 1 (CDRL1) comprising the amino acid sequence QSVSSSY (SEQ ID NO:5), a variable light chain complementary determining region 2 (CDRL2) comprising the amino acid sequence GAS (SEQ ID NO:6), and a variable light chain complementary determining region 3 (CDRL3) comprising the amino acid sequence QQYGSSPLYT (SEQ ID NO:7), iii. the drug is attached to the heavy chain of the antibody via a linker moiety at position 297 when numbered according to EU numbering; and iv. ■ is GlcNAc; △ is Fuc; and □ is GalNAc. 2 . The method of claim 1 , wherein the antibody comprises: a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 9 and a variable light chain comprising the amino acid sequence of SEQ ID NO:

8. 3 . The method of claim 1 , wherein the antibody comprises: a heavy chain comprising the amino acid sequence of SEQ ID NO: 13 and a light chain comprising the amino acid sequence of SEQ ID NO:

12.

4. The method according to any one of claims 1 to 3, wherein the antibody-drug conjugate dosage is: About 6.7 mg / m 2 To about 7.7mg / m 2 , About 13.9 mg / m 2 To about 14.9mg / m 2 , About 21.1 mg / m 2 To about 22.1mg / m 2 , About 28.2 mg / m 2 To about 29.2mg / m 2 , About 37.6 mg / m 2 To about 38.6mg / m 2 , About 50.2 mg / m 2 To about 51.2mg / m 2 , About 66.9 mg / m 2 To about 67.9mg / m 2 ,or About 87.1 mg / m 2 To about 88.1mg / m 2 .

5. The method according to any one of claims 1 to 4, wherein the antibody-drug conjugate dose is about 7.2 mg / m 2 .

6. The method of any one of claims 1-4, wherein the antibody-drug conjugate dose is about 14.4 mg / m 2 .

7. The method of any one of claims 1-4, wherein the antibody-drug conjugate dose is about 21.6 mg / m 2 .

8. The method of any one of claims 1-4, wherein the antibody-drug conjugate dose is about 28.7 mg / m 2 .

9. The method of any one of claims 1-4, wherein the antibody-drug conjugate dose is about 38.1 mg / m 2 .

10. The method of any one of claims 1-4, wherein the antibody-drug conjugate dose is about 50.7 mg / m 2 .

11. The method of any one of claims 1-4, wherein the antibody-drug conjugate dose is about 67.4 mg / m 2 .

12. The method of any one of claims 1-4, wherein the antibody-drug conjugate dose is about 87.6 mg / m 2 .

13. The method of any one of the preceding claims, wherein the cancer is B7-H4 positive.

14. The method of any of the preceding claims, wherein the subject with TNBC has received at least 2 lines of systemic therapy in the setting of locally advanced or metastatic breast cancer.

15. The method according to any of the preceding claims, wherein the subject with HR+ / HER2- breast cancer after CDK 4 / 6 inhibitor and endocrine-based therapy has received at least 1 line of systemic therapy, which must include a CDK 4 / 6 inhibitor and endocrine therapy (ET), in the setting of advanced or metastatic breast cancer.

16. The method of any one of the preceding claims, wherein the subject with endometrial cancer has received at least 1 line of systemic therapy for advanced or metastatic disease including platinum-based chemotherapy.

17. The method of any one of the preceding claims, wherein the subject with ovarian cancer, fallopian tube cancer, or primary peritoneal cancer has received at least 2 lines of systemic therapy for advanced or metastatic disease, which should include platinum-based chemotherapy.

18. The method of any one of the preceding claims, wherein the subject is a human.

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