B7-H4 Targeted Antibody-Drug Conjugates for the Treatment of Cancer

Abstract

Disclosed herein are dosing regimens for B7-H4 targeted antibody-drug conjugates for treating cancer. TIFF2025517340000029.tif115128

Description

[Technical field]

[0001] Related Applications This application claims priority to and 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 which are incorporated herein by reference in their entirety.

[0002] INCORPORATION BY REFERENCE TO SEQUENCE LISTING The sequence listing XML associated with this application has been provided electronically in xml file format and is incorporated herein by reference. The name of the xml file containing the sequence listing is MRSN_038_001WO_SeqList_ST26.xml. The xml file is 13,132 bytes, was created on May 12, 2023, and has been submitted electronically via the USPTO Patent Center.

[0003] FIELD OF THEINVENTION The present disclosure relates generally to dosing regimens for administering B7-H4 targeted antibody-drug conjugates for the treatment of cancer. [Background technology]

[0004] background 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 superfamily of proteins that provides co-signals in conjunction with T cell receptor antigenic signals. B7-H4 is a negative regulator of T cell function, and ligation of T cells inhibits their proliferation, cytokine secretion and cytotoxicity. In mice, elimination of B7-H4 does not affect immune cell homeostasis and results in no signs of autoimmunity. The receptor for B7-H4 is unknown and has not been identified.

[0005] Human B7-H4 is mapped to chromosome 1 and consists of six exons and five introns spanning 66 kb, of which exon 6 is used for alternative splicing to generate two distinct transcripts. Human B7-H4 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.

[0006] Although B7-H4 expression is relatively limited at the protein level in healthy tissues, 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 thought to be expressed on T cells. B7-H4 is thought to directly inhibit T cell activity.

[0007] A wide variety of therapeutic modalities are available for the treatment of advanced cancer, including radiation therapy, conventional chemotherapy with cytotoxic antitumor agents, hormonal 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 therapeutic modalities. Therefore, there is a need to identify new therapeutic agents that target the biological activity of B7-H4.

[0008] Thus, there is a need for therapies that target the biological activity of B7-H4. Summary of the Invention

[0009] The present disclosure provides a method of treating a subject with TNBC, HR+ / HER2- breast cancer, endometrial cancer, ovarian cancer, fallopian tube cancer, or primary peritoneal cancer or solid tumors, comprising administering about 7.0 mg / m 2~about 90mg / m 2 wherein the B7-H4 targeting antibody-drug conjugate is a conjugate of formula (I): TIFF2025517340000002.tif110137, wherein i.d 13 is approximately 2, ii. The ANTIBODY binds to B7-H4 and comprises a variable heavy chain complementarity determining region 1 (CDRH1) comprising the amino acid sequence GFIVSRNY (SEQ ID NO:2), a variable heavy chain complementarity determining region 2 (CDRH2) comprising the amino acid sequence IYGSGRT (SEQ ID NO:3), a variable heavy chain complementarity determining region 3 (CDRH3) comprising the amino acid sequence ARDADYGLDV (SEQ ID NO:4), a variable light chain complementarity determining region 1 (CDRL1) comprising the amino acid sequence QSVSSSY (SEQ ID NO:5), a variable light chain complementarity determining region 2 (CDRL2) comprising the amino acid sequence GAS (SEQ ID NO:6), and a variable light chain complementarity determining region 3 (CDRL3) comprising the amino acid sequence QQYGSSPLYT (SEQ ID NO:7). iii. the drug is attached via a linker moiety to an asparagine of the heavy chain of the antibody at position 297 when numbered according to EU numbering; iv. ■ is GlcNAc, △ is Fuc, and □ is GalNAc.

[0010] 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-tetraol, 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).

[0011] In some embodiments, a subject is administered about 6.7 mg / m once every 3 weeks (i.e., a 21 day cycle) or once every 4 weeks (i.e., a 28 day cycle). 2 ~about 7.7mg / m 2 , 13.9 mg / m 2 ~Approx. 14.9mg / m 2 , 21.1 mg / m 2 ~About 22.1mg / m 2 , 28.2 mg / m 2 ~About 29.2mg / m 2 , 37.6 mg / m 2 ~Approx. 38.6mg / m 2 , 50.2 mg / m 2 ~Approx. 51.2mg / m 2 , 66.9 mg / m 2 ~Approx. 67.9mg / m 2 , or 87.1 mg / m 2 ~about 88.1mg / m 2 In some embodiments, the subject is administered a B7-H4 targeting antibody-drug conjugate by IV infusion at a dose of about 7.2 mg / m once every 3 weeks (i.e., a 21 day cycle) or once every 4 weeks (i.e., a 28 day cycle). 2 , about 14.4mg / m 2 , about 21.6mg / m 2 , about 28.7mg / m 2 , about 38.1mg / m 2, 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 dosage is about 14.4 mg / m 2 In some embodiments, the conjugate dosage is about 21.6 mg / m 2 In some embodiments, the conjugate dosage is about 28.7 mg / m 2 In some embodiments, the conjugate dosage is about 38.1 mg / m 2 In some embodiments, the conjugate dosage is about 50.7 mg / m 2 In some embodiments, the conjugate dosage is about 67.4 mg / m 2 In some embodiments, the conjugate dosage is about 87.6 mg / m 2 It is.

[0012] In some embodiments, subjects are administered a B7-H4 targeting antibody-drug until disease progression, death, unacceptable toxicity, or voluntary discontinuation, whichever occurs first.

[0013] In some embodiments, the subject with unresectable or recurrent / metastatic TNBC has received at least two lines of systemic therapy in the locally advanced or metastatic breast cancer setting.

[0014] In some embodiments, the subject has post-CDK4 / 6 inhibitor HR+ / HER2- breast cancer and has received at least one line of systemic therapy, which endocrine-based therapy must include a CDK 4 / 6 inhibitor and endocrine therapy (ET) in the advanced or metastatic breast cancer setting.

[0015] In some embodiments, the subject with endometrial cancer has received at least one line of systemic therapy including platinum-based chemotherapy for advanced or metastatic disease.

[0016] In some embodiments, subjects with ovarian, fallopian tube, or primary peritoneal cancer have received at least two lines of systemic therapy for advanced or metastatic disease, which should include platinum-based chemotherapy.

[0017] In some embodiments, the cancer is B7-H4 positive.

[0018] In some embodiments, the subject is a human.

[0019] Other features and advantages of the invention will become apparent from the following detailed description, and from the claims. [Brief description of the drawings]

[0020] [Figure 1] FIG. 1 is a graph showing the antitumor efficacy of B7-H4 targeted antibody-drug conjugate (XMT-1660) (4.68 / 0.150 mg / kg) and rituximab isotype control conjugate (4.60 / 0.150 mg / kg) (both doses provided by antibody / payload) in a patient-derived endometrial cancer xenograft mouse model. [Diagram 2] FIG. 2 is a graph showing the antitumor efficacy of B7-H4 targeted antibody-drug conjugate (XMT-1660) (4.61 / 0.150 mg / kg) and palivizumab isotype control (4.54 / 0.150 mg / kg) (both doses provided by antibody / payload) in a patient-derived ovarian cancer xenograft mouse model. [Diagram 3] FIG. 3 is a graph showing the antitumor efficacy of B7-H4 targeted antibody-drug conjugate (XMT-1660) (2.30 / 0.075 or 4.60 / 0.150 mg / kg, DAR 5.96) and palivizumab isotype control ADC (2.27 / 0.075 or 4.54 / 0.150 mg / kg, DAR 5.97) (both doses given by antibody / payload) in the CTG-1692 patient-derived ovarian cancer model. [Figure 4]FIG. 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), B7-H4 targeted antibody-drug conjugate (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 given by antibody / payload) in the mBR9013 syngeneic breast cancer mouse model. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0021] Detailed Description 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 solid tumors thereof, comprising administering to the subject a B7-H4-targeting antibody-drug conjugate (XMT-1660) that specifically binds to the extracellular region of B7-H4. Specifically, the present invention provides a dosing regimen for the treatment of cancer or solid tumors expressing B7-H4 by administration as an intravenous infusion. XMT-1660 is a B7-H4-targeting antibody-drug conjugate that includes a B7-H4 modified antibody and two molecules of a fully synthetic macromolecular linker-payload with 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 enabled by glycan remodeling of the antibody at the asparagine groups 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 B7-H4 targeting antibody-drug conjugate is about 6.

[0022] Patients with TNBC, HR+ / HER2- breast cancer, endometrial cancer, ovarian cancer, fallopian tube cancer, or primary peritoneal cancer are administered XMT-1660 intravenously once every 3 weeks (i.e., 21-day cycle) or once every 4 weeks (i.e., 28-day cycle). Thus, the present invention provides a dose-escalation study in which XMT-1660 is administered intravenously at 7.2 mg / m once every 3 weeks (i.e., 21-day cycle) or once every 4 weeks (i.e., 28-day cycle). 2 , 14.4 mg / m 2 , 21.6 mg / m 2 , 28.7 mg / m 2 , 38.1 mg / m 2 , 50.7 mg / m 2 , 67.4 mg / m 2 or 87.6 mg / m 2 The present invention features a method of treating TNBC, HR+ / HER2- breast cancer, endometrial cancer, ovarian cancer, fallopian tube cancer, or primary peritoneal cancer by administering to a subject, i.e., a human, an injection dose of XMT-1660 of

[0023] In some embodiments, the subject has a B7-H4-expressing cancer. In some aspects, the subject has a B7-H4-expressing tumor.

[0024] In some embodiments, the subject is identified as having B7-H4 expression. In some embodiments, the B7-H4 expression is in the form of a B7-H4 expressing tumor. The B7-H4 expression is detected by methods known in the art. For example, by immunohistochemistry (IHC) analysis, fluorescence in situ hybridization (FISH) assay or RNA expression analysis of B7-H4 transcripts or other genes associated with cancer measured in tumor samples. Blood-based biomarkers, which may include serum cytokines, circulating immune cells and circulating tumor cells, can also be used to determine B7-H4 expression levels.

[0025] B7-H4 antibody The present disclosure provides, for example, isolated antibodies that bind to B7-H4, a type I transmembrane protein found on the surface of antigen-presenting cells (APCs). B7-H4 antibodies include, but are not limited to, humanized antibodies, chimeric antibodies, murine antibodies, human antibodies, and antibodies comprising the heavy and / or light chain CDRs described herein.

[0026] In some embodiments, the B7-H4 antibody of the disclosure has the amino acid sequence: Specifically binds to an epitope on the full-length human B7-H4 protein containing TIFF2025517340000003.tif33166.

[0027] 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) response in a subject to which the antibody is administered. In some embodiments, the B7-H4 antibody does not inhibit the T cell suppressive 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, for example, a Fab fragment, a Fab' fragment, or a Fab'2 fragment, or a single chain antibody (e.g., scFv). In some embodiments, the antibody is an IgG1 antibody.

[0028] 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 comprising a heavy chain variable region and at least a portion of a heavy chain constant region, and at least one 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, each of which comprises a heavy chain variable region and at least a portion of a heavy chain constant region, and two light chains, each of which comprises a light chain variable region and at least a portion of a light chain constant region.

[0029] In some embodiments, the human B7-H4 antibody comprises one or more human constant regions. In some embodiments, the human heavy chain constant region is of an isotype selected from IgA, IgG (e.g., IgG1, IgG2, IgG3 or IgG4), and IgD. In some embodiments, the human light chain constant region is of an isotype selected from kappa (κ) and lambda (λ). In some embodiments, the human antibodies described herein comprise a human IgG constant region. In some embodiments, the human antibodies described herein comprise a human IgG4 heavy chain constant region. In some embodiments, the human antibodies described herein comprise a human IgG4 constant region and a human κ light chain.

[0030] In some embodiments, when effector function is desired, a human B7-H4 antibody is selected that comprises a human IgG1 heavy chain constant region or a human IgG3 heavy chain constant region. In some embodiments, when effector function is not desired, a human B7-H4 antibody is selected that comprises a human IgG4 heavy chain constant region or a human IgG2 heavy chain constant region.

[0031] In some embodiments, the antibodies of the present disclosure are characterized by their particular functional features or functional properties. For example, the antibodies specifically bind to human B7-H4. Typically, the antibodies of the present disclosure bind to B7-H4 with high affinity, e.g., greater than 1×10 -7 K below M D Anti-B7-H4 antibodies of the present disclosure typically exhibit one or more of the following characteristics: (a) 1×10 -7 K below M D binds to human B7-H4; and / or (b) Binds to human CHO cells transfected with B7-H4 (eg, human B7-H4).

[0032] In some embodiments, the antibody is at least 5×10 -8 K below M D binds to human B7-H4 at 2 × 10 -8 K below M Dbinds to human B7-H4 at 5 × 10 -9 K below M D binds to human B7-H4 at 4 × 10 -9 K below M D binds to human B7-H4 at 3 × 10 -9 K below M D binds to human B7-H4 at 2 × 10 -9 K below M D or 1 × 10 -9 K below M D It binds to human B7-H4.

[0033] Standard assays for evaluating the binding ability of an antibody 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 an antibody can also be evaluated by standard assays known in the art, such as ELISA, Scatchard and Biacore® system analysis.

[0034] Potential therapeutic mAbs must not only bind their targets, but also be free of "developability issues", e.g., poor stability or high levels of aggregation. We describe guideline values ​​for five metrics believed to be related to poor developability: total length of the complementarity determining regions (CDRs), degree and magnitude of surface hydrophobicity, positive and negative charges within the CDRs, and asymmetry of net heavy and light chain surface charges. Guideline cutoffs for each property are derived from values ​​found in the CST, and a flagging system is proposed to identify unqualified candidates.

[0035] The nucleic acid and amino acid sequences of the monoclonal B7-H4 antibody of the present disclosure are shown below. In the amino acid sequences shown below, the complementarity determining regions (CDRs) of the heavy and light chains are underlined. The amino acids encompassing the complementarity determining regions (CDRs) shown below are defined according to the IMGT numbering system (see IMGT®, international ImMunoGeneTics information system®, available online at http: / / www.imgt.org / ).

[0036] B7-H4 variable region In some embodiments, a B7-H4 antibody of the disclosure comprises a light chain variable region comprising or consisting of the amino acid sequence of SEQ ID NO:8.

[0037] B7-H 4 _V L V L The chain (SEQ ID NO:8) comprises or consists of the following amino acid sequence: TIFF2025517340000004.tif11165

[0038] 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: TIFF2025517340000005.tif18164

[0039] The B7-H4 variable heavy chain is also referred to herein as the XMT-1604 variable heavy chain.

[0040] In some embodiments, the B7-H4 antibody (XMT-1604) comprises a variable heavy chain complementarity determining region 1 (CDRH1) comprising the amino acid sequence GFIVSRNY (SEQ ID NO:2), a variable heavy chain complementarity determining region 2 (CDRH2) comprising the amino acid sequence IYGSGRT (SEQ ID NO:3), and a variable heavy chain complementarity determining region 3 (CDRH3) comprising the amino acid sequence ARDADYGLDV (SEQ ID NO:4).

[0041] In some embodiments, the B7-H4 antibody (XMT-1604) comprises a variable light chain complementarity determining region 1 (CDRL1) comprising the amino acid sequence QSVSSSY (SEQ ID NO:5), a variable light chain complementarity determining region 2 (CDRL2) comprising the amino acid sequence GAS (SEQ ID NO:6), and a variable light chain complementarity determining region 3 (CDRL3) comprising the amino acid sequence QQYGSSPLYT (SEQ ID NO:7).

[0042] In some embodiments, the B7-H4 antibody of the disclosure has the amino acid sequence: and having a light chain constant region comprising or consisting of TIFF2025517340000006.tif11165.

[0043] The B7-H4 light chain constant region (SEQ ID NO:10) is also referred to herein as B7-H4 LC.

[0044] In some embodiments, the antibody of the 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 disclosure comprises or consists of the amino acid sequence of SEQ ID NO:12.

[0045] The B7-H4 antibody of the disclosure has the amino acid sequence: TIFF2025517340000007.tif40166. 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.

[0046] In some embodiments, an antibody of the disclosure comprises a heavy chain set forth in SEQ ID NO:13, comprising or consisting of a heavy chain variable region amino acid sequence and a heavy chain constant region amino acid sequence.

[0047] In yet another embodiment, the B7-H4 antibody of the present disclosure comprises a heavy chain variable region and a light chain variable region comprising amino acid sequences homologous to the amino acid sequences of the antibodies described herein, and the antibody retains the desired functional properties of the anti-B7-H4 antibody 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, (a) the heavy chain variable region comprises an amino acid sequence at least 80% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:9; (b) the light chain variable region comprises an amino acid sequence at least 80% identical to the amino acid sequence of SEQ ID NO:8; (c) Antibody, 1 × 10 -7 K below M D binds to human B7-H4 at (d) The antibody binds to human CHO cells transfected with B7-H4.

[0048] In various aspects, the antibody can be, for example, a human antibody, a humanized antibody, or a chimeric antibody.

[0049] In other embodiments, the VH and / or VL amino acid sequences can be 85%, 90%, 95%, 96%, 97%, 98% or 99% homologous to the above sequences. B7-H4 antibodies having VH and VL regions highly homologous (i.e., 80% or greater) to the VH and VL regions of the above sequences can be obtained by mutagenesis (e.g., site-directed mutagenesis or PCR-mediated mutagenesis) of a nucleic acid molecule encoding the amino acid sequence set forth in SEQ ID NO:9, followed by testing the encoded altered antibody for retained function (i.e., the functions described in (c) and (d) above) using the functional assays described herein.

[0050] In a preferred embodiment, the heavy chain variable region CDR2 sequence comprises the amino acid sequence SEQ ID NO:3 and its conservative modifications, and the light chain variable region CDR2 sequence comprises the amino acid sequence SEQ ID NO:6 and its conservative modifications. In another preferred embodiment, the heavy chain variable region CDR1 sequence comprises the amino acid sequence SEQ ID NO:2 and its conservative modifications, and the light chain variable region CDR1 sequence comprises the amino acid sequence SEQ ID NO:5 and its conservative modifications. In another preferred embodiment, the heavy chain variable region CDR3 sequence comprises the amino acid sequence SEQ ID NO:4 and its conservative modifications, and the light chain variable region CDR3 sequence comprises the amino acid sequence SEQ ID NO:7 and its conservative modifications.

[0051] In various aspects, the antibody can be, for example, a human antibody, a humanized antibody, or a chimeric antibody.

[0052] In some embodiments, the antibody binds to the same epitope on human B7-H4 as any of the B7-H4 monoclonal antibodies of the present disclosure (i.e., an antibody that has the ability to cross-compete with any of the monoclonal antibodies of the present disclosure for binding to B7-H4).

[0053] Accordingly, another aspect of the disclosure relates to an isolated monoclonal antibody or antigen-binding portion thereof comprising a heavy chain variable region comprising CDR1, CDR2 and CDR3 sequences each comprising the amino acid sequence of SEQ ID NO:9, and a light chain variable region comprising CDR1, CDR2 and CDR3 sequences each comprising the amino acid sequences of SEQ ID NO:5, 6 and 7, respectively.

[0054] Accordingly, in another aspect, 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; and (c) a VH CDR3 region comprising an amino acid sequence selected from the group consisting of 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.

[0055] In some embodiments, the antibodies disclosed herein comprise a heavy chain having an amino acid sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more identical to SEQ ID NO:13, and a light chain having an amino acid sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more identical to SEQ ID NO:12.

[0056] In some embodiments, the antibodies disclosed herein comprise a combination of heavy and light chain amino acid sequences comprising a heavy chain amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more identical to the amino acid sequence of SEQ ID NO:13, and a light chain amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more identical to the amino acid sequence of SEQ ID NO:12.

[0057] In some embodiments, the antibodies disclosed herein comprise a heavy chain amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more identical to the amino acid sequence of SEQ ID NO:13, and a light chain amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more identical to the amino acid sequence of SEQ ID NO:12.

[0058] In some embodiments, the antibodies disclosed herein comprise a heavy chain having the amino acid sequence of SEQ ID NO:13 and a light chain having the amino acid sequence of SEQ ID NO:12.

[0059] In some embodiments, the antibodies disclosed herein comprise a combination of the heavy chain and light chain amino acid sequences of SEQ ID NO:13 and SEQ ID NO:12.

[0060] In some embodiments, the antibody disclosed herein comprises a heavy chain amino acid sequence of SEQ ID NO:13 and a light chain amino acid sequence of SEQ ID NO:12.

[0061] The antibodies disclosed herein comprise a heavy chain variable region having an amino acid sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more identical to SEQ ID NO:9, and a light chain variable region having an amino acid sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more identical to a sequence consisting of SEQ ID NO:8.

[0062] In some embodiments, the three heavy chain CDRs of the antibodies disclosed herein comprise a heavy chain complementarity determining region 1 (CDRH1) comprising an amino acid sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more identical to a sequence comprising SEQ ID NO:2; a heavy chain complementarity determining region 2 (CDRH2) comprising an amino acid sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more identical to a sequence comprising SEQ ID NO:3; and a heavy chain complementarity determining region 3 (CDRH3) that comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more identical to SEQ ID NO:4, as well as a heavy chain amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more identical to the amino acid sequence SEQ ID NO:13.

[0063] The three light chain CDRs of the antibodies disclosed herein include a light chain complementarity determining region 1 (CDRL1) that comprises an amino acid sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more identical to a sequence comprising SEQ ID NO:5; a light chain complementarity determining region 2 (CDRL2) that comprises an amino acid sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more identical to a sequence comprising SEQ ID NO:6; and a light chain complementarity determining region 3 (CDRL4) that comprises an amino acid sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more identical to a sequence comprising SEQ ID NO:7. and a light chain complementarity determining region 3 (CDRL3) comprising an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more identical to a sequence comprising NO:7.

[0064] The antibody may be a CDRH1 comprising an amino acid sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more identical to a sequence comprising SEQ ID NO:2; a CDRH2 comprising an amino acid sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more identical to a sequence comprising SEQ ID NO:3; a CDRH3 comprising an amino acid sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to a sequence comprising SEQ ID NO:4; CDRL1 comprising an amino acid sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more identical to a sequence selected from the group consisting of SEQ ID NO:5; CDRL2 comprising an amino acid sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more identical to a sequence selected from the group consisting of SEQ ID NO:6; and CDRL3 comprising an amino acid sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more identical to a sequence selected from the group consisting of SEQ ID NO:7a. It includes a combination of heavy chain CDR sequences and light chain CDR sequences that contain heavy chain amino acid sequences that are at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more identical to the amino acid sequence of NO:45.

[0065] The three heavy chain CDRs of the antibody disclosed herein include CDRH1 comprising an amino acid sequence selected from the group including SEQ ID NO:2, CDRH2 comprising an amino acid sequence including SEQ ID NO:3, and CDRH3 comprising an amino acid sequence including SEQ ID NO:4, as well as a heavy chain amino acid sequence including SEQ ID NO:13.

[0066] The three light chain CDRs of the antibodies disclosed herein include CDRL1 having the amino acid sequence of SEQ ID NO:5, CDRL2 having the amino acid sequence of SEQ ID NO:6, and CDRL3 having the amino acid sequence of SEQ ID NO:7. The antibodies disclosed herein include CDHR1 comprising a selected amino acid sequence comprising SEQ ID NO:2, CDRH2 comprising an amino acid sequence comprising SEQ ID NO:3, CDRH3 comprising an amino acid sequence comprising SEQ ID NO:4, CDRL1 having the amino acid sequence of SEQ ID NO:5, CDRL2 having the amino acid sequence of SEQ ID NO:6, and CDRL3 having the amino acid sequence of SEQ ID NO:7, as well as a combination of heavy and light chain CDR sequences comprising the heavy chain amino acid sequence of SEQ ID NO:13. The antibodies disclosed herein comprise (i) a combination of a heavy chain complementarity determining region amino acid sequence and a light chain complementarity determining region amino acid sequence selected from the group consisting of 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.

[0067] 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.

[0068] Modified B7-H4 antibody In some embodiments, the B7-H4 antibody is a modified B7-H4 antibody. As used herein, the term "modified B7-H4 antibody" may 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" may be used to describe a B7-H4 antibody having a modification to an amino acid of the antibody, including the covalent attachment of a chemical moiety to the amino acid side. In some embodiments, a "modified B7-H4 antibody" may have a sugar or sugar derivative moiety covalently attached to an amino acid of the antibody. In some embodiments, a "modified B7-H4 antibody" may have a sugar or sugar derivative moiety replaced by a different sugar or sugar derivative moiety.

[0069] In some embodiments of the modified B7-H4 antibody, * indicates a direct or indirect bond to the remainder 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).

[0070] In some embodiments, the modified B7-H4 antibody prior to conjugation comprises: Contains the sugar-derivative portion of TIFF2025517340000008.tif3128.

[0071] 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 well conserved and each amino acid can be identified 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 region 290-305 (e.g., 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 moiety is directly or indirectly attached to the asparagine group (e.g., to N297).

[0072] In some embodiments, an asparagine of the B7-H4 antibody heavy chain constant region set forth in SEQ ID NO:11 is modified. In some embodiments, the modified asparagine (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.

[0073] In some embodiments, an asparagine of the B7-H4 antibody heavy chain set forth in SEQ ID NO: 13 is modified. In some embodiments, the modified asparagine (N297 according to EU numbering) corresponds to position 296 (N296) of SEQ ID NO: 13. In some aspects, a macromolecular linker-payload (e.g., a drug, or linker-drug moiety) of a B7-H4 ADC is attached to N296 of SEQ NO: 13.

[0074] In some embodiments, the modified B7-H4 antibody prior to conjugation comprises a modified GlcNAc moiety. TIFF2025517340000009.tif4128, and GlcNAc is N-acetylglucosamine.

[0075] In some embodiments, the modified GlcNAc moiety is attached to the remainder of the modified B7-H4 antibody via the C1 position of the GlcNAc, hi some embodiments, the modified GlcNAc moiety further comprises a fucose.

[0076] In some embodiments, the modified GlcNAc moiety is directly or indirectly attached to an asparagine residue (eg, to N297).

[0077] In some embodiments, the modified B7-H4 antibody is conjugated to a 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.

[0078] In some aspects, the modified B7-H4 antibodies of the disclosure include (a) contacting a glycoprotein (e.g., a B7-H4 antibody glycan) comprising a B7-H4 antibody and a core-GlcNAc moiety with an endoglycosidase, thereby forming an intermediate antibody comprising the antibody and a terminal GlcNAc moiety, wherein optionally, the terminal GlcNAc moiety further comprises fucose; and (b) isolating the intermediate antibody in the presence of a glycosyltransferase; TIFF2025517340000010.tif3128, thereby contacting the antibody with a compound having the structure and optionally a modified GlcNAc moiety is attached to the remainder of the modified B7-H4 antibody via the C1 position of the GlcNAc; GlcNAc is N-acetylglucosamine, S″ is a sugar or a derivatized sugar, A″ is azido, keto or alkynyl; P″ is uridine diphosphate (UDP), guanosine diphosphate (GDP) or cytidine diphosphate (CDP).

[0079] In some embodiments, steps (a) and (b) are performed sequentially. In some embodiments, steps (a) and (b) are performed simultaneously.

[0080] In some embodiments, the B7-H4 antibody is an IgG1 antibody.

[0081] In some embodiments, the antibody is a full-length antibody and the antibody glycan comprises one or more core-GlcNAc moieties.

[0082] 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.

[0083] In some embodiments, the core-GlcNAc moiety further comprises fucose.

[0084] 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.

[0085] 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.

[0086] In some embodiments, at least one of the two or more Core-GlcNAc moieties further comprises a fucose.

[0087] In some embodiments, each of the two or more Core-GlcNAc moieties further comprises a fucose.

[0088] In some embodiments, the Core-GlcNAc moiety is attached to an antibody at a position such that the Core-GlcNAc moiety does not substantially interfere with the antigen-binding site of the antibody.

[0089] In some embodiments, the Core-GlcNAc moiety is attached to an Fc fragment of an antibody. In some embodiments, the Core-GlcNAc moiety is attached to a CH domain. In some embodiments, the Core-GlcNAc moiety is attached to a Fab fragment or an Fc fragment of an antibody. In some embodiments, the Core-GlcNAc moiety is attached to an antibody via an N-glycosidic bond to the amide nitrogen atom of the side chain of an asparagine amino acid of the antibody. In some embodiments, the Core-GlcNAc moiety is attached to a native N-glycosylation site of the antibody.

[0090] In some embodiments, the antibody is an IgG antibody and the core-GlcNAc moiety is joined to a native N-glycosylation site of IgG.

[0091] In some embodiments, the antibody is an IgG antibody, and the core-GlcNAc moiety is attached to a native N-glycosylation site of IgG (e.g., the 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 IgG antibodies at an asparagine within the region 290-305 (e.g., at N297). In some embodiments, the intermediate antibody has formula (XXII): TIFF2025517340000012.tif21128, wherein Ab is B7-H4 antibody, GlcNAc is N-acetylglucosamine, Fuc is fucose, and u 3 is 0 or 1, and u 4 is an integer in the range of 1 to 16.

[0092] In some embodiments, u 4 is an integer ranging from 1 to 10. In some embodiments, u 4 is an integer ranging from 1 to 4. In some embodiments, u 4 is 1, 2, 3, 4, 5, 6, 7 or 8. In some embodiments, u 4 is 1, 2, 3, 4, 5 or 6. In some embodiments, u 4is 1, 2, 3 or 4. In some embodiments, u 4 is 2 or 4. In some embodiments, u 4 is 1 or 2. In some embodiments, u 4 is 1. In some embodiments, u 4 is 2.

[0093] In some embodiments, the antibody comprises one Core-GlcNAc moiety (e.g., u 4 is 1). In some embodiments, the antibody comprises two Core-GlcNAc moieties (e.g., u 4 is 2).

[0094] In some embodiments, the modified B7-H4 antibody is obtained by the process outlined in Scheme 1. As shown below, an intermediate antibody of formula (XXIII) containing one terminal GlcNAc moiety is synthesized in the presence of a glycosyltransferase. Contact with a compound having the structure of TIFF2025517340000013.tif3128 results in a modified B7-H4 antibody that includes one modified GlcNAc moiety (eg, a modified B7-H4 antibody of formula (XXIIIa)).

[0095] In some embodiments, the modified B7-H4 antibody is prepared by synthesis of an intermediate antibody of formula (XXIV) comprising two terminal GlcNAc moieties in the presence of a glycosyltransferase. and a compound having the structure of TIFF2025517340000014.tif3128, thereby providing a modified B7-H4 antibody comprising two modified GlcNAc moieties (eg, a modified B7-H4 antibody of formula (XXIVa)). TIFF2025517340000015.tif51144, u 3 , Ab, S", A", and P" are as defined herein.

[0096] In some embodiments, the antibody glycan modified by the process according to the present disclosure comprises a glycan that comprises a core-GlcNAc moiety, i.e., a GlcNAc moiety that is present at the non-reducing end of the glycan. In some embodiments, the glycan comprises one or more sugar moieties and can be linear or branched.

[0097] In some embodiments, upon reaction with an endoglycosidase, an intermediate antibody may be formed that includes a terminal GlcNAc moiety (eg, an intermediate antibody of formula (XXIII) or (XXIV)).

[0098] In some embodiments, step (a) of the process (deglycosylation or trimming) comprises deglycosylating or trimming a mixture of antibody glycoforms G2F, GIF, G0F, G2, G1, G0 and M5, which may contain fucose (e.g., u 3 is converted to an intermediate antibody that contains a terminal GlcNAc moiety (wherein GlcNAc is 0 or 1).

[0099] In some embodiments, the endoglycosidase is Endo S or Endo SH, or a combination thereof. In some embodiments, the endoglycosidase is Endo SH.

[0100] In some embodiments, step (b) of the process (forming the modified B7-H4 antibody) wherein the intermediate antibody is a monoclonal antibody (mAb) and a terminal GlcNAc moiety (which may include a fucose (e.g., u 3 is 0 or 1).

[0101] In some embodiments, The compound of TIFF2025517340000016.tif3128 is GalNAz-UDP (e.g., 4-AzGalNAc-UDP). In some embodiments, the terminal GlcNAc moiety is *-GlcNAc-GalNAz or *-GlcNAc(Fuc)-GalNAz, where * represents the linkage to the remainder of the modified B7-H4 antibody.

[0102] In some embodiments, the deglycosylation / trimming step and the step of forming the modified B7-H4 antibody are performed sequentially.

[0103] In some embodiments, the deglycosylation / trimming step and the step of forming the modified B7-H4 antibody are performed simultaneously.

[0104] In some embodiments, the process for preparing the modified B7-H4 antibody is carried out in a suitable buffer, such as buffered saline (e.g., phosphate buffered saline, Tris buffered saline) citrate, HEPES, Tris and glycine. In some embodiments, the buffer is phosphate buffered saline (PBS) or Tris buffered saline. In some embodiments, the buffer is phosphate buffered saline (PBS).

[0105] In some embodiments, the process is carried out at a temperature ranging from about 4 to about 50° C. In some embodiments, the process is carried out at a temperature ranging from about 10 to about 45° C. In some embodiments, the process is carried out at a temperature ranging from about 20 to about 40° C. In some embodiments, the process is carried out at a temperature ranging from about 30 to about 37° C. In some embodiments, the process is carried out at a temperature of about 30° C. In some embodiments, the process is carried out at a temperature of 30° C.

[0106] In some embodiments, the process is carried out at a pH value ranging from about 5 to about 9 (e.g., from about 5.5 to about 8.5, from about 6 to about 8, or from about 7 to about 8). In some embodiments, the process is carried out at a pH value of about 7.4.

[0107] In some embodiments, the process for preparing a modified B7-H4 antibody comprises: (a) contacting a glycoprotein (e.g., a B7-H4 antibody glycan) comprising a B7-H4 antibody and a core-GlcNAc moiety attached to site N297 (according to EU numbering) of the antibody with endoglycosidase Endo SH, thereby forming an intermediate antibody comprising a terminal GlcNAc moiety; and (b) contacting the intermediate antibody with 4-AzGalNAc-UDP in the presence of a β-(1,4)-GalNAcT enzyme, thereby forming a modified B7-H4 antibody that includes a modified GlcNAc moiety; Steps (a) and (b) are carried out simultaneously.

[0108] In some embodiments, the endoglycosidase is Endo SH, which is a fusion of two endoglycosidases, Endo S and Endo H, linked by a Gly-rich spacer containing an internal 6xHis tag, resulting in an overall molecular weight of 139 kDa.

[0109] In some embodiments, the β-(1,4)-GalNAcT enzyme comprises an N-terminal 6xHis tag and has an overall molecular weight of 45.7 kDa. In some embodiments, the β-(1,4)-GalNAcT enzyme comprises an N-terminal 6xHis tag and is derived from Trichopulsia ni.

[0110] In some embodiments, the process is carried out in a PBS buffer at a pH value of about 7.4 and a temperature of about 30°C.

[0111] B7-H4 targeting antibody-drug conjugates The present invention relates to therapeutic methods involving immunoconjugates comprising an antibody conjugated via site-specific conjugation to a cytotoxic agent, such as a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant or animal origin, or a fragment thereof).

[0112] The conjugate described herein is a B7-H4 targeted antibody-drug conjugate comprising a B7-H4 modified antibody and two fully synthetic macromolecular linker-payload molecules with 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 enabled by glycan remodeling of the antibody at the asparagine group at position 297 of the antibody according to EU numbering.

[0113] The average drug-to-antibody ratio (DAR) of the B7-H4 targeting antibody-drug conjugate is about 6. In some embodiments, the DAR of the B7-H4 targeting antibody-drug conjugate is about 2 to about 8. In some embodiments, the DAR of the B7-H4 targeting antibody-drug conjugate is about 5 to about 7. In some embodiments, the DAR of the B7-H4 targeting antibody-drug conjugate 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 number therebetween.

[0114] In some embodiments, the B7-H4 antibody-drug conjugate is a conjugate of formula (I): TIFF2025517340000017.tif110137, wherein i.d 13 is approximately 2, ii. an ANTIBODY that binds to B7-H4 and comprises a variable heavy chain complementarity determining region 1 (CDRH1) that comprises the amino acid sequence GFIVSRNY (SEQ ID NO:2), a variable heavy chain complementarity determining region 2 (CDRH2) that comprises the amino acid sequence IYGSGRT (SEQ ID NO:3), a variable heavy chain complementarity determining region 3 (CDRH3) that comprises the amino acid sequence ARDADYGLDV (SEQ ID NO:4), a variable light chain complementarity determining region 1 (CDRL1) that comprises the amino acid sequence QSVSSSY (SEQ ID NO:5), a variable light chain complementarity determining region 2 (CDRL2) that comprises the amino acid sequence GAS (SEQ ID NO:6), and a variable light chain complementarity determining region 3 (CDRL3) that comprises the amino acid sequence QQYGSSPLYT (SEQ ID NO:7); iii. the drug is attached to the antibody via a linker moiety at position 297, as numbered according to EU numbering; iv. ■ is GlcNAc, △ is Fuc, and □ is GalNAc.

[0115] In some embodiments, d 13 can be about 1 to about 3. In some embodiments, d 13 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.

[0116] GlcNAc refers to N-acetylglucosamine (i.e., β-D-(acetylamino)-2-deoxy-glucopyranose, or N-acetyl-D-glucosamine).

[0117] Fuc refers to fucose (i.e., (2S,3R,4R,5S)-6-methyltetrahydro-2H-pyran-2,3,4,5-tetraol, or 6-deoxy-1-galactose).

[0118] 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).

[0119] In some embodiments, the GlcNAc is attached to the conjugate via a reactive moiety on the GlcNAc.

[0120] In some embodiments, the Fuc is attached to the conjugate via a reactive moiety on the Fuc.

[0121] In some embodiments, the GalNAc is attached to the conjugate via a reactive moiety on the GalNAc.

[0122] In some embodiments, the drug is attached to the antibody at position N180 of SEQ NO:11, which is the position corresponding to N297 (as numbered by EU numbering).

[0123] In some embodiments, the drug is attached to the antibody at position N296 of SEQ NO:13, which is the position corresponding to N297 (as numbered by EU numbering).

[0124] In some embodiments, the process for preparing a modified B7-H4 antibody comprises: (a) contacting a glycoprotein (e.g., a B7-H4 antibody glycan) comprising a B7-H4 antibody and a core-GlcNAc moiety attached to site N297 (according to EU numbering) of the antibody with endoglycosidase Endo SH, thereby forming an intermediate antibody comprising a terminal GlcNAc moiety; and (b) contacting the intermediate antibody with 4-AzGalNAc-UDP in the presence of a β-(1,4)-GalNAcT enzyme, thereby forming a modified B7-H4 antibody that includes a modified GlcNAc moiety; Steps (a) and (b) are carried out simultaneously.

[0125] In some embodiments, the endoglycosidase is Endo SH, which is a fusion of two endoglycosidases, Endo S and Endo H, linked by a Gly-rich spacer containing an internal 6xHis tag, resulting in an overall molecular weight of 139 kDa.

[0126] In some embodiments, the β-(1,4)-GalNAcT enzyme comprises an N-terminal 6xHis tag and has an overall molecular weight of 45.7 kDa. In some embodiments, the β-(1,4)-GalNAcT enzyme comprises an N-terminal 6xHis tag and is derived from Stinging nettle grass.

[0127] In some embodiments, the process is carried out in a PBS buffer at a pH value of about 7.4 and a temperature of about 30°C.

[0128] 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 those described in International Publication No. WO 2018098269 and U.S. Patent No. 17 / 568,378, each of which is incorporated by reference in its entirety.

[0129] In some embodiments, the B7-H4 antibody-drug conjugate of formula (I) has the formula (Ia): TIFF2025517340000018.tif125160, wherein □ is GalNAc, and d 13 is approximately 2.

[0130] In some embodiments, the B7-H4 antibody-drug conjugate of formula (I) has the formula (Ib): TIFF2025517340000019.tif122159, wherein ■ is GlcNAc, and d 13 is approximately 2.

[0131] In some embodiments, the B7-H4 antibody-drug conjugate of formula (I) has the formula (Ic): TIFF2025517340000020.tif124159, wherein △ is Fuc, and d 13 is approximately 2.

[0132] In some embodiments, the B7-H4 antibody-drug conjugate is a conjugate of formula (IA). TIFF2025517340000021.tif119155

[0133] Dosage and Administration The cancer treatment provided herein, including the B7-H4 targeting antibody-drug conjugate, is administered in an amount sufficient to exert therapeutically useful effects.Typically, the active agent is administered in an amount that does not cause undesirable side effects in the treated patient, or minimizes or reduces observed side effects.B7-H4 expressing cancers 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, cholangiocarcinoma, uterine cancer and cholangiocarcinoma.

[0134] It is understood that the exact dosage and duration of treatment is a function of the tissue or tumor being treated and can be determined empirically using known testing protocols or by extrapolation from in vivo or in vitro test data and / or from known dosing regimens of a particular agent. It is also noted that concentration and dosage values ​​can vary according to the age of the individual being treated, the weight of the individual, the route of administration, and / or the extent or severity of the disease, as well as other factors that are within the level of a skilled medical practitioner to consider. In general, the dosage regimen is selected to limit toxicity. It should be noted that the attending physician knows how and when to terminate, interrupt or adjust treatment to lower the dosage due to toxicity or insufficiency of bone marrow, liver or kidney or other tissues. Conversely, the attending physician also knows how and when to adjust treatment to a higher level if the clinical response is not adequate (eliminating toxic side effects). It should be further understood that for any particular subject, specific dosage regimen should be adjusted over time according to individual need and the professional judgment of the person administering or supervising the administration of the formulation, and the concentration ranges described herein are merely exemplary and are not intended to be limiting.For example, the B7-H4 targeting antibody-drug conjugate is administered in a therapeutically effective amount to reduce tumor volume.

[0135] Patients with multiple solid tumors, including breast, ovarian and endometrial cancer, are administered a B7-H4 targeted antibody-drug conjugate (eg, XMT-1660) in an amount sufficient to exert a therapeutically useful effect.

[0136] In some embodiments, the tumor or cancer is positive for B7-H4 expression.

[0137] In some embodiments, the subject has triple negative breast cancer (TNBC). In some embodiments, the subject has HR+ / HER2- breast cancer. In some embodiments, the subject has endometrial cancer. In other embodiments, the subject has ovarian cancer, fallopian tube cancer, or primary peritoneal cancer.

[0138] In some embodiments, subjects with unresectable or recurrent / metastatic TNBC following standard chemotherapy. In some embodiments, subjects with TNBC have received at least two lines of systemic therapy in the locally advanced or metastatic breast cancer setting. In some embodiments, patients have a histologically or cytologically proven diagnosis of breast cancer with evidence of metastatic or locally advanced disease. In some embodiments, patients have estrogen receptor (ER) negative tumors, progesterone receptor (PR) negative tumors and HER2 negative tumors based on local examination of the most recent tumor biopsy. In some embodiments, patients have received 1-3 prior lines of chemotherapy for the metastatic setting. In some embodiments, patients with BRCA mutations have received prior treatment with a PARP inhibitor.

[0139] In some embodiments, the subject has HR+ / HER2- breast cancer after CDK4 / 6 inhibitor and endocrine-based therapy. In some embodiments, the subject with HR+ / HER2- breast cancer has received at least one line of systemic therapy, which must include CDK 4 / 6 inhibitor and endocrine therapy (ET) in the advanced or metastatic breast cancer setting. In some embodiments, the subject with HR+ / HER2- breast cancer has a histologically or cytologically proven diagnosis of breast cancer with evidence of metastatic or locally advanced disease. In some embodiments, the subject has ER-positive and / or PR-positive tumors and HER2-negative tumors based on local examination of the most recent tumor biopsy.

[0140] In some embodiments, the subject with endometrial cancer has received at least one line of systemic therapy, including platinum-based chemotherapy, for advanced or metastatic disease. In some embodiments, the subject for whom experimental therapy is appropriate, with no satisfactory treatment options, has a histologically or cytologically proven diagnosis of endometrial cancer, with evidence of metastatic or locally advanced disease. In some embodiments, the subject has received at least one prior line of platinum-doublet chemotherapy and no more than three prior lines of systemic therapy for recurrent or metastatic cancer, not including hormonal therapy. In some embodiments, the subject with endometrial cancer must not have endometrial sarcoma or carcinosarcoma.

[0141] In some embodiments, subjects with ovarian, fallopian tube, or primary peritoneal cancer have had at least two lines of systemic therapy for progressive or metastatic disease, which should include platinum-based chemotherapy. In some embodiments, subjects have a histological diagnosis of high-grade serous ovarian cancer, including fallopian tube or primary peritoneal cancer that is metastatic or recurrent. In some embodiments, subjects must have completed four or more cycles of platinum-based therapy and have platinum-resistant recurrent disease, defined as radiographic progression within six months of the last platinum-based therapy. In some embodiments, subjects who have had one prior line of chemotherapy must have achieved a complete or partial response to that therapy and have disease progression within three to six months after the last dose of platinum in the first-line setting. In some embodiments, subjects who have had two to four prior lines of chemotherapy have disease progression within six months or less after the last dose of platinum in the most recent chemotherapy. In some embodiments, subjects who have had one to four prior lines of systemic therapy for ovarian cancer, including at least one prior line of platinum-containing regimen.

[0142] In some embodiments, subjects with TNBC, HR+ / HER2- breast cancer, endometrial cancer, ovarian cancer, fallopian tube cancer, or primary peritoneal cancer are administered a body surface area (BSA)-dependent dose of B7-H4 targeting antibody-drug conjugate (e.g., XMT-1660) by injection. BSA-adjusted doses are calculated using the Mosteller formula, if possible. Starting doses are 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). Dose calculations in subsequent cycles are performed for weight changes of ≧5% from the most recent dose calculation. Dose adjustments are made based on subsequent weight measurements. Additional weight measurements are obtained to confirm or change BSA before dosing in cycle 2 and every two cycles thereafter.

[0143] In some embodiments, the administration of the B7-H4 targeting antibody-drug conjugate (e.g., XMT-1660) is by injection. The injection method can include any method known in the art for injecting a therapeutic agent into a subject. In some embodiments, the injection is an intravenous (IV) injection.

[0144] In some embodiments, the infusion of the B7-H4 targeting antibody-drug conjugate (e.g., XMT-1660) is carried out for 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 or subsequent infusions.

[0145] In some embodiments, the initial dose of B7-H4 targeting antibody-drug conjugate (e.g., XMT-1660) for each subject is administered over 90±10 minutes. If no infusion-related reactions occur, all subsequent doses can be administered over 60±10 minutes.

[0146] In some embodiments, subjects with TNBC, HR+ / HER2- breast cancer, endometrial cancer, ovarian cancer, fallopian tube cancer, or primary peritoneal cancer are administered about 5 mg / m by injection. 2 ~about 100mg / m 2 A B7-H4 targeting antibody-drug conjugate (eg, XMT-1660) is administered at a dosage of

[0147] In some embodiments, the dosage of the B7-H4 targeting antibody-drug conjugate is about 5 mg / m 2 ~about 50mg / m 2 , or about 50 mg / m 2 ~about 100mg / m 2 It is.

[0148] In some embodiments, the dose is about 5 mg / m 2 ~about 10mg / m 2 , about 10mg / m 2 ~about 15mg / m 2 , about 15mg / m 2 ~about 20mg / m 2 , about 20mg / m 2 ~about 25mg / m 2 , about 25mg / m 2 ~about 30mg / m 2 , about 30mg / m 2 ~about 35mg / m 2 , about 35mg / m 2 ~about 40mg / m 2 , about 40mg / m 2 ~about 45mg / m 2 , about 45mg / m 2 ~about 50mg / m 2 , about 50mg / m 2 ~about 55mg / m 2 , about 55mg / m 2 ~about 60mg / m 2 , about 60mg / m2 ~about 65mg / m 2 , about 65mg / m 2 ~about 70mg / m 2 , about 75mg / m 2 ~about 80mg / m 2 , about 85mg / m 2 ~about 90mg / m 2 , about 90mg / m 2 ~about 95mg / m 2 , or about 95 mg / m 2 ~about 100mg / m 2 It is.

[0149] In some embodiments, the dosage of the B7-H4 targeting antibody-drug conjugate is about 6.0 mg / m 2 ~about 8.0mg / m 2 In some embodiments, the dosage of the B7-H4 targeting antibody-drug conjugate is about 6.7 mg / m 2 ~about 7.7mg / m 2 In some aspects, the dosage 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 dosage of the B7-H4 targeting antibody-drug conjugate is about 7.2 mg / m 2 It is.

[0150] In some embodiments, the dosage of the B7-H4 targeting antibody-drug conjugate is about 12.0 mg / m 2 ~Approx. 15.0mg / m 2 In some embodiments, the dosage of the B7-H4 targeting antibody-drug conjugate is about 13.9 mg / m 2 ~Approx. 14.9mg / m 2 In some aspects, the dosage 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 dosage of the B7-H4 targeting antibody-drug conjugate is about 14.4 mg / m 2 It is.

[0151] In some embodiments, the dosage of the B7-H4 targeting antibody-drug conjugate is about 20.0 mg / m 2 ~About 23.0mg / m 2 In some embodiments, the dosage of the B7-H4 targeting antibody-drug conjugate is about 21.1 mg / m 2 ~About 22.1mg / m 2In some aspects, the dosage 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 dosage of the B7-H4 targeting antibody-drug conjugate is about 21.6 mg / m 2 It is.

[0152] In some embodiments, the dosage of the B7-H4 targeting antibody-drug conjugate is about 27.0 mg / m 2 ~About 30.0mg / m 2 In some embodiments, the dosage of the B7-H4 targeting antibody-drug conjugate is about 28.2 mg / m 2 ~About 29.2mg / m2 In some aspects, the dosage 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 dosage of the B7-H4 targeting antibody-drug conjugate is about 28.7 mg / m 2 It is.

[0153] In some embodiments, the dosage of the B7-H4 targeting antibody-drug conjugate is about 36.0 mg / m 2 ~about 40.0mg / m 2 In some embodiments, the dosage of the B7-H4 targeting antibody-drug conjugate is about 37.6 mg / m 2~Approx. 38.6mg / m 2 In some aspects, the dosage is about 36.0 mg / m 2 , about 36.1mg / m 2 , 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 2In some embodiments, the dosage of the B7-H4 targeting antibody-drug conjugate is about 38.1 mg / m 2 It is.

[0154] In some embodiments, the dosage of the B7-H4 targeting antibody-drug conjugate is about 49.0 mg / m 2 ~Approx. 52.0mg / m 2 In some embodiments, the dosage of the B7-H4 targeting antibody-drug conjugate is about 50.2 mg / m 2 ~Approx. 51.2mg / m 2 In some aspects, the dosage 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 / m2 In some embodiments, the dosage of the B7-H4 targeting antibody-drug conjugate is about 50.7 mg / m 2 It is.

[0155] In some embodiments, the dosage of the B7-H4 targeting antibody-drug conjugate is about 66.0 mg / m 2 ~about 69.0mg / m 2 In some embodiments, the dosage of the B7-H4 targeting antibody-drug conjugate is about 66.9 mg / m 2 ~Approx. 67.9mg / m 2 In some aspects, the dosage 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 2or about 69.0 mg / m 2 In some embodiments, the dosage of the B7-H4 targeting antibody-drug conjugate is about 67.4 mg / m 2 It is.

[0156] In some embodiments, the dosage of the B7-H4 targeting antibody-drug conjugate is about 86.0 mg / m 2 ~about 89.0mg / m 2 In some embodiments, the dosage of the B7-H4 targeting antibody-drug conjugate is about 87.1 mg / m 2 ~about 88.1mg / m 2 In some aspects, the dosage 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 2or about 89.0 mg / m 2 In some embodiments, the dosage of the B7-H4 targeting antibody-drug conjugate is about 87.6 mg / m 2 In some embodiments, the dosage of the B7-H4 targeting antibody-drug conjugate 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 / m 2 , 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 / m2 , approximately 40.0 mg / m 2 , approximately 41.0 mg / m 2 , approximately 42.0 mg / m 2 , approximately 43.0 mg / m 2 , approximately 44.0 mg / m 2 , approximately 45.0 mg / m 2 , approximately 46.0 mg / m 2 , approximately 47.0 mg / m 2 , approximately 48.0 mg / m 2 , approximately 49.0 mg / m 2 , approximately 50.0 mg / m 2 , approximately 51.0 mg / m 2 , approximately 52.0 mg / m 2 , approximately 53.0 mg / m 2 , approximately 54.0 mg / m 2 , approximately 55.0 mg / m 2 , approximately 56.0 mg / m 2 , approximately 57.0 mg / m 2 , approximately 58.0 mg / m 2 , approximately 59.0 mg / m 2 , approximately 60.0 mg / m 2 , approximately 61.0 mg / m 2 , approximately 62.0 mg / m 2 , approximately 63.0 mg / m 2 , approximately 64.0 mg / m 2 , approximately 65.0 mg / m 2 , approximately 66.0 mg / m 2 , approximately 67.0 mg / m 2 , approximately 68.0 mg / m 2 , approximately 69.0 mg / m 2 , approximately 70.0 mg / m 2 , approximately 71.0 mg / m 2 , approximately 72.0 mg / m 2 , approximately 73.0 mg / m 2 , approximately 74.0 mg / m 2 , approximately 75.0 mg / m 2 , approximately 76.0 mg / m 2 , approximately 77.0 mg / m 2 , approximately 78.0 mg / m 2 , approximately 79.0 mg / m 2 , approximately 80.0 mg / m 2 , approximately 81.0 mg / m 2 , approximately 82.0 mg / 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 / m 2 , 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 It is.

[0157] In some embodiments, subjects with TNBC, HR+ / HER2- breast cancer, endometrial cancer, ovarian cancer, fallopian tube cancer, or primary peritoneal cancer are administered about 7.2 mg / m by injection. 2 ~about 87.6mg / m 2 In some embodiments, the dosage of the B7-H4 targeting antibody-drug conjugate (e.g., XMT-1660) is about 7.2 mg / m 2 In some embodiments, the dosage of the B7-H4 targeting antibody-drug conjugate (e.g., XMT-1660) is about 14.4 mg / m 2 In some embodiments, the dosage of the B7-H4 targeting antibody-drug conjugate (e.g., XMT-1660) is about 21.6 mg / m 2 In other embodiments, the dosage of the B7-H4 targeting antibody-drug conjugate (e.g., XMT-1660) is about 28.7 mg / m 2 In some embodiments, the dosage of the B7-H4 targeting antibody-drug conjugate (e.g., XMT-1660) is about 38.1 mg / m 2In other embodiments, the dosage of the B7-H4 targeting antibody-drug conjugate (e.g., XMT-1660) is about 50.7 mg / m 2 In some embodiments, the dosage of the B7-H4 targeting antibody-drug conjugate (e.g., XMT-1660) is about 67.4 mg / m 2 In some embodiments, the dosage of the B7-H4 targeting antibody-drug conjugate (e.g., XMT-1660) is about 87.6 mg / m 2 It is.

[0158] In some embodiments, the dose is administered intravenously once per week (i.e., a 7 day cycle), once every two weeks (i.e., a 14 day cycle), once every three weeks (i.e., a 21 day cycle), or once every four weeks (i.e., a 28 day cycle).

[0159] In some embodiments, dosages of the B7-H4 targeting antibody-drug conjugate are administered every week (i.e., a 7 day cycle), every 2 weeks (i.e., a 14 day cycle), every 3 weeks (i.e., a 21 day cycle), every 4 weeks (i.e., a 28 day cycle), every 5 weeks (i.e., a 35 day cycle), every 6 weeks (i.e., a 42 day cycle), every 7 weeks (i.e., a 49 day cycle), or every 8 weeks (i.e., a 56 day cycle).

[0160] In some embodiments, about 7.2 mg / m 2 ~about 87.6mg / m 2 is administered as an infusion every week (i.e., 7 day cycle), every 2 weeks (i.e., 14 day cycle), every 3 weeks (i.e., 21 day cycle), every 4 weeks (i.e., 28 day cycle), every 5 weeks (i.e., 35 day cycle), every 6 weeks (i.e., 42 day cycle), every 7 weeks (i.e., 49 day cycle), or every 8 weeks (i.e., 56 day cycle).

[0161] In some embodiments, the subject is administered about 7.2 mg / m 2~about 87.6mg / m 2 In some embodiments, a subject is administered a B7-H4 targeting antibody-drug conjugate (e.g., XMT-1660) at a dose of about 7.2 mg / m 2 ~about 87.6mg / m 2 is administered once every four weeks (ie, a 28 day cycle).

[0162] In some embodiments, the subject is administered about 7.2 mg / m 2 ~about 87.6mg / m 2 is administered over 90 minutes for the first infusion, then over 90 minutes for subsequent infusions, once every three weeks (i.e., a 21 day cycle).

[0163] In some embodiments, the subject is administered about 7.2 mg / m 2 ~about 87.6mg / m 2 is administered over 90 minutes for the first infusion, then over 60 minutes for subsequent infusions, once every three weeks (i.e., a 21 day cycle).

[0164] In some embodiments, the subject is administered about 7.2 mg / m 2 ~about 87.6mg / m 2 is administered over 90 minutes for the first infusion, then over 30 minutes for subsequent infusions, once every 4 weeks (i.e., a 28 day cycle).

[0165] In some embodiments, the subject is administered about 7.2 mg / m 2 A dose of a B7-H4 targeting antibody-drug conjugate (e.g., XMT-1660) is administered over 90 minutes for the first infusion, then over 60 or 90 minutes for subsequent infusions, once every three weeks (i.e., a 21 day cycle).

[0166] In some embodiments, the subject is administered about 14.4 mg / m 2 A dose of a B7-H4 targeting antibody-drug conjugate (eg, XMT-1660) is administered over 90 minutes for the first infusion, then over 60 or 90 minutes for subsequent infusions, once every three weeks.

[0167] In some embodiments, the subject is administered about 21.6 mg / m 2 A dose of a B7-H4 targeting antibody-drug conjugate (eg, XMT-1660) is administered over 90 minutes for the first infusion, then over 60 or 90 minutes for subsequent infusions, once every three weeks.

[0168] In some embodiments, the subject is administered about 28.7 mg / m 2 A dose of a B7-H4 targeting antibody-drug conjugate (eg, XMT-1660) is administered over 90 minutes for the first infusion, then over 60 or 90 minutes for subsequent infusions, once every three weeks.

[0169] In some embodiments, the subject is administered about 38.1 mg / m 2 A dose of a B7-H4 targeting antibody-drug conjugate (eg, XMT-1660) is administered over 90 minutes for the first infusion, then over 60 or 90 minutes for subsequent infusions, once every three weeks.

[0170] In some embodiments, the subject is administered about 50.7 mg / m 2 A dose of a B7-H4 targeting antibody-drug conjugate (eg, XMT-1660) is administered over 90 minutes for the first infusion, then over 60 or 90 minutes for subsequent infusions, once every three weeks.

[0171] In some embodiments, the subject is administered about 67.4 mg / m 2A dose of a B7-H4 targeting antibody-drug conjugate (eg, XMT-1660) is administered over 90 minutes for the first infusion, then over 60 or 90 minutes for subsequent infusions, once every three weeks.

[0172] In some embodiments, the subject is administered about 87.6 mg / m 2 A dose of XMT-1660 will be administered over 90 minutes for the first infusion, then over 60 or 90 minutes for subsequent infusions, once every three weeks.

[0173] In some embodiments, the subject is administered 7.2 mg / m 2 A dose of a B7-H4 targeting antibody-drug conjugate (eg, XMT-1660) is administered over 90 minutes for the first infusion, then over 60 or 90 minutes for subsequent infusions, once every four weeks.

[0174] In some embodiments, the subject is administered about 14.4 mg / m 2 A dose of a B7-H4 targeting antibody-drug conjugate (eg, XMT-1660) is administered over 90 minutes for the first infusion, then over 60 or 90 minutes for subsequent infusions, once every four weeks.

[0175] In some embodiments, the subject is administered about 21.6 mg / m 2 A dose of a B7-H4 targeting antibody-drug conjugate (eg, XMT-1660) is administered over 90 minutes for the first infusion, then over 60 or 90 minutes for subsequent infusions, once every four weeks.

[0176] In some embodiments, the subject is administered about 28.7 mg / m 2 A dose of a B7-H4 targeting antibody-drug conjugate (eg, XMT-1660) is administered over 90 minutes for the first infusion, then over 60 or 90 minutes for subsequent infusions, once every four weeks.

[0177] In some embodiments, the subject is administered about 38.1 mg / m 2A dose of a B7-H4 targeting antibody-drug conjugate (eg, XMT-1660) is administered over 90 minutes for the first infusion, then over 60 or 90 minutes for subsequent infusions, once every four weeks.

[0178] In some embodiments, the subject is administered about 50.7 mg / m 2 A dose of a B7-H4 targeting antibody-drug conjugate (eg, XMT-1660) is administered over 90 minutes for the first infusion, then over 60 or 90 minutes for subsequent infusions, once every four weeks.

[0179] In some embodiments, the subject is administered about 67.4 mg / m 2 A dose of a B7-H4 targeting antibody-drug conjugate (eg, XMT-1660) is administered over 90 minutes for the first infusion, then over 60 or 90 minutes for subsequent infusions, once every four weeks.

[0180] In some embodiments, the subject is administered about 87.6 mg / m 2 A dose of a B7-H4 targeting antibody-drug conjugate (eg, XMT-1660) is administered over 90 minutes for the first infusion, then over 60 or 90 minutes for subsequent infusions, once every four weeks.

[0181] The frequency and timing of administration, as well as the dosage, can be administered periodically over an administration cycle to maintain the continuous and / or long-term effect of the active agent over a desired length of time. The provided compositions of B7-H4 targeting antibody-drug conjugates can be administered hourly, daily, weekly, monthly, yearly, or once. The length of time of an administration cycle can be empirically determined and depends on the disease being treated, the severity of the disease, the particular patient, and other considerations within the skill level of the treating physician. The length of time of treatment with the combination therapy provided herein can be one week, two weeks, one month, several months, one year, several years, or longer.

[0182] For example, the administration frequency of the B7-H4 targeting antibody-drug conjugate is once a day, every other day, twice a week, once a week, once every two weeks, once every three weeks, or once every four weeks. The dosage can be divided into multiple administration cycles during the course of treatment. For example, the B7-H4 targeting antibody-drug conjugate can be administered at a frequency for a period of about one month, about two months, about three months, about four months, about five months, about six months, about one year, or more. The administration frequency can be the same or different throughout the duration of the cycle. For example, an exemplary administration frequency is twice a week for at least 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 once a week or less. It is within the level of one of ordinary skill in the art to determine the specific administration frequency and administration cycle based on the specific dosage administered, the disease or condition being treated, the severity of the disease or condition, the age of the subject, and other similar factors.

[0183] If disease symptoms persist in the absence of discontinued treatment, treatment can be continued for a longer period of time. Over the course of treatment, evidence of disease and / or treatment-related toxicity or side effects can be monitored.

[0184] The administration cycle of the B7-H4 targeting antibody-drug conjugate can be adjusted to add a period of interrupted treatment to provide a rest period from exposure to the agent.The length of time for interrupting treatment can be for a predetermined period of time or can be empirically determined according to how the patient responds or according to observed side effects.For example, treatment can be interrupted for one week, two weeks, three weeks, one month or several months.Generally, the period of interrupted treatment is incorporated into the cycle of the administration regimen for the patient.

[0185] Exemplary dosing regimen is a 21-day or 28-day treatment or administration cycle.Preferably, dosing regimen is a treatment or administration cycle is 28 days.The B7-H4 targeting antibody-drug conjugate disclosed herein is administered on day 1, followed by not administering for 20 days, or administered on day 1, followed by not administering for 27 days.It is within the level of a person skilled in the art to determine the exact dosing cycle and administration schedule.

[0186] As mentioned above, the administration cycle can be for any desired length of time.Therefore, the administration of the 21-day cycle or the 28-day cycle can be repeated for any length of time.For example, the administration of the 21-day cycle or the 28-day 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 more.It is within the level of skill of the treating physician to adopt the administration cycle and administration regimen that meets the needs of the patient, depending on the individual considerations specific to the patient and the disease being treated.

[0187] In some embodiments, subjects are administered a B7-H4 targeting antibody-drug conjugate (e.g., XMT-1660) until disease progression, death, unacceptable toxicity, or voluntary discontinuation, whichever occurs first.

[0188] Dose reduction and delay In some cases, toxicity or adverse reactions may occur in response to the initial dose of B7-H4 targeting antibody-drug conjugate (e.g. XMT-1660) at the dose described herein.Subsequent doses may be reduced, or the time between doses may be delayed or extended.In some embodiments, the toxicity or adverse reactions that may lead to dose reduction or delay include, but are not limited to, elevated ALT or AST levels, liver toxicity, interstitial lung disease (ILD), pneumonitis, hematological toxicity, proteinuria, fatigue, nausea, other clinically significant non-clinical laboratory toxicity, other clinically significant adverse events.

[0189] Measurement of B7-H4 expression In various aspects, the present invention provides methods for identifying cancer patients suitable for B7-H targeted therapy or monitoring treatment regimens by measuring the B7-H4 expression status in tumor samples obtained from the patient.

[0190] In some embodiments, a B7-H4 diagnostic test can be used to identify subjects for treatment with a B7-H4 targeted drug conjugate.

[0191] The sample is derived from a subject with cancer. The sample of cancer cells is examined from tissue removed or obtained from the subject. In some embodiments, the sample is a fresh biopsy sample, a frozen biopsy sample, or an archived biopsy sample.

[0192] 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 from a biopsy or surgical sample, or from ascites or pleural effusion. In some embodiments, the test cell population is derived from 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 specimen. The tissue sample can be frozen or fresh.

[0193] The required level of B7-H4 expression can be identified by any method known in the art, more specifically by the method described herein.For example, the level of B7-H4 expression can be measured by performing known immunological assays such as enzyme immunoassay, radioimmunoassay, competitive immunoassay, double antibody sandwich assay, fluorescent immunoassay, ELISA, Western blotting technique, agglutination assay, cytofluorometry (e.g., flow cytometry), fluorescent in situ hybridization (FISH), colorimetric method or immunohistochemical staining assay (IHC) for protein expression using an antibody that specifically recognizes B7-H4.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 suitable.

[0194] Flow cytometry (FC) may be used to determine cell surface expression of B7-H4 in tumor samples before, during and after treatment with drugs. For example, tumor cells may be analyzed by flow cytometry for B7-H4 expression, and markers that identify cancer cell type, etc., if desired. Flow cytometry may be performed according to standard methods. See, for example, Chow et al., Cytometry (Communications in Clinical Cytometry) 46:72-78 (2001). In summary, as an example, the following protocol for cytometric analysis may be used: fix cells with 2% paraformaldehyde for 10 minutes at 37°C, followed by permeabilization in 90% methanol for 30 minutes on ice. Cells may then be stained with B7-H4 specific antibodies, washed, and labeled with fluorescently labeled secondary antibodies. Cells are then analyzed using a flow cytometer (e.g., Beckman Coulter FC500) according to the specific protocol of the instrument used. Such an analysis identifies the level of expressed B7-H4 within the tumor.

[0195] Immunohistochemistry (IHC) staining may be used to determine the expression of B7-H4 in tumor samples before, during and after treatment with drugs. IHC may be performed according to well-known techniques. For example, see ANTIBODIES; A LABORATORY MANUAL, Chapter 10, Harlow & Lane Eds., Cold Spring Harbor Laboratory (1988). In summary, prepare paraffin-embedded tissue (e.g., tumor tissue from biopsy) for immunohistochemical staining by, for example, deparaffinizing tissue sections with xylene, followed by ethanol, hydrating in water and then in PBS, unmasking antigens by heating slides in sodium citrate buffer, incubating sections in hydrogen peroxide, blocking in blocking solution, incubating slides in primary polypeptide antibody and secondary antibody, and finally detecting using the ABC avidin / biotin method according to the manufacturer's instructions.

[0196] Immunofluorescence (IF) assay may be used to determine the expression of B7-H4 tumor samples before, during and after treatment with drugs. IF may be performed according to well-known techniques. For example, see JM Polak and S. Van Noorden (1997) INTRODUCTION TO IMMUNOCYTOCHEMISTRY, 2nd Ed.; ROYAL MICROSCOPY SOCIETY MICROSCOPY HANDBOOK 37, BioScientific / Springer-Verlag. In summary, as an example, patient samples may be fixed in paraformaldehyde, followed by methanol, blocked with a blocking solution such as horse serum, incubated with a primary antibody against a polypeptide, followed by a secondary antibody labeled with a fluorescent dye such as Alexa 488, and analyzed using an epifluorescence microscope.

[0197] The antibodies used in the above assays may be advantageously conjugated to other labels, such as fluorescent dyes (e.g., Alexa488, PE), or quantum dots, for use in multiparametric analysis together with other signaling (phospho-AKT, phospho-Erk 1 / 2) and / or cell marker (cytokeratin) antibodies.

[0198] In one embodiment, the expression of B7-H4 in a sample from a tumor is determined immunohistochemically using a system, for example, the Leica Bond III Fully Automated Stainer (BOND III) system.

[0199] Alternatively, the assay may include preparing RNA from the sample, optionally for use in PCR (polymerase chain reaction) or other analytical methods.The PCR method may be, for example, RT-PCR (reverse transcription-PCR) or quantitative PCR, such as real-time RT-PCR, RNA seq, etc.Alternatively, the assay may be performed using arrays such as microarrays, such as nanostring technology, as known in the relevant field.

[0200] Patients are identified as responsive to treatment, treatment is monitored, or cancer is detected by detecting and / or measuring the expression level of B7-H4 in tumor cells in a sample.

[0201] The detection / measurement of the expression level of B7-H4 is determined by calculating B7-H4 score.B7-H4 score is quantitative or semi-quantitative.For example, detection is pathologically scored to arrive at pathological score.It is contemplated that any scoring method known in the art may be used in the method of the present invention.In particular, any histological scoring method known in the art.

[0202] The methods for evaluating the measurement results obtained by immunohistochemical staining assay include, for example, the H score method, TPS (tumor proportion score), and PS2+ (percent score) score. 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+); where staining intensity 0 is no staining; staining intensity 1 is weak staining; staining intensity 2 is moderate staining; staining intensity 3 is strong staining. In some embodiments, when TPS is scored as tumor cell membrane reactivity, subjects with a TPS of ≧75 are considered B7-H4 positive (high) in this assay, and a TPS of <75 is considered B7-H4 negative (low).

[0203] Only the cancer cell portion is used for the evaluation by the scoring method. Formalin-fixed paraffin-embedded cell lines or xenografts (lines with known protein expression levels) may be used as negative or positive controls for staining intensity. In the absence of control specimens, multiple specimens may be evaluated simultaneously to confirm the overall staining intensity distribution, and then the staining intensity may be set.

[0204] In addition to the above scoring methods, other scoring methods known in the art can be used, such as the Allred method (Harvey, et al. Journal of Clinical Oncology 17, No. 5 (May 1999) 1474-1474). A cut-off point needs to be set for each method. Allred score = percentage score of positive cells + staining intensity score.

[0205] The present disclosure also provides kits and / or methods for identifying or otherwise refining, e.g., stratifying, patient populations suitable for therapeutic administration of the B7-H4 targeting antibody-drug conjugates disclosed herein by identifying a subject's B7-H4 score prior to treatment with the B7-H4 targeting antibody-drug conjugates 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 from a biopsy or surgical sample or from ascites or pleural effusion. In some embodiments, the test cell population is derived from fixed tissue (e.g., formalin-fixed) from a biopsy or surgical sample. IHC testing measures the amount of B7-H4 receptor protein on the surface of cells in a cancer tissue sample.

[0206] definition As utilized in accordance with the present disclosure, the following terms, unless otherwise specified, shall be understood to have the following meanings:

[0207] As used herein, the terms "anti-B7-H4 antibody," "B7-H4 antibody," and "antibody that binds to B7-H4" refer to an antibody that can bind to B7-H4 with sufficient affinity such that the antibody is useful as a diagnostic and / or therapeutic agent in targeting B7-H4.

[0208] The term "B7-H4" used herein refers to any natural mature B7-H4 that is generated from the processing of B7-H4 precursor protein in cells.Unless otherwise specified, 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).The term also includes naturally occurring variants of B7-H4, such as splice variants or allelic variants.

[0209] The term "B7-H4 positive cancer" refers to a cancer that contains cells that express B7-H4 on their surface. In some embodiments, expression of B7-H4 on the cell surface is determined using an antibody against B7-H4, for example, by immunohistochemistry, FACS, or other methods.

[0210] Alternatively, B7-H4 mRNA expression, which is believed to correlate with B7-H4 expression on the cell surface, can be determined by a method selected from in situ hybridization and RT-PCR (including quantitative RT-PCR).

[0211] The term "B7-H4 positive cells" refers to cells that express B7-H4 on their surface.

[0212] The term "antibody" as used herein is used in the broadest sense and encompasses various antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) and antibody fragments, so long as they exhibit the desired antigen-binding activity. Various methods for numbering the amino acid sequence of an antibody and identifying the complementarity determining region are known in the art. For example, the Kabat numbering system (see Kabat, EA, et al., Sequences of Protein of immunological interest, Fifth Edition, US Department of Health and Human Services, US Government Printing Office (1991)) or the IMGT numbering system (see IMGT®, international ImMunoGeneTics information system®, available online at http: / / www.imgt.org / ). The IMGT numbering system is routinely used and accepted in the art as a reliable and accurate system for determining amino acid positions within coding sequences, alignment of alleles, and easily comparing sequences within immunoglobulins (IG) and T cell receptors (TR) from all vertebrate species. The accuracy and consistency of the IMGT data is based on the IMGT-ONTOLOGY, the first and so far unique ontology for immunogenetics and immunoinformatics (see Lefranc.MP et al., Biomolecules, 2014 Dec;4(4),1102-1139). IMGT tools and databases run against the IMGT reference directory built from a large repository of sequences. In the IMGT system, IG V-DOMAINS and IG C-DOMAINS are delimited, whenever appropriate, taking into account exon delimitations. Thus, as more sequences become available in the IMGT database, the IMGT exon numbering system can and will be used reliably by those skilled in the art to determine amino acid positions within coding sequences and to align alleles.Additionally, the correspondence between IMGT specific numbering and other numbering (i.e., Kabat) is available in the IMGT Scientific chart (see Lefranc.MP et al.,Biomolecules,2014 Dec;4(4),1102-1139). The EU numbering system (see Edelman,GM et al.,Proc.Natl.Acad.USA,63,78-85(1969)) can also be used. In some aspects, the EU numbering system can be used to number the amino acid positions of an antibody. In some aspects, the EU numbering system can be used to determine the position of the constant region of the heavy or light chain of an antibody.

[0213] 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.

[0214] The term "antibody that binds to the same epitope" as a reference antibody as used herein refers to an antibody that blocks the binding of the reference antibody to its antigen by 50% or more in a competitive assay, and conversely, the reference antibody blocks the binding of the antibody to its antigen by 50% or more in a competitive assay. An exemplary competitive assay is provided herein.

[0215] The term "class" of an antibody refers to the type of constant domain or region that its heavy chain possesses. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and some of these have subclasses (isotypes), e.g., IgGi, IgG2, IgG3, IgG 4 The heavy-chain constant domains that correspond to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0216] The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies constituting the population are identical and / or bind the same epitope, except for variant antibodies that may contain naturally occurring mutations or arise during the production of a monoclonal antibody preparation, and such variants are generally present in small amounts. In contrast to polyclonal antibody preparations, which typically contain different antibodies directed to different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed to a single determinant on an antigen. Thus, the modifier "monoclonal" indicates the character of the antibody obtained from a substantially homogeneous population of antibodies and should not be construed as requiring the production of the antibody by any particular method. For example, the monoclonal antibodies used in accordance with the present invention may be produced by a variety of techniques, including, but not limited to, hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin locus, and such methods, as well as other exemplary methods for producing monoclonal antibodies, are described herein.

[0217] The term "naked antibody" refers to an antibody that is not conjugated to a heterologous moiety (e.g., a cytotoxic moiety or a STING agonist drug moiety). Naked antibodies can be present in a pharmaceutical formulation.

[0218] The term "natural antibody" refers to naturally occurring immunoglobulin molecules with various structures. For example, natural IgG antibodies are heterotetrameric glycoproteins of about 150,000 daltons composed of two identical light chains and two identical heavy chains that are disulfide-bonded. From the N-terminus to the C-terminus, each heavy chain has a variable region (VH), also called the variable heavy domain or the heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH3). Similarly, from the -terminus to the C-terminus, each light chain has a variable region (VL), also called the variable light domain or the 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 kappa (κ) and lambda (λ), based on the amino acid sequence of its constant domain.

[0219] An "isolated antibody" is one that has been separated from a component of its natural environment. In some embodiments, the antibody is purified to greater than 95% or greater than 99% purity, for example, as determined by electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion-exchange HPLC or reverse-phase HPLC). For a review of methods for assessing antibody purity, see, for example, Flatman et al., J. Chromatogr. B 848:79-87 (2007).

[0220] The term "epitope" refers to a specific site on an antigen molecule to which an antibody binds.

[0221] The term "humanized antibody" of an antibody refers to an antibody derived from a non-human antibody (e.g., mouse) that retains or substantially retains the antigen-binding properties of the parent antibody, but is less immunogenic in humans. Humanized as used herein is intended to include deimmunized antibodies.

[0222] The term "humanized form" of an antibody, e.g., a non-human antibody, refers to an antibody that has undergone humanization.

[0223] The terms "compete with" or "cross-compete with," when used herein in reference to two or more antibodies, indicate that the two or more antibodies compete for binding to B7-H4, e.g., compete for B7-H4 binding. An antibody "blocks" or "cross-blocks" one or more other antibodies from binding to B7-H4 if it competes with one or more other antibodies by 25% or more, with 25%-74% representing "partial blocking" and 75%-400% representing "full blocking." Unless otherwise defined or contradicted by context, as used herein, the terms "compete with," "cross-compete with," "blocking," or "cross-blocking" are also intended to encompass such pairs of antibodies.

[0224] As used herein, an antibody that "specifically binds to human B7-H4" refers to an antibody that specifically binds to human B7-H4. -7 Below, more typically 5×10 -8 M or less, more typically 3×10 -8 M or less, more typically 1×10 -9 M or less, and even more typically 5×10 -9 K below M D The term "antibody" is intended to refer to an antibody that binds to human B7-H4 at

[0225] The term "does not substantially bind" to a protein or cell, as used herein, means that the antibody does not bind to the protein or cell or does not bind with high affinity, i.e., less than 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, and even more preferably 1×10 -2 K over M D This means that the molecule binds to a protein or cell via the

[0226] 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 sugar, i.e., a monosaccharide sugar that includes a substituent and / or functional group. Examples of sugar derivatives include, but are not limited to, amino sugars and sugar acids. Examples of sugar derivatives also include S'(F') X1 In addition, the present invention also includes compounds represented by the formula: 1 indicates the number of functional groups.

[0227] The term "core-GlcNAc moiety" as used herein refers to a monosaccharide, polysaccharide, or oligosaccharide moiety comprising a GlcNAc (e.g., Core-GlcNAc) bound to an antibody (e.g., via the C1 position of GlcNAc). In some embodiments, the GlcNAc is bound to the antibody via an N-glycosidic bond to the amide nitrogen atom of the side chain of an asparagine amino acid of the antibody. In some embodiments, the Core-GlcNAc moiety is present at a native glycosylation site of the antibody or is introduced into 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, e.g., the Core-GlcNAc moiety is a disaccharide Core-GlcNAc-(α1-6-Fuc) moiety (which may be referred to as GlcNAc(Fuc)). Thus, where an antibody comprises a Core-GlcNAc moiety, the antibody may comprise a monosaccharide or a disaccharide Core-GlcNAc moiety, which may further comprise a fucose (e.g., a disaccharide Core-GlcNAc(Fuc) moiety). Where the Core-GlcNAc moiety further comprises a fucose, the fucose may be linked α-1,6 to O-6 of the Core-GlcNAc moiety. A Core-GlcNAc moiety further comprising a fucose may be referred to as Core-GlcNAc(Fuc).

[0228] The term "core-GlcNAc" refers to an internal GlcNAc that is part of a polysaccharide or oligosaccharide that is attached to the antibody via the internal GlcNAc.

[0229] As used herein, the term "terminal GlcNAc moiety" refers to a moiety that is attached to an antibody and contains a GlcNAc with 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.

[0230] The term "nucleotide" is used in its normal scientific sense to refer to a molecule composed of a nucleobase, a five-carbon sugar (either ribose or 2-deoxyribose), and one, two, or three phosphate groups. Without the phosphate groups, the nucleobase and sugar constitute a nucleoside. Thus, a nucleotide may also be referred to as a nucleoside monophosphate, nucleoside diphosphate, or nucleoside triphosphate. The nucleobase may be adenine, guanine, cytosine, uracil, or thymine.

[0231] The term "protein" is used in its normal scientific sense and includes polypeptides containing about 10 or more amino acids. Proteins may contain natural or unnatural amino acids.

[0232] The term "glycoprotein" is used herein in its ordinary scientific sense to refer to a protein that contains one or more mono- or oligosaccharide chains ("glycans") covalently attached to the protein. The glycans may be attached to hydroxyl groups on the protein (O-linked glycans), amide functions on the protein (N-glycoproteins), or carbons on the protein (C-glycoproteins). A glycoprotein may contain multiple glycans, may contain a combination of one or more monosaccharide glycans and one or more oligosaccharide glycans, and may contain a combination of N-linked, O-linked, and C-linked glycans. It is estimated that over 50% of all proteins have some form of glycosylation and therefore qualify as glycoproteins.

[0233] The term "glycan" is used herein in its normal scientific sense to refer to a monosaccharide or oligosaccharide chain attached to a protein. Thus, a glycan refers to the carbohydrate portion of a glycoprotein. A glycan is attached to a protein through the C-1 carbon of one sugar, which may be without additional substitution (monosaccharide) or may be further substituted at one or more of its hydroxyl groups (oligosaccharide). Naturally occurring glycans typically contain one to about ten sugar moieties. However, when a longer sugar chain is attached to a protein, the sugar chain is also considered a glycan. The glycan of a glycoprotein may be a monosaccharide. The glycan may be an oligosaccharide. The oligosaccharide chain of a glycoprotein may be linear or branched. In an oligosaccharide, the sugar that is directly attached to the protein is called the core sugar. In an oligosaccharide, the sugar that is not directly attached to the protein and is attached to at least two other sugars is called the internal sugar. In oligosaccharides, sugars that are not directly attached to the protein but are attached to a single other sugar, i.e., do not have additional sugar substituents on one or more of the other hydroxyl groups, are called terminal sugars. To avoid confusion, there may be multiple terminal sugars in the oligosaccharides of a glycoprotein, but only one core sugar. Glycans may be O-linked, N-linked or C-linked glycans. In delinked glycans, monosaccharide or oligosaccharide glycans are attached to the C atoms of amino acids of the protein.

[0234] The term "glycosyltransferase" refers to a superfamily of enzymes involved in the synthesis of complex carbohydrates present on glycoproteins and glycolipids.

[0235] The term "N-acetylgalactosaminyltransferase" (GalNAc-T) refers to the N-acetyl-D-galactosamine transferase enzyme, which catalyzes the addition of N-acetyl-D-galactosamine to proteins.

[0236] As used herein, the term "independently" means that when multiple substituents are selected from a number of possible substituents, the substituents may be the same or different.

[0237] As used herein, the term "pharmacologically acceptable" refers to those compounds, conjugates, materials, compositions and dosage forms that are suitable for use in contact with the tissues of human beings and animals, within the scope of sound medical judgment, without undue toxicity, irritation, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0238] As used herein, the term "treating" or "treat" refers to the management and care of a patient to combat a disease, condition, or disorder, and includes the administration of a compound of the present disclosure, or a pharma- ceutically acceptable salt, polymorph, or solvate thereof, to alleviate the symptoms or complications of the disease, condition, or disorder, or to eliminate the disease, condition, or disorder. The term "treat" can also include the treatment of cells in vitro or in an animal model.

[0239] As used herein, the terms "preventing," "prevent" or "protecting against" refer to reducing or eliminating the onset of symptoms or complications of such a disease, condition or disorder.

[0240] A therapeutically "effective amount," as defined herein, is intended to mean an amount of a conjugate sufficient to effectively treat or prevent when administered to a patient in need of such treatment. The amount of a given conjugate that corresponds to such an amount may be determined by the potency (pICso), efficacy (EC) of a particular conjugate, or the like. 50) and biological half-life), the disease state and its severity, the identity of the patient in need of treatment (e.g., age, size and weight), and other factors, but can still be routinely determined by one of skill in the art. Similarly, the treatment period and administration period of the conjugate (the period between doses and the timing of doses, e.g., before / with / after a meal) will vary according to the identity of the mammal in need of treatment (e.g., body weight), the particular conjugate and its characteristics (e.g., pharmacokinetic properties), the disease or disorder and its severity, and the particular compositions and methods used, but can still be determined by one of skill in the art.

[0241] The term "composition" refers to a product containing therapeutically effective amounts of specified ingredients, and any product that results directly or indirectly from the combination of the specified ingredients in the specified amounts.

[0242] As used herein, the term "pharmaceutically acceptable excipient" means an excipient that is generally safe, non-toxic, and not biologically or otherwise undesirable and is useful in preparing pharmaceutical compositions, including excipients that are acceptable for veterinary and human pharmaceutical use. As used herein and in the claims, "pharmaceutically acceptable excipient" includes both one and more than one such excipient.

[0243] The term "conjugate of the disclosure" or "conjugate of the present disclosure" as used herein 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 free base form, or as a salt, particularly a pharma- ceutically acceptable salt thereof) and in 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, certain polymorphic forms, solvate forms, including hydrate forms (e.g., monohydrates, dihydrates and hemihydrates)), as well as mixtures of the various forms.

[0244] Thus, the present disclosure includes the conjugates disclosed herein in any salt or non-salt form and any physical form thereof, as well as mixtures of various forms.While such are included in the present disclosure, it will be understood that the conjugates of the present disclosure in any salt or non-salt form and any physical form thereof may have different levels of activity, different bioavailability, and different handling characteristics for formulation purposes.

[0245] It is understood that throughout this specification, when a composition is described as having, including, or comprising certain components, the composition is also contemplated to consist essentially of or consist of the recited components. Similarly, when a method or process is described as having, including, or comprising certain process steps, the process also consists essentially of or consists of the recited processing steps. Furthermore, it is understood that the order or sequence of steps for performing certain operations is not important so long as the invention remains operable. Moreover, two or more steps or operations may be performed simultaneously.

[0246] The term "competes with" or "cross-competes with" when used herein in reference to two or more antibodies indicates that two or more antibodies compete for binding to B7-H4 in the assays described in Examples 5 or 8, e.g., compete for B7-H4 binding. An antibody "blocks" or "cross-blocks" one or more other antibodies from binding to B7-H4, preferably as determined using the assays of Examples 5 and 8, if the antibody competes with one or more other antibodies by 25% or more, with 25%-74% representing "partial blocking" and 75%-400% representing "full blocking." For some pairs of antibodies, competition or blocking in the assays of Examples 5 or 8 is only observed when one antibody is coated on the plate and the other is used to compete, and not vice versa. Unless otherwise defined or negated by context, as used herein, the terms "competes with," "cross-competes with," "blocks" or "cross-blocks" are also intended to encompass such pairs of antibodies.

[0247] As used herein, "administration regimen" or "dosage regimen" refers to the amount of agent administered, e.g., a composition containing a B7-H4 targeting antibody-drug conjugate, and the frequency of administration. The administration regimen is a function of the disease or condition being treated and can therefore vary.

[0248] As used herein, "frequency" of administration refers to the time between successive administrations of treatment.For example, frequency can be several days, weeks or months.For example, frequency can be more than once a week, for example, twice a week, three times a week, four times a week, five times a week, six times a week or every day.Frequency can also be 1, 2, 3 or 4 weeks.The specific frequency is a function of the specific disease or condition being treated.Generally, frequency is more than once a week, generally twice a week.

[0249] As used herein, "administration cycle" refers to the repeat schedule of the administration regimen of the administration of enzyme and / or second agent, which is repeated over successive administrations.For example, an exemplary administration cycle is a 28-day cycle, which is administered twice a week for 3 weeks, followed by 1 week of withdrawal.Preferred administration cycles are a 7-day cycle, which is administered once every 7 days, a 14-day cycle, which is administered once every 14 days (i.e., 2 weeks), a 21-day cycle, which is administered once every 21 days (i.e., 3 weeks), or a 28-day cycle, which is administered once every 28 days (i.e., 4 weeks).

[0250] As used herein, when referring to dosage amounts based on mg / kg of subject, an average human subject would weigh about 70 kg to 75 kg, e.g., 70 kg, and have a body mass of 1.73 m 2 It is believed that the human body has a body surface area (BSA) of 100 mm.

[0251] As used herein, amelioration of symptoms of a particular disease or disorder by treatment, such as by administration of a pharmaceutical composition or other therapeutic agent, refers to either permanent or temporary, persistent or transient, alleviation of the symptoms, or adverse effects of the pathology, such as a reduction in adverse effects associated with or occurring upon administration of a B7-H4 targeted antibody-drug conjugate.

[0252] As used herein, when referring to a dosage based on "body surface area", (BSA;m 2 ) is the measured or calculated surface area of ​​the human body. For many clinical purposes, BSA is a better indicator of metabolic rate than body weight because it is less affected by abnormal fat mass. Various calculations for arriving at BSA without direct measurement have been published. In the formula below, BSA is m 2 where BSA is the mass in kg and H is the height in cm. The most widely used is the Du Bois formula: BSA = 0.007184 × W 0.425 ×H 0.725Other methods for determining BSA include, for example, the formulas of Mosteller, Haycock, Gehan and George, Boyd, Fujimoto, Takahira, Shuter and Aslani, or Schlich.

[0253] "Subject" includes mammals. The mammal can be, for example, any mammal, such as a human, a primate, a bird, a mouse, a rat, a fowl, a dog, a cat, a cow, a horse, a goat, a camel, a sheep, or a pig. Preferably, the mammal is a human.

[0254] As used herein, "unit dosage form" or "unit dosage form" refers to physically discrete units suitable for human and animal subjects and packaged individually as known in the art.

[0255] As used herein, a single dosage formulation refers to a formulation as a single dose.

[0256] As used herein, "temporal proximity" refers to administration of one therapeutic agent occurring within a period of time before or after administration of another therapeutic agent (e.g., an immune checkpoint inhibitor disclosed herein) such that the therapeutic effect of one therapeutic agent (e.g., a B7-H4 targeting antibody-drug conjugate disclosed herein) overlaps with the therapeutic effect of the other 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, "temporal proximity" means administration of one therapeutic agent occurring within a period of time before or after administration of another therapeutic agent such that there is a synergistic effect between the one therapeutic agent and the other therapeutic agent. "Temporal proximity" may vary according to various factors, including, but not limited to, the age, sex, weight, genetic background, medical condition, medical history, and treatment history of the subject to whom the therapeutic agent is administered; the disease or condition to be treated or ameliorated; the therapeutic result to be achieved; the dosage, frequency, 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, 30 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 18 hours, 24 hours, 36 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 6 weeks, or 8 weeks. In some embodiments, multiple administrations of one therapeutic agent may occur in temporal proximity to a single administration of another therapeutic agent. In some embodiments, temporal proximity may vary during a treatment cycle or administration regimen.

[0257] As used herein, a "kit" refers to a combination of components, such as a composition herein with another item for a purpose including, but not limited to, reconstitution, activation, and instruments / devices for delivery, administration, diagnosis, and evaluation of biological activity or properties. The kit optionally includes instructions for use.

[0258] As used herein, a "prior-line platinum-containing regimen" includes the following: i. Adjuvant ± neoadjuvant considered as first line treatment as long as they are the same regimen (e.g., 4 cycles of platinum / taxane pre-surgery followed by 4 cycles of platinum / taxane post-surgery) ii. Maintenance therapy (e.g., bevacizumab, PARPi or endocrine therapy) is considered part of the prior line of treatment. Substitution of a different platinum or taxane does not count as a new line. iii. Treatment given for only one cycle and discontinued due to toxicity in the absence of progression does not count as a line of treatment. Substitution of a different platinum agent or taxane does not count as a new line. iv. Hormonal therapy (e.g., tamoxifen, letrozole) counts as a separate line of treatment unless administered as maintenance therapy.

[0259] As used herein, "recurrent disease" refers to a subject with disease progression after a partial or complete response to one or more therapeutic agents. In some embodiments, recurrence may be local to the original site of disease (i.e., one or more ovaries), or to a distant or metastatic site.

[0260] The present disclosure is intended to include all isotopes of atoms present in the present compounds. Isotopes include atoms having the same atomic number but different mass numbers. By way of general example and without limitation, isotopes of hydrogen include tritium and deuterium. Isotopes of carbon include C-13 and C-14.

[0261] The present disclosure refers to and is intended to include all isomers of compounds, including optical isomers and tautomers, where optical isomers include enantiomers and diastereomers, chiral isomers and non-chiral isomers, and optical isomers include isolated optical isomers, as well as mixtures of optical isomers, including racemic and non-racemic mixtures, and the isomers may be in isolated form or may be in mixture with one or more other isomers.

[0262] All percentages and ratios used herein are by weight unless otherwise specified. Other features and advantages of the present disclosure are apparent from the various examples. The examples provided show various components and methodologies useful for implementing the present disclosure. The examples do not limit the claimed disclosure. Based on the present disclosure, a person skilled in the art can identify and use other components and methodologies useful for implementing the present disclosure.

[0263] Other Aspects All publications and patent documents cited in this specification are incorporated herein by reference as if each such publication or document was specifically and individually indicated to be incorporated herein by reference. Citation of publications and patent documents is not intended as an admission that they are relevant prior art, and does not constitute any admission as to their contents or date. Having described the invention herein, those skilled in the art will recognize that the invention can be implemented in various embodiments, and the following preceding description and examples are for illustrative purposes, and are not intended to limit the scope of the appended claims. EXAMPLES

[0264] The following examples are illustrative and not intended to be limiting, as one of ordinary skill in the art will readily appreciate that other reagents or methods may be utilized.

[0265] Abbreviation The following abbreviations are used in the reaction schemes and synthetic examples below. This list is not meant to be a comprehensive 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. TIFF2025517340000022.tif36166TIFF2025517340000023.tif212166TIFF2025517340000024.tif191166

[0266] General information B7-H4 targeting antibody-drug conjugates (eg, XMT-1660) were prepared as described in US Patent Application Serial No. 17 / 568,376.

[0267] CDRs were identified according to the Kabat numbering scheme.

[0268] Complementarity determining regions (CDRs) are defined according to the IMGT numbering system (IMGT®, see international ImMunoGeneTics information system®, available online at: http: / / www.imgt.org / ).

[0269] Example 1: Study Design and Methodology The single-arm study presented herein is a first-in-human study of B7-H4 targeting antibody-drug conjugate (XMT-1660) in a Phase 1b open-label study in previously treated participants with metastatic TNBC, HR+ / HER2- breast cancer, endometrial cancer, or ovarian, fallopian tube, or primary peritoneal cancer. The study consists of two parts: a dose escalation part (DES) and an expansion part (EXP). 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 recommended dose for Phase 2 (RP2D). The EXP part of the study will further evaluate 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, ovarian, fallopian tube or primary peritoneal cancer.

[0270] A Bayesian optimal interval (BOIN) design will be used to determine the MTD for the dose escalation portion of the study, with a target DLT rate set at 30%. Seven dose levels are planned to be investigated, with intermediate dose levels that may be added based on emerging data. Dose escalation will be performed according to the dose escalation scheme in Table 1. Initially, XMT-1660 will be administered intravenously every 3 weeks, but alternative cycle lengths (e.g., Q4W) may be introduced based on available data and at the recommendation of the sponsor and safety review committee. Single-participant cohorts will be used for the first dose level and dose level 1.5, if they are published. All cohorts for subsequent dose levels will have three participants.

[0271] [Table 1] a: In DL1, if a non-DLT drug-related grade ≥2 AE (excluding grade 2 nausea or fatigue resolving within 7 days) is observed during the dose-limiting toxicity (DLT) period, increase the dose level of the next cohort by 50% (DL 1.5) instead of 100% (DL 2). b: If published, a single participant will be enrolled at DL1 and DL 1.5, except that if a DLT is observed, two additional participants will be added at the same dose level.

[0272] Eight dose levels are planned to be investigated, with intermediate dose levels that may be added based on observed data.

[0273] Once the MTD dose is determined, additional participants will be enrolled into the MTD and MTD DL-1 cohorts for a total of approximately 15 participants in each cohort.

[0274] The SRC will meet throughout the study to discuss ongoing dose escalation. The SRC will review the data outlined in the Charter, including but not limited to safety, PK, and efficacy for the MTD and MTD DL-1 cohorts to determine the RP2D.

[0275] About 30 or about 35 participants are enrolled in each cohort. Based on new data, the sample size of each cohort may be increased to enroll up to about 100 participants.

[0276] The RP2D will be determined based on the entire clinical data, including safety, preliminary antitumor activity, PK and relevant biomarker data. Thirty or 35 participants will be enrolled in each of the expansion cohort participant populations. Additional participants may be enrolled in each of these populations based on new data.

[0277] In both the DES and EXP segments, blood samples are taken to determine the levels of total antibody (XMT-1604), conjugated and free AF-HPA, and the primary metabolite of AF-HPA (AF). Pharmacokinetic parameters are then determined from these analytes. Anti-drug antibodies (ADA) and neutralizing antibodies (nAb) are tested.

[0278] If new data suggest that q3w dosing results in excessive toxicity, alternative schedules may be evaluated. When evaluating alternative schedules, the starting dose is one that did not exceed the maximum tolerated dose (MTD) using the q3w dosing schedule, with continued dose escalation using the guidelines outlined in Table 1.

[0279] Duration of treatment: Treatment with XMT-1660 given intravenously every 3 weeks may be continued indefinitely unless one of the following occurs: ·Disease progression Unacceptable adverse events Participant withdraws consent to participate in the study · A general or specific change in the Participant's condition that makes the Participant unable to tolerate further treatment.

[0280] Number of participants The DES portion of the study is planned to evaluate eight dose levels, which would require a maximum sample size of 42 participants to determine the MTD. However, the sample size may be increased based on new data and the actual number of dose levels evaluated. Once the MTD is determined, additional participants will be enrolled at the MTD and MTD DL-1 to have approximately 15 participants at each of these dose levels.

[0281] In the expansion (EXP) portion of the study, 30, 35, and 35 study participants with advanced / metastatic (1) TNBC; (2) HR+ / HER2- breast cancer; and (3) endometrial, ovarian, fallopian tube, or primary peritoneal cancer, respectively, will be enrolled and treated at the MTD and / or RP2D determined in the dose escalation portion of the study. Based on new data, the sample size of each cancer population may be increased to enroll up to approximately 100 participants. The decision to further enroll additional participants will be made based on the overall data.

[0282] Further enrollment with different dosing schedules, biomarker enrichment or subpopulations may be considered. Additional expansion cohorts may be initiated based on new data.

[0283] the purpose The primary and secondary objectives for dose escalation and expansion are presented below: Primary objectives of dose escalation: Determine the maximum tolerated dose (MTD) and / or recommended phase 2 dose (RP2D) of XMT-1660 Evaluate the safety and tolerability of XMT-1660 Secondary Objectives of Dose Escalation: Evaluate the preliminary antitumor activity of XMT-1660 Primary objective of dose expansion Further evaluate the safety and tolerability of XMT-1660 at the MTD and / or RP2D To evaluate the preliminary antitumor activity of XMT-1660 at the MTD and / or RP2D by measuring ORR and DOR based on RECIST 1.1 Secondary objectives of dose expansion: Evaluate the PK of XMT-1660, its release products and selected metabolites Evaluate the occurrence of anti-drug antibodies (ADA) and neutralizing antibodies (nAb) against XMT-1660 Research objectives: Evaluate tumor expression of B7-H4 and PD-L1 by IHC, as determined by tumor type To evaluate the association of B7-H4 expression levels with the antitumor activity of XMT-1660 - Assess the expression of other markers related to cancer or inflammation in relation to clinical outcomes To evaluate the population pharmacokinetic (pop PK) profile of XMT-1660 and determine any pop PK exposure / response relationships

[0284] Eligibility To be eligible for enrollment in the study, all participants must meet all of the inclusion criteria defined below and must not meet any of the exclusion criteria:

[0285] Main eligibility criteria: The general entry criteria for dose escalation and dose expansion are listed below. Participants are eligible for the study if they meet all of the following criteria: 1. Participants have proven recurrent or progressive solid tumors and have disease progression following treatment with available anticancer therapies known to provide benefit or are intolerant to treatment. 2. Participants must be at least 18 years of age. 3. Participants must have an ECOG performance status of 0 or 1. 4. Participants must be able to understand the study procedures and agree to participate in the study by providing written informed consent. 5. Participants must have a cardiac left ventricular ejection fraction (LVEF) ≥ 50% or above the lower limit of reference for their institution, as determined by either ECHO or MUGA scan. 6. Participants with toxicity from previous treatments or surgical procedures must have resolved to grade ≤ 1. Participants with stable immune-related toxicity such as alopecia, grade 2 peripheral sensory neuropathy, hypothyroidism during hormone replacement therapy, or adrenal insufficiency treated with ≤ 10 mg / day prednisone (or equivalent) are exceptions to this criterion and may be eligible for the study. 7. Participants must have adequate organ function within 14 days prior to Day 1 of Cycle 1, as defined by the following criteria: TIFF2025517340000026.tif1691618. During the study, women of childbearing potential (WOCBP) must use highly effective contraception (with a failure rate of <1% per year when used correctly and consistently) during study participation and for 6 months after receiving their last dose of XMT-1660. 9. Male study participants must use barrier contraception (condoms) and abstain from sperm donation for the duration of study drug treatment and for at least 6 months after the last dose of study drug. Partners of male study participants who are considered WOCBP must use highly effective contraception for the same period of time. 10. Participants must have at least one measurable lesion as defined by RECIST version 1.1. 11. Participants must be willing to undergo minimally invasive tumor biopsy to obtain tumor tissue for local testing prior to C1D1, if medically feasible. If the Investigator feels that a biopsy is not medically feasible, a waiver request must be submitted to the Study Medical Monitor for approval. Additionally, archival tissue samples should be submitted to a central laboratory, if available, to allow for longitudinal analysis of tumor biomarkers. Note: Confirmation of B7-H4 expression is not required for study enrollment.

[0286] General exclusion criteria for dose escalation and expansion are listed below. Participants are not eligible for the study if they meet any of the following criteria: 1. Participants have received prior treatment with another ADC containing an auristatin or maytansinoid payload (e.g., mirvetuximab) NOTE: Prior treatment with another ADC containing a different payload (non-auristatin or non-maytansinoid) is permitted (e.g., sacituzumab govitecan, fam-trastuzumab deruxtecan-nxki) 2. Participants have had major surgery within 28 days of starting study treatment, have received systemic anticancer therapy within 28 days or 5 half-lives of previous treatment (14 days or 5 half-lives for small molecule targeted therapy), whichever is shorter, before starting study treatment, or have received palliative radiation therapy within 14 days of starting study treatment. 3. Having a diagnosis of an additional malignancy that required 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. 4. Participants have untreated Hepatitis B virus (HBV) or Hepatitis C virus (HCV). In addition, seronegativity during HBV and HCV screening (baseline) is required: · HBV: Participants with HBV infection must have HB s AG(-) serum with a viral load below the limit of quantification and HB c ab(+) serum to be eligible. HCV: Participants with a history of HCV infection should have completed curative antiviral treatment and should have an HCV viral load below the limit of quantification. 5. Participants with human immunodeficiency virus (HIV) infection. 6. Participant has a history of cirrhosis, liver fibrosis, esophageal or gastric varices, or other clinically significant liver disease. Liver function tests beyond those laboratory tests defined otherwise in the eligibility criteria to diagnose potentially clinically significant liver disease based on risk factors, e.g., liver fat, or history of excessive alcohol intake, will be based on the clinical judgment of the investigator. 7. Participants may not receive medications associated with hepatotoxicity concurrently with XMT-1660 administration. Participants may receive acetaminophen / paracetamol for a limited time at a total daily dose of ≤2g per day. Use of NSAIDs or steroids is encouraged for treatment of fever. 8. Participant has current severe uncontrolled systemic disease (e.g., clinically significant cardiovascular, pulmonary or metabolic disease) or intercurrent illness that may preclude per-protocol evaluation. In addition, participants will be excluded by the following characteristics: Marked baseline prolongation of QT / QTcF interval CTCAE grade >1: Repeated demonstration of QTcF interval >480 milliseconds (ms) using Fridericia's QT correction formula. History of additional risk factors for torsades de pointes (e.g., family history of long QT syndrome). 9. Participant is receiving concurrent anticancer therapy (e.g., chemotherapy, radiation therapy, biological therapy, immunotherapy, hormonal therapy, experimental treatment). 10. Participant is unable or unlikely to adhere to the dosing schedule and study assessments. 11. Participant has a history or suspicion of pneumonitis or interstitial lung disease. 12. Participants currently use supplemental oxygen therapy, either continuous or intermittent. 13. Participant's oxygen saturation while breathing room air is <93%. 14. Participant is using a strong CYP450 3A inhibitor or CYP450 3A inducer that cannot be discontinued while receiving study treatment. Participants currently taking these medications must discontinue within 14 days of the first dose of study medication. 15. Participant has a history of untreated CNS metastases (including new and progressive brain metastases), leptomeningeal metastases or carcinomatous meningitis. a. Participants are eligible if CNS metastases have been adequately treated and participants have been neurologically stable for at least 2 weeks prior to enrollment. b. Additionally, participants must be corticosteroid-free or on a stable / tapering dose of ≤10 mg / day prednisone (or equivalent). Anticonvulsants are permitted, except for those associated with hepatotoxicity.

[0287] Disease-specific entry criteria for dose escalation are outlined in Table 2.

[0288] [Table 2]

[0289] Eligibility criteria for hormone receptor positive (HR+), human epidermal growth factor receptor 2 negative (HER2-) breast cancer (HR+ / HER2-BC) for the dose expansion cohort were: 1. Participants for whom experimental therapy is appropriate have a histologically or cytologically proven diagnosis of breast cancer with evidence of progressive or metastatic disease as determined by the treating physician. 2. Participants have evidence of ER-positive and / or PR-positive breast cancer and HER2-negative tumors based on local examination of the most recent tumor biopsy, as defined by relevant American Society of Clinical Oncology (ASCO) / College of American Pathologists (CAP) guidelines. 3. Participants must have received prior treatment with a CDK 4 / 6 inhibitor in combination with endocrine therapy in any setting. 4. Participants have not received more than two prior lines of chemotherapy for advanced or metastatic disease. For participants with BRCA mutations, treatment with PARP inhibitors is permitted and does not count as chemotherapy.

[0290] Eligibility criteria for the triple-negative breast cancer (TNBC) dose expansion cohort are: 1. Participants for whom experimental therapy is appropriate have a histologically or cytologically proven diagnosis of breast cancer with evidence of locally advanced or metastatic disease as determined by the treating physician. 2. Participants will have evidence of estrogen receptor (ER)-negative, progesterone receptor (PR)-negative and HER2-negative breast cancer based on local examination of the most recent tumor biopsy, as defined by the relevant American Society of Clinical Oncology (ASCO) / College of American Pathologists (CAP) guidelines. 3. Participants have received 1-3 prior lines of chemotherapy for metastatic disease. For participants with BRCA mutations, treatment with PARP inhibitors is permitted and does not count as chemotherapy.

[0291] There are no disease-specific exclusion criteria for either the HR+ / HER2-BC cohort or the TNBC cohort.

[0292] Eligibility criteria for the endometrial cancer dose expansion cohort were: 1. Participants for whom experimental therapy is appropriate, who have no satisfactory treatment options, have a histologically or cytologically proven diagnosis of endometrial cancer, with evidence of metastatic or locally advanced disease, as determined by the treating physician 2. Received at least one prior line of platinum-based chemotherapy and ≤3 prior lines of systemic therapy for recurrent or metastatic disease, not including hormonal therapy

[0293] Participants enrolled in the endometrial cancer cohort must not have endometrial sarcoma or carcinosarcoma.

[0294] Entry criteria for the ovarian, fallopian tube, or primary peritoneal cancer dose expansion cohorts are: 1. Histologic diagnosis of high-grade serous ovarian carcinoma, including fallopian tube or primary peritoneal carcinoma that is metastatic or recurrent. 2. Participants must have completed 4 or more cycles of platinum-based therapy and have platinum-resistant recurrent disease defined as radiographic progression within 6 months of the last platinum-based therapy. a. For participants who have received one prior line of chemotherapy, they must have achieved a complete or partial response to that treatment and have had disease progression 3-6 months after their last dose of platinum in the first-line setting. b. For participants who have received 2-4 prior lines of chemotherapy, disease has progressed within 6 months or less after the last dose of platinum in their most recent chemotherapy. 3. 1-4 prior lines of systemic therapy for ovarian cancer, including at least one prior line of platinum-containing regimen. Definition of first line treatment: i. Adjuvant ± neoadjuvant is considered 1 line of treatment as long as they are the same regimen (e.g., 4 cycles of platinum / taxane pre-surgery followed by 4 cycles of platinum / taxane post-surgery) ii. Maintenance therapy (e.g., bevacizumab, PARPi or endocrine therapy) is considered part of the prior line of treatment. Substitution of a different platinum or taxane does not count as a new line. iii. Treatment given for only one cycle and discontinued due to toxicity in the absence of progression does not count as a line of treatment. Substitution of a different platinum agent or taxane does not count as a new line. iv. Hormonal therapy (e.g., tamoxifen, letrozole) counts as a separate line of treatment unless administered as maintenance therapy.

[0295] Exclusion criteria for the ovarian, fallopian tube, or primary peritoneal cancer dose expansion cohorts were: 1. Participants enrolled in the ovarian cancer cohort must not have low-grade clear cell tumors, endometrioid tumors, mucinous tumors, carcinosarcoma tumors, germ cell tumors, or stromal tumors. 2. Participants enrolled in the ovarian cancer cohort must not have primary platinum-resistant disease as defined by lack of response or progression within 3 months after completing frontline platinum-containing therapy.

[0296] Evaluation criteria: safety Frequency and severity of AEs and SAEs based on the National Cancer Institute (NCI) Common Terminology Criteria for Adverse Events, version 5.0 (CTCAE v5.0).

[0297] Changes in clinical laboratory parameters (hematology, chemistry, urinalysis), vital signs, ECOG performance status, ECG parameters, physical examination, and use of concomitant medications.

[0298] clinical activity Tumor responses (ORR, DOR) and disease control rates (DCR) will be assessed by the investigator at the end of cycle 2 and every two cycles thereafter using Response Evaluation Criteria in Solid Tumors (RECIST), version 1.1.

[0299] Pharmacokinetics Pharmacokinetics of XMT-1660 (C max , C trough , t max , AUC, AUC last , AUC ∞ , t1 / 2, CL and V ss ) During both the DES and EXP segments, blood samples will be taken to determine levels of total antibody (XMT-1604), conjugated and free XMT-1267, and the primary metabolite of XMT-1267 (XMT-1521). Pharmacokinetic parameters will then be determined from these analytes. · Anti-drug antibodies (ADA) and neutralizing antibodies (nAb) are tested. Population pharmacokinetic and population PK exposure / response analyses will also be performed.

[0300] Dose-limiting toxicity (DLT) The DLT observation period is the time between the start of 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 a predose assessment before receiving cycle 2. Participants must receive at least 80% of the planned dose and a complete cycle 1 to be considered evaluable for tolerability, unless a dose reduction, interruption, or discontinuation was the result of a DLT.

[0301] A DLT is defined as either disease progression or toxicity not attributable to another clearly identifiable cause detailed in the protocol occurring within the DLT observation period.

[0302] Test rationale: Despite advances in novel therapeutic approaches that benefit progression-free survival (PFS) and overall survival (OS), the majority of patients with unresectable or recurrent / metastatic breast cancer eventually acquire resistance to these treatments and die from their disease. Thus, unresectable or recurrent / metastatic TNBC and HR+ / HER2- breast cancer remain serious, life-threatening, and associated with unmet medical need.

[0303] In particular, B7-H4 protein expression has been observed in various human tumors, such as breast, endometrial and ovarian cancers. Furthermore, B7-H4 expression in tumor cells of these malignancies appears to be associated with disease progression and poor prognosis. B7-H4 is expressed in approximately 77% of TNBCs and approximately 60% of HR+ and HER2+ tumors. B7-H4 expression was high in approximately 45% of breast cancers and was independent of ER / HER2 status. Furthermore, B7-H4 expression was reported to be widely expressed in endometrial and ovarian cancers.

[0304] The hypothesis is that by targeting B7-H4, Dolasynthen ADC (XMT-1660) will confer clinical benefit to patients with unresectable or recurrent / metastatic TNBC after standard chemotherapy, and HR+ / HER2- after CDK4 / 6 inhibitor and endocrine-based therapies. To test this hypothesis, we will initiate a first-in-human study with XMT-1660, a B7-H4-directed antibody conjugated with an auristatin payload, in tumors likely to express B7-H4. The primary objective is to evaluate the safety and tolerability of XMT-1660 as a single agent administered intravenously until disease progression or unacceptable toxicity in previously treated participants with TNBC, HR+ / HER2- breast, endometrial and ovarian, fallopian tube, or primary peritoneal cancer. Secondary objectives are to obtain pharmacokinetics, immunogenicity, and preliminary efficacy with this dosing regimen. Investigational objectives are to investigate the association of target expression within tumors with other markers associated with cancer or inflammation.

[0305] Example 2: Administration of XMT-1660 B7-H4 targeting antibody-drug conjugates (e.g., XMT-1660) are administered according to body surface area (BSA). BSA-adjusted doses are calculated according to each institution's standard practice. If possible, the Mosteller formula is used. 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). Dose calculations in subsequent cycles should be made for ≥5% weight change from the most recent dose calculation. Subsequent dose adjustments based on weight measurements should be made according to each institution's standard practice. If an institution does not have a standard practice, obtain additional weight measurements and confirm or modify BSA prior to dosing in cycle 2 and every two cycles thereafter.

[0306] XMT-1660 will be administered IV via an antecubital catheter or an indwelling venous catheter. Each participant's initial dose will be administered over 90 minutes. If no IRR occurs, all subsequent doses may be administered over 60 (± 10) minutes at the investigator's discretion. Subsequent infusions may be administered on an outpatient basis as tolerated, but participants will be monitored for approximately 6 hours after XMT-1660 administration for safety assessments and PK sampling during cycles 1-3.

[0307] In the EXP part of the study, once a participant completes cycle 1, dose adjustments may be made if, in the opinion of the investigator, the participant experiences toxicity but would benefit from further treatment with XMT-1660.

[0308] Example 3: Analysis The safety analysis set includes all participants who received any XMT-1660 dose (partial or complete) in DES and EXP. All safety analyses will be performed in the safety analysis set, except for the evaluation of tolerability. Participants must receive at least 80% of the planned dose of XMT-1660 and a complete cycle 1 to be considered evaluable for tolerability, unless a dose reduction, interruption, or discontinuation was the result of a DLT.

[0309] Enrolled participants who received any amount of assigned dose of XMT-1660 and have a sufficient number of concentration determinations to allow for PK calculations will constitute the PK analysis set. We will provide analyses of PK concentration data by dose level, indication, and participant outcome.

[0310] The Efficacy Response Assessment Analysis Set (EFREAS) includes a subset of the Efficacy Analysis Set for which a baseline response assessment and at least one post-baseline response assessment were available.

[0311] Safety analysis At the end of the DES phase of the study, the MTD is selected as the dose for which the isotonic estimate of DLT probability is closest to the target DLT rate. The isotonic estimate of DLT probability is obtained using a pooled adjacent violators algorithm.

[0312] The number and percentage of participants who experienced DLTs during the DES phase of the study will be reported by dose level and across all dose levels.

[0313] Participants who completed cycle 1 without DLT were considered tolerant of the XMT-1660 regimen. For evaluation of tolerability in the DES phase of the study, the number and percentage of participants who tolerated the XMT-1660 regimen will be reported by dose level in the evaluable subset of participants and across all dose levels.

[0314] All AEs and adverse events of clinical interest (AECIs) will be reported by dose level and across all dose levels in the DES phase of the study. In the expansion phase, all AEs and AECIs will be reported by cancer population. The severity of the adverse events, CTCAE severity classification, and investigator-reported relationship to study drug will be summarized and listed. Safety laboratory data will be summarized and listed.

[0315] Primary efficacy analysis The primary efficacy analysis will be performed using both DES and EXP data. The primary endpoint is ORR by investigator radiological review, defined as the proportion of participants achieving confirmed PR or CR by RECIST v1.1. The secondary endpoint is DCR, defined as the proportion of participants achieving a complete response, partial response, and / or stable disease of any duration by RECIST v1.1. The numbers and proportions of participants achieving a response or clinical benefit will be summarized and provided with exact 95% confidence intervals (CIs).

[0316] Analyses of other efficacy endpoints, including DOR, will also be reported according to standard response criteria whenever possible. Kaplan-Meier estimates of medians and quartiles with 95% CIs will be reported for these statistics. Efficacy endpoints will be analyzed using both EFAS and EFREAS in each cancer type cohort.

[0317] Pharmacokinetic analysis The PK profile of the active moiety / release products / metabolites of XMT-1660 will be determined for each participant by non-compartmental analysis using validated PK software (i.e., Phoenix WinNonlin). PK parameters for each participant will include time to peak plasma concentration (t max ), maximum concentration (C max ), and the area under the concentration curve for the final measurable concentration (AUC 0-last ) is included. If the terminal phase disappearance stage can be identified, 1 / 2 ), clearance (CL), and volume of distribution (Vss) are determined.

[0318] Details of the handling of missing concentration and covariate data, outliers, and values ​​below the limit of quantification, as well as modeling of dose-response and PK parameter-response relationships will be provided in a separate clinical pharmacology analysis plan.

[0319] Objective response rate and measurement of B7-H4 expression B7-H4 expression status will be confirmed by central laboratory testing. ORR and DOR (with associated CIs) will be reported for participants stratified by B7-H4 status.

[0320] Example 4: Tumor growth response to administration of XMT-1660 in a CTG-1280 patient-derived endometrial cancer xenograft mouse model Female athymic nude mice were subcutaneously implanted with CTG-1280 patient-derived endometrial cancer tumor fragments. Tumor volumes ranged from 168 to 299 mm. 3 (Average=221mm 3Animals were randomized into treatment groups (n=10 / group) when the animals reached 10 mg / kg / day of baseline. Vehicle, Rituximab isotype control conjugate (prepared as described in US Patent Application No. 2022 / 0233707, 4.60 / 0.150 mg / kg), and XMT-1660 (4.68 / 0.150 mg / kg, DAR 6) were administered intravenously on day 1 (all doses given by antibody / payload). No significant weight loss or clinical findings were observed.

[0321] Figure 1 provides the results of tumor volume of CTG-1280 tumor-bearing mice treated with isotype control conjugate and XMT-1660. Treatment with 4.60 / 0.150 mg / kg isotype control conjugate had no significant effect on tumor volume compared to vehicle control at day 17 (14% TGI, adjusted p=0.5839). Treatment with 4.68 / 0.150 mg / kg XMT-1660 significantly reduced tumor volume compared to vehicle control group at day 17 (73% TGI, adjusted p<0.00001).

[0322] Example 5: Tumor growth response to administration of XMT-1660 in the OV2423 patient-derived ovarian cancer xenograft mouse model Female BALB / c nude mice were cultured with OV2423 patient-derived ovarian cancer tumor fragments (approximately 2–3 mm 3 The tumor volumes ranged from 101 to 230 mm. 3 (Average=155mm 3 Animals were randomized into treatment groups (n=8 / group) when the animals reached 10 days of baseline (n=10 / group). Vehicle, palivizumab isotype control conjugate (prepared as described in US Patent Application No. 2022 / 0233707, 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 given by antibody / payload). No significant weight loss or clinical findings were observed.

[0323] Figure 2 provides the results for tumor volume of OV2423 tumor-bearing mice treated with isotype control and XMT-1660. Treatment with palivizumab isotype control conjugate (4.54 / 0.150 mg / kg) had no significant effect on tumor volume compared to vehicle control at day 22 (24.8% TGI, adjusted p=0.977). XMT-1660 administered at 4.61 / 0.150 mg / kg significantly reduced tumor volume compared to vehicle control group at day 22 (124.05% TGI, adjusted p<0.001), resulting in 1 PR and 7 TFS.

[0324] Example 6: Tumor growth response to administration of XMT-1660 cytotoxic drug conjugates in the CTG-1692 patient-derived ovarian cancer xenograft mouse model Female athymic nude mice were subcutaneously implanted with CTG-1692 patient-derived ovarian cancer tumor fragments. Tumor volumes ranged from 131 to 447 mm. 3 (Average=229mm 3 Animals were randomized into treatment groups (n=8 / group) when the animals reached baseline (n=10 / 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) prepared as described in US Patent Application No. 2022 / 0233707 were administered intravenously on day 1 (doses given by antibody / payload). No significant weight loss or clinical findings were observed.

[0325] Figure 3 provides the results for tumor volume of CTG-1692 tumor-bearing mice treated with isotype control conjugate or XMT-1660. Treatment with 2.27 / 0.075 or 4.54 / 0.150 mg / kg isotype control conjugate had no significant effect on tumor volume compared to vehicle control at day 49 (TGI=-48%, adjusted p=0.2638, or TGI=8%, adjusted p=0.9973, respectively). Treatment with 2.30 / 0.075 mg / kg XMT-1660 significantly reduced tumor volume compared to vehicle control group at day 49 (TGI=99%, adjusted p=0.0023), resulting in 1 PR. Treatment with 4.60 / 0.150 mg / kg XMT-1660 significantly reduced tumor volume compared to the vehicle control group at day 49 (TGI=92%, adjusted p=0.0058). Treatment with both dose levels of the isotype control conjugate and with 4.60 / 0.150 mg / kg XMT-1660 did not result in CR, PR or TFS.

[0326] Example 7: Tumor growth response to administration of XMT-1660 cytotoxic drug conjugates in the mBR9013 syngeneic breast cancer mouse model Female FVB / NJ mice were treated with mBR9013 tumor fragments (2–3 mm 3 ) were implanted subcutaneously. The tumor volume was 96-277 mm 3 (Average=148mm 3Animals were randomized into treatment groups (n=8 / group) when the animals reached the target dose (n=10 / group). Vehicle, palivizumab isotype control conjugate (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), 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) were administered on day 1 (all doses given by antibody / payload). Vehicle, isotype control conjugate, and XMT-1660 were administered intravenously. Anti-PD-1 was given as an intraperitoneal dose twice weekly for 3 weeks starting on day 1. No significant weight loss or clinical findings were observed.

[0327] Figure 4 provides results regarding tumor volume for mBR9013 tumor-bearing mice treated with isotype control conjugate, XMT-1660 and anti-PD-1. Treatment with 1.51 / 0.050 or 4.54 / 0.150 mg / kg palivizumab isotype control conjugate had no significant effect on tumor volume compared to vehicle control at day 15 (TGI=-7.91%, adjusted p=0.999, or TGI=32.02%, adjusted p=0.857, respectively). Treatment with 1.53 / 0.050 or 4.60 / 0.150 mg / kg XMT-1660 significantly reduced tumor volume compared to vehicle control group on day 15 (TGI=92.69%, adjusted p=0.000276, or TGI=119.29%, adjusted p=1.45e-08, respectively). Treatment with 10.0 / 0 mg / kg anti-PD-1 had no significant effect on tumor volume compared to vehicle control on day 15 (TGI=-9.10%, adjusted p=0.999). Co-treatment with 1.53 / 0.050+10.0 / 0 mg / kg XMT-1660+anti-PD-1 significantly reduced tumor volume compared to vehicle control on day 15 (TGI=100.99%, adjusted p=2.34e-05).

[0328] Other Aspects While the present invention has been described in conjunction with its detailed description, the foregoing description is intended to be illustrative, and not limiting, of the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the appended claims.

Claims

1. 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, comprising administering about 7.0 mg / m 2 ~About 90mg / m 2 administering to the subject by injection a B7-H4 targeting antibody-drug conjugate at a dose of The B7-H4 targeting antibody-drug conjugate is a conjugate of formula (I): where: i.d 13 is approximately 2, ii. an ANTIBODY that binds to B7-H4 and comprises a variable heavy chain complementarity determining region 1 (CDRH1) that comprises the amino acid sequence GFIVSRNY (SEQ ID NO:2), a variable heavy chain complementarity determining region 2 (CDRH2) that comprises the amino acid sequence IYGSGRT (SEQ ID NO:3), a variable heavy chain complementarity determining region 3 (CDRH3) that comprises the amino acid sequence ARDADYGLDV (SEQ ID NO:4), a variable light chain complementarity determining region 1 (CDRL1) that comprises the amino acid sequence QSVSSSY (SEQ ID NO:5), a variable light chain complementarity determining region 2 (CDRL2) that comprises the amino acid sequence GAS (SEQ ID NO:6), and a variable light chain complementarity determining region 3 (CDRL3) that comprises 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, as numbered according to EU numbering; and iv. ■ is GlcNAc, △ is Fuc, and □ is GalNAc; The method.

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 antibody-drug conjugate dose being About 6.7 mg / m 2 to about 7.7 mg / m 2 , Approximately 13.9 mg / m 2 to approximately 14.9 mg / m 2 , Approximately 21.1 mg / m 2 to approximately 22.1 mg / m 2 , Approximately 28.2 mg / m 2 to approximately 29.2 mg / m 2 , Approximately 37.6 mg / m 2 to approximately 38.6 mg / m 2 , About 50.2 mg / m 2 to about 51.2 mg / m 2 , Approximately 66.9mg / m 2 ~Approx. 67.9mg / m 2 ,or Approximately 87.1mg / m 2 ~Approx. 88.1mg / m 2 That is, The method according to any one of claims 1 to 3.

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

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

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

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

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

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

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

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

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

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

15. The method of any one of the preceding claims, wherein the subject with HR+ / HER2- breast cancer after CDK4 / 6 inhibitor and endocrine-based therapy has received at least one line of systemic therapy, which must include a CDK 4 / 6 inhibitor and endocrine therapy (ET) in the advanced or metastatic breast cancer setting.

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

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

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

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