HER3-binding antibody-drug conjugate

Abstract

A method for treating cancer and a composition comprising an antigen-binding molecule that binds to HER3, the antigen-binding molecule comprising (i) a HER3-binding moiety and (ii) a linker payload moiety comprising exatecan or a derivative thereof, are provided. Cancer remains a leading cause of death worldwide. Chemotherapy has good clinical benefits, but has very serious side effects and a short therapeutic index due to its low specificity. More targeted therapies, such as monoclonal antibody therapy, show good specificity but have lower response rates.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 436,730, filed January 3, 2023, and U.S. Provisional Patent Application No. 63 / 459,061, filed April 13, 2023, the entire contents of each of which are incorporated herein by reference.

[0002] Sequence Listing This application contains a Sequence Listing having filename 1959576-00081_Sequence_Listing.xml, which is 107 KB in size and was created on December 19, 2023. The entire contents of this Sequence Listing are incorporated herein by reference.

[0003] The present disclosure relates to molecular biology, and more particularly to antibody technology. The present disclosure also relates to methods of medical treatment and prophylaxis. [Background technology]

[0004] Cancer remains a leading cause of death worldwide. Chemotherapy has good clinical benefits but suffers from significant side effects and a narrow therapeutic index due to its low specificity. More targeted therapies, such as monoclonal antibody therapy, show good specificity but lower response rates. Antibody-drug conjugates (ADCs), a therapeutic modality that exploits the targeting specificity of antibodies to selectively deliver cytotoxic payloads to tumors, have proven increasingly effective in clinical settings.

[0005] HER3 is a member of the epidermal growth factor receptor (ERBB) family of tyrosine kinase receptors that is overexpressed in a wide range of tumors and is associated with disease progression and poor survival. For example, over 50% of tumors in melanoma, cervical cancer, lung cancer, gastric cancer, colorectal cancer, and ovarian cancer show HER3 overexpression, while overexpression is present in 25-40% of tumors in breast cancer, pancreatic cancer, and prostate cancer. Furthermore, recent studies have shown that HER3 expression is significantly induced in tumors during metastasis of colorectal cancer and acquired resistance to standard treatment in lung cancer. Patritumab-deruxtecan (U3-1402, also referred to herein as patritumab-DXd) is a HER3-targeted ADC that has demonstrated clinical responses in EGFR-mutated and TKI-resistant lung cancers, as well as in a subset of metastatic breast cancers (Jaenne et al., Cancer Discov. (2022) 12(1):74-89; Krop et al., J Clin Oncol (2022) 40(suppl 16):abstr 1002). However, toxicities, including severe interstitial lung disease (ILD) and severe cytopenias, have been observed (Kogawa et al., Journal of Clinical Oncology (2018) 36(15, suppl):2512-2512). [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Jaenne et al., Cancer Discov. (2022)12(1):74-89 [Non-patent document 2] Krop et al.,J Clin Oncol(2022)40(suppl 16):abstr 1002 [Non-patent document 3] Kogawa et al.,Journal of Clinical Oncology(2018)36(15,suppl):2512-2512 Summary of the Invention [Means for solving the problem]

[0007] In a first aspect, the present disclosure provides an antigen-binding molecule that binds to HER3, comprising: (i) a HER3-binding moiety; and (ii) a linker payload moiety comprising exatecan or a derivative thereof.

[0008] In some embodiments, the linker payload moiety comprises the structure (A): [ka]

[0009] In some embodiments, the linker payload moiety and the HER3 binding moiety are connected via a cleavable linker moiety.

[0010] In some embodiments, the antigen binding molecule comprises a cleavable linker moiety comprising the structure (B): [ka]

[0011] In some embodiments, the antigen binding molecule comprises a cleavable linker moiety comprising a polyethylene glycol (PEG) moiety.

[0012] In some embodiments, the antigen binding molecule comprises a cleavable linker moiety comprising the structure (C): [ka]

[0013] In some embodiments, the antigen binding molecule comprises a cleavable linker moiety comprising a p-aminobenzylcarbamate (PABC) group.

[0014] In some embodiments, the antigen binding molecule comprises a cleavable linker moiety comprising the structure (D): [ka] wherein R is selected from CH3 and (CH2)3NHCOCNH2.

[0015] In some embodiments, the antigen binding molecule comprises the structure (E): [ka]

[0016] In some embodiments, the antigen binding molecule comprises SYNtecan E.

[0017] In some embodiments, the HER3 binding moiety comprises an Fc region, and the linker payload moiety is conjugated to the HER3 binding site at an azide group provided on a 6-azido-6-deoxy-N-acetylgalactosamine residue of an N-glycan linked to N297 (EU numbering) of the CH2 domain of the Fc region.

[0018] In some embodiments, the HER3 binding moiety binds to HER3 in the region set forth in SEQ ID NO:77.

[0019] In some embodiments, the HER3 binding moiety comprises: (i) a heavy chain variable (VH) region incorporating the following CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO: 40 HC-CDR2 having the amino acid sequence of SEQ ID NO: 43 HC-CDR3 having the amino acid sequence of SEQ ID NO: 48, and (ii) a light chain variable (VL) region incorporating the following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO: 66 LC-CDR2 having the amino acid sequence of SEQ ID NO: 69 LC-CDR3 having the amino acid sequence of SEQ ID NO: 74.

[0020] In some embodiments, the HER3 binding moiety comprises: (i) a VH region incorporating the following CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO: 38 HC-CDR2 having the amino acid sequence of SEQ ID NO: 42 HC-CDR3 having the amino acid sequence of SEQ ID NO: 45, and (ii) a VL region incorporating the following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO: 63 LC-CDR2 having the amino acid sequence of SEQ ID NO: 67 LC-CDR3 having the amino acid sequence of SEQ ID NO: 70.

[0021] In some embodiments, the antigen binding moiety that binds to HER3 comprises: a VH region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 33; and A VL region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO:58.

[0022] In some embodiments, the antigen binding moiety that binds to HER3 comprises: A polypeptide comprising or consisting of an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 75, and A polypeptide comprising or consisting of an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO:76.

[0023] In some embodiments, the antigen binding moiety that binds to HER3 comprises: A polypeptide comprising or consisting of an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 80, 81, 82 or 91; and A polypeptide comprising or consisting of an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO:76.

[0024] The present disclosure also provides a method for producing an antigen-binding molecule, the method comprising contacting a HER3-binding moiety that includes an azide moiety with a compound having structure (F): [ka]

[0025] The present disclosure also provides antigen-binding molecules obtained or obtainable by the methods of the present disclosure.

[0026] The present disclosure also provides a composition comprising an antigen-binding molecule according to the present disclosure and a pharmaceutically acceptable carrier, diluent, excipient, or adjuvant.

[0027] The present disclosure also provides an antigen-binding molecule or composition according to the present disclosure for use in a method of medical treatment or prevention, or in a method of diagnosis or prognosis.

[0028] The present disclosure also provides an antigen-binding molecule or composition according to the present disclosure for use in the treatment or prevention of cancer.

[0029] The present disclosure also provides the use of an antigen-binding molecule or composition according to the present disclosure in the manufacture of a medicament for the treatment or prevention of cancer.

[0030] The present disclosure also provides a method for treating or preventing cancer, comprising administering to a subject a therapeutically or prophylactically effective amount of an antigen-binding molecule or composition according to the present disclosure.

[0031] In some embodiments, the cancer is selected from the group consisting of cancers comprising cells that express / overexpress an EGFR family member, cancers comprising cells that express / overexpress HER3, cancers comprising cells with a mutation that results in increased expression of a ligand for HER3, cancers comprising cells with an NRG gene fusion, solid tumors, hematological cancers, squamous cell carcinoma, breast cancer, breast carcinoma, invasive breast cancer, ductal carcinoma, metastatic breast cancer, triple negative breast cancer, HER2 positive breast cancer, HER2 negative breast cancer, hormone receptor positive breast cancer, HER2 negative / hormone receptor positive breast cancer, gastric cancer, gastric carcinoma, gastric adenocarcinoma, gastrointestinal adenocarcinoma, colorectal cancer, metastatic colorectal cancer, colon cancer, colorectal cancer colorectal adenocarcinoma, colon adenocarcinoma, head and neck cancer, head and neck squamous cell carcinoma (HNSCC), lung cancer, non-small cell lung cancer, lung adenocarcinoma, invasive mucinous lung adenocarcinoma, lung squamous cell carcinoma (LUSC), ovarian cancer, ovarian cancer ovarian serous adenocarcinoma, ovarian serous cystadenocarcinoma, fallopian tube cancer, renal cancer, renal cell carcinoma, renal clear cell carcinoma, renal cell adenocarcinoma, papillary renal cell carcinoma, pancreatic cancer, pancreatic adenocarcinoma, pancreatic ductal adenocarcinoma, cervical cancer, cervical squamous cell carcinoma, skin cancer, Melanoma, oral cavity cancer, oropharyngeal cancer, esophageal cancer, esophageal squamous cell carcinoma (ESCC), esophageal adenocarcinoma, liver cancer, hepatocellular carcinoma, bile duct cancer, gallbladder cancer, biliary tract cancer, uterine cancer, endometrial cancer, uterine body endometrial cancer, uterine carcinosarcoma, thyroid cancer cancer), thyroid cancer carcinoma), pheochromocytoma, paraganglioma, bladder cancer, bladder epithelial carcinoma, prostate cancer, prostate adenocarcinoma, castration-resistant prostate cancer, metastatic prostate cancer, metastatic castration-resistant prostate cancer, retinoblastoma, sarcoma, soft tissue sarcoma, peritoneal cancer, thymoma, neuroendocrine tumor, nasopharyngeal neuroendocrine tumor, and homologous recombination deficient (HRD) cancer.

[0032] The present disclosure also provides the use of an antigen-binding molecule or composition according to the present disclosure to deplete or increase killing of cells expressing HER3.

[0033] The present disclosure also provides an optionally isolated in vitro complex comprising an antigen-binding molecule according to the present disclosure bound to HER3.

[0034] The present disclosure also provides a method for detecting HER3 in a sample, the method comprising contacting a sample containing or suspected of containing HER3 with an antigen-binding molecule according to the present disclosure, and detecting the formation of a complex between the antigen-binding molecule and HER3.

[0035] The present disclosure also provides a method for selecting or stratifying a subject for treatment with a HER3-targeting agent, the method comprising contacting a sample from the subject in vitro with an antigen-binding molecule according to the present disclosure, and detecting the formation of a complex between the antigen-binding molecule and HER3.

[0036] The present disclosure also provides the use of an antigen-binding molecule according to the present disclosure as an in vitro or in vivo diagnostic or prognostic agent.

[0037] Embodiments and experiments illustrating the principles of the present disclosure will now be discussed with reference to the accompanying drawings. [Brief explanation of the drawings]

[0038] [Figure 1A] Graph showing binding of HER3 binding molecules to cells of different human cancer cell lines. Binding of different molecules to HCT116 cells is shown. [Figure 1B] Graph showing binding of HER3 binding molecules to cells of different human cancer cell lines. Binding of different molecules to DU145 cells. [Figure 1C] Graph showing binding of HER3 binding molecules to cells of different human cancer cell lines. Binding of different molecules to H358 cells is shown. [Figure 1D] Graph showing binding of HER3 binding molecules to cells of different human cancer cell lines. Binding of different molecules to T47D cells is shown. [Figure 1E]Graph showing binding of HER3 binding molecules to cells of different human cancer cell lines. Binding of different molecules to OVCAR8 cells is shown. [Figure 1F] Table showing binding of HER3 binding molecules to cells of different human cancer cell lines. Table showing EC50 values ​​for binding of the indicated HER3 binding molecules to the indicated cell types, derived from binding curves. [Figure 2A] Graph showing binding of HER3-binding molecules to cells of different human cancer cell lines in the presence of increasing concentrations of NRG1. The y-axis shows the fold change in relative mean fluorescence intensity (MFI) compared to cells not treated with NRG1. Binding of different molecules to OVCAR8 cells is shown. [Figure 2B] Graph showing binding of HER3-binding molecules to cells of different human cancer cell lines in the presence of increasing concentrations of NRG1. The y-axis shows the fold change in relative mean fluorescence intensity (MFI) compared to cells not treated with NRG1. Binding of different molecules to T47D cells is shown. [Figure 2C] Graph showing binding of HER3-binding molecules to cells of different human cancer cell lines in the presence of increasing concentrations of NRG1. The y-axis shows the fold change in relative mean fluorescence intensity (MFI) compared to cells not treated with NRG1. Binding of different molecules to H358 cells is shown. [Figure 2D] Graph showing binding of HER3-binding molecules to cells of different human cancer cell lines in the presence of increasing concentrations of NRG1. The y-axis shows the fold change in relative mean fluorescence intensity (MFI) compared to cells not treated with NRG1. Binding of different molecules to DU145 cells is shown. [Figure 3A]Graph showing binding of HER3-binding molecules to Fcγ receptors as determined by surface plasmon resonance analysis. Binding of a human IgG1 isotype-matched positive control (IgG1 control), a human IgG1 containing an Fc-silencing mutation (Fc-silencing), patritumab-DXd, 501b.S8D4, and 501c.S8D4 to human FcγRIA (CD64), human FcγRIIA (CD32a), and human FcγRIIIA (CD16a) is shown. The y-axis represents relative resonance units (RU), which are directly proportional to binding of the molecule to the Fcγ receptor tested, and the x-axis represents increasing concentrations of the test molecule. [Figure 3B] Table showing binding of HER3 binding molecules to Fcγ receptors as determined by surface plasmon resonance analysis. Table showing calculated binding rate constants and affinities derived from the binding data. [Figure 4] Graphs and tables showing the inhibition of proliferation of different human cancer cell lines with varying levels of basal HER3 expression by HER3 binding molecules. (A) and (B) show the inhibition of proliferation of (A) T47D cells and (B) HCT116 cells. IC50 values ​​for each of the test articles are provided in the table below each graph, where applicable (IC50 values ​​may not be for HCT116 cells). [Figure 5] Graphs and tables showing inhibition of T47D cell proliferation by HER3-binding molecules in the presence of NRG1. (A) and (B) show inhibition of T47D cell proliferation in the presence of the indicated molecules in the absence or presence of NRG1 (100 ng / ml). IC50 values ​​for each of the test articles are provided in the tables below each graph. [Figure 6] Graph showing the percentage of species with a drug-to-antibody ratio of 4 (DAR4) after incubation of the indicated HER3-binding molecules in mouse plasma at 37° C. for the indicated number of days. [Figure 7] 1 shows a graph depicting tumor growth inhibition in mice in a xenograft model derived from H358 cells after treatment with a HER3-binding molecule or vehicle control. The graph shows tumor volume over time. [Figure 8]Graphs showing tumor growth inhibition in mice in a T47D cell-derived xenograft model after treatment with different doses of HER3-binding molecules or vehicle control (A) and (B) show tumor volume over time. [Figure 9A] 1 is a bar graph showing the levels of red blood cell indices and anemia parameters in blood samples taken from rats after administration of the indicated molecules. Red blood cell counts are shown. The dotted lines indicate the baseline normal ranges for these indices in healthy female rats, as published by Charles River (Clinical Pathology Data for North American CD® Rat Colonies for January 2006-December 2011). [Figure 9B] Bar graphs showing the levels of red blood cell indices and anemia parameters in blood samples taken from rats after administration of the indicated molecules. Hemoglobin concentrations are shown. The dotted lines indicate the baseline normal ranges for these indices in healthy female rats, as published by Charles River (Clinical Pathology Data for North American CD® Rat Colonies for January 2006-December 2011). [Figure 9C] Bar graphs showing levels of red blood cell indices and anemia parameters in blood samples taken from rats after administration of the indicated molecules. Hematocrit percentages are shown. The dotted lines indicate the baseline normal ranges for these indices in healthy female rats, as published by Charles River (Clinical Pathology Data for North American CD® Rat Colonies for January 2006-December 2011). [Figure 9D]Bar graphs showing levels of red blood cell indices and anemia parameters in blood samples taken from rats after administration of the indicated molecules. Mean corpuscular volume (MCV) is shown. The dotted lines indicate the baseline normal ranges for these indices in healthy female rats, as published by Charles River (Clinical Pathology Data for North American CD® Rat Colonies for January 2006-December 2011). [Figure 9E] Bar graphs showing the levels of red blood cell indices and anemia parameters in blood samples taken from rats after administration of the indicated molecules. Mean corpuscular hemoglobin (MCH) levels are shown. The dotted lines indicate the baseline normal ranges for these indices in healthy female rats, as published by Charles River (Clinical Pathology Data for North American CD® Rat Colonies for January 2006-December 2011). [Figure 9F] Bar graphs showing the levels of red blood cell indices and anemia parameters in blood samples taken from rats after administration of the indicated molecules. Mean corpuscular hemoglobin concentration (MCHC) is shown. The dotted lines indicate the baseline normal ranges for these indices in healthy female rats, as published by Charles River (Clinical Pathology Data for North American CD® Rat Colonies for January 2006-December 2011). [Figure 10A]Bar graphs showing white blood cell indices and platelet levels in blood samples taken from rats 3 days after dose 1 (day 4) and 3 days after dose 2 (day 11) following administration of the indicated molecules. White blood cell counts are shown. The dotted lines indicate the baseline normal ranges for these indices in healthy female rats, as published by Charles River (Clinical Pathology Data for North American CD® Rat Colonies for January 2006-December 2011). [Figure 10B] Bar graphs showing white blood cell indices and platelet levels in blood samples taken from rats 3 days after dose 1 (day 4) and 3 days after dose 2 (day 11) following administration of the indicated molecules. Lymphocyte counts are shown. Dotted lines indicate the baseline normal ranges for these indices in healthy female rats, as published by Charles River (Clinical Pathology Data for North American CD® Rat Colonies for January 2006-December 2011). [Figure 10C] Bar graphs showing white blood cell indices and platelet levels in blood samples taken from rats 3 days after dose 1 (day 4) and 3 days after dose 2 (day 11) following administration of the indicated molecules. Monocyte counts are shown. Dotted lines indicate the baseline normal ranges for these indices in healthy female rats, as published by Charles River (Clinical Pathology Data for North American CD® Rat Colonies for January 2006-December 2011). [Figure 10D]Bar graphs showing white blood cell indices and platelet levels in blood samples taken from rats 3 days after dose 1 (day 4) and 3 days after dose 2 (day 11) following administration of the indicated molecules. Neutrophil counts are shown. Dotted lines indicate the baseline normal ranges for these indices in healthy female rats, as published by Charles River (Clinical Pathology Data for North American CD® Rat Colonies for January 2006-December 2011). [Figure 10E] Bar graphs showing white blood cell indices and platelet levels in blood samples taken from rats 3 days after dose 1 (day 4) and 3 days after dose 2 (day 11) following administration of the indicated molecules. Platelet counts are shown. The dotted lines indicate the baseline normal ranges for these indices in healthy female rats, as published by Charles River (Clinical Pathology Data for North American CD® Rat Colonies for January 2006-December 2011). [Figure 11A] Bar graphs showing levels of liver, kidney, and pancreatic indices in blood samples taken from rats after administration of the indicated molecules, 3 days after dose 1 (day 4) and 3 days after dose 2 (day 11). Alanine aminotransferase (ALT) levels are shown. Dotted lines indicate the baseline normal ranges for these indices in healthy female rats, as published by Charles River (Clinical Pathology Data for North American CD® Rat Colonies for January 2006-December 2011). [Figure 11B]Bar graphs showing levels of liver, kidney, and pancreatic indices in blood samples taken from rats after administration of the indicated molecules, 3 days after dose 1 (day 4) and 3 days after dose 2 (day 11). Aspartate aminotransferase (AST) levels are shown. The dotted lines indicate the baseline normal ranges for these indices in healthy female rats, as published by Charles River (Clinical Pathology Data for North American CD® Rat Colonies for January 2006-December 2011). [Figure 11C] Bar graphs showing levels of liver, kidney, and pancreatic indices in blood samples taken from rats 3 days after dose 1 (day 4) and 3 days after dose 2 (day 11) following administration of the indicated molecules. Albumin levels are shown. Dotted lines indicate the baseline normal ranges for these indices in healthy female rats, as published by Charles River (Clinical Pathology Data for North American CD® Rat Colonies for January 2006-December 2011). [Figure 11D] Bar graphs showing levels of liver, kidney, and pancreatic indices in blood samples taken from rats 3 days after dose 1 (day 4) and 3 days after dose 2 (day 11) following administration of the indicated molecules. Total protein levels are shown. Dotted lines indicate the baseline normal ranges for these indices in healthy female rats, as published by Charles River (Clinical Pathology Data for North American CD® Rat Colonies for January 2006-December 2011). [Figure 11E]Bar graphs showing levels of liver, kidney, and pancreatic indices in blood samples taken from rats after administration of the indicated molecules, 3 days after dose 1 (day 4) and 3 days after dose 2 (day 11). Creatinine levels are shown. Dotted lines indicate the baseline normal ranges for these indices in healthy female rats, as published by Charles River (Clinical Pathology Data for North American CD® Rat Colonies for January 2006-December 2011). [Figure 11F] Bar graphs showing levels of liver, kidney, and pancreatic indices in blood samples taken from rats after administration of the indicated molecules, 3 days after dose 1 (day 4) and 3 days after dose 2 (day 11). Glucose levels are shown. Dotted lines indicate the baseline normal ranges for these indices in healthy female rats, as published by Charles River (Clinical Pathology Data for North American CD® Rat Colonies for January 2006-December 2011). [Figure 12A] Bar graphs showing electrolyte levels in blood samples taken from rats after administration of the indicated molecules, 3 days after dose 1 (day 4) and 3 days after dose 2 (day 11). Sodium levels are shown. Dotted lines indicate the baseline normal range for these indices in healthy female rats, as published by Charles River (Clinical Pathology Data for North American CD® Rat Colonies for January 2006-December 2011). [Figure 12B]Bar graphs showing electrolyte levels in blood samples taken from rats after administration of the indicated molecules, 3 days after dose 1 (day 4) and 3 days after dose 2 (day 11). Potassium levels are shown. Dotted lines indicate the baseline normal ranges for these indices in healthy female rats, as published by Charles River (Clinical Pathology Data for North American CD® Rat Colonies for January 2006-December 2011). [Figure 12C] Bar graphs showing electrolyte levels in blood samples taken from rats after administration of the indicated molecules, 3 days after dose 1 (day 4) and 3 days after dose 2 (day 11). Phosphate levels are shown. Dotted lines indicate the baseline normal ranges for these indices in healthy female rats, as published by Charles River (Clinical Pathology Data for North American CD® Rat Colonies for January 2006-December 2011). [Figure 12D] Bar graphs showing electrolyte levels in blood samples taken from rats after administration of the indicated molecules, 3 days after dose 1 (day 4) and 3 days after dose 2 (day 11). Calcium levels are shown. Dotted lines indicate the baseline normal range for these indices in healthy female rats, as published by Charles River (Clinical Pathology Data for North American CD® Rat Colonies for January 2006-December 2011). [Figure 13] Fluorescence images of APC-labeled 501c.S8D4 incubated with T47D cells for 0.5, 4, and 16 hours at 37°C to measure internalization and trafficking to lysosomes. Cells incubated at 4°C for 1 hour served as a negative control. [Figure 14] Graph showing internalization of Fab fluorescently labeled 501c.S8D4 into lysosomes in T47D cells using live cell imaging. [Figure 15] Graph showing macropinocytosis-dependent uptake of Fab-fluor-labeled 501c.S8D4 or patritumab-DXd by human megakaryocytes. p-value, ***<0.001. [Figure 16] Graph showing Fc-mediated binding of IgG-DXd (positive control), 501c.S8D4, or patritumab-DXd to human PBMCs at concentrations of 30 μg / ml, 10 μg / ml, or 3 μg / ml. p-values, **<0.01, ***<0.001. DETAILED DESCRIPTION OF THE INVENTION

[0039] The present disclosure relates to antigen-binding molecules comprising a HER3-binding moiety and an exatecan payload moiety.

[0040] In the experimental examples of the present disclosure, the inventors demonstrate that such antigen-binding molecules possess unexpected and advantageous properties compared to known anti-HER3 antibody-drug conjugates.

[0041] In particular, the antigen-binding molecules of the present disclosure are demonstrated herein to have similar or improved anti-cancer activity in vitro and in vivo, while possessing a much more favorable toxicological profile compared to patritumab-DXd, a known anti-HER3 antibody-drug conjugate.

[0042] The similar / improved anticancer activity relative to patritumab-DXd is particularly unexpected because the antigen-binding molecules of the present disclosure have an average drug-to-antibody ratio (DAR) of 4, compared to an average DAR of 8 for patritumab-DXd.

[0043] antigen binding part An "antigen-binding molecule" refers to a molecule that binds to a given target antigen. An antigen-binding molecule comprises an antigen-binding portion that binds to its target antigen. The antigen-binding molecule of the present disclosure comprises an antigen-binding portion that binds to HER3 (i.e., a HER3-binding portion).

[0044] Antigen-binding portions may comprise or be derived from antibodies (i.e., immunoglobulins (Ig)) and antigen-binding fragments of antibodies. As used herein, "antibody" includes monoclonal antibodies, polyclonal antibodies, monospecific and multispecific (e.g., bispecific, trispecific, etc.) antibodies, as well as antigen-binding molecules derived from antibodies, such as scFv, scFab, diabodies, triabodies, scFv-Fc, minibodies, single-domain antibodies (e.g., VhH, etc.). Antigen-binding fragments of antibodies include, for example, Fv, Fab, F(ab')2, and F(ab')2 fragments.

[0045] Antigen-binding moieties also include target antigen-binding aptamers, such as nucleic acid aptamers (reviewed, e.g., in Zhou and Rossi, Nat Rev Drug Discov. (2017) 16(3):181-202). In some embodiments, the antigen-binding moiety comprises or consists of an antigen-binding peptide / polypeptide, such as a peptide aptamer, thioredoxin, monobody, anticalin, Kunitz domain, avimer, knottin, fynomer, atrimer, DARPin, affibody, nanobody (i.e., single domain antibody (sdAb), affilin, armadillo repeat protein (ArmRP), OBody, or fibronectin, as reviewed, e.g., in Reverdatto et al., Curr Top Med Chem. 2015;15(12):1082-1101, which is incorporated herein by reference in its entirety (see also, e.g., Boersma et al., J Biol Chem (2011) 286:41273-85 and Emanuel et al., Mabs (2011) 3:38-48).

[0046] In some embodiments, the antigen-binding portion comprises or consists of the antigen-binding region of an antibody (e.g., an antigen-binding fragment of an antibody). The antigen-binding portion of the antigen-binding molecule of the present disclosure preferably comprises the antibody heavy chain variable region (VH) and the antibody light chain variable region (VL) of an antibody that binds to a target antigen. The antigen-binding domain formed by the VH and VL may also be referred to herein as the Fv region.

[0047] In some embodiments, the antigen-binding portion is or comprises an Fv of an antibody (e.g., provided as an scFv). In some embodiments, the antigen-binding portion is or comprises an Fab region of an antibody. In some embodiments, the antigen-binding portion is or comprises a whole antibody (i.e., including variable and constant regions).

[0048] An antigen-binding portion can be or comprise an antigen-binding polypeptide or an antigen-binding polypeptide complex. An antigen-binding portion can comprise multiple polypeptides that together form the antigen-binding portion. The polypeptides can be covalently or non-covalently associated. In some embodiments, a polypeptide forms part of a larger polypeptide that comprises the polypeptide (e.g., in the case of an scFv comprising a VH and a VL, or in the case of an scFab comprising a VH-CH1 and a VL-CL).

[0049] An antigen-binding portion can refer to a non-covalent or covalent complex of multiple polypeptides (e.g., 2, 3, 4, 6, or 8 polypeptides), e.g., an IgG-like antigen-binding molecule comprising two heavy chain polypeptides and two light chain polypeptides.

[0050] The antigen-binding portion of the present disclosure can be designed and prepared using the sequence of a monoclonal antibody (mAb) capable of binding to a given target antigen (e.g., HER3). Antigen-binding regions of antibodies, such as single-chain variable fragments (scFv), Fab and F(ab')2 fragments, can also be used / provided. An "antigen-binding region" is any fragment of an antibody that binds to the target to which a given antibody is specific.

[0051] Antibodies generally contain six complementarity determining region (CDRs): three in the heavy chain variable (VH) region: HC-CDR1, HC-CDR2, and HC-CDR3, and three in the light chain variable (VL) region: LC-CDR1, LC-CDR2, and LC-CDR3. Together, the six CDRs define the antibody paratope, which is the portion of the antibody that binds to the target antigen.

[0052] The VH and VL regions each contain a framework region (FR) on either side of each CDR, providing a scaffold for the CDR. From N- to C-terminus, the VH region comprises the following structure: N-terminus-[HC-FR1]-[HC-CDR1]-[HC-FR2]-[HC-CDR2]-[HC-FR3]-[HC-CDR3]-[HC-FR4]-C-terminus, and the VL region comprises the following structure: N-terminus-[LC-FR1]-[LC-CDR1]-[LC-FR2]-[LC-CDR2]-[LC-FR3]-[LC-CDR3]-[LC-FR4]-C-terminus.

[0053] There are several different conventions for defining antibody CDRs and FRs, see, for example, Kabat et al., Sequences of Proteins of Immunological Interest, 5 th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991), those described in Chothia et al., J. Mol. Biol. 196:901-917 (1987), and VBASE2 as described in Retter et al., Nucl. Acids Res. (2005) 33(suppl 1):D671-D674. The CDRs and FRs of the VH and VL regions of the antibody clones described herein are defined according to the international IMGT (ImMunoGeneTics) information system (LeFranc et al., Nucleic Acids Res. (2015) 43(Database issue):D413-22), which uses the IMGT V-DOMAIN as described in Lefranc et al., Dev. Comp. Immunol. (2003) 27:55-77. In a preferred embodiment, the CDRs and FRs of the antigen-binding molecules referred to herein are defined according to the IMGT information system.

[0054] In some embodiments, an antigen-binding molecule according to the present disclosure comprises or consists of an Fv region that binds to HER3. In some embodiments, the VH and VL regions of the Fv are provided as a single polypeptide, i.e., a single-chain Fv (scFv), connected by a linker sequence.

[0055] The VL and light chain constant (CL) regions, and the VH region and heavy chain constant 1 (CH1) region of the antigen-binding region of an antibody together constitute a Fab region. In some embodiments, the antigen-binding molecule comprises a Fab region comprising a VH, CH1, VL, and CL (e.g., CK or Cλ). In some embodiments, the Fab region comprises a polypeptide comprising a VH and CH1 (e.g., a VH-CH1 fusion polypeptide) and a polypeptide comprising a VL and CL (e.g., a VL-CL fusion polypeptide). In some embodiments, the Fab region comprises a polypeptide comprising a VH and CL (e.g., a VH-CL fusion polypeptide) and a polypeptide comprising a VL and CH1 (e.g., a VL-CH1 fusion polypeptide), i.e., in some embodiments, the Fab region is a CrossFab region. In some embodiments, the VH, CH1, VL, and CL regions of a Fab or CrossFab are provided as a single polypeptide linked by a linker region, i.e., as a single-chain Fab (scFab) or single-chain CrossFab (scCrossFab).

[0056] In some embodiments, the antigen-binding molecules described herein comprise or consist of a whole antibody that binds to HER3. As used herein, "whole antibody" refers to an antibody having a structure substantially similar to that of an immunoglobulin (Ig). Different types of immunoglobulins and their structures are described, for example, in Schroeder and Cavacini J Allergy Clin Immunol. (2010) 125(202):S41-S52, which is incorporated herein by reference in its entirety.

[0057] Type G immunoglobulins (i.e., IgG) are glycoproteins of approximately 150 kDa that contain two heavy chains and two light chains. From the N- to C-terminus, the heavy chain contains a VH followed by a heavy chain constant region containing three constant domains (CH1, CH2, and CH3); similarly, the light chain contains a VL followed by a CL. Depending on the heavy chain, immunoglobulins can be classified as IgG (e.g., IgG1, IgG2, IgG3, IgG4), IgA (e.g., IgA1, IgA2), IgD, IgE, or IgM. The light chain can be kappa (κ) or lambda (λ).

[0058] In some embodiments, the antigen binding molecule comprises or consists of an IgG (e.g., IgG1, IgG2, IgG3, IgG4), IgA (e.g., IgA1, IgA2), IgD, IgE, or IgM that binds to HER3.

[0059] In some embodiments described herein, one or more amino acids of an amino acid sequence referred to herein (e.g., an amino acid sequence of an antigen-binding molecule, e.g., an amino acid sequence of a CDR or VH / VL region) are substituted with another amino acid sequence. Substitution includes replacement of an amino acid residue with a non-identical "replacement" amino acid residue. The replacement amino acid residue for substitutions according to the present disclosure may be a naturally occurring amino acid residue (i.e., encoded by the genetic code) that is not identical to the amino acid residue at the corresponding position in the equivalent unsubstituted amino acid sequence selected from alanine (Ala), arginine (Arg), asparagine (Asn), aspartic acid (Asp), cysteine ​​(Cys), glutamine (Gln), glutamic acid (Glu), glycine (Gly), histidine (His), isoleucine (Ile), leucine (Leu), lysine (Lys), methionine (Met), phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr), tryptophan (Trp), tyrosine (Tyr), and valine (Val). In some embodiments, the replacement amino acid may be a non-naturally occurring amino acid residue, i.e., an amino acid residue other than those listed above. Examples of non-naturally occurring amino acid residues include norleucine, ornithine, norvaline, homoserine, aib, and other amino acid residue analogs, such as those described in Ellman, et al., Meth. Enzym. 202 (1991) 301-336.

[0060] In some embodiments, substitutions can be biochemically conservative. In some embodiments, if the substituted amino acid is provided in one of rows 1-5 of the table below, the replacement amino acid of the substitution is another non-identical amino acid provided in the same row: [Table 1]

[0061] By way of example, in some embodiments where the substitution is of a Met residue, the replacement amino acid may be selected from Ala, Val, Leu, Ile, Trp, Tyr, Phe, and norleucine.

[0062] In some embodiments, the replacement amino acid in the substitution may have the same side chain polarity as the amino acid residue it replaces. In some embodiments, the replacement amino acid in the substitution may have the same side chain charge (pH 7.4) as the amino acid residue it replaces. [Table 2]

[0063] That is, in some embodiments, a nonpolar amino acid is substituted with another non-identical nonpolar amino acid; in some embodiments, a polar amino acid is substituted with another non-identical polar amino acid; in some embodiments, an acidic polar amino acid is substituted with another non-identical acidic polar amino acid; in some embodiments, a basic polar amino acid is substituted with another non-identical basic polar amino acid; in some embodiments, a neutral amino acid is substituted with another non-identical neutral amino acid; in some embodiments, a positive amino acid is substituted with another non-identical positive amino acid; in some embodiments, a negative amino acid is substituted with another non-identical negative amino acid.

[0064] In some embodiments, the substitution(s) may be functionally conservative, i.e., in some embodiments, the substitution may not affect (or may not substantially affect) one or more functional properties (e.g., target binding) of an antigen-binding molecule containing the substitution, compared to a comparable unsubstituted molecule.

[0065] The antigen binding molecules of the present disclosure comprise an antigen binding portion that binds to HER3.

[0066] In some embodiments, the antigen-binding portion comprises the CDRs of an antigen-binding portion capable of binding to HER3. In some embodiments, the antigen-binding portion comprises the FRs of an antigen-binding portion capable of binding to HER3. In some embodiments, the antigen-binding portion comprises the CDRs and FRs of an antibody capable of binding to HER3. That is, in some embodiments, the antigen-binding portion comprises the VH and VL regions of an antibody capable of binding to HER3.

[0067] In some embodiments, the antigen-binding moiety capable of binding to HER3 according to the present disclosure is an antibody described in WO2019 / 185878A1 (herein incorporated by reference in its entirety), 10D1F (e.g., as described in WO2019 / 185878A1), seribantumab (also known as MM-121, e.g., as described in Schoeberl et al., Sci. Signal. (2009) 2(77):ra31; DrugBank Acc. No. DB11857), elgemtumab (also known as LJM-716, e.g., as described in Garner et al., Cancer Res (2013) 73:6024-6035; DrugBank Acc. No. DB15430), patritumab (also known as U-1287 and AMG-888, e.g., as described in Shimizu et al. Cancer Chemotherapist (2014) 74:101-104; DrugBank Acc. No. DB15430), or the like. Pharmacol. (2017) 79(3): 489-495; DrugBank Acc. No. DB12090), GSK2849330 (e.g., described in Clarke et al., Eur J Cancer. (2014) 50: 98-9), lumletuzumab (also known as RG7116 and RO-5479599, e.g., described in Mirschberger et al. Cancer Research (2013) 73(16) 5183-5194; DrugBank Acc. No. DB12683), CDX-3379 (also known as KTN3379, e.g., described in Lee et al., Proc Natl Acad Sci US A. 2015 Oct 27; 112(43): 13225), AV-203 (also known as CAN-017, e.g., described in Meetze et al., Eur J Cancer 2012;48:126), varsetamab (also known as ISU104, e.g., as described in Kim et al., Cancer Res (2018) 78 (13 Suppl): Abstract #830), TK-A3, TK-A4 (e.g., as described in Malm et al., Mabs (2016) 8:1195-209), MP-EV20 (e.g., as described in Sala et al., Transl. Oncol. (2013) 6:676-84), 1A5-3D4 (e.g., as described in Wang et al., Cancer Lett (2016) 380:20-30), 9F7-F11, 16D3-C1 (e.g., as described in Lazrek et al., Neoplasia (2013) 15:335-47), NG33, A5, F4 (e.g., as described in Gaborit et al., PNAS USA (2015) 112:839-44), huHER3-8 (e.g., as described in Kugel et al., Cancer Res. (2014) 74:4122-32), REGN1400 (e.g., as described in Zhang et al., Mol Cancer Ther (2014) 13:1345-1355), and xenoctuzumab (also known as MCLA-128, e.g., as described in de Vries Schultink et al., Clin Pharmacokinet. (2020) 59:875-884; DrugBank Acc. No. DB15559). In some embodiments, the antigen-binding portion is or is derived from 10D1F.

[0068] In some embodiments, the antigen binding moiety binds to the extracellular region of HER3 (e.g., the region set forth in SEQ ID NO: 9). In some embodiments, the antigen binding moiety binds to subdomain II of the extracellular region of HER3 (e.g., the region set forth in SEQ ID NO: 16).

[0069] In some embodiments, the antigen binding portion binds to a region of HER3 set forth in SEQ ID NO: 77. In some embodiments, the antigen binding portion contacts one or more amino acid residues of the region of HER3 set forth in SEQ ID NO: 77. In some embodiments, the antigen binding portion binds to a region of HER3 set forth in SEQ ID NOs: 78 and 79. In some embodiments, the antigen binding portion contacts one or more amino acid residues of the region of HER3 set forth in SEQ ID NOs: 78 and 79. In some embodiments, the antigen binding portion binds to a region of HER3 set forth in SEQ ID NO: 78. In some embodiments, the antigen binding portion contacts one or more amino acid residues of the region of HER3 set forth in SEQ ID NO: 78. In some embodiments, the antigen binding portion binds to a region of HER3 set forth in SEQ ID NO: 79. In some embodiments, the antigen binding portion contacts one or more amino acid residues of the region of HER3 set forth in SEQ ID NO: 79.

[0070] In some embodiments, the antigen-binding portion does not bind to a region of HER3 corresponding to positions 260-279 of SEQ ID NO: 1. In some embodiments, the antigen-binding portion does not contact amino acid residues in a region of HER3 corresponding to positions 260-279 of SEQ ID NO: 1.

[0071] The region of a peptide / polypeptide to which an antibody / antigen-binding moiety binds can be identified by one of skill in the art using a variety of methods well known in the art, including X-ray crystallography of the antibody-antigen complex, peptide scanning, mutagenesis mapping, mass spectrometry hydrogen-deuterium exchange analysis, phage display, competitive ELISA, and proteolysis-based "protection" methods. Such methods are described, for example, in Gershoni et al., BioDrugs, 2007, 21(3):145-156, which is incorporated herein by reference in its entirety.

[0072] In some embodiments, the antigen-binding portion can bind to the same region of HER3, or overlapping regions of HER3, as bound by antigen-binding molecules comprising the VH and VL sequences of one of antibody clones 10D1_c89, 10D1, 10D1_c75, 10D1_c76, 10D1_c77, 10D1_c78v1, 10D1_c78v2, 10D1_11B, 10D1_c85v1, 10D1_c85v2, 10D1_c85o1, 10D1_c85o2, 10D1_c87, 10D1_c90, 10D1_c91, 10D1_c92, and 10D1_c93 described herein. In some embodiments, the antigen-binding portion can bind to the same region of HER3, or overlapping regions of HER3, as bound by an antigen-binding molecule comprising the VH and VL sequences of antibody clone 10D1_c89 described herein.

[0073] In some embodiments, the antigen-binding portion is capable of binding to a polypeptide comprising or consisting of the amino acid sequence of one of SEQ ID NOs: 1, 3, 4, 6, or 8. In some embodiments, the antigen-binding portion is capable of binding to a polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 9. In some embodiments, the antigen-binding portion is capable of binding to a polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 16. In some embodiments, the antigen-binding portion is capable of binding to a peptide / polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 77. In some embodiments, the antigen-binding portion is capable of binding to a peptide / polypeptide comprising or consisting of the amino acid sequence of SEQ ID NOs: 78 and 79. In some embodiments, the antigen-binding portion is capable of binding to a peptide / polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 78. In some embodiments, the antigen-binding portion is capable of binding to a peptide / polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 79.

[0074] In some embodiments, the antigen-binding portion is unable to bind to a peptide consisting of an amino acid sequence corresponding to positions 260-279 of SEQ ID NO:1.

[0075] The ability of an antigen-binding moiety to bind to a given peptide / polypeptide can be analyzed by methods well known to those of skill in the art, including analysis by ELISA, immunoblot (e.g., Western blot), immunoprecipitation, surface plasmon resonance (SPR; see, e.g., Hearty et al., Methods Mol Biol (2012) 907:411-442), or biolayer interferometry (see, e.g., Lad et al., (2015) J Biomol Screen 20(4):498-507).

[0076] In embodiments in which the antigen-binding moiety can bind to a peptide / polypeptide comprising a reference amino acid sequence, the peptide / polypeptide may comprise one or more additional amino acids at one or both termini of the reference amino acid sequence. In some embodiments, the peptide / polypeptide comprises, for example, 1 to 5, 1 to 10, 1 to 20, 1 to 30, 1 to 40, 1 to 50, 5 to 10, 5 to 20, 5 to 30, 5 to 40, 5 to 50, 10 to 20, 10 to 30, 10 to 40, 10 to 50, 20 to 30, 20 to 40, or 20 to 50 additional amino acids at one or both termini of the reference amino acid sequence.

[0077] In some embodiments, the additional amino acid(s) provided at one or both ends (i.e., the N-terminus and C-terminus) of the reference sequence correspond to the position of the ends of the reference sequence in the context of the amino acid sequence of HER3.

[0078] In some embodiments, the antigen-binding portion is capable of binding to a peptide / polypeptide bound by an antibody comprising the VH and VL sequences of one of antibody clones 10D1_c89, 10D1, 10D1_c75, 10D1_c76, 10D1_c77, 10D1_c78v1, 10D1_c78v2, 10D1_11B, 10D1_c85v1, 10D1_c85v2, 10D1_c85o1, 10D1_c85o2, 10D1_c87, 10D1_c90, 10D1_c91, 10D1_c92, and 10D1_c93 described herein. In some embodiments, the antigen-binding portion is capable of binding to a peptide / polypeptide bound by an antibody comprising the VH and VL sequences of antibody clone 10D1_c89.

[0079] In some embodiments, the antigen-binding portion comprises the CDRs of a HER3-binding antibody clone selected from 10D1_c89, 10D1, 10D1_c75, 10D1_c76, 10D1_c77, 10D1_c78v1, 10D1_c78v2, 10D1_11B, 10D1_c85v1, 10D1_c85v2, 10D1_c85o1, 10D1_c85o2, 10D1_c87, 10D1_c90, 10D1_c91, 10D1_c92, and 10D1_c93, or comprises the VH and VL thereof.

[0080] In some embodiments, the antigen-binding portion comprises: (4) a VH region incorporating the following CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO: 40 HC-CDR2 having the amino acid sequence of SEQ ID NO: 43 HC-CDR3 having the amino acid sequence of SEQ ID NO: 48; or a variant thereof in which one, two, or three amino acids in one or more of HC-CDR1, HC-CDR2, or HC-CDR3 are replaced with another amino acid; and A VL region incorporating the following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO: 66 LC-CDR2 having the amino acid sequence of SEQ ID NO: 69 LC-CDR3 having the amino acid sequence of SEQ ID NO: 74; or a variant thereof in which one, two, or three amino acids in one or more of LC-CDR1, LC-CDR2, or LC-CDR3 are replaced with another amino acid.

[0081] (2) a VH region incorporating the following CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO: 38 HC-CDR2 having the amino acid sequence of SEQ ID NO: 41 HC-CDR3 having the amino acid sequence of SEQ ID NO: 44; or a variant thereof in which one, two, or three amino acids in one or more of HC-CDR1, HC-CDR2, or HC-CDR3 are replaced with another amino acid; and A VL region incorporating the following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO: 63 LC-CDR2 having the amino acid sequence of SEQ ID NO: 67 LC-CDR3 having the amino acid sequence of SEQ ID NO: 70; or a variant thereof in which one, two, or three amino acids in one or more of LC-CDR1, LC-CDR2, or LC-CDR3 are replaced with another amino acid.

[0082] (3) a VH region incorporating the following CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO: 38 HC-CDR2 having the amino acid sequence of SEQ ID NO: 41 HC-CDR3 having the amino acid sequence of SEQ ID NO: 44; or a variant thereof in which one, two, or three amino acids in one or more of HC-CDR1, HC-CDR2, or HC-CDR3 are replaced with another amino acid; and A VL region incorporating the following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO: 64 LC-CDR2 having the amino acid sequence of SEQ ID NO: 67 LC-CDR3 having the amino acid sequence of SEQ ID NO: 70; or a variant thereof in which one, two, or three amino acids in one or more of LC-CDR1, LC-CDR2, or LC-CDR3 are replaced with another amino acid.

[0083] (4) a VH region incorporating the following CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO: 38 HC-CDR2 having the amino acid sequence of SEQ ID NO: 41 HC-CDR3 having the amino acid sequence of SEQ ID NO: 44; or a variant thereof in which one, two, or three amino acids in one or more of HC-CDR1, HC-CDR2, or HC-CDR3 are replaced with another amino acid; and A VL region incorporating the following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO: 65 LC-CDR2 having the amino acid sequence of SEQ ID NO: 67 LC-CDR3 having the amino acid sequence of SEQ ID NO: 71; or a variant thereof in which one, two, or three amino acids in one or more of LC-CDR1, LC-CDR2, or LC-CDR3 are replaced with another amino acid.

[0084] (5) A VH region incorporating the following CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO: 38 HC-CDR2 having the amino acid sequence of SEQ ID NO: 42 HC-CDR3 having the amino acid sequence of SEQ ID NO: 45; or a variant thereof in which one, two, or three amino acids in one or more of HC-CDR1, HC-CDR2, or HC-CDR3 are replaced with another amino acid; and A VL region incorporating the following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO: 63 LC-CDR2 having the amino acid sequence of SEQ ID NO: 67 LC-CDR3 having the amino acid sequence of SEQ ID NO: 70; or a variant thereof in which one, two, or three amino acids in one or more of LC-CDR1, LC-CDR2, or LC-CDR3 are replaced with another amino acid.

[0085] (6) A VH region incorporating the following CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO: 39 HC-CDR2 having the amino acid sequence of SEQ ID NO: 42 HC-CDR3 having the amino acid sequence of SEQ ID NO: 45; or a variant thereof in which one, two, or three amino acids in one or more of HC-CDR1, HC-CDR2, or HC-CDR3 are replaced with another amino acid; and A VL region incorporating the following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO: 63 LC-CDR2 having the amino acid sequence of SEQ ID NO: 67 LC-CDR3 having the amino acid sequence of SEQ ID NO: 70; or a variant thereof in which one, two, or three amino acids in one or more of LC-CDR1, LC-CDR2, or LC-CDR3 are replaced with another amino acid.

[0086] (7) A VH region incorporating the following CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO: 38 HC-CDR2 having the amino acid sequence of SEQ ID NO: 42 HC-CDR3 having the amino acid sequence of SEQ ID NO: 44; or a variant thereof in which one, two, or three amino acids in one or more of HC-CDR1, HC-CDR2, or HC-CDR3 are replaced with another amino acid; and A VL region incorporating the following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO: 63 LC-CDR2 having the amino acid sequence of SEQ ID NO: 68 LC-CDR3 having the amino acid sequence of SEQ ID NO: 70; or a variant thereof in which one, two, or three amino acids in one or more of LC-CDR1, LC-CDR2, or LC-CDR3 are replaced with another amino acid.

[0087] (8) A VH region incorporating the following CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO: 38 HC-CDR2 having the amino acid sequence of SEQ ID NO: 42 HC-CDR3 having the amino acid sequence of SEQ ID NO: 46; or a variant thereof in which one, two, or three amino acids in one or more of HC-CDR1, HC-CDR2, or HC-CDR3 are replaced with another amino acid; and A VL region incorporating the following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO: 63 LC-CDR2 having the amino acid sequence of SEQ ID NO: 68 LC-CDR3 having the amino acid sequence of SEQ ID NO: 70; or a variant thereof in which one, two, or three amino acids in one or more of LC-CDR1, LC-CDR2, or LC-CDR3 are replaced with another amino acid.

[0088] (9) A VH region incorporating the following CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO: 38 HC-CDR2 having the amino acid sequence of SEQ ID NO: 42 HC-CDR3 having the amino acid sequence of SEQ ID NO: 47; or a variant thereof in which one, two, or three amino acids in one or more of HC-CDR1, HC-CDR2, or HC-CDR3 are replaced with another amino acid; and A VL region incorporating the following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO: 63 LC-CDR2 having the amino acid sequence of SEQ ID NO: 68 LC-CDR3 having the amino acid sequence of SEQ ID NO: 70; or a variant thereof in which one, two, or three amino acids in one or more of LC-CDR1, LC-CDR2, or LC-CDR3 are replaced with another amino acid.

[0089] (10) A VH region incorporating the following CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO: 38 HC-CDR2 having the amino acid sequence of SEQ ID NO: 42 HC-CDR3 having the amino acid sequence of SEQ ID NO: 45; or a variant thereof in which one, two, or three amino acids in one or more of HC-CDR1, HC-CDR2, or HC-CDR3 are replaced with another amino acid; and A VL region incorporating the following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO: 63 LC-CDR2 having the amino acid sequence of SEQ ID NO: 67 LC-CDR3 having the amino acid sequence of SEQ ID NO: 72; or a variant thereof in which one, two, or three amino acids in one or more of LC-CDR1, LC-CDR2, or LC-CDR3 are replaced with another amino acid.

[0090] (11) A VH region incorporating the following CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO: 38 HC-CDR2 having the amino acid sequence of SEQ ID NO: 41 HC-CDR3 having the amino acid sequence of SEQ ID NO: 44; or a variant thereof in which one, two, or three amino acids in one or more of HC-CDR1, HC-CDR2, or HC-CDR3 are replaced with another amino acid; and A VL region incorporating the following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO: 63 LC-CDR2 having the amino acid sequence of SEQ ID NO: 67 LC-CDR3 having the amino acid sequence of SEQ ID NO: 73; or a variant thereof in which one, two, or three amino acids in one or more of LC-CDR1, LC-CDR2, or LC-CDR3 are replaced with another amino acid.

[0091] In some embodiments, the antigen-binding portion comprises: (12) A VH region comprising an amino acid sequence having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, to the amino acid sequence of SEQ ID NO: 21, and a VL region comprising an amino acid sequence having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, to the amino acid sequence of SEQ ID NO: 49.

[0092] (13) A VH region comprising an amino acid sequence having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, to the amino acid sequence of SEQ ID NO: 22, and a VL region comprising an amino acid sequence having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, to the amino acid sequence of SEQ ID NO: 50.

[0093] (14) A VH region comprising an amino acid sequence having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, to the amino acid sequence of SEQ ID NO: 23, and a VL region comprising an amino acid sequence having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, to the amino acid sequence of SEQ ID NO: 51.

[0094] (15) A VH region comprising an amino acid sequence having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, to the amino acid sequence of SEQ ID NO: 24, and a VL region comprising an amino acid sequence having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, to the amino acid sequence of SEQ ID NO: 52.

[0095] (16) A VH region comprising an amino acid sequence having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, to the amino acid sequence of SEQ ID NO: 25, and a VL region comprising an amino acid sequence having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, to the amino acid sequence of SEQ ID NO: 53.

[0096] (17) A VH region comprising an amino acid sequence having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, to the amino acid sequence of SEQ ID NO: 26, and a VL region comprising an amino acid sequence having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, to the amino acid sequence of SEQ ID NO: 53.

[0097] (18) A VH region comprising an amino acid sequence having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, to the amino acid sequence of SEQ ID NO: 27, and a VL region comprising an amino acid sequence having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, to the amino acid sequence of SEQ ID NO: 53.

[0098] (19) A VH region comprising an amino acid sequence having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, to the amino acid sequence of SEQ ID NO: 28, and a VL region comprising an amino acid sequence having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, to the amino acid sequence of SEQ ID NO: 54.

[0099] (20) A VH region comprising an amino acid sequence having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, to the amino acid sequence of SEQ ID NO: 29, and a VL region comprising an amino acid sequence having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, to the amino acid sequence of SEQ ID NO: 54.

[0100] (21) A VH region comprising an amino acid sequence having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, to the amino acid sequence of SEQ ID NO: 30, and a VL region comprising an amino acid sequence having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, to the amino acid sequence of SEQ ID NO: 55.

[0101] (22) A VH region comprising an amino acid sequence having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, to the amino acid sequence of SEQ ID NO: 31, and a VL region comprising an amino acid sequence having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, to the amino acid sequence of SEQ ID NO: 56.

[0102] (23) A VH region comprising an amino acid sequence having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, to the amino acid sequence of SEQ ID NO: 32, and a VL region comprising an amino acid sequence having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, to the amino acid sequence of SEQ ID NO: 57.

[0103] (24) A VH region comprising an amino acid sequence having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, to the amino acid sequence of SEQ ID NO: 33, and a VL region comprising an amino acid sequence having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, to the amino acid sequence of SEQ ID NO: 58.

[0104] (25) A VH region comprising an amino acid sequence having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, to the amino acid sequence of SEQ ID NO: 34, and a VL region comprising an amino acid sequence having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, to the amino acid sequence of SEQ ID NO: 59.

[0105] (26) A VH region comprising an amino acid sequence having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, to the amino acid sequence of SEQ ID NO: 35, and a VL region comprising an amino acid sequence having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, to the amino acid sequence of SEQ ID NO: 60.

[0106] (27) A VH region comprising an amino acid sequence having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, to the amino acid sequence of SEQ ID NO: 36, and a VL region comprising an amino acid sequence having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, to the amino acid sequence of SEQ ID NO: 61.

[0107] (28) A VH region comprising an amino acid sequence having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, to the amino acid sequence of SEQ ID NO: 37, and a VL region comprising an amino acid sequence having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, to the amino acid sequence of SEQ ID NO: 62.

[0108] In some embodiments, the antigen-binding portion comprises or consists of: (29)(i) one or more (e.g., two) polypeptides comprising or consisting of an amino acid sequence having at least 70%, more preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 75; and (ii) One or more (e.g., two) polypeptides comprising or consisting of an amino acid sequence having at least 70%, more preferably at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 76.

[0109] (30)(i) one or more (e.g., two) polypeptides comprising or consisting of an amino acid sequence having at least 70%, more preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 80; and (ii) One or more (e.g., two) polypeptides comprising or consisting of an amino acid sequence having at least 70%, more preferably at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 76.

[0110] (31)(i) one or more (e.g., two) polypeptides comprising or consisting of an amino acid sequence having at least 70%, more preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 81; and (ii) One or more (e.g., two) polypeptides comprising or consisting of an amino acid sequence having at least 70%, more preferably at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 76.

[0111] (32)(i) one or more (e.g., two) polypeptides comprising or consisting of an amino acid sequence having at least 70%, more preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 82; and (ii) One or more (e.g., two) polypeptides comprising or consisting of an amino acid sequence having at least 70%, more preferably at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 76.

[0112] (33)(i) one or more (e.g., two) polypeptides comprising or consisting of an amino acid sequence having at least 70%, more preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 91; and (ii) One or more (e.g., two) polypeptides comprising or consisting of an amino acid sequence having at least 70%, more preferably at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 76.

[0113] In some embodiments, the antigen-binding portion of the present disclosure comprises an Fc region.

[0114] As used herein, "Fc region" refers to the polypeptide complex formed by the interaction between two polypeptides, each of which contains the CH2-CH3 region of an immunoglobulin (Ig) heavy chain constant sequence.

[0115] As used herein, "CH2 domain" refers to the amino acid sequence corresponding to the CH2 domain of an immunoglobulin (Ig). The CH2 domain is the region of an Ig formed by positions 231 to 340 of the immunoglobulin constant domain according to the EU numbering system described in Edelman et al., Proc Natl Acad Sci USA (1969) 63(1):78-85. "CH3 domain" refers to the amino acid sequence corresponding to the CH3 domain of an immunoglobulin (Ig). The CH3 domain is the region of an Ig formed by positions 341 to 447 of the immunoglobulin constant domain according to the EU numbering system described in Edelman et al., Proc Natl Acad Sci USA (1969) 63(1):78-85. "CH2-CH3 region" refers to the amino acid sequence corresponding to the CH2 and CH3 domains of an immunoglobulin (Ig). The CH2-CH3 region is the region of an Ig formed by positions 231 to 447 of the immunoglobulin constant domain according to the EU numbering system described in Edelman et al., Proc Natl Acad Sci USA (1969) 63(1):78-85.

[0116] In some embodiments, a CH2 domain, a CH3 domain, and / or a CH2-CH3 region according to the present disclosure corresponds to a CH2 domain / CH3 domain / CH2-CH3 region of an IgG (e.g., IgG1, IgG2, IgG3, IgG4), an IgA (e.g., IgA1, IgA2), an IgD, an IgE, or an IgM. In some embodiments, a CH2 domain, a CH3 domain, and / or a CH2-CH3 region corresponds to a CH2 domain / CH3 domain / CH2-CH3 region of a human IgG (e.g., hIgG1, hIgG2, hIgG3, hIgG4), an hIgA (e.g., hIgA1, hIgA2), an hIgD, an hIgE, or an hIgM. In some embodiments, the CH2 domain, CH3 domain, and / or CH2-CH3 region correspond to the CH2 domain / CH3 domain / CH2-CH3 region of a human IgG1 allotype (e.g., G1m1, G1m2, G1m3, or G1m17).

[0117] The Fc region interacts with Fc receptors and other molecules of the immune system to produce functional effects. Fc-mediated effector functions are reviewed, for example, in Jefferis et al., Immunol Rev 1998 163:59-76 (incorporated herein by reference in its entirety), and involve the Fc-mediated recruitment and activation of immune cells (e.g., macrophages, dendritic cells, neutrophils, basophils, eosinophils, platelets, mast cells, NK cells, and T cells) through interaction between the Fc region and Fc receptors expressed by the cells, recruitment of complement pathway components through binding of the Fc region to the complement protein C1q, and the resulting activation of the complement cascade. Fc-mediated functions include Fc receptor binding, antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), complement-dependent cytotoxicity (CDC), formation of the membrane attack complex (MAC), cell degranulation, cytokine and / or chemokine production, and antigen processing and presentation.

[0118] The sequence of the CH2-CH3 region of human IgG1 G1m1 allotype is shown in SEQ ID NO: 83. The sequence of the CH2-CH3 region of human IgG1 G1m3 allotype is shown in SEQ ID NO: 84. The sequence of the CH2-CH3 region of human IgG2 is shown in SEQ ID NO: 85. The sequence of the CH2-CH3 region of human IgG3 is shown in SEQ ID NO: 86. The sequence of the CH2-CH3 region of human IgG4 is shown in SEQ ID NO: 87.

[0119] In some embodiments, an Fc region according to the present disclosure comprises two polypeptides, each comprising a CH2-CH3 region comprising or consisting of an amino acid sequence having at least 70% sequence identity, more preferably at least 75% or more, 80% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 83, 84, 85, 86, or 87. In some embodiments, a reference Fc region according to the present disclosure comprises two polypeptides, each comprising a CH2-CH3 region comprising or consisting of the amino acid sequence of SEQ ID NO: 83, 84, 85, 86, or 87.

[0120] Modifications to antibody Fc regions that affect Fc-mediated function are known in the art, such as those described in Wang et al., Protein Cell (2018) 9(1):63-73, which is incorporated herein by reference in its entirety. Exemplary Fc region modifications known to affect antibody effector function are summarized in Table 1 of Wang et al., Protein Cell (2018) 9(1):63-73. In some embodiments, antigen-binding molecules of the present disclosure comprise an Fc region comprising a modification that increases or decreases an Fc-mediated function compared to an antigen-binding molecule comprising a corresponding unmodified Fc region. When an Fc region / CH2 / CH3 is described as comprising modification(s) "corresponding to" a reference substitution(s), the equivalent substitution(s) in the homologous Fc / CH2 / CH3 are contemplated. For example, the L234A / L235A substitution in human IgG1 (Kabat et al., Sequences of Proteins of Immunological Interest, 5 thThe modifications (numbered according to the EU numbering system as described in Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991) correspond to L to A substitutions at positions 117 and 118 of the mouse Ig gamma-2 A chain C region (UniProtKB: P01863-1, v1). When an Fc region is described as containing a modification, the modification can be present in one or both of the polypeptide chains that together form the Fc region.

[0121] In some embodiments, the antigen binding molecules of the present disclosure comprise an Fc region that comprises a modification, hi some embodiments, the antigen binding molecules of the present disclosure comprise an Fc region that comprises a modification in one or more of the CH2 and / or CH3 regions.

[0122] In some embodiments, the Fc region comprises a modification to increase an Fc-mediated function. In some embodiments, the Fc region comprises a modification to increase ADCC. In some embodiments, the Fc region comprises a modification to increase ADCP. In some embodiments, the Fc region comprises a modification to increase CDC. Antigen binding molecules comprising an Fc region comprising a modification to increase an Fc-mediated function (e.g., ADCC, ADCP, CDC) induce increased levels of such effector function compared to a corresponding antigen binding molecule comprising an unmodified Fc region. In some embodiments, the antibody comprises a modification to increase binding to an Fc receptor. In some embodiments, the antibody comprises a modification to increase binding to an Fcγ receptor. In some embodiments, the Fc region comprises a modification to increase binding to one or more of FcγRI, FcγRIIa, FcγRIIb, FcγRIIc, FcγRIIIa, and FcγRIIIb. In some embodiments, the antibody comprises a modification to increase binding to FcγRIIIa. In some embodiments, the antibody comprises a modification to increase binding to FcγRIIa. In some embodiments, the antibody comprises a modification to increase binding to FcγRIIb. In some embodiments, the antibody comprises a modification to increase binding to FcRn. In some embodiments, the antibody comprises a modification to increase binding to a complement protein. In some embodiments, the antibody comprises a modification to increase binding to C1q. In some embodiments, the Fc region comprises a modification to promote hexamerization of the antigen-binding molecule. In some embodiments, the Fc region comprises a modification to increase the half-life of the antigen-binding molecule. In some embodiments, the Fc region comprises a modification to increase co-binding.

[0123] In some embodiments, the Fc region comprises modifications corresponding to the substitution combination F243L / R292P / Y300L / V305I / P396L as described in Stavenhagen et al. Cancer Res. (2007) 67:8882-8890. In some embodiments, the Fc region comprises modifications corresponding to the substitution combination S239D / I332E or S239D / I332E / A330L as described in Lazar et al., Proc Natl Acad Sci USA. (2006) 103:4005-4010. In some embodiments, the Fc region comprises modifications corresponding to the substitution combination S298A / E333A / K334A as described in Shields et al., J Biol Chem. (2001) 276:6591-6604. In some embodiments, the Fc region comprises modifications to one of the heavy chain polypeptides corresponding to the substitution combination L234Y / L235Q / G236W / S239M / H268D / D270E / S298A, and modifications to the other heavy chain polypeptide corresponding to the substitution combination D270E / K326D / A330M / K334E, as described in Mimoto et al., Mabs. (2013):5:229-236. In some embodiments, the Fc region comprises modifications corresponding to the substitution combination G236A / S239D / I332E, as described in Richards et al., Mol Cancer Ther. (2008)7:2517-2527.

[0124] In some embodiments, the Fc region comprises modifications corresponding to the substitution combination K326W / E333S as described in Idusogie et al. J Immunol. (2001) 166(4):2571-5. In some embodiments, the Fc region comprises modifications corresponding to the substitution combination S267E / H268F / S324T as described in Moore et al. Mabs. (2010) 2(2):181-9. In some embodiments, the Fc region comprises modifications corresponding to the substitution combination E345R / E430G / S440Y as described in Diebolder et al. Science (2014) 343(6176):1260-3.

[0125] In some embodiments, the Fc region comprises modifications corresponding to the substitution combination M252Y / S254T / T256E as described in Dall'Acqua et al. J Immunol. (2002) 169:5171-5180.

[0126] In some embodiments, the Fc region comprises modifications corresponding to the substitution combination M428L / N434S as described in Zalevsky et al. Nat Biotechnol. (2010) 28:157-159.

[0127] In some embodiments, the Fc region comprises modifications corresponding to the substitution combination S267E / L328F as described in Chu et al., Mol Immunol. (2008) 45:3926-3933. In some embodiments, the Fc region comprises modifications corresponding to the substitution combination N325S / L328F as described in Shang et al. Biol Chem. (2014) 289:15309-15318.

[0128] In some embodiments, the Fc region comprises a modification to reduce / prevent an Fc-mediated function. In some embodiments, the Fc region comprises a modification to reduce / prevent ADCC. In some embodiments, the Fc region comprises a modification to reduce / prevent ADCP. In some embodiments, the Fc region comprises a modification to reduce / prevent CDC. Antigen binding molecules comprising an Fc region comprising a modification to reduce / prevent an Fc-mediated function (e.g., ADCC, ADCP, CDC) induce a reduced level of that effector function compared to a corresponding antigen binding molecule comprising an unmodified Fc region. In some embodiments, an antibody comprises a modification to reduce / prevent binding to an Fc receptor. In some embodiments, an antibody comprises a modification to reduce / prevent binding to an Fcγ receptor. In some embodiments, the Fc region comprises a modification to reduce / prevent binding to one or more of FcγRI, FcγRIIa, FcγRIIb, FcγRIIc, FcγRIIIa, and FcγRIIIb. In some embodiments, an antibody comprises a modification to reduce / prevent binding to FcγRIIIa. In some embodiments, the antibody comprises a modification to reduce / prevent binding to FcγRIIa. In some embodiments, the antibody comprises a modification to reduce / prevent binding to FcγRIIb. In some embodiments, the antibody comprises a modification to reduce / prevent binding to a complement protein. In some embodiments, the antibody comprises a modification to reduce / prevent binding to C1q. In some embodiments, the Fc region comprises a modification to reduce / prevent glycosylation of the amino acid residue corresponding to N297.

[0129] In some embodiments, the Fc region is unable to induce one or more Fc-mediated functions (i.e., lacks the ability to trigger the Fc-mediated function(s)). Accordingly, antigen-binding molecules comprising such Fc regions also lack the ability to induce the function(s). Such antigen-binding molecules may be described as lacking the function(s). In some embodiments, the Fc region is unable to induce ADCC. In some embodiments, the Fc region is unable to induce ADCP. In some embodiments, the Fc region is unable to induce CDC. In some embodiments, the Fc region is unable to induce ADCC and / or unable to induce ADCP and / or unable to induce CDC. In some embodiments, the Fc region is unable to bind to an Fc receptor. In some embodiments, the Fc region is unable to bind to an Fcγ receptor. In some embodiments, the Fc region is unable to bind to one or more of FcγRI, FcγRIIa, FcγRIIb, FcγRIIc, FcγRIIIa, and FcγRIIIb. In some embodiments, the Fc region is unable to bind to FcγRIIIa. In some embodiments, the Fc region is unable to bind to FcγRIIa. In some embodiments, the Fc region is unable to bind to FcγRIIb. In some embodiments, the Fc region is unable to bind to FcRn. In some embodiments, the Fc region is unable to bind to complement proteins. In some embodiments, the Fc region is unable to bind to C1q.

[0130] In some embodiments, the Fc region comprises a modification corresponding to N297A or N297Q or N297G as described in Leabman et al., Mabs. (2013) 5:896-903. In some embodiments, the Fc region comprises a modification corresponding to L235E as described in Alegre et al., J Immunol. (1992) 148:3461-3468. In some embodiments, the Fc region comprises a modification corresponding to the substitution combination L234A / L235A or F234A / L235A as described in Xu et al., Cell Immunol. (2000) 200:16-26. In some embodiments, the Fc region comprises a modification corresponding to P329A or P329G as described in Schlothauer et al., Protein Engineering, Design and Selection (2016), 29(10):457-466. In some embodiments, the Fc region comprises modifications corresponding to the substitution combination L234A / L235A / P329G as described in Lo et al. J. Biol. Chem (2017) 292(9):3900-3908. In some embodiments, the Fc region comprises modifications corresponding to the substitution combination S228P / L235E as described in Newman et al. Clin. Immunol. (2001) 98:164-174. In some embodiments, the Fc region comprises modifications corresponding to the substitution combination H268Q / V309L / A330S / P331S as described in An et al. Mabs. (2009) 1:572-579. In some embodiments, the Fc region comprises modifications corresponding to the substitution combination V234A / G237A / P238S / H268A / V309L / A330S / P331S as described in Vafa et al., Methods. (2014) 65:114-126.In some embodiments, the Fc region comprises modifications corresponding to the substitution combination L234A / L235E / G237A / A330S / P331S as described in US2015 / 0044231A1.

[0131] The substitution combination "L234A / L235A" and corresponding substitutions (e.g., F234A / L235A in human IgG4) are known to disrupt Fc binding to Fcγ receptors, inhibit ADCC, ADCP, and reduce C1q binding and, therefore, CDC (Schlothauer et al., Protein Engineering, Design and Selection (2016), 29(10):457-466 (incorporated herein by reference in its entirety)). The substitutions "P329G" and "P329A" reduce C1q binding (and thereby reduce CDC). It is known that substituting "N297" with "A," "G," or "Q" eliminates glycosylation, thereby reducing Fc binding to C1q and Fcγ receptors, and, therefore, CDC and ADCC. Lo et al. J. Biol. Chem (2017) 292(9):3900-3908 (incorporated herein by reference in its entirety) report that the substitution combination L234A / L235A / P329G eliminated complement binding and fixation, as well as Fcγ receptor-dependent, antibody-dependent, cell-mediated cytotoxicity, in both mouse IgG2a and human IgG1.

[0132] The substitution combination L234A / L235E / G237A / A330S / P331S in IgG1 Fc is disclosed in US2015 / 0044231A1 to abolish the induction of phagocytosis, ADCC and CDC.

[0133] In some embodiments, the Fc region comprises a modification corresponding to substitution S228P as described in Silva et al., J Biol Chem. (2015) 290(9):5462-5469. The substitution S228P in an IgG4 Fc reduces Fab arm exchange, which may be undesirable.

[0134] In some embodiments, the Fc domain comprises a CH2-CH3 region that comprises an amino acid difference at one or more of the following positions relative to the amino acid sequence of the CH2-CH3 region of a reference Fc domain: 234, 235, 252, 254, or 256 (according to the EU numbering system). In some embodiments, the Fc domain comprises a CH2-CH3 region that comprises amino acid differences at positions 234 and 235 relative to the amino acid sequence of the CH2-CH3 region of a reference Fc domain. In some embodiments, the Fc domain comprises a CH2-CH3 region that comprises amino acid differences at positions 252, 254, and 256 relative to the amino acid sequence of the CH2-CH3 region of a reference Fc domain. In some embodiments, the Fc domain comprises a CH2-CH3 region that comprises amino acid differences at positions 234, 235, 252, 254, and 256 relative to the amino acid sequence of the CH2-CH3 region of a reference Fc domain.

[0135] In some embodiments, the Fc domain comprises a CH2-CH3 region that includes one or more of the following specific amino acid residues: A234, A235, Y252, T254, or E256 (according to the EU numbering system).

[0136] In some embodiments, the Fc domain comprises a CH2-CH3 region comprising Y252, T254, and E256. These so-called "YTE" modifications, located at the CH2-CH3 interface of the Fc domain, have been shown to increase binding affinity at pH 6.0 to the MHC class I neonatal Fc receptor (FcRn), which is localized within the acidic endosomes of endothelial and hematopoietic cells, thereby improving the efficient recycling of administered mAbs and increasing their plasma half-life.

[0137] Previous studies have shown that Fcγ receptor interactions can lead to uptake of ADCs by normal (i.e., non-cancerous) cells, causing severe cytopenias (e.g., leukopenia) - see Zhao et al., Mol Cancer Ther (2017) 16(9):1866-1876.

[0138] In some embodiments, the Fc domain comprises a CH2-CH3 region that includes A234 and A235. These so-called "LALA" modifications are known to inhibit the interaction of the Fc domain with Fcγ receptors expressed by hematopoietic cells, potentially reducing Fc-Fcγ receptor interaction-mediated toxicity.

[0139] In some embodiments, the Fc region of a HER3 binding moiety according to the present disclosure comprises a CH2 domain comprising A234 and A235 (EU numbering). In some embodiments, the Fc region of a HER3 binding moiety comprises a CH2 domain comprising Y252, T254, and E256 (EU numbering). In some embodiments, the Fc region of a HER3 binding moiety comprises a CH2 domain comprising A234, A235, Y252, T254, and E256 (EU numbering).

[0140] In some embodiments, the Fc domain comprises a CH2-CH3 region comprising A234, A235, Y252, T254 and E256.

[0141] In some embodiments, the Fc domain comprises a CH2-CH3 region that comprises one or more of the following amino acid substitutions relative to the amino acid sequence of the CH2-CH3 region of a reference Fc domain: L234A, L235A, M252Y, S254T, or T256E (according to the EU numbering system). In some embodiments, the Fc domain comprises a CH2-CH3 region that comprises L234A and L235A relative to the amino acid sequence of the CH2-CH3 region of a reference Fc domain. In some embodiments, the Fc domain comprises a CH2-CH3 region that comprises M252Y, S254T, and T256E relative to the amino acid sequence of the CH2-CH3 region of a reference Fc domain. In some embodiments, the Fc domain comprises a CH2-CH3 region that comprises L234A, L235A, M252Y, S254T, and T256E relative to the amino acid sequence of the CH2-CH3 region of a reference Fc domain.

[0142] In some embodiments, an Fc region according to the present disclosure comprises two polypeptides, each polypeptide comprising a CH2-CH3 region that comprises or consists of an amino acid sequence having at least 70% sequence identity, more preferably at least 75% or more, 80% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 88, 89, or 90.

[0143] In some embodiments, an antigen-binding portion according to the present disclosure comprises an amino acid sequence having at least 70% sequence identity, more preferably at least one of 75% or more, 80% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 94, 95, or 96.

[0144] An antigen-binding molecule according to the present disclosure may comprise a HER3-binding moiety according to any of the above embodiments and a linker payload moiety comprising exatecan or a derivative thereof as described below. In a preferred embodiment, the HER3-binding moiety is selected from one of (1) to (33) above.

[0145] Linker payload part The antigen-binding molecules of the present disclosure comprise a linker-payload moiety. As used herein, the term "linker-payload moiety" refers to a moiety comprising a payload moiety and a linker moiety for linking the payload moiety to the antigen-binding moiety (in the present disclosure, the HER3-binding moiety).

[0146] The antigen-binding molecule of the present disclosure comprises a HER3-binding portion and a linker payload portion comprising exatecan or a derivative thereof, i.e., the payload portion of the antigen-binding molecule of the present disclosure is exatecan or a derivative thereof.

[0147] Exatecan (also known as (1S,9S)-1-amino-9-ethyl-5-fluoro-9-hydroxy-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione, DX-8951, DrugBank accession number DB12185) is a DNA topoisomerase I inhibitor derivative of camptothecin and has the following structure: [ka]

[0148] Exatecan is described, for example, in Takiguchi et al., Jpn J Cancer Res. (1997) 88(8):760-769, which is incorporated herein by reference in its entirety. Exatecan and exatecan derivatives have been investigated as payloads for antibody-drug conjugates—see, for example, Nakada et al., Bioorg. Med. Chem. Lett. (2016) 26:1542-1545, which is incorporated herein by reference in its entirety.

[0149] The linker payload moiety of the present disclosure comprises exatecan or a derivative thereof. In some embodiments, the linker payload moiety comprises exatecan or an exatecan derivative described in WO2022 / 058395A1 (incorporated herein by reference in its entirety).

[0150] In some embodiments, the linker payload moiety comprises exatecan, N-glycyl-exatecan, or deruxtecan. In some embodiments, the antigen binding molecule or linker payload moiety of the present disclosure does not comprise deruxtecan. In some embodiments, the linker payload moiety comprises exatecan or N-glycyl-exatecan. In some embodiments, the linker payload moiety comprises exatecan.

[0151] The linker payload moiety includes a linker moiety. As used herein, a linker moiety refers to a moiety that connects two or more elements of a compound. Linker moieties for conjugating a payload moiety to an antigen-binding moiety are described, for example, in Su et al., Acta Pharmaceutica Sinica B (2021) 11(12):3889-3907 and Tsuchikama and An, Protein Cell. (2018) 9(1):33-46, both of which are incorporated herein by reference in their entireties.

[0152] In some embodiments, the linker payload moiety comprises a cleavable linker moiety. In some embodiments, the linker payload moiety or linker moiety comprises a dipeptide that is cleavable by a lysosomal protease. In some embodiments, the linker payload moiety or linker moiety comprises a moiety that is cleavable by a cathepsin (e.g., cathepsin B, K, and / or L). In some embodiments, the linker payload moiety or linker moiety comprises a moiety that is cleavable by cathepsin B.

[0153] In some embodiments, the linker payload moiety or linker moiety comprises the dipeptide valine-alanine or valine-citrulline. In some embodiments, the linker payload moiety or linker moiety comprises the dipeptide valine-alanine.

[0154] In some embodiments, the linker comprises a self-immolative moiety. In some embodiments, the linker payload moiety or linker moiety comprises a p-aminobenzylcarbamate (PABC) group.

[0155] In some embodiments, the linker payload moiety or linker moiety comprises structure (D): [ka] wherein R is selected from CH3 and (CH2)3NHCOCNH2.

[0156] In some embodiments, the linker payload moiety comprises a spacer moiety. As used herein, a spacer moiety refers to a moiety that separates (i.e., provides distance between) and covalently connects two (or more) portions of a linker moiety. Spacer moieties include moieties that comprise or consist of a polyethylene glycol (PEG) moiety, a polar acylsulfamide moiety, a polar carbamoylsulfamide moiety, and / or a HydraSpace moiety.

[0157] In some embodiments, the linker payload or spacer moiety comprises a polyethylene glycol (PEG) moiety. In some embodiments, the linker payload or spacer moiety comprises the structure (G): [ka]

[0158] In some embodiments, the linker payload moiety or spacer moiety comprises a polar acylsulfamide moiety or a polar carbamoylsulfamide moiety. In some embodiments, the linker payload moiety or spacer moiety comprises one or more HydraSpace moieties. As used herein, a HydraSpace moiety refers to a moiety having the structure (H): [ka]

[0159] HydraSpace moieties are described, for example, in Verkade et al., Antibodies (Basel) (2018) 7(1):12 and WO2016 / 053107A1, both of which are incorporated by reference in their entireties. In some embodiments, the linker payload moiety comprises the structure of a compound described in WO2016 / 053107A1.

[0160] In some embodiments, the linker payload moiety or spacer moiety comprises the structure (I): [ka]

[0161] In a preferred embodiment, the linker payload portion of the antigen-binding molecule of the present disclosure is a linker payload described in WO2022 / 058395A1, which is incorporated herein by reference in its entirety.

[0162] In some embodiments, the antigen binding linker payload moiety comprises or consists of the structure (I): [ka] During the ceremony, L 1 and L 2 is a linker moiety, w is 0 or 1, Z is a linking group obtained by metal-free click reaction or by thiol ligation, Each R 17 are independently amino acid side chains, n is an integer ranging from 1 to 5; A is a 5- or 6-membered aromatic or heteroaromatic ring; x is an integer ranging from 1 to 8; R 21 H, R 22 , C(O)OH and C(O)R 22 Selected from R 22 is C1~C 24 (Hetero)alkyl groups, C3-C 10 (Hetero)cycloalkyl groups, C2-C 10 (Hetero)aryl groups, C3-C 10 Alkyl (hetero)aryl groups and C3-C 10 (hetero)arylalkyl groups, which are O, S and NR 23 and optionally substituted and optionally interrupted by one or more heteroatoms selected from R 23 are independently selected from the group consisting of hydrogen and C1-4 alkyl groups.

[0163] In some embodiments, the linker payload moiety comprises the structure (J): [ka] During the ceremony, The wavy bond labeled * is connected to Z, and the wavy bond labeled ** is connected to NH, Sp 1and Sp 2 are each independently a spacer moiety, n, A.R. 17 and R 21 is as defined above in structure (I).

[0164] That is, in some embodiments, L of structure (I) 2 contains a branching moiety, preferably via a nitrogen atom, such that two payload moieties are connected to a single moiety Z. A "branching moiety" refers to a moiety embedded in a linker that connects three moieties. In other words, the branching moiety contains at least three bonds to other moieties, typically one bond to the HER-3 binding moiety that connects to Z, one bond to the payload moiety, and one bond to the second payload moiety. The branching moiety is preferably connected to a linker L 2 is embedded in.

[0165] In some embodiments, Sp 2 In some embodiments, each occurrence of (NH-CR 17 -CO) n In some embodiments, each occurrence of A is the same. In some embodiments, each occurrence of R 21 Each occurrence of is the same.

[0166] In some embodiments, L2 of structure (I) comprises structure (K): [ka]

[0167] The wavy line represents the remainder of the compound, typically Z and (NH-CR), optionally via a spacer. 17 -CO) n Preferably, (O) a The C(O) moiety is connected to Z, and NR 13 The part is (NH-CR 17 -CO) n is connected to the linker L 2 is the spacer part Sp1 When the sulfamide group according to structure (K) is present in the spacer moiety Sp 1 It is preferred that the hydroxyl group is included in the formula (I).

[0168] Thus, in some embodiments, L 2 comprises the structure (K), wherein: a=0 or 1, R 13 is C1~C 24 Alkyl groups, C3-C 24 Cycloalkyl groups, C2-C 24 (Hetero)aryl groups, C3-C 24 Alkyl (hetero)aryl groups and C3-C 24 (hetero)arylalkyl groups, C1-C 24 Alkyl groups, C3-C 24 Cycloalkyl groups, C2-C 24 (Hetero)aryl groups, C3-C 24 Alkyl (hetero)aryl groups and C3-C 24 (Hetero)arylalkyl groups include O, S and NR 14 and optionally substituted and optionally interrupted by one or more heteroatoms selected from R 14 are independently selected from the group consisting of hydrogen and C1-C4 alkyl groups, or R 13 is the second occurrence of C(O)X connected to N via a spacer moiety.

[0169] In some embodiments, L 2 contains two groups of structure (K).

[0170] In some embodiments, the linker L 2 , preferably a spacer Sp 1 comprises the structure (L): (L) (O) a C(O)NHS(O)2NH-(CH2CH2O) m -C(O)-(NHS(O)2)pN* In the formula, a is 0 or 1, m is an integer ranging from 1 to 10, and p is 0 or 1. N* may represent a branched moiety.

[0171] In some embodiments, (NH-CR 17 -CO) n is selected from Val-Cit, Val-Ala, Val-Lys, Val-Arg, AcLys-Val-Cit, AcLys-Val-Ala, Glu-Val-Ala, Asp-Val-Ala, Phe-Cit, Phe-Ala, Phe-Lys, Phe-Arg, Ala-Lys, Leu-Cit, Ile-Cit, Trp-Cit, Ala-Ala-Asn, Ala-Asn, and Lys. In some embodiments, (NH-CR 17 -CO) n is selected from Val-Cit, Val-Ala, Glu-Val-Ala, Val-Lys, Phe-Cit, Phe-Ala, Phe-Lys, Ala-Ala-Asn. In some embodiments, (NH-CR 17 -CO) n Each occurrence of is Val-Cit or Val-Ala.

[0172] In some embodiments, Z in structure (I) has a structure selected from structures (Z1)-(Z21): [ka] [ka] [ka] [ka] During the ceremony, Wavy line bonds marked with * are optional. L1 The other wavy line bond is connected to the HER3 binding moiety via L 2 is connected to The functional group R in (Z3), (Z7) and (Z8) is hydrogen, C1 to C24 Alkyl groups, C2-C 24 Acyl groups, C3-C 24 Cycloalkyl groups, C2-C 24 (Hetero)aryl groups, C3-C 24 Alkyl(hetero)aryl groups, C3-C 24 (Hetero)arylalkyl groups and C1-C 24 sulfonyl groups, each of which is selected from O, S and NR 32 and R 32 are independently selected from the group consisting of hydrogen and C1-C4 alkyl groups; R 24 is H or C1~ 12 alkyl, preferably H or C1-6 alkyl; R 29 is C1~ 12 Alkyl, preferably C1-4 alkyl, most preferably ethyl.

[0173] In some embodiments, Z in structure (I) is according to structure (Z22): [ka] During the ceremony, Wavy line bonds marked with * are optional. L1 The other wavy line bond is connected to the HER3 binding moiety via L 2 is connected to R 15 is hydrogen, halogen, -OR1 6 , a C1-C6 alkyl group, and a C5-C6 (hetero)aryl group; R 16 is hydrogen or C1-C6 alkyl, and more preferably, R 15 are independently selected from the group consisting of hydrogen and C1-C6 alkyl, and most preferably, all R 15 is H, R 18 are independently selected from the group consisting of hydrogen, C1-C6 alkyl groups, and most preferably both R18 is H, R 19 is H, I is 0 or 1, and more preferably I is 1.

[0174] Specific Exemplary Embodiments In some embodiments, the antigen binding molecule of the present disclosure comprises the structure (L): [ka] During the ceremony, L 1 , Z, A, R 21 ,n,R 17 and x is as defined above in structure (I), a and R1 3 is as defined above in structure (K), L 5 is a linker moiety, r is 0 or 1, m is an integer ranging from 1 to 10; q is an integer ranging from 0 to 10; p is 0 or 1.

[0175] In some embodiments, the antigen binding molecule of the present disclosure comprises the structure (M): [ka] In the formula, Z, L 2 , R 17 , A, R 21 , n and x are as defined above in structure (I); e is an integer ranging from 0 to 20; Su is a monosaccharide, G is a monosaccharide moiety, GlcNAc is an N-acetylglucosamine moiety, Fuc is a fucose moiety, d is 0 or 1.

[0176] In some embodiments, the antigen binding molecule of the present disclosure comprises the structure (N): [ka] During the ceremony, e, Su, G, GicNAc, Fuc, and d are as defined above in structure (M); n is 0 or 1.

[0177] In some embodiments, an antigen binding molecule of the present disclosure comprises SYNtecan E. That is, in some embodiments, an antigen binding molecule of the present disclosure comprises a HER3-binding portion and SYNtecan E.

[0178] As used herein, "SYNtecan E" refers to a moiety consisting of the following structure: [ka]

[0179] Functional properties of antigen-binding molecules The antigen-binding molecules described herein may be characterized by certain functional properties. In some embodiments, the antigen-binding molecules described herein may have one or more of the following properties: Binds to cells expressing HER3, binds to cells expressing HER3 in the presence of NRG1, inhibits the proliferation of HER3-expressing cells, Increases killing of HER3-expressing cells, inhibiting tumor growth and / or reducing tumor size / volume (e.g., in HER3-expressing cancers); improving survival of subjects with cancer (e.g., HER3-expressing cancer); does not exhibit substantial Fc-mediated binding to PBMCs (e.g., human PBMCs); does not exhibit substantial binding to human Fcγ receptors (e.g., FcγRIA, FcγRIIA, and / or FcγRIIIA); Internalized by HER3-expressing cells, does not show substantial macropinocytosis-dependent uptake by human megakaryocytes; Does not induce substantial leukopenia, Does not induce substantial neutropenia, Does not induce substantial thrombocytopenia, Does not induce substantial anemia, Does not induce substantial interstitial lung disease Does not substantially impair liver function does not substantially impair renal function, and / or Does not substantially impair lung function.

[0180] It will be understood that a given antigen-binding molecule may exhibit more than one of the properties listed in the previous paragraph. A given antigen-binding molecule can be evaluated for the properties listed in the previous paragraph using a suitable assay. For example, the assay can be, for example, an in vitro assay, optionally a cell-based assay or a cell-free assay. In some embodiments, the assay can be, for example, an in vivo assay, i.e., performed in a non-human animal. In some embodiments, the assay can be, for example, an ex vivo assay, i.e., performed using cells / tissues / organs obtained from a subject.

[0181] When the assay is a cell-based assay, it can include treating cells with antigen-binding molecules to determine whether the antigen-binding molecules exhibit one or more of the listed properties. The assay can use species labeled with detectable entities to facilitate their detection. The assay can include treating cells separately with a range of amounts / concentrations of a given antigen-binding molecule (e.g., a dilution series), and then evaluating the listed properties.

[0182] Analysis of the results of such assays may include identifying the concentration at which 50% of the maximal level of activity of interest is achieved. The concentration of a given drug at which 50% of the maximal level of activity of interest is achieved is sometimes referred to as the "median effective concentration" of the drug associated with the activity of interest, which is also known as the "EC 50 Depending on the characteristics, EC 50 is the "half maximal inhibitory concentration" or "IC 50 ", which is the concentration of an agent at which 50% of the maximal level of inhibition of a given property is observed.

[0183] In some embodiments, the antigen binding molecule of the present disclosure binds to HER3 at a region accessible to antigen binding molecules (i.e., extracellular antigen binding molecules) when HER3 is expressed on the cell surface (i.e., in or on the cell membrane). In some embodiments, the antigen binding molecule binds to HER3 expressed on the cell surface of HER3-expressing cells (e.g., H358, T47D or OVCAR8, HCT116 or DU145 cells).

[0184] The ability of an antigen-binding molecule to bind to a given cell type can be analyzed by contacting the cells with the antigen-binding molecule and detecting the antigen-binding molecule bound to the cells, for example, after a washing step to remove unbound antigen-binding molecules. The ability of an antigen-binding molecule to bind to HER3-expressing cells can be analyzed by methods such as flow cytometry and immunofluorescence microscopy. For example, the ability of an antigen-binding molecule to bind to HER3-expressing cells can be evaluated as described in Example 3.1 herein.

[0185] In some embodiments, the antigen binding molecules described herein have an EC50 of 10 nM or less, preferably one of 5 nM or less, 4 nM or less, 3 nM or less, 2 nM or less, 1 nM or less, 900 pM or less, 800 pM or less, 700 pM or less, 600 pM or less, or 500 pM or less, as determined, for example, by an assay such as that described in Example 3.1. 50It binds to cells expressing human HER3.

[0186] In some embodiments, the antigen binding molecule can bind to HER3 and / or HER3-expressing cells in the presence and / or absence of a HER3 ligand (e.g., NRG, NRG1, and / or NRG2). In some embodiments, the antigen binding molecule can bind to HER3 and / or HER3-expressing cells independently of a HER3 ligand (e.g., NRG, NRG1, and / or NRG2). In some embodiments, the antigen binding molecule does not compete with a HER3 ligand (e.g., NRG, NRG1, and / or NRG2) for binding to HER3 and / or HER3-expressing cells. In some embodiments, the antigen binding molecule does not bind to HER3 at the ligand-binding site. The ability of an antigen binding molecule to bind to HER3-expressing cells can be assessed as described in Example 3.2 herein.

[0187] In some embodiments, the antigen binding molecule inhibits the proliferation of HER3-expressing cells (e.g., HER3-expressing cancer cells). The ability of an antigen binding molecule to inhibit the proliferation of a given cell type can be analyzed by contacting the cells with the antigen binding molecule in the presence of a HER3 ligand (e.g., NRG1) and subsequently assessing cell proliferation (i.e., after a period sufficient to observe an effect on cell proliferation). Cell proliferation can be measured, for example, by detecting a change in cell number over time, or by measuring the number of cells in a cell population, as described, for example, in Fulcher and Wong, Immunol Cell Biol (1999) 77(6):559-564 (incorporated herein by reference in its entirety). 3This can be assessed by in vitro analysis of H-thymidine incorporation or by CFSE dilution assay. For example, the ability of an antigen-binding molecule to inhibit the proliferation of HER3-expressing cells can be assessed as described in Example 3.4 herein. In this example, cell proliferation was measured using Cell Counting Kit-8 (CCK8). CCK8 contains a tetrazolium salt, which is reduced by cellular dehydrogenases to an orange formazan product soluble in tissue culture medium. The amount of formazan produced is directly proportional to the number of viable cells and is measured by absorbance at 460 nm (Li et al., Cell Prolif. (2021) 54(3): e12986; Zhang et al., Genome Biol. (2021) 22(1): 41).

[0188] In some embodiments, the antigen-binding molecules of the present invention can inhibit the proliferation of HER3-expressing cells in a given assay by less than 1-fold the level of proliferation of HER3-expressing cells observed in the absence of the antigen-binding molecule (or in the presence of a suitable control antigen-binding molecule known to have no effect on the proliferation of HER3-expressing cells), e.g., 0.99-fold or less, 0.95-fold or less, 0.9-fold or less, 0.85-fold or less, 0.8-fold or less, 0.75-fold or less, 0.7-fold or less, 0.65-fold or less, 0.6-fold or less, 0.55-fold or less, 0.5-fold or less, 0.45-fold or less, 0.4-fold or less, 0.35-fold or less, 0.3-fold or less, 0.25-fold or less, 0.2-fold or less, 0.15-fold or less, 0.1-fold or less, 0.05-fold or less, or 0.01-fold or less.

[0189] In some embodiments, the antigen binding molecules described herein have an IC of 100 nM or less, preferably one of 50 nM or less, 40 nM or less, 30 nM or less, 20 nM or less, 10 nM or less, 5 nM or less, 4 nM or less, 3 nM or less, 2 nM or less, 1 nM or less, 900 pM or less, 800 pM or less, 700 pM or less, 600 pM or less, or 500 pM or less, as determined, for example, by an assay such as that described in Example 3.4. 50 inhibits the proliferation of cells expressing human HER3.

[0190] In some embodiments, an antigen binding molecule according to the present disclosure enhances (ie, upregulates, promotes) cell killing of cells containing / expressing HER3.

[0191] In some embodiments, antigen binding molecules according to the present disclosure may inhibit the growth of or reduce metastasis of cancers comprising cells containing / expressing HER3. In some embodiments, antigen binding molecules may enhance (i.e., upregulate, promote) cell killing of cells containing / expressing HER3. In some embodiments, antigen binding molecules may inhibit the growth of cells of cancers comprising cells containing / expressing HER3, or may inhibit the growth of tumors comprising such cells. In some embodiments, antigen binding molecules may inhibit metastasis of cancers / tumors comprising cells containing / expressing HER3.

[0192] Cell killing can be investigated using, for example, any of the methods outlined in Zaritskaya et al., Expert Rev Vaccines (2011), 9(6):601-616, which is incorporated herein by reference in its entirety. Examples of in vitro cytotoxicity / cell killing assays include: 51 These assays include the Cr release assay, lactate dehydrogenase (LDH) release assay, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) release assay, and calcein-acetoxymethyl (calcein-AM) release assay, which measure cell killing based on the detection of factors released from lysed cells.

[0193] In some embodiments, antigen binding molecules according to the present disclosure can reduce the number / proportion of cells expressing HER3, hi some embodiments, antigen binding molecules according to the present disclosure can deplete / promote the depletion of such cells.

[0194] In some embodiments, the antigen-binding molecules of the present disclosure exhibit anti-cancer activity. In some embodiments, the antigen-binding molecules increase the killing of cancer cells. In some embodiments, the antigen-binding molecules cause a reduction in the number of cancer cells in vivo, for example, compared to an appropriate control condition. The cancer may be a cancer described herein, for example, a cancer that expresses / overexpresses HER3.

[0195] In some embodiments, antigen binding molecules according to the present disclosure reduce / inhibit cancer and / or cancer tumor growth. In some embodiments, antigen binding molecules reduce tissue invasion by cancer cells. In some embodiments, antigen binding molecules reduce cancer metastasis. In some embodiments, antigen binding molecules exhibit anti-cancer activity. In some embodiments, antigen binding molecules reduce cancer cell growth / proliferation. In some embodiments, antigen binding molecules reduce cancer cell survival. In some embodiments, antigen binding molecules increase cancer cell killing. In some embodiments, antigen binding molecules of the present disclosure result in a reduction in the number of cancer cells, for example, in vivo. The cancer may be a cancer comprising cells expressing HER3.

[0196] The antigen-binding molecules of the present disclosure can be analyzed for the properties described in the previous paragraph in suitable assays. Such assays include, for example, in vivo models. Antigen-binding molecules can be evaluated for such properties in experiments performed essentially as described in Example 4.1 herein.

[0197] In some embodiments, administration of an antigen binding molecule according to the present disclosure may result in one or more of the following: for example, inhibiting the development / progression of cancer, delaying / preventing the onset of cancer, reducing / delaying / preventing tumor growth, reducing / delaying / preventing tissue invasion, reducing / delaying / preventing metastasis, reducing the severity of one or more symptoms of cancer, reducing the number of cancer cells, reducing the cancer burden, reducing the size / volume of the tumor, and / or improving the survival (e.g., progression-free survival or overall survival) of a subject with cancer, as determined in a suitable model.

[0198] The characteristics described in the previous paragraph are evaluated after a period sufficient to observe the effects associated with treatment with the antigen-binding molecule. Tumor growth can be monitored by examining tumor volume over time. Tumor growth can be measured by examining tumor volume (e.g., mm 3 It can be evaluated by measuring the

[0199] In some embodiments, an antigen binding molecule of the present disclosure can reduce tumor size / volume in a given assay (e.g., the mean tumor size / volume of a treatment group in an in vivo model of a HER3-expressing cancer) by less than 1-fold, e.g., 0.99-fold or less, 0.95-fold or less, 0.9-fold or less, 0.85-fold or less, 0.8-fold or less, 0.75-fold or less, 0.7-fold or less, 0.65-fold or less, 0.6-fold or less, 0.55-fold or less, 0.5-fold or less, 0.45-fold or less, 0.4-fold or less, 0.35-fold or less, 0.3-fold or less, 0.25-fold or less, 0.2-fold or less, 0.15-fold or less, 0.1-fold or less, 0.05-fold or less, or 0.01-fold or less, of the tumor size / volume observed at the same time point in the absence of treatment with the antigen binding molecule (or after treatment with a suitable control antigen binding molecule known not to affect tumor growth). In some embodiments, assessment of tumor size / volume for such comparison purposes is performed in the model more than 5 days after administration of the first dose of the antigen-binding molecule, e.g., 10 days or more, 15 days or more, 20 days or more, 25 days or more, 30 days or more, 35 days or more, 40 days or more, 35 days or more, 50 days or more, 55 days or more, 60 days or more, 65 days or more, 70 days or more, 75 days or more, 80 days or more, 85 days or more, 90 days or more, 95 days or more, or 100 days or more.

[0200] In some embodiments, an antigen binding molecule of the disclosure achieves a level of tumor growth inhibition in a given assay that is more than 1-fold, e.g., 1.01-fold or more, 1.02-fold or more, 1.03-fold or more, 1.04-fold or more, 1.05-fold or more, 1.1-fold or more, 1.2-fold or more, 1.3-fold or more, 1.4-fold or more, 1.5-fold or more, 1.6-fold or more, 1.7-fold or more, 1.8-fold or more, 1.9-fold or more, 2-fold or more, 3-fold or more, 4-fold or more, 5-fold or more, 6-fold or more, 7-fold or more, 8-fold or more, 9-fold or more, or 10-fold or more than the level of tumor growth inhibition observed in the absence of treatment with the antigen binding molecule (or after treatment with a suitable control antigen binding molecule known not to affect tumor growth) at the same time point. In some embodiments, assessment of tumor growth inhibition for such comparison purposes is performed in the model more than 5 days after administration of the first dose of the antigen binding molecule, e.g., 10 days or more, 15 days or more, 20 days or more, 25 days or more, 30 days or more, 35 days or more, 40 days or more, 35 days or more, 50 days or more, 55 days or more, 60 days or more, 65 days or more, 70 days or more, 75 days or more, 80 days or more, 85 days or more, 90 days or more, 95 days or more, or 100 days or more.

[0201] In some embodiments, an antigen binding molecule of the present disclosure can increase the median survival of subjects with cancer in a given assay (e.g., in an in vivo model of a HER3-expressing cancer) by more than 1-fold, e.g., 1.01-fold or more, 1.02-fold or more, 1.03-fold or more, 1.04-fold or more, 1.05-fold or more, 1.1-fold or more, 1.2-fold or more, 1.3-fold or more, 1.4-fold or more, 1.5-fold or more, 1.6-fold or more, 1.7-fold or more, 1.8-fold or more, 1.9-fold or more, 2-fold or more, 3-fold or more, 4-fold or more, 5-fold or more, 6-fold or more, 7-fold or more, 8-fold or more, 9-fold or more, or 10-fold or more than the median survival observed at the same time point in the absence of treatment with the antigen binding molecule (or after treatment with a suitable control antigen binding molecule known not to affect survival of subjects with cancer). Median survival of subjects in the treatment group can be expressed in days from the start of the experiment.

[0202] In some embodiments, the antigen-binding molecules of the present disclosure do not exhibit substantial Fc-mediated binding to PBMCs, e.g., human PBMCs. In some embodiments, the antigen-binding molecules of the present disclosure do not exhibit substantial binding to human Fcγ receptors (e.g., FcγRIA, FcγRIIA, and / or FcγRIIIA). Such binding can be determined, for example, by surface plasmon resonance or fluorescence techniques, as described herein.

[0203] In some embodiments, the antigen binding molecules of the present disclosure can be internalized into cells expressing HER3, e.g., into the lysosomes of the cells. Such internalization can be determined, for example, by fluorescence-based imaging methods as described herein.

[0204] In some embodiments, the antigen binding molecules of the present disclosure do not exhibit substantial macropinocytosis-dependent uptake by megakaryocytes. Macropinocytosis-dependent uptake can be determined, for example, by fluorescence-based live cell imaging methods as described herein.

[0205] In some embodiments, an antigen-binding molecule of the present disclosure does not induce substantial leukopenia (i.e., a decrease in the number / proportion of white blood cells in peripheral blood) after administration to a subject. In some embodiments, an antigen-binding molecule of the present disclosure does not induce substantial neutropenia (i.e., a decrease in the number / proportion of neutrophils in peripheral blood) after administration to a subject. In some embodiments, an antigen-binding molecule of the present disclosure does not induce substantial thrombocytopenia (i.e., a decrease in the number / proportion of platelets in peripheral blood) after administration to a subject. In some embodiments, an antigen-binding molecule of the present disclosure does not induce substantial anemia (i.e., a decrease in the number / proportion of red blood cells in peripheral blood) after administration to a subject.

[0206] An antigen-binding molecule can be evaluated to determine whether it induces leukopenia, neutropenia, thrombocytopenia, and / or anemia by measuring the number / proportion of white blood cells / neutrophils / platelets / erythrocytes in the peripheral blood after administration of the antigen-binding molecule to a subject. Such evaluation can include collecting a blood sample from the subject after administration of the antigen-binding molecule to the subject. It will be understood that the evaluation is performed after a period sufficient for leukopenia / neutropenia / thrombocytopenia / anemia to be observed. Measuring the number / proportion of a given cell type can be performed by any suitable means, for example, flow cytometry following antibody-based labeling of cellular biomarkers capable of distinguishing different types of blood cells.

[0207] An antigen-binding molecule that does not substantially induce leukopenia / neutropenia / thrombocytopenia / anemia can be an antigen-binding molecule that does not show a substantial decrease in the number / proportion of leukocytes / neutrophils / platelets / red blood cells in peripheral blood after administration of the antigen-binding molecule, compared to the number / proportion of leukocytes / neutrophils / platelets / red blood cells in peripheral blood of a subject not treated with the antigen-binding molecule or in peripheral blood of a subject treated with a suitable control antigen-binding molecule that is known not to induce leukopenia / neutropenia / thrombocytopenia / anemia.

[0208] In some embodiments, the number / proportion of white blood cells / neutrophils / platelets / red blood cells in the peripheral blood of a subject after administration of an antigen-binding molecule according to the present disclosure is 0.5-fold or more and 2-fold or less, for example, 0.55-fold or more and 1.9-fold or less, 0.6-fold or more and 1.8-fold or less, 0.65-fold or more and 1.7-fold or less, 0.7-fold or more and 1.6-fold or less, 0.75-fold or more and 1.5-fold or less, 0.8-fold or more and 1.4-fold or less, 0.85-fold or more and 1.3-fold or less, 0.9-fold or more and 1.2-fold or less, or 0.95-fold or more and 1.1-fold or less, of the number / proportion of such cells observed in the peripheral blood of a subject not administered the antigen-binding molecule or in the peripheral blood of a subject administered a suitable control antigen-binding molecule known not to induce leukopenia / neutropenia / thrombocytopenia / anemia. In some embodiments, the number / proportion of leukocytes / neutrophils / platelets / red blood cells in the peripheral blood of a subject after administration of an antigen-binding molecule according to the present disclosure is 0.5-fold or more, e.g., 0.55-fold or more, 0.6-fold or more, 0.65-fold or more, 0.7-fold or more, 0.75-fold or more, 0.8-fold or more, 0.85-fold or more, 0.9-fold or more, or 0.95-fold or more, of the number / proportion of such cells observed in the peripheral blood of a subject not administered the antigen-binding molecule or in the peripheral blood of a subject administered a suitable control antigen-binding molecule known not to induce leukopenia / neutropenia / thrombocytopenia / anemia.

[0209] In some embodiments, the antigen binding molecules of the present disclosure do not induce substantial interstitial lung disease after administration to a subject.

[0210] Antigen-binding molecules can be evaluated to determine whether they induce interstitial lung disease by assessing the subject for one or more correlates of interstitial lung disease (e.g., fibrosis of lung tissue, extracellular matrix deposition in lung tissue, expression of fibroinflammatory genes in lung tissue) after administering the antigen-binding molecule to the subject. Such evaluation can include collecting a sample of lung tissue after administering the antigen-binding molecule to the subject. It will be understood that the evaluation is performed after a period sufficient for interstitial lung disease to be induced by administering the antigen-binding molecule.

[0211] An antigen-binding molecule that does not substantially induce interstitial lung disease can be an antigen-binding molecule for which no substantial increase in the level of a correlate of interstitial lung disease is observed after administration of the antigen-binding molecule, compared to the level of the correlate in subjects not treated with the antigen-binding molecule, or compared to the level of the correlate in subjects treated with a suitable control antigen-binding molecule known not to induce interstitial lung disease.

[0212] In some embodiments, the level of a correlate of interstitial lung disease after administration of an antigen-binding molecule according to the present disclosure is 0.5-fold to 2-fold less than the level of the correlate in a subject not administered the antigen-binding molecule or treated with a suitable control antigen-binding molecule known not to induce interstitial lung disease, e.g., 0.55-fold to 1.9-fold, 0.6-fold to 1.8-fold, 0.65-fold to 1.7-fold, 0.7-fold to 1.6-fold, 0.75-fold to 1.5-fold, 0.8-fold to 1.4-fold, 0.85-fold to 1.3-fold, 0.9-fold to 1.2-fold, or 0.95-fold to 1.1-fold less than the level of the correlate in a subject not administered the antigen-binding molecule or treated with a suitable control antigen-binding molecule known not to induce interstitial lung disease.

[0213] In some embodiments, an antigen binding molecule of the present disclosure does not substantially impair liver function after administration to a subject. In some embodiments, an antigen binding molecule does not exhibit substantial hepatotoxicity. In some embodiments, an antigen binding molecule does not cause substantial damage to liver cells (e.g., hepatocytes) and / or liver tissue. In some embodiments, an antigen binding molecule of the present disclosure does not substantially impair kidney function after administration to a subject. In some embodiments, an antigen binding molecule does not exhibit substantial nephrotoxicity. In some embodiments, an antigen binding molecule does not cause substantial damage to kidney cells and / or kidney tissue. In some embodiments, an antigen binding molecule of the present disclosure does not substantially impair lung function after administration to a subject. In some embodiments, an antigen binding molecule does not exhibit substantial lung toxicity. In some embodiments, an antigen binding molecule does not cause substantial damage to lung cells and / or lung tissue.

[0214] An antigen-binding molecule can be evaluated to determine whether it impairs liver / renal / pulmonary function by measuring correlates of liver / renal / pulmonary function after administration of the antigen-binding molecule to a subject. Such evaluation can include collecting a blood sample from the subject after administration of the antigen-binding molecule to the subject. It will be understood that the evaluation is performed after a period sufficient to observe impairment of liver / renal / pulmonary function. Impairment of liver function can be determined by detecting increased levels of liver transaminases (such as aspartate transaminase (AST) and / or alanine transaminase (ALT)) in peripheral blood compared to the levels observed in the peripheral blood of subjects not treated with the antigen-binding molecule or in the peripheral blood of subjects treated with a suitable control antigen-binding molecule known not to impair liver function. Impairment of renal function can be determined by detecting an increase in the level / concentration of blood urea nitrogen (BUN) and / or creatinine in peripheral blood compared to the levels observed in the peripheral blood of subjects not treated with the antigen-binding molecule or in subjects treated with a suitable control antigen-binding molecule known not to impair renal function. Pulmonary function can be assessed, for example, by spirometry, plethysmography, and / or analysis of blood oxygen levels. Impairment of pulmonary function can be determined by detecting a decrease in one or more of the following compared to subjects not treated with the antigen-binding molecule or in subjects treated with a suitable control antigen-binding molecule known not to impair lung function: blood oxygen level, tidal volume (TV), minute ventilation (MV), vital capacity (VC), functional residual capacity (FRC), total lung capacity, forced vital capacity (FVC), forced expiratory volume (FEV), forced expiratory flow rate (FEF), and / or peak expiratory flow rate (PEFR).

[0215] In some embodiments, the level of a correlate of impaired liver function (e.g., AST and / or ALT) in the peripheral blood of a subject after administration of an antigen-binding molecule according to the present disclosure is 0.5-fold to 2-fold, e.g., 0.55-fold to 1.9-fold, 0.6-fold to 1.8-fold, 0.65-fold to 1.7-fold, 0.7-fold to 1.6-fold, 0.75-fold to 1.5-fold, 0.8-fold to 1.4-fold, 0.85-fold to 1.3-fold, 0.9-fold to 1.2-fold, or 0.95-fold to 1.1-fold the level observed in the peripheral blood of a subject not administered the antigen-binding molecule or in the peripheral blood of a subject administered a suitable control antigen-binding molecule known not to impair liver function. In some embodiments, the level of a correlate of impaired liver function (e.g., AST and / or ALT) in the peripheral blood of a subject after administration of an antigen-binding molecule according to the present disclosure is no more than two-fold, e.g., no more than 1.9-fold, no more than 1.8-fold, no more than 1.7-fold, no more than 1.6-fold, no more than 1.5-fold, no more than 1.4-fold, no more than 1.3-fold, no more than 1.2-fold, or no more than 1.1-fold, the level observed in the peripheral blood of a subject not administered the antigen-binding molecule or in the peripheral blood of a subject administered a suitable control antigen-binding molecule known not to impair liver function.

[0216] In some embodiments, the level / concentration of a correlate of impaired renal function (e.g., BUN and / or creatinine) in the peripheral blood of a subject after administration of an antigen-binding molecule according to the present disclosure is 0.5-fold or more and 2-fold or less, e.g., 0.55-fold or more and 1.9-fold or less, 0.6-fold or more and 1.8-fold or less, 0.65-fold or more and 1.7-fold or less, 0.7-fold or more and 1.6-fold or less, 0.75-fold or more and 1.5-fold or less, 0.8-fold or more and 1.4-fold or less, 0.85-fold or more and 1.3-fold or less, 0.9-fold or more and 1.2-fold or less, or 0.95-fold or more and 1.1-fold or less, of the level / concentration observed in the peripheral blood of a subject not administered the antigen-binding molecule or in the peripheral blood of a subject administered a suitable control antigen-binding molecule known not to impair renal function. In some embodiments, the level / concentration of a correlate of impaired renal function (e.g., BUN and / or creatinine) in the peripheral blood of a subject after administration of an antigen-binding molecule according to the present disclosure is two-fold or less, e.g., 1.9-fold or less, 1.8-fold or less, 1.7-fold or less, 1.6-fold or less, 1.5-fold or less, 1.4-fold or less, 1.3-fold or less, 1.2-fold or less, or 1.1-fold or less, of the level observed in the peripheral blood of a subject not administered the antigen-binding molecule or in the peripheral blood of a subject administered a suitable control antigen-binding molecule known not to impair renal function.

[0217] In some embodiments, the level of a correlate of lung function in a subject after administration of an antigen-binding molecule according to the present disclosure is 0.5-fold to 2-fold, e.g., 0.55-fold to 1.9-fold, 0.6-fold to 1.8-fold, 0.65-fold to 1.7-fold, 0.7-fold to 1.6-fold, 0.75-fold to 1.5-fold, 0.8-fold to 1.4-fold, 0.85-fold to 1.3-fold, 0.9-fold to 1.2-fold, or 0.95-fold to 1.1-fold the level of the correlate observed in a subject not administered the antigen-binding molecule or administered a suitable control antigen-binding molecule known not to impair lung function.

[0218] In some embodiments, the antigen-binding molecules disclosed herein have one or more novel, similar, or improved functional properties compared to known anti-HER3 antibody-drug conjugates. In some embodiments, the antigen-binding molecules have one or more novel, similar, or improved functional properties compared to patritumab-DXd (U3-1402). Patritumab-DXd (also known as U3-1402, FDA UNII:3XPI7EG4W8) is described, for example, in Proposed INN:List 121, WHO Drug Information (2019) 33(2):314-316. It comprises the HER3-binding monoclonal antibody patritumab conjugated to the DNA topoisomerase I inhibitor deruxtecan (DX-8951) via a cleavable tetrapeptide maleimide-Gly-Gly-Phe-Gly linker with a drug-to-antibody ratio (DAR) of 8.

[0219] In some embodiments, the antigen-binding molecules described herein may exhibit one or more of the following: similar or increased binding to cells expressing HER3 compared to binding to such cells shown by patritumab-DXd; increased binding to cells expressing HER3 in the presence of NRG1 compared to the binding to such cells shown by patritumab-DXd; Inhibition of proliferation of cells expressing HER3 is similar or increased compared to the inhibition of proliferation of such cells shown by patritumab-DXd; killing of HER3-expressing cells is similar or increased compared to killing of such cells shown by patritumab-DXd; the inhibition of tumor growth and / or reduction in tumor size / volume (e.g., for HER3-expressing cancers) is similar to or increased compared to the tumor growth inhibition / reduction in tumor size / volume shown by patritumab-DXd; and the survival of subjects with cancer (e.g., HER3-expressing cancers) after treatment with the antigen-binding molecule is similar to or improved compared to the survival observed after treatment with patritumab-DXd. Fc-mediated binding to human PBMCs is similar or increased compared to the Fc-mediated binding exhibited by patritumab-DXd; similar or increased internalization into HER3-expressing cells compared to that exhibited by patritumab-DXd; Macropinocytosis-dependent uptake by megakaryocytes is similar to or reduced compared to macropinocytosis-dependent uptake by patritumab-DXd; Similar or reduced toxicity compared to patritumab-DXd; induction of leukopenia is similar or reduced compared to patritumab-DXd; Similar or reduced induction of neutropenia compared to patritumab-DXd; Similar or reduced induction of anemia compared to patritumab-DXd; Similar or reduced induction of thrombocytopenia compared to patritumab-DXd; Similar or reduced induction of interstitial lung disease compared to patritumab-DXd; Similar or reduced liver dysfunction compared to patritumab-DXd; Similar or reduced renal impairment compared to patritumab-DXd, and / or Similar or reduced impairment of lung function compared to patritumab-DXd.

[0220] According to the previous paragraph, a level of a given trait / outcome that is "similar" to a reference level can be one of: 0.5-fold and 2-fold greater than or equal to the reference level, e.g., 0.55-fold and 1.9-fold, 0.6-fold and 1.8-fold, 0.65-fold and 1.7-fold, 0.7-fold and 1.6-fold, 0.75-fold and 1.5-fold, 0.8-fold and 1.4-fold, 0.85-fold and 1.3-fold, 0.9-fold and 1.2-fold, or 0.95-fold and 1.1-fold greater than or equal to the reference level. In some embodiments, the level of a given trait / outcome that is "increased" compared to a reference level can be more than 1-fold the reference level, for example, one of 1.01-fold or more, 1.02-fold or more, 1.03-fold or more, 1.04-fold or more, 1.05-fold or more, 1.1-fold or more, 1.2-fold or more, 1.3-fold or more, 1.4-fold or more, 1.5-fold or more, 1.6-fold or more, 1.7-fold or more, 1.8-fold or more, 1.9-fold or more, 2-fold or more, 3-fold or more, 4-fold or more, or 5-fold or more.

[0221] It will be understood that for purposes of such evaluation, equal amounts of the antigen-binding molecule and patritumab-DXd can be compared. In some embodiments, equal amounts of each payload are compared. That is, in some embodiments, for purposes of such evaluation, the amounts of the antigen-binding molecule and patritumab-DXd are compared so that the amounts of exatecan and deruxtecan are equivalent.

[0222] In some embodiments, the antigen binding molecule of the present disclosure binds to HER3-expressing cells (e.g., in the presence of NRG1) in a given assay, inhibiting the EC of patritumab-DXd binding to the cells. 50 EC equal to or less than 50 In some embodiments, the EC 50 is the EC of patritumab-DXd binding to the same cell type when determined in the same assay. 50In some embodiments, the EC of binding of the antigen-binding molecule to HER3-expressing cells is 0.5-fold or more and 2-fold or less, for example, 0.55-fold or more and 1.9-fold or less, 0.6-fold or more and 1.8-fold or less, 0.65-fold or more and 1.7-fold or less, 0.7-fold or more and 1.6-fold or less, 0.75-fold or more and 1.5-fold or less, 0.8-fold or more and 1.4-fold or less, 0.85-fold or more and 1.3-fold or less, 0.9-fold or more and 1.2-fold or less, or 0.95-fold or more and 1.1-fold or less. 50 is the EC of patritumab-DXd binding to the same cell type when determined in the same assay. 50 or less than 1 times, for example, one of 0.99 times or less, 0.95 times or less, 0.9 times or less, 0.85 times or less, 0.8 times or less, 0.75 times or less, 0.7 times or less, 0.65 times or less, 0.6 times or less, 0.55 times or less, 0.5 times or less, 0.45 times or less, 0.4 times or less, 0.35 times or less, 0.3 times or less, 0.25 times or less, 0.2 times or less, 0.15 times or less, 0.1 times or less, 0.05 times or less, or 0.01 times or less.

[0223] In some embodiments, the antigen binding molecule of the present disclosure inhibits the proliferation of HER3-expressing cells (e.g., HER3-expressing cancer cells) in a given assay, and the IC of patritumab-DXd inhibits the proliferation of the cells. 50 IC similar to or less than 50 In some embodiments, the antigen binding molecule inhibits proliferation of the same type of cells by patritumab-DXd at an IC50 value greater than or equal to the IC50 value determined in the same assay. 50 0.5 times or more and 2 times or less, for example, 0.55 times or more and 1.9 times or less, 0.6 times or more and 1.8 times or less, 0.65 times or more and 1.7 times or less, 0.7 times or more and 1.6 times or less, 0.75 times or more and 1.5 times or less, 0.8 times or more and 1.4 times or less, 0.85 times or more and 1.3 times or less, 0.9 times or more and 1.2 times or less, or 0.95 times or more and 1.1 times or less 50 In some embodiments, the IC of the antigen binding molecule for inhibiting the proliferation of HER3-expressing cells (e.g., HER3-expressing cancer cells) is 50is the IC for inhibition of proliferation of the same cell types by patritumab-DXd when determined in the same assay. 50 or less than 1 times, for example, one of 0.99 times or less, 0.95 times or less, 0.9 times or less, 0.85 times or less, 0.8 times or less, 0.75 times or less, 0.7 times or less, 0.65 times or less, 0.6 times or less, 0.55 times or less, 0.5 times or less, 0.45 times or less, 0.4 times or less, 0.35 times or less, 0.3 times or less, 0.25 times or less, 0.2 times or less, 0.15 times or less, 0.1 times or less, 0.05 times or less, or 0.01 times or less.

[0224] In some embodiments, an antigen binding molecule of the present disclosure increases killing of HER3-expressing cells in a given assay to a level similar to or higher than the level of killing of such cells exhibited by patritumab-DXd. In some embodiments, the antigen binding molecule increases killing of HER3-expressing cells (e.g., HER3-expressing cancer cells) by 0.5-fold or more and 2-fold or less, e.g., one of the following levels: 0.55-fold or more and 1.9-fold or less, 0.6-fold or more and 1.8-fold or less, 0.65-fold or more and 1.7-fold or less, 0.7-fold or more and 1.6-fold or less, 0.75-fold or more and 1.5-fold or less, 0.8-fold or more and 1.4-fold or less, 0.85-fold or more and 1.3-fold or less, 0.9-fold or more and 1.2-fold or less, or 0.95-fold or more and 1.1-fold or less, of the level of cell killing of such cells exhibited by patritumab-DXd, as determined in the same assay. In some embodiments, the antigen binding molecule increases killing of HER3-expressing cells (e.g., HER3-expressing cancer cells) by more than 1-fold the level of cell killing of such cells exhibited by patritumab-DXd, e.g., by one of 1.01-fold or more, 1.02-fold or more, 1.03-fold or more, 1.04-fold or more, 1.05-fold or more, 1.1-fold or more, 1.2-fold or more, 1.3-fold or more, 1.4-fold or more, 1.5-fold or more, 1.6-fold or more, 1.7-fold or more, 1.8-fold or more, 1.9-fold or more, 2-fold or more, 3-fold or more, 4-fold or more, 5-fold or more, 6-fold or more, 7-fold or more, 8-fold or more, 9-fold or more, or 10-fold or more, as determined in the same assay.

[0225] In some embodiments, the antigen binding molecules of the present disclosure reduce tumor size / volume in a given assay to a level similar to or greater than the level of reduction in tumor size / volume observed after treatment with patritumab-DXd. In some embodiments, the antigen binding molecule is capable of reducing tumor size / volume (e.g., the mean tumor size / volume of a treatment group in, e.g., an in vivo model of a HER3-expressing cancer) by one of the following levels: 0.5-fold or more and 2-fold or less, e.g., 0.55-fold or more and 1.9-fold or less, 0.6-fold or more and 1.8-fold or less, 0.65-fold or more and 1.7-fold or less, 0.7-fold or more and 1.6-fold or less, 0.75-fold or more and 1.5-fold or less, 0.8-fold or more and 1.4-fold or less, 0.85-fold or more and 1.3-fold or less, 0.9-fold or more and 1.2-fold or less, or 0.95-fold or more and 1.1-fold or less, of the tumor size / volume observed at the same time point after treatment with patritumab-DXd, as determined in the same assay. In some embodiments, the antigen binding molecule is capable of reducing tumor size / volume (e.g., mean tumor size / volume of a treatment group in, e.g., an in vivo model of a HER3-expressing cancer) by less than 1-fold, e.g., 0.99-fold or less, 0.95-fold or less, 0.9-fold or less, 0.85-fold or less, 0.8-fold or less, 0.75-fold or less, 0.7-fold or less, 0.65-fold or less, 0.6-fold or less, 0.55-fold or less, 0.5-fold or less, 0.45-fold or less, 0.4-fold or less, 0.35-fold or less, 0.3-fold or less, 0.25-fold or less, 0.2-fold or less, 0.15-fold or less, 0.1-fold or less, 0.05-fold or less, or 0.01-fold or less, of the tumor size / volume observed at the same time point following treatment with patritumab-DXd, as determined in the same assay. In some embodiments, assessment of tumor size / volume for such comparison purposes is performed in the model more than 5 days after administration of the first dose of the antigen-binding molecule, e.g., 10 days or more, 15 days or more, 20 days or more, 25 days or more, 30 days or more, 35 days or more, 40 days or more, 35 days or more, 50 days or more, 55 days or more, 60 days or more, 65 days or more, 70 days or more, 75 days or more, 80 days or more, 85 days or more, 90 days or more, 95 days or more, or 100 days or more.

[0226] In some embodiments, the antigen binding molecules of the present disclosure achieve a level of tumor growth inhibition in a given assay that is similar to or greater than that observed after treatment with patritumab-DXd. In some embodiments, the antigen binding molecules achieve a level of tumor growth inhibition (e.g., expressed as % tumor growth inhibition calculated, e.g., relative to the tumor growth observed after treatment with a suitable control antigen binding molecule) that is 0.5-fold or more and 2-fold or less, e.g., 0.55-fold or more and 1.9-fold or less, 0.6-fold or more and 1.8-fold or less, 0.65-fold or more and 1.7-fold or less, 0.7-fold or more and 1.6-fold or less, 0.75-fold or more and 1.5-fold or less, 0.8-fold or more and 1.4-fold or less, 0.85-fold or more and 1.3-fold or less, 0.9-fold or more and 1.2-fold or less, or 0.95-fold or more and 1.1-fold or less, as determined in the same assay, of tumor growth inhibition observed at the same time point after treatment with patritumab-DXd. In some embodiments, the antigen binding molecule achieves a level of tumor growth inhibition (e.g., expressed as % tumor growth inhibition calculated, e.g., relative to the tumor growth observed with treatment with a suitable control antigen binding molecule) that is more than 1-fold the level of tumor growth inhibition observed with treatment with patritumab-DXd at the same time point, e.g., 1.01-fold or more, 1.02-fold or more, 1.03-fold or more, 1.04-fold or more, 1.05-fold or more, 1.1-fold or more, 1.2-fold or more, 1.3-fold or more, 1.4-fold or more, 1.5-fold or more, 1.6-fold or more, 1.7-fold or more, 1.8-fold or more, 1.9-fold or more, 2-fold or more, 3-fold or more, 4-fold or more, 5-fold or more, 6-fold or more, 7-fold or more, 8-fold or more, 9-fold or more, or 10-fold or more, as determined in the same assay. In some embodiments, assessment of tumor size / volume for such comparison purposes is performed in the model more than 5 days after administration of the first dose of the antigen-binding molecule, e.g., 10 days or more, 15 days or more, 20 days or more, 25 days or more, 30 days or more, 35 days or more, 40 days or more, 35 days or more, 50 days or more, 55 days or more, 60 days or more, 65 days or more, 70 days or more, 75 days or more, 80 days or more, 85 days or more, 90 days or more, 95 days or more, or 100 days or more.

[0227] In some embodiments, the antigen binding molecules of the present disclosure achieve an increase in median survival of subjects with cancer in a given assay (e.g., in an in vivo model of a HER3-expressing cancer) that is similar to or greater than the increase in median survival observed after treatment with patritumab-DXd. In some embodiments, the antigen binding molecule achieves an increase in median survival of subjects with cancer (e.g., in an in vivo model of a HER3-expressing cancer) that is greater than or equal to 0.5-fold and less than or equal to 2-fold, e.g., one of: greater than or equal to 0.55-fold and less than or equal to 1.9-fold, greater than or equal to 0.6-fold and less than or equal to 1.8-fold, greater than or equal to 0.65-fold and less than or equal to 1.7-fold, greater than or equal to 0.7-fold and less than or equal to 1.6-fold, greater than or equal to 0.75-fold and less than or equal to 1.5-fold, greater than or equal to 0.8-fold and less than or equal to 1.4-fold, greater than or equal to 0.85-fold and less than or equal to 1.3-fold, greater than or equal to 0.9-fold and less than or equal to 1.2-fold, or greater than or equal to 0.95-fold and less than or equal to 1.1-fold the level of increase in median survival observed following treatment with patritumab-DXd, as determined in the same assay. In some embodiments, the antigen binding molecule achieves an increase in median survival of subjects with cancer (e.g., in an in vivo model of a HER3-expressing cancer) of more than 1-fold, e.g., 1.01-fold or more, 1.02-fold or more, 1.03-fold or more, 1.04-fold or more, 1.05-fold or more, 1.1-fold or more, 1.2-fold or more, 1.3-fold or more, 1.4-fold or more, 1.5-fold or more, 1.6-fold or more, 1.7-fold or more, 1.8-fold or more, 1.9-fold or more, 2-fold or more, 3-fold or more, 4-fold or more, 5-fold or more, 6-fold or more, 7-fold or more, 8-fold or more, 9-fold or more, or 10-fold or more, as determined by the same assay, relative to the median survival observed after treatment with patritumab-DXd. Median survival of subjects in the treatment group may be expressed in days from the start of the experiment.

[0228] In some embodiments, antigen-binding molecules of the present disclosure exhibit similar or reduced Fc-mediated binding to human PBMCs compared to the Fc-mediated binding to human PBMCs exhibited by patritumab-DXd. In some embodiments, antigen-binding molecules of the present disclosure bind to PBMCs, e.g., human PBMCs, in a given assay with a KD value similar to or higher than the KD value with which patritumab-DXd binds to the cells. In some embodiments, the KD for binding of the antigen-binding molecule to PBMCs is 0.5-fold or more and 2-fold or less than the KD for binding of patritumab-DXd to the same type of cells when determined in the same assay, e.g., 0.55-fold or more and 1.9-fold or less, 0.6-fold or more and 1.8-fold or less, 0.65-fold or more and 1.7-fold or less, 0.7-fold or more and 1.6-fold or less, 0.75-fold or more and 1.5-fold or less, 0.8-fold or more and 1.4-fold or less, 0.85-fold or more and 1.3-fold or less, 0.9-fold or more and 1.2-fold or less, or 0.95-fold or more and 1.1-fold or less. In some embodiments, the KD for binding of the antigen-binding molecule to PBMCs is more than 1-fold, e.g., 1.01-fold or more, 1.02-fold or more, 1.03-fold or more, 1.04-fold or more, 1.05-fold or more, 1.1-fold or more, 1.2-fold or more, 1.3-fold or more, 1.4-fold or more, 1.5-fold or more, 1.6-fold or more, 1.7-fold or more, 1.8-fold or more, 1.9-fold or more, 2-fold or more, 3-fold or more, 4-fold or more, 5-fold or more, 6-fold or more, 7-fold or more, 8-fold or more, 9-fold or more, or 10-fold or more, of the KD for binding of patritumab-DXd to PBMCs when determined in the same assay.In some embodiments, the antigen binding molecule binds to PBMCs by up to 95%, up to 94%, up to 93%, up to 92%, up to 91%, 90%, up to 89%, up to 88%, up to 87%, up to 86%, up to 85%, up to 84%, up to 83%, up to 82%, up to 81%, up to 80%, up to 79%, up to 78%, up to 77%, up to 76%, up to 75%, up to 74%, up to 73%, up to 72%, up to 71%, up to 70%, up to 69%, up to 68%, up to 67%, up to 66%, up to 65%, up to 64%, up to 63%, up to 62%, up to 61%, up to 60%, up to 59%, up to 58%, up to 57%, up to 56%, up to 58% or up to 59%, compared to binding of patritumab-DXd to the same type of cells as determined in the same assay. 5%, max 54%, max 53%, max 52%, max 51%, max 50%, max 49%, max 48%, max 47%, max 46%, max 45%, max 44%, max 43%, max 42%, max 41%, max 40%, max 39%, max 38%, max 37%, max 36%, max 35%, max 34%, max 33%, max 32%, max 31%, max 30%, max Larger than 29%, up to 28%, up to 27%, up to 26%, up to 25%, up to 24%, up to 23%, up to 22%, up to 21%, up to 20%, up to 19%, up to 18%, up to 17%, up to 16%, up to 15%, up to 14%, up to 13%, up to 12%, up to 11%, up to 10%, up to 9%, up to 8%, up to 7%, up to 6%, or up to 5% less binding.

[0229] In some embodiments, antigen-binding molecules of the present disclosure exhibit similar or increased internalization into HER3-expressing cells compared to the internalization exhibited by patritumab-DXd. In some embodiments, the antigen-binding molecule is internalized into HER3-expressing cells to a level that is 0.5-fold to 2-fold, e.g., 0.55-fold to 1.9-fold, 0.6-fold to 1.8-fold, 0.65-fold to 1.7-fold, 0.7-fold to 1.6-fold, 0.75-fold to 1.5-fold, 0.8-fold to 1.4-fold, 0.85-fold to 1.3-fold, 0.9-fold to 1.2-fold, or 0.95-fold to 1.1-fold the level of internalization of patritumab-DXd into the cells, as determined in the same assay. In some embodiments, the antigen binding molecule is internalized into cells expressing HER3 to a level that is more than 1-fold, e.g., 1.01-fold or more, 1.02-fold or more, 1.03-fold or more, 1.04-fold or more, 1.05-fold or more, 1.1-fold or more, 1.2-fold or more, 1.3-fold or more, 1.4-fold or more, 1.5-fold or more, 1.6-fold or more, 1.7-fold or more, 1.8-fold or more, 1.9-fold or more, 2-fold or more, 3-fold or more, 4-fold or more, 5-fold or more, 6-fold or more, 7-fold or more, 8-fold or more, 9-fold or more, or 10-fold or more, the level of internalization of patritumab-DXd into the cells as determined in the same assay.

[0230] In some embodiments, antigen binding molecules of the present disclosure exhibit similar or decreased macropinocytosis-dependent uptake by megakaryocytes compared to macropinocytosis-dependent uptake by patritumab-DXd. In some embodiments, the antigen binding molecule exhibits up to 95%, up to 94%, up to 93%, up to 92%, up to 91%, 90%, up to 89%, up to 88%, up to 87%, up to 86%, up to 85%, up to 84%, up to 83%, up to 82%, up to 81%, up to 80%, up to 79%, up to 78%, up to 77%, up to 76%, up to 75%, up to 74%, up to 73%, up to 72%, up to 71%, up to 70%, up to 69%, up to 68%, up to 67%, up to 66%, up to 65%, up to 64%, up to 63%, up to 62%, up to 61%, up to 60%, up to 59%, up to 58%, up to 57%, up to 56%, up to 55%, up to 54%, compared to macropinocytosis-dependent uptake of patritumab-DXd by megakaryocytes as determined in the same assay. %, max 53%, max 52%, max 51%, max 50%, max 49%, max 48%, max 47%, max 46%, max 45%, max 44%, max 43%, max 42%, max 41%, max Large 40%, Max 39%, Max 38%, Max 37%, Max 36%, Max 35%, Max 34%, Max 33%, Max 32%, Max 31%, Max 30%, Max 29%, Max 28%, Max 27 %, up to 26%, up to 25%, up to 24%, up to 23%, up to 22%, up to 21%, up to 20%, up to 19%, up to 18%, up to 17%, up to 16%, up to 15%, up to 14%, up to 13%, up to 12%, up to 11%, up to 10%, up to 9%, up to 8%, up to 7%, up to 6%, or up to 5% less indicating macropinocytosis-dependent uptake by megakaryocytes.

[0231] In some embodiments, antigen-binding molecules of the present disclosure exhibit similar or reduced toxicity to subjects administered the antigen-binding molecule compared to subjects administered patritumab-DXd, hi some embodiments, the antigen-binding molecule has a similar or improved toxicological profile compared to patritumab-DXd.

[0232] In some embodiments, the number / proportion of white blood cells / neutrophils / platelets / red blood cells in the peripheral blood of a subject after administration of an antigen-binding molecule according to the present disclosure is 0.5-fold and 2-fold, e.g., one of 0.55-fold and 1.9-fold, 0.6-fold and 1.8-fold, 0.65-fold and 1.7-fold, 0.7-fold and 1.6-fold, 0.75-fold and 1.5-fold, 0.8-fold and 1.4-fold, 0.85-fold and 1.3-fold, 0.9-fold and 1.2-fold, or 0.95-fold and 1.1-fold the number / proportion of such cells observed after treatment with patritumab-DXd, as determined by the same assay. In some embodiments, the number / proportion of white blood cells / neutrophils / platelets / red blood cells in the peripheral blood of a subject after administration of an antigen binding molecule according to the disclosure is more than 1-fold, e.g., one of 1.01-fold or more, 1.02-fold or more, 1.03-fold or more, 1.04-fold or more, 1.05-fold or more, 1.1-fold or more, 1.2-fold or more, 1.3-fold or more, 1.4-fold or more, 1.5-fold or more, 1.6-fold or more, 1.7-fold or more, 1.8-fold or more, 1.9-fold or more, 2-fold or more, 3-fold or more, 4-fold or more, 5-fold or more, 6-fold or more, 7-fold or more, 8-fold or more, 9-fold or more, or 10-fold or more, the number / proportion of such cells observed after treatment with patritumab-DXd, as determined by the same assay.

[0233] In some embodiments, the level of a correlate of interstitial lung disease after administration of an antigen binding molecule according to the present disclosure is greater than or equal to 0.5-fold and less than or equal to 2-fold, e.g., greater than or equal to 0.55-fold and less than or equal to 1.9-fold, greater than or equal to 0.6-fold and less than or equal to 1.8-fold, greater than or equal to 0.65-fold and less than or equal to 1.7-fold, greater than or equal to 0.7-fold and less than or equal to 1.6-fold, greater than or equal to 0.75-fold and less than or equal to 1.5-fold, greater than or equal to 0.8-fold and less than or equal to 1.4-fold, greater than or equal to 0.85-fold and less than or equal to 1.3-fold, greater than or equal to 0.9-fold and less than or equal to 1.2-fold, or greater than or equal to 0.95-fold and less than or equal to 1.1-fold the level of the correlate in a subject administered patritumab-DXd, as determined by the same assay. In some embodiments, the level of a correlate of interstitial lung disease after administration of an antigen binding molecule according to the disclosure is less than 1-fold the level of the correlate in a subject administered patritumab-DXd, as determined by the same assay, e.g., 0.99-fold or less, 0.95-fold or less, 0.9-fold or less, 0.85-fold or less, 0.8-fold or less, 0.75-fold or less, 0.7-fold or less, 0.65-fold or less, 0.6-fold or less, 0.55-fold or less, 0.5-fold or less, 0.45-fold or less, 0.4-fold or less, 0.35-fold or less, 0.3-fold or less, 0.25-fold or less, 0.2-fold or less, 0.15-fold or less, 0.1-fold or less, 0.05-fold or less, or 0.01-fold or less.

[0234] In some embodiments, the level of a correlate of impaired liver function (e.g., AST and / or ALT) in the peripheral blood of a subject after administration of an antigen binding molecule according to the disclosure is greater than or equal to 0.5-fold and less than or equal to 2-fold, e.g., one of greater than or equal to 0.55-fold and less than or equal to 1.9-fold, greater than or equal to 0.6-fold and less than or equal to 1.8-fold, greater than or equal to 0.65-fold and less than or equal to 1.7-fold, greater than or equal to 0.7-fold and less than or equal to 1.6-fold, greater than or equal to 0.75-fold and less than or equal to 1.5-fold, greater than or equal to 0.8-fold and less than or equal to 1.4-fold, greater than or equal to 0.85-fold and less than or equal to 1.3-fold, greater than or equal to 0.9-fold and less than or equal to 1.2-fold, or greater than or equal to 0.95-fold and less than or equal to 1.1-fold, the level observed in the peripheral blood of the subject after treatment with patritumab-DXd, as determined by the same assay. In some embodiments, the level of a correlate of impaired liver function (e.g., AST and / or ALT) in the peripheral blood of a subject after administration of an antigen binding molecule according to the disclosure is less than 1-fold, e.g., 0.99-fold or less, 0.95-fold or less, 0.9-fold or less, 0.85-fold or less, 0.8-fold or less, 0.75-fold or less, 0.7-fold or less, 0.65-fold or less, 0.6-fold or less, 0.55-fold or less, 0.5-fold or less, 0.45-fold or less, 0.4-fold or less, 0.35-fold or less, 0.3-fold or less, 0.25-fold or less, 0.2-fold or less, 0.15-fold or less, 0.1-fold or less, 0.05-fold or less, or 0.01-fold or less, the level observed in the peripheral blood of the subject after treatment with patritumab-DXd, as determined by the same assay.

[0235] In some embodiments, the level / concentration of a correlate of impaired renal function (e.g., BUN and / or creatinine) in the peripheral blood of a subject after administration of an antigen-binding molecule according to the disclosure is greater than or equal to 0.5-fold and less than or equal to 2-fold, e.g., one of 0.55-fold and less than or equal to 1.9-fold, 0.6-fold and less than or equal to 1.8-fold, 0.65-fold and less than or equal to 1.7-fold, 0.7-fold and less than or equal to 1.6-fold, 0.75-fold and less than or equal to 1.5-fold, 0.8-fold and less than or equal to 1.4-fold, 0.85-fold and less than or equal to 1.3-fold, 0.9-fold and less than or equal to 1.2-fold, or 0.95-fold and less than or equal to 1.1-fold the level / concentration observed in the peripheral blood of the subject after treatment with patritumab-DXd, when determined by the same assay. In some embodiments, the level / concentration of a correlate of impaired renal function (e.g., BUN and / or creatinine) in the peripheral blood of a subject after administration of an antigen binding molecule according to the disclosure is less than 1-fold, e.g., 0.99-fold or less, 0.95-fold or less, 0.9-fold or less, 0.85-fold or less, 0.8-fold or less, 0.75-fold or less, 0.7-fold or less, 0.65-fold or less, 0.6-fold or less, 0.55-fold or less, 0.5-fold or less, 0.45-fold or less, 0.4-fold or less, 0.35-fold or less, 0.3-fold or less, 0.25-fold or less, 0.2-fold or less, 0.15-fold or less, 0.1-fold or less, 0.05-fold or less, or 0.01-fold or less, of the level / concentration observed in the peripheral blood of the subject after treatment with patritumab-DXd, when determined by the same assay.

[0236] In some embodiments, the level of a correlate of pulmonary function in a subject after administration of an antigen-binding molecule according to the disclosure is greater than or equal to 0.5-fold and less than or equal to 2-fold, e.g., greater than or equal to 0.55-fold and less than or equal to 1.9-fold, greater than or equal to 0.6-fold and less than or equal to 1.8-fold, greater than or equal to 0.65-fold and less than or equal to 1.7-fold, greater than or equal to 0.7-fold and less than or equal to 1.6-fold, greater than or equal to 0.75-fold and less than or equal to 1.5-fold, greater than or equal to 0.8-fold and less than or equal to 1.4-fold, greater than or equal to 0.85-fold and less than or equal to 1.3-fold, greater than or equal to 0.9-fold and less than or equal to 1.2-fold, or greater than or equal to 0.95-fold and less than or equal to 1.1-fold the level of the correlate in a subject administered patritumab-DXd, as determined by the same assay. In some embodiments, the level of a correlate of lung function in a subject after administration of an antigen binding molecule according to the disclosure is more than 1-fold, e.g., one of 1.01-fold or more, 1.02-fold or more, 1.03-fold or more, 1.04-fold or more, 1.05-fold or more, 1.1-fold or more, 1.2-fold or more, 1.3-fold or more, 1.4-fold or more, 1.5-fold or more, 1.6-fold or more, 1.7-fold or more, 1.8-fold or more, 1.9-fold or more, 2-fold or more, 3-fold or more, 4-fold or more, 5-fold or more, 6-fold or more, 7-fold or more, 8-fold or more, 9-fold or more, or 10-fold or more, the level of the correlate in a subject administered patritumab-DXd, when determined by the same assay.

[0237] Generation of antigen-binding molecules Antigen-binding molecules according to the present disclosure can be prepared according to methods for the production of antibody-drug conjugates known to those skilled in the art.

[0238] Antigen-binding portions according to the present disclosure can be prepared by chemical synthesis, e.g., liquid phase synthesis or solid phase synthesis. For example, peptides / polypeptides can be synthesized using the methods described in, for example, Chandrudu et al., Molecules (2013), 18:4373-4388 (incorporated herein by reference in its entirety).

[0239] Alternatively, antigen-binding portions according to the present disclosure can be produced by recombinant expression. Molecular biology techniques suitable for the recombinant production of polypeptides are described in Green and Sambrook, Molecular Cloning: A Laboratory Manual (4 thEdition), Cold Spring Harbor Press, 2012 and Nat Methods. (2008); 5(2): 135-146, both of which are incorporated herein by reference in their entireties. Methods for the recombinant production of antigen-binding polypeptides are described in Frenzel et al., Front Immunol. (2013); 4: 217 and Kunert and Reinhart, Appl Microbiol Biotechnol. (2016) 100: 3451-3461, both of which are incorporated herein by reference in their entireties.

[0240] In some cases, the antigen-binding portion of the present disclosure is composed of multiple polypeptide chains. In such cases, production of the antigen-binding portion may involve transcription and translation of multiple polypeptides and subsequent assembly of the polypeptide chains to form the antigen-binding portion.

[0241] Any cell suitable for expressing a polypeptide may be used in recombinant production according to the present disclosure. The cell may be a prokaryotic or eukaryotic cell. In some embodiments, the cell is a prokaryotic cell, such as an archaeal or bacterial cell. In some embodiments, the bacterium may be a gram-negative bacterium, such as a bacterium of the Enterobacteriaceae family, e.g., Escherichia coli. In some embodiments, the cell is a eukaryotic cell, such as a yeast cell, a plant cell, an insect cell, or a mammalian cell, e.g., a cell described herein. In some cases, the cell is not a prokaryotic cell, as some prokaryotic cells do not allow the same folding or post-translational modifications as eukaryotic cells. Furthermore, very high expression levels are possible in eukaryotic organisms, and proteins may be easier to purify from eukaryotic organisms using appropriate tags. Specific plasmids that enhance protein secretion into the culture medium may also be utilized.

[0242] In some embodiments, polypeptides can be prepared by cell-free protein synthesis (CFPS), for example, according to the system described in Zemella et al. Chembiochem (2015) 16(17):2420-2431, which is incorporated herein by reference in its entirety.

[0243] Production of antigen-binding moieties may involve the culture or fermentation of eukaryotic cells modified to express the polypeptide(s) of interest. The culture or fermentation may be carried out in a bioreactor that is appropriately supplied with nutrients, air / oxygen, and / or growth factors. The secreted proteins may be collected by partitioning the medium / fermentation broth from the cells, extracting the protein content, and separating the individual proteins to isolate the secreted polypeptide(s). Culture, fermentation, and separation techniques are well known to those skilled in the art and are described, for example, in Green and Sambrook, Molecular Cloning: A Laboratory Manual (4 th Edition; incorporated herein by reference above). A bioreactor includes one or more vessels in which cells can be cultured. Culturing in a bioreactor can be continuous, with a continuous inflow of reactants into the reactor and a continuous outflow of cultured cells from the reactor. Alternatively, culturing can be performed in batches. Bioreactors monitor and control environmental conditions such as pH, oxygen, inflow and outflow into and out of the vessel, and agitation within the vessel to provide optimal conditions for the cells being cultured.

[0244] After culturing cells expressing a polypeptide(s), the polypeptide(s) of interest can be isolated. Any suitable method known in the art for separating proteins from cells can be used. To isolate the polypeptide, it may be necessary to separate the cells from the nutrient medium. If the polypeptide(s) are secreted from the cells, the cells can be separated from the culture medium containing the secreted polypeptide(s) of interest by centrifugation. If the polypeptide(s) of interest are assembled intracellularly, protein isolation can include centrifugation to separate the cells from the cell culture medium, treatment of the cell pellet with a lysis buffer, and cell disruption, for example, by sonication, rapid freeze-thawing, or osmotic lysis.

[0245] It may then be desirable to isolate the polypeptide(s) of interest from the supernatant or medium, which may contain other proteins and non-protein components. A common approach to separating protein components from the supernatant or medium is by precipitation. Proteins of different solubilities are precipitated with different concentrations of a precipitant (such as ammonium sulfate). For example, low concentrations of the precipitant extract water-soluble proteins. Thus, by adding different, increasing concentrations of the precipitant, proteins of different solubilities can be differentiated. Dialysis can then be used to remove the ammonium sulfate from the separated proteins. Other methods for differentiating different proteins, such as ion exchange chromatography and size chromatography, are known in the art. These can be used instead of precipitation or can follow precipitation.

[0246] After isolating the polypeptide(s) of interest from the culture, it may be desirable or necessary to concentrate the polypeptide(s). Several methods for concentrating proteins are known in the art, such as ultrafiltration or lyophilization.

[0247] The antigen-binding moieties according to the present disclosure can be conjugated to the linker-payload moieties according to the present disclosure by any suitable technique well known and routinely performed by those skilled in the art for producing antigen-binding molecules according to the present disclosure. Such methods are described, for example, in Chudasama et al., Nature Chemistry, (2016), 8:114-119, Baah et al., Molecules. (2021) 26(10):2943, and Walsh et al., Chem. Soc. Rev. (2021) 50:1305-1353, all of which are incorporated herein by reference in their entireties.

[0248] Conjugation of the antigen-binding moiety and the linker-payload moiety, as well as purification of the antigen-binding molecule produced by such conjugation, can be carried out as described, for example, in Beck et al., (2017) Nat Rev Drug Discov 16:315-337; Peters and Brown Biosci Rep (2015) 35:art:e00225; McCombs and Owen, The AAPS Journal (2015) 17:339-351; Jackson, Org Process Res Dev (2016) 20:852-866; or Olivier and Hurvitz, Antibody-Drug Conjugates: Fundamentals, Drug Development, and Clinical, (2016) Wiley. In a preferred embodiment, the antigen-binding molecule according to the present disclosure can be produced using conjugation employing metal-free click chemistry.

[0249] In some embodiments, the linker payload moiety is conjugated to a polypeptide of the antigen-binding moiety, hi some embodiments, the linker payload moiety is conjugated to multiple polypeptides (e.g., two polypeptides) of the antigen-binding moiety.

[0250] In some embodiments, a linker payload moiety is conjugated to the CH2 region of an antigen-binding moiety according to the present disclosure. In some embodiments, a linker payload moiety is conjugated to each CH2 region of the antigen-binding moiety. In some embodiments, a linker payload moiety is conjugated to a CH2-CH3 region of the antigen-binding moiety. In some embodiments, a linker payload moiety is conjugated to each CH2-CH3 region of the antigen-binding moiety. In some embodiments, a linker payload moiety is conjugated to the Fc region of the antigen-binding moiety. In some embodiments, a linker payload moiety is conjugated to each CH2-CH3 region of the Fc region of the antigen-binding moiety.

[0251] In some embodiments, the linker payload moiety is conjugated to the antigen binding moiety via a linkage at N297 (EU numbering) in the Fc region / CH2-CH3 region / CH2 region. In some embodiments, the linker payload moiety is conjugated to the N-glycan via a linkage at N297 (EU numbering) in the Fc region / CH2-CH3 region / CH2 region.

[0252] In some embodiments, conjugation is achieved by reaction of an azide group on the antigen-binding moiety with a bicyclo[6.1.0]nonyne (BCN) moiety on the linker payload moiety.

[0253] In some embodiments, the antigen-binding portion comprises a carbohydrate moiety comprising an azide group. In some embodiments, the antigen-binding portion comprises a glycan (e.g., an N-glycan) comprising an azide group. In some embodiments, the antigen-binding portion comprises a glycan (e.g., an N-glycan) comprising a 6-azido-6-deoxy-N-acetylgalactosamine (i.e., 6-azido-GalNAc) moiety. In some embodiments, the antigen-binding portion comprises a CH2 / CH2-CH3 / Fc region comprising an N-glycan comprising a 6-azido-GalNAc residue. In some embodiments, the antigen-binding portion comprises a CH2 / CH2-CH3 / Fc region comprising an N-glycan at N297 (EU numbering) comprising a 6-azido-GalNAc residue.

[0254] In some embodiments, antigen-binding molecules according to the present disclosure are produced by methods employing GlycoConnect technology, e.g., as described in van Geel et al., Bioconjug Chem. (2015) 26(11): 2233-2242 and WO2021 / 015622A1, both of which are incorporated herein by reference in their entireties. In some embodiments, antigen-binding molecules according to the present disclosure are produced essentially as described in van Geel et al., Bioconjug Chem. (2015) 26(11): 2233-2242.

[0255] In a preferred embodiment, antigen-binding molecules according to the present disclosure can be produced as described in WO2022 / 058395A1, which is incorporated herein by reference in its entirety. In particular, antigen-binding molecules according to the present disclosure can be produced as described in paragraphs

[0137] to

[0174] of WO2022 / 058395A1, which are specifically incorporated by reference. When following such methods described in WO2022 / 058395A1, it will be understood that the "AB" referred to in paragraphs

[0137] to

[0174] of WO2022 / 058395A1 refers to antigen-binding molecules as described herein.

[0256] In some embodiments, the methods include contacting an antigen-binding moiety that includes an azide moiety with a compound having the structure (F). [ka]

[0257] In some embodiments, the method includes: (i) contacting an antigen-binding portion according to the present disclosure comprising a CH2 region / CH2-CH3 region / Fc region with an endoglycosidase, a galactosyltransferase, and a 6-azido-6-deoxy-N-acetylgalactosamine moiety donor; (ii) contacting the product of step (i) with a compound having structure (F).

[0258] In some embodiments, the endoglycosidase is an endoglycosidase described in WO2017 / 137459A1 (herein incorporated by reference in its entirety), for example, EndoSH, EndoS2, or EndoS as described in WO2017 / 137459A1.

[0259] In some embodiments, the galactosyltransferase is a galactosyltransferase described in Boeggeman et al., Protein Expression and Purification (2003), 30(2):219-229 (incorporated herein by reference in its entirety), e.g., a β1,4-galactosyltransferase (EC 2.4.1.38) or a β1,4-galactosyltransferase comprising the amino acid substitution Y289L.

[0260] It is understood that a "6-azido-6-deoxy-N-acetylgalactosamine moiety donor" refers to a compound that can function as a donor of a 6-azido-6-deoxy-N-acetylgalactosamine moiety. In the context of the present disclosure, the 6-azido-6-deoxy-N-acetylgalactosamine moiety donor serves as a donor for generating an antigen-binding molecule containing a 6-azido-6-deoxy-N-acetylgalactosamine moiety for subsequent conjugation of a linker-payload moiety (e.g., via reaction with a linker-payload moiety containing a BCN moiety). In some embodiments, the 6-azido-6-deoxy-N-acetylgalactosamine moiety donor is 6-azido-6-deoxy-UDP-N-acetylgalactosamine (i.e., UDP-6-azido-GalNAc) (see, e.g., Mayer et al., Bioorg Med Chem Lett (2011) 21(4):1199-201).

[0261] In some embodiments, the method includes purifying / isolating the antigen-binding molecule (i.e., from unreacted precursors and / or by-products). In some embodiments, the antigen-binding molecule can be purified / isolated by chromatography, for example, size exclusion chromatography.

[0262] In some embodiments, an antigen-binding molecule according to the present disclosure is produced essentially as described herein in Example 2. In some embodiments, conjugation of an antigen-binding moiety according to the present disclosure to a linker payload moiety according to the present disclosure is performed essentially as described herein in Example 2.3.

[0263] composition The present disclosure provides a composition comprising an antigen-binding molecule according to the present disclosure.

[0264] The antigen-binding molecules described herein can be formulated as pharmaceutical compositions or medicaments for clinical use, and may contain pharmaceutically acceptable carriers, diluents, excipients, or adjuvants. Thus, the present disclosure provides pharmaceutical compositions / medicaments comprising the antigen-binding molecules described herein.

[0265] The pharmaceutical compositions / medicaments of the present disclosure may contain one or more pharmaceutically acceptable carriers (e.g., liposomes, micelles, microspheres, nanoparticles), diluents / excipients (e.g., starch, cellulose, cellulose derivatives, polyols, dextrose, maltodextrin, magnesium stearate), adjuvants, fillers, buffering agents, preservatives (e.g., vitamin A, vitamin E, vitamin C, retinyl palmitate, selenium, cysteine, methionine, citric acid, sodium citrate, methylparaben, propylparaben), antioxidants, or the like. The composition may contain antiperspirants (e.g., vitamin A, vitamin E, vitamin C, retinyl palmitate, selenium), lubricants (e.g., magnesium stearate, talc, silica, stearic acid, vegetable stearin), binders (e.g., sucrose, lactose, starch, cellulose, gelatin, polyethylene glycol (PEG), polyvinylpyrrolidone) (PVP), xylitol, sorbitol, mannitol), stabilizers, solubilizers, surfactants (e.g., wetting agents), masking agents, or colorants (e.g., titanium dioxide).

[0266] The term "pharmaceutically acceptable," as used herein, refers to compounds, ingredients, materials, compositions, dosage forms, etc., that are suitable for use in contact with the tissues of a subject (e.g., a human subject) without undue toxicity, irritation, allergic response, or other problem or complication, within the scope of sound medical judgment and commensurate with a reasonable benefit / risk ratio. Each carrier, diluent, excipient, adjuvant, filler, buffer, preservative, antioxidant, lubricant, binder, stabilizer, solubilizer, surfactant, masking agent, colorant, flavoring, or sweetener of a composition according to the present disclosure must also be "acceptable" in the sense of being compatible with the other ingredients of the formulation. Suitable carriers, diluents, excipients, adjuvants, fillers, buffers, preservatives, antioxidants, lubricants, binders, stabilizers, solubilizers, surfactants, masking agents, colorants, flavorings, or sweetening agents can be found, for example, in standard pharmaceutical texts, e.g., Remington's 'The Science and Practice of Pharmacy' (Ed. A. Adejare), 23 rd Edition(2020), Academic Press.

[0267] The pharmaceutical compositions and medicaments of the present disclosure may be formulated for topical, parenteral, systemic, intracavitary, intravenous, intraarterial, intramuscular, intrathecal, intraocular, intraconjunctival, intratumoral, subcutaneous, intradermal, intrathecal, oral, or transdermal routes of administration. In some embodiments, the pharmaceutical compositions / medicaments may be formulated for administration by injection or infusion, or by oral ingestion.

[0268] Suitable formulations may include antigen-binding molecules provided in a sterile or isotonic medium. Drugs and pharmaceutical compositions may be formulated in fluid form, including gels. Fluid formulations may be formulated for administration by injection or infusion (e.g., via a catheter) to a selected region of the human or animal body.

[0269] In some embodiments, the pharmaceutical composition / medicament is formulated for injection or infusion, for example, into a blood vessel, a tissue / organ of interest, or a tumor.

[0270] The present disclosure also provides methods for the production of pharmaceutically useful compositions, which may include one or more steps selected from the following: Producing an antigen-binding molecule described herein; Isolating / purifying the antigen-binding molecules described herein, and / or Mixing the antigen-binding molecules described herein with a pharmaceutically acceptable carrier, adjuvant, excipient or diluent.

[0271] For example, a further aspect of the present disclosure relates to a method of formulating or producing a medicament or pharmaceutical composition for use in treating a disease / condition (e.g., a disease / condition described herein), the method comprising formulating the pharmaceutical composition or medicament by mixing an antigen-binding molecule described herein with a pharmaceutically acceptable carrier, adjuvant, excipient, or diluent.

[0272] Therapeutic and prophylactic applications The antigen binding molecules and compositions described herein are used for the therapeutic and prophylactic intervention of diseases, such as cancer.

[0273] The antigen-binding molecules and compositions of the present disclosure can be used for the treatment / prevention of any disease / condition that would receive therapeutic or prophylactic benefit from a reduction in the level / activity of HER3, or a reduction in the number or activity of cells containing / expressing HER3.

[0274] For example, the disease / condition is one in which HER3 or cells expressing / overexpressing HER3 are pathologically implicated, e.g., a disease / condition in which an increase in the level / activity of HER3 or an increase in the number / proportion of cells containing / expressing HER3 is positively correlated with the onset, development, or progression of the disease / condition and / or the severity of one or more symptoms of the disease / condition. In some embodiments, an increase in the level / activity of HER3 or an increase in the number / proportion of cells containing / expressing HER3 can be a risk factor for the onset, development, or progression of the disease / condition.

[0275] The present disclosure provides an antigen-binding molecule or composition described herein for use in a method of medical treatment or prevention. Also provided are an antigen-binding molecule or composition described herein for use in a method of treating or preventing cancer (e.g., a cancer described herein). Also provided is the use of an antigen-binding molecule or composition described herein in the manufacture of a medicament for treating or preventing cancer (e.g., a cancer described herein). Also provided is a method of treating or preventing cancer (e.g., a cancer described herein) in a subject, comprising administering to the subject a therapeutically or prophylactically effective amount of an antigen-binding molecule or composition described herein.

[0276] The method may be effective in reducing the development or progression of cancer, alleviating cancer symptoms, or reducing cancer pathology. The method may be effective for preventing cancer progression, e.g., preventing cancer from worsening or slowing the rate of cancer progression. In some embodiments, the method may result in an improvement of cancer, e.g., a reduction in cancer symptoms, or a reduction in some other correlate of cancer severity / activity. In some embodiments, the method may prevent cancer from progressing to a later stage (e.g., chronic or metastatic).

[0277] As used herein, "cancer" can be or include any unwanted cell proliferation (or any disease manifested by unwanted cell proliferation), neoplasm, or tumor. Cancer can be benign or malignant. Cancer can be primary or secondary (metastatic). A neoplasm or tumor can be any abnormal growth or proliferation of cells and can be located in any tissue. The cancer can be of tissue / cells derived from, for example, the adrenal gland, adrenal medulla, anus, appendix, bladder, blood, bone, bone marrow, brain, breast, cecum, central nervous system (including or excluding the brain), cerebellum, cervix, colon, duodenum, endometrium, epithelial cells (e.g., renal epithelium), gallbladder, biliary tract, esophagus, glial cells, heart, ileum, jejunum, kidney, lacrimal gland, larynx, liver, lung, lymph, lymph node, lymphoblast, maxilla, mediastinum, mesentery, uterine muscle, nasopharynx, omentum, oral cavity, ovary, pancreas, parotid gland, peripheral nervous system, peritoneum, pleura, prostate, salivary gland, sigmoid colon, skin, small intestine, soft tissue, spleen, stomach, testis, thymus, thyroid, tongue, tonsils, trachea, uterus, vulva, white blood cells.

[0278] The tumors to be treated may be nervous system tumors or non-nervous system tumors. Nervous system tumors may occur in either the central or peripheral nervous system, such as glioma, medulloblastoma, meningioma, neurofibroma, ependymoma, schwannoma, neurofibrosarcoma, astrocytoma, and oligodendroglioma. Non-nervous system cancers / tumors may occur in any other non-nervous tissue. Examples include melanoma, mesothelioma, lymphoma, myeloma, leukemia, non-Hodgkin's lymphoma (NHL), Hodgkin's lymphoma, chronic myeloid leukemia (CML), acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), cutaneous T-cell lymphoma (CTCL), chronic lymphocytic leukemia (CLL), hepatocellular carcinoma, epidermoid carcinoma, prostate cancer, breast cancer, lung cancer, colon cancer, ovarian cancer, pancreatic cancer, thymic cancer, NSCLC, blood cancer, and sarcoma.

[0279] In some embodiments, the cancer being treated / prevented comprises cells expressing an EGFR family member (e.g., HER3, EGFR, HER2, or HER4) and / or cells expressing a ligand for an EGFR family member. In some embodiments, the cancer being treated / prevented comprises cells expressing a mutant or wild-type form of an EGFR family member (e.g., HER3, EGFR, HER2, or HER4). In some embodiments, the cancer being treated / prevented is a cancer that is positive for an EGFR family member. In some embodiments, the cancer comprises cells that overexpress an EGFR family member and / or a ligand for an EGFR family member. Overexpression can be determined by detecting expression levels that are higher than expression levels by comparable non-cancer cells / non-tumor tissues.

[0280] Expression can be determined by any suitable means.Expression can be gene expression or protein expression.Gene expression can be determined, for example, by detecting the mRNA encoding HER3, for example, by quantitative real-time PCR (qRT-PCR).Protein expression can be determined, for example, by antibody-based methods, for example, by Western blot, immunohistochemistry, immunocytochemistry, flow cytometry or ELISA.

[0281] In some embodiments, the cancer is a cancer in which HER3 is pathologically implicated. That is, in some embodiments, the cancer is a cancer caused or exacerbated by HER3 expression, a cancer in which HER3 expression is a risk factor, and / or a cancer in which HER3 expression is positively correlated with cancer onset, development, progression, severity, or metastasis. The cancer may be characterized by HER3 expression; for example, the cancer may contain cells (e.g., cells in tumor tissue) that express HER3. Such cancers may be referred to as HER3-positive. A cancer that is "positive" for HER3 may be a cancer that contains cells that express HER3 (e.g., on the cell surface). A cancer that is "positive" for HER3 may overexpress HER3.

[0282] In some embodiments, the cancer being treated / prevented comprises cells harboring a genetic variant (e.g., a mutation) that causes increased HER3 (gene and / or protein) expression and / or activity compared to comparable cells harboring a reference allele that does not contain the genetic variant (e.g., a non-mutated or "wild-type" allele). The genetic variant may be or comprise an insertion, deletion, substitution, or larger rearrangement / rearrangement in the nucleotide sequence compared to the reference allele.

[0283] Mutations that "result in" increased HER3 expression may be known, predicted, or associated with increased HER3 gene / protein expression. Mutations that "result in" increased HER3 activity may be known, predicted, or associated with increased HER3-mediated signaling and / or EGFR-mediated signaling. Mutations that result in increased HER3 expression and / or activity may be referred to as "activating" mutations.

[0284] Mutations that cause increased expression of HER3 may result in gene or protein expression of HER3 that is not expressed by comparable cells that do not carry the mutation and / or that is not encoded by their genomic nucleic acid. That is, HER3 may be a neoantigen that arises as a result of the mutation, and therefore "increased expression" may be due to absent expression.

[0285] A mutation that causes increased expression of HER3 can result in increased expression of the HER3 gene or protein expressed by and / or encoded by its genomic nucleic acid by a comparable cell that does not contain the mutation. By way of example, a cell can contain a mutation that results in an increased level of transcription of a nucleic acid encoding HER3 compared to the level of transcription of the nucleic acid encoding HER3 by a comparable cell that does not contain the mutation.

[0286] In some embodiments, a mutation that causes increased expression of HER3 can cause increased gene expression of HER3 compared to a comparable cell that does not contain the mutation. In some embodiments, a mutation that causes increased expression of HER3 can cause increased protein expression of HER3 compared to a comparable cell that does not contain the mutation.

[0287] In some embodiments, a mutation that causes increased expression of HER3 may cause increased levels of HER3 on or at the cell surface of cells that contain the mutation compared to comparable cells that do not contain the mutation.

[0288] Cells that have increased expression of HER3 (e.g., as a result of a mutation) compared to the expression level of HER3 by a reference cell can be described as "overexpressing" HER3 or "having upregulated expression" of HER3. For example, a cancer containing cells that harbor a mutation that results in increased expression of HER3 compared to comparable cells that lack the mutation can be described as a cancer containing cells that exhibit overexpressed / upregulated expression of HER3. In some embodiments, the reference cells that lack the mutation can be non-cancerous cells (e.g., of a comparable cell type) or cancerous cells (e.g., of a comparable cancer type).

[0289] Mutations that cause increased activity of HER3 can result in increased HER3-mediated signaling compared to the level of HER3-mediated signaling by comparable cells that do not contain the mutation.

[0290] In some embodiments, cancers treated / prevented in accordance with the present disclosure may be characterized, for example, by increased HER3 expression and / or activity (i.e., gene and / or protein expression) in organs / tissues / subjects afflicted with the disease / condition compared to normal organs / tissues / subjects (i.e., in the absence of the disease / condition). In some embodiments, cancer cells and / or tumors treated / prevented may be characterized, for example, by increased HER3 expression and / or activity compared to the level of expression and / or activity observed in comparable non-cancer cells / non-tumor tissues.

[0291] HER3-overexpressing cancers can overexpress HER3 as a result of amplification of the HER3 gene.

[0292] In some embodiments, the cancer treated / prevented by the present disclosure is a HER3-amplified cancer.

[0293] HER3 amplification can be identified using techniques well known in the art, such as in situ hybridization. For example, HER3 amplification can be evaluated by fluorescent in situ hybridization, as described in Chung et al., J Gynecol Oncol. (2019) 30 (5): e75. HER3-amplified cancers can include a 12q13.2 to chromosome 12 centromere ratio of 2 or greater, as determined by in situ hybridization.

[0294] HER3 and its association with and role in cancer are described, for example, in Mishra, et al., Oncol Rev. (2018) 12(1):355, Karachaliou et al., BioDrugs. (2017) 31(1):63-73, and Zhang et al., Acta Biochimica et Biophysica Sinica (2016) 48(1):39-48, all of which are incorporated herein by reference in their entireties. Mishra, et al., Oncol Rev. (2018) 12(1):355 also describes interventions that target HER3 for the treatment of cancer, including monoclonal anti-HER3 antibody therapy.

[0295] In some embodiments, the cancer being treated / prevented comprises cells that express a ligand for HER3 (e.g., NRG1 and / or NRG2). In some embodiments, the cancer being treated / prevented comprises cells that express a higher expression level of NRG1 and / or NRG2 than the expression level by comparable non-cancerous cells / non-tumor tissues. The cancer may be described as comprising cells that overexpress NRG1 and / or NRG2.

[0296] The HER3-binding antigen binding molecules described herein bind to HER3 with extremely high affinity when HER3 is bound by NRG (i.e., when HER3 is provided in an "open" conformation) and when HER3 is not bound by NRG (i.e., when HER3 is provided in a "closed" conformation). Therefore, they are particularly useful for treating / preventing cancers characterized by expression / overexpression of a HER3 ligand, for example, cancers / tumors containing cells expressing / overexpressing a HER3 ligand.

[0297] In some embodiments, the cancer being treated / prevented comprises cells harboring a genetic variant (e.g., a mutation) that causes increased (gene and / or protein) expression of a ligand for HER3 compared to comparable cells harboring a reference allele that does not contain the genetic variant (e.g., a non-mutated or "wild-type" allele). The genetic variant may be or comprise an insertion, deletion, substitution, or larger rearrangement / rearrangement of the nucleotide sequence compared to the reference allele.

[0298] Mutations that "result in" increased expression of a HER3 ligand may be known or predicted to cause, or may be associated with, increased gene / protein expression of the HER3 ligand. Mutations that result in increased expression of a HER3 ligand may be referred to as "activating" mutations.

[0299] A mutation that causes increased expression of a HER3 ligand may result in gene or protein expression of a HER3 ligand that is not expressed by comparable cells that do not carry the mutation and / or that are not encoded by their genomic nucleic acid. That is, the HER3 ligand may be a neoantigen that results from the mutation, and thus "increased expression" may be due to absent expression. For example, cells containing a CD74-NRG1 gene fusion exhibit increased expression of the CD74-NRG1 fusion polypeptide encoded by the gene fusion compared to cells lacking the CD74-NRG1 gene fusion.

[0300] A mutation that causes increased expression of a ligand of HER3 can result in increased gene or protein expression of the ligand of HER3 expressed by and / or encoded by its genomic nucleic acid in a comparable cell that does not contain the mutation. By way of example, a cell can contain a mutation that results in an increased level of transcription of a nucleic acid encoding NRG1 compared to the level of transcription of the nucleic acid encoding NRG1 by a comparable cell that does not contain the mutation.

[0301] In some embodiments, a mutation that causes increased expression of a ligand for HER3 may cause increased gene expression of the ligand for HER3 compared to a comparable cell that does not contain the mutation. In some embodiments, a mutation that causes increased expression of a ligand for HER3 may cause increased protein expression of the ligand for HER3 compared to a comparable cell that does not contain the mutation.

[0302] In some embodiments, a mutation that causes increased expression of a HER3 ligand may cause increased levels of the HER3 ligand on or at the cell surface of cells containing the mutation compared to comparable cells that do not contain the mutation. In some embodiments, a mutation that causes increased expression of a HER3 ligand may cause increased levels of secretion of the HER3 ligand from cells containing the mutation compared to comparable cells that do not contain the mutation.

[0303] Cells that express increased levels of HER3 ligand (e.g., as a result of a mutation) compared to the expression level of HER3 ligand by a reference cell can be described as "overexpressing" the HER3 ligand or "having upregulated expression" of the HER3 ligand. For example, a cancer containing cells that harbor a mutation that results in increased expression of the HER3 ligand compared to comparable cells lacking the mutation can be described as a cancer containing cells that exhibit overexpression / upregulated expression of the HER3 ligand. In some embodiments, the reference cells lacking the mutation can be non-cancerous cells (e.g., of a comparable cell type) or cancerous cells (e.g., of a comparable cancer type).

[0304] As used herein, a "ligand of HER3" is generally intended to refer to a molecule that can bind to HER3 via the ligand-binding region of HER3 formed by domains I and III of HER3. In some embodiments, a ligand of HER3 binds to HER3 via interaction with domains I and / or III of HER3. Exemplary ligands of HER3 include neuregulins, such as NRG1 and NRG2, which bind to HER3 via interaction between their EGF-like domains and the ligand-binding region of HER3.

[0305] The HER3 ligand preferably binds to the HER3 receptor and / or a receptor complex containing HER3, and induces signal transduction therethrough. As is apparent from the present disclosure, the receptor complex containing HER3 may further include an interaction partner of HER3 described herein, e.g., HER3, HER2, EGFR, HER4, HGFR, IGF1R, and / or cMet.

[0306] In some embodiments, the HER3 ligand can bind to a HER3 receptor / receptor complex expressed by a cell other than a cell in which expression of a HER3 ligand is increased. For example, in some embodiments, the HER3 ligand can bind to a HER3-expressing cancer cell.

[0307] In some embodiments, the HER3 ligand is capable of binding to a HER3 receptor / receptor complex expressed by cells that have increased expression of a HER3 ligand.

[0308] In some embodiments, the cancer being treated / prevented comprises (i) cells that express HER3 and (ii) cells that express a ligand of HER3 (e.g., cells that have increased expression of a ligand of HER3, e.g., as a result of a mutation that results in increased expression of a ligand of HER3).

[0309] In some embodiments, the cancer being treated / prevented comprises cells that (i) express HER3 and (ii) also express a ligand for HER3 (e.g., have increased expression of a ligand for HER3, e.g., as a result of a mutation that results in increased expression of a ligand for HER3).

[0310] In some embodiments, the ligand of HER3 comprises or consists of the amino acid sequence of a HER3-binding region of a ligand of HER3, or an amino acid sequence derived from a HER3-binding region of a ligand of HER3. The amino acid sequence derived from a HER3-binding region of a ligand of HER3 may comprise at least 60% (e.g., 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) amino acid sequence identity to the amino acid sequence from which it is derived.

[0311] In some embodiments, the HER3 ligand comprises an EGF-like domain capable of binding to HER3, or a HER3-binding fragment thereof. In some embodiments, the HER3-binding EGF-like domain / fragment is or is derived from an EGF family member (e.g., heparin-binding EGF-like growth factor (HB-EGF), transforming growth factor-α (TGF-α), amphiregulin (AR), epiregulin (EPR), epigen, betacellulin (BTC), NRG1, NRG2, NRG3, or NRG4).

[0312] Exemplary HER3 ligands include neuregulins (NRGs). Neuregulins include NRG1 (including their alpha, alpha2b, and alpha3 isoforms), NRG2, NRG3, and NRG4. In some embodiments, NRGs are selected from NRG1, NRG2, NRG3, and NRG4. In some embodiments, NRGs are selected from NRG1 and NRG2.

[0313] The EGF-like domain of human NRG1 (through which it binds to HER3) is formed by positions 178-222 of UniProt:Q02297-1. The EGF-like domain of human NRG2 is formed by positions 341-382 of UniProt:O14511-1. The EGF-like domain of human NRG3 is formed by positions 286-329 of UniProt:B9EGV5-1. The EGF-like domain of human NRG4 is formed by positions 5-46 of UniProt:Q8WWG1-1. In some embodiments, the EGF-like domain / fragment comprises or consists of an amino acid sequence having at least 60% (e.g., 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) amino acid sequence identity to the EGF-like domain of an NRG (NRG1, NRG2, NRG3 or NRG4).

[0314] In some embodiments, the ligand for HER3 is not an EGFR family protein (e.g., HER3, HER2, EGFR, HER4, HGFR, IGF1R, cMet).

[0315] In some embodiments, the mutation that results in increased expression of a HER3 ligand is an NRG gene fusion. In some embodiments, the HER3 ligand is a product of an NRG gene fusion (i.e., a polypeptide encoded by an NRG gene fusion). In some embodiments, the cancer comprises cells that have an NRG gene fusion. As used herein, "NRG gene fusion" refers to a genetic variant that encodes a polypeptide that includes (i) the amino acid sequence of an NRG protein (e.g., NRG1, NRG2, NRG3, or NRG4; e.g., NRG1 or NRG2), and (ii) the amino acid sequence of a protein other than an NRG protein.

[0316] It will be understood that the NRG gene fusion preferably encodes a HER3 ligand as described herein. In some embodiments, the NRG gene fusion encodes a polypeptide comprising a HER3-binding region of the NRG protein. In some embodiments, the NRG gene fusion encodes an EGF-like domain of the NRG protein, or a polypeptide comprising an amino acid sequence capable of binding to HER3 and having at least 60% (e.g., 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) amino acid sequence identity to the EGF-like domain of the NRG protein.

[0317] In some embodiments, the NRG gene fusion encodes a fusion polypeptide that includes a transmembrane domain. In some embodiments, the NRG gene fusion encodes a fusion polypeptide that includes a transmembrane domain of a protein other than an NRG protein.

[0318] In some embodiments, the NRG gene fusion is an NRG1 gene fusion. In some embodiments, the NRG1 gene fusion encodes an EGF-like domain of NRG1 or a polypeptide that is capable of binding to HER3 and that includes an amino acid sequence having at least 60% (e.g., 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) amino acid sequence identity to the EGF-like domain of NRG1.

[0319] NRG1 gene fusions are described, for example, in WO2021 / 048274A1, WO2018 / 182422A1, WO2019 / 051155A1, Dhanasekaran et al., Nat Commun. (2014) 5:5893, Drilon et al., Cancer Discov. (2018) 8(6):686-695, Nagasaka et al., Journal of Thoracic Oncology (2019) 14(8):1354-1359, and Jonna et al., Clin Cancer Res. (2019) 25(16):4966-4972, all of which are incorporated by reference in their entireties. The diversity of NRG1 gene fusions may be due to the fact that NRG1 is located on chromosome 8, which is particularly susceptible to genomic translocation events (Adelaiede et al., Genes Chromosomes Cancer. (2003) 37(4):333-45).

[0320] In some embodiments, the NRG1 gene fusion is selected from the group consisting of CLU-NRG1, CD74-NRG1, DOC4-NRG1, SLC3A2-NRG1, RBPMS-NRG1, WRN-NRG1, SDC4-NRG1, RAB2IL1-NRG1, VAMP2-NRG1, KIF13B-NRG1, THAP7-NRG1, SMAD4-NRG1, MDK-NRG1, TNC-NRG1, DIP2B-NRG1, MRPL1 In some embodiments, the NRG1 gene fusion is selected from 3-NRG1, PARP8-NRG1, ROCK1-NRG1, DPYSL2-NRG1, ATP1B1-NRG1, CDH6-NRG1, APP-NRG1, AKAP13-NRG1, THBS1-NRG1, FOXA1-NRG1, PDE7A-NRG1, RAB3IL1-NRG1, CDK1-NRG1, BMPRIB-NRG1, TNFRSF10B-NRG1, and MCPH1-NRG1. In some embodiments, the NRG1 gene fusion is CLU-NRG1.

[0321] CD74-NRG1 gene fusions are described, for example, in Fernandez-Cuesta et al. Cancer Discov. (2014) 4:415-22 and Nakaoku et al., Clin Cancer Res (2014) 20:3087-93. DOC4-NRG1 gene fusions are described, for example, in Liu et al., Oncogene. (1999) 18(50):7110-4 and Wang et al., Oncogene. (1999) 18(41):5718-21. SLC3A2-NRG1 gene fusions are described, for example, in Nakaoku et al., Clin Cancer Res (2014) 20:3087-93, Shin et al., Oncotarget (2016) 7:69450-65, and Shin et al., Mol Cancer Ther. (2018) 17(9):2024-2033. RBPMS-NRG1, WRN-NRG1, RAB2IL1-NRG1, and SDC4-NRG1 gene fusions are described, for example, in Dhanasekaran et al., Nat Commun. (2014) 5:5893. VAMP2-NRG1 gene fusions are described, for example, in Jung et al., J Thorac Oncol. (2015) 10(7):1107-11, and Shim et al., J Thorac Oncol. (2015) 10(8):1156-62. KIF13B-NRG1 gene fusions are described, for example, in Xia et al., Int J Surg Pathol. (2017) 25(3):238-240. SMAD4-NRG1, AKAP13-NRG1, THBS1-NRG1, FOXA1-NRG1, PDE7A-NRG1, RAB3IL1-NRG1, and THAP7-NRG1 gene fusions are described, for example, in Drilon et al., Cancer Discov. (2018) 8(6):686-695.MDK-NRG1, TNC-NRG1, DIP2B-NRG1, MRPL13-NRG1, PARP8-NRG1, ROCK1-NRG1, and DPYSL2-NRG1 gene fusions are described, for example, in Jonna et al., Clin Cancer Res. (2019) 25(16):4966-4972. ATP1B1-NRG1 gene fusions are described, for example, in Drilon et al., Cancer Discov. (2018) 8(6):686-695 and Jones et al., Annals of Oncology (2017) 28:3092-3097. CLU-NRG1 gene fusions are described, for example, in Drilon et al., Cancer Discov. (2018) 8(6):686-695 and Nagasaka et al., Journal of Thoracic Oncology (2019) 14(8):1354-1359.

[0322] In some embodiments, the NRG gene fusion is an NRG2 gene fusion. In some embodiments, the NRG2 gene fusion encodes an EGF-like domain of NRG2 or a polypeptide that can bind to HER3 and that includes an amino acid sequence having at least 60% (e.g., 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) amino acid sequence identity to the EGF-like domain of NRG2.

[0323] NRG2 gene fusions include, for example, SLC12A2-NRG2, as described in WO2021 / 048274A1 and WO2015 / 093557A1, and ZNF208-NRG2, as described in Dupain et al., Mol Ther. (2019) 27(1):200-218.

[0324] The cancer cells having a mutation that results in increased expression of a ligand for HER3 (e.g., including cells having an NRG gene fusion, e.g., an NRG1 gene fusion or an NRG2 gene fusion) can be any cancer described herein. In some embodiments, such cancers can be of tissue / cells derived from the lung, breast, head and neck, kidney, ovary, pancreas, prostate, uterus, gallbladder, biliary tract, colon, rectum, bladder, soft tissue, or nasopharynx.

[0325] In some embodiments, the cancer comprising cells with a mutation that results in increased expression of a ligand for HER3 (e.g., comprising cells with an NRG gene fusion, e.g., an NRG1 gene fusion or an NRG2 gene fusion) is selected from lung cancer, non-small cell lung cancer, lung adenocarcinoma, invasive mucinous lung adenocarcinoma, lung squamous cell carcinoma, breast cancer, breast carcinoma, invasive breast cancer, head and neck cancer, head and neck squamous cell carcinoma, renal carcinoma, renal clear cell carcinoma, ovarian cancer, ovarian serous cystadenocarcinoma, pancreatic cancer, pancreatic adenocarcinoma, pancreatic ductal adenocarcinoma, prostate cancer, prostate adenocarcinoma, endometrial cancer, uterine carcinosarcoma, gallbladder cancer, biliary tract cancer, bile duct carcinoma, colorectal cancer, metastatic colorectal cancer, bladder cancer, urothelial bladder cancer, sarcoma, soft tissue sarcoma, neuroendocrine tumors, nasopharyngeal neuroendocrine tumors, and homologous recombination deficient (HRD) cancers.

[0326] In some embodiments, the cancer being treated / prevented is a lung cancer (e.g., non-small cell lung cancer, lung adenocarcinoma, invasive mucinous lung adenocarcinoma, or lung squamous cell carcinoma) comprising cells with NRG1 gene fusions.

[0327] It will be understood that in embodiments herein, a cancer that comprises cells with particular characteristics may be or comprise a tumor that comprises cells with those characteristics.

[0328] As is common in the art, a cancer / tumor containing cells with particular characteristics may be referred to herein simply as a cancer / tumor having those characteristics. For example, a cancer / tumor containing cells with an NRG1 gene fusion may be referred to simply as an "NRG1 gene fusion-containing cancer / tumor" or an "NRG1 gene fusion cancer / tumor."

[0329] In some embodiments, the cancer to be treated / prevented contains a mutation that confers resistance to treatment with a BRAF inhibitor. In some embodiments, the mutation is a mutation in BRAF V600. In some embodiments, the mutation is BRAF V600E or V600K. The cancer may be thyroid cancer or colon cancer, e.g., RAS wild-type colorectal cancer. In some embodiments, the cancer to be treated / prevented contains a mutation that confers resistance to treatment with a BRAF inhibitor (e.g., a mutation in BRAF V600), and the treatment includes administration of vemurafenib or darafenib.

[0330] In some embodiments, the cancer may be a recurrent cancer. As used herein, a "recurrent" cancer refers to a cancer that has responded to treatment (e.g., a first-line cancer therapy) but then reappears / progresses, for example, after a period of remission. For example, a recurrent cancer may be a cancer whose growth / progression has been inhibited by treatment (e.g., a first-line cancer therapy) and then grows / progresses.

[0331] In some embodiments, the cancer may be a refractory cancer. As used herein, a "refractory" cancer refers to a cancer that has not responded to treatment (e.g., a first-line cancer therapy). For example, a refractory cancer may be a cancer whose growth / progression has not been inhibited by treatment (e.g., a first-line cancer therapy). In some embodiments, a refractory cancer may be a cancer in which a subject receiving cancer treatment has not shown a partial or complete response to the treatment.

[0332] In some embodiments, the cancer is a cancer comprising cells that express / overexpress an EGFR family member (e.g., HER3, EGFR, HER2, or HER4), a cancer comprising cells that express / overexpress HER3, a cancer comprising cells with a mutation that results in increased expression of a ligand for HER3, a cancer comprising cells with an NRG gene fusion, a solid tumor, a hematological cancer, a squamous cell carcinoma, breast cancer, breast carcinoma, invasive breast cancer, ductal carcinoma, metastatic breast cancer, triple-negative breast cancer, HER2-positive breast cancer, HER2-negative breast cancer, hormone receptor positive breast cancer, HER2-negative / hormone receptor positive breast cancer (e.g., cancers expressing estrogen receptors (ER) and / or progesterone receptors (PR)), gastric cancer, gastric carcinoma, gastric adenocarcinoma, gastrointestinal adenocarcinoma, colorectal cancer, metastatic colorectal cancer, colon cancer, colorectal cancer colorectal adenocarcinoma, colon adenocarcinoma, head and neck cancer, head and neck squamous cell carcinoma (HNSCC), lung cancer, non-small cell lung cancer, lung adenocarcinoma, invasive mucinous lung adenocarcinoma, lung squamous cell carcinoma (LUSC), ovarian cancer, ovarian cancer ovarian serous adenocarcinoma, ovarian serous cystadenocarcinoma, fallopian tube cancer, renal cancer, renal cell carcinoma, renal clear cell carcinoma, renal cell adenocarcinoma, papillary renal cell carcinoma, pancreatic cancer, pancreatic adenocarcinoma, pancreatic ductal adenocarcinoma, cervical cancer, cervical squamous cell carcinoma, skin cancer, Melanoma, oral cavity cancer, oropharyngeal cancer, esophageal cancer, esophageal squamous cell carcinoma (ESCC), esophageal adenocarcinoma, liver cancer, hepatocellular carcinoma, bile duct cancer, gallbladder cancer, biliary tract cancer, uterine cancer, endometrial cancer, uterine body endometrial cancer, uterine carcinosarcoma, thyroid cancer cancer), thyroid cancer carcinoma), pheochromocytoma, paraganglioma, bladder cancer, bladder epithelial carcinoma, prostate cancer, prostate adenocarcinoma, castration-resistant prostate cancer, metastatic prostate cancer, metastatic castration-resistant prostate cancer, retinoblastoma, sarcoma, soft tissue sarcoma, peritoneal cancer, thymoma, neuroendocrine tumor, nasopharyngeal neuroendocrine tumor, and homologous recombination deficient (HRD) cancer (e.g., HRD ovarian cancer, HRD breast cancer, HRD prostate cancer, or HRD pancreatic cancer).

[0333] In some embodiments, the cancer is selected from colorectal cancer, colorectal carcinoma, prostate cancer, prostate carcinoma, lung cancer, lung adenocarcinoma, breast cancer, breast carcinoma, invasive breast cancer, ovarian cancer, ovarian adenocarcinoma, ovarian serous adenocarcinoma, gastric cancer, and melanoma.

[0334] Treating cancer with the methods of the present disclosure achieves one or more of the following treatment effects: reducing the number of cancer cells in a subject, reducing the size of a cancerous tumor / lesion in a subject, inhibiting (e.g., preventing or slowing) the growth of cancer cells in a subject, inhibiting (e.g., preventing or slowing) the growth of a cancerous tumor / lesion in a subject, inhibiting (e.g., preventing or slowing) the development / progression of cancer (e.g., to a later stage or metastasis), reducing the severity of cancer symptoms in a subject, improving the subject's survival (e.g., progression-free survival or overall survival), reducing correlates of cancer cell number or activity in a subject, and / or reducing the cancer burden in a subject.

[0335] To determine the response to treatment, the subject can be evaluated according to the Revised Criteria for Response Assessment: The Lugano Classification (e.g., as described in Cheson et al., J Clin Oncol (2014) 32: 3059-3068, incorporated herein by reference above). In some embodiments, treatment of a subject with the methods of the present disclosure achieves one of the following: complete response, partial response, or stable disease.

[0336] Prevention can refer to preventing the onset of cancer and / or preventing the worsening of cancer, eg, preventing the progression of cancer, eg, to a later stage (eg, metastasis).

[0337] In some embodiments, administration of an antigen binding molecule / composition according to the present disclosure may be associated with one or more of the following: inhibiting the development / progression of cancer, delaying / preventing the onset of cancer, reducing / delaying / preventing tumor growth, reducing / delaying / preventing tissue invasion, reducing / delaying / preventing metastasis, reducing the severity of one or more symptoms of cancer, reducing the number of cancer cells, reducing the cancer burden, reducing the size / volume of the tumor, and / or improving the survival (e.g., progression-free survival or overall survival) of a subject with cancer.

[0338] According to various aspects of the present disclosure, methods of treating and / or preventing cancer according to the present disclosure may include inhibiting tumor growth, reducing tumor size / volume, and / or improving survival of a subject with cancer.

[0339] According to various aspects of the present disclosure, methods are provided for or including (e.g., in the context of treating / preventing cancer, e.g., a cancer described herein) one or more of the following: Binds to cells expressing HER3, inhibits the proliferation of HER3-expressing cells, Kills cells expressing HER3, inhibiting tumor growth and / or reducing tumor size / volume, e.g., in HER3-expressing cancers; and / or Improves survival of subjects with cancer, e.g., HER3-expressing cancers.

[0340] Also provided are antigen-binding molecules and compositions according to the present disclosure for use in such methods, and the use of antigen-binding molecules and compositions according to the present disclosure in the manufacture of compositions (e.g., medicaments) for use in such methods. It will be understood that the methods typically include administering an antigen-binding molecule according to the present disclosure to a subject.

[0341] Similarly, one or more of the following may be observed in a subject after therapeutic or prophylactic intervention according to the present disclosure (e.g., compared to pre-intervention levels / numbers / proportions, etc.): Inhibition of proliferation of HER3-expressing cells, Killing of HER3-expressing cells, Inhibition of tumor growth and / or reduction of tumor size / volume, e.g., in HER3-expressing cancers, and / or Improved survival of subjects with cancer, e.g., HER3-expressing cancer.

[0342] In some embodiments, a therapeutic / prophylactic intervention according to the present disclosure may be described as "associated with" one or more of the effects described in the previous paragraph. Those skilled in the art can readily assess such properties using techniques routinely practiced in the art.

[0343] Administration of the antigen-binding molecules and compositions of the present disclosure is preferably a "therapeutically effective" or "prophylactically effective" amount, which is sufficient to provide a therapeutic or prophylactic benefit to the subject. The actual amount administered, as well as the rate and duration of administration, will depend on the nature and severity of the disease / condition and the specific product being administered. Decisions regarding treatment prescription, e.g., dosage, etc., are within the responsibility of general practitioners and other physicians, and typically take into account the disease / disorder being treated, the condition of the individual subject, the site of delivery, the method of administration, and other factors known to practitioners. Examples of the above techniques and protocols can be found in Remington's "The Science and Practice of Pharmacy" (Ed. A. Adejare), 23 rd Edition(2020), Academic Press.

[0344] Administration of the antigen-binding molecules and compositions of the present disclosure can be, for example, parenteral, systemic, topical, intracavitary, intravascular, intravenous, intraarterial, intramuscular, intrathecal, intraocular, intraconjunctival, intratumoral, subcutaneous, intradermal, oral, or transdermal. Administration can be by injection, infusion, or oral ingestion.

[0345] In some aspects and embodiments, an article of the present disclosure may be administered to a tissue / organ of interest (e.g., a tissue / organ afflicted with a disease / condition affected by the condition (e.g., a tissue / organ where symptoms of the disease / condition are manifested)). In some aspects and embodiments, an article of the present disclosure may be administered into the blood (i.e., intravenous / intra-arterial administration) by injection or infusion (e.g., via a cannula), or may be administered subcutaneously or orally. In some aspects and embodiments, an article of the present disclosure may be administered to a tumor.

[0346] In some embodiments, therapeutic or prophylactic intervention according to the present disclosure may further include administering another agent for the treatment / prevention of the disease / condition. Administration of the antigen-binding molecules and compositions described herein, alone or in combination with other treatments, may be simultaneous or sequential, depending on the condition being treated. Simultaneous administration refers to administration together with another therapeutic agent or immediately after each other (e.g., within 1, 4, 6, 8, or 12 hours), optionally via the same route (e.g., into the same tissue, artery, vein, or other blood vessel), for example, as a pharmaceutical composition containing both agents (combined preparation). Sequential administration refers to administration of one agent followed by separate administration of another agent after a given time interval. The two agents do not need to be administered by the same route, although in some embodiments, this is the case. The time interval may be any time interval.

[0347] Multiple doses of antigen-binding molecules and compositions may be provided. The multiple doses may be separated by a predetermined time interval, which may be selected to be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 days, or 1, 2, 3, 4, 5, or 6 months. For example, a dose may be given once every 7, 14, 21, or 28 days (plus or minus 3, 2, or 1 day).

[0348] Detection Method The present disclosure also provides articles of the present disclosure for use in methods for detecting, identifying, or imaging HER3 or cells expressing HER3. The present disclosure provides antigen-binding molecules or compositions described herein for use in methods for diagnosing or prognosing a disease / condition, e.g., a disease / condition described herein.

[0349] The antigen-binding molecules described herein can be used in methods that include detecting the binding of the antigen-binding molecules to HER3. Such methods can include detecting the bound complex between the antigen-binding molecule and HER3. It will be understood that HER3 can be HER3 expressed by cells, for example, in cells that express HER3 or on the cell surface.

[0350] Thus, a method is provided that includes contacting a sample containing or suspected of containing HER3 and detecting the formation of a complex between the antigen-binding molecule and HER3. Also provided is a method that includes contacting a sample containing or suspected of containing cells expressing HER3 and detecting the formation of a complex between the antigen-binding molecule and the cells expressing HER3.

[0351] Suitable method formats are well known in the art and include sandwich assays, e.g., immunoassays such as ELISA. The method involves labeling the antigen-binding molecule, or the target(s), or both, with a detectable moiety as described herein, such as a fluorescent label, a phosphorescent label, a luminescent label, a label detectable by 75 icrop, a radioactive label, a chemical label, a nucleic acid label, or an enzyme label. Detection techniques are well known to those skilled in the art and can be selected to correspond to the labeling agent.

[0352] Methods that involve detecting HER3, or cells expressing HER3, include methods for diagnosing / prognosing the diseases / conditions described herein.

[0353] Such methods can be performed in vitro on a patient sample or after processing of a patient sample. After sample collection, the patient does not need to be present to perform the in vitro method, and therefore the method can be a method not performed on a human or animal body. In some embodiments, the method is performed in vivo.

[0354] Such methods may include, for example, detecting or quantifying HER3 and / or cells expressing HER3 in a patient sample. Where the method includes quantifying the factor, the method may further include comparing the determined amount to a standard or reference value as part of the diagnostic or prognostic evaluation. Other diagnostic / prognostic tests may be used in combination with those described herein to improve the accuracy of the diagnosis or prognosis or to confirm the results obtained by using the tests described herein.

[0355] Detection in a sample may be used to diagnose a disease / condition (e.g., cancer), a predisposition to a disease / condition, or to provide a prognosis (prognosis) for a disease / condition, such as a disease / condition described herein. The diagnosis or prognosis may relate to an existing (previously diagnosed) disease / condition.

[0356] A sample can be taken from any tissue or bodily fluid. A sample can include or be derived from: a tumor / biopsy thereof, a blood volume, a volume of serum from an individual's blood, which can include the fluid portion of blood obtained after removal of fibrin clots and blood cells, a tissue sample or biopsy, pleural effusion, cerebrospinal fluid (CSF), or cells isolated from the individual. In some embodiments, a sample can be obtained from or derived from a tissue(s) affected by a disease / condition (e.g., a tissue(s) where symptoms of the disease manifest or that are involved in the pathogenesis of the disease / condition). In some embodiments, a sample can be obtained from or derived from a tumor.

[0357] Subjects may be selected for diagnostic / prognostic evaluation based on the presence of symptoms indicative of a disease / condition described herein, or based on the subject being believed to be at risk for developing a disease / condition described herein.

[0358] The present disclosure also provides methods for selecting / stratifying subjects for treatment with HER3-targeted agents. In some embodiments, a subject is selected for treatment / prevention by a method of the present disclosure, or is identified as a subject who would benefit from such treatment / prevention, for example, based on detection / quantification of HER3 or cells expressing HER3 in a sample obtained from the individual.

[0359] subject A subject according to the embodiments described herein can be any animal or human. The subject is preferably a mammal, more preferably a human. The subject can be a non-human mammal, but is more preferably a human. The subject can be male or female. The subject can be a patient. The subject can be diagnosed with a disease or condition (e.g., cancer, e.g., a cancer described herein) requiring treatment, suspected of having such a disease / condition, or at risk of developing / contracting such a disease / condition.

[0360] In some embodiments, the subject treated according to the therapeutic or prophylactic methods disclosed herein is a subject who has or is at risk of developing cancer, e.g., a cancer described herein. In embodiments according to the present disclosure, the subject may be selected for treatment by the method based on the characterization of certain markers for such disease / condition.

[0361] In some embodiments, patients may be selected for the treatments described herein based, for example, on the detection of a HER3-expressing / overexpressing cancer in a sample obtained from the subject (e.g., a tumor biopsy).

[0362] kit The present disclosure also provides kits of parts. Kits according to the present disclosure may include components for performing, in whole or in part, the methods described herein.

[0363] The kit may have at least one container containing a predetermined amount of an antigen-binding molecule or composition described herein.

[0364] In some aspects of the present disclosure, a kit of parts is provided. In some embodiments, the kit may include an antigen-binding molecule or composition described herein, which may be provided in a predetermined amount.

[0365] The kit may be provided with instructions for administering the antigen-binding molecule or composition described herein to a patient to treat a particular disease / condition (e.g., a disease / condition described herein, e.g., cancer).

[0366] The kit may provide an antigen-binding moiety according to the present disclosure and a linker-payload moiety according to the present disclosure. The kit may further include reagents for conjugating the antigen-binding moiety and the linker-payload moiety.

[0367] The kits may further include reagents, buffers, and / or standards necessary for carrying out the methods according to the present disclosure. The kits according to the present disclosure may include instructions for use, for example, in the form of an instruction booklet or leaflet. The instructions may include protocols for carrying out any one or more of the methods described herein.

[0368] Sequence identity As used herein, "sequence identity" refers to the percentage of nucleotides / amino acid residues in a subject sequence that are identical to nucleotides / amino acid residues in a reference sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percentage of sequence identity between the sequences. Pairwise and multiple sequence alignment for determining the percent sequence identity between two or more amino acid or nucleic acid sequences can be achieved in a variety of ways known to those skilled in the art, for example, using publicly available computer software such as ClustalOmega (Soeding, J. 2005, Bioinformatics 21, 951-960), T-coffee (Notredame et al. 2000, J. Mol. Biol. (2000) 302, 205-217), Kalign (Lassmann and Sonnhammer 2005, BMC Bioinformatics, 6 (298)) and MAFFT (Katoh and Standley 2013, Molecular Biology and Evolution, 30 (4) 772-780). When using such software, default parameters, such as gap penalties and extension penalties, are preferably used. [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4] [Table 3-5] [Table 3-6] [Table 3-7] [Table 3-8] [Table 3-9] [Table 3-10] [Table 3-11] [Table 3-12] [Table 3-13] [Table 3-14] [Table 3-15] [Table 3-16] [Table 3-17] [Table 3-18] [Table 3-19] [Table 3-20] [Table 3-21] ***

[0369] The present disclosure includes combinations of the described aspects and preferred features (except where such combinations are clearly impermissible or explicitly avoided).

[0370] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0371] Aspects and embodiments of the present disclosure will now be illustrated, by way of example, with reference to the accompanying drawings. Further aspects and embodiments will be apparent to those skilled in the art. All documents cited in this document are incorporated herein by reference.

[0372] Throughout this specification, including the claims which follow, unless the context otherwise requires, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps, but not the exclusion of any other integer or step or group of integers or steps.

[0373] It should be noted that, as used in the specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Ranges may be expressed herein as from "about" one particular value and / or to "about" another particular value. When such a range is expressed, another embodiment includes from the one particular value and / or to the other particular value. Similarly, when values ​​are expressed as approximations, by use of the antecedent "about," it will be understood that the particular value forms another embodiment.

[0374] As used herein, "peptide" refers to a chain of two or more amino acid monomers linked by peptide bonds. Peptides typically have a length in the region of about 2 to 50 amino acids. A "polypeptide" is a polymeric chain of two or more peptides. Polypeptides typically have a length greater than about 50 amino acids.

[0375] When a nucleic acid sequence is disclosed or referred to herein, its reverse complement is also expressly contemplated.

[0376] The methods described herein may preferably be performed in vitro. The term "in vitro" is intended to encompass procedures performed on cells in culture, while the term "vivo" is intended to encompass procedures using intact multicellular organisms. [Example]

[0377] Example 1: Characterization of 10D1F in WO2019 / 185878A1 and WO2021 / 048274A1 A HER3-binding antibody clone designated 10D1F is described in WO2019 / 185878A1 (hereby incorporated by reference in its entirety).

[0378] 10D1F comprises a heavy chain variable region set forth in SEQ ID NO: 36 of WO2019 / 185878A1 (=SEQ ID NO: 33 of the present disclosure) and a light chain variable region set forth in SEQ ID NO: 83 of WO2019 / 185878A1 (=SEQ ID NO: 58 of the present disclosure). 10D1F is also referred to as "10D1_c89" in WO2019 / 185878A1.

[0379] Example 2.2 of WO2019 / 185878A1 describes a molecule (molecule

[16] ) comprising the VH and VL regions of 10D1F in human IgG1 / Vκ format (10D1F hIgG1), formed from SEQ ID NO: 206 of WO2019 / 185878A1 and SEQ ID NO: 207 of WO2019 / 185878A1.

[0380] Examples 8.1 to 8.3 and Figures 42 to 46 of WO2019 / 185878A1 show that 10D1F hIgG1 binds to human HER3 with high affinity and specificity (no cross-reactivity with other human EGFR family members), while retaining high affinity binding to cynomolgus monkey, mouse, and rat HER3.

[0381] Example 8.6 and Figures 49A and 49B of WO2019 / 185878A1 demonstrate that 10D1F hIgG1 binds to HER3 in a ligand (NRG)-independent manner and binds via topologically distant epitopes on HER3 to the epitope bound by the anti-HER3 antibodies M-05-74 and M-08-11. Example 8.10 and Figure 78 of WO2021 / 048274A1 demonstrate that 10D1F hIgG1 binds to human HER3 with sub-picomolar affinity in the presence or absence of human NRG1.

[0382] WO2021 / 048274A1 also discloses that antibody clone 10D1 and clones derived from 10D1 (including 10D1F) bind to human HER3 in a region corresponding to positions 218-235 of SEQ ID NO: 1 (i.e., SEQ ID NO: 77 of the present disclosure), and that two consensus binding site motifs have been identified in this region (shown in SEQ ID NOs: 78 and 79 of the present disclosure).

[0383] Example 4.1 and Figure 65, as well as Example 8.7 and Figure 52 of WO2019 / 185878A1, demonstrate that 10D1F hIgG1 is highly potent in inhibiting the interaction between HER3 and HER2, and does so in a dose-dependent manner. Example 8.7 and Figure 53 of WO2019 / 185878A1 show that 10D1F hIgG1 inhibits the interaction between HER3 and EGFR in a dose-dependent manner.

[0384] Example 8.8 and Figure 54 of WO2019 / 185878A1 show that 10D1F hIgG1 induces ADCC activity against HER3-overexpressing cells in a dose-dependent manner.

[0385] Example 8.9 and Figures 55, 63 and 64 of WO2019 / 185878A1 demonstrate that 10D1F hIgG1 inhibits HER3-mediated signaling in cells of a HER3-expressing cancer cell line in vitro.

[0386] Example 11 and Figure 71 of WO2019 / 185878A1 show that 10D1F hIgG1 also inhibits HER3-mediated signaling in vivo in xenograft tumors derived from HER3-expressing human cancer cell lines. Example 14 and Figure 79 of WO2021 / 048274A1 demonstrate that 10D1F is highly potent in inhibiting the growth of xenograft tumors derived from human cancer cell lines harboring NRG gene fusions.

[0387] Examples 9.3 and 9.4 and Figures 59, 60, 61, 62, 74, and 77 of WO2019 / 185878A1 demonstrate that 10D1F potently inhibits cancer cell growth in vitro and the growth of xenograft tumors derived from human cancer cell lines in vivo. Example 10 and Figures 67 and 68 of WO2019 / 185878A1 show that 10D1F hIgG1 inhibits the in vitro growth of thyroid cancer cell lines harboring the V600E BRAF mutation.

[0388] Example 13 and Figures 75 and 76 of WO2019 / 185878A1 demonstrate the utility of 10D1F hIgG1 for use in detecting HER3.

[0389] Example 8.4 and Figure 47A of WO2019 / 185878A1 show that 10D1F hIgG1 is thermostable, with a melting temperature of 70.0°C as determined by differential scanning fluorimetry.

[0390] Examples 9.1 and 9.2 and Figures 56, 57, 58 and 69, 70 of WO2019 / 185878A1 show that 10D1F hIgG1 has a favorable pharmacological and toxicological profile.

[0391] Example 2: Production of anti-HER3 antibody drug conjugates 2.1 Antibody generation DNA sequences encoding the heavy and light chain variable regions of the anti-HER3 antibody clone were subcloned into the mammalian multicistronic vectors pDZ1 or pTarget2.2 carrying human IgG1 Fc region mutations, or into a pair of expression vectors encoding the heavy and light chains of the antibody.DNA sequences encoding the heavy and light chain variable regions of the anti-RSV antibody palivizumab were subcloned into the mammalian dual landing multicistronic vector set pTargetF1314 and pTargetF3F carrying mutations in the hIgG1 Fc region.

[0392] Antibodies were expressed using either (i) the ExpiCHO-S transient expression system (Thermo Fisher, USA), (ii) the CHO-k1 site-specific integration system, (iii) the CHO-k1 random integration system, or (iv) the CHO-k1 site-specific double landing pad integration system.

[0393] ExpiCHO-S transient expression system ExpiCHO-S cells were obtained from Thermo Fisher Scientific. Cells were cultured in serum-free, protein-free, animal-origin-free synthetic medium supplemented with Glutamax (ExpiCHO Expression Medium, Thermo Fisher Scientific) at 37°C in an 8% CO2 and 80% humidified incubator equipped with a shaking platform. Cells were collected at 4–6 × 10 6 The cells were subcultured when they reached a density of 100 cells / mL.

[0394] ExpiCHO-S cells were transfected with the expression plasmid using the ExpiFectamine transfection reagent kit (Thermo Fisher Scientific) according to the manufacturer's protocol. The day before transfection, ExpiCHO-S cells were maintained at a cell density of 4–6 × 10. 6 cells / mL) in ExpiCHO expression medium at 4 × 10 6On the day of transfection, cells were seeded at 2000 cells / mL and grown overnight at a viable cell density of 7–10 × 10 6 / ml with viability >95%. For transfection, use fresh ExpiCHO expression medium to transfect 6 × 10 cells. 6 The DNA-ExpiFectamine complex was diluted to 1000 cells / mL of viable cells. The DNA-ExpiFectamine complex was formed in serum-free medium Optipro SFM (Thermo Fisher Scientific) for 1-5 minutes at room temperature before being added to the cells. ExpiFectamine CHO enhancer and ExpiCHO feed (from the ExpiFectamine Reagent Kit) were added to the transfected cells 18-22 hours after transfection. On day 5 post-transfection, a second amount of ExpiCHO feed was added to the cells and they were placed at 32°C. From day 7 onwards, glucose was checked and replenished to 6 g / L.

[0395] Transfectants were harvested on day 14 by centrifugation at 4000 xg for 10 minutes and filtration through a 0.45 μm filter unit followed by a 0.22 μm sterile filter unit.

[0396] CHO-k1 site-specific integration system CHO-k1 cells stably carrying the site-specific recombinase recognition sequences FRT3 and FRT were maintained in a commercially available serum-free synthetic growth medium consisting of 50% HyQ PF CHO medium (Hyclone, USA) and 50% CD CHO medium (Gibco, USA), containing 6 mM Gln, 0.05% Pluronic® F68, and 600 μg / mL G418. The cells were passaged twice a week and cultured at 37°C in a humidified shaker incubator with 8% CO2 and 80% CO2.

[0397] For transfection, 5 μg of the circular delivery plasmid pTarget2.2 carrying the mAb sequence was transfected together with 5 μg of the FLP recombinase expression plasmid into 1 × 10 cells using the 4D-Nucleofector kit (Lonza, Switzerland) with the electroporation program FF-137. 7 100 CHO-k1 cells were co-transfected. Electroporated cells were cultured in a well of a 6-well plate containing 2 ml of transfection medium (growth medium without G418) in a humidified static cell incubator at 37°C with 5% CO2 for 12–24 hours. One day after transfection by centrifugation, the medium was replaced with fresh transfection medium, and the culture was transferred to a shaker incubator. On day 5, the medium was completely replaced with selective medium (transfection medium containing 20 μg / mL puromycin). Once viability improved, the cells were cultured at 5 × 10 in selective medium. 5 Subculture was performed twice a week at a seeding density of 3 × 10 cells / mL, and finally, when the viability exceeded 75%, the cells were cultured at a density of 3 × 10 cells / mL. 5 The cells were passaged at 1000kJ / mL. Stable pools were established when viability recovered to >95%. For mAb expression, the established pools were cultured in fed-batch mode, with media feed (Cytiva, USA) administered at the appropriate ratio every other day from day 3 to day 11. Glucose was checked daily and replenished to 6 g / L if it fell below 6 g / L. Cultures were harvested on day 14–15. The cell culture supernatant, containing the target mAb, was clarified by acid flocculation or depth filtration.

[0398] CHO-k1 Random Embedded System The host cell line CHO-k1 (CCL-61) was purchased from ATCC (US, lot number 58995535). The cells originally grew in an adherent format and required 10% bovine serum for growth. A four-step medium adaptation protocol was applied to adapt CHO-k1 cells to a serum-free and suspension culture format. Subsequently, to further improve cell growth, the cells were adapted to a new commercially available serum-free synthetic medium, EX-CELL Advance CHO Fed-Batch Medium (Merck, USA). The adapted cells were established as transfection hosts and maintained in a 37°C, 8% CO2, 80% humidified incubator equipped with a shaking table.

[0399] For transfection, 1 x 10 7 CHO-k1 cells were electroporated with 5 μg of linearized pDZ1 expression plasmid using a 4D-Nucleofector kit (Lonza, Switzerland) with the electroporation program CA201. Electroporated cells were maintained in wells of a 6-well plate containing 2 ml of growth medium in a humidified static cell incubator at 37°C and 5% CO2 for 24 hours. The medium was replaced with fresh selective medium, and centrifugation was performed on 5 × 10 cells in a static incubator. 5 Once viability was restored, cells were transferred to a shaker incubator and seeded at a density of 5 × 10 cells / mL once a week starting 24 hours after transfection. 5 The cells were passaged twice weekly at 1000 cells / mL. Stable pools were established through a series of selective medium changes in a static incubator and several passages in selective medium in a shaker incubator. The established pools were subjected to a 14-15 day fed-batch culture process with intermediate medium feeding and glucose feeding to promote cell growth and productivity. The cell culture supernatant, containing the target mAb, was clarified by acid flocculation or depth filtration.

[0400] CHO-k1 site-specific double landing pad integration system The host cell clone 2D5 was derived from CHO-K1 and stably harbored the site-specific recombinase recognition sequences F3F and F13F14. Cells were maintained in a commercially available serum-free synthetic growth medium consisting of 50% HyQ PF CHO medium (Hyclone, USA) and 50% CD CHO medium (Gibco, USA), containing 6 mM Gln, 0.05% Pluronic® F68, 20 μg / mL blasticidin, and 600 μg / mL G418. Cells were passaged twice weekly and cultured at 37°C in a humidified shaker incubator with 8% CO2 and 80% CO2.

[0401] For transfection, 2.5 μg of the circular delivery plasmid ptargetF3F carrying the target mAb sequence and 2.5 μg of the circular delivery plasmid ptargetF13F14 carrying the target mAb sequence were transfected with 5 μg of the FLP recombinase expression plasmid into 1 × 10 cells using a 4D-Nucleofector kit (Lonza, Switzerland) with the electroporation program FF-137. 7 100 CHO-k1 2D5 cells were co-transfected. Electroporated cells were maintained in a well of a 6-well plate containing 2 ml of transfection medium (growth medium without blasticidin and G418) in a humidified static cell incubator at 37°C, 5% CO2 for 12–24 hours. One day after transfection by centrifugation, the medium was replaced with fresh transfection medium, and the culture was transferred to a shaker incubator. On day 5, the medium was completely replaced with selective medium (transfection medium containing 5 μg / mL puromycin + 200 μg / mL Zeocin). Once viability improved, the cells were cultured at 5 × 10 in selective medium. 5 Subculture was performed twice a week at a seeding density of 3 × 10 cells / mL, and finally, when the viability exceeded 75%, the cells were cultured at a density of 3 × 10 cells / mL. 5The cells were passaged at 1000kJ / mL. Stable pools were established when viability recovered to >95%. For mAb expression, the established pools were cultured in fed-batch mode, with media feed (Cytiva, USA) administered at the appropriate ratio every other day from day 3 to day 11. Glucose was checked daily and replenished to 6 g / L if it fell below 6 g / L. Cultures were harvested on day 14–15. The cell culture supernatant, containing the target mAb, was clarified by acid flocculation or depth filtration.

[0402] 2.2 Antibody purification and purity analysis Antibodies secreted into the culture supernatant from transfected cells were purified using the AKTA Pure liquid chromatography system (Cytiva, USA). Specifically, the supernatant was loaded onto a mAbselect Sure, mAbselect Sure LX, or mAbselect PrismA column (Cytiva, USA) with a contact time of 5–10 min. The column was then washed with 3–5 column volumes of wash buffer (10 mM sodium phosphate + 1 M NaCl, pH 7.2 and / or 10 mM sodium phosphate + 150 mM NaCl, pH 7.0 and / or 100 mM sodium citrate, pH 5.5). Bound mAbs were eluted with elution buffer (0.1 M sodium citrate, pH 3.5).

[0403] If the aggregate / fragment level was greater than 8% as assessed by SE-HPLC, the eluate was "finally purified" as follows: The eluate was held at low pH for 1 hour for virus inactivation and then neutralized to pH 6.9. The neutralized pool was further diluted with 20 mM MES, pH 6.9 to reach a conductivity of 9.6 mS / cm and then loaded onto the final purification column, Capto Adhere (Cytiva, USA). The mAb was purified by flow-through in a mobile phase of 20 mM MES, 80 mM NaCl, pH 6.9. The product was collected between the 200 mAU A280 rising peak during the sample injection phase and the 135 mAU A280 falling peak during the chase phase.

[0404] Eluates with aggregate / fragment levels below 8% as assessed by SE-HPLC and the final purified antibody preparation were buffer-exchanged into 20 mM histidine, 240 mM sucrose, 0.02% (w / w) polysorbate 80, pH 5.8, or PBS using a 20K MWCO protein concentrator (Thermo Fisher, USA). The monoclonal antibody preparation was sterilized through a 0.1 μm filter, aliquoted, and stored at -80°C before use.

[0405] Antibody purity was analyzed by SE-HPLC using an Acquity UPLC Protein BEH SEC Column 200, 1.7 μm, 4.6 mm × 150 mm column (Waters, USA) with a mobile phase of 250 mM NaCl, 100 mM sodium phosphate, pH 6.8 on a Waters UPLC system (Waters, USA). 50 μg of antibody in 20 mM histidine, 240 mM sucrose, 0.02% (w / w) polysorbate 80, pH 5.8, or PBS was injected onto the column at a flow rate of 0.4 mL / min at room temperature. Proteins were eluted uniformly according to their molecular weight.

[0406] The SE-HPLC results for the different antibodies after purification (and before conjugation) are summarized below: [Table 4]

[0407] To further examine the purity of the antibodies, capillary electrophoresis (CE-SDS) analysis was performed under non-reducing conditions using a PA800 system (Beckman Coulter, USA) in a bare fused silica capillary with an ID of 50 μm and a length of 20 cm to the detector. Antibody samples in low-salt buffer or buffer exchanged into SDS-MW sample buffer were diluted to 1 mg / mL in distilled water. 45 μL of diluted antibody was mixed with 57 μL of a master mix consisting of 5 μL of IAM, 50 μL of SDS-MW sample buffer, and 2 μL of a 10 kDa internal standard. The mixture was heated at 70°C for 10 min and then cooled to room temperature. Each sample was injected into the capillary at 5 kV (reverse polarity) for 20 s, followed by separation in a capillary containing SDS-MW gel buffer at 15 kV (reverse polarity) for 30 min. Data were analyzed using 32Karat series software.

[0408] Product-associated impurity host cell protein levels were analyzed using a CHO HCP ELISA kit (Cygnus, UK) according to the recommended protocol. Briefly, antibody samples or standard proteins were treated with alkaline phosphatase and then coated onto microtiter strips precoated with anti-CHO host cell protein. The reaction was allowed to proceed at room temperature for 2 hours, followed by multiple washes. PNPP substrate was then added, and the reaction was held for 90 minutes before being read on a plate reader at an absorbance of 405 / 492 nm.

[0409] Endotoxin levels in purified antibody samples were analyzed using an Endosafe instrument (Charles River, USA) and Endosafe PTS cartridges (Charles River, USA) with a sensitivity range of 5–0.05 EU / mL according to the manufacturer's protocol.

[0410] 2.3 Generation of antibody-drug conjugates Antibody SYNtecan E antibody-drug conjugates were prepared from antibody preparations generated and purified as described in Examples 2.1 and 2.2 using GlycoConnect technology, as described, for example, in van Geel et al., Bioconjug Chem. (2015) 26(11):2233-2242 and WO2021 / 015622A1, both of which are incorporated herein by reference in their entireties.

[0411] The antibody (see the table in Example 2.2 above) was dialyzed against remodeling buffer (20 mM histidine, 150 mM NaCl, 6 mM MnCl, pH 7.5) and incubated with endoglycosyltransferase SH (e.g., "EndoSH" as described in WO2017 / 137459A1, at a concentration of 0.15 mg / ml), alkaline phosphatase (at a concentration of 0.0015 mg / ml), urinary diphosphate (UDP)-6-azido-GalNAc (at a concentration of 1 mM), and Trichoplusia ni GalNAc transferase (TnGalNAcT, at a concentration of 0.45 mg / ml) at 30°C for 16 hours (at a concentration of 15 mg / ml) to generate an azide-modified antibody in which the N-glycan at N297 in the CH2 domain of the Fc region contains a terminal 6-azido-GalNAc residue. Conversion to the azide-modified antibody was confirmed by mass spectrometry (after treatment with IdeS and detection of the approximately 25 kDa Fc / 2 fragment). The azide-modified antibody was purified by Protein A chromatography and buffer-exchanged into TBS pH 7.4 at a concentration of approximately 20 mg / ml.

[0412] Metal-free click conjugation of SYNtecan E was performed by incubating 10 mg / ml of the azide-modified antibody species (in TBS pH 7.4) with 4 equivalents (to generate 501c.S8D4 and 501b.S8D4) or 5 equivalents (to generate ISO343c.S8D4 and ISO209b.S8D4) of the following compounds overnight at room temperature in the presence of 30% propylene glycol: [ka]

[0413] The conjugation reactions for the production of 501c.S8D4 and 501b.S8D4 further contained 11 mM sodium deoxycholate.

[0414] Conjugation success was assessed by reverse-phase HPLC (RP-HPLC) after DTT reduction. If the expected drug-to-antibody (DAR) ratio of 4 was not achieved, the sample was incubated with an additional 1.5 equivalents of compound for an additional 5 hours.

[0415] The antibody-SYNtecan E conjugate was purified by size-exclusion chromatography on a Superdex 200 column using PBS 7.4 as the mobile phase. The monomer fraction was buffer-exchanged into a buffer containing 20 mM histidine, 6% sucrose, 0.04% Tween® 20, pH 6.0, and filter-sterilized. Samples of the ADC were then aliquoted, flash-frozen, and stored at -80°C.

[0416] The SE-HPLC results for the different antibody drug conjugates after purification are summarized below: [Table 5]

[0417] 2.4 Antibody Drug Conjugates Characterized in Examples 3 and 4 The antibody drug conjugates characterized in Examples 3 and 4 are shown in the table below. [Table 6]

[0418] Additional control molecules were also used in certain experiments, including an antibody drug conjugate comprising an isotype-matched control antibody linked to deruxtecan (IgG-DXd).

[0419] Example 3: In vitro characterization of anti-HER3 antibody drug conjugates 3.1 Binding to HER3 on the surface of different cancer cell lines The binding of various antibody drug conjugates (ADCs) described in Example 2 to different cell lines was assessed by flow cytometry.

[0420] Binding to cells of the following cell lines was assessed: [Table 7]

[0421] Briefly, cells were seeded at 100,000 cells / well in wells of a 96-well round-bottom plate and incubated (1 h at 4°C) with eight different concentrations of antibody and ADC in a 3-fold dilution series (in PBS) starting from a top concentration of 2 μg / ml.

[0422] Afterwards, the cells were washed twice with FACS buffer (PBS + 5 mM EDTA and 0.5% BSA) and then incubated with F(ab')2-goat anti-human IgG Fc secondary antibody, Alexa Fluor 488 (1:2000 dilution in FACS buffer) for 1 hour at 4°C.

[0423] The cells were then washed twice with FACS buffer and analyzed by flow cytometry using a Cytek Northern Light. The mean fluorescence intensity of the Alexa Fluor 488-positive cell population was recorded, and the EC20 values ​​of the binding of different cell lines to the cells were calculated. 50 Values ​​were derived from binding curves.

[0424] The results are shown in Figures 1A-1F. ADCs containing anti-HER3 antibodies demonstrated binding to all HER3-expressing cell lines tested. 501b.S8D4 and 501c.S8D4 showed lower EC 50 As evidenced by the values, it showed better cell surface binding to 4 / 5 cell lines tested compared to patritumab-DXd.

[0425] 3.2 Binding to HER3 on the surface of different cancer cell lines in the presence of NRG1 Binding of the ADC described in Example 2 to various cell lines in the presence of the HER3 ligand NRG1 was assessed by flow cytometry.

[0426] Briefly, cells were seeded at 100,000 cells / well in wells of a 96-well round-bottom plate and incubated (3 hours at 4°C) with eight different concentrations of recombinant human NRG1 in the presence of 2 μg / ml of the antibody-drug conjugate in a two-fold dilution series (in PBS) starting from a top concentration of 640 ng / ml.

[0427] Afterwards, the cells were washed twice with FACS buffer (PBS + 5 mM EDTA and 0.5% BSA) and then incubated with F(ab')2-goat anti-human IgG Fc secondary antibody, Alexa Fluor 488 (1:2000 dilution in FACS buffer) for 1 hour at 4°C.

[0428] The cells were then washed twice with FACS buffer and analyzed by flow cytometry using a Cytek Northern Light. The mean fluorescence intensity of the Alexa Fluor 488-positive cell population was recorded.

[0429] The results are shown in Figures 2A to 2D. It was found that 501c.S8D4 and 501b.S8D4 exhibited significantly higher spontaneous binding to cells of various HER3-expressing cell lines than patritumab-DXd in the presence of NRG1.

[0430] 3.3 Binding to recombinant Fcγ receptors The ADCs described in Example 2 were analyzed for their ability to bind to human Fcγ receptors by surface plasmon resonance using Biacore.

[0431] Briefly, His-tagged recombinant human Fcγ receptor ligands (FcγRIA, FcγRIIA, or FcγRIIIA) were immobilized on the surface of a CM5 sensor chip using a Biacore HIS Capture Kit (Cytivia, catalog no. 28995056). Five concentrations of a half-logarithmic dilution series of various different analytes—patritumab-DXd, 501b.S8D4, 501c.S8D4, a human IgG1 isotype-matched antibody (positive control), and a human IgG1 isotype-matched antibody containing an Fc-silencing mutation (as a negative control)—were applied to the chip at a flow rate of 30 μl / min, with 60 s of association and 600 s of dissociation. Data acquisition and analysis were performed using a Biacore T200 according to the manufacturer's recommendations for analyzing high-affinity antigen-antibody kinetics. Responses from all steps were normalized for systematic disturbances by subtracting reference cells and correcting for zero concentration. The resulting association and dissociation curves were fitted with 1:1 global fitting to obtain values ​​for the association rate (ka), dissociation rate (kd), and equilibrium dissociation constant (KD).

[0432] The results are shown in Figures 3A and 3B. 501c.S8D4 and 501b.S8D4 completely abrogated binding to FcγRIA, FcγRIIA, and FcγRIIIA. Patritumab-DXd significantly bound to all three FcγRs, with KD values ​​comparable to those of the positive control.

[0433] Next, the Fc-mediated binding of 501c.S8D4 to human PBMCs was evaluated in comparison to patritumab-DXd. Fc-mediated binding of ADCs to normal cells, including hematopoietic cells, can cause severe thrombocytopenia, neutropenia, anemia, and many other adverse events.

[0434] ADCs were labeled with the fluorescent dye APC (red) using an APC conjugation kit. PBMCs were obtained from three different donors. 100 μl of Zombie NIR™ Fixable Viability Dye (Biolegend, #423106) was added at a 1:3,000 dilution per million cells using FACS buffer (0.5% BSA and 4 mM EDTA in PBS) and incubated for 30 minutes at room temperature. 100K cells per well were added to a 96-well round-bottom plate and incubated for 1 hour at 4°C in the presence of three different concentrations (30, 10, and 3 μg / ml) of labeled 501c.S8D4, patritumab-DXd, and IgG1-DXd isotype (positive) control. After incubation, cells were washed, and cell surface binding was quantified by flow cytometry using a Cytek Northern Light 3000. Mean fluorescence intensity (MFI) was calculated and plotted using GraphPad Prism.

[0435] Figure 16 shows that IgG-DXd exhibited the highest binding to PBMCs. This is because this isotype control does not bind to any known human cell surface proteins, and any binding observed is expected to be Fc-mediated (as with any IgG1 isotype). 501c.S8D4 exhibited significantly (81%) less binding than patritumab-DXd in PBMCs from all three donors.

[0436] In conclusion, 501c.S8D4 exhibits significantly reduced binding to human PBMCs, indicating reduced Fc-mediated uptake by healthy cells and a reduced risk of associated toxicity.

[0437] 3.4 Effect on proliferation of HER3-expressing cells in a 3D in vitro proliferation assay The effect of various ADCs described in Example 2 on the proliferation of T47D cells (CVCL_0553), H358 cells (CVCL_1559) and HCT116 cells (CVCL_0291) was assessed in a 3D in vitro proliferation assay.

[0438] Briefly, cells were seeded at 2000 cells / well overnight in RPMI medium + 10% FBS at 37°C, 5% CO2. Three days later, 100 ng / ml of recombinant human NRG1 was added to some of the cultures, and the cells were then treated with a 10-fold, 5-fold dilution series starting at 50 μg / ml. The plates were then incubated at 37°C, 5% CO2 for 7 days.

[0439] Cell viability was then assessed using Cell Counting Kit-8 (Dojindo, #SKU-CK04) according to the manufacturer's instructions. 20 μl of CCK8 reagent was added to each well, and plates were read on a Biotek plate reader at 2, 4, and 6 hours after addition of CCK8 reagent. Percentage inhibition of cell proliferation was calculated relative to readings obtained in the presence of the lowest concentration of drug or buffer alone (i.e., in the absence of ADC / antibody). Data points represent the average of three replicates per condition. IC for inhibition of cell proliferation 50 Values ​​were derived from binding curves.

[0440] The results are shown in Figures 4A, 4B, 5A and 5B.

[0441] 501c.S8D4 and 501b.S8D4 were found to inhibit the growth of HER3-expressing T47D cells to a similar extent as patritumab-DXd (Figure 4A). Neither anti-HER3 antibody-drug conjugate substantially inhibited the growth of HCT116 cells, which express low levels of HER3 (Figure 4B).

[0442] In the absence of NRG1, 501b.S8D4(IC 50 =0.45 nM) and 501c.S8D4 (IC 50 =0.59 nM) showed similar levels of inhibition of cell proliferation compared to patritumab-DXd (0.93 nM) (see Figure 5A and Figure 5A).

[0443] In the presence of NRG1, 501c.S8D4(IC 50=0.71 nM) was associated with patritumab-DXd (IC 50 = 2.2 nM) (Figure 5B), whereas 501b.S8D4 showed a similar level of inhibition compared to patritumab-DXd (Figure 5A).

[0444] Taken together, these data demonstrate that 501c.S8D4 and 501b.S8D4 are effective in inhibiting the proliferation of HER3-expressing cells in vitro, with comparable or improved efficacy compared to patritumab-DXd.

[0445] 3.5 Plasma stability The stability of 501c.S8D4 and 501b.S8D4 in plasma was investigated.

[0446] Briefly, 100 μl of 501c.S8D4 or 501b.S8D4 at a concentration of 100 μg / mL in mouse plasma was incubated at 37° C. for days 0, 3, 7, 10, 14, and 21. Samples were taken at regular time points.

[0447] Antibodies were affinity captured from the samples by adding 4 μg of HIS-tagged HER3 (Sino Biological 10201-H08H) to 50 μl of each sample, followed by 40 μL of anti-HIS tagged mAb DPBS-washed magnetic beads (MBL, D291-11). The samples were incubated with rotation at 4°C for 2 hours, and the captured ADC immobilized on the beads was isolated using a magnetic stand. The beads were washed with 100 μl of ice-cold DPBS, and the captured ADC was eluted with 50 μl of a solution containing 30% acetonitrile, 1% formic acid in MilliQ water.

[0448] A 2 μl sample of the eluted ADC was used for evaluation by liquid chromatography mass spectrometry (LCMS) to determine the percentage of detected ADC that retained the correct antibody-to-drug ratio of 4.

[0449] The results are shown in Figure 6. 501c.S8D4 and 501b.S8D4 showed good stability over the test period, with a consistently high proportion of DAR4 species.

[0450] 3.6 Internalization into HER3-expressing cells 501c.S8D4 was evaluated for its ability to be internalized and trafficked to lysosomes in the HER3-positive breast cancer cell line T47D.

[0451] Fluorescence microscopy of T47D cells (30,000 cells / well) treated with APC (red)-labeled 501c.S8D4 was performed at 37°C for 0.5, 4, and 16 hours to study the localization of 501c.S8D4. Because endocytosis does not occur at this temperature, one set of cells was incubated with APC-labeled 501c.S8D4 for 1 hour at 4°C as a negative control. After incubation for the above time points, the cells were washed, fixed, and permeabilized. These cells were then stained with FITC (green fluorescent)-labeled anti-LAMP1 antibody, a lysosomal marker. Cells were imaged at 40x magnification using a Leica fluorescent microscope (DMi8) and processed using a Thunder imager. In this assay, red fluorescence indicates 501c.S8D4 localization, green fluorescence indicates lysosomal localization, and yellow indicates colocalization of 501c.S8D4 with lysosomes.

[0452] The results are shown in Figure 13. The top panel of Figure 13 shows that 501c.S8D4 is localized to the cell surface at 4°C, indicating binding to cell surface HER3 without observable internalization. Upon incubation at 37°C, 501c.S8D4 is rapidly internalized (within 30 minutes) as indicated by punctate staining (indicating localization within endosomal compartments). Furthermore, overlapping analysis of 501c.S8D4 (top) and lysosomal marker (middle) signals indicates that 501c.S8D4 is transported to lysosomes within 4 hours in T47D cells and remains in lysosomes for up to 16 hours, as indicated by the colocalized signal in the merged (bottom) panel.

[0453] Thus, 501c.S8D4 is rapidly internalized (within 30 minutes) upon incubation at 37° C. and is observed in lysosomes for up to 16 hours.

[0454] The internalization rate was then assessed. 501c.S8D4 was labeled using a pH-dependent fluorescent dye (Human Fabfluor-pH Red (Sartorius, #4722)). The dye fluoresces only at low pH (4.5-5.5), which is the pH within intracellular vesicles. Because the pH of the cell surface is approximately 7, the fluorescent signal can only be detected upon internalization of labeled 501c.S8D4 into intracellular vesicles. The maximum signal occurs when the dye reaches lysosomes, where the pH is at a minimum of 4.5. The internalization rate of the antibody or ADC was determined by measuring the fluorescent signal over time.

[0455] Internalization of 501c.S8D4 was measured using live cell imaging in T47D cells treated with Fab-fluor (red fluorescent)-labeled 501c.S8D4. Six thousand T47D cells per well were seeded in RPMI medium + 10% FBS in a 96-well plate at 37°C in the presence of 5% CO2 for 48 hours. 12.5 nM of 501c.S8D4 was conjugated with Fabfluor at a 1:3 ratio (ADC:Fabfluor) for 15 minutes at 37°C. 100 μl of conjugated 501c.S8D4 was added to the cells. The plate was then incubated in an Incucyte™ SX5 Live-Cell Analysis System at 37°C in the presence of 5% CO2 for 2 days. Four images per well were captured every 30 minutes during the incubation period. Images were analyzed using cell-by-cell analysis software (Incucyte) to measure the mean red fluorescence intensity per cell. Values ​​were normalized to day 0, and data were plotted as a percentage of the mean red intensity using GraphPad PRISM.

[0456] The results are shown in Figure 14. 501c.S8D4 is internalized very rapidly, reaching saturation levels within 12 hours. A peak signal is observed when 501c.S8D4 reaches the lysosomes, and the signal begins to decrease after 24 hours due to lysosomal degradation of 501c.S8D4.

[0457] Thus, 501c.S8D4 is rapidly internalized in T47D cells, reaching saturation within 12 hours.

[0458] 3.7 ADC macropinocytosis by megakaryocytes We evaluated the non-target-dependent uptake of 501c.S8D4 by human stem cell (HSC)-derived megakaryocytes (MKs) via macropinocytosis. 501c.S8D4 was labeled using a pH-dependent fluorescent dye (Fab-Fluor, red, Sartorius, #4722), which fluoresces only at low pH (4.5-5.5), i.e., the pH within intracellular vesicles. Because the pH of the cell surface is approximately 7, the fluorescent signal can only be detected upon internalization of labeled 501c.S8D4 into intracellular vesicles of MKs.

[0459] Thrombocytopenia, or platelet loss, is a common side effect reported in cancer patients treated with ADCs, including patritumab-DXd, a clinical-stage HER3 ADC with a similar payload class. Platelets are produced by MKs derived from HSCs in the bone marrow. Macropinocytosis-mediated uptake of ADCs by MKs interferes with platelet production, ultimately leading to thrombocytopenia. Therefore, 501c.S8D4 was tested to determine whether macropinocytosis-mediated uptake occurs with 501c.S8D4 treatment and how it compares to patritumab-DXd.

[0460] HSCs from three different donors were expanded and transdifferentiated into MKs. MKs were then seeded into 96-well plates (20,000 cells / well) in SFEM II medium (STEMcell Technologies, #9655), StemSpan® MK growth supplement (STEMcell Technologies, #2696), 10 ng / ml IL1, and 10 ng / ml IL3, and incubated at 37°C and 5% CO for 24 hours. 100 μl of Fab-Fluor-labeled patritumab-DXd or 501c.S8D4 (test article:Fab-Fluor ratio 1:3) was added to the cells, and red fluorescence was measured in real time using live-cell imaging for 24 hours, capturing four images per well every 30 minutes. The mean red intensity was plotted against time after normalization to time 0 (t=0). These values ​​were plotted as a percentage of the average red intensity for all three donors using GraphPad PRISM to obtain a 24-hour internalization curve. The area under the curve is directly proportional to the total amount of ADC internalized by megakaryocytes.

[0461] As shown in Figure 15, macropinocytosis-dependent uptake of 501c.S8D4 by megakaryocytes is significantly (30%) less than that of patritumab-DXd.

[0462] These data indicate that reduced uptake of 501c.S8D4 could potentially contribute to reduced thrombocytopenia in humans.

[0463] Example 4: In vivo characterization of anti-HER3 antibody drug conjugates 4.1 Antitumor effects in vivo The therapeutic efficacy of the various ADCs described in Example 2 was investigated in vivo in a xenograft model derived from a human cancer cell line, H358, of lung adenocarcinoma.

[0464] Female Ncr nude mice, approximately 6–8 weeks old, were housed under specific pathogen-free conditions and treated in accordance with Institutional Animal Care and Use Committee (IACUC) guidelines. H358 (CVCL_1559) cell-derived tumors were inoculated at 5 × 10 6 The cells were mixed with an equal volume of Matrigel (Corning, USA) and subcutaneously implanted into the right flank of mice. Tumors were established at approximately 150–160 mm. 3 Treatment was initiated when the mice reached 100 mg / kg body weight. On day 1, the mice were intravenously administered a single dose of 3 mg / kg body weight of the antigen-binding molecule. There were 7 mice per treatment group.

[0465] Tumor volumes were measured three times a week using digital calipers and calculated using the formula [L × W / 2]. Control tumors had a volume of 1.5 cm 3 The study endpoint was reached when

[0466] The results of the experiment are shown in Figure 7. 501c.S8D4 and 501b.S8D4 demonstrated significant inhibition of the growth of H358 CDX tumors (which express HER3 at moderate levels). 501b.S8D4 demonstrated a similar level of tumor growth inhibition to patritumab-DXd, and 501c.S8D4 demonstrated an improved level of tumor growth inhibition compared to patritumab-DXd. Thus, despite having a lower drug-to-antibody ratio (DAR) of 4 compared to patritumab-DXd, which has a DAR of 8, 501c.S8D4 and 501b.S8D4 demonstrated similar or improved tumor growth inhibition compared to patritumab-DXd. These data indicate that 501c.S8D4 and 501b.S8D4 have superior efficacy to patritumab-DXd in this H358 CDX model. Furthermore, the tumor inhibition exhibited by 501c.S8D4 and 501b.S8D4 was much longer lasting than that of patritumab-DXd, suggesting that 501c.S8D4 and 501b.S8D4 may be more stable than patritumab-DXd in vivo.

[0467] In another in vivo experiment, tumors derived from T47D cells (CVCL_0553) were cultured at 1 × 10 7 The cells were mixed with an equal volume of Matrigel (Corning, USA) and subcutaneously implanted into the right flank of 6- to 8-week-old female NOD / SCID mice. Because these tumor cells depend on estrogen for growth, a sustained-release (60-day) estrogen pellet was implanted one day before tumor implantation (Ruan et al., Maturitas. (2019) 123:1-8). Tumors were established when the tumors were approximately 150-250 mm. 3 Treatment was initiated when the IgG4-associated antigen-binding molecule (IgG4-associated antigen) reached 100 mg / kg / day. There were 7 mice per treatment group. On day 1, mice were intravenously administered a single dose of the antigen-binding molecule as follows: Group 1: Vehicle only (PBS) Group 2: Patritumab-DXd, 1.5 mg / kg body weight Group 3: Patritumab-DXd, 4.5 mg / kg body weight Group 4:501b.S8D4, 3mg / kg body weight Group 5:501b.S8D4, 9mg / kg body weight Group 6: 501c.S8D4, 3mg / kg body weight Group 7: 501c.S8D4, 9mg / kg body weight

[0468] Tumor volumes were measured three times a week as described above.

[0469] The results are shown in Figures 8A and 8B. 501c.S8D4 and 501b.S8D4 at 3 mg / kg body weight demonstrated significantly greater tumor growth inhibition compared to patritumab-DXd at equivalent payload doses (i.e., patritumab-DXd at 1.5 mg / kg body weight).

[0470] 501c.S8D4 and 501b.S8D4 at 9 mg / kg body weight showed similar tumor growth inhibition as patritumab-DXd at an equivalent payload dose (ie, patritumab-DXd at 4.5 mg / kg body weight).

[0471] Taken together, these data suggest that 501c.S8D4 and 501b.S8D4 have superior efficacy in vivo in a model derived from the T47D cell line.

[0472] 4.2 Toxicity analysis Toxicities, including severe interstitial lung disease (ILD) and severe cytopenias, have been observed after administration of patritumab-DXd (Jaenne et al., Cancer Discov. (2022) 12(1):74-89). These toxicities may result from Fcγ receptor- and / or macropinocytosis-mediated uptake of the ADC by normal, non-cancerous cells (Zhao et al., Mol Cancer Ther (2017) 16(9):1866-1876; Kumagai et al., Cancer Sci. (2020) 111(12):4636-4645; Zhao et al., Mol Cancer Ther (2017) 16(9):1877-1886).

[0473] The toxicity of various ADCs described in Example 2 was evaluated in female Sprague Dawley rats. Treatment groups were as follows: Group 1: Vehicle only Group 2:501b.S8D4 Group 3:501c.S8D4

[0474] ADC or an equivalent volume of vehicle was administered by slow intravenous injection into the tail vein. The first dose of 60 mg / kg body weight was administered on day 1, and the second dose of 53 mg / kg body weight was administered on day 8. There were three mice per treatment group.

[0475] Survival, body weight, and food intake were monitored. Blood samples were taken on days 4 and 11 for analysis, and a complete necropsy was performed on day 11.

[0476] The health status of the treated rats, including clinical signs, body weight, food intake, and mortality, was monitored and recorded daily after treatment. Tissues including liver, kidney, lung, brain, bone marrow, skin, stomach, small intestine, and large intestine, and any tissue with gross lesions, were collected and processed for histopathological analysis of tissue damage.

[0477] All mice in each treatment group survived the entire 11-day experiment, and no significant loss of body weight or change in food intake was observed throughout the treatment period.

[0478] Red blood cell counts, hemoglobin concentrations, hematocrit percentages, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH) levels, and mean corpuscular hemoglobin concentrations (MCHC) were found to be not significantly different among blood samples obtained from rats in groups 1, 2, and 3 (Figures 9A-9F). Rats in groups 2 and 3 did not exhibit significant signs of either neutropenia (Figure 10D) or thrombocytopenia (Figure 10E), as previously reported in clinical trials of patritumab-DXd (Steuer et al., Journal of Clinical Oncology (2022) 40(16_suppl) 9017; Jaenne et al., Cancer Discov (2022) 12(1):74-89). See Figures 10A-10E. Although a significant decrease in white blood cell counts has previously been reported in preclinical studies of patritumab-DXd in rats (Hashimoto et al., Clin Cancer Res. (2019) 25(23):7151-7161), in the present study, an increase in white blood cell counts was observed in groups 2 and 3 compared to group 1 (Figure 10A).

[0479] Assessment of biochemical markers of liver, kidney, and pancreatic injury revealed no significant differences between rats in groups 1, 2, and 3 (Figures 11A-11F). In contrast, preclinical evaluation of patritumab-DXd previously detected significant increases in transaminase levels (a marker of liver injury) (Hashimoto et al., Clin Cancer Res. (2019) 25(23):7151-7161).

[0480] Finally, the levels of various electrolytes were found not to be significantly different between blood samples obtained from rats in groups 1, 2, and 3 (FIGS. 12A-12D).

[0481] Taken together, the data from this toxicology study indicate that 501c.S8D4 and 501b.S8D4 are likely to have improved tolerability compared to patritumab-DXd in rats.

Claims

1. An antigen-binding molecule that binds to HER3, comprising: (i) a HER3-binding portion; and (ii) a linker payload portion comprising exatecan or a derivative thereof.

2. The antigen-binding molecule of claim 1, wherein the linker payload portion comprises the structure (A): 【Chemistry 26】

3. The antigen-binding molecule of claim 1 or claim 2, wherein the linker payload portion and the HER3 binding portion are connected via a cleavable linker portion.

4. The antigen-binding molecule of any one of claims 1 to 3, wherein the antigen-binding molecule comprises a cleavable linker moiety comprising the structure (B): 【Chemistry 27】

5. The antigen-binding molecule of any one of claims 1 to 4, wherein the antigen-binding molecule comprises a cleavable linker moiety comprising a polyethylene glycol (PEG) moiety.

6. The antigen-binding molecule of any one of claims 1 to 5, wherein the antigen-binding molecule comprises a cleavable linker moiety comprising the structure (E): 【Chemistry 28】

7. The antigen-binding molecule of any one of claims 1 to 6, wherein the antigen-binding molecule comprises a cleavable linker moiety comprising a p-aminobenzylcarbamate (PABC) group.

8. The antigen-binding molecule comprises a cleavable linker moiety comprising the structure (D): 【Chemistry 29】 In the formula, R is CH ３ and (CH ２ ) ３ NHCOCNH ２ The antigen-binding molecule of any one of claims 1 to 7, selected from:

9. The antigen-binding molecule of any one of claims 1 to 8, wherein the antigen-binding molecule comprises a structure (E): 【Transformation 30】

10. The antigen-binding molecule of any one of claims 1 to 9, wherein the antigen-binding molecule comprises SYNtecan E.

11. The antigen-binding molecule of any one of claims 1 to 10, wherein the HER3-binding portion comprises an Fc region, and the linker payload portion is conjugated to the HER3-binding site at an azide group provided to a 6-azido-6-deoxy-N-acetylgalactosamine residue of an N-glycan linked to N297 (EU numbering) of the CH2 domain of the Fc region.

12. The antigen-binding molecule of any one of claims 1 to 11, wherein the HER3-binding portion binds to HER3 at the region shown in SEQ ID NO:

77.

13. The antigen-binding molecule of any one of claims 1 to 12, wherein the HER3-binding portion comprises: (i) a heavy chain variable (VH) region incorporating the following CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO: 40 HC-CDR2 having the amino acid sequence of SEQ ID NO: 43 HC-CDR3 having the amino acid sequence of SEQ ID NO: 48, and (ii) a light chain variable (VL) region incorporating the following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO: 66 LC-CDR2 having the amino acid sequence of SEQ ID NO: 69 LC-CDR3 having the amino acid sequence of SEQ ID NO:

74.

14. The antigen-binding molecule of any one of claims 1 to 13, wherein the HER3-binding portion comprises: (i) a VH region incorporating the following CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO: 38 HC-CDR2 having the amino acid sequence of SEQ ID NO: 42 HC-CDR3 having the amino acid sequence of SEQ ID NO: 45, and (ii) a VL region incorporating the following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO: 63 LC-CDR2 having the amino acid sequence of SEQ ID NO: 67 LC-CDR3 having the amino acid sequence of SEQ ID NO:

70.

15. The antigen-binding molecule of any one of claims 1 to 14, wherein the antigen-binding portion that binds to HER3 comprises: a VH region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 33; and A VL region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO:

58.

16. The antigen-binding molecule of any one of claims 1 to 15, wherein the antigen-binding portion that binds to HER3 comprises: A polypeptide comprising or consisting of an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 75; and A polypeptide comprising or consisting of an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO:

76.

17. The antigen-binding molecule of any one of claims 1 to 16, wherein the antigen-binding portion that binds to HER3 comprises: A polypeptide comprising or consisting of an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 80, 81, 82 or 91; and A polypeptide comprising or consisting of an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO:

76.

18. 1. A method for producing an antigen-binding molecule, comprising contacting a HER3 binding moiety comprising an azide moiety with a compound having structure (F): 【Chemistry 31】

19. 19. An antigen-binding molecule obtained or obtainable by the method of claim 18.

20. A composition comprising the antigen-binding molecule of any one of claims 1 to 17 or 19 and a pharmaceutically acceptable carrier, diluent, excipient or adjuvant.

21. An antigen-binding molecule according to any one of claims 1 to 17 or claim 19, or a composition according to claim 20, for use in a method of medical treatment or prevention, or a method of diagnosis or prognosis.

22. An antigen-binding molecule according to any one of claims 1 to 17 or claim 19, or a composition according to claim 20, for use in the treatment or prevention of cancer.

23. Use of the antigen-binding molecule of any one of claims 1 to 17 or claim 19, or the composition of claim 20, in the manufacture of a medicament for the treatment or prevention of cancer.

24. A method for treating or preventing cancer, comprising administering to a subject a therapeutically or prophylactically effective amount of the antigen-binding molecule of any one of claims 1 to 17 or claim 19, or the composition of claim 20.

25. The cancer is selected from the group consisting of cancers comprising cells expressing / overexpressing an EGFR family member, cancers comprising cells expressing / overexpressing HER3, cancers comprising cells with a mutation that results in increased expression of a HER3 ligand, cancers comprising cells with an NRG gene fusion, solid tumors, blood cancers, squamous cell carcinomas, breast cancer, breast carcinoma, invasive breast cancer, ductal carcinoma, metastatic breast cancer, triple-negative breast cancer, HER2-positive breast cancer, HER2-negative breast cancer, hormone receptor-positive breast cancer, HER2-negative / hormone receptor-positive breast cancer, gastric cancer, gastric carcinoma, gastric adenocarcinoma, gastrointestinal adenocarcinoma, colorectal cancer, metastatic colorectal cancer, colon cancer, colorectal cancer colorectal adenocarcinoma, colon adenocarcinoma, head and neck cancer, head and neck squamous cell carcinoma (HNSCC), lung cancer, non-small cell lung cancer, lung adenocarcinoma, invasive mucinous lung adenocarcinoma, lung squamous cell carcinoma (LUSC), ovarian cancer, ovarian cancer carcinoma), ovarian serous adenocarcinoma, ovarian serous cystadenocarcinoma, fallopian tube cancer, renal cancer, renal cell carcinoma, renal clear cell carcinoma, renal cell adenocarcinoma, papillary renal cell carcinoma, pancreatic cancer, pancreatic adenocarcinoma, pancreatic ductal adenocarcinoma, cervical cancer, cervical squamous cell carcinoma, skin cancer, Melanoma, oral cavity cancer, oropharyngeal cancer, esophageal cancer, esophageal squamous cell carcinoma (ESCC), esophageal adenocarcinoma, liver cancer, hepatocellular carcinoma, cholangiocarcinoma, gallbladder cancer, biliary tract cancer, uterine cancer, endometrial cancer, uterine body endometrial cancer, uterine carcinosarcoma, thyroid cancer (thyroid cancer), thyroid cancer 25. The antigen-binding molecule or composition for use according to claim 22, the use according to claim 23, or the method according to claim 24, wherein the cancer is selected from among carcinoma, pheochromocytoma, paraganglioma, bladder cancer, bladder epithelial carcinoma, prostate cancer, prostate adenocarcinoma, castration-resistant prostate cancer, metastatic prostate cancer, metastatic castration-resistant prostate cancer, retinoblastoma, sarcoma, soft tissue sarcoma, peritoneal cancer, thymoma, neuroendocrine tumor, nasopharyngeal neuroendocrine tumor, and homologous recombination deficient (HRD) cancer.

26. Use of an antigen-binding molecule according to any one of claims 1 to 17 or claim 19, or a composition according to claim 20, for depleting or increasing killing of cells expressing HER3.

27. 20. An in vitro complex, optionally isolated, comprising the antigen-binding molecule of any one of claims 1 to 17 or claim 19 bound to HER3.

28. A method for detecting HER3 in a sample, the method comprising: contacting a sample containing or suspected of containing HER3 with the antigen-binding molecule of any one of claims 1 to 17 or claim 19; and detecting the formation of a complex between the antigen-binding molecule and HER3.

29. A method for selecting or stratifying a subject for treatment with a HER3 targeting agent, the method comprising: contacting a sample from the subject in vitro with the antigen-binding molecule of any one of claims 1 to 17 or claim 19; and detecting the formation of a complex between the antigen-binding molecule and HER3.

30. Use of the antigen-binding molecule of any one of claims 1 to 17 or claim 19 as an in vitro or in vivo diagnostic or prognostic agent.

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