Use of anti-TGFbeta R2 / PD-1 bispecific antibodies to treat cancer

Abstract

This disclosure describes bispecific antibodies targeting human programmed death-1 (PD-1) and human transforming growth factor β receptor 2 (TGFβR2) for use in the treatment of disorders such as non-small cell lung cancer (NSCLC), squamous cell carcinoma of the head and neck (SCCHN), squamous cell carcinoma of the cervix and adenocarcinoma of the cervix (CESC), ovarian cancer, breast cancer, bladder cancer, renal cell carcinoma, melanoma, gastric adenocarcinoma, esophageal cancer, gastroesophageal adenocarcinoma, malignant pleural mesothelioma, pancreatic adenocarcinoma, and colorectal cancer (CRC).

Description

[Technical Field]

[0001] Claim of priority This application claims the interests of U.S. Provisional Patent Application No. 63 / 496,205, filed on 14 April 2023, which is incorporated herein by reference in its entirety.

[0002] Sequence List This application includes a sequence listing submitted electronically as an XML file named 20443-0795WO1.XML. The XML file, created on April 4, 2024, is 19,545 bytes in size. The material contained in the XML file is incorporated herein by reference in its entirety.

[0003] This invention aims to treat cancer by administering bispecific antibodies that bind to human TGFβR2 and human PD-1. [Background technology]

[0004] Cancer is one of the leading causes of death worldwide. Many patients are diagnosed with progressive cancer and either do not respond to treatment or their disease progresses after responding to treatment. Therefore, targeted therapies for cancer are needed. [Overview of the project]

[0005] This disclosure is based, at least in part, on the development of cancer therapies using bispecific antibodies that bind to human TGFβR2 and human PD-1.

[0006] Accordingly, aspects of the present disclosure provide a method for treating a disorder in a human subject requiring treatment, the disorder being selected from the group consisting of non-small cell lung cancer (NSCLC), squamous cell carcinoma of the head and neck (SCCHN), squamous cell carcinoma of the cervix and adenocarcinoma of the cervix (CESC), ovarian cancer, breast cancer, bladder cancer, renal cell carcinoma, melanoma, gastric adenocarcinoma, esophageal cancer, gastric esophageal adenocarcinoma, malignant pleural mesothelioma, pancreatic adenocarcinoma, and colorectal cancer (CRC), the method comprising administering to a human subject a therapeutically effective dose of human programmed death-1 (PD-1) and a bispecific antibody that binds to human transforming growth factor β receptor 2 (TGFβR2), the bispecific antibody being, An anti-human PD-1 binding domain comprising a PD-1 heavy chain variable region and a PD-1 light chain variable region, wherein the PD-1 heavy chain variable region comprises a heavy chain CDR1 (HCDR1) containing the amino acid sequence RFALH (SEQ ID NO: 1), a heavy chain CDR2 (HCDR2) containing the amino acid sequence WIDPNTGTPTFAQGVTG (SEQ ID NO: 2), and a heavy chain CDR3 (HCDR3) containing the amino acid sequence SLGYCDSDICYPNWIFDN (SEQ ID NO: 3), and the PD-1 light chain variable region comprises a light chain CDR1 (LCDR1) containing the amino acid sequence QSISSY (SEQ ID NO: 11), a light chain CDR2 (LCDR2) containing the amino acid sequence AAS (SEQ ID NO: 12), and a light chain CDR3 (LCDR3) containing the amino acid sequence QQSYSTPPT (SEQ ID NO: 13), and An anti-human TGFβR2 binding domain comprising a TGFβR2 heavy chain variable region and a TGFβR2 light chain variable region, wherein the TGFβR2 heavy chain variable region comprises HCDR1 containing the amino acid sequence IYAMT (SEQ ID NO: 6), HCDR2 containing the amino acid sequence VISGSGGTTYYADSVKG (SEQ ID NO: 7), and HCDR3 containing the amino acid sequence RGQYRDIVGATDY (SEQ ID NO: 8), and the TGFβR2 light chain variable region comprises LCDR1 containing the amino acid sequence QSISSY (SEQ ID NO: 11), LCDR2 containing the amino acid sequence AAS (SEQ ID NO: 12), and LCDR3 containing the amino acid sequence QQSYSTPPT (SEQ ID NO: 13), and the anti-human TGFβR2 binding domain.

[0007] In some embodiments, the PD-1 heavy chain variable region includes the amino acid sequence QVQLVQSGSELKKPGASVKVSCKASGYTFTRFALHWVRQAPGQGLEWMGWIDPNTGTPTFAQGVTGRFVFSLDTSVTTAYLQISSLKAEDTAVYYCARSLGYCDSDICYPNWIFDNWGQGTLVTVSS (SEQ ID NO: 4), and the TGFβR2 heavy chain variable region includes the amino acid sequence EVQLVESGGGLVQPGGSLRLSCAASGFTFDIYAMTWVRQAPGKGLEWVSVISGSGGTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARRGQYRDIVGATDYWGQGTLVTVSS (SEQ ID NO: 9).

[0008] In some embodiments, the PD-1 light chain variable region and the TGFβR2 light chain variable region each contain the amino acid sequence DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPTFGQGTKVEIK (SEQ ID NO: 14).

[0009] In some embodiments, the bispecific antibody comprises a PD-1 heavy chain, a PD-1 light chain, a TGFβR2 heavy chain, and a TGFβR2 light chain, wherein the PD-1 heavy chain comprises the amino acid sequence represented by SEQ ID NO: 5, the PD-1 light chain comprises the amino acid sequence represented by SEQ ID NO: 15, the TGFβR2 heavy chain comprises the amino acid sequence represented by SEQ ID NO: 10, and the TGFβR2 light chain comprises the amino acid sequence represented by SEQ ID NO: 15.

[0010] In some embodiments, the human subject has NSCLC. In some embodiments, the NSCLC has a squamous or non-squamous histological form. In some embodiments, the NSCLC is progressive or metastatic.

[0011] In some embodiments, the human subject has SCCHN. In some embodiments, SCCHN includes primary squamous epithelial tumors of the oral cavity, oropharynx, hypopharynx, or larynx. In some embodiments, SCCHN is progressive or metastatic.

[0012] In some embodiments, the human subject has CESC. In some embodiments, CESC is progressive or metastatic.

[0013] In some embodiments, the human subject has human papillomavirus (HPV)-positive SCCHN or HPV-positive CESC.

[0014] In some embodiments, the human subject has ovarian cancer. In some embodiments, ovarian cancer includes epithelial ovarian cancer, fallopian tube cancer, primary peritoneal cancer, or carcinosarcoma. In some embodiments, ovarian cancer is progressive or metastatic.

[0015] In some embodiments, the human subject has breast cancer. In some embodiments, breast cancer includes triple-negative breast cancer. In some embodiments, triple-negative breast cancer includes HER2-negative, ER-negative, and PgR-negative breast cancer. In some embodiments, breast cancer is progressive or metastatic.

[0016] In some embodiments, the human subject has bladder cancer. In some embodiments, bladder cancer includes urothelial carcinoma. In some embodiments, bladder cancer is progressive or metastatic.

[0017] In some embodiments, the human subject has renal cell carcinoma. In some embodiments, renal cell carcinoma is progressive or metastatic. [[ID=2^]]

[0018] In some embodiments, the human subject has melanoma. In some embodiments, melanoma is cutaneous malignant melanoma. In some embodiments, melanoma is progressive or metastatic.

[0019] In some embodiments, the human subject has gastric adenocarcinoma. In some embodiments, the gastric adenocarcinoma is advanced or metastatic.

[0020] In some embodiments, the human subject has esophageal cancer. In some embodiments, the esophageal cancer is advanced or metastatic.

[0021] In some embodiments, the human subject has esophagogastric adenocarcinoma. In some embodiments, the esophagogastric adenocarcinoma is advanced or metastatic.

[0022] In some embodiments, the human subject has malignant pleural mesothelioma. In some embodiments, the malignant pleural mesothelioma is progressive or metastatic.

[0023] In some embodiments, the human subject has pancreatic adenocarcinoma. In some embodiments, the pancreatic adenocarcinoma is advanced or metastatic.

[0024] In some embodiments, the human subject has CRC. In some embodiments, the CRC includes microsatellite-stable colorectal cancer (MSS-CRC). In some embodiments, the CRC includes mismatch repair deficiency (dMMR) / high microsatellite instability (MSI-H) colorectal cancer (dMMR / MSI-H CRC). In some embodiments, the CRC is advanced or metastatic.

[0025] In some embodiments, human subjects have experienced disease progression after prior treatment. In some embodiments, prior treatment includes anti-PD-(L)1 therapy and / or anti-CTLA4 therapy.

[0026] In some embodiments, the disorder is unsuitable for curative treatment or intervention.

[0027] In some embodiments, the bispecific antibody is administered intravenously.

[0028] In some embodiments, the bispecific antibody is administered in doses of approximately 100 mg, 300 mg, 900 mg, 1500 mg, or 2000 mg.

[0029] In some embodiments, the bispecific antibody is administered intravenously in doses of approximately 100 mg, 300 mg, 900 mg, 1500 mg, or 2000 mg.

[0030] In some embodiments, the bispecific antibody comprises a PD-1 heavy chain, a PD-1 light chain, a TGFβR2 heavy chain, and a TGFβR2 light chain, wherein the PD-1 heavy chain comprises the amino acid sequence represented by SEQ ID NO: 5, the PD-1 light chain comprises the amino acid sequence represented by SEQ ID NO: 15, the TGFβR2 heavy chain comprises the amino acid sequence represented by SEQ ID NO: 10, and the TGFβR2 light chain comprises the amino acid sequence represented by SEQ ID NO: 15. The bispecific antibody is administered intravenously in doses of approximately 100 mg, 300 mg, 900 mg, 1500 mg, or 2000 mg.

[0031] In some embodiments, the bispecific antibody is administered once every two weeks. In some embodiments, the bispecific antibody is administered intravenously once every two weeks in doses of approximately 100 mg, approximately 300 mg, approximately 900 mg, approximately 1500 mg, or approximately 2000 mg. In some embodiments, the bispecific antibody comprises a PD-1 heavy chain, a PD-1 light chain, a TGFβR2 heavy chain, and a TGFβR2 light chain, wherein the PD-1 heavy chain comprises the amino acid sequence represented by SEQ ID NO: 5, the PD-1 light chain comprises the amino acid sequence represented by SEQ ID NO: 15, the TGFβR2 heavy chain comprises the amino acid sequence represented by SEQ ID NO: 10, and the TGFβR2 light chain comprises the amino acid sequence represented by SEQ ID NO: 15. The bispecific antibody is administered intravenously once every two weeks in doses of approximately 100 mg, approximately 300 mg, approximately 900 mg, approximately 1500 mg, or approximately 2000 mg.

[0032] In some embodiments, the bispecific antibody is administered once every four weeks. In some embodiments, the bispecific antibody is administered intravenously once every four weeks in doses of approximately 100 mg, approximately 300 mg, approximately 900 mg, approximately 1500 mg, or approximately 2000 mg. In some embodiments, the bispecific antibody comprises a PD-1 heavy chain, a PD-1 light chain, a TGFβR2 heavy chain, and a TGFβR2 light chain, wherein the PD-1 heavy chain comprises the amino acid sequence represented by SEQ ID NO: 5, the PD-1 light chain comprises the amino acid sequence represented by SEQ ID NO: 15, the TGFβR2 heavy chain comprises the amino acid sequence represented by SEQ ID NO: 10, and the TGFβR2 light chain comprises the amino acid sequence represented by SEQ ID NO: 15. The bispecific antibody is administered intravenously once every four weeks in doses of approximately 100 mg, approximately 300 mg, approximately 900 mg, approximately 1500 mg, or approximately 2000 mg. [Brief explanation of the drawing]

[0033] [Figure 1A] This graph shows the results obtained from an in vitro cytotoxicity assay using a co-culture of renal cancer cells and T cells cultured in the presence of exogenous TGF-β (0.1 ng / mL). [Figure 1B] This graph shows the results obtained from an in vitro cytotoxicity assay using a co-culture of renal cancer cells and T cells cultured in the presence of exogenous TGF-β (0.1 ng / mL). [Figure 1C] This graph shows the results obtained from an in vitro cytotoxic assay using a co-culture of renal cancer cells and T cells cultured in the absence of exogenous TGF-β. [Figure 1D] This graph shows the results obtained from an in vitro cytotoxic assay using a co-culture of renal cancer cells and T cells cultured in the absence of exogenous TGF-β. [Figure 1E] This graph shows the results obtained from an in vitro cytotoxicity assay using a co-culture of renal cancer cells and T cells cultured in the presence of exogenous TGF-β (1.0 ng / mL). [Figure 1F]This graph shows the results obtained from an in vitro cytotoxicity assay using a co-culture of renal cancer cells and T cells cultured in the presence of exogenous TGF-β (1.0 ng / mL). [Figure 2] Graphs A and B show tumor volume (A) and body weight (B) of mice subcutaneously inoculated with A375 human melanoma cells and subsequently treated with various antibodies. Humanized mice with subcutaneous A375 human melanoma tumors were administered antibodies every 5 days starting on day 8, and tumor volume was measured twice a week. Body weight was also measured twice a week. Data are shown as mean ± SEM. [Figure 3A] This graph shows the tumor volume plotted against stem cell donors for humanized mice with A375 human melanoma tumors. The mice were untreated. [Figure 3B] This graph shows the tumor volume plotted against stem cell donors for humanized mice with A375 human melanoma tumors. The mice were treated with an IgG1 control antibody. [Figure 3C] This graph shows the tumor volume plotted against stem cell donors for humanized mice with A375 human melanoma tumors. The mice were treated with atezolizumab. [Figure 3D] This graph shows the tumor volume plotted against stem cell donors for humanized mice with A375 human melanoma tumors. The mice were treated with pembrolizumab. [Figure 3E] This graph shows the tumor volume plotted against stem cell donors for humanized mice with A375 human melanoma tumors. The mice were treated with TGF1 antibody. [Figure 3F] This graph shows the tumor volume plotted against stem cell donors for humanized mice with A375 human melanoma tumors. The mice were treated with pembrolizumab + TGF1 antibody. [Figure 3G] This graph shows the tumor volume plotted against stem cell donors for humanized mice with A375 human melanoma tumors. The mice were treated with antibody A (Figure 3G). [Figure 3H]This graph plots tumor volume against stem cell donors for humanized mice with A375 human melanoma tumors. The mice were treated with NIS793. Donors are identified by number (0678, 0717, 0731, and 0687). The 750 mm³ dotted line is an optional line for visual aids. [Figure 4] This graph shows the tumor volume in mice subcutaneously inoculated with MDA-MB-231 human triple-negative breast cancer (TNBC) cells and subsequently treated with various antibodies. [Figure 5A] This graph shows the tumor volume in mice that were subcutaneously inoculated with MC38 colorectal cancer cells and subsequently treated with various antibodies. [Figure 5B] The cured mice were re-inoculated with MC38 colorectal cancer cells, and tumor volume was monitored over time. [Figure 6A] This graph shows the results of flow cytometry analysis of CD8 T cells in MC38 colorectal tumors isolated from transgenic hPD-1 / hTGFβR2 mice after treatment with various antibodies. [Figure 6B] This graph shows the results of flow cytometry analysis of Treg cells in MC38 colorectal tumors isolated from transgenic hPD-1 / hTGFβR2 mice after treatment with various antibodies. [Figure 7A] These are representative micrographs from IHC analysis of MC38 colorectal tumors collected from transgenic hPD-1 / hTGFβR2 mice after treatment with antibody A (1 mg / kg or 10 mg / kg) or control antibody RSV. [Figure 7B] This is a representative graph showing the CD8 cell density from IHC analysis of MC38 colorectal tumors isolated from transgenic hPD-1 / hTGFβR2 mice after treatment with antibody A (1 mg / kg or 10 mg / kg) or control antibody RSV. [Figure 7C] This is a representative graph showing the percentage of CD8-positive cells from IHC analysis of MC38 colorectal tumors isolated from transgenic hPD-1 / hTGFβR2 mice after treatment with antibody A (1 mg / kg or 10 mg / kg) or control antibody RSV. [Figure 8] This graph shows IFNγ-producing Ly95 T cells in co-culture with A549-eso cells in the presence of tumor digests (digest) derived from NSCLC patients. The co-culture was treated with the indicated antibody. [Figure 9] Graphs A and B show the IFNγ concentration (pg / mL) in two different ovarian ascites fluid samples treated with various antibodies for four days. A: Ovarian ascites fluid sample 1, B: Ovarian ascites fluid sample 2. [Modes for carrying out the invention]

[0034] This disclosure provides a method for treating cancer by administering bispecific antibodies that target TGFβR2 and PD-1. While we do not wish to be bound by theory, it is thought that simultaneous targeting of TGFβR2 and PD-1 may mitigate immunosuppressive pathways and promote cytotoxic T-lymphocyte function and T-cell memory for effective cancer elimination, while minimizing the toxicity associated with systemic TGFβR2 blockade.

[0035] Anti-TGFβR2 / PD-1 bispecific antibody Transforming growth factor β is a multifunctional cytokine that acts as a tumor promoter or tumor suppressor in a cell-dependent and context-dependent manner. In healthy cells, TGFβ can arrest the cell cycle in phase 1, leading to decreased proliferation and induction of differentiation, while also promoting apoptosis. In cancer cells, the TGFβ signaling pathway is dysregulated or altered, and TGFβ no longer has the ability to regulate cell proliferation. Mammals have three highly homologous TGFβ isoforms: TGFβ1, TGFβ2, and TGFβ3. The most common TGFβ1 is expressed in the majority of human cancer types. Furthermore, TGFβ1 expression is the isoform most closely correlated with TGFβ signaling activation compared to the isoforms TGFβ2 and TGFβ3.

[0036] TGFβ is synthesized as an inactive precursor that needs to be activated to enable the binding of the tetrameric receptor complex composed of TGFβR1 and TGFβR2. Transforming growth factor β receptor 2 is a membrane-bound serine / threonine kinase that binds to TGFβ1 and TGFβ3 with relatively high affinity and to TGFβ2 with lower affinity. Post-binding signaling of the TGFβ ligand requires the formation of a heterodimer complex with TGFβR1. Activated TGFβR2 phosphorylates multiple serine and threonine residues in the intracellular domain of TGFβR1, leading to activation of TGFβR1. Activated TGFβR1 then mediates the activation of downstream signaling pathways involving SMAD proteins that regulate target gene expression.

[0037] Programmed cell death 1 protein (PD-1, also known as CD279) is a cell surface receptor expressed on CD4+ and CD8+ T cells, B cells, NK cells, and myeloid cells. PD-1 binds to two distinct ligands with different expression patterns: PD-L1 and PD-L2. PD-L1 (also known as B7-H1 or CD274) is expressed on hematopoietic cells such as T cells, B cells, dendritic cells, and macrophages, as well as in peripheral tissue arrays, and PD-L1 expression levels are interferon-induced. In contrast, PD-L2 (also known as B7-DC or CD273) expression is generally restricted to specialized APCs and is interferon-induced depending on the lineage subset of the APCs.

[0038] When PD-1 binds to either PD-L1 or PD-L2 on T cells or B cells, it leads to clustering with the TCR or BCR and transient association with SH2 domain-containing tyrosine phosphatase 2. This then induces negative signaling by dephosphorylating effector molecules that drive positive TCR and BCR signaling. This includes CD28-mediated activation of PI3K, followed by Akt, glucose metabolism, and the survival protein Bcl-XL. Overall, this results in suppression of T cell or B cell activation, proliferation, and cytokine secretion. PD-1 expression on T cells and tumor-infiltrating lymphocytes after chronic viral infection has been shown to lead to immunodeficiency characteristic of exhaustion, while blocking PD-1 signaling has been shown to promote T cell proliferation and restore the immune response.

[0039] Antibody A is a human Fc-silencing IgG1 bispecific antibody that can simultaneously bind to both TGFβR2 and PD-1. Antibody A is designed to block the PD-1 axis and selectively targets TGFβ signaling blockade on activated PD-1 expressing T cells.

[0040] The amino acid sequences of the heavy and light chains of antibody A are described below. Antibody A contains two different heavy chains, a TGFβR2 heavy chain (which binds to TGFβR2) and a PD-1 heavy chain (which binds to PD-1), as well as a common light chain that pairs with each of the TGFβR2 and PD-1 heavy chains. The complementarity-determining regions (CDRs) 1, 2, and 3 of the variable heavy chain (VH) domain and variable light chain (VL) domain are shown below (see HCDR defined according to Kabat (Kabat et al., Sequences of Proteins of Immunological Interest, US Dept. of Health and Human Services, Public Health Service, National Institutes of Health, 1991, (OCoLC) 1138727707) and LCDR defined according to IMGT (Giudicelli et al., IMGT / V-QUEST: IMGT standardized analysis of the immunoglobulin (IG) and T cell receptor (TR) nucleotide sequences. Cold Spring Harb Protoc 2011(6):695-715)). An antibody consisting of the TGFβR2 heavy chain amino acid sequence represented by SEQ ID NO: 10, the PD-1 heavy chain amino acid sequence represented by SEQ ID NO: 5, and a common light chain amino acid sequence (one light chain that pairs with each of the heavy chains) represented by SEQ ID NO: 15 is referred to as "Antibody A".

[0041] Table 1 shows the heavy chain (HC) of the anti-PD-1 binding domain and the anti-TGFβR2 binding domain of antibody A, as well as the common light chain amino acid sequence.

[0042] [Table 1]

[0043] Tables 2 and 3 show the VH and VL values ​​for the anti-PD-1 binding domain and anti-TGFβR2 binding domain of antibody A, as well as the respective CDR values ​​for VH and VL.

[0044] [Table 2]

[0045] [Table 3]

[0046] In some embodiments, the anti-human PD-1 binding domain of the bispecific antibody of the Disclosure includes a heavy chain variable region comprising HCDR1 comprising the amino acid sequence represented by SEQ ID NO: 1, HCDR2 comprising the amino acid sequence represented by SEQ ID NO: 2, and HCDR3 comprising the amino acid sequence represented by SEQ ID NO: 3. In some embodiments, the anti-human PD-1 binding domain of the bispecific antibody of the Disclosure includes a heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 4, or having at least 80%, 85%, 90%, or 95% sequence identity thereto. In some embodiments, the anti-human PD-1 binding domain of the bispecific antibody of the Disclosure includes (1) HCDR1 comprising the amino acid sequence represented by SEQ ID NO: 1, HCDR2 comprising the amino acid sequence represented by SEQ ID NO: 2, and HCDR3 comprising the amino acid sequence represented by SEQ ID NO: 3, and (2) a heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 4, or having at least 80%, 85%, 90%, or 95% sequence identity thereto.

[0047] In some embodiments, the anti-human TGFβR2 binding domain of the bispecific antibody of the Disclosure includes a heavy chain variable region comprising HCDR1 comprising the amino acid sequence represented by SEQ ID NO: 6, HCDR2 comprising the amino acid sequence represented by SEQ ID NO: 7, and HCDR3 comprising the amino acid sequence represented by SEQ ID NO: 8. In some embodiments, the anti-human TGFβR2 binding domain of the bispecific antibody of the Disclosure includes a heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 9, or having at least 80%, 85%, 90%, or 95% sequence identity thereto. In some embodiments, the anti-human TGFβR2 binding domain of the bispecific antibody of the Disclosure includes (1) HCDR1 comprising the amino acid sequence represented by SEQ ID NO: 6, HCDR2 comprising the amino acid sequence represented by SEQ ID NO: 7, and HCDR3 comprising the amino acid sequence represented by SEQ ID NO: 8, and (2) a heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 9, or having at least 80%, 85%, 90%, or 95% sequence identity thereto.

[0048] In some embodiments, the anti-human PD-1 binding domain of the bispecific antibody of the Disclosure includes a heavy chain variable region comprising HCDR1 comprising the amino acid sequence represented by SEQ ID NO: 1, HCDR2 comprising the amino acid sequence represented by SEQ ID NO: 2, and HCDR3 comprising the amino acid sequence represented by SEQ ID NO: 3; and the anti-human TGFβR2 binding domain of the bispecific antibody includes a heavy chain variable region comprising HCDR1 comprising the amino acid sequence represented by SEQ ID NO: 6, HCDR2 comprising the amino acid sequence represented by SEQ ID NO: 7, and HCDR3 comprising the amino acid sequence represented by SEQ ID NO: 8. In some embodiments, the anti-human PD-1 binding domain of the bispecific antibody of the Disclosure includes a heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 4, or having at least 80%, 85%, 90%, or 95% sequence identity thereto; and the anti-human TGFβR2 binding domain of the bispecific antibody includes a heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 9, or having at least 80%, 85%, 90%, or 95% sequence identity thereto. In some embodiments, the anti-human PD-1 binding domain of the bispecific antibody of the present disclosure comprises (1) HCDR1 comprising the amino acid sequence represented by SEQ ID NO: 1, HCDR2 comprising the amino acid sequence represented by SEQ ID NO: 2, and HCDR3 comprising the amino acid sequence represented by SEQ ID NO: 3, and (2) a heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 4 or having at least 80%, 85%, 90%, or 95% sequence identity thereto. The anti-human TGFβR2 binding domain of the bispecific antibody comprises (1) HCDR1 comprising the amino acid sequence represented by SEQ ID NO: 6, HCDR2 comprising the amino acid sequence represented by SEQ ID NO: 7, and HCDR3 comprising the amino acid sequence represented by SEQ ID NO: 8, and (2) a heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 9 or having at least 80%, 85%, 90%, or 95% sequence identity thereto.

[0049] In some embodiments, the anti-human PD-1 binding domain of the bispecific antibody of the Disclosure includes (1) a heavy chain variable region including HCDR1 containing the amino acid sequence represented by SEQ ID NO: 1, HCDR2 containing the amino acid sequence represented by SEQ ID NO: 2, and HCDR3 containing the amino acid sequence represented by SEQ ID NO: 3, and (2) a light chain variable region including LCDR1 containing the amino acid sequence represented by SEQ ID NO: 11, LCDR2 containing the amino acid sequence represented by SEQ ID NO: 12, and LCDR3 containing the amino acid sequence represented by SEQ ID NO: 13. In some embodiments, the anti-human PD-1 binding domain of the bispecific antibody of the Disclosure includes (1) a heavy chain variable region including the amino acid sequence represented by SEQ ID NO: 4, or having at least 80%, 85%, 90%, or 95% sequence identity thereto, and (2) a light chain variable region including the amino acid sequence represented by SEQ ID NO: 14, or having at least 80%, 85%, 90%, or 95% sequence identity thereto. In some embodiments, the anti-human PD-1 binding domain of the bispecific antibody of the present disclosure includes a heavy chain variable region comprising (1) HCDR1 comprising the amino acid sequence represented by SEQ ID NO: 1, HCDR2 comprising the amino acid sequence represented by SEQ ID NO: 2, and HCDR3 comprising the amino acid sequence represented by SEQ ID NO: 3, and (2) the amino acid sequence represented by SEQ ID NO: 4, or having at least 80%, 85%, 90%, or 95% sequence identity thereto, and a light chain variable region comprising (1) LCDR1 comprising the amino acid sequence represented by SEQ ID NO: 11, LCDR2 comprising the amino acid sequence represented by SEQ ID NO: 12, and LCDR3 comprising the amino acid sequence represented by SEQ ID NO: 13, and (2) the amino acid sequence represented by SEQ ID NO: 14, or having at least 80%, 85%, 90%, or 95% sequence identity thereto.

[0050] In some embodiments, the anti-human TGFβR2 binding domain of the bispecific antibody of the Disclosure includes (1) a heavy chain variable region including HCDR1 containing the amino acid sequence represented by SEQ ID NO: 6, HCDR2 containing the amino acid sequence represented by SEQ ID NO: 7, and HCDR3 containing the amino acid sequence represented by SEQ ID NO: 8, and (2) a light chain variable region including LCDR1 containing the amino acid sequence represented by SEQ ID NO: 11, LCDR2 containing the amino acid sequence represented by SEQ ID NO: 12, and LCDR3 containing the amino acid sequence represented by SEQ ID NO: 13. In some embodiments, the anti-human TGFβR2 binding domain of the bispecific antibody of the Disclosure includes (1) a heavy chain variable region including the amino acid sequence represented by SEQ ID NO: 9, or having at least 80%, 85%, 90%, or 95% sequence identity thereto, and (2) a light chain variable region including the amino acid sequence represented by SEQ ID NO: 14, or having at least 80%, 85%, 90%, or 95% sequence identity thereto. In some embodiments, the anti-human TGFβR2 binding domain of the bispecific antibody of the present disclosure includes a heavy chain variable region comprising (1) HCDR1 comprising the amino acid sequence represented by SEQ ID NO: 6, HCDR2 comprising the amino acid sequence represented by SEQ ID NO: 7, and HCDR3 comprising the amino acid sequence represented by SEQ ID NO: 8, and (2) the amino acid sequence represented by SEQ ID NO: 9, or having at least 80%, 85%, 90%, or 95% sequence identity thereto, and a light chain variable region comprising (1) LCDR1 comprising the amino acid sequence represented by SEQ ID NO: 11, LCDR2 comprising the amino acid sequence represented by SEQ ID NO: 12, and LCDR3 comprising the amino acid sequence represented by SEQ ID NO: 13, and (2) the amino acid sequence represented by SEQ ID NO: 14, or having at least 80%, 85%, 90%, or 95% sequence identity thereto.

[0051] In some embodiments, the anti-human PD-1 binding domain of the bispecific antibody of the present disclosure comprises (1) a heavy chain variable region comprising HCDR1 comprising the amino acid sequence represented by SEQ ID NO: 1, HCDR2 comprising the amino acid sequence represented by SEQ ID NO: 2, and HCDR3 comprising the amino acid sequence represented by SEQ ID NO: 3, and (2) a light chain variable region comprising LCDR1 comprising the amino acid sequence represented by SEQ ID NO: 11, LCDR2 comprising the amino acid sequence represented by SEQ ID NO: 12, and LCDR3 comprising the amino acid sequence represented by SEQ ID NO: 13, and the anti-human TGFβR2 binding domain of the bispecific antibody comprises (1) a heavy chain variable region comprising HCDR1 comprising the amino acid sequence represented by SEQ ID NO: 6, HCDR2 comprising the amino acid sequence represented by SEQ ID NO: 7, and HCDR3 comprising the amino acid sequence represented by SEQ ID NO: 8, and (2) a light chain variable region comprising LCDR1 comprising the amino acid sequence represented by SEQ ID NO: 11, LCDR2 comprising the amino acid sequence represented by SEQ ID NO: 12, and LCDR3 comprising the amino acid sequence represented by SEQ ID NO: 13. In some embodiments, the anti-human PD-1 binding domain of the bispecific antibody of the present disclosure includes (1) a heavy chain variable region containing the amino acid sequence represented by SEQ ID NO: 4, or having at least 80%, 85%, 90%, or 95% sequence identity thereto, and (2) a light chain variable region containing the amino acid sequence represented by SEQ ID NO: 14, or having at least 80%, 85%, 90%, or 95% sequence identity thereto, and the anti-human TGFβR2 binding domain of the bispecific antibody includes (1) a heavy chain variable region containing the amino acid sequence represented by SEQ ID NO: 9, or having at least 80%, 85%, 90%, or 95% sequence identity thereto, and (2) a light chain variable region containing the amino acid sequence represented by SEQ ID NO: 14, or having at least 80%, 85%, 90%, or 95% sequence identity thereto.In some embodiments, the anti-human PD-1 binding domain of the bispecific antibody of the present disclosure comprises: (1) a heavy chain variable region comprising HCDR1 containing the amino acid sequence represented by SEQ ID NO: 1, HCDR2 containing the amino acid sequence represented by SEQ ID NO: 2, and HCDR3 containing the amino acid sequence represented by SEQ ID NO: 3, and (2) a heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 4, or having at least 80%, 85%, 90%, or 95% sequence identity thereto; and (1) a light chain variable region comprising LCDR1 containing the amino acid sequence represented by SEQ ID NO: 11, LCDR2 containing the amino acid sequence represented by SEQ ID NO: 12, and LCDR3 containing the amino acid sequence represented by SEQ ID NO: 13, and (2) a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 14, or having at least 80%, 85%, 90%, or 95% sequence identity thereto. The anti-human TGFβR2 binding domain of the bispecific antibody comprises (1) a heavy chain variable region containing the amino acid sequence represented by SEQ ID NO: 6, HCDR1 containing the amino acid sequence represented by SEQ ID NO: 7, and HCDR3 containing the amino acid sequence represented by SEQ ID NO: 8, and (2) a heavy chain variable region containing the amino acid sequence represented by SEQ ID NO: 9, or having at least 80%, 85%, 90%, or 95% sequence identity thereto; and (1) a light chain variable region containing the amino acid sequence represented by SEQ ID NO: 11, LCDR1 containing the amino acid sequence represented by SEQ ID NO: 12, and LCDR3 containing the amino acid sequence represented by SEQ ID NO: 13, and (2) a light chain variable region containing the amino acid sequence represented by SEQ ID NO: 14, or having at least 80%, 85%, 90%, or 95% sequence identity thereto.

[0052] In some embodiments, the bispecific antibody comprises a human heavy chain and a light chain constant region. In some embodiments, the heavy chain constant region comprises a CH1 domain and a hinge region. In some embodiments, the heavy chain constant region comprises a CH2 domain. In some embodiments, the heavy chain constant region comprises a CH3 domain. In some embodiments, the heavy chain constant region comprises CH1, CH2, and CH3 domains. If the heavy chain constant region contains substitutions, such substitutions modify the antibody's properties (e.g., increasing or decreasing one or more of the following: Fc receptor binding, antibody glycosylation, number of cysteine ​​residues, effector cell function, or complement function). In some embodiments, the antibody is an IgG antibody. In some embodiments, the antibody is selected from the group consisting of IgG1, IgG2, IgG3, and IgG4.

[0053] Antibodies such as antibody A can be produced, for example, by preparing and expressing synthetic genes encoding the listed amino acid sequences, or by mutating human germline genes to provide genes encoding the listed amino acid sequences. Furthermore, this antibody and other bispecific antibodies can be obtained, for example, by using one or more of the following methods.

[0054] Humanized antibodies can be produced by replacing sequences in the Fv variable region, which are not directly involved in antigen binding, with equivalent sequences derived from the human Fv variable region. General methods for producing humanized antibodies are provided by Morrison, SL, Science, 229:1202-1207 (1985), Oi et al., BioTechniques, 4:214 (1986), and US5,585,089, US5,693,761, US5,693,762, US5,859,205, and US6,407,213. These methods involve isolating, manipulating, and expressing a nucleic acid sequence encoding all or part of the immunoglobulin Fv variable region from at least one of the heavy or light chains. Sources of such nucleic acids are well known to those skilled in the art and can be obtained, for example, from hybridomas producing antibodies against a given target, from germline immunoglobulin genes, or from synthetic constructs, as described above. Next, recombinant DNA encoding a humanized antibody can be cloned into a suitable expression vector.

[0055] Human germline sequences are disclosed, for example, in Tomlinson, IA et al., J. Mol. Bio., 227:776-798 (1992); Cook, GP et al., Immunol. Today, 16:237-242 (1995); Chothia, D. et al., J. Mol. Bio. 227:799-817 (1992); and Tomlinson et al., EMBO J., 14:4628-4638 (1995). The V BASE directory provides a comprehensive directory of human immunoglobulin variable region sequences (compiled by Tomlinson, IA et al., MRC Centre for Protein Engineering, Cambridge, UK). These sequences can be used, for example, as a source of human sequences for framework regions and CDRs. Consensus human framework regions can also be used, for example, as described in U.S. Patent No. 6,300,064.

[0056] Other methods for humanizing antibodies may also be used. For example, other methods can describe the three-dimensional structure of the antibody, the framework position in three-dimensional proximity to the binding determinant, and the immunogenic peptide sequence. See, for example, WO90 / 07861, U.S. Patents 5,693,762, 5,693,761, 5,585,089, 5,530,101, and 6,407,213, and Tempest et al. (1991) Biotechnology 9:266-271. Yet another method is called "humaneering" and is described, for example, in US2005-008625.

[0057] Antibodies disclosed herein may include human Fc regions, e.g., wild-type Fc regions or Fc regions containing one or more modifications. Antibodies may also have mutations that stabilize the disulfide bond between the two heavy chains of the immunoglobulin, such as mutations in the hinge region of IgG4 (e.g., Angal et al. (1993) Mol.Immunol. 30:105-08), as disclosed in the art. See also, for example, US2005 / 0037000.

[0058] This specification provides compositions comprising a mixture of a bispecific antibody and one or more acidic variants thereof, for example, the amount of acidic variant(s) being about 80%, 70%, 60%, 60%, 50%, 40%, 30%, 30%, 20%, 10%, 5%, or less than 1%. Also provided are compositions comprising a bispecific antibody containing at least one deamidation site, with a pH of about 5.0 to about 6.5 such that, for example, at least about 90% of the bispecific antibody is not deamidated (i.e., less than about 10% of the antibody is deamidated). In some embodiments, about 5%, 3%, 2%, or less than 1% of the antibody is deamidated. The pH may be 5.0 to 6.0, such as 5.5 or 6.0. In some embodiments, the pH of the composition is 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, or 6.5.

[0059] An "acidic mutant" is a variant of the target polypeptide that is more acidic than the target polypeptide (determined, for example, by cation exchange chromatography). An example of an acidic mutant is a deamidated mutant.

[0060] A "deamidation" mutant of a polypeptide molecule is a polypeptide in which one or more asparagine residues of the original polypeptide are converted to aspartic acid, that is, the neutral amide side chain is converted to a residue with overall acidic properties.

[0061] As used herein with respect to compositions containing a bispecific antibody, the term “mixture” means the presence of both the desired bispecific antibody and one or more acidic variants thereof. The acidic variants may primarily consist of the deamide bispecific antibody, along with small amounts of other acidic variants.

[0062] In some embodiments, the binding affinity (K) of the antibody is mutated to eliminate deamidation. D ), On Rate (K D On) and / or Off Rate (K D The off-label (off) antibody is similar to that of the wild-type antibody, for example, with differences of approximately 5 times, 2 times, 1 (100%), 50%, 30%, 20%, 10%, 5%, 3%, 2%, or less than 1%.

[0063] The bispecific antibodies of this disclosure can be prepared as full-length antibodies or in their low molecular weight forms (e.g., F(ab')2 bispecific antibodies, sc(Fv)2 bispecific antibodies, diabody bispecific antibodies).

[0064] Conventional production of full-length bispecific antibodies is based on the co-expression of two immunoglobulin heavy-light chain pairs, where these two chains have different specificities (Millstein et al., Nature, 305:537-539 (1983)). According to a different approach, an antibody variable domain with the desired binding specificity is fused to the immunoglobulin constant domain sequence. The immunoglobulin heavy chain fusion, and optionally the DNA encoding the immunoglobulin light chain, is inserted into separate expression vectors and co-transfected into suitable host cells. This provides greater flexibility in adjusting the ratio of the three polypeptide fragments. However, if high yields are obtained by expressing at least two polypeptide chains in equal ratios, it is possible to insert the coding sequences of two or all three polypeptide chains into a single expression vector.

[0065] According to another approach described in U.S. Patent No. 5,731,168, the interface between a pair of antibody molecules may be manipulated to maximize the proportion of heterodimers recovered from recombinant cell cultures. A preferred interface includes at least a portion of the CH3 domain. In this method, one or more smaller amino acid side chains originating from the interface of the first antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan). By replacing the larger amino acid side chains with smaller amino acid side chains (e.g., alanine or threonine), a compensatory "cavity" of the same or similar size as the larger side chain(s) is created on the interface of the second antibody molecule. This provides a mechanism for increasing the yield of heterodimers over other undesirable end products such as homodimers.

[0066] Examples of bispecific antibodies include crosslinked or "heteroconjugated" antibodies. For example, one antibody in a heteroconjugate may be coupled to avidin, while the other is coupled to biotin. Heteroconjugated antibodies can be prepared using any simple crosslinking method.

[0067] The "Diabody" technology provides an alternative mechanism for producing bispecific antibody fragments. These fragments contain a VH domain connected to a VL domain by a linker that is too short to allow pairing between two domains on the same chain. Thus, the VH and VL domains of one fragment are paired with the complementary VL and VH domains of another fragment, thereby forming two antigen-binding sites.

[0068] Methods for producing antibodies Antibodies can be produced in, for example, bacteria or eukaryotic cells. Some antibodies can be produced in bacterial cells, e.g., E. coli cells. Antibodies can also be produced in eukaryotic cells such as transformed cell lines (e.g., CHO, 293E, COS). Furthermore, antibodies can be expressed in yeast cells such as Pichia (e.g., Powers et al., J Immunol Methods. 251:123-35 (2001)), Hanseula, or Saccharomyces. To produce the antibody of interest, the polynucleotide encoding the antibody is constructed, introduced into an expression vector, and then expressed in a suitable host cell. Using standard molecular biology techniques, recombinant expression vectors are prepared, host cells are transfected, transformants are selected, the host cells are cultured, and the antibodies are recovered.

[0069] When antibodies are expressed in bacterial cells (e.g., E. coli), the expression vector is designed to have characteristics that allow for amplification of the vector within the bacterial cell. Furthermore, when using E. coli such as JM109, DH5α, HB101, or XL1-Blue as the host, the vector must have a promoter, e.g., the lacZ promoter (Ward et al., 341:544-546 (1989)), the araB promoter (Better et al., Science, 240:1041-1043 (1988)), or a T7 promoter that can enable efficient expression in E. coli. Examples of such vectors include, for example, the M13 series vectors, the pUC series vectors, pBR322, pBluescript, pCR-Script, pGEX-5X-1 (Pharmacia), "QIAexpress system" (QIAGEN), pEGFP, and pET (when this expression vector is used, the host is preferably BL21 expressing T7 RNA polymerase). The expression vector may contain a signal sequence for antibody secretion. For production into the peripheral plasma of E. coli, a pelB signal sequence (Lei et al.) may be used. The sequence al., J. Bacteriol., 169:4379 (1987) may be used as the signal sequence for antibody secretion. For bacterial expression, the expression vector may be introduced into bacterial cells using the calcium chloride method or electroporation method.

[0070] When expressing antibodies in animal cells such as CHO, COS, and NIH3T3 cells, the expression vector includes promoters necessary for expression in these cells, such as the SV40 promoter (Mulligan et al., Nature, 277:108 (1979)), the MMLV-LTR promoter, the EF1α promoter (Mizushima et al., Nucleic Acids Res., 18:5322 (1990)), or the CMV promoter. In addition to nucleic acid sequences encoding immunoglobulins or their domains, the recombinant expression vector may contain additional sequences, such as sequences that regulate vector replication in host cells (e.g., origin of replication) and selection marker genes. Selection marker genes can facilitate the selection of host cells into which the vector has been introduced (see, for example, U.S. Patents 4,399,216, 4,634,665, and 5,179,017). For example, selection marker genes typically confer resistance to drugs such as G418, hygromycin, and methotrexate to host cells into which the vector has been introduced. Examples of vectors containing selection markers include pMAM, pDR2, pBK-RSV, pBK-CMV, pOPRSV, and pOP13.

[0071] Antibodies for use in the methods described herein can be produced in mammalian cells. An exemplary mammalian host cell for antibody expression is Chinese hamster ovary (CHO) cell (dhfr, as described in Urlaub and Chasin (1980) Proc. Natl. Acad. Sci. USA 77:4216-4220). - Examples include CHO cells (used with a DHFR selection marker, for example, as described in Kaufman and Sharp (1982) Mol. Biol. 159:601-621), human embryonic kidney 293 cells (e.g., 293, 293E, 293T), COS cells, NIH3T3 cells, lymphocyte cell lines, such as NS0 myeloma cells and SP2 cells, and cells derived from transgenic animals, such as transgenic mammals.

[0072] In an exemplary system for antibody expression, a recombinant expression vector (or multiple vectors) encoding the antibody heavy chain and antibody light chain of a bispecific antibody (e.g., antibody A) is transfected via calcium phosphate-mediated transfection (dhfr). - The recombinant expression vector is introduced into CHO cells. Within the recombinant expression vector, the antibody heavy chain and light chain genes are each ligated to enhancer / promoter regulatory elements (e.g., CMV enhancer / AdMLP promoter regulatory elements or SV40 enhancer / AdMLP promoter regulatory elements derived from SV40, CMV, adenovirus, etc.) to drive high levels of gene transcription. The recombinant expression vector also contains a DHFR gene that allows selection of CHO cells transfected with the vector using methotrexate selection / amplification. The selected transformed host cells are cultured to allow expression of the antibody heavy chain and light chain, and the antibodies are recovered from the culture medium.

[0073] Antibodies can also be produced by transgenic animals. For example, U.S. Patent No. 5,849,992 describes a method for expressing antibodies in the mammary glands of a transgenic mammal. A transgene is constructed containing a milk-specific promoter and a nucleic acid encoding the antibody of interest, as well as a signal sequence for secretion. The milk produced by such a female transgenic mammal contains the antibody of interest, which is secreted therein. The antibody can be purified from the milk or used directly for several applications.

[0074] The antibodies of this disclosure can be isolated from inside or outside host cells (e.g., from culture media) and purified as substantially pure and homogeneous antibodies. Isolation and purification methods commonly used for antibody purification may be used for the isolation and purification of antibodies, and are not limited to any particular method. Antibodies can be isolated and purified, for example, by appropriately selecting and combining column chromatography, filtration, ultrafiltration, salting out, solvent precipitation, solvent extraction, distillation, immunoprecipitation, SDS-polyacrylamide gel electrophoresis, isoelectrofocusing, dialysis, and recrystallization. Examples of chromatography include affinity chromatography, ion exchange chromatography, hydrophobic chromatography, gel filtration, reversed-phase chromatography, and adsorption chromatography (Strategies for Protein Purification and Characterization: A Laboratory Course Manual. Ed. Daniel R. Marshak et al., Cold Spring Harbor Laboratory Press, 1996). Chromatography may be performed using liquid-phase chromatography such as HPLC and FPLC. Examples of columns used in affinity chromatography include protein A columns and protein G columns. Examples of columns using Protein A columns include Hyper D, POROS, and Sepharose FF (GE Healthcare Biosciences). This disclosure also includes antibodies highly purified using these purification methods.

[0075] Antibody pharmaceutical compositions and administration The bispecific antibodies described herein can be formulated as pharmaceutical compositions for administration to a subject, for example, to treat the disorders described herein. Typically, the pharmaceutical composition comprises a pharmaceutically acceptable carrier. As used herein, “pharmaceutically acceptable carrier” includes any physiologically compatible solvent, dispersion medium, coating, antimicrobial and antifungal agent, isotonic agent and absorption retardant, etc. The composition may contain a pharmaceutically acceptable salt, such as an acid-added salt or a base-added salt (see, for example, Berge, SM, et al. (1977) J. Pharm. Sci. 66:1-19).

[0076] Bispecific antibodies can be administered by intravenous injection or infusion (IV) to subjects who require them, such as human subjects.

[0077] Bispecific antibodies can be administered as a fixed dose or in doses of mg / kg patient body weight. The dose may also be selected to reduce or avoid antibody production against the bispecific antibody. The drug regimen is adjusted to provide the desired response, e.g., a therapeutic response or a combined therapeutic effect. Generally, the dose of a bispecific antibody may be used to deliver the drug to the subject in an amount available to the organism.

[0078] For example, doses ranging from approximately 0.1 mg / kg to approximately 30 mg / kg can be administered. In some embodiments, subjects are administered antibodies in doses ranging from approximately 0.1 mg / kg to approximately 10 mg / kg (e.g., doses of approximately 0.1 mg / kg, 0.5 mg / kg, 1 mg / kg, 1.5 mg / kg, 2 mg / kg, 3 mg / kg, 5 mg / kg, 6 mg / kg, 7.5 mg / kg, or approximately 10 mg / kg). In other embodiments, subjects are administered antibodies in doses ranging from approximately 1 mg / kg to approximately 3 mg / kg (e.g., doses of approximately 1 mg / kg, 2 mg / kg, or 3 mg / kg). With respect to dose or dosage, the term "approximately" is intended to indicate a range of ±10% of the listed doses, for example, a dose of approximately 3 mg / kg being between 2.7 mg / kg patient weight and 3.3 mg / kg patient weight.

[0079] As used herein, the terms "dosage unit form," "fixed dose," or "uniform dose" refer to physically separate units suitable as a unit dosage for treating a subject, each unit containing a predetermined amount of the active compound calculated to produce the desired therapeutic effect in relation to the required pharmaceutical carrier and, optionally, in relation to other agents. Single or multiple doses may be administered. Alternatively or additionally, antibodies may be administered via serial infusion. For example, uniform doses ranging from approximately 20 mg to 2500 mg may be administered. In some embodiments, subjects are administered antibodies in doses of approximately 20 mg, 50 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 450 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, 1000 mg, 1100 mg, 1200 mg, 1300 mg, 1400 mg, 1500 mg, 1600 mg, 1700 mg, 1800 mg, 1900 mg, 2000 mg, 2100 mg, 2200 mg, 2300 mg, 2400 mg, or 2500 mg. In some embodiments, subjects are administered antibodies in doses of approximately 100 mg, 300 mg, 900 mg, 1500 mg, or 2000 mg.

[0080] The dose of the bispecific antibody can be administered at regular intervals over a sufficient period (course of treatment) to cover, for example, at least two, three, five, ten, or more doses, such as weekly, every other week (once every two weeks), every three weeks, every four weeks, or monthly. Factors that may influence the dosage and timing required to effectively treat the subject include, for example, the severity of the disease or disorder, the formulation, the route of delivery, previous treatments, the subject's general health and / or age, and other pre-existing conditions. Furthermore, treatment of a subject with a therapeutically effective amount of the compound may consist of a single treatment or, preferably, a series of treatments.

[0081] An exemplary uniform-dose regimen includes intravenous administration of a bispecific antibody (e.g., antibody A) described herein, at a dose of approximately 100 mg once every two weeks.

[0082] A more exemplary uniform-dose regimen includes intravenous administration of a bispecific antibody (e.g., antibody A) described herein, once every two weeks, at a dose of approximately 300 mg.

[0083] A more exemplary uniform-dose regimen includes intravenous administration of a bispecific antibody (e.g., antibody A) described herein, once every two weeks, at a dose of approximately 900 mg.

[0084] A more exemplary uniform-dose regimen includes intravenous administration of a bispecific antibody (e.g., antibody A) described herein, once every two weeks, at a dose of approximately 1500 mg.

[0085] A more exemplary uniform-dose regimen includes intravenous administration of a bispecific antibody (e.g., antibody A) described herein, once every two weeks, at a dose of approximately 2000 mg.

[0086] An exemplary uniform-dose regimen includes intravenous administration of a bispecific antibody (e.g., antibody A) described herein, at a dose of approximately 100 mg once every four weeks.

[0087] A more exemplary uniform-dose regimen includes intravenous administration of a bispecific antibody (e.g., antibody A) described herein, once every four weeks, at a dose of approximately 300 mg.

[0088] A more exemplary uniform-dose regimen includes intravenous administration of a bispecific antibody (e.g., antibody A) described herein, once every four weeks, at a dose of approximately 900 mg.

[0089] A more exemplary uniform-dose regimen includes intravenous administration of a bispecific antibody (e.g., antibody A) described herein, once every four weeks, at a dose of approximately 1500 mg.

[0090] A more exemplary uniform-dose regimen includes intravenous administration of a bispecific antibody (e.g., antibody A) described herein, once every four weeks, at a dose of approximately 2000 mg.

[0091] A pharmaceutical composition may contain a “therapeutic dose” of the bispecific antibodies described herein. Such a dose may be determined based on the effect of the administered drug or, if two or more drugs are used, the combined effect of the drugs. The therapeutic dose of a drug may also vary due to factors such as the individual’s disease state, age, sex, and weight, as well as the compound’s ability to elicit a desired response in the individual, e.g., improvement of at least one disorder parameter or improvement of symptoms of at least one disorder. The therapeutic dose is also such that the therapeutically beneficial effect outweighs any toxic or adverse effects of the composition.

[0092] Indications The bispecific antibodies described herein (e.g., antibody A) can be used to treat non-small cell lung cancer (NSCLC). In some embodiments, NSCLC has a squamous or non-squamous histological type. In some embodiments, NSCLC is progressive or metastatic. In some embodiments, the subject has experienced disease progression after prior treatment (e.g., prior anti-PD-(L)1 therapy and / or anti-CTLA4 therapy).

[0093] The bispecific antibodies described herein (e.g., antibody A) can be used to treat squamous cell carcinoma (SCCHN) of the head and neck. In some embodiments, SCCHN includes primary squamous cell tumors of the oral cavity, oropharynx, hypopharynx, or larynx. In some embodiments, SCCHN includes human papillomavirus (HPV)-positive SCCHN. In some embodiments, SCCHN is progressive or metastatic. In some embodiments, the subject has experienced disease progression after prior treatment (e.g., prior anti-PD-(L)1 therapy and / or anti-CTLA4 therapy).

[0094] The bispecific antibodies described herein (e.g., antibody A) can be used to treat squamous cell carcinoma of the cervix and adenocarcinoma of the endocervical cavity (CESC). In some embodiments, CESC includes human papillomavirus (HPV)-positive CESC. In some embodiments, CESC is progressive or metastatic. In some embodiments, the subject has experienced disease progression after prior treatment (e.g., prior anti-PD-(L)1 therapy and / or anti-CTLA4 therapy).

[0095] The bispecific antibodies described herein (e.g., antibody A) can be used to treat ovarian cancer. In some embodiments, ovarian cancer includes epithelial ovarian cancer, fallopian tube cancer, primary peritoneal cancer, or carcinosarcoma. In some embodiments, ovarian cancer is progressive or metastatic. In some embodiments, the subject has experienced disease progression after prior treatment (e.g., prior anti-PD-(L)1 therapy and / or anti-CTLA4 therapy).

[0096] The bispecific antibodies described herein (e.g., antibody A) can be used to treat breast cancer. In some embodiments, breast cancer includes triple-negative breast cancer. In some embodiments, triple-negative breast cancer includes HER2-negative, ER-negative, and PgR-negative breast cancer. In some embodiments, breast cancer is advanced or metastatic. In some embodiments, the subject has experienced disease progression after prior treatment (e.g., prior anti-PD-(L)1 therapy and / or anti-CTLA4 therapy).

[0097] The bispecific antibodies described herein (e.g., antibody A) can be used to treat bladder cancer. In some embodiments, bladder cancer includes urothelial carcinoma. In some embodiments, bladder cancer is progressive or metastatic. In some embodiments, the subject has experienced disease progression after prior treatment (e.g., prior anti-PD-(L)1 therapy and / or anti-CTLA4 therapy).

[0098] The bispecific antibodies described herein (e.g., antibody A) can be used to treat renal cell carcinoma. In some embodiments, the renal cell carcinoma is progressive or metastatic. In some embodiments, the subject has experienced disease progression after prior treatment (e.g., prior anti-PD-(L)1 therapy and / or anti-CTLA4 therapy).

[0099] The bispecific antibodies described herein (e.g., antibody A) can be used to treat melanoma. In some embodiments, the melanoma is cutaneous malignant melanoma. In some embodiments, the melanoma is progressive or metastatic. In some embodiments, the subject has experienced disease progression after prior treatment (e.g., prior anti-PD-(L)1 therapy and / or anti-CTLA4 therapy).

[0100] The bispecific antibodies described herein (e.g., antibody A) can be used to treat gastric adenocarcinoma. In some embodiments, the gastric adenocarcinoma is progressive or metastatic. In some embodiments, the subject has experienced disease progression after prior treatment (e.g., prior anti-PD-(L)1 therapy and / or anti-CTLA4 therapy).

[0101] The bispecific antibodies described herein (e.g., antibody A) can be used to treat esophageal cancer. In some embodiments, the esophageal cancer is progressive or metastatic. In some embodiments, the subject has experienced disease progression after prior treatment (e.g., prior anti-PD-(L)1 therapy and / or anti-CTLA4 therapy).

[0102] The bispecific antibodies described herein (e.g., antibody A) can be used to treat esophageal and gastric adenocarcinoma. In some embodiments, the esophageal and gastric adenocarcinoma is progressive or metastatic. In some embodiments, the subject has experienced disease progression after prior treatment (e.g., prior anti-PD-(L)1 therapy and / or anti-CTLA4 therapy).

[0103] The bispecific antibodies described herein (e.g., antibody A) can be used to treat malignant pleural mesothelioma. In some embodiments, malignant pleural mesothelioma is progressive or metastatic. In some embodiments, the subject has experienced disease progression after prior treatment (e.g., prior anti-PD-(L)1 therapy and / or anti-CTLA4 therapy).

[0104] The bispecific antibodies described herein (e.g., antibody A) can be used to treat pancreatic adenocarcinoma. In some embodiments, the pancreatic adenocarcinoma is progressive or metastatic. In some embodiments, the subject has experienced disease progression after prior treatment (e.g., prior anti-PD-(L)1 therapy and / or anti-CTLA4 therapy).

[0105] The bispecific antibodies described herein (e.g., antibody A) can be used to treat colorectal cancer (CRC). In some embodiments, CRC includes microsatellite-stable colorectal cancer (MSS-CRC). In some embodiments, CRC includes mismatch repair deficiency (dMMR) / high microsatellite instability (MSI-H) colorectal cancer (dMMR / MSI-H CRC). In some embodiments, CRC is advanced or metastatic. In some embodiments, the subject has experienced disease progression after prior treatment (e.g., prior anti-PD-(L)1 therapy and / or anti-CTLA4 therapy).

[0106] Another embodiment involves a bispecific antibody (e.g., antibody A) described herein for use in the treatment of non-small cell lung cancer (NSCLC). In some embodiments, the NSCLC has a squamous or non-squamous histological type. In some embodiments, the NSCLC is progressive or metastatic. In some embodiments, the subject has experienced disease progression after prior treatment (e.g., prior anti-PD-(L)1 therapy and / or anti-CTLA4 therapy).

[0107] Another embodiment involves a bispecific antibody (e.g., antibody A) described herein for use in the treatment of squamous cell carcinoma of the head and neck (SCCHN). In some embodiments, SCCHN includes primary squamous cell tumors of the oral cavity, oropharynx, hypopharynx, or larynx. In some embodiments, SCCHN includes human papillomavirus (HPV)-positive SCCHN. In some embodiments, SCCHN is progressive or metastatic. In some embodiments, the subject has experienced disease progression after prior treatment (e.g., prior anti-PD-(L)1 therapy and / or anti-CTLA4 therapy).

[0108] Another embodiment involves a bispecific antibody (e.g., antibody A) described herein for use in the treatment of cervical squamous cell carcinoma and endocervical adenocarcinoma (CESC). In some embodiments, CESC includes human papillomavirus (HPV)-positive CESC. In some embodiments, CESC is progressive or metastatic. In some embodiments, the subject has experienced disease progression after prior treatment (e.g., prior anti-PD-(L)1 therapy and / or anti-CTLA4 therapy).

[0109] Another embodiment involves a bispecific antibody (e.g., antibody A) described herein for use in the treatment of ovarian cancer. In some embodiments, ovarian cancer includes epithelial ovarian cancer, fallopian tube cancer, primary peritoneal cancer, or carcinosarcoma. In some embodiments, ovarian cancer is advanced or metastatic. In some embodiments, the subject has experienced disease progression after prior treatment (e.g., prior anti-PD-(L)1 therapy and / or anti-CTLA4 therapy).

[0110] Another embodiment involves a bispecific antibody (e.g., antibody A) described herein for use in the treatment of breast cancer. In some embodiments, breast cancer includes triple-negative breast cancer. In some embodiments, triple-negative breast cancer includes HER2-negative, ER-negative, and PgR-negative breast cancer. In some embodiments, breast cancer is advanced or metastatic. In some embodiments, the subject has experienced disease progression after prior treatment (e.g., prior anti-PD-(L)1 therapy and / or anti-CTLA4 therapy).

[0111] Another embodiment involves a bispecific antibody (e.g., antibody A) described herein for use in the treatment of bladder cancer. In some embodiments, bladder cancer includes urothelial carcinoma. In some embodiments, bladder cancer is progressive or metastatic. In some embodiments, the subject has experienced disease progression after prior treatment (e.g., prior anti-PD-(L)1 therapy and / or anti-CTLA4 therapy).

[0112] Another embodiment involves a bispecific antibody (e.g., antibody A) described herein for use in the treatment of renal cell carcinoma. In some embodiments, the renal cell carcinoma is advanced or metastatic. In some embodiments, the subject has experienced disease progression after prior treatment (e.g., prior anti-PD-(L)1 therapy and / or anti-CTLA4 therapy).

[0113] Another embodiment involves a bispecific antibody (e.g., antibody A) described herein for use in the treatment of melanoma. In some embodiments, the melanoma is cutaneous malignant melanoma. In some embodiments, the melanoma is progressive or metastatic. In some embodiments, the subject has experienced disease progression after prior treatment (e.g., prior anti-PD-(L)1 therapy and / or anti-CTLA4 therapy).

[0114] Another embodiment involves a bispecific antibody (e.g., antibody A) described herein for use in the treatment of gastric adenocarcinoma. In some embodiments, the gastric adenocarcinoma is progressive or metastatic. In some embodiments, the subject has experienced disease progression after prior treatment (e.g., prior anti-PD-(L)1 therapy and / or anti-CTLA4 therapy).

[0115] Another embodiment involves a bispecific antibody (e.g., antibody A) described herein for use in the treatment of esophageal cancer. In some embodiments, the esophageal cancer is progressive or metastatic. In some embodiments, the subject has experienced disease progression after prior treatment (e.g., prior anti-PD-(L)1 therapy and / or anti-CTLA4 therapy).

[0116] Another embodiment involves a bispecific antibody (e.g., antibody A) described herein for use in the treatment of esophageal and gastric adenocarcinoma. In some embodiments, the esophageal and gastric adenocarcinoma is progressive or metastatic. In some embodiments, the subject has experienced disease progression after prior treatment (e.g., prior anti-PD-(L)1 therapy and / or anti-CTLA4 therapy).

[0117] Another embodiment involves a bispecific antibody (e.g., antibody A) described herein for use in the treatment of malignant pleural mesothelioma. In some embodiments, the malignant pleural mesothelioma is progressive or metastatic. In some embodiments, the subject has experienced disease progression after prior treatment (e.g., prior anti-PD-(L)1 therapy and / or anti-CTLA4 therapy).

[0118] Another embodiment involves a bispecific antibody (e.g., antibody A) described herein for use in the treatment of pancreatic adenocarcinoma. In some embodiments, the pancreatic adenocarcinoma is advanced or metastatic. In some embodiments, the subject has experienced disease progression after prior treatment (e.g., prior anti-PD-(L)1 therapy and / or anti-CTLA4 therapy).

[0119] Another embodiment involves a bispecific antibody (e.g., antibody A) described herein for use in the treatment of colorectal cancer (CRC). In some embodiments, the CRC includes microsatellite-stable colorectal cancer (MSS-CRC). In some embodiments, the CRC includes mismatch repair deficiency (dMMR) / high microsatellite instability (MSI-H) colorectal cancer (dMMR / MSI-H CRC). In some embodiments, the CRC is advanced or metastatic. In some embodiments, the subject has experienced disease progression after prior treatment (e.g., prior anti-PD-(L)1 therapy and / or anti-CTLA4 therapy).

[0120] Another embodiment involves a bispecific antibody (e.g., antibody A) described herein in the manufacture of a pharmaceutical product for the treatment of non-small cell lung cancer (NSCLC). In some embodiments, NSCLC has a squamous or non-squamous histological type. In some embodiments, NSCLC is progressive or metastatic. In some embodiments, the subject has experienced disease progression after prior treatment (e.g., prior anti-PD-(L)1 therapy and / or anti-CTLA4 therapy).

[0121] Another embodiment involves a bispecific antibody (e.g., antibody A) described herein in the manufacture of a pharmaceutical product for the treatment of squamous cell carcinoma of the head and neck (SCCHN). In some embodiments, SCCHN includes primary squamous cell tumors of the oral cavity, oropharynx, hypopharynx, or larynx. In some embodiments, SCCHN includes human papillomavirus (HPV)-positive SCCHN. In some embodiments, SCCHN is progressive or metastatic. In some embodiments, the subject has experienced disease progression after prior treatment (e.g., prior anti-PD-(L)1 therapy and / or anti-CTLA4 therapy).

[0122] Another embodiment involves a bispecific antibody (e.g., antibody A) described herein in the manufacture of a pharmaceutical product for the treatment of cervical squamous cell carcinoma and endocervical adenocarcinoma (CESC). In some embodiments, CESC includes human papillomavirus (HPV)-positive CESC. In some embodiments, CESC is progressive or metastatic. In some embodiments, the subject has experienced disease progression after prior treatment (e.g., prior anti-PD-(L)1 therapy and / or anti-CTLA4 therapy).

[0123] Another embodiment involves a bispecific antibody (e.g., antibody A) described herein in the manufacture of a pharmaceutical product for the treatment of ovarian cancer. In some embodiments, ovarian cancer includes epithelial ovarian cancer, fallopian tube cancer, primary peritoneal cancer, or carcinosarcoma. In some embodiments, ovarian cancer is progressive or metastatic. In some embodiments, the subject has experienced disease progression after prior treatment (e.g., prior anti-PD-(L)1 therapy and / or anti-CTLA4 therapy).

[0124] Another embodiment involves a bispecific antibody (e.g., antibody A) described herein in the manufacture of a pharmaceutical product for the treatment of breast cancer. In some embodiments, breast cancer includes triple-negative breast cancer. In some embodiments, triple-negative breast cancer includes HER2-negative, ER-negative, and PgR-negative breast cancer. In some embodiments, breast cancer is progressive or metastatic. In some embodiments, the subject has experienced disease progression after prior treatment (e.g., prior anti-PD-(L)1 therapy and / or anti-CTLA4 therapy).

[0125] Another embodiment involves a bispecific antibody (e.g., antibody A) described herein in the manufacture of a pharmaceutical product for the treatment of bladder cancer. In some embodiments, bladder cancer includes urothelial carcinoma. In some embodiments, bladder cancer is progressive or metastatic. In some embodiments, the subject has experienced disease progression after prior treatment (e.g., prior anti-PD-(L)1 therapy and / or anti-CTLA4 therapy).

[0126] Another embodiment involves a bispecific antibody (e.g., antibody A) described herein for use in the treatment of renal cell carcinoma. In some embodiments, the renal cell carcinoma is advanced or metastatic. In some embodiments, the subject has experienced disease progression after prior treatment (e.g., prior anti-PD-(L)1 therapy and / or anti-CTLA4 therapy).

[0127] Another embodiment involves a bispecific antibody (e.g., antibody A) described herein in the manufacture of a pharmaceutical product for the treatment of melanoma. In some embodiments, the melanoma is cutaneous malignant melanoma. In some embodiments, the melanoma is progressive or metastatic. In some embodiments, the subject has experienced disease progression after prior treatment (e.g., prior anti-PD-(L)1 therapy and / or anti-CTLA4 therapy).

[0128] Another embodiment involves a bispecific antibody (e.g., antibody A) described herein in the manufacture of a pharmaceutical product for treating gastric adenocarcinoma. In some embodiments, the gastric adenocarcinoma is progressive or metastatic. In some embodiments, the subject has experienced disease progression after prior treatment (e.g., prior anti-PD-(L)1 therapy and / or anti-CTLA4 therapy).

[0129] Another embodiment involves a bispecific antibody (e.g., antibody A) described herein in the manufacture of a pharmaceutical product for the treatment of esophageal cancer. In some embodiments, the esophageal cancer is progressive or metastatic. In some embodiments, the subject has experienced disease progression after prior treatment (e.g., prior anti-PD-(L)1 therapy and / or anti-CTLA4 therapy).

[0130] Another embodiment involves a bispecific antibody (e.g., antibody A) described herein in the manufacture of a pharmaceutical product for the treatment of esophageal and gastric adenocarcinoma. In some embodiments, the esophageal and gastric adenocarcinoma is progressive or metastatic. In some embodiments, the subject has experienced disease progression after prior treatment (e.g., prior anti-PD-(L)1 therapy and / or anti-CTLA4 therapy).

[0131] Another embodiment involves a bispecific antibody (e.g., antibody A) described herein in the manufacture of a pharmaceutical product for the treatment of malignant pleural mesothelioma. In some embodiments, malignant pleural mesothelioma is progressive or metastatic. In some embodiments, the subject has experienced disease progression after prior treatment (e.g., prior anti-PD-(L)1 therapy and / or anti-CTLA4 therapy).

[0132] Another embodiment involves a bispecific antibody (e.g., antibody A) described herein in the manufacture of a pharmaceutical product for the treatment of pancreatic adenocarcinoma. In some embodiments, the pancreatic adenocarcinoma is progressive or metastatic. In some embodiments, the subject has experienced disease progression after prior treatment (e.g., prior anti-PD-(L)1 therapy and / or anti-CTLA4 therapy).

[0133] Another embodiment involves a bispecific antibody (e.g., antibody A) described herein in the manufacture of a pharmaceutical product for the treatment of colorectal cancer (CRC). In some embodiments, CRC includes microsatellite-stable colorectal cancer (MSS-CRC). In some embodiments, CRC includes mismatch repair deficiency (dMMR) / high microsatellite instability (MSI-H) colorectal cancer (dMMR / MSI-H CRC). In some embodiments, CRC is progressive or metastatic. In some embodiments, the subject has experienced disease progression after prior treatment (e.g., prior anti-PD-(L)1 therapy and / or anti-CTLA4 therapy).

[0134] The following are examples relating to the implementation of the present invention. These examples should not be construed as limiting the scope of the present invention in any way. [Examples]

[0135] The following examples are provided to better illustrate the claimed invention and are not intended to limit the scope of the invention. To the extent that specific materials are mentioned, they are for illustrative purposes only and are not intended to limit the invention. Those skilled in the art can develop equivalent means or reactants without exercising their inventive capacity and without departing from the scope of the invention.

[0136] The reference antibody and control antibody used in this example include the following: • Reference PD-1 antibody pembrolizumab (also known as pembro or anti-PD-1) (manufactured by Merck, marketed by Myonex) • Reference PD-L1 antibody atezolizumab (also known as Atezolizumab) (manufactured by Genentech) • Cetuximab x OKT3 is a bispecific antibody containing cetuximab (anti-EGFR antibody) to target EGFR and OKT3 (anti-CD3 antibody) to target CD3. • Reference antibody NIS793 (manufactured by Novartis) is a fully human anti-TGFβ IgG2 monoclonal antibody designed to inhibit the TGFβ pathway. • A bivalent monospecific analog of TGF1, comprising two heavy chains having the amino acid sequence represented by SEQ ID NO: 16 and two light chains having the amino acid sequence represented by SEQ ID NO: 17, as a reference anti-TGFβR2 antibody TGF1 (also referred to as TGF1, αTGFβR2, anti-TGFBRII, or anti-TGFBR2). • A bivalent, monospecific antibody containing two heavy chains with the amino acid sequence represented by SEQ ID NO: 18 and two light chains with the amino acid sequence represented by SEQ ID NO: 19; a negative control IgG1 antibody (also called RSVxRSV, RSV control, RSV, αRSV, aRSV, or anti-RSV).

[0137] [Table 4]

[0138] Example 1: Antibody A counteracted the immunosuppressive effect of TGF-β on T cell-mediated cytotoxicity in renal cancer cells. This example describes the in vitro evaluation of various antibodies regarding the immunosuppressive effect of TGF-β on T cell-mediated cytotoxicity induced by a bispecific antibody against EGFR and CD3 (cetuximab xCD3).

[0139] In vitro cytotoxicity assay: Adherent 786-O renal cancer cells were treated with trypsin (0.05% trypsin / EDTA, Gibco catalog number 25300-054) and harvested. Cells (1.0 × 10⁶) 4 Cells (per well) were seeded in phenol-free complete RPMI medium in 96-well block-walled transparent-bottom plates (Greiner No. 655090) and incubated overnight at 37°C under a humidified 5% CO2 atmosphere. 786-O renal cancer cells were pre-incubated for 5-10 minutes at room temperature with or without bispecific antibodies and control antibodies, in or without TGF-β. Subsequently, human CD3+ T cells in AIM V medium were added at an E / T ratio of 10:1 and incubated for 70 hours. The release of lactate dehydrogenase (LDH) from membrane-damaged cells into the supernatant was measured using fluorescence assays (560nm Ex, 590nm Em) according to the manufacturer's instructions (CytoTox-ONE® Homogeneous Membrane Integrity Assay, Promega). Relative fluorescence units (RFU) in each treatment group and the multiplier change in RFU compared to the control group were calculated.

[0140] Results: To mimic an immunosuppressive environment, exogenous TGF-β (0.1 ng / mL or 1.0 ng / mL) was added to a co-culture system of renal cancer cells and effector T cells. The cells were treated with 1.0 μg / mL of cetuximab xCD3 bispecific antibody (BsAb) in combination with one of the following antibodies: antibody A, anti-PD-1 antibody (pembrolizumab), anti-TGFβR2 antibody, or bivalent antibody against RSV (RSV x RSV) used as a negative control antibody. Antibodies were added to the cells at concentrations of 0.1–100 μg / mL.

[0141] Antibody A was more effective than anti-PD-1 antibody, anti-TGFβR2 antibody, or RSVxRSV control antibody in counteracting the immunosuppressive effect of TGF-β (0.1 ng / mL) on cetuximab xCD3 BsAb-induced T cell-mediated cytotoxicity (Figure 1A-1B). Antibody A produced similar effects in co-culture systems without TGF-β (Figure 1C-1D) or in co-culture systems containing TGF-β at a concentration of 1.0 ng / mL (Figure 1E-1F), but to a lesser extent than in co-culture systems containing TGF-β at a concentration of 0.1 ng / mL.

[0142] The concentration (1.0 μg / ml) and treatment time (70 hours) of cetuximab xCD3 BsAb selected for this combination study were based on the dose-response and time course of cetuximab xCD3 BsAb alone inducing T cell-mediated target 786-O renal cancer cell killing.

[0143] Example 2: Antibody A inhibited tumor growth in a xenograft mouse model of melanoma. This example describes the in vivo evaluation of various antibodies in A375 human melanoma using a xenograft mouse model.

[0144] Antibodies and formulations: Antibody A was formulated in a pH 6.5 buffer containing 10 mM histidine and 263 mM sucrose. The compound was solubilized in Dulbecco's phosphate-buffered saline (DPBS) (ThermoFisher Scientific, Waltham, MA, catalog no. 14190235). IgG1 control (Fc Silent), TGF1 Fc Silent (TGFβR2 antibody), and NIS793 (TGFβ1, 2, and 3 antibodies) were produced by Incyte (Wilmington, DE). Atezolizumab (anti-PD-L1 antibody) and pembrolizumab (anti-PD-1 antibody) were purchased from RefDrug Inc. (Hillsborough Township, NJ).

[0145] A375 xenograft model: Female huCD34+ NSG mice (Jackson Labs, 19-21 weeks old) were shaved and then suspended in 200 μL of DPBS (ThermoFisher Scientific) and Matrigel (Corning) in a 1:1 mixture, with 5 × 10⁶ mice. 6 Individual A375 cells (ATCC number CRL-1619) were subcutaneously inoculated. Eight days after cell transplantation, the tumor was approximately 160 mm. 3 At that point, mice were randomized into groups of 10 based on tumor size and stem cell donor. NSG mice engrafted with human CD34+ stem cells from four different donors were randomly assigned to each group of 10 mice (2-3 mice per donor within each group). Each group received 10 mg / kg of antibody in DPBS via intraperitoneal injection once every 5 days. Dose administration was continued throughout the study. Plasma and tumor samples were collected from mice 24, 72, or 108 hours after the last antibody dose. Statistical significance between treatment groups was determined using two-way ANOVA.

[0146] Results: To determine the in vivo efficacy of antibody A in A375 human melanoma, huCD34+ NSG mice were treated every 5 days with either 10 mg / kg of RSVxRSV control, atezolizumab, pembrolizumab, TGF1 (Fc Silent), a combination of pembrolizumab and TGF1, antibody A, or NIS793. Antibody A treatment resulted in statistically significant tumor growth inhibition (TGI) compared to the RSVxRSV control antibody (TGI = 75%, p = 0.002) (Figure 2A). The effect of antibody A on tumor volume reduction in mice treated with the pembrolizumab + TGF1 antibody combination (p < 0.05), mice treated with pembrolizumab alone (p < 0.05), and mice treated with TGF1 alone (p < 0.0001) was also statistically significant (Figure 2A). Table 5 shows the TGI values ​​for all groups. No adverse effects on animal body weight were observed with any of the treatments (Figure 2B).

[0147] [Table 5]

[0148] To determine the effect of the stem cell donor on the activity of antibody A, the individual tumor volumes for each treatment group were plotted according to the stem cell donor. The data showed that a response to antibody A was observed in all 4 donors (Figures 3A - 3H). Partial responses were observed in mice administered atezolizumab, TGF1, and pembrolizumab + TGF1.

[0149] Example 3: Antibody A inhibited tumor growth in a xenograft mouse model of triple - negative breast cancer (TNBC) In this example, an in vivo evaluation of various antibodies in MDA - MB - 231 human triple - negative breast cancer (TNBC) using a xenograft mouse model is described.

[0150] Humanized NSG MDA - MB - 231 mouse model: Humanized CD34 NSG mice were subcutaneously inoculated with a total of 3×10 6 individual MDA - MB - 231 tumor cells suspended in 100 μL of serum - free culture medium and Matrigel matrix (Corning) of equal volume. After the tumors became established (80 - 100 mm 3 ), the mice were randomized into the following treatment groups. · Negative control IgG1 antibody (RSV) (10 mg / kg) · Antibody A (1 mg / kg) · Antibody A (10 mg / kg) · Pembrolizumab (1 mg / kg)

[0151] Each group had 8 - 9 mice. The animals were administered intraperitoneally every 5 days for a period of 27 or 30 days. Tumors were measured using calipers, and tumor volumes were calculated by assimilating them to ellipsoids using the formula: l (length)×w 2 (width)×1 / 2. Body weights were also monitored throughout the study. Tumors were harvested after the end of the study for tumor immunoprofiling and receptor occupancy (24 hours after the last administration).

[0152] Results: Antibody A, a bispecific antibody, induced a superior antitumor response compared to the reference PD-1 antibody pembrolizumab and the negative control IgG1 antibody RSV (Figure 4). Antibody A induced a superior antitumor response compared to 10 mg / kg pembrolizumab at both 1 mg / kg and 10 mg / kg dose levels (Figure 4).

[0153] Example 4: Antibody A inhibited tumor growth in a xenograft mouse model of colorectal cancer. This example describes the in vivo evaluation of various antibodies in MC38 human colorectal cancer using a xenograft mouse model.

[0154] Antibodies and formulations: Antibody A was formulated in a pH 6.5 buffer containing 10 mM histidine and 263 mM sucrose. The compound was solubilized in Dulbecco's phosphate-buffered saline (DPBS) (ThermoFisher Scientific, Waltham, MA, catalog no. 14190235). Mice were treated with either negative control IgG1 antibody (RSV), pembrolizumab (anti-PD-1 antibody), reference anti-TGFβR2 antibody (TGF1), pembrolizumab and reference anti-TGFβR2 antibody (TGF1), or antibody A.

[0155] MC38 colorectal cancer xenograft model: Female huCD34+ NSG mice (Jackson Labs, 19-21 weeks old) were shaved and then transplanted 2 × 10⁻¹⁶ times. 6 Individual MC38 cells were subcutaneously inoculated. Nine days after cell transplantation, the tumor was approximately 110 mm. 3 At that point, mice were randomized into groups of nine based on tumor size. Each group received 10 mg / kg of the indicated antibody via intraperitoneal injection. Mice treated with a combination of pembrolizumab and TGF1 were given 10 mg / kg of each antibody. Antibodies were administered twice weekly. Cured mice were re-challenged with MC38 colorectal cancer cells, and tumor volume was monitored over time. Statistical significance between treatment groups was determined using two-way ANOVA.

[0156] Results: Transgenic hPD-1 / hTGFβR2 mice with MC38 colorectal tumors were intraperitoneally treated with various antibodies, and tumor volume was monitored over time. While both pembrolizumab and anti-TGFβR2 had only a slight effect on reducing tumor volume, antibody A significantly inhibited tumor growth and induced a full response in several mice (Figure 5A). Antibody A also showed superior efficacy against tumor volume compared to simultaneous administration of anti-TGFβR2 antibody (TGF1) and anti-PD-1 antibody (pembrolizumab) (Figure 5A). Animals cured with antibody A developed immunity against recurrence of MC38 colorectal cancer (Figure 5B).

[0157] Example 5: Antibody A increased the number of CD8 T cells and decreased the number of CD4 Treg cells in MC38 colorectal tumors. This example describes flow cytometry analysis of MC38 colorectal tumors collected from transgenic hPD-1 / hTGFβR2 mice after treatment with various antibodies.

[0158] Flow cytometry analysis of MC38 colorectal tumors: MC38 tumor cells (2 million cells) were inoculated into the flank of hPD-1 / hTGFβR2 knock-in mice (Biocytogen). The tumor was approximately 100 mm. 3Upon reaching a certain stage, animals were randomized to different treatment groups including anti-RSV, TGF1, pembrolizumab, a combination of pembrolizumab and TGF1, and antibody A. Antibodies were administered twice weekly at 10 mg / kg. Antibody A was also administered twice weekly at 1 mg / kg. Tumors were collected 24 hours after the third dose, i.e., 8 days after the first dose. The collected MC38 tumors were cut into small fragments and digested using a tumor dissociation kit (Miltenyi Biotec, catalog no. 130-096-730) according to the manufacturer's protocol. Samples were filtered through a 70 μm filter (Corning, catalog no. 352350) to form single-cell suspensions for further analysis. Cells were blocked with anti-Fc receptor antibody (Biolegend, catalog number 101320) for 10 minutes, washed with 2% FBS in PBS, and then stained for immunophenotypic analysis with fluorescent dye conjugated antibodies CD4 (BD Biosciences no. 624296), CD8 (eBioscience no. 365-0081-82), and CD25 (BD Biosciences no. 564368). Cell acquisition was performed using DIVA software (BD Biosciences) under a FACSymphony A3 (BD ​​Biosciences) cytometer. A viability indicator dye (Biolegend no. 423114) was included to exclude dead cells during analysis. Data analysis was performed using FlowJo software (BD Biosciences, version 10.8), and statistical analysis was performed using GraphPad Prism (version 9.3.1).

[0159] Results: MC38 colorectal tumors collected from mice treated with antibody A at a dose of 10 mg / kg twice weekly showed a statistically significant increase in the number of CD8 T cells compared to MC38 colorectal tumors collected from mice treated with RSV control antibody (Figure 6A). Furthermore, compared to tumors from RSV-treated mice, MC38 colorectal tumors collected from mice treated with anti-TGFβR2 antibody (TGF1), a combination of TGF1 and pembrolizumab, or antibody A at doses of either 1 mg / kg or 10 mg / kg showed a statistically significant decrease in the number of CD4 Treg cells (Figure 6B). No changes in the number of T cells or Treg cells were observed in MC38 colorectal tumors collected from mice treated with pembrolizumab alone compared to mice treated with the negative control antibody anti-RSV (Figures 6A-6B).

[0160] Example 6: Antibody A increased the number of CD8+ T cells in MC38 colorectal tumors. This example describes the immunohistochemical (IHC) analysis of MC38 colorectal tumors collected from transgenic hPD-1 / hTGFβR2 mice after treatment with various antibodies.

[0161] IHC analysis of MC38 colorectal tumors: MC38 tumors were grown in hPD-1 / hTGFBR2 knock-in mice, and the mice were treated with RSV control antibody, antibody A (1 mg / kg), or antibody A (10 mg / kg) as described in Examples 4-5. Tumors were collected 24 hours after the third dose, fixed overnight in 10% formalin, and embedded in paraffin blocks. For immunohistochemical staining, 5 μm sections were cut, mounted on charged slides, air-dried overnight, and then stained. T lymphocytes were stained using anti-CD8α antibody (clone D4W2Z, 1:400, Cell Signaling Technology, catalog number 98941). Stained slides were digitally scanned and analyzed. Statistical analysis was performed using one-way ANOVA in GraphPad PRISM (version 9.3.1). All data are expressed as mean ± SD. **p<0.01.

[0162] Results: Representative micrographs are shown in Figure 7A (left panel: RSV control, center panel: antibody A 1 mg / kg, right panel: antibody A 10 mg / kg). Graphs showing CD8 cell density and CD8-positive cell percentage in the control and treatment groups are shown in Figures 7B and 7C, respectively. Overall, these results indicate that tumors treated with antibody A showed dose-dependent CD8+ T cell infiltration, which was increased compared to the RSV control.

[0163] Example 7: Antibody A induced IFNγ production in ex vivo samples from non-small cell lung cancer (NSCLC) patients. This example describes IFNγ production in tumor digests derived from NSCLC patients in the presence of various antibodies.

[0164] Study Design: A total of 10 patients with stage I-II lung cancer scheduled for surgical resection agreed to have tumor tissue and / or blood samples taken for research purposes after obtaining informed consent approved by the Institutional Review Board. All patients selected to participate in this study met the following criteria: (i) had histologically confirmed squamous cell carcinoma (SCC) or adenocarcinoma (AC) of the lung, (ii) had not received prior chemotherapy or radiotherapy within the past two years, and (iii) had no other ongoing malignancies.

[0165] Reagents: The enzyme cocktail for tumor digestion consisted of serum-free Hyclone® Leibovitz L-15 medium supplemented with 1% penicillin-streptomycin, collagenase type I and IV (170 mg / L = 45-60 U / mL), collagenase type II (56 mg / L = 15-20 U / mL), DNase-I (25 mg / L), and elastase (25 mg / L) (all manufactured by Worthington Biochemical, NJ). The culture medium DMEM / F-12 1:1 (HyClone, Thermo Scientific) was supplemented with 2.5 mM L-glutamine, 15 mM HEPES buffer, 10% embryonic stem (ES) cell screening FBS(US) (Thermo Scientific® HyClone®), penicillin (100 U / ml), and streptomycin (100 μg / mL) (hereinafter referred to as complete cell culture medium).

[0166] Preparation of single-cell suspension from tumor lung tissue: Fresh lung tumors surgically resected were processed within 20 minutes of removal from the patient. A deaggregation method optimized for human lung tumors to preserve the phenotype and function of immune cells was used. Briefly, under sterile conditions, all areas of tissue necrosis were excised. The tumor lung tissue was cut 1-2 mm using micro-dissection scissors with tungsten carbide insert blades. 3The tumor was sliced ​​into small pieces. For enzymatic digestion, the pieces were incubated in a shaker at 37°C for 45 minutes in serum-free L-15 Leibovitz medium (HyClone) containing enzymes and 1% penicillin-streptomycin (Life Technologies, Carlsbad, CA). The L-15 Leibovitz medium was formulated for use in a carbon dioxide-free system. After 45 minutes, visible tumor fragments were vigorously pipetteed against the side of a 50 mL tube to promote deaggregation, and then incubated for a further 30–50 minutes under the same conditions. Larger pieces of tumor tissue were allowed to settle to the bottom of the tube, and the supernatant was passed through a 70 μM nylon cell strainer (BD Falcon). The remaining fragments in the tube were further pipettered and passed through the same cell strainer. After filtration, erythrocytes were lysed using 1× erythrocyte (RBC) lysis buffer (Santa Cruz, Dallas, TX). The remaining cells were washed twice with RPMI supplemented with 2% FBS and resuspended in cell culture medium. Cell viability, determined by trypan blue exclusion or Fixable Viability Dye eFluor® 450 staining, was typically >90%. If cell viability was less than 80%, dead cells were removed using a "Dead Cell Removal Kit" (Miltenyi Biotec Inc., Germany).

[0167] Flow Cytometry: Flow cytometry analysis was performed according to a standard protocol. Matched isotype antibodies were used as controls. Negative gating was based on a fluorescence minus one (FMO) strategy. To exclude dead cells from the analysis, cells were stained with LIVE / DEAD® fixable dead cell stain (Molecular probes, Life Technologies).

[0168] For intracellular staining, fixed cells stained for surface markers were permeabilized with BD Perm / Wash® buffer (BD Biosciences) and then stained with FITC anti-human IFN-γ (Biolegend, clone: ​​4S.B3) at room temperature for 45 minutes. All data were acquired using a BD LSRFortessa® (BD Bioscience) flow cytometer or CytoFLEX S (Beckman Coulter) and analyzed using FlowJo software (TreeStar Inc.).

[0169] Generation of NY-ESO-1-specific Ly95 T cells and A549 / A2-NY-ESO-1-targeted lung cancer cell lines: The NY-ESO-1-responsive Ly95 TCR construct is an improved-affinity variant of the wild-type IG4 TCR identified from T cells recognizing the HLA-A2-restricted NY-ESO-1:157-165 peptide antigen. Generation of this Ly95 TCR construct and its packaging into lentiviral vectors were carried out as previously described (Moon EK, et al. Blockade of Programmed Death 1 Augments the Ability of Human T Cells Engineered to Target NY-ESO-1 to Control Tumor Growth after Adoptive Transfer. Clin Cancer Res. 2016;22(2):436-47). Human T cells were isolated from PBMCs of healthy volunteer donors by negative selection using the RosetteSep kit (Stem Cell Technologies, Vancouver, Canada). Isolated T cells were stimulated with magnetic beads coated with anti-CD3 / anti-CD28 at a cell-to-bead ratio of 1:3. Lentiviral vectors were transduced into the T cells at approximately 5 multiples of infection (MOI). Cells were counted and supplied with complete cell culture medium every two days. A small portion of the proliferated cells were stained using Vβ13.1 TCR chain antibody (Beckman Coulter: clone IMMU222) to confirm successful Ly95 transduction by flow cytometry. Transduction of human T cells that had been activated by anti-CD3 / CD28 mAb-coated beads with a high-titer lentivirus encoding the NY-ESO-1-recognizing Ly95 TCR resulted in approximately 50% TRVb13.1 transduction. + CD8 + Cells were brought in.

[0170] For the target cells, the A549 human lung adenocarcinoma cell line was genetically modified to express both the NY-ESO-1 protein and HLA-A*02, as described above (Moon EK, et al. Blockade of Programmed Death 1 Augments the Ability of Human T Cells Engineered to Target NY-ESO-1 to Control Tumor Growth after Adoptive Transfer. Clin Cancer Res. 2016;22(2):436-47). Briefly, the A549 cell line was transduced using a retroviral vector encoding NY-ESO-1-T2A-HLA-A*02. The transduced A549 cells were subjected to limiting dilution at 0.5 cells per well in a 96-well plate. The resulting clones were tested for HLA-A*02 expression by flow cytometry using anti-HLA-A2 Ab (Biolegend, clone: ​​bb7.2). HLA-A2-positive clones were selected and tested in co-culture with T cells expressing the NY-ESO-1 Ly95 TCR. Clones expressing HLA-A2 capable of stimulating IFN-γ secretion in NY-ESO-1 Ly95 TCR-expressing T cells were pooled to generate the A549-NY-ESO-1-A2 (A549-A2-ESO) cell line. Flow sorting was performed to enrich tumor cells that highly express HLA-A2. Intracellular NY-ESO expression was analyzed by flow cytometry using the NY-ESO-1(D1Q2U) mAb (Cell Signaling), which recognizes the endogenous level of total NY-ESO-1 protein.

[0171] NY-ESO-Specific T Cell Response: We investigated the regulation of antigen-specific effector T cell responses by tumor digestion in the presence of antibody A, using TCR-transduced T cells (Ly95 T cells) that recognize the HLA-A2-restricted NY-ESO-1:157-165 peptide antigen. Ly95 T cell responses were stimulated using the A549 human lung adenocarcinoma cell line, genetically modified to express both NY-ESO-1 protein and HLA-A2 A549 (A2-NY-ESO-1 tumor cells). To evaluate the effect of lung tumors on antigen-specific T cell responses, Ly95 cells were co-cultured with A549 / A2-NY-ESO-1 tumor cells in a 1:1:3 ratio (Ly95:A549:tumor) for 48 hours, either in or without a single-cell suspension obtained from digested tumors. A concentration of 20 μg / mL of antibody A or a control antibody was present in the cell co-culture for 48 hours from the start of the assay. A matched isotype antibody (20 μg / mL) was used as a control. To evaluate the effect of NY-ESO-specific Ly95 T cell response in lung tumors, IFN-γ production by Ly95 T cells was measured. 1.5 × 10⁻⁶ 5Ly95 T cells at a cell / well (24-well plate) concentration were mixed with A549 A2-NY-ESO-1 tumor cells in the presence of tumor digestate and either antibody A or a control antibody. BD GolgiStop® and BD GolgiPlug® were added to the cell culture during the last 6 hours. Ly95 T cells co-cultured with NY-ESO-1 negative A549 tumor cells were used as a negative control to define the level of allo-stimulation. Cells were collected, washed in Stain Buffer (BD Biosciences), stained for CD8 and Ly95 TCR surface markers using anti-CD8 (Biolegend, clone: ​​HIT8a) and anti-TCRVβ13.1 (Beckman Coulter: clone IMMU 222) antibodies, and then stained for intracellular IFN-γ. Surface-stained cells were fixed in BD Cytofix® Fixation Buffer (BD Biosciences) for 20 minutes. Fixed cells were permeabilized with BD Perm / Wash® buffer (BD Biosciences) and then stained with anti-human IFN-γ (Biolegend, clone: ​​4S.B3). IFN-γ production was assessed by flow cytometry using gated bioCD8 + TCRVβ13.1 + The analysis was performed on cells.

[0172] Results: Samples treated with antibody A showed a significant increase in IFNγ-producing Ly95 T cell percentage compared to samples treated with the negative control antibody RSV or the anti-PD-1 antibody pembrolizumab (Table 6 and Figure 8). These results indicate that antibody A can reactivate T cells to produce cytokines in ex vivo samples from NSCLC patients.

[0173] [Table 6]

[0174] Example 8: Antibody A induced IFNγ production in ex vivo samples from ovarian cancer patients. This example describes IFNγ production in tumor digests derived from ovarian cancer patients in the presence of various antibodies.

[0175] Methods: Frozen ascites samples were obtained from tumor banks from patients with high-grade serous ovarian cancer. Samples were thawed, cells were washed, and tested for viability using acridine orange / propidium iodide (AOPI) staining. Approximately 2 × 10⁻⁶ cells were found. 6 Each viable cell was plated in triplicate in a 6-well plate under all conditions indicated in the data. The drug was added to the appropriate well at a final concentration of 10 mg / mL relative to the plated cells. The plates were incubated at 37°C for 4 days. At the end of the incubation period, the supernatant of the medium was collected from each well and diluted (1:5) for IFN-γ analysis. IFN-γ ELISA was performed using a commercially available kit (Biolegend No. 430104) according to the manufacturer's guidelines. The IFN-γ concentration was calculated using a standard curve. The values ​​were corrected for the dilution factor and plotted using GraphPad Prism. Statistical analysis was performed using one-way ANOVA with multiple comparisons.

[0176] Results: Antibody A significantly increased the induction of IFNγ in ascites samples from ovarian cancer patients compared to untreated samples (Figures 9A-9B). The amount of induced IFNγ was greater in samples treated with antibody A compared to samples treated with the negative control antibody RSV or a reference antibody against TGFβR2 (anti-TGFβR2) or a reference antibody against PD-1 (pembrolizumab) (Figures 9A-9B). These results indicate that antibody A can reactivate T cells to produce cytokines in ex vivo samples from ovarian cancer patients.

[0177] Example 9: Testing of antibody A in selected participants with advanced malignant tumors. This example describes a Phase 1, multicenter, open-label, dose-escalation, and dose-expansion clinical trial to investigate the safety, tolerability, pharmacokinetics (PK), pharmacodynamics, and preliminary clinical efficacy of antibody A in selected participants with advanced malignancies.

[0178] Part 1 of this study involves dose escalation in selected participants with advanced malignancies. Part 1 will evaluate safety and tolerability and identify the maximum tolerated dose (MTD) and / or recommended dose (RDE) for escalation. The selected advanced malignancies included in Part 1 are non-small cell lung cancer (NSCLC), squamous cell carcinoma of the head and neck (SCCHN), squamous cell carcinoma of the cervix and adenocarcinoma of the cervix (CESC), ovarian cancer, breast cancer, bladder cancer, renal cell carcinoma, melanoma, gastric adenocarcinoma, esophageal cancer, gastric esophageal adenocarcinoma, malignant pleural mesothelioma, pancreatic adenocarcinoma, and colorectal cancer (CRC).

[0179] Part 2 of this study is an open-label dose expansion to further evaluate the safety, tolerability, PK, pharmacodynamics, and preliminary antitumor activity of antibody A at selected recommended doses (RDEs) (RDEs(s)) in the following two tumor-specific cohorts.

[0180] Cohort 1, ICI-sensitive: Participants with one of the following selected advanced or metastatic solid tumors that have progressed, are intolerant to, or are ineligible for standard treatment including immune checkpoint inhibitors (ICIs): bladder cancer, cervical squamous cell carcinoma and endocervical adenocarcinoma, esophageal cancer, gastric adenocarcinoma, gastroesophageal junction cancer, melanoma, malignant pleural mesothelioma, non-small cell lung cancer, ovarian cancer, renal cell carcinoma, squamous cell carcinoma of the head and neck, triple-negative breast cancer, or mismatch repair deficiency / high microsatellite instability colorectal cancer.

[0181] Cohort 2, ICI-insensitive: Participants with the following selected advanced or metastatic solid tumors that have progressed, are intolerant to, or are ineligible for standard treatment (ICI is not indicated): pancreatic adenocarcinoma or microsatellite-stable colorectal cancer.

[0182] Part 2 includes previously administered "standard therapies," which are available standard therapies, including anti-PD-(L)1 and anti-CTLA-4 therapies known to provide clinical benefits.

[0183] Dosage justification In Part 1, a starting dose of 100 mg of antibody A administered every two weeks (Q2W) was selected because this dose was predicted to result in serum antibody levels exceeding EC90 against antibody A that binds to PD-1 / TGFβR2-positive circulating T cells throughout the entire treatment period, leading to PD-1-dependent conditional inhibition of TGFβ signaling.

[0184] The proposed safe starting dose (SSD) attempts to minimize exposure to subthermal doses of antibody A in patients with advanced cancer while balancing the safety risks associated with the nonclinical pharmacological and toxicological profiles. Based on this assessment, the SSD is expected to be safe for the following reasons: (1) In cynomolgus monkeys, the binding affinity of antibody A is similar to that in humans (9 nM and 0.6 nM for TGFβR2 and PD-1, respectively). (2) Serum antibody A levels at the total dose in Good Laboratory Practice (GLP) toxicity studies were EC against antibody A binding in PD-1 / TGFβR2-positive circulating T cells throughout the entire treatment period. 90 This value exceeded [a certain threshold], indicating that sustained inhibition of TGFβR2 / PD-1 was not associated with significant toxicity. (3) There is no evidence of cytokine release that would predict cytokine release syndrome in in vitro whole blood cytokine release assays, and there is no significant cytokine release in cynomolgus monkeys after antibody A administration. (4) No unexpected tissue staining occurred in human tissues during the tissue cross-reactivity test. (5) The high dose (75 mg / kg twice weekly) in the GLP toxicity study was determined to be a no-observed-adverse-effect level based on the absence of target organ toxicity, infusion response, cytokine release, and changes in immune cell populations. (6) Antibody A showed no or weak activity in inhibiting TGFβ signaling in human aortic smooth muscle cells (HASMCs) (maximum concentration of 100 μg / mL). Consistent with tissue cross-reactivity assays, antibody A was shown to bind to mononuclear leukocytes but not to other cell types that do not co-express PD-1, such as endothelial cells or cardiomyocytes. (7) The SSD (100 mg Q2W) is below the maximum SSD of 240 mg / dose acceptable in the ICH S9 guidelines (ICH2009) for determining SSD for anticancer drugs. (8) The predicted human exposure to antibody A at 100 mg Q2W is several orders of magnitude lower (235 ×) than the serum concentration at the no-observed-adverse-effect level (NOAEL) in the GLP toxicity study (75 mg / kg, the highest dose in the study). (9) 100 mg Q2W SSD is used in the in vitro mixed lymphocyte reaction (MLR) assay of antibody A. 50 Covering 24 hours, EC 90 It covers approximately 60 hours, but in vitro pSMAD inhibition EC against TGFβR2 monopositive cells 50 It is predicted that the dose will not reach (>100 μg / mL). Based on quantitative systems pharmacology (QSP) modeling and simulation, the predicted TGFβR2 target occupancy within the tumor at steady state is C for the 100 mg Q2W dose regimen. max 30%, C trough The figure is 17%. Therefore, the proposed SSD is expected to provide pharmacological activity without safety concerns. (10) A 100 mg Q2W dose of antibody A is 20 times lower than the QSP model's predicted safe dose, which is at least 2000 mg Q2W.

[0185] Based on preclinical data (e.g., toxicity, pharmacology, and PK data) and predicted human PK, antibody A 100 mg Q2W IV was selected as the SSD for this Phase 1 trial. The antibody A dosing schedule may be changed to Q4W based on new PK and pharmacodynamic data.

[0186] In Part 2, antibody A is administered at the RDE(s) identified in Part 1. If multiple RDEs are selected, the following criteria may be met: (1) the RDEs do not have overlapping PK exposures (e.g., at intervals of 2-3 times), (2) the RDE 低 (3)RDE 高 The MTD must not be exceeded. If two RDEs are selected for evaluation within a particular dose expansion cohort, participants will be randomized to receive one of the RDEs during their participation in the study.

[0187] Antibody A regimen The starting dose of antibody A in Part 1 is 100 mg, administered intravenously at Q2W. During Part 1 of the study, the following additional dose levels will be evaluated: 300 mg, 900 mg, 1500 mg, and 2000 mg. The frequency of administration at Q4W may be investigated during the study.

[0188] Objectives and evaluation items The primary objective of this study is to evaluate the safety and tolerability of antibody A in selected participants with advanced malignancies and to determine the MTD and / or RDE(s). The primary objectives will be evaluated by measuring (1) the occurrence of dose-limiting toxicity (DLT), (2) the incidence of treatment-related adverse events (TEAEs) as assessed by physical examination evaluating changes in vital signs, left ventricular ejection fraction (LVEF), and electrocardiogram (ECG), as well as by evaluation of blood and urine samples from clinical laboratories, and (3) the incidence of TEAEs leading to interruption of treatment with the investigational drug and discontinuation of the investigational drug due to adverse events (AEs).

[0189] The secondary objectives of this study are: (1) to determine the preliminary efficacy of antibody A in terms of objective response rate (ORR), disease control rate (DCR), and duration of response (DOR) in selected participants with advanced malignancies; (2) to evaluate the pharmacokinetics (PK) of antibody A in selected participants with advanced malignancies; (3) to evaluate the pharmacodynamics of antibody A in selected participants with advanced malignancies; (4) to evaluate the immunogenicity of antibody A in selected participants with advanced malignancies; and (5) to evaluate the target binding of antibody A via receptor occupancy in selected participants with advanced malignancies.

[0190] Secondary objectives are evaluated by measuring the following endpoints: (1) Objective response: complete response (CR) or partial response (PR) as determined by the principal investigator by radiographic assessment in accordance with RECIST v1.1; disease control: CR, PR, or stable disease (SD) as determined by the principal investigator by radiographic assessment in accordance with RECIST v1.1; and DOR: time from the earliest disease response (CR or PR) to the earliest disease progression as determined by the principal investigator by radiographic assessment in accordance with RECIST v1.1; (2) C max , t max , C min AUC, CL, V z , and t 1 / 2 (3) PK parameters for antibody A, including (4) pharmacodynamics of antibody A, including changes in T lymphocyte activation and cytokines in the blood and changes in T lymphocytes within tumors, (5) immunogenicity, defined as the development of antibody A-specific ADA, and (6) receptor occupancy in peripheral blood samples.

[0191] Other Embodiments Although the present invention has been described in relation to its detailed description, the foregoing description is intended to illustrate, not to limit, the scope of the invention, which is defined in the appended claims. Other aspects, advantages, and modifications are within the following claims.

Claims

1. A method for treating a disorder in a human subject requiring treatment, wherein the disorder is selected from the group consisting of non-small cell lung cancer (NSCLC), squamous cell carcinoma of the head and neck (SCCHN), squamous cell carcinoma of the cervix and adenocarcinoma of the cervix (CESC), ovarian cancer, breast cancer, bladder cancer, renal cell carcinoma, melanoma, gastric adenocarcinoma, esophageal cancer, esophageal and gastric adenocarcinoma, malignant pleural mesothelioma, pancreatic adenocarcinoma, and colorectal cancer (CRC), and the method comprises administering to the human subject a therapeutically effective dose of a bispecific antibody that binds to human programmed death-1 (PD-1) and human transforming growth factor β receptor 2 (TGFβR2), wherein the bispecific antibody is An anti-human PD-1 binding domain comprising a PD-1 heavy chain variable region and a PD-1 light chain variable region, wherein the PD-1 heavy chain variable region comprises a heavy chain CDR1 (HCDR1) containing the amino acid sequence RFALH (SEQ ID NO: 1), a heavy chain CDR2 (HCDR2) containing the amino acid sequence WIDPNGTPTFAQGVTG (SEQ ID NO: 2), and a heavy chain CDR3 (HCDR3) containing the amino acid sequence SLGYCDSDICYPNWIFDN (SEQ ID NO: 3), and the PD-1 light chain variable region comprises a light chain CDR1 (LCDR1) containing the amino acid sequence QSISSY (SEQ ID NO: 11), a light chain CDR2 (LCDR2) containing the amino acid sequence AAS (SEQ ID NO: 12), and a light chain CDR3 (LCDR3) containing the amino acid sequence QQSYSTPPT (SEQ ID NO: 13), and the anti-human PD-1 binding domain, An anti-human TGFβR2 binding domain comprising a TGFβR2 heavy chain variable region and a TGFβR2 light chain variable region, wherein the TGFβR2 heavy chain variable region comprises HCDR1 containing the amino acid sequence IYAMT (SEQ ID NO: 6), HCDR2 containing the amino acid sequence VISGSGGTTTYYADSVKG (SEQ ID NO: 7), and HCDR3 containing the amino acid sequence RGQYRDIVGATDY (SEQ ID NO: 8), and the TGFβR2 light chain variable region comprises LCDR1 containing the amino acid sequence QSISSY (SEQ ID NO: 11), LCDR2 containing the amino acid sequence AAS (SEQ ID NO: 12), and LCDR3 containing the amino acid sequence QQSYSTPPT (SEQ ID NO: 13), and the anti-human TGFβR2 binding domain, The method, including the method described above.

2. The PD-1 heavy chain variable region includes the amino acid sequence QVQLVQSGSELKKPGASVKVSCKASGYTFTRFALHWVRQAPGQGLEWMGWIDPNTGTPTFAQGVTGRFVFSLDTSVTTAYLQISSLKAEDTAVYYCARSLGYCDSDICYPNWIFDNWGQGTLVTVSS (SEQ ID NO: 4), and the TG The method according to claim 1, wherein the FβR2 double-chain variable region comprises the amino acid sequence EVQLVESGGGLLVQPGGSLRLSCAASGFTFDIYAMTWVRQAPGKGLEWVSVISGSGGTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARRGQYRDIVGATDYWGQGTLVTVSS (SEQ ID NO: 9).

3. The method according to claim 1 or claim 2, wherein the PD-1 light chain variable region and the TGFβR2 light chain variable region each include the amino acid sequence DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPTFGQGTKVEIK (SEQ ID NO: 14).

4. The method according to claim 1, wherein the bispecific antibody comprises a PD-1 heavy chain, a PD-1 light chain, a TGFβR2 heavy chain, and a TGFβR2 light chain, wherein the PD-1 heavy chain comprises the amino acid sequence represented by SEQ ID NO: 5, the PD-1 light chain comprises the amino acid sequence represented by SEQ ID NO: 15, the TGFβR2 heavy chain comprises the amino acid sequence represented by SEQ ID NO: 10, and the TGFβR2 light chain comprises the amino acid sequence represented by SEQ ID NO:

15.

5. The method according to any one of claims 1 to 4, wherein the human subject has NSCLC.

6. The method according to claim 5, wherein the NSCLC has a squamous or non-squamous epithelial tissue type.

7. The method according to claim 5 or 6, wherein the NSCLC is progressive or metastatic.

8. The method according to any one of claims 1 to 4, wherein the human subject has SCCHN.

9. The method according to claim 8, wherein the SCCHN includes a primary squamous cell tumor of the oral cavity, oropharynx, hypopharynx, or larynx.

10. The method according to claim 8 or 9, wherein the SCCHN is progressive or metastatic.

11. The method according to any one of claims 1 to 4, wherein the human subject has CESC.

12. The method according to claim 11, wherein the CESC is progressive or metastatic.

13. The method according to any one of claims 1 to 4, wherein the human subject has human papillomavirus (HPV)-positive SCCHN or HPV-positive CESC.

14. The method according to any one of claims 1 to 4, wherein the human subject has ovarian cancer.

15. The method according to claim 14, wherein the ovarian cancer includes epithelial ovarian cancer, fallopian tube cancer, primary peritoneal cancer, or carcinosarcoma.

16. The method according to claim 14 or claim 15, wherein the ovarian cancer is progressive or metastatic.

17. The method according to any one of claims 1 to 4, wherein the human subject has breast cancer.

18. The method according to claim 17, wherein the breast cancer includes triple-negative breast cancer.

19. The method according to claim 18, wherein the triple-negative breast cancer includes HER2-negative, ER-negative, and PgR-negative breast cancer.

20. The method according to any one of claims 17 to 19, wherein the breast cancer is progressive or metastatic.

21. The method according to any one of claims 1 to 4, wherein the human subject has bladder cancer.

22. The method according to claim 21, wherein the bladder cancer includes urothelial carcinoma.

23. The method according to claim 21 or claim 22, wherein the bladder cancer is progressive or metastatic.

24. The method according to any one of claims 1 to 4, wherein the human subject has renal cell carcinoma.

25. The method according to claim 24, wherein the renal cell carcinoma is progressive or metastatic.

26. The method according to any one of claims 1 to 4, wherein the human subject has melanoma.

27. The method according to claim 26, wherein the melanoma is a malignant cutaneous melanoma.

28. The method according to claim 26 or claim 27, wherein the melanoma is progressive or metastatic.

29. The method according to any one of claims 1 to 4, wherein the human subject has gastric adenocarcinoma.

30. The method according to claim 29, wherein the gastric adenocarcinoma is progressive or metastatic.

31. The method according to any one of claims 1 to 4, wherein the human subject has esophageal cancer.

32. The method according to claim 31, wherein the esophageal cancer is progressive or metastatic.

33. The method according to any one of claims 1 to 4, wherein the human subject has esophageal gastric adenocarcinoma.

34. The method according to claim 33, wherein the esophageal gastric adenocarcinoma is progressive or metastatic.

35. The method according to any one of claims 1 to 4, wherein the human subject has malignant pleural mesothelioma.

36. The method according to claim 35, wherein the malignant pleural mesothelioma is progressive or metastatic.

37. The method according to any one of claims 1 to 4, wherein the human subject has pancreatic adenocarcinoma.

38. The method according to claim 37, wherein the pancreatic adenocarcinoma is progressive or metastatic.

39. The method according to any one of claims 1 to 4, wherein the human subject has CRC.

40. The method according to claim 39, wherein the CRC includes microsatellite-stabilized colorectal cancer (MSS-CRC).

41. The method according to claim 39, wherein the CRC includes mismatch repair mechanism deficiency (dMMR) / high microsatellite instability (MSI-H) colorectal cancer (dMMR / MSI-H CRC).

42. The method according to any one of claims 39 to 41, wherein the CRC is progressive or metastatic.

43. The method according to any one of the prior claims, wherein the human subject has experienced disease progression after prior treatment.

44. The method according to claim 43, wherein the aforementioned prior treatment includes anti-PD-(L)1 therapy and / or anti-CTLA4 therapy.

45. The method according to any one of the prior claims, wherein the aforementioned disorder is unsuitable for curative treatment or procedure.

46. The method according to any one of the prior claims, wherein the bispecific antibody is administered intravenously.

47. The method according to any one of the prior claims, wherein the bispecific antibody is administered in doses of approximately 100 mg, approximately 300 mg, approximately 900 mg, approximately 1500 mg, or approximately 2000 mg.

48. The method according to any one of the prior claims, wherein the bispecific antibody is administered intravenously in doses of approximately 100 mg, approximately 300 mg, approximately 900 mg, approximately 1500 mg, or approximately 2000 mg.

49. The method according to any one of the prior claims, wherein the bispecific antibody comprises a PD-1 heavy chain, a PD-1 light chain, a TGFβR2 heavy chain, and a TGFβR2 light chain, wherein the PD-1 heavy chain comprises the amino acid sequence represented by SEQ ID NO: 5, the PD-1 light chain comprises the amino acid sequence represented by SEQ ID NO: 15, the TGFβR2 heavy chain comprises the amino acid sequence represented by SEQ ID NO: 10, and the TGFβR2 light chain comprises the amino acid sequence represented by SEQ ID NO: 15, and the bispecific antibody is administered intravenously in doses of approximately 100 mg, approximately 300 mg, approximately 900 mg, approximately 1500 mg, or approximately 2000 mg.

50. The method according to any one of the prior claims, wherein the bispecific antibody is administered once every two weeks.

51. The method according to any one of the prior claims, wherein the bispecific antibody is administered intravenously once every two weeks in doses of approximately 100 mg, approximately 300 mg, approximately 900 mg, approximately 1500 mg, or approximately 2000 mg.

52. The method according to any one of the prior claims, wherein the bispecific antibody comprises a PD-1 heavy chain, a PD-1 light chain, a TGFβR2 heavy chain, and a TGFβR2 light chain, wherein the PD-1 heavy chain comprises the amino acid sequence represented by SEQ ID NO: 5, the PD-1 light chain comprises the amino acid sequence represented by SEQ ID NO: 15, the TGFβR2 heavy chain comprises the amino acid sequence represented by SEQ ID NO: 10, and the TGFβR2 light chain comprises the amino acid sequence represented by SEQ ID NO: 15, and the bispecific antibody is administered intravenously once every two weeks in doses of approximately 100 mg, approximately 300 mg, approximately 900 mg, approximately 1500 mg, or approximately 2000 mg.

53. The method according to any one of claims 1 to 49, wherein the bispecific antibody is administered once every four weeks.

54. The method according to claim 53, wherein the bispecific antibody is administered intravenously once every four weeks in doses of approximately 100 mg, approximately 300 mg, approximately 900 mg, approximately 1500 mg, or approximately 2000 mg.

55. The method according to claim 53 or claim 54, wherein the bispecific antibody comprises a PD-1 heavy chain, a PD-1 light chain, a TGFβR2 heavy chain, and a TGFβR2 light chain, wherein the PD-1 heavy chain comprises the amino acid sequence represented by SEQ ID NO: 5, the PD-1 light chain comprises the amino acid sequence represented by SEQ ID NO: 15, the TGFβR2 heavy chain comprises the amino acid sequence represented by SEQ ID NO: 10, and the TGFβR2 light chain comprises the amino acid sequence represented by SEQ ID NO: 15, and the bispecific antibody is administered intravenously once every four weeks in doses of approximately 100 mg, approximately 300 mg, approximately 900 mg, approximately 1500 mg, or approximately 2000 mg.

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