Use of anti-tgfβr2 / pd-1 bispecific antibodies in the treatment of cancer

Abstract

The present invention relates to the use of anti-TGFβR2 / PD-1 bispecific antibodies for the treatment of cancer, describes a bispecific antibody targeting human Programmed Death-1 (PD-1) and human Transforming Growth Factor beta Receptor 2 (TGFβR2) for the treatment of disorders such as non-small cell lung cancer (NSCLC), squamous cell carcinoma of the head and neck (SCCHN), squamous cell carcinoma 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 cancer and colorectal cancer (CRC).

Description

[0001] This application is a divisional application of Chinese invention patent application filed on April 12, 2024, with Chinese application number 202480033643.8 and invention title "Use of anti-TGFβR2 / PD-1 bispecific antibody in cancer treatment".

[0002] Priority Statement

[0003] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 496,205, filed April 14, 2023, which is incorporated herein by reference in its entirety.

[0004] sequence list

[0005] This application contains a sequence list submitted electronically as an XML document named 20443-0795WO1.XML. The XML document was created on April 4, 2024, and is 19,545 bytes in size. The material contained in the XML document is hereby incorporated in its entirety. Technical Field

[0006] This invention relates to the treatment of cancer by administering a bispecific antibody that binds to human TGFβR2 and human PD-1 to a subject. Background Technology

[0007] Cancer is a leading cause of death worldwide. Many patients are diagnosed with advanced-stage disease that does not respond to treatment, or responds to treatment but then the disease progresses. Therefore, targeted therapies for cancer are needed. Summary of the Invention

[0008] 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.

[0009] Therefore, this disclosure provides a method for treating a condition in a human subject in need, wherein the condition is selected from the group consisting of: non-small cell lung cancer (NSCLC), head and neck squamous cell carcinoma (SCCHN), cervical squamous cell carcinoma and cervical adenocarcinoma (CESC), ovarian cancer, breast cancer, bladder cancer, renal cell carcinoma, melanoma, gastric adenocarcinoma, esophageal cancer, gastroesophageal adenocarcinoma, malignant pleural mesothelioma, pancreatic cancer, and colorectal cancer (CRC), wherein the method comprises administering to the human subject a therapeutically effective amount of a bispecific antibody bound to human programmed death-1 (PD-1) and human transforming growth factor β receptor 2 (TGFβR2), wherein the bispecific antibody comprises:

[0010] 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 wherein 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

[0011] The anti-human TGFβR2 binding domain comprises 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 wherein 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).

[0012] In some embodiments, the PD-1 heavy chain variable region contains the amino acid sequence QVQLVQSGSELKKPGASVKVSCKASGYTFTRFALHWVRQAPGQGLEWMGWIDPNTGTPTFAQGVTGRFVFSLDTSVTTAYLQISSLKAEDTAVYYCARSLGYCDSDICYPNWIFDNWGQGTLVTVSS (SEQ ID NO:4) and the TGFβR2 heavy chain variable region contains the amino acid sequence EVQLVESGGGLVQPGGSLRLSCAASGFTFDIYAMTWVRQAPGKGLEWVSVISGSGGTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARRGQYRDIVGATDYWGQGTLVTVSS (SEQ ID NO:9).

[0013] 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).

[0014] 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 listed in SEQ ID NO:5, the PD-1 light chain comprises the amino acid sequence listed in SEQ ID NO:15, the TGFβR2 heavy chain comprises the amino acid sequence listed in SEQ ID NO:10, and the TGFβR2 light chain comprises the amino acid sequence listed in SEQ ID NO:15.

[0015] In some implementations, the human subject has NSCLC. In some implementations, the NSCLC has squamous or non-squamous histology. In some implementations, the NSCLC is advanced or metastatic.

[0016] In some implementations, the human subject has SCCHN. In some implementations, SCCHN comprises a primary squamous cell tumor of the oral cavity, oropharynx, hypopharynx, or larynx. In some implementations, SCCHN is advanced or metastatic.

[0017] In some implementations, the human subjects have CESC. In some implementations, the CESC is advanced or metastatic.

[0018] In some implementations, the human subjects had human papillomavirus (HPV) positive SCCHN or HPV positive CESC.

[0019] In some implementations, the human subject has ovarian cancer. In some implementations, ovarian cancer includes ovarian epithelial cancer, fallopian tube cancer, primary peritoneal cancer, or carcinosarcoma. In some implementations, the ovarian cancer is advanced or metastatic.

[0020] In some implementations, the human subject has breast cancer. In some implementations, the breast cancer includes triple-negative breast cancer. In some implementations, triple-negative breast cancer includes HER2-negative, ER-negative, and PgR-negative breast cancer. In some implementations, the breast cancer is advanced or metastatic.

[0021] In some implementations, the human subject has bladder cancer. In some implementations, the bladder cancer includes urothelial carcinoma. In some implementations, the bladder cancer is advanced or metastatic.

[0022] In some implementations, the human subjects have renal cell carcinoma. In some implementations, the renal cell carcinoma is advanced or metastatic.

[0023] In some implementations, the human subject has melanoma. In some implementations, the melanoma is a cutaneous malignant melanoma. In some implementations, the melanoma is advanced or metastatic.

[0024] In some implementations, the human subjects have gastric adenocarcinoma. In some implementations, the gastric adenocarcinoma is advanced or metastatic.

[0025] In some implementations, the human subjects have esophageal cancer. In some implementations, the esophageal cancer is advanced or metastatic.

[0026] In some implementations, the human subjects had gastroesophageal adenocarcinoma. In some implementations, the gastroesophageal adenocarcinoma was advanced or metastatic.

[0027] In some implementations, the human subjects have malignant pleural mesothelioma. In some implementations, the malignant pleural mesothelioma is advanced or metastatic.

[0028] In some implementations, the human subjects have pancreatic cancer. In some implementations, the pancreatic cancer is advanced or metastatic.

[0029] In some embodiments, the human subject has CRC. In some embodiments, the CRC comprises microsatellite stable colorectal cancer (MSS-CRC). In some embodiments, the CRC comprises mismatch repair deficient (dMMR) / high microsatellite instability (MSI-H) colorectal cancer (dMMR / MSI-H CRC). In some embodiments, the CRC is advanced or metastatic.

[0030] In some implementations, human subjects experienced disease progression following prior treatment. In some implementations, prior treatment included anti-PD-(L)1 therapy and / or anti-CTLA4 therapy.

[0031] In some implementation schemes, the condition is not eligible for curative treatments or procedures.

[0032] In some implementations, the bispecific antibody is administered intravenously.

[0033] In some implementations, the bispecific antibody is administered at doses of about 100 mg, about 300 mg, about 900 mg, about 1500 mg, or about 2000 mg.

[0034] In some implementations, the bispecific antibody is administered intravenously at doses of about 100 mg, about 300 mg, about 900 mg, about 1500 mg, or about 2000 mg.

[0035] 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 listed in SEQ ID NO:5, the PD-1 light chain comprises the amino acid sequence listed in SEQ ID NO:15, the TGFβR2 heavy chain comprises the amino acid sequence listed in SEQ ID NO:10, and the TGFβR2 light chain comprises the amino acid sequence listed in SEQ ID NO:15, and wherein the bispecific antibody is administered intravenously at a dose of about 100 mg, about 300 mg, about 900 mg, about 1500 mg, or about 2000 mg.

[0036] In some embodiments, the bispecific antibody is administered every two weeks. In some embodiments, the bispecific antibody is administered intravenously every two weeks at a dose of about 100 mg, about 300 mg, about 900 mg, about 1500 mg, or about 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 listed in SEQ ID NO:5, the PD-1 light chain comprises the amino acid sequence listed in SEQ ID NO:15, the TGFβR2 heavy chain comprises the amino acid sequence listed in SEQ ID NO:10, and the TGFβR2 light chain comprises the amino acid sequence listed in SEQ ID NO:15, and wherein the bispecific antibody is administered intravenously every two weeks at a dose of about 100 mg, about 300 mg, about 900 mg, about 1500 mg, or about 2000 mg.

[0037] 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 at a dose of about 100 mg, about 300 mg, about 900 mg, about 1500 mg, or about 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 listed in SEQ ID NO:5, the PD-1 light chain comprises the amino acid sequence listed in SEQ ID NO:15, the TGFβR2 heavy chain comprises the amino acid sequence listed in SEQ ID NO:10, and the TGFβR2 light chain comprises the amino acid sequence listed in SEQ ID NO:15, and wherein the bispecific antibody is administered intravenously once every four weeks at a dose of about 100 mg, about 300 mg, about 900 mg, about 1500 mg, or about 2000 mg. Attached Figure Description

[0038] Figures 1A-1F This is a graph showing the results of an in vitro cytotoxicity assay using a co-culture of renal cell carcinoma cells and T cells in the absence of exogenous TGF-β (TGF-β). Figures 1C-1D ) or exist below ( Figure 1A-Figure 1B 0.1 ng / mL; Figures 1E-1F Cultured at 1.0 ng / mL.

[0039] Figures 2A-2B Tumor volume in mice subcutaneously inoculated with A375 human melanoma cells and then treated with various antibodies ( Figure 2A ) and weight ( Figure 2B The chart is shown. Starting from day 8, humanized mice carrying subcutaneous A375 human melanoma tumors were administered antibodies every 5 days, and tumor volume was measured twice weekly. Body weight was measured twice weekly. Data are presented as mean ± SEM.

[0040] Figures 3A-3H A graph showing tumor volume in stem cell donors from humanized mice carrying A375 human melanoma tumors. The mice were not treated. Figure 3A ) or use IgG1 control antibody ( Figure 3B Atezolizumab Figure 3C ), Pembrolizumab ( Figure 3D ), TGF1 antibody ( Figure 3E ), Pembrolizumab + TGF1 antibody ( Figure 3F Antibody A Figure 3G ) or NIS793 ( Figure 3H Treatment. Donors are identified by numbers (0678, 0717, 0731, and 0687). 750 mm 3 The dashed lines at the point are arbitrary straight lines used for visual assistance.

[0041] Figure 4 A graph showing tumor volume in mice that were subcutaneously inoculated with MDA-MB-231 human triple-negative breast cancer (TNBC) cells and then treated with various antibodies.

[0042] Figures 5A-5B A graph showing tumor volume in mice subcutaneously inoculated with MC38 colorectal cancer cells and then treated with various antibodies. Figure 5A ). Re-stimulated cured mice with MC38 colorectal cancer cells and monitored tumor volume over time ( Figure 5B ).

[0043] Figures 6A-6B CD8 T cells from MC38 colorectal tumors harvested from transgenic hPD-1 / hTGFβR2 mice after treatment with various antibodies ( Figure 6A ) and Treg ( Figure 6B ) flow cytometry analysis chart.

[0044] Figures 7A-7C Representative photomicrographs ( Figure 7A ) and displaying CD8 cell density ( Figure 7B ) and percentage of CD8 positive cells ( Figure 7CThe charts are derived from IHC analysis of MC38 colorectal tumors harvested from transgenic hPD-1 / hTGFβR2 mice after treatment with antibody A (1 mg / kg or 10 mg / kg) or control antibody RSV.

[0045] Figure 8 A graph showing Ly95 T cells producing IFNγ in a co-culture with A549-eso cells in the presence of tumor digestate from NSCLC patients. The co-culture was treated with the indicated antibody.

[0046] Figures 9A-9B A graph showing the concentration (pg / mL) of IFNγ in two different ovarian ascites samples treated with various antibodies for 4 days. Figure 9A : 1 sample of ovarian ascites; Figure 9B : 2 samples of ovarian ascites. Detailed Implementation

[0047] This disclosure partially provides a method for treating cancer by administering a bispecific antibody targeting TGFβR2 and PD-1. While not wishing to be bound by theory, it is believed that simultaneous targeting of TGFβR2 and PD-1 can alleviate immunosuppressive pathways to promote cytotoxic T lymphocyte function and T cell memory, thereby effectively eliminating cancer while minimizing toxicities associated with systemic TGFβR2 blockade.

[0048] Anti-TGFβR2 / PD-1 bispecific antibody

[0049] Transforming growth factor β (TGFβ) is a multifunctional cytokine that acts as a tumor initiator or tumor suppressor in both cell-dependent and background-dependent ways. In healthy cells, TGFβ arrests the cell cycle in growth phase 1, leading to reduced proliferation and differentiation induction, and it also promotes apoptosis. In cancer cells, the TGFβ signaling pathway is dysregulated or altered, and TGFβ loses its ability to control cell proliferation. In mammals, three highly homologous TGFβ isoforms exist: TGFβ1, TGFβ2, and TGFβ3. The most prevalent TGFβ1 is expressed in most human cancer types. Furthermore, compared to isoforms TGFβ2 and TGFβ3, TGFβ1 expression is the isoform most closely associated with TGFβ signaling activation.

[0050] TGFβ is synthesized as an inactive precursor and must be activated to bind to the tetrameric receptor complex composed of TGFβR1 and TGFβR2. Transforming growth factor β receptor 2 (TGFβ 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. Formation of a heterodimeric complex with TGFβR1 is essential for signal transduction after TGFβ ligand binding. Activated TGFβR2 phosphorylates multiple serine and threonine residues in the intracellular domain of TGFβR1, leading to TGFβR1 activation. Activated TGFβR1 then mediates the activation of downstream signaling pathways involving SMAD proteins that regulate target gene expression.

[0051] Programmed cell death 1 (PD-1, also known as CD279) is a cell surface receptor expressed on CD4+ and CD8+ T cells, B cells, NK cells, and bone marrow-derived cells. PD-1 binds to two distinct ligands, PD-L1 and PD-L2, which exhibit different expression patterns. PD-L1 (also known as B7-H1 or CD274) is expressed on hematopoietic cells (e.g., T cells, B cells, dendritic cells, and macrophages) and a range of peripheral tissues, and its expression levels are induced by interferon. In contrast, PD-L2 (also known as B7-DC or CD273) expression is typically restricted to professional APCs and can be induced by IL-4 and IL-10, depending on the lineage subset of the APC.

[0052] Binding of PD-1 to PD-L1 or PD-L2 on T cells or B cells leads to aggregation with the TCR or BCR and transient association with tyrosine phosphatase 2 containing the SH2 domain. This, in turn, induces negative signaling by dephosphorylating effector molecules that drive positive TCR and BCR signaling. These include CD28-mediated activation of PI3K and subsequently Akt, glucose metabolism, and the survival protein Bcl-XL. In summary, 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 following chronic viral infection has been shown to lead to immune dysfunction characterized by exhaustion, while blockade of PD-1 signaling has been shown to enhance T cell proliferation and restore immune responses.

[0053] Antibody A is a bispecific human Fc-silencing IgG1 antibody that can bind to both TGFβR2 and PD-1. Antibody A is designed to block the PD-1 axis and selectively target and block TGFβ signaling on activated PD-1-expressing T cells.

[0054] The amino acid sequences of the heavy and light chains of antibody A are shown below. Antibody A contains two distinct 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) and variable light chain (VL) domains (based on the HCDR specified by Kabat (see 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 the LCDR specified by IMGT (see Giudicelli et al., IMGT / V-QUEST: IMGT standardized analysis of the immunoglobulin (IG) and T cell receptor (TR) nucleotide sequences. ColdSpring Harb Protoc 2011(6): 695-715)) are shown below. An antibody consisting of the TGFβR2 heavy chain amino acid sequence listed in SEQ ID NO:10, the PD-1 heavy chain amino acid sequence listed in SEQ ID NO:5, and the common light chain amino acid sequence listed in SEQ ID NO:15 (one light chain paired with each of the heavy chains) is called "antibody A".

[0055] The heavy chain (HC) and common light chain amino acid sequences of the anti-PD-1 binding domain and anti-TGFβR2 binding domain of antibody A are shown in Table 1.

[0056] Table 1: Antibody A Sequence

[0057]

[0058] The VH and VL domains of the anti-PD-1 binding domain and the anti-TGFβR2 binding domain of antibody A, as well as the CDRs of each of VH and VL, are shown in Table 2-3.

[0059] Table 2: Amino acid sequence of the anti-PD-1 binding domain of antibody A

[0060]

[0061] Table 3: Amino acid sequence of the anti-TGFβR2 binding domain of antibody A

[0062]

[0063] In some embodiments, the anti-human PD-1 binding domain of the bispecific antibody of this disclosure includes a heavy chain variable region comprising HCDR1 containing the amino acid sequence listed in SEQ ID NO:1, HCDR2 containing the amino acid sequence listed in SEQ ID NO:2, and HCDR3 containing the amino acid sequence listed in SEQ ID NO:3. In some embodiments, the anti-human PD-1 binding domain of the bispecific antibody of this disclosure includes a heavy chain variable region comprising an amino acid sequence as listed in SEQ ID NO:4 or having at least 80%, 85%, 90%, or 95% sequence identity with it. In some embodiments, the anti-human PD-1 binding domain of the bispecific antibody of this disclosure includes a heavy chain variable region, said heavy chain variable region (1) comprising HCDR1 comprising the amino acid sequence listed in SEQ ID NO:1, HCDR2 comprising the amino acid sequence listed in SEQ ID NO:2 and HCDR3 comprising the amino acid sequence listed in SEQ ID NO:3, and (2) comprising an amino acid sequence as listed in SEQ ID NO:4 or having at least 80%, 85%, 90% or 95% sequence identity with it.

[0064] In some embodiments, the anti-human TGFβR2 binding domain of the bispecific antibody of this disclosure includes a heavy chain variable region comprising HCDR1 containing the amino acid sequence listed in SEQ ID NO:6, HCDR2 containing the amino acid sequence listed in SEQ ID NO:7, and HCDR3 containing the amino acid sequence listed in SEQ ID NO:8. In some embodiments, the anti-human TGFβR2 binding domain of the bispecific antibody of this disclosure includes a heavy chain variable region comprising an amino acid sequence as listed in SEQ ID NO:9 or having at least 80%, 85%, 90%, or 95% sequence identity with it. In some embodiments, the anti-human TGFβR2 binding domain of the bispecific antibody of this disclosure includes a heavy chain variable region, said heavy chain variable region (1) comprising HCDR1 comprising the amino acid sequence listed in SEQ ID NO:6, HCDR2 comprising the amino acid sequence listed in SEQ ID NO:7 and HCDR3 comprising the amino acid sequence listed in SEQ ID NO:8, and (2) comprising an amino acid sequence as listed in SEQ ID NO:9 or having at least 80%, 85%, 90% or 95% sequence identity with it.

[0065] In some embodiments, the anti-human PD-1 binding domain of the bispecific antibody of this disclosure includes a heavy chain variable region comprising HCDR1 containing the amino acid sequence listed in SEQ ID NO:1, HCDR2 containing the amino acid sequence listed in SEQ ID NO:2, and HCDR3 containing the amino acid sequence listed in SEQ ID NO:3; and the anti-human TGFβR2 binding domain of the bispecific antibody includes a heavy chain variable region comprising HCDR1 containing the amino acid sequence listed in SEQ ID NO:6, HCDR2 containing the amino acid sequence listed in SEQ ID NO:7, and HCDR3 containing the amino acid sequence listed in SEQ ID NO:8. In some embodiments, the anti-human PD-1 binding domain of the bispecific antibody of this disclosure includes a heavy chain variable region comprising an amino acid sequence as shown in SEQ ID NO:4 or having at least 80%, 85%, 90%, or 95% sequence identity therewith, and the anti-human TGFβR2 binding domain of the bispecific antibody includes a heavy chain variable region comprising an amino acid sequence as shown in SEQ ID NO:9 or having at least 80%, 85%, 90%, or 95% sequence identity therewith. In some embodiments, the anti-human PD-1 binding domain of the bispecific antibody of this disclosure includes a heavy chain variable region, wherein the heavy chain variable region (1) includes HCDR1 containing the amino acid sequence listed in SEQ ID NO:1, HCDR2 containing the amino acid sequence listed in SEQ ID NO:2, and HCDR3 containing the amino acid sequence listed in SEQ ID NO:3, and (2) includes an amino acid sequence as listed in SEQ ID NO:4 or having at least 80%, 85%, 90%, or 95% sequence identity therewith, and the anti-human TGFβR2 binding domain of the bispecific antibody includes a heavy chain variable region, wherein the heavy chain variable region (1) includes HCDR1 containing the amino acid sequence listed in SEQ ID NO:6, HCDR2 containing the amino acid sequence listed in SEQ ID NO:7, and HCDR3 containing the amino acid sequence listed in SEQ ID NO:8, and (2) includes an amino acid sequence as listed in SEQ ID NO:9 or having at least 80%, 85%, 90%, or 95% sequence identity therewith.

[0066] In some embodiments, the anti-human PD-1 binding domain of the bispecific antibody of this disclosure comprises: (1) a heavy chain variable region comprising HCDR1 comprising the amino acid sequence listed in SEQ ID NO:1, HCDR2 comprising the amino acid sequence listed in SEQ ID NO:2, and HCDR3 comprising the amino acid sequence listed in SEQ ID NO:3; and (2) a light chain variable region comprising LCDR1 comprising the amino acid sequence listed in SEQ ID NO:11, LCDR2 comprising the amino acid sequence listed in SEQ ID NO:12, and LCDR3 comprising the amino acid sequence listed in SEQ ID NO:13. In some embodiments, the anti-human PD-1 binding domain of the bispecific antibody of this disclosure comprises: (1) a heavy chain variable region comprising an amino acid sequence as listed in SEQ ID NO:4 or having at least 80%, 85%, 90% or 95% sequence identity therewith; and (2) a light chain variable region comprising an amino acid sequence as listed in SEQ ID NO:14 or having at least 80%, 85%, 90% or 95% sequence identity therewith. In some embodiments, the anti-human PD-1 binding domain of the bispecific antibody of this disclosure comprises: a heavy chain variable region (1) comprising HCDR1 comprising the amino acid sequence listed in SEQ ID NO:1, HCDR2 comprising the amino acid sequence listed in SEQ ID NO:2, and HCDR3 comprising the amino acid sequence listed in SEQ ID NO:3, and (2) comprising an amino acid sequence as listed in SEQ ID NO:4 or having at least 80%, 85%, 90%, or 95% sequence identity therewith; and a light chain variable region (1) comprising LCDR1 comprising the amino acid sequence listed in SEQ ID NO:11, LCDR2 comprising the amino acid sequence listed in SEQ ID NO:12, and LCDR3 comprising the amino acid sequence listed in SEQ ID NO:13, and (2) comprising an amino acid sequence as listed in SEQ ID NO:14 or having at least 80%, 85%, 90%, or 95% sequence identity therewith.

[0067] In some embodiments, the anti-human TGFβR2 binding domain of the bispecific antibody of this disclosure comprises: (1) a heavy chain variable region comprising HCDR1 comprising the amino acid sequence listed in SEQ ID NO:6, HCDR2 comprising the amino acid sequence listed in SEQ ID NO:7, and HCDR3 comprising the amino acid sequence listed in SEQ ID NO:8; and (2) a light chain variable region comprising LCDR1 comprising the amino acid sequence listed in SEQ ID NO:11, LCDR2 comprising the amino acid sequence listed in SEQ ID NO:12, and LCDR3 comprising the amino acid sequence listed in SEQ ID NO:13. In some embodiments, the anti-human TGFβR2 binding domain of the bispecific antibody of this disclosure comprises: (1) a heavy chain variable region comprising an amino acid sequence as shown in SEQ ID NO:9 or having at least 80%, 85%, 90% or 95% sequence identity therewith; and (2) a light chain variable region comprising an amino acid sequence as shown in SEQ ID NO:14 or having at least 80%, 85%, 90% or 95% sequence identity therewith. In some embodiments, the anti-human TGFβR2 binding domain of the bispecific antibody of this disclosure comprises: a heavy chain variable region (1) comprising HCDR1 comprising the amino acid sequence listed in SEQ ID NO:6, HCDR2 comprising the amino acid sequence listed in SEQ ID NO:7, and HCDR3 comprising the amino acid sequence listed in SEQ ID NO:8, and (2) comprising an amino acid sequence as listed in SEQ ID NO:9 or having at least 80%, 85%, 90%, or 95% sequence identity therewith; and a light chain variable region (1) comprising LCDR1 comprising the amino acid sequence listed in SEQ ID NO:11, LCDR2 comprising the amino acid sequence listed in SEQ ID NO:12, and LCDR3 comprising the amino acid sequence listed in SEQ ID NO:13, and (2) comprising an amino acid sequence as listed in SEQ ID NO:14 or having at least 80%, 85%, 90%, or 95% sequence identity therewith.

[0068] In some embodiments, the anti-human PD-1 binding domain of the bispecific antibody of this disclosure comprises: (1) a heavy chain variable region comprising HCDR1 containing the amino acid sequence listed in SEQ ID NO:1, HCDR2 containing the amino acid sequence listed in SEQ ID NO:2, and HCDR3 containing the amino acid sequence listed in SEQ ID NO:3; and (2) a light chain variable region comprising LCDR1 containing the amino acid sequence listed in SEQ ID NO:11, LCDR2 containing the amino acid sequence listed in SEQ ID NO:12, and LCDR3 containing the amino acid sequence listed in SEQ ID NO:13, and the anti-human TGFβR2 binding domain of the bispecific antibody comprises: (1) a heavy chain variable region comprising HCDR1 containing the amino acid sequence listed in SEQ ID NO:6, HCDR2 containing the amino acid sequence listed in SEQ ID NO:7, and LCDR3 containing the amino acid sequence listed in SEQ ID NO:1; and (2) a light chain variable region comprising LCDR1 containing the amino acid sequence listed in SEQ ID NO:11, LCDR2 containing the amino acid sequence listed in SEQ ID NO:12, and LCDR3 containing the amino acid sequence listed in SEQ ID NO:13, and (3) a light chain variable region comprising: (4) a heavy chain variable region comprising: (5) a heavy chain variable region comprising: (6) a heavy chain variable region comprising: (7) a heavy chain variable region comprising: (8) a heavy chain variable region comprising: (9) a heavy chain variable region comprising: (10) a heavy chain variable region comprising: (11) a heavy chain variable region comprising: (12) a heavy chain variable region comprising: (13) a heavy chain variable region comprising: (14) a heavy chain variable region comprising: (15) a heavy chain variable region comprising: (16) a heavy chain variable region comprising: (17) a heavy chain variable region comprising: (18) a heavy chain variable region comprising: (19) a heavy chain variable region comprising: (10) a heavy chain variable region comprising: (19) a heavy chain variable region comprising: (10) a heavy chain variable HCDR3 of the amino acid sequence listed in NO:8; and (2) a light chain variable region comprising LCDR1 of the amino acid sequence listed in SEQ ID NO:11, LCDR2 of the amino acid sequence listed in SEQ ID NO:12 and LCDR3 of the amino acid sequence listed in SEQ ID NO:13. In some embodiments, the anti-human PD-1 binding domain of the bispecific antibody of this disclosure comprises: (1) a heavy chain variable region comprising an amino acid sequence as shown in SEQ ID NO:4 or having at least 80%, 85%, 90%, or 95% sequence identity therewith, and (2) a light chain variable region comprising an amino acid sequence as shown in SEQ ID NO:14 or having at least 80%, 85%, 90%, or 95% sequence identity therewith, and the anti-human TGFβR2 binding domain of the bispecific antibody comprises: (1) a heavy chain variable region comprising an amino acid sequence as shown in SEQ ID NO:9 or having at least 80%, 85%, 90%, or 95% sequence identity therewith, and (2) a light chain variable region comprising an amino acid sequence as shown in SEQ ID NO:14 or having at least 80%, 85%, 90%, or 95% sequence identity therewith.In some embodiments, the anti-human PD-1 binding domain of the bispecific antibody of this disclosure comprises: a heavy chain variable region, said heavy chain variable region (1) comprising HCDR1 containing the amino acid sequence listed in SEQ ID NO:1, HCDR2 containing the amino acid sequence listed in SEQ ID NO:2, and HCDR3 containing the amino acid sequence listed in SEQ ID NO:3, and (2) comprising an amino acid sequence as listed in SEQ ID NO:4 or having at least 80%, 85%, 90%, or 95% sequence identity therewith; and a light chain variable region, said light chain variable region (1) comprising LCDR1 containing the amino acid sequence listed in SEQ ID NO:11, LCDR2 containing the amino acid sequence listed in SEQ ID NO:12, and LCDR3 containing the amino acid sequence listed in SEQ ID NO:1 ... containing an amino acid sequence as listed in SEQ ID NO:11; and (3) having at least 80%, 85%, 90%, or 95% sequence identity therewith; and a light chain variable region, said light chain variable region (1) comprising LCDR1 containing the amino acid sequence listed in SEQ ID NO:11, LCDR2 containing the amino acid sequence listed in SEQ ID NO:12, and LCDR3 containing the amino acid sequence listed in SEQ ID NO:13, and (2) having at least 80%, 85%, 90%, or 95% sequence identity therewith; and a light chain variable region (2) comprising an amino acid sequence as listed in SEQ ID NO:11; and (3) having at least 80%, 8 The bispecific antibody's anti-human TGFβR2 binding domain comprises: a heavy chain variable region, wherein (1) the heavy chain variable region comprises HCDR1 containing the amino acid sequence listed in SEQ ID NO:6, HCDR2 containing the amino acid sequence listed in SEQ ID NO:7, and HCDR3 containing the amino acid sequence listed in SEQ ID NO:8, and (2) the heavy chain variable region comprises an amino acid sequence as listed in SEQ ID NO:9 or having at least 80%, 85%, 90%, or 95% sequence identity with it; and a light chain variable region, wherein (1) the light chain variable region comprises LCDR1 containing the amino acid sequence listed in SEQ ID NO:11, LCDR2 containing the amino acid sequence listed in SEQ ID NO:12, and LCDR3 containing the amino acid sequence listed in SEQ ID NO:13, and (2) the heavy chain variable region comprises an amino acid sequence as listed in SEQ ID NO:9 or having at least 80%, 85%, 90%, or 95% sequence identity with it; and a light chain variable region, wherein (1) the light chain variable region comprises LCDR1 containing the amino acid sequence listed in SEQ ID NO:11, LCDR2 containing the amino acid sequence listed in SEQ ID NO:12, and LCDR3 containing the amino acid sequence listed in SEQ ID NO:13, and (2) the light chain variable region comprises an amino acid sequence as listed in SEQ ID NO:9. The amino acid sequence listed in NO:14 or having at least 80%, 85%, 90% or 95% sequence identity with it.

[0069] In some embodiments, the bispecific antibody includes a human heavy chain constant region and a light chain constant region. In some embodiments, the heavy chain constant region includes a CH1 domain and a hinge region. In some embodiments, the heavy chain constant region includes a CH2 domain. In some embodiments, the heavy chain constant region includes a CH3 domain. In some embodiments, the heavy chain constant region includes CH1, CH2, and CH3 domains. If the heavy chain constant region includes substitutions, such substitutions alter the properties of the antibody (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.

[0070] For example, antibody A can be prepared, for instance, by generating and expressing a synthetic gene encoding the listed amino acid sequence, or by mutating a human germline gene to provide a gene encoding the listed amino acid sequence. Furthermore, this antibody and other bispecific antibodies can be obtained, for instance, using one or more of the following methods.

[0071] Humanized antibodies can be generated by replacing Fv variable region sequences that do not directly participate in antigen binding with equivalent sequences from human Fv variable regions. General methods for generating humanized antibodies are provided by: Morrison, SL, Science, 229:1202-1207 (1985); Oi et al., BioTechniques, 4:214 (1986); and US 5,585,089, US 5,693,761, US 5,693,762, US 5,859,205, and US 6,407,213. These methods involve isolating, manipulating, and expressing nucleic acid sequences 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 fusion tumors that produce antibodies against a predetermined target (as described above), from germline immunoglobulin genes, or from synthetic constructs. The recombinant DNA encoding the humanized antibody can then be cloned into an appropriate expression vector.

[0072] Human germline sequences are disclosed, for example, in the following: Tomlinson, IA et al., J. Mol. Biol., 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 catalog provides a comprehensive catalog of human immunoglobulin variable region sequences (compiled by Tomlinson, IA et al., MRC Centre for Protein Engineering, Cambridge, UK). These sequences can be used as sources of human sequences, such as frame regions and CDRs. Common human frame regions can also be used, for example, as described in U.S. Patent No. 6,300,064.

[0073] Other methods can also be used to humanize antibodies. For example, other methods can interpret the three-dimensional structure of the antibody, the framework position of the binding determinant in three dimensions, and the immunogenic peptide sequence. See, for example, WO 90 / 07861; U.S. Patent Nos. 5,693,762, 5,693,761, 5,585,089, 5,530,101, and 6,407,213; Tempest et al. (1991) Biotechnology 9:266-271. Yet another method, termed "humanization," is described, for example, in US 2005-008625.

[0074] The antibodies disclosed herein may include a human Fc region, such as a wild-type Fc region, or include one or more altered Fc regions. Antibodies may also have mutations that stabilize the disulfide bonds between the two heavy chains of an immunoglobulin, such as mutations in the hinge region of IgG4, as disclosed in the art (e.g., Angal et al. (1993) Mol. Immunol. 30:105-08). See also, for example, US2005-0037000.

[0075] This document provides compositions comprising a mixture of a bispecific antibody and one or more acidic variants thereof, for example, wherein the amount of the acidic variant is less than about 80%, 70%, 60%, 60%, 50%, 40%, 30%, 30%, 20%, 10%, 5%, or 1%. Compositions comprising a bispecific antibody containing at least one deamide site are also provided, wherein the pH of the composition is from about 5.0 to about 6.5, such that, for example, at least about 90% of the bispecific antibody is not deamided (i.e., less than about 10% of the antibody is deamided). In some embodiments, less than about 5%, 3%, 2%, or 1% of the antibody is deamided. The pH may be from 5.0 to 6.0, for example, 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.

[0076] "Acidic variant" is a variant of the polypeptide of interest that is more acidic than the polypeptide of interest (e.g., as determined by cation exchange chromatography). An example of an acidic variant is a deamidated variant.

[0077] A “deamidized” variant of a polypeptide molecule is a polypeptide in which one or more asparagine residues of the original polypeptide have been converted to aspartate, i.e., the neutral amide side chain has been converted to residues with an overall acidic character.

[0078] As used herein, the term "mixture" in relation to compositions containing bispecific antibodies means the presence of both the desired bispecific antibody and one or more acidic variants thereof. Acidic variants may consist primarily of deamide-containing bispecific antibodies, along with small amounts of other acidic variants.

[0079] In some implementations, mutations occur to eliminate the binding affinity (K) of the deamidated antibody. D ), association rate (K) D Association) and / or dissociation rates (K) D (Dissociated) Similar to wild-type antibodies, for example, with differences of less than about 5, 2, 1 (100%), 50%, 30%, 20%, 10%, 5%, 3%, 2% or 1%.

[0080] The bispecific antibodies disclosed herein can be prepared as full-length antibodies or their low molecular weight forms (e.g., F(ab')2 bispecific antibodies, sc(Fv)2 bispecific antibodies, bivalent bispecific antibodies).

[0081] Traditional generation of full-length bispecific antibodies is based on the co-expression of two immunoglobulin heavy-light chain pairs, where the two chains have different specificities (Millstein et al., Nature 305:537-539 (1983)). In various approaches, the antibody variable domain with the desired binding specificity is fused to an immunoglobulin constant domain sequence. The DNA encoding the immunoglobulin heavy chain fusion and, if desired, the DNA encoding the immunoglobulin light chain, are 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, when expression of at least two polypeptide chains in equal ratios yields high yields, the coding sequences for two or all three polypeptide chains can be inserted into a single expression vector.

[0082] According to another method described in U.S. Patent No. 5,731,168, the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers recovered from recombinant cell cultures. The preferred interface comprises at least a portion of a CH3 domain. In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan). By replacing the large amino acid side chains with smaller ones (e.g., alanine or threonine), a compensating “cavity” of the same or similar size as the large side chain is created at the interface of the second antibody molecule. This provides a mechanism to increase the yield of heterodimers beyond the undesirable end products of other homodimers.

[0083] Bispecific antibodies include cross-linked or "heteroconjugate" antibodies. For example, one antibody in a heteroconjugate may be coupled to avidin, while the other is coupled to biotin. Heteroconjugate antibodies can be prepared using any convenient cross-linking method.

[0084] The "bivalent antibody" technology provides an alternative mechanism for producing bispecific antibody fragments. These fragments contain a VH domain linked to a VL domain via a linker that is too short to allow pairing between the two domains on the same strand. Therefore, the VH and VL domains of one fragment are forced to pair with the complementary VL and VH domains of another fragment, thereby forming two antigen-binding sites.

[0085] Methods of generating antibodies

[0086] Antibodies can be produced in, for example, bacteria or eukaryotic cells. Some antibodies can be produced in bacterial cells, such as *Escherichia coli* cells. Antibodies can also be produced in eukaryotic cells, such as transformed cell lines (e.g., CHO, 293E, COS). Additionally, antibodies can be expressed in yeast cells, such as *Pichia pastoris* (see, for example, Powers et al., *J Immunol Methods.* 251:123-35 (2001)), *Hansenula*, or *Saccharomyces*. To produce antibodies of interest, a polynucleotide encoding the antibody is constructed, introduced into an expression vector, and then expressed in a suitable host cell. Standard molecular biology techniques are used to prepare recombinant expression vectors, transfect host cells, select transformants, culture host cells, and recover antibodies.

[0087] If the antibody is expressed in bacterial cells (e.g., *E. coli*), the expression vector should have features that allow for amplification within bacterial cells. Additionally, when *E. coli* strains such as JM109, DH5α, HB101, or XL1-Blue are used as the host, the vector must have a promoter that allows for efficient expression in *E. coli*, such as the lacZ promoter (Ward et al., 341:544-546 (1989)), the araB promoter (Better et al., Science, 240:1041-1043 (1988)), or the T7 promoter. Examples of such vectors include, for example, the M13 series vectors, the pUC series vectors, pBR322, pBluescript, pCR-Script, pGEX-5X-1 (Pharmacia), the "QIAexpress system" (QIAGEN), pEGFP, and pET (when using this expression vector, the host is preferably BL21 expressing T7 RNA polymerase). The expression vector may contain a signal sequence for antibody secretion. To generate antibodies into the periplasm of *E. coli*, the pelB signal sequence (Lei et al., J. Bacteriol., 169:4379(1987)) can be used as a signal sequence for antibody secretion. For bacterial expression, the expression vector can be introduced into bacterial cells using the calcium chloride method or electroporation.

[0088] If the antibody is expressed 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 the nucleic acid sequence encoding an immunoglobulin or its domain, the recombinant expression vector may carry additional sequences, such as sequences regulating vector replication in host cells (e.g., origin of replication) and selectable marker genes. Selectable marker genes facilitate the selection of host cells in which the vector is introduced (see, for example, U.S. Patent Nos. 4,399,216, 4,634,665, and 5,179,017). For example, selectable biomarker genes can typically confer resistance to drugs such as G418, hygromycin, or methotrexate to host cells into which the vector is introduced. Examples of vectors with selectable biomarkers include pMAM, pDR2, pBK-RSV, pBK-CMV, pOPRSV, and pOP13.

[0089] The antibodies used in the methods described herein can be produced in mammalian cells. Exemplary mammalian host cells for antibody expression include Chinese hamster ovary (CHO) cells (including those described in Urlaub and Chasin (1980) Proc. Natl. Acad. Sci. USA 77:4216-4220 dhfr – CHO cells, used in conjunction with selectable markers of DHFR, such as those 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; lymphocytic cell lines, such as NSO myeloma cells and SP2 cells; and cells derived from transgenic animals, such as transgenic mammals. For example, mammary epithelial cells.

[0090] In an exemplary system for antibody expression, a recombinant expression vector encoding the heavy and light chains of a bispecific antibody (e.g., antibody A) is introduced into dhfr via calcium phosphate-mediated transfection. –In CHO cells, within the recombinant expression vector, the antibody heavy and light chain genes are each operatively linked 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-level transcription of the genes. The recombinant expression vector also carries the DHFR gene, which allows selection of CHO cells transfected with the vector using methotrexate selection / amplification. The selected transformant host cells are cultured to allow expression of both the antibody heavy and light chains, and the antibody is recovered from the culture medium.

[0091] Antibodies can also be produced through transgenic animals. For example, U.S. Patent No. 5,849,992 describes a method for expressing antibodies in the mammary glands of transgenic mammals. A transgene is constructed comprising a milk-specific promoter, a nucleic acid encoding the antibody of interest, and a signaling sequence for secretion. The milk produced by females of such transgenic mammals contains the antibody of interest secreted therein. The antibody can be purified from the milk or, for some applications, used directly.

[0092] The antibodies disclosed herein can be isolated and purified from inside or outside host cells (e.g., from a culture medium) to be substantially pure and homogeneous. Separation and purification methods commonly used for antibody purification can be used to separate and purify antibodies, and are not limited to any particular method. Antibodies can be separated and purified by appropriate selection and combination of methods such as column chromatography, filtration, ultrafiltration, salting out, solvent precipitation, solvent extraction, distillation, immunoprecipitation, SDS-polyacrylamide gel electrophoresis, isoelectric focusing, dialysis, and recrystallization. Chromatographic methods include, for example, 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. Edited by Daniel R. Marshak et al., Cold Spring Harbor Laboratory Press, 1996). Chromatography can be performed using liquid chromatography, such as HPLC and FPLC. Columns used for affinity chromatography include protein A columns and protein G columns. Examples of columns using protein A columns include Hyper D, POROS, and agarose gel FF (GE Healthcare Biosciences). This disclosure also includes antibodies that have been highly purified using these purification methods.

[0093] Antibody drug composition and administration

[0094] The bispecific antibodies described herein can be formulated into pharmaceutical compositions for administration to a subject, for example, to treat the conditions described herein. Typically, pharmaceutical compositions include pharmaceutically acceptable carriers. As used herein, “pharmaceutically acceptable carriers” include any and all physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, and absorption delay agents. Compositions may include pharmaceutically acceptable salts, such as acid addition salts or base addition salts (see, for example, Berge, SM et al. (1977) J. Pharm. Sci. 66:1-19).

[0095] Bispecific antibodies can be administered to subjects via intravenous injection or infusion (IV), such as subjects in need, such as human subjects.

[0096] Bispecific antibodies can be administered at a fixed dose or at a dose per mg / kg of patient body weight. Dosage can also be selected to reduce or avoid the production of antibodies against the bispecific antibody. Dosing regimens are adjusted to provide the desired response, such as a therapeutic response or the effect of combination therapy. Typically, a dose of the bispecific antibody is used to provide a bioavailable amount to the subject.

[0097] For example, a dose ranging from about 0.1 mg / kg to about 30 mg / kg can be administered. In some embodiments, the antibody is administered to the subject at a dose of about 0.1 mg / kg to about 10 mg / kg (e.g., about 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 about 10 mg / kg). In other embodiments, the antibody is administered to the subject at a dose of about 1 mg / kg to about 3 mg / kg (e.g., about 1 mg / kg, 2 mg / kg, or 3 mg / kg). Regarding dose or dosage, the term "about" is intended to mean a range of ±10% of the listed dose, such that a dose of, for example, about 3 mg / kg would fall between 2.7 mg / kg and 3.3 mg / kg of the patient's body weight.

[0098] As used herein, the term "dosage unit" or "fixed dose" or "uniform dose" refers to a physically discrete unit suitable as a unit dose for a subject to be treated; each unit contains a predetermined amount of active compound calculated to produce the desired therapeutic effect, as well as the desired drug delivery system and optionally in combination with other agents. Single or multiple doses may be administered. Alternatively or additionally, antibodies may be administered via continuous infusion. For example, a uniform dose in the range of approximately 20 mg to 2500 mg may be administered. In some embodiments, the antibody is administered to the subject at 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, the antibody is administered to the subject at doses of approximately 100 mg, 300 mg, 900 mg, 1500 mg, or 2000 mg.

[0099] Bispecific antibody doses may be administered, for example, at periodic intervals sufficient to cover at least two, three, five, ten, or more doses (one course of treatment), such as once weekly, once every two weeks, once every three weeks, once every four weeks, or once monthly, for example, for about one to twelve weeks. Factors that may affect the dosage and timing required for effective treatment of a subject include, for example, the severity of the disease or condition, formulation, route of delivery, prior treatment, the subject's general health and / or age, and any other pre-existing conditions. Furthermore, treatment of a subject with a therapeutically effective amount of the compound may comprise monotherapy, or preferably, a series of treatments.

[0100] An example of a uniform dosing regimen involves intravenous administration of the bispecific antibody (e.g., antibody A) described herein at a dose of approximately 100 mg every two weeks.

[0101] Another example of a consistent dosing regimen involves intravenous administration of the bispecific antibody (e.g., antibody A) described herein at a dose of approximately 300 mg every two weeks.

[0102] Another example of a typical uniform dosing regimen involves intravenous administration of the bispecific antibody (e.g., antibody A) described herein at a dose of approximately 900 mg every two weeks.

[0103] Another example of a consistent dosing regimen involves intravenous administration of the bispecific antibody (e.g., antibody A) described herein at a dose of approximately 1500 mg every two weeks.

[0104] Another example of a typical uniform dosing regimen involves intravenous administration of the bispecific antibody (e.g., antibody A) described herein at a dose of approximately 2000 mg every two weeks.

[0105] An illustrative uniform dosing regimen involves intravenous administration of the bispecific antibody (e.g., antibody A) described herein at a dose of approximately 100 mg every four weeks.

[0106] Another example of a typical uniform dosing regimen involves intravenous administration of the bispecific antibody (e.g., antibody A) described herein at a dose of approximately 300 mg every four weeks.

[0107] Another example of a consistent dosing regimen involves intravenous administration of the bispecific antibody (e.g., antibody A) described herein at a dose of approximately 900 mg every four weeks.

[0108] Another example of a typical uniform dosing regimen involves intravenous administration of the bispecific antibody (e.g., antibody A) described herein at a dose of approximately 1500 mg every four weeks.

[0109] Another example of a typical uniform dosing regimen involves intravenous administration of the bispecific antibody (e.g., antibody A) described herein at a dose of approximately 2000 mg every four weeks.

[0110] Pharmaceutical compositions may include a “therapeutic effective amount” of the bispecific antibody described herein. Such effective amounts may be determined based on the effect of the administered agent or the combined effect of agents (if more than one agent is used). Therapeutic effective amounts of agents may also vary based on factors such as an individual’s disease state, age, sex, and weight, and whether the compound elicits a desired response in the individual (e.g., improvement in at least one disease parameter or improvement in at least one symptom of said disease). A therapeutic effective amount is also an amount in which the beneficial therapeutic effect outweighs any toxic or harmful effects of the composition.

[0111] Indications

[0112] The bispecific antibody (e.g., antibody A) described herein may be used to treat non-small cell lung cancer (NSCLC). In some embodiments, the NSCLC has squamous or non-squamous histology. In some embodiments, the NSCLC is advanced or metastatic. In some embodiments, the subject has experienced disease progression following prior treatment (e.g., prior anti-PD-(L)1 therapy and / or anti-CTLA4 therapy).

[0113] The bispecific antibody (e.g., antibody A) described herein may be used to treat squamous cell carcinoma of the head and neck (SCCHN). In some embodiments, SCCHN comprises primary squamous tumors of the oral cavity, oropharynx, hypopharynx, or larynx. In some embodiments, SCCHN comprises human papillomavirus (HPV)-positive SCCHN. In some embodiments, SCCHN is advanced or metastatic. In some embodiments, the subject has experienced disease progression following prior treatment (e.g., prior anti-PD-(L)1 therapy and / or anti-CTLA4 therapy).

[0114] The bispecific antibodies (e.g., antibody A) described herein may be used to treat cervical squamous cell carcinoma and cervical adenocarcinoma (CESC). In some embodiments, the CESC comprises human papillomavirus (HPV) positive CESCs. In some embodiments, the CESC is advanced or metastatic. In some embodiments, the subject has experienced disease progression following prior treatment (e.g., prior anti-PD-(L)1 therapy and / or anti-CTLA4 therapy).

[0115] The bispecific antibodies (e.g., antibody A) described herein 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, the ovarian cancer is advanced or metastatic. In some embodiments, the subject has experienced disease progression following prior treatment (e.g., prior anti-PD-(L)1 therapy and / or anti-CTLA4 therapy).

[0116] The bispecific antibodies (e.g., antibody A) described herein can be used to treat breast cancer. In some embodiments, the 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, the breast cancer is advanced or metastatic. In some embodiments, the subject has experienced disease progression following prior treatment (e.g., prior anti-PD-(L)1 therapy and / or anti-CTLA4 therapy).

[0117] The bispecific antibody (e.g., antibody A) described herein can be used to treat bladder cancer. In some embodiments, the bladder cancer includes urothelial carcinoma. In some embodiments, the bladder cancer is advanced or metastatic. In some embodiments, the subject has experienced disease progression following prior treatment (e.g., prior anti-PD-(L)1 therapy and / or anti-CTLA4 therapy).

[0118] The bispecific antibodies (e.g., antibody A) described herein can be used to treat renal cell carcinoma. In some embodiments, the renal cell carcinoma is advanced or metastatic. In some embodiments, the subject has experienced disease progression following prior treatment (e.g., prior anti-PD-(L)1 therapy and / or anti-CTLA4 therapy).

[0119] The bispecific antibodies (e.g., antibody A) described herein can be used to treat melanoma. In some embodiments, the melanoma is cutaneous malignant melanoma. In some embodiments, the melanoma is advanced or metastatic. In some embodiments, the subject has experienced disease progression following prior treatment (e.g., prior anti-PD-(L)1 therapy and / or anti-CTLA4 therapy).

[0120] The bispecific antibody described herein (e.g., antibody A) may be used to treat gastric adenocarcinoma. In some embodiments, the gastric adenocarcinoma is advanced or metastatic. In some embodiments, the subject has experienced disease progression following prior treatment (e.g., prior anti-PD-(L)1 therapy and / or anti-CTLA4 therapy).

[0121] The bispecific antibodies (e.g., antibody A) described herein can be used to treat esophageal cancer. In some embodiments, the esophageal cancer is advanced or metastatic. In some embodiments, the subject has experienced disease progression following prior treatment (e.g., prior anti-PD-(L)1 therapy and / or anti-CTLA4 therapy).

[0122] The bispecific antibodies described herein (e.g., antibody A) may be used to treat gastroesophageal adenocarcinoma. In some embodiments, the gastroesophageal adenocarcinoma is advanced or metastatic. In some embodiments, the subject has experienced disease progression following prior treatment (e.g., prior anti-PD-(L)1 therapy and / or anti-CTLA4 therapy).

[0123] The bispecific antibody described herein (e.g., antibody A) may be used to treat malignant pleural mesothelioma. In some embodiments, the malignant pleural mesothelioma is advanced or metastatic. In some embodiments, the subject has experienced disease progression following prior treatment (e.g., prior anti-PD-(L)1 therapy and / or anti-CTLA4 therapy).

[0124] The bispecific antibodies (e.g., antibody A) described herein can be used to treat pancreatic cancer. In some embodiments, the pancreatic cancer is advanced or metastatic. In some embodiments, the subject has experienced disease progression following prior treatment (e.g., prior anti-PD-(L)1 therapy and / or anti-CTLA4 therapy).

[0125] The bispecific antibody (e.g., antibody A) described herein can be used to treat colorectal cancer (CRC). In some embodiments, the CRC comprises microsatellite stable colorectal cancer (MSS-CRC). In some embodiments, the CRC comprises mismatch repair deficient (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 following prior treatment (e.g., prior anti-PD-(L)1 therapy and / or anti-CTLA4 therapy).

[0126] On the other hand, it includes the bispecific antibody (e.g., antibody A) described herein for the treatment of non-small cell lung cancer (NSCLC). In some embodiments, the NSCLC has squamous or non-squamous histology. In some embodiments, the NSCLC is advanced or metastatic. In some embodiments, the subject has experienced disease progression following prior treatment (e.g., prior anti-PD-(L)1 therapy and / or anti-CTLA4 therapy).

[0127] On the other hand, it includes the bispecific antibody (e.g., antibody A) described herein for the treatment of squamous cell carcinoma of the head and neck (SCCHN). In some embodiments, SCCHN comprises primary squamous tumors of the oral cavity, oropharynx, hypopharynx, or larynx. In some embodiments, SCCHN comprises human papillomavirus (HPV)-positive SCCHN. In some embodiments, SCCHN is advanced or metastatic. In some embodiments, the subject has experienced disease progression following prior treatment (e.g., prior anti-PD-(L)1 therapy and / or anti-CTLA4 therapy).

[0128] On the other hand, it includes the bispecific antibody (e.g., antibody A) described herein for the treatment of cervical squamous cell carcinoma and cervical adenocarcinoma (CESC). In some embodiments, the CESC includes human papillomavirus (HPV) positive CESCs. In some embodiments, the CESC is advanced or metastatic. In some embodiments, the subject has experienced disease progression following prior treatment (e.g., prior anti-PD-(L)1 therapy and / or anti-CTLA4 therapy).

[0129] On the other hand, it includes the bispecific antibody (e.g., antibody A) described herein 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, the ovarian cancer is advanced or metastatic. In some embodiments, the subject has experienced disease progression following prior treatment (e.g., prior anti-PD-(L)1 therapy and / or anti-CTLA4 therapy).

[0130] On the other hand, it includes a bispecific antibody (e.g., antibody A) as described herein for the treatment of breast cancer. In some embodiments, the 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, the breast cancer is advanced or metastatic. In some embodiments, the subject has experienced disease progression following prior treatment (e.g., prior anti-PD-(L)1 therapy and / or anti-CTLA4 therapy).

[0131] On the other hand, it includes a bispecific antibody (e.g., antibody A) as described herein for the treatment of bladder cancer. In some embodiments, the bladder cancer comprises urothelial carcinoma. In some embodiments, the bladder cancer is advanced or metastatic. In some embodiments, the subject has experienced disease progression following prior treatment (e.g., prior anti-PD-(L)1 therapy and / or anti-CTLA4 therapy).

[0132] On the other hand, it includes the bispecific antibody (e.g., antibody A) described herein for 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 following prior treatment (e.g., prior anti-PD-(L)1 therapy and / or anti-CTLA4 therapy).

[0133] On the other hand, it includes the bispecific antibody (e.g., antibody A) described herein for the treatment of melanoma. In some embodiments, the melanoma is cutaneous malignant melanoma. In some embodiments, the melanoma is advanced or metastatic. In some embodiments, the subject has experienced disease progression following prior treatment (e.g., prior anti-PD-(L)1 therapy and / or anti-CTLA4 therapy).

[0134] On the other hand, it includes the bispecific antibody (e.g., antibody A) described herein for the treatment of gastric adenocarcinoma. In some embodiments, the gastric adenocarcinoma is advanced or metastatic. In some embodiments, the subject has experienced disease progression following prior treatment (e.g., prior anti-PD-(L)1 therapy and / or anti-CTLA4 therapy).

[0135] On the other hand, it includes the bispecific antibody (e.g., antibody A) described herein for the treatment of esophageal cancer. In some embodiments, the esophageal cancer is advanced or metastatic. In some embodiments, the subject has experienced disease progression following prior treatment (e.g., prior anti-PD-(L)1 therapy and / or anti-CTLA4 therapy).

[0136] On the other hand, it includes the bispecific antibody (e.g., antibody A) described herein for the treatment of gastroesophageal adenocarcinoma. In some embodiments, the gastroesophageal adenocarcinoma is advanced or metastatic. In some embodiments, the subject has experienced disease progression following prior treatment (e.g., prior anti-PD-(L)1 therapy and / or anti-CTLA4 therapy).

[0137] On the other hand, it includes the bispecific antibody (e.g., antibody A) described herein for the treatment of malignant pleural mesothelioma. In some embodiments, the malignant pleural mesothelioma is advanced or metastatic. In some embodiments, the subject has experienced disease progression following prior treatment (e.g., prior anti-PD-(L)1 therapy and / or anti-CTLA4 therapy).

[0138] On the other hand, it includes the bispecific antibody (e.g., antibody A) described herein for the treatment of pancreatic cancer. In some embodiments, the pancreatic cancer is advanced or metastatic. In some embodiments, the subject has experienced disease progression following prior treatment (e.g., prior anti-PD-(L)1 therapy and / or anti-CTLA4 therapy).

[0139] On the other hand, it includes the bispecific antibody (e.g., antibody A) described herein for the treatment of colorectal cancer (CRC). In some embodiments, the CRC comprises microsatellite stable colorectal cancer (MSS-CRC). In some embodiments, the CRC comprises mismatch repair deficient (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 following prior treatment (e.g., prior anti-PD-(L)1 therapy and / or anti-CTLA4 therapy).

[0140] On the other hand, it includes the bispecific antibody described herein (e.g., antibody A) used to manufacture a medicament for treating non-small cell lung cancer (NSCLC). In some embodiments, the NSCLC has squamous or non-squamous histology. In some embodiments, the NSCLC is advanced or metastatic. In some embodiments, the subject has experienced disease progression following prior treatment (e.g., prior anti-PD-(L)1 therapy and / or anti-CTLA4 therapy).

[0141] On the other hand, it includes the bispecific antibody described herein (e.g., antibody A) used to manufacture a drug for treating squamous cell carcinoma of the head and neck (SCCHN). In some embodiments, SCCHN comprises primary squamous tumors of the oral cavity, oropharynx, hypopharynx, or larynx. In some embodiments, SCCHN comprises human papillomavirus (HPV)-positive SCCHN. In some embodiments, SCCHN is advanced or metastatic. In some embodiments, the subject has experienced disease progression following prior treatment (e.g., prior anti-PD-(L)1 therapy and / or anti-CTLA4 therapy).

[0142] On the other hand, it includes the bispecific antibody described herein (e.g., antibody A) used to manufacture drugs for treating cervical squamous cell carcinoma and cervical adenocarcinoma (CESC). In some embodiments, the CESC includes human papillomavirus (HPV) positive CESCs. In some embodiments, the CESC is advanced or metastatic. In some embodiments, the subject has experienced disease progression following prior treatment (e.g., prior anti-PD-(L)1 therapy and / or anti-CTLA4 therapy).

[0143] On the other hand, it includes the bispecific antibody described herein (e.g., antibody A) used to manufacture a drug for treating ovarian cancer. In some embodiments, ovarian cancer includes epithelial ovarian cancer, fallopian tube cancer, primary peritoneal cancer, or carcinosarcoma. In some embodiments, the ovarian cancer is advanced or metastatic. In some embodiments, the subject has experienced disease progression following prior treatment (e.g., prior anti-PD-(L)1 therapy and / or anti-CTLA4 therapy).

[0144] On the other hand, it includes the bispecific antibody (e.g., antibody A) described herein, which is used to manufacture a drug for treating breast cancer. In some embodiments, the 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, the breast cancer is advanced or metastatic. In some embodiments, the subject has experienced disease progression following prior treatment (e.g., prior anti-PD-(L)1 therapy and / or anti-CTLA4 therapy).

[0145] On the other hand, it includes the bispecific antibody described herein (e.g., antibody A), which is used to manufacture a drug for treating bladder cancer. In some embodiments, the bladder cancer includes urothelial carcinoma. In some embodiments, the bladder cancer is advanced or metastatic. In some embodiments, the subject has experienced disease progression following prior treatment (e.g., prior anti-PD-(L)1 therapy and / or anti-CTLA4 therapy).

[0146] On the other hand, it includes the bispecific antibody (e.g., antibody A) described herein for 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 following prior treatment (e.g., prior anti-PD-(L)1 therapy and / or anti-CTLA4 therapy).

[0147] On the other hand, it includes the bispecific antibody described herein (e.g., antibody A), which is used to manufacture a drug for treating melanoma. In some embodiments, the melanoma is cutaneous malignant melanoma. In some embodiments, the melanoma is advanced or metastatic. In some embodiments, the subject has experienced disease progression following prior treatment (e.g., prior anti-PD-(L)1 therapy and / or anti-CTLA4 therapy).

[0148] On the other hand, it includes the bispecific antibody described herein (e.g., antibody A), which is used to manufacture a drug for treating gastric adenocarcinoma. In some embodiments, the gastric adenocarcinoma is advanced or metastatic. In some embodiments, the subject has experienced disease progression following prior treatment (e.g., prior anti-PD-(L)1 therapy and / or anti-CTLA4 therapy).

[0149] On the other hand, it includes the bispecific antibody described herein (e.g., antibody A), which is used to manufacture a drug for treating esophageal cancer. In some embodiments, the esophageal cancer is advanced or metastatic. In some embodiments, the subject has experienced disease progression following prior treatment (e.g., prior anti-PD-(L)1 therapy and / or anti-CTLA4 therapy).

[0150] On the other hand, it includes the bispecific antibody described herein (e.g., antibody A), which is used to manufacture a drug for treating gastroesophageal adenocarcinoma. In some embodiments, the gastroesophageal adenocarcinoma is advanced or metastatic. In some embodiments, the subject has experienced disease progression following prior treatment (e.g., prior anti-PD-(L)1 therapy and / or anti-CTLA4 therapy).

[0151] On the other hand, it includes the bispecific antibody described herein (e.g., antibody A), which is used to manufacture a drug for treating malignant pleural mesothelioma. In some embodiments, the malignant pleural mesothelioma is advanced or metastatic. In some embodiments, the subject has experienced disease progression following prior treatment (e.g., prior anti-PD-(L)1 therapy and / or anti-CTLA4 therapy).

[0152] On the other hand, it includes the bispecific antibody described herein (e.g., antibody A) used to manufacture a drug for treating pancreatic cancer. In some embodiments, the pancreatic cancer is advanced or metastatic. In some embodiments, the subject has experienced disease progression following prior treatment (e.g., prior anti-PD-(L)1 therapy and / or anti-CTLA4 therapy).

[0153] On the other hand, it includes the bispecific antibody described herein (e.g., antibody A) used to manufacture a drug for treating colorectal cancer (CRC). In some embodiments, the CRC comprises microsatellite stable colorectal cancer (MSS-CRC). In some embodiments, the CRC comprises mismatch repair deficient (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 following prior treatment (e.g., prior anti-PD-(L)1 therapy and / or anti-CTLA4 therapy).

[0154] The following are embodiments of implementing the present invention. They should not be construed as limiting the scope of the invention in any way.

[0155] Example

[0156] The following examples are provided to better illustrate the claimed invention and should not be construed as limiting the scope of the invention. References to specific materials 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 inventive capacity and without departing from the scope of the invention.

[0157] The reference and control antibodies used in the examples include:

[0158] • Reference PD-1 antibody pembrolizumab (also known as pembro or anti-PD-1) (manufactured by Merck and distributed by Myonex);

[0159] • Refer to the PD-L1 antibody atezolizumab (also known as atezolizumab) (prepared by Genentech);

[0160] • Reference antibody cetuximab × OKT3 is a bispecific antibody that contains a portion of cetuximab (anti-EGFR antibody) for targeting EGFR and a portion of OKT3 (anti-CD3 antibody) for targeting CD3.

[0161] • Reference antibody NIS793 (produced by Novartis) is a fully human anti-TGFβ IgG2 monoclonal antibody designed to inhibit the TGFβ pathway;

[0162] • Referencing anti-TGFβR2 antibody TGF1 (also known as TGF1, αTGFβR2, anti-TGFBRII, or anti-TGFBR2), it is a bivalent monospecific analog of TGF1 and comprises two heavy chains having the amino acid sequence shown in SEQ ID NO:16 and two light chains having the amino acid sequence shown in SEQ ID NO:17; and

[0163] • Negative control IgG1 antibody (also known as RSV×RSV or RSV control or RSV or αRSV or aRSV or antiRSV) is a bivalent monospecific antibody comprising two heavy chains having the amino acid sequence listed in SEQ ID NO:18 and two light chains having the amino acid sequence listed in SEQ ID NO:19.

[0164] Table 4: Reference Antibody Sequence

[0165]

[0166] Example 1: Immunosuppressive effect of antibody A overpowering TGF-β on T cell-mediated cytotoxicity in renal cell carcinoma cells

[0167] This embodiment describes the in vitro assessment of the immunosuppressive effects of various antibodies on TGF-β against T cell-mediated cytotoxicity induced by a bispecific antibody against EGFR and CD3 (cetuximab × CD3).

[0168] In vitro cytotoxicity assay: Adhesive 786-O renal cell carcinoma cells were trypsinized (0.05% trypsin / EDTA; Gibco catalog number 25300-054) and collected. Cells (1.0 × 10⁻⁶) were... 4 Cells per well were seeded in completely phenol-free RPMI medium in 96-well clear plates (Greiner #655090) and incubated overnight at 37°C in a humidified 5% CO2 atmosphere. 786-O renal cell carcinoma cells were pre-incubated at room temperature for 5–10 min with or without bispecific antibody or control antibody in the absence or presence of TGF-β. Human CD3+ T cells were then added to AIM V medium at a 10:1 E / T ratio and incubated for 70 h. Lactate dehydrogenase (LDH) release from damaged cells to the supernatant was measured using fluorescence assays (560 nm Ex; 590 nm Em) according to the manufacturer's instructions (CytoTox-ONE™ Homogenized Membrane Integrity Assay, Promega). Relative fluorescence units (RFU) and fold changes of RFU relative to the control group were calculated for each treatment group.

[0169] Results: To simulate an immunosuppressive environment, exogenous TGF-β (0.1 ng / mL or 1.0 ng / mL) was added to a co-culture system of renal cell carcinoma cells and effector T cells. Cells were treated with 1.0 μg / mL cetuximab × CD3 bispecific antibody (BsAb) in combination with one of the following antibodies: antibody A, anti-PD-1 antibody (pembrolizumab), anti-TGFβR2 antibody, or a bivalent antibody against RSV (RSV × RSV) used as a negative control. Antibodies were added to cells at concentrations ranging from 0.1 to 100 μg / mL.

[0170] Compared with anti-PD-1 antibody, anti-TGFβR2 antibody, or RSV×RSV control antibody, antibody A more effectively counteracted the immunosuppressive effect of TGF-β (0.1 ng / mL) on T cell-mediated cytotoxicity induced by cetuximab×CD3 BsAb. Figure 1A-Figure 1B Antibody A in a co-culture system without TGF-β (). Figures 1C-1D Or a co-culture system with a concentration of 1.0 ng / mL of TGF-β. Figures 1E-1F It produces a similar effect, although to a lesser extent than those produced in a co-culture system with a TGF-β concentration of 0.1 ng / mL.

[0171] The concentration (1.0 μg / ml) and treatment time (70 hours) of cetuximab × CD3 BsAb used in this combination study were selected based on the dose-response and time course of T cell-mediated killing of targeted 786-O renal cancer cells induced by cetuximab × CD3 BsAb alone.

[0172] Example 2: Antibody A inhibits tumor growth in a mouse model of melanoma xenograft.

[0173] This example describes the in vivo evaluation of various antibodies in A375 human melanoma using a xenograft mouse model.

[0174] Antibody and Preparation: Antibody A was prepared in a buffer containing 10 mM histidine, 263 mM sucrose, and pH 6.5. The compound was dissolved in Dulbecco's phosphate-buffered saline (DPBS) (ThermoFisher Scientific, Waltham, MA; catalog number 14190235). IgG1 control (Fc silencing), TGF1Fc silencing (TGFβR2 antibody), and NIS793 (TGFβ1, 2, and 3 antibodies) were manufactured by Incyte (Wilmington, DE). Atezolizumab (anti-PD-L1 antibody) and pembrolizumab (anti-PD-1 antibody) were purchased from RefDrug (Hillsborough Township, NJ).

[0175] A375 xenograft model: Female huCD34+ NSG mice (Jackson Labs, 19-21 weeks old) were shaved and then subcutaneously inoculated with 5 × 10⁻⁶ ppm of a 1:1 mixture of 200 μL DPBS (ThermoFisher Scientific) and Matrigel (Corning). 6 One A375 cell (ATCC #CRL-1619). On day 8 post-cell engraftment, when the tumor was approximately 160 mm... 3 Mice were randomly assigned to groups of 10 mice each, based on tumor size and stem cell donor. NSG mice implanted with human CD34+ stem cells from four different donors were randomly assigned to each group, with 10 mice per group (2-3 mice from each donor within each group). Each group received 10 mg / kg of antibody in DPBS via intraperitoneal injection every 5 days. Dosing was continuous throughout the study. Plasma and tumor samples were collected from mice at 24, 72, or 108 hours after the last antibody dose. Two-way ANOVA was used to determine statistical differences between treatment groups.

[0176] Results: To determine the in vivo efficacy of antibody A against A375 human melanoma, huCD34+ NSG mice were treated with RSV×RSV control, atezolizumab, pembrolizumab, TGF1 (Fc silenced), a combination of pembrolizumab and TGF1, antibody A, or NIS793 at 10 mg / kg every 5 days. Compared with the RSV×RSV control antibody, antibody A treatment produced statistically significant tumor growth inhibition (TGI) (TGI = 75%, p = 0.002). Figure 2AThe effect of antibody A on tumor volume reduction in mice was also statistically significant compared with mice treated with pembrolizumab + TGF1 antibody combination (p < 0.05), mice treated with pembrolizumab alone (p < 0.05), and mice treated with TGF1 alone (p < 0.0001). Figure 2A The TGI values ​​for all groups are shown in Table 5. No adverse effects were observed on the body weight of the animals with any of the treatments. Figure 2B ).

[0177] Table 5: TGI Values

[0178]

[0179] To determine the effect of stem cell donors on antibody A activity, individual tumor volumes were plotted for each treatment group based on stem cell donor. Data showed that a response to antibody A was observed in all four donors. Figures 3A-3H Partial responses were observed in mice receiving atezolizumab, TGF1, and pembrolizumab + TGF1.

[0180] Example 3: Antibody A inhibits tumor growth in a xenograft mouse model of triple-negative breast cancer (TNBC)

[0181] This example describes the in vivo evaluation of various antibodies in MDA-MB-231 human triple-negative breast cancer (TNBC) using a xenograft mouse model.

[0182] Humanized NSG MDA-MB-231 mouse model: Humanized CD34 NSG mice were subcutaneously inoculated with a total of 3 × 10⁻⁶ mg / L serum-free culture medium and Matrigel matrix (Corning) in 100 μL of each medium. 6 One MDA-MB-231 tumor cell. After tumor establishment (80-100 mm) 3 Mice were randomly assigned to the following treatment groups:

[0183] • Negative control IgG1 antibody (RSV) (10 mg / kg);

[0184] • Antibody A (1 mg / kg)

[0185] • Antibody A (10 mg / kg); and

[0186] • Pembrolizumab (10 mg / kg).

[0187] Each group consisted of 8-9 mice. Intraperitoneal administration was performed every five days for a period of 27 or 30 days. Tumors were measured using calipers, and tumor volume was calculated by assimilating it to an ellipsoid using the following formula: l (length) × w 2 (Width) × ½. Body weight was also monitored throughout the study. Tumor samples were harvested (24 hours after the last dose) for tumor immunoanalysis and receptor occupancy after study termination.

[0188] Results: Compared with the reference PD-1 antibody pembrolizumab and the negative control IgG1 antibody RSV, the bispecific antibody antibody A induced a superior antitumor response. Figure 4 Compared with pembrolizumab at 10 mg / kg, antibody A induced superior antitumor responses at both 1 mg / kg and 10 mg / kg dose levels. Figure 4 ).

[0189] Example 4: Antibody A inhibits tumor growth in a mouse model of colorectal cancer xenograft.

[0190] This example describes the in vivo evaluation of various antibodies against MC38 human colorectal cancer using a xenograft mouse model.

[0191] Antibody and formulation: Antibody A was formulated in a buffer containing 10 mM histidine, 263 mM sucrose, and pH 6.5. The compound was dissolved in Duchenne phosphate-buffered saline (DPBS) (ThermoFisher Scientific, Waltham, MA; catalog number 14190235). Mice were treated with 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.

[0192] MC38 colorectal cancer xenograft model: Female huCD34+ NSG mice (Jackson Labs, 19-21 weeks old) were shaved and then subcutaneously inoculated with 2 × 10⁻⁶ xenografts. 6 MC38 cells. On day 9 post-cell engraftment, when the tumor was approximately 110 mm... 3 Mice were randomly assigned to 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 received 10 mg / kg of each antibody. Antibodies were administered twice weekly. Cured mice were rechallenged with MC38 colorectal cancer cells, and tumor volume was monitored over time. Two-way ANOVA was used to determine statistical differences between treatment groups.

[0193] Results: Transgenic hPD-1 / hTGFβR2 mice carrying MC38 colorectal tumors were treated intraperitoneally with various antibodies, and tumor volume was monitored over time. In some mice, pembrolizumab and anti-TGFβR2 had minimal effects on reducing tumor volume, while antibody A significantly inhibited tumor growth and induced a complete response. Figure 5A Antibody A also showed superior effects on tumor volume compared to co-administration of anti-TGFβR2 antibody (TGF1) and anti-PD-1 antibody (pembrolizumab). Figure 5A Animals cured with antibody A develop immunity to recurrent MC38 colorectal cancer. Figure 5B ).

[0194] Example 5: Antibody A increases the number of CD8 T cells and decreases the number of CD4 Tregs in MC38 colorectal tumors.

[0195] This example describes flow cytometry analysis of MC38 colorectal tumors harvested from transgenic hPD-1 / hTGFβR2 mice treated with various antibodies.

[0196] Flow cytometry analysis of MC38 colorectal tumors: 2 million MC38 tumor cells were seeded into the flank of hPD-1 / hTGFβR2 knock-in mice (Biocytogen). When the tumor reached approximately 100 mm... 3Animals were randomly assigned to different treatment groups, including anti-RSV, TGF1, pembrolizumab, a combination of pembrolizumab and TGF1, and antibody A. Antibody A was administered at 10 mg / kg twice weekly. Antibody A was also administered at 1 mg / kg twice weekly. Tumors were harvested 24 hours after the third dose, or 8 days after the first dose. Harvested MC38 tumors were cut into small fragments according to the manufacturer's protocol and digested using a tumor dissociation kit (Miltenyi Biotec; catalog number 130-096-730). Samples were filtered through a 70 μm filter (Corning; catalog number 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 PBS containing 2% FBS, and then stained with fluorescently conjugated antibodies for CD4 (BD Biosciences # 624296), CD8 (eBioscience # 365-0081-82), and CD25 (BDBiosciences # 564368) for immunophenotypic analysis. Cell acquisition was performed using DIVA software (BD Biosciences) under FACSymphony A3 (BD ​​Biosciences) cytometer. Viability dyes (Biolegend #423114) were 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).

[0197] Results: Compared with MC38 colorectal tumors harvested from mice treated with RSV control antibody, MC38 colorectal tumors harvested from mice treated twice weekly with antibody A showed a significantly increased number of CD8 T cells in the tumors. Figure 6A Furthermore, compared with tumors from mice treated with RSV, a statistically significant reduction in the number of CD4 Tregs was observed in MC38 colorectal tumors harvested from mice treated with a combination of anti-TGFβR2 antibody (TGF1), TGF1 and pembro, or antibody A at doses of 1 mg / kg or 10 mg / kg. Figure 6B No changes in the number of T cells and Tregs were observed in MC38 colorectal tumors harvested from mice treated with pembrolizumab alone, compared to mice treated with the negative control antibody anti-RSV. Figures 6A-6B ).

[0198] Example 6: Antibody A increases the number of CD8+ T cells in MC38 colorectal tumors.

[0199] This example describes the immunohistochemical (IHC) analysis of MC38 colorectal tumors harvested from transgenic hPD-1 / hTGFβR2 mice treated with various antibodies.

[0200] IHC analysis of MC38 colorectal tumors: MC38 tumors were grown in hPD-1 / hTGFBR2 knock-in mice and 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 immunohistochemistry, 5 μm sections were cut, mounted on charged slides, and air-dried overnight before staining. T lymphocytes were stained with anti-CD8α antibody (clone D4W2Z, 1:400, Cell Signaling Technology, catalog number 98941). The 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 presented as mean ± SD. **p < 0.01.

[0201] Results: Representative photomicrographs are shown in Figure 7A (Left panel: RSV control; Middle panel: Antibody A 1 mg / kg; Right panel: Antibody A 10 mg / kg). The charts showing the CD8 cell density and CD8-positive cell percentage in the control and treatment groups are shown below. Figure 7B and Figure 7C In summary, these results indicate that tumors treated with antibody A exhibited dose-dependent CD8+ T cell infiltration, which was increased compared to the RSV control.

[0202] Example 7: Antibody A induces IFNγ production in ex vivo samples from non-small cell lung cancer (NSCLC) patients.

[0203] This example describes the production of IFNγ from tumor digests from NSCLC patients in the presence of various antibodies.

[0204] Study Design: A total of 10 patients with stage I-II lung cancer scheduled for surgical resection consented to have a portion of their tumor tissue and / or blood collected for study purposes, subject to approval from the institutional review board. All patients selected for the study met the following criteria: (i) histologically confirmed squamous cell carcinoma (SCC) or adenocarcinoma (AC) of the lung, (ii) no prior chemotherapy or radiation therapy within the past two years, and (iii) no other active malignancies.

[0205] Reagents: The enzyme mixture used for tumor digestion consisted of serum-free Hyclone™ Leibovitz L-15 medium supplemented with 1% penicillin-streptomycin, type I and IV collagenase (170 mg / L = 45-60 U / mL), type II collagenase (56 mg / L = 15-20 U / mL), DNase-I (25 mg / L), and elastase (25 mg / L) (all from Worthington Biochemical, NJ). The 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 selection FBS (US) (Thermo Scientific™ HyClone™), penicillin (100 U / mL), and streptomycin (100 μg / mL), hereinafter referred to as complete cell culture medium.

[0206] Preparation of single-cell suspensions from tumor lung tissue: Freshly removed surgical lung tumors were processed within 20 minutes of removal from the patient. An optimized decomposition method for human lung tumors that preserves the phenotype and function of immune cells was used. Simply put, under sterile conditions, all necrotic areas were trimmed away. The tumor lung tissue was cut into 1-2 mm pieces using microdissecting scissors equipped with tungsten carbide insert blades. 3 Fragments. For enzymatic digestion, fragments were incubated for 45 minutes at 37°C in serum-free L-15 Leibovitz medium (HyClone) containing enzymes and 1% penicillin-streptomycin (Life Technologies, Carlsbad, CA). L-15 Leibovitz medium was prepared for a carbon dioxide-free system. After 45 minutes, any visible tumor fragments were aspirated forcefully against the side of a 50 mL tube to enhance digestion, and then incubated for another 30–50 minutes under the same conditions. Larger tumor tissue fragments were allowed to settle to the bottom of the tube, and the supernatant was passed through a 70 μM nylon cell filter (BD Falcon). The remaining fragments in the tube were aspirated further and passed through the same cell filter. After filtration, red blood cells were lysed using 1x erythrocyte (RBC) lysis buffer (Santa Cruz, Dallas, TX). The remaining cells were washed twice in RPMI supplemented with 2% FBS and resuspended in cell culture medium. Cells were then lysed using trypan blue rejection or the immobilizable viable dye eFluor. ®Cell viability, as determined by 450 staining, is typically >90%. If cell viability is less than 80%, dead cells are removed using a "Dead Cell Removal Kit" (Miltenyi Biotec, Germany).

[0207] Flow cytometry: Flow cytometry analysis was performed according to standard protocols. 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, LIVE / DEAD was used. ® Cells can be stained with molecular probes (Life Technologies) that can fix dead cells.

[0208] 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) for 45 minutes at room temperature. All data were acquired using a BD LSRFortessa™ (BD Biosciences) flow cytometer or a CytoFLEX S (Beckman Coulter) and analyzed using FlowJo software (TreeStar).

[0209] Generation of NY-ESO-1-specific Ly95 T cells and A549 / A2-NY-ESO-1 target lung cancer cell lines: The NY-ESO-1-responsive Ly95 TCR construct is an affinity-enhanced variant of wild-type IG4 TCRs identified from T cells that recognize the HLA-A2-restricted NY-ESO-1:157-165 peptide antigen. The generation of this Ly95 TCR construct and its packaging into a lentiviral vector 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). T cells isolated were stimulated with magnetic beads coated with anti-CD3 / anti-CD28 at a 1:3 cell-to-bead ratio. T cells were transduced with a lentiviral vector at approximately 5 multiples of infection (MOI). Cells were counted and fed with complete cell culture medium every 2 days. A small fraction of the expanded cells were stained for confirmation of successful Ly95 transduction by flow cytometry using a Vβ13.1 TCR chain antibody (Beckman Coulter: clone IMMU 222). Transduction of bead-activated human T cells coated with anti-CD3 / CD28 mAb using a high-titer lentivirus encoding the Ly95 TCR that recognizes NY-ESO-1 produced approximately 50% TCRvb13.1. + CD8 + cell.

[0210] For the target cells, the A549 human lung adenocarcinoma cell line was genetically modified to express both NY-ESO-1 and HLA-A*02, 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). Briefly, the A549 cell line was transduced using a retroviral vector encoding NY-ESO-1-T2A-HLA-A*02. Transduced A549 cells were limited dilutions in 96-well plates at 0.5 cells per well. HLA-A*02 expression in the resulting clones was tested 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 NY-ESO-1Ly95 TCR. Clones expressing HLA-A2 that stimulates IFN-γ secretion from T cells expressing NY-ESO-1Ly95 TCR were pooled to generate the A549-NY-ESO-1-A2 (A549-A2-ESO) cell line. Flow cytometry sorting was performed to enrich tumor cells with high HLA-A2 expression. Intracellular NY-ESO expression was analyzed by flow cytometry using NY-ESO-1(D1Q2U) mAb (Cell Signaling), which recognizes endogenous levels of total NY-ESO-1 protein.

[0211] NY-ESO-Specific T Cell Response: TCR-transduced T cells recognizing the HLA-A2-restricted NY-ESO-1:157-165 peptide antigen (Ly95 T cells) were used to investigate the regulation of antigen-specific effector T cell responses to tumor digests in the presence of antibody A. Genetically modified A549 human lung adenocarcinoma cell line (A2-NY-ESO-1 tumor cells) expressing NY-ESO-1 protein and HLA-A*02 A549 were used to stimulate Ly95 T cell responses. To evaluate the role of lung tumors in antigen-specific T cell responses, Ly95 cells were co-cultured with A549 / A2-NY-ESO-1 tumor cells at a 1:1:3 ratio (Ly95:A549:tumor) for 48 hours in or without a single-cell suspension obtained from digested tumor. Antibody A or a control antibody at a concentration of 20 μg / mL 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 lung tumors on NY-ESO-specific Ly95 T cell responses, IFN-γ production by Ly95 T cells was measured. A concentration of 1.5 × 10⁻⁶ IFN-γ was used. 5 Ly95 T cells per well (24-well plate) were mixed with A549 A2-NY-ESO-1 tumor cells in the presence of tumor digest and either antibody A or a control antibody. During the last 6 hours, BDGolgiStop™ and BD GolgiPlug™ were added to the cell culture. Ly95 T cells co-cultured with NY-ESO-1 negative A549 tumor cells served as a negative control to define the level of allostimulation. Cells were collected, washed in staining buffer (BD Biosciences), and stained against CD8 (Biolegend, clone: ​​HIT8a) and anti-TCRVβ13.1 (Beckman Coulter: clone IMMU 222) antibodies for CD8 and Ly95 TCR surface markers, followed by staining for intracellular IFN-γ. Surface-stained cells were fixed for 20 minutes with BD Cytofix™ fixation buffer (BD Biosciences). Fixed cells were permeabilized with BD Perm / Wash™ buffer (BD Biosciences) and then stained with anti-human IFN-γ (Biolegend, clone: ​​4S.B3). Gated CD8 activity was analyzed by flow cytometry. + TCRVβ13.1 + The production of IFN-γ in cells.

[0212] Results: Compared with samples treated with the negative control antibody RSV or the anti-PD-1 antibody pembrolizumab, samples treated with antibody A showed a significantly increased percentage of Ly95 T cells producing IFNγ (Table 6 and 1). Figure 8 These results indicate that antibody A can reactivate T cells to produce cytokines in ex vivo samples from NSCLC patients.

[0213] Table 6: Percentage of CD8 cells producing IFNγ as analyzed by flow cytometry

[0214]

[0215] Example 8: Antibody A induces IFNγ production in ex vivo samples from ovarian cancer patients.

[0216] This embodiment describes the production of IFNγ from tumor digests from ovarian cancer patients in the presence of various antibodies.

[0217] Methods: Frozen ascites fluid samples were obtained from patients with high-grade serous ovarian cancer from a tumor bank. Samples were thawed, cells were washed, and viability was tested using acridine orange / propidium iodide (AOPI) staining. Approximately 2 × 10⁻⁶ cells were collected for all conditions indicated in the data. 6 Three live cells were plated in triplicate in a 6-well plate. The drug was added to the appropriate wells of the plated cells to a final concentration of 10 mg / mL. The plate was incubated at 37°C for 4 days. At the end of the incubation period, the culture supernatant was collected from each well and diluted (1:5) for IFN-γ analysis. IFN-γ ELISA was performed using a commercially available kit (Biolegend #430104), following the manufacturer's instructions. The concentration of IFN-γ was calculated using a standard curve. The values ​​were corrected for dilution factors and plotted using GraphPad Prism. Statistical analysis was performed using one-way ANOVA and multiple comparisons.

[0218] Results: Compared with untreated samples, antibody A significantly increased the induction of IFNγ in ascites samples from ovarian cancer patients. Figures 9A-9B The amount of IFNγ induced in the sample treated with antibody A was greater than that induced in the sample treated with negative control antibody RSV or reference antibody against TGFβR2 (anti-TGFβR2) or 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.

[0219] Example 9: Study of antibody A in participants with selected advanced malignant tumors

[0220] This embodiment describes a phase 1, multicenter, open-label, dose-escalation and dose-expansion clinical study to investigate the safety, tolerability, pharmacodynamics (PK), pharmacodynamics, and preliminary clinical efficacy of antibody A in participants with selected advanced malignancies.

[0221] Part 1 of the study involves dose escalation in participants with selected advanced malignancies. Part 1 will assess safety and tolerability and identify the maximum tolerated dose (MTD) and / or recommended dose extension (RDE). The selected advanced malignancies included in Part 1 are non-small cell lung cancer (NSCLC), head and neck squamous cell carcinoma (SCCHN), cervical squamous cell carcinoma and cervical adenocarcinoma (CESC), ovarian cancer, breast cancer, bladder cancer, renal cell carcinoma, melanoma, gastric adenocarcinoma, esophageal cancer, gastroesophageal adenocarcinoma, malignant pleural mesothelioma, pancreatic cancer, and colorectal cancer (CRC).

[0222] Part 2 of the study was an open-label dose extension to further evaluate the safety, tolerability, pharmacokinetics, pharmacodynamics, and preliminary antitumor activity of antibody A at the selected recommended extended dose (RDE) in two tumor-specific cohorts:

[0223] Group 1, ICI Sensitive: Participants with the following selected advanced or metastatic solid tumors who have progressed, are intolerant of, or are unsuitable for standard care therapy including immune checkpoint inhibitors (ICIs) after treatment with: bladder cancer, cervical squamous cell carcinoma and cervical adenocarcinoma, esophageal cancer, gastric adenocarcinoma, gastroesophageal junction cancer, melanoma, malignant pleural mesothelioma, non-small cell lung cancer, ovarian cancer, renal cell carcinoma, head and neck squamous cell carcinoma, triple-negative breast cancer, or mismatch repair deficient / high microsatellite instability colorectal cancer.

[0224] Group 2, ICI insensitive: Participants with the following selected advanced or metastatic solid tumors that have progressed, are intolerant of, or are unsuitable for standard care therapy (not indicated by ICI): pancreatic cancer or microsatellite stable colorectal cancer.

[0225] For Part 2, prior treatment using “standard therapy” includes available standard therapies known to provide clinical benefit, including anti-PD-(L)1 therapy and anti-CTLA-4 therapy.

[0226] Rationalization of dosage

[0227] For Part 1, a starting dose of 100 mg of antibody A every 2 weeks (Q2W) was chosen because this dose was predicted to induce serum antibody levels exceeding the EC90 of antibody A binding to PD-1 / TGFβR2-positive circulating T cells throughout the treatment period, thereby conditionally inhibiting TGFβ signaling in a PD-1-dependent manner.

[0228] The proposed safe starting dose (SSD) aims to minimize exposure to subtherapeutic dose levels of antibody A in patients with advanced cancer, while balancing safety risks associated with non-clinical pharmacological and toxicological characteristics. Based on this assessment, the SSD is expected to be safe for the following reasons:

[0229] (1) In cynomolgus monkeys, the binding affinity of antibody A was similar to that in humans (9 nM for TGFβR2 and 0.6 nM for PD-1).

[0230] (2) In Good Laboratory Practice (GLP) toxicology studies, serum antibody A levels at all doses exceeded those of PD-1 / TGFβR2-positive circulating T cells bound to antibody A throughout the entire treatment period. 90 The study indicated that sustained inhibition of TGFβR2 / PD-1 was not associated with significant toxicity.

[0231] (3) In the in vitro whole blood cytokine release assay, there was no evidence of cytokine release that could predict cytokine release syndrome, and no significant cytokine release was observed in cynomolgus monkeys after administration of antibody A.

[0232] (4) No unexpected tissue staining was observed in human tissues during the tissue cross-reactivity study.

[0233] (5) Based on the absence of target organ toxicity, infusion reaction, cytokine release and immune cell population changes, the high dose (75 mg / kg, twice weekly) in the GLP toxicology study was determined to be the level of no observed adverse effects.

[0234] (6) Antibody A showed no activity in inhibiting TGFβ signaling in human aortic smooth muscle cells (HASMCs) or showed weak inhibitory activity (at concentrations up to 100 μg / mL). Consistent with tissue cross-reactivity assays, antibody A bound to monocytes and not to other cell types that do not co-express PD-1, such as endothelial cells or cardiomyocytes.

[0235] (7) The SSD (100 mg Q2W) is lower than the maximum SSD of 240 mg / dose allowed according to the ICH S9 guideline (ICH 2009) for determining the SSD of oncology agents.

[0236] (8) The predicted human exposure to antibody A at 100 mg Q2W was several orders of magnitude (235×) lower than the serum concentration at the level of no observed adverse effects in the GLP toxicology study (75 mg / kg; the highest dose in the study).

[0237] (9) It is expected that 100 mg Q2W SSD can provide all-day coverage for antibody A in vitro mixed lymphocyte reaction (MLR) assay EC. 50 And cover EC 90 Approximately 60 hours, but the in vitro pSMAD inhibition of TGFβR2 single-positive cells did not reach EC 50 (>100 μg / mL). Based on quantitative systems pharmacology (QSP) modeling and simulation, at a dose regimen of 100 mg Q2W, the predicted TGFβR2 target occupancy in tumors at steady state was [value missing]. 最大 At 30% and in C 谷 The percentage was 17%. Therefore, the proposed SSD is expected to produce pharmacological activity without safety issues.

[0238] (10) Antibody A 100 mg Q2W was 20 times lower than the expected safe dose modeled by QSP, with the expected safe dose being at least 2000 mg Q2W.

[0239] Based on preclinical data (e.g., toxicology, pharmacology, and PK data) and predicted human PK, antibody A 100 mg Q2W IV was selected as the SSD for this Phase 1 study. Based on emerging PK and pharmacodynamic data, the antibody A administration schedule may be changed to Q4W.

[0240] In Part 2, antibody A will be administered via the RDE identified in Part 1. If more than one RDE is selected, the following criteria must be met: (1) the RDEs do not have overlapping PK exposures (e.g., spaced 2 to 3 times apart); (2) the RDEs 低 It should not be lower than the minimum dose identified as exhibiting pharmacological activity; and (3) RDE 高 The mean time to dose (MTD) should not be exceeded. If two randomized dementias (RDEs) are selected for evaluation within a specific dose-expanded cohort, participants will be randomized to receive one of the RDEs during study participation.

[0241] Antibody A regimen

[0242] The starting dose of antibody A in Part 1 is 100 mg administered via intravenous infusion every 2 weeks (Q2W). The following additional dose levels will be evaluated during Part 1 of the study: 300 mg, 900 mg, 1500 mg, and 2000 mg. The frequency of Q4W administration may be explored during the study.

[0243] Goals and End Points

[0244] The primary objective of this study was to evaluate the safety and tolerability of antibody A in participants with selected advanced malignancies and to determine the mean time to treatment (MTD) and / or the rate of response to adverse events (RDE). The primary objectives were evaluated by measuring: (1) the incidence of dose-limiting toxicities (DLTs); (2) the incidence of treatment-related adverse events (TEAEs), assessed by physical examination, evaluation of changes in vital signs, left ventricular ejection fraction (LVEF) and electrocardiogram (ECG), and evaluation of clinical laboratory blood and urine samples; and (3) the incidence of TEAEs leading to interruption of study drug treatment and discontinuation of study drug due to adverse events (AEs).

[0245] The secondary objectives of this study were: (1) to determine the preliminary efficacy of antibody A in participants with selected advanced malignancies in terms of objective response rate (ORR), disease control rate (DCR), and duration of response (DOR); (2) to evaluate the pharmacokinetics of antibody A in participants with selected advanced malignancies; (3) to evaluate the pharmacodynamics of antibody A in participants with selected advanced malignancies; (4) to assess the immunogenicity of antibody A in participants with selected advanced malignancies; and (5) to evaluate the target binding of antibody A by means of receptor occupancy in participants with selected advanced malignancies.

[0246] Secondary objectives are evaluated by measuring the following endpoints: (1) Objective response: complete response (CR) or partial response (PR), as determined by the investigator according to RECIST v1.1 using radiographic disease assessment; Disease control: CR, PR, or stable disease (SD), as determined by the investigator according to RECIST v1.1 using radiographic disease assessment; and DOR: the time from the earliest date of disease response (CR or PR) to the earliest date of disease progression, as determined by the investigator according to RECIST v1.1 using radiographic disease assessment; (2) PK parameters of antibody A, including C 最大 t 最大 C 最小 AUC, CL, V z and t ½ (3) Pharmacodynamics of antibody A, including changes in T lymphocyte activation and cytokines in the blood and changes in T lymphocytes within the tumor; (4) Immunogenicity: defined as the incidence of antibody A-specific ADA; and (5) Receptor occupancy in peripheral blood samples.

[0247] This disclosure relates to the following implementation plan.

[0248] 1. A method for treating a disease in a human subject in need, wherein the disease is selected from the group consisting of: non-small cell lung cancer (NSCLC), head and neck squamous cell carcinoma (SCCHN), cervical squamous cell carcinoma and cervical adenocarcinoma (CESC), ovarian cancer, breast cancer, bladder cancer, renal cell carcinoma, melanoma, gastric adenocarcinoma, esophageal cancer, gastroesophageal adenocarcinoma, malignant pleural mesothelioma, pancreatic cancer, and colorectal cancer (CRC), wherein the method comprises administering to the human subject a therapeutically effective amount of a bispecific antibody bound to human programmed death-1 (PD-1) and human transforming growth factor β receptor 2 (TGFβR2), wherein the bispecific antibody comprises:

[0249] 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 wherein 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

[0250] The anti-human TGFβR2 binding domain comprises 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 wherein 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).

[0251] 2. The method of embodiment 1, wherein the PD-1 heavy chain variable region comprises the amino acid sequence QVQLVQSGSELKKPGASVKVSCKASGYTFTRFALHWVRQAPGQGLEWMGWIDPNTGTPTFAQGVTGRFVFSLDTSVTTAYLQISSLKAEDTAVYYCARSLGYCDSDICYPNWIFDNWGQGTLVTVSS (SEQ ID NO:4) and the TGFβR2 heavy chain variable region comprises the amino acid sequence EVQLVESGGGLVQPGGSLRLSCAASGFTFDIYAMTWVRQAPGKGLEWVSVISGSGGTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARRGQYRDIVGATDYWGQGTLVTVSS (SEQ ID NO:9).

[0252] 3. The method as described in embodiment 1 or embodiment 2, wherein the PD-1 light chain variable region and the TGFβR2 light chain variable region each comprise the amino acid sequence DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPTFGQGTKVEIK (SEQ ID NO:14).

[0253] 4. The method of embodiment 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 listed in SEQ ID NO:5, the PD-1 light chain comprises the amino acid sequence listed in SEQ ID NO:15, the TGFβR2 heavy chain comprises the amino acid sequence listed in SEQ ID NO:10, and the TGFβR2 light chain comprises the amino acid sequence listed in SEQ ID NO:15.

[0254] 5. The method as described in any one of embodiments 1-4, wherein the human subject suffers from NSCLC.

[0255] 6. The method of embodiment 5, wherein the NSCLC has squamous or non-squamous histology.

[0256] 7. The method as described in embodiment 5 or embodiment 6, wherein the NSCLC is late-stage or metastatic.

[0257] 8. The method of any one of embodiments 1-4, wherein the human subject suffers from SCCHN.

[0258] 9. The method of embodiment 8, wherein the SCCHN comprises a primary squamous tumor of the oral cavity, oropharynx, hypopharynx, or larynx.

[0259] 10. The method as described in embodiment 8 or embodiment 9, wherein the SCCHN is late or metastatic.

[0260] 11. The method of any one of embodiments 1-4, wherein the human subject suffers from CESC.

[0261] 12. The method as described in embodiment 11, wherein the CESC is late or metastatic.

[0262] 13. The method of any one of embodiments 1-4, wherein the human subject has human papillomavirus (HPV) positive SCCHN or HPV positive CESC.

[0263] 14. The method of any one of embodiments 1-4, wherein the human subject has ovarian cancer.

[0264] 15. The method of embodiment 14, wherein the ovarian cancer includes ovarian epithelial cancer, fallopian tube cancer, primary peritoneal cancer, or carcinosarcoma.

[0265] 16. The method as described in embodiment 14 or 15, wherein the ovarian cancer is advanced or metastatic.

[0266] 17. The method of any one of embodiments 1-4, wherein the human subject has breast cancer.

[0267] 18. The method of embodiment 17, wherein the breast cancer includes triple-negative breast cancer.

[0268] 19. The method of embodiment 18, wherein the triple-negative breast cancer comprises HER2-negative, ER-negative, and PgR-negative breast cancer.

[0269] 20. The method of any one of embodiments 17-19, wherein the breast cancer is advanced or metastatic.

[0270] 21. The method of any one of embodiments 1-4, wherein the human subject has bladder cancer.

[0271] 22. The method of embodiment 21, wherein the bladder cancer comprises urothelial carcinoma.

[0272] 23. The method as described in embodiment 21 or embodiment 22, wherein the bladder cancer is advanced or metastatic.

[0273] 24. The method of any one of embodiments 1-4, wherein the human subject has renal cell carcinoma.

[0274] 25. The method of embodiment 24, wherein the renal cell carcinoma is advanced or metastatic.

[0275] 26. The method of any one of embodiments 1-4, wherein the human subject has melanoma.

[0276] 27. The method of embodiment 26, wherein the melanoma is a cutaneous malignant melanoma.

[0277] 28. The method as described in embodiment 26 or embodiment 27, wherein the melanoma is advanced or metastatic.

[0278] 29. The method of any one of embodiments 1-4, wherein the human subject has gastric adenocarcinoma.

[0279] 30. The method as described in embodiment 29, wherein the gastric adenocarcinoma is advanced or metastatic.

[0280] 31. The method of any one of embodiments 1-4, wherein the human subject has esophageal cancer.

[0281] 32. The method as described in embodiment 31, wherein the esophageal cancer is advanced or metastatic.

[0282] 33. The method of any one of embodiments 1-4, wherein the human subject has gastric or esophageal adenocarcinoma.

[0283] 34. The method of embodiment 33, wherein the gastroesophageal adenocarcinoma is advanced or metastatic.

[0284] 35. The method of any one of embodiments 1-4, wherein the human subject has malignant pleural mesothelioma.

[0285] 36. The method as described in embodiment 35, wherein the malignant pleural mesothelioma is advanced or metastatic.

[0286] 37. The method of any one of embodiments 1-4, wherein the human subject has pancreatic cancer.

[0287] 38. The method as described in embodiment 37, wherein the pancreatic cancer is advanced or metastatic.

[0288] 39. The method of any one of embodiments 1-4, wherein the human subject suffers from CRC.

[0289] 40. The method of embodiment 39, wherein the CRC comprises microsatellite stable colorectal cancer (MSS-CRC).

[0290] 41. The method of embodiment 39, wherein the CRC comprises mismatch repair deficient (dMMR) / high microsatellite instability (MSI-H) colorectal cancer (dMMR / MSI-H CRC).

[0291] 42. The method of any one of embodiments 39-41, wherein the CRC is late or transferable.

[0292] 43. The method as described in any of the preceding embodiments, wherein the human subject has experienced disease progression following prior treatment.

[0293] 44. The method of embodiment 43, wherein the prior treatment comprises anti-PD-(L)1 therapy and / or anti-CTLA4 therapy.

[0294] 45. The method as described in any of the foregoing embodiments, wherein the condition is not suitable for curative treatment or procedures.

[0295] 46. ​​The method as described in any of the preceding embodiments, wherein the bispecific antibody is administered intravenously.

[0296] 47. The method of any of the preceding embodiments, wherein the bispecific antibody is administered at a dose of about 100 mg, about 300 mg, about 900 mg, about 1500 mg, or about 2000 mg.

[0297] 48. The method of any of the preceding embodiments, wherein the bispecific antibody is administered intravenously at a dose of about 100 mg, about 300 mg, about 900 mg, about 1500 mg or about 2000 mg.

[0298] 49. The method of any one of the foregoing embodiments, 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 listed in SEQ ID NO:5, the PD-1 light chain comprises the amino acid sequence listed in SEQ ID NO:15, the TGFβR2 heavy chain comprises the amino acid sequence listed in SEQ ID NO:10, and the TGFβR2 light chain comprises the amino acid sequence listed in SEQ ID NO:15, and wherein the bispecific antibody is administered intravenously at a dose of about 100 mg, about 300 mg, about 900 mg, about 1500 mg, or about 2000 mg.

[0299] 50. The method as described in any of the preceding embodiments, wherein the bispecific antibody is administered once every two weeks.

[0300] 51. The method of any of the preceding embodiments, wherein the bispecific antibody is administered intravenously every two weeks at a dose of about 100 mg, about 300 mg, about 900 mg, about 1500 mg or about 2000 mg.

[0301] 52. The method of any one of the foregoing embodiments, 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 listed in SEQ ID NO:5, the PD-1 light chain comprises the amino acid sequence listed in SEQ ID NO:15, the TGFβR2 heavy chain comprises the amino acid sequence listed in SEQ ID NO:10, and the TGFβR2 light chain comprises the amino acid sequence listed in SEQ ID NO:15, and wherein the bispecific antibody is administered intravenously every two weeks at a dose of about 100 mg, about 300 mg, about 900 mg, about 1500 mg, or about 2000 mg.

[0302] 53. The method of any one of embodiments 1-49, wherein the bispecific antibody is administered once every four weeks.

[0303] 54. The method of embodiment 53, wherein the bispecific antibody is administered intravenously once every four weeks at a dose of about 100 mg, about 300 mg, about 900 mg, about 1500 mg or about 2000 mg.

[0304] 55. The method of embodiment 53 or embodiment 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 listed in SEQ ID NO:5, the PD-1 light chain comprises the amino acid sequence listed in SEQ ID NO:15, the TGFβR2 heavy chain comprises the amino acid sequence listed in SEQ ID NO:10, and the TGFβR2 light chain comprises the amino acid sequence listed in SEQ ID NO:15, and wherein the bispecific antibody is administered intravenously once every four weeks at a dose of about 100 mg, about 300 mg, about 900 mg, about 1500 mg, or about 2000 mg.

[0305] Other implementation plans

[0306] While the invention has been described in conjunction with a detailed description, the foregoing description is intended to illustrate, and not limit, the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

1. Use of a bispecific antibody binding to human programmed death-1 (PD-1) and human transforming growth factor beta receptor 2 (TGFβR2) in the preparation of a medicament for treating a condition in a human subject in need, wherein the condition is selected from the group consisting of: colorectal cancer (CRC), non-small cell lung cancer (NSCLC), squamous cell carcinoma of the head and neck (SCCHN), cervical squamous cell carcinoma and cervical adenocarcinoma (CESC), ovarian cancer, breast cancer, bladder cancer, renal cell carcinoma, melanoma, gastric adenocarcinoma, esophageal cancer, gastroesophageal adenocarcinoma, malignant pleural mesothelioma, and pancreatic cancer, wherein the bispecific antibody comprises: 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 wherein 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 TGFβR2 binding domain comprises 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 wherein 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).

2. The use as claimed in claim 1, wherein the PD-1 heavy chain variable region comprises the amino acid sequence QVQLVQSGSELKKPGASVKVSCKASGYTFTRFALHWVRQAPGQGLEWMGWIDPNTGTPTFAQGVTGRFVFSLDTSVTTAYLQISSLKAEDTAVYYCARSLGYCDSDICYPNWIFDNWGQGTLVTVSS (SEQ ID NO:4) and the TGFβR2 heavy chain variable region comprises the amino acid sequence EVQLVESGGGLVQPGGSLRLSCAASGFTFDIYAMTWVRQAPGKGLEWVSVISGSGGTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARRGQYRDIVGATDYWGQGTLVTVSS (SEQ ID NO:9).

3. The use as described in claim 1 or claim 2, wherein the PD-1 light chain variable region and the TGFβR2 light chain variable region each comprise the amino acid sequence DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPTFGQGTKVEIK (SEQ ID NO:14).

4. The use as claimed in 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 listed in SEQ ID NO:5, the PD-1 light chain comprises the amino acid sequence listed in SEQ ID NO:15, the TGFβR2 heavy chain comprises the amino acid sequence listed in SEQ ID NO:10, and the TGFβR2 light chain comprises the amino acid sequence listed in SEQ ID NO:

15.

5. The use as described in any one of claims 1-4, wherein the human subject suffers from NSCLC.

6. The use as claimed in claim 5, wherein the NSCLC has squamous or non-squamous histology.

7. The use as described in claim 5 or claim 6, wherein the NSCLC is late-stage or metastatic.

8. The use as claimed in any one of claims 1-4, wherein the human subject suffers from SCCHN.

9. The use as claimed in claim 8, wherein the SCCHN comprises a primary squamous tumor of the oral cavity, oropharynx, hypopharynx, or larynx.

10. The use as described in claim 8 or claim 9, wherein the SCCHN is late or metastatic.

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