FAP-binding domain and bispecific binding moiety that binds FAP and TGF-βRII

Abstract

The present disclosure relates to polypeptides, FAP-binding domains comprising such polypeptides, and binding domains comprising such FAP-binding domains. The disclosure further relates to the use of such binding domains or binding moieties in the treatment of cancer. The disclosure further relates to bispecific binding moieties comprising a FAP-binding domain and a TGF-βRII-binding domain. The disclosure further relates to pharmaceutical compositions comprising an effective amount of the bispecific binding moieties, and methods for treating disease in a subject, comprising administering a therapeutically effective amount of the bispecific binding moiety.

Description

[Technical Field]

[0001] The present disclosure relates to the field of antibodies, particularly therapeutic antibodies for the treatment of diseases involving aberrant cells, particularly binding domains that bind to FAPs and binding moieties that include such FAP-binding domains, and further to bispecific binding moieties that include a binding domain that binds to a FAP and a binding domain that binds to TGF-βRII. [Background technology]

[0002] Cancer-associated fibroblasts (CAFs) have been identified as key factors promoting an immunosuppressive tumor microenvironment (TME), which blocks lymphocyte infiltration of tumors and thereby reduces the effectiveness of checkpoint blockade (Calon, A., et al. Dependency of Colorectal Cancer on a TGF-β-Driven Program in Stromal Cells for Metastasis Initiation. Cancer Cell. 2012 Nov13;22(5):571-584). CAFs promote malignant progression by endowing cancer cells with the capabilities for proliferation, migration, survival, and invasion. Upstream TGF-β has been found to be important in CAF activation and TME maintenance.

[0003] TGF-β signaling regulates many normal physiological and pathological processes, including cell cycle arrest in epithelial and hematopoietic cells, control of mesenchymal cell proliferation and differentiation, wound healing, extracellular matrix production, immunosuppression, and carcinogenesis (Massague J. TGFβ signaling in context. Nat Rev Mol Cell Biol. 2012 Oct; 13(10):616-30). Additionally, TGF-β signaling regulates many cancer cell functions, including cell cycle progression, apoptosis, adhesion, and differentiation (Liu S et al., Signal Transduction and Targeted Therapy, 2021). While TGF-β has been reported to primarily act as a tumor suppressor in normal and precancerous cells, it also exhibits biphasic functions in tumor cells, enabling growth-promoting functions, angiogenesis, and epithelial-mesenchymal transition, thereby enabling tumor cell migration, invasion, intravasation, and extravasation.

[0004] There are three types of TGF-β ligands (TGF-β1, 2, and 3), which mediate signaling through binding to TGF-β receptor type 2 (TGF-βRII), leading to dimerization with TGF-βRI and phosphorylation of TGF-βRI. This heterotetrameric complex, consisting of two TGF-βRIIs and two TGF-βRIs, then recruits and phosphorylates SMAD2 and SMAD3, which then recruit and bind the co-SMAD molecule SMAD4 to form a SMAD / co-SMAD complex that translocates to the nucleus and regulates the transcription of TGF-β target genes (Hata A, Chen YG. TGF-β Signaling from Receptors to Smads. Cold Spring Harb Perspect Biol. 2016 Sep 1;8(9):a022061; Vander Ark A et al. TGF-β receptors: In and beyond TGF-β signaling. Cell Signal. 2018 Dec;52:112-120). TGF-βRII belongs to the serine / threonine protein kinase family and the TGF-β receptor subfamily. It is known by various synonyms, including TGFBR2, AAT3, FAA3, LDS1B, LDS2, LDS2B, MFS2, RIIC, TAAD2, TGFR-2, TGFβ-RII, transforming growth factor beta receptor type 2, TBR-ii, and TBRII.

[0005] Specific moieties targeting the TGF-β pathway have been reported to exhibit antitumor activity in vitro and in vivo; however, poor clinical outcomes continue due to low efficacy and unacceptable toxicity, including significant cytokine release syndrome (CRS). Combined targeting of the TGF-β pathway and immune checkpoint blockade has been attempted using Bintrafusp alpha, an anti-PD-L1-TGF-βRII bifunctional fusion protein, but failed to demonstrate robust clinical efficacy and reduced toxicity.

[0006] Fibroblast activation protein (FAP) is a cell surface serine protease involved in the degradation of extracellular matrix. It is known by various synonyms, including Seprase, DPPIV, FAPalpha, SIMP, dipeptidyl peptidase FAP, and FAPA. FAP is not expressed in normal adult tissues; however, its expression is induced in activated fibroblasts during wound healing, stromal cells of epithelial cancers, and some sarcomas (Kelly T. Fibroblast activation protein-alpha and dipeptidyl peptidase IV (CD26): cell-surface proteases that activate cell signaling and are potential targets for cancer therapy. Drug Resist Update. 2005 Feb-Apr;8(1-2):51-8). FAP is strongly overexpressed in CAFs, which are present in the stroma of approximately 90% of all human epithelial cancers, including breast, lung, and colon cancers. FAP is specifically upregulated by TGF-β. FAP degrades gelatin and type I collagen in the extracellular matrix through its dipeptidyl peptidase and collagenolytic activities (Huber MA, et al. Fibroblast activation protein: differential expression and serine protease activity in reactive stromal fibroblasts of melanocytic skin tumors. J Invest Dermatol. 2003 Feb;120(2):182-8). By locally degrading extracellular matrix components, FAP plays an important role in cell migration and matrix invasion that occur during tumor invasion, angiogenesis, and metastasis.

[0007] Several anti-FAP antibodies are being investigated in clinical trials. The scFv M036 was selected from FAP- / - immune mice and is cross-reactive with human FAP. M036 has been used to generate anti-FAP-CAR human T cells in an immunodeficient mouse model of human lung cancer. The anti-FAP antibody sibrotuzumab, a humanized version of the murine monoclonal antibody F19, has been tested in phase II clinical trials for metastatic CRC and NSCLC. Both trials failed due to lack of therapeutic efficacy. FAP5-DM1, a maytansinoid conjugate of the monoclonal antibody FAP5, has been reported to inhibit tumor growth in xenograft models of lung, pancreatic, and head and neck cancer.

[0008] There remains a need for novel therapeutic interventions that selectively target cancer-associated fibroblasts in the tumor microenvironment. Furthermore, there remains a need for novel therapeutic interventions that selectively inhibit TGF-βRII signaling in the tumor microenvironment. Such targeting aims to inhibit metastasis formation and restore tumor immunity while limiting TGF-β pathway blockade in normal tissues. Summary of the Invention

[0009] One objective of the present disclosure is to provide new pharmaceuticals for the treatment of human diseases, particularly cancer. This objective is achieved by providing a FAP-binding domain and a bispecific binding moiety (e.g., a bispecific antibody) that contains such a FAP-binding domain and binds to both a FAP and TGF-βRII. The FAP-binding domain of the bispecific binding moiety drives the specificity of the bispecific binding moiety for cancer-associated fibroblasts (CAFs) in the tumor microenvironment (TME), where the TGF-βRII-binding domain can locally block TGF-β from binding to TGF-βRII in the TME. Furthermore, the bispecific binding moiety is intended to promote cytotoxic T lymphocyte activity in the tumor microenvironment by alleviating TGF-β-mediated immunosuppressive pathways against activated / exhausted effector T cells.

[0010] In certain embodiments, the present disclosure provides FAP binding domains comprising polypeptides as further detailed herein, which are particularly useful for generating binding moieties such as antibodies.

[0011] In certain embodiments, the present disclosure provides a polypeptide selected from: - a polypeptide comprising a heavy chain CDR1 (HCDR1) having the amino acid sequence set forth in SEQ ID NO: 16, a heavy chain CDR2 (HCDR2) having the amino acid sequence set forth in SEQ ID NO: 17, and a heavy chain CDR3 (HCDR3) having the amino acid sequence set forth in SEQ ID NO: 18; - a polypeptide comprising a heavy chain CDR1 (HCDR1) having the amino acid sequence set forth in SEQ ID NO: 20, a heavy chain CDR2 (HCDR2) having the amino acid sequence set forth in SEQ ID NO: 21, and a heavy chain CDR3 (HCDR3) having the amino acid sequence set forth in SEQ ID NO: 22; - a polypeptide comprising a heavy chain CDR1 (HCDR1) having the amino acid sequence set forth in SEQ ID NO: 12, a heavy chain CDR2 (HCDR2) having the amino acid sequence set forth in SEQ ID NO: 13, and a heavy chain CDR3 (HCDR3) having the amino acid sequence set forth in SEQ ID NO: 14; or - a polypeptide comprising a heavy chain CDR1 (HCDR1) having the amino acid sequence set forth in SEQ ID NO: 70, a heavy chain CDR2 (HCDR2) having the amino acid sequence set forth in SEQ ID NO: 71, and a heavy chain CDR3 (HCDR3) having the amino acid sequence set forth in SEQ ID NO: 72.

[0012] In certain embodiments, the present disclosure provides a FAP binding domain comprising a polypeptide described herein.

[0013] In certain embodiments, the present disclosure provides FAP-binding domains that bind to human and mouse FAPs.

[0014] In certain embodiments, the present disclosure provides a binding moiety comprising a polypeptide described herein or a FAP-binding domain.

[0015] In certain embodiments, the present disclosure provides a pharmaceutical composition comprising an effective amount of a polypeptide, or FAP-binding domain, or binding portion described herein, and a pharmaceutically acceptable carrier.

[0016] In certain embodiments, the present disclosure provides a polypeptide, or FAP-binding domain, or binding portion, or pharmaceutical composition described herein for use in therapy.

[0017] In certain embodiments, the present disclosure provides a polypeptide, or FAP-binding domain, or binding portion, or pharmaceutical composition described herein for use in the treatment of cancer.

[0018] In certain embodiments, the present disclosure provides a method for treating a disease, the method comprising administering to an individual in need thereof an effective amount of a polypeptide, or FAP-binding domain, or binding portion, or pharmaceutical composition described herein.

[0019] In certain embodiments, the present disclosure provides a method for treating cancer, comprising administering to an individual in need thereof an effective amount of a polypeptide, or FAP-binding domain, or binding portion, or pharmaceutical composition described herein.

[0020] In certain embodiments, the present disclosure provides a nucleic acid comprising a sequence encoding a polypeptide described herein.

[0021] In certain embodiments, the present disclosure provides a vector comprising a nucleic acid sequence described herein.

[0022] In certain embodiments, the present disclosure provides a cell comprising a nucleic acid described herein.

[0023] In certain embodiments, the present disclosure provides cells that produce a polypeptide, or FAP-binding domain, or binding portion described herein.

[0024] In certain embodiments, the present disclosure provides a bispecific binding moiety comprising a FAP-binding domain and a TGF-βRII-binding domain, wherein the TGF-βRII-binding domain blocks TGF-βRII-mediated signaling.

[0025] In one embodiment, the present disclosure provides a bispecific binding moiety comprising a FAP-binding domain and a TGF-βRII-binding domain, wherein the FAP-binding domain binds to a FAP expressed on a first cell, and the TGF-βRII-binding domain binds to a TGF-βRII expressed on a second cell.

[0026] In one embodiment, the present disclosure provides a bispecific binding moiety comprising a FAP-binding domain and a TGF-βRII-binding domain, wherein the FAP-binding domain comprises a heavy chain variable region comprising CDR1, CDR2, and CDR3 sequences as further described herein.

[0027] In one embodiment, the present disclosure provides a bispecific binding moiety comprising a FAP binding domain and a TGF-βRII binding domain, wherein the TGF-βRII binding domain comprises a heavy chain variable region comprising CDR1, CDR2, and CDR3 sequences as further described herein.

[0028] In certain embodiments, the present disclosure provides a pharmaceutical composition comprising an effective amount of a bispecific binding moiety described herein.

[0029] In certain embodiments, the present disclosure provides bispecific binding moieties described herein, and pharmaceutical compositions described herein, for use in therapy.

[0030] In certain embodiments, the present disclosure provides a bispecific binding moiety as described herein, and a pharmaceutical composition as described herein, for use in the treatment of cancer.

[0031] In certain embodiments, the present disclosure provides a combination of a bispecific binding moiety described herein and a second binding moiety that binds to PD-1 for use in therapy.

[0032] In certain embodiments, the present disclosure provides a combination of a bispecific binding moiety described herein and a second binding moiety that binds to PD-1 for use in treating cancer.

[0033] In certain embodiments, the present disclosure provides a method for treating a disease, the method comprising administering an effective amount of a bispecific binding moiety described herein, or a pharmaceutical composition described herein, to an individual in need thereof.

[0034] In certain embodiments, the present disclosure further provides a method for treating cancer, the method comprising administering an effective amount of a bispecific binding moiety described herein, or a pharmaceutical composition described herein, to an individual in need thereof.

[0035] In certain embodiments, the present disclosure further provides a nucleic acid sequence encoding the heavy chain variable region of a FAP binding domain described herein.

[0036] In certain embodiments, the present disclosure further provides nucleic acid sequences encoding the heavy chain variable region of the FAP binding domain and the heavy chain variable region of the TGF-βRII binding domain described herein.

[0037] In one embodiment, the present disclosure provides a cell comprising a nucleic acid sequence encoding the heavy chain variable region of the FAP binding domain described herein and a nucleic acid sequence encoding the heavy chain variable region of the TGF-βRII binding domain described herein.

[0038] In certain embodiments, the present disclosure further provides cells that produce the bispecific binding moieties described herein.

[0039] In certain embodiments, the present disclosure further provides bispecific binding moieties that compete with the bispecific binding moieties described herein for binding to FAP and TGF-βRII.

[0040] [Brief description of the drawing] As follows, the following naming conventions are used herein: In the figures, a bivalent monospecific antibody is shown in the format SEQ ID NO: A / B, where SEQ ID NO: A refers to the heavy chain of both binding domains and SEQ ID NO: B refers to the light chain of both binding domains. A bivalent bispecific antibody is shown in the format SEQ ID NO: A x B, where SEQ ID NO: A refers to the heavy chain variable region of one binding domain and SEQ ID NO: B refers to the heavy chain variable region of the other binding domain. Both domains contain identical light chains as described herein. [Brief explanation of the drawings]

[0041] [Figure 1] Expression levels of FAP and TGF-βRII are shown on primary CAF cell lines, including primary human bladder CAFs (BLDA), primary human breast CAFs (breast), primary colon carcinoma CAFs (COAD1), primary human head and neck CAFs (HNSC), primary lung adenocarcinoma CAFs (LUAD), primary human lung squamous cell carcinoma CAFs (LUSC), primary melanoma CAFs (MEL), and primary pancreatic stellate cell CAFs (PAAD). [Figure 1A] FAP expression levels expressed as mean fluorescence intensity (MFI). [Figure 1B] TGF-βRII expression levels expressed as mean fluorescence intensity (MFI). [Figure 2] 1 shows inhibition of pSMAD2 and IL-11 by test and control antibodies in primary CAFs. [Figure 2A] Inhibition of pSMAD2 in primary colonic CAFs, expressed as % inhibition measured at different antibody concentrations. The control antibody is a bivalent monospecific TGF-βRII antibody comprising a heavy chain having the amino acid sequence set forth in SEQ ID NO: 1 and a light chain having the amino acid sequence set forth in SEQ ID NO: 2. Test antibodies include: a bivalent bispecific antibody comprising a FAP-binding domain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 11 and a TGF-βRII-binding domain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 23; a bivalent bispecific antibody comprising a FAP-binding domain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 15 and a TGF-βRII-binding domain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 31; and a bivalent bispecific antibody comprising a FAP-binding domain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 19 and a TGF-βRII-binding domain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 27. [Figure 2B]Inhibition of IL-11 in primary colonic CAFs, expressed as % inhibition measured at different antibody concentrations. The control antibody is a bivalent monospecific TGF-βRII antibody comprising a heavy chain having the amino acid sequence set forth in SEQ ID NO: 1 and a light chain having the amino acid sequence set forth in SEQ ID NO: 2. The test antibodies include: a bivalent bispecific antibody comprising a FAP-binding domain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 11 and a TGF-βRII-binding domain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 23; a bivalent bispecific antibody comprising a FAP-binding domain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 15 and a TGF-βRII-binding domain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 31; and a bivalent bispecific antibody comprising a FAP-binding domain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 19 and a TGF-βRII-binding domain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 27. [Figure 2C] Inhibition of pSMAD2 in primary lung adenocarcinoma (LUAD) CAFs, expressed as % inhibition measured at different antibody concentrations. Control antibodies are: a bivalent monospecific RSV-G antibody comprising a heavy chain having the amino acid sequence set forth in SEQ ID NO: 3 and a light chain having the amino acid sequence set forth in SEQ ID NO: 4, and a bivalent monospecific TGF-βRII antibody comprising a heavy chain having the amino acid sequence set forth in SEQ ID NO: 1 and a light chain having the amino acid sequence set forth in SEQ ID NO: 2. Test antibodies include: a bivalent bispecific antibody comprising a FAP binding domain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 11 and a TGF-βRII binding domain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 23; and a bivalent bispecific antibody comprising a FAP binding domain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 15 and a TGF-βRII binding domain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 31. [Figure 2D]Inhibition of IL-11 in primary lung adenocarcinoma (LUAD) CAFs, expressed as % inhibition measured at different antibody concentrations. Control antibodies are: a bivalent monospecific RSV-G antibody comprising a heavy chain having the amino acid sequence set forth in SEQ ID NO: 3 and a light chain having the amino acid sequence set forth in SEQ ID NO: 4, and a bivalent monospecific TGF-βRII antibody comprising a heavy chain having the amino acid sequence set forth in SEQ ID NO: 1 and a light chain having the amino acid sequence set forth in SEQ ID NO: 2. Test antibodies include: a bivalent bispecific antibody comprising a FAP binding domain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 11 and a TGF-βRII binding domain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 23; and a bivalent bispecific antibody comprising a FAP binding domain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 15 and a TGF-βRII binding domain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 31. [Figure 2E]Inhibition of IL-11 in primary colonic CAFs, expressed as the concentration of IL-11 (pg / ml) measured at different antibody concentrations. Control antibodies were: an Fc-enhanced bivalent monospecific RSV-G antibody comprising a heavy chain having the amino acid sequence set forth in SEQ ID NO: 3 and a light chain having the amino acid sequence set forth in SEQ ID NO: 4; an Fc-silenced bivalent monospecific RSV-G antibody comprising a heavy chain having the amino acid sequence set forth in SEQ ID NO: 5 and a light chain having the amino acid sequence set forth in SEQ ID NO: 4; a bivalent monospecific TGF-βRII antibody comprising a heavy chain having the amino acid sequence set forth in SEQ ID NO: 1 and a light chain having the amino acid sequence set forth in SEQ ID NO: 2; and an Fc-enhanced bivalent monospecific FAP antibody comprising a heavy chain having the amino acid sequence set forth in SEQ ID NO: 7 and a light chain having the amino acid sequence set forth in SEQ ID NO: 8. The test antibodies include the following: an Fc-inactivated bivalent bispecific antibody comprising a FAP-binding domain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 15 and a TGF-βRII-binding domain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 31; an Fc-enhanced bivalent bispecific antibody comprising a FAP-binding domain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 15 and a TGF-βRII-binding domain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 31; an Fc-inactivated bivalent bispecific antibody comprising a FAP-binding domain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 15 and a TGF-βRII-binding domain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 35; and an Fc-enhanced bivalent bispecific antibody comprising a FAP-binding domain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 15 and a TGF-βRII-binding domain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 35. [Figure 3] Inhibition of pSMAD2 in A549 parental and A549-FAP+ cells is shown, expressed as % inhibition measured at different antibody concentrations. [Figure 3A]Inhibition of pSMAD2 in A549-FAP+ cells is induced by a bivalent bispecific antibody comprising a FAP-binding domain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 11 and a TGF-βRII-binding domain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 23. [Figure 3B] Inhibition of pSMAD2 in A549 parental cells is induced by a bivalent bispecific antibody comprising a FAP-binding domain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 11 and a TGF-βRII-binding domain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 23. [Figure 3C] Inhibition of pSMAD2 in A549-FAP+ cells is induced by a bivalent bispecific antibody comprising a FAP-binding domain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 15 and a TGF-βRII-binding domain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 31. [Figure 3D] Inhibition of pSMAD2 in A549 parental cells is induced by a bivalent bispecific antibody comprising a FAP-binding domain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 15 and a TGF-βRII-binding domain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 31. [Figure 3E] Inhibition of pSMAD2 in A549-FAP+ cells is induced by a bivalent bispecific antibody comprising a FAP-binding domain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 19 and a TGF-βRII-binding domain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 27. [Figure 3F] Inhibition of pSMAD2 in A549 parental cells is induced by a bivalent bispecific antibody comprising a FAP-binding domain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 19 and a TGF-βRII-binding domain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 27. [Figure 4]1 shows the ADCC activity of antibodies in A549 parental cells versus A549-FAP+ cells. [Figure 4A] ADCC activity of antibodies in A549 parental cells, expressed in RLU measured at different antibody concentrations. Control antibodies are: a bivalent, monospecific RSV-G antibody comprising a heavy chain having the amino acid sequence set forth in SEQ ID NO:3 and a light chain having the amino acid sequence set forth in SEQ ID NO:4; a bivalent, monospecific TGF-βRII antibody comprising a heavy chain having the amino acid sequence set forth in SEQ ID NO:1 and a light chain having the amino acid sequence set forth in SEQ ID NO:2; and cetuximab. The test antibodies include: a bivalent bispecific antibody comprising a FAP-binding domain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 11, and a TGF-βRII-binding domain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 23; a bivalent bispecific antibody comprising a FAP-binding domain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 15, and a TGF-βRII-binding domain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 31; and a bivalent bispecific antibody comprising a FAP-binding domain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 19, and a TGF-βRII-binding domain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 27. [Figure 4B]ADCC activity of antibodies in A549-FAP+ cells, expressed in RLU measured at different antibody concentrations. Control antibodies are: a bivalent, monospecific RSV-G antibody comprising a heavy chain having the amino acid sequence set forth in SEQ ID NO:3 and a light chain having the amino acid sequence set forth in SEQ ID NO:4; a bivalent, monospecific TGF-βRII antibody comprising a heavy chain having the amino acid sequence set forth in SEQ ID NO:1 and a light chain having the amino acid sequence set forth in SEQ ID NO:2; and cetuximab. The test antibodies include: a bivalent bispecific antibody comprising a FAP-binding domain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 11, and a TGF-βRII-binding domain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 23; a bivalent bispecific antibody comprising a FAP-binding domain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 15, and a TGF-βRII-binding domain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 31; and a bivalent bispecific antibody comprising a FAP-binding domain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 19, and a TGF-βRII-binding domain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 27. [Figure 5] ADCC activity of control and test antibodies with and without Fc modifications is shown. [Figure 5A] ADCC activity of control antibodies against primary melanoma CAFs (MELs), expressed as rounded CAFs per well, measured at different antibody concentrations. The control antibodies include: a bivalent monospecific RSV-G antibody comprising a heavy chain having the amino acid sequence set forth in SEQ ID NO: 3 and a light chain having the amino acid sequence set forth in SEQ ID NO: 4; and cetuximab. [Figure 5B]ADCC activity of test antibodies against primary melanoma CAFs (MELs), expressed as rounded CAFs per well, measured at different antibody concentrations. The test antibodies included: an Fc-unmodified bivalent bispecific antibody comprising a FAP-binding domain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 11 and a TGF-βRII-binding domain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 23; an Fc-inactivated bivalent bispecific antibody comprising a FAP-binding domain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 11 and a TGF-βRII-binding domain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 23; and an Fc-enhanced bivalent bispecific antibody comprising a FAP-binding domain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 11 and a TGF-βRII-binding domain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 23. [Figure 5C] ADCC activity of test antibodies against primary melanoma CAFs (MELs), expressed as rounded CAFs per well, measured at different antibody concentrations. The test antibodies include: an Fc-unmodified bivalent bispecific antibody comprising a FAP-binding domain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 15 and a TGF-βRII-binding domain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 31; an Fc-inactivated bivalent bispecific antibody comprising a FAP-binding domain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 15 and a TGF-βRII-binding domain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 31; and an Fc-enhanced bivalent bispecific antibody comprising a FAP-binding domain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 15 and a TGF-βRII-binding domain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 31. [Figure 5D]ADCC activity of control antibodies against primary lung adenocarcinoma CAFs (LUAD), expressed as the number of detectable CAFs per well, measured at different antibody concentrations. The control antibodies include: a bivalent monospecific RSV-G antibody comprising a heavy chain having the amino acid sequence set forth in SEQ ID NO:3 and a light chain having the amino acid sequence set forth in SEQ ID NO:4; and cetuximab. [Figure 5E] ADCC activity of test antibodies against primary lung adenocarcinoma CAFs (LUAD), expressed as the number of detectable CAFs per well, measured at different antibody concentrations. The test antibodies include: an Fc-unmodified bivalent bispecific antibody comprising a FAP-binding domain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 11 and a TGF-βRII-binding domain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 23; an Fc-inactivated bivalent bispecific antibody comprising a FAP-binding domain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 11 and a TGF-βRII-binding domain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 23; and an Fc-enhanced bivalent bispecific antibody comprising a FAP-binding domain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 11 and a TGF-βRII-binding domain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 23. [Figure 5F]ADCC activity of test antibodies against primary lung adenocarcinoma CAFs (LUAD), expressed as the number of detectable CAFs per well, measured at different antibody concentrations. The test antibodies include: an Fc-unmodified bivalent bispecific antibody comprising a FAP-binding domain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 15 and a TGF-βRII-binding domain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 31; an Fc-inactivated bivalent bispecific antibody comprising a FAP-binding domain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 15 and a TGF-βRII-binding domain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 31; and an Fc-enhanced bivalent bispecific antibody comprising a FAP-binding domain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 15 and a TGF-βRII-binding domain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 31. [Figure 5G]ADCC activity of test antibodies against colonic CAFs, expressed as fold induction of luciferase expression measured at different antibody concentrations. Control antibodies include: an Fc-enhanced bivalent monospecific RSV-G antibody comprising a heavy chain having the amino acid sequence set forth in SEQ ID NO: 3 and a light chain having the amino acid sequence set forth in SEQ ID NO: 4; and an Fc-inactivated bivalent monospecific RSV-G antibody comprising a heavy chain having the amino acid sequence set forth in SEQ ID NO: 5 and a light chain having the amino acid sequence set forth in SEQ ID NO: 4. The test antibodies include the following: an Fc-enhanced bivalent bispecific antibody comprising a FAP-binding domain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 15 and a TGF-βRII-binding domain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 31; an Fc-inactivated bivalent bispecific antibody comprising a FAP-binding domain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 15 and a TGF-βRII-binding domain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 31; an Fc-enhanced bivalent bispecific antibody comprising a FAP-binding domain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 15 and a TGF-βRII-binding domain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 35; and an Fc-inactivated bivalent bispecific antibody comprising a FAP-binding domain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 15 and a TGF-βRII-binding domain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 35. [Figure 6] In vivo studies demonstrate selective localization of the antibody and inhibition of TGF-βRII-mediated signaling in tumor cells expressing both FAP and TGF-βRII. [Figure 6A]Staining of tumor cells isolated from mice inoculated with A549 parental cells (left graph) and A549-FAP+ cells (right graph) by control and test antibodies, expressed as IgG+, % viable cells. Control antibodies include: a bivalent, monospecific RSV-G antibody comprising a heavy chain having the amino acid sequence set forth in SEQ ID NO:3 and a light chain having the amino acid sequence set forth in SEQ ID NO:4; and a bivalent, monospecific TGF-βRII antibody comprising a heavy chain having the amino acid sequence set forth in SEQ ID NO:1 and a light chain having the amino acid sequence set forth in SEQ ID NO:2. The test antibodies include: a bivalent bispecific antibody comprising a FAP-binding domain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 19, and a TGF-βRII-binding domain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 27; a bivalent bispecific antibody comprising a FAP-binding domain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 11, and a TGF-βRII-binding domain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 23; and a bivalent bispecific antibody comprising a FAP-binding domain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 15, and a TGF-βRII-binding domain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 31. [Figure 6B]Inhibition of IL-11 by test control and test antibodies in A549 parental cells (left graph) and A549-FAP+ cells (right graph), expressed as mean fluorescence intensity (MFI). Control antibodies include: a bivalent, monospecific RSV-G antibody comprising a heavy chain having the amino acid sequence set forth in SEQ ID NO:3 and a light chain having the amino acid sequence set forth in SEQ ID NO:4; and a bivalent, monospecific TGF-βRII antibody comprising a heavy chain having the amino acid sequence set forth in SEQ ID NO:1 and a light chain having the amino acid sequence set forth in SEQ ID NO:2. The test antibodies include: a bivalent bispecific antibody comprising a FAP-binding domain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 19, and a TGF-βRII-binding domain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 27; a bivalent bispecific antibody comprising a FAP-binding domain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 11, and a TGF-βRII-binding domain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 23; and a bivalent bispecific antibody comprising a FAP-binding domain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 15, and a TGF-βRII-binding domain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 31. [Figure 6C]Inhibition of pSMAD2 by test control and test antibodies in A549 parental cells (left graph) and A549-FAP+ cells (right graph), expressed as mean fluorescence intensity (MFI). Control antibodies include: a bivalent, monospecific RSV-G antibody comprising a heavy chain having the amino acid sequence set forth in SEQ ID NO:3 and a light chain having the amino acid sequence set forth in SEQ ID NO:4; and a bivalent, monospecific TGF-βRII antibody comprising a heavy chain having the amino acid sequence set forth in SEQ ID NO:1 and a light chain having the amino acid sequence set forth in SEQ ID NO:2. The test antibodies include: a bivalent bispecific antibody comprising a FAP-binding domain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 19, and a TGF-βRII-binding domain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 27; a bivalent bispecific antibody comprising a FAP-binding domain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 11, and a TGF-βRII-binding domain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 23; and a bivalent bispecific antibody comprising a FAP-binding domain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 15, and a TGF-βRII-binding domain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 31. [Figure 7] 1 shows in vivo tumor efficacy with control and test antibodies. [Figure 7A]In vivo tumor efficacy with control antibodies: a bivalent, monospecific RSV-G antibody having an enhanced effector function, comprising a heavy chain having the amino acid sequence set forth in SEQ ID NO: 3 and a light chain having the amino acid sequence set forth in SEQ ID NO: 4, administered at 30 mg / kg (Group 1); a bivalent, monospecific TGF-βRII antibody having a heavy chain having the amino acid sequence set forth in SEQ ID NO: 1 and a light chain having the amino acid sequence set forth in SEQ ID NO: 2, administered at 30 mg / kg (Group 2); cetuximab, administered at 30 mg / kg (Group 3); and a bivalent, monospecific FAP antibody having an enhanced Fc effector function, comprising a heavy chain having the amino acid sequence set forth in SEQ ID NO: 7 and a light chain having the amino acid sequence set forth in SEQ ID NO: 8, administered at 3 mg / kg (Group 4) and 30 mg / kg (Group 5). [Figure 7B]In vivo tumor efficacy with control and test antibodies. Control antibodies include: a bivalent, monospecific RSV-G antibody with enhanced Fc effector function, comprising a heavy chain having the amino acid sequence set forth in SEQ ID NO: 3 and a light chain having the amino acid sequence set forth in SEQ ID NO: 4, administered at 30 mg / kg (Group 1); a bivalent, monospecific TGF-βRII antibody with an unmodified Fc format, comprising a heavy chain having the amino acid sequence set forth in SEQ ID NO: 1 and a light chain having the amino acid sequence set forth in SEQ ID NO: 2, administered at 30 mg / kg (Group 2); and cetuximab, administered at 30 mg / kg (Group 3). The test antibodies included: a bivalent bispecific antibody with enhanced Fc effector function, comprising a FAP-binding domain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 15 and a TGF-βRII-binding domain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 31, administered at 3 mg / kg (Group 6); a bivalent bispecific antibody with enhanced Fc effector function, comprising a FAP-binding domain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 15 and a TGF-βRII-binding domain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 31, administered at 30 mg / kg (Group 7); and a bivalent bispecific antibody with unmodified effector function, comprising a FAP-binding domain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 15 and a TGF-βRII-binding domain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 31, administered at 30 mg / kg (Group 8). [Figure 7C] In vivo tumor efficacy of test antibodies. Inhibition of tumor growth is expressed as tumor volume (mm3) measured on different days (graph). Arrows indicate the days on which the test antibodies were administered. Test antibodies included: a bivalent, bispecific antibody with enhanced effector function comprising a FAP-binding domain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 15 and a TGF-βRII-binding domain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 35, administered at 30 mg / kg (Group 9). [Figure 8]Figure 1 shows the efficacy of control and test antibodies as single agents in a trans-activity mouse model. [Figure 8A] Tumor growth inhibition is expressed as tumor volume (mm3) measured at different days (graph) and as tumor growth inhibition (TGI, %) at day 24 (table). Control antibodies include: a bivalent, monospecific RSV-G antibody comprising a heavy chain having the amino acid sequence set forth in SEQ ID NO: 3 and a light chain having the amino acid sequence set forth in SEQ ID NO: 4, administered at 10 mg / kg (open circles); and a bivalent, monospecific TGF-βRII antibody comprising a heavy chain having the amino acid sequence set forth in SEQ ID NO: 1 and a light chain having the amino acid sequence set forth in SEQ ID NO: 2, administered at 10 mg / kg (filled circles). The test antibodies included: a bivalent, bispecific antibody comprising a murine FAP binding domain and a TGF-βRII binding domain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 23, administered at 1 mg / kg (filled squares); and a bivalent, bispecific antibody comprising a murine FAP binding domain and a TGF-βRII binding domain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 23, administered at 10 mg / kg (filled triangles). *P<0.05 compared to a bivalent, monospecific RSV-G antibody comprising a heavy chain having the amino acid sequence set forth in SEQ ID NO: 3 and a light chain having the amino acid sequence set forth in SEQ ID NO: 4. [Figure 8B]Tumor growth inhibition is expressed as tumor volume (mm3) measured at different days (graph) and as tumor growth inhibition (TGI, %) at day 24 (table). Control antibodies include: a bivalent, monospecific RSV-G antibody comprising a heavy chain having the amino acid sequence set forth in SEQ ID NO: 3 and a light chain having the amino acid sequence set forth in SEQ ID NO: 4, administered at 10 mg / kg (open circles); and a bivalent, monospecific TGF-βRII antibody comprising a heavy chain having the amino acid sequence set forth in SEQ ID NO: 1 and a light chain having the amino acid sequence set forth in SEQ ID NO: 2, administered at 10 mg / kg (filled circles). The test antibodies included: a bivalent, bispecific antibody comprising a murine FAP binding domain and a TGF-βRII binding domain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 31, administered at 1 mg / kg (filled squares); and a bivalent, bispecific antibody comprising a murine FAP binding domain and a TGF-βRII binding domain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 31, administered at 10 mg / kg (filled triangles). *P<0.05 compared to a bivalent, monospecific RSV-G antibody comprising a heavy chain having the amino acid sequence set forth in SEQ ID NO: 3 and a light chain having the amino acid sequence set forth in SEQ ID NO: 4. [Figure 9] 1 shows the efficacy of control and test antibodies in combination with pembrolizumab in a transactivation mouse model. [Figure 9A]Tumor growth inhibition is expressed as tumor volume (mm3) measured on different days (graph) and as tumor growth inhibition rate (TGI, %) at day 24 (table). Controls include: a bivalent monospecific RSV-G antibody comprising a heavy chain having the amino acid sequence set forth in SEQ ID NO: 3 and a light chain having the amino acid sequence set forth in SEQ ID NO: 4; a bivalent monospecific TGF-βRII antibody comprising a heavy chain having the amino acid sequence set forth in SEQ ID NO: 1 and a light chain having the amino acid sequence set forth in SEQ ID NO: 2; a combination of a bivalent monospecific TGF-βRII antibody comprising a heavy chain having the amino acid sequence set forth in SEQ ID NO: 1 and a light chain having the amino acid sequence set forth in SEQ ID NO: 2 with pembrolizumab; and pembrolizumab. The test antibodies included: a bivalent, bispecific antibody comprising a murine FAP binding domain and a TGF-βRII binding domain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 23; and a combination of a bivalent, bispecific antibody comprising a murine FAP binding domain and a TGF-βRII binding domain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 23 with pembrolizumab. *P<0.05 compared to a bivalent, monospecific RSV-G antibody comprising a heavy chain having the amino acid sequence set forth in SEQ ID NO: 3 and a light chain having the amino acid sequence set forth in SEQ ID NO: 4. [Figure 9B]Tumor growth inhibition is expressed as tumor volume (mm3) measured on different days (graph) and as tumor growth inhibition rate (TGI, %) at day 24 (table). Control antibodies include: a bivalent monospecific RSV-G antibody comprising a heavy chain having the amino acid sequence set forth in SEQ ID NO: 3 and a light chain having the amino acid sequence set forth in SEQ ID NO: 4; and a bivalent monospecific TGF-βRII antibody comprising a heavy chain having the amino acid sequence set forth in SEQ ID NO: 1 and a light chain having the amino acid sequence set forth in SEQ ID NO: 2; a combination of a bivalent monospecific TGF-βRII antibody comprising a heavy chain having the amino acid sequence set forth in SEQ ID NO: 1 and a light chain having the amino acid sequence set forth in SEQ ID NO: 2 with pembrolizumab; and pembrolizumab. Test antibodies included: a bivalent, bispecific antibody comprising a murine FAP binding domain and a TGF-βRII binding domain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 31; and a combination of a bivalent, bispecific antibody comprising a murine FAP binding domain and a TGF-βRII binding domain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 31 with pembrolizumab. *P<0.05 compared to a bivalent, monospecific RSV-G antibody comprising a heavy chain having the amino acid sequence set forth in SEQ ID NO: 3 and a light chain having the amino acid sequence set forth in SEQ ID NO: 4. [Figure 10] Inhibition of TGF-βRII-mediated signaling by bispecific antibodies in a trans-binding assay is shown and expressed as % inhibition. [Figure 10A] The test antibody is a bivalent, bispecific antibody comprising a FAP-binding domain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 19, and a TGF-βRII-binding domain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 27. [Figure 10B] The control antibody is a bivalent, bispecific antibody comprising a mock-binding domain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 3 and a TGF-βRII-binding domain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 27. [Figure 10C]The test antibody is a bivalent, bispecific antibody comprising a FAP-binding domain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 15, and a TGF-βRII-binding domain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 31. [Figure 10D] The control antibody is a bivalent, bispecific antibody comprising a mock-binding domain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 3 and a TGF-βRII-binding domain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 31. [Figure 10E] The test antibody is a bivalent, bispecific antibody comprising a FAP-binding domain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 11, and a TGF-βRII-binding domain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 23. [Figure 10F] The control antibody is a bivalent, bispecific antibody comprising a mock-binding domain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 3 and a TGF-βRII-binding domain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 23. [Figure 10G] The negative control antibody is a bivalent monospecific RSV-G antibody comprising a heavy chain having the amino acid sequence set forth in SEQ ID NO:3 and a light chain having the amino acid sequence set forth in SEQ ID NO:4. [Figure 11] ADCP activity of control and test antibodies with and without Fc modifications is shown. [Figure 11A]ADCP activity of test antibodies against A549-FAP+ cells, measured using M2c macrophage cells and A549-FAP+ target cells at a ratio of 1:1 (left graph) or 3:1 (right graph), is expressed as % phagocytosis. The control antibody is an IgG1 isotype antibody. Test antibodies include: an Fc-enhanced bivalent bispecific antibody comprising a FAP-binding domain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 15 and a TGF-βRII-binding domain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 35; and an Fc-inactivated bivalent bispecific antibody comprising a FAP-binding domain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 15 and a TGF-βRII-binding domain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 35. [Figure 11B] The ADCP activity of the test antibodies against pulmonary CAF cells was measured using M2c macrophage cells and pulmonary CAF target cells at a ratio of 1:1 (left graph) or 3:1 (right graph) and is expressed as the number of green objects per image. The control antibody is an IgG1 isotype antibody. The test antibodies included: an Fc-enhanced bivalent bispecific antibody comprising a FAP-binding domain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 15 and a TGF-βRII-binding domain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 35; and an Fc-inactivated bivalent bispecific antibody comprising a FAP-binding domain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 15 and a TGF-βRII-binding domain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 35. DETAILED DESCRIPTION OF THE INVENTION

[0042] [FAP-binding domain] One objective of the present disclosure is to provide binding domains that bind to human FAP for use in the development of new pharmaceuticals for the diagnosis and treatment of disease, particularly in humans, particularly for the diagnosis and treatment of cancer. This objective is achieved by providing binding domains that comprise polypeptides identified herein as useful for generating antibodies, particularly bispecific antibodies.

[0043] In certain embodiments, the present disclosure provides a polypeptide selected from: - a polypeptide comprising a heavy chain CDR1 (HCDR1) having the amino acid sequence set forth in SEQ ID NO: 16, a heavy chain CDR2 (HCDR2) having the amino acid sequence set forth in SEQ ID NO: 17, and a heavy chain CDR3 (HCDR3) having the amino acid sequence set forth in SEQ ID NO: 18; - a polypeptide comprising a heavy chain CDR1 (HCDR1) having the amino acid sequence set forth in SEQ ID NO: 20, a heavy chain CDR2 (HCDR2) having the amino acid sequence set forth in SEQ ID NO: 21, and a heavy chain CDR3 (HCDR3) having the amino acid sequence set forth in SEQ ID NO: 22; - a polypeptide comprising a heavy chain CDR1 (HCDR1) having the amino acid sequence set forth in SEQ ID NO: 12, a heavy chain CDR2 (HCDR2) having the amino acid sequence set forth in SEQ ID NO: 13, and a heavy chain CDR3 (HCDR3) having the amino acid sequence set forth in SEQ ID NO: 14; or - a polypeptide comprising a heavy chain CDR1 (HCDR1) having the amino acid sequence set forth in SEQ ID NO: 70, a heavy chain CDR2 (HCDR2) having the amino acid sequence set forth in SEQ ID NO: 71, and a heavy chain CDR3 (HCDR3) having the amino acid sequence set forth in SEQ ID NO: 72.

[0044] In one embodiment, the polypeptide is an immunoglobulin heavy chain or a portion thereof that, when combined with an appropriate light chain or a portion thereof, binds to a FAP. For example, the portion of the immunoglobulin heavy chain can be a heavy chain variable region having a CH1 region, or a heavy chain variable region. The portion of the light chain can be, for example, a light chain variable region.

[0045] In some embodiments, the polypeptide binds to human FAP when combined with an appropriate light chain or a portion thereof. The amino acid sequence of human FAP is set forth in SEQ ID NO: 6. The intracellular and transmembrane domains are shown in bold and underlined therein. In some embodiments, the polypeptide binds to mouse FAP when combined with an appropriate light chain or a portion thereof. The amino acid sequence of mouse FAP is set forth in SEQ ID NO: 73. The intracellular and transmembrane domains are shown in bold and underlined therein. In some embodiments, the polypeptide binds to cynomolgus monkey FAP when combined with an appropriate light chain or a portion thereof. The amino acid sequence of cynomolgus monkey FAP is set forth in SEQ ID NO: 9. The intracellular and transmembrane domains are shown in bold and underlined therein. In some embodiments, the polypeptide binds to human and mouse FAP when combined with an appropriate light chain or a portion thereof. In some embodiments, the polypeptide binds to human and cynomolgus monkey FAP when combined with an appropriate light chain or a portion thereof. In some embodiments, the polypeptide binds to human, mouse, and cynomolgus monkey FAP when combined with an appropriate light chain or a portion thereof.

[0046] Generally, as described herein, antigen binding can be expressed in terms of specificity and affinity. Specificity determines which antigen or epitope a binding domain or binding moiety specifically binds to. Affinity is a measure of the strength of binding to a particular antigen or epitope.

[0047] In some embodiments, the polypeptide of the present disclosure also includes its polypeptide variants, wherein each of HCDR1 or HCDR2 may contain at most three, two, or one amino acid mutations. In some embodiments, only either HCDR1 or HCDR2 may contain at most three, two, or one amino acid mutations. In some embodiments, such variants do not contain amino acid mutations in HCDR3. In some embodiments, the amino acid mutations are conservative amino acid substitutions.

[0048] Generally, as described herein, conservative amino acid substitutions typically involve amino acid mutations with homologous amino acid residues (residues that share similar characteristics or properties). Homologous amino acids are known in the art, as are routine methods for making amino acid substitutions in antibody binding domains without significantly affecting antibody binding or function. For example, see handbooks such as Lehninger (Nelson, David L., and Michael M. Cox. 2017. Lehninger Principles of Biochemistry. 7th ed. New York, NY: WH Freeman) or Stryer (Berg, J., Tymoczko, J., Stryer, L. and Stryer, L., 2007. Biochemistry. New York: WH Freeman), the entire contents of which are incorporated herein. In determining whether an amino acid can be substituted with a conservative amino acid, an evaluation can typically be made of factors such as, but not limited to: (a) the structure of the polypeptide backbone in the area of ​​the substitution, e.g., sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, and / or (c) the size of the side chain(s). If a residue can be substituted with a residue that shares common characteristics, such as a similar side chain or similar charge or hydrophobicity, then such a residue is preferred as a replacement. For example, the following groups can be determined: (1) non-polar: Ala (A), Gly (G), Val (V), Leu (L), Ile (I), Pro (P), Phe (F), Trp (W), Met (M); (2) uncharged polar: Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gln (Q); (3) acidic: Asp (D), Glu (E); and (4) basic: Lys (K), Arg (R), His (H).Alternatively, amino acids can be grouped as follows: (1) aromatic: Phe (F), Trp (W), Tyr (Y); (2) nonpolar: Leu (L), Val (V), Ile (I), Ala (A), Met (M); (3) aliphatic: Ala (A), Val (V), Leu (L), Ile (I); (4) acidic: Asp (D), Glu (E); (5) basic: His (H), Lys (K), Arg (R); and (6) polar: Gln (Q), Asn (N), Ser (S), Thr (T), Tyr (Y). Alternatively, amino acid residues can be divided into groups based on common side chain properties: (1) hydrophobic: Met (M), Ala (A), Val (V), Leu (L), Ile (I); (2) neutral hydrophilic: Cys (C), Ser (S), Thr (T), Asn (N), Gln (Q); (3) acidic: Asp (D), Glu (E); (4) basic: His (H), Lys (K), Arg (R); (5) residues that influence chain orientation: Gly (G), Pro (P); and (6) aromatic: Trp (W), Tyr (Y), Phe (F).

[0049] It is preferred to substitute an amino acid residue with another amino acid residue from the same group. Conservative amino acid substitutions may therefore involve exchanging one member of these classes for another member of the same class. Typically, the mutations do not or do not substantially impair the binding specificity of the binding domain for its intended target.

[0050] Further types of amino acid mutations include mutations resulting from somatic hypermutation or affinity maturation. The binding variants encompassed by the present disclosure include heavy chain variable regions that have been somatically hypermutated or affinity matured, which are heavy chain variable regions derived from the same VH gene segment as the heavy chain variable regions described herein by sequence, and the variants have amino acid mutations, including non-conservative and / or conservative amino acid substitutions in one or two of HCDR1 and HCDR2. Conventional methods for affinity maturation of antibody binding domains are well known in the art, see, for example, Tabasinezhad M, et al. (Trends in therapeutic antibody affinity maturation: From in-vitro towards next-generation sequencing approaches. Immunol Lett. 2019 Aug;212:106-113).

[0051] In certain embodiments, a polypeptide of the present disclosure comprises a heavy chain variable region having an amino acid sequence set forth in SEQ ID NO: 11; 15; 19; or 69, or having at least 80%, or at least 85%, or at least 90%, or at least 95% sequence identity thereto.

[0052] Generally, as used herein, "percent (%) identity" with respect to nucleic acid or amino acid sequences is defined as the percentage of residues in a candidate sequence that are identical to the residues in a selected sequence after aligning the sequences for optimal comparison purposes. Gaps may be introduced into either of the two sequences being compared to optimize alignment between the two sequences. Such alignments may be performed over the entire length of the sequences being compared. Alternatively, alignments may be performed over a shorter length, e.g., over about 20, about 50, about 100, or more nucleic acids / bases or amino acids. Sequence identity is the percentage of identical matches between the two sequences over the reported alignment region.

[0053] Comparison of sequences and determination of the percentage of sequence identity between two sequences can be achieved using a mathematical algorithm. Those skilled in the art will be aware of the fact that several different computer programs are available for aligning two sequences and determining the identity between two sequences (Kruskal, JB (1983) An overview of sequence comparison In D. Sankoff and JB Kruskal, (ed.), Time warps, string edits and macromolecules: the theory and practice of sequence comparison, pp. 1-44 Addison Wesley). The percentage of sequence identity between two amino acid or nucleic acid sequences can be determined using the Needleman-Wunsch algorithm for aligning two sequences (Needleman, SB and Wunsch, CD (1970) J. Mol. Biol. 48, 443-453). The Needleman-Wunsch algorithm is implemented in the computer program NEEDLE. For the purposes of this disclosure, the NEEDLE program from the EMBOSS package is used to determine percent identity of amino acid and nucleic acid sequences (version 2.8.0, EMBOSS: The European Molecular Biology Open Software Suite (2000) Rice, P. Longden J. and Bleasby, A. Trends in Genetics 16, (6) pp276-277, http: / / emboss.bioinformatics.nl / ). For protein sequences, EBLOSUM62 is used as the substitution matrix. For DNA sequences, DNAFULL is used. The parameters used are a gap-open penalty of 10 and a gap extension penalty of 0.5.

[0054] After alignment by the above-mentioned program NEEDLE, the percentage of sequence identity between the query sequence and a sequence of the invention is calculated as follows: the number of corresponding positions in the alignment that show identical amino acids or identical nucleotides in both sequences divided by the total length of the alignment after subtracting the total number of gaps in the alignment.

[0055] In some embodiments, the polypeptides of the present disclosure also include polypeptide variants, which contain one or more mutations in the framework regions in addition to the above-mentioned mutations in HCDR1 and / or HCDR2. The mutations can be any type of amino acid mutation described herein, such as conservative or non-conservative amino acid substitutions resulting from somatic hypermutation or affinity maturation. In some embodiments, the polypeptides of the present disclosure do not contain mutations in the CDR regions but contain one or more mutations in the framework regions. Such variants have at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the sequences disclosed herein. Such variants are expected to retain FAP binding specificity. Thus, in some embodiments, the polypeptides of the present disclosure include: - a polypeptide having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 15, which polypeptide comprises an HCDR1 amino acid sequence set forth in SEQ ID NO: 16; an HCDR2 amino acid sequence set forth in SEQ ID NO: 17; and an HCDR3 amino acid sequence set forth in SEQ ID NO: 18; - a polypeptide having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 19, which polypeptide comprises an HCDR1 amino acid sequence set forth in SEQ ID NO: 20; an HCDR2 amino acid sequence set forth in SEQ ID NO: 21; and an HCDR3 amino acid sequence set forth in SEQ ID NO: 22; - a polypeptide having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 11, which polypeptide comprises the HCDR1 amino acid sequence set forth in SEQ ID NO: 12; the HCDR2 amino acid sequence set forth in SEQ ID NO: 13; and the HCDR3 amino acid sequence set forth in SEQ ID NO: 14; or - a polypeptide having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 69, which polypeptide comprises an HCDR1 amino acid sequence set forth in SEQ ID NO: 70; an HCDR2 amino acid sequence set forth in SEQ ID NO: 71; and an HCDR3 amino acid sequence set forth in SEQ ID NO: 72.

[0056] In one embodiment, a polypeptide of the present disclosure is produced using the light chain VK1-39 / JK1. The polypeptide of the present disclosure may be paired with any suitable light chain. In one embodiment, a suitable light chain is the light chain VK1-39 / JK1. The light chain comprises a light chain variable region including light chain CDR1 (LCDR1), light chain CDR2 (LCDR2), and light chain CDR3 (LCDR3) having the amino acid sequences set forth in SEQ ID NO:53, SEQ ID NO:54, and SEQ ID NO:55. In one embodiment, a suitable light chain comprises a light chain variable region having the amino acid sequence set forth in SEQ ID NO:52.

[0057] In certain embodiments, the polypeptides of the present disclosure further comprise a CH1 region. In certain embodiments, the polypeptides of the present disclosure further comprise a CH1 region, a hinge, a CH2 region, and a CH3 region. Suitable CH1 regions include, but are not limited to, the CH1 region whose amino acid sequence is set forth in SEQ ID NO: 39. Suitable hinges include, but are not limited to, the hinge whose amino acid sequence is set forth in SEQ ID NO: 40. Suitable CH2 and CH3 regions include, but are not limited to, the CH2 region whose amino acid sequence is set forth in SEQ ID NO: 41 (WT) or 42 (DM), and the CH3 region whose amino acid sequence is set forth in SEQ ID NO: 43 (WT), or 44 (DE) and 45 (KK).

[0058] In certain embodiments, the present disclosure provides a FAP binding domain comprising a polypeptide described herein.

[0059] In certain embodiments, the present disclosure provides a FAP binding domain that binds to a human FAP and a mouse FAP, i.e., a FAP binding domain that is cross-reactive to a human and a mouse FAP. In certain embodiments, the human / mouse cross-reactive FAP binding domain has a binding affinity for a human FAP that is at least 2-3 times higher than the background signal of the assay. In certain embodiments, the human / mouse cross-reactive FAP binding domain has a binding affinity for a mouse FAP that is at least 2-3 times higher than the background signal of the assay. In certain embodiments, the human / mouse cross-reactive FAP binding domain has a binding affinity for a human FAP that is at least 2-fold higher than the background signal of the assay. In certain embodiments, the human / mouse cross-reactive FAP binding domain has a binding affinity for a mouse FAP that is at least 2-fold higher than the background signal of the assay.

[0060] For purposes of this disclosure, in certain embodiments, binding to human FAP (huFAP) or mouse FAP (moFAP) is determined using the assay described in Example 2. In certain embodiments, binding to huFAP is determined using a FACS assay with cells expressing human FAP. In certain embodiments, binding to moFAP is determined using a FACS assay with cells expressing mouse FAP.

[0061] In one embodiment, the human / mouse cross-reactive FAP binding domain comprises a polypeptide comprising a heavy chain CDR1 (HCDR1) having the amino acid sequence set forth in SEQ ID NO: 70, a heavy chain CDR2 (HCDR2) having the amino acid sequence set forth in SEQ ID NO: 71, and a heavy chain CDR3 (HCDR3) having the amino acid sequence set forth in SEQ ID NO: 72.

[0062] In one embodiment, a human / mouse cross-reactive FAP binding domain of the present disclosure comprises a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 69, or having at least 80%, or at least 85%, or at least 90%, or at least 95% sequence identity thereto.

[0063] In certain embodiments, the human / mouse FAP binding domains of the present disclosure also include polypeptide variants, including polypeptides containing mutations in the HCDR1, HCDR2, and / or framework regions described herein.

[0064] In certain embodiments, the human / mouse FAP binding domain of the present disclosure further comprises a light chain, as defined herein.

[0065] In some embodiments, the human / mouse FAP binding domain of the present disclosure further comprises a CH1 region as defined herein. In some embodiments, the human / mouse FAP binding domain of the present disclosure further comprises a CH1 region, hinge, CH2 region, and CH3 region as defined herein.

[0066] The CL, CH1, hinge, CH2, and / or CH3 regions can be modified according to methods known in the art to obtain favorable antibody characteristics, including, for example, promoting heterodimerization of different heavy chains, improving heavy-light chain pairing, and enhancing or reducing immune cell effector function. The CH3 region can include a terminal lysine residue, or can lack a terminal lysine residue to improve manufacturability.

[0067] In certain embodiments, the present disclosure provides a binding moiety comprising a polypeptide or FAP-binding domain described herein, which binding moiety is also referred to herein as a FAP-binding moiety.

[0068] Generally, as used herein, a "binding moiety" refers to a proteinaceous molecule, including all antibody formats available in the art, such as, for example, full-length IgG antibodies, immunoconjugates, diabodies, BiTEs, Fab fragments, scFv, tandem scFv, single domain antibodies (such as VHH and VH), minibodies, scFab, scFv-zippers, nanobodies, DART molecules, TandAb, Fab-scFv, F(ab)', F(ab)'-scFv, and intrabodies, and any other format known to one of skill in the art.

[0069] In some embodiments, the binding moiety of the present disclosure is a monospecific binding moiety, specifically a monospecific antibody. A monospecific antibody according to the present disclosure is an antibody, in any antibody format, comprising one or more binding domains with specificity for a single target. In some embodiments, the monospecific binding moiety of the present disclosure may further comprise an Fc region or a portion thereof. In some embodiments, the monospecific binding moiety of the present disclosure is an IgG1 antibody. In some embodiments, the binding moiety of the present disclosure is a bivalent monospecific antibody.

[0070] Generally, as described herein, an "Fc region" typically comprises a hinge, CH2, and CH3 region. Suitable hinge, CH2, and CH3 regions are as described herein. The Fc region mediates antibody effector functions, such as complement-dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC), and antibody-dependent cellular phagocytosis (ADCP). Depending on the use of a therapeutic antibody or Fc fusion protein, it may be desirable to reduce or enhance effector functions.

[0071] In some embodiments, a binding moiety comprising a polypeptide or binding domain of the present disclosure has Fc effector function. In some embodiments, a binding moiety comprising a polypeptide or binding domain of the present disclosure has enhanced Fc effector function. In some embodiments, a binding moiety comprising a polypeptide or binding domain of the present disclosure exhibits antibody-dependent cell-mediated cytotoxicity (ADCC).

[0072] Generally, as described herein, binding moieties such as antibodies can be modified to enhance ADCC activity (for a review, see Kubota T et al. Cancer Sci. 2009; 100(9):1566-72). For example, if the antibody itself has low ADCC activity, slight modifications to the antibody's constant region can improve the antibody's ADCC activity (Junttila TT. et al. Cancer Res. 2010; 70(11):4481-9). Alterations can also be made to improve storage or production or to remove C-terminal lysines (Kubota T et al. Cancer Sci. 2009; 100(9):1566-72). Another method for improving antibody ADCC activity is to reduce fucose by enzymatically intervening in the glycosylation pathway (von Horsten HH. et al. Glycobiology. 2010; 20(12):1607-18). Alternatively, or in addition, several other strategies can be used to achieve enhanced ADCC, including, for example, glycoengineering (Kyowa Hakko / Biowa, GlycArt (Roche), and Eureka Therapeutics) and mutagenesis, all aimed at improving Fc binding to the low-affinity activating FcγRIIIa and / or reducing binding to the low-affinity inhibitory FcγRIIb. In certain embodiments, the binding moieties of the present disclosure exhibit enhanced antibody-dependent cell-mediated cytotoxicity (ADCC).

[0073] In certain embodiments, the FAP-binding moieties of the present disclosure are defucosylated.

[0074] Generally, as described herein, antibody defucosylation can be achieved using various methods known in the art. Fc-enhanced variants of antibodies can be produced using FUT8 knockout CHO cells, which generate defucosylated antibodies (Zong H, et al. Producing defucosylated antibodies with enhanced in vitro antibody-dependent cellular cytotoxicity via FUT8 knockout CHO-S cells. Eng Life Sci. 2017 Apr 18; 17(7):801-808). Defucosylation of antibodies can also be achieved using CHO cells expressing the GDP-6-deoxy-D-lyxo-4-hexulose reductase (RMD) enzyme (Roy G, et. al., A novel bicistronic gene design couples stable cell line selection with a fucose switch in a designer CHO host to produce native and afucosylated glycoform antibodies, MAbs, 2018 Apr; 10(3):416-430).

[0075] [Bispecific binding moiety that binds to FAP and TGF-βRII] Another object of the present disclosure is to provide new pharmaceutical agents for the diagnosis and treatment of diseases (e.g., in humans), particularly cancer. This object is achieved by providing a bispecific binding moiety (e.g., a bispecific antibody) that binds to a FAP and TGF-βRII. The FAP-binding domain of the bispecific binding moiety drives the specificity of the bispecific binding moiety for cancer-associated fibroblasts (CAFs) in the tumor microenvironment, where the TGF-βRII-binding domain can locally block TGF-β from binding to TGF-βRII. Furthermore, the bispecific binding moiety promotes cytotoxic T lymphocyte activity in the tumor microenvironment by alleviating TGF-β-mediated immunosuppressive pathways in activated / exhausted effector T cells.

[0076] In one embodiment, the present disclosure provides a bispecific binding moiety comprising a FAP binding domain and a TGF-βRII binding domain, wherein the TGF-βRII binding domain blocks TGF-βRII from binding to a TGF-βRII ligand.

[0077] In some embodiments, the bispecific binding moiety of the present disclosure is a bispecific antibody. A bispecific antibody of the present disclosure is an antibody comprising at least two binding domains with specificity for at least two different targets or epitopes. In some embodiments, the bispecific antibody of the present disclosure is a bivalent bispecific antibody. In some embodiments, the bispecific antibody of the present disclosure further comprises an Fc region or a portion thereof. In some embodiments, the bispecific binding moiety of the present disclosure is an IgG1 antibody. The constant region of the binding moiety of the present disclosure may contain one or more mutations that modulate a property of the binding moiety other than binding to the target antigen. For example, the constant region may contain one or more mutations that promote heterodimerization of a FAP heavy chain and a TGF-βRII heavy chain over homodimerization of two FAP heavy chains and / or two TGF-βRII heavy chains, one or more mutations that improve heavy chain-light chain pairing in CH1 and / or CL, and / or one or more mutations that reduce or improve effector function, particularly one or more mutations that reduce effector function.

[0078] In one embodiment, the FAP-binding domain and / or the TGF-βRII-binding domain is a Fab domain, also referred to herein as "Fab." For purposes of this disclosure, "Fab" refers to a binding domain that includes a heavy chain variable region, a light chain variable region, a CH1 region, and a CL region.

[0079] In certain embodiments, a bispecific binding moiety of the present disclosure comprises a single Fab domain that binds a FAP, a single Fab domain that binds TGF-βRII, and an Fc region. In certain embodiments, a bispecific binding moiety of the present disclosure consists of a single Fab domain that binds a FAP, a single Fab domain that binds TGF-βRII, and an Fc region.

[0080] For purposes of this disclosure, an "Fc region" includes hinge, CH2, and CH3 regions. Suitable hinges include, but are not limited to, the hinge whose amino acid sequence is set forth in SEQ ID NO: 40. Suitable CH2 and CH3 regions include, but are not limited to, the CH2 region whose amino acid sequence is set forth in SEQ ID NO: 41 or 42, and the CH3 region whose amino acid sequence is set forth in SEQ ID NO: 43, or 44 and 45. The CH3 region may include a terminal lysine residue, or may lack a terminal lysine residue to improve manufacturability.

[0081] In certain embodiments, the bispecific binding moieties of the present disclosure bind to human FAP, the amino acid sequence of which is set forth in SEQ ID NO: 6. In certain embodiments, the bispecific binding moieties of the present disclosure have a binding affinity for human FAP that is at least 2-fold higher than the background signal of the assay.

[0082] For purposes of the present disclosure, in one embodiment, binding to huFAP is determined using the assay described in Example 2. In one embodiment, binding to huFAP is determined using a FACS assay using cells expressing human FAP.

[0083] In certain embodiments, the FAP-binding domain of a bispecific binding moiety disclosed herein binds to a human FAP and has cross-reactivity with cynomolgus monkey FAP (cyFAP). The amino acid sequence of cyFAP is set forth in SEQ ID NO: 9. In certain embodiments, a bispecific binding moiety of the present disclosure has a binding affinity for cynomolgus monkey FAP that is at least 2-fold higher than the background signal of the assay.

[0084] For purposes of the present disclosure, in one embodiment, binding to cyFAP is determined using the assay described in Example 2. In one embodiment, binding to cyFAP is determined using a FACS assay using cells expressing cynomolgus monkey FAP.

[0085] In some embodiments, the FAP-binding domain of the bispecific binding moiety disclosed herein does not bind to human CD26 (huCD26). The amino acid sequence of huCD26 is set forth in SEQ ID NO: 10. In some embodiments, the bispecific binding moiety of the present disclosure has a binding affinity for human CD26 that is equal to or less than the background signal of the assay. In some embodiments, binding to huCD26 is determined using a FACS assay using cells expressing huCD26.

[0086] In certain embodiments, the bispecific binding moiety of the present disclosure binds to human TGF-βRII. Human TGF-βRII is a transmembrane protein that exists in various isoforms. The amino acid sequence of human TGF-βRII isoform A is set forth in SEQ ID NO: 46; the amino acid sequence of the extracellular domain of human TGF-βRII isoform A is set forth in SEQ ID NO: 47. Human TGF-βRII isoform B is a splice variant that encodes a longer isoform due to an insertion in the extracellular domain. The amino acid sequence of human TGF-βRII isoform B is set forth in SEQ ID NO: 48; the amino acid sequence of the extracellular domain of human TGF-βRII isoform B is set forth in SEQ ID NO: 49. In certain embodiments, the bispecific binding moiety of the present disclosure binds to human TGF-βRII isoform A. In certain embodiments, the bispecific binding moiety of the present disclosure has a binding affinity for human TGF-βRII that is at least two-fold higher than the background signal of the assay. In one embodiment, binding to human TGF-βRII is determined by a FACS assay using cells that express human TGF-βRII (e.g., cells that endogenously express human TGF-βRII, e.g., CCD18Co cells).

[0087] In one embodiment, the TGF-βRII ligand is TGF-β1.

[0088] In certain embodiments, the TGF-βRII binding domain of the bispecific binding moiety of the present disclosure blocks TGF-βRII from binding to the TGF-βRII ligand TGF-βI.

[0089] As used herein, "blocking TGF-βRII from binding to a TGF-βRII ligand" or "blocking TGF-βRII from binding to a TGF-βRII ligand" means preventing or modulating the interaction between a ligand of TGF-βRII and a TGF-βRII receptor. This occurs when the TGF-βRII binding domain of the bispecific binding moiety targets an epitope on TGF-βRII and competes with TGF-β1 for binding to human TGF-βRII. In certain embodiments, blocking TGF-βRII from binding to a TGF-βRII ligand is determined using an ELISA assay described in the art, for example, in WO2021 / 133167.

[0090] In certain embodiments, the TGF-βRII binding domain of the bispecific binding moiety blocks TGF-βRII-mediated signaling in cells expressing a FAP and TGF-βRII.

[0091] As used herein, "blocking TGF-βRII-mediated signaling" or "blocking TGF-βRII-mediated signaling" means causing a complete or partial reduction in the signaling cascade. For purposes of the present disclosure, in certain embodiments, blocking TGF-βRII-mediated signaling is determined by using the TGF-βRII signaling inhibition assay described in Example 7. TGF-βRII-mediated signaling inhibition data for the bispecific binding moieties provided herein is obtained using the assay described in Example 7.

[0092] Briefly, the TGF-βRII signaling inhibition assay of Example 7 is performed using primary CAF cells, which are trypsinized and resuspended in an appropriate buffer. The cells are preincubated with the test bispecific binding moiety, then incubated with recombinant human TGF-β1, and then assayed for pSMAD2 expression. Potency in blocking TGF-βRII-mediated signaling is determined as IC50 (µg / ml).

[0093] In one embodiment, the reduction in pSMAD2 expression is determined as an IC50 (ug / ml), where pSMAD2 expression in the presence of the bispecific binding moiety is compared to pSMAD2 expression in the absence of the bispecific binding moiety in a TGF-βRII signaling inhibition assay.

[0094] In one embodiment, the cells expressing both FAP and TGF-βRII are fibroblasts, specifically primary cancer-associated fibroblasts (CAFs), such as primary human lung squamous cell carcinoma CAFs, primary human bladder CAFs, primary human breast CAFs, primary human head and neck CAFs, primary colon cancer CAFs, primary pancreatic stellate cell CAFs, primary melanoma CAFs, primary lung adenocarcinoma CAFs, primary colorectal adenocarcinoma CAFs, primary ovarian serous CAFs, or primary glioblastoma CAFs. Primary CAF cells expressing FAP and TGF-βRII are commercially available, for example, from BioIVT or Neuromics, as described in Table 3 and Example 6. Alternatively, methods for producing cells expressing FAP and TGF-βRII are known to those skilled in the art. Methods for determining the expression of FAP and TGF-βRII on cells are known to those skilled in the art. In one embodiment, the expression of FAP and TGF-βRII on CAF cells is determined as mean fluorescence intensity (MFI) by using the FACS assay described in Example 6. In one embodiment, the MFI of FAP and TGF-βRII expression is at least two-fold or three-fold higher than the background MFI obtained using only a secondary antibody, as shown in Example 6.

[0095] In Example 6, CAF cells are cultured, resuspended in an appropriate buffer, and stained with a primary antibody. To detect huFAP, a mouse anti-FAP antibody is used as the primary antibody, and to detect huTGF-βRII, an analog reference TGF1 antibody is used as the primary antibody. After washing the primary antibody, the cells are stained with an appropriate secondary antibody, for example, an FITC-labeled goat anti-mouse antibody and an Alexa Fluor 647-labeled goat anti-human antibody. The stained cells are analyzed by FACS, and the expression level is determined as MFI.

[0096] In certain embodiments, the potency of a bispecific binding moiety of the present disclosure in blocking TGF-βRII-mediated signaling is 2.0 to 500 times greater than the potency of a reference anti-TGF-βRII antibody in cells expressing FAP and TGF-βRII.

[0097] In one embodiment, potency in blocking TGF-βRII-mediated signaling is determined by measuring the reduction in pSMAD2 expression at IC50 (ug / ml), as described in Example 7. In one embodiment, potency in blocking TGF-βRII-mediated signaling is determined as the reduction in pSMAD2 expression at IC50 (ug / ml).

[0098] In certain embodiments, a bispecific binding moiety of the present disclosure has a potency in blocking TGF-βRII-mediated signaling that is about 2.0-500 fold higher, or about 2.0-300 fold higher, than the potency of a reference anti-TGF-βRII antibody in cells expressing a FAP and TGF-βRII, as measured as a reduction in pSMAD2 expression, as described in Example 7. In certain embodiments, a bispecific binding moiety of the present disclosure has a potency in blocking TGF-βRII-mediated signaling that is about 2.0-5 fold higher, or about 2 fold higher, than the potency of a reference anti-TGF-βRII antibody in cells expressing a FAP and TGF-βRII, as measured as a reduction in pSMAD2 expression, as described in Example 7. In certain embodiments, bispecific binding moieties of the present disclosure have potency in blocking TGF-βRII-mediated signaling that is in the range of about 50-100 fold, or about 80 fold greater than the potency of a reference anti-TGF-βRII antibody in cells expressing a FAP and TGF-βRII, as measured as a reduction in pSMAD2 expression, as described in Example 7. In certain embodiments, bispecific binding moieties of the present disclosure have potency in blocking TGF-βRII-mediated signaling that is in the range of about 100-500 fold, or about 100-300 fold, or about 200 fold greater than the potency of a reference anti-TGF-βRII antibody in cells expressing a FAP and TGF-βRII, as measured as a reduction in pSMAD2 expression, as described in Example 7.

[0099] In one embodiment, the reference anti-TGF-βRII antibody is a bivalent monospecific antibody comprising a heavy chain having the amino acid sequence set forth in SEQ ID NO:1 and a light chain having the amino acid sequence set forth in SEQ ID NO:2.

[0100] In certain embodiments, the bispecific binding moieties of the present disclosure are more potent at blocking TGF-βRII-mediated signaling in cells that express a FAP and TGF-βRII than in cells that express TGF-βRII and do not express, or express only undetectable levels of, a FAP.

[0101] In one embodiment, cells expressing both FAP and TGF-βRII are designated A549-FAP, e.g., A549 parental cells overexpressing human FAP, as described in Example 8. + In one embodiment, the cell is an A549-FAP cell. + The cells should be at least about 1 x 10 cells on the cell surface. 5 ~1×10 6 In one embodiment, A549-FAP expresses a FAP molecule in the range of + The cells should be at least about 1 x 10 cells on the cell surface. 6 In one embodiment, the FAP molecule of A549-FAP is expressed. + The cells express at least about 5,000 to 10,000 TGF-βRII molecules on the cell surface. + The cells express at least about 10,000 TGF-βRII molecules on the cell surface. In one embodiment, the levels of FAP and TGF-βRII are measured using the quantibrite bead method described in Example 8.

[0102] In one embodiment, the cells that express TGF-βRII and do not express FAP or express FAP at undetectable levels are the A549 parent cells described herein. A549 parent cells are commercially available, for example, from ATCC (Catalog No. CCL-185). In one embodiment, not expressing FAP or expressing FAP at undetectable levels refers to having fewer than about 300 FAP molecules on the cell surface. In one embodiment, the A549 parent cells express fewer than about 200 FAP molecules on the cell surface. In one embodiment, the A549 parent cells express at least about 7,000 TGF-βRII molecules on the cell surface. In one embodiment, the levels of FAP and TGF-βRII are measured using the quantibrite bead method described in Example 8.

[0103] In one embodiment, A549-FAP +The fold difference in FAP receptors on the cells compared to the A549 parent cells is at least in the range of 500-5000 fold, specifically in the range of 1000-5000 fold, specifically in the range of 4000-5000 fold. In one embodiment, A549-FAP + The fold difference in FAP receptors on the cells compared to the A549 parental cells is at least 4500-fold. + The expression of TGF-βRII receptors on the cells and A549 parental cells was comparable, with a difference in the range of 1-2 fold.

[0104] For purposes of this disclosure, determining whether a bispecific binding moiety has greater potency in blocking TGF-βRII-mediated signaling in cells that express both a FAP and TGF-βRII than in cells that express TGF-βRII and no or only undetectable levels of a FAP is done by using the mixed culture pSMAD2 assay described in Example 9. Thus, in one embodiment, potency in blocking TGF-βRII-mediated signaling is measured in the mixed culture pSMAD2 assay described in Example 9.

[0105] Briefly, the mixed culture pSMAD2 assay described in Example 9 involves the use of A549 parental cells and A549-FAP cells cultured in an appropriate buffer. + The cells were trypsinized, washed, and transfected with A549-FAP. + Cells are labeled with CFSE. A549 parental cells and A549-FAP + The cells were then mixed 1:1 and incubated with the test antibody and recombinant human TGF-β1. The washed and fixed cells were then stained for pSMAD2 and acquired by flow cytometry. The efficacy of the antibodies in blocking TGF-βRII-mediated signaling was assessed using A549 parental cells and A549-FAP. + Determined as IC50 (ug / ml) against cells.

[0106] In certain embodiments, the bispecific binding moieties of the present disclosure are at least about 100-fold, or about 100-20,000-fold more potent in blocking TGF-βRII-mediated signaling in cells expressing both a FAP and TGF-βRII, compared to their potency in cells expressing TGF-βRII and either not expressing or expressing only undetectable levels of a FAP. In certain embodiments, the bispecific binding moieties of the present disclosure are at least about 600-700-fold, or at least about 3,000-4,000-fold, or at least about 18,000-20,000-fold more potent in blocking TGF-βRII-mediated signaling in cells expressing both a FAP and TGF-βRII, compared to their potency in cells expressing TGF-βRII and either not expressing or expressing only undetectable levels of a FAP. In certain embodiments, the potency in blocking TGF-βRII-mediated signaling is determined as IC50 (µg / ml) in a mixed culture pSMAD2 assay.

[0107] In one embodiment, determining whether a bispecific binding moiety has greater efficacy in blocking TGF-βRII-mediated signaling in cells that express both a FAP and TGF-βRII than in cells that express TGF-βRII and do not express, or express only undetectable levels of, a FAP is performed in an in vivo study by using the NSG mouse model described in Example 12.

[0108] Briefly, A549 parental cells or A549-FAP + Cells are inoculated into the flanks of NSG mice. After tumor formation, the bispecific antibody is administered. The mice are sacrificed, and single cells are obtained from the tumors harvested. Cells are stained for IL-11, pSMAD2, and anti-human IgG.

[0109] In some embodiments, the bispecific binding moieties of the present disclosure target FAP on specific CAF cells and simultaneously target TGF-βRII on the same CAF cells. This is referred to as cis-mode activity. Thus, the bispecific binding moieties mediate inhibition of TGF-β-induced immunomodulation of CAFs. Furthermore, the bispecific binding moieties can mediate the cytotoxic activity of immune cells against CAFs through Fc-mediated effector function.

[0110] In some embodiments, the bispecific binding moieties of the present disclosure target a FAP expressed on a CAF cell and simultaneously target TGF-βRII expressed on another cell. In some embodiments, the bispecific binding moieties of the present disclosure target a FAP expressed on a CAF cell and simultaneously target TGF-βRII expressed on an immune effector cell. This is referred to as trans-mode activity, whereby the bispecific binding moieties prevent immune cell suppressive signaling in TGF-βRII-expressing immune effector cells in the tumor microenvironment.

[0111] Accordingly, the present disclosure also provides a bispecific binding moiety comprising a FAP-binding domain and a TGF-βRII-binding domain, wherein the FAP-binding domain binds to a FAP expressed on a first cell, and the TGF-βRII-binding domain binds to a TGF-βRII expressed on a second cell.

[0112] In one embodiment, when the FAP binding domain binds to a FAP expressed on the first cell and the TGF-βRII binding domain binds to TGF-βRII expressed on the second cell, the TGF-βRII binding domain blocks TGF-βRII-mediated signaling in the second cell.

[0113] In some embodiments, the first cell and the second cell are different types of cells. In some embodiments, the first cell is a fibroblast. In some embodiments, the second cell is a non-fibroblast. In some embodiments, the second cell is an immune effector cell or a tumor cell. In some embodiments, the immune effector cell is an NK cell, a T cell, a B cell, a monocyte, a macrophage, a dendritic cell, or a neutrophil granulocyte.

[0114] In one embodiment, blockage of TGF-βRII-mediated signaling in the second cell by trans-modulation activity is measured in a TGF-βRII reporter assay as described in Example 15.

[0115] Briefly, the TGF-βRII reporter assay described in Example 15 is performed by using recombinant human TGF-β1, which binds to TGF-βRII expressed on HEK-Blue-TGF-βRII reporter cells and MRC-5 cells. A bispecific binding moiety of the present disclosure is added, and disruption of binding between TGF-βRII and its ligand is measured by detecting secreted alkaline phosphatase (SEAP) levels using an appropriate substrate, such as QUANTI-Blue™ substrate.

[0116] In some embodiments, a bispecific binding moiety of the present disclosure has greater activity in reducing tumor volume than a reference anti-TGF-βRII antibody, in some embodiments, the reference antibody is a bivalent monospecific antibody targeting TGF-βRII, comprising a heavy chain having the amino acid sequence set forth in SEQ ID NO: 1 and a light chain having the amino acid sequence set forth in SEQ ID NO: 2.

[0117] Accordingly, the present disclosure also provides a bispecific binding moiety comprising a FAP-binding domain and a TGF-βRII-binding domain, wherein the bispecific binding moiety has greater activity in reducing tumor volume than a reference anti-TGF-βRII antibody. In one embodiment, the bispecific binding moiety is administered with a 2-fold lower to up to 20-fold lower number of TGF-βRII-binding domains compared to a bivalent monospecific anti-TGF-βRII reference antibody. For example, a bispecific binding moiety that is monovalent for binding to a FAP and monovalent for binding to TGF-βRII, when administered at 3 mg / kg, has greater activity in reducing tumor volume compared to a reference antibody that is bivalent for binding to TGF-βRII and administered at 30 mg / kg. Additionally, a bispecific binding moiety that is monovalent for binding to FAP and monovalent for binding to TGF-βRII has greater activity in reducing tumor volume when administered at 30 mg / kg compared to a reference antibody that is bivalent for binding to TGF-βRII and administered at 30 mg / kg.

[0118] In one embodiment, activity in reducing tumor volume is measured in an in vivo mouse study, specifically, A549-FAP tumors implanted in BALB / c nu / nu mice, as described in Example 13. + It is determined by measuring the reduction in tumor volume in a cell-based in vivo mouse study.

[0119] In certain embodiments, the bispecific binding moieties of the present disclosure exhibit a tumor volume reduction that is at least 1.5-fold, or 1.5-2-fold, that of the reference antibody.

[0120] In one embodiment, the reference antibody is a bivalent, monospecific antibody targeting TGF-βRII, and comprises a heavy chain having the amino acid sequence set forth in SEQ ID NO: 1 and a light chain having the amino acid sequence set forth in SEQ ID NO: 2.

[0121] In certain embodiments, the bispecific binding moieties of the present disclosure reduce tumor volume in an in vivo mouse model compared to untreated mice.

[0122] In certain embodiments, the bispecific binding moieties of the present disclosure reduce tumor volume when administered as a single agent.

[0123] Accordingly, the present disclosure also provides a bispecific binding moiety comprising a FAP-binding domain and a TGF-βRII-binding domain, wherein the bispecific binding moiety induces tumor volume reduction as a single agent.

[0124] In one embodiment, the FAP binding domain of a bispecific binding moiety of the present disclosure comprises a heavy chain variable region comprising: a) a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 14, respectively; b) a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18, respectively; or c) Heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 22, respectively.

[0125] The heavy chain variable region of the FAP binding domain of the bispecific binding moieties of the present disclosure may include a limited number of non-conservative amino acid substitutions, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, or an unlimited number of conservative amino acid substitutions.

[0126] In some embodiments, the FAP-binding domain of the bispecific binding moiety of the present disclosure also includes a variant thereof, in which each of HCDR1 or HCDR2 may contain at most three, two, or one amino acid mutations. In some embodiments, only either HCDR1 or HCDR2 may contain at most three, two, or one non-conservative amino acid mutations. In some embodiments, such a variant does not include an amino acid mutation in HCDR3. In some embodiments, the amino acid mutations are conservative amino acid substitutions.

[0127] In certain embodiments, the FAP-binding domain of a bispecific binding moiety of the present disclosure comprises a heavy chain variable region having the amino acid sequence set forth in any one of SEQ ID NOs: 11, 15, or 19, or a variant thereof. In certain embodiments, the FAP-binding domain of a bispecific binding moiety of the present disclosure comprises a heavy chain variable region having the amino acid sequence set forth in any one of SEQ ID NOs: 11, 15, or 19, or a variant having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity thereto.

[0128] In some embodiments, the FAP-binding domain of the bispecific binding moiety of the present disclosure also includes a FAP-binding domain variant, which contains one or more mutations in the framework regions in addition to the mutations in HCDR1 and HCDR2 described above. The mutations may be any type of amino acid mutation described herein, such as conservative or non-conservative amino acid substitutions resulting from somatic hypermutation or affinity maturation. In some embodiments, the FAP-binding domain variant of the bispecific binding moiety of the present disclosure does not contain a mutation in the CDR region, but contains one or more mutations in the framework region. Such variants are expected to have at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the sequences disclosed herein and retain FAP-binding specificity. Thus, in some embodiments, the FAP-binding domain of the bispecific binding moiety of the present disclosure comprises: - a heavy chain variable region having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 11, wherein the heavy chain variable region comprises an HCDR1 amino acid sequence set forth in SEQ ID NO: 12; an HCDR2 amino acid sequence set forth in SEQ ID NO: 13; and an HCDR3 amino acid sequence set forth in SEQ ID NO: 14; - a heavy chain variable region having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 15, wherein the heavy chain variable region comprises an HCDR1 amino acid sequence set forth in SEQ ID NO: 16; an HCDR2 amino acid sequence set forth in SEQ ID NO: 17; and an HCDR3 amino acid sequence set forth in SEQ ID NO: 18; or - a heavy chain variable region having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 19, wherein the heavy chain variable region comprises an HCDR1 amino acid sequence set forth in SEQ ID NO: 20; an HCDR2 amino acid sequence set forth in SEQ ID NO: 21; and an HCDR3 amino acid sequence set forth in SEQ ID NO: 22.

[0129] The binding domains of the polypeptides and bispecific binding moieties of this disclosure are generated using a common light chain, specifically a common light chain designated VK1-39 / JK1. The binding domains of the polypeptides and bispecific binding moieties of this disclosure can comprise any suitable light chain, including, but not limited to, common light chains known in the art. In certain embodiments, the binding domains of the polypeptides and bispecific binding moieties of this disclosure comprise the common light chain VK1-39 / JK1 or variants thereof with a limited number of non-conservative amino acid substitutions, such as one, two, or three, or an unlimited number of conservative amino acid substitutions.

[0130] In certain embodiments, the FAP-binding domain of a bispecific binding moiety of the present disclosure comprises a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 52, or a variant thereof. In certain embodiments, the FAP-binding domain of a bispecific binding moiety of the present disclosure comprises a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 52, or a variant having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity thereto.

[0131] In certain embodiments, the FAP-binding domain of a bispecific binding moiety of the present disclosure comprises a light chain variable region comprising a light chain CDR1 (LCDR1), a light chain CDR2 (LCDR2), and a light chain CDR3 (LCDR3) having the amino acid sequences set forth in SEQ ID NO: 53, SEQ ID NO: 54, and SEQ ID NO: 55. In certain embodiments, the light chain variable region of the FAP-binding domain of a bispecific binding moiety of the present disclosure also comprises variants thereof, wherein each of the LCDRs may comprise at most three, two, or one amino acid mutations. In certain embodiments, the amino acid mutations are conservative amino acid substitutions.

[0132] In some embodiments, the FAP-binding domain of the bispecific binding moiety of the present disclosure also comprises a FAP-binding domain variant, which, in addition to the above-described mutations in the LCDR, comprises one or more mutations in the framework region. The mutations are preferably conservative amino acid substitutions. In some embodiments, the FAP-binding domain variant of the bispecific binding moiety of the present disclosure does not comprise a mutation in the LCDR region, but comprises one or more mutations in the framework region. Such variants have at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the sequences disclosed herein. Thus, in some embodiments, the FAP-binding domain of the bispecific binding moiety of the present disclosure comprises: - a light chain variable region having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 52, wherein the light chain variable region comprises the LCDR1 amino acid sequence set forth in SEQ ID NO: 53; the LCDR2 amino acid sequence set forth in SEQ ID NO: 54; and the LCDR3 amino acid sequence set forth in SEQ ID NO: 55.

[0133] The light chain or light chain variable region containing these LCDRs and / or light chain variable regions may be, for example, a light chain referred to in the art as VK1-39 / JK1. This is a common light chain. The term "common light chain" described in this disclosure refers to a light chain that can pair with multiple different heavy chains, such as heavy chains with different antigen or epitope binding specificities. Common light chains are particularly useful, for example, in the production of bispecific or multispecific antibodies, where antibody production is more efficient when all binding domains contain the same light chain. The term "common light chain" encompasses light chains that are identical or have some differences in amino acid sequence but do not affect the binding specificity of the full-length antibody. For example, it is possible to prepare or find light chains that are not identical but are still functionally equivalent by using well-established mutations that introduce conservative amino acid changes, i.e., amino acid changes in regions known or shown to not contribute or only partially contribute to binding specificity when paired with a heavy chain, within the scope of the definition of a common light chain used herein.

[0134] Apart from the common light chains comprising the LCDRs and / or light chain variable regions described above, other common light chains known in the art can be used. Examples of such common light chains include, but are not limited to: VK1-39 / JK5 comprises a light chain variable region comprising a light chain CDR1 (LCDR1), a light chain CDR2 (LCDR2), and a light chain CDR3 (LCDR3) of the light chain variable region having the amino acid sequence set forth in SEQ ID NO: 56. In one embodiment, the light chain comprises a light chain variable region comprising a light chain CDR1 (LCDR1), a light chain CDR2 (LCDR2), and a light chain CDR3 (LCDR3) of the light chain variable region having the amino acid sequence set forth in SEQ ID NO: 56, wherein each of the LCDRs may comprise at most three, two, or one amino acid mutation, e.g., substitution. In one embodiment, the light chain comprises a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 56, or having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity thereto. In one embodiment, the light chain comprises a light chain CDR1 (LCDR1), a light chain CDR2 (LCDR2), and a light chain CDR3 (LCDR3) having the amino acid sequences set forth in SEQ ID NO:53, SEQ ID NO:54, and SEQ ID NO:59; VK3-15 / JK1 comprises a light chain variable region comprising a light chain CDR1 (LCDR1), a light chain CDR2 (LCDR2), and a light chain CDR3 (LCDR3) of a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 61. In one embodiment, the light chain comprises a light chain variable region comprising a light chain CDR1 (LCDR1), a light chain CDR2 (LCDR2), and a light chain CDR3 (LCDR3) of a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 61, wherein each of the LCDRs may comprise at most three, two, or one amino acid mutation, e.g., substitution. In one embodiment, the light chain comprises a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 61, or having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity thereto. In one embodiment, the light chain comprises a light chain CDR1 (LCDR1), a light chain CDR2 (LCDR2), and a light chain CDR3 (LCDR3) having the amino acid sequences set forth in SEQ ID NO: 62, SEQ ID NO: 63, and SEQ ID NO: 64; VK3-20 / JK1 comprises a light chain variable region comprising a light chain CDR1 (LCDR1), a light chain CDR2 (LCDR2), and a light chain CDR3 (LCDR3) of a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 66. In one embodiment, the light chain comprises a light chain variable region comprising a light chain CDR1 (LCDR1), a light chain CDR2 (LCDR2), and a light chain CDR3 (LCDR3) of a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 66, wherein each of the LCDRs may comprise at most three, two, or one amino acid mutation, e.g., substitution. In one embodiment, the light chain comprises a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 66, or having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity thereto. In one embodiment, the light chain comprises a light chain CDR1 (LCDR1), a light chain CDR2 (LCDR2), and a light chain CDR3 (LCDR3) having the amino acid sequences set forth in SEQ ID NO:67, SEQ ID NO:63, and SEQ ID NO:68; and VL3-21 / JL3 comprises a light chain variable region comprising a light chain CDR1 (LCDR1), a light chain CDR2 (LCDR2), and a light chain CDR3 (LCDR3) of a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 32. In one embodiment, the light chain comprises a light chain variable region comprising a light chain CDR1 (LCDR1), a light chain CDR2 (LCDR2), and a light chain CDR3 (LCDR3) of a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 32, wherein each of the LCDRs may comprise at most three, two, or one amino acid mutation, e.g., substitution. In one embodiment, the light chain comprises a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 32, or having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity thereto. In one embodiment, the light chain comprises a light chain CDR1 (LCDR1), a light chain CDR2 (LCDR2), and a light chain CDR3 (LCDR3) having the amino acid sequences set forth in SEQ ID NO:36, SEQ ID NO:38, and SEQ ID NO:57.

[0135] VK1-39 is an abbreviation for immunoglobulin variable kappa 1-39 gene. This gene is also known as immunoglobulin kappa variable 1-39; IGKV139; IGKV1-39; IgVκ1-39. The external IDs of this gene are HGNC:5740; Entrez Gene:28930; Ensembl:ENSG00000242371. The amino acid sequence of VK1-39 is shown in SEQ ID NO:60. This is the sequence of the V region. The V region can be combined with one of five J regions. Suitable VJ region sequences are designated as VK1-39 / JK1 (SEQ ID NO:52) and VK1-39 / JK5 (SEQ ID NO:56); alternative names are IgVκ1-39*01 / IGJκ1*01 or IgVκ1-39*01 / IGJκ5*01 (nomenclature according to the IMGT database (on the world wide web at imgt.org)). These names are exemplary and encompass allelic variants of the gene segments.

[0136] VK3-15 is an abbreviation for immunoglobulin variable kappa 3-15 gene. This gene is also known as immunoglobulin kappa variable 3-15; IGKV315; IGKV3-15; IgVκ3-15. The external IDs of this gene are HGNC:5816; Entrez Gene:28913; Ensembl:ENSG00000244437. The amino acid sequence of VK3-15 is shown in SEQ ID NO:65. This is the sequence of the V region. A V region can be combined with one of five J regions. A suitable VJ region sequence is shown as VK3-15 / JK1 (SEQ ID NO:61); another name is Vκ3-15*01 / IGJκ1*01 (nomenclature according to the IMGT database (world wide web imgt.org)). This name is exemplary and encompasses allelic variants of the gene segment.

[0137] VK3-20 is an abbreviation for immunoglobulin variable kappa 3-20 gene. This gene is also known as immunoglobulin kappa variable 3-20; IGKV320; IGKV3-20; IgVκ3-20. The external IDs of this gene are HGNC:5817; Entrez Gene:28912; Ensembl:ENSG00000239951. The amino acid sequence of VK3-20 is shown in SEQ ID NO:28. This is the sequence of the V region. A V region can be combined with one of five J regions. A suitable VJ region sequence is shown as VK3-20 / JK1 (SEQ ID NO:66); another name is IgVκ3-20*01 / IGJκ1*01 (nomenclature according to the IMGT database (world wide web imgt.org)). This name is exemplary and encompasses allelic variants of the gene segment.

[0138] VL3-21 is an abbreviation for immunoglobulin variable lambda 3-21 gene. This gene is also known as immunoglobulin lambda variable 3-21; IGLV321; IGLV3-21; IgVλ3-21. The external IDs of this gene are HGNC:5905; Entrez Gene:28796; Ensembl:ENSG00000211662. The amino acid sequence of VL3-21 is shown in SEQ ID NO:58. This is the sequence of the V region. The V region can be combined with one of five J regions. A suitable VJ region sequence is shown as VL3-21 / JL3 (SEQ ID NO:32); another name is IgVλ3-21 / IGJλ3 (nomenclature according to the IMGT database (world wide web imgt.org)). This name is exemplary and encompasses allelic variants of the gene segment.

[0139] Furthermore, any light chain variable region of a FAP antibody available in the art can be used, as can any other light chain variable region that is readily available, e.g., from an antibody display library, by exhibiting antigen-binding activity when paired with the FAP-binding domain of a bispecific binding moiety of the present disclosure.

[0140] In certain embodiments, the FAP-binding domain of the bispecific binding moiety of the present disclosure may further comprise a CH1 and a CL region. Any CH1 domain, particularly a human CH1 domain, may be used. An example of a suitable CH1 domain is provided by the amino acid sequence set forth in SEQ ID NO: 39. Any CL domain, particularly a human CL, may be used. An example of a suitable CL domain is provided by the amino acid sequence set forth in SEQ ID NO: 51.

[0141] In certain embodiments, the TGF-βRII binding domain of a bispecific binding moiety of the present disclosure that blocks TGF-βRII from binding to a TGF-βRII ligand is a TGF-βRII binding moiety described in WO2021 / 133167, specifically from page 54, line 11 to page 55, line 1; page 59, line 3 to page 60, line 5, and Figure 6. In certain embodiments, the TGF-βRII binding domain of a bispecific binding moiety of the present disclosure that blocks TGF-βRII from binding to a TGF-βRII ligand is described in WO2021 / 133167 as any of SEQ ID NOs: 10-12; SEQ ID NOs: 22-91, and SEQ ID NOs: 93-96.

[0142] In certain embodiments, the TGF-βRII binding domain of a bispecific binding moiety of the present disclosure comprises a heavy chain variable region comprising: a) a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 24, SEQ ID NO: 25, and SEQ ID NO: 26, respectively; b) a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 24, SEQ ID NO: 29, and SEQ ID NO: 30, respectively; c) a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 24, SEQ ID NO: 33, and SEQ ID NO: 34, respectively; or d) Heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO:24, SEQ ID NO:37, and SEQ ID NO:34, respectively.

[0143] The heavy chain variable region of the TGF-βRII binding domain of the bispecific binding moieties of the present disclosure may include a limited number of non-conservative amino acid substitutions, such as one, two, or three, or an unlimited number of conservative amino acid substitutions.

[0144] In some embodiments, the TGF-βRII binding domain of the bispecific binding moiety of the present disclosure also includes its TGF-βRII binding domain variant, wherein each of HCDR1 or HCDR2 may contain at most three, two, or one amino acid mutations. In some embodiments, only either HCDR1 or HCDR2 may contain at most three, two, or one amino acid mutations. In some embodiments, such variants do not contain amino acid mutations in HCDR3. In some embodiments, the amino acid mutations are conservative amino acid substitutions. Conservative amino acid substitutions are further described herein.

[0145] In certain embodiments, the TGF-βRII binding domain of a bispecific binding moiety of the present disclosure comprises a heavy chain variable region having the amino acid sequence set forth in any one of SEQ ID NOs: 23, 27, 31, or 35, or a variant thereof. In certain embodiments, the TGF-βRII binding domain of a bispecific binding moiety of the present disclosure comprises a heavy chain variable region having the amino acid sequence set forth in any one of SEQ ID NOs: 23, 27, 31, or 35, or having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity thereto.

[0146] In certain embodiments, the TGF-βRII binding domain of the bispecific binding moiety of the present disclosure also comprises a TGF-βRII binding domain variant, which comprises one or more mutations in the framework regions in addition to the mutations in HCDR1 and HCDR2 described above. The mutations can be any type of amino acid mutation described herein, such as conservative or non-conservative amino acid substitutions resulting from somatic hypermutation or affinity maturation. In certain embodiments, the TGF-βRII binding domain variant of the bispecific binding moiety of the present disclosure does not comprise a mutation in the CDR region but comprises one or more mutations in the framework region. Such variants are expected to have at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the sequences disclosed herein and retain TGF-βRII binding specificity. Thus, in certain embodiments, the TGF-βRII binding domain of the bispecific binding moiety of the present disclosure comprises: - a heavy chain variable region having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 23, wherein the heavy chain variable region comprises an HCDR1 amino acid sequence set forth in SEQ ID NO: 24; an HCDR2 amino acid sequence set forth in SEQ ID NO: 25; and an HCDR3 amino acid sequence set forth in SEQ ID NO: 26; - a heavy chain variable region having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 27, wherein the heavy chain variable region comprises an HCDR1 amino acid sequence set forth in SEQ ID NO: 24; an HCDR2 amino acid sequence set forth in SEQ ID NO: 29; and an HCDR3 amino acid sequence set forth in SEQ ID NO: 30; - a heavy chain variable region having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 31, wherein the heavy chain variable region comprises an HCDR1 amino acid sequence set forth in SEQ ID NO: 24; an HCDR2 amino acid sequence set forth in SEQ ID NO: 33; and an HCDR3 amino acid sequence set forth in SEQ ID NO: 34; or - a heavy chain variable region having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 35, wherein the heavy chain variable region comprises an HCDR1 amino acid sequence set forth in SEQ ID NO: 24; an HCDR2 amino acid sequence set forth in SEQ ID NO: 37; and an HCDR3 amino acid sequence set forth in SEQ ID NO: 34.

[0147] Any light chain variable region of a TGF-βRII antibody available in the art can be used, e.g., as described herein, as can any other light chain variable region that can be readily obtained from, e.g., an antibody display library, by exhibiting antigen-binding activity when paired with a TGF-βRII binding domain of a bispecific binding moiety of the present disclosure. In one embodiment, the TGF-βRII binding domain of a bispecific binding moiety of the present disclosure comprises a light chain that is identical or substantially identical to the FAP binding domain.

[0148] In certain embodiments, the TGF-βRII binding domain of a bispecific binding moiety of the present disclosure comprises a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 52, or a variant thereof. In certain embodiments, the TGF-βRII binding domain of a bispecific binding moiety of the present disclosure comprises a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 52, or a variant having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity thereto.

[0149] In certain embodiments, the TGF-βRII binding domain of the bispecific binding moiety of the present disclosure comprises a light chain variable region comprising a light chain CDR1 (LCDR1), a light chain CDR2 (LCDR2), and a light chain CDR3 (LCDR3) having the amino acid sequences set forth in SEQ ID NO: 53, SEQ ID NO: 54, and SEQ ID NO: 55. In certain embodiments, the light chain variable region of the TGF-βRII binding domain of the bispecific binding moiety of the present disclosure also comprises variants thereof, wherein each of the LCDRs may comprise at most three, two, or one conservative or non-conservative amino acid mutations. In certain embodiments, the amino acid mutations are conservative amino acid substitutions.

[0150] In certain embodiments, the TGF-βRII binding domain of the bispecific binding moiety of the present disclosure may further comprise a CH1 and a CL region. Any CH1 domain, particularly a human CH1 domain, may be used. An example of a suitable CH1 domain is provided by the amino acid sequence set forth in SEQ ID NO: 39. Any CL domain, particularly a human CL, may be used. An example of a suitable CL domain is provided by the amino acid sequence set forth in SEQ ID NO: 51.

[0151] Thus, the present invention also provides a bispecific binding moiety comprising a FAP-binding domain and a TGF-βRII-binding domain, wherein the FAP-binding domain comprises a heavy chain variable region, and optionally a light chain variable region and CH1 and CL regions, as described herein. In one embodiment, the bispecific binding moiety further comprises a TGF-βRII-binding domain, which comprises a heavy chain variable region, and optionally a light chain variable region and CH1 and CL regions, as described herein.

[0152] Thus, the present invention also provides a bispecific binding moiety comprising a FAP-binding domain and a TGF-βRII-binding domain, wherein the TGF-βRII-binding domain comprises a heavy chain variable region, and optionally a light chain variable region and CH1 and CL regions, as described herein. In one embodiment, the bispecific binding moiety further comprises a FAP-binding domain, which comprises a heavy chain variable region, and optionally a light chain variable region and CH1 and CL regions, as described herein.

[0153] In certain embodiments, any FAP binding domain disclosed herein can be combined with any TGF-βRII binding domain disclosed herein to produce a bispecific binding moiety of the present disclosure. Accordingly, the present disclosure provides exemplary bispecific binding moieties PB1-PB12, as shown in Table 2.

[0154] In one embodiment, the present disclosure provides a bispecific binding moiety comprising: - a FAP binding domain as described herein, comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 14, respectively; and - a TGF-βRII binding domain as described herein, comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 24, SEQ ID NO: 25, and SEQ ID NO: 26, respectively; Here, each of HCDR1 and HCDR2 may contain at most three, two, or one amino acid mutations, e.g., substitutions. In some embodiments, HCDR3 does not contain any amino acid mutations. In some embodiments, no HCDR contains any amino acid mutations.

[0155] In one embodiment, the present disclosure provides a bispecific binding moiety comprising: - a FAP binding domain as described herein, comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18, respectively; and - a TGF-βRII binding domain as described herein, comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 24, SEQ ID NO: 33, and SEQ ID NO: 34, respectively; Here, each of HCDR1 and HCDR2 may contain at most three, two, or one amino acid mutations, e.g., substitutions. In some embodiments, HCDR3 does not contain any amino acid mutations. In some embodiments, no HCDR contains any amino acid mutations.

[0156] In one embodiment, the present disclosure provides a bispecific binding moiety comprising: - a FAP binding domain as described herein, comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 22, respectively; and - a TGF-βRII binding domain as described herein, comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 24, SEQ ID NO: 29, and SEQ ID NO: 30, respectively; Here, each of HCDR1 and HCDR2 may contain at most three, two, or one amino acid mutations, e.g., substitutions. In some embodiments, HCDR3 does not contain any amino acid mutations. In some embodiments, no HCDR contains any amino acid mutations.

[0157] In one embodiment, the present disclosure provides a bispecific binding moiety comprising: - a FAP binding domain as described herein, comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18, respectively; and - a TGF-βRII binding domain as described herein, comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 24, SEQ ID NO: 37, and SEQ ID NO: 34, respectively; Here, each of HCDR1 and HCDR2 may contain at most three, two, or one amino acid mutations, e.g., substitutions. In some embodiments, HCDR3 does not contain any amino acid mutations. In some embodiments, no HCDR contains any amino acid mutations.

[0158] In one embodiment, the present disclosure provides a bispecific binding moiety comprising: - a FAP binding domain as described herein, comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 14, respectively; and - a TGF-βRII binding domain as described herein, comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 24, SEQ ID NO: 25, and SEQ ID NO: 26, respectively; wherein the FAP-binding domain and the TGF-βRII-binding domain comprise a light chain CDR1 (LCDR1) having the amino acid sequence set forth in SEQ ID NO: 53, a light chain CDR2 (LCDR2) having the amino acid sequence set forth in SEQ ID NO: 54, and a light chain CDR3 (LCDR3) having the amino acid sequence set forth in SEQ ID NO: 55; and Here, each of HCDR1, HCDR2, LCDR1, LCDR2, and LCDR3 may contain at most three, two, or one amino acid mutations, such as substitutions. In some embodiments, HCDR3 does not contain any amino acid mutations. In some embodiments, HCDR and / or LCDR do not contain any amino acid mutations.

[0159] In one embodiment, the present disclosure provides a bispecific binding moiety comprising: - a FAP binding domain as described herein, comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18, respectively; and - a TGF-βRII binding domain as described herein, comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 24, SEQ ID NO: 33, and SEQ ID NO: 34, respectively; wherein the FAP-binding domain and the TGF-βRII-binding domain comprise a light chain CDR1 (LCDR1) having the amino acid sequence set forth in SEQ ID NO: 53, a light chain CDR2 (LCDR2) having the amino acid sequence set forth in SEQ ID NO: 54, and a light chain CDR3 (LCDR3) having the amino acid sequence set forth in SEQ ID NO: 55; and Here, each of HCDR1, HCDR2, LCDR1, LCDR2, and LCDR3 may contain at most three, two, or one amino acid mutations, such as substitutions. In some embodiments, HCDR3 does not contain any amino acid mutations. In some embodiments, HCDR and / or LCDR do not contain any amino acid mutations.

[0160] In one embodiment, the present disclosure provides a bispecific binding moiety comprising: - a FAP binding domain as described herein, comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 22, respectively; and - a TGF-βRII binding domain as described herein, comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 24, SEQ ID NO: 29, and SEQ ID NO: 30, respectively; wherein the FAP-binding domain and the TGF-βRII-binding domain comprise a light chain CDR1 (LCDR1) having the amino acid sequence set forth in SEQ ID NO: 53, a light chain CDR2 (LCDR2) having the amino acid sequence set forth in SEQ ID NO: 54, and a light chain CDR3 (LCDR3) having the amino acid sequence set forth in SEQ ID NO: 55; and Here, each of HCDR1, HCDR2, LCDR1, LCDR2, and LCDR3 may contain at most three, two, or one amino acid mutations, such as substitutions. In some embodiments, HCDR3 does not contain any amino acid mutations. In some embodiments, HCDR and / or LCDR do not contain any amino acid mutations.

[0161] In one embodiment, the present disclosure provides a bispecific binding moiety comprising: - a FAP binding domain as described herein, comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18, respectively; and - a TGF-βRII binding domain as described herein, comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 24, SEQ ID NO: 37, and SEQ ID NO: 34, respectively; wherein the FAP-binding domain and the TGF-βRII-binding domain comprise a light chain CDR1 (LCDR1) having the amino acid sequence set forth in SEQ ID NO: 53, a light chain CDR2 (LCDR2) having the amino acid sequence set forth in SEQ ID NO: 54, and a light chain CDR3 (LCDR3) having the amino acid sequence set forth in SEQ ID NO: 55; and Here, each of HCDR1, HCDR2, LCDR1, LCDR2, and LCDR3 may contain at most three, two, or one amino acid mutations, such as substitutions. In some embodiments, HCDR3 does not contain any amino acid mutations. In some embodiments, HCDR and / or LCDR do not contain any amino acid mutations.

[0162] In one embodiment, the present disclosure provides a bispecific binding moiety comprising: - a FAP-binding domain as described herein, comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 11, or a heavy chain variable region that is at least 80%, at least 85%, at least 90%, or at least 95% sequence identical thereto; and - a TGF-βRII binding domain described herein comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 23, or a heavy chain variable region that is at least 80%, at least 85%, at least 90%, or at least 95% sequence identical thereto. In certain embodiments, each of the heavy chain variable regions comprises an HCDR that does not contain an amino acid mutation. In certain embodiments, each of the heavy chain variable regions does not contain an amino acid mutation.

[0163] In one embodiment, the present disclosure provides a bispecific binding moiety comprising: - a FAP-binding domain as described herein, comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 15, or a heavy chain variable region that is at least 80%, at least 85%, at least 90%, or at least 95% sequence identical thereto; and - a TGF-βRII binding domain described herein comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 31, or a heavy chain variable region that is at least 80%, at least 85%, at least 90%, or at least 95% sequence identical thereto. In certain embodiments, each of the heavy chain variable regions comprises an HCDR that does not contain an amino acid mutation. In certain embodiments, each of the heavy chain variable regions does not contain an amino acid mutation.

[0164] In one embodiment, the present disclosure provides a bispecific binding moiety comprising: - a FAP-binding domain as described herein, comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 19, or a heavy chain variable region that is at least 80%, at least 85%, at least 90%, or at least 95% sequence identical thereto; and - a TGF-βRII binding domain described herein comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 27, or a heavy chain variable region that is at least 80%, at least 85%, at least 90%, or at least 95% sequence identical thereto. In certain embodiments, each of the heavy chain variable regions comprises an HCDR that does not contain an amino acid mutation. In certain embodiments, each of the heavy chain variable regions does not contain an amino acid mutation.

[0165] In one embodiment, the present disclosure provides a bispecific binding moiety comprising: - a FAP-binding domain as described herein, comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 15, or a heavy chain variable region that is at least 80%, at least 85%, at least 90%, or at least 95% sequence identical thereto; and - A TGF-βRII binding domain described herein comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 35, or a heavy chain variable region that is at least 80%, at least 85%, at least 90%, or at least 95% sequence identical thereto. In certain embodiments, each of the heavy chain variable regions comprises an HCDR that does not contain an amino acid mutation. In certain embodiments, each of the heavy chain variable regions does not contain an amino acid mutation.

[0166] In one embodiment, the present disclosure provides a bispecific binding moiety comprising: - a FAP-binding domain as described herein, comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 11, or a heavy chain variable region that is at least 80%, at least 85%, at least 90%, or at least 95% sequence identical thereto; and - a TGF-βRII binding domain as described herein, comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 23, or a heavy chain variable region which is at least 80%, at least 85%, at least 90%, or at least 95% sequence identity thereto; wherein the FAP-binding domain and the TGF-βRII-binding domain comprise a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 52, or a light chain variable region having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity thereto. In some embodiments, the heavy chain variable region and the light chain variable region each comprise an HCDR and an LCDR, respectively, that do not contain an amino acid mutation. In some embodiments, the heavy chain variable region and the light chain variable region each do not contain an amino acid mutation.

[0167] In one embodiment, the present disclosure provides a bispecific binding moiety comprising: - a FAP-binding domain as described herein, comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 15, or a heavy chain variable region that is at least 80%, at least 85%, at least 90%, or at least 95% sequence identical thereto; and - a TGF-βRII binding domain as described herein comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 31, or a heavy chain variable region which is at least 80%, at least 85%, at least 90%, or at least 95% sequence identity thereto; wherein the FAP-binding domain and the TGF-βRII-binding domain comprise a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 52, or a light chain variable region having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity thereto. In some embodiments, the heavy chain variable region and the light chain variable region each comprise an HCDR and an LCDR, respectively, that do not contain an amino acid mutation. In some embodiments, the heavy chain variable region and the light chain variable region each do not contain an amino acid mutation.

[0168] In one embodiment, the present disclosure provides a bispecific binding moiety comprising: - a FAP-binding domain as described herein, comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 19, or a heavy chain variable region that is at least 80%, at least 85%, at least 90%, or at least 95% sequence identical thereto; and - a TGF-βRII binding domain as described herein comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 27, or a heavy chain variable region which is at least 80%, at least 85%, at least 90%, or at least 95% sequence identity thereto; wherein the FAP-binding domain and the TGF-βRII-binding domain comprise a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 52, or a light chain variable region having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity thereto. In some embodiments, the heavy chain variable region and the light chain variable region each comprise an HCDR and an LCDR, respectively, that do not contain an amino acid mutation. In some embodiments, the heavy chain variable region and the light chain variable region each do not contain an amino acid mutation.

[0169] In one embodiment, the present disclosure provides a bispecific binding moiety comprising: - a FAP-binding domain as described herein, comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 15, or a heavy chain variable region that is at least 80%, at least 85%, at least 90%, or at least 95% sequence identical thereto; and - a TGF-βRII binding domain as described herein comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 35, or a heavy chain variable region which is at least 80%, at least 85%, at least 90%, or at least 95% sequence identity thereto; wherein the FAP-binding domain and the TGF-βRII-binding domain comprise a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 52, or a light chain variable region having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity thereto. In some embodiments, the heavy chain variable region and the light chain variable region each comprise an HCDR and an LCDR, respectively, that do not contain an amino acid mutation. In some embodiments, the heavy chain variable region and the light chain variable region each do not contain an amino acid mutation.

[0170] The present invention also provides a bispecific binding moiety comprising a FAP-binding domain and a TGF-βRII-binding domain, wherein the affinity of the FAP-binding domain for human FAP is 25-50 times higher than the affinity of the TGF-βRII-binding domain for human TGF-βRII. In one embodiment, the affinity is determined by the equilibrium dissociation constant (K D In one embodiment, the K of the FAP-binding domain for a human FAP is determined as D is the K of the TGF-βRII binding domain for human TGF-βRII. D In one embodiment, the K D is measured using surface plasmon resonance (SPR).

[0171] SPR is an assay that measures binding affinity using a biosensor system such as Biacore® or Solution Equilibrium Titration (SET) (see Friguet B et al. (1985) J. Immunol Methods; 77(2): 305-319, 25 and Hanel C et al. (2005) Anal Biochem; 339(1): 182-184). In one embodiment, K D is measured using SPR as described in Example 14.

[0172] In one embodiment, the affinity of the FAP binding domain of the bispecific binding moiety described herein is in the range of about 0.1-0.2 nM, as measured by SPR as described in Example 14. In one embodiment, the affinity of the TGF-βRII binding domain of the bispecific binding moiety is in the range of about 3.8-5 nM, as measured by SPR as described in Example 14.

[0173] In one embodiment, binding affinity is measured using a FAP x TGF-βRII bispecific binding moiety of the present disclosure in a bivalent, bispecific format. Thus, the binding affinity of the bispecific binding moiety for human FAP and human TGF-βRII exhibits monovalent binding affinity.

[0174] In certain embodiments, the present disclosure also provides a bispecific binding moiety comprising a FAP-binding domain and a TGF-βRII-binding domain, wherein the FAP-binding domain binds to human FAP (huFAP) and is cross-reactive with cynomolgus monkey FAP (cyFAP). In certain embodiments, the present disclosure provides a bispecific binding moiety comprising a FAP-binding domain and a TGF-βRII-binding domain, wherein the TGF-βRII-binding domain binds to human TGF-βRII (huTGF-βRII) and is cross-reactive with cynomolgus monkey TGF-βRII (cyTGF-βRII).

[0175] In certain embodiments, a bispecific binding moiety of the present disclosure comprises a Fab domain that binds to a FAP, a Fab domain that binds to TGF-βRII, and an Fc region.

[0176] In certain embodiments, a polypeptide or binding moiety comprising a binding domain of the present disclosure has an Fc effector function.

[0177] In some embodiments, the Fc region of the bispecific binding moiety has antibody-dependent cellular cytotoxicity (ADCC) activity. In some embodiments, the Fc region of the bispecific binding moiety has antibody-dependent cellular phagocytosis (ADCP) activity. In some embodiments, the Fc region of the bispecific binding moiety has ADCC activity and ADCP activity.

[0178] In some embodiments, the bispecific binding moieties of the present disclosure have unmodified immune cell effector function or modified immune cell effector function, hi some embodiments, the unmodified immune effector function is mediated by a bispecific binding moiety comprising an Fc region comprising the hinge, CH2, and CH3 regions of an IgG1 isotype according to SEQ ID NOs: 40, 41, and 43, respectively.

[0179] In certain embodiments, the bispecific binding moieties of the present disclosure have altered immune cell effector function, e.g., enhanced or reduced immune cell effector function, hi certain embodiments, the altered immune effector function is caused by one or more mutations in the hinge, CH2 and / or CH3 regions of SEQ ID NOs: 40, 41, 43, respectively.

[0180] In certain embodiments, the Fc region of the bispecific binding moieties of the present disclosure has an enhanced or reduced immune effector function.

[0181] In certain embodiments, the Fc region of the bispecific binding moiety has enhanced immune cell effector function, specifically enhanced ADCC activity. Bispecific binding moieties comprising an Fc with enhanced immune effector function are referred to herein as "Fc-enhanced variants." The immune cell effector function exhibited by the Fc-enhanced variants is enhanced compared to the immune cell effector function exhibited by the unmodified bispecific binding moiety.

[0182] In certain embodiments, the Fc region of the bispecific binding moiety has enhanced ADCC activity.

[0183] In certain embodiments, the Fc region of the bispecific binding moiety has ADCP activity and enhanced ADCC activity.

[0184] In certain embodiments, the Fc region of the bispecific binding moiety has enhanced ADCP activity.

[0185] In certain embodiments, the Fc region of the bispecific binding moiety has enhanced ADCP activity and enhanced ADCC activity.

[0186] Bispecific binding moieties, such as antibodies, can be modified to enhance ADCC activity. Methods for doing so are well known to those skilled in the art (for a review, see Kubota T et al. Cancer Sci. 2009; 100(9):1566-72). For example, minor modifications to the antibody's constant region can improve the antibody's ADCC activity (Junttila TT. et al. Cancer Res. 2010; 70(11):4481-9). Alterations can also be made to improve storage or production or to remove C-terminal lysines (Kubota T et al. Cancer Sci. 2009; 100(9):1566-72). Another method for improving antibody ADCC activity is to reduce fucose by enzymatically intervening in the glycosylation pathway (von Horsten HH. et al. Glycobiology. 2010; 20(12):1607-18). Alternatively, or in addition, several other strategies can be used to achieve enhanced ADCC, including, for example, glycoengineering (Kyowa Hakko / Biowa, GlycArt (Roche), and Eureka Therapeutics) and mutagenesis, all aimed at improving Fc binding to the low-affinity activating FcγRIIIa and / or reducing binding to the low-affinity inhibitory FcγRIIb. In certain embodiments, the bispecific binding moieties of the present disclosure exhibit enhanced ADCC.

[0187] Thus, the present disclosure provides a bispecific binding moiety comprising a FAP-binding domain and a TGF-βRII-binding domain, wherein the bispecific binding moiety has enhanced immune cell effector function. In one embodiment, the bispecific binding moiety is defucosylated.

[0188] In certain embodiments, the immune cell with effector function is an NK cell, a T cell, a B cell, a monocyte, a macrophage, a dendritic cell, or a neutrophil granulocyte.

[0189] In certain embodiments, the Fc region of the bispecific binding moiety has reduced immune cell effector function, specifically reduced ADCC and / or ADCP activity. Bispecific binding moieties comprising an Fc with reduced immune effector function are referred to herein as "Fc-silenced variants." The immune cell effector function exhibited by the Fc-silenced variants is reduced compared to the immune cell effector function exhibited by the unmodified bispecific binding moiety.

[0190] In some embodiments, the bispecific binding moieties of the present disclosure have reduced Fc receptor interaction or reduced C1q binding. In some embodiments, the bispecific binding moieties of the present disclosure exhibit reduced ADCC and / or ADCP. Bispecific binding moieties, such as antibodies, can be modified to reduce ADCC and / or ADCP activity (Liu R, et. al. Fc-Engineering for Modulated Effector Functions-Improving Antibodies for Cancer Treatment. Antibodies (Basel). 2020 Nov 17;9(4):64). For example, the ADCC and / or ADCP activity of an antibody can be reduced by modifying the CH2 and / or lower hinge region of an IgG antibody to reduce the antibody's interaction with Fcγ receptors. The CH2 and / or lower hinge region of such a variant IgG1 comprises amino acid substitutions at positions 235 and / or 236 (EU numbering), such as L235G and / or G236R substitutions.

[0191] Thus, the present disclosure also provides a bispecific binding moiety comprising a FAP-binding domain and a TGF-βRII-binding domain, wherein the bispecific binding moiety has reduced immune cell effector function. In one embodiment, the bispecific binding moiety has an L235G and / or G236R mutation in the CH2 domain of the Fc region (SEQ ID NO: 42) (EU numbering).

[0192] Several in vitro methods exist for determining the effectiveness of antibodies or effector cells in inducing ADCC. These include the chromium 51 [Cr51] release assay, europium [Eu] release assay, and sulfur 35 [S35] release assay. Typically, a labeled target cell line expressing a particular surface-exposed antigen is incubated with an antibody specific for that antigen. After washing, effector cells expressing the Fc receptor CD16 are co-incubated with the antibody-labeled target cells. Target cell lysis is then measured by the release of intracellular label using a scintillation counter or spectrophotometrically.

[0193] In one embodiment, the efficacy of the bispecific binding moiety in inducing ADCC is determined by the methods described in Examples 10 or 11.

[0194] In one embodiment, the efficacy of a bispecific binding moiety in inducing ADCP is determined by the method described in Example 17. Briefly, the method described in Example 17 involves the differentiation of human peripheral blood monocytes into M0 / M2C macrophages, which are confirmed to express M1 / M2 markers. An ADCP assay is then performed in the presence of a bispecific binding moiety of the present disclosure, using the differentiated macrophages as effector cells and A549-FAP+ cells or lung CAFs as target cells. The ability of the bispecific binding moiety to mediate ADCP to target cells can be measured by flow cytometry or imaging.

[0195] In certain embodiments, the present disclosure provides bispecific binding moieties that compete with the bispecific binding moieties described herein for binding to huFAP and huTGF-βRII.

[0196] For purposes of this disclosure, "compete," "competes," or "competing" refers to the activity of a bispecific binding moiety to displace a bispecific binding moiety described herein from its target antigen in a cross-blocking assay. Thus, in some embodiments, a binding moiety that competes for binding with a bispecific binding moiety described herein binds to huFAP and huTGF-βRII and displaces the bispecific binding moiety described herein in a cross-blocking assay. In some embodiments, the cross-blocking assay is a competitive ELISA. Methods for performing competitive ELISAs are known to those skilled in the art.

[0197] Briefly, in a competitive ELISA, antigen is coated onto the wells of a microtiter plate and preincubated with or without a competing binding moiety. This is followed by the addition of a biotin-labeled bispecific binding moiety disclosed herein. The amount of labeled bispecific binding moiety that binds to the antigen in the well is measured using an avidin-peroxidase conjugate and an appropriate substrate. The amount of labeled bispecific binding moiety that binds to the antigen is negatively correlated with the ability of the competing binding moiety to compete for binding to the same antigen. That is, the higher the affinity of the competing binding moiety for the same antigen, the less labeled bispecific binding moiety will bind to the antigen-coated well. A candidate competing binding moiety is considered to compete for binding to an antigen as a bispecific binding moiety of the present disclosure if it can block binding of the bispecific binding moiety to its respective target antigen by at least 20%, specifically at least 20-50%, specifically at least 50%, compared to a parallel control run without the candidate competing binding moiety.

[0198] [Nucleic acids, vectors, and cells] Further provided herein are nucleic acids useful for producing the polypeptides, binding domains, or binding moieties of the present disclosure. In certain embodiments, such nucleic acids comprise nucleic acid sequences that encode the polypeptides described herein.

[0199] Further provided herein is a nucleic acid useful for producing the bispecific binding moiety of the present disclosure.In some embodiments, such a nucleic acid comprises a nucleic acid sequence encoding the heavy chain variable region of the FAP binding domain described herein.In some embodiments, such a nucleic acid comprises a nucleic acid sequence encoding the heavy chain variable region of the FAP binding domain described herein and the heavy chain variable region of the TGF-βRII binding domain described herein.

[0200] In some embodiments, the nucleic acid of the present disclosure may further comprise a nucleic acid sequence encoding a CH1 region, and preferably a hinge, CH2, and CH3 region. In some embodiments, the nucleic acid of the present disclosure may further comprise at least one nucleic acid sequence encoding a light chain variable region, and preferably a CL region. In some embodiments, the light chain variable region may be the light chain variable region described herein. In some embodiments, the light chain variable region is a light chain variable region capable of pairing with multiple heavy chains having different epitope specificities.

[0201] Further provided herein are vectors comprising the nucleic acids of the present disclosure useful for producing the binding domains or binding moieties of the present disclosure. In certain embodiments, such vectors comprise a nucleic acid sequence encoding a polypeptide described herein.

[0202] Furthermore, the present specification provides a vector comprising the nucleic acid of the present disclosure, which is useful for producing the bispecific binding moiety of the present disclosure.In some embodiments, such a vector comprises the nucleic acid sequence encoding the heavy chain variable region of the FAP binding domain described herein.In some embodiments, such a vector comprises the nucleic acid sequence encoding the heavy chain variable region of the FAP binding domain described herein and the heavy chain variable region of the TGF-βRII binding domain described herein.

[0203] In some embodiments, the vector of the present disclosure may further comprise a nucleic acid sequence encoding a CH1 region, and preferably a hinge, CH2, and CH3 region. In some embodiments, the vector of the present disclosure may further comprise at least one nucleic acid sequence encoding a light chain variable region, and preferably a CL region. In some embodiments, the light chain variable region may be the common light chain variable region described herein. In some embodiments, the light chain variable region is a light chain variable region of a light chain that can pair with multiple heavy chains having different specificities.

[0204] The present disclosure also provides cells that contain (eg, as part of a vector) a nucleic acid that includes a sequence encoding a polypeptide described herein.

[0205] The present disclosure also provides a cell comprising a nucleic acid sequence (e.g., as part of a vector) encoding the heavy chain variable region of the FAP binding domain described herein and a nucleic acid sequence encoding the heavy chain variable region of the TGF-βRII binding domain described herein.

[0206] In some embodiments, the cells of the present disclosure may further comprise (e.g., as part of a vector) a nucleic acid sequence encoding a CH1 region, and preferably a hinge, CH2, and CH3 region. In some embodiments, the cells of the present disclosure may further comprise (e.g., as part of a vector) at least one nucleic acid sequence encoding a light chain variable region, and preferably a CL region. In some embodiments, the light chain variable region may be the common light chain variable region described herein.

[0207] The present disclosure also provides cells that produce the polypeptides, binding domains, or binding moieties described herein. The present disclosure also provides cells that produce the bispecific binding moieties described herein. In some embodiments, such cells may be recombinant cells containing the nucleic acids (e.g., vectors) of the present disclosure. In some embodiments, the cells of the present disclosure contain a nucleic acid sequence (e.g., vector) that includes a sequence encoding a polypeptide described herein. In some embodiments, the cells of the present disclosure contain a nucleic acid sequence (e.g., vector) encoding the heavy chain variable region of the FAP binding domain described herein and a nucleic acid sequence encoding the heavy chain variable region of the TGF-βRII binding domain described herein. In some embodiments, the cells of the present disclosure further contain a nucleic acid sequence (e.g., vector) encoding a CH1 region, and preferably a hinge, CH2, and CH3 region. In some embodiments, the cells of the present disclosure further contain at least one nucleic acid sequence (e.g., vector) encoding a light chain variable region, specifically a light chain variable region described herein, and preferably a CL region.

[0208] Pharmaceutical Compositions and Methods of Use In certain embodiments, the present disclosure provides a pharmaceutical composition comprising an effective amount of a polypeptide described herein, or a FAP binding domain described herein, or a binding moiety described herein, and a pharmaceutically acceptable carrier.

[0209] In certain embodiments, the present disclosure provides a pharmaceutical composition comprising an effective amount of a bispecific binding moiety described herein and, optionally, a pharmaceutically acceptable carrier.

[0210] In certain embodiments, the present disclosure provides a polypeptide described herein, or a FAP binding domain described herein, or a binding moiety described herein, or a pharmaceutical composition described herein, for use in therapy.

[0211] In certain embodiments, the present disclosure provides bispecific binding moieties described herein, and pharmaceutical compositions described herein, for use in therapy.

[0212] In certain embodiments, the present disclosure provides a polypeptide described herein, or a FAP binding domain described herein, or a binding moiety described herein, or a pharmaceutical composition described herein, for use in the treatment of cancer.

[0213] In certain embodiments, the present disclosure provides a bispecific binding moiety as described herein, and a pharmaceutical composition as described herein, for use in the treatment of cancer.

[0214] In certain embodiments, the present disclosure provides a method for treating a disease, the method comprising administering to an individual in need thereof an effective amount of a polypeptide described herein, or a FAP binding domain described herein, or a binding moiety described herein, or a pharmaceutical composition described herein.

[0215] In certain embodiments, the present disclosure provides a method for treating a disease, comprising administering an effective amount of a bispecific binding moiety described herein, or a pharmaceutical composition described herein, to an individual in need thereof.

[0216] In certain embodiments, the present disclosure provides a method for treating cancer, comprising administering to an individual in need thereof an effective amount of a polypeptide described herein, or a FAP binding domain described herein, or a binding moiety described herein, or a pharmaceutical composition described herein.

[0217] In certain embodiments, the present disclosure provides a method for treating cancer, comprising administering to an individual in need thereof an effective amount of a bispecific binding moiety described herein, or a pharmaceutical composition described herein.

[0218] [Combination therapy] The bispecific binding moieties of the present disclosure may be particularly effective when used in combination with a programmed cell death protein 1 (PD-1) inhibitor. The combination of a bispecific binding moiety described herein with a PD-1 inhibitor allows for inhibition of the PD-1 / PD-L1 axis in addition to alleviating immune cell suppression mediated by TGF-βRII signaling in T cells. Thus, the present disclosure also provides a combination of a bispecific binding moiety described herein with a PD-1 inhibitor. The PD-1 inhibitor may be any PD-1 inhibitor. In some embodiments, the PD-1 inhibitor is an anti-PD-1 binding moiety. In some embodiments, the PD-1 inhibitor is an anti-PD-1 antibody. In some embodiments, the anti-PD-1 antibody is a full-length antibody, Fab, modified Fab, or scFv. The anti-PD-1 antibody may be a commercially available antibody, such as pembrolizumab, retifanlimab, nivolumab, cemiplimab, or dostarlimab, or an analog or variant thereof. In one embodiment, the PD-1 antibody is pembrolizumab. In one embodiment, the PD-1 antibody is retifanlimab.

[0219] PD-1 is a cell surface receptor belonging to the CD28 family of receptors and is expressed on T cells and pro-B cells. PD-1 is currently known to bind two ligands, PD-L1 and PD-L2. PD-1 functions as an immune checkpoint, playing a key role in downregulating the immune system by inhibiting T cell activation, thereby reducing autoimmunity and promoting self-tolerance. PD-1's inhibitory effects are thought to be achieved through a dual mechanism: promoting apoptosis (programmed cell death) in antigen-specific T cells in lymph nodes while simultaneously reducing apoptosis in regulatory T cells (suppressor T cells). PD-1 is also known by many different names, including PDCD1; programmed cell death 1; systemic lupus erythematosus susceptibility 2; protein PD-1; HPD-1; PD1; programmed cell death 1 protein; CD279 antigen; CD279; HPD-L; HSLE1; SLEB2; and PD-1. The external IDs for PD-1 are HGNC:8760; Entrez Gene:5133; Ensembl:ENSG00000188389; OMIM:600244; and UniProtKB:Q15116. The amino acid sequence of human PD-1 is set forth in SEQ ID NO:50. PD-1 inhibitors, a new class of drugs that block the activity of PD-1, have been used with some success to activate the immune system to attack tumors and thus treat certain cancers.

[0220] The present disclosure further provides a kit of parts comprising a bispecific binding moiety described herein and a PD-1 inhibitor. The present disclosure also provides a kit of parts comprising a bispecific binding moiety described herein and instructions for using the bispecific binding moiety in combination with a PD-1 inhibitor.

[0221] The bispecific binding moiety and the PD-1 inhibitor may be formulated and / or administered together or separately, simultaneously or sequentially.

[0222] The present disclosure further provides a combination of a bispecific binding moiety described herein with a PD-1 inhibitor for reducing inhibitory signals in T cells. The present disclosure further provides a combination of a bispecific binding moiety described herein with a PD-1 inhibitor for use in therapy. The present disclosure further provides a combination of a bispecific binding moiety described herein with a PD-1 inhibitor for use in treating a subject in need thereof, particularly for use in treating cancer. The bispecific binding moiety described herein and the PD-1 inhibitor may be administered simultaneously or sequentially, with the PD-1 inhibitor preceding or following administration of the bispecific binding moiety.

[0223] The present disclosure further provides the bispecific binding moieties and PD-1 inhibitors described herein for use in therapy.The present disclosure further provides the bispecific binding moieties and PD-1 inhibitors described herein for use in the treatment of cancer.

[0224] The present disclosure further provides a bispecific binding moiety described herein for use in therapy, wherein the therapy further comprises administering a PD-1 inhibitor. The present disclosure further provides a bispecific binding moiety described herein for use in therapy, wherein the therapy further comprises administering a PD-1 inhibitor. In certain embodiments, the present disclosure provides a method for treating a disease, particularly cancer, comprising administering to an individual in need thereof an effective amount of a combination of a bispecific binding moiety described herein and a PD-1 inhibitor.

[0225] In certain embodiments, the present disclosure provides a method for treating a disease, particularly cancer, comprising administering to an individual in need thereof an effective amount of a bispecific binding moiety described herein and a PD-1 inhibitor.

[0226] In certain embodiments, the present disclosure provides use of a bispecific binding moiety disclosed herein and a PD-1 inhibitor in the manufacture of a medicament for treating a disease in a subject. In certain embodiments, the bispecific binding moiety disclosed herein and the PD-1 inhibitor are administered in separate dosage forms. In certain embodiments, the bispecific binding moiety disclosed herein and the PD-1 inhibitor are administered simultaneously or sequentially.

[0227] As used herein, the terms "individual," "subject," and "patient" are used interchangeably and refer to mammals such as humans, mice, rats, hamsters, guinea pigs, rabbits, cats, dogs, monkeys, cows, horses, pigs, etc., particularly human subjects with cancer.

[0228] As used herein, the terms "treat," "treating," and "treatment" refer to any type of intervention or process performed on a subject, or the administration of an active agent or combination of active agents to a subject, with the intent of curing or ameliorating a disease or its symptoms, or producing a positive therapeutic response. As used herein, a "positive therapeutic response" refers to a treatment that produces a beneficial effect, such as an effect that reverses, alleviates, improves, inhibits, or slows a symptom, complication, condition, or biochemical manifestation associated with a disease, and an effect that prevents the onset, progression, development, severity, or recurrence of a symptom, complication, condition, or biochemical manifestation associated with a disease, e.g., an improvement in at least one symptom of a disease or disorder, such as cancer. A beneficial effect can take the form of an improvement over baseline, including an improvement over measurements or observations made before initiation of treatment according to the method. For example, a beneficial effect can take the form of slowing, stabilizing, halting, or reversing the progression of cancer in a subject at any clinical stage, as evidenced by a reduction or elimination of clinical or diagnostic symptoms of the disease or of a marker of cancer. Effective treatment can be, for example, a reduction in tumor size, a reduction in the presence of circulating tumor cells, a reduction or prevention of tumor metastasis, a delay or arrest of tumor growth, and / or a prevention or delay of tumor recurrence or relapse.

[0229] The term "therapeutic amount" or "effective amount" refers to an amount of a drug or combination of drugs to treat a disease, such as cancer. In some embodiments, a therapeutic amount is an amount sufficient to delay tumor progression. In some embodiments, a therapeutic amount is an amount sufficient to prevent or delay tumor recurrence.

[0230] As used herein, an effective amount of an agent or composition is one that can, for example: (i) reduce the number of cancer cells; (ii) reduce tumor size; (iii) inhibit, hinder, to some extent slow, and stop the invasion of cancer cells into peripheral organs; (iv) inhibit tumor metastasis; (v) inhibit tumor growth; (vi) prevent or delay the onset and / or recurrence of tumors; and / or (vii) relieve to some extent one or more symptoms associated with cancer.

[0231] The effective amount may vary according to factors such as the disease state, age, sex, and weight of the individual being treated, and the ability of the agent or combination of agents to elicit a desired response in the individual, which can be readily assessed by a physician or other medical professional of ordinary skill.

[0232] An effective amount can be administered to a subject in one or more administrations.

[0233] An effective amount may also include an amount that balances any toxic or detrimental effects of the agent or combination of agents with the beneficial effects.

[0234] The term "agent" refers to a therapeutically active substance, in this case a polypeptide, binding domain, binding moiety, or bispecific binding moiety of the disclosure, or a pharmaceutical composition of the disclosure.

[0235] As used herein, the word "to comprise" and its conjugations are used in an open-ended sense, meaning that items following the word are included, but items not specifically mentioned are not excluded.

[0236] The articles "a" and "an" are used herein to refer to one or to more than one of the grammatical object of the article. By way of example, "an element" means one or more elements.

[0237] Reference in this specification to a patent document or other matter should not be taken as an admission that the document or matter was publicly known or that the information it contains was part of the common general knowledge at the priority date of any of the claims.

[0238] All patents and references cited herein are incorporated by reference in their entirety.

[0239] Unless otherwise specified, amino acid positions assigned to CDRs and frameworks in the variable region of an antibody or antibody fragment are identified according to Kabat numbering (see Sequences of Proteins of Immunological Interest (National Institute of Health, Bethesda, Md., 1987 and 1991)). Amino acids in the constant region are indicated according to the EU numbering system.

[0240] Accession numbers are provided primarily to provide an additional method of target identification; the actual sequence of the binding protein may vary due to mutations in the encoding gene, such as those that occur in some cancers. The antigen-binding site of a binding domain, binding moiety, or bispecific binding moiety of the present disclosure may bind to an antigen and its various variants, such as those expressed by some antigen-positive immune cells or tumor cells. HGNC stands for HUGO Gene Nomenclature Committee. The number following the abbreviation is the accession number, by which information about the gene and the protein encoded by the gene can be retrieved from the HGNC database. Entrez Gene provides the accession number or gene ID, by which information about the gene or the protein encoded by the gene can be retrieved from the NCBI (National Center for Biotechnology Information) database. Ensembl provides the accession number, by which information about the gene or the protein encoded by the gene can be obtained from the Ensemble database. Ensembl is a collaborative project between EMBL-EBI and the Wellcome Trust Sanger Institute developing a software system to create and maintain automated annotations of selected eukaryotic genomes.

[0241] Reference herein to a gene or protein preferably refers to the human form of that gene or protein. Reference herein to a gene or protein refers to both the native gene or protein and to variant forms of the gene or protein that may be detected in tumors, cancers, etc., preferably human tumors, cancers, etc. [Example]

[0242] In an example used to illustrate, but not intended to limit the present disclosure in any way, a FAP-binding domain comprises a heavy chain variable region, as further specified herein, and a CH1 region having the amino acid sequence set forth in SEQ ID NO: 39. The FAP-binding domain further comprises a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 52, and a light chain constant region having the amino acid sequence set forth in SEQ ID NO: 51.

[0243] When screened in IgG1 format, the IgG comprises a hinge region having the amino acid sequence set forth in SEQ ID NO:40, a CH2 region having the amino acid sequence set forth in SEQ ID NO:41, and a CH3 region having the amino acid sequence set forth in SEQ ID NO:43.

[0244] In an example used to illustrate, but not intended to limit in any way, the present disclosure, each binding domain of the bispecific antibody comprises a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 52 and a light chain constant region having the amino acid sequence set forth in SEQ ID NO: 51.

[0245] The bispecific antibody is preferably an IgG1 antibody, comprising a CH1, hinge, CH2, and CH3. The Fc region of the IgG1 antibody may be modified to reduce or enhance the ADCC and / or CDC activity of the antibody.

[0246] In an example used to illustrate the present disclosure, but not intended to limit the disclosure in any way, bispecific antibodies were screened in an IgG1 format, wherein the FAP-binding heavy chain comprises a CH1 having the amino acid sequence set forth in SEQ ID NO: 39, a CH2 having the amino acid sequence set forth in SEQ ID NO: 41 or 42, and a CH3 having the amino acid sequence set forth in SEQ ID NO: 44; and the TGF-βRII-binding heavy chain comprises a CH1 having the amino acid sequence set forth in SEQ ID NO: 39, a CH2 having the amino acid sequence set forth in SEQ ID NO: 41 or 42, and a CH3 having the amino acid sequence set forth in SEQ ID NO: 45.

[0247] Reference antibodies and molecules used in the examples include: - Analog Reference TGF-βRII Antibody TGF1 (herein referred to as Analog Reference TGF1) is a bivalent, monospecific analog of TGF1 and comprises two heavy chains having the amino acid sequences set forth in SEQ ID NO:1 and two light chains having the amino acid sequences set forth in SEQ ID NO:2. - The analog reference FAP antibody sibrotuzumab (referred to herein as analog reference sibrotuzumab or sibrotuzumab analog) is a bivalent, monospecific analog of sibrotuzumab and comprises two heavy chains having the amino acid sequences set forth in SEQ ID NO:7 and two light chains having the amino acid sequences set forth in SEQ ID NO:8. The negative control IgG1 antibody (RSV-G) (herein referred to as the negative control RSV-G antibody or negative control RSV antibody) is a bivalent monospecific antibody that comprises two heavy chains having the amino acid sequence set forth in SEQ ID NO:3 and two light chains having the amino acid sequence set forth in SEQ ID NO:4, or two heavy chains having the amino acid sequence set forth in SEQ ID NO:5 and two light chains having the amino acid sequence set forth in SEQ ID NO:4. - The positive control antibody cetuximab is an anti-EGFR monoclonal antibody obtained from Refdrug, Inc. (NDC#66733-958-23). - Pembrolizumab is a bivalent anti-PD-1 antibody available from Refdrug, Inc. (NDC#0006-3026-02). - An analog of the reference antibody ESC11 (herein referred to as ESC11 analog) is a bivalent monospecific analog of ESC11 and comprises two heavy chains having the amino acid sequences set forth in SEQ ID NO: 74 and two light chains having the amino acid sequences set forth in SEQ ID NO: 75. - A positive control IgG1 antibody against human and mouse FAP (herein referred to as the positive control hu / moFAP antibody) is a bivalent monospecific antibody, comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO: 76 and two light chains having the amino acid sequences set forth in SEQ ID NO: 77. - A negative control IgG1 antibody against tetanus toxoid (TT) (herein referred to as the negative control TT antibody) is a bivalent monospecific antibody, comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO: 78 and two light chains having the amino acid sequences set forth in SEQ ID NO: 4. - Ab0625(R&D Systems, FAB1180P). - The small molecule inhibitor talabostat (Gentaur, GEN2327500).

[0248] Example 1 - Generation of FAP-binding domains and antibodies containing such binding domains Binding domains, antibodies, and heavy chain variable regions with binding specificity for human FAP were obtained by immunizing transgenic mice (MeMo® mice) containing the common IGKV1-39 light chain with human FAP antigenic portions (including using various forms of DNA-, protein-, and cell-based antigen delivery).

[0249] A phage display library was constructed and human FAP binders were selected by recombinant protein panning and cell selection. The binders were reformatted into a bivalent, monospecific IgG format for subsequent screening and characterization in binding and functional assays.

[0250] The binding domain sequences herein may be obtained by any method known in the art after being characterized and sequenced through the techniques provided herein.

[0251] Example 2 - Characterization of anti-human FAP bivalent monospecific IgG A large and diverse panel of anti-human FAP bivalent monospecific IgGs was screened in a FACS assay for cross-reactivity to cynomolgus monkey FAP (cyFAP), binding to mouse FAP (moFAP), and binding to human CD26 (huCD26); and for binding to WI-38 cells endogenously expressing human FAP (huFAP). The IgGs were also screened for their ability to inhibit the catalytic activity of FAP. Binning experiments were performed to bin the FAP-binding domains into groups that differentially bind to human FAP.

[0252] [FACS] FACS analysis was performed to examine the specificity, species cross-reactivity, and CD26 cross-reactivity of the anti-human FAP panel.

[0253] In this assay, 293FF-huFAP cells (stably expressing huFAP) and 2833FF-cyFAP cells (stably expressing cyFAP) were used. Additionally, 293FF cells transiently transfected with the following constructs were used: moFAP and huCD26. Mock-transfected 293FF cells were used to analyze background antibody binding.

[0254] The following control antibodies were included: sibrotuzumab analogue (as a positive control for huFAP and cyFAP), positive control hu / moFAP antibody (as a positive control for huFAP and moFAP), negative control TT antibody (as a negative control), and Ab0625 (as a positive control for huCD26).

[0255] All test and control antibodies were tested for cell binding at a single concentration of 2.5 μg / ml. Goat anti-human IgG F(ab′)2-PE (Invitrogen, H10104) was used as the secondary antibody (1:100 in FACS buffer). Ab0625 was used at 10 μg / ml, and its binding was detected using Ab0250 (Becton Dickinson, 550767) (1:100 in FACS buffer). FACS buffer containing PBS (Gibco, Cat. No. 10010-015), 0.5% BSA (Thermo Scientific, Cat. No. 37525), and 2 mM EDTA (Invitrogen, Cat. No. 15575-020) was kept ice-cold throughout the assay.

[0256] Target cells were harvested, counted, and centrifuged at 300g for 5 minutes at 4°C. Cells were then resuspended in FACS buffer at a concentration of 1x106 cells / ml and transferred to a U-bottom 96-well FACS plate (BD, catalog no. 353910) (200,000 cells / well). Cells were then centrifuged at 300g for 3 minutes at 4°C, and the supernatant was removed. 50µl of primary antibody solution was added to the cells, mixed, and incubated in the dark for 30 minutes at 2-8°C. Cells were washed by adding 150µl of FACS buffer and centrifuged at 300g for 3 minutes at 4°C. The supernatant was removed, and the cells were washed again by adding 200µl of FACS buffer to the plate. Cells were then centrifuged at 300g for 3 minutes at 4°C, and the supernatant was removed. 50 μl of secondary antibody solution was added to the cells, mixed, and incubated for 30 minutes at 2–8°C in the dark. The cells were then washed by adding 150 μl of FACS buffer to the plate and centrifuged at 300 g for 3 minutes at 4°C. The supernatant was removed, and the cells were washed again by adding 200 μl of FACS buffer to the plate and centrifuged at 300 g for 3 minutes at 4°C. The supernatant was removed, and the cells were fixed by adding 100 μl / well of ice-cold fixation buffer (2% formaldehyde solution in PBS, Sigma-Aldrich, catalog no. 47608-250ML-F). The cells were incubated on ice in the dark for 15 minutes. Then, 100 μl of ice-cold PBS was added to the cells and centrifuged at 300 g for 3 minutes at 4°C. The supernatant was removed, and the cells were resuspended in 120 μl of FACS buffer. The plates were then sealed with EASYseal (Greiner, Cat. No. 67600) and stored in the dark at 4°C until measurement. The fluorescence intensity of the cells was then measured using an iQue VBR (Intellicyt).

[0257] All IgGs screened showed binding to 293FF cells stably transfected with huFAP and to 293FF cells stably transfected with cyFAP (data not shown). The IgGs showed a wide range in binding activity.

[0258] Of the IgGs screened, only a few showed binding to 293FF cells stably transfected with moFAP (data not shown). An example of an IgG that binds to moFAP is one that contains a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO:69.

[0259] None of the screened IgGs showed binding to cells expressing huCD26 (data not shown).

[0260] [Binding to WI-38 cells endogenously expressing human FAP] A panel of human anti-FAP antibodies was tested on WI-38 cells (which endogenously express huFAP) and binding was measured by FACS.

[0261] Target cells were harvested, counted, and centrifuged at 300g for 5 minutes at 4°C. Cells were then resuspended in FACS buffer at a concentration of 1 x 106 cells / ml and transferred to a U-bottom 96-well FACS plate (BD, catalog no. 353910) (200,000 cells / well). Cells were washed with 200 μl of PBS and centrifuged at 300g for 3 minutes at 4°C. Cells were then centrifuged at 300g for 3 minutes at 4°C, and the supernatant was removed. Antibodies were diluted in FACS buffer (50 μl / well) in eight half-log dilutions ranging from 10 μg / ml to 3.16 ng / ml, mixed, and incubated for 30 minutes at 2-8°C in the dark. A titration of sibrotuzumab analogs was included as a positive control and used to normalize the data. A negative control TT antibody was included as a negative control. The cells were washed by adding 150 μl of FACS buffer and centrifuged at 300 g for 3 minutes at 4°C. The supernatant was removed, and the cells were washed again by adding 200 μl of FACS buffer to the plate. The cells were then centrifuged at 300 g for 3 minutes at 4°C, after which the supernatant was removed. Goat anti-human IgG F(ab′)2-PE (Invitrogen, H10104) was used as the secondary antibody (1:100 in FACS buffer). 50 μl of the secondary antibody solution was added to the cells, mixed, and incubated for 30 minutes at 2–8°C in the dark. The cells were then washed by adding 150 μl of FACS buffer to the plate and centrifuged at 300 g for 3 minutes at 4°C. The supernatant was removed, and the cells were washed again by adding 200 μl of FACS buffer to the plate and centrifuged at 300 g for 3 minutes at 4°C. The supernatant was removed, and the cells were fixed by adding 100 μl / well of ice-cold fixation buffer (2% formaldehyde solution in PBS, Sigma-Aldrich, Cat. No. 47608-250ML-F). The cells were incubated on ice in the dark for 15 minutes. Then, 100 μl of ice-cold PBS was added to the cells, and the cells were centrifuged at 300 g for 3 minutes at 4°C. The supernatant was removed, and the cells were resuspended in 120 μl of FACS buffer. The plates were then sealed with EASYseal (Greiner, Cat. No. 67600) and stored in the dark at 4°C until measurement.The fluorescence intensity of the cells was then measured by iQue VBR (Intellicyt).

[0262] All screened IgGs showed binding to WI-38 cells that endogenously express human FAP (data not shown). Some IgGs (including bivalent, bispecific antibodies containing a FAP-binding domain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 19) showed binding equal to or greater than that of the positive control.

[0263] [Characterization of domain specificity] FACS analysis was performed to examine the domain specificity of anti-human FAP antibodies. The same FACS assay as described above was used in this assay; however, instead of using 293FF cells transiently transfected with moFAP or huCD26, 293FF cells transiently transfected with human FAP and chicken FAP chimeric constructs were used. The chimeric constructs used were the huFAP Dom1-chFAP Dom2 chimeric construct and the chFAP Dom1-huFAP Dom2 chimeric construct. Human FAP domain 1 is a β-propeller domain, while human FAP domain 2 is an αβ-hydrolase domain.

[0264] All antibodies in the panel bound to the FAP β propeller domain, and none bound to the αβ hydrolase domain (data not shown).

[0265] [Inhibition of FAP catalytic activity] The Fluorogenic FAP Assay Kit (Bio-connect, catalog no. 80210) was used to test the ability of IgG to inhibit the catalytic activity of FAP. A two-fold dilution series of fluorescent AMC standard was made in DPP buffer as follows: 1.25 μM, 0.625 μM, 0.312 μM, 0.156 μM, 0.078 μM, and 0.039 μM (100 μl per well in a 96-well assay plate). Recombinant FAP protein (2 ng / μl, 25 μl per well) (Fluorogenic FAP Assay Kit, Bio-connect, catalog no. 80210) was added to the 96-well assay plate, followed by the addition of IgG at a single concentration of 25 μg / ml (50 μl / well). An ESC11 analog and the small molecule inhibitor talabostat (at a concentration of 1 μM) (Gentaur, GEN2327500) were included as positive controls. A negative control TT antibody was included as a negative control. The assay plate was incubated at 22°C for 15 minutes. The fluorescent substrate Ala-Pro-AMC (2.5 μM, 25 μl / well) was then added. After 6 hours of incubation at 22°C, fluorescence (produced by cleaved fluorophore AMC) was measured using a fluorescence plate reader (excitation 380 nm and emission 460 nm).

[0266] The small molecule inhibitor talabostat demonstrated potent inhibition of the catalytic activity of FAP. In this assay, none of the IgGs inhibited the catalytic activity of FAP as potently as the small molecule inhibitor talabostat (data not shown).

[0267] [Competitive ELISA] An ELISA was used to examine the competitive binding of a panel of human anti-FAP antibodies and Fab fragments generated from sibrotuzumab analogs to huFAP-His protein (R&D Systems, catalog number 3715-SE).

[0268] The huFAP-His protein was coated (0.5 μg / ml, 50 μl / well) onto two 96-well ELISA plates (Certified Nunc-Immuno Maxisorp F96, Greiner Bio-One, Catalog No. 655061). The plates were sealed (EASYseal, Greiner, Catalog No. 676001) and incubated overnight at 4°C. The plates were washed with PBS / 0.05% Tween 20 (PBS, Gibco, Catalog No. 10010-015) (Tween 20, Merck, Catalog No. 8.22184.0500) using an ELISA plate washer (BioTek 405 TS). The plates were then emptied. The Fab fragment of a sibrotuzumab analog (25 μg / ml) was incubated on one plate for 30 minutes at room temperature, while the second plate was incubated with 300 μl / well of PBS / 2% BSA blocking buffer (BSA, Sigma, catalog no. A3294-500g) for 1 hour at room temperature. The plates were then emptied. Human anti-FAP panel antibodies (0.05 μg / ml, 50 μl / well) were added, the plates were covered with EASYseal, and incubated for 1 hour at room temperature. The plates were then washed three times with wash buffer using an ELISA washer and emptied. HRP-conjugated anti-human Fc detection antibody (1:2000 dilution, Ab#0074, Bethyl labs, A80-104P) was added to the wells (50 μl / well), covered with EASYseal, and incubated for 60 minutes at room temperature. The plate was then washed three times with wash buffer using an ELISA washer and emptied. BD OptEIATMB Substrate Reagent Set (BD, Cat. No. 555214) was then added to the wells (50 μl / well) and allowed to develop for up to 10 minutes. 1M H2SO4 (50 μl / well) was then added to the wells to stop the staining reaction (color changed from blue to yellow). The plate was then measured using an ELISA plate reader (BioTek ELx808).

[0269] In the presence of the Fab fragment of the sibrotuzumab analog, the anti-FAP IgGs in the panel exhibited varying degrees of binding (data not shown). Some IgGs fully competed with the Fab of the sibrotuzumab analog for binding to the huFAP protein, including an IgG whose FAP-binding domain comprised a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 15 and an IgG whose FAP-binding domain comprised a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 19; some IgGs partially competed; and some IgGs did not compete, including an IgG whose FAP-binding domain comprised a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 11. Interestingly, the degree of competition between the IgGs and the Fab of the sibrotuzumab analog did not correlate with their affinity.

[0270] Example 3 - Binning Binning was performed using an AR2G biosensor (Pall Forte Bio, Cat. No. 18-5092) and a 96-well black microplate. In this experiment, the FAP binding domain was reformatted into an IgG1 bispecific antibody containing a second binding domain targeting TGF-βRII, as described in Example 5.

[0271] The FAP x TGF-βRII bispecific antibody, sibrotuzumab analog, and negative control TT antibody were diluted to 10 μg / ml (66.7 nM, 200 μl / well) in 1x PBS. The negative control TT antibody and acetate buffer pH 6 (used as mock immobilization) were used to identify nonspecific interference during the binning assay. Each antibody immobilized in the AR2G biosensor was incubated with 5.7 μg / ml (66.7 nM, 200 μl / well) of huFAP-His recombinant protein (R&D Systems, catalog number 3715-SE-010) and then sandwiched with each of the antibodies used for immobilization (10 μg / ml, 66.7 nM, 200 μl / well).

[0272] The binning assay was performed as follows: First, the AR2G biosensor was equilibrated by immersion in ultrapure water for 60 seconds. The sensor was then activated for 300 seconds in an activation reagent (a mixture of 20 nM EDC (Pall ForteBio, Cat. No. 18-1033) and 10 nM S-NHS (Pall ForteBio, Cat. No. 18-1067) in ultrapure water (200 μl / well). Next, the anti-huFAP:huTGF-βRII bispecific antibody was immobilized on the sensor for 1200 seconds, followed by quenching with 1 M ethanolamine pH 8.5 (Pall Forte Bio, Cat. No. 18-1071, 200 μl / well) for 300 seconds. The sensor was then immersed in ultrapure water (200 μl / well) for 120 seconds and 1× PBS (200 μl / well) for 300 seconds. Next, binding of the huFAP-His recombinant protein was carried out for 300 seconds, followed by dissociation of the sensor by immersion in 1x PBS for 600 seconds. The sensor was then immersed in regeneration buffer (10 mM glycine pH 2.5) for 5 seconds, followed by 7 cycles of 5 seconds in 1x PBS. The sensor was then immersed in 1x PBS for 120 seconds, followed by 120 seconds of binding with the huFAP-His recombinant protein. The sensor was then immersed in 1x PBS for 60 seconds, followed by 120 seconds of binding with the anti-huFAP:huTGF-βRII bispecific antibody, sibrotuzumab analog, and negative control TT antibody. Finally, the sensor was immersed in regeneration buffer (10 mM glycine pH 2.5) for 5 seconds, followed by 7 cycles of 5 seconds in 1x PBS.

[0273] The results are shown in Table 1. Sibrotuzumab analogs are included in bin G, subbin G1 (data not shown). Bivalent bispecific antibodies whose FAP-binding domains comprise heavy chain variable regions having the amino acid sequence set forth in SEQ ID NO: 19 are included in the same bin (bin G1) as sibrotuzumab analogs, and bivalent bispecific antibodies whose FAP-binding domains comprise heavy chain variable regions having the amino acid sequence set forth in SEQ ID NO: 15 or SEQ ID NO: 11 are included in bins different from sibrotuzumab analogs (bins F1 and B2, respectively).

[0274] From this data it can be concluded that a bivalent, bispecific antibody whose FAP-binding domain comprises a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 15 competes with sibrotuzumab for binding to human FAP, although binning experiments indicate that the binding of this antibody to human FAP differs from that of sibrotuzumab.

[0275] Example 4 - Selection of FAP-binding domains for use in bispecific IgG formats From the large and diverse panel of anti-human FAP bivalent monospecific IgGs generated, three FAP-binding domains were identified as being particularly useful for generating bispecific antibodies: FAP-binding domains comprising heavy chain variable regions having the amino acid sequences set forth in SEQ ID NOs: 15; 19; and 11. These three FAP-binding domains were combined with different TGF-βRII-binding domains, as described in Example 5, and the resulting FAP×TGF-βRII bispecific antibodies were screened in an in vitro mixed-culture pSMAD2 assay and in an in vivo tumor-targeting study.

[0276] JPEG2025542209000001.jpg79166

[0277] Example 5: Preparation of FAP x TGF-βRII bispecific antibody Binding domains, antibodies, and heavy chain variable regions with binding specificity for human FAP and heavy chain variable regions with binding specificity for human TGF-βRII were obtained by immunizing transgenic mice (MeMo® mice) containing the common IGKV1-39 light chain with human FAP or TGF-βRII antigenic portions (including using various forms of DNA-, protein-, and cell-based antigen delivery).

[0278] Heavy chain variable regions with binding specificity for TGF-βRII are also described in WO2021 / 133167 as SEQ ID NOs: 10-12; SEQ ID NOs: 22-91; and SEQ ID NOs: 93-96.

[0279] Heavy chain variable regions with binding specificity for human FAP, having the amino acid sequences set forth in SEQ ID NOs: 11, 15, and 19, and heavy chain variable regions with binding specificity for human TGF-βRII, having the amino acid sequences set forth in SEQ ID NOs: 23, 27, 31, and 35, were selected for production of bispecific antibodies. The binding domain sequences herein may be characterized and sequenced via the techniques provided herein and then obtained by any method known in the art.

[0280] JPEG2025542209000002.jpg56166

[0281] Bispecific IgG antibodies were generated by transiently cotransfecting two plasmid vectors, one encoding an IgG heavy chain with a FAP-binding VH region and the other encoding an IgG heavy chain with a TGF-βRII-binding VH region. The CH3 modification technique described in WO2013 / 157954 and WO2013 / 157953 was used to ensure efficient heterodimerization and bispecific antibody formation. Both vectors further encode a common light chain containing the IGKV1-39 / Jk1 light chain variable region. Cell transfection, cell culture, and antibody harvesting and purification were performed using methods known in the art. Furthermore, the CH3 modification technique described, for example, in WO2021 / 235936, can be used to ensure efficient dimerization and bispecific antibody formation.

[0282] [Antibodies with engineered effector functions:] The bispecific antibody with unmodified effector function comprises a CH2 region having the amino acid sequence set forth in SEQ ID NO: 41. An Fc-inactivated variant of the bispecific antibody was produced by introducing L235G and G236R mutations into the CH2 domain (SEQ ID NO: 42). An Fc-enhanced variant of the bispecific antibody was produced using FUT8 knockout CHO cells, which generate defucosylated antibodies (Zong H, et al. Producing defucosylated antibodies with enhanced in vitro antibody-dependent cellular cytotoxicity via FUT8 knockout CHO-S cells. Eng Life Sci. 2017 Apr 18;17(7):801-808). An Fc-enhanced variant of the analog reference FAP antibody sibrotuzumab was produced using the method described in Roy G, et. al., A novel bicistronic gene design couples stable cell line selection with a fucose switch in a designer CHO host to produce native and afucosylated glycoform antibodies, MAbs, 2018 Apr;10(3):416-430.

[0283] Example 6: Primary CAF cell lines and expression Primary cancer-associated fibroblasts (CAFs) were obtained and validated for use in characterizing the bispecific antibodies.

[0284] JPEG2025542209000003.jpg149166

[0285] CAF cells (Table 3) were obtained from BioIVT or Neuromics and shown to express both human FAP and human TGF-βRII.

[0286] huFAP was detected using a primary mouse anti-FAP antibody (R&D Systems, catalog no. MAB3715) and a secondary FITC-conjugated goat anti-mouse antibody (Jackson IR, catalog no. 115-095-062). huTGF-βRII was detected using an analog reference TGF1 antibody and a secondary Alexa fluor F647-conjugated goat anti-human antibody (Jackson IR, catalog no. 109-605 003). Cells were cultured in Pen / Strep-free DMEM + 10% FBS. On the day of the assay, cells were trypsinized, washed in growth medium, pelleted, and resuspended in FACS buffer (PBS / 1% FBS). One million cells per well were resuspended in 100 μl of primary antibody solution (in PBS / 1% FBS). Cells were stained for 30 minutes in the dark. The cells were then washed twice with FACS buffer, pelleted, and stained with a viability dye (Zombie Violet™ Fixable Viability Kit, BioLegend, Cat. No. 423114) and secondary antibody IgG for 25 minutes in the dark. The cells were washed twice with FACS buffer, pelleted, and fixed in paraformaldehyde on ice for 15 minutes. The cells were then washed in PBS, resuspended in 200 μl of PBS, and analyzed by flow cytometry. Graphs were plotted as mean fluorescence intensity (MFI).

[0287] Figure 1 shows the MFI obtained by FACS for the detection of FAP (1A) and TGF-βRII (1B). All CAFs tested were found to express both FAP and TGF-βRII. The MFI for both FAP and TGF-βRII expression was at least 2-3 times higher than the background MFI obtained with secondary antibody staining alone.

[0288] Example 7: TGF-βRII signaling inhibition assay in primary CAF cells expressing FAP and TGF-βRII The bispecific antibodies were characterized in a TGF-βRII signaling inhibition assay to determine their ability to inhibit TGF-βRII-mediated signaling. TGF-β-induced IL-11 and pSMAD2 expression were used as readouts of TGF-βRII signaling. The assay was performed using primary colonic CAFs (BioIVT) previously validated using FACS to express both FAP and TGF-βRII, as described in Example 6.

[0289] Multiple FAP x TGF-βRII bispecific antibodies were tested. The reference antibody included in the assay was the analog reference antibody TGF1 described herein.

[0290] IL-11 inhibition Primary colonic CAFs or other primary CAFs described herein were cultured in antibiotic-free DMEM + 10% FBS medium. When cells reached 80-90% confluence, they were trypsinized with TrypLE (Gibco, 12604-013), pelleted at 200 g, and washed once with DMEM + 0.2% FBS. Cells were cultured at 1 x 10 6 cells / ml (primary colonic CAFs) or 5 × 10 5 CAFs were resuspended at 10000 cells / ml (other CAFs) and seeded at 50µl / well (50,000 or 2,500 cells) in DMEM + 0.2% FBS into 96-well flat plates. Cells were preincubated with bispecific or reference antibodies for 1 hour at room temperature. Antibodies were serially diluted 5- or 8-fold and added to wells starting at 400µg / ml (final concentration 100µg / ml). After 1 hour, 100µl of 2ng / ml (2x) recombinant human TGF-β1 (R&D #7754-BH) was added to the cells (total volume 200µl / well) and incubated at 37°C for 48 or 72 hours. Plates were then centrifuged at 300g for 3 minutes, and the supernatant was assayed for IL-11 expression using the Ella platform.

[0291] pSMAD2 inhibitionPrimary colonic CAFs or other primary CAFs described herein were cultured in antibiotic-free DMEM + 10% FBS medium. When cells reached 80-90% confluence, they were trypsinized with TrypLE (Gibco, 12604-013), pelleted at 200 g, and washed once with DMEM + 0.2% FBS. Cells were resuspended and seeded at 120,000 cells / well in DMEM + 0.2% FBS. Cells were preincubated with bispecific or reference antibodies for 1 hour at room temperature. Antibodies were serially diluted 5-fold and added to wells starting at 400 μg / ml (final concentration 100 μg / ml). After 1 hour, 100 μl of 2 ng / ml (2x) recombinant human TGF-β1 was added to the wells and incubated for 2 hours. Cells were washed in PBS and resuspended in 50 μl of 1:300 diluted dead cell staining dye (Zombie Violet, BioLegend, Catalog No. 423113) in 1% FBS / PBS for 10 minutes. Cells were then washed and fixed and permeabilized using 200 μl of buffer according to the TFP protocol (Transcription Factor Phospho Buffer Set, BD Biosciences, Catalog No. 563239). Cells were stained with rabbit anti-human pSMAD2 antibody (Cell Signaling, Catalog No. 18338) for 1 hour on ice. Cells were then washed and stained with anti-rabbit IgG secondary antibody (JAX Immuno, Catalog No. 611-605-215) for 30 minutes on ice in the dark. After staining, cells were washed three times in wash buffer, resuspended in 200 μl of PBS, and acquired on a Fortessa flow cytometer.

[0292] Data were plotted in GraphPad Prism, and IC50 values ​​were calculated using nonlinear regression. Inhibition of IL-11 / pSMAD2 expression in CAFs incubated with TGF-β1 and test antibodies was compared to expression in the presence of TGF-β1 alone.

[0293] The results are shown in Table 4 and Figure 2. All bispecific antibodies inhibited TGF-βRII-mediated signaling. Figure 2A shows the % inhibition of pSMAD2 expression, and Figure 2B shows the % inhibition of IL-11 expression for some antibodies in primary colonic CAFs. The bispecific antibodies shown below are monovalent for binding to TGF-βRII and demonstrate superior inhibition of TGF-βRII signaling compared to the bivalent monospecific analog reference antibody TGF1. The range of IL-11 inhibition by the bispecific antibodies is 2-2700-fold higher when compared to the inhibition achieved by the analog reference antibody TGF1 in this assay. The range of pSMAD2 inhibition by the bispecific antibodies is 2-300-fold higher when compared to the inhibition achieved by the analog reference antibody TGF1 in this assay.

[0294] JPEG2025542209000004.jpg93166

[0295] In addition to primary colonic CAFs, inhibition of TGF-βRII signaling by bispecific antibodies was also tested in various primary CAF cell lines. Details of the various CAFs used are shown in Table 3. The FAP×TGF-βRII bispecific antibodies tested were those shown in SEQ ID NO:15×SEQ ID NO:31 and SEQ ID NO:11×SEQ ID NO:23.

[0296] The FAP x TGF-βRII bispecific antibody effectively inhibited TGF-βRII-mediated signaling expression in 10 different primary human CAF cell lines. Figure 2C shows pSMAD2 inhibition and Figure 2D shows IL-11 inhibition for CAFs derived from lung adenocarcinoma (LUAD). Table 5 shows the inhibition of IL-11 and pSMAD2 for all CAFs tested.

[0297] JPEG2025542209000005.jpg145166

[0298] Additional bispecific antibodies, designated SEQ ID NO: 15 × SEQ ID NO: 31 (Fc-inactivated), SEQ ID NO: 15 × SEQ ID NO: 31 (Fc-enhanced), SEQ ID NO: 15 × SEQ ID NO: 35 (Fc-inactivated), and SEQ ID NO: 15 × SEQ ID NO: 35 (Fc-enhanced), were tested in a TGF-βRII-mediated signaling inhibition assay from MSD (MSD, Catalog No. K151E2R-2) using IL-11 as a readout. Primary colon CAFs (BioIVT, Lot 427650A1-6037, 373853A2-6020, passage 5) were used, and the assay was performed as described above. A negative control IgG1 antibody (RSV-G) in Fc-inactivated and Fc-enhanced formats was used as the control antibody; the analog reference FAP antibody sibrotuzumab and analog reference TGF-βRII antibody TGF1 in Fc-enhanced formats were used as reference antibodies. The control antibody and the analogue reference FAP antibody sibrotuzumab did not affect IL-11 expression in primary CAFs, confirming the validity of the assay.

[0299] All bispecific antibodies effectively inhibited TGF-βRII-mediated signaling in primary colonic CAF cells, as shown in Figure 2E. Similar results were obtained in lung and additional colonic CAFs, as shown in Table 6.

[0300] JPEG2025542209000006.jpg95166

[0301] Example 8: Receptor density of huFAP and huTGF-βRII The FAP x TGF-βRII bispecific antibody was characterized in various assays to determine its ability to mediate its function in correlation with FAP expression. For this purpose, two cell lines were generated. Parental A549 cells were obtained from ATCC and designated A549 parental cells. These parental A549 cells were modified to stably express FAP (A549-FAP). + cell).

[0302] The receptor densities of huFAP and huTGF-βRII were measured in parental A549 cells and A549-FAP cells. + Cells were assessed using PE beads and flow cytometry. Both BD Quantibrite PE beads and Bangs Quantum MESF PE beads were used to quantify receptor density according to the manufacturer's instructions provided with the kit. Cells were thawed, washed, and counted using the Guava ViaCount assay on a Guava easyCyte instrument. Cells were plated evenly across all wells of an ultra-low attachment U-bottom plate for immediate antibody staining. A dead cell staining dye (BD FVS780, catalog no. 565388) was added to all samples to distinguish between live and dead cells. PE-labeled FAP and huTGF-βRII antibodies (described in Example 6) were used for cell staining. PE-labeled isotype controls (isotype mIgG1, isotype hIgG1) were also used as negative controls.

[0303] JPEG2025542209000007.jpg34166

[0304] A549 parental cells and A549-FAP + The expression levels of huFAP and huTGF-βRII for the cells are shown in Table 7. A549-FAP + The cells express higher levels of huFAP compared to the A549 parental cells, by a fold difference of approximately 4500-fold.

[0305] Example 9: Inhibition of TGF-βRII-mediated signaling in a mixed culture pSMAD2 assay The FAP x TGF-βRII bispecific antibody was characterized in a mixed culture pSMAD2 assay to determine its ability to block TGF-βRII-mediated signaling, which correlated with FAP expression. In the mixed culture pSMAD2 assay, A549 parental cells and A549-FAP cells were cultured in 10% of the control cells. +We compared the inhibition of TGF-βRII signaling by the bispecific antibody in A549 parental cells, which express human TGF-βRII but do not express or express only undetectable levels of FAP; + The cells are A549 cells that have been modified to overexpress human FAP.

[0306] A549 parental cells were cultured in DMEM + 10% FBS, and A549-FAP + (Example 8) Cells were cultured in DMEM + 10% FBS supplemented with 5 μg / mL puromycin until 80-90% confluent. Cells were trypsinized, washed three times in medium, trypsinized, and centrifuged at 200 g for 10 minutes. Cells were then washed twice in PBS. A549-FAP + Cells were labeled with 1 μM CFSE by resuspending the cells in prewarmed PBS and mixing with an equal volume of warmed 2 μM CFSE (BD Bioscience). The cells were incubated at 37°C for 20 minutes with occasional mixing. The CFSE reaction was stopped by adding chilled FBS / medium followed by centrifugation. The labeled cells were then washed three times in medium. A549 parental cells and A549-FAP + The cells were then mixed 1:1, 1.2 x 10 5 50ul of cells / well were transferred to a 96 well plate.

[0307] Antibodies were serially diluted 5-fold and added to wells at 11 concentration points, starting at 400 μg / ml (4x). 50 μl of antibody was added to the wells, mixed, and incubated at room temperature for 1 hour. 100 μl of 2 ng / ml (2x) recombinant human TGF-β1 was added to the wells and incubated for 2 hours. Cells were washed in PBS and resuspended in 50 μl of 300x diluted dead cell staining dye (Zombie Violet, BioLegend, #423113) in 1% FBS / PBS and incubated for 10 minutes. Cells were then washed and fixed and permeabilized using 200 μl of buffer according to the BD TFP protocol. Cells were stained with anti-pSMAD2 antibody (Cell Signaling, #E8F3R) on ice for 1 hour. The cells were then washed and stained with anti-rabbit IgG secondary antibody (JAX, #611-605-215) for 30 minutes on ice in the dark. After staining, the cells were washed three times in wash buffer, resuspended in 200 μl of PBS, and acquired using a Fortessa flow cytometer.

[0308] The results are shown in Table 8 and Figure 3. Table 8 shows the results of the A549 parental cells versus A549-FAP for the exemplary bispecific antibodies. + IC50 values ​​and fold difference in potency in inhibiting TGF-βRII signaling in A549-FAP cells are shown. + The fold difference in pSMAD2 inhibition by bispecific antibodies in cells is 600-19,000 times higher compared to the fold difference in A549 parental cells in this assay.

[0309] All bispecific antibodies were synthesized using A549 parental cells and A549-FAP. + The bispecific antibody inhibited TGF-βRII-mediated signaling in both A549-FAP and A549-FAP cells more than in parental A549 cells. + In cells, they were more potent at inhibiting TGF-βRII signaling, indicating that they inhibit TGF-β-induced SMAD2 signaling in a manner that correlates with FAP expression.

[0310] The results of the mixed culture pSMAD2 assay demonstrate the FAP of the bispecific antibody. + This indicates that the cell specificity is due to the FAP-binding domain, which is further supported by the in vivo tumor targeting studies described in Example 12.

[0311] JPEG2025542209000008.jpg60166

[0312] Example 10: FAP + Cell killing: ADCC assay] ADCC assays were performed to test the ability of bispecific antibodies to mediate immune effector cell killing of antibody-coated target cells via Fc receptors that recognize the constant region of the antibody.

[0313] Several bispecific antibodies were tested in the unmodified format in the ADCC assay. The technical controls for the assay were a negative control RSV-G antibody and cetuximab. The target cells were A549 parental cells and A549-FAP. + Cells were obtained as described in Example 8.

[0314] For ADCC detection, Eurofins' KILR® detection kit (#97-0001M) was used. +The cells and A549 parental cells were engineered to express a protein with an enhanced version of ProLabel (β-Gal reporter fragment). Upon target cell killing by CD16 effector cells, the reporter fragment can be detected in the supernatant using the enzyme acceptor fragment of the β-Gal enzyme. CD16 KILR effector cells were cultured in AssayComplete cell culture medium supplemented with 600 IU / ml recombinant human IL-2 according to Eurofins' protocol. Cells were fed with fresh medium and 600 IU / ml recombinant human IL-2 every two days. A549 parental cells were cultured in DMEM supplemented with 10% HI-FBS, 1x glutamine, and 500 μg / ml G418. A549-FAP + Target pool cells were cultured in DMEM supplemented with 10% HI-FBS, 1x glutamine, 5 μg / mL puromycin, and 500 μg / mL G418. + After the cells were established, they were maintained in a medium containing 500 μg / ml of G418. + Target cells, either A549 or parental A549 cells, were cultured in antibiotic-free medium for 48 hours and then harvested. Cells were washed twice with medium and resuspended in medium at a concentration of 200,000 cells / mL. Target cells were seeded in 50 μL of medium and incubated for 30 minutes. Antibodies were added to the target cells and incubated at 37°C, 5% CO2 for 30 minutes. CD16 effector cells were cultured at 1.6 × 10 6 The KILR-100001 was resuspended at a concentration of 1000 / mL and added to the target cells at an E:T ratio of 10:1. The cells were incubated at 37°C, 5% CO2 for 3 hours. KILR detection solution was added and incubated at room temperature in the dark for 1 hour. After incubation, the chemiluminescent signal was detected.

[0315] Results for exemplary antibodies are shown in Figure 4 and Table 9. The FAP x TGF-βRII bispecific antibody significantly reduced the proliferation of A549-FAP cells compared to parental A549 cells. +The FAP×TGF-βRII bispecific antibody exhibited high ADCC killing activity in cells. The ADCC activity of the FAP×TGF-βRII bispecific antibody was higher than that of cetuximab.

[0316] JPEG2025542209000009.jpg55166

[0317] Example 11: ADCC activity mediated by different Fc formats The FAP x TGF-βRII bispecific antibody was tested in Fc-inactivated, unmodified, and Fc-enhanced IgG1 formats to compare the effectiveness of different Fc backbones in mediating ADCC killing. The bispecific antibodies shown in SEQ ID NO: 15 x SEQ ID NO: 31 and SEQ ID NO: 11 x SEQ ID NO: 23 were tested for ADCC activity using primary CAF cells (melanoma CAFs and lung adenocarcinoma CAFs described in Example 6) as target cells and human NK cells as immune effector cells. The technical controls for the assay were a negative control RSV-G antibody and a positive control cetuximab, both in unmodified Fc format.

[0318] Two days before co-culture with CAFs, human NK cells were thawed, washed, and resuspended in RPMI + 10% FBS containing 50 IU / ml rhIL-2 for 2 days. On the day of co-culture, NK cells were centrifuged, washed, and resuspended at 2 × 10 in phenol red-free assay medium. 6 CAFs were cultured in T150 flasks until ~90% confluent. CAFs were washed and stained with a total of 20 ml of complete medium containing a 1:500 dilution of Nuclight Rapid Red reagent. CAFs were left to incubate overnight at 37°C, 5% CO2. The next morning, cells were washed with PBS, trypsinized, and resuspended at 1 × 10 cells / mL in phenol red-free assay medium. 5The CAFs were resuspended at 100µL / mL. On the morning of assay setup, 100µL of stained CAFs were added to each well of a 96-well flat-bottom plate. The plate was incubated at room temperature for 30 minutes, then at 37°C with 5% CO2 for 6 hours. Antibody dilutions were prepared as a 12-point concentration curve in phenol red-free medium. After 6 hours, 50µL of NK cells and 50µL of antibody dilution were added to each appropriate well. The plate was incubated at room temperature for 30 minutes. The plate was scanned at 3-hour intervals using the Incucyte instrument in phase contrast and red channels using the "adherent cell by cell module" and stopped after 15 hours. For melanoma CAFs, visually dead cells that retained red staining were counted. For lung adenocarcinoma CAFs, visually viable cells were counted. NK cells were excluded from the cell count by size filtering. The number of live or dead cells was plotted against antibody concentration, and the EC50 for live cells was calculated in GraphPad Prism. Figure 5 shows the killing activity of the antibodies as the number of rounded CAFs per well (A-C), which indicates killed CAFs that have detached from the adhesive surface, or as the number of detectable CAFs (D-F).

[0319] The results are shown in Table 10 and Figures 5A-F. The FAPxTGF-βRII bispecific antibodies shown in SEQ ID NO:15 x SEQ ID NO:31 and SEQ ID NO:11 x SEQ ID NO:23 exhibited ADCC killing activity in different primary human CAF cell lines. Both antibodies were more potent in ADCC killing when used in an Fc-enhanced format compared to an Fc-unmodified format.

[0320] JPEG2025542209000010.jpg70166

[0321] Additional ADCC reporter assays were performed using the following antibodies: SEQ ID NO: 15 x SEQ ID NO: 31 (Fc-inactivated), SEQ ID NO: 15 x SEQ ID NO: 31 (Fc-enhanced), SEQ ID NO: 15 x SEQ ID NO: 35 (Fc-inactivated), and SEQ ID NO: 15 x SEQ ID NO: 35 (Fc-enhanced). Negative control RSV-G antibodies in Fc-enhanced and Fc-inactivated formats were used as reference antibodies.

[0322] The Promega ADCC Reporter Bioassay Kit (Cat. No. G7018) was used, and the assay was performed according to the manufacturer's protocol. Briefly, colonic CAFs were plated at 20,000 cells per well in 100 μL per well of a 96-well plate and incubated overnight. The following day, 25 μL of assay buffer per well was added. Antibodies were then added at 25 μL per well in a 1:5 serial dilution starting at 10 μg / ml, resulting in the following concentrations (μg / ml): 10, 2, 0.4, 0.08, 0.016, 0.0032, 0.00064, 0.000128, and 0.0000256. FcyRIIIa effector cells were thawed and immediately diluted at 7.5 × 10 in 25 μL per well. 4 Cells were added one at a time to the CAFs. The plates were covered and incubated at 37°C in a humidified CO2 incubator for 6 hours. The plates were then equilibrated to ambient temperature for 15 minutes. Bio-Glo Luciferase Assay Reagent was added at 75 μl / well. The plates were incubated for 20 minutes. Luminescence was measured using a plate reader. Fold of induction was calculated as follows: Fold induction = RLU (induction - background) / RLU (no antibody control - background)

[0323] The results are shown in Figure 5G and Table 11. The negative control antibodies RSV-G (Fc-enhanced) and RSV-G (Fc-inactivated) did not mediate ADCC killing of primary CAFs, confirming the validity of the assay. The Fc-enhanced bispecific antibody mediated ADCC killing more effectively than the Fc-inactivated bispecific antibody.

[0324] JPEG2025542209000011.jpg32166

[0325] Example 12: FAP x TGF-βRII antibody-mediated in vivo tumor targeting The bispecific antibodies were characterized in vivo in the NSG mouse model to determine their ability to selectively localize and inhibit TGF-βRII-mediated signaling in tumor cells expressing both FAP and TGF-βRII. To this end, NSG mice were transfected with either A549 parental cells or A549-FAP. + Mice were inoculated with either tumor cells or the bispecific antibody and treated with the bispecific antibody. The model was validated using the following antibodies: a negative control RSV-G antibody, an experimental control FAP × TGF-βRII antibody, and an analog reference antibody TGF1 (data not shown).

[0326] Several bispecific antibodies were tested in Fc-inactivated or Fc-unmodified formats.

[0327] Approximately 5 million A549 parental cells or A549-FAP + Cells were resuspended in 200 ul of a 1:1 mixture of PBS and Matrigel (VWR, Cat. No. 47743-706) and inoculated into the flanks of NSG mice. After tumor formation, mice were randomized based on body weight into the following treatment groups: 1) Negative control RSV-G antibody 10 mg / kg 2) Analogue reference TGF1 antibody 10 mg / kg 3) SEQ ID NO: 19 × SEQ ID NO: 27 1 mg / kg 4) SEQ ID NO: 19 x SEQ ID NO: 27 10 mg / kg 5) SEQ ID NO: 11 × SEQ ID NO: 23 1 mg / kg 6) SEQ ID NO: 11 × SEQ ID NO: 23 10 mg / kg 7) SEQ ID NO: 15 × SEQ ID NO: 31 1 mg / kg 8) SEQ ID NO: 15 × SEQ ID NO: 31 10 mg / kg

[0328] Bispecific, control, and reference antibodies were administered on days 17 and 20 after cell inoculation. Mice were dosed on days 3 and 4 after inoculation and sacrificed on day 5 for tumor harvest.

[0329] Tumors were harvested and placed on ice. Single cells were generated in a 50 ml Falcon tube by squeezing the tumor through a 100-micron filter using a 3 ml syringe plunger. The filter was rinsed with DMEM + 10% FBS, and the single cells were cast into the Falcon tube. The cells were then pelleted by centrifugation at 300 g for 10 minutes. The cell pellet was washed once with 1% FBS / PBS FACS medium and counted. One to two million cells were transferred to a 96-well plate and stained in 100 μl of diluted surface primary antibody solution in FACS buffer, protected from light, for 30 minutes on ice.

[0330] For IL-11 staining, cells were incubated in medium containing 1x monensin (eBioscience™, catalog number 00-4505-51, ThermoFisher Scientific) for 5 hours before FACS staining. Cells were then washed three times by centrifugation in FACS buffer. For pSMAD2 staining (Cell Signaling, catalog number E8F3R), cells were washed, fixed, and permeabilized using 200 ul of buffer according to the BD TFP protocol (catalog number 563239). For IL-11 staining (ThermoFisher, catalog number 551691AP), cells were fixed / permeabilized using 200 ul of buffer according to the eBioscience Foxp3 protocol (ThermoFisher, catalog number 00-5523-00). For human IgG staining, Jackson Laboratories antibody (catalog number 109-605 003) was used. Cells were then resuspended in 200 μl of 1:400 diluted primary antibody solution and stained for 1 hour on ice in the dark. Cells were washed twice with wash buffer and then stained with 1:400 diluted secondary antibody for 30 minutes on ice in the dark. After staining, cells were washed three times in wash buffer, resuspended in 200 μl of PBS, and acquired on a BD Fortessa flow cytometer.

[0331] The results are shown in Figure 6. Data for pSMAD2 staining are not shown.

[0332] Figure 6A shows A549 parental cells versus A549-FAP. + Figure 1 shows IgG staining of tumor cells isolated from inoculated mice. All FAP x TGF-βRII bispecific antibodies, when administered at 1 mg / ml (mpk) and 10 mg / ml (mpk), significantly reduced the tumor size of A549-FAP compared to A549 parental tumors. + It localized preferentially to the tumor, in contrast to the analogous reference TGF1 antibody.

[0333] Figure 6B shows the inhibition of TGF-βRII-induced IL-11 expression in tumor cells. + In mice inoculated with tumor cells, A549-FAP effectively reduced IL-11 levels compared with parental A549 cells. The extent of IL-11 inhibition was significantly greater in A549-FAP than in parental A549 cells. + A549-FAP in tumor-bearing mice treated with bispecific antibodies + This was greater than the IL-11 inhibition in tumor-bearing mice treated with the analog reference TGF1 antibody TGF1. The negative control RSV-G antibody did not suppress IL-11 expression in this assay.

[0334] Figure 6C shows inhibition of TGF-βRII-induced pSMAD2 expression in tumor cells. + In mice inoculated with tumor cells, pSMAD2 levels were effectively reduced compared with parental A549 cells. The extent of pSMAD2 inhibition was significantly reduced in A549-FAP cells. + A549-FAP in tumor-bearing mice treated with bispecific antibodies + This was compared to the pSMAD2 inhibition in tumor-bearing mice treated with the analog reference TGF1 antibody TGF1. The negative control RSV-G antibody did not suppress pSMAD2 expression in this assay.

[0335] Taken together, these results demonstrate that the FAP×TGF-βRII bispecific antibody selectively localizes to tumors expressing both FAP and TGF-βRII in an in vivo mouse model and inhibits TGF-βRII-mediated signaling in a manner that correlates with FAP expression.

[0336] Thus, bispecific antibodies comprising a FAP-binding domain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 15, SEQ ID NO: 19, or SEQ ID NO: 11 exhibit selective functional activity against cells that express both FAP and TGF-βRII, compared to cells that express TGF-βRII but only express undetectable levels of FAP. Thus, these FAP-binding domains and their variants are useful for the development of FAP×TGF-βRII bispecific antibodies for the treatment of cancer.

[0337] Example 13: FAP x TGF-βRII antibody-mediated in vivo tumor efficacy The bispecific antibody is A549-FAP + It was characterized in vivo in a tumor-bearing athymic nude mouse model to determine its efficacy in reducing tumor volume.

[0338] Female BALB / c nu / nu mice (5–7 weeks old, Charles River Laboratories or Taconic Biosciences) received 1 × 10 IgG in 0.2 mL of sterile PBS. 7 tumor cells (A549-FAP + Cells (described in Example 8) and matrigel (BD Biosciences, #354234) were inoculated into the flank subcutaneously. Treatment of tumor-bearing mice was initiated 12 or 13 days after cell inoculation, when the mean tumor volume reached 135 mm. 3 or 143mm 3 The study began with 2 weeks of treatment. The study treatment was administered to mice via intraperitoneal injection (IP). Cetuximab (Erbitux, NDC: 66733-958-23; Lot No. C2100112) was purchased from RefDrug. The treatment frequency in this study was twice weekly for 7 or 5 weeks. The treatment groups are shown in Table 12.

[0339] JPEG2025542209000012.jpg144166

[0340] Subcutaneous tumor size was measured twice weekly using digital calipers. Tumor volume was calculated by measuring the tumor in two dimensions and using the following formula: volume = [length × (width)]. 2 )] / 2, where the larger value was the length and the smaller value was the width. The effect on tumor growth was reported as the percentage tumor growth inhibition (%TGI), which was calculated using the following formula: (1-(Tx volume / control volume)) x 100, where control volume is the administration vehicle or untreated tumor. No significant weight loss was observed in any treatment group (data not shown).

[0341] The results are shown in Figure 7. The bispecific antibody SEQ ID NO: 15 x SEQ ID NO: 31 induced an anti-tumor response comparable to that induced by the positive control antibody cetuximab (Figure 7B). 3 mg / kg of the bispecific antibody SEQ ID NO: 15 x SEQ ID NO: 31 (Fc-enhanced) was equally effective in inducing an anti-tumor response as 30 mg / kg of the antibody SEQ ID NO: 15 x SEQ ID NO: 31 (non-Fc-modified) (Figure 7B). The bispecific antibody SEQ ID NO: 15 x SEQ ID NO: 35 (Fc-inactivated) induced greater in vivo tumor regression compared to untreated mice (Figure 7C).

[0342] Example 14: Binding affinity The binding affinity of a bispecific antibody comprising a FAP-binding domain according to SEQ ID NO: 15 and a TGF-βRII-binding domain according to SEQ ID NO: 31 was determined for binding to human FAP and human TGF-βRII. SPR experiments were performed on a Biacore T200 controlled by T200 control software.

[0343] All test antibodies were captured by an anti-human IgG antibody (Biacore®) immobilized on a CM5 chip surface, followed by the addition of human TGF-βRII (R&D, Cat. No. 241-R2 / CF) or human FAP (R&D, Cat. No. 3715-SE). Measurements were performed at 25°C. Binding data were analyzed using double-reference subtraction (0 concentration and reference flow cell 1 with no antibody captured) using Biacore T200 Evaluation Software.

[0344] The results for exemplary antibodies tested are shown in Table 13. The affinity of the FAP-binding domain in the FAP×TGF-βRII bispecific antibody is in the range of 0.1-0.2 nM. The affinity of the TGF-βRII binding domain in the FAP×TGF-βRII bispecific antibody is in the range of 3.8-5 nM. As shown in Table 13, the affinity of the TGF-βRII binding domain is in the range of 25-50 times lower than the affinity of the FAP-binding domain.

[0345] JPEG2025542209000013.jpg113166

[0346] Example 15: TGF-β reporter assay - trans TGF-βRII inhibition assay The bispecific antibodies were characterized in a TGF-β reporter assay to determine their ability to inhibit TGF-βRII-mediated signaling by trans-activation (trans-binding) in HEK-Blue-TGF-βRII reporter cells (HEK-Blue cells). The assay was performed using co-cultures of MRC5 cells obtained from ATCC (#CCL171) and confirmed to express FAP and TGF-βRII, with HEK-Blue cells purchased from InVivogen (#hkb-tgfbv2) known to express TGF-βRII.

[0347] Recombinant human TGF-β1 (rhTGF-β1; R&D, #240-B) was prepared in duplicate at an initial concentration of 40 ng / ml (2x) with two-fold serial dilutions in medium containing 0.1% FBS. Eighty percent confluent HEK-Blue and MRC-5 cells were trypsinized, washed twice, and diluted to 2 × 10 6 Cells were resuspended at 100 μl / ml, mixed 1:1, and transferred at 25 μl (25,000 cells) per well. Serially diluted bispecific antibodies were added to the cell mixture, mixed gently, and incubated at room temperature for 2 hours. Next, 100 μl of rhTGF-β1 dilutions (2 ng / mL) were transferred to the cells, mixed gently, and cultured at 37°C for 24 hours. Next, Quanti-Blue™ substrate was transferred to a flat 96-well plate along with 40 μl of cell culture supernatant, mixed thoroughly, and incubated at 37°C for 1 hour. Secreted alkaline phosphatase (SEAP) levels in the supernatant were measured spectrophotometrically at optical density (OD) at 650 nm.

[0348] The results are shown in Table 14 and Figure 10 and are expressed as EC50. The reference antibody used was a negative control RSV-G antibody. The FAPxTGF-βRII bispecific antibodies set forth in SEQ ID NO: 15 x SEQ ID NO: 31 and SEQ ID NO: 11 x SEQ ID NO: 23 inhibited TGF-βRII signaling in HEK-Blue-TGF-βRII reporter cells when these cells were incubated with MRC5 cells expressing FAP. The FAPxTGF-βRII bispecific antibody inhibited TGF-βRII signaling to a greater extent than the control RSV-GxTGF-βRII antibody comprising a heavy chain having the amino acid sequence set forth in SEQ ID NO: 79 and a heavy chain having the amino acid sequence set forth in SEQ ID NO: 31 or 23. These results indicate that the FAPxTGF-βRII bispecific antibody binds to HEK-Blue cells via the TGF-βRII binding domain and to MRC5 cells via the FAP binding domain, inhibiting TGF-βRII-mediated signaling in HEK-Blue cells.

[0349] JPEG2025542209000014.jpg71166

[0350] Example 16: moFAP x huTGF-βRII antibody-mediated antitumor efficacy as a single agent and in combination with a PD-1 inhibitor The bispecific antibody was characterized in vivo in a transgenic mouse model harboring immune cells expressing human TGF-βRII and tumors expressing murine FAP (moFAP), and its ability to inhibit TGF-βRII signaling in non-fibroblast cells through transactivation was determined. To this end, CD34 + Humanized NSG mice were inoculated with MDA-MB-231 tumor cells and administered the bispecific antibody or a reference antibody.

[0351] The bispecific antibody (moFAP x TGF-βRII) tested for transactivation activity was produced in an Fc-inactivated format according to methods known in the art and comprises a murine FAP binding domain (moFAP) and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 52, and a TGF-βRII binding domain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 31 or SEQ ID NO: 23 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 52. The bispecific antibodies are designated moFAP x SEQ ID NO: 31 and moFAP x SEQ ID NO: 23. The reference antibodies used were a negative control RSV-G antibody and an analog reference TGF-βRII antibody TGF1.

[0352] female CD34 + Humanized mice (JAX) were inoculated with approximately 3 million MDA-MB-231 cells in 50% Matrigel. Seven days after tumor inoculation, mice were randomized based on tumor volume and stem cell donor and received intraperitoneal injections of the indicated agents twice weekly. Mice received a total of seven antibody injections.

[0353] TGI (tumor growth inhibition rate) was calculated from the mean tumor volume on day 24 using the formula (1-(VT / VC)) x 100, where VT is the tumor volume of the treatment group on the last day of treatment, and VC is the tumor volume of the control group on the last day of treatment. Statistical analysis was performed using two-way analysis of variance (ANOVA) with Dunnett's multiple comparison test.

[0354] In addition to its ability to inhibit TGF-βRII signaling in T cells, the moFAP×TGF-βRII bispecific antibody was also characterized for its ability to inhibit the PD-1 / PD-L1 axis in T cells. This was achieved by combining the moFAP×TGF-βRII bispecific antibody with the anti-PD-1 antibody pembrolizumab. Details regarding treatment groups and dosing are shown in Table 15.

[0355] JPEG2025542209000015.jpg126166

[0356] The results are shown in Figure 8. When administered as single agents, the moFAP x TGF-βRII bispecific antibody in treatment groups 3, 5, and 6 induced anti-tumor responses comparable to those induced by the analog reference TGF1 antibody (group 2). With single-agent treatment, all groups except group 4 showed significant reductions in tumor volume compared to the negative control RSV-G antibody (p<0.05).

[0357] The results for the combination treatment are shown in Figure 9. When tested in combination with pembrolizumab, the moFAP x TGF-βRII bispecific antibody resulted in a reduction in tumor volume that was comparable to that achieved with the analog reference TGF1 antibody and pembrolizumab alone (Groups 2 and 7, respectively), and in combination (Group 8).

[0358] It was noted that the bispecific antibody, monovalent for binding to TGF-βRII, induced similar anti-tumor responses at the same dose as the bivalent monospecific analog reference antibody TGF1 in both single-agent and combination treatment studies.

[0359] Example 17: Macrophage effector function assay The FAP x TGF-βRII bispecific antibody was tested for its ability to mediate macrophage effector function in an antibody-dependent cellular phagocytosis (ADCP) assay. The bispecific antibody shown in SEQ ID NO: 15 x SEQ ID NO: 35 was used in both Fc-inactivated and Fc-enhanced formats to mediate macrophage effector function in A549-FAP cells as target cells. + ADCP activity was tested using cells or lung CAFs and M0 / M2c macrophages as effector cells. An isotype IgG1 control antibody served as a negative control for the assay. A549-FAP + It was confirmed that cells and lung CAFs express both TGF-βRII and FAP proteins (data not shown).

[0360] Macrophage differentiation protocol: Human peripheral blood monocytes were differentiated into M0 / M2c macrophages as follows: CD16-undepleted monocytes (CD14+CD16+) were isolated from human peripheral blood using the EasySep Human Monocyte Isolation Kit (Stemcell technologies, Cat. No. 19058) according to the manufacturer's instructions and resuspended in XVIVO10 medium (Lonza, Cat. No. BEBP02-055Q) containing 10% FBS. Cells were counted using a cellometer and plated at 15 x 10 cells per plate in two 150 mm Petri dishes. 6The cells were plated in 30 mL of medium and maintained at 37°C in a 5% CO2 incubator with M-CSF (25 ng / mL, R&D systems, catalog no. 216-MC-025 / CF) for 3 days (72 hours). On day 4, after half of the medium was replaced, IL-10 (10 ng / mL; R&D systems, catalog no. 217-IL / CF) was added to one Petri dish for 48 hours to differentiate into M2c macrophages, while the other Petri dish was added with the same volume of medium without IL-10 to differentiate into M0 macrophages. After washing the Petri dish with sterile PBS, the macrophages were harvested by incubating with Accutase™ (20 mL; Millipore, catalog no. SCR005) for 10 minutes in a 37°C incubator. The cells were counted and used for staining with macrophage marker antibodies and ADCP assay.

[0361] Differentiated macrophages were stained with live / dead Fix Aqua dye (100 μL of a 1:1000 dilution in 1x DPBS; Invitrogen, Catalog No. L34957) for 15 minutes at room temperature. Cells were washed with BSA staining buffer (300 μL; BD, Catalog No. 554657) by centrifugation at 500 × g for 5 minutes at room temperature, and the supernatant was removed. 100 μL of Fc block (1:20 in staining buffer) was added and incubated for 10 minutes at room temperature, followed by another wash step with BSA staining buffer. 50 μL of an antibody mixture containing CD11b BC421, CD163 APC, CD206 BUV737, CD80 BV650, HLA-DR PerCP Cy5.5, PD1 PE, and TGFβR2 was added to the sample tube and incubated on ice for 30 minutes. Flow cytometry minus one (FMO) controls for CD163, CD206, CD80, HLADR, PD1, and TGF-βRII were included for interpretation of flow cytometry data. Cells were washed with BSA staining buffer (300 μL), centrifuged at 500 × g for 5 minutes at room temperature, and resuspended in 300 μL of BSA staining buffer. Data acquisition was performed on an LSR Fortessa X-20.

[0362] M2c macrophages were confirmed to specifically express M1 / M2 markers. M2c macrophages also expressed TGF-βRII but not FAP (data not shown).

[0363] FAP×TGF-βRII bispecific antibody A549-FAP + The ability to mediate macrophage effector functions on cells was tested by flow cytometry, and on lung CAFs by IncuCyte live cell imaging.

[0364] ADCP assay: target cells (A549-FAP + Cells and lung CAFs) were trypsinized, washed, and cultured at 1 × 10 6 The cells were resuspended at 1000 cells / mL and labeled with 2 μM CFSE (Invitrogen, Cat. No. C34554) according to the manufacturer's instructions. 50 μL of labeled target cells were added to a 96-well U-bottom polypropylene plate at a predetermined concentration (a cell concentration of 1 × 10 cells was required to achieve 50,000 target cells in 50 μL). 6 100 μL of the bispecific antibody (SEQ ID NO: 15 × SEQ ID NO: 35) (both Fc-inactivated and Fc-enhanced formats) and isotype control antibody were added at concentrations ranging from 10 μg / mL to 0.001 μg / mL (10-fold dilutions) and incubated at room temperature for 30 minutes. A no-antibody control (0 μg / mL) was also included. Macrophages harvested as described above were resuspended at the indicated concentrations in 50 μL / well and added to a 96-well U-bottom polypropylene plate (effector:target cell ratio of 1:1 for all target cell types). A549-FAP + Cells and lung CAFs were tested at both 1:1 and 3:1 effector:target cell ratios.

[0365] Flow cytometry: The cell / antibody suspension was mixed and centrifuged at 800 rpm for 3 minutes to concentrate the cells to the bottom. The plate was incubated at 37°C, 5% CO2 for 24 hours and then washed by centrifugation at 500 x g for 5 minutes. The cell pellet was stained with live dead aqua dye (100 μL of a 1:1000 dilution in 1x DPBS) and incubated at room temperature for 15 minutes. 100 μL of BSA staining buffer was added, and the cells were centrifuged at 500 x g for 5 minutes at room temperature. The supernatant was removed, and 100 μL of Fc block (1:20 in staining buffer) was added and incubated at room temperature for 10 minutes. 300 μL of BSA staining buffer was added again, and the cells were centrifuged at 500 x g for 5 minutes at room temperature, and the supernatant was removed. Cells were stained with 50 μL of anti-CD11b APC-Cy7 (2 μL antibody + 48 μL BSA staining buffer; BD Biosciences, catalog no. 557754) and kept on ice for 30 minutes. Cells were washed again with 300 μL of BSA staining buffer, centrifuged at 500 × g for 5 minutes at room temperature, and the supernatant was removed. Data acquisition was performed on an LSR Fortessa X-20.

[0366] Live-cell imaging: Macrophages were labeled with 3 μM Cell Tracker Red CMPTX dye (Invitrogen, catalog no. C34552) according to the manufacturer's recommendations, and a mixture of effector and target cells (based on a predetermined ratio) was added to a 96-well polystyrene flat-bottom plate. The cell / antibody suspension was mixed, and the plate was centrifuged at 800 rpm for 3 minutes to concentrate the cells at the bottom. The plate was then placed in an IncuCyte live-cell imaging incubator (Leica microsystems) for data acquisition.

[0367] The results are shown in Figure 11. The FAPxTGF-βRII Fc-enhanced antibody inhibited A549-FAP compared to the FAPxTGF-βRII Fc-inactivated antibody at both effector:target cell ratios of 1:1 and 3:1. +A dose-dependent increase in phagocytosis of A549-FAP cells was observed (Figure 11A), as measured by flow cytometry. Lung CAF killing by M2c macrophages was also observed with the Fc-enhanced FAP x TGF-βRII bispecific antibody (Figure 11B), but not with the Fc-inactivated FAP x TGF-βRII bispecific antibody, as measured by IncuCyte live-cell imaging. A negative control IgG1 isotype antibody significantly increased the phagocytosis of A549-FAP cells (Figure 11B). + or did not mediate ADCP killing of pulmonary CAFs.

[0368] [Sequence table] SEQ ID NO: 1 Heavy chain reference anti-TGF-βRII antibody TGF1 analog QLQVQESGPGLVKPSETLSLTCTVSGGSISNSYFSWGWIRQPPGKGLEWIGSFYYGEKTYYNPSLKSRATISIDTSKSQFSLKLSSVTAADTAVYYCPRGPTMIRGVIDSWG QGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCD KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0369] SEQ ID NO: 2 Light chain reference anti-TGF-βRII antibody TGF1 analog EIVLTQSPATLSLSPGERATLSCRASQSVRSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0370] SEQ ID NO: 3 Heavy chain negative control RSV IgG1 antibody EVQLVESGGGVVQPGRSLRLSCAASGFTFSNYGMHWVRQAPGKGLEWVAVISYDGSTKYSADSLKGRFTISRDNSKNTLYLQMNSLRADDTTAVYYCAKEGWSFDSSGYRSWFD SWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKS CDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPI EKTISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0371] SEQ ID NO: 4 Light chain DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0372] SEQ ID NO: 5 Heavy chain negative control RSV IgG1 antibody EVQLVESGGGVVQPGRSLRLSCAASGFTFSNYGMHWVRQAPGKGLEWVAVISYDGSTKYSADSLKGRFTISRDNSKNTLYLQMNSLRADDTTAVYYCAKEGWSFDSSGYRSWFD SWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKS CDKTHTCPPCPAPELGRGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPI EKTISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0373] SEQ ID NO: 6 huFAP MKTWVKIVFGVATSAVLALLVMCIVLRPSRVHNSEENTMRALTLKDILNGTFSYKTFFPNWISGQEYLHQSADNNIVLYNIETGQSYTILSNRTMKSVNASNYGLSPDRQFVYLESDYSKLWRYSYTATYYIYDLSNGEFVRGNELPRPIQYLCWSPVGSKLAYVYQNNIYLKQRPGDPPFQITFNGRENKIFNGIPDWVYEEEMLAT KYALWWSjpgKFLAYAEFNDTDIPVIAYSYYGDEQYPRTINIPYPKAGAKNPVVRIFIIDTTYPAYVGPQEVPVPAMIASSDYYFSWLTWVTDERVCLQWLKRVQNVSVLSICDFREDWQTWDCPKTQEHIEESRTGWAGGFFVSTPVFSYDAISYYKIFSDKDGYKHIHYIKDTVENAIQITS GKWEAINIFRVTQDSLFYSNEFEEYPGRRNIYRISIGSYPPSKKCVTCHLRKERCQYYTASFSDYAKYYALVCYGPGIPISTLHDGRTDQEIKILEENKELENALKNIQLPKEEIKKLEVDEITLWYKMILPPQFDRSKKYPLLIQVYGGPCSQSVRSVFAVNWISYLASKEGMVIALVDGRG TAFQGDKLLYAVYRKLGVYEVEDQITAVRKFIEMGFIDEKRIAIWGWSYGGYVSSLALASGTGLFKCGIAVAPVSSWEYYASVYTERFMGLPTKDDNLEHYKNSTVMARAEYFRNVDYLLIHGTADDNVHFQNSAQIAKALVNAQVDFQAMWYSDQNHGLSGLSTNHLYTHMTHFLKQCFSLSD

[0374] SEQ ID NO: 7 Heavy chain analogue reference FAP antibody sibrotuzumab QVQLVQSGAEVKKPGASVKVSCKTSRYTFTEYTIHWVRQAPGQRLEWIGGINPNNGIPNYNQKFKGRVTITVDTSASTAYMELSSLRSEDTAVYYCARRRIAYGYDEGHAMDY WGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0375] SEQ ID NO: 8 Light chain analogue reference FAP antibody sibrotuzumab DIVMTQSPDSLAVSLGERATINCKSSQSLLYSRNQKNYLAWYQQKPGQPPKLLIFWASTRESGVPDRFSGSGFGTDFTLTISSLQAEDVAVYYCQQYFSYPLTFGQGTKV EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0376] SEQ ID NO: 9 cyFAP MKTWVKIVFGVATSAVLALLVMCIVLRPPRVHNSEENTMRALTLKDILNGTFSYKTFFPNWISGQEYLHQSADNNIVLYNIETGQSYTILSNRTMKSVNASNYGLSPDRQFVYLESDYSKLWRYSYTATYYIYDLSNGEFVRGNELPRPIQYLCWSPVGSKLAYVYQNNIYLKQRPGDPPFQITFNGRENKIFNGIPDWVYEEEMLAT KYALWWSjpgKFLAYAEFNDTDIPVIAYSYYGDEQYPRTINIPYPKAGAKNPFVRIFIIDTTYPAYVGPQEVPVPAMIASSDYYFSWLTWVTDERVCLQWLKRVQNVSVLSICDFREDWQTWDCPKTQEHIEESRTGWAGGFFVSTPVFSYDAISYYKIFSDKDGYKHIHYIKDTVENAIQITS GKWEAINIFRVTQDSLFYSSNEFEDYPGRRNIYRISIGSYPPSKKCVTCHLRKERCQYYTASFSDYAKYYALVCYGPGIPISTLHDGRTDQEIKILEENKELENALKNIQLPKEEIKKLEVDEITLWYKMILPPQFDRSKKYPLLIQVYGGPCSQSVRSVFAVNWISYLASKEGMVIALVDGRG TAFQGDKLLYAVYRKLGVYEVEDQITAVRKFIEMGFIDEKRIAIWGWSYGGYVSSLALASGTGLFKCGIAVAPVSSWEYYASVYTERFMGLPTKDDNLEHYKNSTVMARAEYFRNVDYLLIHGTADDNVHFQNSAQIAKALVNAQVDFQAMWYSDQNHGLSGLSTNHLYTHMTHFLKQCFSLSD

[0377] SEQ ID NO: 10 huCD26 MKTPWKVLLGLLGAAALVTIITVPVVLLNKGTDDATADSRKTYTLTDYLKNTYRLKLYSLRWISDHEYLYKQENNILVFNAEYGNSSVFLENSTFDEFGHSINDYSISPDGQFILLEYNYVKQWRHSYTASYDIYDLNKRQLITEERIPNNTQWVTWSPVGHKLAYVWNNDIYVKIEPNLPSYRITWTGKE DIIYNGITDWVYEEEVFSAYSALWWSjpgTFLAYAQFNDTEVPLIEYSFYSDESLQYPKTVRVPYPKAGAVNPTVKFFVVNTDSLSSVTNATSIQITAPASMLIGDHYLCDVTWATQERISLQWLRRIQNYSVMDICDYDESSGRWNCLVARQHIEMSTTGWVGRFRPSEPHFTLDGNSFYKIISNEEGYRH ICYFQIDKKDCTFITKGTWEVIGIEALTSDYLYYISNEYKGMPGGRNLYKIQLSDYTKVTCLSCELNPERCQYYSVSFSKEAKYYQLRCSGPGLPLYTLHSSVNDKGLRVLEDNSALDKMLQNVQMPSKKLDFIILNETKFWYQMILPPHFDKSKKYPLLLDVYAGPCSQKADTVFRLNWATYLASTENII VASFDGRGSGYQGDKIMHAINRRLGTFEVEDQIEAARQFSKMGFVDNKRIAIWGWSYGGYVTSMVLGSGSGVFKCGIAVAPVSRWEYYDSVYTERYMGLPTPEDNLDHYRNSTVMSRAENFKQVEYLLIHGTADDNVHFQQSAQISKALVDVGVDFQAMWYTDEDHGIASSTAHQHIYTHMSHFIKQCFSLP

[0378] SEQ ID NO: 11 Heavy chain variable region EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMIWVRQAPGKGLEWVSAISGSGGYTFNADSVKGRFTMSRDNSKNTLYLQMNSLRAEDTAVYYCAKRDGGYEGGAFDIWGQGTLVTVSS

[0379] SEQ ID NO: 12 Heavy chain CDR1 SYAMI

[0380] SEQ ID NO: 13 Heavy chain CDR2 AISGSGGYTFNADSVKG

[0381] SEQ ID NO: 14 Heavy chain CDR3 RDGGYEGGAFDI

[0382] SEQ ID NO: 15 Heavy chain variable region QLQLQESGPGLVKPSETLSLTCTVSGGSISSSSYFWAWIRQPPGKGLEFIGNIYYSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARPSYYGSGNYYFAFWGQGTLVTVSS

[0383] SEQ ID NO: 16 Heavy chain CDR1 SSSYFWA

[0384] SEQ ID NO: 17 Heavy chain CDR2 NIYYSGSTYYNPSLKS

[0385] SEQ ID NO: 18 Heavy chain CDR3 PSYYGSGNYYFAF

[0386] SEQ ID NO: 19 Heavy chain variable region EVQLVESGGGLVKPGGSLRLSCAASGFPFNYAWMSWVRQAPGKGLEWVGRIKPKTSGGATDYAAPVKDRFTISRDSRNTLYLQMNSLKTEDTAVYYCSAREDIWNFFFDLWGQGTLVTVSS

[0387] SEQ ID NO: 20 Heavy chain CDR1 YAWMS

[0388] SEQ ID NO: 21 Heavy chain CDR2 RIKPKTSGGATDYAAPVKD

[0389] SEQ ID NO: 22 Heavy chain CDR3 REDIWNFFFDL

[0390] SEQ ID NO: 23 Heavy chain variable region QVQLVESGGGLVQPGGSLRLSCAVSGFTFRRYAMSWVRQAPGKGLEWVSAISASGDRTHNTDSVKGRFSISRDNSKNTLYLQMNSLRAEDTAVYFCAKGIAASGKNYFDPWGQGTLVTVSS

[0391] SEQ ID NO: 24 Heavy chain CDR1 RYAMS

[0392] SEQ ID NO: 25 Heavy chain CDR2 AISASGDRTHNTDSVKG

[0393] SEQ ID NO: 26 Heavy chain CDR3 GIAASGKNYFDP

[0394] SEQ ID NO: 27 Heavy chain variable region QVQLVESGGGLVQPGGSLRLSCAVSGFTFSRYAMSWVRQAPGKGLEWVSAISASGDRTKNTDSVKGRFSISRDNSKNTLYLQMNSLRAEDTAVYFCAKGTAAAGKNYFDPWGQGTLVTVSS

[0395] SEQ ID NO: 28 Light chain V region EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSP

[0396] SEQ ID NO: 29 Heavy chain CDR2 AISASGDRTKNTDSVKG

[0397] SEQ ID NO: 30 Heavy chain CDR3 GTAAAGKNYFDP

[0398] SEQ ID NO: 31 Heavy chain variable region QVQLVESGGGLVQPGGSLRLSCAVSGFTFERYAMSWVRQAPGKGLEWVSAISASGDRTQNTDSVKGRFSISRDNSKNTLYLQMNSLRAEDTAVYFCAKGTAASGRNYFDPWGQGTLVTVSS

[0399] SEQ ID NO: 32 Light chain variable region SYVLTQPPSVSVAPGETARITCGGDNIGRKSVYWYQQKSGQAPVLVIYYDSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDGSSDHWVFGGGTKLTVL

[0400] SEQ ID NO: 33 Heavy chain CDR2 AISASGDRTQNTDSVKG

[0401] SEQ ID NO: 34 Heavy chain CDR3 GTAASGRNYFDP

[0402] SEQ ID NO: 35 Heavy chain variable region QVQLVESGGGLVQPGGSLRLSCAVSGFTFERYAMSWVRQAPGKGLEWVSAISASGDRTQYTDSVKGRFSISRDNSKNTLYLQMNSLRAEDTAVYFCAKGTAASGRNYFDPWGQGTLVTVSS

[0403] SEQ ID NO: 36 LCDR1 shown according to IMGT NIGRKS

[0404] SEQ ID NO: 37 Heavy chain CDR2 AISASGDRTQYTDSVKG

[0405] SEQ ID NO: 38 LCDR2 shown according to IMGT YDS

[0406] SEQ ID NO: 39 CH1 region ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRV

[0407] Sequence number 40: Hinge EPKSCDKTHTCPPCP

[0408] SEQ ID NO: 41 CH2 region Unmodified effector function APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK

[0409] SEQ ID NO: 42 CH2-DM region APELGRGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK

[0410] SEQ ID NO: 43 CH3 region GQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0411] SEQ ID NO: 44 CH3-DE region GQPREPQVYTDPPSREEMTKNQVSLTCEVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0412] SEQ ID NO: 45 CH3-KK region GQPREPQVYTKPPSREEMTKNQVSLKCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0413] SEQ ID NO: 46 Human TGF-βRII isoform A MGRGLLRGLWPLHIVLWTRIASTIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSD ECNDNIIFSEEYNTSNPDLLLVIFQVTGISLLPPLGVAISVIIIFYCYRVNRQQKLSSTWETGKTRKLMEFSEHCAIILEDDRSDISSTCANNINHNTELLPIELDTLVGKGRFAEVYKAKLKQNTSEQFETVAVKIFPYEE YASWKTEKDIFSDINLKHENILQFLTAEERKTELGKQYWLITAFHAKGNLQEYLTRHVISWEDLRKLGSSLARGIAHLHSDHTPCGRPKMPIVHRDLKSSNILVKNDLTCCLCDFGLSLRLDPTLSVDDLANSGQVGTARYM APEVLESRMNLENVESFKQTDVYSMALVLWEMTSRCNAVGEVKDYEPPFGSKVREHPCVESMKDNVLRDRGRPEIPSFWLNHQGIQMVCETLTECWDHDPEARLTAQCVAERFSELEHLDRLSGRSCSEEKIPEDGSLNTTK

[0414] SEQ ID NO: 47 Extracellular domain of human TGF-βRII isoform A TIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDLLLVIFQ

[0415] SEQ ID NO: 48 Human TGF-βRII isoform B MGRGLLRGLWPLHIVLWTRIASTIPPHVQKSDVEMEAQKDEIICPSCNRTAHPLRHINNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIM KEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDLLLVIFQVTGISLLPPLGVAISVIIIFYCYRVNRQQKLSSTWETGKTRKLMEFSEHCAIILEDDRSDISSTCANNINHNTELLPIELDTLVGKGRFAEVYKAKLKQNTSEQF ETVAVKIFPYEEYASWKTEKDIFSDINLKHENILQFLTAEERKTELGKQYWLITAFHAKGNLQEYLTRHVISWEDLRKLGSSLARGIAHLHSDHTPCGRPKMPIVHRDLKSSNILVKNDLTCCLCDFGLSLRLDPTLSVDDLANSGQV GTARYMAPEVLESRMNLENVESFKQTDVYSMALVLWEMTSRCNAVGEVKDYEPPFGSKVREHPCVESMKDNVLRDRGRPEIPSFWLNHQGIQMVCETLTECWDHDPEARLTAQCVAERFSELEHLDRLSGRSCSEEKIPEDGSLNTTK

[0416] SEQ ID NO: 49 Extracellular domain of isoform B of human TGF-βRII TIPPHVQKSDVEMEAQKDEIICPSCNRTAHPLRHINNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDLLLVIFQ

[0417] SEQ ID NO: 50 huPD-1 MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLjpgRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRV TERRAEVPTAHPSPSPRPAGQFQTLVVGVVGGLLGSLVLLVWVLAVICSRAARGTIGARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTPEPPVPCVPEQTEYATIVFPSGMGTSPARRGSADGPRSAQPLRPEDGHCSWPL

[0418] SEQ ID NO: 51 Light chain constant region RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0419] SEQ ID NO: 52 Light chain variable region DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSR FSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPTFGQGTKVEIK

[0420] SEQ ID NO: 53 LCDR1 shown according to IMGT QSISSY

[0421] SEQ ID NO: 54 LCDR2 shown according to IMGT AAS

[0422] SEQ ID NO: 55 LCDR3 shown according to IMGT QQSYSTPPT

[0423] SEQ ID NO: 56 Light chain variable region DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPITFGQGTRLEIK

[0424] SEQ ID NO: 57 LCDR3 shown according to IMGT QVWDGSSDHWV

[0425] SEQ ID NO: 58 Light chain V region SYVLTQPPSVSVAPGETARITCGGDNIGRKSVYWYQQKSGQAPVLVIYYDSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDGSSDH

[0426] SEQ ID NO: 59 LCDR3 shown according to IMGT QQSYSTPPIT

[0427] SEQ ID NO: 60 Light chain V region DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTP

[0428] SEQ ID NO: 61 Light chain variable region EIVMTQSPATLSVSPGERATLSCRASQSVSSNLAWYQQKPGQAPRLLIYGASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYNNWPWTFGQGTKVEIK

[0429] SEQ ID NO: 62 LCDR1 shown according to IMGT QSVSSN

[0430] SEQ ID NO: 63 LCDR2 shown according to IMGT GAS

[0431] LCDR3 shown according to SEQ ID NO: 64 IMGT QQYNNWPWT

[0432] SEQ ID NO: 65 Light chain V region EIVMTQSPATLSVSPGERATLSCRASQSVSSNLAWYQQKPGQAPRLLIYGASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYNNWP

[0433] SEQ ID NO: 66 Light chain variable region EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPD RFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPWTFGQGTKVEIK

[0434] SEQ ID NO: 67 LCDR1 shown according to IMGT QSVSSSY

[0435] SEQ ID NO: 68 LCDR3 shown according to IMGT QQYGSSPWT

[0436] SEQ ID NO: 69 - Heavy chain variable region EVQLVETGGGLIQPGGSLRLSCAASGFTVSSNYMSWVRQAPGKGLEWVSVIYSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGPTDAYPYLDYLWGQGTLVTVSS

[0437] SEQ ID NO: 70 - Heavy chain CDR1 SNYMS

[0438] SEQ ID NO: 71 - Heavy chain CDR2 VIYSGGSTYYADSVKG

[0439] SEQ ID NO: 72 - Heavy chain CDR3 GPTDAYPYLDYL

[0440] SEQ ID NO: 73 - Mouse FAP MKTWLKTVFGVTTLAALALVVICIV LRPSRVYKPEGNTKRALTLKDILNGTFSYKTYFPNWISEQEYLHQSEDDNIVFYNIETRESYIILSNSTMKSVNATDYGLSPDRQFVYLESDYSKLWRYSYTATYYIYDLQNGEFVRGYELPRPIQYLCWSPVGSKLAYVYQNNIYLKQRPGDPPFQITYTGRENRIFNGIPDWVYEEEMLATK YALWWSPDGKFLAYVEFNDSDIPIIAYSYYGDGQYPRTINIPYPKAGAKNPVVRVFIVDTTYPHHVGPMEVPVPEMIASSDYYFSWLTWVSSERVCLQWLKRVQNVSVLSICDFREDWHAWECPKNQEHVEESRTGWAGGFFVSTPAFSQDATSYYKIFSDKDGYKHIHYIKDTVENAIQITSG KWEAIYIFRVTQDSLFYSSNEFEGYPGRRNIYRISIGNSPPSKKCVTCHLRKERCQYYTASFSYKAKYYALVCYGPGLPISTLHDGRTDQEIQVLEENKELENSLRNIQLPKVEIKKLKDGGLTFWYKMILPPQFDRSKKYPLLIQVYGGPCSQSVKSVFAVNWITYLASKEGIVIALVDGRGT AFQGDKFLHAVYRKLGVYEVEDQLTAVRKFIEMGFIDEERIAIWGWSYGGYVSSLALASGTGLFKCGIAVAPVSSWEYYASIYSERFMGLPTKDDNLEHYKNSTVMARAEYFRNVDYLLIHGTADDNVHFQNSAQIAKALVNAQVDFQAMWYSDQNHGISSGRSQNHLYTHMTHFLKQCFSLSD

[0441] SEQ ID NO:74 - Heavy chain ESC11 analog QVQLQESGPGLVKPSETLSLTCTVSGGSISSNNYYWGWIRQTPGKGLEWIGSIYYSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARGARWQARPATRIDGV AFDIWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEP KSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPI EKTISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0442] SEQ ID NO:75 - Light chain ESC11 analog EIVLTQSPGTLSLSPGERATLSCRASQTVTRNYLAWYQQKPGQAPRLLMYGASNRAAGVPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQFGSPYTFGQGTVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0443] SEQ ID NO: 76 - Heavy chain IgG1 hu / moFAP EVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEWMGWISAYNGNTNYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARDWSRSGYYLPDYWG QGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCD KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0444] SEQ ID NO:77 - Light chain IgG1 hu / moFAP DVVMTQSPLSLPVTLGQPASISCRSSQSLLHSNGYNYLDWYLQRPGQSPHLLIFLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGIYYCMQALQTPPTFGQGTKV EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0445] SEQ ID NO: 78 - Heavy chain IgG1 negative control TT EVQLVETGAEVKKPGASVKVSCKASDYIFTKYDINWVRQAPGQGLEWMGWMSANTGNTGYAQKFQGRVTMTRDTSINTAYMELSSLTSGDTAVYFCARSSLFKTETAPYYHFAL DVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPK SCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPI EKTISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0446] SEQ ID NO: 79 - Heavy chain variable region negative control RSV IgG1 antibody EVQLVESGGGVVQPGRSLRLSCAASGFTFSNYGMHWVRQAPGKGLEWVAVISYDGSTKYSADSLKGRFTISRDNSKNTLYLQMNSLRADDTAVYYCAKEGWSFDSSGYRSWFDSWGQGTLVTVSS

[0447] [Provisions] 1. A bispecific binding moiety comprising a FAP-binding domain and a TGF-βRII-binding domain, the TGF-βRII binding domain blocks TGF-βRII from binding to a TGF-βRII ligand; Bispecific binding moieties.

[0448] 2. A bispecific binding moiety as described in clause 1, the bispecific binding moiety blocks TGF-βRII-mediated signaling in cells expressing FAP and TGF-βRII; Bispecific binding moieties.

[0449] 3. A bispecific binding moiety according to any of the preceding clauses, wherein the potency of the bispecific binding moiety in blocking TGF-βRII-mediated signaling is 2.0 to 500 times greater than the potency of a reference anti-TGF-βRII antibody in cells expressing FAP and TGF-βRII; the reference anti-TGF-βRII antibody is a bivalent monospecific antibody comprising a heavy chain having the amino acid sequence set forth in SEQ ID NO: 1 and a light chain having the amino acid sequence set forth in SEQ ID NO: 2; Bispecific binding moieties.

[0450] 4. A bispecific binding moiety according to any of the preceding clauses, wherein the blockade of TGF-βRII-mediated signaling is measured as a decrease in pSMAD2 expression in a TGF-βRII signaling inhibition assay, Bispecific binding moieties.

[0451] 5. A bispecific binding moiety according to any of the preceding clauses, The cells expressing FAP and TGF-βRII are human fibroblasts. Bispecific binding moieties.

[0452] 6. A bispecific binding moiety according to any of the preceding clauses, The cells expressing FAP and TGF-βRII are primary cancer-associated fibroblasts (CAFs); Bispecific binding moieties.

[0453] 7. A bispecific binding moiety according to any of the preceding clauses, the bispecific binding moiety has greater potency in blocking TGF-βRII-mediated signaling in cells that express a FAP and TGF-βRII than in cells that express TGF-βRII and do not express, or express only undetectable levels of, a FAP; Bispecific binding moieties.

[0454] 8. A bispecific binding moiety according to any of the preceding clauses, The cells expressing FAP and TGF-βRII are A549-FAP + cells, and The cells that express TGF-βRII and do not express or express FAP at undetectable levels are A549 parental cells. Bispecific binding moieties.

[0455] 9. A bispecific binding moiety according to any of the preceding clauses, wherein said efficacy in blocking TGF-βRII-mediated signaling is measured as a reduction in pSMAD2 levels in a mixed culture pSMAD2 assay. Bispecific binding moieties.

[0456] 10. A bispecific binding moiety according to any of the preceding clauses, wherein the potency of the bispecific binding moiety in blocking TGF-βRII-mediated signaling in cells expressing a FAP and TGF-βRII is 100 to 20,000 times greater than in cells expressing TGF-βRII and not expressing or expressing only undetectable levels of a FAP; Bispecific binding moieties.

[0457] 11. A bispecific binding moiety according to any of the preceding clauses, the FAP-binding domain binds to a FAP expressed on a first cell; and the TGF-βRII binding domain binds to TGF-βRII expressed on a second cell; Bispecific binding moieties.

[0458] 12. A bispecific binding moiety according to any of the preceding clauses, when the FAP-binding domain binds to a FAP expressed on the first cell and the TGF-βRII-binding domain binds to a TGF-βRII expressed on the second cell, the TGF-βRII-binding domain blocks TGF-βRII-mediated signaling in the second cell. Bispecific binding moieties.

[0459] 13. A bispecific binding moiety according to any of the preceding clauses, the first cell is a fibroblast; Bispecific binding moieties.

[0460] 14. A bispecific binding moiety according to any of the preceding clauses, the second cell is a non-fibroblast cell; Bispecific binding moieties.

[0461] 15. A bispecific binding moiety according to any of the preceding clauses, the second cell is an immune effector cell or a tumor cell. Bispecific binding moieties.

[0462] 16. A bispecific binding moiety according to any of the preceding clauses, the second cell is a T cell; Bispecific binding moieties.

[0463] 17. A bispecific binding moiety according to any of the preceding clauses, The blockage of TGF-βRII-mediated signaling in the second cell is measured in a TGF-β reporter assay described in Example 15. Bispecific binding moieties.

[0464] 18. A bispecific binding moiety according to any of the preceding clauses, the first cells used in the TGF-β reporter assay are MRC5 cells expressing FAP; and the second cell is a HEK-Blue cell expressing TGF-βRII; Bispecific binding moieties.

[0465] 19. A bispecific binding moiety according to any of the preceding clauses, the affinity of the FAP-binding domain for human FAP is 25 to 50 times higher than the affinity of the TGF-βRII-binding domain for human TGF-βRII; Bispecific binding moieties.

[0466] 20. A bispecific binding moiety according to any of the preceding clauses, the affinity of the FAP-binding domain is 0.1 to 0.2 nM; and The affinity of the TGF-βRII binding domain is 3.8 to 5 nM. Bispecific binding moieties.

[0467] 21. A bispecific binding moiety according to any of the preceding clauses, The affinity is determined by the equilibrium dissociation constant K D is determined as Bispecific binding moieties.

[0468] 22. A bispecific binding moiety according to any of the preceding clauses, The FAP binding domain comprises a heavy chain variable region comprising: heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 14, respectively; heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18, respectively; or heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 22, respectively; wherein each of said HDCR1 or HCDR2 may contain at most three, two, or one conservative amino acid mutations; Bispecific binding moieties.

[0469] 23. A bispecific binding moiety according to any of the preceding clauses, the FAP-binding domain comprises a heavy chain variable region having an amino acid sequence set forth in any one of SEQ ID NOs: 11, 15, or 19, or having at least 80%, 85%, 90%, or 95% sequence identity thereto within the framework regions; Bispecific binding moieties.

[0470] 24. A bispecific binding moiety according to any of the preceding clauses, The FAP-binding domain a light chain variable region comprising a light chain CDR1 (LCDR1), a light chain CDR2 (LCDR2), and a light chain CDR3 (LCDR3) having the amino acid sequences set forth in SEQ ID NO: 53, SEQ ID NO: 54, and SEQ ID NO: 55, respectively; or variants thereof, which contain at most three, two, or one amino acid mutation in each LCDR; Including, Bispecific binding moieties.

[0471] 25. A bispecific binding moiety according to any of the preceding clauses, the FAP-binding domain comprises a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 52, or having at least 80%, 85%, 90%, or 95% sequence identity thereto; Bispecific binding moieties.

[0472] 26. A bispecific binding moiety according to any of the preceding clauses, The TGF-βRII binding domain comprises a heavy chain variable region comprising: heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 24, SEQ ID NO: 25, and SEQ ID NO: 26, respectively; heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 24, SEQ ID NO: 29, and SEQ ID NO: 30, respectively; heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 24, SEQ ID NO: 33, and SEQ ID NO: 34, respectively; or heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO:24, SEQ ID NO:37, and SEQ ID NO:34, respectively; wherein each of said HDCR1 or HCDR2 may contain at most three, two, or one conservative amino acid mutations; Bispecific binding moieties.

[0473] 27. A bispecific binding moiety according to any of the preceding clauses, The TGF-βRII binding domain comprises a heavy chain variable region having an amino acid sequence set forth in any one of SEQ ID NOs: 23, 27, 31, or 35, or having at least 80%, 85%, 90%, or 95% sequence identity thereto within the framework regions; Bispecific binding moieties.

[0474] 28. A bispecific binding moiety according to any of the preceding clauses, The TGF-βRII binding domain is a light chain variable region comprising a light chain CDR1 (LCDR1), a light chain CDR2 (LCDR2), and a light chain CDR3 (LCDR3) having the amino acid sequences set forth in SEQ ID NO: 53, SEQ ID NO: 54, and SEQ ID NO: 55, respectively; or variants thereof, which contain at most three, two, or one amino acid mutation in each LCDR; Including, Bispecific binding moieties.

[0475] 29. A bispecific binding moiety according to any of the preceding clauses, the TGF-βRII binding domain comprises a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 52, or having at least 80%, 85%, 90%, or 95% sequence identity thereto; Bispecific binding moieties.

[0476] 30. A bispecific binding moiety according to any of the preceding clauses, The TGF-βRII binding domain comprises a heavy chain variable region comprising: heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 24, SEQ ID NO: 25, and SEQ ID NO: 26, respectively; heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 24, SEQ ID NO: 29, and SEQ ID NO: 30, respectively; heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 24, SEQ ID NO: 33, and SEQ ID NO: 34, respectively; or heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO:24, SEQ ID NO:37, and SEQ ID NO:34, respectively; wherein each of said HDCR1 or HCDR2 may contain at most three, two, or one conservative amino acid mutations; Bispecific binding moieties.

[0477] 31. A bispecific binding moiety according to any of the preceding clauses, The TGF-βRII binding domain comprises a heavy chain variable region having an amino acid sequence set forth in any one of SEQ ID NOs: 23, 27, 31, or 35, or having at least 80%, 85%, 90%, or 95% sequence identity thereto within the framework regions; Bispecific binding moieties.

[0478] 32. A bispecific binding moiety according to any of the preceding clauses, The TGF-βRII binding domain is a light chain variable region comprising a light chain CDR1 (LCDR1), a light chain CDR2 (LCDR2), and a light chain CDR3 (LCDR3) having the amino acid sequences set forth in SEQ ID NO: 53, SEQ ID NO: 54, and SEQ ID NO: 55, respectively; or variants thereof, which contain at most three, two, or one amino acid mutation in each LCDR; Including, Bispecific binding moieties.

[0479] 33. A bispecific binding moiety according to any of the preceding clauses, the TGF-βRII binding domain comprises a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 52, or having at least 80%, 85%, 90%, or 95% sequence identity thereto; Bispecific binding moieties.

[0480] 34. A bispecific binding moiety according to any of the preceding clauses, The FAP binding domain comprises a heavy chain variable region comprising: heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 14, respectively; heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18, respectively; or heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 22, respectively; wherein each of said HDCRs may contain at most three, two, or one conservative amino acid mutation. Bispecific binding moieties.

[0481] 35. A bispecific binding moiety according to any of the preceding clauses, the FAP-binding domain comprises a heavy chain variable region having an amino acid sequence set forth in any one of SEQ ID NOs: 11, 15, or 19, or having at least 80%, 85%, 90%, or 95% sequence identity thereto within the framework regions; Bispecific binding moieties.

[0482] 36. A bispecific binding moiety according to any of the preceding clauses, The FAP-binding domain a light chain variable region comprising a light chain CDR1 (LCDR1), a light chain CDR2 (LCDR2), and a light chain CDR3 (LCDR3) having the amino acid sequences set forth in SEQ ID NO: 53, SEQ ID NO: 54, and SEQ ID NO: 55, respectively; or variants thereof, which contain at most three, two, or one amino acid mutation in each LCDR; Including, Bispecific binding moieties.

[0483] 37. A bispecific binding moiety according to any of the preceding clauses, the FAP-binding domain comprises a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 52, or having at least 80%, 85%, 90%, or 95% sequence identity thereto; Bispecific binding moieties.

[0484] 38. A bispecific binding moiety according to any of the preceding clauses, the bispecific binding moiety comprises a Fab domain that binds to a FAP, a Fab domain that binds to TGF-βRII, and an Fc region; Bispecific binding moieties.

[0485] 39. A bispecific binding moiety according to any of the preceding clauses, the Fc region has enhanced or reduced immune effector function; Bispecific binding moieties.

[0486] 40. A bispecific binding moiety according to any of the preceding clauses, The Fc region has an enhanced immune cell effector function, specifically, an enhanced ADCC activity. Bispecific binding moieties.

[0487] 41. A bispecific binding moiety according to any of the preceding clauses, the bispecific binding moiety is defucosylated; Bispecific binding moieties.

[0488] 42. A bispecific binding moiety according to any of the preceding clauses, the Fc region has reduced immune cell effector function, specifically reduced ADCC and / or ADCP activity; Bispecific binding moieties.

[0489] 43. A bispecific binding moiety according to any of the preceding clauses, the Fc region has a L235G and / or G236R mutation in the CH2 domain (EU numbering); Bispecific binding moieties.

[0490] 44. A bispecific binding moiety according to any of the preceding clauses, the bispecific binding moiety is a bispecific antibody; Bispecific binding moieties.

[0491] 45. A method of treating a rheumatoid arthritis (HVA) comprising administering to a subject a therapeutically effective amount of a bispecific binding moiety as described in any of the preceding clauses and a pharmaceutically acceptable carrier; Pharmaceutical compositions.

[0492] 46. ​​A bispecific binding moiety or pharmaceutical composition according to any of the preceding clauses for use in therapy.

[0493] 47. A bispecific binding moiety or pharmaceutical composition according to any of the preceding clauses for use in treating cancer.

[0494] 48. A bispecific binding moiety described in any of the preceding clauses, and a second binding moiety that binds to PD-1, for use in therapy.

[0495] 49. A bispecific binding moiety according to any of the preceding clauses, and a second binding moiety that binds to PD-1, for use in treating cancer.

[0496] 50. A bispecific binding moiety according to any of the preceding clauses for use in therapy, the treatment further comprises administration of a PD-1 inhibitor. Bispecific binding moieties.

[0497] 51. A bispecific binding moiety according to any of the preceding clauses for use in treating cancer, the treatment further comprises administration of a PD-1 inhibitor. Bispecific binding moieties.

[0498] 52. A bispecific binding moiety according to any of the preceding clauses, wherein the second binding moiety that binds to PD-1 is pembrolizumab. Bispecific binding moieties.

[0499] 53. A method for treating a disease in a subject, comprising: administering to said subject in need thereof a therapeutically effective amount of the bispecific binding moiety or pharmaceutical composition of any one of the preceding clauses. method.

[0500] 54. A method for treating cancer in a subject, comprising: administering to said subject in need thereof a therapeutically effective amount of the bispecific binding moiety or pharmaceutical composition of any one of the preceding clauses. method.

[0501] 55. A method of treatment as described in any one of the preceding clauses, comprising: The method further comprises administering an effective amount of a second binding moiety that binds to PD-1. method.

[0502] 56. A method of treatment as described in any of the preceding clauses, wherein the second binding moiety that binds to PD-1 is pembrolizumab. method.

[0503] 57. A nucleic acid sequence encoding a heavy chain variable region described in any one of the preceding clauses.

[0504] 58. A vector comprising a nucleic acid sequence described in any one of the preceding clauses.

[0505] 59. A vector according to any one of the preceding clauses, The vector further comprises a nucleic acid sequence encoding a CH1 region, and preferably a hinge, CH2 and CH3 region. vector.

[0506] 60. A vector according to any one of the preceding clauses, The vector further comprises at least one nucleic acid sequence encoding a light chain variable region, and preferably a CL region. vector.

[0507] 61. A vector as described in any one of the preceding clauses, The light chain variable region is a light chain variable region capable of pairing with multiple heavy chains having different epitope specificities. vector.

[0508] 62. A nucleic acid sequence encoding a heavy chain variable region of a FAP binding domain described in any one of the preceding clauses, and a heavy chain variable region of a TGF-βRII binding domain described in any one of the preceding clauses, cell.

[0509] 63. A cell described in any of the preceding clauses: the one or more nucleic acids further encode a CH1 region, and preferably a hinge, CH2 and CH3 region; cell.

[0510] 64. A cell described in any of the preceding clauses: the one or more nucleic acids further encode a light chain variable region, particularly a light chain variable region as described in any one of the preceding clauses, and preferably a CL region; cell.

[0511] 65. A cell that produces a bispecific binding moiety described in any one of the preceding clauses.

[0512] 66. Competes with a bispecific binding moiety described in any of the preceding clauses for binding to FAP and TGF-βRII; Bispecific binding moieties.

[0513] 67. A polypeptide comprising: - a polypeptide comprising a heavy chain CDR1 (HCDR1) having the amino acid sequence set forth in SEQ ID NO: 16, a heavy chain CDR2 (HCDR2) having the amino acid sequence set forth in SEQ ID NO: 17, and a heavy chain CDR3 (HCDR3) having the amino acid sequence set forth in SEQ ID NO: 18; - a polypeptide comprising a heavy chain CDR1 (HCDR1) having the amino acid sequence set forth in SEQ ID NO: 20, a heavy chain CDR2 (HCDR2) having the amino acid sequence set forth in SEQ ID NO: 21, and a heavy chain CDR3 (HCDR3) having the amino acid sequence set forth in SEQ ID NO: 22; - a polypeptide comprising a heavy chain CDR1 (HCDR1) having the amino acid sequence set forth in SEQ ID NO: 12, a heavy chain CDR2 (HCDR2) having the amino acid sequence set forth in SEQ ID NO: 13, and a heavy chain CDR3 (HCDR3) having the amino acid sequence set forth in SEQ ID NO: 14; or - a polypeptide comprising a heavy chain CDR1 (HCDR1) having the amino acid sequence set forth in SEQ ID NO: 70, a heavy chain CDR2 (HCDR2) having the amino acid sequence set forth in SEQ ID NO: 71, and a heavy chain CDR3 (HCDR3) having the amino acid sequence set forth in SEQ ID NO: 72; Selected from: Polypeptide.

[0514] 68. A polypeptide according to clause 67, The polypeptide comprises a heavy chain variable region having an amino acid sequence set forth in SEQ ID NO: 15; 19; 11; 69, or having at least 80%, or at least 85%, or at least 90%, or at least 95% sequence identity thereto; Polypeptide.

[0515] 69. A polypeptide according to clause 67 or 68, Further comprising a CH1 region, Polypeptide.

[0516] 70. A FAP-binding domain comprising a polypeptide according to any one of clauses 67 to 69.

[0517] 71. A FAP-binding domain according to clause 70, The FAP-binding domain further comprises a polypeptide comprising a light chain CDR1 (LCDR1), a light chain CDR2 (LCDR2), and a light chain CDR3 (LCDR3) having the amino acid sequences set forth in SEQ ID NO: 53, SEQ ID NO: 54, and SEQ ID NO: 55, respectively; FAP-binding domain.

[0518] 72. A FAP-binding domain according to clause 70 or 71, The FAP-binding domain comprises a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 52, or having at least 80%, or at least 85%, or at least 90%, or at least 95% sequence identity thereto; FAP-binding domain.

[0519] 73. A FAP-binding domain according to any one of clauses 70 to 72, Further comprising a CL region, FAP-binding domain.

[0520] 74. FAP-binding domain that binds to human and mouse FAP.

[0521] 75. A FAP-binding domain according to clause 74, The FAP-binding domain comprises a polypeptide comprising a heavy chain CDR1 (HCDR1) having the amino acid sequence set forth in SEQ ID NO: 70, a heavy chain CDR2 (HCDR2) having the amino acid sequence set forth in SEQ ID NO: 71, and a heavy chain CDR3 (HCDR3) having the amino acid sequence set forth in SEQ ID NO: 72, FAP-binding domain.

[0522] 76. A FAP-binding domain according to clause 75, The polypeptide comprises a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 69, or having at least 80%, or at least 85%, or at least 90%, or at least 95% sequence identity thereto; FAP-binding domain.

[0523] 77. A FAP-binding domain according to clause 75 or 76, the polypeptide further comprises a CH1 region. FAP-binding domain.

[0524] 78. A FAP-binding domain according to any one of clauses 75 to 77, The FAP-binding domain further comprises a polypeptide comprising a light chain CDR1 (LCDR1), a light chain CDR2 (LCDR2), and a light chain CDR3 (LCDR3) having the amino acid sequences set forth in SEQ ID NO: 53, SEQ ID NO: 54, and SEQ ID NO: 55, respectively, or variants thereof; FAP-binding domain.

[0525] 79. A FAP-binding domain according to clause 78, The FAP-binding domain comprises a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 52, or having at least 80%, or at least 85%, or at least 90%, or at least 95% sequence identity thereto; FAP-binding domain.

[0526] 80. A FAP-binding domain according to any one of clauses 75 to 79, Further comprising a CL region, FAP-binding domain.

[0527] 81. A binding moiety comprising a polypeptide according to any one of clauses 67-69 or a FAP binding domain according to any one of clauses 70-80.

[0528] 82. A connecting part as described in Article 81, the binding moiety is a monospecific binding moiety, in particular a bivalent monospecific antibody; joining part.

[0529] 83. A method of treating a leukemia comprising administering to a subject an effective amount of a polypeptide according to any one of clauses 67-69, or a FAP binding domain according to any one of clauses 70-80, or a binding moiety according to clause 81 or 82, and a pharmaceutically acceptable carrier; Pharmaceutical compositions.

[0530] 84. A polypeptide according to any one of clauses 67 to 69, or a FAP binding domain according to any one of clauses 70 to 80, or a binding moiety according to clause 81 or 82, or a pharmaceutical composition according to clause 83, for use in therapy.

[0531] 85. A polypeptide according to any one of clauses 67 to 69, or a FAP binding domain according to any one of clauses 70 to 80, or a binding moiety according to clause 81 or 82, or a pharmaceutical composition according to clause 83, for use in the treatment of cancer.

[0532] 86. A method for treating a disease, comprising: administering to an individual in need thereof an effective amount of a polypeptide according to any one of clauses 67 to 69, or a FAP binding domain according to any one of clauses 70 to 80, or a binding moiety according to clause 81 or 82, or a pharmaceutical composition according to clause 83, method.

[0533] 87. A method for treating cancer, comprising: administering to an individual in need thereof an effective amount of a polypeptide according to any one of clauses 67 to 69, or a FAP binding domain according to any one of clauses 70 to 80, or a binding moiety according to clause 81 or 82, or a pharmaceutical composition according to clause 83, method.

[0534] 88. A nucleic acid comprising a sequence encoding a polypeptide according to any one of clauses 67 to 69.

[0535] 89. A vector comprising a nucleic acid as described in clause 88.

[0536] 90. A vector as described in Article 89, The vector further comprises a nucleic acid sequence encoding a CH1 region, and preferably a hinge, CH2 and CH3 region. vector.

[0537] 91. A vector as described in clause 89 or 90, The vector further comprises at least one nucleic acid sequence encoding a light chain variable region, and preferably a CL region. vector.

[0538] 92. A vector as described in Article 91, the light chain variable region is a light chain variable region comprising the light chain CDRs described in clause 78, or is a light chain variable region described in clause 79. vector.

[0539] 93. A cell comprising a nucleic acid according to clause 88 or a vector according to any one of clauses 89 to 92.

[0540] 94. Cells as described in article 93, the cell further comprises a nucleic acid comprising a sequence encoding a CH1 region, and preferably a hinge, CH2 and CH3 region; cell.

[0541] 95. Cells as described in clauses 93 or 94, the cells further comprise at least one nucleic acid comprising a sequence encoding a light chain variable region, and preferably a CL region; cell.

[0542] 96. A cell producing a polypeptide according to any one of clauses 67-69, or a FAP binding domain according to any one of clauses 70-80, or a binding moiety according to clause 81 or 82.

[0543] 97. Cells as described in article 96, the cell is a recombinant cell comprising a vector according to any one of clauses 89 to 92; cell.

[0544] 98. A bispecific binding moiety according to any of the preceding clauses, Here, the Fc region has immune cell effector function, specifically ADCP activity. Bispecific binding moieties.

Claims

1. A bispecific binding moiety comprising a FAP-binding domain and a TGF-βRII-binding domain, the TGF-βRII binding domain blocks TGF-βRII from binding to a TGF-βRII ligand; Bispecific binding moieties.

2. 2. A bispecific binding moiety according to claim 1, the bispecific binding moiety blocks TGF-βRII-mediated signaling in cells expressing FAP and TGF-βRII; Bispecific binding moieties.

3. 3. A bispecific binding moiety according to claim 2, wherein the potency of the bispecific binding moiety in blocking TGF-βRII-mediated signaling is 2.0 to 500 times greater than the potency of a reference anti-TGF-βRII antibody in cells expressing FAP and TGF-βRII; the reference anti-TGF-βRII antibody is a bivalent, monospecific antibody comprising a heavy chain having the amino acid sequence set forth in SEQ ID NO: 1 and a light chain having the amino acid sequence set forth in SEQ ID NO: 2; Bispecific binding moieties.

4. 4. A bispecific binding moiety according to claim 2 or 3, The cells expressing FAP and TGF-βRII are fibroblasts. Bispecific binding moieties.

5. A bispecific binding moiety according to any one of claims 2 to 4, The cells expressing FAP and TGF-βRII are primary cancer-associated fibroblasts (CAFs). Bispecific binding moieties.

6. A bispecific binding moiety according to any one of claims 2 to 5, wherein the blockade of TGF-βRII-mediated signaling is measured as a reduction in pSMAD2 expression in a TGF-βRII signaling inhibition assay. Bispecific binding moieties.

7. 7. A bispecific binding moiety according to any one of claims 1 to 6, the bispecific binding moiety has greater potency in blocking TGF-βRII-mediated signaling in cells that express FAP and TGF-βRII than in cells that express TGF-βRII and do not express, or express only undetectable levels of, FAP; The cells expressing FAP and TGF-βRII are A549-FAP ＋ cells, and The cells that express TGF-βRII and do not express or express only undetectable levels of FAP are A549 parental cells. Bispecific binding moieties.

8. 8. A bispecific binding moiety according to claim 7, wherein said efficacy in blocking TGF-βRII-mediated signaling is measured as a reduction in pSMAD2 levels in a mixed culture pSMAD2 assay. Bispecific binding moieties.

9. 9. A bispecific binding moiety according to claim 7 or 8, wherein the potency of the bispecific binding moiety in blocking TGF-βRII-mediated signaling in cells expressing FAP and TGF-βRII is about 100 to 20,000 times greater than in cells expressing TGF-βRII and no or only undetectable levels of FAP; Bispecific binding moieties.

10. A bispecific binding moiety comprising a FAP-binding domain and a TGF-βRII-binding domain, the FAP-binding domain binds to FAP expressed on a first cell; and the TGF-βRII binding domain binds to TGF-βRII expressed on a second cell; Bispecific binding moieties.

11. 11. A bispecific binding moiety according to claim 10, when the FAP-binding domain binds to FAP expressed on the first cell and the TGF-βRII-binding domain binds to TGF-βRII expressed on the second cell, the TGF-βRII-binding domain blocks TGF-βRII-mediated signaling in the second cell. Bispecific binding moieties.

12. 12. A bispecific binding moiety according to claim 10 or 11, the first cell is a fibroblast; Bispecific binding moieties.

13. 13. A bispecific binding moiety according to any one of claims 10 to 12, the second cell is a non-fibroblast cell; Bispecific binding moieties.

14. 14. A bispecific binding moiety according to claim 13, the second cell is an immune effector cell or a tumor cell. Bispecific binding moieties.

15. 15. A bispecific binding moiety according to claim 14, the second cell is a T cell; Bispecific binding moieties.

16. 16. A bispecific binding moiety according to any one of claims 11 to 15, The blockage of TGF-βRII-mediated signaling in the second cell is measured in a TGF-β reporter assay. Bispecific binding moieties.

17. 17. A bispecific binding moiety according to claim 16, the first cells used in the TGF-β reporter assay are MRC5 cells expressing FAP; and the second cell is a HEK-Blue cell expressing TGF-βRII; Bispecific binding moieties.

18. A bispecific binding moiety comprising a FAP-binding domain and a TGF-βRII-binding domain, The FAP binding domain comprises a heavy chain variable region comprising: a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 14, respectively; a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18, respectively; or a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO:20, SEQ ID NO:21, and SEQ ID NO:22, respectively; wherein each of said HDCR1 or HCDR2 may contain at most three, two, or one amino acid mutations; Bispecific binding moieties.

19. 19. A bispecific binding moiety according to claim 18, the FAP-binding domain comprises a heavy chain variable region having an amino acid sequence set forth in any one of SEQ ID NOs: 11, 15, or 19, or having at least 80%, 85%, 90%, or 95% sequence identity thereto; Bispecific binding moieties.

20. 20. A bispecific binding moiety according to claim 18 or 19, The FAP-binding domain a light chain variable region comprising a light chain CDR1 (LCDR1), a light chain CDR2 (LCDR2), and a light chain CDR3 (LCDR3) having the amino acid sequences set forth in SEQ ID NO:53, SEQ ID NO:54, and SEQ ID NO:55, respectively; or variants thereof, which contain at most three, two, or one amino acid mutation in each LCDR; Including, Bispecific binding moieties.

21. 21. A bispecific binding moiety according to any one of claims 18 to 20, the FAP-binding domain comprises a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 52, or having at least 80%, 85%, 90%, or 95% sequence identity thereto; Bispecific binding moieties.

22. 22. A bispecific binding moiety according to any one of claims 18 to 21, The TGF-βRII binding domain comprises a heavy chain variable region comprising: a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO:24, SEQ ID NO:25, and SEQ ID NO:26, respectively; a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO:24, SEQ ID NO:29, and SEQ ID NO:30, respectively; a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO:24, SEQ ID NO:33, and SEQ ID NO:34, respectively; or a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO:24, SEQ ID NO:37, and SEQ ID NO:34, respectively; wherein each of said HDCR1 or HCDR2 may contain at most three, two, or one amino acid mutations; Bispecific binding moieties.

23. 23. A bispecific binding moiety according to any one of claims 18 to 22, the TGF-βRII binding domain comprises a heavy chain variable region having an amino acid sequence set forth in any one of SEQ ID NOs: 23, 27, 31, or 35, or having at least 80%, 85%, 90%, or 95% sequence identity thereto; Bispecific binding moieties.

24. 24. A bispecific binding moiety according to any one of claims 18 to 23, The TGF-βRII binding domain comprises: a light chain variable region comprising a light chain CDR1 (LCDR1), a light chain CDR2 (LCDR2), and a light chain CDR3 (LCDR3) having the amino acid sequences set forth in SEQ ID NO:53, SEQ ID NO:54, and SEQ ID NO:55, respectively; or variants thereof, which contain at most three, two, or one amino acid mutation in each LCDR; Including, Bispecific binding moieties.

25. 25. A bispecific binding moiety according to any one of claims 18 to 24, the TGF-βRII binding domain comprises a light chain variable region having the amino acid sequence set forth in SEQ ID NO:52, or having at least 80%, 85%, 90%, or 95% sequence identity thereto; Bispecific binding moieties.

26. A bispecific binding moiety comprising a FAP-binding domain and a TGF-βRII-binding domain, The TGF-βRII binding domain comprises a heavy chain variable region comprising: a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO:24, SEQ ID NO:25, and SEQ ID NO:26, respectively; a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO:24, SEQ ID NO:29, and SEQ ID NO:30, respectively; a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO:24, SEQ ID NO:33, and SEQ ID NO:34, respectively; or a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO:24, SEQ ID NO:37, and SEQ ID NO:34, respectively; wherein each of said HDCR1 or HCDR2 may contain at most three, two, or one amino acid mutations; Bispecific binding moieties.

27. 27. A bispecific binding moiety according to claim 26, the TGF-βRII binding domain comprises a heavy chain variable region having an amino acid sequence set forth in any one of SEQ ID NOs: 23, 27, 31, or 35, or having at least 80%, 85%, 90%, or 95% sequence identity thereto; Bispecific binding moieties.

28. 28. A bispecific binding moiety according to claim 26 or 27, The TGF-βRII binding domain comprises: a light chain variable region comprising a light chain CDR1 (LCDR1), a light chain CDR2 (LCDR2), and a light chain CDR3 (LCDR3) having the amino acid sequences set forth in SEQ ID NO:53, SEQ ID NO:54, and SEQ ID NO:55, respectively; or variants thereof, which contain at most three, two, or one amino acid mutation in each LCDR; Including, Bispecific binding moieties.

29. 29. A bispecific binding moiety according to any one of claims 26 to 28, the TGF-βRII binding domain comprises a light chain variable region having the amino acid sequence set forth in SEQ ID NO:52, or having at least 80%, 85%, 90%, or 95% sequence identity thereto; Bispecific binding moieties.

30. 30. A bispecific binding moiety according to any one of claims 26 to 29, The FAP binding domain comprises a heavy chain variable region comprising: a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 14, respectively; a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18, respectively; or a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO:20, SEQ ID NO:21, and SEQ ID NO:22, respectively; wherein each of said HDCR1 or HCDR2 may contain at most three, two, or one amino acid mutations; Bispecific binding moieties.

31. 31. A bispecific binding moiety according to any one of claims 26 to 30, comprising: the FAP-binding domain comprises a heavy chain variable region having an amino acid sequence set forth in any one of SEQ ID NOs: 11, 15, or 19, or having at least 80%, 85%, 90%, or 95% sequence identity thereto; Bispecific binding moieties.

32. 32. A bispecific binding moiety according to any one of claims 26 to 31, The FAP-binding domain a light chain variable region comprising a light chain CDR1 (LCDR1), a light chain CDR2 (LCDR2), and a light chain CDR3 (LCDR3) having the amino acid sequences set forth in SEQ ID NO:53, SEQ ID NO:54, and SEQ ID NO:55, respectively; or variants thereof, which contain at most three, two, or one amino acid mutation in each LCDR; Including, Bispecific binding moieties.

33. 33. A bispecific binding moiety according to any one of claims 26 to 32, comprising: the FAP-binding domain comprises a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 52, or having at least 80%, 85%, 90%, or 95% sequence identity thereto; Bispecific binding moieties.

34. 34. A bispecific binding moiety according to any one of claims 1 to 33, the bispecific binding moiety comprises a Fab domain that binds to FAP, a Fab domain that binds to TGF-βRII, and an Fc region; Bispecific binding moieties.

35. 35. A bispecific binding moiety according to claim 34, the Fc region has enhanced or reduced immune effector functions; Bispecific binding moieties.

36. 36. A bispecific binding moiety according to claim 34 or 35, The Fc region has an enhanced immune cell effector function, specifically, an enhanced ADCC activity. Bispecific binding moieties.

37. 37. A bispecific binding moiety according to claim 36, the bispecific binding moiety is defucosylated; Bispecific binding moieties.

38. 36. A bispecific binding moiety according to claim 34 or 35, the Fc region has reduced immune cell effector function, specifically reduced ADCC and / or ADCP activity; Bispecific binding moieties.

39. 39. The bispecific binding moiety of claim 38, the Fc region has a L235G and / or G236R mutation in the CH2 domain (EU numbering); Bispecific binding moieties.

40. 40. A method for the preparation of a bispecific binding moiety comprising administering to a subject an effective amount of a bispecific binding moiety according to any one of claims 1 to 39 and a pharmaceutically acceptable carrier. Pharmaceutical compositions.

41. A bispecific binding moiety according to any one of claims 1 to 39 or a pharmaceutical composition according to claim 40 for use in therapy.

42. A bispecific binding moiety according to any one of claims 1 to 39 or a pharmaceutical composition according to claim 40 for use in the treatment of cancer.

43. 40. A combination of a bispecific binding moiety according to any one of claims 1 to 39 and a second binding moiety that binds to PD-1 for use in therapy.

44. 40. A combination of a bispecific binding moiety according to any one of claims 1 to 39 and a second binding moiety that binds to PD-1 for use in the treatment of cancer.

45. 45. A combination according to claim 43 or 44, wherein the second binding moiety that binds to PD-1 is pembrolizumab. combination.

46. 1. A method for treating a disease in a subject, comprising: administering to said subject in need thereof a therapeutically effective amount of a bispecific binding moiety according to any one of claims 1 to 39 or a pharmaceutical composition according to claim 40. method.

47. 1. A method for treating cancer in a subject, comprising: administering to said subject in need thereof a therapeutically effective amount of a bispecific binding moiety according to any one of claims 1 to 39 or a pharmaceutical composition according to claim 40. method.

48. 48. A method of treatment according to claim 46 or 47, comprising: The method further comprises administering an effective amount of a second binding moiety that binds to PD-1. method.

49. 49. A method of treatment according to claim 48, comprising: wherein the second binding moiety that binds to PD-1 is pembrolizumab. method.

50. A nucleic acid sequence encoding the heavy chain variable region of any one of claims 18 to 33.

51. 51. A vector comprising the nucleic acid sequence of claim 50.

52. 52. The vector of claim 51 , the vector further comprises a nucleic acid sequence encoding a CH1 region, and preferably a hinge, CH2 and CH3 region; vector.

53. 53. The vector of claim 52, The vector further comprises at least one nucleic acid sequence encoding a light chain variable region, in particular a light chain variable region according to any one of claims 20, 21, 24, 25, 28, 29, 32, or 33, and preferably a CL region. vector.

54. 54. The vector of claim 53, The light chain variable region is a light chain variable region capable of pairing with multiple heavy chains having different epitope specificities. vector.

55. The invention comprises one or more nucleic acids encoding the heavy chain variable region of the FAP-binding domain of any one of claims 18 to 25 and the heavy chain variable region of the TGF-βRII-binding domain of any one of claims 26 to 33. cell.

56. 56. The cell of claim 55, the one or more nucleic acids further encode a CH1 region, and preferably a hinge, CH2 and CH3 region; cell.

57. 57. The cell of claim 55 or 56, The one or more nucleic acids further encode a light chain variable region, specifically a light chain variable region according to any one of claims 20, 21, 24, 25, 28, 29, 32, or 33, and preferably a CL region. cell.

58. A cell producing the bispecific binding moiety of any one of claims 1 to 39.

59. competes with a bispecific binding moiety according to any one of claims 1 to 39 for binding to FAP and TGF-βRII. Bispecific binding moieties.

60. A polypeptide comprising: - a polypeptide comprising a heavy chain CDR1 (HCDR1) having the amino acid sequence set forth in SEQ ID NO: 16, a heavy chain CDR2 (HCDR2) having the amino acid sequence set forth in SEQ ID NO: 17, and a heavy chain CDR3 (HCDR3) having the amino acid sequence set forth in SEQ ID NO: 18; - a polypeptide comprising a heavy chain CDR1 (HCDR1) having the amino acid sequence set forth in SEQ ID NO: 20, a heavy chain CDR2 (HCDR2) having the amino acid sequence set forth in SEQ ID NO: 21, and a heavy chain CDR3 (HCDR3) having the amino acid sequence set forth in SEQ ID NO: 22; - a polypeptide comprising a heavy chain CDR1 (HCDR1) having the amino acid sequence set forth in SEQ ID NO: 12, a heavy chain CDR2 (HCDR2) having the amino acid sequence set forth in SEQ ID NO: 13, and a heavy chain CDR3 (HCDR3) having the amino acid sequence set forth in SEQ ID NO: 14; - a polypeptide comprising a heavy chain CDR1 (HCDR1) having the amino acid sequence set forth in SEQ ID NO: 70, a heavy chain CDR2 (HCDR2) having the amino acid sequence set forth in SEQ ID NO: 71, and a heavy chain CDR3 (HCDR3) having the amino acid sequence set forth in SEQ ID NO: 72; Selected from: Polypeptide.

61. 61. The polypeptide of claim 60, The polypeptide comprises a heavy chain variable region having an amino acid sequence set forth in SEQ ID NO: 15; 19; 11; 69, or having at least 80%, or at least 85%, or at least 90%, or at least 95% sequence identity thereto. Polypeptide.

62. 62. A FAP binding domain comprising a polypeptide according to claim 60 or 61.

63. FAP-binding domain that binds to human and mouse FAP.

64. 64. A FAP binding domain according to claim 63, The FAP-binding domain comprises a polypeptide comprising a heavy chain CDR1 (HCDR1) having the amino acid sequence set forth in SEQ ID NO: 70, a heavy chain CDR2 (HCDR2) having the amino acid sequence set forth in SEQ ID NO: 71, and a heavy chain CDR3 (HCDR3) having the amino acid sequence set forth in SEQ ID NO: 72; FAP binding domain.

65. 65. A FAP binding domain according to claim 64, The polypeptide comprises a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO:69, or having at least 80%, or at least 85%, or at least 90%, or at least 95% sequence identity thereto; FAP binding domain.

66. 66. A binding moiety comprising a polypeptide according to any one of claims 60 to 61 or a FAP binding domain according to any one of claims 62 to 65.

67. 67. A method for treating a leukemia comprising administering to a subject therapies comprising administering to said subject an effective amount of a polypeptide according to any one of claims 60 to 61, or a FAP binding domain according to any one of claims 62 to 65, or a binding moiety according to claim 66, and a pharmaceutically acceptable carrier. Pharmaceutical compositions.

68. 68. A polypeptide according to any one of claims 60 to 61, or a FAP binding domain according to any one of claims 62 to 65, or a binding moiety according to claim 66, or a pharmaceutical composition according to claim 67, for use in therapy.

69. 68. A polypeptide according to any one of claims 60 to 61, or a FAP binding domain according to any one of claims 62 to 65, or a binding moiety according to claim 66, or a pharmaceutical composition according to claim 67, for use in the treatment of cancer.

70. 1. A method for treating a disease, comprising: Administering to an individual in need thereof an effective amount of a polypeptide according to any one of claims 60 to 61, or a FAP binding domain according to any one of claims 62 to 65, or a binding moiety according to claim 66, or a pharmaceutical composition according to claim 67. method.

71. 1. A method for treating cancer, comprising: Administering to an individual in need thereof an effective amount of a polypeptide according to any one of claims 60 to 61, or a FAP binding domain according to any one of claims 62 to 65, or a binding moiety according to claim 66, or a pharmaceutical composition according to claim 67. method.

72. A nucleic acid comprising a sequence encoding a polypeptide according to any one of claims 60 to 61.

73. 73. A vector comprising the nucleic acid of claim 72.

74. 74. A cell comprising the nucleic acid of claim 72 or the vector of claim 73.

75. 67. A cell producing a polypeptide according to any one of claims 60 to 61, or a FAP binding domain according to any one of claims 62 to 65, or a binding moiety according to claim 66.

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