Multispecific binding site containing PD-1 and TGF-BRII binding domains

Abstract

The present disclosure relates to multispecific binding sites comprising a PD-1 binding domain and a TGF-βRII binding domain, wherein the PD-1 binding domain inhibits PD-1-mediated signaling and the TGF-βRII binding domain inhibits TGF-βRII-mediated signaling. The present disclosure further relates to pharmaceutical compositions comprising such multispecific binding sites, methods of treatment using such multispecific binding sites, and cells that produce such multispecific binding sites.

Description

[Technical Field]

[0001] The present disclosure relates to the field of antibodies, particularly therapeutic antibodies for the treatment of diseases involving aberrant cells, and more particularly to multispecific binding sites that include a binding domain that binds to PD-1 and a binding domain that binds to TGF-βRII. [Background technology]

[0002] Although the role of T lymphocytes in tumor immune surveillance is well known, cancer cells can escape immune control by inducing inhibitory immune pathways. Consequently, immune checkpoint blockade (ICB), in which antibodies are used to block inhibitory immune pathways, has emerged as a promising therapeutic option and has been demonstrated in preclinical and clinical trials to enhance and maintain endogenous immunity against certain cancers.

[0003] Programmed death 1 (PD-1) and programmed death-ligand 1 (PD-L1) are components of an immunosuppressive network that attenuates T cell activity in normal physiology but can be exploited by tumors to suppress T cell-mediated antitumor immune responses. Antibodies directed against PD-1 and PD-L1 have improved the response and survival of some patients with several different cancers. However, despite promising clinical activity, only a small number of patients respond to anti-PD-1 / PD-L1 therapy, and the response is limited in durability. Therefore, there is an urgent need to develop new, safe, and effective therapies to treat cancer.

[0004] The programmed cell death 1 protein (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 when present on somatic cells. The inhibitory effect of PD-1 is 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. PD-1 inhibitors, a new class of drugs that block the activity of PD-1, activate the immune system to attack tumors and are therefore used to treat certain cancers.

[0005] Monoclonal antibodies targeting PD-1 have been approved for the treatment of various malignancies. For example, the response rate in melanoma patients treated with the anti-PD-1 antibody (pembrolizumab) is 33% at 3 years, despite an initial response rate of 70–80% (Ribas A. et al. Association of Pembrolizumab With Tumor Response and Survival Among Patients With Advanced Melanoma. JAMA. 2016 Apr 19; 315(15):1600-9).

[0006] TGF-β signaling regulates many 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, including growth promotion and epithelial-mesenchymal transition, which allows tumor cell migration, invasion, intravasation, and extravasation.

[0007] TGF-βRII is a member of the serine / threonine protein kinase family and the TGFβ 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 2, TBR-ii, and TBRII. TGF-βRII forms a heterodimeric complex with another receptor protein and binds to TGF-β. This receptor / ligand complex phosphorylates proteins, which then enter the nucleus and regulate the transcription of a subset of genes related to cell proliferation.

[0008] Several inhibitors targeting the TGF-β pathway are in preclinical and clinical development, inhibiting TGF-β at various levels: at the ligand, ligand-receptor, or intracellular levels. These include, for example, TGF-β neutralizing monoclonal antibodies, anti-TGF-βRII antibodies, soluble receptors, antibody ligand traps (e.g., anti-PD-L1-TGF-βRIIECD), antisense oligonucleotides that block TGF-β synthesis, TGF-β2 antisense gene-modified allogeneic cancer cell vaccines, and small molecules that target the kinase domain of TGF-βRI.

[0009] Targeting the TGF-β pathway with monospecific antibodies has been reported to demonstrate antitumor activity in vitro and in vivo; however, poor clinical outcomes have continued due to low efficacy and unacceptable toxicity, including significant cytokine release syndrome (CRS). Combined targeting of the TGF-β pathway with immune checkpoint blockade has been attempted using Bintrafusp alpha, an anti-PD-L1-TGFBRII bifunctional fusion protein, but no robust clinical efficacy was demonstrated.

[0010] Novel therapeutic interventions that selectively inhibit TGF-β signaling in locally activated tumor-specific PD-1-expressing T cells in the tumor microenvironment remain needed to promote T cell tumor infiltration and restore and sustain T cell antitumor effector activity. Such targeting would alleviate immunosuppressive pathways and potently promote CTL function and T cell memory for effective and durable cancer elimination while minimizing the toxicity associated with systemic TGF-β inhibition. Summary of the Invention [Problem to be solved by the invention]

[0011] One objective of the present disclosure is to provide new pharmaceuticals for the treatment of human diseases, particularly cancer. This objective is met by providing a multispecific binding site, e.g., a bispecific antibody, that binds to PD-1 and TGF-βRII. The PD-1-binding domain of the multispecific binding site is intended to drive the specificity of the multispecific binding site for activated / exhausted effector T cells in tumors and tumor-draining lymph nodes, where the TGF-βRII-binding domain can locally inhibit TGF-β binding to TGF-βRII, thereby reducing the systemic toxicity of TGF-β inhibition to non-T cells. Furthermore, the multispecific binding site alleviates both PD1- and TGF-β-mediated immunosuppressive pathways in the tumor microenvironment, promoting cytotoxic T lymphocyte activity. [Means for solving the problem]

[0012] The present disclosure provides a multispecific binding site comprising a PD-1-binding domain and a TGF-βRII-binding domain, wherein the PD-1-binding domain inhibits PD-1-mediated signaling, and the TGF-βRII-binding domain inhibits TGF-βRII-mediated signaling.

[0013] The present disclosure also provides a multispecific binding site comprising a PD-1-binding domain and a TGF-βRII-binding domain, wherein the PD-1-binding domain comprises a heavy chain variable region comprising CDR1, CDR2, and CDR3 sequences as further described herein.

[0014] The present disclosure also provides a multispecific binding site comprising a PD-1-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.

[0015] The present disclosure further provides pharmaceutical compositions comprising an effective amount of the multispecific binding sites described herein.

[0016] The present disclosure further provides the multispecific binding sites described herein, and the pharmaceutical compositions described herein, for use in therapy.

[0017] The present disclosure further provides the multispecific binding sites described herein, and the pharmaceutical compositions described herein, for use in the treatment of cancer.

[0018] The present disclosure further provides a method for treating a disease, the method comprising administering to an individual in need thereof an effective amount of a multispecific binding site described herein, or a pharmaceutical composition described herein.

[0019] The present disclosure further provides a method for treating cancer, the method comprising administering to an individual in need thereof an effective amount of a multispecific binding site described herein, or a pharmaceutical composition described herein.

[0020] The present disclosure further provides a cell comprising a nucleic acid sequence encoding a heavy chain variable region of the PD-1 binding domain described herein and a nucleic acid sequence encoding a heavy chain variable region of the TGF-βRII binding domain described herein.

[0021] The present disclosure further provides cells that produce the multispecific binding sites described herein. [Brief explanation of the drawings]

[0022] [Figure 1]Figure 1 shows the percentage inhibition of PD-1-mediated SHP recruitment by bispecific antibodies and control antibodies as measured in a PD-1-SHP recruitment assay. A) The bispecific antibodies are: SEQ ID NO:39 x SEQ ID NO:9 and SEQ ID NO:35 x SEQ ID NO:5. The control antibodies are: pembrolizumab analog - SEQ ID NO:78 / SEQ ID NO:79; PD-L1-TGF-β TRAP molecule analog - SEQ ID NO:80 / SEQ ID NO:81, and TGF1 analog - SEQ ID NO:76 / SEQ ID NO:77. B) The bispecific antibodies are: SEQ ID NO:23 x SEQ ID NO:18; SEQ ID NO:47 x SEQ ID NO:13; SEQ ID NO:88 x SEQ ID NO:13; SEQ ID NO:89 x SEQ ID NO:13; SEQ ID NO:23 x SEQ ID NO:14; and SEQ ID NO:43 x SEQ ID NO:9. The control antibodies are: pembrolizumab analog - SEQ ID NO:78 / SEQ ID NO:79; and RSV IgG1 - SEQ ID NO:86 / SEQ ID NO:87. [Figure 2] Figure 1 shows the fold induction of T cell activation by bispecific and control antibodies as measured in a PD-1-NFAT reporter assay. A) The bispecific antibodies are: SEQ ID NO:39 x SEQ ID NO:9 and SEQ ID NO:35 x SEQ ID NO:5. The control antibodies are: pembrolizumab analog - SEQ ID NO:78 / SEQ ID NO:79; PD-L1-TGF-β TRAP molecule analog - SEQ ID NO:80 / SEQ ID NO:81, and TGF1 analog - SEQ ID NO:76 / SEQ ID NO:77. B) The bispecific antibodies are: SEQ ID NO:47 x SEQ ID NO:13; SEQ ID NO:88 x SEQ ID NO:13; and SEQ ID NO:89 x SEQ ID NO:13. The control antibodies are: pembrolizumab analog - SEQ ID NO:78 / SEQ ID NO:79; and RSV IgG1 - SEQ ID NO:86 / SEQ ID NO:87. C) The bispecific antibodies are: SEQ ID NO:23 x SEQ ID NO:18; SEQ ID NO:23 x SEQ ID NO:14; and SEQ ID NO:43 x SEQ ID NO:9. Control antibodies are: pembrolizumab analogue - SEQ ID NO: 78 / SEQ ID NO: 79; and RSV IgG1 - SEQ ID NO: 86 / SEQ ID NO: 87. [Figure 3]Inhibition of phosphorylated SMAD2 / 3 by bispecific and control antibodies in Jurkat-PD-1 null (A-G) and Jurkat-PD-1 + cells (H-N). These graphs show phosphorylated SMAD2 / 3 levels in lysates of Jurkat-PD-1 null and Jurkat-PD-1 + cells incubated with bispecific or control antibodies. "No TGF-β1" indicates background phosphorylated SMAD2 / 3 levels measured in the absence of bispecific antibody when no TGF-β ligand is added; "10 ng / ml TGF-β1" indicates maximal phosphorylated SMAD2 / 3 levels when 10 ng / ml TGF-β ligand is added in the absence of bispecific antibody. The control antibodies were: RSV IgG1-SEQ ID NO:86 / SEQ ID NO:87; TGF1 analog-SEQ ID NO:76 / SEQ ID NO:77; and a TGF-βRII x RSV antibody comprising a TGF-βRII binding domain comprising a heavy chain variable region amino acid sequence set forth in SEQ ID NO:23, 31, 39, 27, 35, or 43; an RSV binding domain comprising a heavy chain variable region amino acid sequence set forth in SEQ ID NO:86; and a common light chain comprising a light chain variable region amino acid sequence set forth in SEQ ID NO:48 and a light chain constant region amino acid sequence set forth in SEQ ID NO:75. Each data point represents the average absorbance of the corresponding replicates.A and H: a bispecific antibody comprising a PD-1-binding domain having a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 1, 5, 9, 14, or 19, and a TGF-βRII-binding domain having a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 23; B and I: a bispecific antibody comprising a PD-1-binding domain having a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 1, 5, 9, 14, or 19, and a TGF-βRII-binding domain having a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 31; C and J: a bispecific antibody comprising a PD-1-binding domain having a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 1, 5, 9, 14, or 19, and a TGF-βRII-binding domain having a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 39; D and K: a PD-1-binding domain having a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 1, 5, 9, 14, or 19, and a TGF-βRII-binding domain having a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: and a TGF-βRII-binding domain having a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO:27; E and L: a bispecific antibody comprising a PD-1-binding domain having a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO:1, 5, 9, 14, or 19, and a TGF-βRII-binding domain having a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO:35; F and M: a bispecific antibody comprising a PD-1-binding domain having a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO:1, 5, 9, 14, or 19, and a TGF-βRII-binding domain having a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO:43; G and N: the bispecific antibodies are: SEQ ID NO:23 x SEQ ID NO:9; SEQ ID NO:23 x SEQ ID NO:14; SEQ ID NO:43 x SEQ ID NO:9; SEQ ID NO:23 x SEQ ID NO:13; SEQ ID NO:23 x SEQ ID NO:18; SEQ ID NO:47 x SEQ ID NO:13; SEQ ID NO:88 x SEQ ID NO:13; and SEQ ID NO:89 x SEQ ID NO:13. Control antibodies are: RSV IgG1 - SEQ ID NO: 86 / SEQ ID NO: 87; TGF1 analog - SEQ ID NO: 76 / SEQ ID NO: 77; and pembrolizumab - SEQ ID NO: 78 / SEQ ID NO: 79. [Figure 4]Measurement of intracellular phosphorylated SMAD2 by flow cytometry. These graphs show the intracellular phosphorylated SMAD2 levels of stimulated and unstimulated CD4+ and CD8+ T cells incubated with bispecific antibodies or control antibodies. The bispecific antibodies are: SEQ ID NO: 23 x SEQ ID NO: 18 and SEQ ID NO: 47 x SEQ ID NO: 13. The control antibodies are: RSV IgG1-SEQ ID NO: 86 / SEQ ID NO: 87; TGF1 analog-SEQ ID NO: 76 / SEQ ID NO: 77; and pembrolizumab-SEQ ID NO: 78 / SEQ ID NO: 79. A: Stimulated CD4+ T cells from donor A; B: Stimulated CD8+ T cells from donor A; C: Unstimulated CD4+ T cells from donor A; D: Unstimulated CD8+ T cells from donor A; E: Stimulated CD4+ T cells from donor B; F: Stimulated CD8+ T cells from donor B; G: Unstimulated CD4+ T cells from donor B; H: Unstimulated CD8+ T cells from donor B. [Figure 5]Measurement of cytokine production induced by bispecific antibodies or control antibodies in an exhaustion MLR assay. The bispecific antibodies tested were: SEQ ID NO:23 x SEQ ID NO:9; SEQ ID NO:23 x SEQ ID NO:14; SEQ ID NO:23 x SEQ ID NO:19; SEQ ID NO:31 x SEQ ID NO:14; SEQ ID NO:39 x SEQ ID NO:9; SEQ ID NO:35 x SEQ ID NO:9; SEQ ID NO:35 x SEQ ID NO:14; SEQ ID NO:35 x SEQ ID NO:19; SEQ ID NO:27 x SEQ ID NO:9; SEQ ID NO:43 x SEQ ID NO:9; SEQ ID NO:43 x SEQ ID NO:19; SEQ ID NO:23 x SEQ ID NO:13; SEQ ID NO:23 x SEQ ID NO:18; SEQ ID NO:47 x SEQ ID NO:13; SEQ ID NO:88 x SEQ ID NO:13; and SEQ ID NO:89 x SEQ ID NO:13. Control antibodies were: RSV IgG1 - SEQ ID NO: 86 / SEQ ID NO: 87; TGF1 analog - SEQ ID NO: 76 / SEQ ID NO: 77; pembrolizumab and TGF1 analog combination - SEQ ID NO: 78 / SEQ ID NO: 79 + SEQ ID NO: 76 / SEQ ID NO: 77; PD-L1-TGF-β TRAP molecule analog - SEQ ID NO: 80 / SEQ ID NO: 81; and pembrolizumab - SEQ ID NO: 78 / SEQ ID NO: 79. A: This graph shows the induction of IFN-γ cytokine secretion by exhausted T cells from one representative donor. B: This graph shows the induction of IFN-γ cytokine secretion by exhausted T cells from one representative donor. C: This graph shows the induction of IL-2 cytokine secretion by exhausted T cells from one representative donor. D: This graph shows the induction of TNF-α cytokine secretion by exhausted T cells from one representative donor. E: This graph shows the induction of TNF-α cytokine secretion by exhausted T cells from one representative donor. [Figure 6]Measurement of the percentage inhibition of TGF-β-induced signal transduction induced by bispecific antibodies or control antibodies. Bispecific antibodies are: SEQ ID NO:23 x SEQ ID NO:14; SEQ ID NO:23 x SEQ ID NO:19; SEQ ID NO:39 x SEQ ID NO:9; SEQ ID NO:35 x SEQ ID NO:9; SEQ ID NO:35 x SEQ ID NO:14; SEQ ID NO:35 x SEQ ID NO:19; SEQ ID NO:27 x SEQ ID NO:9; SEQ ID NO:43 x SEQ ID NO:9; and SEQ ID NO:43 x SEQ ID NO:19. Control antibodies are: RSV IgG1-SEQ ID NO:86 / SEQ ID NO:87; TGF1 analog-SEQ ID NO:76 / SEQ ID NO:77; and pembrolizumab-SEQ ID NO:78 / SEQ ID NO:79. A and B: These graphs show the inhibition of TGF-β signaling by bispecific antibodies or control antibodies in HEK-Blue™ TGF-β cells. C and D: These graphs show the inhibition of TGF-βR signaling by bispecific antibodies or control antibodies in HEK-Blue™ TGF-β-PD-1+ cells. [Figure 7] Cytokine production induced by bispecific antibodies or control antibodies was measured in a Treg suppression assay. The bispecific antibodies were: SEQ ID NO:23 x SEQ ID NO:14; SEQ ID NO:23 x SEQ ID NO:19; SEQ ID NO:31 x SEQ ID NO:14; SEQ ID NO:39 x SEQ ID NO:9; SEQ ID NO:35 x SEQ ID NO:9; SEQ ID NO:35 x SEQ ID NO:14; SEQ ID NO:35 x SEQ ID NO:19; SEQ ID NO:27 x SEQ ID NO:9; SEQ ID NO:43 x SEQ ID NO:9; and SEQ ID NO:43 x SEQ ID NO:19. The control antibodies were: RSV IgG1-SEQ ID NO:86 / SEQ ID NO:87; TGF1 analog-SEQ ID NO:76 / SEQ ID NO:77; pembrolizumab and TGF1 analog combination-SEQ ID NO:78 / SEQ ID NO:79 + SEQ ID NO:76 / SEQ ID NO:77; and pembrolizumab-SEQ ID NO:78 / SEQ ID NO:79. A: This graph shows the induction of IFN-γ cytokine secretion in co-culture with PBMCs of Tregs from one representative donor. B: The graph shows the induction of TNF-α cytokine secretion in co-cultures of Tregs with PBMCs from one representative donor. [Figure 8]Measurement of cytokine production induced by bispecific antibodies or control antibodies in a macrophage suppression assay. The bispecific antibodies are: SEQ ID NO:35 x SEQ ID NO:9; SEQ ID NO:23 x SEQ ID NO:14; SEQ ID NO:23 x SEQ ID NO:19; SEQ ID NO:43 x SEQ ID NO:9; SEQ ID NO:39 x SEQ ID NO:9; SEQ ID NO:43 x SEQ ID NO:19; SEQ ID NO:35 x SEQ ID NO:14; SEQ ID NO:35 x SEQ ID NO:19; SEQ ID NO:31 x SEQ ID NO:14; and SEQ ID NO:27 x SEQ ID NO:9. The control antibodies are: RSV IgG1-SEQ ID NO:86 / SEQ ID NO:87; TGF1 analog-SEQ ID NO:76 / SEQ ID NO:77; Opdivo; LILRB2; PD-L1-TGF-β TRAP molecule analog-SEQ ID NO:80 / SEQ ID NO:81; pembrolizumab-SEQ ID NO:78 / SEQ ID NO:79; and nivolumab analog-SEQ ID NO:96 / SEQ ID NO:97. A: These graphs show the expression of CD163, CD209, CD206, and CD86 on M2 macrophages obtained from PBMCs from three different donors. B: These graphs show the induction of IFN-γ cytokine secretion by CD4+ T cells in the presence of M2 macrophages obtained from PBMCs from three different donors. [Figure 9]Figure 1 shows the in vivo efficacy of bispecific antibodies. A: This graph shows the reduction in tumor volume (mm3) induced by a control antibody and a reference antibody. B-E: These graphs show the reduction in tumor volume (mm3) induced by bispecific antibodies compared to a control antibody and a reference antibody. The bispecific antibodies are: SEQ ID NO:43 x SEQ ID NO:9; SEQ ID NO:43 x SEQ ID NO:19; SEQ ID NO:23 x SEQ ID NO:14; SEQ ID NO:23 x SEQ ID NO:19; SEQ ID NO:39 x SEQ ID NO:9; SEQ ID NO:27 x SEQ ID NO:9; SEQ ID NO:23 x SEQ ID NO:18; and SEQ ID NO:47 x SEQ ID NO:13. The control antibodies are: RSV IgG1 - SEQ ID NO:86 / SEQ ID NO:87; TGF1 analog - SEQ ID NO:76 / SEQ ID NO:77; pembrolizumab - SEQ ID NO:78 / SEQ ID NO:79; PD-L1-TGF-β TRAP molecule analog - SEQ ID NO:80 / SEQ ID NO:81; and the combination of pembrolizumab and TGF1 analog - SEQ ID NO:78 / SEQ ID NO:79 + SEQ ID NO:76 / SEQ ID NO:77. Bispecific antibodies were administered at 1 mg / kg (1) and / or 10 mg / kg (10) (A-C), and at 10 mg / kg alone (D-F). F: Left graph shows TGF-βRII receptor occupancy, and right graph shows PD-1 receptor occupancy after treatment with bispecific antibodies or control antibodies. [Figure 10] Vector Map [Figure 11] In vivo efficacy of bispecific antibodies. This graph shows the reduction in tumor volume (mm3) induced by an exemplary bispecific antibody at two different dose levels: 1 mg / kg and 10 mg / kg, compared to a control antibody at 10 mg / kg. The bispecific antibody is: SEQ ID NO:23 x SEQ ID NO:18, and the control antibody is: SEQ ID NO:86 / SEQ ID NO:87. DETAILED DESCRIPTION OF THE INVENTION

[0023] In certain embodiments, the present disclosure provides a multispecific binding moiety comprising a PD-1-binding domain and a TGF-βRII-binding domain, wherein the PD-1-binding domain inhibits PD-1-mediated signaling, and the TGF-βRII-binding domain inhibits TGF-βRII-mediated signaling.

[0024] As used herein, "blocks" or "blocking" means to prevent or modify the interaction between a ligand and a receptor, or to cause a total or partial decrease in a signal transduction cascade. For purposes of this disclosure, in one embodiment, potency in inhibiting ligand-induced PD-1 signaling is determined by using the SHP recruitment assay described in Example 2 or the NFAT reporter assay described in Example 3. In one embodiment, potency in inhibiting ligand-induced PD-1 signaling is determined by using the SHP recruitment assay described in Example 2. In one embodiment, potency in inhibiting ligand-induced PD-1 signaling is determined by using the NFAT reporter assay described in Example 3. For purposes of this disclosure, in one embodiment, potency in inhibiting ligand-induced TGF-βRII signaling is determined by using the SMAD assay described in Example 4 or 5. In one embodiment, potency in inhibiting ligand-induced TGF-βRII signaling is determined by using the SMAD assay described in Example 4. In one embodiment, potency in inhibiting ligand-induced TGF-βRII signaling is determined by using the SMAD assay described in Example 5.

[0025] In certain embodiments, the multispecific binding site of the present disclosure binds to human PD-1. In certain embodiments, the PD-1-binding domain of the multispecific binding site of the present disclosure inhibits the binding of PD-L1 to PD-1, e.g., as measured in the assay described in Example 2 or 3.

[0026] In one embodiment, the multispecific binding site 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 provided as SEQ ID NO: 82; the amino acid sequence of the extracellular domain of human TGF-βRII isoform A is provided as SEQ ID NO: 83. 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 provided as SEQ ID NO: 84; the amino acid sequence of the extracellular domain of human TGF-βRII isoform B is set forth in SEQ ID NO: 85.

[0027] "Binding moiety" refers to a proteinaceous molecule and includes 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-zipper, nanobodies, DART molecules, TandAb, Fab-scFv, F(ab)'2, F(ab)'2-scFv2, and intrabodies.

[0028] In some embodiments, the multispecific binding site is a multispecific antibody. The multispecific antibodies described herein are antibodies comprising at least two binding domains with specificity for at least two different targets or epitopes. In some embodiments, the multispecific antibody of the present disclosure is a bispecific antibody. In some embodiments, the multispecific antibody of the present disclosure may further comprise an Fc region or a portion thereof. In some embodiments, the multispecific binding site of the present disclosure is an IgG1 antibody. The constant region of the binding site of the present disclosure may contain one or more variations that modulate properties of the binding site other than binding to the target antigen. For example, the constant region may contain one or more variations that promote heterodimerization of PD-1 and TGF-βRII heavy chains over homodimerization of two PD-1 heavy chains and / or two TGF-βRII heavy chains, and / or the constant region may contain one or more variations that reduce or improve effector function, particularly one or more variations that reduce effector function.

[0029] "Fab" refers to a binding domain that typically includes a heavy chain variable region, a light chain variable region, a CH1 and a CL region.

[0030] In one embodiment, a multispecific binding site of the present disclosure comprises a single Fab domain that binds PD-1, a single Fab domain that binds TGF-βRII, and an Fc region. In one embodiment, a multispecific binding site of the present disclosure consists of a single Fab domain that binds PD-1, a single Fab domain that binds TGF-βRII, and an Fc region. In one embodiment, a multispecific binding site of the present disclosure consists essentially of a single Fab domain that binds PD-1, a single Fab domain that binds TGF-βRII, and an Fc region.

[0031] The "Fc region" typically comprises a 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: 68. 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: 70 or 71, and the CH3 region whose amino acid sequence is set forth in SEQ ID NO: 72, or 73 and 74.

[0032] The CL, CH1, CH2, and / or CH3 regions may be modified according to methods known in the art to obtain preferred antibody properties, including, for example, to promote heterodimerization of different heavy chains, to improve heavy chain-light chain pairing, and to increase or decrease immune cell effector function.

[0033] In one embodiment, the PD-1 binding domain of the multispecific binding site inhibits PD-1-mediated signaling and the TGF-βRII binding domain of the multispecific binding site inhibits TGF-βRII-mediated signaling in activated T cells, particularly activated tumor-specific T cells.

[0034] In certain embodiments, the multispecific binding sites of the present disclosure are more potent in inhibiting TGF-βRII-mediated signaling in cells that express both PD-1 and TGF-βRII than in cells that express TGF-βRII and no, substantially no, or low levels of PD-1.

[0035] Accordingly, the present disclosure also provides a multispecific binding moiety comprising a PD-1 binding domain and a TGF-βRII binding domain, wherein the multispecific binding moiety has greater potency in inhibiting TGF-βRII-mediated signaling in cells that express both PD-1 and TGF-βRII than in cells that express TGF-βRII and no, substantially no, or low levels of PD-1.

[0036] In one embodiment, the cells expressing both PD-1 and TGF-βRII are Jurkat-PD-1 + Cells, such as Jurkat T cells, that express human PD-1 and a luciferase reporter driven by the NFAT response element (NFAT-RE), and that express TGF-βRII but not PD-1, are referred to as Jurkat-PD-1 null cells, e.g., Jurkat-PD-1 null Jurkat T cells expressing human PD-1 and a luciferase reporter driven by NFAT-RE are commercially available, for example, from Promega (catalog number CS187105 - part of kits CS187106 and CS187107); Jurkat-PD-1 null The cells are publicly available, for example, from the ATCC (catalog number TIB-152).

[0037] In one embodiment, cells expressing both PD-1 and TGF-βRII are stimulated CD4 + or CD8 + cells, expressing TGF-βRII and low levels of PD-1, are expressed by unstimulated CD4 + or CD8 + In one embodiment, stimulated CD4 + or CD8 + Cells are stimulated with recombinant human TGF-β1.

[0038] In one embodiment, cells expressing both PD-1 and TGF-βRII are expressed using HEK-Blue™ TGF-β-PD-1, e.g., as described in Example 7. +The cells expressing TGF-βRII but not PD-1 are HEK-Blue™ TGF-β cells, such as HEK-Blue™ TGF-β cells. HEK-Blue™ TGF-β cells are commercially available, for example, from Invivogen (catalog number hkb-tgfb), and are stably transfected human embryonic kidney HEK293 cells containing the human TGFBR1, Smad3, and Smad4 genes. They further express a Smad3 / 4 binding element (SBE)-inducible SEAP reporter gene.

[0039] In one embodiment, no, substantially no, or low levels of PD-1 refer to levels of PD-1 on the cell surface that are undetectable in a suitable assay such as those provided herein. In one embodiment, low levels of PD-1 refer to fewer than 100 PD-1 molecules present on the cell surface. In one embodiment, the level of PD-1 is measured using quantibrite bead methodology.

[0040] For purposes of this disclosure, determining whether a multispecific binding site has greater potency in inhibiting TGF-βRII-mediated signaling in cells that express both PD-1 and TGF-βRII than in cells that express TGF-βRII and no, substantially no, or low levels of PD-1 is conducted by using one of the assays described herein.

[0041] Potency data for the multispecific binding sites provided herein are obtained using the phospho-SMAD2 / 3 assay described in Examples 4 and 5, and the isogenic HEK-BLUE-PD-1 TGF-β reporter assay described in Example 7. Thus, in certain embodiments, potency in inhibiting TGF-βRII-mediated signaling is measured in the phospho-SMAD2 / 3 assay described in Example 4 or Example 5.

[0042] Briefly, the phosphorylated SMAD2 / 3 assay described in Example 4 was performed using Jurkat-PD-1 cells cultured in RPMI / 10% FBS. null Jurkat-PD-1 cells + The assay is performed using cells. Cells are incubated with test or control antibodies at six serial dilutions (100 μg / ml to 0.001 μg / ml) for 1 hour at 37°C / 5% CO2. After 1 hour, human recombinant TGF-β1 is added to a final concentration of 10 ng / ml, and the cells are incubated for an additional 2 hours at 37°C / 5% CO2. After incubation, cells are gently washed with PBS. Cell lysates are prepared using a lysis buffer containing phosphatase inhibitors and protease inhibitors. Phosphorylated SMAD2 / 3 levels are determined using ELISA.

[0043] Briefly, the phosphorylated SMAD2 / 3 assay described in Example 5 was performed using PBMCs from healthy donors, which were stimulated with 1 μg / ml anti-CD3 for 48 hours, followed by serum deprivation in 0.1% FBS for 16 hours. Stimulated and unstimulated PBMCs were incubated with test and control antibodies for 30 minutes at room temperature. Recombinant human TGF-β1 was added at a final concentration of 1 ng / ml, and the cells were incubated for an additional 30 minutes. Finally, the cells were washed twice with PBS and stained for cell surface markers, followed by intracellular phosphorylated SMAD2 staining. Flow cytometry was performed to measure the expression of CD4 + and CD8 + This is done to gate the cells, and the phosphorylated SMAD2 signal is measured as geometric mean fluorescence intensity (GMFI) on these cells.

[0044] In one embodiment, potency in inhibiting TGF-βRII-mediated signaling is measured in an isogenic HEK-BLUE-PD-1 TGF-β reporter assay as described in Example 7.

[0045] Briefly, the HEK-BLUE-PD-1 TGF-β reporter assay utilizes HEK-BLUE™ TGF-β cells and HEK-BLUE™ TGF-β-PD-1 + The incubation is performed at 25,000 cells per well using QUANTI-Blue™ solution. Serial dilutions of test and control antibodies are added and the cells are incubated for 1 hour at room temperature, followed by the addition of human recombinant TGF-β1 at a final concentration of 1 ng / ml. The cells are incubated overnight at 37°C / 5% CO2. After incubation, 40 ul of the supernatant and 160 ul of resuspended QUANTI-Blue™ solution are incubated for 40 minutes at 37°C / 5% CO2. The amount of SEAP secreted into the supernatant is assessed using QUANTI-Blue™ solution. SEAP levels are determined at 650 nm using a spectrophotometer.

[0046] In certain embodiments, a multispecific binding site of the present disclosure is at least about 10-fold, preferably about 10-100,000-fold more potent in inhibiting TGF-βRII-mediated signaling in cells expressing both PD-1 and TGF-βRII than in cells expressing TGF-βRII but not PD-1. In certain embodiments, a multispecific binding site of the present disclosure is at least 10-fold, preferably about 10-100,000-fold more potent in inhibiting TGF-βRII-mediated signaling in cells expressing both PD-1 and TGF-βRII than in cells expressing TGF-βRII but not PD-1. In certain embodiments, potency in inhibiting TGF-βRII-mediated signaling is determined by IC50 (μg / ml) in a phosphorylated SMAD2 / 3 assay or a HEK-BLUE-PD-1 TGF-β reporter assay. In one embodiment, potency in inhibiting TGF-βRII-mediated signaling is determined by IC50 (μg / ml) in a phosphorylated SMAD2 / 3 assay. In one embodiment, potency in inhibiting TGF-βRII-mediated signaling is determined by IC50 (μg / ml) in a HEK-BLUE-PD-1 TGF-β reporter assay.

[0047] In certain embodiments, the potency of a multispecific binding site of the present disclosure in inhibiting TGF-βRII-mediated signaling in cells that express TGF-βRII and no, substantially no, or low levels of PD-1 is lower than the potency of a reference anti-TGF-βRII antibody targeting the same cells, and the potency of a multispecific binding site in inhibiting TGF-βRII-mediated signaling in cells that express both TGF-βRII and PD-1 is higher than the potency of a reference anti-TGF-βRII antibody targeting the same cells, wherein 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:76 and a light chain having the amino acid sequence set forth in SEQ ID NO:77.

[0048] In one embodiment, the cells expressing both PD-1 and TGF-βRII are Jurkat-PD-1 + cells, stimulated CD4 + or CD8 + cells, or HEK-Blue™ TGF-β-PD-1 + cells that express TGF-βRII and do not express or substantially do not express PD-1; null cells or HEK-Blue™ TGF-β cells; and cells expressing TGF-βRII and low levels of PD-1 are unstimulated CD4 + or CD8 + cells, as further described herein.

[0049] In certain embodiments, the potency of a multispecific binding site of the present disclosure in inhibiting TGF-βRII-mediated signaling in cells expressing both TGF-βRII and PD-1 is at least 10-fold, preferably 10-100,000-fold, higher than that of a reference anti-TGF-βRII antibody. In certain embodiments, the potency of a multispecific binding site of the present disclosure in inhibiting TGF-βRII-mediated signaling in cells expressing both TGF-βRII and PD-1 is at least about 10-fold, preferably about 10-100,000-fold higher than that of a reference anti-TGF-βRII antibody. In certain embodiments, the reference TGF-βRII antibody is a bivalent monospecific antibody comprising a heavy chain having the amino acid sequence set forth in SEQ ID NO: 76 and a light chain having the amino acid sequence set forth in SEQ ID NO: 77. In certain embodiments, potency in inhibiting TGF-βRII-mediated signaling is determined as IC50 (μg / ml) in a phosphorylated SMAD2 / 3 assay.

[0050] In certain embodiments, the multispecific binding site of the present disclosure has greater activity in reducing tumor volume than a reference antibody combination. In certain embodiments, the reference antibody combination is two bivalent monospecific antibodies targeting PD-1 and TGF-βRII, wherein the bivalent monospecific antibody targeting PD-1 comprises a heavy chain having the amino acid sequence set forth in SEQ ID NO:78 and a light chain having the amino acid sequence set forth in SEQ ID NO:79, and the bivalent monospecific antibody targeting TGF-βRII comprises a heavy chain having the amino acid sequence set forth in SEQ ID NO:76 and a light chain having the amino acid sequence set forth in SEQ ID NO:77.

[0051] Accordingly, the present disclosure also provides a multispecific binding site comprising a PD-1-binding domain and a TGF-βRII-binding domain, wherein the multispecific binding site has greater activity in reducing tumor volume than a reference antibody combination. In one embodiment, the multispecific binding site is administered with a number of PD-1 and TGF-βRII binding domains that is 2-fold lower and up to 20-fold lower than each of the bivalent monospecific antibodies of the reference antibody combination. For example, a multispecific binding site that is monovalent for binding to PD-1 and monovalent for binding to TGF-βRII, when administered at 1 mg / kg, has greater activity in reducing tumor volume than a reference antibody combination that is each bivalent for binding to PD-1 or TGF-βRII and each administered at 10 mg / kg. Additionally, a multispecific binding site that is monovalent for binding to PD-1 and monovalent for binding to TGF-βRII, when administered at 10 mg / kg, has greater activity in reducing tumor volume than a combination of reference antibodies that are each bivalent for binding to PD-1 or TGF-βRII and each administered at 10 mg / kg.

[0052] In one embodiment, the reference antibody combination is two bivalent monospecific antibodies targeting PD-1 and TGF-βRII, where the bivalent monospecific antibody targeting PD-1 comprises a heavy chain having the amino acid sequence set forth in SEQ ID NO: 78 and a light chain having the amino acid sequence set forth in SEQ ID NO: 79, and the bivalent monospecific antibody targeting TGF-βRII comprises a heavy chain having the amino acid sequence set forth in SEQ ID NO: 76 and a light chain having the amino acid sequence set forth in SEQ ID NO: 77.

[0053] In one embodiment, activity in reducing tumor volume is determined by measuring the reduction in tumor volume in an in vivo mouse study, specifically in an in vivo mouse study using MDA-MB-231 xenograft huCD34 NSG mice.

[0054] In certain embodiments, the multispecific binding sites of the present disclosure result in a reduction in tumor volume that is at least 1.5-fold, preferably between 1.5-100-fold, or 2-80-fold, or 5-80-fold, or 10-80-fold, or 15-80-fold, or 20-80-fold, or 30-80-fold, or 40-80-fold, or 50-80-fold, or 2-60-fold, or 5-60-fold, or 10-60-fold, or 15-60-fold, or 20-60-fold, or 30-60-fold, or 40-60-fold, that of a reference antibody combination. In certain embodiments, the multispecific binding sites of the present disclosure provide a tumor volume reduction that is at least about 1.5-fold, preferably between approximately 1.5-100-fold, or 2-80-fold, or 5-80-fold, or 10-80-fold, or 15-80-fold, or 20-80-fold, or 30-80-fold, or 40-80-fold, or 50-80-fold, or 2-60-fold, or 5-60-fold, or 10-60-fold, or 15-60-fold, or 20-60-fold, or 30-60-fold, or 40-60-fold, that of the reference antibody combination. In other words, the multispecific binding sites of the present disclosure provide a tumor volume reduction that is greater than the reference antibody combination. In one embodiment, the reference antibody combination is two bivalent monospecific antibodies targeting PD-1 and TGF-βRII, where the bivalent monospecific antibody targeting PD-1 comprises a heavy chain having the amino acid sequence set forth in SEQ ID NO: 78 and a light chain having the amino acid sequence set forth in SEQ ID NO: 79, and the bivalent monospecific antibody targeting TGF-βRII comprises a heavy chain having the amino acid sequence set forth in SEQ ID NO: 76 and a light chain having the amino acid sequence set forth in SEQ ID NO: 77.

[0055] In certain embodiments, the multispecific binding sites of the present disclosure reduce tumor volume when administered as a single agent.

[0056] Accordingly, the present disclosure also provides a multispecific binding moiety comprising a PD-1-binding domain and a TGF-βRII-binding domain, wherein the multispecific binding moiety induces a reduction in tumor volume as a single agent.

[0057] In certain embodiments, the present disclosure provides several PD-1 x TGF-βRII bispecific antibodies as exemplary multispecific binding sites, wherein the PD-1 binding domain comprises a heavy chain variable region having an amino acid sequence selected from SEQ ID NOs: 1; 5; 9; 13; 14; 18, and 19, the TGF-βRII binding domain comprises a heavy chain variable region having an amino acid sequence selected from SEQ ID NOs: 23; 27; 31; 35; 39; 43; 47; 88; and 89, and both the PD-1 and TGF-βRII binding domains comprise the same light chain.

[0058] In one embodiment, the PD-1 binding domain of a multispecific binding site 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:2, SEQ ID NO:3, and SEQ ID NO:4, 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:6, SEQ ID NO:7, and SEQ ID NO:8, 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: 10, SEQ ID NO: 11, and SEQ ID NO: 12, respectively; d) 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: 15, SEQ ID NO: 16, and SEQ ID NO: 17, respectively; or e) 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.

[0059] The heavy chain variable region of the PD-1 binding domain of a multispecific binding site of the 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.

[0060] In some embodiments, the PD-1-binding domain of the multispecific binding site of the present disclosure also includes variants thereof, in which each of the HCDRs may contain at most three, two, or one amino acid variations. In some embodiments, only one or two HCDRs may contain at most three, two, or one non-conservative amino acid variations. In some embodiments, such variants do not include amino acid variations in HCDR3. In some embodiments, the amino acid variations are conservative amino acid substitutions.

[0061] Typically, conservative amino acid substitutions involve amino acid variations with homologous amino acid residues (residues that share similar characteristics or properties). Homologous amino acids are known in the art, as are conventional 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), which are incorporated herein in their entirety. 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).

[0062] 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 variation does not, or does not substantially, impair the binding specificity of the binding domain for its intended target.

[0063] Further types of amino acid variations include those resulting from somatic hypermutation or affinity maturation. PD-1-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 which have amino acid variations, including non-conservative and / or conservative amino acid substitutions, in one, two, or all three HCDRs. Routine 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).

[0064] Examples of suitable positions for introducing an amino acid variation include, but are not limited to, the first, second, and / or fourth amino acid of HCDR1; the third, seventh, eighth, ninth, tenth, eleventh, thirteenth, fourteenth, and / or sixteenth amino acid of HCDR2; and / or the sixth and / or thirteenth amino acid of HCDR3.

[0065] In certain embodiments, the present disclosure therefore also provides a multispecific binding site, the PD-1 binding domain of which comprises: - an HCDR1 having the amino acid sequence X1X2FX3S, X1 may be F, Y, T, or H; X2 may be Y, Q, E, H, or D; X3 may be W or Y; and / or - an HCDR2 having the amino acid sequence YIX1YSGX2X3X4X5X6PX7X8KX9, X1 may be Y, V, or I; X2 may be S or G; X3 may be T, Y, S, H, N, W, L, or Q; X4 may be S or N; X5 may be F, V, or L; X6 may be N or S; X7 may be S or A; X8 may be F or L; X9 may be S, T, G, D, R, or N; and / or - an HCDR3 having the amino acid sequence GGYTGX1GGDWFDX2, X1 may be Y, H, V, or A; X2 may be P, V, Y, W, F, T, Q, H, or S.

[0066] Other suitable positions for introducing an amino acid variation include, but are not limited to, the second, third, fourth, and / or fifth amino acids of HCDR1; the third, fourth, fifth, sixth, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth, sixteenth, and / or seventeenth amino acids of HCDR2; and / or the first, second, sixth, seventh, ninth, tenth, fourteenth, fifteenth, sixteenth, and / or eighteenth amino acids of HCDR3.

[0067] In certain embodiments, the present disclosure therefore also provides a multispecific binding site, the PD-1 binding domain of which comprises: - an HCDR1 having the amino acid sequence RX1X2X3X4, X1 may be F or Y; X2 may be T, A, or V; X3 may be M, L, or V; X4 may be S, H, N, V, or T; and / or - Amino acid sequence WIX1X2X3X4GX5X6X7X8X9X 10 X11 X 12 X 13 X 14 and an HCDR2 having X1 may be N or D; X2 may be P, S, or T; X3 may be N or Q; X4 may be T or D; X5 may be N, S, T, K, L, or E; X6 may be P, Y, A, H, or F; X7 may be T or S; X8 may be Y, F, or H; X9 may be A, G, V, or F; X 10 may be Q, R, N, L, T, or S; X 11 may be D, A, G, or S; X 12 may be F, V, or A; X 13 may be T, K, H, G; X 14 may be G, N, E, or D; and / or an HCDR3 having the amino acid sequence X1X2GYCX3X4DX5CYPNX6X7X8DX9, X1 may be I, S, or V; X2 may be L, Q, or N; X3 may be N, G, S, or D; X4 may be T, S, P, N, or E; X5 may be N or I; X6 may be W, G, Q, H, W, A, or L; X7 may be I, V, or L; X8 may be F, L, or I; X9 may be Y, S, N, I, R, H, V, T, K, A, or L.

[0068] In certain embodiments, the PD-1-binding domain of a multispecific binding site of the present disclosure comprises a heavy chain variable region having the amino acid sequence set forth in any one of SEQ ID NOs: 1; 5; 9; 13; 14; 18; 19, or a variant thereof. In certain embodiments, the PD-1-binding domain of a multispecific binding site of the present disclosure comprises a heavy chain variable region having the amino acid sequence set forth in any one of SEQ ID NOs: 1; 5; 9; 13; 14; 18; 19, or a variant having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity thereto.

[0069] 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 can be introduced into either of the two sequences being compared to optimize alignment between the two sequences. Such alignments can be performed over the entire length of the sequences being compared. Alternatively, alignments can be performed over shorter lengths, 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.

[0070] 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 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 the present invention, 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.

[0071] 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.

[0072] In certain embodiments, the PD-1-binding domain of a multispecific binding site of the present disclosure also comprises a PD-1-binding domain variant, which comprises one or more variations in the framework regions in addition to the variations in the HCDRs described above. The variations can be any type of amino acid variation described herein, such as conservative or non-conservative amino acid substitutions resulting from somatic hypermutation or affinity maturation. In certain embodiments, the PD-1-binding domain variant of a multispecific binding site of the present disclosure does not comprise variations in the CDR regions but comprises one or more variations in the framework regions. 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 PD-1 binding specificity. Thus, in certain embodiments, the PD-1-binding domain of a multispecific binding site 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: 1; The heavy chain variable region comprises the HCDR1 amino acid sequence set forth in SEQ ID NO:2; the HCDR2 amino acid sequence set forth in SEQ ID NO:3; and the HCDR3 amino acid sequence set forth in SEQ ID NO:4; - 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: 5; The heavy chain variable region comprises the HCDR1 amino acid sequence set forth in SEQ ID NO:6; the HCDR2 amino acid sequence set forth in SEQ ID NO:7; and the HCDR3 amino acid sequence set forth in SEQ ID NO:8; - 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: 9; The heavy chain variable region comprises the HCDR1 amino acid sequence set forth in SEQ ID NO: 10; the HCDR2 amino acid sequence set forth in SEQ ID NO: 11; and the HCDR3 amino acid sequence set forth in SEQ ID NO: 12; - 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: 13; The heavy chain variable region comprises the HCDR1 amino acid sequence set forth in SEQ ID NO: 10; the HCDR2 amino acid sequence set forth in SEQ ID NO: 11; and the HCDR3 amino acid sequence set forth in SEQ ID NO: 12; - 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: 14; The heavy chain variable region comprises the HCDR1 amino acid sequence set forth in SEQ ID NO: 15; the HCDR2 amino acid sequence set forth in SEQ ID NO: 16; and the HCDR3 amino acid sequence set forth in SEQ ID NO: 17; - 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: 18; The heavy chain variable region comprises the HCDR1 amino acid sequence set forth in SEQ ID NO: 15; the HCDR2 amino acid sequence set forth in SEQ ID NO: 16; and the HCDR3 amino acid sequence set forth in SEQ ID NO: 17; 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; The heavy chain variable region comprises the HCDR1 amino acid sequence shown in SEQ ID NO:20; the HCDR2 amino acid sequence shown in SEQ ID NO:21; and the HCDR3 amino acid sequence shown in SEQ ID NO:22.

[0073] The binding domains of the multispecific binding sites of the present disclosure are generated using a common light chain, specifically a common light chain designated VK1-39 / JK1. The binding domains of the multispecific binding sites of the present 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 multispecific binding sites of the present disclosure comprise the common light chain VK1-39 / JK1 or variants thereof harboring a limited number of non-conservative amino acid substitutions, e.g., one, two, or three, or an unlimited number of conservative amino acid substitutions.

[0074] In certain embodiments, the PD-1-binding domain of a multispecific binding site of the present disclosure comprises a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 48, or a variant thereof. In certain embodiments, the PD-1-binding domain of a multispecific binding site of the present disclosure comprises a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 48, or a variant having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity thereto.

[0075] In one embodiment, the PD-1-binding domain of a multispecific binding site 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:49, SEQ ID NO:50, and SEQ ID NO:51. In one embodiment, the light chain variable region of the PD-1-binding domain of a multispecific binding site of the present disclosure also comprises variants thereof, wherein each of the LCDRs may comprise at most three, two, or one amino acid variations. In one embodiment, the amino acid variations are conservative amino acid substitutions.

[0076] In certain embodiments, the PD-1-binding domain of a multispecific binding site of the present disclosure also comprises a PD-1-binding domain variant, which, in addition to the variations in the LCDRs described above, comprises one or more variations in the framework regions. The variations are preferably conservative amino acid substitutions. In certain embodiments, the PD-1-binding domain variant of a multispecific binding site of the present disclosure does not comprise variations in the LCDR regions but comprises one or more variations 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. Thus, in certain embodiments, the PD-1-binding domain of a multispecific binding site 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: 48; The light chain variable region comprises the LCDR1 amino acid sequence set forth in SEQ ID NO:49; the LCDR2 amino acid sequence set forth in SEQ ID NO:50; and the LCDR3 amino acid sequence set forth in SEQ ID NO:51.

[0077] 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 within the definition of a common light chain used herein to prepare or find light chains that are not identical but are still functionally equivalent by using well-established variations 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.

[0078] 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 a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 52. 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: 52, wherein each of the LCDRs may contain at most three, two, or one amino acid variations, e.g., substitutions. In one embodiment, the light chain comprises a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 52, 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:55; 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: 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 a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 56, wherein each of the LCDRs may contain at most three, two, or one amino acid variations, e.g., substitutions. 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:57, SEQ ID NO:58, and SEQ ID NO:59; 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: 60. 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: 60, wherein each of the LCDRs may comprise at most three, two, or one amino acid variations, e.g., substitutions. In one embodiment, the light chain comprises a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 60, 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: 61, SEQ ID NO: 62, and SEQ ID NO: 63; 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: 64. 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: 64, wherein each of the LCDRs may contain at most three, two, or one amino acid variations, e.g., substitutions. In one embodiment, the light chain comprises a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 64, 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:65, SEQ ID NO:66, and SEQ ID NO:67.

[0079] 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 given as SEQ ID NO:93. 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:94) and VK1-39 / JK5 (SEQ ID NO:95); 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.

[0080] 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 given as SEQ ID NO:98. 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 designated as VK3-15 / JK1 (SEQ ID NO:99); 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.

[0081] 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 as SEQ ID NO:100. 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:101); 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.

[0082] 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 ID of this gene is HGNC:5905; Entrez Gene:28796; Ensembl:ENSG00000211662.2. The amino acid sequence of VL3-21 is given as SEQ ID NO:102. 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 designated as VL3-21 / JL3 (SEQ ID NO:103); 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.

[0083] Furthermore, any light chain variable region of a PD-1 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 virtue of exhibiting antigen-binding activity when paired with the PD-1 binding domain of a multispecific binding site of the present disclosure.

[0084] In certain embodiments, the PD-1 binding domain of the multispecific binding site 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 provided as SEQ ID NO: 69. Any CL domain, particularly a human CL domain, may be used. An example of a suitable CL domain is provided by the amino acid sequence provided as SEQ ID NO: 75.

[0085] In certain embodiments, the TGF-βRII binding domain of the multispecific binding site 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: 28, 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: 32, SEQ ID NO: 33, and SEQ ID NO: 34, respectively; 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: 36, SEQ ID NO: 37, and SEQ ID NO: 38, respectively; e) heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 40, SEQ ID NO: 41, and SEQ ID NO: 42, respectively; f) 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: 44, SEQ ID NO: 45, and SEQ ID NO: 46, respectively; or g) Heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 90, SEQ ID NO: 91, and SEQ ID NO: 92, respectively.

[0086] The heavy chain variable region of the TGF-βRII binding domain of the multispecific binding site 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.

[0087] In some embodiments, the TGF-βRII binding domain of the multispecific binding site of the present disclosure also includes its TGF-βRII binding domain variant, wherein each of the HCDRs may contain at most three, two, or one amino acid variation. In some embodiments, only one or two HCDRs may contain at most three, two, or one amino acid variation. In some embodiments, such variants do not contain amino acid variation in HCDR3. In some embodiments, the amino acid variation is a conservative amino acid substitution. Conservative amino acid substitutions are further described herein.

[0088] TGF-βRII binding variants encompassed by the present disclosure include heavy chain variable regions that have been somatically hypermutated or affinity matured, which heavy chain variable regions are derived from the same VH gene segment as the heavy chain variable regions described by sequence herein, and which variants have amino acid variations, including non-conservative and / or conservative amino acid substitutions, in one, two, or all three HCDRs.

[0089] In certain embodiments, the TGF-βRII binding domain of a multispecific binding site 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; 35; 39; 43; 47; 88; 89, or a variant thereof. In certain embodiments, the TGF-βRII binding domain of a multispecific binding site 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; 35; 39; 43; 47; 88; 89, or having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity thereto.

[0090] In some embodiments, the TGF-βRII binding domain of the multispecific binding site of the present disclosure also comprises a TGF-βRII binding domain variant, which comprises one or more variations in the framework regions in addition to the variations in the HCDRs described above. The variations can be any type of amino acid variation described herein, such as conservative or non-conservative amino acid substitutions resulting from somatic hypermutation or affinity maturation. In some embodiments, the TGF-βRII binding domain variant of the multispecific binding site of the present disclosure does not comprise variations in the CDR regions but comprises one or more variations in the framework regions. 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 some embodiments, the TGF-βRII binding domain of the multispecific binding site 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; The heavy chain variable region comprises the HCDR1 amino acid sequence set forth in SEQ ID NO:24; the HCDR2 amino acid sequence set forth in SEQ ID NO:25; and the 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; The heavy chain variable region comprises the HCDR1 amino acid sequence set forth in SEQ ID NO:28; the HCDR2 amino acid sequence set forth in SEQ ID NO:29; and the 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; The heavy chain variable region comprises the HCDR1 amino acid sequence set forth in SEQ ID NO: 32; the HCDR2 amino acid sequence set forth in SEQ ID NO: 33; and the HCDR3 amino acid sequence set forth in SEQ ID NO: 34; - 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; The heavy chain variable region comprises the HCDR1 amino acid sequence set forth in SEQ ID NO:36; the HCDR2 amino acid sequence set forth in SEQ ID NO:37; and the HCDR3 amino acid sequence set forth in SEQ ID NO:38; - 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: 39; The heavy chain variable region comprises the HCDR1 amino acid sequence set forth in SEQ ID NO:40; the HCDR2 amino acid sequence set forth in SEQ ID NO:41; and the HCDR3 amino acid sequence set forth in SEQ ID NO:42; - 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: 43; The heavy chain variable region comprises the HCDR1 amino acid sequence set forth in SEQ ID NO:44; the HCDR2 amino acid sequence set forth in SEQ ID NO:45; and the HCDR3 amino acid sequence set forth in SEQ ID NO:46; - 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: 47; The heavy chain variable region comprises the HCDR1 amino acid sequence set forth in SEQ ID NO:90; the HCDR2 amino acid sequence set forth in SEQ ID NO:91; and the HCDR3 amino acid sequence set forth in SEQ ID NO:92; - 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: 88; The heavy chain variable region comprises the HCDR1 amino acid sequence set forth in SEQ ID NO:44; the HCDR2 amino acid sequence set forth in SEQ ID NO:45; and the HCDR3 amino acid sequence set forth in SEQ ID NO:46; 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: 89; The heavy chain variable region comprises the HCDR1 amino acid sequence set forth in SEQ ID NO:90; the HCDR2 amino acid sequence set forth in SEQ ID NO:91; and the HCDR3 amino acid sequence set forth in SEQ ID NO:92.

[0091] 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 is readily available from, e.g., an antibody display library, by exhibiting antigen-binding activity when paired with a TGF-βRII binding domain of a multispecific binding site of the present disclosure. In one embodiment, the TGF-βRII binding domain of a multispecific binding site of the present disclosure comprises a light chain that is identical or substantially identical to the PD-1 binding domain.

[0092] In certain embodiments, the TGF-βRII binding domain of a multispecific binding site of the present disclosure comprises a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 48, or a variant thereof. In certain embodiments, the TGF-βRII binding domain of a multispecific binding site of the present disclosure comprises a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 48, or a variant having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity thereto.

[0093] In certain embodiments, the TGF-βRII binding domain of a multispecific binding site 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: 49, SEQ ID NO: 50, and SEQ ID NO: 51, respectively. In certain embodiments, the light chain variable region of the TGF-βRII binding domain of a multispecific binding site 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 variations. In certain embodiments, the amino acid variations are conservative amino acid substitutions.

[0094] In certain embodiments, the TGF-βRII binding domain of the multispecific binding site 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 provided as SEQ ID NO: 69. Any CL domain, particularly a human CL, may be used. An example of a suitable CL domain is provided by the amino acid sequence provided as SEQ ID NO: 75.

[0095] Accordingly, the present invention also provides a multispecific binding site comprising a PD-1-binding domain and a TGF-βRII-binding domain, wherein the PD-1-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 multispecific binding site 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.

[0096] Accordingly, the present invention also provides a multispecific binding site comprising a PD-1-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 multispecific binding site further comprises a PD-1-binding domain, which comprises a heavy chain variable region, and optionally a light chain variable region and CH1 and CL regions, as described herein.

[0097] In certain embodiments, any PD-1 binding domain disclosed herein can be combined with any TGF-βRII binding domain disclosed herein to produce a multispecific binding site of the present disclosure. Accordingly, the present disclosure provides exemplary multispecific binding sites PB1-PB18, as shown in Table 1.

[0098] In one embodiment, the present disclosure provides a multispecific binding site comprising: - a PD-1 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: 10, SEQ ID NO: 11, and SEQ ID NO: 12, 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 each of the HCDRs may contain at most three, two, or one amino acid variations, e.g., substitutions. In certain embodiments, the HCDRs contain no amino acid variations.

[0099] In one embodiment, the present disclosure provides a multispecific binding site comprising: - a PD-1 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: 15, SEQ ID NO: 16, and SEQ ID NO: 17, 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 each of the HCDRs may contain at most three, two, or one amino acid variations, e.g., substitutions. In certain embodiments, the HCDRs contain no amino acid variations.

[0100] In one embodiment, the present disclosure provides a multispecific binding site comprising: - a PD-1 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: 25, and SEQ ID NO: 26, respectively; wherein each of the HCDRs may contain at most three, two, or one amino acid variations, e.g., substitutions. In certain embodiments, the HCDRs contain no amino acid variations.

[0101] In one embodiment, the present disclosure provides a multispecific binding site comprising: - a PD-1 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: 15, SEQ ID NO: 16, and SEQ ID NO: 17, 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: 32, SEQ ID NO: 33, and SEQ ID NO: 34, respectively; wherein each of the HCDRs may contain at most three, two, or one amino acid variations, e.g., substitutions. In certain embodiments, the HCDRs contain no amino acid variations.

[0102] In one embodiment, the present disclosure provides a multispecific binding site comprising: - a PD-1 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: 10, SEQ ID NO: 11, and SEQ ID NO: 12, 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: 40, SEQ ID NO: 41, and SEQ ID NO: 42, respectively; wherein each of the HCDRs may contain at most three, two, or one amino acid variations, e.g., substitutions. In certain embodiments, the HCDRs contain no amino acid variations.

[0103] In one embodiment, the present disclosure provides a multispecific binding site comprising: - a PD-1 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: 10, SEQ ID NO: 11, and SEQ ID NO: 12, 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: 36, SEQ ID NO: 37, and SEQ ID NO: 38, respectively; wherein each of the HCDRs may contain at most three, two, or one amino acid variations, e.g., substitutions. In certain embodiments, the HCDRs contain no amino acid variations.

[0104] In one embodiment, the present disclosure provides a multispecific binding site comprising: - a PD-1 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: 15, SEQ ID NO: 16, and SEQ ID NO: 17, 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: 36, SEQ ID NO: 37, and SEQ ID NO: 38, respectively; wherein each of the HCDRs may contain at most three, two, or one amino acid variations, e.g., substitutions. In certain embodiments, the HCDRs contain no amino acid variations.

[0105] In one embodiment, the present disclosure provides a multispecific binding site comprising: - a PD-1 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: 36, SEQ ID NO: 37, and SEQ ID NO: 38, respectively; wherein each of the HCDRs may contain at most three, two, or one amino acid variations, e.g., substitutions. In certain embodiments, the HCDRs contain no amino acid variations.

[0106] In one embodiment, the present disclosure provides a multispecific binding site comprising: - a PD-1 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: 10, SEQ ID NO: 11, and SEQ ID NO: 12, 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: 28, SEQ ID NO: 29, and SEQ ID NO: 30, respectively; wherein each of the HCDRs may contain at most three, two, or one amino acid variations, e.g., substitutions. In certain embodiments, the HCDRs contain no amino acid variations.

[0107] In one embodiment, the present disclosure provides a multispecific binding site comprising: - a PD-1 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: 10, SEQ ID NO: 11, and SEQ ID NO: 12, 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: 90, SEQ ID NO: 91, and SEQ ID NO: 92, respectively; wherein each of the HCDRs may contain at most three, two, or one amino acid variations, e.g., substitutions. In certain embodiments, the HCDRs contain no amino acid variations.

[0108] In one embodiment, the present disclosure provides a multispecific binding site comprising: - a PD-1 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: 44, SEQ ID NO: 45, and SEQ ID NO: 46, respectively; wherein each of the HCDRs may contain at most three, two, or one amino acid variations, e.g., substitutions. In certain embodiments, the HCDRs contain no amino acid variations.

[0109] In one embodiment, the present disclosure provides a multispecific binding site comprising: - a PD-1 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: 10, SEQ ID NO: 11, and SEQ ID NO: 12, 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 PD-1-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: 49, a light chain CDR2 (LCDR2) having the amino acid sequence set forth in SEQ ID NO: 50, and a light chain CDR3 (LCDR3) having the amino acid sequence set forth in SEQ ID NO: 51; and wherein each of the HCDRs and / or LCDRs may contain at most three, two, or one amino acid variations, such as substitutions. In some embodiments, the HCDRs and / or LCDRs contain no amino acid variations.

[0110] In one embodiment, the present disclosure provides a multispecific binding site comprising: - a PD-1 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: 15, SEQ ID NO: 16, and SEQ ID NO: 17, 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 PD-1-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: 49, a light chain CDR2 (LCDR2) having the amino acid sequence set forth in SEQ ID NO: 50, and a light chain CDR3 (LCDR3) having the amino acid sequence set forth in SEQ ID NO: 51; and wherein each of the HCDRs and / or LCDRs may contain at most three, two, or one amino acid variations, such as substitutions. In some embodiments, the HCDRs and / or LCDRs contain no amino acid variations.

[0111] In one embodiment, the present disclosure provides a multispecific binding site comprising: - a PD-1 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: 25, and SEQ ID NO: 26, respectively; wherein the PD-1-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: 49, a light chain CDR2 (LCDR2) having the amino acid sequence set forth in SEQ ID NO: 50, and a light chain CDR3 (LCDR3) having the amino acid sequence set forth in SEQ ID NO: 51; and wherein each of the HCDRs and / or LCDRs may contain at most three, two, or one amino acid variations, such as substitutions. In some embodiments, the HCDRs and / or LCDRs contain no amino acid variations.

[0112] In one embodiment, the present disclosure provides a multispecific binding site comprising: - a PD-1 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: 15, SEQ ID NO: 16, and SEQ ID NO: 17, 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: 32, SEQ ID NO: 33, and SEQ ID NO: 34, respectively; wherein the PD-1-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: 49, a light chain CDR2 (LCDR2) having the amino acid sequence set forth in SEQ ID NO: 50, and a light chain CDR3 (LCDR3) having the amino acid sequence set forth in SEQ ID NO: 51; and wherein each of the HCDRs and / or LCDRs may contain at most three, two, or one amino acid variations, such as substitutions. In some embodiments, the HCDRs and / or LCDRs contain no amino acid variations.

[0113] In one embodiment, the present disclosure provides a multispecific binding site comprising: - a PD-1 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: 10, SEQ ID NO: 11, and SEQ ID NO: 12, 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: 40, SEQ ID NO: 41, and SEQ ID NO: 42, respectively; wherein the PD-1-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: 49, a light chain CDR2 (LCDR2) having the amino acid sequence set forth in SEQ ID NO: 50, and a light chain CDR3 (LCDR3) having the amino acid sequence set forth in SEQ ID NO: 51; and wherein each of the HCDRs and / or LCDRs may contain at most three, two, or one amino acid variations, such as substitutions. In some embodiments, the HCDRs and / or LCDRs contain no amino acid variations.

[0114] In one embodiment, the present disclosure provides a multispecific binding site comprising: - a PD-1 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: 10, SEQ ID NO: 11, and SEQ ID NO: 12, 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: 36, SEQ ID NO: 37, and SEQ ID NO: 38, respectively; wherein the PD-1-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: 49, a light chain CDR2 (LCDR2) having the amino acid sequence set forth in SEQ ID NO: 50, and a light chain CDR3 (LCDR3) having the amino acid sequence set forth in SEQ ID NO: 51; and wherein each of the HCDRs and / or LCDRs may contain at most three, two, or one amino acid variations, such as substitutions. In some embodiments, the HCDRs and / or LCDRs contain no amino acid variations.

[0115] In one embodiment, the present disclosure provides a multispecific binding site comprising: - a PD-1 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: 15, SEQ ID NO: 16, and SEQ ID NO: 17, 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: 36, SEQ ID NO: 37, and SEQ ID NO: 38, respectively; wherein the PD-1-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: 49, a light chain CDR2 (LCDR2) having the amino acid sequence set forth in SEQ ID NO: 50, and a light chain CDR3 (LCDR3) having the amino acid sequence set forth in SEQ ID NO: 51; and wherein each of the HCDRs and / or LCDRs may contain at most three, two, or one amino acid variations, such as substitutions. In some embodiments, the HCDRs and / or LCDRs contain no amino acid variations.

[0116] In one embodiment, the present disclosure provides a multispecific binding site comprising: - a PD-1 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: 36, SEQ ID NO: 37, and SEQ ID NO: 38, respectively; wherein the PD-1-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: 49, a light chain CDR2 (LCDR2) having the amino acid sequence set forth in SEQ ID NO: 50, and a light chain CDR3 (LCDR3) having the amino acid sequence set forth in SEQ ID NO: 51; and wherein each of the HCDRs and / or LCDRs may contain at most three, two, or one amino acid variations, such as substitutions. In some embodiments, the HCDRs and / or LCDRs contain no amino acid variations.

[0117] In one embodiment, the present disclosure provides a multispecific binding site comprising: - a PD-1 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: 10, SEQ ID NO: 11, and SEQ ID NO: 12, 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: 28, SEQ ID NO: 29, and SEQ ID NO: 30, respectively; wherein the PD-1-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: 49, a light chain CDR2 (LCDR2) having the amino acid sequence set forth in SEQ ID NO: 50, and a light chain CDR3 (LCDR3) having the amino acid sequence set forth in SEQ ID NO: 51; and wherein each of the HCDRs and / or LCDRs may contain at most three, two, or one amino acid variations, such as substitutions. In some embodiments, the HCDRs and / or LCDRs contain no amino acid variations.

[0118] In one embodiment, the present disclosure provides a multispecific binding site comprising: - a PD-1 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: 10, SEQ ID NO: 11, and SEQ ID NO: 12, 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: 90, SEQ ID NO: 91, and SEQ ID NO: 92, respectively; wherein the PD-1-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: 49, a light chain CDR2 (LCDR2) having the amino acid sequence set forth in SEQ ID NO: 50, and a light chain CDR3 (LCDR3) having the amino acid sequence set forth in SEQ ID NO: 51; and wherein each of the HCDRs and / or LCDRs may contain at most three, two, or one amino acid variations, such as substitutions. In some embodiments, the HCDRs and / or LCDRs contain no amino acid variations.

[0119] In one embodiment, the present disclosure provides a multispecific binding site comprising: - a PD-1 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: 44, SEQ ID NO: 45, and SEQ ID NO: 46, respectively; wherein the PD-1-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: 49, a light chain CDR2 (LCDR2) having the amino acid sequence set forth in SEQ ID NO: 50, and a light chain CDR3 (LCDR3) having the amino acid sequence set forth in SEQ ID NO: 51; and wherein each of the HCDRs and / or LCDRs may contain at most three, two, or one amino acid variations, such as substitutions. In some embodiments, the HCDRs and / or LCDRs contain no amino acid variations.

[0120] In one embodiment, the present disclosure provides a multispecific binding site comprising: - a PD-1-binding domain described herein comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO:9, or a heavy chain variable region having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity 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 having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity thereto. In certain embodiments, each of the heavy chain variable regions comprises an HCDR that does not contain an amino acid variation. In certain embodiments, each of the heavy chain variable regions does not contain an amino acid variation.

[0121] In one embodiment, the present disclosure provides a multispecific binding site comprising: - a PD-1-binding domain described herein comprising a heavy chain variable region having the 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 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 having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity thereto. In certain embodiments, each of the heavy chain variable regions comprises an HCDR that does not contain an amino acid variation. In certain embodiments, each of the heavy chain variable regions does not contain an amino acid variation.

[0122] In one embodiment, the present disclosure provides a multispecific binding site comprising: - a PD-1-binding domain 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 having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity 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 having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity thereto. In certain embodiments, each of the heavy chain variable regions comprises an HCDR that does not contain an amino acid variation. In certain embodiments, each of the heavy chain variable regions does not contain an amino acid variation.

[0123] In one embodiment, the present disclosure provides a multispecific binding site comprising: - a PD-1-binding domain described herein comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 14, or a heavy chain variable region having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity 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 having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity thereto. In certain embodiments, each of the heavy chain variable regions comprises an HCDR that does not contain an amino acid variation. In certain embodiments, each of the heavy chain variable regions does not contain an amino acid variation.

[0124] In one embodiment, the present disclosure provides a multispecific binding site comprising: - a PD-1-binding domain described herein comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO:9, or a heavy chain variable region having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity 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: 39, or a heavy chain variable region having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity thereto. In certain embodiments, each of the heavy chain variable regions comprises an HCDR that does not contain an amino acid variation. In certain embodiments, each of the heavy chain variable regions does not contain an amino acid variation.

[0125] In one embodiment, the present disclosure provides a multispecific binding site comprising: - a PD-1-binding domain described herein comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO:9, or a heavy chain variable region having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity 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 having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity thereto. In certain embodiments, each of the heavy chain variable regions comprises an HCDR that does not contain an amino acid variation. In certain embodiments, each of the heavy chain variable regions does not contain an amino acid variation.

[0126] In one embodiment, the present disclosure provides a multispecific binding site comprising: - a PD-1-binding domain described herein comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 14, or a heavy chain variable region having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity 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 having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity thereto. In certain embodiments, each of the heavy chain variable regions comprises an HCDR that does not contain an amino acid variation. In certain embodiments, each of the heavy chain variable regions does not contain an amino acid variation.

[0127] In one embodiment, the present disclosure provides a multispecific binding site comprising: - a PD-1-binding domain 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 having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity 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 having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity thereto. In certain embodiments, each of the heavy chain variable regions comprises an HCDR that does not contain an amino acid variation. In certain embodiments, each of the heavy chain variable regions does not contain an amino acid variation.

[0128] In one embodiment, the present disclosure provides a multispecific binding site comprising: - a PD-1-binding domain described herein comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO:9, or a heavy chain variable region having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity 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 having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity thereto. In certain embodiments, each of the heavy chain variable regions comprises an HCDR that does not contain an amino acid variation. In certain embodiments, each of the heavy chain variable regions does not contain an amino acid variation.

[0129] In one embodiment, the present disclosure provides a multispecific binding site comprising: - a PD-1 binding domain as described herein comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 13, or a heavy chain variable region having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity 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: 47, or a heavy chain variable region having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity thereto. In certain embodiments, each of the heavy chain variable regions comprises an HCDR that does not contain an amino acid variation. In certain embodiments, each of the heavy chain variable regions does not contain an amino acid variation.

[0130] In one embodiment, the present disclosure provides a multispecific binding site comprising: - a PD-1-binding domain 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 having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity 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: 43, or a heavy chain variable region having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity thereto. In certain embodiments, each of the heavy chain variable regions comprises an HCDR that does not contain an amino acid variation. In certain embodiments, each of the heavy chain variable regions does not contain an amino acid variation.

[0131] In one embodiment, the present disclosure provides a multispecific binding site comprising: - a PD-1-binding domain described herein comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO:9, or a heavy chain variable region having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity 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 having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity thereto; wherein the PD-1-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:48, 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 contain no amino acid variation. In some embodiments, the heavy chain variable region and the light chain variable region each contain no amino acid variation.

[0132] In one embodiment, the present disclosure provides a multispecific binding site comprising: - a PD-1-binding domain described herein comprising a heavy chain variable region having the 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 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 having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity thereto; wherein the PD-1-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:48, 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 contain no amino acid variation. In some embodiments, the heavy chain variable region and the light chain variable region each contain no amino acid variation.

[0133] In one embodiment, the present disclosure provides a multispecific binding site comprising: - a PD-1-binding domain 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 having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity 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 having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity thereto; wherein the PD-1-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:48, 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 contain no amino acid variation. In some embodiments, the heavy chain variable region and the light chain variable region each contain no amino acid variation.

[0134] In one embodiment, the present disclosure provides a multispecific binding site comprising: - a PD-1-binding domain described herein comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 14, or a heavy chain variable region having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity 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 having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity thereto; wherein the PD-1-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:48, 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 contain no amino acid variation. In some embodiments, the heavy chain variable region and the light chain variable region each contain no amino acid variation.

[0135] In one embodiment, the present disclosure provides a multispecific binding site comprising: - a PD-1-binding domain described herein comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO:9, or a heavy chain variable region having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity 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: 39, or a heavy chain variable region having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity thereto; wherein the PD-1-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:48, 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 contain no amino acid variation. In some embodiments, the heavy chain variable region and the light chain variable region each contain no amino acid variation.

[0136] In one embodiment, the present disclosure provides a multispecific binding site comprising: - a PD-1-binding domain described herein comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO:9, or a heavy chain variable region having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity 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 having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity thereto; wherein the PD-1-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:48, 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 contain no amino acid variation. In some embodiments, the heavy chain variable region and the light chain variable region each contain no amino acid variation.

[0137] In one embodiment, the present disclosure provides a multispecific binding site comprising: - a PD-1-binding domain described herein comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 14, or a heavy chain variable region having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity 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 having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity thereto; wherein the PD-1-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:48, 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 contain no amino acid variation. In some embodiments, the heavy chain variable region and the light chain variable region each contain no amino acid variation.

[0138] In one embodiment, the present disclosure provides a multispecific binding site comprising: - a PD-1-binding domain 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 having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity 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 having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity thereto; wherein the PD-1-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:48, 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 contain no amino acid variation. In some embodiments, the heavy chain variable region and the light chain variable region each contain no amino acid variation.

[0139] In one embodiment, the present disclosure provides a multispecific binding site comprising: - a PD-1-binding domain described herein comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO:9, or a heavy chain variable region having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity 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 having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity thereto; wherein the PD-1-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:48, 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 contain no amino acid variation. In some embodiments, the heavy chain variable region and the light chain variable region each contain no amino acid variation.

[0140] In one embodiment, the present disclosure provides a multispecific binding site comprising: - a PD-1 binding domain as described herein comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 13, or a heavy chain variable region having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity 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: 47, or a heavy chain variable region having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity thereto; wherein the PD-1-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:48, 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 contain no amino acid variation. In some embodiments, the heavy chain variable region and the light chain variable region each contain no amino acid variation.

[0141] In one embodiment, the present disclosure provides a multispecific binding site comprising: - a PD-1-binding domain 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 having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity 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: 43, or a heavy chain variable region having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity thereto; wherein the PD-1-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:48, 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 contain no amino acid variation. In some embodiments, the heavy chain variable region and the light chain variable region each contain no amino acid variation.

[0142] Also provided herein are nucleic acids useful for producing the multispecific binding sites of the present disclosure. In some embodiments, such nucleic acids comprise a nucleic acid sequence encoding a heavy chain variable region of the PD-1-binding domain described herein and a nucleic acid sequence encoding a heavy chain variable region of the TGF-βRII-binding domain described herein. In some embodiments, the nucleic acids 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 acids 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 consensus light chain variable region described herein.

[0143] Also provided herein are vectors containing the nucleic acids of the present disclosure, which are useful for producing the multispecific binding sites of the present disclosure. In some embodiments, such vectors contain a nucleic acid sequence encoding a heavy chain variable region of the PD-1-binding domain described herein and a nucleic acid sequence encoding a heavy chain variable region of the TGF-βRII-binding domain described herein. In some embodiments, the vectors of the present disclosure may further contain a nucleic acid sequence encoding a CH1 region, and preferably a hinge, CH2, and CH3 region. In some embodiments, the vectors of the present disclosure may further contain 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.

[0144] The present disclosure also provides cells comprising a nucleic acid sequence, e.g., a vector, encoding the heavy chain variable region of the PD-1-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 may further comprise a nucleic acid sequence, e.g., a vector, encoding a CH1 region, and preferably a hinge, CH2, and CH3 region. In some embodiments, the cells of the present disclosure may further comprise at least one nucleic acid sequence, e.g., a vector, encoding a light chain variable region, and preferably a CL region. In some embodiments, the light chain variable region may be the consensus light chain variable region described herein.

[0145] The present disclosure also provides cells that produce the multispecific binding sites described herein. In some embodiments, such cells may be recombinant cells transformed with nucleic acids, e.g., vectors, of the present disclosure. In some embodiments, the cells of the present disclosure comprise a nucleic acid sequence, e.g., a vector, encoding the heavy chain variable region of the PD-1-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 comprise a nucleic acid sequence, e.g., a vector, encoding a CH1 region, and preferably a hinge, CH2, and CH3 region. In some embodiments, the cells of the present disclosure further comprise at least one nucleic acid sequence, e.g., a vector, encoding a light chain variable region, specifically a light chain variable region described herein, and preferably a CL region.

[0146] The present disclosure further provides cells that produce the multispecific binding sites described herein.

[0147] In certain embodiments, the present disclosure provides a pharmaceutical composition comprising an effective amount of a multispecific binding site described herein and, optionally, a pharmaceutically acceptable carrier.

[0148] In certain embodiments, the present disclosure provides the multispecific binding sites described herein, and the pharmaceutical compositions described herein, for use in therapy.

[0149] In certain embodiments, the present disclosure provides a multispecific binding site as described herein, or a pharmaceutical composition as described herein, for use in the treatment of cancer.

[0150] In certain embodiments, the present disclosure provides a method for treating a disease, comprising administering to an individual in need thereof an effective amount of a multispecific binding site described herein, or a pharmaceutical composition described herein.

[0151] 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 multispecific binding site described herein, or a pharmaceutical composition described herein.

[0152] 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., and specifically to human subjects with cancer.

[0153] 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 treatment that produces a beneficial effect, such as reversing, alleviating, ameliorating, inhibiting, or slowing a symptom, complication, condition, or biochemical manifestation associated with a disease, and preventing the onset, progression, development, severity, or recurrence of a symptom, complication, condition, or biochemical manifestation associated with a disease, e.g., amelioration of 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 improvement over measurements or observations made prior to the 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 disappearance 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.

[0154] 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.

[0155] 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 cancer cell invasion into peripheral organs; (iv) inhibit tumor metastasis; (v) inhibit tumor growth; (vi) prevent or delay tumor onset and / or recurrence; and / or (vii) relieve to some extent one or more symptoms associated with cancer.

[0156] 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.

[0157] An effective amount can be administered to a subject in one or more administrations.

[0158] 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.

[0159] The term "agent" refers to a therapeutically active substance, in this case a multispecific binding site of the present disclosure, or a pharmaceutical composition of the present disclosure.

[0160] 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.

[0161] 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.

[0162] Any 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.

[0163] All patents and literature references cited herein are incorporated by reference in their entirety.

[0164] Unless otherwise specified, the amino acid positions assigned to CDRs and frameworks in the variable regions of antibodies or antibody fragments are identified according to the Kabat numbering system (see Sequences of Proteins of Immunological Interest (National Institute of Health, Bethesda, Md., 1987 and 1991)). Amino acids in the constant regions are indicated according to the EU numbering system.

[0165] 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 the multispecific 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.

[0166] 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.

[0167] [Provisions] 1. A multispecific binding site comprising a PD-1 binding domain and a TGF-βRII binding domain, the PD-1 binding domain inhibits PD-1-mediated signaling; and the TGF-βRII binding domain inhibits TGF-βRII-mediated signal transduction; Multispecific binding site.

[0168] 2. A multispecific binding site according to clause 1, In activated T cells, the PD-1 binding domain inhibits PD-1-mediated signaling; and the TGF-βRII binding domain inhibits TGF-βRII-mediated signal transduction; Multispecific binding site.

[0169] 3. A multispecific binding site according to clause 1 or 2, In activated tumor-specific T cells, the PD-1 binding domain inhibits PD-1-mediated signaling; and the TGF-βRII binding domain inhibits TGF-βRII-mediated signal transduction; Multispecific binding site.

[0170] 4. A Fab domain that specifically binds to PD-1 and a Fab domain that specifically binds to TGF-βRII; Multispecific binding site.

[0171] 5. A multispecific binding site according to any one of the preceding clauses, the multispecific binding site consists of a single Fab domain that specifically binds to PD-1, a single Fab domain that specifically binds to TGF-βRII, and an Fc region; Multispecific binding site.

[0172] 6. A multispecific binding site according to any one of the preceding clauses, the multispecific binding site has greater potency in inhibiting TGF-βRII-mediated signaling in cells that express both PD-1 and TGF-βRII than in cells that express TGF-βRII and no, substantially no, or low levels of PD-1; Multispecific binding site.

[0173] 7. A multispecific binding site comprising a PD-1-binding domain and a TGF-βRII-binding domain, the multispecific binding site has greater potency in inhibiting TGF-βRII-mediated signaling in cells that express both PD-1 and TGF-βRII than in cells that express TGF-βRII and no, substantially no, or low levels of PD-1; Multispecific binding site.

[0174] 8. A multispecific binding site comprising a PD-1-binding domain and a TGF-βRII-binding domain, the multispecific binding site has greater activity in reducing tumor volume than a combination of reference antibodies; said combination of reference antibodies is two bivalent monospecific antibodies targeting PD-1 and TGF-βRII; the bivalent monospecific antibody targeting PD-1 comprises a heavy chain having the amino acid sequence set forth in SEQ ID NO: 78 and a light chain having the amino acid sequence set forth in SEQ ID NO: 79; and The bivalent monospecific antibody targeting TGF-βRII comprises a heavy chain having the amino acid sequence set forth in SEQ ID NO: 76 and a light chain having the amino acid sequence set forth in SEQ ID NO: 77. Multispecific binding site.

[0175] 9. A multispecific binding site according to clause 8, The activity in reducing tumor volume is determined by measuring the reduction in tumor volume in an in vivo mouse study, specifically in an in vivo mouse study using MDA-MB-231 xenograft huCD34 NSG mice. Multispecific binding site.

[0176] 10. A multispecific antibody or variant thereof according to clause 8 or 9, Here, a higher activity in reducing tumor volume refers to a reduction in tumor volume that is at least about 1.5 times, preferably about 1.5 to 100 times, the reduction in tumor volume of the combination of reference antibodies. Multispecific binding site.

[0177] 11. A multispecific binding site according to any one of the preceding clauses, The cells that express both PD-1 and TGF-βRII are activated T cells. Multispecific binding site.

[0178] 12. A multispecific binding site according to any one of the preceding clauses, The cells that express both PD-1 and TGF-βRII are activated tumor-specific T cells. Multispecific binding site.

[0179] 13. A multispecific binding site according to any one of the preceding clauses, The cells expressing both PD-1 and TGF-βRII are called Jurkat-PD-1 + cells, and The cells that express TGF-βRII and do not express or substantially do not express PD-1 are called Jurkat-PD-1 null The cells, Multispecific binding site.

[0180] 14. A multispecific binding site according to any one of the preceding clauses, These cells express both PD-1 and TGF-βRII, and are associated with activated CD4 + and / or CD8 + cells, and The cells that express TGF-βRII and do not express, or substantially do not express, or express low levels of PD-1 are non-activated CD4 + and / or CD8 + The cells, Multispecific binding site.

[0181] 15. A multispecific binding site according to any one of the preceding clauses, The cells expressing both PD-1 and TGF-βRII were identified as HEK-Blue TGF-β-PD-1 + cells, and The cells that express TGF-βRII and do not express, or substantially do not express, or express low levels of PD-1 are HEK-Blue TGF-β cells. Multispecific binding site.

[0182] 16. A multispecific binding site according to any one of the preceding clauses, The efficacy in inhibiting TGF-βRII-mediated signaling is measured in a phosphorylated SMAD2 / 3 assay. Multispecific binding site.

[0183] 17. A multispecific binding site according to any one of the preceding clauses, The efficacy in inhibiting TGF-βRII-mediated signaling is measured in an isogenic PD-1-TGF-β reporter assay. Multispecific binding site.

[0184] 18. A multispecific binding site according to any one of the preceding clauses, the potency in inhibiting TGF-βRII-mediated signaling in cells expressing both PD-1 and TGF-βRII is at least about 200-fold, or 500-fold, or 1000-fold, or 5000-fold, or 10,000-fold, or 15,000-fold, or 20,000-fold, or 30,000-fold, or about 200-30,000-fold, or 500-30,000-fold, or 1000-30,000-fold, or 5,000-30,000-fold, or 10,000-30,000-fold, or 200-20,000-fold, or 200-15,000-fold greater than in cells expressing TGF-βRII and no, or substantial, or low levels of PD-1; Multispecific binding site.

[0185] 19. A multispecific binding site according to any one of the preceding clauses, the potency of the multispecific binding site in inhibiting TGF-βRII-mediated signaling in cells that express TGF-βRII and do not express PD-1 at all is less than the potency of a reference anti-TGF-βRII antibody; and the potency of the multispecific binding site in inhibiting TGF-βRII-mediated signaling in cells expressing both PD-1 and TGF-βRII is greater than the potency of the reference anti-TGF-βRII antibody; 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: 76 and a light chain having the amino acid sequence set forth in SEQ ID NO: 77; Multispecific binding site.

[0186] 20. A multispecific binding site according to any one of the preceding clauses, the potency of the multispecific binding site in inhibiting TGF-βRII-mediated signaling in cells expressing both PD-1 and TGF-βRII is at least about 100-fold or 200-fold, preferably about 100-20,000-fold, or 100-15,000-fold, or 100-12,000-fold, or 200-20,000-fold, or 200-15,000-fold, or 200-12,000-fold greater than the potency of the reference anti-TGF-βRII antibody; Multispecific binding site.

[0187] 21. A multispecific binding site according to any one of the preceding clauses, The PD-1 binding domain and the TGF-βRII binding domain are Fab domains. Multispecific binding site.

[0188] 22. A multispecific binding site according to any one of the preceding clauses, The multispecific binding moiety is a bispecific antibody Multispecific binding site.

[0189] 23. A multispecific binding site according to any one of the preceding clauses, The multispecific binding site is an IgG1 bispecific antibody Multispecific binding site.

[0190] 24. A multispecific binding site according to any one of the preceding clauses, the PD-1-binding domain and the TGF-βRII-binding domain each comprise a light chain comprising a light chain variable region capable of pairing with multiple heavy chains having different epitope specificities; Multispecific binding site.

[0191] 25. A multispecific binding site according to any one of the preceding clauses, The PD-1 binding domain and the TGF-βRII binding domain contain the same light chain Multispecific binding site.

[0192] 26. A multispecific binding site according to any one of the preceding clauses, The PD-1 binding domain 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:2, SEQ ID NO:3, and SEQ ID NO:4, 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:6, SEQ ID NO:7, and SEQ ID NO:8, 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: 10, SEQ ID NO: 11, and SEQ ID NO: 12, respectively; d) 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: 15, SEQ ID NO: 16, and SEQ ID NO: 17, respectively; or e) 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 amino acid variation; Multispecific binding site.

[0193] 27. A multispecific binding site according to any one of the preceding clauses, The TGF-βRII binding domain 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: 28, 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: 32, SEQ ID NO: 33, and SEQ ID NO: 34, respectively; 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: 36, SEQ ID NO: 37, and SEQ ID NO: 38, respectively; e) heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 40, SEQ ID NO: 41, and SEQ ID NO: 42, respectively; f) 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: 44, SEQ ID NO: 45, and SEQ ID NO: 46, respectively; or g) 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: 90, SEQ ID NO: 91, and SEQ ID NO: 92, respectively; wherein each of said HDCRs may contain at most three, two, or one amino acid variation; Multispecific binding site.

[0194] 28. A multispecific binding site comprising a PD-1 binding domain and a TGF-βRII binding domain, The PD-1 binding domain 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:2, SEQ ID NO:3, and SEQ ID NO:4, 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:6, SEQ ID NO:7, and SEQ ID NO:8, 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: 10, SEQ ID NO: 11, and SEQ ID NO: 12, respectively; d) 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: 15, SEQ ID NO: 16, and SEQ ID NO: 17, respectively; or e) 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 amino acid variation; Multispecific binding site.

[0195] 29. A multispecific binding site comprising a PD-1 binding domain and a TGF-βRII binding domain, The TGF-βRII binding domain 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: 28, 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: 32, SEQ ID NO: 33, and SEQ ID NO: 34, respectively; 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: 36, SEQ ID NO: 37, and SEQ ID NO: 38, respectively; e) heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 40, SEQ ID NO: 41, and SEQ ID NO: 42, respectively; f) 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: 44, SEQ ID NO: 45, and SEQ ID NO: 46, respectively; or g) 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: 90, SEQ ID NO: 91, and SEQ ID NO: 92, respectively; wherein each of said HDCRs may contain at most three, two, or one amino acid variation; Multispecific binding site.

[0196] 30. A multispecific binding site according to any one of the preceding clauses, the PD-1 binding domain comprises a heavy chain variable region having an amino acid sequence set forth in any one of SEQ ID NOs: 1; 5; 9; 13; 14; 18; 19, or having at least 80%, preferably 85%, more preferably 90%, or most preferably 95% sequence identity thereto; Multispecific binding site.

[0197] 31. A multispecific binding site according to any one of the preceding clauses, the PD-1 binding domain comprises a light chain variable region including 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:49, SEQ ID NO:50, and SEQ ID NO:51, respectively; wherein each of said LDCRs may contain at most three, two, or one amino acid variation; Multispecific binding site.

[0198] 32. A multispecific binding site according to any one of the preceding clauses, the PD-1 binding domain comprises a light chain variable region having the amino acid sequence set forth in SEQ ID NO:48, or having at least 80%, preferably 85%, more preferably 90%, or most preferably 95% sequence identity thereto; Multispecific binding site.

[0199] 33. A multispecific binding site according to any one of the preceding clauses, The TGF-βRII binding domain 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: 28, 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: 32, SEQ ID NO: 33, and SEQ ID NO: 34, respectively; 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: 36, SEQ ID NO: 37, and SEQ ID NO: 38, respectively; e) heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 40, SEQ ID NO: 41, and SEQ ID NO: 42, respectively; f) 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: 44, SEQ ID NO: 45, and SEQ ID NO: 46, respectively; or g) 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: 90, SEQ ID NO: 91, and SEQ ID NO: 92, respectively; wherein each of said HDCRs may contain at most three, two, or one amino acid variation; Multispecific binding site.

[0200] 34. A multispecific binding site according to any one 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; 35; 39; 43; 47; 88; 89, or having at least 80%, preferably 85%, more preferably 90%, or most preferably 95% sequence identity thereto. Multispecific binding site.

[0201] 35. A multispecific binding site according to any one of the preceding clauses, The TGF-βRII binding domain comprises a light chain variable region including 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: 49, SEQ ID NO: 50, and SEQ ID NO: 51, respectively; wherein each of said LDCRs may contain at most three, two, or one amino acid variation; Multispecific binding site.

[0202] 36. A multispecific binding site according to any one 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: 48, or having at least 80%, preferably 85%, more preferably 90%, or most preferably 95% sequence identity thereto; Multispecific binding site.

[0203] 37. A multispecific binding site comprising a PD-1 binding domain and a TGF-βRII binding domain, competes for binding to PD-1 and / or TGF-βRII with the multispecific binding site of any one of the preceding clauses. Multispecific binding site.

[0204] 38. A method of treating a pulmonary arthritis with a pulmonary arthritis comprising administering to a patient a therapeutically effective amount of a multispecific binding site according to any one of the preceding clauses and a pharmaceutically acceptable carrier. Pharmaceutical compositions.

[0205] 39. A multispecific binding site according to any one of clauses 1 to 37, or a pharmaceutical composition according to clause 38, for use in therapy.

[0206] 40. A multispecific binding site according to any one of clauses 1 to 37, or a pharmaceutical composition according to clause 38, for use in treating a disease associated with a suppressed immune system.

[0207] 41. A multispecific binding site according to any one of clauses 1 to 37, or a pharmaceutical composition according to clause 38, for use in the treatment of cancer.

[0208] 42. Use of a multispecific binding moiety according to any one of clauses 1 to 37, or a pharmaceutical composition according to clause 38, for the manufacture of a medicament for use in the treatment of a disease associated with a suppressed immune system.

[0209] 43. Use of a multispecific binding moiety according to any one of clauses 1 to 37, or a pharmaceutical composition according to clause 38, for the manufacture of a medicament for use in the treatment of cancer.

[0210] 44. A method for treating a disease, comprising: administering to a human subject in need thereof an effective amount of a multispecific binding site according to any one of clauses 1 to 37, or a pharmaceutical composition according to clause 38. method.

[0211] 45. A method for treating a disease associated with a suppressed immune system, comprising: administering to a human subject in need thereof an effective amount of a multispecific binding site according to any one of clauses 1 to 37, or a pharmaceutical composition according to clause 38. method.

[0212] 46. ​​A method for treating cancer, comprising: administering to a human subject in need thereof an effective amount of a multispecific binding site according to any one of clauses 1 to 37, or a pharmaceutical composition according to clause 38. method.

[0213] 47. A nucleic acid sequence encoding a heavy chain variable region of a PD-1 binding domain defined in clause 28 or 30, and a nucleic acid sequence encoding a heavy chain variable region of a TGF-βRII binding domain defined in clause 29 or 34, cell.

[0214] 48. Cells as described in clause 47, The cell further comprises a nucleic acid sequence encoding a CH1 region, and preferably a hinge, CH2 and CH3 region. cell.

[0215] 49. A cell according to clause 47 or 48, said cell further comprising at least one nucleic acid sequence encoding a light chain variable region, in particular a light chain variable region as defined in clause 34 or 35, and preferably a CL region. cell.

[0216] 50. A cell producing a multispecific binding site according to any one of clauses 1 to 37.

[0217] [Brief description of the drawing] The following naming conventions are used herein as follows: In the figures, bivalent monospecific antibodies are shown in the format SEQ ID NO:A / SEQ ID NO:B, where SEQ ID NO:A refers to the heavy chains of both binding domains and SEQ ID NO:B refers to the light chains of both binding domains.

[0218] A bivalent, bispecific antibody is shown in the format SEQ ID NO: A x SEQ ID NO: B, where both SEQ ID NO: A and B refer to heavy chain variable sequences. Each binding domain of the bispecific antibody contains the same light chain.

[0219] The bivalent monospecific reference antibodies pembrolizumab, nivolumab, and TGF1 analogue are shown in the format SEQ ID NO:A / SEQ ID NO:B, where SEQ ID NO:A refers to the respective heavy chain sequence and SEQ ID NO:B refers to the respective light chain sequence. The combination of pembrolizumab and TGF1 analogue is shown in the format SEQ ID NO:A / SEQ ID NO:B+SEQ ID NO:C / SEQ ID NO:D, where SEQ ID NO:A refers to the heavy chain sequence of either pembrolizumab or TGF1 analogue, SEQ ID NO:B refers to the light chain sequence of either pembrolizumab or TGF1 analogue, SEQ ID NO:C refers to the other heavy chain sequence, and SEQ ID NO:D refers to the other light chain sequence.

[0220] The reference PD-L1-TGF-β TRAP molecule analogue is an analogue of Bintrafusp alfa and is shown in the format SEQ ID NO:A / SEQ ID NO:B, where SEQ ID NO:A refers to the heavy chain sequence comprising the (G4S)4G linker and extracellular domain of TGF-βRII, and SEQ ID NO:B refers to the light chain sequence. The reference PD-L1-TGF-β TRAP molecule analogue contains two PD-L1 binding domains and two TGF-βRII extracellular domains. [Example]

[0221] In an example used to illustrate, but not intended to limit, the present disclosure in any way, each binding domain of the bispecific antibody comprises a light chain variable region having the amino acid sequence set forth in SEQ ID NO:48 and a light chain constant region having the amino acid sequence set forth in SEQ ID NO:75. The bispecific antibody is preferably an IgG1 antibody comprising a CH1, hinge, CH2, and CH3. In an example used to illustrate, but not intended to limit, the present disclosure, the bispecific antibody is screened in an IgG1 format, and the PD-1-binding heavy chain comprises a CH1 having the amino acid sequence set forth in SEQ ID NO:69, a CH2 having the amino acid sequence set forth in SEQ ID NO:71, and a CH3 having the amino acid sequence set forth in SEQ ID NO:73; and the TGF-RII-binding heavy chain comprises a CH1 having the amino acid sequence set forth in SEQ ID NO:69, a CH2 having the amino acid sequence set forth in SEQ ID NO:71, and a CH3 having the amino acid sequence set forth in SEQ ID NO:74.

[0222] Reference antibodies and molecules, and control antibodies used in the examples include: - The reference PD-1 antibody pembrolizumab analogue 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: 79. - Reference PD-1 antibody pembrolizumab (manufactured by Merck, distributed by Myonex). - the reference TGF-βRII antibody TGF1 is a bivalent monospecific analogue of TGF1 and comprises 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. - The reference PD-L1-TGF-β TRAP molecule is a bivalent, monospecific analog of Bintrafusp alfa for binding to PD-L1, and comprises two heavy chains having the amino acid sequence set forth in SEQ ID NO: 80 and two light chains having the amino acid sequence set forth in SEQ ID NO: 81, with the extracellular domain of TGF-βRII, having the amino acid sequence set forth in SEQ ID NO: 104, linked to the C-terminus of each heavy chain via a (G4S)4G linker. - The negative control IgG1 antibody (RSV-G) is a bivalent monospecific antibody comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO: 86 and two light chains having the amino acid sequences set forth in SEQ ID NO: 87. The control TGF-βRII x RSV antibody is a bivalent, bispecific antibody comprising: a TGF-βRII binding domain comprising a heavy chain variable region amino acid sequence set forth in SEQ ID NO: 23, 31, 39, 27, 35, or 43; a RSV binding domain comprising a heavy chain variable region amino acid sequence set forth in SEQ ID NO: 86; and a common light chain comprising a light chain variable region amino acid sequence set forth in SEQ ID NO: 48 and a light chain constant region amino acid sequence set forth in SEQ ID NO: 75. - Positive control LILRB2 (BioLegend; Cat. No. 338714). - The commercially available reference PD-1 antibody Opdivo (manufactured by Bristol Myers Squibb (BMS)).

[0223] Example 1 - Generation of PD-1 x TGF-βRII Bispecific Antibody Binding domains, antibodies, and heavy chain variable regions with binding specificity for human PD-1 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 PD-1 or TGF-βRII antigenic sites (including using various forms of DNA-, protein-, and cell-based antigen delivery).

[0224] Heavy chain variable regions with binding specificity for human PD-1 having the amino acid sequences set forth in SEQ ID NOs: 1, 5, 9, 13, 14, 18, 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, 35, 39, 43, 47, 88, and 89 were selected for production of bispecific antibodies. Once characterized and sequenced through the techniques provided herein, the binding domain sequences herein may thereafter be obtained by any method known in the art.

[0225] JPEG2024542879000001.jpg113166

[0226] Bispecific IgG antibodies were generated by transient cotransfection of two plasmid vectors, one encoding an IgG heavy chain with a PD-1-binding VH region and the other encoding an IgG heavy chain with a TGF-βRII-binding VH region. CH3 engineering techniques described in WO2013 / 157954 and WO2013 / 157953 were employed to ensure efficient heterodimerization and bispecific antibody formation. Both vectors further encode a common light chain comprising 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.

[0227] Example 2 - PD-1-SHP Recruitment Assay The bispecific antibodies were characterized in a PD-1-SHP recruitment assay to determine their ability to inhibit ligand binding to PD-1, thereby inhibiting PD-1 / PD-L1 signaling in T cells. The PD-1-SHP recruitment assay involves two cell lines: U2OS cells engineered to express an enzyme donor (ED)-tagged PD-1 receptor (e.g., PD-L1 or PD-L2) and Jurkat T cells engineered to express PD-1 and enzyme acceptor (EA)-fused SHP1. Activation of Jurkat T cells by ligand or agonist antibody-induced PD-1 recruits SHP1, allowing the ED and EA to interact and reconstitute active β-gal enzyme. The reconstituted β-gal generates a chemiluminescent signal in the presence of substrate.

[0228] The antibody samples included several bispecific antibodies, a negative control IgG1 antibody (RSV), an analog of the reference PD-1 antibody pembrolizumab, an analog of the reference TGF-βRII antibody TGF1, and an analog of the reference PD-L1-TGF-β TRAP molecule.

[0229] U2OS / PD-L1 cells (DiscoveRx Corporation) were maintained in McCoy's 5A medium (Thermo Fisher Scientific) supplemented with 10% FBS and 0.25 μg / ml puromycin (Thermo Fisher Scientific). Jurkat-PD-1-SHP cells (Src homology region 2 domain-containing phosphatase; DiscoveRx Corporation) were cultured in RPMI 1640 medium (Thermo Fisher Scientific) supplemented with 10% FBS, 250 μg / ml hygromycin B (Thermo Fisher Scientific), and 500 μg / ml G418 (Thermo Fisher Scientific). Both U2OS / PD-L1 and Jurkat-PD-1-SHP cells were first centrifuged in a conical tube to remove the culture medium, then washed and resuspended in assay medium (RPMI 1640 medium containing 1% FBS) before cell plating. U2OS / PD-L1 cells were plated in 20μL of assay medium at 5000 cells per well in a 384-well black, clear-bottom assay plate (CELLCOAT® Tissue Culture Plates, Greiner Bio-One). Antibody samples were serially diluted in phosphate-buffered saline (PBS) containing 1% FBS, and 5μL / well was transferred to the cell plate and incubated at 37°C and 5% CO2 for 1 hour. Jurkat-PD-1-SHP cells were then plated in 20μL of assay medium at 5000 cells per well in the cell plate and incubated at 37°C and 5% CO2 for 2 hours, after which 2.5μL of PathHunter Reagent 1 (DiscoveRx Corporation) was added to each well. The assay plate was then shaken at 350 rpm for 1 min and kept in the dark at room temperature for 15 min before adding 10 μL of PathHunter Reagent 2 (DiscoveRx Corporation). Chemiluminescence signals were recorded using a TopCount reader (Perkin Elmer) after 1 h of incubation at room temperature.Wells containing PBS only served as positive controls, and wells without cells served as negative controls. IC. 50 Determination of β was performed by fitting a curve of percent control activity versus log compound concentration using GraphPad Prism 7.0 software.

[0230] The results are shown in Table 2 and Figure 1. All bispecific antibodies inhibited PD-1-mediated SHP recruitment. Many bispecific antibodies that are monovalent for PD-1 binding are equipotent in the PD-1-SHP recruitment assay to the bivalent monospecific reference PD-1 antibody pembrolizumab analog and the bivalent reference PD-L1-TGF-β TRAP molecule for PD-L1 binding.

[0231] JPEG2024542879000002.jpg62166

[0232] Example 3 - PD-1-NFAT Reporter Assay The bispecific antibodies were characterized in a PD-1-NFAT reporter assay to determine their ability to inhibit PD-1 / PD-L1 signaling in activated T cells. The PD-1-NFAT reporter assay detects PD-L1 signaling in cells co-expressing the TCR cognate protein. + aAPC / CHO-K1 cells and PD-1 driving a luciferase reporter under the control of the NFAT-RE cis-element + The transgenic cell line system includes two transgenic cell lines harboring effector Jurkat T cells and effector Jurkat T cells. The effector Jurkat T cells activate TCR signaling in an antigen-independent manner. PD-1 / PD-L1 interaction inhibits TCR-mediated luminescence. Inhibition of PD-1 / PD-L1 interaction allows TCR signaling, thereby inducing luminescence that can be detected by adding a substrate.

[0233] The antibody samples included several bispecific antibodies, a negative control IgG1 antibody (RSV), an analog of the reference PD-1 antibody pembrolizumab, an analog of the reference TGF-βRII antibody TGF1, and an analog of the reference PD-L1-TGF-β TRAP molecule.

[0234] PD-L1 aAPC / CHO-K1 cells (artificial antigen-presenting cells) / CHO (Chinese hamster ovary)-K1 cells (Promega) were maintained in F-12 medium (Thermo Fisher Scientific) supplemented with 10% FBS, 200 μg / mL hygromycin B (Thermo Fisher Scientific), and 250 μg / mL Geneticin (G418; Thermo Fisher Scientific). Jurkat-PD-1-NFAT effector cells (nuclear factor of activated T cells; Promega) were cultured in RPMI 1640 medium (Thermo Fisher Scientific) supplemented with 10% FBS, 100 μg / mL hygromycin B (Thermo Fisher Scientific), and 500 μg / mL G418 (Thermo Fisher Scientific). Both PD-L1 aAPC / CHO-K1 cells and Jurkat-PD-1-NFAT effector cells were first centrifuged to remove the culture medium, then washed and resuspended in assay medium (RPMI1640 medium containing 1% FBS) before cell plating. PD-L1 aAPC / CHO-K1 cells were added to a 384-well white, clear-bottom assay plate (CELLCOAT® Tissue Culture Plates, Greiner Bio-One) at 8,000 cells per well in 10 μL of assay medium. Antibody samples were prepared by serial dilution in phosphate-buffered saline (PBS) containing 1% FBS, and 5 μL per well was transferred to the cell plate. Jurkat-PD-1-NFAT effector cells were then dispensed into each well at 10,000 cells per well in 5 μL of assay medium. The assay plate was incubated at 37°C, 5% CO2 for 24 hours. The assay plate was equilibrated to room temperature for 15 minutes before adding 20 μL / well of Bio-Glo™ reagent (Promega). After an 8-minute incubation at room temperature, luminescence was read using a Pherastar microplate reader (BMG Labtech).Wells containing PBS were used as negative controls (0% induction) and wells containing 12.5 μg / mL of pembrolizumab analogue were used as positive controls (100% induction). EC. 50 Determination of β was performed by fitting a curve of percent control activity versus log compound concentration using GraphPad Prism 7.0 software.

[0235] The results are shown in Table 3 and Figure 2. All bispecific antibodies inhibited PD-1-mediated T cell inhibition. Many bispecific antibodies that are monovalent for PD-1 binding are equipotent in the PD-1-NFAT reporter assay to the bivalent monospecific reference PD-1 antibody pembrolizumab analog.

[0236] JPEG2024542879000003.jpg65166

[0237] Example 4 - Jurkat Phosphorylated SMAD2 / 3 Assay The bispecific antibodies were characterized in the Jurkat phospho-SMAD2 / 3 assay to determine their ability to inhibit TGF-βRII signaling in T cells. null Cells and Jurkat-PD-1 + The activity of the bispecific antibodies in cells was compared to determine whether the bispecific antibodies inhibited TGF-β-induced SMAD2 / 3 phosphorylation in a manner correlated with PD-1. An analog of the reference TGF-βRII antibody TGF1, a TGF-βRII x RSV bispecific antibody, and a negative control bivalent monospecific IgG1 antibody comprising a heavy chain having the amino acid sequence set forth in SEQ ID NO: 86 and a light chain having the amino acid sequence set forth in SEQ ID NO: 87 were included as control antibodies.

[0238] Jurkat-PD-1 in RPMI / 10% FBS null cells (ATCC catalog number TIB-152) and Jurkat-PD-1 +Cells (Promega Cat. No. CS187105) were seeded in 96-well flat-bottom plates. PD-1 expression was measured using Jurkat-PD-1 null was not detected in Jurkat-PD-1 cells; + The number of PD-1 molecules on cells was determined to be approximately 4,000 using the quantibrite bead method. Bispecific and control antibodies were added at six serial dilutions (100 μg / ml to 0.001 μg / ml), and the cells were incubated for 1 hour at 37°C / 5% CO2. After 1 hour, human recombinant TGF-β1 (R&D Systems catalog number 7754-BH) was added to a final concentration of 10 ng / ml, and the cells were incubated for an additional 2 hours at 37°C / 5% CO2. After incubation, the cells were gently washed with PBS. Cell lysates were prepared using lysis buffer (MSD# R60TX-2) containing phosphatase inhibitors (Sigma# P0044 and P-5726) and protease inhibitor (Pierce Biotechnology# 87785). Samples were normalized to total protein using the BCA protein assay kit (Pierce# 23227). Phosphorylated SMAD2 / 3 levels were determined using ELISA (Cell Signaling #12001) according to the manufacturer's instructions. GraphPad Prism (8.2.0) was used to plot the graphs.

[0239] The results are shown in Tables 4, 5, and 6 and Figure 3. Table 6 shows the Jurkat-PD-1 antibody binding of the 10 bispecific antibodies. null Jurkat-PD-1 vs. cells + The fold difference in potency in inhibiting TGF-βRII signaling in cells is shown, with the bispecific antibodies having a fold difference between 200- and 11,000-fold higher than the fold difference of the reference antibody TGF1 analog in this assay.

[0240] All bispecific antibodies were designed to target Jurkat-PD-1 null Jurkat-PD-1 cells +The bispecific antibody inhibited TGF-βRII signaling in both Jurkat-PD-1 and Jurkat-PD-1 cells. + Jurkat-PD-1 inhibits TGF-βRII signaling in cells null The bispecific antibodies were more potent than in Jurkat-PD-1 cells, demonstrating that they inhibit TGF-β-induced SMAD2 / 3 phosphorylation in a manner that correlates with PD-1 expression. null Many bispecific antibodies that are monovalent for binding to TGF-βRII require higher concentrations to inhibit TGF-βRII signaling in cells than analogs of TGF1. + It is superior to bivalent monospecific analogs of the reference TGF-βRII antibody TGF1 in inhibiting TGF-βRII signaling in cells.

[0241] JPEG2024542879000004.jpg70166

[0242] JPEG2024542879000005.jpg69166

[0243] JPEG2024542879000006.jpg122166

[0244] Example 5 - Stimulated and unstimulated CD4 + and CD8 + Phosphorylated SMAD2 assay using T cells The bispecific antibodies were characterized in a phospho-SMAD2 assay to determine their specificity in inhibiting TGF-βRII signaling in PD-1-positive T cells. The phospho-SMAD2 assay compared the activity of the bispecific antibodies in unstimulated T cells expressing low levels of PD-1 with stimulated T cells expressing high levels of PD-1 to determine whether the bispecific antibodies inhibited TGF-β-induced SMAD2 phosphorylation in a manner that correlated with PD-1 expression. An analog of the reference TGF-βRII antibody TGF1, a reference PD-1 antibody pembrolizumab, and a negative control bivalent monospecific IgG1 antibody comprising a heavy chain having the amino acid sequence set forth in SEQ ID NO: 86 and a light chain having the amino acid sequence set forth in SEQ ID NO: 87 were included as positive and negative controls, respectively.

[0245] PBMCs from healthy donors were stimulated with 1 μg / ml anti-CD3 antibody (BD Biosciences # 555336) for 48 hours, followed by serum deprivation (0.1% FBS) for 16 hours. + and CD8 + The number of PD-1 molecules on T cells was determined to be approximately 1,000–2,000 using the quantibrite bead method. Stimulated and unstimulated PBMCs were then incubated with the bispecific antibody and control antibody for 30 minutes at room temperature. Recombinant human TGF-β1 (Cell Signaling #7754-BH) was added at a final concentration of 1 ng / ml, and the cells were incubated for an additional 30 minutes. Finally, the cells were washed twice with PBS and stained for cell surface markers, followed by intracellular phosphorylated SMAD2 staining.

[0246] The following antibodies were used for flow cytometry staining: human CD45 (clone: ​​HI30; catalog #557748, BD Biosciences), human CD11b (clone: ​​M1 / 70; catalog #563015, BD Biosciences), human CD3 (clone: ​​UCHT1; catalog #565491, BD Biosciences), human CD4 (clone: ​​SK3; catalog #563550, BD Biosciences), human CD8 (clone #SK1; catalog #344714, Biolegend), and human phosphorylated SMAD2 (catalog #56532, Cell Signaling). A viability dye (Biolegend #423114) was used to exclude dead cells during analysis. Cell acquisition was performed under a FACSymphony A3 using DIVA software.

[0247] PBMCs were gated based on size and granularity using FSC-A vs. SSC-A to exclude debris. Then, dead cells were excluded using a fixable dead cell staining dye. CD45-positive cells were selected, followed by CD11b-negative and CD3-positive T cells. Finally, CD4 and CD8-positive subsets were gated, and the phospho-SMAD2 signal was measured as geometric mean fluorescence intensity (GMFI) on these subsets. Data analysis was performed using FlowJo software, and GraphPad Prism (8.2.0) was used to plot graphs.

[0248] The results are shown in Figure 4. All bispecific antibodies stimulated CD4 + and CD8 + In one donor (donor B), the bispecific antibody inhibited TGF-βRII signaling in unstimulated CD4 T cells. + and CD8 +In the other donor, a bispecific antibody comprising a PD-1 heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 18 and a TGF-βRII heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 23 inhibited unstimulated CD4 T cells. + and CD8 + A bispecific antibody that does not inhibit TGF-βRII signaling in T cells and that comprises a PD-1 heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 13 and a TGF-βRII heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 47 inhibits non-stimulatory CD4 + and CD8 + In T cells, stimulated CD4 + T and CD8 + Inhibition of TGF-βRII signaling was significantly less potent in T cells than in PD-1 T cells. These data confirm the findings in Example 4 that the bispecific antibody inhibits TGF-β-induced SMAD phosphorylation in a manner that correlates with PD-1 expression.

[0249] Example 6 - Exhausted Mixed Lymphocyte Reaction (MLR) Assay The bispecific antibodies were characterized in exhausted mixed lymphocyte reaction (MLR) assays to determine their potency in inducing cytokine production by exhausted T cells.

[0250] The antibody samples included several bispecific antibodies, a negative control IgG1 antibody (RSV), the reference PD-1 antibody pembrolizumab, an analog of the reference TGF-βRII antibody TGF1, and a combination of the reference PD-1 antibody pembrolizumab and an analog of the reference TGF-βRII antibody TGF1.

[0251] Human PBMCs were isolated from healthy donors. In vitro T cell exhaustion was performed by repeatedly activating PBMCs with Staphylococcal enterotoxin B (SEB) for 6 days. Total T cells were isolated using a T cell isolation kit (StemCell Technologies, catalog #17951) according to the manufacturer's instructions. T cells were mixed with dendritic cells (DCs) from different donors at a T cell / DC ratio of 10:1 in a 96-well U-bottom plate. Serial dilutions of antibody samples were added, and the plates were incubated at 37°C / 5% CO2 for an additional 6 days. After 6 days, IFN-γ, IL-2, or TNF-α cytokine levels in the supernatants were measured using custom MSD kits. GraphPad Prism (8.2.0) was used to plot graphs.

[0252] Representative results from two experiments with five donors each are shown in Figure 5A, C, and D. Results from an additional experiment with three donors are shown in Figure 5B and E. Most bispecific antibodies induced similar levels of IFN-γ, IL-2, or TNF-α as the reference PD-1 antibody pembrolizumab, an analog of the reference PD-L1-TGF-β TRAP molecule, or a combination of the reference PD-1 antibody pembrolizumab and the reference TGF-βRII antibody TGF1. The IC of many bispecific antibodies from the initial studies 50 The values ​​are shown in Table 7.

[0253] JPEG2024542879000007.jpg99166

[0254] Example 7 - HEK-BLUE-PD-1 TGF-β Reporter Assay The bispecific antibodies were characterized in a HEK-BLUE-PD-1 TGF-β reporter assay to determine their potency in inhibiting TGF-β-induced signaling in T cells. HEK-Blue™ TGF-β cells or HEK-Blue™ TGF-β-PD-1 +Stimulation of cells with TGF-β induces activation of the TGF-β / Smad signaling pathway, leading to the formation of the Smad3 / Smad4 complex, which induces the production of SEAP.

[0255] Reagents used: Recombinant human TGF-beta 1 (human cell expressed) protein, R&D, Catalog # 7754-BH. Growth medium: DMEM, 4.5 g / l glucose, 2 mM L-glutamine, 10% heat-inactivated fetal bovine serum (FBS; 56°C for 30 min), 100 μg / ml Normocin™, Pen-Strep (100 U / ml-100 μg / ml). HEK-Blue™ TGF-β-PD-1 + Growth medium for cells containing puromycin 0.4ug / mL. Test medium: DMEM 4.5g / l glucose, 2mM L-glutamine, 0.1% heat-inactivated FBS, Pen-Strep (100U / ml-100µg / ml). No Normocin™, blasticidin, hygromycin B, or Zeocin™.

[0256] A stable PD-1-expressing HEK-Blue™ TGF-β cell line was generated as follows: the full-length cds of PD-1 were inserted into the mammalian expression vector pD2529-EFM(ATUM), which contains a puromycin resistance gene. The promoter is a modified EF1a. Vector construction was performed by ATUM, and the sequence was verified. See Figure 10 for the vector map. HEK-Blue™ TGF-β cells (Invivogen), which lack detectable PD-1 expression, were transfected using TransIT-293 transfection reagent (Mirus Bio) according to the manufacturer's protocol. Stably transfected cells were selected in HEK-Blue™ TGF-β medium containing 0.4 μg / ml puromycin. Clones were isolated by limiting dilution and characterized for PD-1 expression by Western blot. One clone was selected based on: 1) its stable and uniform surface PD-1 expression (single peak in the histogram plot); 2) similar surface TGF-βRII expression compared to the parental HEK-Blue™ cell line; and 3) a similar EC50 to the reference antibody TGF1 in the reporter assay. The GMFI of PD-1 in the selected clone was 3272, compared to 5 for the parental cell line and 8 for the isotype control. The number of PD-1 molecules on these cells was determined to be approximately 20,000 using the quantibrite bead method.

[0257] Antibody samples included several bispecific antibodies, a negative control IgG1 antibody (RSV), the reference PD-1 antibody pembrolizumab, and an analogue of the reference TGF-βRII antibody TGF1.

[0258] HEK-Blue™ TGF-β cells (Invivogen, catalog code: hkb-tgfb) expressing TGF-βRII, or HEK-Blue™ TGF-β-PD-1 +Cells were seeded in test medium at 25,000 cells per well in a 96-well flat-bottom plate. Serial dilutions of antibody samples were added, and the cells were incubated at room temperature for 1 hour; human recombinant TGF-β1 was then added to a final concentration of 1 ng / ml. The cells were incubated overnight at 37°C / 5% CO2. After incubation, 40 μl of supernatant was transferred from each well to a new flat-bottom 96-well plate; 160 μl of resuspended QUANTI-Blue™ solution was added to the supernatant. The plate was incubated at 37°C / 5% CO2 for 40 minutes. The amount of SEAP secreted into the supernatant was assessed using QUANTI-Blue™ solution, a SEAP detection reagent. SEAP levels were determined at 650 nm using a spectrophotometer. GraphPad Prism (8.2.0) was used to plot the graphs.

[0259] The results are shown in Figure 6 and Table 8. Many bispecific antibodies demonstrate potent inhibition of TGF-β-induced signaling in a manner that correlates with PD-1 expression.

[0260] JPEG2024542879000008.jpg111166

[0261] Example 8 - Treg suppression assay The bispecific antibodies were characterized in a Treg suppression assay to determine their ability to eliminate or reduce the suppressive effect of regulatory T cells, thereby inducing cytokine production by T cells.

[0262] The antibody samples included several bispecific antibodies, a negative control IgG1 antibody (RSV), the reference PD-1 antibody pembrolizumab, an analog of the reference TGF-βRII antibody TGF1, and a combination of the reference PD-1 antibody pembrolizumab and an analog of the reference TGF-βRII antibody TGF1.

[0263] Human PBMCs were isolated from healthy donors by Ficoll-Paque gradient centrifugation. Tregs were isolated using the EasySep Treg Isolation Kit (StemCell Technologies, #18063) according to the manufacturer's instructions. Tregs were mixed with PBMCs from the same donors. Anti-CD3 antibody (BD Biosciences, #555336) and anti-CD28 antibody (BD Biosciences, #555725) were added to the coculture. Finally, serial dilutions of the bispecific antibodies were added, and the plates were incubated at 37°C / 5% CO2 for 3 days. After 3 days of incubation, IFN-γ and TNF-α cytokine levels in the supernatants were measured using an MSD kit (Mesoscale). GraphPad Prism (8.2.0) was used to plot the graphs.

[0264] The results are shown in Figure 7 and Table 9. Most bispecific antibodies induced similar levels of IFN-γ or TNF-α as the reference PD-1 antibody pembrolizumab, or the combination of the reference PD-1 antibody pembrolizumab with an analog of the reference TGF-βRII antibody TGF1.

[0265] JPEG2024542879000009.jpg93166

[0266] Example 9 - Macrophage suppression assay Tumor-associated macrophages of the M2 phenotype inhibit T cell proliferation and cytokine production. Bispecific antibodies were characterized in an M2 macrophage suppression assay to test whether they could reverse the inhibitory effects of M2 macrophages on T cell proliferation and IFN-γ production.

[0267] The bispecific antibodies were tested alongside a negative control IgG1 antibody (RSV), an analog of the reference TGF-βRII antibody TGF1, an analog of the reference PD-L1-TGF-β TRAP molecule, an analog of the reference PD-1 antibody nivolumab, the commercially available reference PD-1 antibody Opdivo (BMS), a positive control antibody anti-LILRB2 (Biolegend), a rat IgG2a isotype control (Biolegend), and a huIgG4 isotype control (Biolegend). In addition, stimulated CD4+ cells without test or control antibodies were tested. + Co-culture of T cells with M2 macrophages or CD4 + T cells alone (unstimulated vs. stimulated) were included as a control condition in the assay.

[0268] PBMC-isolated monocytes from three different healthy donors were differentiated into macrophages using M-CSF for 6 days and polarized using a specific cocktail of cytokines: IL-4 (20 ng / ml), IL-10 (20 ng / ml), and TGF-β (20 ng / ml) (+M-CSF) to obtain M2 macrophages. To confirm the phenotype, expression of CD163 (Miltenye Biotec), CD209 (Miltenye Biotec), CD206 (BD Bioscience), and CD86 (Miltenye Biotec) was measured by flow cytometry (Figure 8). CD163 is expressed by M2-like macrophages in all donors. CD209 and CD206 are highly expressed by M2 macrophages in all donors. CD86 is expressed at low levels by M2-like macrophages. M2-like macrophages exhibit the expected phenotype in all three donors.

[0269] M2 macrophages were then activated with LPS (100 ng / ml) for 4 hours. Macrophages were harvested, washed, and transfected with autologous CD4 +Activated T cells (activated with CD3 / CD28 ImmunoCult™ from StemCell technologies) were seeded at a 1:5 ratio in the presence of test or control antibodies at a concentration of 10 μg / ml in five replicates. On day 5 of co-culture, the concentration of secreted IFN-γ was measured by ELISA (LEGEND MAX™ Human IFN-γ ELISA Kit, Biolegend). Data were analyzed using multi-way analysis of variance (ANOVA) in GraphPad Prism 7.0.

[0270] The results are shown in Figure 8. Many bispecific antibodies induced similar or higher levels of IFN-γ compared to analogs of the reference PD-1 antibody nivolumab, analogs of the reference TGF-βRII antibody TGF1, or analogs of the reference PD-L1-TGF-β TRAP molecule.

[0271] Example 10 - In vivo humanized NSG MDA-MB-231 mouse model The bispecific antibody was characterized in vivo in a humanized NSG MDA-MB-231 mouse model to determine its efficacy in reducing tumor volume. This mouse model was tested using 10 mg / kg of a negative control bivalent monospecific IgG1 antibody comprising a heavy chain having the amino acid sequence set forth in SEQ ID NO: 86 and a light chain having the amino acid sequence set forth in SEQ ID NO: 87; a reference TGF-βRII antibody TGF1 analog (10 mg / kg); a reference PD-1 antibody pembrolizumab (10 mg / kg); a combination of a reference TGF-βRII antibody TGF1 analog (10 mg / kg) and a reference PD-1 antibody pembrolizumab (10 mg / kg); and a reference PD-L1-TGF-β TRAP molecule analog (10 mg / kg). The results are shown in Figure 9A.

[0272] Humanized CD34 NSG mice were cultured in a total of 3 × 10 cells suspended in equal volumes in 100 μl of serum-free culture medium and Matrigel matrix (Corning). 6 MDA-MB-231 tumor cells were inoculated subcutaneously. After tumor establishment (80-100 mm3 ), mice were randomly assigned to the following treatment groups: 1) a negative control bivalent monospecific IgG1 antibody (10 mg / kg) comprising a heavy chain having the amino acid sequence set forth in SEQ ID NO: 86 and a light chain having the amino acid sequence set forth in SEQ ID NO: 87; 2) reference TGF-βRII antibody TGF1 analog (10 mg / kg); 3) reference PD-1 antibody pembrolizumab (10 mg / kg); 4) reference TGF-βRII antibody TGF1 analog (10 mg / kg) + reference PD-1 antibody pembrolizumab (10 mg / kg); 5) a bispecific antibody (1 mg / kg) comprising a TGF-βRII-binding domain having a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 43 and a PD-1-binding domain having a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 9; 6) a bispecific antibody (10 mg / kg) comprising a TGF-βRII-binding domain having a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 43 and a PD-1-binding domain having a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 9; 7) a bispecific antibody (1 mg / kg) comprising a TGF-βRII-binding domain having a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 43 and a PD-1-binding domain having a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 19; 8) a bispecific antibody (10 mg / kg) comprising a TGF-βRII-binding domain having a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 43 and a PD-1-binding domain having a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 19; 9) A bispecific antibody (1 mg / kg) comprising a TGF-βRII-binding domain having a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 23 and a PD-1-binding domain having a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 14; 10) A bispecific antibody (10 mg / kg) comprising a TGF-βRII-binding domain having a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 23 and a PD-1-binding domain having a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 14; 11) A bispecific antibody (1 mg / kg) comprising a TGF-βRII-binding domain having a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 23 and a PD-1-binding domain having a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 19; 12) A bispecific antibody (10 mg / kg) comprising a TGF-βRII-binding domain having a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 23 and a PD-1-binding domain having a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 19; 13) A bispecific antibody (10 mg / kg) comprising a TGF-βRII-binding domain having a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 39 and a PD-1-binding domain having a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 9; 14) A bispecific antibody (10 mg / kg) comprising a TGF-βRII-binding domain having a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 27 and a PD-1-binding domain having a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 9; 15) A bispecific antibody (10 mg / kg) comprising a TGF-βRII-binding domain having a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 23 and a PD-1-binding domain having a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 18; 16) A bispecific antibody (10 mg / kg) comprising a TGF-βRII-binding domain having a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 47, and a PD-1-binding domain having a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 13.

[0273] Each group had 8-9 mice. Animals were injected intraperitoneally every 5 days for a period of 27 or 30 days. Tumors were measured using calipers and calculated using the formula: l (length) × w 2Tumor volumes were calculated by fitting to an ellipsoid using (width) x 1 / 2. Body weight was also monitored throughout the study. Tumors were harvested at the end of the study (24 hours after the last dose) for tumor immune profiling and receptor occupancy.

[0274] The results are shown in Figures 9B-E. All bispecific antibodies induced superior anti-tumor responses than the combination of the reference PD-1 antibody pembrolizumab and the reference TGF-βRII antibody TGF1 analog. The bispecific antibodies induced superior anti-tumor responses at both the 1 mg / kg and 10 mg / kg dose levels, while the reference antibody combination included a 10 mg / kg dose of each reference antibody.

[0275] Along with efficacy, receptor coverage of both PD-1 and TGF-βRII receptors with the bispecific antibody treatment was observed in T cells analyzed 24 hours after the last dose, similar to that observed with the combination treatment of the reference TGF-βRII antibody TGF1 analog with the reference PD-1 antibody pembrolizumab (Figure 9F).

[0276] Example 11 - Testing Different Doses in an In Vivo Humanized NSG MDA-MB-231 Mouse Model A bispecific antibody comprising a TGF-βRII-binding domain having a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 23 and a PD-1-binding domain having a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 18 was characterized in vivo in a humanized NSG MDA-MB-231 mouse model at two different dose levels, 1 mg / kg and 10 mg / kg, as described in Example 10. A negative control bivalent monospecific IgG1 antibody comprising a heavy chain having the amino acid sequence set forth in SEQ ID NO: 86 and a light chain having the amino acid sequence set forth in SEQ ID NO: 87 was included at 10 mg / kg.

[0277] The results are shown in Figure 11. The bispecific antibody induced significant anti-tumor responses at both dose levels.

[0278] [Sequence table] SEQ ID NO: 1 - Heavy chain variable region EVQLVQSGAEVKKPGSSMKVSCKASGGTFSSYVISWVRQAPGQGLEWMGMIIPVFDTSSYEKKFQGRITIIADKSTSTVYLELSSLRSEDAAVYYCARGTVEATLLFDFWGQGTLVTVSS

[0279] SEQ ID NO:2 - Heavy chain CDR1 SYVIS

[0280] SEQ ID NO:3 - Heavy chain CDR2 MIIPVFDTSSYEKKFQG

[0281] SEQ ID NO:4 - Heavy chain CDR3 GTVEATLLFDF

[0282] SEQ ID NO:5 - Heavy chain variable region QVQLQESGPGLVKPSETLSLTCTVSNGSLGFDFWSWIRQPPGRGLEWIGYIYYSGSWSLNPSFKGRVTMSVDTSKNQFSLNLRSVTAADTAVYYCARGGYTGYGGDWFDPWGQGTLVTVSS

[0283] SEQ ID NO:6 - Heavy chain CDR1 FDFWS

[0284] SEQ ID NO:7 - Heavy chain CDR2 YIYYSGSWSLNPSFKG

[0285] SEQ ID NO:8 - Heavy chain CDR3 GGYTGYGGDWFDP

[0286] SEQ ID NO:9 - Heavy chain variable region QVQLQESGPGLVKPSETLSLTTCTVSDGSIGYHFWSWIRQPPGRGLEWIGYIVYSGSYNVNPSLKTRVTMSVDTSKNQFSLNLRSVTAADTAVYYCARGGYTGYGGDWFDPWGQGTLVTVSS

[0287] SEQ ID NO: 10 - Heavy chain CDR1 YHFWS

[0288] SEQ ID NO: 11 - Heavy chain CDR2 YIVYSGSYNVNPSLKT

[0289] SEQ ID NO: 12 - Heavy chain CDR3 GGYTGYGGDWFDP

[0290] SEQ ID NO: 13 - Heavy chain variable region QVQLQESGPGLVKPSETLSLTTCTVSEGSIGYHFWSWIRQPPGRGLEWIGYIVYSGSYNVNPSLKTRVTMSVDTSKNQFSLNLRSVTAADTAVYYCARGGYTGYGGDWFDPWGQGTLVTVSS

[0291] SEQ ID NO: 14 - Heavy chain variable region QVQLVQSGSELKKPGASVKVSCKASGYTFTRFALHWVRQAPGQGLEWMGWIDPNTGTPTFAQGVTGRFVFSLDTSVTTAYLQISSLKAEDTAVYYCARSLGYCDSDICYPNWIFDNWGQGTLVTVSS

[0292] SEQ ID NO: 15 - Heavy chain CDR1 RFALH

[0293] SEQ ID NO: 16 - Heavy chain CDR2 WIDPNTGTPTFAQGVTG

[0294] SEQ ID NO: 17 - Heavy chain CDR3 SLGYCDSDICYPNWIFDN

[0295] SEQ ID NO: 18 - Heavy chain variable region QVQLVQSGSELKKPGASVKVSCKASGYTFTRFALHWVRQAPGQGLEWMGWIDPNTGTPTFAQGVTGRFVFSLDTSVTTAYLQISSLKAEDTAVYYCARSLGYCDSDICYPNWIFDNWGQGTLVTVSS

[0296] SEQ ID NO: 19 - Heavy chain variable region QVQLVQSGSELKKPGASVKVSCKASGYTFTRFALSWVRQAPGQGLEWMGWIDPNTGTPTYAQDFTGRFVFSLDTSVTTAYLQISSLKAEDTAVYYCARSLGYCGSDICYPNGILDNWGQGTLVTVSS

[0297] SEQ ID NO:20 - Heavy chain CDR1 RFALS

[0298] SEQ ID NO:21 - Heavy chain CDR2 WIDPNTGTPTYAQDFTG

[0299] SEQ ID NO: 22 - Heavy chain CDR3 SLGYCGSDICYPNGILDN

[0300] SEQ ID NO:23 - Heavy chain variable region EVQLVESGGGLVQPGGSLRLSCAASGFTFDIYAMTWVRQAPGKGLEWVSVISGSGGTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARRGQYRDIVGATDYWGQGTLVTVSS

[0301] SEQ ID NO:24 - Heavy chain CDR1 IYAMT

[0302] SEQ ID NO:25 - Heavy chain CDR2 VISGSGGTTYYADSVKG

[0303] SEQ ID NO:26 - Heavy chain CDR3 RGQYRDIVGATDY

[0304] SEQ ID NO:27 - Heavy chain variable region EVQLVESGGGLVQPGGSLRLSCAASGFTFDINAMTWVRQAPGKGLEWVSVISGSGGTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARRGQYRDIVGATDYWGQGTLVTVSS

[0305] SEQ ID NO:28 - Heavy chain CDR1 INAMT

[0306] SEQ ID NO:29 - Heavy chain CDR2 VISGSGGTTYYADSVKG

[0307] SEQ ID NO: 30 - Heavy chain CDR3 RGQYRDIVGATDY

[0308] SEQ ID NO:31 - Heavy chain variable region QVQLVESGGGLVEPGGSLRLSCAASGFTFSNAWMSWVRQAPGKGLEWVGRIKTTISGGATDFAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTLDLRDYWGQGTLVTVSS

[0309] SEQ ID NO: 32 - Heavy chain CDR1 NAWMS

[0310] SEQ ID NO: 33 - Heavy chain CDR2 RIKTTISGGATDFAAPVKG

[0311] SEQ ID NO: 34 - Heavy chain CDR3 DLRDY

[0312] SEQ ID NO: 35 - Heavy chain variable region QVQLVESGGGLVEPGGSLRLSCAASGFKFSNAWMSWVRQAPGKGLEWVGRIKTTISGGATQFAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTLDLRDYWGQGTLVTVSS

[0313] SEQ ID NO: 36 - Heavy chain CDR1 NAWMS

[0314] SEQ ID NO: 37 - Heavy chain CDR2 RIKTTISGGATQFAAPVKG

[0315] SEQ ID NO: 38 - Heavy chain CDR3 DLRDY

[0316] SEQ ID NO: 39 - Heavy chain variable region QVQLVESGGGLVQPGGSLRLSCAVSGFTFRRYAMSWVRQAPGKGLEWVSAISASGDRTHNTDSVKGRFSISRDNSKNTLYLQMNSLRAEDTAVYFCAKGIAASGKNYFDPWGQGTLVTVSS

[0317] SEQ ID NO: 40 - Heavy chain CDR1 RYAMS

[0318] SEQ ID NO: 41 - Heavy chain CDR2 AISASGDRTHNTDSVKG

[0319] SEQ ID NO: 42 - Heavy chain CDR3 GIAASGKNYFDP

[0320] SEQ ID NO: 43 - Heavy chain variable region QVQLVESGGGLVQPGGSLRLSCAVSGFTFSRYAMSWVRQAPGKGLEWVSAISASGDRTKNTDSVKGRFSISRDNSKNTLYLQMNSLRAEDTAVYFCAKGTAAAGKNYFDPWGQGTLVTVSS

[0321] SEQ ID NO: 44 - Heavy chain CDR1 RYAMS

[0322] SEQ ID NO: 45 - Heavy chain CDR2 AISASGDRTKNTDSVKG

[0323] SEQ ID NO: 46 - Heavy chain CDR3 GTAAAGKNYFDP

[0324] SEQ ID NO: 47 - Heavy chain variable region QVQLVESGGGLVQPGGSLRLSCAVSGFTFSRYAMSWVRQAPGKGLEWVSAISASGDRTKYTDSVKGRFSISRDNSKNTLYLQMNSLRAEDTAVYFCAKGTAAAGKNYFDPWGQGTLVTVSS

[0325] SEQ ID NO: 48 - Light chain variable region DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPTFGQGTKVEIK

[0326] Light chain CDR1 according to SEQ ID NO: 49-IMGT QSISSY

[0327] Light chain CDR2 according to SEQ ID NO: 50-IMGT AAS

[0328] Light chain CDR3 according to SEQ ID NO: 51-IMGT QQSYSTPPT

[0329] SEQ ID NO: 52 - Light chain variable region DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPITFGQGTRLEIK

[0330] Light chain CDR1 according to SEQ ID NO: 53-IMGT QSISSY

[0331] Light chain CDR2 according to SEQ ID NO: 54-IMGT AAS

[0332] Light chain CDR3 according to SEQ ID NO: 55-IMGT QQSYSTPPIT

[0333] SEQ ID NO:56 - Light chain variable region EIVMTQSPATLSVSPGERATLSCRASQSVSSNLAWYQQKPGQAPRLLIYGASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYNNWPWTFGQGTKVEIK

[0334] Light chain CDR1 according to SEQ ID NO: 57-IMGT QSVSSN

[0335] Light chain CDR2 according to SEQ ID NO: 58-IMGT GAS

[0336] Light chain CDR3 according to SEQ ID NO: 59-IMGT QQYNNWPWT

[0337] SEQ ID NO: 60 - Light chain variable region EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPWTFGQGTKVEIK

[0338] Light chain CDR1 according to SEQ ID NO: 61-IMGT QSVSSSY

[0339] Light chain CDR2 according to SEQ ID NO: 62-IMGT GAS

[0340] Light chain CDR3 according to SEQ ID NO: 63-IMGT QQYGSSPWT

[0341] SEQ ID NO: 64 - Light chain variable region SYVLTQPPSVSVAPGETARITCGGDNIGRKSVYWYQQKSGQAPVLVIYYDSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDGSSDHWVFGGGTKLTVL

[0342] Light chain CDR1 according to SEQ ID NO: 65-IMGT NIGRKS

[0343] Light chain CDR2 according to SEQ ID NO: 66-IMGT YDS

[0344] Light chain CDR3 according to SEQ ID NO: 67-IMGT QVWDGSSDHWV

[0345] SEQ ID NO: 68 - Hinge region EPKSCDKTHTCPPCP

[0346] SEQ ID NO: 69 - CH1 region ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRV

[0347] SEQ ID NO: 70 - CH2 region APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK

[0348] SEQ ID NO: 71 - CH2-DM region APELGRGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK

[0349] SEQ ID NO: 72 - CH3 region GQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0350] SEQ ID NO: 73 - CH3-DE region GQPREPQVYTDPPSREEMTKNQVSLTCEVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0351] SEQ ID NO: 74-CH3-KK region GQPREPQVYTKPPSREEMTKNQVSLKCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0352] SEQ ID NO: 75 - CL region RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0353] SEQ ID NO: 76 - Heavy chain reference anti-TGF-βRII antibody TGF1 analog QLQVQESGPGLVKPSETLSLTCTVSGGSISNSYFSWGWIRQPPGKGLEWIGSFYYGEKTYYNPSLKSRATISIDTSKSQFSLKLSSVTAADTAVYYCPRGPTMIRGVIDSWG QGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCD KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0354] SEQ ID NO: 77 - Light chain reference anti-TGF-βRII antibody TGF1 analog EIVLTQSPATLSLSPGERATLSCRASQSVRSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0355] SEQ ID NO: 78 - Heavy chain reference PD-1 antibody pembrolizumab QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGINPSNGGTNFNEKFKNRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWG QGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYG PPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKT ISKAKGQPREPQVYTLPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0356] SEQ ID NO: 79 - Light chain reference PD-1 antibody pembrolizumab EIVLTQSPATLLSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQAPRLLIYLASYLESGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLTFGGGGTKVE IKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0357] SEQ ID NO: 80 - Heavy chain reference PD-L1-TGF-β TRAP molecule analogue EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYIMMWVRQAPGKGLEWVSSIYPSGGITFYADTVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARIKLGTVTTVDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKD YFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGAGGGGS GGGGSGGGGSGGGGSGIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD

[0358] SEQ ID NO: 81 - Light chain reference PD-L1-TGF-β TRAP molecule analogue QSALTQPASVGSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYDVSNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYTSSSTRVFGTGTKVT VLGQPKANTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS

[0359] SEQ ID NO:82 - Human TGF-βRII isoform A MGRGLLRGLWPLHIVLWTRIASTIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSD ECNDNIIFSEEYNTSNPDLLLVIFQVTGISLLPPLGVAISVIIIFYCYRVNRQQKLSSTWETGKTRKLMEFSEHCAIILEDDRSDISSTCANNINHNTELLPIELDTLVGKGRFAEVYKAKLKQNTSEQFETVAVKIFPYEE YASWKTEKDIFSDINLKHENILQFLTAEERKTELGKQYWLITAFHAKGNLQEYLTRHVISWEDLRKLGSSLARGIAHLHSDHTPCGRPKMPIVHRDLKSSNILVKNDLTCCLCDFGLSLRLDPTLSVDDLANSGQVGTARYM APEVLESRMNLENVESFKQTDVYSMALVLWEMTSRCNAVGEVKDYEPPFGSKVREHPCVESMKDNVLRDRGRPEIPSFWLNHQGIQMVCETLTECWDHDPEARLTAQCVAERFSELEHLDRLSGRSCSEEKIPEDGSLNTTK

[0360] SEQ ID NO: 83 - Extracellular domain of human TGF-βRII isoform A TIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDLLLVIFQ

[0361] SEQ ID NO:84 - Human TGF-βRII isoform B MGRGLLRGLWPLHIVLWTRIASTIPPHVQKSDVEMEAQKDEIICPSCNRTAHPLRHINNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIM KEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDLLLVIFQVTGISLLPPLGVAISVIIIFYCYRVNRQQKLSSTWETGKTRKLMEFSEHCAIILEDDRSDISSTCANNINHNTELLPIELDTLVGKGRFAEVYKAKLKQNTSEQF ETVAVKIFPYEEYASWKTEKDIFSDINLKHENILQFLTAEERKTELGKQYWLITAFHAKGNLQEYLTRHVISWEDLRKLGSSLARGIAHLHSDHTPCGRPKMPIVHRDLKSSNILVKNDLTCCLCDFGLSLRLDPTLSVDDLANSGQV GTARYMAPEVLESRMNLENVESFKQTDVYSMALVLWEMTSRCNAVGEVKDYEPPFGSKVREHPCVESMKDNVLRDRGRPEIPSFWLNHQGIQMVCETLTECWDHDPEARLTAQCVAERFSELEHLDRLSGRSCSEEKIPEDGSLNTTK

[0362] SEQ ID NO: 85 - Extracellular domain of isoform B of human TGF-βRII TIPPHVQKSDVEMEAQKDEIICPSCNRTAHPLRHINNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDLLLVIFQ

[0363] SEQ ID NO: 86 - Heavy chain negative control RSV IgG1 antibody EVQLVESGGGVVQPGRSLRLSCAASGFTFSNYGMHWVRQAPGKGLEWVAVISYDGSTKYSADSLKGRFTISRDNSKNTLYLQMNSLRADDTTAVYYCAKEGWSFDSSGYRSWFD SWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKS CDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPI EKTISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0364] SEQ ID NO:87 - Light chain DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0365] SEQ ID NO: 88 - Heavy chain variable region QVQLVESGGGLVQPGGSLRLSCAVSGFTFSRYAMSWVRQAPGKGLEWVSAISASGDRTKNTDSVKGRFSISRDNSKNTLYLQMNSLRAEDTAVYYCAKGTAAAGKNYFDPWGQGTLVTVSS

[0366] SEQ ID NO: 89 - Heavy chain variable region QVQLVESGGGLVQPGGSLRLSCAVSGFTFSRYAMSWVRQAPGKGLEWVSAISASGDRTKYTDSVKGRFSISRDNSKNTLYLQMNSLRAEDTAVYYCAKGTAAAGKNYFDPWGQGTLVTVSS

[0367] SEQ ID NO: 90 - Heavy chain CDR1 RYAMS

[0368] SEQ ID NO: 91 - Heavy chain CDR2 AISASGDRTKYTDSVKG

[0369] SEQ ID NO: 92 - Heavy chain CDR3 GTAAAGKNYFDP

[0370] SEQ ID NO: 93 - V region VK1-39 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTP

[0371] SEQ ID NO: 94-VK1-39 / JK1 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPTFGQGTKVEIK

[0372] SEQ ID NO: 95-VK1-39 / JK5 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPITFGQGTRLEIK

[0373] SEQ ID NO: 96 - Heavy chain reference nivolumab analog antibody QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKGLEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAEDTAVYYCATNDDYWGQGTLVT VSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCP PCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTIS KAKGQPREPQVYTLPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0374] SEQ ID NO: 97 - Light chain reference nivolumab analog antibody EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWPRTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0375] SEQ ID NO: 98 - V region VK3-15 EIVMTQSPATLSVSPGERATLSCRASQSVSSNLAWYQQKPGQAPRLLIYGASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYNNWP

[0376] SEQ ID NO: 99-VK3-15 / JK1 EIVMTQSPATLSVSPGERATLSCRASQSVSSNLAWYQQKPGQAPRLLIYGASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYNNWPWTFGQGTKVEIK

[0377] SEQ ID NO: 100 - V region VK3-20 EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSP

[0378] SEQ ID NO: 101-VK3-20 / JK1 EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPWTFGQGTKVEIK

[0379] SEQ ID NO: 102 - V region VL3-21 SYVLTQPPSVSVAPGETARITCGGDNIGRKSVYWYQQKSGQAPVLVIYYDSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDGSSDH

[0380] SEQ ID NO: 103-VL3-21 / JL3 SYVLTQPPSVSVAPGETARITCGGDNIGRKSVYWYQQKSGQAPVLVIYYDSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDGSSDHWVFGGGTKLTVL

[0381] SEQ ID NO: 104 - Extracellular domain TGF-βRII IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD

Claims

1. A multispecific binding site comprising a PD-1 binding domain and a TGF-βRII binding domain, The PD-1 binding domain 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:2, SEQ ID NO:3, and SEQ ID NO:4, 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:6, SEQ ID NO:7, and SEQ ID NO:8, 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:10, SEQ ID NO:11, and SEQ ID NO:12, respectively; d) 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:15, SEQ ID NO:16, and SEQ ID NO:17, respectively; or e) 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 HDCRs may contain at most three, two, or one amino acid variation; Multispecific binding site.

2. 2. A multispecific binding site according to claim 1, The PD-1 binding domain comprises a heavy chain variable region having an amino acid sequence set forth in any one of SEQ ID NOs: 1; 5; 9; 13; 14; 18; 19, or having at least 80%, preferably 85%, more preferably 90%, or most preferably 95% sequence identity thereto. Multispecific binding site.

3. 2. A multispecific binding site according to claim 1, the PD-1 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 NOs:49, 50, and 51, respectively, or variants thereof; Multispecific binding site.

4. 4. A multispecific binding site according to claim 3, comprising: The PD-1 binding domain comprises a light chain variable region having the amino acid sequence set forth in SEQ ID NO:48, or having at least 80%, preferably 85%, more preferably 90%, or most preferably 95% sequence identity thereto. Multispecific binding site.

5. A multispecific binding site according to any one of claims 1 to 4, The TGF-βRII binding domain 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:28, 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:32, SEQ ID NO:33, and SEQ ID NO:34, respectively; d) 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:36, SEQ ID NO:37, and SEQ ID NO:38, respectively; e) 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:40, SEQ ID NO:41, and SEQ ID NO:42, respectively; f) 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:44, SEQ ID NO:45, and SEQ ID NO:46, respectively; or g) 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:90, SEQ ID NO:91, and SEQ ID NO:92, respectively; wherein each of said HDCRs may contain at most three, two, or one amino acid variation; Multispecific binding site.

6. 6. A multispecific binding site according to claim 5, comprising: The TGF-βRII binding domain comprises a heavy chain variable region having an amino acid sequence as set forth in any one of SEQ ID NOs: 23; 27; 31; 35; 39; 43; 47; 88; 89, or having at least 80%, preferably 85%, more preferably 90%, or most preferably 95% sequence identity thereto. Multispecific binding site.

7. 6. A multispecific binding site according to claim 5, comprising: 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:49, SEQ ID NO:50, and SEQ ID NO:51, respectively, or variants thereof; Multispecific binding site.

8. 8. A multispecific binding site according to claim 7, comprising: The TGF-βRII binding domain comprises a light chain variable region having the amino acid sequence set forth in SEQ ID NO:48, or having at least 80%, preferably 85%, more preferably 90%, or most preferably 95% sequence identity thereto; Multispecific binding site.

9. A multispecific binding site comprising a PD-1 binding domain and a TGF-βRII binding domain, The TGF-βRII binding domain 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:28, 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:32, SEQ ID NO:33, and SEQ ID NO:34, respectively; d) 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:36, SEQ ID NO:37, and SEQ ID NO:38, respectively; e) 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:40, SEQ ID NO:41, and SEQ ID NO:42, respectively; f) 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:44, SEQ ID NO:45, and SEQ ID NO:46, respectively; or g) 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:90, SEQ ID NO:91, and SEQ ID NO:92, respectively; wherein each of said HDCRs may contain at most three, two, or one amino acid variation; Multispecific binding site.

10. 10. A multispecific binding site according to claim 9, comprising: The TGF-βRII binding domain comprises a heavy chain variable region having an amino acid sequence as set forth in any one of SEQ ID NOs: 23; 27; 31; 35; 39; 43; 47; 88; 89, or having at least 80%, preferably 85%, more preferably 90%, or most preferably 95% sequence identity thereto. Multispecific binding site.

11. 10. A multispecific binding site according to claim 9, comprising: 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:49, SEQ ID NO:50, and SEQ ID NO:51, respectively, or variants thereof; Multispecific binding site.

12. 12. A multispecific binding site according to claim 11, The TGF-βRII binding domain comprises a light chain variable region having the amino acid sequence set forth in SEQ ID NO:48, or having at least 80%, preferably 85%, more preferably 90%, or most preferably 95% sequence identity thereto; Multispecific binding site.

13. A multispecific binding site according to any one of claims 9 to 12, The PD-1 binding domain 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:2, SEQ ID NO:3, and SEQ ID NO:4, 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:6, SEQ ID NO:7, and SEQ ID NO:8, 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:10, SEQ ID NO:11, and SEQ ID NO:12, respectively; d) 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:15, SEQ ID NO:16, and SEQ ID NO:17, respectively; or e) 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 HDCRs may contain at most three, two, or one amino acid variation; Multispecific binding site.

14. 14. A multispecific binding site according to claim 13, comprising: The PD-1 binding domain comprises a heavy chain variable region having an amino acid sequence set forth in any one of SEQ ID NOs: 1; 5; 9; 13; 14; 18; 19, or having at least 80%, preferably 85%, more preferably 90%, or most preferably 95% sequence identity thereto. Multispecific binding site.

15. 14. A multispecific binding site according to claim 13, comprising: the PD-1 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 NOs:49, 50, and 51, respectively, or variants thereof; Multispecific binding site.

16. 16. A multispecific binding site according to claim 15, The PD-1 binding domain comprises a light chain variable region having the amino acid sequence set forth in SEQ ID NO:48, or having at least 80%, preferably 85%, more preferably 90%, or most preferably 95% sequence identity thereto. Multispecific binding site.

17. A multispecific binding site comprising a PD-1-binding domain and a TGF-βRII-binding domain, the PD-1 binding domain inhibits PD-1 mediated signaling, and the TGF-βRII binding domain inhibits TGF-βRII mediated signaling; The PD-1 binding domain may inhibit PD-1 mediated signaling and the TGF-βRII binding domain may inhibit TGF-βRII mediated signaling, particularly in activated T cells, particularly in activated tumor-specific T cells; Multispecific binding site.

18. A multispecific binding site according to claim 17, comprising: The multispecific binding site comprises a single Fab domain that binds PD-1, a single Fab domain that binds TGF-βRII, and an Fc region. Multispecific binding site.

19. A multispecific binding site according to claim 17 or 18, comprising: the multispecific binding site has greater potency in inhibiting TGF-βRII-mediated signaling in cells that express both PD-1 and TGF-βRII than in cells that express TGF-βRII and no, substantially no, or low levels of PD-1; Specifically, (i) the cells expressing both PD-1 and TGF-βRII may be Jurkat-PD-1 + cells, and the cells expressing TGF-βRII and not expressing or substantially not expressing PD-1 may be Jurkat-PD-1 null cells, (ii) the cells expressing both PD-1 and TGF-βRII may be activated CD4 + and / or CD8 + cells, and the cells expressing TGF-βRII and not expressing PD-1 at all may be non-activated CD4 + and / or CD8 + cells, or (iii) the cells expressing both PD-1 and TGF-βRII may be HEK-Blue TGF-β-PD-1 + cells. and the cell expressing TGF-βRII and no expression of PD-1 may be a HEK-Blue TGF-β cell; Multispecific binding site.

20. A multispecific binding site according to claim 19, comprising: The potency in inhibiting TGF-βRII-mediated signaling is measured in a phosphorylated SMAD2 / 3 assay. Multispecific binding site.

21. A multispecific binding site according to claim 19, comprising: The potency in inhibiting TGF-βRII-mediated signaling is measured in an isogenic PD-1-TGF-β reporter assay. Multispecific binding site.

22. A multispecific binding site according to claim 19, comprising: said potency in inhibiting TGF-βRII-mediated signaling in cells expressing both PD-1 and TGF-βRII is at least about 200-fold, preferably about 200-30,000-fold, greater than in cells expressing TGF-βRII and no, substantially no, or low levels of PD-1; Multispecific binding site.

23. A multispecific binding site according to claim 19, comprising: the potency of the multispecific binding site in inhibiting TGF-βRII-mediated signaling in cells that express TGF-βRII and do not express PD-1 at all is less than the potency of a reference anti-TGF-βRII antibody; and the potency of the multispecific binding site in inhibiting TGF-βRII-mediated signaling in cells expressing both PD-1 and TGF-βRII is greater than the potency of the reference anti-TGF-βRII antibody; said 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: 76 and a light chain having the amino acid sequence set forth in SEQ ID NO: 77; Specifically, the potency of the multispecific binding site in inhibiting TGF-βRII-mediated signaling in cells expressing both PD-1 and TGF-βRII may be at least about 100-fold, preferably about 100-20,000-fold, greater than the potency of the reference anti-TGF-βRII antibody; Multispecific binding site.

24. A multispecific binding site according to any one of claims 17, comprising: said multispecific binding site has greater activity in reducing tumor volume than a combination of reference antibodies; said combination of reference antibodies are two bivalent monospecific antibodies targeting PD-1 and TGF-βRII; The bivalent monospecific antibody targeting PD-1 comprises a heavy chain having the amino acid sequence set forth in SEQ ID NO: 78 and a light chain having the amino acid sequence set forth in SEQ ID NO: 79, and the bivalent monospecific antibody targeting TGF-βRII comprises a heavy chain having the amino acid sequence set forth in SEQ ID NO: 76 and a light chain having the amino acid sequence set forth in SEQ ID NO: 77; In particular, said activity in reducing tumor volume may be determined by measuring the reduction in tumor volume in an in vivo mouse study, in particular in an in vivo mouse study using MDA-MB-231 xenograft huCD34 NSG mice, Multispecific binding site.

25. A multispecific binding site according to claim 24, comprising: wherein the higher activity in reducing tumor volume is a reduction in tumor volume of at least about 1.5 times, preferably about 1.5 to 100 times, of the reduction in tumor volume of said combination of reference antibodies; Multispecific binding site.

26. A multispecific binding site comprising a PD-1 binding domain and a TGF-βRII binding domain, competes for binding to PD-1 and / or TGF-βRII with a multispecific binding site according to claim 1, 9 or 17; Multispecific binding site.

27. 20. A method for the preparation of a multispecific binding site comprising administering to a subject an effective amount of a multispecific binding site according to claim 1, 9, or 17, and a pharma- ceutically acceptable carrier. Pharmaceutical compositions.

28. 23. A multispecific binding site according to claim 1, 9 or 17 for use in therapy.

29. A pharmaceutical composition as described in claim 27 for use in treatment.

30. 20. A multispecific binding site as claimed in claim 1, 9 or 17 for use in the treatment of a disease associated with a suppressed immune system, in particular cancer.

31. A pharmaceutical composition as claimed in claim 27 for use in the treatment of diseases associated with a suppressed immune system, particularly cancer.

32. A cell comprising a nucleic acid sequence encoding a heavy chain variable region of a PD-1 binding domain and a nucleic acid sequence encoding a heavy chain variable region of a TGF-βRII binding domain, In particular, the cell may further comprise a nucleic acid sequence encoding a CH1 region, and preferably a hinge, CH2 and CH3 region, The PD-1 binding domain 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:2, SEQ ID NO:3, and SEQ ID NO:4, 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:6, SEQ ID NO:7, and SEQ ID NO:8, 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:10, SEQ ID NO:11, and SEQ ID NO:12, respectively; d) 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:15, SEQ ID NO:16, and SEQ ID NO:17, respectively; or e) 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; The TGF-βRII binding domain 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:28, 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:32, SEQ ID NO:33, and SEQ ID NO:34, respectively; d) 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:36, SEQ ID NO:37, and SEQ ID NO:38, respectively; e) 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:40, SEQ ID NO:41, and SEQ ID NO:42, respectively; f) 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:44, SEQ ID NO:45, and SEQ ID NO:46, respectively; or g) 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:90, SEQ ID NO:91, and SEQ ID NO:92, respectively; wherein each of said HDCRs may contain at most three, two, or one amino acid variation; cell.

33. The cell of claim 32, The PD-1 binding domain comprises a heavy chain variable region having an amino acid sequence set forth in any one of SEQ ID NOs: 1; 5; 9; 13; 14; 18; 19, or having at least 80%, preferably 85%, more preferably 90%, or most preferably 95% sequence identity thereto; The TGF-βRII binding domain comprises a heavy chain variable region having an amino acid sequence as set forth in any one of SEQ ID NOs: 23; 27; 31; 35; 39; 43; 47; 88; 89, or having at least 80%, preferably 85%, more preferably 90%, or most preferably 95% sequence identity thereto. cell.

34. 34. The cell of claim 32 or 33, The cell further comprises at least one nucleic acid sequence encoding a light chain variable region, in particular a light chain variable region as defined in claim 3, and preferably a CL region. cell.

35. The cell of claim 34, The cell further comprises at least one nucleic acid sequence encoding a light chain variable region, in particular a light chain variable region as defined in claim 4, and preferably a CL region. cell.

36. A cell producing a multispecific binding site according to claim 1, 9 or 17.

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