Multiple specific binding sites including PD-1 and TGF-BRII binding domains

Abstract

To provide new medicines for the treatment of cancer. [Solution] 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. Disclosed are a pharmaceutical composition comprising such a multispecific binding site, a therapeutic method using such a multispecific binding site, and cells producing such a multispecific binding site.

Description

Technical Field

[0001] The present disclosure relates to the field of antibodies. Specifically, it relates to the field of therapeutic antibodies for the treatment of diseases involving aberrant cells. More specifically, it relates to a multispecific binding site comprising a binding domain that binds to PD-1 and a binding domain that binds to TGF-βRII.

Background Art

[0002] The role of T lymphocytes in tumor immune surveillance is well known, but cancer cells can evade immune regulation by inducing inhibitory immune pathways. As a result, immune checkpoint blockade (ICB), which uses antibodies to inhibit 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) attenuate T cell activity in normal physiological function, but are components of an immunosuppressive network that can be co-opted by tumors to suppress T cell-mediated anti-tumor immune responses. Antibodies directed against PD-1 and PD-L1 have produced improvements in response and survival for some patients with several different cancers. However, despite promising clinical activity, only a minority of patients respond to anti-PD-1 / PD-L1 therapy, and the durability of response is also limited. Therefore, there is an urgent need for the development of new, safe and effective therapies for the treatment of cancer.

[0004] 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 to two ligands, PD-L1 and PD-L2. PD-1 functions as an immune checkpoint, playing a crucial role in downregulating the immune system by inhibiting T cell activation. Consequently, when present on somatic cells, it reduces autoimmunity and promotes self-tolerance. 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 aliases, 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. A new class of drugs that inhibit the activity of PD-1, PD-1 inhibitors, activate the immune system to attack tumors and are therefore used to treat certain types of cancer.

[0005] Monoclonal antibodies targeting PD-1 have been approved for the treatment of various malignancies. The response rate in melanoma patients treated with an anti-PD-1 antibody (pembrolizumab) is, for example, 33% at 3 years, even though 70-80% of patients initially responded (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 controls many physiological and pathological processes, including cell cycle arrest in epithelial and hematopoietic cells, regulation of mesenchymal cell proliferation and differentiation, wound healing, extracellular matrix production, immunosuppression, and carcinogenesis (Massague J. TGFβ signalling in context. Nat Rev Mol Cell Biol. 2012 Oct; 13(10):616-30). In addition, TGF-β signaling controls 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 act primarily as a tumor suppressor in normal and precancerous cells, it exhibits a biphasic function in tumor cells, enabling proliferation and epithelial-mesenchymal transition, thereby allowing tumor cell migration, invasion, intravascular invasion, and extravasation.

[0007] TGF-βRII is a member of the serine / threonine protein kinase family and the TGFB receptor subfamily. It is known by various synonyms, including TGFBR2, AAT3, FAA3, LDS1B, LDS2, LDS2B, MFS2, RIIC, TAAD2, TGFR-2, TGF-beta-RII, transforming growth factor beta receptor 2, TBR-ii, and TBRII. TGF-βRII forms a heterodimer complex with another receptor protein and binds to TGF-β. This receptor / ligand complex phosphorylates the protein, which then enters the nucleus and regulates 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 stages, inhibiting TGF-β at various levels; i.e., ligand, ligand-receptor, or intracellular levels. Inhibitors 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 interfere with 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 exhibit antitumor activity in vitro and in vivo; however, poor clinical outcomes persist due to low efficacy and unacceptable toxicity, including severe cytokine release syndrome (CRS). Combined targeting of the TGF-β pathway with immune checkpoint inhibition has been attempted using Bintrafusp alpha, a bifunctional anti-PD-L1-TGFBRII fusion protein, but has not demonstrated strong clinical efficacy.

[0010] Novel therapeutic interventions that selectively inhibit TGF-β signaling in locally activated tumor-specific PD-1-expressing T cells within the tumor microenvironment remain necessary to promote T cell tumor invasion and restore and sustain T cell antitumor effector activity. Such targeting mitigates immunosuppressive pathways and powerfully promotes CTL function and T cell memory for effective and durable cancer removal while minimizing the toxicity associated with systemic TGF-β inhibition. [Overview of the project] [Problems that the invention aims to solve]

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

[0012] This 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 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] This 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] This disclosure further provides pharmaceutical compositions comprising an effective amount of the multispecific binding sites described herein.

[0016] This disclosure further provides multispecific binding sites described herein and pharmaceutical compositions described herein for use in therapeutic purposes.

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

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

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

[0020] This disclosure further provides cells comprising a nucleic acid sequence 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.

[0021] This disclosure further provides cells that produce the multispecific binding sites described herein. [Brief explanation of the drawing]

[0022] [Figure 1]Percentage inhibition of PD-1-mediated SHP recruitment by bispecific antibodies and control antibodies, as measured by the PD-1-SHP recruitment assay. A) Bispecific antibodies: SEQ ID NO: 39 × SEQ ID NO: 9 and SEQ ID NO: 35 × SEQ ID NO: 5. Control antibodies: Pembrolizumab analog - SEQ ID NO: 78 / 79; PD-L1-TGF-β TRAP molecular analog - SEQ ID NO: 80 / 81; and TGF1 analog - SEQ ID NO: 76 / 77. B) Bispecific antibodies: SEQ ID NO: 23 × SEQ ID NO: 18; SEQ ID NO: 47 × SEQ ID NO: 13; SEQ ID NO: 88 × SEQ ID NO: 13; SEQ ID NO: 89 × SEQ ID NO: 13; SEQ ID NO: 23 × SEQ ID NO: 14; and SEQ ID NO: 43 × SEQ ID NO: 9. Control antibodies: Pembrolizumab analog - SEQ ID NO: 78 / 79; and RSV IgG1 - SEQ ID NO: 86 / 87. [Figure 2] Fold induction of T cell activation by bispecific antibodies and control antibodies as measured by the PD-1-NFAT reporter assay. A) Bispecific antibodies: SEQ ID NO: 39 × SEQ ID NO: 9 and SEQ ID NO: 35 × SEQ ID NO: 5. Control antibodies: Pembrolizumab analog - SEQ ID NO: 78 / 79; PD-L1-TGF-β TRAP molecular analog - SEQ ID NO: 80 / 81; and TGF1 analog - SEQ ID NO: 76 / 77. B) Bispecific antibodies: SEQ ID NO: 47 × SEQ ID NO: 13; SEQ ID NO: 88 × SEQ ID NO: 13; and SEQ ID NO: 89 × SEQ ID NO: 13. Control antibodies: Pembrolizumab analog - SEQ ID NO: 78 / 79; and RSV IgG1 - SEQ ID NO: 86 / 87. C) Bispecific antibodies: SEQ ID NO: 23 × SEQ ID NO: 18; SEQ ID NO: 23 × SEQ ID NO: 14; and SEQ ID NO: 43 × SEQ ID NO: 9. The control antibodies are: pembrolizumab analog - SEQ ID NO: 78 / 79; and RSV IgG1 - SEQ ID NO: 86 / 87. [Figure 3]Inhibition of phosphorylated SMAD2 / 3 in Jurkat-PD-1 null (A-G) cells and Jurkat-PD-1+ cells (H-N) by bispecific and control antibodies. These graphs show the levels of phosphorylated SMAD2 / 3 in lysates of Jurkat-PD-1 null and Jurkat-PD-1+ cells incubated with either a bispecific or control antibody. "No TGF-β1" shows the background phosphorylated SMAD2 / 3 level without TGF-β ligand, measured in the absence of the bispecific antibody; "10 ng / ml TGF-β1" shows the maximum phosphorylated SMAD2 / 3 level when 10 ng / ml TGF-β ligand is added in the absence of the bispecific antibody. The control antibody is a TGF-βRII×RSV antibody containing a TGF-βRII binding domain with the heavy chain variable region amino acid sequence shown in RSV IgG1-SEQ ID NO: 86 / 87; TGF1 analog-SEQ ID NO: 76 / 77; and SEQ ID NO: 23, 31, 39, 27, 35, or 43; an RSV binding domain with the heavy chain variable region amino acid sequence shown in SEQ ID NO: 86; and a common light chain containing the light chain variable region amino acid sequence shown in SEQ ID NO: 48 and the light chain constant region amino acid sequence shown in SEQ ID NO: 75. Each data point represents the average absorbance of the corresponding replica.A and H: Bispecific antibodies comprising a PD-1 binding domain having a heavy chain variable region having the amino acid sequence shown in SEQ ID NOs: 1, 5, 9, 14, or 19, and a TGF-βRII binding domain having a heavy chain variable region having the amino acid sequence shown in SEQ ID NOs: 23; B and I: Bispecific antibodies comprising a PD-1 binding domain having a heavy chain variable region having the amino acid sequence shown in SEQ ID NOs: 1, 5, 9, 14, or 19, and a TGF-βRII binding domain having a heavy chain variable region having the amino acid sequence shown in SEQ ID NOs: 31; C and J: Bispecific antibodies comprising a PD-1 binding domain having a heavy chain variable region having the amino acid sequence shown in SEQ ID NOs: 1, 5, 9, 14, or 19, and a TGF-βRII binding domain having a heavy chain variable region having the amino acid sequence shown in SEQ ID NOs: 39; D and K: Bispecific antibodies comprising a PD-1 binding domain having a heavy chain variable region having the amino acid sequence shown in SEQ ID NOs: 1, 5, 9, 14, or 19, and SEQ ID NOs: Bispecific antibodies comprising: E ​​and L: a PD-1 binding domain having a heavy chain variable region having the amino acid sequence shown in 27, and a TGF-βRII binding domain having a heavy chain variable region having the amino acid sequence shown in SEQ ID NOs: 1, 5, 9, 14, or 19, and a TGF-βRII binding domain having a heavy chain variable region having the amino acid sequence shown in SEQ ID NOs: 35; F and M: bispecific antibodies comprising a PD-1 binding domain having a heavy chain variable region having the amino acid sequence shown in SEQ ID NOs: 1, 5, 9, 14, or 19, and a TGF-βRII binding domain having a heavy chain variable region having the amino acid sequence shown in SEQ ID NOs: 43; G and N: The bispecific antibodies are: SEQ ID NOs: 23×SEQ ID NOs: 9; SEQ ID NOs: 23×SEQ ID NOs: 14; SEQ ID NOs: 43×SEQ ID NOs: 9; SEQ ID NOs: 23×SEQ ID NOs: 13; SEQ ID NOs: 23×SEQ ID NOs: 18; SEQ ID NOs: 47×SEQ ID NOs: 13; SEQ ID NOs: 88×SEQ ID NOs: 13; and SEQ ID NOs: 89×SEQ ID NOs: 13. The control antibodies are: RSV IgG1-SEQ ID NO: 86 / 87; TGF1 analog-SEQ ID NO: 76 / 77; and pembrolizumab-SEQ ID NO: 78 / 79. [Figure 4]Measurement of intracellular phosphorylated SMAD2 by flow cytometry. These graphs show intracellular phosphorylated SMAD2 levels in stimulated and unstimulated CD4+ and CD8+ T cells incubated with bispecific antibodies or control antibodies. Bispecific antibodies are: SEQ ID NO: 23 × SEQ ID NO: 18 and SEQ ID NO: 47 × SEQ ID NO: 13. Control antibodies are: RSV IgG1 - SEQ ID NO: 86 / 87; TGF1 analog - SEQ ID NO: 76 / 77; and pembrolizumab - SEQ ID NO: 78 / 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 exhausted MLR assay. The bispecific antibodies tested were: SEQ ID NO: 23×SEQ ID NO: 9; SEQ ID NO: 23×SEQ ID NO: 14; SEQ ID NO: 23×SEQ ID NO: 19; SEQ ID NO: 31×SEQ ID NO: 14; SEQ ID NO: 39×SEQ ID NO: 9; SEQ ID NO: 35×SEQ ID NO: 9; SEQ ID NO: 35×SEQ ID NO: 14; SEQ ID NO: 35×SEQ ID NO: 19; SEQ ID NO: 27×SEQ ID NO: 9; SEQ ID NO: 43×SEQ ID NO: 9; SEQ ID NO: 43×SEQ ID NO: 19; SEQ ID NO: 23×SEQ ID NO: 13; SEQ ID NO: 23×SEQ ID NO: 18; SEQ ID NO: 47×SEQ ID NO: 13; SEQ ID NO: 88×SEQ ID NO: 13; and SEQ ID NO: 89×SEQ ID NO: 13. The control antibodies are: RSV IgG1 - SEQ ID NO: 86 / 87; TGF1 analog - SEQ ID NO: 76 / 77; combination of pembrolizumab and TGF1 analog - SEQ ID NO: 78 / 79 + SEQ ID NO: 76 / 77; PD-L1-TGF-β TRAP molecular analog - SEQ ID NO: 80 / 81; and pembrolizumab - SEQ ID NO: 78 / 79. A: This graph shows the induction of IFN-γ cytokine secretion by exhausted T cells in one representative donor. B: This graph shows the induction of IFN-γ cytokine secretion by exhausted T cells in one representative donor. C: This graph shows the induction of IL-2 cytokine secretion by exhausted T cells in one representative donor. D: This graph shows the induction of TNF-α cytokine secretion by exhausted T cells in one representative donor. E: This graph shows the induction of TNF-α cytokine secretion by exhausted T cells in one representative donor. [Figure 6]Measurement of the percentage of inhibition of TGF-β-induced signaling induced by bispecific antibodies or control antibodies. The bispecific antibodies are: SEQ ID NO: 23 × SEQ ID NO: 14; SEQ ID NO: 23 × SEQ ID NO: 19; SEQ ID NO: 39 × SEQ ID NO: 9; SEQ ID NO: 35 × SEQ ID NO: 9; SEQ ID NO: 35 × SEQ ID NO: 14; SEQ ID NO: 35 × SEQ ID NO: 19; SEQ ID NO: 27 × SEQ ID NO: 9; SEQ ID NO: 43 × SEQ ID NO: 9; and SEQ ID NO: 43 × SEQ ID NO: 19. 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 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-β® signaling by bispecific antibodies or control antibodies in HEK-Blue® TGF-β-PD-1+ cells. [Figure 7] Measurement of cytokine production induced by bispecific antibodies or control antibodies in a Treg suppression assay. Bispecific antibodies are: SEQ ID NO: 23 × SEQ ID NO: 14; SEQ ID NO: 23 × SEQ ID NO: 19; SEQ ID NO: 31 × SEQ ID NO: 14; SEQ ID NO: 39 × SEQ ID NO: 9; SEQ ID NO: 35 × SEQ ID NO: 9; SEQ ID NO: 35 × SEQ ID NO: 14; SEQ ID NO: 35 × SEQ ID NO: 19; SEQ ID NO: 27 × SEQ ID NO: 9; SEQ ID NO: 43 × SEQ ID NO: 9; and SEQ ID NO: 43 × SEQ ID NO: 19. Control antibodies are: RSV IgG1 - SEQ ID NO: 86 / 87; TGF1 analog - SEQ ID NO: 76 / 77; combination of pembrolizumab and TGF1 analog - SEQ ID NO: 78 / 79 + SEQ ID NO: 76 / 77; and pembrolizumab - SEQ ID NO: 78 / 79. A: This graph shows the induction of IFN-γ cytokine secretion in co-culture of Tregs from one representative donor with PBMCs. B: This graph shows the induction of TNF-α cytokine secretion in co-culture of Treg cells from one representative donor with PBMCs. [Figure 8]Measurement of cytokine production induced by bispecific antibodies or control antibodies in a macrophage suppression assay. Bispecific antibodies are: SEQ ID NO: 35 × SEQ ID NO: 9; SEQ ID NO: 23 × SEQ ID NO: 14; SEQ ID NO: 23 × SEQ ID NO: 19; SEQ ID NO: 43 × SEQ ID NO: 9; SEQ ID NO: 39 × SEQ ID NO: 9; SEQ ID NO: 43 × SEQ ID NO: 19; SEQ ID NO: 35 × SEQ ID NO: 14; SEQ ID NO: 35 × SEQ ID NO: 19; SEQ ID NO: 31 × SEQ ID NO: 14; and SEQ ID NO: 27 × SEQ ID NO: 9. Control antibodies are: RSV IgG1 - SEQ ID NO: 86 / 87; TGF1 analog - SEQ ID NO: 76 / 77; Opdivo; LILRB2; PD-L1-TGF-β TRAP molecular analog - SEQ ID NO: 80 / 81; Pembrolizumab - SEQ ID NO: 78 / 79; and Nivolumab analog - SEQ ID NO: 96 / 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]In vivo efficacy of bispecific antibodies. A: This graph shows the reduction in tumor volume (mm3) induced by the control antibody and the reference antibody. B-E: These graphs show the reduction in tumor volume (mm3) induced by bispecific antibodies compared to the control antibody and the reference antibody. The bispecific antibodies are: SEQ ID NO: 43 × SEQ ID NO: 9; SEQ ID NO: 43 × SEQ ID NO: 19; SEQ ID NO: 23 × SEQ ID NO: 14; SEQ ID NO: 23 × SEQ ID NO: 19; SEQ ID NO: 39 × SEQ ID NO: 9; SEQ ID NO: 27 × SEQ ID NO: 9; SEQ ID NO: 23 × SEQ ID NO: 18; and SEQ ID NO: 47 × SEQ ID NO: 13. The control antibodies are: RSV IgG1 - SEQ ID NO: 86 / 87; TGF1 analog - SEQ ID NO: 76 / 77; Pembrolizumab - SEQ ID NO: 78 / 79; PD-L1-TGF-β TRAP molecular analog - SEQ ID NO: 80 / 81; and combination of pembrolizumab and TGF1 analog - SEQ ID NO: 78 / 79 + SEQ ID NO: 76 / 77. The bispecific antibody was administered at 1 mg / kg (1) and / or 10 mg / kg (10) (A-C), and at 10 mg / kg only (D-F). F: After treatment with the bispecific antibody or control antibody, the left graph shows the occupancy rate of TGF-βRII receptors, and the right graph shows the occupancy rate of PD-1 receptors. [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 × SEQ ID NO: 18, and the control antibody is: SEQ ID NO: 86 / SEQ ID NO: 87. [Modes for carrying out the invention]

[0023] In one embodiment, 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.

[0024] As used herein, “blocks” or “blocking” means interfering with or modifying the interaction between a ligand and a receptor, or causing a total or partial reduction of the signaling cascade. For the purposes of this disclosure, in some embodiments, the potency of 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. For the purposes of this disclosure, in some embodiments, the potency of inhibiting ligand-induced PD-1 signaling is determined by using the SHP recruitment assay described in Example 2. In some embodiments, the potency of inhibiting ligand-induced PD-1 signaling is determined by using the NFAT reporter assay described in Example 3. In one embodiment, the efficacy of inhibiting ligand-induced TGF-βRII signaling is determined by using the SMAD assay described in Example 5.

[0025] In one embodiment, the multispecific binding site of the Disclosure binds to human PD-1. In one embodiment, the PD-1 binding domain of the multispecific binding site of the Disclosure inhibits the binding of PD-L1 to PD-1, as measured, for example, in the assay described in Example 2 or 3.

[0026] In one embodiment, the multispecific binding site of this disclosure binds to human TGF-βRII. Human TGF-βRII is a transmembrane protein with 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 by 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 shown as SEQ ID NO: 85.

[0027] "Binding moiety" refers to a proteinaceous molecule and includes all antibody formats available in the art, such as: full-length IgG antibodies, immune complexes, diabodies, BiTE, Fab fragments, scFv, tandem scFv, single-domain antibodies (such as VHH and VH), minibodies, scFab, scFv-zippers, nanobodies, DART molecules, TandAb, Fab-scFv, F(ab)'2, F(ab)'2-scFv2, and intrabodies.

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

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

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

[0031] The “Fc region” typically includes the hinge, CH2, and CH3 regions. Suitable hinges include, but are not limited to, the hinge whose amino acid sequence is shown in SEQ ID NO: 68. Suitable CH2 and CH3 regions include, but are not limited to, the CH2 region whose amino acid sequence is shown in SEQ ID NO: 70 or 71, and the CH3 regions whose amino acid sequences are shown 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 desirable antibody properties, including, for example, to promote heterodimerization of different heavy chains, to improve heavy-light chain pairing, and to increase or decrease immune cell effector function.

[0033] In one embodiment, in activated T cells, specifically activated tumor-specific T cells, 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.

[0034] In one embodiment, the multispecific binding sites of the present disclosure are 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 at all, substantially not, or expressing low levels of PD-1.

[0035] Accordingly, the disclosure also provides a multispecific binding site comprising a PD-1 binding domain and a TGF-βRII binding domain, wherein the multispecific binding site is 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 expressing PD-1 at all, substantially not expressing it, or expressing 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 expressing human PD-1 and a luciferase reporter driven by an NFAT response element (NFAT-RE), and the cells expressing TGF-βRII but not PD-1 are Jurkat-PD-1 null cells, such as Jurkat-PD-1 null cells. Jurkat T cells expressing human PD-1 and a luciferase reporter driven by an NFAT-RE are commercially available, for example, from Promega (catalog number CS187105 - part of kits CS187106 and CS187107); Jurkat-PD-1 null cells are publicly available, for example, from ATCC (catalog number TIB-152).

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

[0038] In one embodiment, the cells expressing both PD-1 and TGF-βRII are HEK-Blue™ TGF-β-PD-1, for example as described in Example 7 +Cells that express TGF-βRII and do not express PD-1 include 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 human TGFBRI, Smad3, and Smad4 genes. These also express the Smad3 / 4 binding element (SBE)-inducible SEAP reporter gene.

[0039] In some embodiments, PD-1 being absent, substantially absent, or at low levels refers to a level of PD-1 on the cell surface that is undetectable by suitable assays such as those presented herein. In some embodiments, low levels of PD-1 refer to fewer than 100 PD-1 molecules present on the cell surface. In some embodiments, the level of PD-1 is measured using the quantibrite bead methodology.

[0040] For the purposes of this disclosure, determining whether the multispecific binding site is 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 at all, substantially not, or expressing low levels of PD-1 is done by using one of the assays described herein.

[0041] The efficacy data of the multispecific binding sites provided herein are obtained using the phosphorylated SMAD2 / 3 assays described in Examples 4 and 5, and the isogenic HEK-BLUE-PD-1 TGF-β reporter assay described in Example 7. Thus, in a given embodiment, the efficacy in inhibiting TGF-βRII-mediated signaling is measured using the phosphorylated SMAD2 / 3 assay described in Example 4 or Example 5.

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

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

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

[0045] Simply put, the HEK-BLUE-PD-1 TGF-β reporter assay uses HEK-BLUE® TGF-β cells and HEK-BLUE® TGF-β-PD-1 + The test is performed using 25,000 cells per well. Serial dilutions of the test and control antibodies are added, and the cells are incubated at room temperature for 1 hour, 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 μl of supernatant and 160 μl of resuspended QUANTI-Blue® solution are incubated at 37°C / 5% CO2 for 40 minutes. 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 one embodiment, the multispecific binding site of the Disclosure has at least about 10 times, preferably about 10 to 100,000 times, higher efficacy 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 one embodiment, the efficacy in inhibiting TGF-βRII-mediated signaling is determined by the IC50 (μg / ml) in a phosphorylated SMAD2 / 3 assay or a HEK-BLUE-PD-1 TGF-β reporter assay. In one embodiment, the efficacy of inhibiting TGF-βRII-mediated signaling is determined by the IC50 (μg / ml) in a phosphorylated SMAD2 / 3 assay. In another embodiment, the efficacy of inhibiting TGF-βRII-mediated signaling is determined by the IC50 (μg / ml) in a HEK-BLUE-PD-1 TGF-β reporter assay.

[0047] In one embodiment, the potency of the multispecific binding site of this disclosure in inhibiting TGF-βRII-mediated signaling in cells expressing TGF-βRII but not expressing PD-1 at all, substantially not expressing PD-1, or expressing PD-1 at low levels is lower than the potency of a reference anti-TGF-βRII antibody targeting the same cells, and the potency of the multispecific binding site in inhibiting TGF-βRII-mediated signaling in cells expressing 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 shown in SEQ ID NO: 76 and a light chain having the amino acid sequence shown in SEQ ID NO: 77.

[0048] In one embodiment, cells expressing both PD-1 and TGF-βRII are Jurkat-PD-1 + cells, stimulated CD4 + or CD8 + Cells, or HEK-Blue(trademark) TGF-β-PD-1 + Cells that express TGF-βRII and do not express PD-1 at all or substantially do not express it are Jurkat-PD-1 null Cells that are either TGF-β cells or HEK-Blue® TGF-β cells; and cells that express TGF-βRII and low levels of PD-1 are unstimulated CD4 + or CD8 + These are cells, which will be further described herein.

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

[0050] In one embodiment, the multispecific binding sites of the present disclosure have higher activity than a combination of reference antibodies in reducing tumor volume. In one embodiment, the combination of reference antibodies 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 shown in SEQ ID NO: 78 and a light chain having the amino acid sequence shown in SEQ ID NO: 79, and the bivalent monospecific antibody targeting anti-TGF-βRII comprises a heavy chain having the amino acid sequence shown in SEQ ID NO: 76 and a light chain having the amino acid sequence shown in SEQ ID NO: 77.

[0051] Accordingly, this 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 higher activity in reducing tumor volume than a combination of reference antibodies. In one embodiment, the multispecific binding site is administered with 2 to 20 times fewer PD-1 and TGF-βRII binding domains 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 has higher activity in reducing tumor volume when administered at 1 mg / kg than a combination of reference antibodies that are bivalent for binding to either PD-1 or TGF-βRII and administered at 10 mg / kg each. Furthermore, the multispecific binding site, which is monovalent for binding to PD-1 and monovalent for binding to TGF-βRII, exhibits higher activity in reducing tumor volume when administered at 10 mg / kg compared to a combination of reference antibodies that are bivalent for binding to either PD-1 or TGF-βRII and administered at 10 mg / kg each.

[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 shown in SEQ ID NO: 78 and a light chain having the amino acid sequence shown in SEQ ID NO: 79, and the bivalent monospecific antibody targeting TGF-βRII comprises a heavy chain having the amino acid sequence shown in SEQ ID NO: 76 and a light chain having the amino acid sequence shown in SEQ ID NO: 77.

[0053] In one embodiment, 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.

[0054] In one embodiment, the multispecific binding sites of the present disclosure result in a tumor volume reduction of at least 1.5 times, preferably between 1.5 to 100 times, or 2 to 80 times, or 5 to 80 times, or 10 to 80 times, or 15 to 80 times, or 20 to 80 times, or 30 to 80 times, or 40 to 80 times, or 50 to 80 times, or 2 to 60 times, or 5 to 60 times, or 10 to 60 times, or 15 to 60 times, or 20 to 60 times, or 30 to 60 times, or 40 to 60 times, compared to the tumor volume reduction of the reference antibody combination. In one embodiment, the multispecific binding sites of the Disclosure result in a tumor volume reduction of at least about 1.5 times, preferably between approximately 1.5 to 100 times, or 2 to 80 times, or 5 to 80 times, or 10 to 80 times, or 15 to 80 times, or 20 to 80 times, or 30 to 80 times, or 40 to 80 times, or 50 to 80 times, or 2 to 60 times, or 5 to 60 times, or 10 to 60 times, or 15 to 60 times, or 20 to 60 times, or 30 to 60 times, or 40 to 60 times, compared to the tumor volume reduction of the reference antibody combination. In other words, the multispecific binding sites of the Disclosure result in a greater tumor volume reduction 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 shown in SEQ ID NO: 78 and a light chain having the amino acid sequence shown in SEQ ID NO: 79, and the bivalent monospecific antibody targeting TGF-βRII comprises a heavy chain having the amino acid sequence shown in SEQ ID NO: 76 and a light chain having the amino acid sequence shown in SEQ ID NO: 77.

[0055] In one embodiment, the multispecific binding sites of the present disclosure reduce tumor volume when administered as a monotherapy.

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

[0057] In one embodiment, the Disclosure provides several PD-1 × 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 the multispecific binding site of this disclosure includes a heavy chain variable region comprising: a) Heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), each having the amino acid sequences shown in SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4, respectively; b) Heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), each having the amino acid sequences shown in SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8, respectively; c) Heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), each having the amino acid sequences shown in SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12, respectively; d) Heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), having the amino acid sequences shown 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 shown 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 the multispecific binding site of the present disclosure may include a limited number of non-conservative amino acid substitutions, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, or an unlimited number of conservative amino acid substitutions.

[0060] In some embodiments, the PD-1 binding domain of the multispecific binding site of the present disclosure also includes variants of that PD-1 binding domain, where each HCDR 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 non-conservative 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 conserved amino acid substitution.

[0061] Typically, conservative amino acid substitutions involve variations of amino acids that have homologous amino acid residues (residues that share similar characteristics or properties). Homologous amino acids are known in the art, as are standard methods for making amino acid substitutions in antibody-binding domains without significantly affecting antibody binding or function. See, for example, 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 entirely herein. When determining whether an amino acid can be substituted with a conserved amino acid, the evaluation may typically be based on the following factors, but is not limited to these: (a) the structure of the polypeptide backbone in the region of substitution, e.g., sheet-like or helical conformation; (b) the molecular charge or hydrophobicity at the target site; and / or (c) the size of the side chain(s). If a residue can be substituted with a residue that has similar side chains or common characteristics such as similar charge or hydrophobicity, such residues are preferred as substituents. For example, the following groups can be determined: (1) Nonpolar: Ala(A), Gly(G), Val(V), Leu(L), Ile(I), Pro(P), Phe(F), Trp(W), Met(M); (2) Uncharged: 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 grouped 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 affecting chain orientation: Gly(G), Pro(P); and (6) aromatic: Trp(W), Tyr(Y), Phe(F).

[0062] It is preferable to substitute one amino acid residue with another amino acid residue belonging to the same group. Therefore, conservative amino acid substitutions may include exchanging one member of these classes with another member of the same class. Typically, the variations do not impair, or substantially impair, the binding specificity of the binding domain to the intended target.

[0063] Further types of amino acid variations include variations resulting from somatic hypermutation or affinity maturation. PD-1 binding variants included in this disclosure include somatic hypermutation or affinity-matured heavy chain variable regions, which are heavy chain variable regions derived from the same VH gene segment as the heavy chain variable regions described herein by sequence, and the variants have amino acid variations, including non-conservative and / or conserved amino acid substitutions in one, two, or all three HCDRs. Standard 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 locations for introducing amino acid variations include, but are not limited to, the first, second, and / or fourth amino acids of HCDR1; the third, seventh, eighth, ninth, tenth, eleventh, thirteenth, fourteenth, and / or sixteenth amino acids of HCDR2; and / or the sixth and / or thirteenth amino acids of HCDR3.

[0065] In one embodiment, the present disclosure also provides a multispecific binding site, the PD-1 binding domain comprising: - 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 - 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 - 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 amino acid variations 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 one embodiment, the present disclosure also provides a multispecific binding site, the PD-1 binding domain comprising: - 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 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 also be T or S; X8 may be Y, F, or H; X9 may be A, G, V, or F; X 10 This may be Q, R, N, L, T, or S; X 11 This may be D, A, G, or S; X 12 This may be F, V, or A; X 13 This can also be T, K, H, or G; X 14 This may be G, N, E, or D; and / or - 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 one embodiment, the PD-1 binding domain of the multispecific binding site of the Disclosure comprises a heavy chain variable region having the amino acid sequence shown in any one of SEQ ID NOs: 1;5;9;13;14;18;19, or a variant thereof. In one embodiment, the PD-1 binding domain of the multispecific binding site of the Disclosure comprises a heavy chain variable region having the amino acid sequence shown 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 to these.

[0069] In this specification, “percent (%) identity” of nucleic acid or amino acid sequences is defined as the percentage of residues in a candidate sequence that are identical to residues in a selected sequence after the sequences have been aligned for the purpose of optimal comparison. To optimize the alignment between two sequences, gaps may be introduced in either of the two sequences being compared. Such alignment may be performed over the entire length of the sequences being compared. Alternatively, the alignment may be performed over a shorter length, for example, about 20, about 50, about 100 or more nucleic acids / bases or amino acids. Sequence identity is the percentage of identical matching between two sequences across the reported alignment region.

[0070] The comparison of sequences and the determination of the percentage of sequence identity between two sequences can be achieved using mathematical algorithms. Those skilled in the art will know that several different computer programs are available for aligning two sequences and determining their identity (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 sequences or nucleic acid sequences can be determined using the Needleman-Wunsch algorithm for aligning two sequences (Needleman, SB and Wunsch, CD (1970) J. Mol. Biol. 48, 443-453). The Needleman-Wunsch algorithm is implemented in the computer program NEEDLE. For the purposes of this invention, the NEEDLE program from the EMBOSS package is used to determine the identity percentage of amino acids 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 were a gap-open penalty of 10 and a gap extension penalty of 0.5.

[0071] After alignment using the NEEDLE program described above, the percentage of sequence identity between the query sequence and the sequence of the present invention is calculated as follows: the number of corresponding positions in the alignment that show the same amino acid or the same nucleotide in both sequences, divided by the total length of the alignment after subtracting the total number of gaps in the alignment.

[0072] In some embodiments, the PD-1 binding domain of the multispecific binding site of the Disclosure also comprises a PD-1 binding domain variant, which includes one or more variations in the framework region in addition to the variations in the HCDR described above. The variations may be any type of amino acid variation described herein, such as conserved or non-conserved amino acid substitutions resulting from somatic hypermutation or affinity maturation. In some embodiments, the PD-1 binding domain variant of the multispecific binding site of the Disclosure does not include variations in the CDR region but includes one or more variations in the framework region. Such variants are expected to have at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with respect to the sequences disclosed herein and to retain PD-1 binding specificity. Accordingly, in some embodiments, the PD-1 binding domain of the multispecific binding site of the Disclosure includes: - Heavy chain variable regions having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with respect to the amino acid sequence shown in Sequence ID No. 1; This heavy chain variable region includes the HCDR1 amino acid sequence shown in SEQ ID NO: 2; the HCDR2 amino acid sequence shown in SEQ ID NO: 3; and the HCDR3 amino acid sequence shown in SEQ ID NO: 4; - Heavy chain variable regions having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with respect to the amino acid sequence shown in Sequence ID No. 5; This heavy chain variable region includes the HCDR1 amino acid sequence shown in SEQ ID NO: 6; the HCDR2 amino acid sequence shown in SEQ ID NO: 7; and the HCDR3 amino acid sequence shown in SEQ ID NO: 8. - Heavy chain variable regions having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with respect to the amino acid sequence shown in Sequence ID No. 9; This heavy chain variable region includes the HCDR1 amino acid sequence shown in SEQ ID NO: 10; the HCDR2 amino acid sequence shown in SEQ ID NO: 11; and the HCDR3 amino acid sequence shown in SEQ ID NO: 12; - Heavy chain variable regions having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with respect to the amino acid sequence shown in SEQ ID NO: 13; This heavy chain variable region includes the HCDR1 amino acid sequence shown in SEQ ID NO: 10; the HCDR2 amino acid sequence shown in SEQ ID NO: 11; and the HCDR3 amino acid sequence shown in SEQ ID NO: 12; - Heavy chain variable regions having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with respect to the amino acid sequence shown in SEQ ID NO: 14; This heavy chain variable region includes the HCDR1 amino acid sequence shown in SEQ ID NO: 15; the HCDR2 amino acid sequence shown in SEQ ID NO: 16; and the HCDR3 amino acid sequence shown in SEQ ID NO: 17. - Heavy chain variable regions having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with respect to the amino acid sequence shown in SEQ ID NO: 18; This heavy chain variable region includes the HCDR1 amino acid sequence shown in SEQ ID NO: 15; the HCDR2 amino acid sequence shown in SEQ ID NO: 16; and the HCDR3 amino acid sequence shown in SEQ ID NO: 17. or - Heavy chain variable regions having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO: 19; This heavy chain variable region includes 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 this disclosure are constructed using a common light chain, specifically a common light chain called VK1-39 / JK1. The binding domains of the multispecific binding sites of this disclosure may include any suitable light chain, including but not limited to common light chains known in the art. In some embodiments, the binding domains of the multispecific binding sites of this disclosure include the common light chain VK1-39 / JK1, or variants thereof that harbor a limited number of non-conservative amino acid substitutions, such as one, two, or three, or an unlimited number of conservative amino acid substitutions.

[0074] In one embodiment, the PD-1 binding domain of the multispecific binding site of the Disclosure comprises a light chain variable region having the amino acid sequence shown in SEQ ID NO: 48, or a variant thereof. In one embodiment, the PD-1 binding domain of the multispecific binding site of the Disclosure comprises a light chain variable region having the amino acid sequence shown 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 the multispecific binding site of the Disclosure includes a light chain variable region comprising light chain CDR1 (LCDR1), light chain CDR2 (LCDR2), and light chain CDR3 (LCDR3), having the amino acid sequences shown in SEQ ID NOs. 49, SEQ ID NOs. 50, and SEQ ID NOs. In one embodiment, the light chain variable region of the PD-1 binding domain of the multispecific binding site of the Disclosure also includes variants thereof, where each LCDR may contain at most three, two, or one amino acid variation. In one embodiment, the amino acid variation is a conservative amino acid substitution.

[0076] In some embodiments, the PD-1 binding domain of the multispecific binding site of the Disclosure also comprises a PD-1 binding domain variant, which includes one or more variations in the framework region in addition to the variations in the LCDR described above. The variations are preferably conservative amino acid substitutions. In some embodiments, the PD-1 binding domain variant of the multispecific binding site of the Disclosure does not include variations in the LCDR region but includes one or more variations in the framework region. Such variants have at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with respect to the sequences disclosed herein. Accordingly, in some embodiments, the PD-1 binding domain of the multispecific binding site of the Disclosure comprises: - Light chain variable regions having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with respect to the amino acid sequence shown in Sequence ID No. 48; This light chain variable region includes the LCDR1 amino acid sequence shown in SEQ ID NO: 49; the LCDR2 amino acid sequence shown in SEQ ID NO: 50; and the LCDR3 amino acid sequence shown in SEQ ID NO: 51.

[0077] These LCDRs and / or light chain variable regions, including the light chain or light chain variable region, may be a light chain referred to, for example, in the art as VK1-39 / JK1. This is a common light chain. The term “common light chain” as used herein refers to a light chain that can pair with multiple different heavy chains, such as heavy chains having different antigen or epitope binding specificities. Common light chains are particularly useful, for example, in the production of bispecific or multispecific antibodies, as antibody production becomes 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 their amino acid sequence but do not affect the binding specificity of the full-length antibody. Within the scope of the definition of a common light chain as used herein, it is possible, for example, to prepare or find a light chain that is not identical but is still functionally equivalent by using well-established variations, such as introducing conservative amino acid changes, i.e., changes in amino acids in regions that are known or have been shown to contribute little to or no to binding specificity when paired with a heavy chain.

[0078] Apart from the common light chains including the aforementioned LCDR and / or light chain variable regions, other common light chains known in the art may be used. Examples of such common light chains include, but are not limited to, the following: VK1-39 / JK5 includes a light chain variable region comprising light chain CDR1 (LCDR1), light chain CDR2 (LCDR2), and light chain CDR3 (LCDR3), having the amino acid sequence shown in SEQ ID NO: 52. In one embodiment, the light chain includes a light chain variable region comprising light chain CDR1 (LCDR1), light chain CDR2 (LCDR2), and light chain CDR3 (LCDR3), having the amino acid sequence shown in SEQ ID NO: 52, where each LCDR may contain at most three, two, or one amino acid variation, such as substitution. In one embodiment, the light chain includes a light chain variable region having the amino acid sequence shown 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 light chain CDR1 (LCDR1), light chain CDR2 (LCDR2), and light chain CDR3 (LCDR3), having the amino acid sequences shown in SEQ ID NOs. 53, 54, and 55; VK3-15 / JK1 includes a light chain variable region comprising light chain CDR1 (LCDR1), light chain CDR2 (LCDR2), and light chain CDR3 (LCDR3), having the amino acid sequence shown in SEQ ID NO: 56. In one embodiment, the light chain includes a light chain variable region comprising light chain CDR1 (LCDR1), light chain CDR2 (LCDR2), and light chain CDR3 (LCDR3), having the amino acid sequence shown in SEQ ID NO: 56, where each LCDR may contain at most three, two, or one amino acid variation, such as substitution. In one embodiment, the light chain includes a light chain variable region having the amino acid sequence shown 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 light chain CDR1 (LCDR1), light chain CDR2 (LCDR2), and light chain CDR3 (LCDR3), having the amino acid sequences shown in SEQ ID NOs. 57, 58, and 59; VK3-20 / JK1 includes a light chain variable region comprising light chain CDR1 (LCDR1), light chain CDR2 (LCDR2), and light chain CDR3 (LCDR3), having the amino acid sequence shown in SEQ ID NO: 60. In one embodiment, the light chain includes a light chain variable region comprising light chain CDR1 (LCDR1), light chain CDR2 (LCDR2), and light chain CDR3 (LCDR3), having the amino acid sequence shown in SEQ ID NO: 60, where each LCDR may contain at most three, two, or one amino acid variation, such as substitution. In one embodiment, the light chain includes a light chain variable region having the amino acid sequence shown 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 light chain CDR1 (LCDR1), light chain CDR2 (LCDR2), and light chain CDR3 (LCDR3), having the amino acid sequences shown in SEQ ID NOs. 61, 62, and 63; and VL3-21 / JL3 includes a light chain variable region comprising light chain CDR1 (LCDR1), light chain CDR2 (LCDR2), and light chain CDR3 (LCDR3), having the amino acid sequence shown in SEQ ID NO: 64. In one embodiment, the light chain includes a light chain variable region comprising light chain CDR1 (LCDR1), light chain CDR2 (LCDR2), and light chain CDR3 (LCDR3), having the amino acid sequence shown in SEQ ID NO: 64, where each LCDR may contain at most three, two, or one amino acid variation, such as substitution. In one embodiment, the light chain includes a light chain variable region having the amino acid sequence shown 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 light chain CDR1(LCDR1), light chain CDR2(LCDR2), and light chain CDR3(LCDR3), having the amino acid sequences shown in SEQ ID NOs. 65, 66, and 67.

[0079] VK1-39 is an abbreviation for the immunoglobulin variable kappa 1-39 gene. This gene is also known as immunoglobulin variable kappa 1-39;IGKV139;IGKV1-39;IgVκ1-39. The external ID of this gene is 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 bind to one of five J regions. Appropriate VJ region sequences are indicated 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 (World Wide Web imgt.org)). These names are illustrative and encompass allele variants of the gene segment.

[0080] VK3-15 is an abbreviation for the immunoglobulin variable kappa 3-15 gene. This gene is also known as immunoglobulin variable kappa 3-15; IGKV315; IGKV3-15; IgVκ3-15. The external ID of this gene is 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. The V region can be bound to one of five J regions. The appropriate VJ region sequence is shown as VK3-15 / JK1 (SEQ ID NO: 99); alternative names are Vκ3-15*01 / IGJκ1*01 (nomenclature according to the IMGT database (imgt.org)). This name is illustrative and encompasses allele variants of the gene segment.

[0081] VK3-20 is an abbreviation for the immunoglobulin variable kappa 3-20 gene. This gene is also known as immunoglobulin variable kappa 3-20; IGKV320; IGKV3-20; IgVκ3-20. The external ID of this gene is 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. The V region can be bound to one of five J regions. The appropriate VJ region sequence is shown as VK3-20 / JK1 (SEQ ID NO: 101); alternative names are IgVκ3-20*01 / IGJκ1*01 (nomenclature according to the IMGT database (imgt.org)). This name is illustrative and encompasses allele variants of the gene segment.

[0082] VL3-21 is an abbreviation for the immunoglobulin variable lambda 3-21 gene. This gene is also known as immunoglobulin variable lambda 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 bound to one of five J regions. The appropriate VJ region sequence is shown as VL3-21 / JL3 (SEQ ID NO: 103); alternative names are IgVλ3-21 / IGJλ3 (nomenclature according to the IMGT database (World Wide Web imgt.org)). This name is illustrative and encompasses allele variants of the gene segment.

[0083] Furthermore, any light chain variable region of a PD-1 antibody available in the art can be used, in the same way as any other light chain variable region readily available from, for example, an antibody display library, as it exhibits antigen-binding activity when paired with the PD-1 binding domain of the multispecific binding site of this disclosure.

[0084] In one embodiment, the PD-1 binding domain of the multispecific binding site of the present disclosure may further include CH1 and CL regions. Any CH1 domain, specifically 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, specifically 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.

[0085] In one embodiment, the TGF-βRII binding domain of the multispecific binding site of this disclosure includes a heavy chain variable region comprising: a) Heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), having the amino acid sequences shown in SEQ ID NO: 24, SEQ ID NO: 25, and SEQ ID NO: 26, respectively; b) Heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), having the amino acid sequences shown in SEQ ID NO: 28, SEQ ID NO: 29, and SEQ ID NO: 30, respectively; c) Heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), having the amino acid sequences shown 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 shown 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 shown in SEQ ID NO: 40, SEQ ID NO: 41, and SEQ ID NO: 42, respectively; f) Heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), having the amino acid sequences shown in SEQ ID NOs. 44, 45, and 46, respectively; or g) Heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), each having the amino acid sequences shown 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 variants of that TGF-βRII binding domain, where each HCDR 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] The TGF-βRII binding variants included in this disclosure include somatic hypermutant or affinity-matured heavy chain variable regions, which are heavy chain variable regions derived from the same VH gene segment as the heavy chain variable regions described herein by sequence, and the variants have amino acid variations, including non-conserved and / or conserved amino acid substitutions in one, two, or all three HCDRs.

[0089] In one embodiment, the TGF-βRII binding domain of the multispecific binding site of the Disclosure comprises a heavy chain variable region having the amino acid sequence shown in any one of SEQ ID NOs: 23;27;31;35;39;43;47;88;89, or a variant thereof. In one embodiment, the TGF-βRII binding domain of the multispecific binding site of the Disclosure comprises a heavy chain variable region having the amino acid sequence shown 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 to these.

[0090] In some embodiments, the TGF-βRII binding domain of the multispecific binding site of the Disclosure also comprises a TGF-βRII binding domain variant, which includes one or more variations in the framework region in addition to the variations in the HCDR described above. The variations may be any type of amino acid variation described herein, such as conserved or non-conserved 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 Disclosure does not include variations in the CDR region but includes one or more variations in the framework region. Such variants are expected to have at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with respect to the sequences disclosed herein and to retain TGF-βRII binding specificity. Accordingly, in some embodiments, the TGF-βRII binding domain of the multispecific binding site of the Disclosure comprises: - Heavy chain variable regions having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with respect to the amino acid sequence shown in SEQ ID NO: 23; This heavy chain variable region includes the HCDR1 amino acid sequence shown in SEQ ID NO: 24; the HCDR2 amino acid sequence shown in SEQ ID NO: 25; and the HCDR3 amino acid sequence shown in SEQ ID NO: 26. - Heavy chain variable regions having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with respect to the amino acid sequence shown in SEQ ID NO: 27; This heavy chain variable region includes the HCDR1 amino acid sequence shown in SEQ ID NO: 28; the HCDR2 amino acid sequence shown in SEQ ID NO: 29; and the HCDR3 amino acid sequence shown in SEQ ID NO: 30; - Heavy chain variable regions having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with respect to the amino acid sequence shown in SEQ ID NO: 31; This heavy chain variable region includes the HCDR1 amino acid sequence shown in SEQ ID NO: 32; the HCDR2 amino acid sequence shown in SEQ ID NO: 33; and the HCDR3 amino acid sequence shown in SEQ ID NO: 34; - Heavy chain variable regions having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with respect to the amino acid sequence shown in SEQ ID NO: 35; This heavy chain variable region includes the HCDR1 amino acid sequence shown in SEQ ID NO: 36; the HCDR2 amino acid sequence shown in SEQ ID NO: 37; and the HCDR3 amino acid sequence shown in SEQ ID NO: 38. - Heavy chain variable regions having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO: 39; This heavy chain variable region includes the HCDR1 amino acid sequence shown in SEQ ID NO: 40; the HCDR2 amino acid sequence shown in SEQ ID NO: 41; and the HCDR3 amino acid sequence shown in SEQ ID NO: 42; - Heavy chain variable regions having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with respect to the amino acid sequence shown in SEQ ID NO: 43; This heavy chain variable region includes the HCDR1 amino acid sequence shown in SEQ ID NO: 44; the HCDR2 amino acid sequence shown in SEQ ID NO: 45; and the HCDR3 amino acid sequence shown in SEQ ID NO: 46. - Heavy chain variable regions having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO: 47; This heavy chain variable region includes the HCDR1 amino acid sequence shown in SEQ ID NO: 90; the HCDR2 amino acid sequence shown in SEQ ID NO: 91; and the HCDR3 amino acid sequence shown in SEQ ID NO: 92; - Heavy chain variable regions having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with respect to the amino acid sequence shown in Sequence ID No. 88; This heavy chain variable region includes the HCDR1 amino acid sequence shown in SEQ ID NO: 44; the HCDR2 amino acid sequence shown in SEQ ID NO: 45; and the HCDR3 amino acid sequence shown in SEQ ID NO: 46. or - Heavy chain variable regions having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with respect to the amino acid sequence shown in Sequence ID No. 89; This heavy chain variable region includes the HCDR1 amino acid sequence shown in SEQ ID NO: 90; the HCDR2 amino acid sequence shown in SEQ ID NO: 91; and the HCDR3 amino acid sequence shown in SEQ ID NO: 92.

[0091] Any light chain variable region of a TGF-βRII antibody available in the art may be used, like any other light chain variable region readily available from, for example, an antibody display library, by exhibiting antigen-binding activity when paired with the TGF-βRII binding domain of the multispecific binding site of this disclosure, as described herein, for example. In one embodiment, the TGF-βRII binding domain of the multispecific binding site of this disclosure comprises a light chain identical or substantially identical to that of the PD-1 binding domain.

[0092] In one embodiment, the TGF-βRII binding domain of the multispecific binding site of the Disclosure comprises a light chain variable region having the amino acid sequence shown in SEQ ID NO: 48, or a variant thereof. In one embodiment, the TGF-βRII binding domain of the multispecific binding site of the Disclosure comprises a light chain variable region having the amino acid sequence shown 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 one embodiment, the TGF-βRII binding domain of the multispecific binding site of the Disclosure includes a light chain variable region comprising light chain CDR1 (LCDR1), light chain CDR2 (LCDR2), and light chain CDR3 (LCDR3), having the amino acid sequences shown in SEQ ID NOs. 49, SEQ ID NOs. 50, and SEQ ID NOs. In one embodiment, the light chain variable region of the TGF-βRII binding domain of the multispecific binding site of the Disclosure also includes variants thereof, where each LCDR may contain at most three, two, or one conserved or non-conserved amino acid variation. In one embodiment, the amino acid variation is a conserved amino acid substitution.

[0094] In one embodiment, the TGF-βRII binding domain of the multispecific binding site of the present disclosure may further include CH1 and CL regions. Any CH1 domain, specifically 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, specifically 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 may also comprise 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 may also comprise 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 may also comprise 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 comprising a heavy chain variable region and may also comprise a light chain variable region and CH1 and CL regions, as described herein.

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

[0098] In one embodiment, the present disclosure provides a multispecific binding site including: - PD-1 binding domains as described herein, comprising heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), each having the amino acid sequences shown in SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12, respectively; and - TGF-βRII binding domains as described herein, comprising heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), having the amino acid sequences shown in SEQ ID NOs. 24, 25, and 26, respectively; Here, each HCDR may contain at most three, two, or one amino acid variation, such as a substitution. In one embodiment, the HCDR does not contain any amino acid variation.

[0099] In one embodiment, the present disclosure provides a multispecific binding site including: - PD-1 binding domains as described herein, comprising heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), having the amino acid sequences shown in SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 17, respectively; and - TGF-βRII binding domains as described herein, comprising heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), having the amino acid sequences shown in SEQ ID NOs. 24, 25, and 26, respectively; Here, each HCDR may contain at most three, two, or one amino acid variation, such as a substitution. In one embodiment, the HCDR does not contain any amino acid variation.

[0100] In one embodiment, the present disclosure provides a multispecific binding site including: - PD-1 binding domains as described herein, comprising heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), each having the amino acid sequences shown in SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 22, respectively; and - TGF-βRII binding domains as described herein, comprising heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), having the amino acid sequences shown in SEQ ID NOs. 24, 25, and 26, respectively; Here, each HCDR may contain at most three, two, or one amino acid variation, such as a substitution. In one embodiment, the HCDR does not contain any amino acid variation.

[0101] In one embodiment, the present disclosure provides a multispecific binding site including: - PD-1 binding domains as described herein, comprising heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), having the amino acid sequences shown in SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 17, respectively; and - TGF-βRII binding domains as described herein, comprising heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), having the amino acid sequences shown in SEQ ID NOs. 32, 33, and 34, respectively; Here, each HCDR may contain at most three, two, or one amino acid variation, such as a substitution. In one embodiment, the HCDR does not contain any amino acid variation.

[0102] In one embodiment, the present disclosure provides a multispecific binding site including: - PD-1 binding domains as described herein, comprising heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), each having the amino acid sequences shown in SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12, respectively; and - TGF-βRII binding domains as described herein, comprising heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), having the amino acid sequences shown in SEQ ID NOs. 40, 41, and 42, respectively; Here, each HCDR may contain at most three, two, or one amino acid variation, such as a substitution. In one embodiment, the HCDR does not contain any amino acid variation.

[0103] In one embodiment, the present disclosure provides a multispecific binding site including: - PD-1 binding domains as described herein, comprising heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), each having the amino acid sequences shown in SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12, respectively; and - TGF-βRII binding domains as described herein, comprising heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), having the amino acid sequences shown in SEQ ID NO: 36, SEQ ID NO: 37, and SEQ ID NO: 38, respectively; Here, each HCDR may contain at most three, two, or one amino acid variation, such as a substitution. In one embodiment, the HCDR does not contain any amino acid variation.

[0104] In one embodiment, the present disclosure provides a multispecific binding site including: - PD-1 binding domains as described herein, comprising heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), having the amino acid sequences shown in SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 17, respectively; and - TGF-βRII binding domains as described herein, comprising heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), having the amino acid sequences shown in SEQ ID NO: 36, SEQ ID NO: 37, and SEQ ID NO: 38, respectively; Here, each HCDR may contain at most three, two, or one amino acid variation, such as a substitution. In one embodiment, the HCDR does not contain any amino acid variation.

[0105] In one embodiment, the present disclosure provides a multispecific binding site including: - PD-1 binding domains as described herein, comprising heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), each having the amino acid sequences shown in SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 22, respectively; and - TGF-βRII binding domains as described herein, comprising heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), having the amino acid sequences shown in SEQ ID NO: 36, SEQ ID NO: 37, and SEQ ID NO: 38, respectively; Here, each HCDR may contain at most three, two, or one amino acid variation, such as a substitution. In one embodiment, the HCDR does not contain any amino acid variation.

[0106] In one embodiment, the present disclosure provides a multispecific binding site including: - PD-1 binding domains as described herein, comprising heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), each having the amino acid sequences shown in SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12, respectively; and - TGF-βRII binding domains as described herein, comprising heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), having the amino acid sequences shown in SEQ ID NOs. 28, 29, and 30, respectively; Here, each HCDR may contain at most three, two, or one amino acid variation, such as a substitution. In one embodiment, the HCDR does not contain any amino acid variation.

[0107] In one embodiment, the present disclosure provides a multispecific binding site including: - PD-1 binding domains as described herein, comprising heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), each having the amino acid sequences shown in SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12, respectively; and - TGF-βRII binding domains as described herein, comprising heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), having the amino acid sequences shown in SEQ ID NOs. 90, 91, and 92, respectively; Here, each HCDR may contain at most three, two, or one amino acid variation, such as a substitution. In one embodiment, the HCDR does not contain any amino acid variation.

[0108] In one embodiment, the present disclosure provides a multispecific binding site including: - PD-1 binding domains as described herein, comprising heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), each having the amino acid sequences shown in SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 22, respectively; and - TGF-βRII binding domains as described herein, comprising heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), having the amino acid sequences shown in SEQ ID NOs. 44, 45, and 46, respectively; Here, each HCDR may contain at most three, two, or one amino acid variation, such as a substitution. In one embodiment, the HCDR does not contain any amino acid variation.

[0109] In one embodiment, the present disclosure provides a multispecific binding site including: - PD-1 binding domains as described herein, comprising heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), each having the amino acid sequences shown in SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12, respectively; and - TGF-βRII binding domains as described herein, comprising heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), having the amino acid sequences shown in SEQ ID NOs. 24, 25, and 26, respectively; Here, the PD-1 binding domain and the TGF-βRII binding domain include a light chain CDR1 (LCDR1) having the amino acid sequence shown in SEQ ID NO: 49, a light chain CDR2 (LCDR2) having the amino acid sequence shown in SEQ ID NO: 50, and a light chain CDR3 (LCDR3) having the amino acid sequence shown in SEQ ID NO: 51. and Here, each of the HCDR and / or LCDR may contain at most three, two, or one amino acid variation, such as substitutions. In one embodiment, the HCDR and / or LCDR do not contain any amino acid variations.

[0110] In one embodiment, the present disclosure provides a multispecific binding site including: - PD-1 binding domains as described herein, comprising heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), having the amino acid sequences shown in SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 17, respectively; and - TGF-βRII binding domains as described herein, comprising heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), having the amino acid sequences shown in SEQ ID NOs. 24, 25, and 26, respectively; Here, the PD-1 binding domain and the TGF-βRII binding domain include a light chain CDR1 (LCDR1) having the amino acid sequence shown in SEQ ID NO: 49, a light chain CDR2 (LCDR2) having the amino acid sequence shown in SEQ ID NO: 50, and a light chain CDR3 (LCDR3) having the amino acid sequence shown in SEQ ID NO: 51. and Here, each of the HCDR and / or LCDR may contain at most three, two, or one amino acid variation, such as substitutions. In one embodiment, the HCDR and / or LCDR do not contain any amino acid variations.

[0111] In one embodiment, the present disclosure provides a multispecific binding site including: - PD-1 binding domains as described herein, comprising heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), each having the amino acid sequences shown in SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 22, respectively; and - TGF-βRII binding domains as described herein, comprising heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), having the amino acid sequences shown in SEQ ID NOs. 24, 25, and 26, respectively; Here, the PD-1 binding domain and the TGF-βRII binding domain include a light chain CDR1 (LCDR1) having the amino acid sequence shown in SEQ ID NO: 49, a light chain CDR2 (LCDR2) having the amino acid sequence shown in SEQ ID NO: 50, and a light chain CDR3 (LCDR3) having the amino acid sequence shown in SEQ ID NO: 51. and Here, each of the HCDR and / or LCDR may contain at most three, two, or one amino acid variation, such as substitutions. In one embodiment, the HCDR and / or LCDR do not contain any amino acid variations.

[0112] In one embodiment, the present disclosure provides a multispecific binding site including: - PD-1 binding domains as described herein, comprising heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), having the amino acid sequences shown in SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 17, respectively; and - TGF-βRII binding domains as described herein, comprising heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), having the amino acid sequences shown in SEQ ID NOs. 32, 33, and 34, respectively; Here, the PD-1 binding domain and the TGF-βRII binding domain include a light chain CDR1 (LCDR1) having the amino acid sequence shown in SEQ ID NO: 49, a light chain CDR2 (LCDR2) having the amino acid sequence shown in SEQ ID NO: 50, and a light chain CDR3 (LCDR3) having the amino acid sequence shown in SEQ ID NO: 51. and Here, each of the HCDR and / or LCDR may contain at most three, two, or one amino acid variation, such as substitutions. In one embodiment, the HCDR and / or LCDR do not contain any amino acid variations.

[0113] In one embodiment, the present disclosure provides a multispecific binding site including: - PD-1 binding domains as described herein, comprising heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), each having the amino acid sequences shown in SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12, respectively; and - TGF-βRII binding domains as described herein, comprising heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), having the amino acid sequences shown in SEQ ID NOs. 40, 41, and 42, respectively; Here, the PD-1 binding domain and the TGF-βRII binding domain include a light chain CDR1 (LCDR1) having the amino acid sequence shown in SEQ ID NO: 49, a light chain CDR2 (LCDR2) having the amino acid sequence shown in SEQ ID NO: 50, and a light chain CDR3 (LCDR3) having the amino acid sequence shown in SEQ ID NO: 51. and Here, each of the HCDR and / or LCDR may contain at most three, two, or one amino acid variation, such as substitutions. In one embodiment, the HCDR and / or LCDR do not contain any amino acid variations.

[0114] In one embodiment, the present disclosure provides a multispecific binding site including: - PD-1 binding domains as described herein, comprising heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), each having the amino acid sequences shown in SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12, respectively; and - TGF-βRII binding domains as described herein, comprising heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), having the amino acid sequences shown in SEQ ID NO: 36, SEQ ID NO: 37, and SEQ ID NO: 38, respectively; Here, the PD-1 binding domain and the TGF-βRII binding domain include a light chain CDR1 (LCDR1) having the amino acid sequence shown in SEQ ID NO: 49, a light chain CDR2 (LCDR2) having the amino acid sequence shown in SEQ ID NO: 50, and a light chain CDR3 (LCDR3) having the amino acid sequence shown in SEQ ID NO: 51. and Here, each of the HCDR and / or LCDR may contain at most three, two, or one amino acid variation, such as substitutions. In one embodiment, the HCDR and / or LCDR do not contain any amino acid variations.

[0115] In one embodiment, the present disclosure provides a multispecific binding site including: - PD-1 binding domains as described herein, comprising heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), having the amino acid sequences shown in SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 17, respectively; and - TGF-βRII binding domains as described herein, comprising heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), having the amino acid sequences shown in SEQ ID NO: 36, SEQ ID NO: 37, and SEQ ID NO: 38, respectively; Here, the PD-1 binding domain and the TGF-βRII binding domain include a light chain CDR1 (LCDR1) having the amino acid sequence shown in SEQ ID NO: 49, a light chain CDR2 (LCDR2) having the amino acid sequence shown in SEQ ID NO: 50, and a light chain CDR3 (LCDR3) having the amino acid sequence shown in SEQ ID NO: 51. and Here, each of the HCDR and / or LCDR may contain at most three, two, or one amino acid variation, such as substitutions. In one embodiment, the HCDR and / or LCDR do not contain any amino acid variations.

[0116] In one embodiment, the present disclosure provides a multispecific binding site including: - PD-1 binding domains as described herein, comprising heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), each having the amino acid sequences shown in SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 22, respectively; and - TGF-βRII binding domains as described herein, comprising heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), having the amino acid sequences shown in SEQ ID NO: 36, SEQ ID NO: 37, and SEQ ID NO: 38, respectively; Here, the PD-1 binding domain and the TGF-βRII binding domain include a light chain CDR1 (LCDR1) having the amino acid sequence shown in SEQ ID NO: 49, a light chain CDR2 (LCDR2) having the amino acid sequence shown in SEQ ID NO: 50, and a light chain CDR3 (LCDR3) having the amino acid sequence shown in SEQ ID NO: 51. and Here, each of the HCDR and / or LCDR may contain at most three, two, or one amino acid variation, such as substitutions. In one embodiment, the HCDR and / or LCDR do not contain any amino acid variations.

[0117] In one embodiment, the present disclosure provides a multispecific binding site including: - PD-1 binding domains as described herein, comprising heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), each having the amino acid sequences shown in SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12, respectively; and - TGF-βRII binding domains as described herein, comprising heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), having the amino acid sequences shown in SEQ ID NOs. 28, 29, and 30, respectively; Here, the PD-1 binding domain and the TGF-βRII binding domain include a light chain CDR1 (LCDR1) having the amino acid sequence shown in SEQ ID NO: 49, a light chain CDR2 (LCDR2) having the amino acid sequence shown in SEQ ID NO: 50, and a light chain CDR3 (LCDR3) having the amino acid sequence shown in SEQ ID NO: 51. and Here, each of the HCDR and / or LCDR may contain at most three, two, or one amino acid variation, such as substitutions. In one embodiment, the HCDR and / or LCDR do not contain any amino acid variations.

[0118] In one embodiment, the present disclosure provides a multispecific binding site including: - PD-1 binding domains as described herein, comprising heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), each having the amino acid sequences shown in SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12, respectively; and - TGF-βRII binding domains as described herein, comprising heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), having the amino acid sequences shown in SEQ ID NOs. 90, 91, and 92, respectively; Here, the PD-1 binding domain and the TGF-βRII binding domain include a light chain CDR1 (LCDR1) having the amino acid sequence shown in SEQ ID NO: 49, a light chain CDR2 (LCDR2) having the amino acid sequence shown in SEQ ID NO: 50, and a light chain CDR3 (LCDR3) having the amino acid sequence shown in SEQ ID NO: 51. and Here, each of the HCDR and / or LCDR may contain at most three, two, or one amino acid variation, such as substitutions. In one embodiment, the HCDR and / or LCDR do not contain any amino acid variations.

[0119] In one embodiment, the present disclosure provides a multispecific binding site including: - PD-1 binding domains as described herein, comprising heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), each having the amino acid sequences shown in SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 22, respectively; and - TGF-βRII binding domains as described herein, comprising heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), having the amino acid sequences shown in SEQ ID NOs. 44, 45, and 46, respectively; Here, the PD-1 binding domain and the TGF-βRII binding domain include a light chain CDR1 (LCDR1) having the amino acid sequence shown in SEQ ID NO: 49, a light chain CDR2 (LCDR2) having the amino acid sequence shown in SEQ ID NO: 50, and a light chain CDR3 (LCDR3) having the amino acid sequence shown in SEQ ID NO: 51. and Here, each of the HCDR and / or LCDR may contain at most three, two, or one amino acid variation, such as substitutions. In one embodiment, the HCDR and / or LCDR do not contain any amino acid variations.

[0120] In one embodiment, the present disclosure provides a multispecific binding site including: - A PD-1 binding domain as described herein, comprising a heavy chain variable region having the amino acid sequence shown in Sequence 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 shown 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 one embodiment, each of the heavy chain variable regions comprises an HCDR that does not contain amino acid variations. In one embodiment, each of the heavy chain variable regions does not contain amino acid variations.

[0121] In one embodiment, the present disclosure provides a multispecific binding site including: - A PD-1 binding domain as described herein, comprising a heavy chain variable region having the amino acid sequence shown in Sequence 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 shown 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 one embodiment, each of the heavy chain variable regions comprises an HCDR that does not contain amino acid variations. In one embodiment, each of the heavy chain variable regions does not contain amino acid variations.

[0122] In one embodiment, the present disclosure provides a multispecific binding site including: - A PD-1 binding domain as described herein, comprising a heavy chain variable region having the amino acid sequence shown in Sequence 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 shown 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 one embodiment, each of the heavy chain variable regions comprises an HCDR that does not contain amino acid variations. In one embodiment, each of the heavy chain variable regions does not contain amino acid variations.

[0123] In one embodiment, the present disclosure provides a multispecific binding site including: - A PD-1 binding domain as described herein, comprising a heavy chain variable region having the amino acid sequence shown in Sequence 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 shown 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 one embodiment, each of the heavy chain variable regions comprises an HCDR that does not contain amino acid variations. In one embodiment, each of the heavy chain variable regions does not contain amino acid variations.

[0124] In one embodiment, the present disclosure provides a multispecific binding site including: - A PD-1 binding domain as described herein, comprising a heavy chain variable region having the amino acid sequence shown in Sequence 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 shown 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 one embodiment, each of the heavy chain variable regions comprises an HCDR that does not contain amino acid variations. In one embodiment, each of the heavy chain variable regions does not contain amino acid variations.

[0125] In one embodiment, the present disclosure provides a multispecific binding site including: - A PD-1 binding domain as described herein, comprising a heavy chain variable region having the amino acid sequence shown in Sequence 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 shown 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 one embodiment, each of the heavy chain variable regions comprises an HCDR that does not contain amino acid variations. In one embodiment, each of the heavy chain variable regions does not contain amino acid variations.

[0126] In one embodiment, the present disclosure provides a multispecific binding site including: - A PD-1 binding domain as described herein, comprising a heavy chain variable region having the amino acid sequence shown in Sequence 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 shown 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 one embodiment, each of the heavy chain variable regions comprises an HCDR that does not contain amino acid variations. In one embodiment, each of the heavy chain variable regions does not contain amino acid variations.

[0127] In one embodiment, the present disclosure provides a multispecific binding site including: - A PD-1 binding domain as described herein, comprising a heavy chain variable region having the amino acid sequence shown in Sequence 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 shown 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 one embodiment, each of the heavy chain variable regions comprises an HCDR that does not contain amino acid variations. In one embodiment, each of the heavy chain variable regions does not contain amino acid variations.

[0128] In one embodiment, the present disclosure provides a multispecific binding site including: - A PD-1 binding domain as described herein, comprising a heavy chain variable region having the amino acid sequence shown in Sequence 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 shown 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 one embodiment, each of the heavy chain variable regions comprises an HCDR that does not contain amino acid variations. In one embodiment, each of the heavy chain variable regions does not contain amino acid variations.

[0129] In one embodiment, the present disclosure provides a multispecific binding site including: - A PD-1 binding domain as described herein, comprising a heavy chain variable region having the amino acid sequence shown in Sequence 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 shown in Sequence 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 one embodiment, each of the heavy chain variable regions comprises an HCDR that does not contain amino acid variations. In one embodiment, each of the heavy chain variable regions does not contain amino acid variations.

[0130] In one embodiment, the present disclosure provides a multispecific binding site including: - A PD-1 binding domain as described herein, comprising a heavy chain variable region having the amino acid sequence shown in Sequence 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 shown in Sequence 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 one embodiment, each of the heavy chain variable regions comprises an HCDR that does not contain amino acid variations. In one embodiment, each of the heavy chain variable regions does not contain amino acid variations.

[0131] In one embodiment, the present disclosure provides a multispecific binding site including: - A PD-1 binding domain as described herein, comprising a heavy chain variable region having the amino acid sequence shown in Sequence 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 shown in Sequence 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; Here, the PD-1 binding domain and the TGF-βRII binding domain include a light chain variable region having the amino acid sequence shown 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 one embodiment, each of the heavy chain variable region and the light chain variable region includes an HCDR and an LCDR that do not contain amino acid variations, respectively. In another embodiment, each of the heavy chain variable region and the light chain variable region does not contain amino acid variations.

[0132] In one embodiment, the present disclosure provides a multispecific binding site including: - A PD-1 binding domain as described herein, comprising a heavy chain variable region having the amino acid sequence shown in Sequence 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 shown in Sequence 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; Here, the PD-1 binding domain and the TGF-βRII binding domain include a light chain variable region having the amino acid sequence shown 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 one embodiment, each of the heavy chain variable region and the light chain variable region includes an HCDR and an LCDR that do not contain amino acid variations, respectively. In another embodiment, each of the heavy chain variable region and the light chain variable region does not contain amino acid variations.

[0133] In one embodiment, the present disclosure provides a multispecific binding site including: - A PD-1 binding domain as described herein, comprising a heavy chain variable region having the amino acid sequence shown in Sequence 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 shown in Sequence 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; Here, the PD-1 binding domain and the TGF-βRII binding domain include a light chain variable region having the amino acid sequence shown 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 one embodiment, each of the heavy chain variable region and the light chain variable region includes an HCDR and an LCDR that do not contain amino acid variations, respectively. In another embodiment, each of the heavy chain variable region and the light chain variable region does not contain amino acid variations.

[0134] In one embodiment, the present disclosure provides a multispecific binding site including: - A PD-1 binding domain as described herein, comprising a heavy chain variable region having the amino acid sequence shown in Sequence 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 shown in Sequence 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; Here, the PD-1 binding domain and the TGF-βRII binding domain include a light chain variable region having the amino acid sequence shown 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 one embodiment, each of the heavy chain variable region and the light chain variable region includes an HCDR and an LCDR that do not contain amino acid variations, respectively. In another embodiment, each of the heavy chain variable region and the light chain variable region does not contain amino acid variations.

[0135] In one embodiment, the present disclosure provides a multispecific binding site including: - A PD-1 binding domain as described herein, comprising a heavy chain variable region having the amino acid sequence shown in Sequence 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 shown in Sequence 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; Here, the PD-1 binding domain and the TGF-βRII binding domain include a light chain variable region having the amino acid sequence shown 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 one embodiment, each of the heavy chain variable region and the light chain variable region includes an HCDR and an LCDR that do not contain amino acid variations, respectively. In another embodiment, each of the heavy chain variable region and the light chain variable region does not contain amino acid variations.

[0136] In one embodiment, the present disclosure provides a multispecific binding site including: - A PD-1 binding domain as described herein, comprising a heavy chain variable region having the amino acid sequence shown in Sequence 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 shown in Sequence 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; Here, the PD-1 binding domain and the TGF-βRII binding domain include a light chain variable region having the amino acid sequence shown 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 one embodiment, each of the heavy chain variable region and the light chain variable region includes an HCDR and an LCDR that do not contain amino acid variations, respectively. In another embodiment, each of the heavy chain variable region and the light chain variable region does not contain amino acid variations.

[0137] In one embodiment, the present disclosure provides a multispecific binding site including: - A PD-1 binding domain as described herein, comprising a heavy chain variable region having the amino acid sequence shown in Sequence 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 shown in Sequence 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; Here, the PD-1 binding domain and the TGF-βRII binding domain include a light chain variable region having the amino acid sequence shown 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 one embodiment, each of the heavy chain variable region and the light chain variable region includes an HCDR and an LCDR that do not contain amino acid variations, respectively. In another embodiment, each of the heavy chain variable region and the light chain variable region does not contain amino acid variations.

[0138] In one embodiment, the present disclosure provides a multispecific binding site including: - A PD-1 binding domain as described herein, comprising a heavy chain variable region having the amino acid sequence shown in Sequence 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 shown in Sequence 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; Here, the PD-1 binding domain and the TGF-βRII binding domain include a light chain variable region having the amino acid sequence shown 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 one embodiment, each of the heavy chain variable region and the light chain variable region includes an HCDR and an LCDR that do not contain amino acid variations, respectively. In another embodiment, each of the heavy chain variable region and the light chain variable region does not contain amino acid variations.

[0139] In one embodiment, the present disclosure provides a multispecific binding site including: - A PD-1 binding domain as described herein, comprising a heavy chain variable region having the amino acid sequence shown in Sequence 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 shown in Sequence 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; Here, the PD-1 binding domain and the TGF-βRII binding domain include a light chain variable region having the amino acid sequence shown 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 one embodiment, each of the heavy chain variable region and the light chain variable region includes an HCDR and an LCDR that do not contain amino acid variations, respectively. In another embodiment, each of the heavy chain variable region and the light chain variable region does not contain amino acid variations.

[0140] In one embodiment, the present disclosure provides a multispecific binding site including: - A PD-1 binding domain as described herein, comprising a heavy chain variable region having the amino acid sequence shown in Sequence 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 shown in Sequence 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; Here, the PD-1 binding domain and the TGF-βRII binding domain include a light chain variable region having the amino acid sequence shown 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 one embodiment, each of the heavy chain variable region and the light chain variable region includes an HCDR and an LCDR that do not contain amino acid variations, respectively. In another embodiment, each of the heavy chain variable region and the light chain variable region does not contain amino acid variations.

[0141] In one embodiment, the present disclosure provides a multispecific binding site including: - A PD-1 binding domain as described herein, comprising a heavy chain variable region having the amino acid sequence shown in Sequence 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 shown in Sequence 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; Here, the PD-1 binding domain and the TGF-βRII binding domain include a light chain variable region having the amino acid sequence shown 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 one embodiment, each of the heavy chain variable region and the light chain variable region includes an HCDR and an LCDR that do not contain amino acid variations, respectively. In another embodiment, each of the heavy chain variable region and the light chain variable region does not contain amino acid variations.

[0142] Further provided herein are nucleic acids useful for producing the multispecific binding sites of the Disclosure. In some embodiments, such nucleic acids include a nucleic acid sequence 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 nucleic acids of the Disclosure may further include nucleic acid sequences encoding the CH1 region and, preferably, the hinge, CH2, and CH3 regions. In some embodiments, the nucleic acids of the Disclosure may further include at least one nucleic acid sequence encoding the light chain variable region and, preferably, the CL region. In some embodiments, the light chain variable region may be a common light chain variable region described herein.

[0143] Further provided herein are vectors comprising nucleic acids of the Disclosure useful for producing the multispecific binding sites of the Disclosure. In some embodiments, such a vector comprises a nucleic acid sequence encoding a heavy chain variable region of a PD-1 binding domain as described herein and a nucleic acid sequence encoding a heavy chain variable region of a TGF-βRII binding domain as described herein. In some embodiments, the vector of the Disclosure may further comprise nucleic acid sequences encoding a CH1 region and, preferably, a hinge, CH2, and CH3 region. In some embodiments, the vector of the 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 a common light chain variable region as described herein.

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

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

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

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

[0148] In one embodiment, the present disclosure provides a multispecific binding site described herein and a pharmaceutical composition described herein for therapeutic use.

[0149] In one embodiment, this disclosure provides a multispecific binding site described herein, or a pharmaceutical composition described herein, for use in the treatment of cancer.

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

[0151] In one embodiment, the present disclosure provides a method for treating cancer, comprising the step of administering an effective amount of a multispecific binding site described herein, or a pharmaceutical composition described herein, to an individual in need.

[0152] As used herein, the terms “individual,” “subject,” and “patient” are interchangeable and refer to mammals such as humans, mice, rats, hamsters, guinea pigs, rabbits, cats, dogs, monkeys, cattle, horses, and pigs, specifically 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 for the purpose of curing or improving a disease or its symptoms, or for the purpose of producing a positive therapeutic response, or the administration of an active agent or combination of active agents to a subject. As used herein, “positive therapeutic response” refers to a treatment that produces a beneficial effect, such as an effect of reversing, alleviating, improving, inhibiting, or slowing disease-related symptoms, complications, conditions, or biochemical signs, or an effect of preventing the onset, progression, advancement, worsening, or recurrence of disease-related symptoms, complications, conditions, or biochemical signs, such as an improvement in at least one symptom of a disease or disorder, such as cancer. The beneficial effect may take the form of an improvement over baseline and includes an improvement over measurements or observations made before the commencement of treatment according to this method. For example, beneficial effects can take the form of slowing, stabilizing, stopping, 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 markers of cancer. Effective treatments may include, 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 inhibition of tumor growth, and / or the prevention or delay of tumor recurrence or relapse.

[0154] The terms "therapeutic amount" or "effective amount" refer to the quantity of a drug or combination of drugs used to treat a disease such as cancer. In some embodiments, the therapeutic amount is sufficient to slow the progression of a tumor. In some embodiments, the therapeutic amount is sufficient to prevent or delay the recurrence of a tumor.

[0155] An effective amount of a drug or composition used herein may, for example, do the following: (i) reduce the number of cancer cells; (ii) reduce the size of a tumor; (iii) inhibit, interfere with, to some extent delay and halt the invasion of cancer cells into peripheral organs; (iv) inhibit tumor metastasis; (v) inhibit tumor growth; (vi) prevent or delay the onset and / or recurrence of a tumor; and / or (vii) to some extent eliminate one or more symptoms associated with cancer.

[0156] The effective dose may vary depending on factors such as the disease state, age, sex, and weight of the individual being treated, and the ability of the drug or combination of drugs to induce the desired response in the individual, which can be easily assessed by a physician or other healthcare professional with the usual skills.

[0157] The effective dose can be administered to the subject in one or more doses.

[0158] The effective dose may also include the amount that balances any toxic or harmful effects of the drug or combination of drugs with the beneficial effects.

[0159] The term "agent" refers to a therapeutically active substance, and in this case, to the multispecific binding sites of the Disclosure or the pharmaceutical compositions of the Disclosure.

[0160] As used herein, "to comprise" and its conjugations are used in a non-restrictive sense, meaning that the item following the word is included, but not excluded from items not specifically mentioned.

[0161] The articles "a" and "an" are used herein to refer to one or more grammatical objects of the article. For example, "an element" means one or more elements.

[0162] References to patent documents or other matters in the present specification shall not be construed as an admission that such documents or matters were publicly known, or that the information contained therein forms part of the common general knowledge as of the priority date of any of the claims.

[0163] All patents and references cited in the present specification are hereby incorporated by reference in their entirety into the present specification.

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

[0165] Accession numbers are provided primarily to offer an additional method of identifying a target; the actual sequence of the binding protein may vary, for example, due to mutations in the coding gene as occurs in some cancers. The antigen-binding sites of the multispecific binding sites of the present disclosure are capable of binding antigens and various variants thereof, such as those expressed by certain antigen-positive immune cells or tumor cells. HGNC is the abbreviation for HUGO Gene Nomenclature Committee. The number following the abbreviation is an accession number, with which information about the gene and the protein encoded by the gene can be retrieved from the HGNC database. Entrez Gene provides an accession number or gene ID, with 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 an accession number, with which information about the gene or the protein encoded by the gene can be obtained from the Ensembl database. Ensembl is a joint project of EMBL-EBI and the Wellcome Trust Sanger Institute, which develops software systems for generating and maintaining automatic annotations for selected eukaryotic genomes.

[0166] Where reference is made herein to a gene or protein, reference is preferably made to the human form of said gene or protein. Where reference is made herein to a gene or protein, reference is made to both the native gene or protein and variant forms of the gene or protein that can be detected in tumors, cancers and the like, preferably that can be detected in human tumors, cancers and the like.

[0167] [Clause] 1. A multispecific binding site comprising a PD-1 binding domain and a TGF-βRII binding domain, wherein The aforementioned PD-1 binding domain inhibits PD-1-mediated signaling, and The TGF-βRII binding domain inhibits TGF-βRII-mediated signaling. Multispecific binding site.

[0168] 2. The multiple specific binding sites described in Clause 1, In activated T cells, The aforementioned PD-1 binding domain inhibits PD-1-mediated signaling, and The TGF-βRII binding domain inhibits TGF-βRII-mediated signaling. Multispecific binding site.

[0169] 3. A multispecific binding site as described in Clause 1 or 2, In activated tumor-specific T cells, The aforementioned PD-1 binding domain inhibits PD-1-mediated signaling, and The TGF-βRII binding domain inhibits TGF-βRII-mediated signaling. 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 described in 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 described in any one of the preceding clauses, The multispecific binding site exhibits higher efficacy 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 at all, substantially not at all, or expressing 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 exhibits higher efficacy 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 at all, substantially not at all, or expressing 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 aforementioned multispecific binding site exhibits higher activity than the reference antibody combination when reducing tumor volume. The aforementioned combination of reference antibodies consists of 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 shown in SEQ ID NO: 78 and a light chain having the amino acid sequence shown in SEQ ID NO: 79. and The bivalent monospecific antibody targeting TGF-βRII comprises a heavy chain having the amino acid sequence shown in SEQ ID NO: 76 and a light chain having the amino acid sequence shown in SEQ ID NO: 77. Multispecific binding site.

[0175] 9. A multispecific binding site as described in Clause 8, Said activity in reducing tumor volume is determined by measuring reduction in tumor volume in an in vivo mouse test, specifically in an in vivo mouse test using MDA-MB-231 xenograft huCD34 NSG mice, Multispecific binding site.

[0176] 10. The multispecific antibody or a variant thereof according to clause 8 or 9, wherein higher activity in reducing tumor volume is 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 achieved by the combination of reference antibodies, Multispecific binding site.

[0177] 11. The multispecific binding site according to any one of the preceding clauses, said cell expressing both PD-1 and TGF-βRII is an activated T cell, Multispecific binding site.

[0178] 12. The multispecific binding site according to any one of the preceding clauses, said cell expressing both PD-1 and TGF-βRII is an activated tumor-specific T cell, Multispecific binding site.

[0179] 13. The multispecific binding site according to any one of the preceding clauses, said cell expressing both PD-1 and TGF-βRII is Jurkat-PD-1 + cells, and and said cell that expresses TGF-βRII and expresses no or substantially no PD-1 is Jurkat-PD-1 null cells, Multispecific binding site.

[0180] 14. The multispecific binding site according to any one of the preceding clauses, The cells expressing both PD-1 and TGF-βRII are activated CD4 + and / or CD8 + It is a cell, and Cells expressing TGF-βRII and not expressing PD-1 at all, substantially not expressing it, or expressing low levels of PD-1 are inactivated CD4 + and / or CD8 + Cells Multispecific binding site.

[0181] 15. A multispecific binding site described in any one of the preceding clauses, The cells expressing both PD-1 and TGF-βRII are HEK-Blue TGF-β-PD-1 + It is a cell, and Cells that express TGF-βRII and do not express PD-1 at all, substantially do not express it, or express it at a low level are HEK-Blue TGF-β cells. Multispecific binding site.

[0182] 16. A multispecific binding site described in 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 described in 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 as described in any one of the preceding clauses, In cells expressing both PD-1 and TGF-βRII, the efficacy in inhibiting TGF-βRII-mediated signaling is at least about 200 times, or 500 times, or 1,000 times, or 5,000 times, or 10,000 times, or 15,000 times, or 20,000 times, or 30,000 times, or about 200-30,000 times, or 500-30,000 times, or 1,000-30,000 times, or 5,000-30,000 times, or 10,000-30,000 times, or 200-20,000 times, or 200-15,000 times, compared to cells expressing TGF-βRII and expressing PD-1 not at all, substantially, or at low levels of PD-1. Multispecific binding site.

[0185] 19. A multispecific binding site described in any one of the preceding clauses, In cells expressing TGF-βRII but not expressing PD-1 at all, the efficacy of the multispecific binding site in inhibiting TGF-βRII-mediated signaling is lower than that of the reference anti-TGF-βRII antibody. and In cells expressing both PD-1 and TGF-βRII, the efficacy of the multispecific binding site in inhibiting TGF-βRII-mediated signaling is higher than that of the reference anti-TGF-βRII antibody. The aforementioned reference anti-TGF-βRII antibody is a bivalent monospecific antibody comprising a heavy chain having the amino acid sequence shown in SEQ ID NO: 76 and a light chain having the amino acid sequence shown in SEQ ID NO: 77. Multispecific binding site.

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

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

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

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

[0190] 24. A multispecific binding site described in any one of the preceding clauses, The PD-1 binding domain and the TGF-βRII binding domain each include a light chain containing a light chain variable region, which is capable of pairing with multiple heavy chains having different epitope specificities. Multispecific binding site.

[0191] 25. A multispecific binding site described in 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 described in any one of the preceding clauses, The PD-1 binding domain includes a heavy chain variable region comprising: a) Heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), each having the amino acid sequences shown in SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4, respectively; b) Heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), each having the amino acid sequences shown in SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8, respectively; c) Heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), each having the amino acid sequences shown in SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12, respectively; d) Heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), having the amino acid sequences shown 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 shown in SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 22, respectively; Here, each of the HCDRs may contain at most three, two, or one amino acid variation; Multispecific binding site.

[0193] 27. A multispecific binding site described in any one of the preceding clauses, The TGF-βRII binding domain includes a heavy chain variable region comprising: a) Heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), having the amino acid sequences shown in SEQ ID NO: 24, SEQ ID NO: 25, and SEQ ID NO: 26, respectively; b) Heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), having the amino acid sequences shown in SEQ ID NO: 28, SEQ ID NO: 29, and SEQ ID NO: 30, respectively; c) Heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), having the amino acid sequences shown 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 shown 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 shown in SEQ ID NO: 40, SEQ ID NO: 41, and SEQ ID NO: 42, respectively; f) Heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), having the amino acid sequences shown in SEQ ID NOs. 44, 45, and 46, respectively; or g) Heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), each having the amino acid sequences shown in SEQ ID NO: 90, SEQ ID NO: 91, and SEQ ID NO: 92, respectively; Here, each of the HCDRs 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 includes a heavy chain variable region comprising: a) Heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), each having the amino acid sequences shown in SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4, respectively; b) Heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), each having the amino acid sequences shown in SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8, respectively; c) Heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), each having the amino acid sequences shown in SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12, respectively; d) Heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), having the amino acid sequences shown 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 shown in SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 22, respectively; Here, each of the HCDRs 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 includes a heavy chain variable region comprising: a) Heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), having the amino acid sequences shown in SEQ ID NO: 24, SEQ ID NO: 25, and SEQ ID NO: 26, respectively; b) Heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), having the amino acid sequences shown in SEQ ID NO: 28, SEQ ID NO: 29, and SEQ ID NO: 30, respectively; c) Heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), having the amino acid sequences shown 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 shown 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 shown in SEQ ID NO: 40, SEQ ID NO: 41, and SEQ ID NO: 42, respectively; f) Heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), having the amino acid sequences shown in SEQ ID NOs. 44, 45, and 46, respectively; or g) Heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), each having the amino acid sequences shown in SEQ ID NO: 90, SEQ ID NO: 91, and SEQ ID NO: 92, respectively; Here, each of the HCDRs may contain at most three, two, or one amino acid variation; Multispecific binding site.

[0196] 30. A multispecific binding site described in any one of the preceding clauses, The PD-1 binding domain includes a heavy chain variable region having an amino acid sequence shown 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 to these sequences. Multispecific binding site.

[0197] 31. A multispecific binding site described in any one of the preceding clauses, The PD-1 binding domain includes a light chain variable region comprising light chain CDR1 (LCDR1), light chain CDR2 (LCDR2), and light chain CDR3 (LCDR3), each having the amino acid sequences shown in SEQ ID NOs. 49, SEQ ID NOs. 50, and SEQ ID NOs. 51, respectively; Here, each of the LDCRs may contain at most three, two, or one amino acid variation; Multispecific binding site.

[0198] 32. A multispecific binding site described in any one of the preceding clauses, The PD-1 binding domain includes a light chain variable region having the amino acid sequence shown 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 described in any one of the preceding clauses, The TGF-βRII binding domain includes a heavy chain variable region comprising: a) Heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), having the amino acid sequences shown in SEQ ID NO: 24, SEQ ID NO: 25, and SEQ ID NO: 26, respectively; b) Heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), having the amino acid sequences shown in SEQ ID NO: 28, SEQ ID NO: 29, and SEQ ID NO: 30, respectively; c) Heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), having the amino acid sequences shown 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 shown 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 shown in SEQ ID NO: 40, SEQ ID NO: 41, and SEQ ID NO: 42, respectively; f) Heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), having the amino acid sequences shown in SEQ ID NOs. 44, 45, and 46, respectively; or g) Heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), each having the amino acid sequences shown in SEQ ID NO: 90, SEQ ID NO: 91, and SEQ ID NO: 92, respectively; Here, each of the HCDRs may contain at most three, two, or one amino acid variation; Multispecific binding site.

[0200] 34. A multispecific binding site described in any one of the preceding clauses, The TGF-βRII binding domain includes a heavy chain variable region having an amino acid sequence represented by 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 to these sequences. Multispecific binding site.

[0201] 35. A multispecific binding site described in any one of the preceding clauses, The TGF-βRII binding domain includes a light chain variable region comprising light chain CDR1 (LCDR1), light chain CDR2 (LCDR2), and light chain CDR3 (LCDR3), each having the amino acid sequences shown in SEQ ID NOs. 49, SEQ ID NOs. 50, and SEQ ID NOs. 51, respectively; Here, each of the LDCRs may contain at most three, two, or one amino acid variation; Multispecific binding site.

[0202] 36. A multispecific binding site described in any one of the preceding clauses, The TGF-βRII binding domain includes a light chain variable region having the amino acid sequence shown 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, With respect to binding to PD-1 and / or TGF-βRII, the multiple specific binding sites described in any one of the preceding clauses compete with the following: Multispecific binding site.

[0204] 38. A compound comprising an effective amount of a multispecific binding site as described in any one of the preceding clauses and a pharmaceutically acceptable carrier, Pharmaceutical composition.

[0205] 39. A multispecific binding site as described in any one of Clauses 1 to 37, or a pharmaceutical composition as described in Clause 38, for use in therapeutic purposes.

[0206] 40. A multispecific binding site as described in any one of Clauses 1 to 37, or a pharmaceutical composition as described in Clause 38, for use in the treatment of diseases associated with a suppressed immune system.

[0207] 41. A multispecific binding site as described in any one of Clauses 1 to 37, or a pharmaceutical composition as described in Clause 38, for use in the treatment of cancer.

[0208] 42. Use of a multispecific binding site as described in any one of Clauses 1 to 37, or a pharmaceutical composition as described in Clause 38, for the manufacture of a drug used for the treatment of a disease associated with a suppressed immune system.

[0209] 43. Use of a multispecific binding site as described in any one of Clauses 1 to 37, or a pharmaceutical composition as described in Clause 38, for the manufacture of a drug used for the treatment of cancer.

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

[0211] 45. Methods for treating diseases associated with a suppressed immune system, The procedure includes administering an effective amount of a multispecific binding site described in any one of clauses 1 to 37, or a pharmaceutical composition described in clause 38, to a human subject in need. method.

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

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

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

[0215] 49. Cells described in Article 47 or 48, The cell further comprises at least one nucleic acid sequence encoding a light chain variable region, specifically a light chain variable region as defined in clause 34 or 35, and preferably a CL region. cell.

[0216] 50. Cells that produce a multispecific binding site as described in any one of clauses 1 to 37.

[0217] [Brief description of the drawing] The following nomenclature rules are used in this specification. In the figures, bivalent monospecific antibodies are indicated in the format SEQ ID NO: A / SEQ ID NO: B, where SEQ ID NO: A refers to the heavy chain of the bibinding domain and SEQ ID NO: B refers to the light chain of the bibinding domain.

[0218] Bivalent bispecific antibodies are indicated in the format SEQ ID NO: A × SEQ ID NO: B, where both SEQ ID NOs A and B refer to heavy chain variable sequences. Each binding domain of a bispecific antibody contains the same light chain.

[0219] The bivalent monospecificity reference antibodies pembrolizumab, nivolumab, and TGF1 analogs are indicated 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 analogs is indicated 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 the TGF1 analog, SEQ ID NO: B refers to the light chain sequence of either pembrolizumab or the TGF1 analog, SEQ ID NO: C refers to the heavy chain sequence of the other, and SEQ ID NO: D refers to the light chain sequence of the other.

[0220] The reference PD-L1-TGF-β TRAP molecular analog is an analog of Bintrafusp alfa and is indicated in the format SEQ ID NO: A / SEQ ID NO: B, where SEQ ID NO: A refers to the heavy chain sequence containing 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 molecular analog contains two PD-L1 binding domains and two TGF-βRII extracellular domains. [Examples]

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

[0222] The reference antibody and molecules, and control antibody used in the examples include: - The reference PD-1 antibody pembrolizumab analog is a bivalent, monospecific antibody containing two heavy chains having the amino acid sequence shown in SEQ ID NO: 78 and two light chains having the amino acid sequence shown in SEQ ID NO: 79. - Reference PD-1 antibody pembrolizumab (Merck, distributed by Myonex). - The reference TGF-βRII antibody TGF1 is a bivalent, monospecific analog of TGF1, comprising two heavy chains having the amino acid sequence shown in SEQ ID NO: 76 and two light chains having the amino acid sequence shown 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, comprising two heavy chains having the amino acid sequence shown in SEQ ID NO: 80 and two light chains having the amino acid sequence shown in SEQ ID NO: 81, with the extracellular domain of TGF-βRII having the amino acid sequence shown 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 containing two heavy chains having the amino acid sequence shown in SEQ ID NO: 86 and two light chains having the amino acid sequence shown in SEQ ID NO: 87. - The control TGF-βRII×RSV antibody is a bivalent, bispecific antibody comprising: a TGF-βRII binding domain containing the heavy chain variable region amino acid sequence shown in SEQ ID NOs. 23, 31, 39, 27, 35, or 43; an RSV binding domain containing the heavy chain variable region amino acid sequence shown in SEQ ID NOs. 86; and a common light chain containing the light chain variable region amino acid sequence shown in SEQ ID NOs. 48 and the light chain constant region amino acid sequence shown in SEQ ID NOs. 75. - Positive control LILRB2 (BioLegend; catalog number 338714). - A commercially available reference PD-1 antibody, Opdivo (manufactured by Bristol Myers Squibb (BMS)).

[0223] [Example 1 - Preparation of PD-1 × TGF-βRII bispecific antibody] Binding domains, antibodies, and heavy chain variable regions with binding specificity to human PD-1, and heavy chain variable regions with binding specificity to human TGF-βRII, were obtained by immunizing transgenic mice (MeMo® mice) containing a common IGKV1-39 light chain with human PD-1 or TGF-βRII antigen sites (including the use of various forms of DNA, protein, and cell-based antigen delivery).

[0224] A heavy chain variable region having binding specificity to human PD-1 with the amino acid sequences shown in SEQ ID NOs: 1;5;9;13;14;18; and 19, and a heavy chain variable region having binding specificity to human TGF-βRII with the amino acid sequences shown in SEQ ID NOs: 23;27;31;35;39;43;47;88; and 89, were selected for the production of bispecific antibodies. Once the binding domain sequences described herein are characterized and sequenced through the techniques provided herein, they can then be obtained by any method known in the art.

[0225] JPEG2026143542000001.jpg113166

[0226] Bispecific IgG antibodies were prepared by transiently co-transfecting 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. The CH3 engineering techniques described in WO2013 / 157954 and WO2013 / 157953 were employed to ensure efficient heterodimerization and the formation of bispecific antibodies. Both vectors further encode a common light chain containing the IGKV1-39 / Jk1 light chain variable region. Cell transfection, cell culture, and antibody collection and purification were performed by methods known in the art.

[0227] [Example 2 - PD-1-SHP Recruitment Assay] 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: U20S 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 receptor (EA)-fused SHP1. Activation of Jurkat T cells with ligand or agonist antibody-induced PD-1 recruits SHP1, leading to interaction between ED and EA and reconstitution of the active β-gal enzyme. The reconstituted β-gal generates a chemiluminescent signal in the presence of a substrate.

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

[0229] U2OS / PD-L1 cells (DiscoveRx Corporation) were maintained in McCoy's 5A medium (Thermo Fisher Scientific) supplemented with 10% FBS + 0.25 μg / ml puromycin (Thermo Fisher Scientific). Jurkat-PD-1-SHP cells (Src homologous region 2-domain phosphatase; DiscoveRx Corporation) were cultured in RPMI1640 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 cells and Jurkat-PD-1-SHP cells were first centrifuged in conical tubes to remove the culture medium, then washed, and resuspended in assay medium (RPMI1640 medium with 1% FBS) before cell plating. U2OS / PD-L1 cells were added to 384-well black transparent-bottom assay plates (CELLCOAT® Tissue Culture Plates, Greiner Bio-One) at a rate of 5000 cells per well in 20 μL of assay medium. Antibody samples were prepared by serial dilution in phosphate-buffered saline (PBS) containing 1% FBS, and 5 μL / well was transferred to the cell plates and incubated at 37°C and 5% CO2 for 1 hour. Subsequently, Jurkat-PD-1-SHP cells were added to the cell plates at a rate of 5000 cells per well in 20 μL of assay medium and incubated at 37°C and 5% CO2 for 2 hours, after which 2.5 μL of PathHunter Reagent 1 (DiscoveRx Corporation) was added to each well. The assay plate was then shaken at 350 rpm for 1 minute, kept in the dark at room temperature for 15 minutes, and then 10 μL of PathHunter Reagent 2 (DiscoveRx Corporation) was added. The chemiluminescence signal was recorded using a TopCount reader (Perkin Elmer) after incubation at room temperature for 1 hour.Wells containing only PBS were used as positive controls, and wells without cells were used as negative controls. IC. 50 The determination was made by fitting a logarithmic curve of control activity percentage versus 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 equivalent in potency to a bivalent monospecific reference PD-1 antibody pembrolizumab analog and a bivalent reference PD-L1-TGF-β TRAP molecule for PD-L1 binding in the PD-1-SHP recruitment assay.

[0231] JPEG2026143542000002.jpg62166

[0232] [Example 3 - PD-1-NFAT Reporter Assay] The bispecific antibodies were characterized using 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 is used to inhibit PD-L1 co-expressing TCR homologous proteins. + aAPC / CHO-K1 cells and PD-1 that drives a luciferase reporter under the control of the NFAT-RE cis element + The system includes two transgenic cell lines, one containing effector Jurkat T cells. 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 enables TCR signaling, thereby inducing detectable luminescence upon substrate addition.

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

[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 (activated T cell nuclear factor; Promega) were cultured in RPMI1640 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 384-well white clear-bottom assay plates (CELLCOAT® Tissue Culture Plates, Greiner Bio-One) at a rate of 8000 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 / well was transferred to the cell plates. Subsequently, Jurkat-PD-1-NFAT effector cells were dispensed into each well at a rate of 10000 cells per well in 5 μL of assay medium. The assay plates were incubated at 37°C and 5% CO2 for 24 hours. The assay plate was equilibrated at room temperature for 15 minutes, after which 20 μL / well of Bio-Glo® reagent (Promega) was added. After incubation at room temperature for 8 minutes, 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 ug / mL of pembrolizumab analog were used as positive controls (100% induction). EC. 50 The determination was made by fitting a logarithmic curve of control activity percentage versus 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 equivalent in potency to the bivalent monospecific reference PD-1 antibody pembrolizumab analog in the PD-1-NFAT reporter assay.

[0236] JPEG2026143542000003.jpg65166

[0237] [Example 4 - Jurkat Phosphorylated SMAD2 / 3 Assay] The bispecific antibodies were characterized in the Jurkat phosphorylated SMAD2 / 3 assay to determine their ability to inhibit TGF-βRII signaling in T cells. In the Jurkat phosphorylated SMAD2 / 3 assay, Jurkat-PD-1 null Cells and Jurkat-PD-1 + The activity of 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. The control antibodies included a reference TGF-βRII antibody, an analog of TGF1, a TGF-βRII × RSV bispecific antibody, and a negative control bivalent monospecific IgG1 antibody containing a heavy chain with the amino acid sequence shown in SEQ ID NO: 86 and a light chain with the amino acid sequence shown in SEQ ID NO: 87.

[0238] Jurkat-PD-1 in RPMI / 10% FBS null Cells (ATCC catalog number TIB-152) and Jurkat-PD-1 +Cells (Promega catalog number CS187105) were seeded in a 96-well flat-bottom plate. PD-1 expression was controlled by Jurkat-PD-1 null Not detected in cells; Jurkat-PD-1 + The number of PD-1 molecules in cells was determined to be approximately 4000 using the quantibrite bead method. Bispecific antibodies and control antibodies were added in six serial dilutions (100 μg / ml to 0.001 μg / ml), and the cells were incubated at 37°C / 5% CO2 for 1 hour. After 1 hour, human recombinant TGF-β1 (R&D Systems catalog no. 7754-BH) was added at a final concentration of 10 ng / ml, and the cells were incubated for a further 2 hours at 37°C / 5% CO2. After incubation, the cells were gently washed with PBS. Cell lysates were prepared using a lysis buffer (MSD# R60TX-2) containing phosphatase inhibitors (Sigma# P0044 and P-5726) and a 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 results for 10 bispecific antibodies against Jurkat-PD-1 null Cell vs. Jurkat-PD-1 + This assay demonstrates the difference in potency when inhibiting TGF-βRII signaling in cells. In this assay, the potency difference of the bispecific antibodies is 200 to 11,000 times higher than that of the reference antibody TGF1 analog.

[0240] All bispecific antibodies are Jurkat-PD-1 null Cells and Jurkat-PD-1 +In both cells, TGF-βRII signaling was inhibited. The bispecific antibody was Jurkat-PD-1 + Jurkat-PD-1 inhibits TGF-βRII signaling in cells. null They exhibit higher efficacy in cells than in cells, and have been shown to inhibit TGF-β-induced SMAD2 / 3 phosphorylation in a manner correlated with PD-1 expression. All bispecific antibodies are Jurkat-PD-1 null To inhibit TGF-βRII signaling in cells, a higher concentration is required than that of a reference TGF-βRII antibody or TGF1 analog. Many bispecific antibodies that are monovalent in binding to TGF-βRII are Jurkat-PD-1 + It is superior to a bivalent monospecific analog of the reference TGF-βRII antibody TGF1 in inhibiting TGF-βRII signaling in cells.

[0241] JPEG2026143542000004.jpg70166

[0242] JPEG2026143542000005.jpg69166

[0243] JPEG2026143542000006.jpg122166

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

[0245] PBMCs from healthy donors were stimulated for 48 hours with 1 μg / ml anti-CD3 antibody (BD Biosciences # 555336), followed by 16 hours of serum depletion (0.1% FBS). Activated CD4 + and CD8 + The number of PD-1 molecules in T cells was determined to be approximately 1000–2000 using the quantibrite bead method. Subsequently, stimulated and unstimulated PBMCs were incubated with bispecific and control antibodies at room temperature for 30 minutes. Recombinant human TGF-β1 (Cell Signaling #7754-BH) was added at a final concentration of 1 ng / ml, and the cells were incubated for a further 30 minutes. Finally, the cells were washed twice with PBS, stained for cell surface markers, and subsequently stained for intracellular phosphorylated SMAD2.

[0246] For staining for flow cytometry, the following antibodies were used: 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 FACSymphony A3 using DIVA software.

[0247] PBMCs were gated based on size and particle size using FSC-A versus SSC-A, and debris was excluded. Dead cells were then excluded using fixable dead cell staining dyes. CD45-positive cells were selected, followed by CD11b-negative and CD3-positive T cells. Finally, CD4 and CD8-positive subsets were gated, and phosphorylated SMAD2 signals were measured by 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 were stimulated by CD4 + and CD8 + In T cells, TGF-βRII signaling was inhibited. In the other donor (Donor B), the bispecific antibody inhibited unstimulated CD4 + and CD8 +In T cells, it did not inhibit TGF-βRII signaling. In the other donor, a bispecific antibody containing the PD-1 heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 18 and the TGF-βRII heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 23 was unstimulated in CD4 + and CD8 + In T cells, a bispecific antibody that does not inhibit TGF-βRII signaling and contains a PD-1 heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 13 and a TGF-βRII heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 47 is unstimulated CD4 + and CD8 + In T cells, stimulated CD4 + T and CD8 + The antibody inhibited TGF-βRII signaling with significantly lower efficacy than in T cells. These data confirm the findings of Example 4, which indicated that the bispecific antibody inhibited TGF-β-induced SMAD phosphorylation in a manner correlated with PD-1 expression.

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

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

[0251] Human PBMCs were isolated from healthy donors. In vitro T cell exhaustion was performed by repeating activation of PBMCs with Staphylococcus 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 in a 10:1 T-to-DC ratio in 96-well U-bottom plates. Serial dilutions of antibody samples were added, and the plates were incubated at 37°C / 5% CO2 for a further 6 days. After 6 days, IFN-γ, IL-2, or TNF-α cytokine levels in the supernatant were measured using a custom MSD kit. GraphPad Prism (8.2.0) was used to plot graphs.

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

[0253] JPEG2026143542000007.jpg99166

[0254] [Example 7 - HEK-BLUE-PD-1 TGF-β Reporter Assay] The bispecific antibodies were characterized using the HEK-BLUE-PD-1 TGF-β reporter assay to determine their efficacy in inhibiting TGF-β-induced signaling in T cells. (HEK-Blue® TGF-β cells or HEK-Blue® TGF-β-PD-1) +When cells are stimulated with TGF-β, activation of the TGF-β / Smad signaling pathway is induced, leading to the formation of the Smad3 / Smad4 complex, which induces SEAP production.

[0255] Reagents used: Recombinant human TGF-beta-1 (human cell expression) 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; 30 min at 56°C), 100 μg / ml Normocin®, Pen-Strep (100 U / ml-100 μg / ml). HEK-Blue® TGF-β-PD-1 + Cell growth medium containing 0.4 ug / mL puromycin. Test medium: DMEM 4.5 g / l glucose, 2 mM L-glutamine, 0.1% thermoactivated FBS, Pen-Strep (100 U / ml-100 μg / ml). Does not contain Normocin®, blastocydin, hygromycin B, or Zeocin®.

[0256] Stable PD-1-expressing HEK-Blue® TGF-β cell lines were constructed as follows: Full-length PD-1 cds were inserted into the mammalian expression vector pD2529-EFM (ATUM), which contains the puromycin resistance gene. The promoter was modified EF1a. Vector construction was performed using ATUM and sequence verification was confirmed. See Figure 10 for the vector map. HEK-Blue® TGF-β cells (Invivogen) in which PD-1 expression was not detected were transfected using TransIT-293 transfection reagent (Mirus Bio) according to the manufacturer's protocol. Stable transfected cells were selected in HEK-Blue® TGF-β medium containing 0.4 ug / ml puromycin. Clones were isolated by limiting dilution and characterized for PD-1 expression by Western blotting. One clone was selected based on the following criteria: 1) its stable and uniform surface PD-1 expression (one peak in the histogram plot); 2) similar surface TGF-βRII expression compared to the parental HEK-Blue® cell line; and 3) 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 in the parental cell line and 8 in 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] The antibody sample included several bispecific antibodies, a negative control IgG1 antibody (RSV), a reference PD-1 antibody pembrolizumab, and a reference TGF-βRII antibody TGF1 analog.

[0258] HEK-Blue® TGF-β cells expressing TGF-βRII (Invivogen, catalog code: hkb-tgfb), or HEK-Blue® TGF-β-PD-1 +Cells were seeded in test medium at a rate of 25,000 cells per well in a 96-well flat-bottom plate. Serial dilutions of the antibody sample were added, and the cells were incubated at room temperature for 1 hour; subsequently, recombinant human TGF-β1 was added at 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 into 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 exhibit potent inhibitory activity against TGF-β-induced signaling in a manner correlated with PD-1 expression.

[0260] JPEG2026143542000008.jpg111166

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

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

[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. The Tregs were mixed with PBMCs from the same donor. Anti-CD3 ab (BD Biosciences, #555336) and anti-CD28 an (BD Biosciences, #555725) were added to the co-culture. 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 supernatant were measured using the 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 reference PD-1 antibody pembrolizumab, or a combination of reference PD-1 antibody pembrolizumab and reference TGF-βRII antibody TGF1 analog.

[0265] JPEG2026143542000009.jpg93166

[0266] [Example 9 - Macrophage Suppression Assay] Tumor-associated macrophages with 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] Bispecific antibodies were tested with negative control IgG1 antibody (RSV), reference TGF-βRII antibody TGF1 analog, reference PD-L1-TGF-β TRAP molecule analog, reference PD-1 antibody nivolumab analog, commercially available reference PD-1 antibody Opdivo (BMS), positive control antibody anti-LILRB2 (Biolegend), rat IgG2a isotype control (Biolegend), and huIgG4 isotype control (Biolegend). In addition, stimulated CD4 without test antibody or control antibody was tested. + Co-culture of T cells and M2 macrophages, or CD4 + T cells alone (non-stimulated vs. stimulated) were included as a control condition in the assay.

[0268] PBMC-isolated monocytes from three different healthy donors were differentiated into macrophages for 6 days using M-CSF and polarized using a specificity 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, the expression of CD163 (Miltenye Biotec), CD209 (Miltenye Biotec), CD206 (BD Bioscience), and CD86 (Miltenye Biotec) was measured by flow cytometry (Figure 8). CD163 was expressed by Ms-like macrophages in all donors. CD209 and CD206 were highly expressed by M2 macrophages in all donors. CD86 was expressed at low levels by M2-like macrophages. The M2-like macrophages exhibited the expected phenotype in all three donors.

[0269] Next, M2 macrophages were activated with LPS (100 ng / ml) for 4 hours. The macrophages were collected, washed, and placed in a 96-well plate with autologous CD4 +Activated T cells (activated with CD3 / CD28 ImmunoCult™ from StemCell Technologies) were seeded in a 1:5 ratio at a concentration of 10 μg / ml in the presence of a test antibody or control antibody, in 5 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) on 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, the reference TGF-βRII antibody TGF1, or the reference PD-L1-TGF-β TRAP molecule.

[0271] [Example 10 - In vivo humanized NSG MDA-MB-231 mouse model] Bispecific antibodies were characterized in vivo in a humanized NSG MDA-MB-231 mouse model to determine their efficacy in reducing tumor volume. This mouse model was validated using a negative control bivalent monospecific IgG1 antibody (10 mg / kg) containing a heavy chain with the amino acid sequence shown in SEQ ID NO: 86 and a light chain with the amino acid sequence shown 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 suspended in equal volumes in 100 μl of serum-free culture medium and Matrigel matrix (Corning) for a total of 3 × 10⁶ mice. 6 MDA-MB-231 tumor cells were subcutaneously inoculated. After the tumor was established (80-100 mm)3 ), the 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 shown in SEQ ID NO: 86 and a light chain having the amino acid sequence shown in SEQ ID NO: 87; 2) Reference TGF-βRII antibody, analog of TGF1 (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 with the amino acid sequence shown in SEQ ID NO: 43, and a PD-1 binding domain having a heavy chain variable region with the amino acid sequence shown in SEQ ID NO: 9; 6) A bispecific antibody (10 mg / kg) comprising a TGF-βRII binding domain having a heavy chain variable region with the amino acid sequence shown in SEQ ID NO: 43, and a PD-1 binding domain having a heavy chain variable region with the amino acid sequence shown in SEQ ID NO: 9; 7) A bispecific antibody (1 mg / kg) comprising a TGF-βRII binding domain having a heavy chain variable region with the amino acid sequence shown in SEQ ID NO: 43, and a PD-1 binding domain having a heavy chain variable region with the amino acid sequence shown in SEQ ID NO: 19; 8) A bispecific antibody (10 mg / kg) comprising a TGF-βRII binding domain having a heavy chain variable region with the amino acid sequence shown in SEQ ID NO: 43, and a PD-1 binding domain having a heavy chain variable region with the amino acid sequence shown in SEQ ID NO: 19; 9) A bispecific antibody (1 mg / kg) comprising a TGF-βRII binding domain having a heavy chain variable region with the amino acid sequence shown in SEQ ID NO: 23, and a PD-1 binding domain having a heavy chain variable region with the amino acid sequence shown in SEQ ID NO: 14; 10) A bispecific antibody (10 mg / kg) comprising a TGF-βRII binding domain having a heavy chain variable region with the amino acid sequence shown in SEQ ID NO: 23, and a PD-1 binding domain having a heavy chain variable region with the amino acid sequence shown in SEQ ID NO: 14; 11) A bispecific antibody (1 mg / kg) comprising a TGF-βRII binding domain having a heavy chain variable region with the amino acid sequence shown in SEQ ID NO: 23, and a PD-1 binding domain having a heavy chain variable region with the amino acid sequence shown in SEQ ID NO: 19; 12) A bispecific antibody (10 mg / kg) comprising a TGF-βRII binding domain having a heavy chain variable region with the amino acid sequence shown in SEQ ID NO: 23, and a PD-1 binding domain having a heavy chain variable region with the amino acid sequence shown in SEQ ID NO: 19; 13) A bispecific antibody (10 mg / kg) comprising a TGF-βRII binding domain having a heavy chain variable region with the amino acid sequence shown in SEQ ID NO: 39, and a PD-1 binding domain having a heavy chain variable region with the amino acid sequence shown in SEQ ID NO: 9; 14) A bispecific antibody (10 mg / kg) comprising a TGF-βRII binding domain having a heavy chain variable region with the amino acid sequence shown in SEQ ID NO: 27, and a PD-1 binding domain having a heavy chain variable region with the amino acid sequence shown in SEQ ID NO: 9; 15) A bispecific antibody (10 mg / kg) comprising a TGF-βRII binding domain having a heavy chain variable region with the amino acid sequence shown in SEQ ID NO: 23, and a PD-1 binding domain having a heavy chain variable region with the amino acid sequence shown in SEQ ID NO: 18; 16) A bispecific antibody (10 mg / kg) comprising a TGF-βRII binding domain having a heavy chain variable region with the amino acid sequence shown in SEQ ID NO: 47, and a PD-1 binding domain having a heavy chain variable region with the amino acid sequence shown in SEQ ID NO: 13.

[0273] Each group consisted of 8-9 mice. The animals were administered intraperitoneally every 5 days for a period of 27 or 30 days. Tumor size was measured using a caliper, formula: l(length) × w 2Tumor volume was calculated by assimilating (width) × 1 / 2 into an ellipsoid. Body weight was also monitored throughout the study. After the study, tumors were collected (24 hours after the final dose) for tumor immunoprofiling and receptor occupancy.

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

[0275] Along with efficacy, the receptor coverage of both PD-1 and TGF-βRII receptors with bispecific antibody therapy was observed in T cells analyzed 24 hours after the final dose, similar to the combination therapy with a reference TGF-βRII antibody TGF1 analog and a 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 with the amino acid sequence shown in SEQ ID NO: 23 and a PD-1-binding domain having a heavy chain variable region with the amino acid sequence shown 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 with the amino acid sequence shown in SEQ ID NO: 86 and a light chain with the amino acid sequence shown in SEQ ID NO: 87 was included at 10 mg / kg.

[0277] The results are shown in Figure 11. The bispecific antibody induced a significant antitumor response at both dose levels.

[0278] [Sequence List] Sequence ID 1 - Heavy Chain Variable Region EVQLVQSGAEVKKPGSSMKVSCKASGGTFSSYVISWVRQAPGQGLEWMGMIIPVFDTSSYEKKFQGRITIIADKSTSTVYLELSSLRSEDAAVYYCARGTVEATLLFDFWGQGTLVTVSS

[0279] Sequence ID 2 - Heavy Chain CDR1 SYVIS

[0280] Sequence ID 3 - Heavy Chain CDR2 MIIPVFDTSSYEKKFQG

[0281] Sequence ID 4 - Heavy Chain CDR3 GTVEATLLFDF

[0282] Sequence ID 5 - Heavy Chain Variable Region QVQLQESGPGLVKPSETLSLTCTVSNGSLGFDFWSWIRQPPGRGLEWIGYIYYSGSWSLNPSFKGRVTMSVDTSKNQFSLNLRSVTAADTAVYYCARGGYTGYGGDWFDPWGQGTLVTVSS

[0283] Sequence ID 6 - Heavy Chain CDR1 FDFWS

[0284] Sequence ID 7 - Heavy Chain CDR2 YIYYSGSWSLNPSFKG

[0285] Sequence ID 8 - Heavy Chain CDR3 GGYTGYGGDWFDP

[0286] Sequence ID 9 - Heavy Chain Variable Region QVQLQESGPGLVKPSETLSLTTCTVSDGSIGYHFWSWIRQPPGRGLEWIGYIVYSGSYNVNPSLKTRVTMSVDTSKNQFSLNLRSVTAADTAVYYCARGGYTGYGGDWFDPWGQGTLVTVSS

[0287] Sequence ID 10 - Heavy Chain CDR1 YHFWS

[0288] Sequence ID 11 - Heavy Chain CDR2 YIVYSGSYNVNPSLKT

[0289] Sequence ID 12 - Heavy Chain CDR3 GGYTGYGGDWFDP

[0290] Sequence ID 13 - Heavy Chain Variable Region QVQLQESGPGLVKPSETLSLTTCTVSEGSIGYHFWSWIRQPPGRGLEWIGYIVYSGSYNVNPSLKTRVTMSVDTSKNQFSLNLRSVTAADTAVYYCARGGYTGYGGDWFDPWGQGTLVTVSS

[0291] Sequence ID 14 - Heavy Chain Variable Region QVQLVQSGSELKKPGASVKVSCKASGYTFTRFALHWVRQAPGQGLEWMGWIDPNTGTPTFAQGVTGRFVFSLDTSVTTAYLQISSLKAEDTAVYYCARSLGYCDSDICYPNWIFDNWGQGTLVTVSS

[0292] Sequence ID 15 - Heavy Chain CDR1 RFALH

[0293] Sequence ID 16 - Heavy Chain CDR2 WIDPNTGTPTFAQGVTG

[0294] Sequence ID 17 - Heavy Chain CDR3 SLGYCDSDICYPNWIFDN

[0295] Sequence ID 18 - Heavy Chain Variable Region QVQLVQSGSELKKPGASVKVSCKASGYTFTRFALHWVRQAPGQGLEWMGWIDPNTGTPTFAQGVTGRFVFSLDTSVTTAYLQISSLKAEDTAVYYCARSLGYCDSDICYPNWIFDNWGQGTLVTVSS

[0296] Sequence ID 19 - Heavy Chain Variable Region QVQLVQSGSELKKPGASVKVSCKASGYTFTRFALSWVRQAPGQGLEWMGWIDPNTGTPTYAQDFTGRFVFSLDTSVTTAYLQISSLKAEDTAVYYCARSLGYCGSDICYPNGILDNWGQGTLVTVSS

[0297] Sequence ID 20 - Heavy Chain CDR1 RFALS

[0298] Sequence ID 21 - Heavy Chain CDR2 WIDPNTGTPTYAQDFTG

[0299] Sequence ID 22 - Heavy Chain CDR3 SLGYCGSDICYPNGILDN

[0300] Sequence ID 23 - Heavy Chain Variable Region EVQLVESGGGLVQPGGSLRLSCAASGFTFDIYAMTWVRQAPGKGLEWVSVISGSGGTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARRGQYRDIVGATDYWGQGTLVTVSS

[0301] Sequence ID 24 - Heavy Chain CDR1 IYAMT

[0302] Sequence ID 25 - Heavy Chain CDR2 VISGSGGTTYYADSVKG

[0303] Sequence ID 26 - Heavy Chain CDR3 RGQYRDIVGATDY

[0304] Sequence ID 27 - Heavy Chain Variable Region EVQLVESGGGLVQPGGSLRLSCAASGFTFDINAMTWVRQAPGKGLEWVSVISGSGGTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARRGQYRDIVGATDYWGQGTLVTVSS

[0305] Sequence ID 28 - Heavy Chain CDR1 INAMT

[0306] Sequence ID 29 - Heavy Chain CDR2 VISGSGGTTYYADSVKG

[0307] Sequence ID 30 - Heavy Chain CDR3 RGQYRDIVGATDY

[0308] Sequence ID 31 - Heavy Chain Variable Region QVQLVESGGGLVEPGGSLRLSCAASGFTFSNAWMSWVRQAPGKGLEWVGRIKTTISGGATDFAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTLDLRDYWGQGTLVTVSS

[0309] Sequence ID 32 - Heavy Chain CDR1 NAWMS

[0310] Sequence ID 33 - Heavy Chain CDR2 RIKTTISGGATDFAAPVKG

[0311] Sequence ID 34 - Heavy Chain CDR3 DLRDY

[0312] Sequence ID 35 - Heavy Chain Variable Region QVQLVESGGGLVEPGGSLRLSCAASGFKFSNAWMSWVRQAPGKGLEWVGRIKTTISGGATQFAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTLDLRDYWGQGTLVTVSS

[0313] Sequence ID 36 - Heavy Chain CDR1 NAWMS

[0314] Sequence ID 37 - Heavy Chain CDR2 RIKTTISGGATQFAAPVKG

[0315] Sequence ID 38 - Heavy Chain CDR3 DLRDY

[0316] Sequence ID 39 - Heavy Chain Variable Region QVQLVESGGGLVQPGGSLRLSCAVSGFTFRRYAMSWVRQAPGKGLEWVSAISASGDRTHNTDSVKGRFSISRDNSKNTLYLQMNSLRAEDTAVYFCAKGIAASGKNYFDPWGQGTLVTVSS

[0317] Sequence ID 40 - Heavy Chain CDR1 RYAMS

[0318] Sequence ID 41 - Heavy Chain CDR2 AISASGDRTHNTDSVKG

[0319] Sequence ID 42 - Heavy Chain CDR3 GIAASGKNYFDP

[0320] Sequence ID 43 - Heavy Chain Variable Region QVQLVESGGGLVQPGGSLRLSCAVSGFTFSRYAMSWVRQAPGKGLEWVSAISASGDRTKNTDSVKGRFSISRDNSKNTLYLQMNSLRAEDTAVYFCAKGTAAAGKNYFDPWGQGTLVTVSS

[0321] Sequence ID 44 - Heavy Chain CDR1 RYAMS

[0322] Sequence ID 45 - Heavy Chain CDR2 AISASGDRTKNTDSVKG

[0323] Sequence ID 46 - Heavy Chain CDR3 GTAAAGKNYFDP

[0324] Sequence ID 47 - Heavy Chain Variable Region QVQLVESGGGLVQPGGSLRLSCAVSGFTFSRYAMSWVRQAPGKGLEWVSAISASGDRTKYTDSVKGRFSISRDNSKNTLYLQMNSLRAEDTAVYFCAKGTAAAGKNYFDPWGQGTLVTVSS

[0325] Sequence ID 48 - Light chain variable region DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPTFGQGTKVEIK

[0326] Light chain CDR1 according to sequence number 49-IMGT QSISSY

[0327] Light chain CDR2 according to sequence number 50-IMGT AAS

[0328] Light chain CDR3 according to sequence number 51-IMGT QQSYSTPPT

[0329] Sequence ID 52 - Light chain variable region DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPITFGQGTRLEIK

[0330] Light chain CDR1 according to sequence number 53-IMGT QSISSY

[0331] Light chain CDR2 according to sequence number 54-IMGT AAS

[0332] Light chain CDR3 according to sequence number 55-IMGT QQSYSTPPIT

[0333] Sequence ID 56 - Light chain variable region EIVMTQSPATLSVSPGERATLSCRASQSVSSNLAWYQQKPGQAPRLLIYGASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYNNWPWTFGQGTKVEIK

[0334] Light chain CDR1 according to sequence number 57-IMGT QSVSSN

[0335] Light chain CDR2 according to sequence number 58-IMGT GAS

[0336] Light chain CDR3 according to sequence number 59-IMGT QQYNNWPWT

[0337] Sequence ID 60 - Light chain variable region EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPWTFGQGTKVEIK

[0338] Light chain CDR1 according to sequence number 61-IMGT QSVSSSY

[0339] Light chain CDR2 according to sequence number 62-IMGT GAS

[0340] Light chain CDR3 according to sequence number 63-IMGT QQYGSSPWT

[0341] Sequence ID 64 - Light chain variable region SYVLTQPPSVSVAPGETARITCGGDNIGRKSVYWYQQKSGQAPVLVIYYDSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDGSSDHWVFGGGTKLTVL

[0342] Light chain CDR1 according to sequence number 65-IMGT NIGRKS

[0343] Light chain CDR2 according to sequence number 66-IMGT YDS

[0344] Light chain CDR3 according to sequence number 67-IMGT QVWDGSSDHWV

[0345] Sequence ID 68 - Hinge Region EPKSCDKTHTCPPCP

[0346] Sequence ID 69-CH1 region ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRV

[0347] Sequence ID 70-CH2 region APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK

[0348] Sequence ID 71-CH2-DM region APELGRGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK

[0349] Sequence ID 72-CH3 region GQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0350] Sequence ID 73-CH3-DE region GQPREPQVYTDPPSREEMTKNQVSLTCEVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0351] Sequence ID 74-CH3-KK region GQPREPQVYTKPPSREEMTKNQVSLKCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0352] Sequence ID 75-CL region RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0353] SEQ ID NO: 76 - Heavy Chain Reference Anti-TGF-βRII Antibody TGF1 Analogue QLQVQESGPGLVKPSETLSLTCTVSGGSISNSYFSWGWIRQPPGKGLEWIGSFYYGEKTYYNPSLKSRATISIDTSKSQFSLKLSSVTAADTAVYYCPRGPTMIRGVIDSWG QGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCD KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0354] Sequence ID 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 EIVLTQSPATLSLSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQAPRLLIYLASYLESGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLTFGGGGTKVE IKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0357] SEQ ID NO: 80 - Heavy Chain Reference PD-L1-TGF-β TRAP Molecular Analog EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYIMMWVRQAPGKGLEWVSSIYPSGGITFYADTVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARIKLGTVTTVDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKD YFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGAGGGGS GGGGSGGGGSGGGGSGIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD

[0358] Sequence ID 81 - Light chain reference PD-L1-TGF-β TRAP molecular analog QSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYDVSNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYTSSSTRVFGTGTKVT VLGQPKANTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS

[0359] Sequence ID No. 82 - Human TGF-βRII isoform A MGRGLLRGLWPLHIVLWTRIASTIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSD ECNDNIIFSEEYNTSNPDLLLVIFQVTGISLLPPLGVAISVIIIFYCYRVNRQQKLSSTWETGKTRKLMEFSEHCAIILEDDRSDISSTCANNINHNTELLPIELDTLVGKGRFAEVYKAKLKQNTSEQFETVAVKIFPYEE YASWKTEKDIFSDINLKHENILQFLTAEERKTELGKQYWLITAFHAKGNLQEYLTRHVISWEDLRKLGSSLARGIAHLHSDHTPCGRPKMPIVHRDLKSSNILVKNDLTCCLCDFGLSLRLDPTLSVDDLANSGQVGTARYM APEVLESRMNLENVESFKQTDVYSMALVLWEMTSRCNAVGEVKDYEPPFGSKVREHPCVESMKDNVLRDRGRPEIPSFWLNHQGIQMVCETLTECWDHDPEARLTAQCVAERFSELEHLDRLSGRSCSEEKIPEDGSLNTTK

[0360] Sequence ID 83 - Extracellular domain of human TGF-βRII isoform A TIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDLLLVIFQ

[0361] Sequence ID 84 - Human TGF-βRII isoform B MGRGLLRGLWPLHIVLWTRIASTIPPHVQKSDVEMEAQKDEIICPSCNRTAHPLRHINNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIM KEKKKPGETFFMCCSSDECNDNIIFSEEYNTSNPDLLLVIFQVTGISLLPPLGVAISVIIIFYCYRVNRQQKLSSTWETGKTRKLMEFSEHCAIILEDDRSDISSTCANNINHNTELLPIELDTLVGKGRFAEVYKAKLKQNTSEQF ETVAVKIFPYEEYASWKTEKDIFSDINLKHENILQFLTAEERKTELGKQYWLITAFHAKGNLQEYLTRHVISWEDLRKLGSSLARGIAHLHSDHTPCGRPKMPIVHRDLKSSNILVKNDLTCCLCDFGLSLRLDPTLSVDDLANSGQV GTARYMAPEVLESRMNLENVESFKQTDVYSMALVLWEMTSRCNAVGEVKDYEPPFGSKVREHPCVESMKDNVLRDRGRPEIPSFWLNHQGIQMVCETLTECWDHDPEARLTAQCVAERFSELEHLDRLSGRSCSEEKIPEDGSLNTTK

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

[0363] SEQ ID NO: 86 - Heavy Chain Negative Control RSV IgG1 Antibody EVQLVESGGGVVQPGRSLRLSCAASGFTFSNYGMHWVRQAPGKGLEWVAVISYDGSTKYSADSLKGRFTISRDNSKNTLYLQMNSLRADDDTAVYYCAKEGWSFDSSGYRSWFD SWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKS CDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPI EKTISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0364] Sequence ID 87 - Light Chain DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0365] Sequence ID 88 - Heavy Chain Variable Region QVQLVESGGGLVQPGGSLRLSCAVSGFTFSRYAMSWVRQAPGKGLEWVSAISASGDRTKNTDSVKGRFSISRDNSKNTLYLQMNSLRAEDTAVYYCAKGTAAAGKNYFDPWGQGTLVTVSS

[0366] Sequence ID 89 - Heavy Chain Variable Region QVQLVESGGGLVQPGGSLRLSCAVSGFTFSRYAMSWVRQAPGKGLEWVSAISASGDRTKYTDSVKGRFSISRDNSKNTLYLQMNSLRAEDTAVYYCAKGTAAAGKNYFDPWGQGTLVTVSS

[0367] Sequence ID 90 - Heavy Chain CDR1 RYAMS

[0368] Sequence ID 91 - Heavy Chain CDR2 AISASGDRTKYTDSVKG

[0369] Sequence ID 92 - Heavy Chain CDR3 GTAAAGKNYFDP

[0370] Sequence ID 93-V region VK1-39 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTP

[0371] Sequence ID 94-VK1-39 / JK1 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPTFGQGTKVEIK

[0372] Sequence ID 95-VK1-39 / JK5 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPITFGQGTRLEIK

[0373] SEQ ID NO: 96 - Heavy Chain Reference Nivolumab Analogue Antibody QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKGLEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAEDTAVYYCATNDDYWGQGTLVT VSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCP PCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTIS KAKGQPREPQVYTLPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

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

[0375] Sequence ID 98-V region VK3-15 EIVMTQSPATLSVSPGERATLSCRASQSVSSNLAWYQQKPGQAPRLLIYGASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYNNWP

[0376] Sequence ID 99-VK3-15 / JK1 EIVMTQSPATLSVSPGERATLSCRASQSVSSNLAWYQQKPGQAPRLLIYGASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYNNWPWTFGQGTKVEIK

[0377] Sequence ID 100-V region VK3-20 EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSP

[0378] Sequence ID 101-VK3-20 / JK1 EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPWTFGQGTKVEIK

[0379] Sequence ID 102-V region VL3-21 SYVLTQPPSVSVAPGETARITCGGDNIGRKSVYWYQQKSGQAPVLVIYYDSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDGSSDH

[0380] Sequence ID 103-VL3-21 / JL3 SYVLTQPPSVSVAPGETARITCGGDNIGRKSVYWYQQKSGQAPVLVIYYDSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDGSSDHWVFGGGTKLTVL

[0381] Sequence ID 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 inhibits PD-1-mediated signaling, and The TGF-βRII binding domain inhibits TGF-βRII-mediated signaling. Multispecific binding site.

2. A multispecific binding site as described in claim 1, In activated T cells, specifically 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 signaling. Multispecific binding site.

3. A multispecific binding site according to claim 1 or 2, The multispecific binding site includes a single Fab domain that binds to PD-1, a single Fab domain that binds to TGF-βRII, and an Fc region. Multispecific binding site.

4. A multispecific binding site as described in any one of claims 1 to 3, The multispecific binding site exhibits higher potency 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 at all, not substantially, or expressing low levels of PD-1. Multispecific binding site.

5. A multispecific binding site as described in claim 4, The cells expressing both PD-1 and TGF-βRII are Jurkat-PD-1 ＋ It is a cell, and Cells that express TGF-βRII and do not express PD-1 at all or substantially do not express it are Jurkat-PD-1 ｎｕｌｌ Cells Multispecific binding site.

6. A multispecific binding site as described in claim 4, The cells expressing both PD-1 and TGF-βRII are activated CD4 ＋ and / or CD8 ＋ It is a cell, and The cells expressing TGF-βRII and not expressing PD-1 at all are inactivated CD4 ＋ and / or CD8 ＋ Cells Multispecific binding site.

7. A multispecific binding site as described in claim 4, The cells expressing both PD-1 and TGF-βRII are HEK-Blue TGF-β-PD-1 ＋ It is a cell, and The cells that express TGF-βRII and do not express PD-1 at all are HEK-Blue TGF-β cells. Multispecific binding site.

8. A multispecific binding site as described in any one of claims 4 to 6, The efficacy in inhibiting TGF-βRII-mediated signaling is measured in a phosphorylated SMAD2 / 3 assay. Multispecific binding site.

9. A multispecific binding site according to claim 4 or 7, The efficacy in inhibiting TGF-βRII-mediated signaling is measured in an isogenic PD-1-TGF-β reporter assay. Multispecific binding site.

10. A multispecific binding site as described in any one of claims 4 to 9, In cells expressing both PD-1 and TGF-βRII, the efficacy in inhibiting TGF-βRII-mediated signaling is at least about 200 times, preferably about 200 to 30,000 times higher, than in cells expressing TGF-βRII but not expressing PD-1 at all, substantially not expressing it, or expressing it at a low level. Multispecific binding site.

11. A multispecific binding site as described in any one of claims 4 to 10, In cells expressing TGF-βRII but not expressing PD-1 at all, the efficacy of the multispecific binding site in inhibiting TGF-βRII-mediated signaling is lower than that of the reference anti-TGF-βRII antibody. and In cells expressing both PD-1 and TGF-βRII, the efficacy of the multispecific binding site in inhibiting TGF-βRII-mediated signaling is higher than that of the reference anti-TGF-βRII antibody. The aforementioned reference anti-TGF-βRII antibody is a bivalent monospecific antibody comprising a heavy chain having the amino acid sequence shown in SEQ ID NO: 76 and a light chain having the amino acid sequence shown in SEQ ID NO:

77. Multispecific binding site.

12. A multispecific binding site as described in claim 11, In cells expressing both PD-1 and TGF-βRII, the efficacy of the multispecific binding site in inhibiting TGF-βRII-mediated signaling is at least about 100 times, preferably about 100 to 20,000 times, higher than the efficacy of the reference anti-TGF-βRII antibody. Multispecific binding site.

13. A multispecific binding site according to any one of claims 1 to 12, The aforementioned multispecific binding site exhibits higher activity than the reference antibody combination when reducing tumor volume. The aforementioned combination of reference antibodies consists of 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 shown in SEQ ID NO: 78 and a light chain having the amino acid sequence shown in SEQ ID NO:

79. and The bivalent monospecific antibody targeting TGF-βRII comprises a heavy chain having the amino acid sequence shown in SEQ ID NO: 76 and a light chain having the amino acid sequence shown in SEQ ID NO:

77. Multispecific binding site.

14. A multispecific binding site as described in claim 13, 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.

15. A multispecific binding site according to claim 13 or 14, Here, higher activity in reducing tumor volume means a reduction in tumor volume of at least about 1.5 times, preferably about 1.5 to 100 times, compared to the reduction in tumor volume of the aforementioned combination of reference antibodies. Multispecific binding site.

16. A multispecific binding site comprising a PD-1 binding domain and a TGF-βRII binding domain, The PD-1 binding domain includes a heavy chain variable region comprising: a) Heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), each having the amino acid sequences shown in SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4, respectively; b) Heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), each having the amino acid sequences shown in SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8, respectively; c) Heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), each having the amino acid sequences shown in SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12, respectively; d) Heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), each having the amino acid sequences shown 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), each having the amino acid sequences shown in SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 22, respectively; Here, each of the HCDRs may contain at most three, two, or one amino acid variation; Multispecific binding site.

17. A multispecific binding site as described in claim 16, The PD-1 binding domain includes a heavy chain variable region having an amino acid sequence shown 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 to these sequences. Multispecific binding site.

18. A multispecific binding site according to claim 16 or 17, The PD-1 binding domain includes a light chain variable region comprising light chain CDR1 (LCDR1), light chain CDR2 (LCDR2), and light chain CDR3 (LCDR3), or variants thereof, each having the amino acid sequences shown in SEQ ID NO: 49, SEQ ID NO: 50, and SEQ ID NO: 51, respectively. Multispecific binding site.

19. A multispecific binding site as described in any one of claims 16 to 18, The PD-1 binding domain includes a light chain variable region having the amino acid sequence shown in Sequence ID No. 48, or having at least 80%, preferably 85%, more preferably 90%, or most preferably 95% sequence identity thereto. Multispecific binding site.

20. A multispecific binding site as described in any one of claims 16 to 17, The TGF-βRII binding domain includes a heavy chain variable region comprising: a) Heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), each having the amino acid sequences shown in SEQ ID NO: 24, SEQ ID NO: 25, and SEQ ID NO: 26, respectively; b) Heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), each having the amino acid sequences shown in SEQ ID NO: 28, SEQ ID NO: 29, and SEQ ID NO: 30, respectively; c) Heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), each having the amino acid sequences shown 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), each having the amino acid sequences shown 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), each having the amino acid sequences shown in SEQ ID NO: 40, SEQ ID NO: 41, and SEQ ID NO: 42, respectively; f) Heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), each having the amino acid sequences shown 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), each having the amino acid sequences shown in SEQ ID NO: 90, SEQ ID NO: 91, and SEQ ID NO: 92, respectively; Here, each of the HCDRs may contain at most three, two, or one amino acid variation; Multispecific binding site.

21. A multispecific binding site as described in any one of claims 16 to 20, The TGF-βRII-binding domain includes a heavy chain variable region having an amino acid sequence represented by 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 to these sequences. Multispecific binding site.

22. A multispecific binding site as described in any one of claims 16 to 21, The TGF-βRII-binding domain includes a light chain variable region comprising light chain CDR1 (LCDR1), light chain CDR2 (LCDR2), and light chain CDR3 (LCDR3), or variants thereof, each having the amino acid sequences shown in SEQ ID NO: 49, SEQ ID NO: 50, and SEQ ID NO: 51, respectively. Multispecific binding site.

23. A multispecific binding site as described in any one of claims 16 to 22, The TGF-βRII-binding domain includes a light chain variable region having the amino acid sequence shown 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.

24. A multispecific binding site comprising a PD-1 binding domain and a TGF-βRII binding domain, The TGF-βRII binding domain includes a heavy chain variable region comprising: a) Heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), each having the amino acid sequences shown in SEQ ID NO: 24, SEQ ID NO: 25, and SEQ ID NO: 26, respectively; b) Heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), each having the amino acid sequences shown in SEQ ID NO: 28, SEQ ID NO: 29, and SEQ ID NO: 30, respectively; c) Heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), each having the amino acid sequences shown 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), each having the amino acid sequences shown 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), each having the amino acid sequences shown in SEQ ID NO: 40, SEQ ID NO: 41, and SEQ ID NO: 42, respectively; f) Heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), each having the amino acid sequences shown 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), each having the amino acid sequences shown in SEQ ID NO: 90, SEQ ID NO: 91, and SEQ ID NO: 92, respectively; Here, each of the HCDRs may contain at most three, two, or one amino acid variation; Multispecific binding site.

25. A multispecific binding site as described in claim 24, The TGF-βRII-binding domain includes a heavy chain variable region having an amino acid sequence represented by 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 to these sequences. Multispecific binding site.

26. A multispecific binding site as described in claim 24 or 25, The TGF-βRII-binding domain includes a light chain variable region comprising light chain CDR1 (LCDR1), light chain CDR2 (LCDR2), and light chain CDR3 (LCDR3), or variants thereof, each having the amino acid sequences shown in SEQ ID NO: 49, SEQ ID NO: 50, and SEQ ID NO: 51, respectively. Multispecific binding site.

27. A multispecific binding site as described in any one of claims 24 to 26, The TGF-βRII-binding domain includes a light chain variable region having the amino acid sequence shown 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.

28. A multispecific binding site as described in any one of claims 24 to 27, The PD-1 binding domain includes a heavy chain variable region comprising: a) Heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), each having the amino acid sequences shown in SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4, respectively; b) Heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), each having the amino acid sequences shown in SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8, respectively; c) Heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), each having the amino acid sequences shown in SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12, respectively; d) Heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), each having the amino acid sequences shown 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), each having the amino acid sequences shown in SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 22, respectively; Here, each of the HCDRs may contain at most three, two, or one amino acid variation; Multispecific binding site.

29. A multispecific binding site as described in any one of claims 24 to 28, The PD-1 binding domain includes a heavy chain variable region having an amino acid sequence shown 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 to these sequences. Multispecific binding site.

30. A multispecific binding site as described in any one of claims 24 to 29, The PD-1 binding domain includes a light chain variable region comprising light chain CDR1 (LCDR1), light chain CDR2 (LCDR2), and light chain CDR3 (LCDR3), or variants thereof, each having the amino acid sequences shown in SEQ ID NO: 49, SEQ ID NO: 50, and SEQ ID NO: 51, respectively. Multispecific binding site.

31. A multispecific binding site as described in any one of claims 24 to 30, The PD-1 binding domain includes a light chain variable region having the amino acid sequence shown in Sequence ID No. 48, or having at least 80%, preferably 85%, more preferably 90%, or most preferably 95% sequence identity thereto. Multispecific binding site.

32. A multispecific binding site comprising a PD-1 binding domain and a TGF-βRII binding domain, With respect to binding to PD-1 and / or TGF-βRII, the multiple specific binding sites described in any one of claims 1 to 31 compete with the following: Multispecific binding site.

33. A multispecific binding site described in any one of claims 1 to 32 in an effective amount, comprising a pharmaceutically acceptable carrier, Pharmaceutical composition.

34. A multispecific binding site according to any one of claims 1 to 32, or a pharmaceutical composition according to claim 33, for use in therapeutic purposes.

35. A multispecific binding site according to any one of claims 1 to 32, or a pharmaceutical composition according to claim 33, for use in the treatment of diseases associated with a suppressed immune system, specifically cancer.

36. A method for treating a disease, The process includes the step of administering an effective amount of a multispecific binding site described in any one of claims 1 to 32, or the pharmaceutical composition described in claim 33, to a subject requiring it. method.

37. A method for treating diseases associated with a suppressed immune system, specifically cancer. The process includes the step of administering an effective amount of a multispecific binding site described in any one of claims 1 to 32, or the pharmaceutical composition described in claim 33, to a subject requiring it. method.

38. A nucleic acid sequence comprising a nucleic acid sequence encoding the heavy chain variable region of a PD-1 binding domain as defined in claim 16 or 17, and a nucleic acid sequence encoding the heavy chain variable region of a TGF-βRII binding domain as defined in claim 20 or 21, cell.

39. A cell according to claim 38, The cell further comprises nucleic acid sequences encoding a CH1 region and, preferably, a hinge, CH2, and CH3 region. cell.

40. A cell according to claim 38 or 39, The cell further comprises at least one nucleic acid sequence encoding a light chain variable region, specifically a light chain variable region as defined in claim 18 or 19, and preferably a CL region. cell.

41. A cell that produces a multispecific binding site as described in any one of claims 1 to 32.

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