Multi-specific binding moieties comprising PD-1 and TGF-beta RII binding domains

Abstract

The present disclosure relates to multispecific binding moieties comprising a PD-1 and a TGF-[beta] RII binding domain, where the PD-1 binding domain blocks PD-1 mediated signaling, and the TGF-[beta] RII binding domain blocks TGF-[beta] RII mediated signaling. The present disclosure also relates to a pharmaceutical composition comprising such multispecific binding moieties, a method of treatment using such multispecific binding moieties, and a cell producing such multispecific binding moieties.

Description

[0001] This application is a divisional application of a patent application with an application date of November 18, 2022, application number 202280076849X, and invention name “Multi-specific binding portion comprising PD-1 and TGF-βRII binding domains”. Technical Field

[0002] The present disclosure relates to the field of antibodies. Specifically, the present disclosure relates to the field of therapeutic antibodies for treating diseases involving abnormal cells. More specifically, the present disclosure relates to multispecific binding portions comprising a binding domain that binds to PD-1 and a binding domain that binds to TGF-βRII. Background Art

[0003] Despite the known role of T lymphocytes in tumor immunosurveillance, cancer cells are able to evade immune control by inducing inhibitory immune pathways. Therefore, immune checkpoint blockade (ICB), in which antibodies are used to block inhibitory immune pathways, has become a promising therapeutic option and has been shown in preclinical and clinical studies to enhance and maintain endogenous immunity against certain cancers.

[0004] Programmed death 1 (PD-1) and programmed death ligand 1 (PD-L1) are components of an immunosuppressive network that inhibits T cell activity in normal physiology but can be exploited by tumors to suppress T cell-mediated antitumor immune responses. Antibodies against PD-1 and PD-L1 have improved responses and survival in 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 persistence of responses is limited. Therefore, there is an urgent need to develop new, safe, and effective therapies for the treatment of cancer.

[0005] Programmed cell death 1 protein (PD-1) is a cell surface receptor that belongs to the CD28 receptor family and is expressed on T cells and pre-B cells. It is currently known that PD-1 binds to two ligands, PD-L1 and PD-L2. As an immune checkpoint, PD-1 plays an important role in downregulating the immune system by inhibiting the activation of T cells, and when PD-1 is present on somatic cells, it reduces autoimmunity and promotes self-tolerance. The inhibitory effect of PD-1 is believed to be achieved by promoting apoptosis (programmed cell death) of antigen-specific T cells in lymph nodes while reducing the apoptosis of regulatory T cells (suppressor T cells) through a dual mechanism. It is also known that PD-1 has many different aliases, such as 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. Newer drugs that block the activity of PD-1, PD-1 inhibitors, activate the immune system to attack tumors and are therefore used to treat some types of cancer.

[0006] Monoclonal antibodies targeting PD-1 have been approved for the treatment of various malignancies. For example, although 70% to 80% of patients initially responded, the response rate of melanoma patients treated with the anti-PD-1 antibody (pembrolizumab) was 33% at 3 years (Ribas A. et al., Association of Pembrolizumab With Tumor Response and Survival Among Patients With Advanced Melanoma, JAMA, April 19, 2016, Vol. 315 No. 15: pp. 1600-1609).

[0007] TGF-β signaling regulates a plethora of physiological and pathological processes, including cell cycle arrest of epithelial and hematopoietic cells, control of mesenchymal cell proliferation and differentiation, wound healing, extracellular matrix production, immunosuppression, and carcinogenesis (Massagué J., TGFβsignalling in context, Nat Rev Mol Cell Biol, October 2012, Vol. 13, No. 10: pp. 616-630). In addition, TGF-β signaling regulates many cancer cell functions, including cell cycle progression, apoptosis, adhesion, and differentiation (Liu S et al., Signal Transduction and targeted Therapy, 2021). TGF-β exhibits biphasic functions, so that in normal cells and precancerous cells, TGF-β is mainly reported to be used as a tumor suppressor; while in tumor cells, TGF-β allows growth-promoting functions and the transformation of epithelial cells to mesenchymal cells, thereby allowing tumor cell migration, invasion, intravasation, and extravasation.

[0008] 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, TGFβ-RII, transforming growth factor beta receptor 2, TBR-ii, and TBRII. TGF-βRII forms a heterodimeric complex with another receptor protein and binds TGF-β. This receptor / ligand complex phosphorylates proteins that then enter the nucleus and regulate the transcription of a subset of genes associated with cell proliferation.

[0009] Several inhibitors targeting the TGF-β pathway are in preclinical and clinical development and inhibit TGF-β at different levels (i.e., at the ligand level, ligand-receptor level, or intracellular level). These 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 block TGF-β synthesis, TGF-β2 antisense gene-modified allogeneic cancer cell vaccines, and small molecules that target the kinase domain of TGF-βRI.

[0010] Targeting the TGF-β pathway with monospecific antibodies has been reported to show antitumor activity in vitro and in vivo; however, poor clinical outcomes with low efficacy and unacceptable toxicity, including major cytokine release syndrome (CRS), persist. Bintrafuspα, an anti-PD-L1–TGFBRII bifunctional fusion protein, has been tried in combination with targeting the TGF-β pathway and immune checkpoint inhibition, but clinical efficacy has been modest.

[0011] There remains a need for new therapeutic interventions that selectively inhibit TGF-β signaling in activated, tumor-specific PD-1-expressing T cells localized in the tumor microenvironment in order to promote T cell tumor infiltration and restore and maintain T cell antitumor effector activity. Such targeting would relieve immunosuppressive pathways to effectively promote CTL function and T cell memory, thereby effectively and durable elimination of cancer, while minimizing the toxicities associated with systemic TGF-β blockade. Summary of the invention

[0012] One of the purposes of the present disclosure is to provide a novel pharmaceutical formulation for treating human diseases, particularly for treating cancer. This purpose is achieved by providing a multispecific binding portion that binds to PD-1 and TGF-βRII, such as a bispecific antibody. The PD-1 binding domain of the multispecific binding portion is intended to drive the specificity of the multispecific binding portion to activated / exhausted effector T cells in tumors and tumor-draining lymph nodes, wherein the TGF-βRII binding domain can locally block the binding of TGF-β to TGF-βRII, thereby reducing the systemic toxicity of TGF-β inhibition to non-T cells. In addition, the multispecific binding portion alleviates both PD1- and TGF-β-mediated immunosuppressive pathways to enhance the activity of cytotoxic T lymphocytes in the tumor microenvironment.

[0013] The present disclosure provides a multispecific binding portion comprising a PD-1 binding domain and a TGF-βRII binding domain, wherein the PD-1 binding domain blocks PD-1 mediated signaling and the TGF-βRII binding domain blocks TGF-βRII mediated signaling.

[0014] The present disclosure also provides a multispecific binding portion 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.

[0015] The present disclosure also provides a multispecific binding portion 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.

[0016] The present disclosure also provides a pharmaceutical composition comprising an effective amount of a multispecific binding moiety as described herein.

[0017] The present disclosure also provides a multispecific binding moiety as described herein and a pharmaceutical composition as described herein for use in therapy.

[0018] The present disclosure also provides a multispecific binding moiety as described herein and a pharmaceutical composition as described herein for use in treating cancer.

[0019] The present disclosure also provides a method for treating a disease, comprising administering to an individual in need thereof an effective amount of a multispecific binding moiety as described herein or a pharmaceutical composition as described herein.

[0020] The present disclosure also provides a method for treating cancer, comprising administering to an individual in need thereof an effective amount of a multispecific binding moiety as described herein or a pharmaceutical composition as described herein.

[0021] The present disclosure also provides a cell comprising 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.

[0022] The present disclosure also provides a cell that produces a multispecific binding moiety as described herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] The following naming conventions are used herein as follows: In the figures, bivalent monospecific antibodies are represented in the format of SEQ ID NO: A / SEQ ID NO: B, wherein SEQ ID NO: A refers to the heavy chains of the two binding domains and SEQ ID NO: B refers to the light chains of the two binding domains.

[0024] The bivalent bispecific antibody is represented in the format of SEQ ID NO: A x SEQ ID NO: B, wherein both SEQ ID NO: A and SEQ ID NO: B refer to the heavy chain variable sequence. Each binding domain of the bispecific antibody comprises the same light chain.

[0025] The bivalent monospecific reference antibodies pembrolizumab, nivolumab, and TGF1 analogs are represented in the format of SEQ ID NO: A / SEQ ID NO: B, wherein SEQ ID NO: A refers to the corresponding heavy chain sequence, and SEQ ID NO: B refers to the corresponding light chain sequence. The combination of pembrolizumab and a TGF1 analog is represented in the format of SEQ ID NO: A / SEQ ID NO: B+SEQ ID NO: C / SEQ ID NO: D, wherein SEQ ID NO: A refers to the heavy chain sequence of pembrolizumab or a TGF1 analog, and SEQ ID NO: B refers to the light chain sequence of pembrolizumab or a TGF1 analog, and SEQ ID NO: C refers to the heavy chain sequence of the other of pembrolizumab or a TGF1 analog, and SEQ ID NO: D refers to the light chain sequence of the other of pembrolizumab or a TGF1 analog.

[0026] Reference PD-L1-TGF-βTRAP molecule analogs, which are analogs of Bintrafuspα, are represented in the form of SEQ ID NO: A / SEQ ID NO: B, wherein SEQ ID NO: A refers to a heavy chain sequence comprising a (G4S)4G linker and an extracellular domain of TGF-βRII, and SEQ ID NO: B refers to a light chain sequence. The reference PD-L1-TGF-βTRAP molecule analogs comprise two PD-L1 binding domains and two TGF-βRII extracellular domains.

[0027] Figure 1A-1B Percent inhibition of PD-1-mediated SHP recruitment by bispecific antibodies and control antibodies as measured in the PD-1-SHP recruitment assay. Figure 1A The bispecific antibodies are: SEQ ID NO: 39×SEQ ID NO: 9 and SEQ ID NO: 35×SEQ ID NO: 5. The control antibodies are: pembrolizumab analogs-SEQ ID NO: 78 / SEQ ID NO: 79, PD-L1-TGF-βTRAP molecule analogs-SEQ ID NO: 80 / SEQ ID NO: 81 and TGF1 analogs-SEQ ID NO: 76 / SEQ ID NO: 77.

[0028] Figure 1BThe bispecific antibodies were: 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. The control antibodies were: pembrolizumab analogue - SEQ ID NO: 78 / SEQ ID NO: 79 and RSV IgG1 - SEQ ID NO: 86 / SEQ ID NO: 87.

[0029] Figure 2A-2C Fold induction of T cell activation by bispecific and control antibodies as measured in a PD-1-NFAT reporter assay. Figure 2A The bispecific antibodies are: SEQ ID NO: 39×SEQ ID NO: 9 and SEQ ID NO: 35×SEQ ID NO: 5. The control antibodies are: pembrolizumab analogs-SEQ ID NO: 78 / SEQ ID NO: 79, PD-L1-TGF-βTRAP molecule analogs-SEQ ID NO: 80 / SEQ ID NO: 81 and TGF1 analogs-SEQ ID NO: 76 / SEQ ID NO: 77.

[0030] Figure 2B The bispecific antibodies were: 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 were: pembrolizumab analogue - SEQ ID NO: 78 / SEQ ID NO: 79 and RSV IgG1 - SEQ ID NO: 86 / SEQ ID NO: 87.

[0031] Figure 2C The bispecific antibodies were: 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 were: pembrolizumab analogue-SEQ ID NO: 78 / SEQ ID NO: 79 and RSV IgG1-SEQ ID NO: 86 / SEQ ID NO: 87.

[0032] Figure 3 – In Jurkat-PD-1 null(AG) cells and Jurkat-PD-1 + Inhibition of phosphorylated SMAD2 / 3 by bispecific and control antibodies in cells (HN). These figures show Jurkat-PD-1 incubated with bispecific or control antibodies. null Cells and Jurkat-PD-1 +Phosphorylated SMAD2 / 3 levels in cell lysates. "No TGF-β1" indicates background phosphorylated SMAD2 / 3 levels when no TGF-β ligand is added as measured in the absence of bispecific antibodies; "10 ng / mL TGF-β1" indicates maximum phosphorylated SMAD2 / 3 levels when 10 ng / mL of TGF-β ligand is added in the absence of bispecific antibodies. The control antibodies were: RSV IgG1-SEQ ID NO:86 / SEQ ID NO:87, TGF1 analog-SEQ ID NO:76 / SEQ ID NO:77, and TGF-βRII×RSV antibody, which comprises a TGF-βRII binding domain, a RSV binding domain, and a common light chain, wherein the TGF-βRII binding domain comprises a heavy chain variable region amino acid sequence as shown in SEQ ID NO:23, 31, 39, 27, 35 or 43; the RSV binding domain comprises a heavy chain variable region amino acid sequence as shown in SEQ ID NO:86; the common light chain comprises a light chain variable region amino acid sequence as shown in SEQ ID NO:48 and a light chain constant region amino acid sequence as shown in SEQ ID NO:75. Each data point represents the average absorbance of the corresponding replicates. A and H: bispecific antibodies comprising a PD-1 binding domain and a TGF-βRII binding domain, wherein the PD-1 binding domain comprises a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 1, 5, 9, 14 or 19, and the TGF-βRII binding domain comprises a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 23; B and I: bispecific antibodies comprising a PD-1 binding domain and a TGF-βRII binding domain, wherein the PD-1 binding domain comprises a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 1, 5, 9, 14 or 19, and the TGF-βRII binding domain comprises a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 31; C and J: bispecific antibodies comprising a PD-1 binding domain and a TGF-βRII binding domain, wherein the PD-1 binding domain comprises a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: NO:1, 5, 9, 14 or 19, the TGF-βRII binding domain comprising a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO:39; D and K: bispecific antibodies comprising a PD-1 binding domain and a TGF-βRII binding domain, the PD-1 binding domain comprising a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO:1, 5, 9, 14 or 19, the TGF-βRII binding domain comprising a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO:27;E and L: bispecific antibodies comprising a PD-1 binding domain and a TGF-βRII binding domain, wherein the PD-1 binding domain comprises a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 1, 5, 9, 14 or 19, and the TGF-βRII binding domain comprises a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 35; F and M: bispecific antibodies comprising a PD-1 binding domain and a TGF-βRII binding domain, wherein the PD-1 binding domain comprises a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 1, 5, 9, 14 or 19, and the TGF-βRII binding domain comprises a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 43; G and N: bispecific antibodies are: SEQ ID NO: 23 × SEQ ID NO: 9; SEQ ID NO: 23 × SEQ ID NO: 14; SEQ ID NO: 43 × SEQ ID NO: 9; 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. Control antibodies were: 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. ;

[0033] Figure 4 – Measurement of intracellular phosphorylated SMAD2 by flow cytometry. These graphs show stimulated and unstimulated CD4 + and CD8 + Intracellular phosphorylated SMAD2 levels in T cells. 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 / SEQ ID NO:87, TGF1 analog-SEQ ID NO:76 / SEQ ID NO:77, and pembrolizumab-SEQ ID NO:78 / SEQ ID NO:79.

[0034] A: CD4 stimulated by donor A + T cells, B: CD8 stimulated by donor A +T cells, C: CD4 not stimulated by donor A + T cells, D: CD8 not stimulated by donor A + T cells, E: CD4 stimulated by donor B + T cells, F: CD8 stimulated by donor B + T cells, G: CD4 not stimulated by donor B + T cells, H: CD8 not stimulated by donor B + T cells.

[0035] Figure 5- Measurement of cytokine production induced by bispecific antibodies or control antibodies in a depletion 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. Control antibodies were: RSV IgG1 – SEQ ID NO:86 / SEQ ID NO:87, TGF1 analogue – SEQ ID NO:76 / SEQ ID NO:77, combination of pembrolizumab and TGF1 analogue – SEQ ID NO:78 / SEQ ID NO:79 + SEQ ID NO:76 / SEQ ID NO:77, PD-L1-TGF-βTRAP molecule analogue – SEQ ID NO:80 / SEQ ID NO:81 and pembrolizumab – SEQ ID NO:78 / SEQ ID NO:79. A: The graph shows the induction of IFN-γ cytokine secretion by exhausted T cells of a representative donor. B: The graph shows the induction of IFN-γ cytokine secretion by exhausted T cells of a representative donor. C: The graph shows the induction of IL-2 cytokine secretion by exhausted T cells in a representative donor. D: The graph shows the induction of TNF-α cytokine secretion by exhausted T cells in a representative donor. E: The figure shows the induction of TNF-α cytokine secretion by exhausted T cells in one representative donor.

[0036] Figure 6- Measurement of % inhibition of TGF-β-induced signaling induced by bispecific antibodies or control antibodies. 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. Control antibodies are: RSV IgG1–SEQ ID NO:86 / SEQ ID NO:87, TGF1 analogue-SEQ ID NO:76 / SEQ ID NO:77 and pembrolizumab-SEQ ID NO:78 / SEQ ID NO:79. A and B: These figures show the inhibition of TGF-β-induced signaling in HEK-Blue TM Inhibition of TGF-β signaling by bispecific antibodies or control antibodies in TGF-β cells. C and D: These figures show the inhibition of TGF-β signaling by bispecific antibodies or control antibodies in HEK-Blue TM TGF-β-PD-1 + Inhibition of TGF-βR signaling in cells by bispecific antibodies or control antibodies.

[0037] 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 x SEQ ID NO: 14, SEQ ID NO: 23 x SEQ ID NO: 19, SEQ ID NO: 31 x SEQ ID NO: 14, SEQ ID NO: 39 x SEQ ID NO: 9, SEQ ID NO: 35 x SEQ ID NO: 9, SEQ ID NO: 35 x SEQ ID NO: 14, SEQ ID NO: 35 x SEQ ID NO: 19, SEQ ID NO: 27 x SEQ ID NO: 9; SEQ ID NO: 43 x SEQ ID NO: 9; and SEQ ID NO: 43 x SEQ ID NO: 19. Control antibodies were: RSV IgG1 - SEQ ID NO:86 / SEQ ID NO:87; TGF1 analog - SEQ ID NO:76 / SEQ ID NO:77; combination of pembrolizumab and TGF1 analog - SEQ ID NO:78 / SEQ ID NO:79 + SEQ ID NO:76 / SEQ ID NO:77; and pembrolizumab - SEQ ID NO:78 / SEQ ID NO:79. A: The graph shows the induction of IFN-γ cytokine secretion in co-cultures of Tregs and PBMCs from one representative donor. B: The graph shows the induction of TNF-α cytokine secretion in co-cultures of Tregs and PBMCs from one representative donor.

[0038] Figure 8- Measurement of cytokine production induced by bispecific antibodies or control antibodies in a macrophage inhibition assay. Bispecific antibodies are: SEQ ID NO: 35 x SEQ ID NO: 9, SEQ ID NO: 23 x SEQ ID NO: 14, SEQ ID NO: 23 x SEQ ID NO: 19, SEQ ID NO: 43 x SEQ ID NO: 9, SEQ ID NO: 39 x SEQ ID NO: 9, SEQ ID NO: 43 x SEQ ID NO: 19, SEQ ID NO: 35 x SEQ ID NO: 14, SEQ ID NO: 35 x SEQ ID NO: 19, SEQ ID NO: 31 x SEQ ID NO: 14, and SEQ ID NO: 27 x SEQ ID NO: 9. The control antibodies were: RSV IgG1 – SEQ ID NO:86 / SEQ ID NO:87, TGF1 analogue – SEQ ID NO:76 / SEQ ID NO:77, Opdivo, LILRB2, PD-L1-TGF-βTRAP molecule analogue – SEQ ID NO:80 / SEQ ID NO:81, Pembrolizumab – SEQ ID NO:78 / SEQ ID NO:79 and Nivolumab analogue – SEQ ID NO:96 / SEQ ID NO:97. A: These figures show the expression of CD163, CD209, CD206 and CD86 on M2 macrophages obtained from PBMCs of three different donors. B: These figures show the expression of CD4 in the presence of M2 macrophages obtained from PBMCs of three different donors. + Induction of IFN-γ cytokine secretion by T cells.

[0039] Fig. 9 - In vivo efficacy of bispecific antibodies. A: The figure shows the reduction in tumor volume (in mm) induced by the control and reference antibodies. 3 BE: These graphs show the reduction in tumor volume (in mm) induced by the bispecific antibody compared to the control antibody and the reference antibody. 3The 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. Control antibodies were: RSV IgG1 - SEQ ID NO:86 / SEQ ID NO:87, TGF1 analog - SEQ ID NO:76 / SEQ ID NO:77, pembrolizumab - SEQ ID NO:78 / SEQ ID NO:79, PD-L1-TGF-βTRAP molecule analog - SEQ ID NO:80 / SEQ ID NO:81 and combination of pembrolizumab and TGF1 analog - SEQ ID NO:78 / SEQ ID NO:79 + SEQ ID NO:76 / SEQ ID NO:77. Bispecific antibodies were dosed at 1 mg / kg (1) and / or 10 mg / kg (10) (AC) and only 10 mg / kg (DF). F: The left panel shows TGF-βRII receptor occupancy after treatment with bispecific antibodies or control antibodies, and the right panel shows PD-1 receptor occupancy.

[0040] Fig.10 – Vector map.

[0041] Fig.11 - In vivo efficacy of bispecific antibodies. The figure shows the reduction in tumor volume (in mm) induced by the exemplary bispecific antibodies at two different dose levels (1 mg / kg and 10 mg / kg) compared to the control antibody at 10 mg / kg. 3 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. DETAILED DESCRIPTION

[0042] In certain embodiments, the present disclosure provides a multispecific binding moiety comprising a PD-1 binding domain and a TGF-βRII binding domain, wherein the PD-1 binding domain blocks PD-1 mediated signaling and the TGF-βRII binding domain blocks TGF-βRII mediated signaling.

[0043] As used herein, "blocking" refers to interfering with or altering the interaction between a ligand and a receptor, or causing a total or partial reduction in a signal transduction cascade. For the purposes of the present disclosure, in certain embodiments, the effectiveness of blocking ligand-induced PD-1 signaling is determined by using a SHP recruitment assay as described in Example 2 or a NFAT reporter gene assay as described in Example 3. In certain embodiments, the effectiveness of blocking ligand-induced PD-1 signaling is determined by using a SHP recruitment assay as described in Example 2. In certain embodiments, the effectiveness of blocking ligand-induced PD-1 signaling is determined by using a NFAT reporter gene assay as described in Example 3. For the purposes of the present disclosure, in certain embodiments, the effectiveness of blocking ligand-induced TGF-βRII signaling is determined by using a SMAD assay as described in Example 4 or 5. In certain embodiments, the effectiveness of blocking ligand-induced TGF-βRII signaling is determined by using a SMAD assay as described in Example 4. In certain embodiments, the effectiveness of blocking ligand-induced TGF-βRII signaling is determined by using a SMAD assay as described in Example 5.

[0044] In certain embodiments, the multispecific binding moieties of the present disclosure bind to human PD-1. In certain embodiments, the PD-1 binding domain of the multispecific binding moieties of the present disclosure blocks the binding of PD-L1 to PD-1, e.g., as measured in an assay described in Example 2 or 3.

[0045] In certain embodiments, the multispecific binding moieties of the present disclosure bind to human TGF-βRII. Human TGF-βRII is a transmembrane protein that has different isoforms. The amino acid sequence of human TGF-βRII isoform A is provided as SEQ ID NO: 82; the amino acid sequence of the extracellular domain of human TGF-βRII isoform A is provided as SEQ ID NO: 83. Human TGF-βRII isoform B is a splice variant that encodes a longer isoform due to the insertion of 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 in SEQ ID NO: 85.

[0046] "Binding moiety" refers to a protein molecule and includes, for example, all antibody formats available in the art, such as, for example, full-length IgG antibodies, immunoconjugates, diabodies, BiTEs, Fab fragments, scFv, tandem scFv, single domain antibodies (such as VHH and VH), miniantibodies, scFab, scFv-zipper, nanobodies, DART molecules, TandAb, Fab-scFv, F(ab)'2, F(ab)'2-scFv2 and intrabody.

[0047] In certain embodiments, the multispecific binding moiety is a multispecific antibody. The multispecific antibody according to the present disclosure is an antibody comprising at least two binding domains, and the at least two binding domains are specific for at least two different targets or epitopes. In certain embodiments, the multispecific antibody of the present disclosure is a bispecific antibody. In certain embodiments, the multispecific antibody of the present disclosure may also include an Fc region or a portion thereof. In certain embodiments, the multispecific binding moiety of the present disclosure is an IgG1 antibody. The constant region of the binding moiety of the present disclosure may include one or more variations that regulate the properties of the binding moiety rather 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 instead of 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, particularly one or more variations that reduce effector function.

[0048] "Fab" generally refers to the binding domain comprising the heavy chain variable region, the light chain variable region, the CH1 region, and the CL region.

[0049] In certain embodiments, the multispecific binding moieties of the present disclosure comprise a single Fab domain that binds to PD-1, a single Fab domain that binds to TGF-βRII, and an Fc region. In certain embodiments, the multispecific binding moieties of the present disclosure consist of a single Fab domain that binds to PD-1, a single Fab domain that binds to TGF-βRII, and an Fc region. In certain embodiments, the multispecific binding moieties of the present disclosure consist essentially of a single Fab domain that binds to PD-1, a single Fab domain that binds to TGF-βRII, and an Fc region.

[0050] The "Fc region" generally comprises a hinge, a CH2 region and a CH3 region. Suitable hinges include, but are not limited to, hinges whose amino acid sequences are shown in SEQ ID NO: 68. Suitable CH2 and CH3 regions include, but are not limited to, CH2 regions whose amino acid sequences are shown in SEQ ID NO: 70 or 71, and CH3 regions whose amino acid sequences are shown in SEQ ID NO: 72 or 73 and 74.

[0051] The CL region, CH1 region, CH2 region and / or CH3 region can be modified according to methods known in the art to obtain favorable antibody characteristics, including, for example, promoting heterodimerization of different heavy chains, improving heavy-light chain pairing, and enhancing or reducing immune cell effector function.

[0052] In certain embodiments, in activated T cells, particularly activated tumor-specific T cells, the PD-1 binding domain of the multispecific binding moiety blocks PD-1-mediated signaling, and the TGF-βRII binding domain of the multispecific binding moiety blocks TGF-βRII-mediated signaling.

[0053] In certain embodiments, the potency of the multispecific binding portions of the present disclosure in blocking TGF-βRII-mediated signaling is greater in cells that express both PD-1 and TGF-βRII than in cells that express TGF-βRII and do not express, do not substantially express, or express low levels of PD-1.

[0054] Thus, the present disclosure also provides a multispecific binding portion comprising a PD-1 binding domain and a TGF-βRII binding domain, wherein the potency of the multispecific binding portion in blocking TGF-βRII-mediated signaling is greater in cells that express both PD-1 and TGF-βRII than in cells that express TGF-βRII and do not express, do not substantially express, or express low levels of PD-1.

[0055] In certain embodiments, the cell expressing both PD-1 and TGF-βRII is Jurkat-PD-1. + Cells such as, for example, Jurkat T cells that express human PD-1 and a luciferase reporter gene driven by a NFAT response element (NFAT-RE), and cells that express TGF-βRII and do not express PD-1 are Jurkat-PD-1 null Cells, such as Jurkat-PD-1 null Jurkat T cells expressing human PD-1 and a luciferase reporter gene driven by NFAT-RE are commercially available, for example, from Promega (Catalog No. CS187105, part of kits CS187106 and CS187107); Jurkat-PD-1 null Cells are publicly available, for example, from ATCC (Catalog No. TIB-152).

[0056] In certain embodiments, cells expressing both PD-1 and TGF-βRII are stimulated CD4 + or CD8 +cells, and cells expressing TGF-βRII and low levels of PD-1 are unstimulated CD4 + or CD8 + In certain embodiments, the stimulated CD4 + or CD8 + Cells were stimulated with recombinant human TGF-β1.

[0057] In certain embodiments, cells expressing both PD-1 and TGF-βRII are HEK-Blue TM TGF-β-PD-1 + Cells, such as described in Example 7, and cells expressing TGF-βRII and not expressing PD-1 are HEK-Blue TM TGF-β cells, such as HEK-Blue TM TGF-β cells. HEK-Blue TM TGF-β cells, such as those commercially available from Invivogen (Catalog No. hkb-tgfb), are stably transfected human embryonic kidney HEK 293 cells containing human TGFBRI, Smad3 and Smad4 genes. They also express a Smad3 / 4-binding element (SBE)-inducible SEAP reporter gene.

[0058] In certain embodiments, not expressing, substantially not expressing, or expressing low levels of PD-1 refers to undetectable levels of PD-1 on the cell surface in a suitable assay as described herein. In certain embodiments, low levels of PD-1 refer to less than 100 PD-1 molecules present on the cell surface. In certain embodiments, the level of PD-1 is measured using the Quantibrite bead method.

[0059] For purposes of the present disclosure, whether the potency of a multispecific binding moiety in blocking TGF-βRII-mediated signaling is greater in cells expressing both PD-1 and TGF-βRII than in cells expressing TGF-βRII and not expressing, not expressing substantially, or expressing low levels of PD-1 is determined by using one of the assays described herein.

[0060] The efficacy data for the multispecific binding moieties as provided herein were obtained using the phosphorylated SMAD2 / 3 assay as described in Examples 4 and 5 and the isogenic HEK-BLUE-PD-1 TGF-β reporter assay as described in Example 7. Thus, in certain embodiments, the efficacy of blocking TGF-βRII-mediated signaling is measured in the phosphorylated SMAD2 / 3 assay as described in Example 4 or Example 5.

[0061] Briefly, the phosphorylated SMAD2 / 3 assay as described in Example 4 was performed using Jurkat-PD-1 cultured in RPMI / 10% FBS. null and Jurkat-PD-1 + Cells were incubated with test antibodies or control antibodies in 6-step serial dilutions (100 μg / mL to 0.001 μg / mL) at 37°C / 5% CO2 for one hour. After one hour, human recombinant TGF-β1 was added at a final concentration of 10 ng / mL, and the cells were incubated for another two hours at 37°C / 5% CO2. After incubation, the cells were gently washed with PBS. Cell lysates were prepared using a lysis buffer containing phosphatase inhibitors and protease inhibitors. Phosphorylated SMAD2 / 3 levels were determined using ELISA.

[0062] Briefly, the phosphorylated SMAD2 / 3 assay as described in Example 5 was performed using PBMCs from healthy donors, which were stimulated with 1 μg / mL anti-CD3 for 48 hours and then serum deprived for 16 hours in 0.1% FBS. Stimulated and unstimulated PBMCs were incubated with test and control antibodies for 30 minutes at room temperature. Recombinant human TGF-β1 was added at a final concentration of 1 ng / mL and the cells were incubated for an additional 30 minutes. Finally, the cells were washed twice with PBS and stained for cell surface markers followed by intracellular phosphorylated SMAD2 staining. Flow cytometry was performed to gate CD4 + and CD8 + The mean geometric fluorescence intensity (GMFI) of the phosphorylated SMAD2 signal was measured on these cells.

[0063] In certain embodiments, the efficacy of blocking TGF-βRII-mediated signaling is measured in a isogenic HEK-BLUE-PD-1 TGF-β reporter assay as described in Example 7.

[0064] Briefly, the HEK-BLUE-PD-1TGF-β reporter gene assay was performed using HEK-Blue TM TGF-β cells and HEK-Blue TM TGF-β-PD-1 + Cells were plated at 25,000 cells / well. Serial dilutions of the test and control antibodies were added and the cells were incubated for one hour at room temperature before adding human recombinant TGF-β1 at a final concentration of 1 ng / mL. The cells were incubated overnight at 37°C / 5% CO2. After incubation, 40 μl of the supernatant and 160 μl of the resuspended QUANTI-Blue TM The solution was incubated at 37°C / 5% CO2 for 40 minutes.TM The solution was evaluated for the amount of secreted SEAP in the supernatant. SEAP levels were measured using a spectrophotometer at 650 nm.

[0065] In certain embodiments, the potency of the multispecific binding moieties of the present disclosure in blocking TGF-βRII-mediated signaling is at least about 10 times higher in cells expressing both PD-1 and TGF-βRII than in cells expressing TGF-βRII and not expressing PD-1, preferably between about 10 times and 100,000 times. In certain embodiments, the potency of the multispecific binding moieties of the present disclosure in blocking TGF-βRII-mediated signaling is at least 10 times higher in cells expressing both PD-1 and TGF-βRII than in cells expressing TGF-βRII and not expressing PD-1, preferably between 10 times and 100,000 times. In certain embodiments, the potency of blocking TGF-βRII-mediated signaling is determined in a phosphorylated SMAD2 / 3 assay or a HEK-BLUE-PD-1 TGF-β reporter assay as IC50 (μg / mL). In certain embodiments, the efficacy of blocking TGF-βRII-mediated signaling is determined in a phosphorylated SMAD2 / 3 assay as IC50 (μg / mL). In certain embodiments, the efficacy of blocking TGF-βRII-mediated signaling is determined in a HEK-BLUE-PD-1 TGF-β reporter gene assay as IC50 (μg / mL).

[0066] In certain embodiments, the multispecific binding moieties of the present disclosure block TGF-βRII-mediated signaling in cells that express TGF-βRII and do not express, do not substantially express, or express low levels of PD-1 with less potency than a reference anti-TGF-βRII antibody targeted to the same cells, and block TGF-βRII-mediated signaling in cells that express both TGF-βRII and PD-1 with greater potency than a reference anti-TGF-βRII antibody targeted to the same cells, wherein the reference anti-TGF-βRII antibody is a bivalent monospecific antibody comprising a heavy chain having the amino acid sequence set forth in SEQ ID NO:76 and a light chain having the amino acid sequence set forth in SEQ ID NO:77.

[0067] In certain embodiments, the cell expressing both PD-1 and TGF-βRII is Jurkat-PD-1. + cells, stimulated CD4 + or CD8 + cells, or HEK-Blue TM TGF-β-PD-1 +cells; cells that express TGF-βRII and do not express, or substantially do not express, PD-1 are Jurkat-PD-1 null Cells or HEK-Blue TM TGF-β cells; and cells expressing TGF-βRII and low levels of PD-1 are unstimulated CD4 + or CD8 + Cells, as further described herein.

[0068] In certain embodiments, the multispecific binding moieties of the present disclosure block TGF-βRII-mediated signaling in cells expressing both TGF-βRII and PD-1 at least 10 times more potent than a reference anti-TGF-βRII antibody, preferably between 10 and 100,000 times. In certain embodiments, the multispecific binding moieties of the present disclosure block TGF-βRII-mediated signaling in cells expressing both TGF-βRII and PD-1 at least about 10 times more potent than a reference anti-TGF-βRII antibody, preferably between about 10 and 100,000 times. In certain embodiments, the reference TGF-βRII antibody is a bivalent monospecific antibody comprising a heavy chain having an amino acid sequence as shown in SEQ ID NO: 76 and a light chain having an amino acid sequence as shown in SEQ ID NO: 77. In certain embodiments, the potency of blocking TGF-βRII-mediated signaling is determined in a phosphorylated SMAD2 / 3 assay as IC50 (μg / mL).

[0069] In certain embodiments, the multispecific binding moieties of the present disclosure have greater activity in reducing tumor volume than a combination of reference antibodies. In certain embodiments, the combination of reference antibodies is two bivalent monospecific antibodies targeting PD-1 and TGF-βRII, wherein the bivalent monospecific antibody targeting PD-1 comprises a heavy chain having an amino acid sequence as shown in SEQ ID NO: 78 and a light chain having an amino acid sequence as shown in SEQ ID NO: 79, and the bivalent monospecific antibody targeting anti-TGF-βRII comprises a heavy chain having an amino acid sequence as shown in SEQ ID NO: 76 and a light chain having an amino acid sequence as shown in SEQ ID NO: 77.

[0070] Thus, the present disclosure also provides a multispecific binding portion comprising a PD-1 binding domain and a TGF-βRII binding domain, wherein the multispecific binding portion has a higher activity in reducing tumor volume than a combination of reference antibodies. In certain embodiments, the multispecific binding portion is administered with a number of PD-1 and TGF-βRII binding domains that is two-fold to up to twenty-fold lower than each of the bivalent monospecific antibodies of the combination of reference antibodies. For example, when administered at 1 mg / kg, a multispecific binding portion (which is monovalent for binding to PD-1 and monovalent for binding to TGF-βRII) has a higher activity in reducing tumor volume than a combination of reference antibodies (each of which is bivalent for binding to PD-1 or TGF-βRII, and each of which is administered at 10 mg / kg). In addition, when dosed at 10 mg / kg, the multispecific binding portion (which is monovalent for binding to PD-1 and monovalent for binding to TGF-βRII) has greater activity in reducing tumor volume than a combination of reference antibodies (each of which is bivalent for binding to PD-1 or TGF-βRII, and each of which is dosed at 10 mg / kg).

[0071] In certain embodiments, the combination of reference antibodies is two bivalent monospecific antibodies targeting PD-1 and TGF-βRII, wherein the bivalent monospecific antibody targeting PD-1 comprises a heavy chain having the amino acid sequence shown in SEQ ID NO:78 and a light chain having the amino acid sequence shown in SEQ ID NO:79, and wherein 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.

[0072] In certain embodiments, the activity in reducing tumor volume is determined by measuring reduction in tumor volume in in vivo studies in mice, particularly in in vivo studies in mice using MDA-MB-231 xenograft huCD34 NSG mice.

[0073] In certain embodiments, the reduction in tumor volume of the multispecific binding portions of the present disclosure is at least 1.5-fold greater than the reduction in tumor volume of the combination of reference antibodies, and preferably between 1.5-fold to 100-fold, or 2-fold to 80-fold, or 5-fold to 80-fold, or 10-fold to 80-fold, or 15-fold to 80-fold, or 20-fold to 80-fold, or 30-fold to 80-fold, or 40-fold to 80-fold, or 50-fold to 80-fold, or 2-fold to 60-fold, or 5-fold to 60-fold, or 10-fold to 60-fold, or 15-fold to 60-fold, or 20-fold to 60-fold, or 30-fold to 60-fold, or 40-fold to 60-fold. In certain embodiments, the reduction in tumor volume of the multispecific binding portions of the present disclosure is at least about 1.5-fold greater than the reduction in tumor volume of the combination of reference antibodies, preferably between about 1.5-fold to 100-fold, or 2-fold to 80-fold, or 5-fold to 80-fold, or 10-fold to 80-fold, or 15-fold to 80-fold, or 20-fold to 80-fold, or 30-fold to 80-fold, or 40-fold to 80-fold, or 50-fold to 80-fold, or 2-fold to 60-fold, or 5-fold to 60-fold, or 10-fold to 60-fold, or 15-fold to 60-fold, or 20-fold to 60-fold, or 30-fold to 60-fold, or 40-fold to 60-fold. In other words, the multispecific binding portions of the present disclosure provide greater reduction in tumor volume than the combination of reference antibodies. In certain embodiments, the combination of reference antibodies is two bivalent monospecific antibodies targeting PD-1 and TGF-βRII, wherein the bivalent monospecific antibody targeting PD-1 comprises a heavy chain having the amino acid sequence 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.

[0074] In certain embodiments, the multispecific binding moieties of the present disclosure reduce tumor volume when administered as a single agent.

[0075] Thus, the present disclosure also provides a multispecific binding moiety comprising a PD-1 binding domain and a TGF-βRII binding domain, wherein the multispecific binding moiety induces a reduction in tumor volume as a single agent.

[0076] In certain embodiments, the present disclosure provides several PD-1×TGF-βRII bispecific antibodies as exemplary multispecific binding portions, whose 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, whose 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.

[0077] In certain embodiments, the PD-1 binding domain of the multispecific binding moieties of the present disclosure comprises a heavy chain variable region comprising:

[0078] a) heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2) and heavy chain CDR3 (HCDR3), which have the amino acid sequences shown in SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4, respectively;

[0079] b) heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2) and heavy chain CDR3 (HCDR3), which have the amino acid sequences shown in SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, respectively;

[0080] c) heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2) and heavy chain CDR3 (HCDR3), which have the amino acid sequences shown in SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, respectively;

[0081] d) heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2) and heavy chain CDR3 (HCDR3), which have the amino acid sequences shown in SEQ ID NO: 15, SEQ ID NO: 16 and SEQ ID NO: 17, respectively; or

[0082] e) heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2) and heavy chain CDR3 (HCDR3), which have the amino acid sequences shown in SEQ ID NO: 20, SEQ ID NO: 21 and SEQ ID NO: 22, respectively.

[0083] The heavy chain variable region of the PD-1 binding domain of the multispecific binding moieties of the present disclosure may comprise a limited number, such as, for example, one, two, three, four, five, six, seven, eight, nine or ten non-conservative amino acid substitutions, or an unlimited number of conservative amino acid substitutions.

[0084] In certain embodiments, the PD-1 binding domain of the multispecific binding moiety of the present disclosure also includes a PD-1 binding domain variant thereof, wherein each of the HCDRs can comprise up to three, two, or one amino acid variation. In certain embodiments, only one or two HCDRs can comprise up to three, two, or one non-conservative amino acid variation. In certain embodiments, such variants do not comprise an amino acid variation in HCDR3. In certain embodiments, the amino acid variation is a conservative amino acid substitution.

[0085] Generally, conservative amino acid substitutions involve the variation of amino acids with homologous amino acid residues, which are residues with similar characteristics or properties. Homologous amino acids are known in the art, such as conventional methods for amino acid substitutions in antibody binding domains without significantly affecting the binding or function of antibodies, see, for example, manuals, such as Lehninger (Nelson, David L. and Michael M. Cox., 2017, Lehninger Principles of Biochemistry, 7th Edition, New York, NY: WH Freeman) or Stryer (Berg J., Tymoczko J., Stryer L. and Stryer L., 2007, Biochemistry, New York: WH Freeman), the entire contents of which are incorporated herein. When determining whether an amino acid can be replaced by a conservative amino acid, factors such as, but not limited to, (a) the structure of the polypeptide backbone in the replacement region, such as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, and / or (c) the body of the side chain can generally be evaluated. If a residue can be substituted with a residue having common features (such as a similar side chain or similar charge or hydrophobicity), such a residue is preferred as a substituent. For example, the following groups can be identified: (1) nonpolar: Ala (A), Gly (G), Val (V), Leu (L), Ile (I), Pro (P), Phe (F), Trp (W), Met (M); (2) uncharged polar: Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gln (Q); (3) acidic: Asp (D), Glu (E); and (4) basic: Lys (K), Arg (R), His (H). Alternatively, amino acids can be grouped as follows: (1) aromatic: Phe (F), Trp (W), Tyr (Y); (2) non-polar: Leu (L), Val (V), Ile (I), Ala (A), Met (M); (3) aliphatic: Ala (A), Val (V), Leu (L), Ile (I); (4) acidic: Asp (D), Glu (E); (5) basic: His (H), Lys (K), Arg (R); and (6) polar: Gln (Q), Asn (N), Ser (S), Thr (T), Tyr (Y).Alternatively, amino acid residues can be divided into the following groups based on common side chain properties: (1) hydrophobic: Met (M), Ala (A), Val (V), Leu (L), Ile (I); (2) neutral hydrophilic: Cys (C), Ser (S), Thr (T), Asn (N), Gln (Q); (3) acidic: Asp (D), Glu (E); (4) basic: His (H), Lys (K), Arg (R); (5) residues affecting chain orientation: Gly (G), Pro (P); and (6) aromatic: Trp (W), Tyr (Y), Phe (F).

[0086] Preferably, the amino acid residue is substituted with another amino acid residue present in the same group. Thus, conservative amino acid substitutions may involve exchanging a member of one of these classes with another member of the same class. Typically, the variation does not result in, or substantially does not result in, a loss of binding specificity of the binding domain to its intended target.

[0087] Other types of amino acid variations include variations caused by somatic hypermutation or affinity maturation. The PD-1 binding variants included in the present disclosure include somatic hypermutation or affinity maturation heavy chain variable regions, which are derived from the heavy chain variable regions of the same VH gene fragments as described in the sequences herein, and these variants have amino acid variations, including non-conservative and / or conservative amino acid substitutions in one, two or all three HCDRs. Conventional methods for affinity maturation antibody binding domains are widely 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., August 2019; Vol. 212: Pages 106-113).

[0088] Examples of suitable positions for introducing amino acid variations include, but are not limited to, the first, second and / or fourth amino acid of HCDR1; the third, seventh, eighth, ninth, tenth, eleventh, thirteenth, fourteenth and / or sixteenth amino acid of HCDR2; and / or the sixth and / or thirteenth amino acid of HCDR3.

[0089] In certain embodiments, the present disclosure therefore also provides multispecific binding moieties, the PD-1 binding domain of which comprises:

[0090] - HCDR1 having the amino acid sequence X1X2FX3S, wherein

[0091] X1 can be F, Y, T or H;

[0092] X2 can be Y, Q, E, H or D;

[0093] X3 may be W or Y; and / or

[0094] - HCDR2 having the amino acid sequence YIX1YSGX2X3X4X5X6PX7X8KX9, wherein

[0095] X1 can be Y, V or I;

[0096] X2 can be S or G;

[0097] X3 can be T, Y, S, H, N, W, L, or Q;

[0098] X4 can be S or N;

[0099] X5 can be F, V or L;

[0100] X6 can be N or S;

[0101] X7 can be S or A;

[0102] X8 can be F or L;

[0103] X9 can be S, T, G, D, R or N; and / or

[0104] - HCDR3 having the amino acid sequence GGYTGX1GGDWFDX2, wherein

[0105] X1 can be Y, H, V or A;

[0106] X2 can be P, V, Y, W, F, T, Q, H or S.

[0107] Other suitable positions for introducing amino acid variations include, but are not limited to, the second, third, fourth and / or fifth amino acid of HCDR1; the third, fourth, fifth, sixth, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth, sixteenth and / or seventeenth amino acid of HCDR2; and / or the first, second, sixth, seventh, ninth, tenth, fourteenth, fifteenth, sixteenth and / or eighteenth amino acid of HCDR3.

[0108] In certain embodiments, the present disclosure therefore also provides multispecific binding moieties, the PD-1 binding domain of which comprises:

[0109] - HCDR1 having the amino acid sequence RX1X2X3X4, wherein

[0110] X1 can be F or Y;

[0111] X2 can be T, A or V;

[0112] X3 can be M, L or V;

[0113] X4 may be S, H, N, V ​​or T; and / or

[0114] - HCDR2, which has the amino acid sequence WIX1X2X3X4GX5X6X7X8X9X 10 X 11 X 12 X 13 X 14 ,in

[0115] X1 can be N or D;

[0116] X2 can be P, S or T;

[0117] X3 can be N or Q;

[0118] X4 can be T or D;

[0119] X5 can be N, S, T, K, L or E;

[0120] X6 can be P, Y, A, H or F;

[0121] X7 can be T or S;

[0122] X8 can be Y, F or H;

[0123] X9 can be A, G, V or F;

[0124] X 10 Can be Q, R, N, L, T or S;

[0125] X 11 It can be D, A, G or S;

[0126] X 12 It can be F, V or A;

[0127] X 13 It can be T, K, H, G;

[0128] X 14 Can be G, N, E or D; and / or

[0129] - HCDR3 having the amino acid sequence X1X2GYCX3X4DX5CYPNX6X7X8DX9, wherein

[0130] X1 can be I, S or V;

[0131] X2 can be L, Q or N;

[0132] X3 can be N, G, S or D;

[0133] X4 can be T, S, P, N or E;

[0134] X5 can be N or I;

[0135] X6 can be W, G, Q, H, W, A, or L;

[0136] X7 can be I, V or L;

[0137] X8 can be F, L or I;

[0138] X9 can be Y, S, N, I, R, H, V, T, K, A or L.

[0139] In certain embodiments, the PD-1 binding domain of the multispecific binding moiety of the present disclosure comprises a heavy chain variable region having an amino acid sequence as set forth in any one of SEQ ID NOs: 1, 5, 9, 13, 14, 18, and 19, or a variant thereof. In certain embodiments, the PD-1 binding domain of the multispecific binding moiety of the present disclosure comprises a heavy chain variable region having an amino acid sequence as set forth in any one of SEQ ID NOs: 1, 5, 9, 13, 14, 18, and 19, or a variant thereof having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity thereto.

[0140] "Percentage (%) identity" refers to nucleic acid or amino acid sequences in this article and is defined as the percentage of residues in a candidate sequence that are identical to the residues in a selected sequence after the sequences are aligned for optimal comparison purposes. In order to optimize the alignment between the two sequences, a gap may be introduced in either of the two sequences being compared. This alignment may be performed over the full length of the sequences being compared. Alternatively, the alignment may be performed over a shorter length, such as about 20, about 50, about 100 or more nucleic acids / bases or amino acids. Sequence identity is the percentage of identity matches between the two sequences over the reported alignment region.

[0141] The comparison of sequences and the determination of the percent sequence identity between two sequences can be accomplished using a mathematical algorithm. The skilled person will be aware of the fact that several different computer programs can be used to compare two sequences and determine the identity between two sequences (Kruskal, JB (1983) An overview of sequence comparison, in D. Sankoff and JB Kruskal, (eds.), Time warps, string edits and macromolecules: the theory and practice of sequence comparison, pp. 1-44, Addison Wesley). The percent sequence identity between two amino acid sequences or nucleic acid sequences can be determined using the Needleman and Wunsch algorithm for the comparison of two sequences. (Needleman, SB and Wunsch, CD (1970) J. Mol. Biol, Vol. 48, pp. 443-453). The Needleman-Wunsch algorithm has been implemented in the computer program NEEDLE. For the purpose of the present invention, the NEEDLE program from the EMBOSS package is used to determine the identity percentage of amino acid and nucleic acid sequences (version 2.8.0, EMBOSS: The European Molecular Biology Open Software Suite (2000), Rice, P. Longden J. and Bleasby A., Trends in Genetics, Vol. 16, No. 6, pp. 276-277, http: / / emboss.bioinformatics.nl / ). For protein sequences, EBLOSUM62 is used to replace the matrix. For DNA sequences, DNAFULL is used. The parameters used are a gap opening penalty of 10 and a gap extension penalty of 0.5.

[0142] After alignment by the program NEEDLE as described above, the percentage of sequence identity between the query sequence and the sequence of the invention is calculated as follows: the number of corresponding positions in the alignment showing the same amino acid or the same nucleotide in both sequences is divided by the total length of the alignment after subtracting the total number of gaps in the alignment.

[0143] In certain embodiments, the PD-1 binding domain of the multispecific binding portion of the present disclosure further comprises a PD-1 binding domain variant, which, in addition to the variations in the HCDR mentioned above, also comprises one or more variations in the framework region. The variation can be any type of amino acid variation described herein, such as, for example, a conservative amino acid substitution or a non-conservative amino acid substitution caused by somatic hypermutation or affinity maturation. In certain embodiments, the PD-1 binding domain variant of the multispecific binding portion of the present disclosure does not comprise a variation in the CDR region, but comprises 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 the sequences disclosed herein, and are expected to retain PD-1 binding specificity. Therefore, in certain embodiments, the PD-1 binding domain of the multispecific binding portion of the present disclosure comprises:

[0144] - a heavy chain variable region having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the amino acid sequence shown in SEQ ID NO: 1, the heavy chain variable region comprising 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;

[0145] - a heavy chain variable region having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the amino acid sequence shown in SEQ ID NO:5, the heavy chain variable region comprising 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;

[0146] - a heavy chain variable region having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the amino acid sequence shown in SEQ ID NO:9, the heavy chain variable region comprising 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;

[0147] - a heavy chain variable region having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the amino acid sequence shown in SEQ ID NO: 13, the heavy chain variable region comprising 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;

[0148] - a heavy chain variable region having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the amino acid sequence shown in SEQ ID NO: 14, the heavy chain variable region comprising 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;

[0149] - a heavy chain variable region having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the amino acid sequence shown in SEQ ID NO: 18, the heavy chain variable region comprising 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

[0150] - a heavy chain variable region having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the amino acid sequence shown in SEQ ID NO: 19, the heavy chain variable region comprising 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.

[0151] The binding domains of the multispecific binding moieties of the present disclosure have been generated with a common light chain, in particular with a common light chain called VK1-39 / JK1. The binding domains of the multispecific binding moieties of the present disclosure may comprise any suitable light chain, including but not limited to common light chains known in the art. In certain embodiments, the binding domains of the multispecific binding moieties of the present disclosure comprise the common light chain VK1-39 / JK1, or variants thereof containing a limited number, such as, for example, one, two or three non-conservative amino acid substitutions, or an unlimited number of conservative amino acid substitutions.

[0152] In certain embodiments, the PD-1 binding domain of the multispecific binding moiety of the present disclosure comprises a light chain variable region having an amino acid sequence as set forth in SEQ ID NO: 48, or a variant thereof. In certain embodiments, the PD-1 binding domain of the multispecific binding moiety of the present disclosure comprises a light chain variable region having an amino acid sequence as set forth in SEQ ID NO: 48, or a variant thereof having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity thereto.

[0153] In certain embodiments, the PD-1 binding domain of the multispecific binding moiety of the present disclosure comprises a light chain variable region comprising a light chain CDR1 (LCDR1), a light chain CDR2 (LCDR2), and a light chain CDR3 (LCDR3) having an amino acid sequence as shown in SEQ ID NO: 49, SEQ ID NO: 50, and SEQ ID NO: 51. In certain embodiments, the light chain variable region of the PD-1 binding domain of the multispecific binding moiety of the present disclosure also includes variants thereof, wherein each of the LCDRs can comprise up to three, two, or one amino acid variation. In certain embodiments, the amino acid variation is a conservative amino acid substitution.

[0154] In certain embodiments, the PD-1 binding domain of the multispecific binding portion of the present disclosure also includes a PD-1 binding domain variant, which includes one or more variations in the framework region in addition to the variations in the LCDR mentioned above. The variation is preferably a conservative amino acid substitution. In certain embodiments, the PD-1 binding domain variant of the multispecific binding portion of the present 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 the sequences disclosed herein. Therefore, in certain embodiments, the PD-1 binding domain of the multispecific binding portion of the present disclosure includes:

[0155] - a light chain variable region having at least 80%, at least 85%, at least 90% or at least 95% sequence identity to the amino acid sequence shown in SEQ ID NO:48, the light chain variable region comprising 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.

[0156] The light chain or light chain variable region comprising these LCDRs and / or light chain variable regions can be, for example, a light chain referred to as VK1-39 / JK1 in the art. This is a common light chain. According to the term "common light chain" disclosed herein, it refers to a light chain that can be paired with a plurality of different heavy chains, such as, for example, a heavy chain with different antigen or epitope binding specificity. Common light chains can be particularly useful for generating, for example, bispecific antibodies or multispecific antibodies, wherein when all binding domains comprise the same light chain, antibody production is more effective. The term "common light chain" encompasses light chains that are identical or have some amino acid sequence differences, but the binding specificity of the full-length antibody is not affected. For example, within the definition of a common light chain as used herein, light chains that are not identical but still functionally equivalent can be prepared or found, for example, by using established variations that introduce conservative amino acid changes, known or shown to not contribute or only partially contribute to the changes in amino acids in the region of binding specificity when paired with the heavy chain, and the like.

[0157] In addition to the common light chains comprising the LCDRs and / or light chain variable regions mentioned above, other common light chains known in the art can be used. Examples of these common light chains include, but are not limited to, VK1-39 / JK5, ​​which comprises a light chain variable region comprising a light chain CDR1 (LCDR1), a light chain CDR2 (LCDR2), and a light chain CDR3 (LCDR3) of a light chain variable region having an amino acid sequence as shown in SEQ ID NO: 52. In certain embodiments, the light chain comprises a light chain variable region comprising a light chain CDR1 (LCDR1), a light chain CDR2 (LCDR2), and a light chain CDR3 (LCDR3) of a light chain variable region having an amino acid sequence as shown in SEQ ID NO: 52, wherein each of the LCDRs can comprise up to three, two, or one amino acid variation, such as a substitution. In certain embodiments, the light chain comprises a light chain variable region having an amino acid sequence as shown in SEQ ID NO: 52, or an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity thereto. In certain embodiments, the light chain comprises a light chain CDR1 (LCDR1), a light chain CDR2 (LCDR2), and a light chain CDR3 (LCDR3) having an amino acid sequence as shown in SEQ ID NO: 53, SEQ ID NO: 54, and SEQ ID NO: 55; VK3-15 / JK1, which comprises a light chain variable region comprising a light chain CDR1 (LCDR1), a light chain CDR2 (LCDR2), and a light chain CDR3 (LCDR3) having an amino acid sequence as shown in SEQ ID NO: 56. In certain embodiments, the light chain comprises a light chain variable region comprising a light chain CDR1 (LCDR1), a light chain CDR2 (LCDR2), and a light chain CDR3 (LCDR3) of a light chain variable region having an amino acid sequence as shown in SEQ ID NO: 56, wherein each of the LCDRs can comprise up to three, two, or one amino acid variation, e.g., a substitution. In certain embodiments, the light chain comprises a light chain variable region having an amino acid sequence as shown in SEQ ID NO: 56, or an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity thereto.In certain embodiments, the light chain comprises a light chain CDR1 (LCDR1), a light chain CDR2 (LCDR2), and a light chain CDR3 (LCDR3) having an amino acid sequence as shown in SEQ ID NO: 57, SEQ ID NO: 58, and SEQ ID NO: 59; VK3-20 / JK1, which comprises a light chain variable region comprising a light chain CDR1 (LCDR1), a light chain CDR2 (LCDR2), and a light chain CDR3 (LCDR3) having an amino acid sequence as shown in SEQ ID NO: 60. In certain embodiments, the light chain comprises a light chain variable region comprising a light chain CDR1 (LCDR1), a light chain CDR2 (LCDR2), and a light chain CDR3 (LCDR3) having an amino acid sequence as shown in SEQ ID NO: 60, wherein each of the LCDRs can comprise up to three, two, or one amino acid variation, such as a substitution. In certain embodiments, the light chain comprises a light chain variable region having an amino acid sequence as shown in SEQ ID NO: 60, or an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity thereto. In certain embodiments, the light chain comprises a light chain CDR1 (LCDR1), a light chain CDR2 (LCDR2), and a light chain CDR3 (LCDR3) having an amino acid sequence as shown in SEQ ID NO: 61, SEQ ID NO: 62, and SEQ ID NO: 63; VL3-21 / JL3, which comprises a light chain variable region comprising a light chain CDR1 (LCDR1), a light chain CDR2 (LCDR2), and a light chain CDR3 (LCDR3) having an amino acid sequence as shown in SEQ ID NO: 64. In certain embodiments, the light chain comprises a light chain variable region comprising a light chain CDR1 (LCDR1), a light chain CDR2 (LCDR2), and a light chain CDR3 (LCDR3) of a light chain variable region having an amino acid sequence as shown in SEQ ID NO: 64, wherein each of the LCDRs can comprise up to three, two, or one amino acid variation, e.g., a substitution. In certain embodiments, the light chain comprises a light chain variable region having an amino acid sequence as shown in SEQ ID NO: 64, or an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity thereto. In certain embodiments, the light chain comprises a light chain CDR1 (LCDR1), a light chain CDR2 (LCDR2), and a light chain CDR3 (LCDR3) having an amino acid sequence as shown in SEQ ID NO: 65, SEQ ID NO: 66, and SEQ ID NO: 67.

[0158] VK1-39 is the abbreviation of immunoglobulin variable kappa 1-39 gene. The gene is also called immunoglobulin kappa variable 1-39; IGKV139; IGKV1-39; IgVκ1-39. The external ID of the 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 be combined with one of the five J regions. Suitable VJ region sequences are represented as VK1-39 / JK1 (SEQ ID NO: 94) and VK1-39 / JK5 (SEQ ID NO: 95); aliases are IgVκ1-39*01 / IGJκ1*01 or IgVκ1-39*01 / IGJκ5*01 (named according to the global network of IMGT databases, imgt.org). These designations are exemplary and include allelic variants of the gene segments.

[0159] VK3-15 is the abbreviation of immunoglobulin variable κ3-15 gene. The gene is also called immunoglobulin κ variable 3-15, IGKV315, IGKV3-15, IgVκ3-15. The external ID of the 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 combined with one of the five J regions. A suitable VJ region sequence is represented as VK3-15 / JK1 (SEQ ID NO: 99); the alias is Vκ3-15*01 / IGJκ1*01 (named according to the global network of the IMGT database, imgt.org). The name is exemplary and includes allelic variants of the gene fragment.

[0160] VK3-20 is the abbreviation of immunoglobulin variable kappa 3-20 gene. The gene is also known as immunoglobulin kappa variable 3-20; IGKV320; IGKV3-20; IgVκ3-20. The external ID of the gene is HGNC: 5817; Entrez gene: 28912; Ensembl: ENSG00000239951. The amino acid sequence of VK3-20 is represented by SEQ ID NO: 100. This is the sequence of the V region. The V region can be combined with one of the five J regions. A suitable VJ region sequence is represented by VK3-20 / JK1 (SEQ ID NO: 101); the alias is IgVκ3-20*01 / IGJκ1*01 (named according to the global network of the IMGT database, imgt.org). The name is exemplary and includes allelic variants of the gene fragment.

[0161] VL3-21 is the abbreviation of immunoglobulin variable λ3-21 gene. The gene is also called immunoglobulin λ variable 3-21, IGLV321, IGLV3-21, IgVλ3-21. The external ID of the gene is HGNC: 5905; Entrez gene: 28796; Ensembl: ENSG00000211662.2. The amino acid sequence of VL3-21 is given as SEQ ID NO: 102. This is the sequence of the V region. The V region can be combined with one of the five J regions. A suitable VJ region sequence is represented as VL3-21 / JL3 (SEQ ID NO: 103); the alias is IgVλ3-21 / IGJλ3 (named according to the global network of the IMGT database, imgt.org). The name is exemplary and includes allelic variants of the gene fragment.

[0162] Furthermore, any light chain variable region of a PD-1 antibody available in the art may be used, as may any other light chain variable region that can be readily obtained, such as, for example, a light chain variable region obtained from an antibody display library by showing antigen binding activity when paired with a PD-1 binding domain of a multispecific binding moiety of the present disclosure.

[0163] In certain embodiments, the PD-1 binding domain of the multispecific binding moiety of the present disclosure may also comprise a CH1 region and a CL region. Any CH1 domain may be used, in particular a human CH1 domain. An example of a suitable CH1 domain is provided by the amino acid sequence provided as SEQ ID NO: 69. Any CL domain may be used, in particular a human CL. An example of a suitable CL domain is provided by the amino acid sequence provided as SEQ ID NO: 75.

[0164] In certain embodiments, the TGF-βRII binding domain of the multispecific binding moieties of the present disclosure comprises a heavy chain variable region comprising:

[0165] a) heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2) and heavy chain CDR3 (HCDR3), which have the amino acid sequences shown in SEQ ID NO: 24, SEQ ID NO: 25 and SEQ ID NO: 26, respectively;

[0166] b) heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2) and heavy chain CDR3 (HCDR3), which have the amino acid sequences shown in SEQ ID NO: 28, SEQ ID NO: 29 and SEQ ID NO: 30, respectively;

[0167] c) heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2) and heavy chain CDR3 (HCDR3), which have the amino acid sequences shown in SEQ ID NO: 32, SEQ ID NO: 33 and SEQ ID NO: 34, respectively;

[0168] d) heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2) and heavy chain CDR3 (HCDR3), which have the amino acid sequences shown in SEQ ID NO: 36, SEQ ID NO: 37 and SEQ ID NO: 38, respectively;

[0169] e) heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2) and heavy chain CDR3 (HCDR3), which have the amino acid sequences shown in SEQ ID NO:40, SEQ ID NO:41 and SEQ ID NO:42, respectively;

[0170] f) heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2) and heavy chain CDR3 (HCDR3), which have the amino acid sequences shown in SEQ ID NO:44, SEQ ID NO:45 and SEQ ID NO:46, respectively; or

[0171] g) heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2) and heavy chain CDR3 (HCDR3), which have the amino acid sequences shown in SEQ ID NO:90, SEQ ID NO:91 and SEQ ID NO:92, respectively.

[0172] The heavy chain variable region of the TGF-βRII binding domain of the multispecific binding moieties of the disclosure may comprise a limited number, such as, for example, one, two or three, non-conservative amino acid substitutions, or an unlimited number of conservative amino acid substitutions.

[0173] In certain embodiments, the TGF-βRII binding domain of the multispecific binding moiety of the present disclosure also includes a TGF-βRII binding domain variant thereof, wherein each of the HCDRs can comprise up to three, two, or one amino acid variation. In certain embodiments, only one or two HCDRs can comprise up to three, two, or one amino acid variation. In certain embodiments, such variants do not comprise an amino acid variation in HCDR3. In certain embodiments, the amino acid variation is a conservative amino acid substitution. Conservative amino acid substitutions are as further described herein.

[0174] TGF-βRII binding variants encompassed by the present disclosure include somatically hypermutated or affinity matured heavy chain variable regions, which are heavy chain variable regions derived from the same VH gene fragment as the heavy chain variable region described in the sequence herein, and these variants have amino acid variations, including non-conservative and / or conservative amino acid substitutions in one, two or all three HCDRs.

[0175] In certain embodiments, the TGF-βRII binding domain of the multispecific binding moieties of the present disclosure comprises a heavy chain variable region having an amino acid sequence as set forth in any one of SEQ ID NOs: 23, 27, 31, 35, 39, 43, 47, 88, and 89, or a variant thereof. In certain embodiments, the TGF-βRII binding domain of the multispecific binding moieties of the present disclosure comprises a heavy chain variable region having an amino acid sequence as set forth in any one of SEQ ID NOs: 23, 27, 31, 35, 39, 43, 47, 88, and 89, or an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity thereto.

[0176] In certain embodiments, the TGF-βRII binding domain of the multispecific binding portion of the present disclosure also includes a TGF-βRII binding domain variant, which, in addition to the variations in the HCDR mentioned above, also includes one or more variations in the framework region. The variation can be any type of amino acid variation described herein, such as, for example, conservative amino acid substitutions or non-conservative amino acid substitutions caused by somatic hypermutation or affinity maturation. In certain embodiments, the TGF-βRII binding domain variant of the multispecific binding portion of the present disclosure does not include variations in the CDR 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 the sequences disclosed herein, and are expected to retain TGF-βRII binding specificity. Therefore, in certain embodiments, the TGF-βRII binding domain of the multispecific binding portion of the present disclosure comprises:

[0177] - a heavy chain variable region having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the amino acid sequence shown in SEQ ID NO:23, the heavy chain variable region comprising 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;

[0178] - a heavy chain variable region having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the amino acid sequence shown in SEQ ID NO: 27, the heavy chain variable region comprising 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;

[0179] - a heavy chain variable region having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the amino acid sequence shown in SEQ ID NO:31, the heavy chain variable region comprising 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;

[0180] - a heavy chain variable region having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the amino acid sequence shown in SEQ ID NO:35, the heavy chain variable region comprising 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;

[0181] - a heavy chain variable region having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the amino acid sequence shown in SEQ ID NO:39, the heavy chain variable region comprising 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;

[0182] - a heavy chain variable region having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the amino acid sequence shown in SEQ ID NO:43, the heavy chain variable region comprising 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;

[0183] - a heavy chain variable region having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the amino acid sequence shown in SEQ ID NO:47, the heavy chain variable region comprising 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;

[0184] - a heavy chain variable region having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the amino acid sequence shown in SEQ ID NO:88, the heavy chain variable region comprising 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

[0185] - a heavy chain variable region having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the amino acid sequence shown in SEQ ID NO:89, the heavy chain variable region comprising 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.

[0186] Any light chain variable region of a TGF-βRII antibody available in the art may be used, e.g., as described herein, as may any other light chain variable region that can be readily obtained, such as, for example, a light chain variable region obtained from an antibody display library by showing antigen binding activity when paired with a TGF-βRII binding domain of a multispecific binding moiety of the present disclosure. In certain embodiments, the TGF-βRII binding domain of a multispecific binding moiety of the present disclosure comprises a light chain that is identical or substantially identical to a PD-1 binding domain.

[0187] In certain embodiments, the TGF-βRII binding domain of the multispecific binding moieties of the present disclosure comprises a light chain variable region having an amino acid sequence as set forth in SEQ ID NO: 48, or a variant thereof. In certain embodiments, the TGF-βRII binding domain of the multispecific binding moieties of the present disclosure comprises a light chain variable region having an amino acid sequence as set forth in SEQ ID NO: 48, or a variant thereof having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity thereto.

[0188] In certain embodiments, the TGF-βRII binding domain of the multispecific binding moieties of the present disclosure comprises a light chain variable region comprising a light chain CDR1 (LCDR1), a light chain CDR2 (LCDR2), and a light chain CDR3 (LCDR3) having the amino acid sequences shown in SEQ ID NO: 49, SEQ ID NO: 50, and SEQ ID NO: 51, respectively. In certain embodiments, the light chain variable region of the TGF-βRII binding domain of the multispecific binding moieties of the present disclosure also includes variants thereof, wherein each of the LCDRs can comprise up to three, two, or one conservative or non-conservative amino acid variation. In certain embodiments, the amino acid variation is a conservative amino acid substitution.

[0189] In certain embodiments, the TGF-βRII binding domain of the multispecific binding moiety of the present disclosure may also comprise a CH1 region and a CL region. Any CH1 domain may be used, in particular a human CH1 domain. An example of a suitable CH1 domain is provided by the amino acid sequence provided as SEQ ID NO: 69. Any CL domain may be used, in particular a human CL. An example of a suitable CL domain is provided by the amino acid sequence provided as SEQ ID NO: 75.

[0190] Thus, the present invention also provides a multispecific binding moiety comprising a PD-1 binding domain and a TGF-βRII binding domain, wherein the PD-1 binding domain comprises a heavy chain variable region, and optionally a light chain variable region, and a CH1 region and a CL region, as described herein. In certain embodiments, the multispecific binding moiety further comprises a TGF-βRII binding domain, which comprises a heavy chain variable region, and optionally a light chain variable region, and a CH1 region and a CL region, as described herein.

[0191] Therefore, the present invention also provides a multispecific binding portion comprising a PD-1 binding domain and a TGF-βRII binding domain, wherein the TGF-βRII binding domain comprises a heavy chain variable region, and optionally a light chain variable region and a CH1 region and a CL region, as described herein. In certain embodiments, the multispecific binding portion further comprises a PD-1 binding domain, which comprises a heavy chain variable region, and optionally a light chain variable region and a CH1 region and a CL region, as described herein.

[0192] In certain embodiments, any PD-1 binding domain disclosed herein can be combined with any TGF-βRII binding domain disclosed herein to generate a multispecific binding moiety of the present disclosure. Accordingly, the present disclosure provides exemplary multispecific binding moieties PB1-PB18 as presented in Table 1.

[0193] In one embodiment, the disclosure provides a multispecific binding moiety comprising:

[0194] - a PD-1 binding domain as described herein, comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2) and a heavy chain CDR3 (HCDR3) having the amino acid sequences as described in SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, respectively; and

[0195] - a TGF-βRII binding domain as described herein, comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2) and a heavy chain CDR3 (HCDR3) having the amino acid sequences shown in SEQ ID NO: 24, SEQ ID NO: 25 and SEQ ID NO: 26, respectively,

[0196] Each of the HCDRs can comprise up to three, two or one amino acid variation, such as a substitution. In certain embodiments, the HCDRs do not comprise an amino acid variation.

[0197] In one embodiment, the disclosure provides a multispecific binding moiety comprising:

[0198] - a PD-1 binding domain as described herein, comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) having the amino acid sequences as described in SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 17, respectively; and

[0199] - a TGF-βRII binding domain as described herein, comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2) and a heavy chain CDR3 (HCDR3) having the amino acid sequences shown in SEQ ID NO: 24, SEQ ID NO: 25 and SEQ ID NO: 26, respectively,

[0200] Each of the HCDRs can comprise up to three, two or one amino acid variation, such as a substitution. In certain embodiments, the HCDRs do not comprise an amino acid variation.

[0201] In one embodiment, the disclosure provides a multispecific binding moiety comprising:

[0202] - a PD-1 binding domain as described herein, comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) having the amino acid sequences of SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 22, respectively; and

[0203] - a TGF-βRII binding domain as described herein, comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2) and a heavy chain CDR3 (HCDR3) having the amino acid sequences shown in SEQ ID NO: 24, SEQ ID NO: 25 and SEQ ID NO: 26, respectively,

[0204] Each of the HCDRs can comprise up to three, two or one amino acid variation, such as a substitution. In certain embodiments, the HCDRs do not comprise an amino acid variation.

[0205] In one embodiment, the disclosure provides a multispecific binding moiety comprising:

[0206] - a PD-1 binding domain as described herein, comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) having the amino acid sequences as described in SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 17, respectively; and

[0207] - a TGF-βRII binding domain as described herein, comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2) and a heavy chain CDR3 (HCDR3) having the amino acid sequences shown in SEQ ID NO: 32, SEQ ID NO: 33 and SEQ ID NO: 34, respectively,

[0208] Each of the HCDRs can comprise up to three, two or one amino acid variation, such as a substitution. In certain embodiments, the HCDRs do not comprise an amino acid variation.

[0209] In one embodiment, the disclosure provides a multispecific binding moiety comprising:

[0210] - a PD-1 binding domain as described herein, comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2) and a heavy chain CDR3 (HCDR3) having the amino acid sequences as described in SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, respectively; and

[0211] - a TGF-βRII binding domain as described herein, comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2) and a heavy chain CDR3 (HCDR3) having the amino acid sequences shown in SEQ ID NO: 40, SEQ ID NO: 41 and SEQ ID NO: 42, respectively,

[0212] Each of the HCDRs can comprise up to three, two or one amino acid variation, such as a substitution. In certain embodiments, the HCDRs do not comprise an amino acid variation.

[0213] In one embodiment, the disclosure provides a multispecific binding moiety comprising:

[0214] - a PD-1 binding domain as described herein, comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2) and a heavy chain CDR3 (HCDR3) having the amino acid sequences as described in SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, respectively; and

[0215] - a TGF-βRII binding domain as described herein, comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2) and a heavy chain CDR3 (HCDR3) having the amino acid sequences shown in SEQ ID NO: 36, SEQ ID NO: 37 and SEQ ID NO: 38, respectively,

[0216] Each of the HCDRs can comprise up to three, two or one amino acid variation, such as a substitution. In certain embodiments, the HCDRs do not comprise an amino acid variation.

[0217] In one embodiment, the disclosure provides a multispecific binding moiety comprising:

[0218] - a PD-1 binding domain as described herein, comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) having the amino acid sequences as described in SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 17, respectively; and

[0219] - a TGF-βRII binding domain as described herein, comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2) and a heavy chain CDR3 (HCDR3) having the amino acid sequences shown in SEQ ID NO: 36, SEQ ID NO: 37 and SEQ ID NO: 38, respectively,

[0220] Each of the HCDRs can comprise up to three, two or one amino acid variation, such as a substitution. In certain embodiments, the HCDRs do not comprise an amino acid variation.

[0221] In one embodiment, the disclosure provides a multispecific binding moiety comprising:

[0222] - a PD-1 binding domain as described herein, comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) having the amino acid sequences of SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 22, respectively; and

[0223] - a TGF-βRII binding domain as described herein, comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2) and a heavy chain CDR3 (HCDR3) having the amino acid sequences shown in SEQ ID NO: 36, SEQ ID NO: 37 and SEQ ID NO: 38, respectively,

[0224] Each of the HCDRs can comprise up to three, two or one amino acid variation, such as a substitution. In certain embodiments, the HCDRs do not comprise an amino acid variation.

[0225] In one embodiment, the disclosure provides a multispecific binding moiety comprising:

[0226] - a PD-1 binding domain as described herein, comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2) and a heavy chain CDR3 (HCDR3) having the amino acid sequences as described in SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, respectively; and

[0227] - a TGF-βRII binding domain as described herein, comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2) and a heavy chain CDR3 (HCDR3) having the amino acid sequences shown in SEQ ID NO: 28, SEQ ID NO: 29 and SEQ ID NO: 30, respectively,

[0228] Each of the HCDRs can comprise up to three, two or one amino acid variation, such as a substitution. In certain embodiments, the HCDRs do not comprise an amino acid variation.

[0229] In one embodiment, the disclosure provides a multispecific binding moiety comprising:

[0230] - a PD-1 binding domain as described herein, comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2) and a heavy chain CDR3 (HCDR3) having the amino acid sequences as described in SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, respectively; and

[0231] - a TGF-βRII binding domain as described herein, comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2) and a heavy chain CDR3 (HCDR3) having the amino acid sequences shown in SEQ ID NO: 90, SEQ ID NO: 91 and SEQ ID NO: 92, respectively,

[0232] Each of the HCDRs can comprise up to three, two or one amino acid variation, such as a substitution. In certain embodiments, the HCDRs do not comprise an amino acid variation.

[0233] In one embodiment, the disclosure provides a multispecific binding moiety comprising:

[0234] - a PD-1 binding domain as described herein, comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) having the amino acid sequences of SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 22, respectively; and

[0235] - a TGF-βRII binding domain as described herein, comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2) and a heavy chain CDR3 (HCDR3) having the amino acid sequences shown in SEQ ID NO: 44, SEQ ID NO: 45 and SEQ ID NO: 46, respectively,

[0236] Each of the HCDRs can comprise up to three, two or one amino acid variation, such as a substitution. In certain embodiments, the HCDRs do not comprise an amino acid variation.

[0237] In one embodiment, the disclosure provides a multispecific binding moiety comprising:

[0238] - a PD-1 binding domain as described herein, comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2) and a heavy chain CDR3 (HCDR3) having the amino acid sequences as described in SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, respectively; and

[0239] - a TGF-βRII binding domain as described herein, comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2) and a heavy chain CDR3 (HCDR3) having the amino acid sequences shown in SEQ ID NO: 24, SEQ ID NO: 25 and SEQ ID NO: 26, respectively,

[0240] wherein the PD-1 binding domain and the TGF-βRII binding domain comprise a light chain CDR1 (LCDR1) having an amino acid sequence as shown in SEQ ID NO:49, a light chain CDR2 (LCDR2) having an amino acid sequence as shown in SEQ ID NO:50, and a light chain CDR3 (LCDR3) having an amino acid sequence as shown in SEQ ID NO:51, and

[0241] Each of the HCDRs and / or LCDRs may comprise up to three, two or one amino acid variations, such as substitutions. In certain embodiments, the HCDRs and / or LCDRs do not comprise amino acid variations.

[0242] In one embodiment, the disclosure provides a multispecific binding moiety comprising:

[0243] - a PD-1 binding domain as described herein, comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) having the amino acid sequences as described in SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 17, respectively; and

[0244] - a TGF-βRII binding domain as described herein, comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2) and a heavy chain CDR3 (HCDR3) having the amino acid sequences shown in SEQ ID NO: 24, SEQ ID NO: 25 and SEQ ID NO: 26, respectively,

[0245] wherein the PD-1 binding domain and the TGF-βRII binding domain comprise a light chain CDR1 (LCDR1) having an amino acid sequence as shown in SEQ ID NO:49, a light chain CDR2 (LCDR2) having an amino acid sequence as shown in SEQ ID NO:50, and a light chain CDR3 (LCDR3) having an amino acid sequence as shown in SEQ ID NO:51, and

[0246] Each of the HCDRs and / or LCDRs may comprise up to three, two or one amino acid variations, such as substitutions. In certain embodiments, the HCDRs and / or LCDRs do not comprise amino acid variations.

[0247] In one embodiment, the disclosure provides a multispecific binding moiety comprising:

[0248] - a PD-1 binding domain as described herein, comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) having the amino acid sequences of SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 22, respectively; and

[0249] - a TGF-βRII binding domain as described herein, comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2) and a heavy chain CDR3 (HCDR3) having the amino acid sequences shown in SEQ ID NO: 24, SEQ ID NO: 25 and SEQ ID NO: 26, respectively,

[0250] wherein the PD-1 binding domain and the TGF-βRII binding domain comprise a light chain CDR1 (LCDR1) having an amino acid sequence as shown in SEQ ID NO:49, a light chain CDR2 (LCDR2) having an amino acid sequence as shown in SEQ ID NO:50, and a light chain CDR3 (LCDR3) having an amino acid sequence as shown in SEQ ID NO:51, and

[0251] Each of the HCDRs and / or LCDRs may comprise up to three, two or one amino acid variations, such as substitutions. In certain embodiments, the HCDRs and / or LCDRs do not comprise amino acid variations.

[0252] In one embodiment, the disclosure provides a multispecific binding moiety comprising:

[0253] - a PD-1 binding domain as described herein, comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) having the amino acid sequences as described in SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 17, respectively; and

[0254] - a TGF-βRII binding domain as described herein, comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2) and a heavy chain CDR3 (HCDR3) having the amino acid sequences shown in SEQ ID NO: 32, SEQ ID NO: 33 and SEQ ID NO: 34, respectively,

[0255] wherein the PD-1 binding domain and the TGF-βRII binding domain comprise a light chain CDR1 (LCDR1) having an amino acid sequence as shown in SEQ ID NO:49, a light chain CDR2 (LCDR2) having an amino acid sequence as shown in SEQ ID NO:50, and a light chain CDR3 (LCDR3) having an amino acid sequence as shown in SEQ ID NO:51, and

[0256] Each of the HCDRs and / or LCDRs may comprise up to three, two or one amino acid variations, such as substitutions. In certain embodiments, the HCDRs and / or LCDRs do not comprise amino acid variations.

[0257] In one embodiment, the disclosure provides a multispecific binding moiety comprising:

[0258] - a PD-1 binding domain as described herein, comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2) and a heavy chain CDR3 (HCDR3) having the amino acid sequences as described in SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, respectively; and

[0259] - a TGF-βRII binding domain as described herein, comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2) and a heavy chain CDR3 (HCDR3) having the amino acid sequences shown in SEQ ID NO: 40, SEQ ID NO: 41 and SEQ ID NO: 42, respectively,

[0260] wherein the PD-1 binding domain and the TGF-βRII binding domain comprise a light chain CDR1 (LCDR1) having an amino acid sequence as shown in SEQ ID NO:49, a light chain CDR2 (LCDR2) having an amino acid sequence as shown in SEQ ID NO:50, and a light chain CDR3 (LCDR3) having an amino acid sequence as shown in SEQ ID NO:51, and

[0261] Each of the HCDRs and / or LCDRs may comprise up to three, two or one amino acid variations, such as substitutions. In certain embodiments, the HCDRs and / or LCDRs do not comprise amino acid variations.

[0262] In one embodiment, the disclosure provides a multispecific binding moiety comprising:

[0263] - a PD-1 binding domain as described herein, comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2) and a heavy chain CDR3 (HCDR3) having the amino acid sequences as described in SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, respectively; and

[0264] - a TGF-βRII binding domain as described herein, comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2) and a heavy chain CDR3 (HCDR3) having the amino acid sequences shown in SEQ ID NO: 36, SEQ ID NO: 37 and SEQ ID NO: 38, respectively,

[0265] wherein the PD-1 binding domain and the TGF-βRII binding domain comprise a light chain CDR1 (LCDR1) having an amino acid sequence as shown in SEQ ID NO:49, a light chain CDR2 (LCDR2) having an amino acid sequence as shown in SEQ ID NO:50, and a light chain CDR3 (LCDR3) having an amino acid sequence as shown in SEQ ID NO:51, and

[0266] Each of the HCDRs and / or LCDRs may comprise up to three, two or one amino acid variations, such as substitutions. In certain embodiments, the HCDRs and / or LCDRs do not comprise amino acid variations.

[0267] In one embodiment, the disclosure provides a multispecific binding moiety comprising:

[0268] - a PD-1 binding domain as described herein, comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) having the amino acid sequences as described in SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 17, respectively; and

[0269] - a TGF-βRII binding domain as described herein, comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2) and a heavy chain CDR3 (HCDR3) having the amino acid sequences shown in SEQ ID NO: 36, SEQ ID NO: 37 and SEQ ID NO: 38, respectively,

[0270] wherein the PD-1 binding domain and the TGF-βRII binding domain comprise a light chain CDR1 (LCDR1) having an amino acid sequence as shown in SEQ ID NO:49, a light chain CDR2 (LCDR2) having an amino acid sequence as shown in SEQ ID NO:50, and a light chain CDR3 (LCDR3) having an amino acid sequence as shown in SEQ ID NO:51, and

[0271] Each of the HCDRs and / or LCDRs may comprise up to three, two or one amino acid variations, such as substitutions. In certain embodiments, the HCDRs and / or LCDRs do not comprise amino acid variations.

[0272] In one embodiment, the disclosure provides a multispecific binding moiety comprising:

[0273] - a PD-1 binding domain as described herein, comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) having the amino acid sequences of SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 22, respectively; and

[0274] - a TGF-βRII binding domain as described herein, comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2) and a heavy chain CDR3 (HCDR3) having the amino acid sequences shown in SEQ ID NO: 36, SEQ ID NO: 37 and SEQ ID NO: 38, respectively,

[0275] wherein the PD-1 binding domain and the TGF-βRII binding domain comprise a light chain CDR1 (LCDR1) having an amino acid sequence as shown in SEQ ID NO:49, a light chain CDR2 (LCDR2) having an amino acid sequence as shown in SEQ ID NO:50, and a light chain CDR3 (LCDR3) having an amino acid sequence as shown in SEQ ID NO:51, and

[0276] Each of the HCDRs and / or LCDRs may comprise up to three, two or one amino acid variations, such as substitutions. In certain embodiments, the HCDRs and / or LCDRs do not comprise amino acid variations.

[0277] In one embodiment, the disclosure provides a multispecific binding moiety comprising:

[0278] - a PD-1 binding domain as described herein, comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2) and a heavy chain CDR3 (HCDR3) having the amino acid sequences as described in SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, respectively; and

[0279] - a TGF-βRII binding domain as described herein, comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2) and a heavy chain CDR3 (HCDR3) having the amino acid sequences shown in SEQ ID NO: 28, SEQ ID NO: 29 and SEQ ID NO: 30, respectively,

[0280] wherein the PD-1 binding domain and the TGF-βRII binding domain comprise a light chain CDR1 (LCDR1) having an amino acid sequence as shown in SEQ ID NO:49, a light chain CDR2 (LCDR2) having an amino acid sequence as shown in SEQ ID NO:50, and a light chain CDR3 (LCDR3) having an amino acid sequence as shown in SEQ ID NO:51, and

[0281] Each of the HCDRs and / or LCDRs may comprise up to three, two or one amino acid variations, such as substitutions. In certain embodiments, the HCDRs and / or LCDRs do not comprise amino acid variations.

[0282] In one embodiment, the disclosure provides a multispecific binding moiety comprising:

[0283] - a PD-1 binding domain as described herein, comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2) and a heavy chain CDR3 (HCDR3) having the amino acid sequences as described in SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, respectively; and

[0284] - a TGF-βRII binding domain as described herein, comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2) and a heavy chain CDR3 (HCDR3) having the amino acid sequences shown in SEQ ID NO: 90, SEQ ID NO: 91 and SEQ ID NO: 92, respectively,

[0285] wherein the PD-1 binding domain and the TGF-βRII binding domain comprise a light chain CDR1 (LCDR1) having an amino acid sequence as shown in SEQ ID NO:49, a light chain CDR2 (LCDR2) having an amino acid sequence as shown in SEQ ID NO:50, and a light chain CDR3 (LCDR3) having an amino acid sequence as shown in SEQ ID NO:51, and

[0286] Each of the HCDRs and / or LCDRs may comprise up to three, two or one amino acid variations, such as substitutions. In certain embodiments, the HCDRs and / or LCDRs do not comprise amino acid variations.

[0287] In one embodiment, the disclosure provides a multispecific binding moiety comprising:

[0288] - a PD-1 binding domain as described herein, comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) having the amino acid sequences of SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 22, respectively; and

[0289] - a TGF-βRII binding domain as described herein, comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2) and a heavy chain CDR3 (HCDR3) having the amino acid sequences shown in SEQ ID NO: 44, SEQ ID NO: 45 and SEQ ID NO: 46, respectively,

[0290] wherein the PD-1 binding domain and the TGF-βRII binding domain comprise a light chain CDR1 (LCDR1) having an amino acid sequence as shown in SEQ ID NO:49, a light chain CDR2 (LCDR2) having an amino acid sequence as shown in SEQ ID NO:50, and a light chain CDR3 (LCDR3) having an amino acid sequence as shown in SEQ ID NO:51, and

[0291] Each of the HCDRs and / or LCDRs may comprise up to three, two or one amino acid variations, such as substitutions. In certain embodiments, the HCDRs and / or LCDRs do not comprise amino acid variations.

[0292] In one embodiment, the disclosure provides a multispecific binding moiety comprising:

[0293] - a PD-1 binding domain as described herein, comprising a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 9, or a heavy chain variable region having at least 80%, at least 85%, at least 90% or at least 95% sequence identity thereto; and

[0294] - A TGF-βRII binding domain as described herein, comprising a heavy chain variable region having an amino acid sequence as 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 certain embodiments, each of the heavy chain variable regions comprises a HCDR that does not comprise an amino acid variation. In certain embodiments, each of the heavy chain variable regions does not comprise an amino acid variation.

[0295] In one embodiment, the disclosure provides a multispecific binding moiety comprising:

[0296] - a PD-1 binding domain as described herein, comprising a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 18, or a heavy chain variable region having at least 80%, at least 85%, at least 90% or at least 95% sequence identity thereto; and

[0297] - A TGF-βRII binding domain as described herein, comprising a heavy chain variable region having an amino acid sequence as 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 certain embodiments, each of the heavy chain variable regions comprises a HCDR that does not comprise an amino acid variation. In certain embodiments, each of the heavy chain variable regions does not comprise an amino acid variation.

[0298] In one embodiment, the disclosure provides a multispecific binding moiety comprising:

[0299] - a PD-1 binding domain as described herein, comprising a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 19, or a heavy chain variable region having at least 80%, at least 85%, at least 90% or at least 95% sequence identity thereto; and

[0300] - A TGF-βRII binding domain as described herein, comprising a heavy chain variable region having an amino acid sequence as 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 certain embodiments, each of the heavy chain variable regions comprises a HCDR that does not comprise an amino acid variation. In certain embodiments, each of the heavy chain variable regions does not comprise an amino acid variation.

[0301] In one embodiment, the disclosure provides a multispecific binding moiety comprising:

[0302] - a PD-1 binding domain as described herein, comprising a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 14, or a heavy chain variable region having at least 80%, at least 85%, at least 90% or at least 95% sequence identity thereto; and

[0303] - A TGF-βRII binding domain as described herein, comprising a heavy chain variable region having an amino acid sequence as 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 certain embodiments, each of the heavy chain variable regions comprises a HCDR that does not comprise an amino acid variation. In certain embodiments, each of the heavy chain variable regions does not comprise an amino acid variation.

[0304] In one embodiment, the disclosure provides a multispecific binding moiety comprising:

[0305] - a PD-1 binding domain as described herein, comprising a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 9, or a heavy chain variable region having at least 80%, at least 85%, at least 90% or at least 95% sequence identity thereto; and

[0306] - A TGF-βRII binding domain as described herein, comprising a heavy chain variable region having an amino acid sequence as 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 certain embodiments, each of the heavy chain variable regions comprises a HCDR that does not comprise an amino acid variation. In certain embodiments, each of the heavy chain variable regions does not comprise an amino acid variation.

[0307] In one embodiment, the disclosure provides a multispecific binding moiety comprising:

[0308] - a PD-1 binding domain as described herein, comprising a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 9, or a heavy chain variable region having at least 80%, at least 85%, at least 90% or at least 95% sequence identity thereto; and

[0309] - A TGF-βRII binding domain as described herein, comprising a heavy chain variable region having an amino acid sequence as 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 certain embodiments, each of the heavy chain variable regions comprises a HCDR that does not comprise an amino acid variation. In certain embodiments, each of the heavy chain variable regions does not comprise an amino acid variation.

[0310] In one embodiment, the disclosure provides a multispecific binding moiety comprising:

[0311] - a PD-1 binding domain as described herein, comprising a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 14, or a heavy chain variable region having at least 80%, at least 85%, at least 90% or at least 95% sequence identity thereto; and

[0312] - A TGF-βRII binding domain as described herein, comprising a heavy chain variable region having an amino acid sequence as 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 certain embodiments, each of the heavy chain variable regions comprises a HCDR that does not comprise an amino acid variation. In certain embodiments, each of the heavy chain variable regions does not comprise an amino acid variation.

[0313] In one embodiment, the disclosure provides a multispecific binding moiety comprising:

[0314] - a PD-1 binding domain as described herein, comprising a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 19, or a heavy chain variable region having at least 80%, at least 85%, at least 90% or at least 95% sequence identity thereto; and

[0315] - A TGF-βRII binding domain as described herein, comprising a heavy chain variable region having an amino acid sequence as 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 certain embodiments, each of the heavy chain variable regions comprises a HCDR that does not comprise an amino acid variation. In certain embodiments, each of the heavy chain variable regions does not comprise an amino acid variation.

[0316] In one embodiment, the disclosure provides a multispecific binding moiety comprising:

[0317] - a PD-1 binding domain as described herein, comprising a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 9, or a heavy chain variable region having at least 80%, at least 85%, at least 90% or at least 95% sequence identity thereto; and

[0318] - A TGF-βRII binding domain as described herein, comprising a heavy chain variable region having an amino acid sequence as 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 certain embodiments, each of the heavy chain variable regions comprises a HCDR that does not comprise an amino acid variation. In certain embodiments, each of the heavy chain variable regions does not comprise an amino acid variation.

[0319] In one embodiment, the disclosure provides a multispecific binding moiety comprising:

[0320] - a PD-1 binding domain as described herein, comprising a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 13, or a heavy chain variable region having at least 80%, at least 85%, at least 90% or at least 95% sequence identity thereto; and

[0321] - A TGF-βRII binding domain as described herein, comprising a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 47, or a heavy chain variable region having at least 80%, at least 85%, at least 90% or at least 95% sequence identity thereto. In certain embodiments, each of the heavy chain variable regions comprises a HCDR that does not comprise an amino acid variation. In certain embodiments, each of the heavy chain variable regions does not comprise an amino acid variation.

[0322] In one embodiment, the disclosure provides a multispecific binding moiety comprising:

[0323] - a PD-1 binding domain as described herein, comprising a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 19, or a heavy chain variable region having at least 80%, at least 85%, at least 90% or at least 95% sequence identity thereto; and

[0324] - A TGF-βRII binding domain as described herein, comprising a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 43, or a heavy chain variable region having at least 80%, at least 85%, at least 90% or at least 95% sequence identity thereto. In certain embodiments, each of the heavy chain variable regions comprises a HCDR that does not comprise an amino acid variation. In certain embodiments, each of the heavy chain variable regions does not comprise an amino acid variation.

[0325] In one embodiment, the disclosure provides a multispecific binding moiety comprising:

[0326] - a PD-1 binding domain as described herein, comprising a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 9, or a heavy chain variable region having at least 80%, at least 85%, at least 90% or at least 95% sequence identity thereto; and

[0327] - a TGF-βRII binding domain as described herein, comprising a heavy chain variable region having an amino acid sequence as 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,

[0328] Wherein the PD-1 binding domain and the TGF-βRII binding domain comprise a light chain variable region having an amino acid sequence as 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 certain embodiments, each of the heavy chain variable region and the light chain variable region comprises a HCDR and a LCDR, respectively, and does not comprise amino acid variation. In certain embodiments, each of the heavy chain variable region and the light chain variable region does not comprise amino acid variation.

[0329] In one embodiment, the disclosure provides a multispecific binding moiety comprising:

[0330] - a PD-1 binding domain as described herein, comprising a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 18, or a heavy chain variable region having at least 80%, at least 85%, at least 90% or at least 95% sequence identity thereto; and

[0331] - a TGF-βRII binding domain as described herein, comprising a heavy chain variable region having an amino acid sequence as 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,

[0332] Wherein the PD-1 binding domain and the TGF-βRII binding domain comprise a light chain variable region having an amino acid sequence as 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 certain embodiments, each of the heavy chain variable region and the light chain variable region comprises a HCDR and a LCDR, respectively, and does not comprise amino acid variation. In certain embodiments, each of the heavy chain variable region and the light chain variable region does not comprise amino acid variation.

[0333] In one embodiment, the disclosure provides a multispecific binding moiety comprising:

[0334] - a PD-1 binding domain as described herein, comprising a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 19, or a heavy chain variable region having at least 80%, at least 85%, at least 90% or at least 95% sequence identity thereto; and

[0335] - a TGF-βRII binding domain as described herein, comprising a heavy chain variable region having an amino acid sequence as 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,

[0336] Wherein the PD-1 binding domain and the TGF-βRII binding domain comprise a light chain variable region having an amino acid sequence as 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 certain embodiments, each of the heavy chain variable region and the light chain variable region comprises a HCDR and a LCDR, respectively, and does not comprise amino acid variation. In certain embodiments, each of the heavy chain variable region and the light chain variable region does not comprise amino acid variation.

[0337] In one embodiment, the disclosure provides a multispecific binding moiety comprising:

[0338] - a PD-1 binding domain as described herein, comprising a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 14, or a heavy chain variable region having at least 80%, at least 85%, at least 90% or at least 95% sequence identity thereto; and

[0339] - a TGF-βRII binding domain as described herein, comprising a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 31, or a heavy chain variable region having at least 80%, at least 85%, at least 90%, at least 95% sequence identity thereto,

[0340] Wherein the PD-1 binding domain and the TGF-βRII binding domain comprise a light chain variable region having an amino acid sequence as 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 certain embodiments, each of the heavy chain variable region and the light chain variable region comprises a HCDR and a LCDR, respectively, and does not comprise amino acid variation. In certain embodiments, each of the heavy chain variable region and the light chain variable region does not comprise amino acid variation.

[0341] In one embodiment, the disclosure provides a multispecific binding moiety comprising:

[0342] - a PD-1 binding domain as described herein, comprising a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 9, or a heavy chain variable region having at least 80%, at least 85%, at least 90% or at least 95% sequence identity thereto; and

[0343] - a TGF-βRII binding domain as described herein, comprising a heavy chain variable region having an amino acid sequence as 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,

[0344] Wherein the PD-1 binding domain and the TGF-βRII binding domain comprise a light chain variable region having an amino acid sequence as 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 certain embodiments, each of the heavy chain variable region and the light chain variable region comprises a HCDR and a LCDR, respectively, and does not comprise amino acid variation. In certain embodiments, each of the heavy chain variable region and the light chain variable region does not comprise amino acid variation.

[0345] In one embodiment, the disclosure provides a multispecific binding moiety comprising:

[0346] - a PD-1 binding domain as described herein, comprising a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 9, or a heavy chain variable region having at least 80%, at least 85%, at least 90% or at least 95% sequence identity thereto; and

[0347] - a TGF-βRII binding domain as described herein, comprising a heavy chain variable region having an amino acid sequence as 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,

[0348] Wherein the PD-1 binding domain and the TGF-βRII binding domain comprise a light chain variable region having an amino acid sequence as 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 certain embodiments, each of the heavy chain variable region and the light chain variable region comprises a HCDR and a LCDR, respectively, and does not comprise amino acid variation. In certain embodiments, each of the heavy chain variable region and the light chain variable region does not comprise amino acid variation.

[0349] In one embodiment, the disclosure provides a multispecific binding moiety comprising:

[0350] - a PD-1 binding domain as described herein, comprising a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 14, or a heavy chain variable region having at least 80%, at least 85%, at least 90% or at least 95% sequence identity thereto; and

[0351] - a TGF-βRII binding domain as described herein, comprising a heavy chain variable region having an amino acid sequence as 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,

[0352] Wherein the PD-1 binding domain and the TGF-βRII binding domain comprise a light chain variable region having an amino acid sequence as 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 certain embodiments, each of the heavy chain variable region and the light chain variable region comprises a HCDR and a LCDR, respectively, and does not comprise amino acid variation. In certain embodiments, each of the heavy chain variable region and the light chain variable region does not comprise amino acid variation.

[0353] In one embodiment, the disclosure provides a multispecific binding moiety comprising:

[0354] - a PD-1 binding domain as described herein, comprising a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 19, or a heavy chain variable region having at least 80%, at least 85%, at least 90% or at least 95% sequence identity thereto; and

[0355] - a TGF-βRII binding domain as described herein, comprising a heavy chain variable region having an amino acid sequence as 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,

[0356] Wherein the PD-1 binding domain and the TGF-βRII binding domain comprise a light chain variable region having an amino acid sequence as 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 certain embodiments, each of the heavy chain variable region and the light chain variable region comprises a HCDR and a LCDR, respectively, and does not comprise amino acid variation. In certain embodiments, each of the heavy chain variable region and the light chain variable region does not comprise amino acid variation.

[0357] In one embodiment, the disclosure provides a multispecific binding moiety comprising:

[0358] - a PD-1 binding domain as described herein, comprising a heavy variable region having an amino acid sequence as shown in SEQ ID NO: 9, or a heavy chain variable region having at least 80%, at least 85%, at least 90% or at least 95% sequence identity thereto; and

[0359] - a TGF-βRII binding domain as described herein, comprising a heavy chain variable region having an amino acid sequence as 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,

[0360] Wherein the PD-1 binding domain and the TGF-βRII binding domain comprise a light chain variable region having an amino acid sequence as 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 certain embodiments, each of the heavy chain variable region and the light chain variable region comprises a HCDR and a LCDR, respectively, and does not comprise amino acid variation. In certain embodiments, each of the heavy chain variable region and the light chain variable region does not comprise amino acid variation.

[0361] In one embodiment, the disclosure provides a multispecific binding moiety comprising:

[0362] - a PD-1 binding domain as described herein, comprising a heavy variable region having an amino acid sequence as shown in SEQ ID NO: 13, or a heavy chain variable region having at least 80%, at least 85%, at least 90% or at least 95% sequence identity thereto; and

[0363] - a TGF-βRII binding domain as described herein, comprising a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 47, or a heavy chain variable region having at least 80%, at least 85%, at least 90% or at least 95% sequence identity thereto,

[0364] Wherein the PD-1 binding domain and the TGF-βRII binding domain comprise a light chain variable region having an amino acid sequence as 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 certain embodiments, each of the heavy chain variable region and the light chain variable region comprises a HCDR and a LCDR, respectively, and does not comprise amino acid variation. In certain embodiments, each of the heavy chain variable region and the light chain variable region does not comprise amino acid variation.

[0365] In one embodiment, the disclosure provides a multispecific binding moiety comprising:

[0366] - a PD-1 binding domain as described herein, comprising a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 19, or a heavy chain variable region having at least 80%, at least 85%, at least 90% or at least 95% sequence identity thereto; and

[0367] - a TGF-βRII binding domain as described herein, comprising a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 43, or a heavy chain variable region having at least 80%, at least 85%, at least 90% or at least 95% sequence identity thereto,

[0368] Wherein the PD-1 binding domain and the TGF-βRII binding domain comprise a light chain variable region having an amino acid sequence as 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 certain embodiments, each of the heavy chain variable region and the light chain variable region comprises a HCDR and a LCDR, respectively, and does not comprise amino acid variation. In certain embodiments, each of the heavy chain variable region and the light chain variable region does not comprise amino acid variation.

[0369] Also provided herein are nucleic acids that can be used to generate multi-specific binding portions of the present disclosure. In certain embodiments, such nucleic acids include nucleic acid sequences encoding the heavy chain variable region of the PD-1 binding domain as described herein and nucleic acid sequences encoding the heavy chain variable region of the TGF-βRII binding domain as described herein. In certain embodiments, the nucleic acid of the present disclosure may also include nucleic acid sequences encoding the CH1 region and preferably the hinge, CH2 region, and CH3 region. In certain embodiments, the nucleic acid of the present disclosure may also include at least one nucleic acid sequence encoding the light chain variable region and preferably the CL region. In certain embodiments, the light chain variable region may be a common light chain variable region as described herein.

[0370] Also provided herein are vectors comprising nucleic acids of the present disclosure that can be used to produce multi-specific binding portions of the present disclosure. In certain embodiments, such vectors comprise nucleic acid sequences encoding heavy chain variable regions of PD-1 binding domains as described herein and nucleic acid sequences encoding heavy chain variable regions of TGF-βRII binding domains as described herein. In certain embodiments, the vectors of the present disclosure may also include nucleic acid sequences encoding CH1 regions and preferably hinge, CH2 regions, and CH3 regions. In certain embodiments, the vectors of the present disclosure may also include at least one nucleic acid sequence encoding a light chain variable region and preferably a CL region. In certain embodiments, the light chain variable region may be a common light chain variable region as described herein.

[0371] The present disclosure also provides a cell comprising a nucleic acid sequence (e.g., a vector) 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 certain embodiments, the cell of the present disclosure may also include a nucleic acid sequence encoding a CH1 region and preferably encoding a hinge, CH2 region, and CH3 region, such as a vector. In certain embodiments, the cell of the present disclosure may also include at least one nucleic acid sequence encoding a light chain variable region and preferably encoding a CL region, such as a vector. In certain embodiments, the light chain variable region may be a common light chain variable region as described herein.

[0372] The present disclosure also provides a cell that produces a multi-specific binding portion as described herein. In certain embodiments, such a cell may be a recombinant cell that has been transformed with a nucleic acid such as a vector disclosed herein. In certain embodiments, the cell disclosed herein comprises a nucleic acid sequence (e.g., a vector) 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 certain embodiments, the cell disclosed herein further comprises a nucleic acid sequence encoding a CH1 region and preferably encoding a hinge, CH2 region, and CH3 region, such as a vector. In certain embodiments, the cell disclosed herein further comprises a light chain variable region, particularly a light chain variable region as described herein, and preferably at least one nucleic acid sequence encoding a CL region, such as a vector.

[0373] The present disclosure also provides a cell that produces a multispecific binding moiety as described herein.

[0374] In certain embodiments, the present disclosure provides a pharmaceutical composition comprising an effective amount of a multispecific binding moiety as described herein and optionally a pharmaceutically acceptable carrier.

[0375] In certain embodiments, the disclosure provides a multispecific binding moiety as described herein and a pharmaceutical composition as described herein for use in therapy.

[0376] In certain embodiments, the disclosure provides a multispecific binding moiety as described herein or a pharmaceutical composition as described herein for use in treating cancer.

[0377] In certain embodiments, the present disclosure provides a method for treating a disease, the method comprising administering to an individual in need thereof an effective amount of a multispecific binding moiety as described herein or a pharmaceutical composition as described herein.

[0378] In certain embodiments, the present disclosure provides a method for treating cancer, the method comprising administering to an individual in need thereof an effective amount of a multispecific binding moiety as described herein or a pharmaceutical composition as described herein.

[0379] As used herein, the terms "individual," "subject," and "patient" are used interchangeably and refer to mammals, such as humans, mice, rats, hamsters, guinea pigs, rabbits, cats, dogs, monkeys, cows, horses, pigs, etc., and particularly to human subjects suffering from cancer.

[0380] As used herein, the term "treatment" refers to any type of intervention or process performed on a subject or administered to a subject with an active agent or combination of active agents, with the purpose of curing or improving its disease or symptom or producing a positive therapeutic response. As used herein, "positive therapeutic response" refers to a treatment that produces a beneficial effect, such as reversing, alleviating, improving, inhibiting or slowing down symptoms, complications, conditions or biochemical indicators associated with the disease, and preventing the onset, progression, development, severity or recurrence of symptoms, complications, conditions or biochemical indicators associated with the disease, such as, for example, the improvement of at least one symptom of a disease or disorder (e.g., cancer). The beneficial effect can take the form of an improvement over a baseline, including an improvement over a measurement or observation made before treatment is started according to the method. For example, a beneficial effect can take the form of slowing down, stabilizing, stopping or reversing cancer progression in a subject at any clinical stage, as demonstrated by the reduction or elimination of clinical or diagnostic symptoms of the disease or cancer markers. Effective treatment can, for example, reduce tumor size, reduce the presence of circulating tumor cells, reduce or prevent the metastasis of a tumor, slow down or stop tumor growth and / or prevent or delay tumor recurrence or re-exacerbation.

[0381] The term "therapeutic amount" or "effective amount" refers to the amount of an agent or combination of agents that treats a disease, such as cancer. In some embodiments, a therapeutic amount is an amount sufficient to delay tumor development. In some embodiments, a therapeutic amount is an amount sufficient to prevent or delay tumor recurrence.

[0382] As used herein, an effective amount of an agent or composition is, for example, an amount of an agent or composition that can: (i) reduce the number of cancer cells; (ii) reduce tumor size; (iii) inhibit, delay, slow down, and prevent to some extent the infiltration of cancer cells into peripheral organs; (iv) inhibit tumor metastasis; (v) inhibit tumor growth; (vi) prevent or delay the occurrence and / or recurrence of tumors; and / or (vii) alleviate to some extent one or more symptoms associated with cancer.

[0383] The effective amount may vary according to factors such as the disease state, age, sex, and weight of the individual being treated, and the ability of the agent or combination of agents to elicit a desired response in the individual, and can be readily assessed by an ordinarily skilled physician or other health care practitioner.

[0384] An effective amount can be administered to a subject in one or more administrations.

[0385] An effective amount may also include an amount that balances any toxic or detrimental effects of the agent or combination of agents with the beneficial effects.

[0386] The term "agent" refers to a therapeutically active substance, which in the present case refers to a multispecific binding moiety of the disclosure, or a pharmaceutical composition of the disclosure.

[0387] As used herein, "comprise," "comprises," and its conjugations are used in their non-limiting sense, meaning that items following the word are included, but items not specifically mentioned are not excluding.

[0388] The articles "a" and "an" are used herein to refer to one or more of the grammatical object of the article. By way of example, "an element" means one or more elements.

[0389] The reference herein to a patent document or other matter is not to be taken as an admission that the document or matter was known at the priority date of any claim, or that the information it contains was part of the common general knowledge.

[0390] All patents and literature references cited in this specification are incorporated herein by reference in their entirety.

[0391] Note that in this specification, unless otherwise specified, the amino acid positions assigned to the CDR and framework in the variable region of an antibody or antibody fragment are designated according to the numbering of Kabat (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.

[0392] Accession numbers are given primarily to provide a further means of identifying the target, the actual sequence of the bound protein may vary, for example, because of mutations in the encoding gene, such as those occurring in certain cancers, etc. The antigen binding site of the multispecific binding portion of the present disclosure can bind to antigens and their various variants, such as those expressed by some antigen-positive immune or tumor cells and their various variants. HGNC stands for HUGO Gene Nomenclature Committee. The number following the abbreviation is the 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 accession numbers or gene IDs, 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 accession numbers, 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 between EMBL-EBI and the Wellcome Trust Sanger Institute to develop a software system that generates and maintains automatic annotations of selected eukaryotic genomes.

[0393] When a gene or protein is mentioned herein, it is preferably a human form of the gene or protein. When a gene or protein is mentioned herein, it refers to both the native gene or protein and variant forms of the gene or protein as can be detected in tumors, cancers, etc., preferably as can be detected in human tumors, cancers, etc.

[0394] Terms

[0395] 1. A multispecific binding portion comprising a PD-1 binding domain and a TGF-βRII binding domain, wherein the PD-1 binding domain blocks PD-1 mediated signaling and the TGF-βRII binding domain blocks TGF-βRII mediated signaling.

[0396] 2. The multispecific binding portion of clause 1, wherein in activated T cells, the PD-1 binding domain blocks PD-1 mediated signaling and the TGF-βRII binding domain blocks TGF-βRII mediated signaling.

[0397] 3. The multispecific binding portion of clause 1 or 2, wherein in activated tumor-specific T cells, the PD-1 binding domain blocks PD-1-mediated signaling and the TGF-βRII binding domain blocks TGF-βRII-mediated signaling.

[0398] 4. A multispecific binding portion comprising a Fab domain that specifically binds to PD-1 and a Fab domain that specifically binds to TGF-βRII.

[0399] 5. The multispecific binding moiety according to any of the preceding clauses, wherein the multispecific binding moiety 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.

[0400] 6. A multispecific binding portion according to any of the preceding clauses, wherein the potency of the multispecific binding portion in blocking TGF-βRII-mediated signaling is higher in cells expressing both PD-1 and TGF-βRII than in cells expressing TGF-βRII and not expressing, not substantially expressing, or expressing low levels of PD-1.

[0401] 7. A multispecific binding portion comprising a PD-1 binding domain and a TGF-βRII binding domain, wherein the potency of the multispecific binding portion in blocking TGF-βRII-mediated signaling is higher in cells expressing both PD-1 and TGF-βRII than in cells expressing TGF-βRII and not expressing, not substantially expressing, or expressing low levels of PD-1.

[0402] 8. A multispecific binding portion comprising a PD-1 binding domain and a TGF-βRII binding domain, wherein the multispecific binding portion has greater activity in reducing tumor volume than a combination of reference antibodies, wherein the combination of reference antibodies is two bivalent monospecific antibodies targeting PD-1 and TGF-βRII, wherein the bivalent monospecific antibody targeting PD-1 comprises a heavy chain having an amino acid sequence as shown in SEQ ID NO:78 and a light chain having an amino acid sequence as shown in SEQ ID NO:79, and the bivalent monospecific antibody targeting TGF-βRII comprises a heavy chain having an amino acid sequence as shown in SEQ ID NO:76 and a light chain having an amino acid sequence as shown in SEQ ID NO:77.

[0403] 9. The multispecific binding portion of clause 8, wherein the activity of reducing tumor volume is determined by measuring tumor volume reduction in in vivo studies in mice, particularly in in vivo studies in mice using MDA-MB-231 xenograft huCD34 NSG mice.

[0404] 10. The multispecific antibody or variant thereof according to clause 8 or 9, wherein the higher activity in reducing tumor volume is a tumor volume reduction of at least about 1.5 times, preferably between about 1.5 times and 100 times, greater than the tumor volume reduction of the combination of the reference antibodies.

[0405] 11. The multispecific binding moiety according to any of the preceding clauses, wherein the cells expressing both PD-1 and TGF-βRII are activated T cells.

[0406] 12. The multispecific binding moiety according to any of the preceding clauses, wherein the cells expressing both PD-1 and TGF-βRII are activated tumor-specific T cells.

[0407] 13. The multispecific binding moiety according to any of the preceding clauses, wherein the cells expressing both PD-1 and TGF-βRII are Jurkat-PD-1 + cells, and these cells that express TGF-βRII and do not express or substantially do not express PD-1 are Jurkat-PD-1 null cell.

[0408] 14. The multispecific binding moiety according to any of the preceding clauses, wherein the cells expressing both PD-1 and TGF-βRII are activated CD4 + and / or CD8 + cells, and these cells expressing TGF-βRII and no, substantially no, or low levels of PD-1 are non-activated CD4 + and / or CD8 + cell.

[0409] 15. The multispecific binding moiety according to any of the preceding clauses, wherein the cells expressing both PD-1 and TGF-βRII are HEK-Blue TGF-β-PD-1 + Cells, and these cells expressing TGF-βRII and not expressing, substantially not expressing, or expressing low levels of PD-1 are HEK-Blue TGF-β cells.

[0410] 16. The multispecific binding portion of any preceding clause, wherein the potency to block TGF-βRII mediated signaling is measured in a phosphorylated SMAD2 / 3 assay.

[0411] 17. The multispecific binding portion of any preceding clause, wherein the potency to block TGF-βRII-mediated signaling is measured in a syngeneic PD-1-TGF-β reporter gene assay.

[0412] 18. A multispecific binding portion according to any of the preceding clauses, wherein the potency of blocking TGF-βRII-mediated signaling is at least about 200-fold or 500-fold or 1000-fold or 5000-fold or 10000-fold or 15000-fold or 20000-fold or 30000-fold, or between about 200-fold and 30000-fold, or between 500-fold and 30000-fold, or between 1000-fold and 30000-fold, or between 5000-fold and 30000-fold, or between 1000-fold and 30000-fold, or between 1000-fold and 30000-fold, or between 200-fold and 20000-fold, or between 200-fold and 15000-fold higher in cells expressing both PD-1 and TGF-βRII than in cells expressing TGF-βRII and not expressing, or substantially not expressing, or expressing low levels of PD-1.

[0413] 19. The multispecific binding moiety of any of the preceding clauses, wherein the potency of the multispecific binding moiety in blocking TGF-βRII mediated signaling in cells that express TGF-βRII and do not express PD-1 is lower than the potency of a reference anti-TGF-βRII antibody, and the potency of the multispecific binding moiety in blocking TGF-βRII mediated signaling in cells that express both TGF-βRII and PD-1 is higher than the potency of the reference anti-TGF-βRII antibody, wherein the reference anti-TGF-βRII antibody is a bivalent monospecific antibody comprising a heavy chain having the amino acid sequence as shown in SEQ ID NO:76 and a light chain having the amino acid sequence as shown in SEQ ID NO:77.

[0414] 20. The multispecific binding portion of any of the preceding clauses, wherein the potency of the multispecific binding portion in blocking TGF-βRII-mediated signaling in cells expressing both TGF-βRII and PD-1 is at least about 100-fold or 200-fold greater than the potency of the reference anti-TGF-βRII antibody, preferably between about 100-fold and 20,000-fold, or between 100-fold and 15,000-fold, or between 100-fold and 12,000-fold, or between 200-fold and 20,000-fold, or between 200-fold and 15,000-fold, or between 200-fold and 12,000-fold.

[0415] 21. The multispecific binding moiety of any of the preceding clauses, wherein the PD-1 binding domain and the TGF-βRII binding domain are Fab domains.

[0416] 22. The multispecific binding moiety of any of the preceding clauses, wherein the multispecific binding moiety is a bispecific antibody.

[0417] 23. The multispecific binding moiety of any preceding clause, wherein the multispecific binding moiety is an IgG1 bispecific antibody.

[0418] 24. The multispecific binding portion of any of the preceding clauses, wherein the PD-1 binding domain and the TGF-βRII binding domain each comprise a light chain comprising a light chain variable region capable of pairing with a plurality of heavy chains having different epitope specificities.

[0419] 25. The multispecific binding portion of any of the preceding clauses, wherein the PD-1 binding domain and the TGF-βRII binding domain comprise the same light chain.

[0420] 26. The multispecific binding moiety of any of the preceding clauses, wherein the PD-1 binding domain comprises a heavy chain variable region comprising:

[0421] a) heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2) and heavy chain CDR3 (HCDR3), which have the amino acid sequences shown in SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4, respectively;

[0422] b) heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2) and heavy chain CDR3 (HCDR3), which have the amino acid sequences shown in SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, respectively;

[0423] c) heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2) and heavy chain CDR3 (HCDR3), which have the amino acid sequences shown in SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, respectively;

[0424] d) heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2) and heavy chain CDR3 (HCDR3), which have the amino acid sequences shown in SEQ ID NO: 15, SEQ ID NO: 16 and SEQ ID NO: 17, respectively; or

[0425] e) heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2) and heavy chain CDR3 (HCDR3), which have the amino acid sequences shown in SEQ ID NO: 20, SEQ ID NO: 21 and SEQ ID NO: 22, respectively;

[0426] Each of these HCDRs can contain up to three, two or one amino acid variation.

[0427] 27. The multispecific binding moiety of any of the preceding clauses, wherein the TGF-βRII binding domain comprises a heavy chain variable region comprising:

[0428] a) heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2) and heavy chain CDR3 (HCDR3), which have the amino acid sequences shown in SEQ ID NO: 24, SEQ ID NO: 25 and SEQ ID NO: 26, respectively;

[0429] b) heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2) and heavy chain CDR3 (HCDR3), which have the amino acid sequences shown in SEQ ID NO: 28, SEQ ID NO: 29 and SEQ ID NO: 30, respectively;

[0430] c) heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2) and heavy chain CDR3 (HCDR3), which have the amino acid sequences shown in SEQ ID NO: 32, SEQ ID NO: 33 and SEQ ID NO: 34, respectively;

[0431] d) heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2) and heavy chain CDR3 (HCDR3), which have the amino acid sequences shown in SEQ ID NO: 36, SEQ ID NO: 37 and SEQ ID NO: 38, respectively;

[0432] e) heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2) and heavy chain CDR3 (HCDR3), which have the amino acid sequences shown in SEQ ID NO:40, SEQ ID NO:41 and SEQ ID NO:42, respectively;

[0433] f) heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2) and heavy chain CDR3 (HCDR3), which have the amino acid sequences shown in SEQ ID NO:44, SEQ ID NO:45 and SEQ ID NO:46, respectively; or

[0434] g) heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2) and heavy chain CDR3 (HCDR3), which have the amino acid sequences shown in SEQ ID NO:90, SEQ ID NO:91 and SEQ ID NO:92, respectively,

[0435] Each of these HCDRs can contain up to three, two or one amino acid variation.

[0436] 28. A multispecific binding moiety 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:

[0437] a) heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2) and heavy chain CDR3 (HCDR3), which have the amino acid sequences shown in SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4, respectively;

[0438] b) heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2) and heavy chain CDR3 (HCDR3), which have the amino acid sequences shown in SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, respectively;

[0439] c) heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2) and heavy chain CDR3 (HCDR3), which have the amino acid sequences shown in SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, respectively;

[0440] d) heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2) and heavy chain CDR3 (HCDR3), which have the amino acid sequences shown in SEQ ID NO: 15, SEQ ID NO: 16 and SEQ ID NO: 17, respectively; or

[0441] e) heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2) and heavy chain CDR3 (HCDR3), which have the amino acid sequences shown in SEQ ID NO: 20, SEQ ID NO: 21 and SEQ ID NO: 22, respectively;

[0442] Each of these HCDRs can contain up to three, two or one amino acid variation.

[0443] 29. A multispecific binding moiety 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:

[0444] a) heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2) and heavy chain CDR3 (HCDR3), which have the amino acid sequences shown in SEQ ID NO: 24, SEQ ID NO: 25 and SEQ ID NO: 26, respectively;

[0445] b) heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2) and heavy chain CDR3 (HCDR3), which have the amino acid sequences shown in SEQ ID NO: 28, SEQ ID NO: 29 and SEQ ID NO: 30, respectively;

[0446] c) heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2) and heavy chain CDR3 (HCDR3), which have the amino acid sequences shown in SEQ ID NO: 32, SEQ ID NO: 33 and SEQ ID NO: 34, respectively;

[0447] d) heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2) and heavy chain CDR3 (HCDR3), which have the amino acid sequences shown in SEQ ID NO: 36, SEQ ID NO: 37 and SEQ ID NO: 38, respectively;

[0448] e) heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2) and heavy chain CDR3 (HCDR3), which have the amino acid sequences shown in SEQ ID NO:40, SEQ ID NO:41 and SEQ ID NO:42, respectively;

[0449] f) heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2) and heavy chain CDR3 (HCDR3), which have the amino acid sequences shown in SEQ ID NO:44, SEQ ID NO:45 and SEQ ID NO:46, respectively; or

[0450] g) heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2) and heavy chain CDR3 (HCDR3), which have the amino acid sequences shown in SEQ ID NO:90, SEQ ID NO:91 and SEQ ID NO:92, respectively,

[0451] Each of these HCDRs can contain up to three, two or one amino acid variation.

[0452] 30. The multispecific binding portion according to any of the preceding clauses, wherein the PD-1 binding domain comprises a heavy chain variable region having an amino acid sequence as shown in any one of SEQ ID NOs: 1, 5, 9, 13, 14, 18 and 19, or an amino acid sequence having at least 80%, preferably 85%, more preferably 90%, or most preferably 95% sequence identity thereto.

[0453] 31. The multispecific binding portion of any of the preceding clauses, wherein the PD-1 binding domain comprises a light chain variable region comprising a light chain CDR1 (LCDR1), a light chain CDR2 (LCDR2) and a light chain CDR3 (LCDR3) having amino acid sequences as shown in SEQ ID NO:49, SEQ ID NO:50 and SEQ ID NO:51, respectively, wherein each of these LCDRs can comprise up to three, two or one amino acid variation.

[0454] 32. A multispecific binding portion according to any of the preceding clauses, wherein the PD-1 binding domain comprises a light chain variable region having an amino acid sequence as shown in SEQ ID NO:48 or an amino acid sequence having at least 80%, preferably 85%, more preferably 90%, or most preferably 95% sequence identity thereto.

[0455] 33. The multispecific binding moiety of any of the preceding clauses, wherein the TGF-βRII binding domain comprises a heavy chain variable region comprising:

[0456] a) heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2) and heavy chain CDR3 (HCDR3), which have the amino acid sequences shown in SEQ ID NO: 24, SEQ ID NO: 25 and SEQ ID NO: 26, respectively;

[0457] b) heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2) and heavy chain CDR3 (HCDR3), which have the amino acid sequences shown in SEQ ID NO: 28, SEQ ID NO: 29 and SEQ ID NO: 30, respectively;

[0458] c) heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2) and heavy chain CDR3 (HCDR3), which have the amino acid sequences shown in SEQ ID NO: 32, SEQ ID NO: 33 and SEQ ID NO: 34, respectively;

[0459] d) heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2) and heavy chain CDR3 (HCDR3), which have the amino acid sequences shown in SEQ ID NO: 36, SEQ ID NO: 37 and SEQ ID NO: 38, respectively;

[0460] e) heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2) and heavy chain CDR3 (HCDR3), which have the amino acid sequences shown in SEQ ID NO:40, SEQ ID NO:41 and SEQ ID NO:42, respectively;

[0461] f) heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2) and heavy chain CDR3 (HCDR3), which have the amino acid sequences shown in SEQ ID NO:44, SEQ ID NO:45 and SEQ ID NO:46, respectively; or

[0462] g) heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2) and heavy chain CDR3 (HCDR3), which have the amino acid sequences shown in SEQ ID NO:90, SEQ ID NO:91 and SEQ ID NO:92, respectively,

[0463] Each of these HCDRs can contain up to three, two or one amino acid variation.

[0464] 34. A multispecific binding portion according to any of the preceding clauses, wherein the TGF-βRII binding domain comprises a heavy chain variable region having an amino acid sequence as shown in any one of SEQ ID NO: 23, 27, 31, 35, 39, 43, 47, 88 and 89, or an amino acid sequence having at least 80%, preferably 85%, more preferably 90%, or most preferably 95% sequence identity thereto.

[0465] 35. The multispecific binding portion of any of the preceding clauses, wherein the TGF-βRII binding domain comprises a light chain variable region comprising a light chain CDR1 (LCDR1), a light chain CDR2 (LCDR2) and a light chain CDR3 (LCDR3) having amino acid sequences as shown in SEQ ID NO:49, SEQ ID NO:50 and SEQ ID NO:51, respectively, wherein each of these LCDRs can comprise up to three, two or one amino acid variation.

[0466] 36. A multispecific binding portion according to any of the preceding clauses, wherein the TGF-βRII binding domain comprises a light chain variable region having an amino acid sequence as shown in SEQ ID NO:48 or an amino acid sequence having at least 80%, preferably 85%, more preferably 90%, or most preferably 95% sequence identity thereto.

[0467] 37. A multispecific binding moiety comprising a PD-1 binding domain and a TGF-βRII binding domain, which competes with the multispecific binding moiety according to any of the preceding clauses for binding to PD-1 and / or TGF-βRII.

[0468] 38. A pharmaceutical composition comprising an effective amount of a multispecific binding moiety according to any of the preceding clauses, and a pharmaceutically acceptable carrier.

[0469] 39. The multispecific binding moiety according to any one of clauses 1 to 37 or the pharmaceutical composition according to clause 38 for use in therapy.

[0470] 40. The multispecific binding moiety according to any one of clauses 1 to 37 or the pharmaceutical composition according to clause 38 for use in treating a disease associated with a suppressed immune system.

[0471] 41. The multispecific binding moiety according to any one of clauses 1 to 37 or the pharmaceutical composition according to clause 38 for use in treating cancer.

[0472] 42. Use of a multispecific binding moiety according to any one of clauses 1 to 37 or a pharmaceutical composition according to clause 38 for the manufacture of a medicament for treating a disease associated with a suppressed immune system.

[0473] 43. Use of a multispecific binding moiety according to any one of clauses 1 to 37 or a pharmaceutical composition according to clause 38 for the manufacture of a medicament for treating cancer.

[0474] 44. A method for treating a disease, the method comprising administering to a human subject in need thereof an effective amount of a multispecific binding moiety according to any one of clauses 1 to 37 or a pharmaceutical composition according to clause 38.

[0475] 45. A method for treating a disease associated with a suppressed immune system, the method comprising administering to a human subject in need thereof an effective amount of a multispecific binding moiety according to any one of clauses 1 to 37 or a pharmaceutical composition according to clause 38.

[0476] 46. ​​A method for treating cancer, the method comprising administering to a human subject in need thereof an effective amount of the multispecific binding moiety according to any one of clauses 1 to 37 or the pharmaceutical composition according to clause 38.

[0477] 47. A cell comprising a nucleic acid sequence encoding a heavy chain variable region of a PD-1 binding domain as defined in clause 28 or 30 and a nucleic acid sequence encoding a heavy chain variable region of a TGF-βRII binding domain as defined in clause 29 or 34.

[0478] 48. The cell according to clause 47, wherein the cell further comprises a nucleic acid sequence encoding a CH1 region and preferably encoding a hinge, a CH2 region and a CH3 region.

[0479] 49. The cell according to clause 47 or 48, wherein the cell further comprises at least one nucleic acid sequence encoding a light chain variable region, in particular a light chain variable region as defined in clause 34 or 35, and preferably encoding a CL region.

[0480] 50. A cell which produces a multispecific binding moiety according to any one of clauses 1 to 37.

[0481] Example

[0482] In an embodiment used to illustrate the present disclosure but not intended to limit the present disclosure in any way, each binding domain of the bispecific antibody comprises a light chain variable region having an amino acid sequence as shown in SEQ ID NO: 48 and a light chain constant region having an amino acid sequence as shown in SEQ ID NO: 75. The bispecific antibody is preferably an IgG1 antibody comprising CH1, hinge, CH2 and CH3. In an embodiment used to illustrate the present disclosure but not intended to limit the present disclosure in any way, bispecific antibodies are screened in IgG1 format, wherein the PD-1 binding heavy chain comprises CH1 having an amino acid sequence as shown in SEQ ID NO: 69, CH2 having an amino acid sequence as shown in SEQ ID NO: 71, and CH3 having an amino acid sequence as shown in SEQ ID NO: 73; and the TGF-βRII binding heavy chain comprises CH1 having an amino acid sequence as shown in SEQ ID NO: 69, CH2 having an amino acid sequence as shown in SEQ ID NO: 71, and CH3 having an amino acid sequence as shown in SEQ ID NO: 74.

[0483] Reference antibodies and molecules and control antibodies used in the examples include:

[0484] - Reference to the PD-1 antibody pembrolizumab analog, which is a bivalent monospecific antibody comprising 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.

[0485] - Reference PD-1 antibody pembrolizumab (manufactured by Merck and distributed by Myonex).

[0486] - With reference to the TGF-βRII antibody TGF1, which is a bivalent monospecific analog of TGF1 and comprises 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.

[0487] - Reference PD-L1-TGF-βTRAP molecule, which is a bivalent monospecific analog of Bintrafusa alpha for binding to PD-L1 and comprises 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, wherein the extracellular domain of TGF-βRII has the amino acid sequence shown in SEQ ID NO: 104, which is linked to the C-terminus of each heavy chain via a (G4S)4G linker.

[0488] - A negative control IgG1 antibody (RSV-G), which is a bivalent monospecific antibody comprising 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.

[0489] - A control TGF-βRII×RSV antibody, which is a bivalent bispecific antibody, comprising a TGF-βRII binding domain, a RSV binding domain and a common light chain, wherein the TGF-βRII binding domain comprises a heavy chain variable region amino acid sequence as shown in SEQ ID NO:23, 31, 39, 27, 35 or 43; the RSV binding domain comprises a heavy chain variable region amino acid sequence as shown in SEQ ID NO:86; the common light chain comprises a light chain variable region amino acid sequence as shown in SEQ ID NO:48 and a light chain constant region amino acid sequence as shown in SEQ ID NO:75.

[0490] - Positive control LILRB2 (BioLegend; Cat. No. 338714).

[0491] - Commercial reference PD-1 antibody Opdivo (made by Bristol Myers Squibb (BMS)).

[0492] Example 1 - Generation of PD-1×TGF-βRII bispecific antibody

[0493] By immunizing transgenic mice containing the common IGKV1-39 light chain with human PD-1 or TGF-βRII antigen ( mice) (including using different forms of DNA, protein, and cell-based antigen delivery) to obtain 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.

[0494] Heavy chain variable regions with binding specificity to human PD-1 having amino acid sequences as shown in SEQ ID NOs: 1, 5, 9, 13, 14, 18 and 19, and heavy chain variable regions with binding specificity to human TGF-βRII having amino acid sequences as shown in SEQ ID NOs: 23, 27, 31, 35, 39, 43, 47, 88 and 89 were selected for generating bispecific antibodies. The binding domain sequences herein, once characterized and sequenced by the techniques provided herein, can then be obtained by any method known in the art.

[0495] Table 1. Combinations of PD-1 heavy chain variable regions and TGF-βRII heavy chain variable regions that can be used to generate bispecific antibodies.

[0496]

[0497] Bispecific IgG antibodies were generated by transient co-transfection of two plasmid vectors: one encoding an IgG heavy chain with a PD-1 binding VH region and the other encoding an IgG heavy chain with a TGF-βRII binding VH region. The CH3 engineering technology described in WO2013 / 157954 and WO2013 / 157953 was used to ensure efficient heterodimerization and formation of bispecific antibodies. The two vectors further encoded a common light chain comprising the IGKV1-39 / Jk1 light chain variable region. Cell transfection, cell culture, and antibody harvesting and purification were performed by methods known in the art.

[0498] Example 2 - PD-1-SHP recruitment assay

[0499] Bispecific antibodies were characterized in a PD-1-SHP recruitment assay to determine their ability to block ligand binding to PD-1 and thereby inhibit PD-1 / PD-L1 signaling in T cells. The PD-1SHP recruitment assay involves two cell systems, including U20S cells engineered to express an enzyme donor (ED)-tagged PD-1 receptor (e.g., PD-L1 or PD-L2) and Jurkat T cells expressing PD-1 and engineered to express SHP1 fused to an enzyme receptor (EA). PD-1 activation of Jurkat T cells induced by ligand or agonistic antibodies leads to SHP1 recruitment, forcing ED and EA to interact and reconstruct active β-gal enzyme. In the presence of substrate, the reconstructed β-gal produces a chemiluminescent signal.

[0500] The antibody samples included several bispecific antibodies, a negative control IgG1 antibody (RSV), an analog of the reference PD-1 antibody pembrolizumab, an analog of the reference TGF-βRII antibody TGF1, and an analog of the reference PD-L1-TGF-βTRAP molecule.

[0501] U2OS / PD-L1 cells (DiscoveRx) 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 (phosphatase containing the Src homology region 2 domain; DiscoveRx) 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 containing 1% FBS) before cell plating. U2OS / PD-L1 cells were plated at 5000 cells per well in 20 μL of assay medium in a 384-well black clear bottom assay plate ( Tissue culture plates, Greiner Bio-One). Antibody samples were prepared by serial dilution in phosphate buffered saline (PBS) with 1% FBS and transferring 5 μL / well to the cell plate and incubating at 37°C, 5% CO2 for one hour. Jurkat-PD-1-SHP cells were then added to the cell plate at a rate of 5000 cells per well in 20 μL of assay medium and incubated at 37°C, 5% CO2 for two hours, followed by the addition of 2.5 μL of PathHunter reagent 1 (DiscoveRx) to each well. The assay plate was then shaken at 350 rpm for 1 minute and kept in the dark at room temperature for 15 minutes, followed by the addition of 10 μL of PathHunter reagent 2 (DiscoveRx). After incubation for one hour at room temperature, the chemiluminescent signal was recorded using a TopCount reader (PerkinElmer). Wells with PBS alone were used as positive controls, and wells without cells were used as negative controls. IC 50 The assay was performed by fitting a curve of percent control activity versus the logarithm of compound concentration using GraphPad Prism 7.0 software.

[0502] The results are shown in Table 2 and Figure 1A and Figure 1BAll bispecific antibodies inhibited PD-1-mediated SHP recruitment. In the PD-1-SHP recruitment assay, many bispecific antibodies that were monovalent for PD-1 binding were equivalent to the bivalent monospecific reference PD-1 antibody pembrolizumab analog and the reference PD-L1-TGF-βTRAP molecule that was bivalent for PD-L1 binding.

[0503] Table 2. Results of PD-1-SHP recruitment assay.

[0504]

[0505] Example 3 - PD-1-NFAT reporter gene assay

[0506] The bispecific antibodies were characterized in a PD-1-NFAT reporter gene assay to determine their ability to block PD-1 / PD-L1 signaling in activated T cells. The PD-1-NFAT reporter gene assay involves two transgenic cell line systems in which PD-L1 + aAPC / CHO-K1 cells co-express TCR homologous proteins and PD-1 + Effector Jurkat T cells drive a luciferase reporter gene under the control of the NFAT-RE cis-element. Effector Jurkat T cells activate TCR signaling in an antigen-independent manner. PD-1 / PD-L1 interaction inhibits TCR-mediated luminescence. Blocking PD-1 / PD-L1 interaction allows TCR signaling, thereby inducing luminescence that can be detected by adding substrate.

[0507] The antibody samples included several bispecific antibodies, a negative control IgG1 antibody (RSV), an analog of the reference PD-1 antibody pembrolizumab, an analog of the reference TGF-βRII antibody TGF1, and an analog of the reference PD-L1-TGF-βTRAP molecule.

[0508] PD-L1 aAPC / CHO-K1 cells ((artificial antigen presenting cells) / CHO (Chinese hamster ovary)-K1 cells; Promega) were maintained in F-12 medium (Thermo Fisher Scientific) supplemented with 10% FBS, 200 μg / mL hygromycin B (Thermo Fisher Scientific) and 250 μg / mL geneticin (G418; Thermo Fisher Scientific). Jurkat-PD-1-NFAT effector cells (nuclear factor of activated T cells; Promega) were cultured in RPMI 1640 medium (Thermo Fisher Scientific) supplemented with 10% FBS, 100 μg / mL hygromycin B (Thermo Fisher Scientific) and 500 μg / mL G418 (Thermo Fisher Scientific). Both PD-L1 aAPC / CHO-K1 cells and Jurkat-PD-1-NFAT effector cells were first centrifuged to remove the culture medium, then washed and suspended in assay medium (RPMI1640 medium containing 1% FBS) before cell seeding. PD-L1 aAPC / CHO-K1 cells were plated in 10 μL assay medium at 8,000 cells per well in a 384-well white clear bottom assay plate ( Tissue Culture Plates, Greiner Bio One). Antibody samples were prepared by serial dilution in phosphate buffered saline (PBS) with 1% FBS and 5 μL / well were transferred to the cell plate. Jurkat-PD-1-NFAT effector cells were then distributed to each well at a rate of 10,000 cells per well in 5 μL of assay medium. The assay plates were incubated for 24 hours at 37°C, 5% CO2. After the assay plates were equilibrated to room temperature for 15 minutes, 20 μL / well of Bio-Glo TM Reagent (Promega). After incubation for 8 minutes at room temperature, luminescence was read using a Phrastar microplate reader (BMG Labtech). Wells containing PBS served as negative controls (0% induction), and wells containing 12.5 μg / mL pembrolizumab analogs served as positive controls (100% induction). EC 50 The assay was performed by fitting a curve of percent control activity versus the logarithm of compound concentration using GraphPad Prism 7.0 software.

[0509] The results are shown in Table 3 and Figure 2A-2C All bispecific antibodies inhibited PD-1-mediated T cell suppression. Many bispecific antibodies that were monovalent for PD-1 binding were equivalent to the bivalent monospecific reference PD-1 antibody pembrolizumab analog in the PD-1-NFAT reporter assay.

[0510] Table 3. Results of PD-1-NFAT reporter gene assay.

[0511]

[0512] Example 4 - Jurkat phosphorylation SMAD2 / 3 assay

[0513] The bispecific antibodies were characterized in the Jurkat phosphorylated SMAD2 / 3 assay to determine their ability to block TGF-βRII signaling in T cells. null Cells and Jurkat-PD-1 + Comparison of cell activity to determine whether the bispecific antibody inhibits TGF-β-induced SMAD2 / 3 phosphorylation in a PD-1-dependent manner. An analog of the reference TGF-βRII antibody TGF1, a TGF-βRII×RSV bispecific antibody, and a negative control bivalent monospecific IgG1 antibody comprising a heavy chain having an amino acid sequence as shown in SEQ ID NO: 86 and a light chain having an amino acid sequence as shown in SEQ ID NO: 87 were included as control antibodies.

[0514] Jurkat-PD-1 was added to RPMI / 10% FBS. null Cells (ATCC catalog number TIB-152) and Jurkat-PD-1 + Cells (Promega catalog number CS187105) were seeded in 96-well flat-bottom plates. null No PD-1 expression can be detected in the cells; Jurkat-PD-1 +The number of PD-1 molecules on the cells was determined to be approximately 4000 using the Quantibrite bead method. Bispecific antibodies and control antibodies were added in a 6-step serial dilution (100 μg / mL to 0.001 μg / mL), and the cells were incubated for one hour at 37°C / 5% CO2. One hour later, human recombinant TGF-β1 (R&D system catalog number 7754-BH) was added at a final concentration of 10 ng / mL, and the cells were incubated for another two 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 standardized to total protein using a BCA protein assay kit (Pierce#23227). Phosphorylated SMAD2 / 3 levels were determined using ELISA (Cell Signaling #12001) according to the manufacturer's instructions. Graphs were plotted using GraphPad Prism (8.2.0).

[0515] The results are shown in Tables 4, 5 and 6 and Figure 3 Table 6 shows the bioavailability of 10 bispecific antibodies in Jurkat-PD-1 null With Jurkat-PD-1 + Fold difference in potency for inhibiting TGF-βRII signaling in cells. In this assay, the fold difference of the bispecific antibodies was between 200-fold and 11,000-fold higher than the fold difference of the reference antibody TGF1 analog.

[0516] All bispecific antibodies in Jurkat-PD-1 null Cells and Jurkat-PD-1 + The bispecific antibody inhibited TGF-βRII signaling in both cells. + In Jurkat-PD-1 null All bispecific antibodies required higher concentrations to inhibit Jurkat-PD-1 compared with the reference TGF-βRII antibody TGF1. null TGF-βRII signaling in cells. Many bispecific antibodies that bind to TGF-βRII as a monovalent antibody are effective in inhibiting Jurkat-PD-1 +A bivalent monospecific analog of the reference TGF-βRII antibody TGF1 that outperforms the reference TGF-βRII antibody in TGF-β signaling in cells.

[0517] Table 4. Jurkat-PD-1 null IC of Jurkat phosphorylated SMAD2 / 3 assay in cells 50 value.

[0518]

[0519] Table 5. Jurkat-PD-1 + IC of Jurkat phosphorylated SMAD2 / 3 assay in cells 50 value.

[0520]

[0521] Table 6. In Jurkat-PD-1 null With Jurkat-PD-1 + Fold difference in the potency of inhibiting TGF-βRII signaling in cells.

[0522]

[0523] Example 5 - Using stimulated and unstimulated CD4 + and CD8 + Phosphorylation of SMAD2 in T cells

[0524] Characterize bispecific antibodies in a phosphorylated SMAD2 assay to determine their specificity for blocking TGF-βRII signaling in PD-1-positive T cells. The phosphorylated SMAD2 assay involves a comparison of the activity of bispecific antibodies on 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 inhibit TGF-β-induced SMAD2 phosphorylation in a manner associated with PD-1 expression. An analog of the reference TGF-βRII antibody TGF1, a reference PD-1 antibody pembrolizumab, and a negative control bivalent monospecific IgG1 antibody are included as positive and negative controls, respectively, and the negative control bivalent monospecific IgG1 antibody comprises a heavy chain having an amino acid sequence as shown in SEQ ID NO: 86 and a light chain having an amino acid sequence as shown in SEQ ID NO: 87.

[0525] PBMCs from healthy donors were stimulated with 1 μg / mL anti-CD3 antibody (BD Biosciences #555336) for 48 hours and then serum deprived (0.1% FBS) for 16 hours. +and CD8 + The number of PD-1 molecules on T cells was determined to be approximately 1000 to 2000 using the Quantibrite bead method. Stimulated and unstimulated PBMCs were then incubated with bispecific antibodies and control antibodies for 30 minutes at room temperature. Recombinant human TGF-β1 (CellSignaling #7754-BH) was added at a final concentration of 1 ng / mL, and the cells were incubated for another 30 minutes. Finally, the cells were washed twice with PBS and stained for cell surface markers, followed by intracellular phosphorylated SMAD2 staining.

[0526] The following antibodies were used for flow cytometry staining: anti-human CD45 (clone: ​​HI30; catalog number 557748, BD Biosciences), anti-human CD11b (clone: ​​M1 / 70; catalog number 563015, BD Biociences), anti-human CD3 (clone: ​​UCHT1; catalog number 565491, BD Biosciences), anti-human CD4 (clone; SK3; catalog number 563550, BD Biosciences), anti-human CD8 (clone #SK1, catalog number 344714, BioLegend) and human phosphorylated SMAD2 (catalog number 56532, Cell Signaling) antibody. Active dye (BioLegend #423114) was used to exclude dead cells during analysis. Cell acquisition was performed under FACSymphony A3 using DIVA software.

[0527] PBMCs were gated based on size and granularity using FSC-A and SSC-A to exclude debris. Dead cells were then excluded using a fixable active dye. CD45-positive cells were selected, followed by CD11b-negative T cells and CD3-positive T cells. Finally, CD4 and CD8-positive subpopulations were gated, and phosphorylated SMAD2 signals were measured on these subpopulations as geometric mean fluorescence intensity (GMFI). Data analysis was performed using FlowJo software and graphed using GrapPad Prism (8.2.0).

[0528] The results are Figure 4 All bispecific antibodies expressed in stimulated CD4 + and CD8 + Inhibition of TGF-βRII signaling in T cells. In one donor (donor B), the bispecific antibody was expressed in unstimulated CD4 + and CD8 +In another donor, the bispecific antibody comprising 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 did not inhibit unstimulated CD4 + and CD8 + TGF-βRII signaling in T cells, and a bispecific antibody comprising a PD-1 heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 13 and a TGF-βRII heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 47 in unstimulated CD4 + and CD8 + The efficacy of inhibiting TGF-βRII signaling in T cells was significantly lower than that in stimulated CD4 + T cells and CD8 + Efficacy in T cells. These data confirm the findings in Example 4 that the bispecific antibody inhibits TGF-β-induced SMAD phosphorylation in a manner that correlates with PD-1 expression.

[0529] Example 6 - Depletion Mixed Lymphocyte Reaction (MLR) Assay

[0530] The bispecific antibodies were characterized in a depletion mixed lymphocyte reaction (MLR) assay to determine their potency in inducing cytokine production by exhausted T cells.

[0531] The antibody samples included several bispecific antibodies, a negative control IgG1 antibody (RSV), a reference PD-1 antibody pembrolizumab, an analog of the reference TGF-βRII antibody TGF1, and a combination of the reference PD-1 antibody pembrolizumab and the analog of the reference TGF-βRII antibody TGF1.

[0532] Human PBMCs were isolated from healthy donors. In vitro T cell depletion was performed by repeated activation of PBMCs with staphylococcal enterotoxin B (SEB) for 6 days. Total T cells were separated using a T cell separation kit (StemCell Technologies, catalog number 17951) according to the manufacturer's instructions. T cells were mixed with dendritic cells (DCs) from different donors in a 96-well U-bottom plate with a T cell to DC ratio of 10:1. Serial dilutions of antibody samples were added, and the plate was incubated for another six days at 37°C / 5% CO2. After six days, the IFN-γ, IL-2 or TNF-α cytokine levels in the supernatant were measured using a customized MSD kit. GraphPad Prism (8.2.0) was used for mapping.

[0533] Representative results from two experiments performed with five donors each are shown in Figure 5 A. Figure 5 C and Figure 5 The results of further experiments with three donors are shown in D. Figure 5 B and Figure 5 Most bispecific antibodies induced IFN-γ, IL-2, or TNF-α levels similar to those of the reference PD-1 antibody pembrolizumab, an analog of the reference PD-L1-TGF-βTRAP molecule, or a combination of the reference PD-1 antibody pembrolizumab and the reference TGF-βRII antibody TGF1. IC values ​​for many of the bispecific antibodies from the initial study were 50 The values ​​are shown in Table 7.

[0534] Table 7. IC of depletion MLR assay 50 value

[0535]

[0536] Example 7 - HEK-BLUE-PD-1 TGF-β reporter gene assay

[0537] The bispecific antibodies were characterized in a HEK-BLUE-PD-1TGF-β reporter assay to determine their potency in inhibiting TGF-β-induced signaling in T cells. TM TGF-β cells or HEK-Blue TM TGF-β-PD-1 + Cells can induce activation of the TGF-β / Smad signaling pathway, leading to the formation of the Smad3 / Smad4 complex that induces SEAP production.

[0538] Reagents used: Recombinant human TGF-β1 (human cell expressed) protein, R&D, catalog number 7754-BH. Growth medium: DMEM, 4.5 g / L glucose, 2 mM L-glutamine, 10% heat-inactivated fetal bovine serum (FBS; 56°C for 30 minutes), 100 μg / mL Normocin TM , Pen-Strep (100 U / mL to 100 μg / mL). For HEK-Blue TM TGF-β-PD-1 + Cells, use growth medium containing 0.4μg / mL puromycin. Test medium: DMEM 4.5g / L glucose, 2mM L-glutamine, 0.1% heat-inactivated FBS, without Normocin TM , Blasticidin, Hygromycin B and ZeocinTM of Pen-Strep (100 U / mL to 100 μg / mL).

[0539] Stable HEK-Blue expressing PD-1 was generated as follows TM TGF-β cell line: The full-length cd of PD-1 was inserted into the mammalian expression vector pD2529-EFM (ATUM) containing the puromycin resistance gene. The promoter is a modified EF1a. The vector was constructed by ATUM and the sequence was confirmed. Fig.10 HEK-Blue cells that could not detect PD-1 expression were transfected using TransIT-293 transfection reagent (Mirus Bio) according to the manufacturer's protocol. TM TGF-β cells (Invivogen). HEK-Blue containing 0.4 μg / mL puromycin TM Stably transfected cells were selected in TGF-β medium. Clones were isolated by limiting dilution and characterized by Western blot for PD-1 expression. One clone was selected based on: 1) its stable and uniform surface PD-1 expression (one peak in the histogram); 2) its homology to the parental HEK-Blue TM The GMFI of PD-1 in the selected clones was 3272 (compared to 5 for the parental cell line and 8 for the isotype control). The number of PD-1 molecules on these cells was determined to be approximately 20,000 using the Quantibrite bead method.

[0540] The antibody samples included several bispecific antibodies, a negative control IgG1 antibody (RSV), a reference PD-1 antibody pembrolizumab, and an analog of the reference TGF-βRII antibody TGF1.

[0541] HEK-Blue expressing TGF-βRII TM TGF-β cells (Invivogen, catalog code: hkb-tgfb) or HEK-Blue TM TGF-β-PD-1 + Cells were seeded in 96-well flat-bottom plates at 25,000 cells per well in assay medium. Serial dilutions of the antibody samples were added and the cells were incubated at room temperature for one hour; then human recombinant TGF-β1 was added at a final concentration of 1 ng / mL. The cells were incubated overnight at 37°C / 5% CO2. After incubation, 40ul of supernatant was transferred from each well to a fresh flat-bottom 96-well plate; and 160ul of resuspended QUANTI-Blue was added to the supernatant. TMSolution. Incubate the plate at 37°C / 5% CO2 for 40 minutes. TM Solution (SEAP detection reagent) was used to evaluate the amount of secreted SEAP in the supernatant. SEAP levels were measured using a spectrophotometer at 650 nm. Graphs were plotted using GraphPad Prism (8.2.0).

[0542] The results are Figure 6 and are shown in Table 8. Many bispecific antibodies showed potent inhibition of TGF-β-induced signaling in a manner correlated with PD-1 expression.

[0543] Table 8. Results of HEK-BLUE-PD-1 TGF-β reporter assay.

[0544]

[0545] Example 8 - Treg suppression assay

[0546] The bispecific antibodies were characterized in a Treg suppression assay to determine their ability to abrogate or reduce the suppressive effects of regulatory T cells and thereby induce cytokine production by T cells.

[0547] The antibody samples included several bispecific antibodies, a negative control IgG1 antibody (RSV), a reference PD-1 antibody pembrolizumab, an analog of the reference TGF-βRII antibody TGF1, and a combination of the reference PD-1 antibody pembrolizumab and the analog of the reference TGF-βRII antibody TGF1.

[0548] Human PBMCs were isolated from healthy donors by Ficoll-Paque gradient centrifugation. Tregs were isolated using the EasySep Treg isolation kit (StemCell Technologies, #18063) according to the manufacturer's instructions. Tregs were mixed with PBMCs from the same donor. Anti-CD3 ab (BD Biosciences#555336) and anti-CD28 ab (BD Biosciences#555725) were added to the co-culture. Finally, serial dilutions of bispecific antibodies were added, and the plates were incubated at 37°C / 5% CO2 for three days. After three 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 for mapping.

[0549] The results are Figure 7and are shown in Table 9. Most bispecific antibodies induced IFN-γ or TNF-α levels similar to those of the reference PD-1 antibody pembrolizumab or a combination of the reference PD-1 antibody pembrolizumab and the analog of the reference TGF-βRII antibody TGF1.

[0550] Table 9. Results of Treg suppression assay.

[0551]

[0552] Example 9 - Macrophage Inhibition Assay

[0553] Tumor-associated macrophages of the M2 phenotype inhibit T cell proliferation and cytokine production. Bispecific antibodies were characterized in an M2 macrophage inhibition assay to test whether they could reverse the inhibitory effects of M2 macrophages on T cell proliferation and IFN-γ production.

[0554] The bispecific antibodies were tested with a negative control IgG1 antibody (RSV), an analog of the reference TGF-βRII antibody TGF1, an analog of the reference PD-L1-TGF-βTRAP molecule, an analog of the reference PD-1 antibody nivolumab, the commercial reference PD-1 antibody Opdivo (BMS), a positive control antibody anti-LILRB2 (BioLegend), a rat IgG2a isotype control (BioLegend), and a huIgG4 isotype control (BioLegend). In addition, CD4 T cells stimulated without the addition of test or control antibodies were tested. + Co-cultures of T cells and M2 macrophages or CD4 + T cells (unstimulated and stimulated) were included in the assay as control conditions.

[0555] Monocytes isolated from PBMCs from three different healthy donors were differentiated into macrophages with M-CSF over six days and polarized to obtain M2 macrophages using a specific cocktail of cytokines IL-4 (20 ng / mL), IL-10 (20 ng / mL) and TGF-β (20 ng / mL) (+M-CSF). To confirm this 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. M2-like macrophages showed the expected phenotype in all three donors.

[0556] These M2 macrophages were then activated with LPS (100 ng / mL) for 4 hours. These macrophages were harvested, washed, and seeded in a 1:5 ratio in the presence of test or control antibodies at a concentration of 10 μg / mL in cells with autologous CD4 + Activated T cells (CD3 / CD28 ImmunoCult from StemCell technologies TM Five replicate wells were set in a 96-well plate with 1% activated IFN-γ. On the fifth day of co-culture, the concentration of secreted IFN-γ was measured by ELISA (LEGEND MAXTM human IFN-γ ELISA kit, BioLegend). Data were analyzed using multi-factor ANOVA in GraphPad Prism 7.0.

[0557] The results are Figure 8 Many bispecific antibodies induced similar or higher levels of IFN-γ compared to analogs of the reference PD-1 antibody nivolumab, analogs of the reference TGF-βRII antibody TGF1, or analogs of the reference PD-L1-TGF-βTRAP molecule.

[0558] Example 10 - In vivo humanized NSG MDA-MB-231 mouse model

[0559] The bispecific antibodies were characterized in vivo in a humanized NSG MDA-MB-231 mouse model to determine their efficacy in reducing tumor volume. The mouse model was validated using 10 mg / kg of a negative control bivalent monospecific IgG1 antibody (the antibody comprising a heavy chain having an amino acid sequence as shown in SEQ ID NO:86 and a light chain having an amino acid sequence as shown in SEQ ID NO:87); an analog of the reference TGF-βRII antibody TGF1 (10 mg / kg); a reference PD-1 antibody pembrolizumab (10 mg / kg); a combination of an analog of the reference TGF-βRII antibody TGF1 (10 mg / kg) and a reference PD-1 antibody pembrolizumab (10 mg / kg); and an analog of the reference PD-L1-TGF-βTRAP molecule (10 mg / kg). Results were shown in Fig. 9 As shown in A.

[0560] A total of 3 × 10 cells / ml of cells were suspended in 100 μl of serum-free medium and an equal volume of matrigel matrix (Corning). 6 MDA-MB-231 tumor cells were subcutaneously inoculated into humanized CD34 NSG mice. 3 Up to 100mm 3 ), the mice were randomly divided into the following treatment groups:

[0561] 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;

[0562] 2) an analog of the reference TGF-βRII antibody TGF1 (10 mg / kg);

[0563] 3) Reference PD-1 antibody pembrolizumab (10 mg / kg);

[0564] 4) analog of reference TGF-βRII antibody TGF1 (10 mg / kg) + reference PD-1 antibody pembrolizumab (10 mg / kg);

[0565] 5) a bispecific antibody (1 mg / kg) comprising a TGF-βRII binding domain and a PD-1 binding domain, wherein the TGF-βRII binding domain comprises a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 43, and the PD-1 binding domain comprises a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 9;

[0566] 6) a bispecific antibody (10 mg / kg) comprising a TGF-βRII binding domain and a PD-1 binding domain, wherein the TGF-βRII binding domain comprises a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 43, and the PD-1 binding domain comprises a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 9;

[0567] 7) a bispecific antibody (1 mg / kg) comprising a TGF-βRII binding domain and a PD-1 binding domain, wherein the TGF-βRII binding domain comprises a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 43, and the PD-1 binding domain comprises a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 19;

[0568] 8) a bispecific antibody (10 mg / kg) comprising a TGF-βRII binding domain and a PD-1 binding domain, wherein the TGF-βRII binding domain comprises a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 43, and the PD-1 binding domain comprises a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 19;

[0569] 9) a bispecific antibody (1 mg / kg) comprising a TGF-βRII binding domain and a PD-1 binding domain, wherein the TGF-βRII binding domain comprises a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 23, and the PD-1 binding domain comprises a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 14;

[0570] 10) a bispecific antibody (10 mg / kg) comprising a TGF-βRII binding domain and a PD-1 binding domain, wherein the TGF-βRII binding domain contains a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 23, and the PD-1 binding domain contains a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 14.

[0571] 11) a bispecific antibody (1 mg / kg) comprising a TGF-βRII binding domain and a PD-1 binding domain, wherein the TGF-βRII binding domain comprises a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 23, and the PD-1 binding domain comprises a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 19;

[0572] 12) a bispecific antibody (10 mg / kg) comprising a TGF-βRII binding domain and a PD-1 binding domain, wherein the TGF-βRII binding domain comprises a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 23, and the PD-1 binding domain comprises a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 19;

[0573] 13) a bispecific antibody (10 mg / kg) comprising a TGF-βRII binding domain and a PD-1 binding domain, wherein the TGF-βRII binding domain comprises a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 39, and the PD-1 binding domain comprises a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 9;

[0574] 14) a bispecific antibody (10 mg / kg) comprising a TGF-βRII binding domain and a PD-1 binding domain, wherein the TGF-βRII binding domain comprises a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 27, and the PD-1 binding domain comprises a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 9;

[0575] 15) A bispecific antibody (10 mg / kg) comprising a TGF-βRII binding domain and a PD-1 binding domain, wherein the TGF-βRII binding domain contains a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 23, and the PD-1 binding domain contains a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 18.

[0576] 16) A bispecific antibody (10 mg / kg) comprising a TGF-βRII binding domain and a PD-1 binding domain, wherein the TGF-βRII binding domain contains a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO:47, and the PD-1 binding domain contains a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO:13.

[0577] There were 8-9 mice per group. Animals were dosed intraperitoneally every five days for a period of 27 or 30 days. Tumors were measured using calipers and tumor volumes were calculated by assimilating them to ellipsoids using the following formula: l(length)×w 2 (width) x 1 / 2. Body weight was also monitored throughout the study. Tumors were harvested (24 hours after the last dose) for post-study tumor immune analysis and receptor occupancy.

[0578] Results are shown in Fig. 9 B to Fig. 9 All bispecific antibodies induced better antitumor responses than the combination of the reference PD-1 antibody pembrolizumab and the reference TGF-βRII antibody TGF1 analog. The bispecific antibodies induced good antitumor responses even at two dose levels of 1 mg / kg and 10 mg / kg, while the combination of reference antibodies included a dose of 10 mg / kg of each reference antibody.

[0579] In addition to efficacy, similar receptor coverage of both PD-1 and TGF-βRII receptors was observed with the bispecific antibody treatment, as was seen with the combination treatment of the reference TGF-βRII antibody TGF1 analog and the reference PD-1 antibody pembrolizumab on T cells analyzed 24 hours after the last dose ( Fig. 9 F).

[0580] Example 11 - Testing different doses in the in vivo humanized NSG MDA-MB-231 mouse model

[0581] As described in Example 10, a bispecific antibody comprising a TGF-βRII binding domain comprising a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 23 and a PD-1 binding domain comprising a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 18 was characterized in vivo at two different dose levels of 1 mg / kg and 10 mg / kg in the humanized NSG MDA-MB-231 mouse model. A negative control bivalent monospecific IgG1 antibody comprising a heavy chain having an amino acid sequence as shown in SEQ ID NO: 86 and a light chain having an amino acid sequence as shown in SEQ ID NO: 87 was included at 10 mg / kg.

[0582] The results are Fig.11 The bispecific antibody induced significant anti-tumor responses at both dose levels.

[0583] Sequence Listing:

[0584] SEQ ID NO: 1—Heavy chain variable region

[0585] EVQLVQSGAEVKKPGSSMKVSCKASGGTFSSYVISWVRQAPGQGLEWMGMIIPVFDTSSYEKK FQGRITIIADKSTSTVYLELSSLRSEDAAVYYCARGTVEATLLFDFWGQGTLVTVSS

[0586] SEQ ID NO:2—Heavy chain CDR1

[0587] SYVIS

[0588] SEQ ID NO:3—Heavy chain CDR2

[0589] MIIPVFDTSSYEKKFQG

[0590] SEQ ID NO:4—Heavy chain CDR3

[0591] GTVEATLLFDF

[0592] SEQ ID NO:5—Heavy chain variable region

[0593] QVQLQESGPGLVKPSETLSLTCTVSNGSLGFDFWSWIRQPPGRGLEWIGYIYYSGSWSLNPSFK GRVTMSVDTSKNQFSLNLRSVTAADTAVYYCARGGYTGYGGDWFDPWGQGTLVTVSS

[0594] SEQ ID NO:6 - Heavy chain CDR1

[0595] FDFWS

[0596] SEQ ID NO:7 - Heavy chain CDR2

[0597] YIYYSGSWSLNPSFKG

[0598] SEQ ID NO:8 - Heavy chain CDR3

[0599] GGYTGYGGDWFDP

[0600] SEQ ID NO:9 - Heavy chain variable region

[0601] QVQLQESGPGLVKPSETLSLTCTVSDGSIGYHFWSWIRQPPGRGLEWIGYIVYSGSYNVNPSLKTRVTMSVDTSKNQFSLNLRSVTAADTAVYYCARGGYTGYGGDWFDPWGQGTLVTVSS

[0602] SEQ ID NO:10 - Heavy chain CDR1

[0603] YHFWS

[0604] SEQ ID NO:11 - Heavy chain CDR2

[0605] YIVYSGSYNVNPSLKT

[0606] SEQ ID NO:12 - Heavy chain CDR3

[0607] GGYTGYGGDWFDP

[0608] SEQ ID NO:13 - Heavy chain variable region

[0609] QVQLQESGPGLVKPSETLSLTCTVSEGSIGYHFWSWIRQPPGRGLEWIGYIVYSGSYNVNPSLKTRVTMSVDTSKNQFSLNLRSVTAADTAVYYCARGGYTGYGGDWFDPWGQGTLVTVSS

[0610] SEQ ID NO: 14 - Heavy chain variable region

[0611] QVQLVQSGSELKKPGASVKVSCKASGYTFTRFALHWVRQAPGQGLEWMGWIDPNTGTPTFAQGVTGRFVFSLDTSVTTAYLQISSLKAEDTAVYYCARSLGYCDSDICYPNWIFDNWGQGTLVTVSS

[0612] SEQ ID NO: 15 - Heavy chain CDR1

[0613] RFALH

[0614] SEQ ID NO: 16 - Heavy chain CDR2

[0615] WIDPNTGTPTFAQGVTG

[0616] SEQ ID NO: 17 - Heavy chain CDR3

[0617] SLGYCDSDICYPNWIFDN

[0618] SEQ ID NO: 18 - Heavy chain variable region

[0619] QVQLVQSGSELKKPGASVKVSCKASGYTFTRFALHWVRQAPGQGLEWMGWIDPNTGTPTFAQGVTGRFVFSLDTSVTTAYLQISSLKAEDTAVYYCARSLGYCDSDICYPNWIFDNWGQGTLVTVSS

[0620] SEQ ID NO: 19 - Heavy chain variable region

[0621] QVQLVQSGSELKKPGASVKVSCKASGYTFTRFALSWVRQAPGQGLEWMGWIDPNTGTPTYAQDFTGRFVFSLDTSVTTAYLQISSLKAEDTAVYYCARSLGYCGSDICYPNGILDNWGQGTLVTVSS

[0622] SEQ ID NO: 20 - Heavy chain CDR1

[0623] RFALS

[0624] SEQ ID NO: 21 - Heavy chain CDR2

[0625] WIDPNTGTPTYAQDFTG

[0626] SEQ ID NO:22—Heavy chain CDR3

[0627] SLGYCGSDICYPNGILDN

[0628] SEQ ID NO:23—Heavy chain variable region

[0629] EVQLVESGGGLVQPGGSLRLSCAASGFTFDIYAMTWVRQAPGKGLEWVSVISGSGGTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARRGQYRDIVGATDYWGQGTLVTVSS

[0630] SEQ ID NO:24—Heavy chain CDR1

[0631] IYAMT

[0632] SEQ ID NO:25—Heavy chain CDR2

[0633] VISGSGGTTYYADSVKG

[0634] SEQ ID NO:26—Heavy chain CDR3

[0635] RGQYRDIVGATDY

[0636] SEQ ID NO:27—Heavy chain variable region

[0637] EVQLVESGGGLVQPGGSLRLSCAASGFTFDINAMTWVRQAPGKGLEWVSVISGSGGTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARRGQYRDIVGATDYWGQGTLVTVSS

[0638] SEQ ID NO:28—Heavy chain CDR1

[0639] INAMT

[0640] SEQ ID NO:29—Heavy chain CDR2

[0641] VISGSGGTTYYADSVKG

[0642] SEQ ID NO:30—Heavy chain CDR3

[0643] RGQYRDIVGATDY

[0644] SEQ ID NO:31 - Heavy chain variable region

[0645] QVQLVESGGGLVEPGGSLRLSCAASGFTFSNAWMSWVRQAPGKGLEWVGRIKTTISGGATDFAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTLDLRDYWGQGTLVTVSS

[0646] SEQ ID NO:32—Heavy chain CDR1

[0647] NAWMS

[0648] SEQ ID NO:33—Heavy chain CDR2

[0649] RIKTTISGGATDFAAPVKG

[0650] SEQ ID NO:34—Heavy chain CDR3

[0651] DLRDY

[0652] SEQ ID NO:35—Heavy chain variable region

[0653] QVQLVESGGGLVEPGGSLRLSCAASGFKFSNAWMSWVRQAPGKGLEWVGRIKTTISGGATQFAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTLDLRDYWGQGTLVTVSS

[0654] SEQ ID NO:36—Heavy chain CDR1

[0655] NAWMS

[0656] SEQ ID NO:37—Heavy chain CDR2

[0657] RIKTTISGGATQFAAPVKG

[0658] SEQ ID NO:38—Heavy chain CDR3

[0659] DLRDY

[0660] SEQ ID NO:39—Heavy chain variable region

[0661] QVQLVESGGGLVQPGGSLRLSCAVSGFTFRRYAMSWVRQAPGKGLEWVSAISASGDRTHNTDS VKGRFSISRDNSKNTLYLQMNSLRAEDTAVYFCAKGIAASGKNYFDPWGQGTLVTVSS

[0662] SEQ ID NO:40 - Heavy chain CDR1

[0663] RYAMS

[0664] SEQ ID NO:41 - Heavy chain CDR2

[0665] AISASGDRTHNTDSVKG

[0666] SEQ ID NO:42 - Heavy chain CDR3

[0667] GIAASGKNYFDP

[0668] SEQ ID NO:43 - Heavy chain variable region

[0669] QVQLVESGGGLVQPGGSLRLSCAVSGFTFSRYAMSWVRQAPGKGLEWVSAISASGDRTKNTDS VKGRFSISRDNSKNTLYLQMNSLRAEDTAVYFCAKGTAAAGKNYFDPWGQGTLVTVSS

[0670] SEQ ID NO:44 - Heavy chain CDR1

[0671] RYAMS

[0672] SEQ ID NO:45 - Heavy chain CDR2

[0673] AISASGDRTKNTDSVKG

[0674] SEQ ID NO:46 - Heavy chain CDR3

[0675] GTAAAGKNYFDP

[0676] SEQ ID NO:47 - Heavy chain variable region

[0677] QVQLVESGGGLVQPGGSLRLSCAVSGFTFSRYAMSWVRQAPGKGLEWVSAISASGDRTKYTDS VKGRFSISRDNSKNTLYLQMNSLRAEDTAVYFCAKGTAAAGKNYFDPWGQGTLVTVSS

[0678] SEQ ID NO:48—Light chain variable region

[0679] DIQMTQSPSSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGS GSGTDFTLTISSLQPEDFATYYCQQSYSTPPTFGQGTKVEIK

[0680] SEQ ID NO:49—Light chain CDR1 according to IMGT

[0681] QSISSY

[0682] SEQ ID NO:50—Light chain CDR2 according to IMGT

[0683] AAS

[0684] SEQ ID NO:51—Light chain CDR3 according to IMGT

[0685] QQSYSTPPT

[0686] SEQ ID NO:52—Light chain variable region

[0687] DIQMTQSPSSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGS GSGTDFTLTISSLQPEDFATYYCQQSYSTPPITFGQGTRLEIK

[0688] SEQ ID NO:53—Light chain CDR1 according to IMGT

[0689] QSISSY

[0690] SEQ ID NO:54—Light chain CDR2 according to IMGT

[0691] AAS

[0692] SEQ ID NO:55—Light chain CDR3 according to IMGT

[0693] QQSYSTPPIT

[0694] SEQ ID NO:56—Light chain variable region

[0695] EIVMTQSPATLSVSPGERATLSCRASQSVSSNLAWYQQKPGQAPRLLIYGASTRATGIPARFSGS GSGTEFTLTISSLQSEDFAVYYCQQYNNWPWTFGQGTKVEIK

[0696] SEQ ID NO:57—Light chain CDR1 according to IMGT

[0697] QSVSSN

[0698] SEQ ID NO:58—Light chain CDR2 according to IMGT

[0699] GAS

[0700] SEQ ID NO:59—Light chain CDR3 according to IMGT

[0701] QQYNNWPWT

[0702] SEQ ID NO:60—Light chain variable region

[0703] EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPWTFGQGTKVEIK

[0704] SEQ ID NO:61—Light chain CDR1 according to IMGT

[0705] QSVSSSY

[0706] SEQ ID NO:62—Light chain CDR2 according to IMGT

[0707] GAS

[0708] SEQ ID NO:63—Light chain CDR3 according to IMGT

[0709] QYGSSPWT

[0710] SEQ ID NO:64—Light chain variable region

[0711] SYVLTQPPSVSVAPGETARITCGGDNIGRKSVYWYQQKSGQAPVLVIYYDSDRPSGIPERFSGSN SGNTATLTISRVEAGDEADYYCQVWDGSSDHWVFGGGTKLTVL

[0712] SEQ ID NO:65—Light chain CDR1 according to IMGT

[0713] NIGRKS

[0714] SEQ ID NO:66—Light chain CDR2 according to IMGT

[0715] YDS

[0716] SEQ ID NO:67—Light chain CDR3 according to IMGT

[0717] QVWDGSSDHWV

[0718] SEQ ID NO:68—Hinge region

[0719] EPKSCDKTHTCPPCP

[0720] SEQ ID NO:69—CH1 region

[0721] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL SSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRV

[0722] SEQ ID NO:70—CH2 region

[0723] APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK

[0724] SEQ ID NO:71 - CH2-DM region

[0725] APELGRGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK

[0726] SEQ ID NO:72—CH3 region

[0727] GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0728] SEQ ID NO:73 – CH3 - DE region

[0729] GQPREPQVYTDPPSREEMTKNQVSLTCEVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0730] SEQ ID NO:74 – CH3 - KK region

[0731] GQPREPQVYTKPPSREEMTKNQVSLKCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0732] SEQ ID NO:75 – CL region

[0733] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKD STYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0734] SEQ ID NO:76 – Heavy chain reference anti - TGF - βRII antibody TGF1 analogue

[0735] QLQVQESGPGLVKPSETLSLTCTVSGGSISNSYFSWGWIRQPPGKGLEWIGSFYYGEKTYYNPSLKSRATISIDTSKSQFSLKLSSVTAADTAVYYCPRGPTMIRGVIDSWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0736] SEQ ID NO:77 - Light chain reference anti - TGF - βRII antibody TGF1 analogue

[0737] EIVLTQSPATLSLSPGERATLSCRASQSVRSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0738] SEQ ID NO:78 - Heavy chain reference PD - 1 antibody pembrolizumab

[0739] QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGINPSNGGTNFNEKFKNRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0740] SEQ ID NO:79 - Light chain reference PD - 1 antibody pembrolizumab

[0741] EIVLTQSPATLSLSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQAPRLLIYLASYLESGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0742] SEQ ID NO:80 - Heavy chain reference PD - L1 - TGF - βTRAP molecular analog

[0743] EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYIMMWVRQAPGKGLEWVSSIYPSGGITFYADTVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARIKLGTVTTVDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGAGGGGSGGGGSGGGGSGGGGSGIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD

[0744] SEQ ID NO:81 - Light chain reference PD - L1 - TGF - βTRAP molecular analog

[0745] QSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYDVSNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYTSSSTRVFGTGTKVTVLGQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS

[0746] SEQ ID NO:82 - Human TGF - βRII isotype A

[0747] MGRGLLRGLWPLHIVLWTRIASTIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDLLLVIFQVTGISLLPPLGVAISVIIIFYCYRVNRQQKLSSTWETGKTRKLMEFSEHCAIILEDDRSDISSTCANNINHNTELLPIELDTLVGKGRFAEVYKAKLKQNTSEQFETVAVKIFPYEEYASWKTEKDIFSDINLKHENILQFLTAEERKTELGKQYWLITAFHAKGNLQEYLTRHVISWEDLRKLGSSLARGIAHLHSDHTPCGRPKMPIVHRDLKSSNILVKNDLTCCLCDFGLSLRLDPTLSVDDLANSGQVGTARYMAPEVLESRMNLENVESFKQTDVYSMALVLWEMTSRCNAVGEVKDYEPPFGSKVREHPCVESMKDNVLRDRGRPEIPSFWLNHQGIQMVCETLTECWDHDPEARLTAQCVAERFSELEHLDRLSGRSCSEEKIPEDGSLNTTK

[0748] SEQ ID NO:83 - Extracellular domain of human TGF-βRII isoform A

[0749] TIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDLLLVIFQ

[0750] SEQ ID NO:84 - Human TGF-βRII isoform B

[0751] MGRGLLRGLWPLHIVLWTRIASTIPPHVQKSDVEMEAQKDEIICPSCNRTAHPLRHINNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDLLLVIFQVTGISLLPPLGVAISVIIIFYCYRVNRQQKLSSTWETGKTRKLMEFSEHCAIILEDDRSDISSTCANNINHNTELLPIELDTLVGKGRFAEVYKAKLKQNTSEQFETVAVKIFPYEEYASWKTEKDIFSDINLKHENILQFLTAEERKTELGKQYWLITAFHAKGNLQEYLTRHVISWEDLRKLGSSLARGIAHLHSDHTPCGRPKMPIVHRDLKSSNILVKNDLTCCLCDFGLSLRLDPTLSVDDLANSGQVGTARYMAPEVLESRMNLENVESFKQTDVYSMALVLWEMTSRCNAVGEVKDYEPPFGSKVREHPCVESMKDNVLRDRGRPEIPSFWLNHQGIQMVCETLTECWDHDPEARLTAQCVAERFSELEHLDRLSGRSCSEEKIPEDGSLNTTK

[0752] SEQ ID NO:85 - Extracellular domain of human TGF-βRII isotype B

[0753] TIPPHVQKSDVEMEAQKDEIICPSCNRTAHPLRHINNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDLLLVIFQ

[0754] SEQ ID NO:86 - Heavy chain negative control RSV IgG1 antibody

[0755] EVQLVESGGGVVQPGRSLRLSCAASGFTFSNYGMHWVRQAPGKGLEWVAVISYDGSTKYSADSLKGRFTISRDNSKNTLYLQMNSLRADDTAVYYCAKEGWSFDSSGYRSWFDSWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0756] SEQ ID NO:87 - Light chain

[0757] DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0758] SEQ ID NO:88 - Heavy chain variable region

[0759] QVQLVESGGGLVQPGGSLRLSCAVSGFTFSRYAMSWVRQAPGKGLEWVSAISASGDRTKNTDS VKGRFSISRDNSKNTLYLQMNSLRAEDTAVYYCAKGTAAAGKNYFDPWGQGTLVTVSS

[0760] SEQ ID NO:89 - Heavy chain variable region

[0761] QVQLVESGGGLVQPGGSLRLSCAVSGFTFSRYAMSWVRQAPGKGLEWVSAISASGDRTKYTDS VKGRFSISRDNSKNTLYLQMNSLRAEDTAVYYCAKGTAAAGKNYFDPWGQGTLVTVSS

[0762] SEQ ID NO:90 - Heavy chain CDR1

[0763] RYAMS

[0764] SEQ ID NO:91 - Heavy chain CDR2

[0765] AISASGDRTKYTDSVKG

[0766] SEQ ID NO:92 - Heavy chain CDR3

[0767] GTAAAGKNYFDP

[0768] SEQ ID NO:93 - V region VK1 - 39

[0769] DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTP

[0770] SEQ ID NO:94 - VK1 - 39 / JK1

[0771] DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGS GSGTDFTLTISSLQPEDFATYYCQQSYSTPPTFGQGTKVEIK

[0772] SEQ ID NO:95 - VK1 - 39 / JK5

[0773] DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGS GSGTDFTLTISSLQPEDFATYYCQQSYSTPPITFGQGTRLEIK

[0774] SEQ ID NO:96: - Heavy chain reference nivolumab - like antibody

[0775] QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKGLEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAEDTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0776] SEQ ID NO: 97 - Light chain reference nivolumab - like antibody

[0777] EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWPRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0778] SEQ ID NO: 98 - V region VK3 - 15

[0779] EIVMTQSPATLSVSPGERATLSCRASQSVSSNLAWYQQKPGQAPRLLIYGASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYNNWP

[0780] SEQ ID NO: 99 - VK3 - 15 / JK1

[0781] EIVMTQSPATLSVSPGERATLSCRASQSVSSNLAWYQQKPGQAPRLLIYGASTRATGIPARFSGS GSGTEFTLTISSLQSEDFAVYYCQQYNNWPWTFGQGTKVEIK

[0782] SEQ ID NO:100 - V region VK3 - 20

[0783] EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSP

[0784] SEQ ID NO:101 - VK3 - 20 / JK1

[0785] EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGS GSGTDFTLTISRLEPEDFAVYYCQQYGSSPWTFGQGTKVEIK

[0786] SEQ ID NO:102 - V region VL3 - 21

[0787] SYVLTQPPSVSVAPGETARITCGGDNIGRKSVYWYQQKSGQAPVLVIYYDSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDGSSDH

[0788] SEQ ID NO:103 - VL3 - 21 / JL3

[0789] SYVLTQPPSVSVAPGETARITCGGDNIGRKSVYWYQQKSGQAPVLVIYYDSDRPSGIPERFSGSN SGNTATLTISRVEAGDEADYYCQVWDGSSDHWVFGGGTKLTVL

[0790] SEQ ID NO:104 – TGF - βRII extracellular domain

[0791] IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAV WRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNT SNPD。

Claims

1. A multispecific binding moiety comprising a PD-1 binding domain and a TGF-βRII binding domain, characterized in that The PD-1 binding domain blocks PD-1 mediated signaling, and the TGF-βRII binding domain blocks TGF-βRII mediated signaling.

2. The multispecific binding moiety according to claim 1, characterized in that In activated T cells, particularly activated tumor-specific T cells, the PD-1 binding domain blocks PD-1-mediated signaling, and the TGF-βRII binding domain blocks TGF-βRII-mediated signaling.

3. The multispecific binding moiety according to claim 1 or 2, characterized in that The multispecific binding moiety comprises a single Fab domain that binds to PD-1, a single Fab domain that binds to TGF-βRII, and an Fc region.

4. The multispecific binding moiety according to any one of claims 1 to 3, characterized in that The multispecific binding moieties block TGF-βRII-mediated signaling with greater potency in cells expressing both PD-1 and TGF-βRII than in cells expressing TGF-βRII and not expressing, not expressing substantially, or expressing low levels of PD-1.

5. The multispecific binding moiety of claim 4, wherein: The cells expressing both PD-1 and TGF-βRII are Jurkat-PD-1 + Cells, and the cells expressing TGF-βRII and not expressing or substantially not expressing PD-1 are Jurkat-PD-1 null cell.

6. The multispecific binding moiety of claim 4, wherein: The cells expressing both PD-1 and TGF-βRII are activated CD4 + and / or CD8 + cells, and the cells expressing TGF-βRII and not expressing PD-1 are unactivated CD4 + and / or CD8 + cell.

7. The multispecific binding moiety of claim 4, wherein: The cells expressing both PD-1 and TGF-βRII are HEK-Blue TGF-β-PD-1 + Cells, and the cells expressing TGF-βRII and not expressing PD-1 are HEK-Blue TGF-β cells.

8. The multispecific binding moiety according to any one of claims 4 to 6, characterized in that The efficacy of blocking TGF-βRII-mediated signaling was measured in a phosphorylated SMAD2 / 3 assay.

9. The multispecific binding moiety of claim 4 or 7, wherein: The efficacy of blocking TGF-βRII-mediated signaling was measured in a syngeneic PD-1-TGF-β reporter assay.

10. The multispecific binding moiety according to any one of claims 4 to 9, characterized in that The potency of blocking TGF-βRII-mediated signaling is at least about 200-fold higher in cells expressing both PD-1 and TGF-βRII than in cells expressing TGF-βRII and not expressing, not expressing substantially, or expressing low levels of PD-1; preferably, between about 200-fold and 30,000-fold higher.

11. The multispecific binding moiety according to any one of claims 4 to 10, characterized in that The potency of the multispecific binding portion in blocking TGF-βRII-mediated signaling in cells that express TGF-βRII and do not express PD-1 is lower than the potency of a reference anti-TGF-βRII antibody, and the potency of the multispecific binding portion in blocking TGF-βRII-mediated signaling in cells that express both TGF-βRII and PD-1 is higher than the potency of the reference anti-TGF-βRII antibody, wherein the reference anti-TGF-βRII antibody is a bivalent monospecific antibody comprising a heavy chain having the amino acid sequence as shown in SEQ ID NO:76 and a light chain having the amino acid sequence as shown in SEQ ID NO:

77.

12. The multispecific binding moiety of claim 11, wherein: The potency of the multispecific binding moiety in blocking TGF-βRII-mediated signaling in cells expressing both TGF-βRII and PD-1 is at least about 100-fold greater than the potency of the reference anti-TGF-βRII antibody; preferably, between about 100-fold and 20,000-fold greater.

13. The multispecific binding moiety according to any one of claims 1 to 12, characterized in that The multispecific binding portion has higher activity in reducing tumor volume than a combination of reference antibodies, wherein the combination of reference antibodies is two bivalent monospecific antibodies targeting PD-1 and TGF-βRII, wherein the bivalent monospecific antibody targeting PD-1 comprises a heavy chain having the amino acid sequence as shown in SEQ ID NO:78 and a light chain having the amino acid sequence as shown in SEQ ID NO:79, and the bivalent monospecific antibody targeting TGF-βRII comprises a heavy chain having the amino acid sequence as shown in SEQ ID NO:76 and a light chain having the amino acid sequence as shown in SEQ ID NO:

77.

14. The multispecific binding moiety of claim 13, wherein: Said activity in reducing tumor volume is determined by measuring the reduction of tumor volume in in vivo studies in mice, particularly in in vivo studies in mice using MDA-MB-231 xenograft huCD34 NSG mice.

15. The multispecific antibody according to claim 13 or 14, characterized in that: A higher activity in reducing tumor volume is a reduction in tumor volume of at least about 1.5 times the reduction in tumor volume of the combination of reference antibodies; preferably, between about 1.5-fold and 100-fold.

16. A multispecific binding moiety comprising a PD-1 binding domain and a TGF-βRII binding domain, characterized in that The PD-1 binding domain comprises a heavy chain variable region, and the heavy chain variable region comprises: a) heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2) and heavy chain CDR3 (HCDR3), which have 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), which have 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), which have 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), which have 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), which have the amino acid sequences shown in SEQ ID NO: 20, SEQ ID NO: 21 and SEQ ID NO: 22, respectively; Wherein, each of the HCDRs can contain up to three, two or one amino acid variation.

17. The multispecific binding moiety of claim 16, wherein: The PD-1 binding domain comprises a heavy chain variable region having an amino acid sequence as shown in any one of SEQ ID NOs: 1, 5, 9, 13, 14, 18 and 19, or an amino acid sequence having at least 80%, preferably 85%, more preferably 90%, or most preferably 95% sequence identity thereto.

18. The multispecific binding moiety of claim 16 or 17, wherein: The PD-1 binding domain comprises a light chain variable region, which comprises a light chain CDR1 (LCDR1), a light chain CDR2 (LCDR2) and a light chain CDR3 (LCDR3) having an amino acid sequence as shown in SEQ ID NO:49, SEQ ID NO:50 and SEQ ID NO:51 or a variant thereof, respectively.

19. The multispecific binding moiety according to any one of claims 16 to 18, characterized in that The PD-1 binding domain comprises a light chain variable region having an amino acid sequence as shown in SEQ ID NO: 48 or an amino acid sequence having at least 80%, preferably 85%, more preferably 90%, or most preferably 95% sequence identity thereto.

20. The multispecific binding moiety of claim 16 or 17, wherein: The TGF-βRII binding domain comprises a heavy chain variable region, wherein the heavy chain variable region comprises: a) heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2) and heavy chain CDR3 (HCDR3), which have 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), which have 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), which have 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), which have 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), which have 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), which have 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), which have the amino acid sequences shown in SEQ ID NO:90, SEQ ID NO:91 and SEQ ID NO:92, respectively, Wherein, each of the HCDRs can contain up to three, two or one amino acid variation.

21. The multispecific binding moiety according to any one of claims 16 to 20, characterized in that The TGF-βRII binding domain comprises a heavy chain variable region having an amino acid sequence as shown in any one of SEQ ID NOs: 23, 27, 31, 35, 39, 43, 47, 88 and 89, or an amino acid sequence having at least 80%, preferably 85%, more preferably 90%, or most preferably 95% sequence identity thereto.

22. The multispecific binding moiety according to any one of claims 16 to 21, characterized in that The TGF-βRII binding domain comprises a light chain variable region, which comprises a light chain CDR1 (LCDR1), a light chain CDR2 (LCDR2) and a light chain CDR3 (LCDR3) having the amino acid sequences shown in SEQ ID NO:49, SEQ ID NO:50 and SEQ ID NO:51 or variants thereof, respectively.

23. The multispecific binding moiety according to any one of claims 16 to 22, characterized in that The TGF-βRII binding domain comprises a light chain variable region having an amino acid sequence as shown in SEQ ID NO: 48 or an amino acid sequence having at least 80%, preferably 85%, more preferably 90%, or most preferably 95% sequence identity thereto.

24. A multispecific binding moiety comprising a PD-1 binding domain and a TGF-βRII binding domain, characterized in that The TGF-βRII binding domain comprises a heavy chain variable region, wherein the heavy chain variable region comprises: a) heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2) and heavy chain CDR3 (HCDR3), which have 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), which have 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), which have 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), which have 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), which have 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), which have 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), which have the amino acid sequences shown in SEQ ID NO:90, SEQ ID NO:91 and SEQ ID NO:92, respectively, Wherein, each of the HCDRs can contain up to three, two or one amino acid variation.

25. The multispecific binding moiety of claim 24, wherein: The TGF-βRII binding domain comprises a heavy chain variable region having an amino acid sequence as shown in any one of SEQ ID NOs: 23, 27, 31, 35, 39, 43, 47, 88 and 89, or an amino acid sequence having at least 80%, preferably 85%, more preferably 90%, or most preferably 95% sequence identity thereto.

26. The multispecific binding moiety of claim 24 or 25, wherein: The TGF-βRII binding domain comprises a light chain variable region, which comprises a light chain CDR1 (LCDR1), a light chain CDR2 (LCDR2) and a light chain CDR3 (LCDR3) having the amino acid sequences shown in SEQ ID NO:49, SEQ ID NO:50 and SEQ ID NO:51 or variants thereof, respectively.

27. The multispecific binding moiety according to any one of claims 24 to 26, characterized in that The TGF-βRII binding domain comprises a light chain variable region having an amino acid sequence as shown in SEQ ID NO: 48 or an amino acid sequence having at least 80%, preferably 85%, more preferably 90%, or most preferably 95% sequence identity thereto.

28. The multispecific binding moiety according to any one of claims 24 to 27, characterized in that The PD-1 binding domain comprises a heavy chain variable region, and the heavy chain variable region comprises: a) heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2) and heavy chain CDR3 (HCDR3), which have 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), which have 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), which have 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), which have 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), which have the amino acid sequences shown in SEQ ID NO: 20, SEQ ID NO: 21 and SEQ ID NO: 22, respectively; Wherein, each of the HCDRs can contain up to three, two or one amino acid variation.

29. The multispecific binding moiety according to any one of claims 24 to 28, characterized in that The PD-1 binding domain comprises a heavy chain variable region having an amino acid sequence as shown in any one of SEQ ID NOs: 1, 5, 9, 13, 14, 18 and 19, or an amino acid sequence having at least 80%, preferably 85%, more preferably 90%, or most preferably 95% sequence identity thereto.

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

31. The multispecific binding moiety of any one of claims 24 to 30, characterized in that The PD-1 binding domain comprises a light chain variable region having an amino acid sequence as shown in SEQ ID NO: 48 or an amino acid sequence having at least 80%, preferably 85%, more preferably 90%, or most preferably 95% sequence identity thereto.

32. A multispecific binding moiety comprising a PD-1 binding domain and a TGF-βRII binding domain, which competes with the multispecific binding moiety of any one of claims 1 to 31 for binding to PD-1 and / or TGF-βRII.

33. A pharmaceutical composition comprising an effective amount of a multispecific binding moiety according to any one of claims 1 to 32, and a pharmaceutically acceptable carrier.

34. The multispecific binding moiety of any one of claims 1 to 32 or the pharmaceutical composition of claim 33 for use in therapy.

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

36. A method for treating a disease, the method comprising administering to a subject in need thereof an effective amount of a multispecific binding moiety according to any one of claims 1 to 32 or a pharmaceutical composition according to claim 33.

37. A method for treating a disease associated with a suppressed immune system, in particular cancer, comprising administering to a subject in need thereof an effective amount of a multispecific binding moiety according to any one of claims 1 to 32 or a pharmaceutical composition according to claim 33.

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

39. The cell according to claim 38, characterized in that The cell further comprises a nucleic acid sequence encoding a CH1 region and preferably encoding a hinge, a CH2 region and a CH3 region.

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

41. A cell producing a multispecific binding moiety according to any one of claims 1 to 32.

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