MULTISPECIFIC BINDING MOIETIES COMPRISING PD-1 AND TGF-BRII BINDING DOMAINS

REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

The contents of the electronic sequence listing (AttachC_SeqListingST26-2189A.xml; Size: 149 kilobytes; and Date of Creation: Nov. 16, 2022) is herein incorporated by reference in its entirety.

TECHNICAL FIELD

The present disclosure relates to the field of antibodies. In particular it relates to the field of therapeutic antibodies for the treatment of diseases involving aberrant cells. More particularly it relates to multispecific binding moieties comprising a binding domain that binds to PD-1 and a binding domain that binds to TGF-βRII.

BACKGROUND

Although T lymphocytes are known for their role in immunosurveillance of tumors, cancer cells are capable of escaping immune control by induction of inhibitory immune pathways. Consequently, immune checkpoint blockade (ICB) in which antibodies are used to block inhibitory immune pathways, has emerged as a promising therapeutic option and has been demonstrated in preclinical and clinical studies to enhance and sustain endogenous immunity against certain cancers.

Programmed death 1 (PD-1) and programmed death ligand 1 (PD-L1) are components of an immunosuppressive network that dampens T cell activity in normal physiology but can be exploited by tumors to suppress T cell-mediated antitumor immune responses. Antibodies directed against PD-1 and PD-L1 have effected improvements in responses and survival for some patients suffering from several different cancers. However, despite promising clinical activity, only a minority of patients respond to anti-PD-1/PD-L1 therapies, with limited durability. Thus, there is an urgent need to develop new, safe and effective therapies to treat cancer.

Programmed Cell Death 1 protein (PD-1) is a cell surface receptor that belongs to the CD28 family of receptors and is expressed on T cells and pro-B cells. PD-1 is presently known to bind two ligands, PD-L1 and PD-L2. PD-1, functioning as an immune checkpoint, plays an important role in downregulating the immune system by inhibiting the activation of T-cells, which in turn, when present on somatic cells, reduces autoimmunity and promotes self-tolerance. The inhibitory effect of PD-1 is thought to be accomplished through a dual mechanism of promoting apoptosis (programmed cell death) in antigen specific T-cells in lymph nodes while simultaneously reducing apoptosis in regulatory T cells (suppressor T cells). PD-1 is also known under a number of 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. External Ids for PD-1 are HGNC: 8760; Entrez Gene: 5133; Ensembl: ENSG00000188389; OMIM: 600244; and UniProtKB: Q15116. New classes of drugs that block the activity of PD-1, the PD-1 inhibitors, activate the immune system to attack tumors and are therefore used to treat some types of cancer.

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

TGF-β signaling regulates a plethora of physiological and pathological processes including cell cycle arrest in epithelial and hematopoietic cells, control of mesenchymal cell proliferation and differentiation, wound healing, extracellular matrix production, immunosuppression and carcinogenesis (Massagué J. TGF-β signalling in context. Nat Rev Mol Cell Biol. 2012 October; 13(10):616-30). In addition, TGF-β signaling regulates numerous cancer cell functions, including cell cycle progression, apoptosis, adhesion and differentiation (Liu S et al, Signal Transduction and targeted Therapy, 2021). TGF-β exhibits a biphasic function such that in normal and premalignant cells, it predominantly has been reported to act as a tumor suppressor, whereas in tumor cells it permits growth promoting functions and epithelial-to-mesenchymal transition, which in turn permits tumor cell migration, invasion, intravasation and extravasation.

TGF-βRII is a member of the serine/threonine protein kinase family and the TGFB receptor subfamily. It is known under various synonyms, including TGFBR2, AAT3, FAA3, LDS1B, LDS2, LDS2B, MFS2, RIIC, TAAD2, TGFR-2, TGFbeta-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, which then enter the nucleus and regulate the transcription of a subset of genes related to cell proliferation.

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, ligand-receptor level or intracellular level. The inhibitors include for instance TGF-β-neutralizing monoclonal antibodies, an anti-TGF-βRII antibody, soluble receptors, antibody ligand traps (e.g. anti-PD-L1-TGF-βRIIECD), antisense oligonucleotides to prevent TGF-β synthesis, a TGF-β2 antisense gene-modified allogeneic cancer cell vaccine, and small molecules targeting the kinase domain of TGF-βRI.

Targeting of the TGF-β pathway with a monospecific antibody is reported to show anti-tumor activity in vitro and in vivo; however, poor clinical outcomes persist with low efficacy and unacceptable toxicity, including major cytokine release syndrome (CRS). Combined targeting of the TGF-β pathway and immune checkpoint inhibition has been attempted with Bintrafusp alpha, an anti-PD-L1-TGFBRII bifunctional fusion protein, without demonstrating a strong clinical effect.

There remains a need for novel therapeutic interventions that selectively inhibit TGF-β signaling in activated tumor-specific PD-1-expressing T cells locally in the tumor microenvironment, in order to promote T cell tumor infiltration, and restore and sustain T cell antitumor effector activity. Such targeting will alleviate immunosuppressive pathways to potently promote CTL function and T cell memory for effective durable cancer elimination while minimizing toxicity associated with systemic TGF-β blockade.

SUMMARY

One of the objects of the present disclosure is to provide a new pharmaceutical agent for the treatment of human disease, in particular for the treatment of cancer. This object is met by the provision of multispecific binding moieties, for example bispecific antibodies, that bind PD-1 and TGF-βRII. The PD-1 binding domain of the multispecific binding moieties aims at driving the specificity of the multispecific binding moiety to activated/exhausted effector T cells in tumors and tumor-draining lymph nodes, where the TGF-βRII binding domain can locally block TGF-β from binding to TGF-βRII, so as to reduce systemic toxicity of TGF-β inhibition on non-T cells. Further, the multispecific binding moieties alleviate both the PD1- and TGF-β-mediated immunosuppressive pathways to promote cytotoxic T lymphocyte activity in the tumor microenvironment.

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.

The present disclosure 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 comprising CDR1, CDR2, and CDR3 sequences as described further herein.

The present disclosure also provides 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 CDR1, CDR2, and CDR3 sequences as described further herein.

The present disclosure further provides a pharmaceutical composition comprising an effective amount of the multispecific binding moiety as described herein.

The present disclosure further provides a multispecific binding moiety as described herein, and a pharmaceutical composition as described herein, for use in therapy.

The present disclosure further provides a multispecific binding moiety as described herein, and a pharmaceutical composition as described herein, for use in the treatment of cancer.

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

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

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

The present disclosure further provides a cell producing a multispecific binding moiety as described herein.

DETAILED DESCRIPTION

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.

As used herein, “blocks” or “blocking” means interfering or modifying the interaction between a ligand and a receptor, or causing a total or partial reduction of the signal transduction cascade. For the purposes of the present disclosure, in certain embodiments, potency in blocking ligand induced PD-1 signaling is determined by using a SHP recruitment assay as described in Example 2 or a NFAT reporter assay as described in Example 3. In certain embodiments, potency in blocking ligand induced PD-1 signaling is determined by using a SHP recruitment assay as described in Example 2. In certain embodiments, potency in blocking ligand induced PD-1 signaling is determined by using a NFAT reporter assay as described in Example 3. For the purposes of the present disclosure, in certain embodiments, potency in blocking ligand induced TGF-βRII signaling is determined by using a SMAD assay as described in Example 4 or 5. In certain embodiments, potency in blocking ligand induced TGF-βRII signaling is determined by using a SMAD assay as described in Example 4. In certain embodiments, potency in blocking ligand induced TGF-βRII signaling is determined by using a SMAD assay as described in Example 5.

In certain embodiments, a multispecific binding moiety of the present disclosure binds to human PD-1. In certain embodiments, the PD-1 binding domain of the multispecific binding moiety of the present disclosure blocks binding of PD-L1 to PD-1, for instance as measured in an assay as described in Example 2 or 3.

In certain embodiments, a multispecific binding moiety of the present disclosure binds to human TGF-βRII. Human TGF-βRII is a transmembrane protein of which there are 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-isoform B is a splice variant encoding a longer isoform due to an insertion in the extracellular domain. The amino acid sequence of human TGF-βRII isoform B is provided as SEQ ID NO: 84; the amino acid sequence of the extracellular domain of isoform B of human TGF-βRII is as set forth in SEQ ID NO: 85.

A “binding moiety” refers to a proteinaceous molecule and includes for instance all antibody formats available in the art, such as for example a full length IgG antibody, immunoconjugates, diabodies, BiTEs, Fab fragments, scFv, tandem scFv, single domain antibody (like VHH and VH), minibodies, scFab, scFv-zipper, nanobodies, DART molecules, TandAb, Fab-scFv, F(ab)′2, F(ab)′2-scFv2, and intrabodies.

In certain embodiments, the multispecific binding moiety is a multispecific antibody. A multispecific antibody according to the present disclosure is an antibody that comprises at least two binding domains which have specificity for at least two different targets or epitopes. In certain embodiments, a multispecific antibody of the present disclosure is a bispecific antibody. In certain embodiments, a multispecific antibody of the present disclosure may further comprise an Fc region or a part thereof. In certain embodiments, a multispecific binding moiety of the present disclosure is an IgG1 antibody. Constant regions of a binding moiety of the present disclosure may comprise one or more variations that modulate properties of the binding moiety other than its binding properties to the target antigens. For instance, the constant regions may comprise one or more variations that promote heterodimerization of the PD-1 and TGF-βRII heavy chains over homodimerization of two PD-1 heavy chains and/or two TGF-βRII heavy chains, and/or the constant regions may comprise one or more variations that reduce or improve effector function, in particular one or more variations that reduce effector function.

A “Fab” typically means a binding domain comprising a heavy chain variable region, a light chain variable region, a CH1 and a CL region.

In certain embodiments, a multispecific binding moiety of the present disclosure comprises a single Fab domain that binds to PD-1, a single Fab domain that binds to TGF-βRII, and an Fc region. In certain embodiments, a multispecific binding moiety of the present disclosure consists 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, a multispecific binding moiety of the present disclosure consists essentially of a single Fab domain that binds to PD-1, a single Fab domain that binds to TGF-βRII, and an Fc region.

An “Fc region” typically comprises a hinge, CH2, and CH3 region. A suitable hinge includes, but is not limited to, the hinge of which the amino acid sequence is set forth in SEQ ID NO: 68. Suitable CH2 and CH3 regions include, but are not limited to, the CH2 region of which the amino acid sequence is set forth in SEQ ID NO: 70 or 71, and the CH3 region of which the amino acid sequence is set forth in SEQ ID NO: 72, or 73 and 74.

A CL, CH1, CH2, and/or CH3 region may be modified according to methods known in the art in order to obtain favorable antibody characteristics, including for instance to promote heterodimerization of different heavy chains, to improve heavy-light chain pairing, and to enhance or reduce immune cell effector function.

In certain embodiments, 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 in activated T cells, in particular activated tumor-specific T cells.

In certain embodiments, a multispecific binding moiety of the present disclosure has a higher potency in blocking TGF-βRII-mediated signaling in cells expressing both PD-1 and TGF-βRII than in cells expressing TGF-βRII and no, substantially no, or low levels of, PD-1.

The present disclosure therefore also provides a multispecific binding moiety comprising a PD-1 binding domain and a TGF-βRII binding domain, wherein the multispecific binding moiety has a higher potency in blocking TGF-βRII-mediated signaling in cells expressing both PD-1 and TGF-βRII than in cells expressing TGF-βRII and no, substantially no, or low levels of, PD-1.

In certain embodiments, cells expressing both PD-1 and TGF-βRII are Jurkat-PD-1⁺ cells, such as for instance Jurkat T cells expressing human PD-1 and a luciferase reporter driven by an NFAT response element (NFAT-RE), and the cells expressing TGF-βRII and no PD-1 are Jurkat-PD-1^nullcells, such as for instance Jurkat-PD-1^nullcells. Jurkat T cells expressing human PD-1 and a luciferase reporter driven by an NFAT-RE are commercially available, for instance from Promega (cat. no. CS187105—part of kit CS187106 and CS187107); Jurkat-PD-1^nullcells are publicly available, for instance from the ATCC (cat. no. TIB-152).

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

In certain embodiments, cells expressing both PD-1 and TGF-βRII are HEK-Blue™ TGF-β-PD-1⁺ cells, such as for instance described in Example 7, and the cells expressing TGF-βRII and no PD-1 are HEK-Blue™ TGF-β cells, such as for instance HEK-Blue™ TGF-β cells. HEK-Blue™ TGF-β cells, commercially available from for instance Invivogen (cat. no. hkb-tgfb), are stably transfected human embryonic kidney HEK 293 cells comprising the human TGFBRI, Smad3, and Smad4 genes. They further express a Smad3/4-binding elements (SBE)-inducible SEAP reporter gene.

In certain embodiments no, substantially no, or a low level of PD-1 refers to a level of PD-1 on the cell surface that is undetectable in a suitable assay as set out herein. In certain embodiments, a low level of PD-1 refers to less than 100 PD-1 molecules present on the cell surface. In certain embodiments, the level of PD-1 is measured using quantibrite bead methodology.

For the purposes of the present disclosure, determining if a multispecific binding moiety has a higher potency in blocking TGF-βRII-mediated signaling in cells expressing both PD-1 and TGF-βRII than in cells expressing TGF-βRII and no, substantially no, or low levels of PD-1, is done by using one of the assays described herein.

The potency data of the multispecific binding moieties as provided herein is obtained with the phospho-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. Therefore, in certain embodiments, the potency in blocking TGF-βRII-mediated signaling is measured in a phospho-SMAD2/3 assay as described in Example 4 or Example 5.

In brief, the phospho-SMAD2/3 assay as described in Example 4 is performed using Jurkat-PD-1^nulland Jurkat-PD-1⁺ cells cultured in RPMI/10% FBS. Cells are incubated with test or control antibodies in 6-step serial dilutions (100 μg/ml to 0.001 μg/ml) for one hour at 37° C./5% CO₂. After one hour, human recombinant TGF-β1 is added at a final concentration of 10 ng/ml and the cells are incubated for two more hours at 37° C./5% CO₂. After incubation, cells are washed gently with PBS. Cell lysates are prepared using lysis buffer containing phosphatase inhibitors and protease inhibitors. PhosphoSMAD2/3 levels are determined using ELISA.

In brief, the phospho-SMAD2/3 assay as described in Example 5 is performed using PBMCs from healthy donors, which are stimulated with 1 μg/ml anti-CD3 for 48 hours followed by 16 hour serum deprivation in 0.1% FBS. Stimulated and unstimulated PBMCs are incubated with test and control antibodies for 30 minutes at room temperature. Recombinant human TGF-β1 is added at a final concentration of 1 ng/ml and the cells are incubated for another 30 minutes. Finally, cells are washed twice with PBS and stained for cell surface markers followed by intracellular phosphoSMAD2 staining. Flow cytometry is performed to gate CD4⁺ and CD8+ cells. Phospho-SMAD2 signal is measured in geo mean fluorescence intensity (GMFI) on these cells.

In certain embodiments, the potency in blocking TGF-βRII-mediated signaling is measured in an isogenic HEK-BLUE-PD-1 TGF-β reporter assay as described in Example 7.

In brief, the HEK-BLUE-PD-1 TGF-β reporter assay is performed using HEK-Blue™ TGF-β cells and HEK-Blue™ TGF-β-PD-1⁺ cells at 25000 cells per well. Serial dilutions of test and control antibodies are added and the cells are incubated for one hour at room temperature, followed by the addition of human recombinant TGF-β1 at a final concentration of 1 ng/ml. Cells are incubated at 37° C./5% CO₂overnight. After incubation, 40 ul of supernatants and 160 ul of re-suspended QUANTI-Blue™ Solution are incubated at 37° C./5% CO₂for 40 minutes. The quantity of SEAP secreted in the supernatant is assessed using QUANTI-Blue™ Solution. SEAP levels are determined using a spectrophotometer at 650 nm.

In certain embodiments, a multispecific binding moiety of the present disclosure has a potency in blocking TGF-βRII-mediated signaling in cells expressing both PD-1 and TGF-βRII of at least about 10 fold, preferably between about 10-100000 fold, higher than in cells expressing TGF-βRII and no PD-1. In certain embodiments, a multispecific binding moiety of the present disclosure has a potency in blocking TGF-βRII-mediated signaling in cells expressing both PD-1 and TGF-βRII of at least 10 fold, preferably between 10-100000 fold, higher than in cells expressing TGF-βRII and no PD-1. In certain embodiments, the potency in blocking TGF-βRII-mediated signaling is determined in IC50 (μg/ml) in a phospho-SMAD2/3 assay or HEK-BLUE-PD-1 TGF-β reporter assay. In certain embodiments, the potency in blocking TGF-βRII-mediated signaling is determined in IC50 (μg/ml) in a phospho-SMAD2/3 assay. In certain embodiments, the potency in blocking TGF-βRII-mediated signaling is determined in IC50 (μg/ml) in a HEK-BLUE-PD-1 TGF-β reporter assay.

In certain embodiments, the potency of a multispecific binding moiety of the present disclosure in blocking TGF-βRII-mediated signaling in cells expressing TGF-βRII and no, substantially no, or low levels of, PD-1 is lower than the potency of a reference anti-TGF-βRII antibody targeting the same cells, and the potency of the multispecific binding moiety in blocking TGF-βRII-mediated signaling in cells expressing both TGF-βRII and PD-1 is higher than the potency of the reference anti-TGF-βRII antibody targeting the same cells, wherein the reference anti-TGF-βRII antibody is a bivalent monospecific antibody comprising a heavy chain having an amino acid sequence as set forth in SEQ ID NO: 76 and a light chain having an amino acid sequence as set forth in SEQ ID NO: 77.

In certain embodiments, cells expressing both PD-1 and TGF-βRII are Jurkat-PD-1⁺ cells, stimulated CD4⁺ or CD8⁺ cells, or HEK-Blue™ TGF-β-PD-1⁺ cells; the cells expressing TGF-βRII and no, or substantially no, PD-1 are Jurkat-PD-1^nullcells or are HEK-Blue™ TGF-β cells; and the cells expressing TGF-βRII and low levels of PD-1 are unstimulated CD4⁺ or CD8⁺ cells, as described further herein.

In certain embodiments, the potency of a multispecific binding moiety of the present disclosure in blocking TGF-βRII-mediated signaling in cells expressing both TGF-βRII and PD-1 is at least 10 fold, preferably between 10-100000 fold, higher than the potency of a reference anti-TGF-βRII antibody. In certain embodiments, the potency of a multispecific binding moiety of the present disclosure in blocking TGF-βRII-mediated signaling in cells expressing both TGF-13RII and PD-1 is at least about 10 fold, preferably between about 10-100000 fold, higher than the potency of a reference anti-TGF-βRII antibody. In certain embodiments, the reference TGF-βRII antibody is a bivalent monospecific antibody comprising a heavy chain having an amino acid sequence as set forth in SEQ ID NO: 76 and a light chain having an amino acid sequence as set forth in SEQ ID NO: 77. In certain embodiments, the potency in blocking TGF-βRII-mediated signaling is determined in IC50 (μg/nil) in a phospho-SMAD2/3 assay.

In certain embodiments, a multispecific binding moiety of the present disclosure has a higher activity in reducing tumor volume than a combination of reference antibodies. In certain embodiments, the combination of reference antibodies are 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 set forth in SEQ ID NO: 78 and a light chain having an amino acid sequence as set forth in SEQ ID NO: 79 and the bivalent monospecific antibody targeting anti-TGF-βRII comprises a heavy chain having an amino acid sequence as set forth in SEQ ID NO: 76 and a light chain having an amino acid sequence as set forth in SEQ ID NO: 77.

The present disclosure therefore also provides a multispecific binding moiety comprising a PD-1 binding domain and a TGF-βRII binding domain, wherein the multispecific binding moiety has a higher activity in reducing tumor volume than a combination of reference antibodies. In certain embodiments, the multispecific binding moiety is dosed with a two-fold lower to up to twenty-fold lower number of PD-1 and TGF-βRII binding domains than each of the bivalent monospecific antibodies of the combination of reference antibodies. For example, a multispecific binding moiety, which is monovalent for binding to PD-1 and monovalent for binding to TGF-βRII, when dosed at 1 mg/kg 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 dosed at 10 mg/kg. Also, a multispecific binding moiety, which is monovalent for binding to PD-1 and monovalent for binding to TGF-βRII, when dosed at 10 mg/kg 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 dosed at 10 mg/kg.

In certain embodiments, the combination of reference antibodies are 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 set forth in SEQ ID NO: 78 and a light chain having an amino acid sequence as set forth in SEQ ID NO: 79, and wherein the bivalent monospecific antibody targeting TGF-βRII comprises a heavy chain having an amino acid sequence as set forth in SEQ ID NO: 76 and a light chain having an amino acid sequence as set forth in SEQ ID NO: 77.

In certain embodiments, the activity in reducing tumor volume is determined by measuring tumor volume reduction in an in vivo mouse study, in particular in an in vivo mouse study using MDA-MB-231 xenograft huCD34 NSG mice.

In certain embodiments, a multispecific binding moiety of the present disclosure has a tumor volume reduction that is at least 1.5 fold, preferably between 1.5-100 fold or 2-80 fold or 5-80 fold or 10-80 fold or 15-80 fold or 20-80 fold or 30-80 fold or 40-80 fold or 50-80 fold or 2-60 fold or 5-60 fold or 10-60 fold or 15-60 fold or 20-60 fold or 30-60 fold or 40-60 fold, of the tumor volume reduction of a combination of reference antibodies. In certain embodiments, a multispecific binding moiety of the present disclosure has a tumor volume reduction that is at least about 1.5 fold, preferably about between 1.5-100 fold or 2-80 fold or 5-80 fold or 10-80 fold or 15-80 fold or 20-80 fold or 30-80 fold or 40-80 fold or 50-80 fold or 2-60 fold or 5-60 fold or 10-60 fold or 15-60 fold or 20-60 fold or 30-60 fold or 40-60 fold, of the tumor volume reduction of a combination of reference antibodies. In other words, the multispecific binding moiety of the present disclosure provides a greater reduction in tumor volume than the combination of reference antibodies. In certain embodiments, the combination of reference antibodies are 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 set forth in SEQ ID NO: 78 and a light chain having an amino acid sequence as set forth in SEQ ID NO: 79, and the bivalent monospecific antibody targeting TGF-βRII comprises a heavy chain having an amino acid sequence as set forth in SEQ ID NO: 76 and a light chain having an amino acid sequence as set forth in SEQ ID NO: 77.

In certain embodiments, a multispecific binding moiety of the present disclosure reduces tumor volume when administered as a single agent.

In certain embodiments, the present disclosure provides several PD-1xTGF-βRII bispecific antibodies as exemplary multispecific binding moieties, the PD-1 binding domains of which comprise a heavy chain variable region having an amino acid sequence selected from SEQ ID NO: 1; 5; 9; 13; 14; 18 and 19, the TGF-βRII binding domains of which comprise a heavy chain variable region having an amino acid sequence selected from SEQ ID NO: 23; 27; 31; 35; 39; 43; 47; 88; and 89, and both the PD-1 and TGF-βRII binding domains comprising the same light chain.

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

a) heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), having an amino acid sequence as set forth 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), having an amino acid sequence as set forth 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), having an amino acid sequence as set forth in SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12, respectively;

d) heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), having an amino acid sequence as set forth in SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 17, respectively; or

e) heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), having an amino acid sequence as set forth in SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 22, respectively.

The heavy chain variable regions of the PD-1 binding domains of a multispecific binding moiety of the present disclosure may comprise a limited number, such as for instance 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.

In certain embodiments, the PD-1 binding domain of a multispecific binding moiety of the present disclosure also includes PD-1 binding domain variants thereof, wherein each of the HCDRs may comprise at most three, two, or one amino acid variations. In certain embodiments, only one or two HCDRs may comprise at most three, two, or one non-conservative amino acid variations. In certain embodiments, such variants do not comprise amino acid variations in HCDR3. In certain embodiments, the amino acid variation is a conservative amino acid substitution.

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

The substitution of an amino acid residue with another present in the same group would be preferred. Accordingly, conservative amino acid substitution can involve exchanging a member of one of these classes for another member of that same class. Typically, the variation results in no, or substantially no, loss in binding specificity of the binding domain to its intended target.

Additional types of amino acid variations include variations resulting from somatic hypermutation or affinity maturation. PD-1 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 segments as the heavy chain variable regions described by sequence herein, the variants having amino acid variations, including non-conservative and/or conservative amino acid substitutions in one, two, or all three HCDRs. Routine methods for affinity maturing antibody binding domains are widely known in the art, see for instance Tabasinezhad M, et al. (Trends in therapeutic antibody affinity maturation: From in-vitro towards next-generation sequencing approaches. Immunol Lett. 2019 August; 212:106-113).

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

In certain embodiments, the present disclosure thus also provides a multispecific binding moiety, the PD-1 binding domain of which comprising:

- HCDR1 having amino acid sequence X₁X₂FX₃S, wherein
  - X₁can be F, Y, T, or H;
  - X₂can be Y, Q, E, H, or D;
  - X₃can be W, or Y; and/or
- HCDR2 having amino acid sequence YIX₁YSGX₂X₃X₄X₅X₆PX₇X₈KX₉, wherein
  - X₁can be Y, V, or I;
  - X₂can be S, or G;
  - X₃can be T, Y, S, H, N, W, L, or Q;
  - X₄can be S, or N;
  - X₅can be F, V, or L;
  - X₆can be N, or S;
  - X₇can be S or A;
  - X₈can be F or L;
  - X₉can be S, T, G, D, R, or N; and/or
- HCDR3 having amino acid sequence GGYTGX₁GGDWFDX₂, wherein
  - X₁can be Y, H, V, or A;
  - X₂can be P, V, Y, W, F, T, Q, H, or S.

Other suitable positions for introducing an amino acid variation 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.

In certain embodiments, the present disclosure thus also provides a multispecific binding moiety, the PD-1 binding domain of which comprising:

- HCDR1 having amino acid sequence RX₁X₂X₃X₄, wherein
  - X₁can be F, or Y;
  - X₂can be T, A, or V;
  - X₃can be M, L, or V;
  - X₄can be S, H, N, V, or T; and/or
- HCDR2 having amino acid sequence WIX₁X₂X₃X₄GX₅X₆X₇X₈X₉X₁₀X₁₁X₁₂X₁₃X₁₄, wherein
  - X₁can be N, or D;
  - X₂can be P, S, or T;
  - X₃can be N, or Q;
  - X₄can be T, or D;
  - X₅can be N, S, T, K, L, or E;
  - X₆can be P, Y, A, H, or F;
  - X₇can be T, or S;
  - X₈can be Y, F, or H;
  - X₉can be A, G, V, or F;
  - X₁₀can be Q, R, N, L, T, or S;
  - X₁₁can be D, A, G, or S;
  - X₁₂can be F, V, or A;
  - X₁₃can be T, K, H, G;
  - X₁₄can be G, N, E, or D; and/or
- HCDR3 having amino acid sequence X₁X₂GYCX₃X₄DX₅CYPNX₆X₇X₈DX₉, wherein
  - X₁can be I, S, or V;
  - X₂can be L, Q, or N;
  - X₃can be N, G, S, or D;
  - X₄can be T, S, P, N, or E;
  - X₅can be N, or I;
  - X₆can be W, G, Q, H, W, A, or L;
  - X₇can be I, V, or L;
  - X₈can be F, L, or I;
  - X₉can be Y, S, N, I, R, H, V, T, K, A, or L.

In certain embodiments, a PD-1 binding domain of a 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 NO: 1; 5; 9; 13; 14; 18; 19, or a variant thereof. In certain embodiments, a PD-1 binding domain of a 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 NO: 1; 5; 9; 13; 14; 18; 19, or a variant having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity thereto.

“Percent (%) identity” as referring to nucleic acid or amino acid sequences herein is defined as the percentage of residues in a candidate sequence that are identical with the residues in a selected sequence, after aligning the sequences for optimal comparison purposes. In order to optimize the alignment between the two sequences gaps may be introduced in any of the two sequences that are compared. Such alignment can be carried out over the full length of the sequences being compared. Alternatively, the alignment may be carried out over a shorter length, for example over about 20, about 50, about 100 or more nucleic acids/based or amino acids. The sequence identity is the percentage of identical matches between the two sequences over the reported aligned region.

A comparison of sequences and determination of percentage of 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 are available to align two sequences and determine the identity between two sequences (Kruskal, J. B. (1983) An overview of sequence comparison In D. Sankoff and J. B. Kruskal, (ed.), 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 may be determined using the Needleman and Wunsch algorithm for the alignment of two sequences. (Needleman, S. B. and Wunsch, C. D. (1970) J. Mol. Biol. 48, 443-453). The Needleman-Wunsch algorithm has been implemented in the computer program NEEDLE. For the purpose of this invention the NEEDLE program from the EMBOSS package is used to determine percent identity of amino acid and nucleic acid sequences (version 2.8.0, EMBOSS: The European Molecular Biology Open Software Suite (2000) Rice, P. LongdenJ. and Bleasby, A. Trends in Genetics 16, (6) pp276-277, http://emboss.bioinformatics.n1/). For protein sequences, EBLOSUM62 is used for the substitution matrix. For DNA sequences, DNAFULL is used. The parameters used are a gap-open penalty of 10 and a gap extension penalty of 0.5.

After alignment by the program NEEDLE as described above the percentage of sequence identity between a query sequence and a sequence of the invention is calculated as follows: Number of corresponding positions in the alignment showing an identical amino acid or identical nucleotide in both sequences divided by the total length of the alignment after subtraction of the total number of gaps in the alignment.

In certain embodiments, a PD-1 binding domain of a multispecific binding moiety of the present disclosure also comprises PD-1 binding domain variants, which, in addition to the variations in the HCDRs referred to above, comprise one or more variations in the framework regions. A variation can be any type of amino acid variation described herein, such as for instance a conservative amino acid substitution or non-conservative amino acid substitution resulting from somatic hypermutation or affinity maturation. In certain embodiments, a PD-1 binding domain variant of a multispecific binding moiety of the present disclosure comprises no variations in the CDR regions but comprises one or more variations in the framework regions. Such variants have at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the sequences disclosed herein, and are expected to retain PD-1 binding specificity. Thus, in certain embodiments, a PD-1 binding domain of a multispecific binding moiety of the present disclosure comprises:

- a heavy chain variable region having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 1, which heavy chain variable region comprises a HCDR1 amino acid sequence as set forth in SEQ ID NO: 2; a HCDR2 amino acid sequence as set forth in SEQ ID NO: 3; and a HCDR3 amino acid sequence as set forth in SEQ ID NO: 4;
- a heavy chain variable region having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 5, which heavy chain variable region comprises a HCDR1 amino acid sequence as set forth in SEQ ID NO: 6; a HCDR2 amino acid sequence as set forth in SEQ ID NO: 7; and a HCDR3 amino acid sequence as set forth in SEQ ID NO: 8;
- a heavy chain variable region having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 9, which heavy chain variable region comprises a HCDR1 amino acid sequence as set forth in SEQ ID NO: 10; a HCDR2 amino acid sequence as set forth in SEQ ID NO: 11; and a HCDR3 amino acid sequence as set forth in SEQ ID NO: 12;
- a heavy chain variable region having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 13, which heavy chain variable region comprises a HCDR1 amino acid sequence as set forth in SEQ ID NO: 10; a HCDR2 amino acid sequence as set forth in SEQ ID NO: 11; and a HCDR3 amino acid sequence as set forth in SEQ ID NO: 12;
- a heavy chain variable region having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 14, which heavy chain variable region comprises a HCDR1 amino acid sequence as set forth in SEQ ID NO: 15; a HCDR2 amino acid sequence as set forth in SEQ ID NO: 16; and a HCDR3 amino acid sequence as set forth in SEQ ID NO: 17;
- a heavy chain variable region having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 18, which heavy chain variable region comprises a HCDR1 amino acid sequence as set forth in SEQ ID NO: 15; a HCDR2 amino acid sequence as set forth in SEQ ID NO: 16; and a HCDR3 amino acid sequence as set forth in SEQ ID NO: 17; or
- a heavy chain variable region having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 19, which heavy chain variable region comprises a HCDR1 amino acid sequence as set forth in SEQ ID NO: 20; a HCDR2 amino acid sequence as set forth in SEQ ID NO: 21; and a HCDR3 amino acid sequence as set forth in SEQ ID NO: 22.

The binding domains of a multispecific binding moiety of the present disclosure have been generated with a common light chain, in particular with a common light chain referred to as VK1-39/JK1. The binding domains of a multispecific binding moiety of the present disclosure can comprise any suitable light chain, including but not limited to common light chains known in the art. In certain embodiments, the binding domains of a multispecific binding moiety of the present disclosure comprise common light chain VK1-39/JK1, or a variant thereof harboring a limited number, such as for instance one, two, or three, non-conservative amino acid substitutions, or an unlimited number of conservative amino acid substitutions.

In certain embodiments, a PD-1 binding domain of a 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, a PD-1 binding domain of a 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 having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity thereto.

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

In certain embodiments, a PD-1 binding domain of a multispecific binding moiety of the present disclosure also includes PD-1 binding domain variants, which, in addition to the variations in the LCDRs referred to above, comprise one or more variations in the framework regions. A variation is preferably a conservative amino acid substitution. In certain embodiments, a PD-1 binding domain variant of a multispecific binding moiety of the present disclosure comprises no variations in the LCDR regions but comprises one or more variations in the framework regions. Such variants have at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the sequences disclosed herein. Thus, in certain embodiments, a PD-1 binding domain of a multispecific binding moiety of the present disclosure comprises:

- a light chain variable region having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 48, which light chain variable region comprises a LCDR1 amino acid sequence as set forth in SEQ ID NO: 49; a LCDR2 amino acid sequence as set forth in SEQ ID NO: 50; and a LCDR3 amino acid sequence as set forth in SEQ ID NO: 51.

A light chain or light chain variable region comprising these LCDRs and/or light chain variable region can be, for example, the light chain referred to in the art as VK1-39/JK1. This is a common light chain. The term ‘common light chain’ according to the present disclosure refers to a light chain that is capable of pairing with multiple different heavy chains, such as for instance heavy chains having different antigen or epitope binding specificities. A common light chain is particularly useful in the generation of, for instance, bispecific or multispecific antibodies, where antibody production is more efficient when all binding domains comprise the same light chain. The term “common light chain” encompasses light chains that are identical or have some amino acid sequence differences while the binding specificity of the full length antibody is not affected. It is for instance possible within the scope of the definition of common light chains as used herein, to prepare or find light chains that are not identical but still functionally equivalent, e.g., by using well established variations that introduce conservative amino acid changes, changes of amino acids in regions that are known to or are shown to not or only partly contribute to binding specificity when paired with the heavy chain, and the like.

Apart from a common light chain comprising the LCDRs and/or light chain variable region referred to above, other common light chains known in the art may be used. Examples of such common light chains include, but are not limited to: VK1-39/JK5, comprising a light chain variable region comprising a light chain CDR1 (LCDR1), light chain CDR2 (LCDR2), and light chain CDR3 (LCDR3), of a light chain variable region having an amino acid sequence as set forth in SEQ ID NO: 52. In certain embodiments, the light chain comprises a light chain variable region comprising a light chain CDR1 (LCDR1), light chain CDR2 (LCDR2), and light chain CDR3 (LCDR3), of a light chain variable region having an amino acid sequence as set forth in SEQ ID NO: 52, wherein each of the LCDRs may comprise at most three, two, or one amino acid variations, for example substitutions. In certain embodiments, the light chain comprises a light chain variable region having an amino acid sequence as set forth in SEQ ID NO: 52, or having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity thereto. In certain embodiments, the light chain comprises a light chain CDR1 (LCDR1), light chain CDR2 (LCDR2), and light chain CDR3 (LCDR3) having an amino acid sequence as set forth in SEQ ID NO: 53, SEQ ID NO: 54, and SEQ ID NO: 55; VK3-15/JK1, comprising a light chain variable region comprising a light chain CDR1 (LCDR1), light chain CDR2 (LCDR2), and light chain CDR3 (LCDR3), of a light chain variable region having an amino acid sequence as set forth in SEQ ID NO: 56. In certain embodiments, the light chain comprises a light chain variable region comprising a light chain CDR1 (LCDR1), light chain CDR2 (LCDR2), and light chain CDR3 (LCDR3), of a light chain variable region having an amino acid sequence as set forth in SEQ ID NO: 56, wherein each of the LCDRs may comprise at most three, two, or one amino acid variations, for example substitutions. In certain embodiments, the light chain comprises a light chain variable region having an amino acid sequence as set forth in SEQ ID NO: 56, or having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity thereto. In certain embodiments, the light chain comprises a light chain CDR1 (LCDR1), light chain CDR2 (LCDR2), and light chain CDR3 (LCDR3) having an amino acid sequence as set forth in SEQ ID NO: 57, SEQ ID NO: 58, and SEQ ID NO: 59; VK3-20/JK1, comprising a light chain variable region comprising a light chain CDR1 (LCDR1), light chain CDR2 (LCDR2), and light chain CDR3 (LCDR3), of a light chain variable region having an amino acid sequence as set forth in SEQ ID NO: 60. In certain embodiments, the light chain comprises a light chain variable region comprising a light chain CDR1 (LCDR1), light chain CDR2 (LCDR2), and light chain CDR3 (LCDR3), of a light chain variable region having an amino acid sequence as set forth in SEQ ID NO: 60, wherein each of the LCDRs may comprise at most three, two, or one amino acid variations, for example substitutions. In certain embodiments, the light chain comprises a light chain variable region having an amino acid sequence as set forth in SEQ ID NO: 60, or having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity thereto. In certain embodiments, the light chain comprises a light chain CDR1 (LCDR1), light chain CDR2 (LCDR2), and light chain CDR3 (LCDR3) having an amino acid sequence as set forth in SEQ ID NO: 61, SEQ ID NO: 62, and SEQ ID NO: 63; and VL3-21/JL3, comprising a light chain variable region comprising a light chain CDR1 (LCDR1), light chain CDR2 (LCDR2), and light chain CDR3 (LCDR3), of a light chain variable region having an amino acid sequence as set forth in SEQ ID NO: 64. In certain embodiments, the light chain comprises a light chain variable region comprising a light chain CDR1 (LCDR1), light chain CDR2 (LCDR2), and light chain CDR3 (LCDR3), of a light chain variable region having an amino acid sequence as set forth in SEQ ID NO: 64, wherein each of the LCDRs may comprise at most three, two, or one amino acid variations, for example substitutions. In certain embodiments, the light chain comprises a light chain variable region having an amino acid sequence as set forth in SEQ ID NO: 64, or having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity thereto. In certain embodiments, the light chain comprises a light chain CDR1 (LCDR1), light chain CDR2 (LCDR2), and light chain CDR3 (LCDR3) having an amino acid sequence as set forth in SEQ ID NO: 65, SEQ ID NO: 66, and SEQ ID NO: 67.

VK1-39 is short for Immunoglobulin Variable Kappa 1-39 Gene. The gene is also known as Immunoglobulin Kappa Variable 1-39; IGKV139; IGKV1-39; IgVκ1-39. External Ids for the gene are HGNC: 5740; Entrez Gene: 28930; Ensembl: ENSG00000242371. An amino acid sequence for VK1-39 is given as SEQ ID NO: 93. This is the sequence of the V-region. The V-region can be combined with one of five J-regions. Suitable VJ-region sequences are indicated as VK1-39/JK1 (SEQ ID NO: 94) and VK1-39/JK5 (SEQ ID NO: 95); alternative names are IgVκ1-39*01/IGJκ1*01 or IgVκ1-39*01/IGJκ5*01 (nomenclature according to the IMGT database worldwide web at imgt.org). These names are exemplary and encompass allelic variants of the gene segments.

VK3-15 is short for Immunoglobulin Variable Kappa 3-15 Gene. The gene is also known as Immunoglobulin Kappa Variable 3-15; IGKV315; IGKV3-15; IgVκ3-15. External Ids for the gene are HGNC: 5816; Entrez Gene: 28913; Ensembl: ENSG00000244437. An amino acid sequence for 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 five J-regions. A suitable VJ-region sequence is indicated as VK3-15/JK1 (SEQ ID NO: 99); alternative name is Vκ3-15*01/IGJκ1*01 (nomenclature according to the IMGT database worldwide web at imgt.org). This name is exemplary and encompasses allelic variants of the gene segments.

VK3-20 is short for Immunoglobulin Variable Kappa 3-20 Gene. The gene is also known as Immunoglobulin Kappa Variable 3-20; IGKV320; IGKV3-20; IgVκ3-20. External Ids for the gene are HGNC: 5817; Entrez Gene: 28912; Ensembl: ENSG00000239951. An amino acid sequence for VK3-20 is indicated as SEQ ID NO: 100. This is the sequence of the V-region. The V-region can be combined with one of five J-regions. A suitable VJ-region sequence is indicated as VK3-20/JK1 (SEQ ID NO: 101); alternative name is IgVκ3-20*01/IGJκ1*01 (nomenclature according to the IMGT database worldwide web at imgt.org). This name is exemplary and encompasses allelic variants of the gene segments.

VL3-21 is short for Immunoglobulin Variable Lambda 3-21 Gene. The gene is also known as Immunoglobulin Lambda Variable 3-21; IGLV321; IGLV3-21; IgVλ3-21. External Ids for the gene are HGNC: 5905; Entrez Gene: 28796; Ensembl: ENSG00000211662.2. An amino acid sequence for VL3-21 is given as SEQ ID NO: 102. This is the sequence of the V-region. The V-region can be combined with one of five J-regions. A suitable VJ-region sequence is indicated as VL3-21/JL3 (SEQ ID NO: 103); alternative name is IgVλ3-21/IGJλ3 (nomenclature according to the IMGT database worldwide web at imgt.org). This name is exemplary and encompasses allelic variants of the gene segments.

Further, 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 readily be obtained, such as from, for instance, 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.

In certain embodiments, a PD-1 binding domain of a multispecific binding moiety of the present disclosure may further comprise a CH1 and 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.

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

a) heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), having an amino acid sequence as set forth in SEQ ID NO: 24, SEQ ID NO: 25, and SEQ ID NO: 26, respectively;

b) heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), having an amino acid sequence as set forth in SEQ ID NO: 28, SEQ ID NO: 29, and SEQ ID NO: 30, respectively;

c) heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), having an amino acid sequence as set forth in SEQ ID NO: 32, SEQ ID NO: 33, and SEQ ID NO: 34, respectively;

d) heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), having an amino acid sequence as set forth in SEQ ID NO: 36, SEQ ID NO: 37, and SEQ ID NO: 38, respectively;

e) heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), having an amino acid sequence as set forth in SEQ ID NO: 40, SEQ ID NO: 41, and SEQ ID NO: 42, respectively;

f) heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), having an amino acid sequence as set forth in SEQ ID NO: 44, SEQ ID NO: 45, and SEQ ID NO: 46, respectively; or

g) heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), having an amino acid sequence as set forth in SEQ ID NO: 90, SEQ ID NO: 91, and SEQ ID NO: 92, respectively.

The heavy chain variable regions of the TGF-βRII binding domains of a multispecific binding moiety of the present disclosure may comprise a limited number, such as for instance one, two, or three, non-conservative amino acid substitutions, or an unlimited number of conservative amino acid substitutions.

In certain embodiments, the TGF-βRII binding domain of a multispecific binding moiety of the present disclosure also includes TGF-βRII binding domain variants thereof, wherein each of the HCDRs may comprise at most three, two, or one amino acid variations. In certain embodiments, only one or two HCDRs may comprise at most three, two, or one amino acid variations. In certain embodiments, such variants do not comprise amino acid variations in HCDR3. In certain embodiments, the amino acid variation is a conservative amino acid substitution. A conservative amino acid substitution is as described further herein.

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 segment as the heavy chain variable regions described by sequence herein, the variants having amino acid variations, including non-conservative and/or conservative amino acid substitutions in one, two, or all three HCDRs.

In certain embodiments, a TGF-βRII binding domain of a 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 NO: 23; 27; 31; 35; 39; 43; 47; 88; 89, or a variant thereof. In certain embodiments, a TGF-βRII binding domain of a 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 NO: 23; 27; 31; 35; 39; 43; 47; 88; 89, or having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity thereto.

In certain embodiments, a TGF-βRII binding domain of a multispecific binding moiety of the present disclosure also includes TGF-βRII binding domain variants, which, in addition to the variations in the HCDRs referred to above, comprise one or more variations in the framework regions. A variation can be any type of amino acid variation described herein, such as for instance a conservative amino acid substitution or non-conservative amino acid substitution resulting from somatic hypermutation or affinity maturation. In certain embodiments, a TGF-βRII binding domain variant of a multispecific binding moiety of the present disclosure comprises no variations in the CDR regions but comprises one or more variations in the framework regions. Such variants have at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the sequences disclosed herein, and are expected to retain TGF-βRII binding specificity. Thus, in certain embodiments, a TGF-βRII binding domain of a multispecific binding moiety of the present disclosure comprises:

- a heavy chain variable region having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 23, which heavy chain variable region comprises a HCDR1 amino acid sequence as set forth in SEQ ID NO: 24; a HCDR2 amino acid sequence as set forth in SEQ ID NO: 25; and a HCDR3 amino acid sequence as set forth in SEQ ID NO: 26;
- a heavy chain variable region having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 27, which heavy chain variable region comprises a HCDR1 amino acid sequence as set forth in SEQ ID NO: 28; a HCDR2 amino acid sequence as set forth in SEQ ID NO: 29; and a HCDR3 amino acid sequence as set forth in SEQ ID NO: 30;
- a heavy chain variable region having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 31, which heavy chain variable region comprises a HCDR1 amino acid sequence as set forth in SEQ ID NO: 32; a HCDR2 amino acid sequence as set forth in SEQ ID NO: 33; and a HCDR3 amino acid sequence as set forth in SEQ ID NO: 34;
- a heavy chain variable region having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 35, which heavy chain variable region comprises a HCDR1 amino acid sequence as set forth in SEQ ID NO: 36; a HCDR2 amino acid sequence as set forth in SEQ ID NO: 37; and a HCDR3 amino acid sequence as set forth in SEQ ID NO: 38;
- a heavy chain variable region having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 39, which heavy chain variable region comprises a HCDR1 amino acid sequence as set forth in SEQ ID NO: 40; a HCDR2 amino acid sequence as set forth in SEQ ID NO: 41; and a HCDR3 amino acid sequence as set forth in SEQ ID NO: 42;
- a heavy chain variable region having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 43, which heavy chain variable region comprises a HCDR1 amino acid sequence as set forth in SEQ ID NO: 44; a HCDR2 amino acid sequence as set forth in SEQ ID NO: 45; and a HCDR3 amino acid sequence as set forth in SEQ ID NO: 46;
- a heavy chain variable region having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 47, which heavy chain variable region comprises a HCDR1 amino acid sequence as set forth in SEQ ID NO: 90; a HCDR2 amino acid sequence as set forth in SEQ ID NO: 91; and a HCDR3 amino acid sequence as set forth in SEQ ID NO: 92;
- a heavy chain variable region having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 88, which heavy chain variable region comprises a HCDR1 amino acid sequence as set forth in SEQ ID NO: 44; a HCDR2 amino acid sequence as set forth in SEQ ID NO: 45; and a HCDR3 amino acid sequence as set forth in SEQ ID NO: 46; or
- a heavy chain variable region having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 89, which heavy chain variable region comprises a HCDR1 amino acid sequence as set forth in SEQ ID NO: 90; a HCDR2 amino acid sequence as set forth in SEQ ID NO: 91; and a HCDR3 amino acid sequence as set forth in SEQ ID NO: 92.

Any light chain variable region of a TGF-βRII antibody available in the art may be used, for example as described herein, as may any other light chain variable region that can readily be obtained, such as from, for instance, 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 the same or substantially the same light chain as the PD-1 binding domain.

In certain embodiments, the TGF-βRII binding domain of a 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 TGF-βRII binding domain of a 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 having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity thereto.

In certain embodiments, the TGF-βRII binding domain of a multispecific binding moiety of the present disclosure comprises a light chain variable region comprising light chain CDR1 (LCDR1), light chain CDR2 (LCDR2), and light chain CDR3 (LCDR3), having an amino acid sequence as set forth in SEQ ID NO: 49, SEQ ID NO: 50, and SEQ ID NO: 51, respectively. In certain embodiments, the light chain variable region of a TGF-βRII binding domain of a multispecific binding moiety of the present disclosure also includes variants thereof, wherein each of the LCDRs may comprise at most three, two, or one conservative or non-conservative amino acid variations. In certain embodiments, the amino acid variation is a conservative amino acid substitution.

In certain embodiments, a TGF-βRII binding domain of a multispecific binding moiety of the present disclosure may further comprise a CH1 and 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.

The present invention thus 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 CH1 and CL regions, as described herein. In certain embodiments, the multispecific binding moiety further comprises a TGF-βRII binding domain that comprises a heavy chain variable region, and optionally a light chain variable region and CH1 and CL regions, as described herein.

The present invention thus also provides 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, and optionally a light chain variable region and CH1 and CL regions, as described herein. In certain embodiments, the multispecific binding moiety further comprises a PD-1 binding domain that comprises a heavy chain variable region, and optionally a light chain variable region and CH1 and CL regions, as described herein.

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